Myelinated and unmyelinated axons of the corpus callosum differ in

vulnerability and functional recovery following traumatic brain injury

Thomas M. Reeves
*
, Linda L. Phillips, John T. Povlishock
Department of Anatomy and Neurobiology, Medical College of Virginia Campus, Virginia Commonwealth University,
1217 E. Marshall Street, Room 740, MCV Campus Box 980709, Richmond, VA 23298, USA
Received 6 June 2005; revised 12 July 2005; accepted 20 July 2005
Available online 18 August 2005
Abstract
Traumatic axonal injury (TAI), a common feature of traumatic brain injury, is associated with postinjury morbidity and mortality.
However, TAI is not uniformly expressed in all axonal populations, with fiber caliber and anatomical location influencing specific TAI
pathology. To study differential axonal vulnerability to brain injury, axonal excitability and integrity were assessed in the corpus callosum
following fluid percussion injury in the rat. In brain slice electrophysiological recordings, compound action potentials (CAPs) were evoked in
the corpus callosum, and injury effects were quantified separately for CAP waveform components generated by myelinated axons (N
1
wave)
and by unmyelinated axons (N
2
wave). Ultrastructural analyses were also conducted of TAI-induced morphological changes in these axonal
populations. The two populations of axons differed in response to brain injury, and in their functional recovery, during the first week
postinjury. Amplitudes of N
1
and N
2
were significantly depressed at 3 h, 1 day, and 3 days survival. N
1
amplitudes exhibited a recovery to
control levels by 7 days postinjury. In contrast, N
2
amplitudes were persistently suppressed through 7 days postinjury. Strength-duration
properties of evoked CAPs further differentiated the effects of injury in these axonal populations, with N
2
exhibiting an elevated strength-
duration time constant postinjury. Ultrastructural observations revealed degeneration of myelinated axons consistent with diffuse injury
sequelae, as well as previously undocumented pathology within the unmyelinated fiber population. Collectively, these findings demonstrate
differential vulnerabilities of axons to brain injury and suggest that damage to unmyelinated fibers may play a significant role in morbidity
associated with brain injury.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Traumatic axonal injury; Brain injury; Compound action potentials; Corpus callosum; Axonal excitability; Recovery of function
Introduction
Traumatic axonal injury (TAI) is a common feature of
traumatic brain injury (TBI) and underlies much of the
resulting morbidity and mortality (Gennarelli et al., 1982;
Graham et al., 1988; Povlishock, 1992; Smith and
Meaney, 2000). Although isolated axons may be torn
by shearing forces during severe injuries, TAI is usually
manifested as a multiphasic pathology, with axolemmal
and cytoskeletal alterations evolving, over the course of
hours to days, to result in axonal swelling and/or
disconnection (Fitzpatrick et al., 1998; Maxwell et al.,
1997; Povlishock et al., 1997). Key features of this
pathology have been described, including deleterious
proteolysis, ionic dysregulation, and mitochondrial failure
(Banik et al., 1987; Buki et al., 1999, 2000; Iwata et al.,
2004; Povlishock et al., 1983). However, questions
remain regarding whether distinct populations of axons
exhibit different responses to injury, and therefore
necessitate specific therapeutic strategies.
Most prior studies of in vivo TAI have emphasized the
role of larger myelinated axons, with relatively little
attention to the pathophysiology of small caliber unmye-
linated axons. However, evidence indicates that TAI is
0014-4886/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.expneurol.2005.07.014
* Corresponding author. Fax: +1 804 828 3276.
E-mail address: tmreeves@vcu.edu (T.M. Reeves).
Experimental Neurology 196 (2005) 126 137
www.elsevier.com/locate/yexnr
not uniformly expressed across axonal populations. In
some injured axons, neurofilament compaction has been
observed independently of impaired fast transport (Stone
et al., 2001). The degree to which these two pathological
components were conjointly expressed in individual axons
was partly determined by axon caliber and anatomical
location, with larger medial lemniscal fibers exhibiting both
pathologies concurrently to a greater extent than smaller
corticospinal axons. Other workers, using an in vivo optic-
nerve stretch model of injury, demonstrated that caliber of
the myelinated axons was associated with several TAI
parameters, including the degree of neurofilament and
microtubule damage (Jafari et al., 1997, 1998). To date,
there have been no in vivo descriptions of traumatic
damage in unmyelinated fiber populations: a consequence,
most likely, of their small size which has precluded their
inclusion in routine analysis.
Despite progress in the structural and molecular
characterization of cerebral TAI, few studies have
addressed electrophysiological aspects of this pathology.
TAI typically occurs diffusely in central white matter,
complicating electrophysiological measures based on
single units or individual axons. Baker et al. (2002)
reported that TBI in rats suppressed compound action
potentials (CAPs) evoked in corpus callosum. The current
study employed the methodology first reported by Baker
and colleagues, now modified to enable separate quanti-
fication of CAPs generated by two populations of callosal
axons: relatively fast conducting fibers, corresponding to
large, myelinated axons, and slower conducting unmyeli-
nated, small-caliber fibers. This electrophysiological
assessment was combined with an ultrastructural exami-
nation of callosal axons in the same and parallel
populations of animals. The current results indicated that
these two axonal populations differed in their response to
TBI, and in their capacities for functional recovery, with
the small fibers exhibiting more dramatic and persistent
electrophysiological change. The novel finding, of a
preferential vulnerability of the small unmyelinated axon
population, distinguishes the current results from prior
studies of TAI which have generally emphasized the
pathophysiological role of large myelinated axons.
