Title

Magnetic resonance imaging investigation of normal and altered

brain functions and metabolisms

Advisor(s)

Wu, EX

Author(s)

Zhou, Yuwen;

Citation

Issued Date

URL

Rights

2012

http://hdl.handle.net/10722/173928

The author retains all proprietary rights, (such as patent rights)

and the right to use in future works.

Magnetic Resonance Imaging Investigation of Normal

and Altered Brain Functions and Metabolisms

by

Zhou Yuwen
B. Eng. (Southeast University)

A thesis submitted in partial fulfillment of the requirements

for the degree of Doctor of Philosophy
at the University of Hong Kong.
August 2012

Abstract of thesis entitled

Magnetic Resonance Imaging Investigation of Normal

and Altered Brain Functions and Metabolisms
submitted by

Zhou Yuwen
for the degree of Doctor of Philosophy
at The University of Hong Kong
in August 2012

Benefiting from higher SNR as well as better spatial, temporal and spectral
resolution, magnetic resonance imaging (MRI) at high field has proved to be a
valuable neuroimaging modality which provides comprehensive evaluation of the
central nervous system non-invasively. The objectives of this doctoral work were
to develop MRI methodologies and to assess the functional, metabolic and
structural alterations in rodent brains under normal and manipulated conditions.

Firstly, to improve the functional sensitivity and spatial precision, a novel

functional MRI (fMRI) method using balanced steady state free precession with
intravascular susceptibility contrast agent was proposed and its feasibility was
evaluated in rat visual system. This new approach was sensitized to cerebral blood

volume (CBV) changes. It provided comparable sensitivity to conventional CBVweighted fMRI using echo planar imaging but with no severe image distortion and
signal dropout. Robust negative responses during visual stimulation were
observed and activation patterns were in excellent agreement with known
neuroanatomy. As a promising alternative to conventional CBV-weighted fMRI,
it was particularly suited for fMRI investigation of animal models at high field.

Secondly, the relationship between anatomical connections and resting-state

fMRI connectivity was explored using a well-controlled animal model of corpus
callosotomy. Both complete and partial callosotomy resulted in significant loss of
interhemispheric

connectivity

in

the

cortical

areas

whose

primary

interhemispheric connections via corpus callosum (CC) were severed. Partial

restoration of interhemispheric connectivity and increased intrahemispheric
connectivity were also observed. The experimental findings directly supported
that anatomical connections via CC play a primary and indispensable role in
resting-state connectivity, and that resting-state networks could be dynamically
reorganized or acquired directly or indirectly through the remaining anatomical
connections.
Thirdly, proton magnetic resonance spectroscopy (1H MRS) was employed
to monitor the longitudinal metabolic alterations elicited by exogenous
stimulation and endogenous modification, respectively. Significantly lower
hippocampal N-acetylaspartate (NAA) was observed in fear conditioning animals,
indicating reduced neuronal dysfunction and/or integrity, which contributed to the
trauma-related symptoms. Meanwhile, pregnant animals exhibited prominently
higher hippocampal NAA level, reflecting the increased density of neurons in this

region, which might facilitate supporting behaviors that involving learning and
memory. The 1H MRS detection of ongoing neurochemical changes induced by
fear conditioning and pregnancy, especially in the hippocampus, can shed light on
the mechanisms of learning and memory and the neurochemical underpinnings of
behavioral improvement in pregnant animals.

Lastly, manganese-enhanced MRI (MEMRI) was employed to investigate

the hypoxic-ischemic (HI) injury in the late phase and the neural response to
conditioned fear. Significantly higher enhancement in T1-weighted images was
found in the peri-lesional region 24 hours after manganese administration and it
colocalized with the increase in glial cell density in histological staining,
demonstrating the existence of reactive gliosis in the late phase after HI injury. In
fear conditioned animals, higher manganese uptake was observed in amygdala,
hippocampus, paraventricular nucleus of hypothalamus and cingulate cortex,
which were all highly-involved in the process of fear. These findings suggested
MEMRI approach were useful in investigation of post-injury cellular events and
functional reorganization as well as for in vivo mapping of neuronal activity.

(500 words)

DECLARATION
I declare that the thesis represents my own work, except where due
acknowledgement is made, and that is it has not been previously included in a
thesis, dissertation or report submitted to this University or to any other institution
for a degree, diploma or other qualifications.

Signed

Zhou Yuwen

ACKNOWLEDGEMENTS

It is an honor to express my sincere thanks to all of those who supported and

helped me in any way during my doctoral study. First and foremost, I owe my
deepest gratitude to my supervisor, Prof. Ed Wu, for his guidance, encouragement,
and support throughout the years. I truly admire him for being an inspiring mentor
and an insightful scientist with enthusiasm. He generously shares his immense
knowledge, skills and experiences, which have benefited me both professionally
and personally. I am also grateful for the time and availability on discussion and
consultation whenever I encounter problems. Talking with him always motivates
me, broadens my vision and refreshes my mind. It is my privilege to be his
student and my growth would not be so rewarding without him.

I am indebted to many of my colleagues from the Laboratory of Biomedical

Imaging and Signal Processing, who have supported and helped me over the years.
My gratitude goes to Dr. Kevin Chan, Dr. Matthew Cheung and Dr. Condon Lau
for the knowledgeable advice and inspiring discussions. Special thanks to Miss
Abby Ding and Miss Shujuan Fan for their technical contribution towards the
work that has been included in this thesis and for being supportive friends.
Deepest thanks to Dr. Kexia Cai, Dr. Kannie Chan, Dr. April Chow and Mr. Frank
Lee for their friendship, care and encouragement. Many thanks to Dr. Hua Guo,
Dr. Edward Hui, Dr. Jerry Cheung, Dr. Li Xiao, Dr. Zhongwei Qiao, Mr. Peng
Cao, Mr. Kyle Xing, Miss Darwin Gao, Miss Joe Cheng, Mr. Jevin Zhang, Mr.
II

Russell Chan, Mr. Patrick Gao, Mr. Leon Ho, Miss Anna Wang and Mr. Victor
Xie for their generous supports and assistance. I would also like to thank Prof.
Kwok-Fai So, Prof. Grainne McAlonan, Prof. Yong Hu, Dr. Yu-Xiang Liang and
Dr. Qi Li at The University of Hong Kong for their collaboration and helpful
discussions.

It is a pleasure to acknowledge The University of Hong Kong for offering

me a postgraduate studentship throughout my study (2008-2012). I am also
grateful to the conference grants that enabled me to participate in several
international conferences, especially the International Society for Magnetic
Resonance in Medicine Annual Meetings (2010-2012), which widened my
horizon and inspired my work.

I would also like to thank my fianc, Alvin Yang and my dear friends for
their company and tremendous supports throughout my 4-year Ph.D. study.

Lastly, and most importantly, I would like to express my deepest gratitude

to my parents, Meizhen Chen and Renming Zhou. They bore me, raised me,
supported me, taught me, and loved me. To them I dedicate this thesis.

III

CONTENTS
DECLARATION ................................................................................................... I
ACKNOWLEDGEMENTS ................................................................................. II
CONTENTS ........................................................................................................ IV
LIST OF FIGURES ......................................................................................... VIII
LIST OF TABLES ........................................................................................... XIV
LIST OF ABBREVIATIONS AND SYMBOLS ........................................... XVI
CHAPTER 1 INTRODUCTION .......................................................................... 1
1.1 Magnetic Resonance Imaging ............................................................................ 1
1.1.1 General Overview .................................................................................. 1
1.1.2 Basic Principles of MRI ......................................................................... 2
1.1.3 Image Contrast in MRI ........................................................................... 3
1.1.4 MRI Techniques ...................................................................................... 4
1.2 Functional Magnetic Resonance Imaging.......................................................... 5
1.2.1 Blood oxygenation level dependent contrast ........................................... 5
1.2.2 Resting-state functional MRI ................................................................... 7
1.3 Magnetic Resonance Spectroscopy ................................................................... 8
1.4 Manganese-enhance Magnetic Resonance Imaging .......................................... 9
1.5 Thesis outline and contribution........................................................................ 10
CHAPTER 2 BALANCED STEADY STATE FREE PRECESSION FMRI
WITH INTRAVASCULAR SUSCEPTIBILITY CONTRAST AGENT ....... 16
2.1 Introduction...................................................................................................... 16

IV

2.2 Materials and Methods..................................................................................... 18

2.2.1 Animal Procedure .................................................................................. 18
2.2.2 MRI Protocols ........................................................................................ 20
2.2.3 Data Analysis ......................................................................................... 22
2.3 Results.............................................................................................................. 23
2.4 Discussion ........................................................................................................ 34
2.5 Conclusion ....................................................................................................... 39
CHAPTER
3
ALTERATIONS
IN
RESTING-STATE
FMRI
CONNECTIVITY AFTER COMPLETE AND PARTIAL CORPUS
CALLOSOTOMY ............................................................................................... 41
3.1 Introduction...................................................................................................... 41
3.2 Materials and Methods..................................................................................... 43
3.2.1 Animal Preparation ................................................................................ 43
3.2.2 MRI Protocols ........................................................................................ 45
3.2.3 Data Analysis ......................................................................................... 46
3.3 Results.............................................................................................................. 48
3.3.1 Effects of Complete and Partial Callosotomy on Interhemispheric
Connectivity ......................................................................................... 49
3.3.2 Interhemispheric Connectivity Changes Long after Corpus Callosotomy
.............................................................................................................. 56
3.3.3 Effects of Corpus Callosotomy on Intrahemispheric Connectivity ....... 59
3.4 Discussion ........................................................................................................ 59
3.4.1 Anatomical Connections vs. Dynamical Organization in rsfMRI ......... 60
3.4.2 The Role of Callosal Connections in Interhemispheric Connectivity ... 62
3.4.3 Post-callosotomy Plasticity of Interhemispheric and Intrahemispheric
Connectivity ......................................................................................... 64
3.4.4 Limitations and Future Work................................................................. 65
V

3.5 Conclusion ....................................................................................................... 67

CHAPTER 4 PROTON MAGNETIC RESONANCE SPECTROSCOPY OF
ALTERED NEUROCHEMICAL PROFILES ................................................. 69
4.1 Proton MRS Reveals N-acetylaspartate Reduction in Hippocampus and
Cingulate Cortex after Fear Conditioning ............................................................. 69
4.1.1 Introduction............................................................................................ 69
4.1.2 Materials and Methods .......................................................................... 71
4.1.3 Results.................................................................................................... 75
4.1.4 Discussion .............................................................................................. 79
4.1.5 Conclusion ............................................................................................. 84
4.2 Proton MRS Reveals Regional Metabolic Changes in Rat Brain during
Pregnancy and Motherhood ................................................................................... 85
4.2.1 Introduction............................................................................................ 85
4.2.2 Materials and Methods .......................................................................... 87
4.2.4 Discussion and Conclusion .................................................................... 92
CHAPTER 5 MANGANESE ENHANCED MAGNETIC RESONANCE
IMAGING OF MORPHOLOGICAL AND FUNCTIONAL BRAIN
CHANGES............................................................................................................ 96
5.1 MEMRI Study of Neonatal Hypoxic-ischemic Injury in the Late Stage......... 96
5.1.1 Introduction............................................................................................ 96
5.1.2 Materials and Methods .......................................................................... 97
5.1.3 Results.................................................................................................. 100
5.1.4 Discussion and Conclusion .................................................................. 103
5.2 In Vivo Manganese-enhanced MRI of Conditioned Fear Response ............. 105
5.2.1 Introduction.......................................................................................... 105
5.2.2 Methods ............................................................................................... 106

VI

5.2.3 Results.................................................................................................. 108

5.2.4 Discussion and Conclusion .................................................................. 112
CHAPTER 6 GENERAL CONCLUSIONS AND FUTURE STUDIES ...... 115
REFERENCES .................................................................................................. 119
LIST OF PUBLICATIONS .............................................................................. 142

VII

LIST OF FIGURES
Figure 1.1 A schematic diagram showing the source of BOLD signal in detecting neural activity. 6

Figure 2.1 Typical images acquired with conventional T2-weighted spin-echo (a), conventional
T2*-weighted gradient-echo (b), bSSFP (c), GE-EPI (d) and SE-EPI (e) at the identical
slice location from a rat brain before and after intravenous injection of intravascular
susceptibility contrast agent MION (15 mg/kg). Note that all bSSFP, GE-EPI and SEEPI images were acquired with the same spatial resolution and matrix size (6464) and
reconstructed without any post-processing. ................................................................. 25

Figure 2.2 Post-MION bSSFP fMRI yielded good agreement between the activation patterns and
known neuroanatomy during unilateral 20-s block-design stimulations in a normal
adult SD rats (a). The activation map was computed by correlating the fMRI timecourse with the stimulation paradigm and overlaid on the post-MION bSSFP image.
Only the voxels with cross-correlation coefficient (cc) < -0.35 were color coded. For
each activation cluster, the time-courses from the single voxel with the strongest cc
value (as marked by the yellow crosses in the small inset) (b) and all activated voxels
within the ROI (as defined by the green lines in the small inset) (c) were plotted in
mean SD. These time-courses were computed by averaging across all blocks, trials
and animals studied (n = 9). The strongest activation was observed in the superficial
layers of contralateral SC. Note that bSSFP images were acquired in a 6464 matrix
and reconstructed to 128128 by zero padding. The shaded area indicates the
stimulation period. ....................................................................................................... 26

Figure 2.3 Typical activation patterns observed by post-MION bSSFP (a), GE-EPI (b) and SEEPI (c) fMRI methods from an animal. The imaging slice was located at Bregma 7.2mm as shown in the coronal T2W image (d). For each method, the time-course

VIII

depicts the raw (colored) and low-pass filtered (black) signal changes in the voxel with
the strongest cc value in the SC (indicated by the green squares in the activation maps).
..................................................................................................................................... 28

Figure 2.4 Average post-MION bSSFP (a), GE-EPI (b) and SE-EPI (c) time-courses in all
activated voxels (cc < -0.35) within the entire slice in the animals studied (n = 5). The
shaded areas indicate the stimulation periods. ............................................................. 31

Figure 2.5 Average post-MION bSSFP signal time-course from the entire brain during the 4-min
hypercapnia challenge using 5% CO2 inhalation in the animals studied (n = 3).
Measurement ROIs were shown in the small inset, and covered all cortical and
subcortical regions except those severely darkened by MION because of large blood
vessels. The shaded area identifies the period of hypercapnia challenge. .................... 32

Figure 2.6 Apparent tissue longitudinal relaxation rate R1 (=1/ T1) (a), transverse relaxation rate
R2 (=1/T2) (b) and bSSFP signal (c) as a function of MION concentration in different
brain regions of one animal. ......................................................................................... 33

Figure 3.1 Representative T2-weighted (T2W) images and fractional anisotropy (FA) maps from
the animals with complete (a), anterior partial (b) and posterior partial (c) corpus
callosotomy and sham surgery (d). The transected part of the corpus callosum (CC) is
indicated in red color in the sagittal planes (left panel) and by yellow arrows in the
T2W images. The blue lines indicate the corresponding locations of T2W and FA slices
in the right panel. Distance to Bregma for each slice is given at the bottom. .............. 49

Figure 3.2 Typical resting-state connectivity maps from individual animals with complete (a),
anterior partial (b) and posterior partial (c) callosotomy and sham surgery (d) at postsurgery day 7. Independent component analysis (ICA) was performed. Spatial ICA
maps of independent components were scaled to z-scores (z>1.5) and overlaid on the

IX

EPI images. The color bars display z-scores with a higher z-score (yellow)
representing a stronger correlation between the time course of that voxel and the mean
time course of this component. The CC is organized in a rostrocaudal topographical
manner with anterior fibers connecting frontal areas of the two hemispheres and
posterior fibers connecting caudal cortical structures. The components shown in this
figure correspond to five brain areas ranging from the anterior to posterior part of the
brain. They are caudate putamen (CPu), secondary somatosensory cortex (S2), primary
somatosensory cortex (S1), auditory cortex (AC) and visual cortex (VC), respectively.
..................................................................................................................................... 51

Figure 3.3 Localization of the seeds and corresponding regions of interest (ROIs) for seed-based
cross-correlation analysis (SBA). Five brain areas were selected based on the ICA
results. Seeds and ROIs were overlaid on EPI images with slices located from Bregma
1.2mm to -7.2mm (slice 1-9). Color code: CPu (red), S2 (yellow), S1 (green), AC
(purple) and VC (blue). ................................................................................................ 53

Figure 3.4 Typical histograms showing the distribution of numbers of voxels within the ROIs (as
illustrated in Figure 3.3) across all correlation coefficient (cc) values. The data was
from the SBA results of one sham animal at post-surgery day 7. For each brain area,
the results of the seeds in both left (in red) and right (in blue) hemispheres and the
ROIs ipsilateral (a) and contralateral (b) to the seeds are presented here. ................... 54

Figure 3.5 Scatter plots of the mean value of the cc distribution in S2 and AC ROIs ipsilateral (a)
and contralateral (b) to the seeds for all animals at post-surgery day 7. ...................... 54

Figure 3.6 Typical resting-state connectivity maps from individual animals with complete (a),
anterior partial (b) and posterior partial (c) callosotomy and sham surgery (d) at postsurgery day 28. Spatial ICA maps of independent components were scaled to z-scores

(z>1.5) and overlaid on the EPI images. The color bars display z-scores with a higher
z-score (yellow) representing a stronger correlation between the time course of that
voxel and the mean time course of this component. The ICA components shown in this
figure correspond to secondary somatosensory cortex and auditory cortex. ................ 57

Figure 4.1 Freezing responses measured during the initial 6 minutes (pre-shock, free exploring)
and the following 6 minutes (fear conditioning) of training session as well as
contextual and cued test sessions, respectively. One-way ANOVA followed by
Bonferroni multiple comparison post-test was performed with * P < 0.05, ** P < 0.01,
*** P < 0.001. Data were presented as mean standard deviation. ............................ 76

Figure 4.2 Typical localization of voxel of interest (VOI) in the hippocampus (a), cingulate cortex
(b) and thalamus (c) (solid-line boxes) on coronal and axial slices of T2-weighted
images for proton magnetic resonance spectroscopy measurements (L-left; R-right; Aanterior; P-posterior). The size of VOI in the left hippocampus, the cingulate cortex
and the left thalamus was 1.22.51.6 mm3, 1.21.52.5 mm3 and 222 mm3,
respectively. ................................................................................................................. 77

Figure 4.3 Representative raw spectra (black) along with QUEST fitting (red) of the VOIs in the
hippocampus, cingulate cortex and thalamus, respectively. The spectra were acquired
from the same mouse before fear conditioning. Residuals of QUEST quantitation are
shown in the top entry. Abbreviations: NAA, N-acetylaspartate; Glu, glutamate; Cr,
creatine; Cho, choline; Tau, taurine; m-Ins, myo-inositol. .......................................... 78

Figure 4.4 A schematic diagram summarizing the timeline of experimental procedures. 1H MRS
measurements were performed on pregnant primiparous rats (N=15) at 3 days before
mating (Baseline), gestational day 17 (G17), lactation day 7 (L7) and post-weaning
day 7 (PW7). Age-matched nulliparous female rats (N=9) which served as non-

XI

pregnant controls were examined at the same timepoints with the pregnant rats. ....... 88

Figure 4.5 Typical in vivo 1H MRS spectra of voxel of interest (VOI) in the hippocampus (a) and
thalamus (b) (solid-line boxes) on coronal slices of T2-weighted images for 1H MRS
measurement (L-left; R-right) from the same rat. ........................................................ 91

Figure 4.6 Comparisons of metabolite to Cr ratios between the pregnant and non-pregnant rats in
the hippocampus (left column) and thalamus (right column) at each timepoint. Mean
SD presented. Unpaired t-tests were performed with * P<0.05, ** P <0.01. .............. 92

Figure 5.1 A schematic diagram showing the timeline of experimental procedures. Animals in
Group 1 (N=6) were subjected to hypoxic-ischemic (HI) insult at postnatal day 7 while
animals from Group 2 (N=6) were served as controls. Manganese-enhanced MRI
(MEMRI) measurements were performed prior to Mn2+ administration and at 1, 7 days
after the injection. ........................................................................................................ 99

Figure 5.2 Representative T1W images of Group 1 (HI) and Group 2 (normal control) before and
after Mn2+ administration. The yellow lines indicate the manually defined regions of
interest that were used for comparisons of signal intensity........................................ 100

Figure 5.3 Percentage change maps (from pre-injection) at day 1 and day 7 computed from
coregistered images, directly illustrating the significant SI increase in peri-lesional
region at day 1 (white arrows) and relatively slow clearance in contra-lesional
thalamus (black arrow). ............................................................................................. 101

Figure 5.4 Mean SD illustrates SI changes in different regions after Mn2+ administration in
peri-lesional area: ipsi-lateral thalamus (top left), ipsi-lateral cingulate cortex (top right)
and contra-lateral thalamus (bottom). All the post-injection SI was normalized by the
pre-injection SI within the same animal, respectively, before calculating mean SD.

