Screening for novel central nervous system biomarkers in veterans with Gulf War Illness.

Gulf War illness (GWI) is primarily diagnosed by symptom report; objective biomarkers are needed that distinguish those with GWI. Prior chemical exposures during deployment have been associated in epidemiologic studies with altered central nervous system functioning in veterans with GWI. Previous studies from our group have demonstrated the presence of autoantibodies to essential neuronal and glial proteins in patients with brain injury and autoantibodies have been identified as candidate objective markers that may distinguish GWI. Here, we screened the serum of 20 veterans with GWI and 10 non-veteran symptomatic (low back pain) controls for the presence of such autoantibodies using Western blot analysis against the following proteins: neurofilament triplet proteins (NFP), tubulin, microtubule associated tau proteins (Tau), microtubule associated protein-2 (MAP-2), myelin basic protein (MBP), myelin associated glycoprotein (MAG), glial fibrillary acidic protein (GFAP), calcium-calmodulin kinase II (CaMKII) and glial S-100B protein. Serum reactivity was measured as arbitrary chemiluminescence units. As a group, veterans with GWI had statistically significantly higher levels of autoantibody reactivity in all proteins examined except S-100B. Fold increase of the cases relative to controls in descending order were: CaMKII 9.27, GFAP 6.60, Tau 4.83, Tubulin 4.41, MAG 3.60, MBP 2.50, NFP 2.45, MAP-2 2.30, S-100B 1.03. These results confirm the continuing presence of neuronal injury/gliosis in these veterans and are in agreement with the recent reports indicating that 25years after the war, the health of veterans with GWI is not improving and may be getting worse. Such serum autoantibodies may prove useful as biomarkers of GWI, upon validation of the findings using larger cohorts.

Primary Blast Injury Depressed Hippocampal Long-Term Potentiation through Disruption of Synaptic Proteins.

Blast-induced traumatic brain injury (bTBI) is a major threat to United States service members in military conflicts worldwide. The effects of primary blast, caused by the supersonic shockwave interacting with the skull and brain, remain unclear. Our group has previously reported that in vitro primary blast exposure can reduce long-term potentiation (LTP), the electrophysiological correlate of learning and memory, in rat organotypic hippocampal slice cultures (OHSCs) without significant changes to cell viability or basal, evoked neuronal function. We investigated the time course of primary blast-induced deficits in LTP and the molecular mechanisms that could underlie these deficits. We found that pure primary blast exposure induced LTP deficits in a delayed manner, requiring longer than 1 hour to develop, and that these deficits spontaneously recovered by 10 days following exposure depending on blast intensity. Additionally, we observed that primary blast exposure reduced total α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor 1 (GluR1) subunit expression and phosphorylation of the GluR1 subunit at the serine-831 site. Blast also reduced the expression of postsynaptic density protein-95 (PSD-95) and phosphorylation of stargazin protein at the serine-239/240 site. Finally, we found that modulation of the cyclic adenosine monophosphate (cAMP) pathway ameliorated electrophysiological and protein-expression changes caused by blast. These findings could inform the development of novel therapies to treat blast-induced loss of neuronal function.

Did a "lucky shot" sink the submarine H.L. Hunley?

The H.L. Hunley was the first submarine to be successful in combat, sinking the Union vessel Housatonic outside Charleston Harbor in 1864 during the Civil War. However, despite marking a milestone in military history, little is known about this vessel or why it sank. One popular theory is the "lucky shot" theory: the hypothesis that small arms fire from the crew of the Housatonic may have sufficiently damaged the submarine to sink it. However, ballistic experiments with cast iron samples, analysis of historical experiments firing Civil War-era projectiles at cast iron samples, and calculation of the tidal currents and sinking trajectory of the submarine indicate that this theory is not likely. Based on our results, the "lucky shot" theory does not explain the sinking of the world's first successful combat submarine.

Primary blast injury causes cognitive impairments and hippocampal circuit alterations.

Blast-induced traumatic brain injury (bTBI) and its long term consequences are a major health concern among veterans. Despite recent work enhancing our knowledge about bTBI, very little is known about the contribution of the blast wave alone to the observed sequelae. Herein, we isolated its contribution in a mouse model by constraining the animals' heads during exposure to a shockwave (primary blast). Our results show that exposure to primary blast alone results in changes in hippocampus-dependent behaviors that correspond with electrophysiological changes in area CA1 and are accompanied by reactive gliosis. Specifically, five days after exposure, behavior in an open field and performance in a spatial object recognition (SOR) task were significantly different from sham. Network electrophysiology, also performed five days after injury, demonstrated a significant decrease in excitability and increase in inhibitory tone. Immunohistochemistry for GFAP and Iba1 performed ten days after injury showed a significant increase in staining. Interestingly, a threefold increase in the impulse of the primary blast wave did not exacerbate these measures. However, we observed a significant reduction in the contribution of the NMDA receptors to the field EPSP at the highest blast exposure level. Our results emphasize the need to account for the effects of primary blast loading when studying the sequelae of bTBI.