Methods
The procedures for this study followed all national
guidelines for the care and use of experimental animals,
and the experimental protocol was approved by the Medical
College of Virginia Animal Research Committee. Male
SpragueDawley rats (n = 48) weighing 300350 g, at the
start of the study, were used in these experiments. Animals
were housed in individual cages in a temperature- (22-C)
and humidity-controlled (50% relative) animal facility on a
12-h light/dark cycle. Rat chow and water were continually
available.
Fluid percussion traumatic brain injury (TBI) procedure
Rats were anesthetized with sodium pentobarbital (54
mg/kg, i.p.), and a 4.8 mm skull craniotomy was prepared
over the midline, centered between bregma and lambda. A
Leur-Loc syringe hub was cemented with cyanoacrylate to
the skull surrounding the craniotomy, and dental acrylic was
poured around the syringe hub and two small screws placed
in the skull for implant rigidity. Bacitracin was applied to
the incision, and the animal returned to its home cage.
Twenty-four hours following implantation of the syringe
hub, rats were anesthetized with isoflurane (4% in carrier
gas of 70% N
2
O and 30% O
2
), and immediately subjected to
TBI. The fluid percussion device used to produce TBI is
described in greater detail elsewhere (Dixon et al., 1987).
Briefly, the device consisted of a 60 4.5 cm Plexiglas
water-filled cylinder, fitted at one end with a piston mounted
on O-rings, with the opposite end housing a pressure
transducer (Entran Devices, Inc.; EPN-0300A). At the time
of injury, the Leur-Loc fitting, filled with saline, was
attached to the transducer housing. The injury was produced
by a metal pendulum that struck the piston, injecting a small
volume of saline into the cranial cavity, briefly deforming
the brain tissue (20 ms pulse duration). The resulting
pressure pulse was recorded extracranially by the transducer
and expressed in atmospheres (atm) of pressure. Injury
magnitude was controlled by setting the height from which
the pendulum was released. Following injury, all animals
were promptly ventilated with room air until spontaneous
breathing resumed. The duration of suppression of the
righting reflex was used as an index of traumatic uncon-
sciousness. For control rats administered sham injuries, TBI
procedures were as above except the intracranial pressure
pulse was not injected.
Electrophysiological recording procedures
Following the fluid percussion injury, rats were allowed
to survive for the following intervals prior to electro-
physiological recording: 3 h (n = 5), 1 day (n = 10), 3 days
(n = 8), or 7 days (n = 8). Sham-injured cases (n = 11) were
recorded at 1 day (n = 7) or at 3 days (n = 4) following the
sham injury procedure. All of these rats were used for
measurements of CAP amplitude, and some of these animals
were also used for tests of refractoriness or threshold
excitability.
Each rat was anesthetized with isoflurane, decapitated,
and the brain rapidly removed. Coronal slices of 450 Am
thickness were cut in ice cold (4 to 6-C) artificial
cerebrospinal fluid (ACSF) with a vibrating-knife micro-
tome (Electron Microscopic Sciences model OTS-3000-
03). Those slices were saved which contained midline-
crossing segments of the corpus callosum, overlying the
mid-dorsal hippocampus, which yielded 4 to 6 slices per
brain. Slices were then transferred to a holding chamber
containing oxygenated ACSF at room temperature, and
T.M. Reeves et al. / Experimental Neurology 196 (2005) 126137 127
were allowed to equilibrate under these conditions for at
least 1 h prior to recording. For recording, a slice was
transferred to a submersion-type chamber, and perfused at
a rate of 12 ml/min with ACSF. The ACSF contained (in
mM): NaCl 124, KCl 5, NaH
2
PO
4
1.25, NaHCO
3
26,
MgSO
4
1.3, CaCl
2
2, glucose 10; pH 7.4; saturated with a
95% O
2
/5% CO
2
gas mixture. Where indicated, the ACSF
was supplemented with 4-aminopyridine (4-AP, 10 AM),
or was adjusted to a low Ca
2+
ion concentration by using
modified ACSF containing NaCl 124, KCl 5, NaH
2
PO
4
1.25, NaHCO
3
26, MgSO
4
1.3, MgCl
2
3.7, CaCl
2
0.5,
glucose 10. When switching from a normal ACSF, a 30
min equilibration time was allowed prior to the resumption
of recording.
A bipolar stimulating electrode (teflon-insulated tung-
sten; 0.30.4 mm intertip distance) was lowered into the
corpus callosum, at approximately 0.5 mm lateral to
midline, and a recording electrode (ACSF-filled glass
micropipette; resistance 68 MV) was also placed in the
corpus callosum of the contralateral hemisphere at a
distance of approximately 1.0 mm from the stimulating
electrode (Figs. 1A,B). The initial depth of electrodes was
200 Am below the surface of the slice; however, fine
adjustments were made in the depths of both stimulating and
recording electrodes to optimize the signal amplitude.
Evoked callosal CAP field potentials were amplified
(bandpass = DC to 10 kHz), digitized at 25 kHz, and stored
on disk for offline analysis.
Measurements of CAP amplitude may reflect TBI-
induced changes in the functional properties of individual
axons, as well as in the total number of axons recruited into
the CAP field potential. Accordingly, assessments of CAP
amplitude were supplemented with strength-duration anal-
yses and refractoriness testing, both of which reflect
intrinsic functional properties of axons and are less sensitive
to the absolute numbers of responsive fibers.