XII

T-tests between Group 1: HI (N=6) and Group 2: normal control (N=6), *P<0.05, ** P
<0.01 and ++ P=0.09. ................................................................................................ 102

Figure 5.5 GFAP staining, illustrating the hyper cellular density of astrocytes in the peri-lesional
region in thalamus (middle column) and cingulate cortex (right column) as compared
to the contra-lesional hemisphere (left column). ........................................................ 103

Figure 5.6 Schematic diagrams showing the timeline of experimental procedures (a), the fear
conditioning paradigm (b) and setup (c). ................................................................... 108

Figure 5.7 Freezing response and locomotor activity measured during the initial habituation
period and the following fear conditioning (FC) period. Significantly increased
freezing duration and decreased locomotor activity (expressed as total distance
traveled in cm) confirmed that the mice acquired associative learning with the electric
footshock. Two-tailed Students t-tests were performed between the habituation period
and fear conditioning period: * P<0.05 ** P <0.005. ................................................ 109

Figure 5.8 Averaged post-Mn T1W images from Group 1 (FC) and Group 2 (control) with the
ratio maps showing the percentage signal differences between them. Enhanced Mnuptake was observed in amygdala (yellow arrows), hippocampus (red arrows),
paraventricular nucleus of hypothalamus (green arrows) and cingulate cortex (purple
arrows) in FC animals. ............................................................................................... 110

Figure 5.9 T1W signal intensity changes (Mean SD) before and after Mn injection were
compared between the two groups using ROIs covering amygdala (Amyg),
hippocampus (Hip), paraventricular nucleus of hypothalamus (PVH), cingulate cortex
(Cg) and the entire brain. Two-tailed t-test was performed with * P < 0.05, ** P <0.01
................................................................................................................................... 111

XIII

LIST OF TABLES
Table 1.1 Principal metabolites observed in 1H MRS ...................................................................... 8

Table 2.1 Comparison of post-MION bSSFP, GE-EPI and SE-EPI fMRI methods as determined
from activated voxels (cc < -0.35) within the entire slice in all five animals studied. ... 30

Table 3.1 Mean value and standard deviation of the cc values within the ROIs in hemispheres
ipsilateral and contralateral to the seeds at post-surgery day 7. The results are presented
in mean standard deviation. Statistical comparisons between different groups were
performed using one-way ANOVA. and denote decrease and increase, respectively,
with the significance level indicated by , (P<0.05, P<0.01 compared to anterior
partial transection, respectively), , (P<0.05, P<0.01 compared to posterior partial
transection, respectively) and *, **, *** (P<0.05, P<0.01, P<0.005 compared to sham
control, respectively). .................................................................................................... 55

Table 3.2 Mean value and standard deviation of the cc values within the ROIs in hemispheres
ipsilateral and contralateral to the seeds at post-surgery day 28. The results are
presented in mean standard deviation. Statistical comparisons between different
groups was performed using one-way ANOVA. and denote decrease and increase,
respectively, with the significance level indicated by , (P<0.05, P<0.01 compared
to anterior partial transection, respectively), , , (P<0.05, P<0.01, P<0.005
compared to posterior partial transection, respectively) and *, **, *** (P<0.05, P<0.01,
P<0.005 compared to sham control, respectively). ........................................................ 58

Table 4.1 Metabolite to Cr ratios and corresponding Cramr-Rao lower bounds (CRLBs) at 1 day
before, 1 day and 1 week after the fear conditioning experiment in the hippocampus,
cingulate cortex and thalamus, respectively. Data from all animals studied (N = 12)

XIV

were presented as mean standard deviation (SD). ...................................................... 79

XV

LIST OF ABBREVIATIONS AND SYMBOLS

1

H MRS

proton magnetic resonance spectroscopy

2D

two-dimensional

3D

three-dimensional

AC

auditory cortex

ANOVA

analysis of variance

BOLD

blood oxygenation level dependent

bSSFP

balanced steady state free precession

CBV

cerebral blood volume

cc

cross-correlation coefficient

CC

corpus callosum

Cho

choline

CNR

contrast to noise ratio

CO2

carbon dioxide

CPu

caudate putamen

Cr

creatine

CRLB

Cramr-Rao lower bound

CS

conditioned stimulus

DTI

diffusion tensor imaging

EPI

echo planner imaging

FA

fractional anisotropy

XVI

FLASH

fast low angle shot

fMRI

functional magnetic resonance imaging

FOV

field of view

FWHM

full width at half maximum

GE

gradient echo

Glu

glutamate

ICA

independent component analysis

Lac

lactate

LFF

low-frequency fluctuation

MEMRI

manganese-enhanced magnetic resonance imaging

m-Ins

myo-inositol

MION

monocrystalline iron oxide nanoparticle

MRI

magnetic resonance imaging

NAA

N-acetylaspartate

NEX

number of excitations

NMR

nuclear magnetic resonance

PRESS

point-resolved spectroscopy

PTSD

post-traumatic stress disorder

R1

longitudinal relaxation rate

R2

transverse relaxation rate

R2*

effective transverse relaxation rate

RARE

rapid acquisition with relaxation enhancement

XVII

RF

radiofrequency

ROI

region of interest

rsfMRI

resting-state functional MRI

RSN

resting-state network

S1

primary somatosensory cortex

S2

secondary somatosensory cortex

SBA

seed-based analysis

SC

superior colliculus

SD

standard deviation

SE

spin echo

SEM

standard error of mean

SI

signal intensity

SNR

signal to noise ratio

SpO2

oxygen saturation

T1

longitudinal relaxation time

T1W

T1-weighted

T2

transverse relaxation time

T2W

T2-weighted

T2*

effective transverse relaxation time

T2*W

T2*-weighted

Tau

taurine

TE

echo time

XVIII

TR

repetition time

TrueFISP

true fast imaging with steady state precession

tSNR

temporal signal to noise ratio

US

unconditioned stimulus

USPIO

ultrasmall superparamagnetic iron oxide

VC

visual cortex

VOI

voxel of interest

flip angle

successive phase advance per repetition time

duration of diffusion gradient pulse

diffusion time

standard deviation

mean value

XIX

CHAPTER 1 INTRODUCTION
1.1 Magnetic Resonance Imaging
1.1.1 General Overview

Magnetic resonance imaging (MRI) is one of the most significant

developments in medical imaging in the twentieth century. Although the physical
phenomenon of nuclear magnetic resonance (NMR) has been known since the
early 1940s (1, 2), it has not been applied to the field of medical imaging until
1973 when Paul C. Lauterbur made the first NMR image by introducing gradients
in the magnetic field (3). In 1974 Peter Mansfield presented the mathematical
theory for rapid imaging and image reconstruction, which were found much
needed in clinical practice. Since then, though only developed within decades,
MRI has become one of the most clinically used diagnosis and assessment tools,
largely due to its non-invasive nature and its sensitivity to a broad range of tissue
properties. In addition to routine clinical diagnosis, MRI is widely used for in vivo
biomedical research. Due to its excellent capability of soft-tissue characterization,
MRI offers great promises in neurological imaging, especially in understanding
the brain, both its structure and its functions. Today, MRI has evolved into an
extremely versatile modality for evaluating many different parameters of
anatomical, physiological and metabolic interest.

In parallel to the rapid growth of MR techniques, there is a drive towards

high-field MRI. The principal advantage of MRI at high field is the increase in
signal to noise ratio, contrast to noise ratio and spectral resolution. Such increases
have been largely demonstrated experimentally, which lead to significant
improvement in both diagnostic imaging and biomedical research.

1.1.2 Basic Principles of MRI

The proton possesses a property called spin which can be considered as a

small magnetic field. Under normal circumstances these tiny magnets are
randomly distributed in space and thus the net magnetic vector is zero. When an
external magnetic field B0 is present, the spin nuclei align parallel or antiparallel
to the external field B0. The parallel orientation is the low energy state while
antiparallel orientation is the high-energy state. The number of spins in the low
energy state excesses the number in the high-energy state. A net magnetization is
produced by the difference between the two populations and thus the tissue placed
in the magnetic field becomes magnetized. Nuclei do not line up with the
magnetic field but wobble or precess around the direction of the external field.
The Larmor equation gives the relationship between the strength of a magnetic
field, B0, and the precessional frequency, 0, of an individual spin:

0 = B0
where is the gyromagnetic ratio.
The hydrogen nucleus contains one proton and possesses a significant

magnetic moment. Hydrogen is extremely abundant in biological tissues because

hydrogen nuclei exist in water and fat molecules. The tissue (hydrogen) will be
magnetized in a large external magnetic field, preparing it for the MR imaging
process.
When a radiofrequency (RF) energy pulse is oriented perpendicular to B0 at
the Larmor frequency, the individual spins begin to precess in phase, as will the
net magnetization vector. Magnetic resonance occurs when some of the spins in
the lower energy state absorb energy from the RF field and make a transition into
the higher energy state. This has the effect of tipping the net magnetization away
from B0 at the flip angle and toward the transverse plane. As the transverse
magnetization precesses through the receiver coil, a current or a signal is induced
in the coil. When the RF pulse is discontinued, the signal in the coil begins at a
given amplitude (determined by the amount of magnetization precessing in the
transverse plane and the precessional frequency) and fades away rapidly. This
initial signal is referred to as the free induction decay or FID. Spatial information
was resolved by applying slice selection, frequency encoding and phase encoding.
Fourier transform is then performed to convert the detected signals and
reconstruct the image in the spatial domain.

1.1.3 Image Contrast in MRI

In MRI, image contrast is determined by the imaging protocols and the tissue
properties, including proton density (PD), longitudinal relaxation time (T1) and
transverse relaxation time (T2). PD refers to the amount of protons that could

contribute to the MR signal within a voxel. T1 is defined as the time required for
the longitudinal magnetization to return to the equilibrium state after a RF pulse.
T2 reflects the time required for the transverse magnetization to decay to 1/e of its
original amplitude after disturbed by a RF pulse. However, in the presence of the
inhomogeneity in the external magnetic field, the time required for the transverse
magnetization to decay is named as effective transverse relaxation time (T2*),
which is usually smaller than T2.

The image contrast of MRI also depends on the selecting of pulse sequences
and imaging parameters. Variations in the value of TR (repetition time), TE (echo
time) and flip angle can affect the image contrast characteristics. For example, if
short values of TR and TE are used, images will exhibit T1 contrast while long
values of TR and TE result in a T2-dependent contrast. Middle TR values and
middle TE values are common for density weighted contrast.

One may also manipulates the MR image contrast by the use of exogenous
contrast agents, such as paramagnetic ions (e.g. Mn2+) and ferromagnetic particles
(e.g. iron oxide). In Chapter 2 and 5, these contrast agents have been used to
enhance the contrast and improve the sensitivity of detection.

1.1.4 MRI Techniques

Since its introduction, MRI has become one of the fastest expanding and
most active modality in the imaging field. There are a large number of MRI

techniques, each of which provides a particular type of information about the

subject tissues. According to the tissues properties they sensitized to, the MRI
techniques for brain imaging can be categorized into three main types, including
functional, metabolic and anatomical MRI. Functional MRI measures blood
oxygenation level dependent (BOLD) signal, cerebral blood flow and cerebral
blood volume, providing information of neuronal activity and tissue perfusion.
Metabolic MRI mainly refers to magnetic resonance spectroscopy which provides
information about chemical composition of the tissues and changes in chemical
composition. Anatomical MRI includes basic T1-, T2-, PD- and T2*-weighted
MRI scans as well as diffusion MRI, which measures the diffusion characteristics
of the water molecules in brain tissues and the connectivity of white matter axons
in the central nervous system. MRI can add valuable information by multiparametric and longitudinal assessments of the functional, metabolic and
structural changes in the central nervous system (CNS).

1.2 Functional Magnetic Resonance Imaging

1.2.1 Blood oxygenation level dependent contrast
Functional magnetic resonance imaging (fMRI) can be used to map the
neuronal activity by using blood oxygenation level dependent (BOLD) contrast
(4). The BOLD mechanism refers to the phenomenon that increases in neural
activity cause changes in the MR signal via T2* changes (Figure 1.1). Increased
neural activity leads to an increased demand for oxygen. The vascular system

overcompensates for this demand, resulting in the larger amount of

oxyhemoglobin relative to deoxyhemoglobin. Deoxyhemoglobin [dHb], as an
endogenous paramagnetic contrast, attenuates the MR signal. BOLD fMRI takes
advantage of the magnetic susceptibility difference of oxyhemoglobin and
deoxyhemoglobin

and

measures the

neuronal

responses

indirectly

via

hemodynamic changes. It has shown better correlation with synaptic and

postsynaptic activity than the spiking activity (5).

Figure 1.1 A schematic diagram showing the source of BOLD signal in detecting neural
activity.

Based on the rapid echo planar imaging (EPI) sequence, fMRI provides a
good compromise between spatial and temporal resolution. However, severe
image distortion and signal dropout at air/tissue interface due to the susceptibility
and field inhomogeneities deteriorates the quality of EPI images (6). The other
major limiting factor in EPI fMRI is the constraint on achievable spatial resolution.
At high field, EPI spatial resolution is limited intrinsically by T2* that can be short
and vary across image due to field inhomogeneity (7, 8). Furthermore, several

physiological factors can also adversely affect EPI-based fMRI, including motion
artifacts and noises from cardiac and respiratory pulsations (9). These limitations
must be addressed to achieve accurate and high resolution mapping of brain
activities.

1.2.2 Resting-state functional MRI

Since its introduction of BOLD contrast (10), BOLD fMRI has been widely
applied to study the functions and connectivity of the CNS due to its
noninvasiveness, large field-of-view and 3D imaging capabilities. The majority of
fMRI studies have focused on studying neuronal activities associated with stimuli
or tasks. It is not until recently that investigating the resting brain by fMRI
became of immense interest. The motivations mainly arise from two aspects. First,
most of the brains energy is consumed at rest by spontaneous neuronal activity
(20% of bodys energy) while the task-related increases in energy metabolism are
usually small (<5%) (11). Second, low-frequency fluctuations (LFFs) (<0.1 Hz) of
resting-state fMRI (rsfMRI) signals were found to be coherent among brain areas
with similar functions and known anatomical interconnections (12, 13). Therefore,
efforts have been made to examine the coherence in LFFs, or resting-state
connectivity, providing not only new insights into the functional organization of
the brain (14-16), but also a better understanding of brain functional plasticity
during disease, aging and learning (17-19). Despite the wide interest in mapping
resting-state connectivity, the underlying physiological mechanisms remain to be
fully understood, and thus hinders the interpretation of rsfMRI data.
7

1.3 Magnetic Resonance Spectroscopy

Magnetic resonance spectroscopy (MRS) is one of the few available
techniques that can assess the neurochemistry non-invasively. Though MRS can
be performed using a variety of nuclei such as carbon (13C), nitrogen (15N),
fluorine (19F), and sodium (23Na), the nuclei phosphorus (31P) and hydrogen (1H),
which have high concentrations in vivo, are more frequently used. Proton (1H)
MRS has become popular due to the high natural abundance of protons and their
high absolute sensitivity to magnetic manipulation and better spectral resolution
(20). In the brain, the principle molecules that can be detected by 1H MRS are
listed in Table 1.1 with chemical shifts and physiological significance (21).

Table 1.1 Principal metabolites observed in 1H MRS

Metabolites
m-Ins
Tau
Cho
Cr
PCr
Glx

NAA

Myo-inositol
Taurine
Choline
Creatine,
Phosphocreatine
GABA,
Glutamate,
Glutamine
N-acetylaspartate

Ala
Lac
Lip

Alanine
Lactate
Free lipids

Chemical
shift (ppm)
3.6
3.4
3.2
3.0

Physiological significance
Glial marker and cerebral osmolyte
Pediatric tumors
Cell membrane metabolism marker
Energy metabolism marker, internal
reference peak
Intracellular neurotransmitter marker

2.1-2.5
2.0
1.5
1.3
0.9, 1.4

Marker for neuronal density, integrity and

health
Meningioma, Abscess
Anaerobic glycolysis
Tissue necrosis

The current impetus for higher field strengths benefits 1H MRS in terms of
better SNR and increased spectral dispersion, favoring the detection of many
overlapping resonances. Chapter 4 has demonstrated the usage of 1H MRS at 7T

to monitor longitudinal neurochemical alterations.

1.4 Manganese-enhance Magnetic Resonance Imaging

As previously mentioned, tissue T1, T2 or T2* can be manipulated by the use
of MR contrast agents, and thus differentiates the targeted cells from surrounding
tissues. Paramagnetic ion Mn2+ is one of the most widely used positive MRI
contrast agents. After the administration of manganese, the MRI signal intensity is
altered due to more changes in T1 than T2, thus providing increased signal in T1weighted MRI. Based on the increasing number of applications, manganeseenhance MRI (MEMRI) has proved to be a valuable tool for molecular imaging
(22). There are three major types of applications using MEMRI. First, systemic
administration, such as intraperitoneal, intravenous or subcutaneous injection of
Mn2+, can be used for enhanced visualization of the cerebral neuroarchitecture,
providing unique contrast in specific areas of the brain (23-27). Second, Mn2+ has
shown the capability of tracing neuronal pathways in an anterograde manner when
injected to specific brain regions. Therefore, MEMRI has been increasingly used
to map neuronal tracts in the living brain (28-30). Third, due to the factor that
Mn2+ can enter synaptically activated neurons through voltage-gated calcium
channels, MEMRI has been also used to map brain activities (23, 31-33). This
technique is also referred to as activation-induced MEMRI (AIM-MRI) (34, 35).
Comparing to fMRI, MEMRI maps the neuronal activations independently of the
surrogate hemodynamic responses. MEMRI also provides higher SNR and spatial

resolution. However, the major limitation of using Mn2+ is its cellular toxicity.
Therefore, it is critical to achieve desired contrast with as low a dose as possible.

1.5 Thesis outline and contribution

This thesis focuses on the development and applications of several MRI
methods, for in vivo assessments of the function, metabolism and structure of the
rodent brains at 7 Tesla. Briefly, a new fMRI technique was introduced and
demonstrated on rat brain in Chapter 2. The mechanism of resting-state fMRI was
explored in Chapter 3. Chapter 4 used proton MRS to investigate the
neurochemical alterations in mouse brain elicited by exogenous stimulation and
endogenous modification, respectively. In Chapter 5, manganese-enhanced MRI
was applied to investigate the cellular alterations after brain injury and neural
responses to conditioned fear. The organization of this thesis is described as
follows:
In Chapter 2, a new CBV-weighted fMRI technique using distortion-free
balanced steady-state free precession (bSSFP) sequence was proposed and its
feasibility was investigated in rat brain at 7 Tesla. After intravascular
susceptibility contrast agent administration (MION at 15 mg/kg), unilateral visual
stimulation was presented using a block-design paradigm. With TR/TE = 3.8/1.9
ms and =18o, bSSFP fMRI was performed and compared with the conventional
CBV-weighted fMRI using post-MION GE- and SE-EPI. The results showed that
post-MION bSSFP fMRI provides comparable sensitivity but with no severe

10

image distortion and signal dropout. Robust negative responses were observed
during stimulation and activation patterns were in excellent agreement with
known neuroanatomy. Furthermore, the post-MION bSSFP signal was observed
to decrease significantly during hypercapnia challenge, indicating its sensitivity to
CBV changes. These findings demonstrated that post-MION bSSFP fMRI is a
promising alternative to conventional CBV-weighted fMRI. This technique is
particularly suited for fMRI investigation of animal models at high field. This
work was published in Magnetic Resonance in Medicine 68(1):65-73.

In Chapter 3, an experimental model of corpus callosotomy was employed to

investigate the relationship between anatomical connections and resting-state
fMRI connectivity. Complete, anterior and posterior mid-sagittal corpus callosum
(CC) transections were performed in normal adult Sprague-Dawley rats. Restingstate fMRI was performed in these animals and sham controls at post-surgery day
7 and day 28. Five resting-state networks, including caudate putamen (CPu),
secondary somatosensory cortex (S2), primary somatosensory cortex (S1),
auditory cortex (AC) and visual cortex (VC) were examined using both
independent component analysis and seed-based analysis. Complete callosotomy
resulted in loss of interhemispheric connectivity in all cortical areas examined,
including S2, S1, AC and VC, at day 7 and day 28. Partial callosotomy led to
significantly decreased interhemispheric connectivity at day 7 in the cortical areas
whose primary interhemispheric connections via CC were severed, namely S2 and
S1 after anterior transection and S1, AC and VC after posterior transection. At

11

day 28, some of these connectivity reductions were restored. In addition,

intrahemispheric connectivity was found to generally increase in areas where
interhemispheric connectivity reductions sustained at day 28 after partial and
complete callosotomy. These experimental findings directly support that
anatomical connections via CC play a primary and indispensable role in restingstate connectivity, and that resting-state networks can be dynamically reorganized
or acquired directly or indirectly through the remaining anatomical connections.
This work provides direct evidence of the relationship between anatomical and
functional connectivity, contributing to a better understanding of the biological
mechanisms of rsfMRI. This work was collaborated with Prof. KF So and Dr.
Yuxiang Liang in Department of Anatomy in The University of Hong Kong who
prepared the animal model and performed the surgery.
In Chapter 4, proton MRS was employed to monitor the longitudinal
metabolic alterations in animal brains elicited by exogenous stimulation and
endogenous modification, respectively. First, longitudinal neurochemical changes
underlying fear conditioning was characterized by 1H MRS, aiming to contribute
towards a clear understanding of the neurobiological mechanisms of fear learning
and memory. The fear conditioning in rodents provides a valuable translational
tool to investigate the neural basis of learning and memory and potentially the
neurobiology of post-traumatic stress disorder (PTSD). Neurobiological changes
induced by fear conditioning have largely been examined ex vivo while
progressive real-time changes in vivo remain underexplored. Single voxel proton
magnetic resonance spectroscopy of the hippocampus, cingulate cortex and

12

thalamus of adult male C57BL/6N mice (N=12) was performed at 1 day before, 1
day and 1 week after, fear conditioning training using a 7T scanner. Nacetylaspartate (NAA), a marker for neuronal integrity and viability, significantly
decreased in the hippocampus at 1 day and 1 week post-conditioning. Significant
NAA reduction was also observed in the cingulate cortex at 1 day postconditioning. These findings of hippocampal NAA decrease indicate reduced
neuronal dysfunction and/or neuronal integrity, contributing to the trauma-related
PTSD-like symptoms. The neurochemical changes characterized by 1H MRS can
shed light on the biochemical mechanisms of learning and memory. Moreover,
such information can potentially facilitate prompt intervention for patients with
psychiatric disorders. This work was collaborated with Prof. GM McAlonan and
Dr. Qi Li in Department of Psychiatry in The University of Hong Kong who
provided the experimental setup for fear conditioning. This work has been
submitted to Psychiatry Research: Neuroimaging and it is under revision at the
time this thesis is submitted. Second, longitudinal 1H MRS during pregnancy and
motherhood was performed to evaluate the regional metabolic changes in the
hippocampus and thalamus of maternal brain. Pregnant primiparous rats (N=15)
were studied at 3 days before mating, gestational day 17, lactation day 7 and postweaning day 7. Age-matched nulliparous female rats (N=9) served as nonpregnant controls. Single voxel 1H MRS of the hippocampus and thalamus was
performed using a 7T scanner. Significantly higher NAA level observed in the
hippocampus of late pregnant rats level of hippocampal NAA of pregnant rats
indicates the increased density of neurons in this region, facilitating supporting

13

behaviors that involving learning and memory. Reduced level of choline in the
maternal brain reflects high fetal demands. The raised lactate level in thalamus
might be related to the hyperventilation of pregnancy. The results of this study
provide neurochemical evidence of the behavioral changes associated with
pregnancy.