Impact responses of the cervical spine: A computational study of the effects of muscle activity, torso constraint, and pre-flexion.

Cervical spine injuries continue to be a costly societal problem. Future advancements in injury prevention depend on improved physical and computational models, which are predicated on a better understanding of the neck response during dynamic loading. Previous studies have shown that the tolerance of the neck is dependent on its initial position and its buckling behavior. This study uses a computational model to examine three important factors hypothesized to influence the loads experienced by vertebrae in the neck under compressive impact: muscle activation, torso constraints, and pre-flexion angle of the cervical spine. Since cadaver testing is not practical for large scale parametric analyses, these factors were studied using a previously validated computational model. On average, simulations with active muscles had 32% larger compressive forces and 25% larger shear forces-well in excess of what was expected from the muscle forces alone. In the short period of time required for neck injury, constraints on torso motion increased the average neck compression by less than 250N. The pre-flexion hypothesis was tested by examining pre-flexion angles from neutral (0°) to 64°. Increases in pre-flexion resulted in the largest increases in peak loads and the expression of higher-order buckling modes. Peak force and buckling modality were both very sensitive to pre-flexion angle. These results validate the relevance of prior cadaver models for neck injury and help explain the wide variety of cervical spine fractures that can result from ostensibly similar compressive loadings. They also give insight into the mechanistic differences between burst fractures and lower cervical spine dislocations.

Isolated Primary Blast Inhibits Long-Term Potentiation in Organotypic Hippocampal Slice Cultures.

Over the last 13 years, traumatic brain injury (TBI) has affected over 230,000 U.S. service members through the conflicts in Iraq and Afghanistan, mostly as a result of exposure to blast events. Blast-induced TBI (bTBI) is multi-phasic, with the penetrating and inertia-driven phases having been extensively studied. The effects of primary blast injury, caused by the shockwave interacting with the brain, remain unclear. Earlier in vivo studies in mice and rats have reported mixed results for primary blast effects on behavior and memory. Using a previously developed shock tube and in vitro sample receiver, we investigated the effect of isolated primary blast on the electrophysiological function of rat organotypic hippocampal slice cultures (OHSC). We found that pure primary blast exposure inhibited long-term potentiation (LTP), the electrophysiological correlate of memory, with a threshold between 9 and 39 kPa·ms impulse. This deficit occurred well below a previously identified threshold for cell death (184 kPa·ms), supporting our previously published finding that primary blast can cause changes in brain function in the absence of cell death. Other functional measures such as spontaneous activity, network synchronization, stimulus-response curves, and paired-pulse ratios (PPRs) were less affected by primary blast exposure, as compared with LTP. This is the first study to identify a tissue-level tolerance threshold for electrophysiological changes in neuronal function to isolated primary blast.

Isolated primary blast alters neuronal function with minimal cell death in organotypic hippocampal slice cultures.

An increasing number of U.S. soldiers are diagnosed with traumatic brain injury (TBI) subsequent to exposure to blast. In the field, blast injury biomechanics are highly complex and multi-phasic. The pathobiology caused by exposure to some of these phases in isolation, such as penetrating or inertially driven injuries, has been investigated extensively. However, it is unclear whether the primary component of blast, a shock wave, is capable of causing pathology on its own. Previous in vivo studies in the rodent and pig have demonstrated that it is difficult to deliver a primary blast (i.e., shock wave only) without rapid head accelerations and potentially confounding effects of inertially driven TBI. We have previously developed a well-characterized shock tube and custom in vitro receiver for exposing organotypic hippocampal slice cultures to pure primary blast. In this study, isolated primary blast induced minimal hippocampal cell death (on average, below 14% in any region of interest), even for the most severe blasts tested (424 kPa peak pressure, 2.3 ms overpressure duration, and 248 kPa*ms impulse). In contrast, measures of neuronal function were significantly altered at much lower exposures (336 kPa, 0.84 ms, and 86.5 kPa*ms), indicating that functional changes occur at exposures below the threshold for cell death. This is the first study to investigate a tolerance for primary blast-induced brain cell death in response to a range of blast parameters and demonstrate functional deficits at subthreshold exposures for cell death.

Blood-brain barrier dysfunction after primary blast injury in vitro.