Stimulation parameters
Stimulation used for evoked CAPs was constant current
stimulus-isolated square wave pulses, with stimulus pulse
duration and intensity adjusted for specific recording proto-
cols. For analyses of TBI-related changes in CAP amplitude,
standardized input output functions were generated, for
each slice, by varying the intensity of stimulus pulses (200 As
duration, delivered at 0.1 Hz) in 10 equal steps from
approximately threshold level to an asymptotic maximum
for the short-latency negative CAP component (FN
1
_, gray
shaded wave component in Fig. 1C). In refractoriness testing,
pairs of pulses were presented with interpulse interval
increased, in 0.5 ms steps, from 1.5 ms to 6.0 ms, using the
maximum current level established during input output
testing for that slice. For strength-duration analyses, stimulus
pulses were presented using a graded series of durations (in
As): 50, 60, 80, 100, 120, 140, 160, 200, 300, 400, 500, and
600. At each duration, stimulus current was adjusted to evoke
a response which was 30% of the maximum response
amplitude observed in input output testing.
Statistical analysis
All quantitative electrophysiological analyses were con-
ducted on waveforms which were the average of four
successive sweeps, for enhanced signal-to-noise ratio. The
Fig. 1. Placement of electrodes for callosal CAP recording, and responses of
N
1
and N
2
CAP components to temperature and ionic manipulations. (A, B)
Stimulating and recording electrodes were positioned in midline corpus
callosum separated by approximately 1.0 mm. (C) Decreasing bath
temperature slowed conduction time, enabling quantification of N
1
(gray
shading) and N
2
(black shading) waveforms. (D) Recording in low [Ca
2+
]
o
did not significantly alter evoked CAPs, while bath application of the K
+
channel blocker (4-AP) selectively prolonged the N
2
CAP component,
predominantly generated by unmyelinated axons.
T.M. Reeves et al. / Experimental Neurology 196 (2005) 126137 128
amplitude of two components of the CAP waveform (N
1
and
N
2
) was routinely quantified as the maximum negative
deflection measured relative to a tangent connecting the
preceding and following positivities (see insets in Fig. 3 for
graphical illustrations of this measurement). For quantitative
evaluation of injury effects, the unit of analysis was the rat,
and not the slice. Accordingly, if more than one slice was used
from a single rat, those multiple-slice data were averaged for
that rat and used for the analysis. The effect of injury on
callosal CAP amplitude was evaluated using mixed-design
ANOVA (SPSS v11.5) with injury condition as between-
subjects factor and normalized stimulus intensity as repeated-
measures factor. Planned pairwise comparisons (SPSS
MANOVA CONTRASTsyntax) were implemented between
control CAP amplitudes vs. CAP amplitudes measured at
each postinjury survival period. In analyses of refractoriness,
ratios of CAP
2
/CAP
1
(obtained in paired stimulus presenta-
tions) were plotted as a function of interpulse interval, and the
significance of shifts in this relationship due to injury was
tested with the MannWhitney U statistic.
Strength-duration data were fitted to Weiss formula
(Bostock et al., 1983; Burke et al., 2000; Mogyoros et al.,
1998):
Q I
rh
T s
SD
;
where Q is the stimulus charge, I
rh
is the rheobasic current,
s
SD
the strength-duration time constant, and T is stimulus
duration. In Weiss formulation, there is a linear relationship
between stimulus charge (equal to threshold stimulus
current multiplied by its duration) and stimulus duration.
Accordingly, I
rh
and s
SD
were estimated by performing a
linear regression of Q on T, wherein s
SD
(corresponding to
chronaxie) is given by the negative intercept of the
regression line on the duration axis. Rheobase (I
rh
) is the
threshold current for an infinitely long stimulus and is
estimated by the slope of the regression line. The effect of
injury on strength-duration relationships was evaluated
statistically using mixed-design ANOVA with injury con-
dition as between-subjects factor and stimulus duration as
repeated-measures factor, and planned comparisons (MAN-
OVA contrasts) used to test injury effects at specific survival
intervals. In addition, the average group estimates of I
rh
and
s
SD
, generated with the charge-duration regression, were
evaluated with ANOVA. The significance level, a = 0.05,
was used for all inferential statistics, and averaged values
are expressed as mean T SEM.
Electron microscopic studies
In order to document myelinated or unmyelinated fiber
damage in the corpus callosum after TBI, two different
sampling approaches were used. Survival intervals of 1 h, 3
h, and 1 day were selected for the purpose of examining
both the initial stages of fiber response and the later, more
progressive phases of injury, which directly correlate with
the periods of significant functional change. In a parallel
cohort of TBI animals, two subgroups were subjected to
cardiac perfusion with 4% paraformaldehyde + 2.5%
glutaraldehyde in a 0.1 M sodium phosphate buffer at
either 1 h or 3 h after injury (n = 3 per time point) and the
brains blocked to include regions of the corpus callosum
matching those used for electrophysiological assessment.
For the 1 day time point, 3 pairs of sham and injured in vitro
brain slices were harvested from regions adjacent to those
analyzed electrophysiologically. These brain slices were
fixed by immersion in the same buffered aldehyde solution
as the in vivo cases. Blocked tissues were then embedded in
agar and 40 Am Vibratome sections generated for each
sample. All sections were osmicated, en bloc stained in
uranyl acetate, dehydrated and embedded in Medcast Resin,
using the flat mounting technique with plastic slides and
coverslips. Regions of interest within the corpus callosum
were then identified, cut out, mounted on plastic studs, and
serially sectioned with a diamond knife. The resulting thin
sections were collected on Formvar coated slotted grids and
stained with lead citrate. Using a JEOL 1230 electron
microscope, sections were screened and representative
images digitally acquired at 5000 with a Gatan Digital
Camera. Regional montages were generated and transferred
via DVD to a computer station for analysis at 100,000. In
this fashion, broad areas of the corpus callosum could be
assessed for axonal damage. For the myelinated axons,
multiple, well established criteria, including cytoskeletal
disruption and organelle pooling or redistribution, were used
to classify axonal damage after TBI (Povlishock and
Christman, 1995). Given that no specific criteria have been
established to describe the pathological sequelae in unmye-
linated axons after TBI, these fibers were assessed for
evidence of any structural/subcellular perturbation over time.