In Chapter 5, manganese-enhanced MRI was applied to investigate the

cellular alterations after neonatal hypoxic-ischemic injury and after fear
conditioning training, respectively. First, in vivo MEMRI was employed to
investigate the hypoxic-ischemic injury in the late phase. Mn2+ induced signal
changes were examined using SPM coregistration and ROI analysis. T1W images
SI increase was detected in the peri-lesional region 24 hours after Mn2+
administration and it colocalized with the increase in glial cell density in GFAP
staining, demonstrating the existence of reactive gliosis in the late phase after H-I
injury. The results suggest such MEMRI approach may be useful in investigation
of post-injury cellular events and functional reorganization. Second, to study the
neurocircuitry behind this paradigm, in vivo MEMRI was employed to investigate
the neural response after subjection to fear conditioning in mice. Fear
conditioning is a widely used procedure to study the neural basis of learning and
memory. Compared to controls, fear conditioned animals exhibited higher Mn
uptake in amygdala, hippocampus, paraventricular nucleus of hypothalamus and
cingulate cortex, which are all highly-involved in the process of fear. The results
provide insights to neurocircuitry involved in fear-conditioning and consolidate

14

the capability of MEMRI as an in vivo probe for mapping neural activity.

In Chapter 6, potential applications and expansion of the investigated MRI

methods for future studies were discussed.

15

CHAPTER 2 BALANCED STEADY STATE

FREE PRECESSION FMRI WITH
INTRAVASCULAR SUSCEPTIBILITY
CONTRAST AGENT
2.1 Introduction
Since the introduction of blood oxygenation level dependent (BOLD)
contrast, fMRI has assumed an invaluable role in mapping brain functions due to
its noninvasiveness, large field-of-view and 3D imaging capabilities compared
with other imaging modalities (10). fMRI is often conducted with echo planar
imaging (EPI), which goes through the entire frequency space from one single
shot, providing a good compromise between spatial and temporal resolution. With
the availability of high field scanners, fMRI has also been expanded from humans
to small animal models for various neuroscience applications (36). High field
improves fMRI by increasing signal to noise ratio (SNR) and sensitivity (37).
However, it also increases the susceptibility and field inhomogeneities that give
rise to severe image distortion and signal dropout at air/tissue interface in EPI
images (6). The other major limiting factor in EPI fMRI is the constraint on
achievable spatial resolution. At high field, EPI spatial resolution is limited
intrinsically by T2* that can be short and vary across image due to field
inhomogeneity (7, 8). Furthermore, several physiological factors can also
adversely affect EPI-based fMRI, including motion artifacts and noises from

16

cardiac and respiratory pulsations (9). These limitations must be addressed to

achieve accurate and high resolution mapping of brain activities.

Balanced steady-state free precession (bSSFP) imaging has been proposed as

a promising alternative to EPI for fMRI (38, 39). bSSFP is a technique that uses
fully balanced gradients in each repetition time. The image contrast of bSSFP is
determined by T2/T1 (40). Its short repetition time and readout duration, together
with high signal efficiency, allow the fast, distortion-free and high resolution
imaging that is highly desirable for functional imaging. The original bSSFP fMRI
studies using the steep magnitude/phase transition in the bSSFP off-resonance
profile (38, 39, 41) were limited by the need for multi-frequency acquisitions to
find the narrow range transition band. Later, it was demonstrated that the
functional contrast can be achieved by utilizing the relatively large flat portion of
the bSSFP profile (42-45). The contrast mechanism of this pass-band bSSFP is
similar to that of conventional spin-echo BOLD but is less sensitive to motion and
physiological noises (40, 44, 46).

Signal contrast-to-noise ratio (CNR) measured during activation is another

key factor in fMRI. Using an intravascular susceptibility contrast agent of long
blood half-life such as monocrystalline iron oxide nanoparticles (MIONs) or other
ultrasmall superparamagnetic iron oxides (USPIOs), larger CNR and more robust
signal changes can be achieved (47-49). The negative post-contrast fMRI signal
changes primarily reflect the increase in cerebral blood volume (CBV) that
reduces the apparent T2 and T2* significantly (50). Signals from large blood
17

vessels are effectively suppressed in CBV-weighted images because of the

relatively high intravascular concentration of contrast agent (51). However, the
strong susceptibility contrast agent causes further image distortion and severe
signal loss in highly vascularized regions, which worsen at high field.

In this study, we investigated the feasibility of bSSFP imaging in

combination with intravascular susceptibility contrast agent MION as a new fMRI
approach. Brain responses to unilateral visual stimulation were examined at 7T
using this approach. The results were compared with those obtained using postMION GE- and SE-EPI fMRI. To support our hypothesis that this approach is
primarily sensitized to CBV changes, we qualitatively examined the post-MION
bSSFP signal changes during hypercapnia. Apparent tissue T1 and T2 changes
were also measured at varying MION concentrations to assess the post-MION
bSSFP signal properties.

2.2 Materials and Methods

2.2.1 Animal Procedure

All experiments were approved by the Institutional Animal Care and Use
Committee. Normal adult SpragueDawley rats (220~250g) were initially
anesthetized with 3% isoflurane and then maintained with 1.5% isoflurane during
the left femoral vein catheterization surgery. Animals were then placed on a
plastic cradle and maintained with 1% isoflurane during imaging. To minimize

18

head motion, the head was fixed with a tooth bar and plastic screws were inserted
into the ear canals. Animals were kept warm by a circulating water pad at 37oC.
Respiration rate, heart rate, oxygenation saturation and rectal temperature were
continuously monitored and maintained within normal physiological ranges (52,
53).

Before fMRI experiments, MION (MGH Center of Molecular Imaging

Research, MA) with approximately 4-hr blood half-life (54, 55) was intravenously
injected via the femoral vein catheter at a dosage of 15 Fe mg/kg. This dosage was
shown to be sufficient to overwhelm the positive BOLD contribution during
functional activations (56, 57). The fMRI scans were initiated 15 min after the
injection, which was substantially longer than the recirculation time (on order of
seconds) in adult rats, to ensure steady-state MION distribution in the vasculature.

Using unilateral visual stimulation, nine rats were examined by post-MION

bSSFP fMRI. Among them, five were also examined by conventional CBVweighed fMRI using post-MION GE- and SE-EPI methods for comparison. For
visual stimulation, an optical fiber with one end connected to a green lightemitting diode (LED) was placed 5 mm in front of the left eye. The LED was
flashed at 1 Hz with 5% duty cycle. One trial of the simulation paradigm
consisted of four blocks of 40-s rest and 20-s stimulation. Stimuli were
synchronized with the scanner under computerized control using LabVIEW v8.0
(National Instruments Corporation, Austin, TX). All animals were allowed to rest
for 10 minutes between stimulation trials. Two to five trials were recorded for
19

every fMRI method in each animal.

The effect of hypercapnia challenge on post-MION bSSFP signals was

examined in three animals. The gas conditioning paradigm consisted of 2-min
baseline followed by 4-min 5% CO2 exposure and 4-min recovery. In addition, T1
and T2 mapping and bSSFP imaging were performed on one animal to document
the longitudinal and transverse relaxation rates (R1 = 1/T1 and R2 = 1/T2) and
bSSFP signal as a function of accumulated MION dosage ranging from 0 mg/kg
(before injection) to 30 mg/kg in 5 mg/kg increment.

2.2.2 MRI Protocols

All MRI experiments were performed on a 7 T Bruker scanner with a

maximum gradient of 360 mT/m (70/16 PharmaScan, Bruker Biospin GmbH,
Germany) using a 72 mm birdcage transmit-only RF coil and an actively
decoupled receive-only quadrature surface coil. Scout T2-weighted (T2W) images
were first acquired in three planes with a rapid acquisition with relaxation
enhancement (RARE) sequence to guide the positioning of the subsequent fMRI
slice at the standard coronal orientation covering Bregma -6.7 to -7.7 mm. To
avoid the narrow transition band and minimize banding artifacts in bSSFP, local
shimming was performed with a FieldMap based procedure prior to fMRI scans
(58).

All bSSFP fMRI images were acquired with alternating RF pulse ( = ),

20

TR/TE = 3.8/1.9 ms, FOV = 3232 mm2, acquisition matrix = 6464 (zero-filled
to 128128 during reconstruction unless when compared with EPI results), slice
thickness = 1 mm, number of slices = 1, NEX = 4 and temporal resolution of 1 s.
A relatively small flip angle of 18o was estimated from the apparent T1 and T2

cosopt =
values measured at 15 mg/kg MION by

T1 / T2 1
T1 / T2 + 1 to provide maximum

flat pass-band region in the bSSFP profile (59). For comparison, post-MION
single-shot SE-EPI fMRI with TR/TE = 1000/21 ms and GE-EPI fMRI with
TR/TE = 1000/18 ms and = 30o were performed with identical slice orientation,
spatial geometry and visual stimulation paradigm. The total acquisition time for
bSSFP, GE-EPI and SE-EPI is identical, which is 5 minutes each. For anatomical
referencing, a 2D RARE T2W image was acquired at the same slice location with
TR/TE = 4200/36 ms, acquisition matrix = 256256, echo train length = 4 and
NEX = 2. To depict brain vasculature, a high resolution 2D T2*-weighted (T2*W)
image was also acquired using a FLASH sequence with TR/TE = 250/10 ms,
acquisition matrix = 512512, = 15o and NEX = 2.

For T1 mapping, a saturation recovery method, using rapid acquisition with

relaxation enhancement with variable repetition time (RAREVTR) sequence (60,
61), was employed with TR = 60, 120, 240, 480, 960, 1920, 3840 and 7680 ms
and TE = 7.5 ms. T2 mapping was performed using a CarrPurcellMeiboomGill
(CPMG) imaging sequence (62, 63) with TR = 4000 ms, 12 echoes, first TE of 7.5
ms and echo spacing of 7.5 ms.

21

2.2.3 Data Analysis

All fMRI data was realigned to the mean image of the time series using the
2D rigid-body transformation with AIR v5.2.5 (Roger Woods, UCLA, USA). The
first 5 images of each fMRI trial were discarded to eliminate possible nonequilibrium effects in dynamic bSSFP or EPI series. A linear detrending with
least-square estimation was performed on the time-course of each voxel to
eliminate the baseline drift caused by physiological noises and system instability.
Cross-correlation coefficient (cc) activation maps were generated by calculating
the correlation between the measured time-course and the box-car function
representing the stimulation paradigm on a voxel-by-voxel basis using the
STIMULATE software package (Center for Magnetic Resonance Research,
University of Minnesota). To identify areas showing strong activation, cc
threshold of -0.35 and cluster of 2 voxels were applied.

bSSFP signal time-courses were collected from every activated voxel within
three regions of interest (ROIs) covering the contralateral (right) superior
colliculus (SC), the contralateral visual cortex (VC) and ipsilateral (left) VC.
After temporal low-pass filtering (<0.1 Hz), the time-courses were transformed to
signal percentage changes by normalizing to the baseline signal (mean of first 40
time points). Within each ROI, the time-courses for the voxel with the strongest
cc value as well as all activated voxels were computed by averaging all blocks,
trials and nine animals studied. They were presented as mean standard deviation
(SD).
22

For comparison of post-MION bSSFP, GE-EPI and SE-EPI fMRI methods,

the single voxel with the strongest cc value for each method was selected. Their
raw time-courses were normalized and plotted together with the temporally lowpass filtered ones. Temporal SNR (tSNR) and CNR were measured for all the raw
time-courses. tSNR was calculated by tSNR =

, where was the mean of the

non-stimuli-related time-courses and its SD. CNR was calculated by

CNR =

, where S was the average signal change during activation (44). The

time-courses averaged over all visually activated voxels (cc<-0.35) within the
entire brain region were also plotted for each method by averaging the five
animals studied (mean SD).

For hypercapnia data, the bSSFP time-courses were computed in all three
animals from an ROI covering the entire brain except the regions that were
severely darkened by MION due to large blood vessels. For T1 and T2 mapping,
T1-weighted and T2W signals in each voxel were fitted with the monoexponential
relaxation and decay functions, respectively, using a nonlinear least square
algorithm provided by ParaVision 5.03 (Bruker Biospin GmbH, Germany). ROI
measurements were performed using ImageJ v1.40g (Wayne Rasband, NIH,
USA).

2.3 Results
Figure 2.1 shows that bSSFP images exhibited better spatial conformity to
23

anatomical T2W images than GE- and SE-EPI ones. The severe image distortion
seen in EPI images was not observed in bSSFP images. Note that all images were
acquired from the same animal at identical slice location during the same scan
session. bSSFP, GE-EPI and SE-EPI images had the same spatial resolution (500
500 m2). Before MION injection, certain brain regions (e.g. those close to the
ear canal) in EPI images suffered from signal dropout and image distortion, which
were absent in bSSFP images within the locally shimmed region (Figure 2.1 left
column). After 15 mg/kg MION injection, the T2W signal was seen to decrease in
the area between cortical and subcortical regions that contain mainly large blood
vessels (arrow in Figure 2.1a right). Such intravascular MION susceptibility effect
became more pronounced in the T2*W image as both blood vessels and their
surrounding tissue were dramatically darkened (arrow in Figure 2.1b right). More
importantly, severe signal loss and distortion occurred in GE-EPI (Figure 2.1d
right) and SE-EPI (Figure 2.1e right) after MION injection. In contrast, postMION bSSFP image exhibited less signal loss and no apparent distortion (Figure
2.1c right) and was in good agreement with post-MION T2W images.

24

Figure 2.1 Typical images acquired with conventional T2-weighted spin-echo (a),
conventional T2*-weighted gradient-echo (b), bSSFP (c), GE-EPI (d) and SE-EPI (e) at
the identical slice location from a rat brain before and after intravenous injection of
intravascular susceptibility contrast agent MION (15 mg/kg). Note that all bSSFP, GEEPI and SE-EPI images were acquired with the same spatial resolution and matrix size
(6464) and reconstructed without any post-processing.

25

Figure 2.2 Post-MION bSSFP fMRI yielded good agreement between the activation
patterns and known neuroanatomy during unilateral 20-s block-design stimulations in a
normal adult SD rats (a). The activation map was computed by correlating the fMRI
time-course with the stimulation paradigm and overlaid on the post-MION bSSFP image.
Only the voxels with cross-correlation coefficient (cc) < -0.35 were color coded. For each
activation cluster, the time-courses from the single voxel with the strongest cc value (as
marked by the yellow crosses in the small inset) (b) and all activated voxels within the

26

ROI (as defined by the green lines in the small inset) (c) were plotted in mean SD.
These time-courses were computed by averaging across all blocks, trials and animals
studied (n = 9). The strongest activation was observed in the superficial layers of
contralateral SC. Note that bSSFP images were acquired in a 6464 matrix and
reconstructed to 128128 by zero padding. The shaded area indicates the stimulation
period.

With post-MION bSSFP fMRI, unilateral visual stimulation produced robust

activations in contralateral SC and bilateral VC (Figure 2.2a). Without any spatial
co-registration, the activation map derived from bSSFP data yielded excellent
agreement between the activation patterns and known neuroanatomy such as the
superficial layers of contralateral SC, monocular area of primary VC (V1M), and
secondary VC (V2) of the contralateral cortical region, and binocular area (V1B)
of ipsilateral primary VC. Within each clustered region, the average time-course
from the single voxel with the strongest cc value (Figure 2.2b) and that from all
activated voxels (Figure 2.3c) showed robust bSSFP signal decrease in all animals
(n = 9) with small SDs. Contralateral SC, especially its superficial layers which
receives direct inputs from the retina (29, 64, 65), exhibited the highest percentage
signal changes. Contralateral and ipsilateral cortical activations exhibited similar
changes but contralateral activations were more extensive, covering the entire V1
and neighboring part of V2.

27

Figure 2.3 Typical activation patterns observed by post-MION bSSFP (a), GE-EPI (b)
and SE-EPI (c) fMRI methods from an animal. The imaging slice was located at Bregma
-7.2mm as shown in the coronal T2W image (d). For each method, the time-course
depicts the raw (colored) and low-pass filtered (black) signal changes in the voxel with
the strongest cc value in the SC (indicated by the green squares in the activation maps).

28

Figure 2.3 compares the typical post-MION bSSFP, GE- and SE-EPI fMRI
results obtained in one animal. All showed strong activations in SC and bilateral
VC (Figure 2.3 left column). For each method, the voxel with the strongest cc
value in the SC region was selected (as marked by green squares in Figure 2.3a-c).
Its raw time-course and filtered one were plotted for comparison (Figure 2.3 right).
Robust signal decrease in response to stimulation was observed by all three
methods from all five animals studied. The percentage bSSFP signal change was
between those for SE-EPI and GE-EPI. The tSNRs were 42.8 4.7, 32.4 3.4
and 53.6 6.2 for bSSFP, GE- and SE-EPI methods, respectively, while CNRs
were 1.6 0.3, 2.2 0.5, 1.4 0.4.

Table 2.1 summarizes the strongest voxel-wise cc value, mean cc value of all
activated voxels and number of activated voxels within the entire brain region in
all five animals studied. There was no significant difference in the strongest
voxel-wise cc value between bSSFP and GE- or SE-EPI fMRI results. The mean
cc value of bSSFP method was significantly weaker than that of GE-EPI (P <
0.001), but not significantly different from that of SE-EPI method. Note that
bSSFP yielded more activated voxels than GE- and SE-EPI methods (P < 0.01).
Figure 2.4 depicts the mean time-courses of all activated voxels (cc value < -0.35)
for post-MION bSSFP, GE-EPI, and SE-EPI methods by averaging the data from
all animals (n = 5). Robust signal decrease in response to four 20-s stimulations
was observed for all methods. Although the mean bSSFP signal percentage
change was smaller than that of EPI methods, bSSFP yielded smaller SD.

29

Table 2.1 Comparison of post-MION bSSFP, GE-EPI and SE-EPI fMRI methods as
determined from activated voxels (cc < -0.35) within the entire slice in all five animals
studied.

Strongest cc

Mean cc

No. of Activated
Voxels

bSSFP

-0.69 0.05 *

-0.46 0.02

29 7

GE-EPI

-0.73 0.06

-0.51 0.06

22 9

SE-EPI

-0.64 0.04

-0.49 0.06

20 11

No significant difference between bSSFP and GE- or SE-EPI fMRI method.

Significantly weaker than that of GE-EPI method (P < 0.001) but not significantly

different from that of SE-EPI method.

Significantly more than those by GE- and SE-EPI methods (P < 0.01).

Statistical analysis was performed using two-tailed paired Students t-test with P < 0.05
considered as significant.

30

Figure 2.4 Average post-MION bSSFP (a), GE-EPI (b) and SE-EPI (c) time-courses in
all activated voxels (cc < -0.35) within the entire slice in the animals studied (n = 5). The
shaded areas indicate the stimulation periods.

Figure 2.5 shows that the average post-MION bSSFP signal decreased
significantly in the entire brain in response to the 4-min 5% CO2 challenge in all
three animals studied. Such global signal reduction was observed in each

31

individual animal. Figure 2.6a shows that R1 increased substantially at small

MION concentration ( 5 mg/kg) in various brain regions. However, such R1
increase reached a plateau at high concentration ( 10 mg/kg), which was similar
to previous studies (66, 67). On the other hand, a strong and approximately linear
dependence (e.g., R2 = 0.98 for the whole brain ROI) of R2 on MION
concentration was observed (Figure 2.6b) in agreement with the earlier reports (68,
69). Figure 2.6c shows that bSSFP signal decreased monotonically with
increasing MION concentration.

Figure 2.5 Average post-MION bSSFP signal time-course from the entire brain during
the 4-min hypercapnia challenge using 5% CO2 inhalation in the animals studied (n = 3).
Measurement ROIs were shown in the small inset, and covered all cortical and
subcortical regions except those severely darkened by MION because of large blood
vessels. The shaded area identifies the period of hypercapnia challenge.

32

Figure 2.6 Apparent tissue longitudinal relaxation rate R1 (=1/ T1) (a), transverse
relaxation rate R2 (=1/T2) (b) and bSSFP signal (c) as a function of MION concentration
in different brain regions of one animal.

33

2.4 Discussion
The present study demonstrated the robustness of post-MION bSSFP fMRI
approach for detecting brain activity. The negative signal change mainly arises
from its CBV sensitivity although the detailed mechanism is complex and remains
to be elucidated. Conventional CBV-weighted fMRI relies on the increased
susceptibility effect of intravascular contrast agent caused by local CBV increase
upon activation, which manifests as the apparent R2* or R2 increase in GE- or SEEPI. At steady-state MION concentration, the relationship between relative CBV
change and R2* or R2 change is approximately linear (68). Consequently, the
conventional CBV-weighted fMRI is capable of quantitatively estimating the
relative CBV increases upon stimulation. In contrast, the bSSFP signal in
biological tissues is determined by multiple factors such as T1, T2, diffusion,
actual sequence parameters and susceptibility contributions. For simplicity, it is
often regarded to provide the T2/T1- or R1/R2-weighted contrast (59). With the
high intravascular MION dosage used in the current study, bSSFP signal from
intravascular spins becomes negligible and extravascular spins are subject to the
strong microscopically inhomogeneous magnetic fields around capillary vessels.
Given the small capillary vessel size (on order of few microns), short TR (of 3.8
ms) and high intravascular MION concentration (of 15 mg/kg), the mechanistic
effect of intravascular MION on bSSFP signal will likely be dominated by the
transition regime (between diffusion narrowing and static dephasing regimes) (46).
Therefore, the susceptibility-induced bSSFP signal decrease during activation

34

could be primarily attributed to the apparent R2 increase.

In this study, the linear dependency of apparent R2 increase on MION

concentration was observed (Figure 2.6b) although the actual R2 measurement
could be TE and sequence dependent. Such apparent R2 increase was a direct
result of the microscopic susceptibility effect, arising from diffusion decay in the
randomized magnetic susceptibility field created by intravascular contrast agent.
During functional activation, local CBV increase will augment the local
intravascular susceptibility effect and cause further bSSFP signal decrease. On the
other hand, one cannot completely ignore the MION T1 shortening effect on the
post-MION bSSFP fMRI contrast although the effect diminishes at high MION
concentration. Our experimental observation of the apparent tissue R1 saturation
at high MION concentration (Figure 2.6a) was consistent with the previous animal
studies in the brain and kidney (70, 71). It was also in good agreement with the
two-compartment model analysis (66, 72, 73) where the effect of intravascular T1
contrast

agent

on

apparent

tissue

R1

is

ultimately

limited

by

the

intravascular/extravascular water exchange rate and blood volume fraction. Thus,

the MION T1 effect on bSSFP signal changes during activation was likely
minimal at the high concentration used in the present study. In addition, the
bSSFP signal was observed to generally decrease with MION concentration
(Figure 2.6c), supporting the hypothesis that post-MION bSSFP fMRI is likely
dominated by the apparent R2 increase caused by CBV increase.

