Modeling traumatic brain injury in vitro:

functional changes in the

absence of cell death

Barclay Morrison III, Ph.D.

Columbia University
Biomedical Engineering
Traumatic Brain Injury
• “Silent epidemic”

50,000
Deaths
80,000
Disabilities
235,000
Hospitalizations

1,111,000
• 5.3 million Americans
Emergency Room Visits currently live with
1,400,000? long-term disability
Total

(Langlois et al., 2006) www.cdc.gov

Traumatic Brain Injury Simulation
• Finite element models of TBI
• Predict brain deformation
• Mechanical basis for TBI
• Initiator for the bio-response
• Require critical information
• Geometry
• Material properties
• Tolerance criteria

Wayne State
Tolerance Criteria
• To quantify brain tissue’s bio-response to
controlled mechanical stimuli Mechanical
• Cell damage / death Input
• Neuronal dysfunction 
• Requirements:
Bio
Output
• Precise control of tissue strain with verification
• Very difficult in vivo
• Quantification of the living tissue response
• Tissue must be alive
Cater HL, Sundstrom LE, Morrison III B. J.Biomech. 39:2810-2818, 2006.
Elkin BS, Morrison III B. Stapp Car Crash J. 51:127-138, 2007.
Morrison III B, et al. Stapp Car Crash J. 47:93-105, 2003.
In Vitro Injury Model
• 2 Components
• Tissue cultures of hippocampus and cortex
• Organotypic brain slice cultures
• Maintain anatomical structures and organization
• Possible to quantify bio-response in anatomical regions
• Injury device
• Precise control over biomechanics
• Strain and strain rate
• Sterile injury of the cultures
• Measure tissue response at different time points

Morrison III B, et al. J.Neurosci.Methods 150: 192-201, 2006

In Vitro Model of TBI
• Organotypic hippocampal brain slice cultures
• Complex tissue culture In Vivo
• Maintains in vivo anatomy
CA1

• Functionally active
S

H
CA3
DG

Nissl
CA1
DG
CA3
MAP-2 H&E Transverse
In Vitro Model of TBI
• Precise control of
• Injury biomechanics
• Appropriate loading for TBI
• E >10% ; E’ >10 s-1
• Extracellular environment
• Increased access to tissue

Tissue
Well Membrane

Indenter
Motivation
• Previously determined cell death tolerance
• Hippocampus and cortex
• Want to determine functional tolerance
• Can the neuronal network function of the
hippocampus be disrupted by mechanical
deformation in the absence of cell death?
• Experimental design
• Injure cultures at 5% and 10%
• Quantify changes in function at 6 days post-injury
• Microelectrode arrays – 60 electrodes simultaneously
Stimulus Response Curves
• Quantify neuronal functionality
• Plot evoked response magnitude
vs. stimulus magnitude
• Fit sigmoid function for comparison
Evoked Response

Rmax
R( S )  m  I 50  S 
1 e
 Decreased Rmax
CA1

• Maximum evoked response DG

• Was decreased 6 days after injury CA3

• Decrease was correlated with injury severity

1000 1000 1000
800 800 800
Response (uV)

Response (uV)
Response (uV)

600 600 600

400 400 400
200 200 200
0 0 0
0% 5% 10% 0% 5% 10% 0% 5% 10%
Strain Strain Strain

CA1 CA3 DG
 Increased I50
• Stimulus required for half maximal response
• Was increased 6 days after injury
• Increase was correlated with injury severity
150 150 150
Stimulus (uA)

Stimulus (uA)
Stimulus (uA)

100 100 100

50 50 50

0 0
0
0% 5% 10% 0% 5% 10%
0% 5% 10%
Strain
Strain Strain

CA1 CA3 DG
Discussion: Rmax
• Decreased Rmax
• Fewer neurons were firing
or fewer firing in a coordinated fashion
• These low strain levels induce minimal cell death
• Not simply a loss of cells leading to a reduced response
• There is cell loss at higher levels of strain
• Would expect a reduction of Rmax with cell loss


Discussion: I50
• Increased I50
• The firing neurons were less excitable
• Required larger stimuli for half-maximal response
• Measure is independent of Rmax
• Independent of the number of neurons firing
• Suggests that cellular machinery required for
neurotransmission was damaged by tissue deformation
• Voltage sensitive channels
• Calcium, sodium 
• Vesicular release machinery
• Synaptotagmin, SNAPs
Discussion: In Vivo Findings
• In vivo studies report decreased Rmax in CA1
• Models are accompanied by substantial cell loss
• Unknown levels of tissue deformation
• In vivo studies report increased excitability of DG
• In response to perforant path stimulation
• Limitation of the slice model – no perforant path
• Attributed to selective loss of inhibitory inter-neurons
• Functional changes may depend on injury severity
• May see similar excitability with greater stretch
• No reports of CA3 function
Future Directions
• Examine response to different injury levels
• Additional strains
• Additional strain rates
• Explore cellular mechanisms of dysfunction
• Identify therapeutic targets
• Proteolysis of neuronal machinery
• Production of free radicals
• Test therapies to preserve function
Acknowledgements
Columbia University
• Ben Elkin
• Zhe Yu

Zhe Barclay Mike Ben Shamik Melissa