Results
Consistency in injury severity was assured by including
only animals with injury magnitudes in the range 2.0 T 0.1
atm. In addition, the duration of suppression of the righting
reflex, used as an index of traumatic unconsciousness, was
compared among the analytical groups used in the study.
The overall mean latency to regain the righting reflex, for
all animals given fluid percussion injury, was 8.7 T 0.8
min. None of the injury groups used in the study differed
significantly from this overall mean righting latency ( F
(3,17)
=
1.42; P = 0.278).
During initial recording sessions, it was determined that
the short-latency component (N
1
) in the biphasic callosal
CAP was obscured by the stimulus artifact, when the bath
temperature was near physiological (36-C) temperature.
Quantification of N
1
was facilitated by lowering the bath
temperature to 23-C, slowing conduction sufficiently to
allow separation of this waveform component from the
stimulus artifact (Fig. 1C). Accordingly, the subsequent
T.M. Reeves et al. / Experimental Neurology 196 (2005) 126137 129
evaluation of the effects of injury on the callosal CAPs was
conducted at 23-C. The biphasic CAP was reliably obtained
and was not affected by recording in low Ca
2+
, indicating the
signal was generated axonally, without synaptic involvement
(Fig. 1D), a finding consistent with prior observations
(Swanson et al., 1998). N
1
was not affected by bath
application of 4-AP, but this potassium channel blocker
selectively increased the duration of N
2
(example in Fig. 1D).
In a subset of rats (n = 9), 4-AP application prolonged N
2
by
47% ( F
(1,8)
= 28.722; P < 0.001), although the degree of 4-
AP-induced increases did not differ between control and
injured rats ( F
(1,7)
= 3.525; P = 0.103). Previous work has
reported similar 4-AP effects (Preston et al., 1983; Swanson
et al., 1998), and it was argued that N
2
represented CAPs
generated in unmyelinated axons with 4-AP-sensitive K
+
channels along the entire axolemma, whereas N
1
was
generated by heavily myelinated axons, where 4-AP-sensi-
tive K
+
channels are present only sparsely (Waxman, 1995).
Injury effects on CAP amplitude
Quantitative analyses revealed that the two CAP compo-
nents (N
1
, N
2
) were differentially affected by injury, and ex-
hibited divergent courses of postinjury recovery. The injury
resulted in reductions in amplitude of N
2
to a greater extent
than of N
1
, and only the N
1
component exhibited a significant
time-dependent recovery during the first postinjury week.
Current intensities used in the evaluation of CAP
amplitudes were selected to range from approximately
threshold level up to an asymptotic level for the N
1
CAP,
although the N
2
CAP required comparatively more current
to reach asymptote. This conservative stimulation approach
avoided extensive stimulation at supramaximal levels for
only one of the axonal subpopulations (i.e., the faster
conducting N
1
generating fibers), yet still permitted the
recording of the effective linear portion of the N
2
input
output range. It is important to note that the mean current
levels required to evoke the asymptotic N
1
field component
did not differ significantly among the analytic groups
( F
(4,33)
= 0.07; P = 0.991), and were as follows (in mA):
control (0.367), and for postinjury groups, 3 h (0.400), 1 day
(0.390), 3 days (0.386), and 7 days (0.393).
Examples of evoked CAPs are shown in Fig. 2 from
sham-injured rats, and from injured rats recorded at 3 h
to 7 days postinjury survival intervals, in each case
displaying a response evoked with the maximum stimulus
level used for that rat. As indicated by the waveforms of
Fig. 2, both N
1
and N
2
field components were present
after injury, although the amplitude of N
2
was markedly
reduced. The most extreme example of postinjury N
2
suppression was in a rat recorded at 3 h postinjury, in
which the N
2
component was entirely absent (illustrated
in middle column of Fig. 2).
The curves in Figs. 3B and C illustrate the mean injury-
induced suppression of N
1
and N
2
, respectively, over the full
range of stimulation current used. For both N
1
and N
2
, this
injury effect was greatest at 1 day. ANOVA contrasts
between the amplitudes of control CAPs and the amplitudes
at the various post-TBI survival intervals revealed that the N
1
amplitudes were significantly depressed at postinjury times 3
h ( P < 0.05), 1 day ( P < 0.001), and 3 days ( P < 0.01), but
were no longer significantly different from control levels at 7
days postinjury. N
2
amplitudes remained significantly below
control levels at all postinjury survival intervals ( P < 0.001
for 3 h, 1 day, 3 days, and 7 days); no significant time-
dependent recovery was observed for N
2
amplitudes.
If TBI impairs unmyelinated axons to a greater extent
than myelinated axons, then post-TBI amplitude measure-
ments of N
1
and N
2
should reflect this differential vulner-
ability. An analysis of the postinjury percent decreases,
observed in N
1
and N
2
amplitudes, did support such a
differential injury effect. Considering the amplitude of CAPs
evoked with the highest stimulus current (100% normalized
current in Fig. 3B) N
2
was reduced to a minimum of 31% of
control levels at 1 day, which was significantly lower than N
1
amplitudes at 1 day (53% of control levels) ( F
(1,18)
= 6.65;
P = 0.019). Thus, at 1 day, the injury effect to N
2
was
22% more severe than for N
1
, and this disparity remained
significant at 3 days when the N
2
reduction was 34%
Fig. 2. Examples of CAPs evoked from control rats (sham-injured) and
from injured rats recorded at 3 h, 1 day, 3 days, or 7 days postinjury. All
CAPs in this figure were evoked with the maximum stimulus level used for
that rat (100% normalized current level). Note comparatively large
amplitude of the slower component (N
2
) in the control examples, and the
suppression of this field potential in CAPs following injury. An extreme
case of this injury effect is evident in middle trace at 3 h, where the
unmyelinated wave component is entirely suppressed.
