Rodent Blast Injury Models – Good "Bang for the Buck"? Ibolja Cernak, M.D., M.E., Ph.D. Biomedicine Business Area; National Security Technology Department
2 Complex Mechanisms of Injuries
Cernak I & Noble-Hauesslein L. Journal of Cerebral Blood Flow & Metabolism (2010) 30, 255–266 Complex Array of Injuries
FREQUENCY high frequency (0.5-1.5 kHz) low-amplitude stress waves: target mostly organs that contain abrupt density changes from one medium to other (for example, air/blood interface in the lungs or blood/parenchyma interface in the brain); low-frequency (<0.5 kHz) high-amplitude shear waves: disrupt tissue structures by generating local motions that overcome natural tissue elasticity (for example, at the contact of gray and white brain matter). OVERPRESSURE Peak; Duration of the peak; Number of peaks; Distance between peaks; Interaction between stress, expansion and refraction waves. BLAST WAVE COMPOUNDS EM Pulse
5 Cernak et al; J Trauma 47(1): 96-104, 1999. Research Challenge: Unique and Complex Ways of Interaction Between Blast and Body/Brain DIRECT INDIRECT
Transfer of the kinetic energy of the blast wave via oscillating pressures in fluid phase (large blood vessels) to the CNS
Direct interaction with the skull
Direct interaction with the spine
Blast-induced neurological deficits and psychological impairments
In Vivo Models of Traumatic Brain Injury UNCLASSIFIED 7 CERNAK, I. Experimental models of traumatic brain injury. NeuroRx, 2(3): 410-422, 2005.
UNCLASSIFIED 8 Requirements for a Reliable Experimental Model of Traumatic Brain Injury The mechanical force used to induce injury is controlled, reproducible, and quantifiable; The inflicted injury is reproducible, quantifiable, and mimics components of human conditions; The injury outcome, measured by morphological, physiological, biochemical, or behavioral parameters, is related to the mechanical force causing the injury; The intensity of the mechanical force used to inflict injury should predict the outcome severity. Cernak I, 2005, NeuroRx 2: 410-422 MODELING BLAST INJURIES AND BLAST-INDUCED NEUROTRAUMA
Mechanical factors inducing injury, based on physics of field explosions;
Essential hallmarks of blast injuries and BINT:
Position-dependent injury severity and injury pattern;
Graded functional (motor, cognitive, and behavioral) deficits;
Graded induction of molecular response in the brain;
Importance of systemic response in BINT.
What to Measure? UNCLASSIFIED 9 Reneer DV, Hisel RD, Hoffman JM, Kryscio RJ, Lusk BT, Geddes JW: J Neurotrauma JOURNAL 28:95–104 (January 2011 T0 - the time at which the pressure began to rise above ambient pressure. Positive magnitude (Pos. Mag.) – the difference between peak pressure and ambient pressure. Positive duration (Pos. Dur.) - the time between T0 and when the pressure goes below ambient pressure. Positive impulse (Pos. Impulse) - the integral of the pressure-time trace during the positive phase. Negative magnitude (Neg. Mag.) - the difference between ambient and peak negative pressure. The Friedlander wave describes an ideal blast from a spherical source in an open environment.
certain properties of the resultant shockwave are modifiable;
the pressure-time trace from air-driven shock tubes is flatter than the peaked waves resulting from high explosives;
compressed air fails to expand as quickly as would an ideal gas when the membrane is ruptured (due in part to intermolecular forces strengthened during compression;
do not model other components of a chemical blast, including acoustic, thermal, optical, and electromagnetic components;
Compressed helium-driven shock tubes: PROS:
improves the performance of shock tubes due to the increased speed of sound in helium compared to air, resulting in a lower driver- to driven-tube ratio;
produce a sharper overpressure peak and shorter overpressure duration;
The variability (standard deviation) of the peak overpressures can be greater than that generated by compressed air;
large amounts required to pressurize the driver;
Elements of a Shock Tube UNCLASSIFIED 14 Cernak I et al. Neurobiology of Disease 41(2011): 538-551 Diaphragm(s) [Mylar; Kapton; Polyethylene) inserted between the two sections, creates a closed volume driver section that allows pressurization. The driver section, which initially contains ambient air, is pressurized with helium (or other gas) until the pressure differential between the driver and driven sections exceeded the material failure threshold of the diaphragm thus initiating rupture. Upon diaphragm rupture, a shock wave propagates down the length of the shock tube. Side mounted pressure sensors, used to record the static pressure and wave propagation While a side mounted pressure sensor can only measure static pressures, a pressure probe pointed into the flow can be used to measure the total pressure. The total pressure is the stagnation pressure for compressible flow where the flow is brought to rest adiabatically, and it more closely represents the pressure that the specimen experiences when facing the shock wave. To measure the total pressure for each test condition, shock tube tests were conducted with a pressure probe inserted in place of the small animal specimen.
