This document summarizes research on diverse mechanisms of blast-induced neurotrauma in rat models. It finds that exposure to a single "composite" blast, which includes head acceleration, results in cerebrovascular damage, astrocyte activation (glyosis), and neuronal injury. A single "primary" blast exposure instigated predominantly systemic/vascular changes and glyosis. The positional orientation of the animal relative to the shock wave influences whether a composite or primary blast is experienced. Responses depend on the type and magnitude of blast exposure.

1. DIVERSE MECHANISMS OF BLAST INDUCED NEUROTRAUMA IN RAT MODELS Stanislav Svetlov, MD, PhD Animal procedures employed in this work have been approved by the University of Florida/VA IACUC protocols # E491 and 201005207 Body/Brain Responses Genomic/Proteomic/ Metabolomic Analyses

2. 7/11/2011 2 BLAST WAVE Blast wave: positive pressure rise and flow resulting from deposition of a large amount of energy in a localized volume Peak overpressure decays exponentially from origin Duration of positive phase increases away from origin Total impulse  mechanical energy transferred to target Primary Blast Injury: Overpressure shock wave Tertiary Blast Injury: ‘Axial load’ injuries/concussion Secondary Blast Injury: Injury from flying debris

3. Blast Generator Facility at Banyan Biomarkers •Principle: sudden exposure of a low pressure gas to a gas at significantly higher pressure that will result in the formation of a shock wave •Driver section is initialized to a pressure of 750 psi. When the diaphragm ruptures, the driver gas, acting as an impulsively started piston, sets up a series of pressure waves •Data acquisition: PCB piezoelectric transducers and LabView 8.2 software; a National Instruments 1.25 Msamples/sec data acquisition card multiple channels. •The rat head images during the blast event were captured at >25,000 frames/sec using a high speed video camera and Schlieren optics. Svetlov et al. J. Trauma, 2010

4. Components of shock tube-generated blast wave: Peak Overpressure and VentingGas ‘Composite’ blast: peak overpressure force + head acceleration ‘Primary blast: peak overpressure wave flows inside brain Level 1 –moderate/severe Level 2a-moderate Level 2b-mild/moderate Level 3 –mild

5. Blast OP Wave Off-axis exposure: avoids gas venting On-axis exposure: blast gas venting Gas Venting ‘Composite’ Blast Primary Blast Different rat positions toward shock tube exit nozzle: on-axis (A) and off-axis (B). Placing rats outside axis avoids gas venting effect and exposes rat to a pure primary blast.

6. Banyan Biomarkers Blast Injury Pre-Clinical Program Paradigm: blast-induced neuroinjury is mediated via overlapping sequence of pathophysiological responses depending on the type of blast exposure. Single Blast Exposure Systemic/Vascular responses: Interleukins, ICAM, L, E-selectins VCAM composite vs. primary Neuroinflammation: CD11b, Iba-1, MMPs TBI signatures Glyosis: GFAP, CNPase, S100b Systems Biology Analysis Neural control of metabolism/ adipogenesis: Orexin A,Resistin Neuronal injury/Neurodegeneration: NSE, UCH-L1 Neuroregeneration: Neuropilin-2, CRMP-2 TBI pathology: Biomarkers/Diagnostics

7. Brain Pathomorphology/Silver Staining Head-Directed Severe Blast Exposure (52.6 psi/10 msec) Cortex, 7 days Sham (noise exposure) Hippocampus, 7 days 48 hours post-blast 5 days post-blast Thalamus, 7 days Gross pathology: Focal intracranial hematomas. Histopathology: diffuse and local silver accumulation in Caudal Diencephalon section (B2 and C2). Black arrows indicate strong silver staining in Nucleus Subthalamicus. (1.5x, A2-C2). Red arrows point to silver accumulation in perivascular and periventricular tissue zone. (10x, A3-C3)

8. Brain Pathomorphology/Silver Staining Head-Directed Severe Blast Exposure (52.6/10 msec) Head-Directed Severe Blast Exposure (33.9/113.8 µsec) Cortex, 7 days CCI Primary, off axis On-axis Hippocampus, 7 days On-axis Primary, off axis CCI

9. Accumulation of NSE and UCH-L1 in blood after blast exposure: Unpaired t-test was employed to analyze statistical significance of values. On-axis ‘composite’ blast Serum .6 .5 .4 UCH-L1, ng/ml .3 NSE, ng/ml .2 .1 Post-blast Period Off axis primary blast

10. Banyan Biomarkers Blast Injury Pre-Clinical Program Paradigm: blast-induced neuroinjury is mediated via overlapping sequence of pathophysiological responses depending on the type of blast exposure. Blast Exposure Systemic/Vascular responses: Interleukins, ICAM, L, E-selectins VCAM composite vs. primary Neuroinflammation: CD11b, Iba-1, MMPs TBI signatures Glyosis: GFAP, CNPase, S100b Systems Biology Analysis Neural control of metabolism/ adipogenesis: Orexin A,Resistin Neuronal injury/Neurodegeneration: NSE, UCH-L1 Neuroregeneration: Neuropilin-2, CRMP-2 TBI pathology: Biomarkers/Diagnostics