T.M. Reeves et al. / Experimental Neurology 196 (2005) 126137 130
greater than N
1
( F
(1,14)
= 7.63; P = 0.015) and at 7 days
when this difference was at 37% ( F
(1,14)
= 6.83; P =
0.020). Only at 3 h postinjury, when N
2
amplitudes were
measured as 25% lower than in N
1
, did this differential
injury effect fail to reach statistical significance ( F
(1,7)
=
1.83; P = 0.218).
Refractoriness testing
The refractoriness of the callosal fibers was analyzed by
quantifying the suppression of the second CAP response in
paired stimulus trials. This analysis was completed for a
subset of 23 rats as follows: control (n = 6), and injured
groups at 3 h (n = 4), 1 day (n = 5), 3 days (n = 4), and 7 days
(n = 4). Figs. 4A and B show example series of the second
response evoked in paired stimulus presentations, at the
indicated interpulse intervals (IPI, 1.5 to 6.0 ms), after
subtracting out the responses to the conditioning pulse, for a
control slice (Fig. 4A) and for a slice recorded at 1 day
postinjury (Fig. 4B). Fig. 4C plots the CAP amplitude
elicited by the second pulse in each paired stimulation (C
2
)
divided by the CAP amplitude to single pulse stimulation
(C
1
). These C
2
/C
1
ratios are averaged for each analytic group
and plotted for N
1
(left panel of Fig. 4C) and for N
2
(right
panel). These mean values were fitted to Boltzmann sigmoid
curves (lines in Fig. 4C), with average adjusted R
2
values for
N
1
= 0.993 and for N
2
= 0.938. Rightward shifts in these
curves correspond to increases in the refractory recovery
cycle in the callosal axons, indicative of axonal damage.
These curves show the sham control N
1
waveform compo-
nent, evoked by the second of a pair of pulses, achieved 50%
of the amplitude of a single pulse presentation, when the
interpulse interval was approximately 3.7 ms, and this
parameter for the N
2
field component was 4.5 ms. Averaged
over all postinjury survival periods, the fluid percussion
injury degraded the refractory performance for these fibers
by approximately 0.5 ms for both N
1
and N
2
field
components, with the largest injury effects noted at 3 h,
with a 0.78 ms increase for N
1
and a 1.3 ms increase for N
2
refractoriness. A time-dependent recovery was evident in
this functional parameter, as measured by the significance of
the shifts in these refractory curves. Refractory curves were
significantly right-shifted at 3 h, 1 day, and 3 days, for the N
1
fibers, and at 3 h and 1 day for the slower N
2
fibers (Mann
Whitney U, P < 0.05). At 7 days postinjury, refractory curves
were not statistically significantly different from control
curves for either the N
1
or N
2
wave component.
Strength-duration properties
An assessment of axonal excitability, which included an
evaluation of the parameters of rheobase and the strength-
duration time constant, was conducted at the early survival
intervals when functional deficits were at a maximum. This
analysis used a subset of 15 rats, including sham-injured
controls (n = 6), and injured rats recorded at 3 h (n = 4), or 1
day (n = 5). The current threshold data conformed well to
the hyperbolic function given by Weiss law (Bostock et al.,
1983; Burke et al., 2000; Mogyoros et al., 1998), and
average adjusted R
2
values were high, with mean values
Fig. 3. Differential effects of TBI on callosal N
1
and N
2
waveform
components. (A) Recruitment of field potential with graduated series of
stimulus pulses (in examples, current was stepped from 10% to 100% of
maximum) for a sham control rat and for a rat recorded at 1 day postinjury.
Open arrow = time of stimulus onset. (B) Average amplitude of N
1
over the
full range of stimulation current used, for shamand TBI groups. N
1
amplitude
was significantly decreased at 3 h ( P < 0.05), 1 day ( P < 0.001), and 3 days
( P < 0.01), but was not different from control levels at 7 days postinjury. (C)
Average amplitude of N
2
over the full stimulus range, for sham and TBI
groups. More persistent injury-induced suppression of average response
amplitude was observed for the N
2
, which remained significantly below
control levels at all postinjury survival intervals (3 h, 1 day, 3 days, and 7
days; P < 0.001). Inserts in panels B and C show CAP amplitude
measurement technique for N
1
and N
2
components (height of vertical line*).
T.M. Reeves et al. / Experimental Neurology 196 (2005) 126137 131
ranging from 0.924 to 0.990. Fig. 5A [left panel] shows
averaged strength-duration curves for N
1
, plotting the
threshold current required to evoke a CAP of criterion
amplitude (30% of maximum established in input output
relationships) as a function of stimulus pulse duration. Fluid
percussion injury significantly altered the N
1
strength-
duration relationship, when measured at 3 h postinjury
( F
(1,10)
= 19.03; P < 0.01), indicating a depression of
axonal excitability at that postinjury time. At 1 day
postinjury, the N
1
strength-duration curve was not signifi-
cantly different from controls ( P = 0.446). The trans-
formation of the N
1
strength-duration data, which relates
threshold stimulus charge to stimulus pulse duration,
provides estimates of rheobasic current (I
rh
, slope of the
regression line) and strength-duration time constant, s
SD
, as
the (negative) x-intercept where threshold charge is zero
(Fig. 5A [right panel]). Rheobase current was significantly
elevated above control levels at 3 h postinjury ( F
(1,8)
=
18.28; P < 0.01), but was not different when tested at 1 day
postinjury. The average value of the N
1
s
SD
, was 245 As for
control rats, and 430 As and 342 As when measured at 3 h and
1 day postinjury, respectively. However, these injury-related
changes in N
1
s
SD
did not reach statistical significance. Fig.