Hypercapnia challenge is known to induce vasodilation and CBV increase

35

(74). It is commonly employed to examine the brain hemodynamic regulation in

the absence of neuronal activation (50, 75). In the present study, continuous
physiological recordings showed that SpO2 and respiration rate increased while
heart rate slightly decreased upon inhaling CO2 (data not shown), which was in
agreement with the previous hypercapnia study (76). More importantly, the robust
post-MION bSSFP signal decrease during hypercapnia challenge (Figure 2.5)
further supported our hypothesis that post-MION bSSFP provides a CBV
sensitive fMRI approach. Approximately 35% CBV changes were reported
previously based on the post-MION SE T2W signal decrease during similar
hypercapnia challenge (74). However, the post-MION bSSFP signal decrease
during hypercapnia observed in the present study may not allow the direct
quantitation of such relative CBV increase because post-MION bSSFP signal is
not related to R2 in a purely exponential manner. In this regard, it remains
imperative to formulate a comprehensive description in order to understand postMION bSSFP signal change during functional activation.

Post-MION bSSFP fMRI provides better functional mapping quality than

conventional post-MION EPI-based fMRI. As shown in Figure 2.1, post-MION
EPI images suffered from signal dropout and severe distortion, dramatically
limiting their true spatial resolution and fidelity when mapping brain activities. In
contrast, post-MION bSSFP images showed good agreement with high resolution
anatomical images. The high image fidelity provided by post-MION bSSFP not
only alleviates difficulties in post-processing, but also facilitates accurate

36

localization of brain activities. As seen in Figure 2.3, the activation patterns in

both SE- and GE-EPI results were displaced to varying extents due to the
inhomogeneity and susceptibility induced image distortion. For example, the
ipsilateral cortical activation locations were shifted approximately by one to two
voxels when compared to those determined by bSSFP. Such region specific
distortion can affect the fMRI interpretation and is difficult to correct by postprocessing. This issue together with poor spatial resolution, strong susceptibility
and physiological noises, have severely hampered the conventional EPI-based
CBV-weighted fMRI study of small animals at high field. However, these
limitations can be mitigated by using the post-MION bSSFP fMRI demonstrated
in the present study. Rapid refocusing and short readout duration in bSSFP
sequence provide high signal efficiency with no image distortion (45) while the
CBV sensitivity incurred by intravascular susceptibility contrast agent minimizes
the contamination from remote site activations and physiological noises.

Robust tSNR and CNR enable post-MION bSSFP fMRI to capture

activations with sensitivity comparable to EPI methods. On the first order, bSSFPbased fMRI contrast can be considered to be similar to that of post-MION SE-EPI,
which is mainly sensitive to changes in microvascular blood volume at capillary
level (44). Our experimental findings showed that post-MION bSSFP activation
pattern was more similar to that of SE-EPI (Figure 2.3 left). However, the
percentage change of bSSFP signal as determined from single voxel time-courses
was higher than that of SE-EPI during visual stimulation (Figure 2.3 right). This

37

might partly arise from the differences between bSSFP and SE-EPI signal
properties such as the prominent role of stimulated echo pathways in bSSFP
signal formation. Post-MION GE-EPI fMRI exhibited much larger percentage
signal change since it is sensitized to the relatively large R2* change during
activation, which is less influenced by vessel size and more sensitive to total CBV
change. Lastly, post-MION bSSFP fMRI yielded more activated voxels than EPI
methods (Table 2.1), likely a consequence of less signal loss and image distortion.

Several limitations exist for the fMRI approach proposed in this study. First,
use of intravascular susceptibility limits its application in humans. Nevertheless,
given that certain USPIOs such as MION have long blood half-life and induce no
apparent physiological and functional perturbations, this new approach can greatly
facilitate the fMRI study in animals such as rodents and monkeys. Second, the
present study demonstrated the post-MION bSSFP fMRI with single slice
acquisition. However, fast 3D bSSFP can be achieved for fMRI as shown in a
recent study using various 3D k-space trajectories (45). Such high resolution 3D
acquisition can be readily adopted to post-MION fMRI. Doing so may prolong the
acquisition time and affect temporal resolution, but it gains full brain coverage
and SNR. Third, the post-MION bSSFP fMRI requires good shimming to avoid
the banding artifacts in bSSFP image particularly at high field. Moreover, this
fMRI approach is largely based on the large flat portion of the off-resonance
profile that is sensitive to shimming. In this study, localized shimming was
carefully performed within the single image slab prior to fMRI acquisition to

38

minimize the banding artifacts and avoid the narrow transition band.

In this study, the distortion-free technique was demonstrated in the visual

area which was severely distorted in conventional EPI-based fMRI methods. It not
only enables precise localization of the activations to neuroanatomy in living
subjects, but also promotes better understandings of neuronal pathways of
different sensory systems. The visual region is not strongly affected by
susceptibility problems that causing signal dropouts, therefore the benefit in this
aspect from the new fMRI technique was not significant. However, it can be used
to investigate the brain regions that are vulnerable to the susceptibility in EPI
images. For example, the deep brain structures located in the posterior portion of
the brain, the auditory cortex and the olfactory bulbs which are close to air
cavities and suffer from significant signal loss in EPI images. With this regard,
post-MION bSSFP fMRI can be employed to examine the responses under
various types of functional manipulation such as auditory or pain stimulation.

2.5 Conclusion
In conclusion, the proposed bSSFP fMRI with intravascular susceptibility
contrast agent provides sensitivity comparable to conventional CBV-weighted
EPI-based fMRI but with no severe image distortion and signal dropout. Robust
negative responses during visual stimulation were observed and activation
patterns were in excellent agreement with known neuroanatomy. In addition, postMION bSSFP signal was observed to decrease significantly during hypercapnia
39

challenge, indicating its sensitivity to CBV changes. These findings demonstrated

that post-MION bSSFP fMRI is a promising alternative to conventional CBVweighted fMRI. It is particularly suited for fMRI investigation of animal models
at high field.

40

CHAPTER 3 ALTERATIONS IN RESTINGSTATE FMRI CONNECTIVITY AFTER

COMPLETE AND PARTIAL CORPUS
CALLOSOTOMY
3.1 Introduction
Since the introduction of blood oxygenation level-dependent (BOLD)
contrast (10), functional MRI (fMRI) has offered a powerful approach to study
brain functions due to its noninvasiveness, large field-of-view and 3D imaging
capabilities compared with other imaging modalities. The majority of fMRI
studies have focused on examining the changes in neuronal activities associated
with stimuli or tasks. It is not until recently that studying the resting brain by
fMRI became of immense interest. The motivations mainly arise from two
aspects. First, most of the brains energy is consumed at rest by spontaneous
neuronal activity (20% of bodys energy) while the task-related increases in
energy metabolism are usually small (<5%) (11). Second, low-frequency
fluctuations (LFFs) (<0.1 Hz) of resting-state fMRI (rsfMRI) signals were found
to be coherent among brain areas with similar functions and known anatomical
interconnections (12, 13). Therefore, efforts have been made to examine the
coherence in LFFs, or resting-state connectivity, providing not only new insights
into the functional organization of the brain (14-16), but also a better
understanding of brain functional plasticity during disease, aging and learning
41

(17-19).

Despite the wide interest in mapping resting-state connectivity, the

physiological mechanisms underlying coherent LFFs remain to be fully explored.
The interpretation of rsfMRI data is therefore hindered. Given the similarity
between the spatial organization of resting-state networks (RSNs) and
neuroanatomy, one common hypothesis is that resting-state connectivity is based
on anatomical connections. Anatomically, the hemispheres are interconnected by
axonal projections through midline commissural structures such as the corpus
callosum (CC), anterior commissure and posterior commissure. The largest
among them is the CC, which connects most areas of the cerebral cortex to
contralateral homologous areas that share similar functions (77, 78). Considering
the primary role of CC in interhemispheric communication, the relationship
between callosal connections and resting-state connectivity naturally is an issue of
interest. Previously, human studies have demonstrated the effects of the absence
of callosal connections on resting-state connectivity. Two rsfMRI studies on
callosal agenesis (79) and complete corpus callosotomy (in a single patient) (80)
showed significantly diminished and complete loss of interhemispheric
connectivity, respectively. These results support anatomical connections as key
constraints on resting-state connectivity. However, two other studies reported
predominately bilateral RSNs in a patient after complete transection of forebrain
commissures (81) and in patients with congenital callosal agenesis (82). These
findings favor another possibility that resting-state connectivity emerges flexibly

42

and is not limited by direct anatomical connections. While the interpretations of

the above studies seem to be contradictory, it may be due to the limited number of
subjects, large difference in subject age and diversity in remaining anatomical
connections. Therefore the role of CC in resting-state connectivity is still open to
debate and well controlled animal models are valuable in this regard.

In this study, we employed a simple and direct animal model of corpus

callosotomy to evaluate the relationship between anatomical connections and
resting-state connectivity. Complete or partial transection of the CC was
performed in adult rats. The RSNs were examined at post-surgery day 7 and day
28 using both independent component analysis and seed-based cross-correlation
analysis and the results were compared to those from sham controls.

3.2 Materials and Methods

3.2.1 Animal Preparation

All experiments were approved by the local institutional animal care and use
committee. A total of 34 adult Sprague-Dawley rats weighing 220~250 g were
used in this study. Animals were divided into four groups and subjected to
complete callosotomy (N = 8), anterior partial callosotomy (N = 8), posterior
partial callosotomy (N = 10) and sham surgery (N = 8). For the surgery, animals
were first anesthetized with intramuscular injection of mixture of ketamine (80
mg/kg) /xylazine (8 mg/kg), after the scalp fur was shaved animals were

43

restrained with a stereotaxic frame. Then the skin was sterilized and a midline
incision was made along the top of the head, exposing the skull. Areas of the skull
above brain regions of interest were opened with a drill to expose the brain (83,
84). Care was taken to avoid avulsion of the superior sagittal sinus. A U-shape cut
along the edge of the opened skull was made on the dura of one side, and then the
dura was lifted to expose the midline. With the help of a thin blunt spatula and a
fine micro knife, animals in the first group underwent a transection of the entire
CC, from Bregma 2 mm to -6 mm. Animals in the second group received a
transection of the anterior CC, from Bregma 2 mm to -4 mm. In the third group,
animals underwent a transection of the posterior CC, from Bregma -2 mm to -6
mm. The animals in the sham group had their skulls opened but received no
further surgery. Extremely care was taken to avoid injury to the superior sagittal
sinus. Bleeding from the cerebral veins was stopped immediately with cold gel
foam, which was removed when homeostasis was satisfactory. After surgery, the
animals were returned to their home cages under warm condition to allow them to
recover, and housed under a 12:12 hour light/dark cycle in a temperaturecontrolled room and had ad libitum access to food and water, anti-inflammatory
drug were supplied in the water for a week. MRI was performed on all animals at
post-surgery day 7 and day 28. After MRI acquisition, one animal from each
group was sacrificed for hematoxylin and eosin staining to confirm the complete
disruption of callosal fibers at the transection sites.

44

3.2.2 MRI Protocols

All MRI measurements were acquired utilizing a 7 T Bruker scanner with a

maximum gradient of 360 mT/m (70/16 PharmaScan, Bruker Biospin GmbH,
Germany), a 72 mm birdcage transmit-only RF coil and an actively decoupled
receive-only quadrature surface coil. Animals were initially anaesthetized with
3% isoflurane and then orally intubated. During MRI, animals were placed on a
plastic cradle with the head fixed with a tooth bar and plastic screws in the ear
canals. Animal was mechanically ventilated with 1.2-1.5% isoflurane and kept
warm with circulating water at 37 oC. Continuous monitoring of the respiration
rate, heart rate, oxygenation saturation and rectal temperature was performed and
vital signs were maintained within normal physiological ranges (85-88). Scout T2weighted (T2W) images were first acquired in three planes with a rapid acquisition
with relaxation enhancement (RARE) pulse sequence to position the subsequent
multi-parametric MR images along standard anatomical orientations in a
reproducible manner. For rsfMRI, a single-shot gradient-echo echo-planarimaging (EPI) sequence was used with repetition time/echo time (TR/TE) =
1000/18ms, flip angle = 30o, field of view = 3232mm2, 6464 matrix, nine 1 mm
thick contiguous slices and 280 repetitions. It resulted in a 5 minute scan time for
each rsfMRI dataset and 4-6 datasets were acquired from each animal. RARE
T2W images were acquired with the same spatial dimensions with TR/TE =
4200/36ms, 256256 matrix as an anatomic reference for rsfMRI data. In
addition, diffusion tensor imaging (DTI) was performed to assess and depict CC

45

integrity in all animals. Diffusion images were acquired with a 4-shot spin-echo
EPI sequence using TR/TE = 3000/28.8ms, / = 5/17ms, 9696 matrix (zerofilled to 128 128) and an encoding scheme of 15 gradient directions at b-value =
1000s/mm2. Five additional images with b = 0 s/mm2 were also acquired (89).

3.2.3 Data Analysis

For each rsfMRI dataset, all images were first corrected for slice timing
differences using SPM5 and then realigned to the mean image of the series using
2D rigid-body transformation with AIR v5.2.5 (Roger Woods, UCLA, USA). A
voxel-wise linear detrending with least-squares estimation was performed
temporally to eliminate the baseline drift caused by physiological noises and
system instability. In-plane smoothing was done using a Gaussian kernel with full
width at half maximum of 0.5 mm (86, 87, 90). Finally, a temporal band-pass
filter (0.005-0.1Hz) was applied to all voxels to extract the LFFs (91).

Independent component analysis (ICA) was performed on the preprocessed

data using GIFT v2.0d Toolbox (http://www.nitrc.org/projects/gift/) (92, 93). The
estimated number of components was found to be 37 by the minimum description
length (MDL) criterion. The Infomax algorithm was used and the spatial ICA
maps of independent RSNs were scaled to z-scores with a threshold of z>1.5
(correlation coefficient >0.25). The ICA maps were visually compared to the
neuroanatomic atlas (94). The components located at anatomically defined regions
were considered as meaningful. Seed-based analysis (SBA) was also performed

46

on the preprocessed data (16, 95). Five brain areas where bilateral RSNs were
commonly observed in sham animals were examined by SBA. They were caudate
putamen (CPu), secondary somatosensory cortex (S2), primary somatosensory
cortex (S1), auditory cortex (AC) and visual cortex (VC). For each brain area, a
22-voxel region on each hemisphere was chosen as the seed where high z-score
was generally seen in the corresponding ICA maps of all animals. Correlation
coefficients (cc) were calculated between the average time course of the four
voxels within the seed and every other voxel time course. Then two symmetric
sets of regions of interest (ROIs) covering the entire left or right parts of the
functional area were defined according to the visual correlation between ICA
results and rat brain atlas (94). The distribution of cc values within each set of
ROIs ipsilateral or contralateral to the corresponding seeds was displayed in a
histogram. For each animal, the histograms from different datasets taken within
the same MRI session and based on the same pair of seed and ROIs were
averaged. The average histogram was then fitted with a Gaussian function using
the least-squares method.

Mean value and standard deviation of the

distribution were obtained. Statistical evaluation of and among different

groups was performed using one-way ANOVA. Results were considered
significant when P<0.05.

For DTI analysis, the fractional anisotropy (FA) map was calculated using
DTIStudio v3.02 (Johns Hopkins University, Baltimore, MD) for each animal
(89).

47

3.3 Results
Figure 3.1 shows the T2W images and FA maps from the representative animals
that had complete, anterior partial or posterior partial corpus callosotomy and
sham surgery. The transected part of CC is indicated in red color in the sagittal
planes (Figure 3.1, left panel) as well as by yellow arrows in the T2W images.
Lower signal intensity in FA maps depicts the fiber disruption in the
corresponding CC regions. The middle part of the CC body, which was
approximately 1 mm in length along the sagittal direction, was severed in animals
of both partial callosotomy groups. All animals survived after the surgery and
animals within each group had similar surgical outcome as shown in Figure 3.1.
Histological examinations of the animals from the present study and additional
animals at post-surgery day 7 confirmed the disruption of callosal connections at
the locations of transection (data not shown). Moreover, no apparent reconnection
of the callosal fibers was observed in the histological results at day 28.

48

Figure 3.1 Representative T2-weighted (T2W) images and fractional anisotropy (FA)
maps from the animals with complete (a), anterior partial (b) and posterior partial (c)
corpus callosotomy and sham surgery (d). The transected part of the corpus callosum (CC)
is indicated in red color in the sagittal planes (left panel) and by yellow arrows in the T2W
images. The blue lines indicate the corresponding locations of T2W and FA slices in the
right panel. Distance to Bregma for each slice is given at the bottom.

3.3.1

Effects

of

Complete

and

Partial

Callosotomy

on

Interhemispheric Connectivity

The CC is organized in a rostrocaudal topographical manner with anterior

fibers connecting frontal areas of the two hemispheres and posterior fibers
connecting caudal cortical structures. Therefore, to compare the effects of
transecting location on different RSNs, five areas ranging from the anterior to the
posterior part of the brain, which are CPu, S2, S1, AC and VC, were examined.
Figure 3.2 shows the ICA maps covering these areas from one representative

49

animal in each group at post-surgery day 7. Consistent ICA maps were observed
in all rats from the same group. The components covering bilateral homologous in
all cortical areas, namely S2, S1, AC and VC, of sham controls were prominently
absent in the complete callosotomy group (Figure 3.2a and 3.2d). Instead, two
unilateral independent components were observed for each cortical area,
indicating the loss of interhemispheric resting-state connectivity. The same result
was found in S2 of the anterior partial callosotomy group (Figure 3.2b) and in AC
and VC of the posterior partial callosotomy group (Figure 3.2c). Apart from the
above differences, both partial callosotomy groups exhibited the loss of
interhemispheric resting-state connectivity in S1 (Figure 3.2b and 3.2c).
Meanwhile, the subcortical component located in CPu remained bilateral in all
groups of animals except that in the complete transection group, where the map
became more asymmetric.

50

51

auditory cortex (AC) and visual cortex (VC), respectively.

to posterior part of the brain. They are caudate putamen (CPu), secondary somatosensory cortex (S2), primary somatosensory cortex (S1),

fibers connecting caudal cortical structures. The components shown in this figure correspond to five brain areas ranging from the anterior

CC is organized in a rostrocaudal topographical manner with anterior fibers connecting frontal areas of the two hemispheres and posterior

score (yellow) representing a stronger correlation between the time course of that voxel and the mean time course of this component. The

independent components were scaled to z-scores (z>1.5) and overlaid on the EPI images. The color bars display z-scores with a higher z-

callosotomy and sham surgery (d) at post-surgery day 7. Independent component analysis (ICA) was performed. Spatial ICA maps of

Figure 3.2 Typical resting-state connectivity maps from individual animals with complete (a), anterior partial (b) and posterior partial (c)

Figure 3.3 shows the definition of seeds and ROIs in CPu, S2, S1, AC and
VC that were used for SBA. In Figure 3.4, typical histograms from a sham animal
at post-surgery day 7 show the distribution of cc values within the ROIs ipsilateral
or contralateral to the seeds in each area. All the histograms followed bell shape,
indicating Gaussian distribution. In the histograms of ROIs ipsilateral to the seed,
there was a second peak at high cc values with much lower magnitude compared
to that of the peak at mean. It resulted from the high cc values of the voxels within
the seed as the ipsilateral ROIs also covered the seed. The mean value and
standard deviation of the distribution were obtained by Gaussian fitting and the
results from different groups at post-surgery day 7 are shown in Table 3.1.
Regarding the ROIs contralateral to the seeds, the varied among different groups
while the remained similar. In the complete callosotomy group, significantly
smaller was found in S2, S1, AC and VC compared to that of sham control,
indicating significantly reduced interhemispheric resting-state connectivity. In
addition, the was also significantly smaller in AC and VC compared to the
anterior partial callosotomy group and in CPu and S2 compared to the posterior
partial callosotomy group. In the anterior partial callosotomy group, was found
to be lower in S2 and S1 than that of sham control but higher in AC and VC than
that of the posterior partial callosotomy group. Significantly lower was observed
in S1, AC and VC of the posterior partial callosotomy group with respect to sham
controls. Figure 3.5 shows the scatter plots between the of cc values in S2 and
AC for all animals at post-surgery day 7. For the ROIs contralateral to the seeds
(Figure 3.5b), the plots of different groups can be seen as clearly isolated clusters.

52

In brief, both ICA and SBA results showed that callosotomy disrupted
interhemispheric resting-state connectivity. Specifically, complete callosotomy
resulted in loss of interhemispheric connectivity in all cortical areas examined
while partial callosotomy led to significantly decreased connectivity in those
cortical areas whose primary interhemispheric connections via CC were severed.

Figure 3.3 Localization of the seeds and corresponding regions of interest (ROIs) for
seed-based cross-correlation analysis (SBA). Five brain areas were selected based on the
ICA results. Seeds and ROIs were overlaid on EPI images with slices located from
Bregma 1.2mm to -7.2mm (slice 1-9). Color code: CPu (red), S2 (yellow), S1 (green),
AC (purple) and VC (blue).

53

Figure 3.4 Typical histograms showing the distribution of numbers of voxels within the
ROIs (as illustrated in Figure 3.3) across all correlation coefficient (cc) values. The data
was from the SBA results of one sham animal at post-surgery day 7. For each brain area,
the results of the seeds in both left (in red) and right (in blue) hemispheres and the ROIs
ipsilateral (a) and contralateral (b) to the seeds are presented here.

Figure 3.5 Scatter plots of the mean value of the cc distribution in S2 and AC ROIs
ipsilateral (a) and contralateral (b) to the seeds for all animals at post-surgery day 7.

54

Table 3.1 Mean value and standard deviation of the cc values within the ROIs
in hemispheres ipsilateral and contralateral to the seeds at post-surgery day 7. The
results are presented in mean standard deviation. Statistical comparisons
between different groups were performed using one-way ANOVA. and denote
decrease and increase, respectively, with the significance level indicated by ,
(P<0.05, P<0.01 compared to anterior partial transection, respectively), ,
(P<0.05, P<0.01 compared to posterior partial transection, respectively) and *, **,
*** (P<0.05, P<0.01, P<0.005 compared to sham control, respectively).