The incidence of blast-induced traumatic brain injury (bTBI) has increased substantially in recent military conflicts. However, the consequences of bTBI on the blood-brain barrier (BBB), a specialized cerebrovascular structure essential for brain homeostasis, remain unknown. In this study, we utilized a shock tube driven by compressed gas to generate operationally relevant, ideal pressure profiles consistent with improvised explosive devices (IEDs). By multiple measures, the barrier function of an in vitro BBB model was disrupted following exposure to a range of controlled blast loading conditions. Trans-endothelial electrical resistance (TEER) decreased acutely in a dose-dependent manner that was most strongly correlated with impulse, as opposed to peak overpressure or duration. Significantly increased hydraulic conductivity and solute permeability post-injury further confirmed acute alterations in barrier function. Compromised ZO-1 immunostaining identified a structural basis for BBB breakdown. After blast exposure, TEER remained significantly depressed 2 days post-injury, followed by spontaneous recovery to pre-injury control levels at day 3. This study is the first to report immediate disruption of an in vitro BBB model following primary blast exposure, which may be important for the development of novel helmet designs to help mitigate the effects of blast on the BBB.

Tensile failure properties of the perinatal, neonatal, and pediatric cadaveric cervical spine.

STUDY DESIGN: Biomechanical tensile testing of perinatal, neonatal, and pediatric cadaveric cervical spines to failure. OBJECTIVE: To assess the tensile failure properties of the cervical spine from birth to adulthood. SUMMARY OF BACKGROUND DATA: Pediatric cervical spine biomechanical studies have been few due to the limited availability of pediatric cadavers. Therefore, scaled data based on human adult and juvenile animal studies have been used to augment the limited pediatric cadaver data. Despite these efforts, substantial uncertainty remains in our understanding of pediatric cervical spine biomechanics. METHODS: A total of 24 cadaveric osteoligamentous head-neck complexes, 20 weeks gestation to 18 years, were sectioned into segments (occiput-C2 [O-C2], C4-C5, and C6-C7) and tested in tension to determine axial stiffness, displacement at failure, and load-to-failure. RESULTS: Tensile stiffness-to-failure (N/mm) increased by age (O-C2: 23-fold, neonate: 22 ± 7, 18 yr: 504; C4-C5: 7-fold, neonate: 71 ± 14, 18 yr: 509; C6-C7: 7-fold, neonate: 64 ± 17, 18 yr: 456). Load-to-failure (N) increased by age (O-C2: 13-fold, neonate: 228 ± 40, 18 yr: 2888; C4-C5: 9-fold, neonate: 207 ± 63, 18 yr: 1831; C6-C7: 10-fold, neonate: 174 ± 41, 18 yr: 1720). Normalized displacement at failure (mm/mm) decreased by age (O-C2: 6-fold, neonate: 0.34 ± 0.076, 18 yr: 0.059; C4-C5: 3-fold, neonate: 0.092 ± 0.015, 18 yr: 0.035; C6-C7: 2-fold, neonate: 0.088 ± 0.019, 18 yr: 0.037). CONCLUSION: Cervical spine tensile stiffness-to-failure and load-to-failure increased nonlinearly, whereas normalized displacement at failure decreased nonlinearly, from birth to adulthood. Pronounced ligamentous laxity observed at younger ages in the O-C2 segment quantitatively supports the prevalence of spinal cord injury without radiographic abnormality in the pediatric population. This study provides important and previously unavailable data for validating pediatric cervical spine models, for evaluating current scaling techniques and animal surrogate models, and for the development of more biofidelic pediatric crash test dummies.

Brain injury risk from primary blast.

BACKGROUND: Military service members are often exposed to at least one explosive event, and many blast-exposed veterans present with symptoms of traumatic brain injury. However, there is little information on the intensity and duration of blast necessary to cause brain injury. METHODS: Varying intensity shock tube blasts were focused on the head of anesthetized ferrets, whose thorax and abdomen were protected. Injury evaluations included physiologic consequences, gross necropsy, and histologic diagnosis. The resulting apnea, meningeal bleeding, and fatality were analyzed using logistic regressions to determine injury risk functions. RESULTS: Increasing severity of blast exposure demonstrated increasing apnea immediately after the blast. Gross necropsy revealed hemorrhages, frequently near the brain stem, at the highest blast intensities. Apnea, bleeding, and fatality risk functions from blast exposure to the head were determined for peak overpressure and positive-phase duration. The 50% risk of apnea and moderate hemorrhage were similar, whereas the 50% risk of mild hemorrhage was independent of duration and required lower overpressures (144 kPa). Another fatality risk function was determined with existing data for scaled positive-phase durations from 1 millisecond to 20 milliseconds. CONCLUSION: The first primary blast brain injury risk assessments for mild and moderate/severe injuries in a gyrencephalic animal model were determined. The blast level needed to cause a mild/moderate brain injury may be similar to or less than that needed for pulmonary injury. The risk functions can be used in future research for blast brain injury by providing realistic injury risks to guide the design of protection or evaluate injury.

Porcine head response to blast.