5B shows the results of the same analysis for N
2
. As was the
case for N
1
, the postinjury N
2
strength-duration relationship
showed a significant suppression of excitability at 3 h ( F
(1,9)
=
13.37; P < 0.01), but no significant differences at 1 day
postinjury. Average N
2
rheobase current was higher at 3 h
( F
(1,7)
= 6.68; P < 0.05), but not different from controls at 1
day. Inspection of Fig. 5B [right panel] suggested an N
2
s
SD
elevation above the control level of 501 As to 697 As at 3 h
and extending to 914 As at 1 day postinjury. This increase in
time constant was significant at 1 day postinjury ( F
(1,8)
=
18.78; P < 0.01), although this effect did not reach statistical
significance in the 3 h sample, possibly due to greater
variability observed in this wave component (for example,
the N
2
wave component was not measurable in one of the
rats at 3 h postinjury).
Ultrastructural findings
Ultrastructural analysis of the injured corpus callosum
showed a progression of pathological change over the
period between 1 h and 1 day postinjury. At 1 h, the
callosum contained fields of mixed myelinated and unmye-
linated fibers, the majority of which appeared intact (Figs.
6A and C). Pathology was exhibited in scattered fibers of
each type, often seen adjacent to profiles of axons with
normal morphology. The primary signs of axonal degener-
ation in the myelinated population included mitochondrial
Fig. 4. Effect of TBI on callosal CAP refractoriness. Example waveforms at top show second potentials in paired responses, after subtraction of response to the
conditioning pulse, from a sham control rat (A) and from a rat recorded at 1 day post-TBI (B), at indicated interpulse intervals from 1.5 to 6.0 ms. (C) Plots of
mean CAP amplitude elicited by the second pulse in each paired stimulation (C
2
) divided by the CAP amplitude to single pulse stimulation (C
1
), for control and
injured groups. Average C
2
/C
1
ratios were fitted to Boltzmann sigmoid curves, and showed significant increases in refractoriness (rightward curve shifts) at 3 h,
1 day, and 3 days for N
1
[left panel] and at 3 h and 1 day N
2
[right panel].
T.M. Reeves et al. / Experimental Neurology 196 (2005) 126137 132
swelling and cytoskeletal disorganization (Fig. 6B), often
accompanied by disrupted myelin and increased electron
density of the axoplasm. This rapid injury response was also
evident in the unmyelinated axons (Fig. 6D). Typically,
unmyelinated profiles in the range of 0.4 to 0.5 Am in
diameter revealed focal axonal swelling which was often
associated with axolemmal irregularity, the intraaxonal
accumulation of vesicles, and tubulo-vesicular profiles and
local mitochondrial disruption.
By 3 h postinjury, both populations of axons showed
significant pathology, consistent with a progressive fiber
degeneration and irreversible disconnection (Fig. 6E). Multi-
ple myelinated fiber profiles exhibited microtubule loss and
neurofilament compaction, as well as the accumulation of
organelles reflecting impaired axonal transport (Fig. 6F). As
in the 1 h group, these injured axons were scattered among
numerous uninjured axonal profiles, although the proportion
of injured axons appeared to be increased. Progressive
degeneration was also visible in the unmyelinated population
(Figs. 6G and H), where total breakdown of the axolemma
and cytoskeletal network was seen.
At 1 day, the corpus callosum exhibited advanced
pathological change in both myelinated and unmyelinated
fiber populations (Fig. 7A). These pathological changes
were not observed in sections from parallel sham control
cases (Fig. 7B). Many myelinated axons revealed mature
reactive axonal swellings, suggesting frank detachment
from downstream axonal segments (Fig. 7C). Multiple sites
of Wallerian degeneration were also clearly visible. Perhaps
most striking, was the significant evolution of unmyelinated
fiber pathology. At this time, remnants of damaged axons
were observed, consisting of membrane and cytoskeletal
debris interspersed between myelinated and unmyelinated
fiber profiles (Figs. 7D and E). Other unmyelinated fibers
were either swollen and disorganized or shrunken and
electron dense, suggesting irreversible damage.
Discussion
This is the first study to directly investigate electro-
physiological and structural evidence of selective vulner-
abilities among CNS axonal populations following traumatic
brain injury. We examined injury-induced changes in func-
tional properties of corpus callosum fibers using recording
conditions that facilitate separate quantification of two
Fig. 5. Strength-duration and charge-duration curves for N
1
and N
2
CAP components, measured in sham-injured control rats, and in rats recorded at 3 h and 1
day postinjury. (A) [left panel] Threshold data are plotted for N
1
waves, and fitted to Weiss formula (curves). [right panel] Charge-duration transformation of
the same data provides a graphical estimate of the strength-duration time constant (s
SD
) as an extrapolation of the regression line to the zero charge axis.
Rheobase (I
rh
) is estimated as the slope of the regression line. (B) Equivalent analysis of N
2
threshold data. These data suggest a transient decrease in
excitability of corpus callosum axons, affecting both N
1
and N
2
wave components at 3 h postinjury. At 1 day after injury, N
1
threshold functioning exhibited
some degree of recovery, while deficits persisted in fibers generating the N
2
CAP component.
