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” “Silent epidemic” 5.3 million Americans currently live with long-term disability 5.3 million Americans currently live with long-term disability (Langlois et al., 2006) 1,400,000? Total 1,111,000 Emergency Room Visits 235,000 Hospitalizations 50,000 Deaths 80,000 Disabilities

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

Tolerance Criteria To quantify brain tissue’s bio-response to controlled mechanical stimuli To quantify brain tissue’s bio-response to controlled mechanical stimuli Cell damage / death Cell damage / death Neuronal dysfunction Neuronal dysfunction Requirements: Requirements: Precise control of tissue strain with verification Precise control of tissue strain with verification Very difficult in vivo Very difficult in vivo Quantification of the living tissue response Quantification of the living tissue response Tissue must be alive Tissue must be alive Mechanical Input Bio Output  Cater HL, Sundstrom LE, Morrison III B. J.Biomech. 39: , Elkin BS, Morrison III B. Stapp Car Crash J. 51: , Morrison III B, et al. Stapp Car Crash J. 47:93-105, 2003.

In Vitro Injury Model 2 Components 2 Components Tissue cultures of hippocampus and cortex Tissue cultures of hippocampus and cortex Organotypic brain slice cultures Organotypic brain slice cultures Maintain anatomical structures and organization Maintain anatomical structures and organization Possible to quantify bio-response in anatomical regions Possible to quantify bio-response in anatomical regions Injury device Injury device Precise control over biomechanics Precise control over biomechanics Strain and strain rate Strain and strain rate Sterile injury of the cultures Sterile injury of the cultures Measure tissue response at different time points Measure tissue response at different time points Morrison III B, et al. J.Neurosci.Methods 150: , 2006

In Vitro Model of TBI Organotypic hippocampal brain slice cultures Organotypic hippocampal brain slice cultures Complex tissue culture Complex tissue culture Maintains in vivo anatomy Maintains in vivo anatomy Functionally active Functionally active CA1 CA3 DG Nissl MAP-2 H&E Transverse In Vivo

In Vitro Model of TBI Precise control of Precise control of Injury biomechanics Injury biomechanics Appropriate loading for TBI Appropriate loading for TBI E >10% ; E’ >10 s -1 E >10% ; E’ >10 s -1 Extracellular environment Extracellular environment Increased access to tissue Increased access to tissue

Motivation Previously determined cell death tolerance Previously determined cell death tolerance Hippocampus and cortex Hippocampus and cortex Want to determine functional tolerance Want to determine functional tolerance Can the neuronal network function of the hippocampus be disrupted by mechanical deformation in the absence of cell death? Can the neuronal network function of the hippocampus be disrupted by mechanical deformation in the absence of cell death? Experimental design Experimental design Injure cultures at 5% and 10% Injure cultures at 5% and 10% Quantify changes in function at 6 days post-injury Quantify changes in function at 6 days post-injury Microelectrode arrays – 60 electrodes simultaneously Microelectrode arrays – 60 electrodes simultaneously

Stimulus Response Curves Quantify neuronal functionality Quantify neuronal functionality Plot evoked response magnitude vs. stimulus magnitude Plot evoked response magnitude vs. stimulus magnitude Fit sigmoid function for comparison Fit sigmoid function for comparison Evoked Response

Decreased R max Maximum evoked response Maximum evoked response Was decreased 6 days after injury Was decreased 6 days after injury Decrease was correlated with injury severity Decrease was correlated with injury severity CA1CA3DG CA1 CA3 DG

Increased I 50 Stimulus required for half maximal response Stimulus required for half maximal response Was increased 6 days after injury Was increased 6 days after injury Increase was correlated with injury severity Increase was correlated with injury severity CA1CA3DG

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

Discussion: I 50 Increased I 50 Increased I 50 The firing neurons were less excitable The firing neurons were less excitable Required larger stimuli for half-maximal response Required larger stimuli for half-maximal response Measure is independent of R max Measure is independent of R max Independent of the number of neurons firing Independent of the number of neurons firing Suggests that cellular machinery required for neurotransmission was damaged by tissue deformation Suggests that cellular machinery required for neurotransmission was damaged by tissue deformation Voltage sensitive channels Voltage sensitive channels Calcium, sodium Calcium, sodium Vesicular release machinery Vesicular release machinery Synaptotagmin, SNAPs Synaptotagmin, SNAPs 

Discussion: In Vivo Findings In vivo studies report decreased R max in CA1 In vivo studies report decreased R max in CA1 Models are accompanied by substantial cell loss Models are accompanied by substantial cell loss Unknown levels of tissue deformation Unknown levels of tissue deformation In vivo studies report increased excitability of DG In vivo studies report increased excitability of DG In response to perforant path stimulation In response to perforant path stimulation Limitation of the slice model – no perforant path Limitation of the slice model – no perforant path Attributed to selective loss of inhibitory inter-neurons Attributed to selective loss of inhibitory inter-neurons Functional changes may depend on injury severity Functional changes may depend on injury severity May see similar excitability with greater stretch May see similar excitability with greater stretch No reports of CA3 function No reports of CA3 function

Future Directions Examine response to different injury levels Examine response to different injury levels Additional strains Additional strains Additional strain rates Additional strain rates Explore cellular mechanisms of dysfunction Explore cellular mechanisms of dysfunction Identify therapeutic targets Identify therapeutic targets Proteolysis of neuronal machinery Proteolysis of neuronal machinery Production of free radicals Production of free radicals Test therapies to preserve function Test therapies to preserve function

Acknowledgements Ben Elkin Ben Elkin Zhe Yu Zhe Yu Columbia University ZheMikeBenShamikMelissa Barclay