Svetlov, Stanislav

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Svetlov, Stanislav

  1. 1. DIVERSE MECHANISMS OF BLAST INDUCED NEUROTRAUMA <br />IN RAT MODELS<br />Stanislav Svetlov, MD, PhD<br />Animal procedures employed in this work have been approved by the University of Florida/VA IACUC protocols # E491 and 201005207<br />Body/Brain Responses<br />Genomic/Proteomic/<br />Metabolomic Analyses<br />
  2. 2. 7/11/2011<br />2<br />BLAST WAVE<br />Blast wave: positive pressure rise and flow resulting from deposition of a large amount of energy in a localized volume<br />Peak overpressure decays exponentially from origin<br />Duration of positive phase increases away from origin<br />Total impulse  mechanical energy transferred to target<br />Primary Blast Injury: <br />Overpressure shock wave<br />Tertiary Blast Injury:<br />‘Axial load’ injuries/concussion<br />Secondary Blast Injury:<br />Injury from flying debris<br />
  3. 3. Blast Generator Facility at Banyan Biomarkers<br />•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 <br />•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 <br />•Data acquisition: PCB piezoelectric transducers and LabView 8.2 software; a National Instruments 1.25 Msamples/sec data acquisition card multiple channels. <br />•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<br />
  4. 4. Components of shock tube-generated blast wave: Peak Overpressure and VentingGas<br />‘Composite’ blast: peak overpressure force + head acceleration <br />‘Primary blast: peak overpressure wave flows inside brain <br />Level 1 –moderate/severe<br />Level 2a-moderate<br />Level 2b-mild/moderate<br />Level 3 –mild <br />
  5. 5. Blast OP Wave<br />Off-axis exposure: <br />avoids gas venting<br />On-axis exposure: <br />blast gas venting<br />Gas Venting<br />‘Composite’ Blast<br />Primary Blast<br />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. <br />
  6. 6. Banyan Biomarkers Blast Injury Pre-Clinical Program<br />Paradigm: blast-induced neuroinjury is mediated via overlapping sequence of<br />pathophysiological responses depending on the type of blast exposure. <br />Single Blast Exposure<br />Systemic/Vascular responses:<br />Interleukins, ICAM, L, E-selectins VCAM<br />composite vs. primary <br />Neuroinflammation:<br />CD11b, Iba-1, MMPs <br />TBI signatures<br />Glyosis: GFAP, CNPase, S100b<br />Systems Biology Analysis<br /> Neural control of <br />metabolism/ adipogenesis:<br />Orexin A,Resistin<br />Neuronal injury/Neurodegeneration:<br />NSE, UCH-L1<br />Neuroregeneration: <br />Neuropilin-2, CRMP-2<br />TBI pathology: <br />Biomarkers/Diagnostics<br />
  7. 7. Brain Pathomorphology/Silver Staining<br />Head-Directed Severe Blast Exposure (52.6 psi/10 msec)<br />Cortex, 7 days<br />Sham (noise exposure)<br />Hippocampus, 7 days<br />48 hours <br />post-blast<br />5 days<br /> post-blast<br />Thalamus, 7 days<br />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)<br />
  8. 8. Brain Pathomorphology/Silver Staining<br />Head-Directed Severe Blast Exposure (52.6/10 msec)<br />Head-Directed Severe Blast Exposure (33.9/113.8 µsec)<br />Cortex, 7 days<br />CCI<br />Primary, off axis<br />On-axis<br />Hippocampus, 7 days<br />On-axis<br />Primary, off axis<br />CCI<br />
  9. 9. Accumulation of NSE and UCH-L1 in blood after blast exposure: Unpaired t-test was employed to analyze statistical significance of values. <br />On-axis ‘composite’ blast<br />Serum<br />.6<br />.5<br />.4<br />UCH-L1, ng/ml<br />.3<br />NSE, ng/ml<br />.2<br />.1<br />Post-blast Period <br />Off axis primary blast<br />