T.M. Reeves et al. / Experimental Neurology 196 (2005) 126137 133
distinct axonal populations: relatively fast-conducting fibers,
corresponding to larger, myelinated axons, and a population
of slower-conducting unmyelinated, small-caliber callosal
fibers. Among the measures of axonal function used in this
study, the amplitude of evoked CAPs appeared most
profoundly affected by the fluid percussion injury, although
mechanisms underlying the discharge recovery cycle and
threshold-level responses were also clearly affected. An
additional finding, of central importance in the issue of
selective axonal vulnerabilities, is a strikingly different
degree of functional recovery exhibited by these fiber types.
Companion ultrastructural studies confirmed distinct differ-
ences in the level of pathological change between myelinated
and unmyelinated fiber populations. While these morpho-
logical observations cannot fully explain the mechanism
underlying differential physiological recovery, they do
support significant, irreparable damage within the unmyeli-
nated fiber group. Taken together, the electrophysiological
and morphological results indicated that TAI did not induce a
generalized deficit, affecting all axons equally. Rather,
factors unique to the individual axon subpopulations
appeared to confer a differential vulnerability and influence
potential for functional recovery.
Multiple lines of evidence indicate that the two CAP
components arise from distinct populations of faster-
conducting myelinated fibers (N
1
) and slower unmyelinated
fibers (N
2
) (Preston et al., 1983; Swanson et al., 1998). The
consistent biphasic negative CAP indicates a bimodal
distribution of conduction velocities, and not a unitary
population varying along a single continuous distribution.
Blockade of potassium channels with 4-AP prolongs only
the N
2
wave component, consistent with an unmyelinated
status in fibers generating that field component. The corpus
callosum has been the focus of quantitative ultrastructural
studies of axonal caliber, and findings from a variety of
species demonstrate the callosum has a rich mixture of
myelinated and unmyelinated fibers (Lamantia and Rakic,
1990; Sturrock, 1980; Swadlow et al., 1980; Waxman and
Swadlow, 1976). These prior studies have shown a
consistency in caliber measurements, converging on a range
Fig. 7. Ultrastructural view of corpus callosum at 1 day following central
fluid percussion injury. Low magnification panel of in vitro immersion
fixed injured case (A) shows extensive fiber degeneration relative to paired
sham control callosal slice in panel B. Multiple profiles of myelinated axons
with electron dense axoplasm and disrupted sheaths were visible (arrows),
as well as evidence of swollen and degenerated unmyelinated fibers
(arrowheads). In some regions, myelinated fiber breakdown had progressed
to axonal separation stages (C), with sites of unmyelinated fiber lysis and
phagocytosis clearly visible (arrows in panels D, E). Scale bars in panels
AC = 1 Am; D, E = 0.5 Am.
Fig. 6. Ultrastructural view of corpus callosum at 1 h (AD) and 3 h (EH)
following central fluid percussion injury. Low magnification panels (A, E)
of in vivo perfused cases show evolution of pathological change between 1
h and 3 h postinjury. At 1 h, the majority of both fiber populations appeared
intact, with some early evidence of electron dense axoplasm visible in select
myelinated profiles (arrowheads in panel A). Panel B illustrates initial
phases of myelin pathology emerging at 1 h (arrowhead). A field of normal
unmyelinated fibers with intact axonal membranes and cytoarchitecture is
shown in panel C. Other regions exhibited early phases of degenerative
change in the unmyelinated fiber population (D), with disrupted axonal
membranes and disorganized cytoskeleton (outlined areas). By 3 h
postinjury, a large number of axons showed more advanced stages of
degenerative response (E). Dense axoplasm and neurofilament compaction
was common in myelinated axons (arrowheads), with vesicular profiles
seen in disrupted unmyelinated fibers (arrows). Organelle accumulation in
swollen myelinated axons was also observed (F). Overall, the unmyelinated
fiber population showed a greater extent of axolemmal breakdown and
cytoskeletal disorganization at 3 h after injury (outlined areas in panels G
and H). In some regions, groups of intact unmyelinated fibers remained
visible (arrows in panel G). Scale bars in panels A, E = 2 Am; B, F = 1 Am;
C, D, G, H = 0.5 Am.
T.M. Reeves et al. / Experimental Neurology 196 (2005) 126137 134
of 0.60.7 Am median diameter for myelinated fibers, and
approximately 0.20.25 Am median diameter for the
unmyelinated subpopulation. These estimates of fiber
diameters would lead to predictions of conduction velocities
that accord well with actual velocity measurements (Preston
et al., 1983; Swadlow and Waxman, 1976).
The posttraumatic decrease in CAP amplitude,
observed in this study, may reflect alterations to axons
such as pathological depolarization, as well as reduced
numbers of responsive axons. Mounting evidence has
identified influxes of calcium (Banik et al., 1987; Buki et
al., 1999, 2000; Povlishock, 1992; Wolf et al., 2001) and
sodium (Iwata et al., 2004; Wolf et al., 2001) as early
pathogenic events in TAI, and such postinjury cationic
fluxes should be associated with decreased amplitude of
evoked CAPs. Alternatively, the pathological axonal
profiles observed ultrastructurally strongly suggest that
some callosal fibers are injured sufficiently to prevent
their recruitment into the CAP field potential. However,
the strength-duration and refractoriness analyses reported
in the current study suggest that the postinjury CAPs
reflect, at least in part, injury-induced alterations in the
functional properties of individual axons, such as level of
depolarization or pathological changes to sodium chan-
nels. Prior work has extensively documented the strength-
duration parameters of s
SD
and I
rh
for peripheral
myelinated axons (Bostock et al., 1983; Bostock and
Rothwell, 1997; Mogyoros et al., 1998), and comparable
properties have been reported for human corticospinal
axons (Burke et al., 2000). s
SD
appears to be strongly
dependent on a noninactivating Na
+
conductance due to
Fpersistent_ Na
+
channels, and increases in s
SD
have been
reported when this conductance is activated by depolari-
zation (Bostock and Rothwell, 1997). In the present study,
s
SD
for the N
2
wave was elevated at 1 day, whereas I
rh
was increased at 3 h for both N
1
and N
2
. This dissimilar
pattern further substantiates that the N
1
and N
2
compo-
nents arise from distinct fiber populations with different
responses to injury. These pathological changes are also
consistent with injury-induced axonal depolarization or
alterations in the function of sodium channels. A rapidly
activated tetrodotoxin (TTX)-sensitive sodium influx has
been observed in vitro as an initiating pathology in TAI,
and was linked to subsequent proteolytic damage to
sodium channel a-subunits (Iwata et al., 2004). The
primary ionic event, generating the callosal CAP, is influx
through voltage gated sodium channels, and TTX has
been reported to entirely suppress callosal CAPs in brain
slices (Swanson et al., 1998).