15 Cernak I et al. Neurobiology of Disease 41(2011): 538-551 Sensor Locations DRIVEN DRIVER Example: JHU/APL Mouse BINT Model
UNCLASSIFIED 16 Reneer DV, Hisel RD, Hoffman JM, Kryscio RJ, Lusk BT, Geddes JW: J Neurotrauma JOURNAL 28:95–104 (January 2011 Example: The McMillan blast device (MBD) The Mylar membrane The reflected (face-on) pressure sensor (embedded in the dorsal surface of the polyurethane rat model The anesthetized rat is fitted with a Kevlar vest (not shown) and inserted into a mesh netting support. This is then loaded into a shock tube insert. A test article (i.e., animal) should present less than 5% area blockage in order to replicate free-field conditions
How to Measure Blast Injury Severity UNCLASSIFIED 17 Bowen’s Injury Risk Curve (number of animals in parentheses) (adapted from “Estimate of man’s tolerance to the direct effects of air blast,” Technical Progress Report, DASA-2113, Defense Atomic Support Agency, Department of Defense, Washington, DC, October 1968).
Blast Injury Severity Measurement UNCLASSIFIED 18 Problem: The Bowen criteria used lethality and lung (i.e.organ) damages as outcome measures in function of pressure and duration of the blast. As such, they are less reliable and/or useful for predicting functional deficits (such as memory deficits) as outcomes; The physiological and biological responses to injurious environment are specific and unique among the different animal species. Thus, physical scaling based on animals’ size and using pressure-duration factors will give unreliable and inconsistent information about functional changes due to blast.
19 Koliatsos V, Cernak I, et al. Journal of Neuropathology and Experimental Neurology, 70(5): 399-416, 2011 Type, Severity and Frequency of Blast-induced Microscopic Lesions in Key Thoracic and Abdominal Organs Supine > Prone Prone > Supine
UNCLASSIFIED Cernak I et al. Neurobiology of Disease 41(2011): 538-551 Functional Deficits in BINT: Motor * * ** ** * *** * *** *** ** *** *** *** *** ** *** 20
UNCLASSIFIED Cernak I et al. Neurobiology of Disease 41(2011): 538-551 Functional Deficits in BINT: Cognitive * * ** ** ** *** *** *** *** *** *** *** *** *** *** *** *** 21
UNCLASSIFIED Cernak I et al. Neurobiology of Disease 41(2011): 538-551 Post-traumatic Depression: Open Field Test Pre-blast Post-blast (14 d) Post-blast (30 d) 22
Take Home Message #1: BINT is not “just” a blunt traumatic brain injury UNCLASSIFIED 23
Importance of Blast Transmission Pathways 24 Cernak I Frontiers in Neurology (2010); 1: 1-9 Head protection & Torso exposure Torso protection & Head exposure Whole-body exposure UNCLASSIFIED
25 Cernak I Frontiers in Neurology (2010); 1: 1-9 In Vivo Imaging of Inflammation
XenoLight Rediject Inflammation probe is a chemiluminescent reagent for in vivo monitoring of inflammation using the IVIS bioluminescence / fluorescence camera;
This probe is offered in a ready-to-use format and measures myeloperoxidase (MPO) activity of activated phagocytes allowing for longitudinal tracking of MPO level and inflammation status in vivo;
Intraperitoneal (i.p.) injection at 200 mg/kg (150 μL /mouse*) and imaging 10 minutes post i.p. injection of the probe with exposure time of 5 minutes for better sensitivity.
Cernak I Frontiers in Neurology (2010); 1: 1-9 Blast with No Protection The injected bioluminescent marker labels myeloperoxidase in activated macrophages, and is imaged in real-time using the IVIS bioluminescent camera in mice after mild intensity blast . 1 Day 3 Days 7 Days 14 Days 30 Days Blast with Head Protection Blast with Body Protection
27 Koliatsos V, Cernak I, et al. Journal of Neuropathology and Experimental Neurology, 70(5): 399-416, 2011 Axonal Pathology in Distinct CNS Tracts Based on Silver Degeneration Staining 0 – no pathology 1 – mild pathology (scattered axons) 2 – moderate pathology 3 – severe pathology (confluent axons) CC – corpus callosum Cing – cingulum AC – anterior commissure Frnx – fornix SM – stria medullaris MTT – mammilothalamic tract IC – internal capsule Low CST – low corticospinal tract Olf – olfactory tract Optic – optic tract ML – medial lemniscus LL – lateral lemniscus Crbl WM – cerebellar white matter Crbl Pedn – cerebellar peduncles Spt V – spinal tract of trigeminal nucleus VSCT – ventral spinocerebellar tract Whole-body exposure Torso protection & head exposure Head protection & torso exposure
UNCLASSIFIED 28 Take Home Message #2 Well characterized experimental models reproducing military relevant conditions and related symptoms should be used to develop reliable diagnosis, therapy, and prevention. vs