  10. 10. Banyan Biomarkers Blast Injury Pre-Clinical Program<br />Paradigm: blast-induced neuroinjury is mediated via overlapping sequence of<br />pathophysiological responses depending on the type of blast exposure. <br />Blast Exposure<br />Systemic/Vascular responses:<br />Interleukins, ICAM, L, E-selectins VCAM<br />composite vs. primary <br />Neuroinflammation:<br />CD11b, Iba-1, MMPs <br />TBI signatures<br />Glyosis: GFAP, CNPase, S100b<br />Systems Biology Analysis<br /> Neural control of <br />metabolism/ adipogenesis:<br />Orexin A,Resistin<br />Neuronal injury/Neurodegeneration:<br />NSE, UCH-L1<br />Neuroregeneration: <br />Neuropilin-2, CRMP-2<br />TBI pathology: <br />Biomarkers/Diagnostics<br />
  11. 11. Astrocytosis<br />Expression of GFAP in cortex and hippocampus of rats after severe head-directed blast exposure<br />Hippocampus <br />Cortex<br />Blast<br />Blast<br />Sham<br />Sham<br />24h 7-d 14-d 30-d<br />24h 7-d 14-d 30-d<br />GFAP<br />GFAP<br />Note : moderate astroglyosis in hippocampus 7 days after blast<br />
  12. 12. Astrocytosis<br />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.<br />B<br />C<br />A<br />D<br />E<br />7/11/2011<br />12<br />Banyan Biomarkers, Confidential<br />
  13. 13. 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase)<br />Human tissue panel<br />Blast<br />M.W.<br />Off-axis<br />On-axis<br />M.W.<br />Naive<br />1d<br />1d<br />1d<br />7d<br />7d<br />7d<br />50<br />Rat Hippocampus<br />36<br />50<br />Whole Brain<br />36<br />50<br />6h 1d<br />7d<br />1d<br />7d<br />Sham<br />Naive<br />On-axis<br />Off-axis<br />36<br />Rat Cerebellum <br />Lung<br />Liver<br />Heart<br />Testes<br />Spleen<br />Muscles<br />Kidney<br />Skin<br />Rat tissue panel<br />
  14. 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.<br />A<br />B<br />C<br />D<br />E<br />7/11/2011<br />14<br />Banyan Biomarkers, Confidential<br />
  15. 15. On axis ‘composite’ blast<br />GFAP, ng/ml<br />Off axis primary blast<br />CSF<br />Serum<br />GFAP, ng/ml<br />On axis: Unpaired t-test was employed to analyze statistical significance of values. (*-p<0.05; **-p<0.01). <br />Off axis: t-test with Welch correction was done. (*-p<0.05)<br />
  16. 16. Banyan Biomarkers Blast Injury Pre-Clinical Program<br />Paradigm: blast-induced neuroinjury is mediated via overlapping sequence of<br />pathophysiological responses depending on the type of blast exposure. <br />Blast Exposure<br />Systemic/Vascular responses:<br />Interleukins, ICAM, L, E-selectins VCAM<br />composite vs. primary <br />Neuroinflammation:<br />CD11b, Iba-1, MMPs <br />TBI signatures<br />Glyosis: GFAP, CNPase, S100b<br />Systems Biology Analysis<br /> Neural control of <br />metabolism/ adipogenesis:<br />Orexin A,Resistin<br />Neuronal injury/Neurodegeneration:<br />NSE, UCH-L1<br />Neuroregeneration: <br />Neuropilin-2, CRMP-2<br />TBI pathology: <br />Biomarkers/Diagnostics<br />
  17. 17. Serum levels of Pro- and Anti-inflammatory cytokines IL-1 and IL-10.<br />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.<br />
  18. 18. Serum levels of Integrin alpha/beta – a complement receptor <br />composed of CD11c and CD18<br />
  19. 19. **<br /> **<br />sICAM in rat CSF<br />* * <br />* * <br />On axis, head<br />Levels sICAM in CSF and serum after different types of <br /> blast exposure. sICAM was determined by ELISA Kit <br />