Refractoriness, long recognized to depend largely on
recovery of Na
+
channels from inactivation (Hodgkin and
Huxley, 1952), was altered after TAI, though less strikingly
than was CAP amplitude or axonal excitability. This change
was also consistent with an axonal depolarization persisting
after injury, or to altered sodium channel functioning.
Refractoriness is sensitive to changes in membrane potential
(Burke et al., 1998); a depolarizing shift will increase (and a
hyperpolarizing shift will decrease) the extent of Na
+
channel inactivation.
To our knowledge, the present study is the first to
electrophysiologically address selective fiber vulnerabilities
following TBI, although one prior morphological study of
corpus callosum in the primate did report axolemmal tearing
at the nodes of small, thinly myelinated axons within 35 min
after injury (Maxwell et al., 1993). In contrast to this earlier
study, our ultrastructural analysis at 13 h postinjury failed
to detect any signs of acute axolemmal shearing in the
callosum. We attribute this difference in outcome to
dissimilarity of injury model and severity, as well as
subregion of callosum examined. In the present diffuse
injury model, myelinated fibers showed temporal patholog-
ical sequelae similar to that previously described for
brainstem descending fiber systems (Singleton et al.,
2002; Stone et al., 2001). These changes were consistent
with a local impairment of axonal transport, resulting in
organelle accumulation, cytoskeletal compaction, fiber
swelling, and ultimate disconnection. Notably, significant
myelinated fiber pathology was observed 3 h1 day
postinjury, the interval exhibiting more profound CAP
signal deficits.
Perhaps the most interesting result from the current
morphological assessment was the finding of rapid unmye-
linated fiber degeneration within the injured corpus cal-
losum, an observation not previously reported. While the
molecular events underlying this response are unknown, the
observed sequence of axolemmal breakdown, vesicular
pooling and cytoskeletal alteration are all consistent with
the hypothesis that injury compromises the local environ-
ment responsible for maintaining axolemmal integrity,
thereby permitting focal Na
+
/Ca
++
dysregulation and its
pathological consequences. This possibility is supported by
recent in vitro studies which show that injured small caliber
axons exhibit Na
+
and Ca
++
channel pathology (Iwata et al.,
2004), and that CNS extracellular proteins such as tenascin
can modulate the function of the Na
+
channel beta subunit
(Xiao et al., 1999). Obviously, these issues will require
further investigation of in vivo unmyelinated nerve fiber
damage, including detailed stereological assessment to
determine the magnitude of such damage, an effort clearly
beyond the scope of the current study.
A novel finding, in the present study, was a differential
functional recovery exhibited by the two fiber populations;
N
1
wave amplitudes, demonstrated a significant recovery by
7 days postinjury, whereas N
2
amplitudes did not recover
during this interval. This result must be interpreted in the
context of the ultrastructural finding that scattered axons in
both fiber populations showed progressive pathological
change consistent with irreversible damage rather than
recovery. One possibility is that some axons may be
functionally impaired, and yet appear to have a normal
ultrastructure. These axons may undergo a time-dependent
functional restitution and are recruited into the evoked CAP
T.M. Reeves et al. / Experimental Neurology 196 (2005) 126137 135
signal by 7 days postinjury. If these recoverable axons are
preferentially the larger, myelinated, fibers, this could
account for the selective improvements in the N
1
wave. In
this regard, freeze fracture studies have suggested that the
myelin sheath itself is not immediately damaged by the
forces of injury, and it was thought that TAI acted first on
the axolemma or axoplasm while sparing the myelin sheath
(Maxwell et al., 1988). Accordingly, one explanation may
be that unmyelinated fibers are intrinsically more suscep-
tible to concussive injury, and conversely that myelin may
confer some degree of protection to axons.
New information regarding how subpopulations of axons
are selectively affected by neurotrauma may contribute to an
understanding of the pathobiology of injury, and may
suggest potential strategies for therapeutic intervention.
Differential pathologies for fiber subtypes, for example,
may prove critical in the area of postinjury neuroplasticity.
The common clinical observation of diffuse axonal injury
necessarily indicates a diffuse synaptic terminal loss that
will initiate a plasticity response involving the removal of
degenerating terminals and reactive synaptogenesis (Phillips
and Reeves, 2001; Phillips et al., 1994; Povlishock, 2000;
Reeves et al., 2003). Whether such postinjury neuro-
plasticity is adaptive, and contributes to functional recovery,
or maladaptive may be conditioned, in part, by the specific
types and numbers of fibers involved.
Acknowledgments
The authors wish to thank Susan Walker, Nancy Lee,
Tom Coburn, Lesley Harris, and Raiford Black for technical
assistance, and Dr. Jonathan Lifshitz for helpful comments
on the manuscript. Supported by Virginia Commonwealth
Initiative Award 04-091 (TMR) and NS20193 (JTP).
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