Complete
callosotomy
(N = 8)
Seed-ROI

Anterior partial
callosotomy
(N = 8)

Posterior partial
callosotomy
(N = 10)

Sham control
(N = 8)

CPu
ipsilateral

0.360.08

0.260.04

0.350.06

0.260.03

0.350.08

0.250.04

0.330.08

0.260.03

contralateral

0.210.07
()

0.180.02

0.260.08

0.180.02

0.290.07

0.190.02

0.280.08

0.190.02

ipsilateral

0.420.10

0.250.05

0.400.08

0.250.03

0.340.08

0.270.05

0.410.11

0.270.05

contralateral

0.200.06
()(***)

0.200.03

0.220.09
(***)

0.200.02

0.300.08

0.220.06

0.360.11

0.210.03

ipsilateral

0.450.09

0.250.04

0.430.07

0.240.05

0.380.08

0.250.07

0.430.11

0.260.06

contralateral

0.220.05
(***)

0.210.03

0.260.09
(**)

0.230.04

0.290.09
(*)

0.220.07

0.390.09

0.230.06

ipsilateral

0.330.07

0.240.03

0.330.05

0.250.03

0.310.08

0.250.03

0.300.11

0.260.03

contralateral

0.210.05
()(*)

0.200.03

0.280.06
()

0.200.02

0.210.08
(*)

0.200.02

0.300.10

0.210.03

ipsilateral

0.360.09

0.310.05

0.360.07

0.290.05

0.330.09

0.320.05

0.330.11

0.330.07

contralateral

0.230.06
() (*)

0.240.04

0.300.07
()

0.260.04

0.220.07
(*)

0.270.06

0.300.08

0.270.06

S2

S1

AC

VC

55

3.3.2 Interhemispheric Connectivity Changes Long after Corpus

Callosotomy

Figure 3.6 shows the ICA maps corresponding to S2 and AC respectively

from one representative animal in each group at post-surgery day 28. Similar to
that of post-surgery day 7, the complete callosotomy group showed two unilateral
RSNs in S2 and AC whereas sham control group had bilateral components (Figure
3.6a and 3.6d). In contrast, the two unilateral RSNs that were observed in S2 of
the anterior partial callosotomy group at post-surgery day 7 were replaced by one
RSN covering bilateral sides (Figure 3.6b). The same result was also found in AC
of the posterior partial callosotomy group (Figure 3.6c). These findings indicate
the disrupted resting-state connectivity in these areas was restored at day 28 after
partial callosotomy. Meanwhile, for each group, the ICA maps in CPu, S1 and VC
remained the same as those of post-surgery day 7 (data not shown).

Table 3.2 shows the and obtained from SBA at post-surgery day 28. In
the ROIs contralateral to the seeds, the varied among different groups while the
remained similar. In the complete callosotomy group, significant lower was
found in all cortical areas examined compared to sham, indicating the loss of
interhemispheric resting-state connectivity was persistent to post-surgery day 28.
In both groups of partial callosotomy, was still lower in S1 compared to sham.
Interestingly, the significantly smaller that was observed in S2 of the anterior
partial callosotomy group and in AC of the posterior partial callosotomy group at
56

post-surgery day 7 was no longer seen at day 28. This is in line with the ICA
results showing the restoration of bilateral RSNs in these areas long after partial
callosotomy (Figure 3.6b and 3.6c).

Figure 3.6 Typical resting-state connectivity maps from individual animals with
complete (a), anterior partial (b) and posterior partial (c) callosotomy and sham surgery
(d) at post-surgery day 28. Spatial ICA maps of independent components were scaled to
z-scores (z>1.5) and overlaid on the EPI images. The color bars display z-scores with a
higher z-score (yellow) representing a stronger correlation between the time course of
that voxel and the mean time course of this component. The ICA components shown in
this figure correspond to secondary somatosensory cortex and auditory cortex.

57

Table 3.2 Mean value and standard deviation of the cc values within the ROIs in
hemispheres ipsilateral and contralateral to the seeds at post-surgery day 28. The results
are presented in mean standard deviation. Statistical comparisons between different
groups was performed using one-way ANOVA. and denote decrease and increase,
respectively, with the significance level indicated by , (P<0.05, P<0.01 compared to
anterior partial transection, respectively), , , (P<0.05, P<0.01, P<0.005 compared
to posterior partial transection, respectively) and *, **, *** (P<0.05, P<0.01, P<0.005
compared to sham control, respectively).

Complete
callosotomy
(N = 7)
Seed-ROI

Anterior partial
callosotomy
(N = 7)

Posterior partial
callosotomy
(N = 9)

Sham control
(N = 6)

CPu
0.300.06

0.280.04 0.280.04 0.280.03 0.300.08 0.260.03 0.280.05 0.270.02

contralateral 0.190.06

0.200.03 0.190.04 0.200.03 0.220.07 0.190.03 0.190.06 0.190.01

ipsilateral

S2
0.340.06
0.270.03 0.300.03 0.290.02 0.270.05 0.290.04 0.250.05 0.310.03
()(***)
()(*)
()(**)
0.200.03 0.200.05 0.210.02 0.230.04 0.220.03 0.230.05 0.210.02
contralateral 0.150.06
()()(**)

ipsilateral

S1
0.380.07
(*)
contralateral 0.200.08
(*)

ipsilateral

0.280.04 0.360.06 0.280.04 0.360.05 0.250.03 0.310.06 0.270.03

(*)
(*)
0.240.04 0.220.06 0.250.03 0.210.07 0.230.05 0.270.06 0.240.03
(*)
(*)

AC
0.310.05
() (*)
contralateral 0.160.05
()()(*)

ipsilateral

0.260.05 0.260.06 0.260.03 0.290.06 0.270.05 0.260.07 0.270.03

0.230.06 0.230.04 0.210.03 0.220.06 0.230.05 0.210.06 0.210.02

VC
0.320.05
(*)
contralateral 0.170.05
(*)

ipsilateral

0.320.06 0.290.05 0.330.07 0.300.07 0.350.05 0.260.05 0.320.04

(*)
0.270.03 0.210.05 0.290.07 0.170.05 0.300.06 0.220.05 0.250.03
(*)

58

3.3.3 Effects of Corpus Callosotomy on Intrahemispheric

Connectivity

As shown in Table 3.1, no significant difference in and for ROIs

ipsilateral to the seeds was observed between different groups at post-surgery day
7. The scatter plots for the ROIs ipsilateral to the seeds (Figure 3.4a) show that the
clusters of different groups overlapped with each other. Therefore, for animals
that underwent complete or partial callosotomy, intrahemispheric connectivity
was preserved and appeared to be comparable to that of sham control at postsurgery day 7. However, at post-surgery day 28, the of the callosotomy groups
were significantly altered from those of sham control (Table 3.2). In both
callosotomy groups, significantly larger was generally found, not just limited to
the areas where disrupted interhemispheric connectivity was still observed.
Significantly larger was also seen in S2 of the anterior partial callosotomy
group, where restored interhemispheric connectivity was found. These results
indicated that alterations in intrahemispheric connectivity occurred long after
callosotomy and were usually found in areas with altered interhemispheric
connectivity.

3.4 Discussion
In this study, an experimental model of corpus callosotomy was employed to
investigate the role of callosal connections in rsfMRI connectivity. Longitudinal

59

rsfMRI was performed on animals that underwent complete, anterior partial and
posterior partial callosotomy at post-surgery day 7 and day 28. Complete
callosotomy resulted in loss of interhemispheric connectivity in all cortical areas
examined at both day 7 and day 28. Partial callosotomy led to significantly
decreased interhemispheric connectivity at day 7 in the cortical areas whose
primary interhemispheric connections via CC were severed. However, some of the
disrupted connectivity was restored at day 28. On the other hand, intrahemispheric
connectivity was generally found to increase at day 28 in areas where reduced
interhemispheric connectivity was still observed.

3.4.1 Anatomical Connections vs. Dynamical Organization in

rsfMRI

Though our understanding of the brains intrinsic activity is expanding

rapidly, the biological mechanisms underlying rsfMRI connectivity is still under
debate. Given the similarity between the spatial patterns of coherence in rsfMRI
and neuroanatomy, one common hypothesis is that resting-state connectivity is
organized on the basis of anatomical connections. Early electroencephalogram
(EEG) studies showed decreased interhemispheric coherence in patients after
corpus callosotomy or with callosal agenesis (96-98). Recently, a case study on a
6-year-old child before and after complete corpus callosotomy for the treatment of
intractable epilepsy showed loss of interhemispheric connectivity in rsfMRI with
preserved intrahemispheric connectivity (80). Another study reported significantly

60

reduced interhemispheric functional connectivity in motor and auditory cortices in

three patients with callosal agenesis (79). These findings suggest the absence of
callosal connections would lead to diminished interhemispheric connectivity,
indicating the indispensable role of CC in resting-state connectivity. Meanwhile,
studies using rsfMRI in combination with diffusion MRI demonstrated that
resting-state connectivity and structural connectivity are strongly correlated (99101). All these findings support the fundamental role of anatomical connections in
resting-state connectivity.

The other major hypothesis is that resting-state networks are dynamic and
not limited to anatomical connections. It arises from the observations that the
strength of resting-state connectivity varies with different developmental stages
(102), diseases (103, 104) and exercises (105, 106). One early EEG study showed
no substantial difference in the interhemispheric correlation between a
callosotomized patient and matched controls (107). A recent rsfMRI report
showed the presence of bilateral networks in a patient 45 years after complete
transection of forebrain commissures (81). Moreover, predominately bilateral
symmetric resting-state networks were observed in a group of people with
congenital callosal agenesis (82). All these results indicate that the absence of
anatomical connections does not preclude resting-state connectivity. However, the
possibility that resting-state networks may be restored through other anatomical
connections is not excluded in these interpretations.

61

3.4.2 The Role of Callosal Connections in Interhemispheric

Connectivity

Corpus callosotomy is originally a surgical treatment for intractable

generalized seizures (108). The underlying rationale is to prevent electrical
activity from propagating interhemispherically through the CC during epileptic
seizure (109). In other words, the CC not only anatomically connects the
homotopic cortical areas of the cerebral hemispheres, but is also considered as the
major pathway for mutual functional communication between hemispheres.
Therefore, examining resting-state connectivity after complete, anterior and
posterior partial corpus callosotomy would provide direct evidence concerning the
role of callosal connection in the genesis of resting-state connectivity with
minimum effects from development, epilepsy or medication.

Independent component analysis (ICA) can identify the resting-state

networks among the noise without requiring priori defined seed region (93). In the
present study, ICA was employed to determine the spatial distribution of restingstate networks. The ICA results were also used as references for a less subjective
selection of seed regions for seed-based analysis (SBA). As shown in Figure 3.2
and Table 3.1, both ICA and SBA results show a striking loss of interhemispheric
correlations in the cortical areas of complete corpus callosotomy at post-surgery
day 7, indicating the important role of callosal connections in resting-state
connectivity. Since the CC is the major commissural structure interconnecting

62

cortical homologues of the two hemispheres in the mammalian brain (77, 110),
the findings further suggest the coherent LFFs in cortical rsfMRI signal is largely
organized on the basis of anatomical connections via the CC. At post-surgery day
28, the loss of bilateral ICA components (Figure 3.6) and significantly reduced
(Table 3.2) were still seen in animals with complete corpus callosotomy.
Therefore, the results observed at day 7 are mainly attributed to the complete
disruption of callosal connections that interhemispheric resting-state connectivity
relies on rather than acute post-surgical effects.

The CC is organized in a rostrocaudal topographical manner with anterior

fibers connecting frontal areas of the two hemispheres and posterior fibers
connecting caudal cortical structures. All the cortical areas examined in this study,
namely S2, S1, AC and VC, send axonal projections to the contralateral
homologues via different sections of the CC (111-114). For example, S1 sends
callosal projections to the contralateral cortex via the midbody of the CC while
fibers from the VC are confined to the caudal section of the CC as well as the
splenium. In this study, animals with anterior partial callosotomy showed the loss
of bilateral ICA components and significantly reduced in S2 and S1 at postsurgery day 7, where the corresponding callosal fibers were transected. Similar
results were seen in S1, AC and VC of posterior partial callosotomy group. These
findings reveal the strong dependence of resting-state networks on their callosal
connections, further validating the crucial role of anatomical connections.

The reduced functional connectivity in the CPu of the complete callosotomy

63

group was found at post-surgery day 7. Anatomically, all major cortical areas send
projections to the CPu (or striatum) in a bilateral fashion. It has been suggested
that these corticostriatal connections may provide the pathway for the
interhemispheric coordination of striatal activity in mammalian brain (115). In
this study, it might be possible that the disruption of cortical interhemispheric
connectivity by the callosotomy indirectly affect the CPu network via the
corticostriatal projections.

3.4.3

Post-callosotomy

Plasticity

of

Interhemispheric

and

Intrahemispheric Connectivity

At 28 days after the partial callosotomy, the bilateral ICA components were
restored in S2 of the anterior partial callosotomy group and in the AC of the
posterior partial callosotomy group (Figure 3.6). The appeared to be comparable
to that of sham control (Table 3.2). In addition, for the areas still showing two
unilateral ICA components, SBA revealed that the differences compared to sham
control was not as large as that at post-surgery day 7. These results indicate that
the disrupted interhemispheric connectivity in partial callosotomy groups had
recovered, at least partially at the chronic stage of callosotomy. Recently, a
pediatric case report also showed the restoration of bilateral resting-state networks
after partial corpus callosotomy (116). One possibility was that the compensation
might occur through the spared pathways in the remaining callosal fibers since the
restoration was not observed in the complete corpus callosotomy group.

64

Considering the propagation of electrical activity through the CC during epileptic

seizure, anatomical connections, or more specifically axonal connections, are
essentially the pathways for electrical signals or so-called functional activity. In
other words, while anatomical connections are the indispensable framework for
resting-state connectivity, the latter could be mediated by activity on top of the
former.

From the seed-based analysis results at post-surgery day 28 (Table 3.2), the
increased intrahemispheric connectivity was commonly found in terms of
increased and sometimes also decreased in all the cortical areas of animals
with complete callosotomy. In the present study of callosotomy in adult rat brains,
extensive anatomical reorganization may not take place as observed in the
developing brain (117). An early study using horseradish peroxidase technique
reported that the overall pattern of intrahemispheric anatomical connections in the
brain of mice with congenital callosal agenesis is not different from that of normal
mice (118). Therefore, the increased intrahemispheric resting-state connectivity
shall again be predominantly attributed to the activity-induced reorganization.
This finding further substantiates the hypothesis that the resting-state connectivity
can be dynamically organized on the basis of anatomical connections.

3.4.4 Limitations and Future Work

Our preliminary findings suggest the crucial role of anatomical connections

in the genesis of resting-state connectivity. However, the biological mechanisms

65

of spontaneous neuronal activity are complex and further exploration in the

following directions would lead to a better understanding. First, the vasculature
within and around the CC was unavoidably damaged during the callosotomy in
the current study, and this may affect the spontaneous neuronal activity and its
regional connectivity. However, it is almost impossible to transect the axonal
connections without affecting vasculature during callosotomy. Therefore, in order
to further understand the role of anatomical connections in resting-state
connectivity, future work on the effect of altered vasculature on RSNs is
necessary. On the other hand, electrophysiological recording measures the
electrical activity of neurons rather than the hemodynamics in BOLD fMRI.
Therefore, electrophysiology can be used to complement the observations in
rsfMRI after callosotomy without the effect from compromised vasculature.
Second, assessment of the behavioral consequences after corpus callosotomy is
desired to assess the surgical outcomes. It was not performed in the current study
because behavioral tasks may complicate the rsfMRI results. Recent studies
suggest the crucial role of spontaneous activity in mediating behavioral responses
by providing top-down modulation of sensory processing (119, 120). Moreover,
our findings showed that the extent of restoration in different resting-state
networks varied after partial callosotomy. Therefore, future study of the
relationship between the behavioral performance and alterations in resting-state
connectivity after callosotomy may provide new insights into the role of
spontaneous neuronal activity in brain functions. Third, detailed histological
examination will be desired to examine whether any regeneration such as axonal

66

sprouting occurs after callosotomy. Given that the regenerative sprouting period
in the central nervous system is known to be very short and abortive (121), it was
likely not the primary reason that led to the restoration of RSNs at 28 days after
partial transection in the present study. Lastly, our quantitative measurement of
the RSNs was performed using the traditional seed-based analysis, which is
dependent on the seed placement and leads to more diffusive connectivity patterns
than ICA (93). Registering the brains of all animals and performing group ICA
would give more accurate measurement of the resting-state connectivity
differences between different groups. However, such a registration procedure
would be difficult to carry out in the present study due to the limited spatial
resolution, EPI related image distortions and differences in image appearances
between animals due to different transections and subsequent healings.

3.5 Conclusion
In the present study, a significant loss of interhemispheric resting-state
connectivity was observed at both day 7 and 28 after complete corpus callosotomy
compared to that of sham, providing direct evidence of the crucial role of callosal
connections in resting-state connectivity. Meanwhile, partial callosotomy lead to
significantly decreased interhemispheric connectivity at day 7 in the cortical areas
whose primary interhemispheric connections via CC were severed, further
reinforcing the indispensable role of anatomical connections via CC in restingstate connectivity. However, some of the disrupted connectivity in animals with

67

partial CC transection was found to restore at day 28. Such restoration might
result from the compensation that occurred through the spared pathways in the
remaining callosal fibers since the restoration was not observed in the complete
corpus callosotomy group. On the other hand, intrahemispheric connectivity was
generally found to increase at day 28 in areas where reduced interhemispheric
connectivity was still observed. These experimental findings directly support that
anatomical connections via CC play a primary and indispensable role in restingstate connectivity, and that resting-state networks can be dynamically reorganized
or acquired through the remaining anatomical connections.

68

CHAPTER 4 PROTON MAGNETIC

RESONANCE SPECTROSCOPY OF ALTERED
NEUROCHEMICAL PROFILES
4.1 Proton MRS Reveals N-acetylaspartate Reduction in
Hippocampus

and

Cingulate

Cortex

after

Fear

Conditioning
4.1.1 Introduction

Post-traumatic stress disorder (PTSD) is a highly prevalent and severe

anxiety disorder triggered when a vulnerable individual experiences a highly
traumatic event. In the past decade, a number of neuroimaging techniques have
been employed to investigate the underlying functional and structural brain
abnormalities in PTSD patients (122-124). In general, the findings include hyperresponsivity of amygdala (125), hypofunction of anterior cingulate (126), and
hippocampal dysfunction and volume reduction (127, 128) in patients with PTSD
compared to healthy controls.

Proton magnetic resonance spectroscopy (1H MRS) is a non-invasive method

used to assess metabolic changes in living brain and provides biochemical clue to
underlying neural pathology, even when morphological changes are not apparent.
Previously, 1H MRS has been used to evaluate the neurochemical changes in
hippocampus and anterior cingulate cortex in PTSD patients (129, 130). A

69

reduction of the neuronal marker N-acetylaspartate (NAA) in terms of a lower

NAA/creatine ratio or NAA concentration in the hippocampus has been reported
in patients with PTSD both with and without hippocampal volume changes
compared to healthy control subjects (131-133). Decreased NAA level has also
been reported in the anterior cingulate cortex of subjects with PTSD. However,
these changes were mainly observed in the chronic stage of disorder and
compared to control groups. The pathological mechanisms of PTSD are still
poorly understood. In particular, the acute changes occurring following trauma
exposure and development of PTSD cannot easily be captured due to its
complexity and diversity in humans (134). Therefore, animal models are
indispensable in understanding the basic mechanisms of this disorder (135).

The fear conditioning paradigm involves the learned association of an

initially neutral conditioned stimulus (CS), such as a tone or light, with an
aversive unconditioned stimulus (US), usually footshock. After a few such parings,
the CS alone comes to elicit physiological and behavioral fear reactions. Because
fear conditioning has a rapid and long-lasting behavioral effect, it has been
considered as a simple yet valuable model to investigate the neurobiological
mechanisms of learning and memory and to understand the pathological
mechanisms of fear-related disorders, such as PTSD (136, 137). Behavioral
studies indicate that lesions of the amygdala or hippocampus interfere with the
acquisition of conditioned fear (138, 139). Consistent with this, functional
neuroimaging studies have reported altered activation in amygdala and inter-

70

connected regions such as hippocampus, anterior cingulate cortex and insular

cortex are associated with fear conditioning (140, 141). These brain regions are
considered to be key components in a neurocircuitry of fear conditioning (142).
However, to date, acute in vivo neurochemical changes in these structures
precipitated by fear conditioning have not been examined.

In this study, in vivo 1H MRS was employed to investigate the metabolic

changes in hippocampus, cingulate cortex and thalamus of mouse brain before and
after fear conditioning. This study aimed to characterize the longitudinal
neurochemical changes underlying fear conditioning by 1H MRS and to contribute
towards a clear understanding of the neurobiological mechanisms of fear learning
and memory.

4.1.2 Materials and Methods

4.1.2.1 Animals

All experiments were approved by the Institutional Animal Care and Use
Committee. Adult male C57BL/6N mice (N = 12) weighing 23-28g were used in
this study. Animals were housed in groups of four under a 12:12 hour light/dark
cycle and had ad libitum access to food and water. 1H MRS measurements were
performed 1 day prior to fear-conditioning training, 1 day and 1 week after the
training. During the imaging experiments, each mouse was anesthetized with
isoflurane (with 3% induction and 1.0-1.5% maintenance) and kept warm with

71

circulating water at 37oC while under respiratory monitoring.

4.1.2.2 Behavioral procedure

The experimental setup for fear conditioning is custom-made according to

the previous description (143). Briefly, the fear conditioning apparatus comprised
a conditioning chamber (252525 cm) with a grid floor made of 4 mm diameter
stainless steel rods spaced at 8.9 mm apart. The chamber was entirely encased
within a sound attenuating box and masking noise was provided by an extractor
fan. An infrared digital camera was mounted 50 cm directly on the roof of a sound
proof box above the area of interest in each chamber. On the training day, mice
were placed individually into the conditioning chamber for 6-minute acclimation,
followed by 3 paired presentations of a clicker (CS) and footshock (US). A clicker
(30 sec, 4Hz, 80 dB) presented through a speaker initiated each trial. The clicker
co-terminated with an electric footshock onset (2 sec, 0.5 mA) delivered through
the grid floor. The inter-trial interval was 2 minutes. After the final clicker/shock
pairing, the mice remained in the chamber for an additional 2 minutes without
clicker or shock stimuli. The chambers were cleaned with 70% alcohol between
each training session. Contextual and cued tests were performed at one month
after fear conditioning using the method described previously (143). A videotracking system EthoVision XT7 (Noldus, Wageningen, The Netherlands) was
used for monitoring and recording. A freezing response (i.e., absence of
movement except respiratory movement) was measured during the initial 6
minutes (pre-shock, free exploring) and the following 6 minutes (fear
72

conditioning, US and CS paring) of training session as well as contextual and

cued test sessions, respectively. Percentage of freezing duration in each session
were analyzed by one-way ANOVA followed by Bonferroni multiple comparison
post-test in Prism 5.00 (GraphPad Software Inc., California, USA). P < 0.05 was
considered as statistically significant. Behavioral data were presented as mean
standard deviation.