  20. 20. Levels of L-selectin and E-selectin in serum after different types of<br /> blast exposure. L- and E-selectins were determined using antibody arrays<br />B<br />A<br />L-Selectin<br />by ELISA:<br />
  21. 21. Banyan Biomarkers Blast Injury Pre-Clinical Program<br />Paradigm: blast-induced neuroinjury is mediated via overlapping sequence of<br />pathophysiological responses depending on the type of blast exposure. <br />Blast Exposure<br />Systemic/Vascular responses:<br />Interleukins, ICAM, L, E-selectins VCAM<br />composite vs. primary <br />Neuroinflammation:<br />CD11b, Iba-1, MMPs <br />TBI signatures<br />Glyosis: GFAP, CNPase, S100b<br />Systems Biology Analysis<br /> Neural control of <br />metabolism/ adipogenesis:<br />Orexin A,Resistin<br />Neuronal injury/Neurodegeneration:<br />NSE, UCH-L1<br />Neuroregeneration: <br />Neuropilin-2, b-NGF, CRMP-2<br />TBI pathology: <br />Biomarkers/Diagnostics<br />
  22. 22. Neuropilin-2 (NRP-2)-receptor for VEGF and semaphorins.<br />Links vascular growth factors and axonal growth cone guidance in CNS. <br />Rat Hippocampus<br />Blast<br />On-axis<br />Off-axis<br />M.W.<br />Sham<br />Naive<br />1d<br />1d<br />1d<br />7d<br />7d<br />7d<br />Open <br />body<br />Protected <br />body<br />On-axis, Composite<br />Off-axis, Primary<br />(protected body)<br />Off-axis, Primary<br />(open body)<br />
  23. 23. Levels of soluble Neuropilin-2 in Rat Serum <br />(Western blot and quantitative protein array<br />On-axis<br />Off-axis<br />Naive<br />Sham<br />1d<br />1d<br />1d<br />7d<br />7d<br />7d<br />98<br />Open <br />body<br />Protected <br />body<br />Serum NRP-2, Arb. U/ml) <br />
  24. 24. Systemic/Vascular responses:<br />Interleukins, ICAM, L, E-selectins<br />Awaits Systems Biology Analyses:<br />BLAST-omics (?) <br />Neuroinflammation:<br />CD11b, integrins, MMPs <br />Single Blast Exposure<br />Glyosis: GFAP, CNPase, S100b<br />composite<br />vs. <br /> primary <br /> Neural control of <br />metabolism/ adipogenesis:<br />Orexin A,Resistin<br />Neuronal injury/Neurodegeneration:<br />NSE, UCH-L1<br />Neuroregeneration: <br />Neuropilin-2, CRMP-2, b-NGF<br />Summary<br /><ul><li>pathophysiological responses to blast delivered via shock tube depend on </li></ul> the positional orientation of animal to the shock wave <br /><ul><li> exposure to a single ‘composite’ blast is accompanied by head acceleration </li></ul> and resulted in cerebrovascular damage, glyosis and neuronal injury<br /><ul><li> single primary blast exposure instigated predominantly systemic/vascular </li></ul> changes and glyosis<br />? Responses to multiple blast of ‘low’ magnitude<br />? Behavioral studies of mild blast<br />? Are there any specific biomarkers for primary blast injuries?<br />
  25. 25. Center of Innovative Research <br />(CoIR):<br />Mechanical and Aerospace Engineering Department<br />Florida Institute of Technology<br />Victor Prima, PhD<br />Kevin Wang, PhD<br />Ronald Hayes, PhD<br />OlenaGlushakova, M.S.<br />Quishi Tang, M.S.<br />Archie Svetlov<br />Daniel Kirk, PhD<br />Hector Gutierrez, Ph.D.<br />Joseph Atkinson, M.S.<br />Mark Pereira, Graduate Student<br />Supported by N14-06-1-1029 (Hayes), W81XWH-8-1-0376 (Hayes/Wang) and W81XWH-07-01-0701 (Svetlov) from Department of Defense<br />

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