11. Astrocytosis Expression of GFAP in cortex and hippocampus of rats after severe head-directed blast exposure Hippocampus Cortex Blast Blast Sham Sham 24h 7-d 14-d 30-d 24h 7-d 14-d 30-d GFAP GFAP Note : moderate astroglyosis in hippocampus 7 days after blast

12. Astrocytosis Expression of GFAP in Hippocampus (CA1 region ) of OBI injured rats. A, control naïve; B on axis 1 day; C, on axis 7 days; D, off axis 1 day and E on axis 7 days. Low magnification 5 x and high magnification 20x of CA1 region boxed in top of panels are shown. B C A D E 7/11/2011 12 Banyan Biomarkers, Confidential

13. 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase) Human tissue panel Blast M.W. Off-axis On-axis M.W. Naive 1d 1d 1d 7d 7d 7d 50 Rat Hippocampus 36 50 Whole Brain 36 50 6h 1d 7d 1d 7d Sham Naive On-axis Off-axis 36 Rat Cerebellum Lung Liver Heart Testes Spleen Muscles Kidney Skin Rat tissue panel

14. Expression of CNPase in Hippocampus (DG region ) of OBI injured rats. A, control naïve; B on axis 1 day; C, on axis 7 days; D, off axis 1 day and E off axis 7 days. Low magnification 5 x and high magnification 20x of DG region boxed in top of panels are shown. A B C D E 7/11/2011 14 Banyan Biomarkers, Confidential

15. On axis ‘composite’ blast GFAP, ng/ml Off axis primary blast CSF Serum GFAP, ng/ml On axis: Unpaired t-test was employed to analyze statistical significance of values. (*-p<0.05; **-p<0.01). Off axis: t-test with Welch correction was done. (*-p<0.05)

16. Banyan Biomarkers Blast Injury Pre-Clinical Program Paradigm: blast-induced neuroinjury is mediated via overlapping sequence of pathophysiological responses depending on the type of blast exposure. Blast Exposure Systemic/Vascular responses: Interleukins, ICAM, L, E-selectins VCAM composite vs. primary Neuroinflammation: CD11b, Iba-1, MMPs TBI signatures Glyosis: GFAP, CNPase, S100b Systems Biology Analysis Neural control of metabolism/ adipogenesis: Orexin A,Resistin Neuronal injury/Neurodegeneration: NSE, UCH-L1 Neuroregeneration: Neuropilin-2, CRMP-2 TBI pathology: Biomarkers/Diagnostics

17. Serum levels of Pro- and Anti-inflammatory cytokines IL-1 and IL-10. Rats were subjected to on axis or off-axis head + total body blast: 33.9psi, 113 µsec with body armored or uncovered. Blood was collected and cytokines were assayed in serum using RayBiotech L-arrays.

18. Serum levels of Integrin alpha/beta – a complement receptor composed of CD11c and CD18

19. ** ** sICAM in rat CSF * * * * On axis, head Levels sICAM in CSF and serum after different types of blast exposure. sICAM was determined by ELISA Kit

20. Levels of L-selectin and E-selectin in serum after different types of blast exposure. L- and E-selectins were determined using antibody arrays B A L-Selectin by ELISA:

21. Banyan Biomarkers Blast Injury Pre-Clinical Program Paradigm: blast-induced neuroinjury is mediated via overlapping sequence of pathophysiological responses depending on the type of blast exposure. Blast Exposure Systemic/Vascular responses: Interleukins, ICAM, L, E-selectins VCAM composite vs. primary Neuroinflammation: CD11b, Iba-1, MMPs TBI signatures Glyosis: GFAP, CNPase, S100b Systems Biology Analysis Neural control of metabolism/ adipogenesis: Orexin A,Resistin Neuronal injury/Neurodegeneration: NSE, UCH-L1 Neuroregeneration: Neuropilin-2, b-NGF, CRMP-2 TBI pathology: Biomarkers/Diagnostics

22. Neuropilin-2 (NRP-2)-receptor for VEGF and semaphorins. Links vascular growth factors and axonal growth cone guidance in CNS. Rat Hippocampus Blast On-axis Off-axis M.W. Sham Naive 1d 1d 1d 7d 7d 7d Open body Protected body On-axis, Composite Off-axis, Primary (protected body) Off-axis, Primary (open body)

23. Levels of soluble Neuropilin-2 in Rat Serum (Western blot and quantitative protein array On-axis Off-axis Naive Sham 1d 1d 1d 7d 7d 7d 98 Open body Protected body Serum NRP-2, Arb. U/ml)

25. Center of Innovative Research (CoIR): Mechanical and Aerospace Engineering Department Florida Institute of Technology Victor Prima, PhD Kevin Wang, PhD Ronald Hayes, PhD OlenaGlushakova, M.S. Quishi Tang, M.S. Archie Svetlov Daniel Kirk, PhD Hector Gutierrez, Ph.D. Joseph Atkinson, M.S. Mark Pereira, Graduate Student Supported by N14-06-1-1029 (Hayes), W81XWH-8-1-0376 (Hayes/Wang) and W81XWH-07-01-0701 (Svetlov) from Department of Defense