4.1.2.3 1H MRS acquisition

Single voxel 1H MRS experiments were acquired on a 7 T MRI (70/16

PharmaScan, Bruker Biospin GmbH, Germany) using a 23-mm birdcage
quadrature RF coil for both transmitting and receiving. Three T2-weighted (T2W)
scout images were first acquired with a rapid acquisition relaxation enhanced
(RARE) sequence (TR/TE = 4200/36 ms, RARE factor = 8, spatial resolution =
0.1090.1090.48 mm3) for the localization of the voxel of interest (VOI). The
size of VOI in the left hippocampus, the cingulate cortex and the left thalamus
was 1.22.51.6 mm3, 1.21.52.5 mm3 and 222 mm3, respectively. After
first- and second-order localized shimming with a FieldMap based procedure
(144), a full-width half-maximum linewidth of water signal of 15 Hz was
achieved. The water signal was suppressed by variable RF pulses with optimized
relaxation delays (VAPOR). A point-resolved spectroscopy (PRESS) sequence
combined with outer volume suppression (OVS) was used for spectrum
acquisition using TR/TE = 2500/17 ms, spectral bandwidth = 3 kHz, 2048 data
points and 256 averages.
73

4.1.2.4 1H MRS spectral analysis

MR

spectra

were

processed

using

the

jMRUI

software

(http://www.mrui.uab.es/mrui/) as previously described (145). In brief, the raw

data was apodized with a 15-Hz Gaussian filter and phase corrected. The residual
water signal was filtered out using the Hackel-Lanczos singular value
decomposition (HLSVD) algorithm. Chemical shifts of peaks were assigned with
reference to the CH3-group of NAA at 2.02 ppm. Metabolite area under the peak
was quantified by quantum estimation (QUEST) method with subtraction
approach for background modeling. The algorithm QUEST, for optimal fitting of
metabolite basis-set signals to data is based on a semi-parametric approach.
QUEST sequentially uses (1) untangling of the background from the metabolite
signal, (2) separate modeling and (3) a parametric nonlinear least-squares fitting
of the untangled metabolite signal knowing the background (146, 147). The
metabolite parameters were decorrelated from the background with truncation of
initial data points given that macromolecules and lipids signals decay rapidly in
time-domain. The numerical time-domain model functions of twelve metabolites,
including alanine (Ala), aspartate (Asp), choline (Cho), total creatine (Cr), aminobutyrate (GABA), glutamate (Glu), glutamine (Gln), glycine (Gly), lactate
(Lac), myo-inositol (m-Ins), NAA and taurine (Tau), were used as prior
knowledge in QUEST. These metabolite model signals were quantum
mechanically simulated in NMR spectra calculation using operators (NMRSCOPE) for the in vivo experimental protocol. Errors in measurement of noise

74

and inadequate modeling of the overlapping background signal were calculated by

the Cramr-Rao lower bounds (CRLBs), which were used to assess the reliability
of metabolite quantitation. The quantification was considered as relevant only
when the corresponding bound was below 25%. Cr was used as the internal
spectral reference. Differences of NAA/Cr, Cho/Cr, Glu/Cr, Lac/Cr, m-Ins/Cr and
Tau/Cr ratios at different time-points were statistically evaluated using repeated
measures ANOVA test followed by Tukeys multiple comparison post-test with P
< 0.05 considered as significant. All data were presented as mean standard
deviation.

4.1.3 Results

Figure 4.1 shows the freezing responses measured during the initial 6-minute
period (pre-shock) and the following 6-minute period (fear conditioning) of
training session as well as contextual and cued test sessions, respectively.
Significantly enhanced freezing responses (P < 0.05) were observed during the 6minute fear conditioning period compared to the 6-minute pre-shock period for
acclimation, indicating that all mice quickly acquired fear memory. Subsequent
contextual and cued tests showed the freezing duration significantly increased
compared to the pre-shock period (P < 0.001 for both comparisons). These
behavioral results confirm the long-term fear memories have been established
successfully one month after fear conditioning. In addition, statistical evaluation
reveals the freezing responses during the contextual and cued tests were also
significantly elevated with regard to the fear conditioning period of the training
75

session with P < 0.001and P < 0.01, respectively. This finding of exaggerated
trauma-related fear memories in mice suggests the development of PTSD-like
symptoms.

Figure 4.1 Freezing responses measured during the initial 6 minutes (pre-shock,
free exploring) and the following 6 minutes (fear conditioning) of training session
as well as contextual and cued test sessions, respectively. One-way ANOVA
followed by Bonferroni multiple comparison post-test was performed with * P <
0.05, ** P < 0.01, *** P < 0.001. Data were presented as mean standard
deviation.

Figure 4.2 illustrates the typical localization of the VOIs in the left
hippocampus, the cingulate cortex and the left thalamus (solid-line boxes) during
1

H MRS experiment. The VOIs were positioned based on T2-weighted images of

coronal and axial planes. Figure 4.3 shows the representative raw 1H MRS spectra
with QUEST fitting within each VOI from the same mouse before fear
conditioning. Table 4.1 compares the mean metabolite to Cr ratios from all
76

animals (N = 12) at each time-point in the left hippocampus, cingulate cortex and
the left thalamus, respectively. The average CRLBs of Cho, Glu, m-Ins, NAA and
Tau were less than 15%, indicating reliable peak quantitation of these metabolites.
In the hippocampus, significant reduction of NAA/Cr was observed at 1 day postconditioning experiment (P < 0.001) compared to pre-conditioning. At 1 week
post-conditioning, NAA/Cr was still significantly lower (P < 0.05). Cho/Cr
decreased significantly (P < 0.01) at 1 day post-conditioning, but not at 1 week
later. In the cingulate cortex, significant reduction in NAA/Cr (P < 0.01) was
observed at 1 day post-conditioning. In contrast, no significant changes of
metabolites were found in the thalamus except for decreased Glu/Cr (P < 0.05) at
1 day after the fear-conditioning.

Figure 4.2 Typical localization of voxel of interest (VOI) in the hippocampus (a),
cingulate cortex (b) and thalamus (c) (solid-line boxes) on coronal and axial slices of T2weighted images for proton magnetic resonance spectroscopy measurements (L-left; Rright; A-anterior; P-posterior). The size of VOI in the left hippocampus, the cingulate
cortex and the left thalamus was 1.22.51.6 mm3, 1.21.52.5 mm3 and 222 mm3,
respectively.

77

78

inositol.

shown in the top entry. Abbreviations: NAA, N-acetylaspartate; Glu, glutamate; Cr, creatine; Cho, choline; Tau, taurine; m-Ins, myo-

thalamus, respectively. The spectra were acquired from the same mouse before fear conditioning. Residuals of QUEST quantitation are

Figure 4.3 Representative raw spectra (black) along with QUEST fitting (red) of the VOIs in the hippocampus, cingulate cortex and

Table 4.1 Metabolite to Cr ratios and corresponding Cramr-Rao lower bounds (CRLBs)
at 1 day before, 1 day and 1 week after the fear conditioning experiment in the
hippocampus, cingulate cortex and thalamus, respectively. Data from all animals studied
(N = 12) were presented as mean standard deviation (SD).
Pre-conditioning (-1 day)
Metabolites

Post-conditioning (+1 day)

Post-conditioning (+1 week)

Metabolite/Cr

CRLB (%)

Metabolite/Cr

CRLB (%)

Metabolite/Cr

CRLB (%)

Hippocampus
Cho

0.30 0.08

11.27 2.95

0.21 0.07

11.03 2.87

0.28 0.08

10.86 1.95

Glu

0.84 0.28

8.98 2.36

1.00 0.27

6.90 2.64

0.87 0.28

8.56 2.66

m-Ins
NAA

0.61 0.14
1.32 0.30

8.26 1.59
7.19 1.58

0.49 0.15
0.83 0.21

7.23 1.97
7.17 1.57

0.60 0.16
0.98 0.33

7.74 2.51
7.44 1.75

Tau

1.06 0.35

5.94 1.41

0.86 0.32

5.76 1.06

1.14 0.17

6.32 1.18

Cingulate Cortex
Cho
0.33 0.05

12.14 1.72

0.34 0.07

10.90 1.75

0.39 0.03

12.18 2.36

Glu

1.24 0.23

10.11 2.15

1.31 0.25

9.55 2.75

1.12 0.21

9.09 2.05

m-Ins
NAA

1.03 0.31
1.65 0.34

10.67 1.56
10.49 1.66

0.73 0.28
1.43 0.31

10.02 2.13
9.99 2.96

1.06 0.48
1.71 0.37

12.74 3.50
10.62 2.55

Tau

1.50 0.33

9.73 1.65

1.35 0.40

8.88 1.61

1.56 0.31

10.45 2.38

Thalamus
Cho

0.25 0.08

10.01 1.93

0.19 0.07

10.99 3.19

0.25 0.07

9.40 1.92

6.93 1.89

0.87 0.31

6.68 1.35

Glu

1.00 0.29

6.82 2.28

0.79 0.22

m-Ins
NAA

0.50 0.14
1.05 0.22

8.42 2.42
6.98 1.81

0.62 0.15
1.02 0.19

7.45 2.41
6.80 1.38

0.54 0.12
1.04 0.23

6.96 1.43
5.97 0.80

Tau

1.30 0.24

7.52 1.90

1.33 0.26

6.76 1.79

1.37 0.21

5.47 1.15

Repeated measures ANOVA were performed among three time-points with P <0.05,

P <0.01 and P <0.001 compared to pre-conditioning.

4.1.4 Discussion

This study investigates the neurochemical alterations elicited by fear

conditioning in the hippocampus, cingulate cortex and thalamus using in vivo 1H
MRS. It is generally believed that the hippocampus, as part of its functions in
spatial or configural information processing, is involved in contextual fear

79

conditioning (148, 149). In behavioral studies, hippocampal lesions indicate that

the hippocampus plays an important role in mediating the acquisition and
consolidation of memory for the conditioning context (138, 150-152). In rats, the
medial prefrontal cortex consists of four main divisions, which from dorsal to
ventral are the medial agranular, anterior cingulate, prelimbic, and infralimbic
cortex (153). The cingulate cortex is a region of the limbic system reciprocally
connected to the hippocampus (154). In rodents, the cingulate cortex is located in
the dorsal portion of medial prefrontal cortex. Early studies suggested a key
function of cingulate cortex in modulating conditioned fear responses (155).
Therefore, as the key components of the fear circuitry, hippocampus and cingulate
cortex often exhibit functional abnormalities in the in the anxiety disorders (141).
Consistent with this, neurochemical changes in hippocampus and cingulate cortex
are also characteristic of PTSD (130). The thalamus, whose function in the
neurocircuitry of fear conditioning is to relay sensory inputs to amygdala and
cortex (137), was also examined in this study. Metabolic changes induced by fear
conditioning in these regions would not only provide neurobiological information
related to learning and memory, but also give insights into the pathological
mechanisms of neuropsychiatric disorders such as PTSD.

NAA, the major peak seen in 1H MRS spectrum, is a marker of neuronal

density, integrity and health (53). Regional reduction in NAA potentially indicates
diminished neuronal density, neuronal loss or partially reversible cellular
dysfunction (20, 156). Our findings of significantly reduced hippocampal

80

NAA/Cr at 1 day post-conditioning could be explained by neuronal dysfunction at

1 day post-conditioning. Previous studies of patients with multiple sclerosis (157),
focal ischemia (158) and schizophrenia (159) showed that transitory NAA
decrease can reflect a decrease in neuronal viability rather than neuronal loss. In
the present study, the reversible hippocampal dysfunction might be a result of
acute footshock stress. Stressful events, such as inescapable electric footshock, are
known to cause deficits in hippocampal function (160, 161). More importantly, it
is possible that the hippocampal dysfunction induced by acute stress may affect
initial encoding of contextual information associated with a traumatic experience.
This could possibly underlie our observation of exaggerated responses during the
contextual and cued test sessions at one month after fear conditioning. Such
behavioral deficits in identifying safe contexts and explicit memory difficulties
are phenomena paralleled between fear conditioning and PTSD (140). Meanwhile,
significantly reduced hippocampal NAA/Cr at 1 week post-conditioning possibly
indicates reduced neuronal integrity and/or neuronal dysfunction. Previous 1H
MRS studies of PTSD patients showed NAA decrease in the hippocampus with or
without volumetric reduction, suggesting decreased hippocampal neuronal
integrity (132, 162). Recently, investigation of a mouse model of PTSD by means
of Manganese-enhanced MRI demonstrated that traumatic experience lead to
volume loss in the hippocampus, possibly due to shrinkage of axonal protrusions
(163). By applying the similar stimulus to theirs, our results of hippocampal NAA
decrease may therefore reflect reduced neuronal integrity and contribute to
trauma-related PTSD-like symptoms. Moreover, hippocampal cell dysfunction

81

may also contribute to the decreased NAA level at 1week post-conditioning.

Functional imaging studies showed diminished activation of hippocampus in
PTSD patients compared to healthy controls during explicit learning paradigms
(127, 142).

NAA decrease was reported in the cingulate cortex of abused children with
PTSD (162, 164). Moreover, functional imaging studies provide additional
evidence for functional deficiency of anterior cingulate cortex in PTSD patients
(165, 166). However, our findings of decreased NAA/Cr at 1 day postconditioning in cingulate cortex may mainly arise from neuronal dysfunction
induced by acute stress because the NAA level was normalized at 1 week postconditioning. Previous study on a rat model of depression demonstrated acute
stress inhibits long-term potentiation (LTP) at synapses and causes anterior
cingulate cortex dysfunction (167). In the present study, NAA/Cr in thalamus
remained unchanged, possibly because thalamus is merely to relay sensory inputs
during fear conditioning. This also indicates that the NAA changes were specific
to the hippocampus and cingulate cortex.

Cho is involved in phospholipid metabolism and thus acts as a marker for

cellular membrane turnover (168). In this study, decreased hippocampal Cho/Cr at
1 day post-conditioning but not 1 week later may be associated with the acute
traumatic stress induced by the electrical footshock. Cho decrease can be a result
of reduced phospholipase C activity and protein synthesis in the hippocampus
after inescapable shock (169). Previous studies of stress models have shown
82

consistent Cho level reduction (170, 171). However, its role in PTSD is
controversial as both decreased and increased Cho have been reported in clinical
studies (130).

Glu is the major excitatory neurotransmitter in the mammalian central

nervous system. It is recognized as a potential excitotoxin and decreased levels
have been found in many neurodegenerative conditions (172, 173). However, the
Glu decrease detected in thalamus at 1 day post-conditioning is transient since this
change was not seen at 1 week later. Besides, decrease of NAA, the neuronal
marker, was not observed and therefore the Glu decrease might not be related to
neuronal degeneration. Though the concentration of Glu in brain is high, the Glu
signal detected by 1H MRS is low (174, 175). A peak overlap with other
metabolites further limits the accuracy of Glu quantification. Therefore, the
findings of Glu/Cr decrease in thalamus reported here should be regarded as
preliminary and needs further investigation with the help of postmortem
examination.

One limitation to the current study is the lack of measurement of absolute

concentration. The NAA/Cr decrease in hippocampus and cingulate cortex
reported here might imply Cr increase. However, absolute quantification requires
more time than does relative quantification, and one can benefit from absolute
quantification only if all additional referencing steps to identify each metabolite
are executed properly; otherwise, unwanted additional errors may be introduced.
Nevertheless, the reduction was not observed for other metabolites normalized to
83

Cr or in thalamus. Note that such ratio analysis has been employed in many 1H
MRS studies of PTSD, yielding consistent results. Another limitation is that the
amygdala, a critical structure in fear conditioning, was not examined. The small
size of amygdala in mouse makes it difficult to acquire reliable spectrum with
sufficient SNR to detect metabolic changes. As metabolic changes following fear
conditioning would certainly be predicted in this region, future study will be
carried out with more efficient sequences such as chemical shift imaging (CSI). In
the current study, only the left hippocampus was examined. According to the
previous work by Siegmund et al. (176), the left hippocampal NAA level could be
used to predict the susceptibility to trauma which the right hippocampal NAA
level was only partially predictive. Moreover, the lateralized decrease of
hippocampal NAA was also observed in human with PTSD (177). Therefore,
further investigation of NAA change in the right hippocampus with comparison to
that of left hippocampus should also be carried out.

4.1.5 Conclusion
In conclusion, this study investigates the longitudinal metabolic changes
induced by fear conditioning in the hippocampus, cingulate cortex and thalamus
using in vivo 1H MRS. The major findings of significantly lower NAA level in the
hippocampus at both 1 day and 1 week post-conditioning indicate reduced
neuronal dysfunction and/or neuronal integrity, contributing to the trauma-related
PTSD-like symptoms. Significant reduction of NAA was also observed in the
cingulate cortex at 1 day post-conditioning but not at 1 week post-conditioning,

84

possibly due to the transitory neuronal dysfunction induced by acute stress. The
1

H MRS detection of ongoing neurochemical changes elicited by fear

conditioning can shed light on the mechanisms of learning and memory.

Moreover, as a valuable animal model of studying the pathological mechanisms of
PTSD, MRS in conjunction with the fear conditioning paradigm in rodents will
find much needed application to the search for causal mechanisms and novel
interventions in patients with psychiatric disorders. Due to the non-invasiveness
of MRS, this early measurement can be easily expanded to human applications in
the future.

4.2 Proton MRS Reveals Regional Metabolic Changes in

Rat Brain during Pregnancy and Motherhood
4.2.1 Introduction
Pregnancy initiates a series of fundamental behavioral changes, which are socalled maternal behaviors, in virtually all female mammals. These behavioral
modifications are elicited by the dramatic hormonal and neurochemical changes
that accompany the onset of motherhood. The reproductive hormones,
prominently estrogen and progesterone, are known to remodel the neuronal
structure and function in many regions of the female brain (178). The affected
regions are not limited to those involved in regulating basic maternal behaviors,
such as the medial preoptic area of the hypothalamus (179, 180). Pregnancy also

85

initiates changes in the brain regions that control learning, memory and responses
to fear and stress (181). For example, late-pregnant and lactating rats showed
increased hippocampal dendritic spine density than that of the nulliparous females
(182). Consistently, previous behavioral experiments also demonstrated that
reproductive and/or maternal experience enhances spatial learning and memory in
rats (183) while alleviating fear and stress (184). The improvement of these
support behaviors provides the offspring a better chance of survival. More
importantly, the cognitive benefits appear to be long-lasting, which has been
suggested to be one of the key factors driving the evolution of the mammalian
brain (185). Therefore, the neurochemical underpinnings of the maternal
behaviors are of immense interest.
Previous MRI investigation of pregnant patients has focused on evaluating
obstetrical, placental, or fetal abnormalities. There is limited number of
neuroimaging studies on maternal brain largely due to the potential safety issues.
Therefore, animal models are indispensable in understanding the basic
mechanisms of the behavioral changes associated with pregnancy and motherhood.
Proton magnetic resonance spectroscopy is a powerful tool that provides
noninvasive quantification of brain metabolites, and offers biochemical
information from distinct brain regions. Therefore, it is often used to monitor
neural processes such as development (186, 187), brain disorders (188)and their
treatment (20).
In this study, we aim to investigate the neurobiological alterations underlying
behavioral changes during pregnancy and motherhood, especially in regions

86

related to learning and memory such as hippocampus and in the structures

involved in alertness and attention such as thalamus. Longitudinal 1H MRS during
pregnancy and motherhood was performed to evaluate the regional metabolic
changes in the hippocampus and thalamus of maternal brain.

4.2.2 Materials and Methods

All procedures were approved by the Institutional Animal Care and Use
Committee. A total of 24 adult female Sprague-Dawley rats weighing 250-300g
initially were used in this study. The animals were housed in a temperaturecontrolled room with a 12:12 hour light/dark cycle and had ad libitum access to
food and water. Pregnant primiparous rats (N=15) were studied at 3 days before
mating (Baseline), gestational day 17 (G17), lactation day 7 (L7) and postweaning day 7 (PW7). Age-matched nulliparous female rats (N=9) served as nonpregnant controls which were examined at the same timepoints with the pregnant
rats. A schematic diagram showing the timeline of experimental procedures is
illustrated in Figure 4.4.

87

Figure 4.4 A schematic diagram summarizing the timeline of experimental procedures.

1

H MRS measurements were performed on pregnant primiparous rats (N=15) at 3 days

before mating (Baseline), gestational day 17 (G17), lactation day 7 (L7) and postweaning day 7 (PW7). Age-matched nulliparous female rats (N=9) which served as nonpregnant controls were examined at the same timepoints with the pregnant rats.

Single voxel 1H MRS experiments were performed on a 7 T MRI scanner

(70/16 PharmaScan, Bruker Biospin GmbH, Germany) using a 72 mm birdcage
transmit-only RF coil and an actively decoupled receive-only quadrature surface
coil. Animals were anesthetized with isoflurane (with 3% induction and 1.2-1.5%
maintenance) and kept warm with circulating water at 37oC. Continuous
monitoring of the respiration rate, heart rate, oxygenation saturation and rectal
temperature was performed and vital signs were maintained within normal
physiological ranges. Three T2-weighted (T2W) scout images were first acquired
with a rapid acquisition relaxation enhanced (RARE) sequence (TR/TE = 4200/36
ms, RARE factor = 8, spatial resolution = 0.1090.1090.48 mm3) for the
localization of the voxel of interest (VOI). A 242 mm3 VOI was placed over
the left hippocampus and another 333 mm3 VOI was centered at the left
thalamus. After first- and second-order localized shimming with a FieldMap
based procedure (144), a full-width half-maximum linewidth of water signal of
15 Hz was achieved. The water signal was suppressed by variable RF pulses with
optimized relaxation delays (VAPOR). A point-resolved spectroscopy (PRESS)
sequence combined with outer volume suppression (OVS) was used for spectrum
acquisition using TR/TE = 2500/20 ms, spectral bandwidth = 3 kHz, 2048 data

88

points and 256 averages.

MR

spectra

were

processed

using

the

jMRUI

software

(http://www.mrui.uab.es/mrui/) (189). The raw data was apodized with a 15-Hz

Gaussian filter and phase corrected. The residual water signal was filtered out
using the Hackel-Lanczos singular value decomposition (HLSVD) algorithm.
Chemical shifts of peaks were assigned with reference to the CH3-group of Nacetylaspartate (NAA) at 2.02 ppm. Metabolite area under the peak was quantified
by quantum estimation (QUEST) method with subtraction approach for
background modeling. The metabolite parameters were decorrelated from the
background with truncation of initial data points given that macromolecules and
lipids signals decay rapidly in time-domain. The numerical time-domain model
functions of twelve metabolites, including alanine (Ala), aspartate (Asp), choline
(Cho), creatine (Cr), -aminobutyrate (GABA), glutamate (Glu), glutamine (Gln),
glycine (Gly), lactate (Lac), myo-inositol (m-Ins), NAA and taruine (Tau), were
used as prior knowledge in QUEST. These metabolite model signals were
quantum mechanically simulated in NMR spectra calculation using operators
(NMR-SCOPE) for the in vivo experimental protocol. Errors in measurement of
noise and inadequate modeling of the overlapping background signal were
calculated by the Cramr-Rao lower bounds (CRLBs), which were used to assess
the reliability of metabolite quantitation. The quantification was considered as
relevant only when the corresponding bound was below 25%. Total Cr (creatine
and phosphocreatine) was used as the internal spectral reference. Longitudinal
comparison within the same group was performed using repeated measures

89

ANOVA test. Differences between the two groups were evaluated using twotailed Students t-test. In all cases, differences were considered statistically
significant if P < 0.05. All data were presented as mean standard deviation.

4.2.3 Results
Figure 4.5 illustrates the typical 1H MRS spectra of the VOIs in the
hippocampus and thalamus, respectively. Figure 4.6 compares the mean
metabolite to Cr ratios from animals in the pregnant and non-pregnant groups at
each time-point in hippocampus and thalamus. No significant differences were
observed between the two groups at baseline. At gestational day 17 (G17), higher
NAA/Cr was observed in both the hippocampus (P<0.01) and thalamus (P<0.01)
of the pregnant rats compared to that of non-pregnant controls. Meanwhile,
hippocampal Tau level was significantly lower (P<0.01) in pregnant group than
that of non-pregnant group. This decrease was also observed (P<0.05) at lactation
day 7 (L7). At post-weaning day 7, animals in the pregnant group showed a lower
Cho/Cr in the hippocampus. In thalamus, Lac level was found to be lower (p<0.05)
in non-pregnant group at G17. At both G17 and PW7, pregnant rats showed
increased

thalamic

m-Ins/Cr

ratio

(P<0.01

and

P<0.05,

respectively).

Longitudinal comparisons of the metabolite to Cr ratios at different timepoints

from the same group showed consistent results (Figure 4.7). No significant
changes were detected in the non-pregnant group.

90

Figure 4.5 Typical in vivo 1H MRS spectra of voxel of interest (VOI) in the hippocampus
(a) and thalamus (b) (solid-line boxes) on coronal slices of T2-weighted images for 1H
MRS measurement (L-left; R-right) from the same rat.

91

Figure 4.6 Comparisons of metabolite to Cr ratios between the pregnant and

non-pregnant rats in the hippocampus (left column) and thalamus (right column)
at each timepoint. Mean SD presented. Unpaired t-tests were performed with *
P<0.05, ** P <0.01.

4.2.4 Discussion and Conclusion

This study monitored the neurochemical alterations accompany pregnancy

and motherhood in the hippocampus and thalamus longitudinally by in vivo 1H

92

MRS. The hippocampus is the key structure that governs learning and memory.
The thalamus, as the brains relay center, is not only an important part of the
neural pathways of maternal behavior system, but also involved in regulating
alertness and attention. Metabolic changes in these regions would provide
neurobiological information related to the maternal behaviors brought out by the
hormonal fluctuations.

NAA, the major peak seen in 1H MRS spectrum, is a marker of neuronal

density, integrity and health (53). Previously, it has been shown that the steroid
hormones of pregnancy, particularly estradiol, can alter the hippocampal neurons,
leading to significantly more neuronal dendrite spines in the female rat (178).
Further, estrogen and progesterone stimulate the cell proliferation rate in
hippocampal region (190). In this study, the significantly higher NAA level
observed in the hippocampus of late pregnant rats (G17) may result from the
increased neuronal density in this region. This neuronal modification in the
hippocampus of pregnant rats may therefore contribute to better navigation skills
involved in supporting behaviors such as foraging.

Tau is the major osmolyte in the brain. Previous ex vivo measurements

showed pregnancy was associated with a fall in taurine concentration in all tissues,
resulting from an effect of steroid hormones.(191). A lower Tau/Cr in
hippocampus of pregnant rats was also found in this study. This difference was
not detected in thalamus possibly due to the low concentration of Tau in this
region compared to hippocampus. Cho is involved in phospholipid metabolism
93

and thus acts as a marker for cellular membrane turnover (168). Reduced level of
Cho in the maternal brain during pregnancy has been observed in humans (192),
reflecting high fetal demands. Previous studies of stress models have shown
consistent Cho level reduction (170, 171). A decreased level of hippocampal
Cho/Cr has also been reported in patients with depression in previous 1H MRS
studies (193, 194). Therefore, lower Cho/Cr at PW7 observed in this work might
potentially arise from the anxiety of separation from pups (195). Lac is a substrate
for energy which is responsible for anaerobic metabolism, interruption of
oxidative phosphorlyation and anaerobic glycolysis (196). It has been reported
that possible source of increased Lac concentrations is hyperventilation (197).
Therefore, the raised Lac level in thalamus might be related to the
hyperventilation of pregnancy due to high oxygen consumption (198).

m-Ins is considered to be a marker for gliosis (199). It is also involved in

osmoregulation (200). Previous 1H MRS studies showed m-Ins may play an
essential role in the mechanisms of brain adaptation and plasticity (201, 202).
Sustained higher level of m-Ins/Cr in thalamus may provide new insights into the
plasticity of maternal brain.
In conclusion, this study shows the metabolic differences between pregnant
and non-pregnant rats using in vivo 1H MRS. This non-invasive approach enables
longitudinal monitoring of biochemical alterations during different stages of
pregnancy and motherhood. The findings of this study provide neurochemical
evidence of the behavioral changes associated with pregnancy and motherhood.

94

Such detailed information may be important when assessing cerebral pathology

during pregnancy with MRS.

95

CHAPTER 5 ANGANESE ENHANCED

MAGNETIC RESONANCE IMAGING OF
MORPHOLOGICAL AND FUNCTIONAL
BRAIN CHANGES
5.1 MEMRI Study of Neonatal Hypoxic-ischemic Injury
in the Late Stage
5.1.1 Introduction

Neonatal hypoxic-ischemic (HI) cerebral injury is a major cause of neuronal

damage which is linked to chronic neurological disorders associated with
developmental deficits in motor, sensory, visual or cognitive functions (203-205).
Significant recovery is known to occur weeks after the initial H-I insult and last
for at least 6 months (206), indicating that the regenerative processes toward the
functional restitution continues late after the HI insult when the infarct lesion
stabilizes. These processes include hyper proliferation of astrocytes, neurogenesis
and regeneration of synapses (207, 208). Understanding the long-term
consequences of chronic neonatal HI insults is particularly useful for monitoring
the vulnerability and progression of lesions, and for promoting a better
understanding of the recovery process after neonatal brain injury.
Divalent manganese ion (Mn2+), as a calcium analog, has been introduced as
a valuable cellular contrast agent for tracing neuronal pathways, for the
enhancement of neural architecture and for brain function (22, 23, 209). Several

96

previous studies have reported the use of manganese-enhanced MRI (MEMRI) to

detect neurodegeneration during the acute-phase of neonatal HI cerebral insult (26,
27). However, the long-term outcomes of severe neonatal hypoxic-ischemic brain
injury in both ipsi-lesional and contra-lesional hemispheres was not explored.
This study aims to employ in vivo MEMRI to investigate the cellular alterations at
the late stage after HI insult.

5.1.2 Materials and Methods

5.1.2.1 Animal Preparation

All animal experiments were approved by the local institutional animal ethics
committee. Sprague-Dawley rats (7-day-old, 12-16 g, N=13) were divided into 2
groups. Animals in Group 1 (N=7) were subjected to HI injury while animals in
Group 2 (N=6) were age-matched normal controls. Unilateral common carotid
artery occlusion combined with hypoxia was used to produce neonatal HI cerebral
injury. Specifically, pregnant Sprague-Dawley rats were first obtained
approximately 2 d before parturition, and their litters were culled to nine to 12
pups. Neonatal rats were kept with their dams in a regular light/dark cycle (lights
on from 8 AM to 8 PM) till day 7 (P7) after birth. The P7 rats, each weighing 12
16 g, then underwent unilateral ligation of the left common carotid artery under
isoflurane anesthesia. The surgery required 5-6 min for each pup. Following 10 15 min for observation in an incubator at 34C, the pups were returned to their
dams for 1.5 - 2 h nursing, once regaining normal movement. Thereafter, they

97

were placed in a hypoxic chamber with 8% O2 and 92% N2, maintained at an

ambient temperature of 3637C for 2 h. Among a total of seven P7 rats
underwent HI insult, one showed mild cerebral HI injury and was excluded from
the study. MEMRI was performed at 5 months old for both groups. MnCl2
solution (60 mg/kg, 100 mM) was slowly infused intraperitoneally at a rate of 15
l/min.

5.1.2.2 MRI Protocols

All MRI experiments were conducted using a 7 T Bruker scanner with a 38

mm volume coil. During the MRI scan, rats were anaesthetized with isoflurane
(3% induction and 1.5% maintenance) with respiratory monitoring and kept warm
under circulating water at 37 oC. Scout images were first acquired in three planes
with a RARE sequence to position the subsequent high-resolution MR images
along standard anatomical orientations in a reproducible manner. 2D T1-weighted
images and 2D T2-weighted images were acquired prior to Mn2+ administration
and at 1, 7 days after the injection. T1W images were collected with a RARE
sequence using FOV = 3.2 x 3.2 cm2, matrix resolution = 256 x 256, slice
thickness = 1 mm, number of slices = 10, TR/TE = 400/7.5 ms, RARE factor = 4
and NEX = 16; T2W images were acquired using the same voxel dimensions and
slice geometry with TR/TE = 4200/38.7ms, RARE factor = 8 and NEX = 2. A
schematic diagram showing the timeline of experimental procedures is illustrated
in Figure 5.1.

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Figure 5.1 A schematic diagram showing the timeline of experimental procedures.

Animals in Group 1 (N=6) were subjected to hypoxic-ischemic (HI) insult at postnatal
day 7 while animals from Group 2 (N=6) were served as controls. Manganese-enhanced
MRI (MEMRI) measurements were performed prior to Mn2+ administration and at 1, 7
days after the injection.

5.1.2.3 Data Analysis

SPM5 (Statistical Parametric Mapping, Wellcome Department of Imaging

Neuroscience, Functional Imaging Laboratory, London, UK) was utilized for
coregistration of brain images at different time points within the same animal.
Percentage change maps were computed using the coregistrated T1W image sets
for visualization and quantification of the signal intensity (SI) changes in the
brains. ROIs were manually defined and SI was measured using ImageJ v1.40g
(Wayne Rasband, NIH, USA). Mean SI values from each ROI were compared
between the two groups using two-tailed Students t-test.

5.1.2.4 Histology

In addition, two H-I rat was sacrificed for histological analysis after MEMRI
investigation. It was transcardially perfused with 4% paraformaldehyde. 10 m
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sections were prepared and stained with glial fibrillary acidic protein (GFAP) as
markers for gliosis and with hematoxylin and eosin (H&E) to detect general
morphological abnormalities.

5.1.3 Results

Figure 5.2 Representative T1W images of Group 1 (HI) and Group 2 (normal control)
before and after Mn2+ administration. The yellow lines indicate the manually defined
regions of interest that were used for comparisons of signal intensity.

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Figure 5.2 shows typical T1W images acquired prior to Mn2+ administration
and at 1, 7 days after the injection from one animal in each group, respectively.
One day after Mn2+ injection, conspicuous Mn-induced SI enhancement in T1W
images was observed in the peri-lesional regions in Group 1 (HI), especially in the
remaining thalamus (red arrows), cingulate cortex (yellow arrows) and
retrosplenial cortex (green arrows) ipsi-lateral to the lesioned hemisphere.
Meanwhile, neither the contra-lesional brain nor the normal brain (Group 2)
exhibited the same contrast enhancement in the same area.

Figure 5.3 Percentage change maps (from pre-injection) at day 1 and day 7 computed
from coregistered images, directly illustrating the significant SI increase in peri-lesional
region at day 1 (white arrows) and relatively slow clearance in contra-lesional thalamus
(black arrow).

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Figure 5.4 Mean SD illustrates SI changes in different regions after Mn2+

administration in peri-lesional area: ipsi-lateral thalamus (top left), ipsi-lateral cingulate
cortex (top right) and contra-lateral thalamus (bottom). All the post-injection SI was
normalized by the pre-injection SI within the same animal, respectively, before
calculating mean SD. T-tests between Group 1: HI (N=6) and Group 2: normal control
(N=6), *P<0.05, ** P <0.01 and ++ P=0.09.

Figure 5.3 illustrates percentage change maps (from pre-injection) at day 1

and day 7. In addition to the greater enhancement in the peri-lesional rim at day 1
(white arrows), contra-lesional thalamus demonstrated a delayed clearance at day
7 (black arrow), compared to normal brain. These Mn-induced SI changes were
quantified and compared the two groups and shown in Figure 5.4. Mn-induced
enhancemanet was significantly higher at 1 day after injection in the thalamus
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(P<0.05) and cingulate cortex (P<0.01) ipsi-lateral to the lesion site compared to
that of the normal animals. Contra-lateral thalamus exhibited a relative slower
clearance of Mn2+ enhancement at day 7 with marginal significance P=0.09. The
higher T1W SI enhancement in the peri-lesional rim was spatially correlated with
the hyperproliferation of astrocytes as observed in GFAP staining (Figure 5.5).

Figure 5.5 GFAP staining, illustrating the hyper cellular density of astrocytes in the perilesional region in thalamus (middle column) and cingulate cortex (right column) as
compared to the contra-lesional hemisphere (left column).

5.1.4 Discussion and Conclusion

In the present study we demonstrated the enhanced detection of cellular

alterations long after neonatal HI injury using longitudinal MEMRI. It has been
suggested that HI injury in the neonatal brain will lead to both acute and delayed
neurodegeneration in which the morphological changes of cells are determined by
time, region, and cell types (210). Determining the long-term consequences is of

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great importance in understanding and promoting the functional outcome of the

brain lesion even after the initial compensatory and reparative phases. The
increased Mn enhancement pattern was observed 5 months after HI insult, which
may attribute to lasting consequences of remodeling following neonatal HI insults.

Manganese ion, which can be taken by biological cells via voltage gated
calcium channels, has been introduced as a valuable cellular contrast agent for
tracing neuronal pathways, for the enhancement of neural architecture and for
brain function (23). Several previous studies have reported the use of MEMRI to
detect longitudinal neurodegeneration such as brain ischemia (27, 211). In this
study, significantly increased Mn uptake in the peri-lesional rim was found and
such MRI enhancement spatially correlated with the hyperproliferation of
astrocytes as observed in GFAP staining. Previous MEMRI study of ischemic
brain also showed that areas with increased Mn enhancement corresponded best
with areas with high concentrations of activated microglia (26). Therefore, the
MEMRI hyper-intensity in peri-lesional region at day 1 indicates the existence of
reactive gliosis and microglial activation in the late phase after HI injury. In
addition, it was found that astroglial cells would secrete reactive oxygen species,
resulting in an upregulation of Mn-superoxide dismutase (MnSOD) and glutamine
synthetase (GS) (27, 212), which are enzymes that Mn binds to. The upregulation
of MnSOD and GS as well as other cellular alterations in the peri-lesional areas,
such as cell proliferation and neurogenesis in the subventricular zone and periinfarct striatum (207, 208) and the regeneration of synapses and axonal sprouting

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(213-215), may also contribute to the Mn-induced enhancement observed in MRI.

Contra-lesional thalamus exhibited a slower clearance of Mn enhancement, which
might result from the sustained Mn accumulation in contra-lesional thalamus as a
result of reinnervation after injury (30, 216).

In summary, the experimental findings provide information regarding the

cellular alterations after chronic neonatal hypoxic-ischemic injury. Mn2+ might be
a useful in vivo MRI probe for understanding the long-term effects of cerebral
injury. Such MEMRI approach may be useful in investigation of post-injury
cellular events and functional reorganization.

5.2 In Vivo Manganese-enhanced MRI of Conditioned

Fear Response
5.2.1 Introduction

Various neuroimaging techniques have been employed to measure structural

and functional brain alterations in Post-traumatic stress disorder (PTSD) (139).
However, the changes can hardly be fully captured due to its complexity and high
diversity in human (134). Fear conditioning (FC) paradigm is widely used to
reveal the pathomechanisms of PTSD (139) and to study the neural basis of
learning and memory (217). It allows an organism to quickly assess and react to

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stimuli and predict danger. However, it was mostly restricted to behavioral or

histological investigation of brain regions associated with conditioned fear (218,
219). Recent studies have been using BOLD-fMRI to detect stimulus-related
neural activity in living animals (36). However it is technically difficult to study
conditioned fear responses with BOLD-fMRI due to subject motion and poor
spatial resolution. Paramagnetic manganese ion (Mn2+) is known to enter
synaptically activated neurons through voltage-gated calcium channels. Therefore,
it is a valuable cellular MRI contrast agent for visualization of neural architecture
(23) and for detection of brain activities (209). Based on this, manganeseenhanced MRI (MEMRI) can be used to assess the neural circuitry involved in
sensory paradigms (33) and to investigate more complex cognitive and emotional
processes such as fear conditioning longitudinally with higher spatial resolution.
Previously, MEMRI was used to assess the neural circuitry involved in
unconditioned sensory paradigms (31). In this study, we aim to use in vivo
MEMRI to detect the acute neuronal response after fear conditioning.

5.2.2 Methods

Animal Preparation: Adult C57BL/6N mice (N=16, 90-100 days old,

weighing 23-28g) were divided into 2 groups. Animals in Group 1 (N=8) were
subjected to fear conditioning (FC), followed immediately by intraperitoneal
injection of a 100 mM solution of MnCl2 in isotonic saline at a dose of 45 mg/kg.
Group 2 (N=8) served as controls with no fear conditioned stimulus but the same
Mn administration dosage. MRI scans were performed on all the mice before and
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24 hours after Mn injection.

Conditioning protocol: On the training day, mice were placed individually

into a conditioning chamber (252525cm3) for 6-minute acclimation, followed
by 3 paired presentations of a clicker (CS) (30 sec, 4Hz, 80 dB) and footshock
(US) (2 sec, 0.5 mA). The inter-trial interval was 2 min and an additional 2-min
rest after the final clicker/shock pairing in the chamber, yielding a total training
time of 13min30s. The chambers were cleaned with 70% alcohol between each
training session.

MRI Protocols: All MRI experiments were performed on a Bruker 7 T

scanner with a mouse brain coil. During the MRI scan, mice were anaesthetized
with isoflurane (2.5% induction and 1.5% maintenance) with respiratory
monitoring and kept warm under circulating water at 37 oC. T1W images were
collected with a RARE sequence using FOV = 25.625.6mm, MTX = 256256,
slice thickness = 0.5 mm, number of slices = 10, TR/TE = 420/7.5 ms, RARE
factor = 4 and NEX = 64; T2WIs were acquired using the same voxel dimensions
and slice geometry with TR/TE = 4200/36ms, RARE factor = 8 and NEX = 2.

Data Analysis: Pre- and post-injection T1W images from different animals
were first co-registered together with T2WIs as references using AIR5.2.5.
Averaged ratio maps between the two groups were computed using the
coregistrated image sets by Matlab7 for visualization and quantification of the
signal intensity (SI) differences. ROIs were manually defined according to mouse

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brain atlas and signal intensity was measured using ImageJ. Total distance and
freezing behavior (absence of movement except respiration) were automatically
measured using EthoVision XT7. Two-tailed unpaired t-test was performed
between the two groups with P< 0.05 which could be considered as statistically
significant.

Figure 5.6 Schematic diagrams showing the timeline of experimental procedures (a), the
fear conditioning paradigm (b) and setup (c).

5.2.3 Results

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Figure 5.7 Freezing response and locomotor activity measured during the initial
habituation period and the following fear conditioning (FC) period. Significantly
increased freezing duration and decreased locomotor activity (expressed as total distance
traveled in cm) confirmed that the mice acquired associative learning with the electric
footshock. Two-tailed Students t-tests were performed between the habituation period
and fear conditioning period: * P<0.05 ** P <0.005.

Figure 5.7 shows decreased locomotor movement and increased freezing

duration during FC training confirmed the mice acquired associative learning with
aversive stimulus quickly. Cued and contextual fear memories were confirmed
latterly (data not shown). With the present dosage of Mn administration, no
animals were found to have significant Mn-induced behavioral disturbances due
to its toxicity. The Mn-enhanced T1W images in Figure 5.8 show that the animals
in Group 1 (FC) exhibited a higher Mn uptake in several brain regions as
indicated

by

the

pointing

arrows,

including

amygdala,

hippocampus,

paraventricular nucleus of hypothalamus (PVH), and cingulate cortex comparing

to the normal controls. The color-coded ratio map between the two groups further
delineated the differences of Mn-enhancement in these regions.

109

Figure 5.8 Averaged post-Mn T1W images from Group 1 (FC) and Group 2 (control)
with the ratio maps showing the percentage signal differences between them. Enhanced
Mn-uptake was observed in amygdala (yellow arrows), hippocampus (red arrows),
paraventricular nucleus of hypothalamus (green arrows) and cingulate cortex (purple
arrows) in FC animals.

110

Figure 5.9 compares the signal intensity of T1W images before and after Mn
injection between the two groups using ROIs covering amygdala (Amyg),
hippocampus (Hip), paraventricular nucleus of hypothalamus (PVH), cingulate
cortex (Cg), and whole brain. A significantly higher Mn-uptake was found in FC
animals in amygdala, hippocampus and PVH region. Cingulate cortex also had
relatively higher post-Mn signal intensity with marginal significance (P =0.058).
No distinct signal difference was found either prior to Mn-injection or in global
enhancement after Mn-injection between the FC animals and controls, indicating
the regional enhancement was specific to fear-conditioning.

Figure 5.9 T1W signal intensity changes (Mean SD) before and after Mn injection were
compared between the two groups using ROIs covering amygdala (Amyg), hippocampus
(Hip), paraventricular nucleus of hypothalamus (PVH), cingulate cortex (Cg) and the
entire brain. Two-tailed t-test was performed with * P < 0.05, ** P <0.01

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5.2.4 Discussion and Conclusion

This study examines the neuronal activation induced by fear conditioning

using in vivo MEMRI. Mn2+ is known to enter synaptically activated neurons
through the voltage-gated calcium channels (23). Higher post-Mn signal intensity
in amygdala, hippocampus, paraventricular nucleus of hypothalamus, cingulate
cortex of FC animals compared to controls indicates additional activation-induced
Mn-accumulation in neural cells of these regions. All these structures were closely
related to the process of conditioned fear (220). The amygdala is a key component
in the neurocircuitry underlying fear conditioning where crucial neural changes
occur. Pharmacological studies has shown that amygdala is involved in mediating
the acquisition of fear associations and the formation and maintenance of longterm memories of those associations, especially when an auditory CS is presented
(137). Meanwhile, the fear memory is stored in the hippocampus. It is generally
believed that the hippocampus, as part of its functions in spatial or configural
information processing, is involved in contextual fear conditioning (148, 149). In
behavioral studies, hippocampal lesions indicate that the hippocampus plays an
important role in mediating the acquisition and consolidation of memory for the
conditioning context (138, 150-152). The cingulate cortex is a region of the limbic
system reciprocally connected to the hippocampus (154). Early studies suggested
a key function of cingulate cortex in modulating conditioned fear responses (155).
Therefore, as the key components of the fear circuitry, amygdala, hippocampus
and cingulate cortex are expected to exhibit functional activations elicited by FC

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paradigm. Paraventricular nucleus of hypothalamus regulates the autonomic and

neuroendocrine responses to stress. The significantly higher Mn-uptake in FC
animals in amygdala, hippocampus and PVH may due to the fear-induced cellular
hyperactivity in these regions. Exceeding the pain threshold (221) with the
stimulation current during footshock may also result in the greater Mnenhancement in PVH (222) in the FC Group. Previous immunohistochemistry
studies also showed high density c-Fos immunoreactivity in the above regions
after FC paradigm (220, 223), indicating increased neural activity in response to
conditioned-fear.

One potential limitation of the present study is the relatively narrow window
between the contrast agent dosage and the sensitivity of detection due to the
toxicity of Mn. Systemic administration of Mn provides reasonable contrast
without disrupting hippocampus-dependent behavior (24, 25). Recent MEMRI
study of freely behaving rats has showed promising results using systemic
administration combined an infusion produce (32). This approach can minimize
adverse locomotor effects, while achieving a sufficient contrast agent
concentration for functional mapping. Currently, the achievable enhancement is
also limited by the ability of Mn across the blood-brain barrier (BBB) into the
central nervous system (224). Disrupting the BBB will enable a rapid uptake of
Mn to the whole brain (225), especially to the cortical regions whose neuronal
activity is of interest. This method can provide a more accurate mapping of
functional changes with efficiency. Further studies using MEMRI with an

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optimized manganese delivery that avoids toxic effects can be applied for
longitudinal studies in behaving animals.

In conclusion, this study demonstrated that the neural response after the
induction of conditioned-fear can be detected in most brain regions that are highly
related to the process of fear by in vivo MEMRI. The results provide insights to
neurocircuitry involved in fear conditioning and consolidate the capability of
MEMRI as an in vivo probe for mapping neural activity following fear
conditioning. The MEMRI findings in conjunction with the results from MRS
study in Chapter 4 could further provide a comprehensive understanding in the
mechanisms of fear learning and memory.

114

CHAPTER 6 GENERAL CONCLUSIONS AND

FUTURE STUDIES
In this thesis, several MRI techniques and their applications were
investigated in the rodent brains at 7 Tesla. Benefiting from the higher SNR as
well as better spatial, temporal and spectral resolution provided by high field, in
vivo assessments of the functional, metabolic and structural changes using fMRI,
rsfMRI, 1H MRS and MEMRI were successfully demonstrated. The multiparametric MRI has proved to be a valuable neuroimaging tool and provided
comprehensive information regarding the alterations in rodent brains under
normal and manipulated conditions.

To improve the functional sensitivity and spatial precision, a novel fMRI

method was proposed using bSSFP imaging technique with intravascular
susceptibility contrast agent MION in Chapter 2. This new approach provides
sensitivity comparable to conventional CBV-weighted EPI-based fMRI but with
no severe image distortion and signal dropout. Robust negative responses during
visual stimulation were observed and activation patterns were in excellent
agreement with known neuroanatomy. The signal was sensitized to CBV changes.
As a promising alternative to conventional CBV-weighted fMRI, it is particularly
suited for fMRI investigation of animal models at high field. The feasibility of this
new fMRI method was demonstrated with single slice acquisition. Further
improvement by adopting fast 3D bSSFP (45) will gain full brain coverage and
SNR. This technique can be used to investigate the brain regions that are

115

vulnerable to the susceptibility and have severe image distortion in EPI images.

In Chapter 3, the relationship between anatomical connections and restingstate fMRI connectivity has been explored using a well-controlled animal model
of corpus callosotomy. The experimental findings directly support that anatomical
connections via CC play a primary and indispensable role in resting-state
connectivity, and that resting-state networks can be dynamically reorganized or
acquired directly or indirectly through the remaining anatomical connections.
However, the underlying biological mechanisms are complex and further
exploration in the following directions would lead to a better understanding. First,
assessment of the behavioral consequences after corpus callosotomy is desired to
assess the surgical outcomes. It was not performed in the current study because
behavioral tasks may complicate the rsfMRI results. Recent studies suggest the
crucial role of spontaneous activity in mediating behavioral responses by
providing top-down modulation of sensory processing (119, 120). Moreover, our
findings showed that the extent of restoration in different resting-state networks
varied after partial callosotomy. Therefore, future study of the relationship
between the behavioral performance and alterations in resting-state connectivity
after callosotomy may provide new insights into the role of spontaneous neuronal
activity in brain functions. In addition, electrophysiological examination of the
coherence in the studied brain regions with or without callosotomy will
complement the rsfMRI findings and further reveal the origins of rsfMRI signal.
Chapter 4 investigates the longitudinal metabolic changes induced by fear

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conditioning and pregnancy using in vivo 1H MRS. Exposure to the fear

conditioning paradigm, significantly lower NAA level in the hippocampus at both
1 day and 1 week post-conditioning indicate reduced neuronal dysfunction and/or
neuronal integrity, contributing to the trauma-related PTSD-like symptoms.
Significant reduction of NAA was also observed in the cingulate cortex at 1 day
post-conditioning but not at 1 week post-conditioning, possibly due to the
transitory neuronal dysfunction induced by acute stress. The 1H MRS detection of
ongoing neurochemical changes elicited by fear conditioning can shed light on the
mechanisms of learning and memory. Moreover, as a valuable animal model of
studying the pathological mechanisms of PTSD, MRS in conjunction with the fear
conditioning paradigm in rodents will find much needed application to the search
for causal mechanisms and novel interventions in patients with psychiatric
disorders. Due to the non-invasiveness of MRS, this early measurement can be
easily expanded to human applications in the future. In this work, the amygdala, a
critical structure in fear conditioning, was not examined. The small size of
amygdala in mouse makes it difficult to acquire reliable spectrum with sufficient
SNR to detect metabolic changes. As metabolic changes following fear
conditioning would certainly be predicted in this region, future study will be
carried out with more efficient sequences such as chemical shift imaging (CSI).
The second study shows the metabolic differences between pregnant and nonpregnant rats using in vivo 1H MRS. The neurochemical findings are important in
understanding the neurochemical modification of the maternal brain and in
assessing cerebral pathology during pregnancy with 1H MRS. Future extension of

117

this study by correlating the neurochemical findings with histological and

behavioral results will contribute to a better understanding of the neuronal
modifications associated with pregnancy. Relaxometry measurements of the
metabolites might also be needed to exclude any potential effects from the
alteration in water content of the maternal brain. Further translation of MRS
approach to human application is of interest.

In Chapter 5, in vivo MEMRI was employed to investigate the hypoxicischemic injury in the late phase and the neural response to conditioned fear. T1W
images SI increase was detected in the peri-lesional region 24 hours after Mn2+
administration and it colocalized with the increase in glial cell density in GFAP
staining, demonstrating the existence of reactive gliosis in the late phase after H-I
injury. Previously, MEMRI has been used to detect neurodegeneration during the
acute-phase of neonatal HI cerebral insult (26, 27). The findings of this work
suggest such MEMRI approach may be also useful in investigation of post-injury
cellular events and functional reorganization. In fear conditioned animals, higher
Mn uptake was observed in amygdala, hippocampus, paraventricular nucleus of
hypothalamus and cingulate cortex, which are all highly-involved in the process
of fear. The results provide insights to neurocircuitry involved in fearconditioning and consolidate the capability of MEMRI as an in vivo probe for
mapping neural activity. With optimized dosage and delivery method, MEMRI
can be potentially used as an alternative for functional MRI, providing unique
contrast for mapping the neural activity.

118

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LIST OF PUBLICATIONS

Journal Papers
1.

Zhou IY, Liang YX, Cheng JS, Chan RW, Chan KC, So KF, Wu EX.
Resting-State Functional Connectivity Altered by Complete and Partial
Corpus Callosotomy in Rats. (In preparation)

2.

Zhou IY, Chan RW, Ho LC, Wu EX. Proton Magnetic Resonance

Spectroscopy of Regional Metabolic Changes in Rat Brain During
Pregnancy and Mother hood. (In preparation)

3.

Zhou IY, Ding AY, Lee FY, Wu EX. Resting-State Functional

Connectivity fMRI and Proton Magnetic Resonance Spectroscopy of Sleep
Deprivation. (In preparation)

4.

Zhou IY, Ding AY, Li Q, McAlonan GM, Wu EX. (2012) Magnetic

resonance spectroscopy reveals N-acetylaspartate reduction in hippocampus
and cingulate cortex after fear conditioning. Psychiatry Research:
Neuroimaging (Under revision).

5.

Zhou IY, Cheung MM, Lau C, Chan KC, Wu EX. (2012) Balanced steadystate free precession fMRI with intravascular susceptibility contrast agent.
Magn Reson Med 68(1):65-73.

6.

Cheung MM, Lau C, Zhou IY, Chan KC, Zhang JW, Fan SJ, Wu EX.
(2012) High fidelity tonotopic mapping using swept source functional
magnetic resonance imaging. Neuroimage 61(4):978-986.

7.

Cheung MM, Lau C, Zhou IY, Chan KC, Cheng JS, Zhang JW, Ho LC, Wu
EX. (2012) BOLD fMRI investigation of the rat auditory pathway and
tonotopic organization. Neuroimage 60(2):1205-1211.

8.

Chan KC, Cheng JS, Fan S, Zhou IY, Yang J, Wu EX. (2012) In vivo
evaluation of retinal and callosal projections in early postnatal development
and plasticity using manganese-enhanced MRI and diffusion tensor imaging.
Neuroimage 59(3):2274-2283.

142

9.

Lau C, Zhou IY, Cheung MM, Chan KC, Wu EX. (2011) BOLD temporal
dynamics of rat superior colliculus and lateral geniculate nucleus following
short duration visual stimulation. PLoS One 6(4):e18914.

10.

Lau C, Zhang JW, Xing KK, Zhou IY, Cheung MM, Chan KC, Wu EX.
(2011) BOLD responses in the superior colliculus and lateral geniculate
nucleus of the rat viewing an apparent motion stimulus. Neuroimage
58(3):878-884.

11.

Chow AM, Zhou IY, Fan SJ, Chan KW, Chan KC, Wu EX. (2011)
Metabolic changes in visual cortex of neonatal monocular enucleated rat: a
proton magnetic resonance spectroscopy study. Int J Dev Neurosci
29(1):25-30.

12.

Cheung JS, Au WY, Ha SY, Kim D, Jensen JH, Zhou IY, Cheung MM, Wu
Y, Guo H, Khong PL, Brown TR, Brittenham GM, Wu EX. (2011) Reduced
transverse relaxation rate (RR2) for improved sensitivity in monitoring
myocardial iron in thalassemia. J Magn Reson Imaging 33(6):1510-1516.

13.

Chan KC, Li J, Kau P, Zhou IY, Cheung MM, Lau C, Yang J, So KF, Wu
EX. (2011) In vivo retinotopic mapping of superior colliculus using
manganese-enhanced magnetic resonance imaging. Neuroimage 54(1):389395.

14.

Chan KC, Fan SJ, Zhou IY, Wu EX. (2011) In vivo chromium-enhanced
MRI of the retina. Magn Reson Med.

15.

Chan KC, Xing KK, Cheung MM, Zhou IY, Wu EX. (2010) Functional
MRI of postnatal visual development in normal and hypoxic-ischemicinjured superior colliculi. Neuroimage 49(3):2013-2020.

Conference Proceedings
1.

Zhou IY, Liang YX, Cheng JS, Chan RW, Chan KC, So KF, Wu EX. (2012)
Resting-State Functional Connectivity Altered by Complete and Partial
Corpus Callosotomy in Rats. Proceedings of 20th Scientific Meeting,
International Society for Magnetic Resonance in Medicine, p 2874.

2.

Zhou IY, Ding AY, Lee FY, Wu EX. (2012) Metabolic Changes in
Hippocampus and Thalamus After Sleep Deprivation: An Experimental
Proton MRS Study. Proceedings of 20th Scientific Meeting, International
Society for Magnetic Resonance in Medicine, p 3093.

143

3.

Zhou IY, Chan RW, Ho LC, Gao PP, Wu EX. (2012) Proton Magnetic
Resonance Spectroscopy of Regional Metabolic Changes in Rat Brain
During Pregnancy. Proceedings of 20th Scientific Meeting, International
Society for Magnetic Resonance in Medicine, p 304.

4.

Zhou IY, Chan RW, Ding AY, Lee FY, Wu EX. (2012) Resting-State
Functional Connectivity Changes Induced by Sleep Deprivation.
Proceedings of 20th Scientific Meeting, International Society for Magnetic
Resonance in Medicine, p 3134.

5.

Zhang JW, Lau C, Cheng JS, Xing KK, Zhou IY, Cheung MM, Wu EX.
(2012) fMRI Study of Sound Pressure Level Processing in the Central
Auditory System. Proceedings of 20th Scientific Meeting, International
Society for Magnetic Resonance in Medicine, p 2898.

6.

Qiao ZW, Cao P, Zhou IY, Fan S, Cheung MM, Zhang JW, Wu EX. (2012)
Dynamic 1H-MRS Revealed Muscle Type Dependent IMCL Storage at
Resting State. Proceedings of 20th Scientific Meeting, International Society
for Magnetic Resonance in Medicine, p 438.

7.

Lee FY, Zhou IY, Fan S, Ding AY, Wu EX. (2012) MEMRI Reveals
Neuronal Changes in Specific Hippocampal Substructures Following Sleep
Deprivation. Proceedings of 20th Scientific Meeting, International Society
for Magnetic Resonance in Medicine, p 943.

8.

Lau C, Zhang JW, Cheng JS, Xing KK, Zhou IY, Cheung MM, Wu EX.
(2012) Noninvasive fMRI Investigation of Interaural Level Difference
Processing in the Rat Subcortex. Proceedings of 20th Scientific Meeting,
International Society for Magnetic Resonance in Medicine, p 2180.

9.

Ding AY, Zhou IY, Lee FY, Wu EX. (2012) DTI Detection of
Microstructural Changes Induced by Sleep Deprivation. Proceedings of 20th
Scientific Meeting, International Society for Magnetic Resonance in
Medicine, p 910.

10.

Ding AY, Li Q, Zhou IY, McAlonan GM, Wu EX. (2012) DTI

Investigation of Short-Term Plasticity in Amygdala and Hippocampus
Induced by Fear Conditioning. Proceedings of 20th Scientific Meeting,
International Society for Magnetic Resonance in Medicine, p 878.

11.

Cheung MM, Lau C, Cheng JS, Zhou IY, Chan KC, Zhang JW, Wu EX.
(2012) BOLD fMRI Investigation of Tonotopic Changes in Normal and
Injured Auditory Systems. Proceedings of 20th Scientific Meeting,
International Society for Magnetic Resonance in Medicine, p 659.

12.

Cheng JS, Gao PP, Lau C, Zhang JW, Cheung MM, Zhou IY, Wu EX.
(2012) Amplitude Modulation Frequency and Duty Cycle Processing in the
144

Auditory System: An fMRI Investigation. Proceedings of 20th Scientific

Meeting, International Society for Magnetic Resonance in Medicine, p 2178.
13.

Chan RW, Zhou IY, Ho LC, Wu EX. (2012) Resting-State Functional

Connectivity During Pregnancy. Proceedings of 20th Scientific Meeting,
International Society for Magnetic Resonance in Medicine, p 2122.

14.

Chan KC, Li J, Zhou IY, Kau P, So KF, Wu EX. (2012) Differentiation of

Primary and Secondary Degeneration in the Visual Pathway Using In Vivo
Mn-Enhanced MRI. Proceedings of 20th Scientific Meeting, International
Society for Magnetic Resonance in Medicine, p 949.

15.

Chan KC, Fan S, Zhou IY, Wu EX. (2012) In Vivo Chromium-Enhanced

MRI of Normal and Injured Retinas. Proceedings of 20th Scientific Meeting,
International Society for Magnetic Resonance in Medicine, p 890.

16.

Zhou IY, Liang YX, Chan KC, Cheung MM, Lau C, So KF, Wu EX. (2011)
Resting-state Functional Connectivity Alterations after Corpus Callosotomy
in Rats. Proceedings of 19th Scientific Meeting, International Society for
Magnetic Resonance in Medicine, p 3686.

17.

Zhou IY, Ding AY, Li Q, Lee FY, Fan S, Chan KC, McAlonan GM, Wu
EX. (2011) In Vivo Manganese-enhanced MRI of Conditioned Fear
Response. Proceedings of 19th Scientific Meeting, International Society for
Magnetic Resonance in Medicine, p 234.

18.

Zhou IY, Ding AY, Li Q, Fan S, Chan KC, Cao P, Chow AM, McAlonan
GM, Wu EX. (2011) Early Metabolic Changes in Hippocampus and
Cingulate Cortex after Fear Conditioning. Proceedings of 19th Scientific
Meeting, International Society for Magnetic Resonance in Medicine, p 4180.

19.

Zhou IY, Cheung MM, Chan KC, Lau C, Wu EX. (2011) Balanced Steady
State Free Precession fMRI Using Intravascular Susceptibility Contrast
Agent. Proceedings of 19th Scientific Meeting, International Society for
Magnetic Resonance in Medicine, p 3619.

20.

Zhang JW, Lau C, Cheung MM, Xing KK, Zhou IY, Chan KC, Wu EX.
(2011) BOLD Response Dependence on the Stimulation Light Intensity in
the Rat Superior Colliculus. Proceedings of 19th Scientific Meeting,
International Society for Magnetic Resonance in Medicine, p 3677.

21.

Lau C, Zhang JW, Cheung MM, Xing KK, Zhou IY, Chan KC, Wu EX.
(2011) Spin-Echo BOLD Temporal Dynamics in the Rat Superior
Colliculus and Lateral Geniculate Nucleus. Proceedings of 19th Scientific
Meeting, International Society for Magnetic Resonance in Medicine, p 3650.

145

22.

Lau C, Zhang JW, Cheung MM, Xing KK, Zhou IY, Chan KC, Wu EX.
(2011) BOLD fMRI Study of the Rat Superior Colliculus Responding to a
Moving Visual Stimulus. Proceedings of 19th Scientific Meeting,
International Society for Magnetic Resonance in Medicine, p 3678.

23.

Ding AY, Zhou IY, Li Q, McAlonan GM, Wu EX. (2011) DTI detection of
fear conditioning induced microstructural plasticity. Proceedings of 19th
Scientific Meeting, International Society for Magnetic Resonance in
Medicine, p 4342.

24.

Cheung MM, Zhou IY, Chan KC, Ho LC, Lau C, Wu EX. (2011) BOLD
fMRI investigation of rat auditory system. Proceedings of 19th Scientific
Meeting, International Society for Magnetic Resonance in Medicine, p 3676.

25.

Cheung MM, Cheng JS, Zhou IY, Chan KC, Lau C, Wu EX. (2011)
Tonotopic Mapping in Inferior Colliculus using bSSFP fMRI and Sweeping
Frequency Auditory Stimulation. Proceedings of 19th Scientific Meeting,
International Society for Magnetic Resonance in Medicine, p 638.

26.

Cheng JS, Chan KC, Zhou IY, Cheung MM, Lau C, Wu EX. (2011)
BOLD-fMRI study of Effect of Dark-rearing on Postnatal Visual
Development. Proceedings of 19th Scientific Meeting, International Society
for Magnetic Resonance in Medicine, p 3681.

27.

Chan KC, Zhou IY, Fan S, Cheng JS, Wu EX. (2011) In vivo MEMRI of
Neuronal Plasticity in Retinocollicular Projection. Proceedings of 19th
Scientific Meeting, International Society for Magnetic Resonance in
Medicine, p 4256.

28.

Chan KC, Cheng JS, Zhou IY, Lau C, So KF, Wu EX. (2011) In vivo
Mapping of Retinal Projections in Rat, Gerbil and Mouse Brains using
MEMRI. Proceedings of 19th Scientific Meeting, International Society for
Magnetic Resonance in Medicine, p 2408.

29.

Chan KC, Cheng JS, Fan S, Zhou IY, Wu EX. (2011) In vivo manganeseenhanced MRI and diffusion tensor imaging of developing and impaired
visual brains. Conf Proc IEEE Eng Med Biol Soc 2011:7005-7008.

30.

Zhou IY, Chow AM, Fan S, Wu EX. (2010) Manganese-enhanced MRI

detection of neural compensatory after neonatal monocular enucleation.
Proceedings of 18th Scientific Meeting, International Society for Magnetic
Resonance in Medicine, p 4505.

31.

Zhou IY, Chan KC, Lau C, Wu EX. (2010) MEMRI study of cerebellar
activation after voluntary wheel running in mice. Proceedings of 18th
Scientific Meeting, International Society for Magnetic Resonance in
Medicine, p 4504.
146

32.

Chow AM, Zhou IY, Fan S, Chan KW, Chan KC, Wu EX. (2010) Taurine
change in visual cortex of neonatal monocular enucleated rat: a proton MRS
study. Proceedings of 18th Scientific Meeting, International Society for
Magnetic Resonance in Medicine, p 4530.

33.

Chow AM, Zhou IY, Fan S, Chan KW, Chan KC, Wu EX. (2010) Proton
MRS in the late stage of neonatal hypoxic-ischemic cerebral injury.
Proceedings of 18th Scientific Meeting, International Society for Magnetic
Resonance in Medicine, p 4527.

34.

Cheung JS, Au WY, Ha SY, Jensen JH, Kim D, Ding AY, Zhou IY, Guo H,
Brown TR, Chu WC, Rasalkar DD, Khong PL, Brittenham GM, E.X. W.
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