Recent studies have shown an increase in the frequency of traumatic brain injuries related to blast exposure. However, the mechanisms that cause blast neurotrauma are unknown. Blast neurotrauma research using computational models has been one method to elucidate that response of the brain in blast, and to identify possible mechanical correlates of injury. However, model validation against experimental data is required to ensure that the model output is representative of in vivo biomechanical response. This study exposes porcine subjects to primary blast overpressures generated using a compressed-gas shock tube. Shock tube blasts were directed to the unprotected head of each animal while the lungs and thorax were protected using ballistic protective vests similar to those employed in theater. The test conditions ranged from 110 to 740 kPa peak incident overpressure with scaled durations from 1.3 to 6.9 ms and correspond approximately with a 50% injury risk for brain bleeding and apnea in a ferret model scaled to porcine exposure. Instrumentation was placed on the porcine head to measure bulk acceleration, pressure at the surface of the head, and pressure inside the cranial cavity. Immediately after the blast, 5 of the 20 animals tested were apneic. Three subjects recovered without intervention within 30 s and the remaining two recovered within 8 min following respiratory assistance and administration of the respiratory stimulant doxapram. Gross examination of the brain revealed no indication of bleeding. Intracranial pressures ranged from 80 to 390 kPa as a result of the blast and were notably lower than the shock tube reflected pressures of 300-2830 kPa, indicating pressure attenuation by the skull up to a factor of 8.4. Peak head accelerations were measured from 385 to 3845 G's and were well correlated with peak incident overpressure (R(2) = 0.90). One SD corridors for the surface pressure, intracranial pressure (ICP), and head acceleration are presented to provide experimental data for computer model validation.

Primary blast survival and injury risk assessment for repeated blast exposures.

BACKGROUND: The widespread use of explosives by modern insurgents and terrorists has increased the potential frequency of blast exposure in soldiers and civilians. This growing threat highlights the importance of understanding and evaluating blast injury risk and the increase of injury risk from exposure to repeated blast effects. METHODS: Data from more than 3,250 large animal experiments were collected from studies focusing on the effects of blast exposure. The current study uses 2,349 experiments from the data collection for analysis of the primary blast injury and survival risk for both long- and short-duration blasts, including the effects from repeated exposures. A piecewise linear logistic regression was performed on the data to develop survival and injury risk assessment curves. RESULTS: New injury risk assessment curves uniting long- and short-duration blasts were developed for incident and reflected pressure measures and were used to evaluate the risk of injury based on blast over pressure, positive-phase duration, and the number of repeated exposures. The risk assessments were derived for three levels of injury severity: nonauditory, pulmonary, and fatality. The analysis showed a marked initial decrease in injury tolerance with each subsequent blast exposure. This effect decreases with increasing number of blast exposures. CONCLUSIONS: The new injury risk functions showed good agreement with the existing experimental data and provided a simplified model for primary blast injury risk. This model can be used to predict blast injury or fatality risk for single exposure and repeated exposure cases and has application in modern combat scenarios or in setting occupational health limits.

Evaluation of pediatric skull fracture imaging techniques.

Radiologic imaging is crucial in the diagnosis of skull fracture, but there is some doubt as to whether different imaging modalities can accurately identify fractures present on a human skull. While studies have been performed to evaluate the efficacy of radiologic imaging at other anatomical locations, there have been no systematic studies comparing various CT techniques, including high resolution imaging with and without 3D reconstructions to conventional radiologic imaging in children, we investigated which imaging modalities: high-resolution CT scan with 3D projections, clinical-resolution CT scans or X-rays, best showed fracture occurrence in a pediatric human cadaver skull by having an expert pediatric radiologist examine radiologic images from fractured skulls. The skulls used were taken from pediatric cadavers ranging in age from 5 months to 16 years. We evaluated the sensitivity and specificity for the imaging modalities using dissection findings as the gold standard. We found that high-resolution CT scans with 3D projections and conventional CT provided the most accurate fracture diagnosis (single-fracture sensitivity of 71%) followed by X-rays (single-fracture sensitivity of 63%). Linear fractures outsider the region of the sutures were more identifiable than diastatic fractures, though the incidence of false positives was greater for linear fractures. In the two cases where multiple fractures were present on the same anatomical skull location, the radiologist was less likely to identify the presence of additional fractures than a single fracture. Overall, the high-resolution and clinical-resolution CT scans had the similar accuracy for detecting skull fractures while the use of the X-ray was both less accurate and had a lower specificity.

Survival risk assessment for primary blast exposures to the head.

Many soldiers returning from the current conflicts in Iraq and Afghanistan have had at least one exposure to an explosive event and a significant number have symptoms consistent with traumatic brain injury. Although blast injury risk functions have been determined and validated for pulmonary injury, there is little information on the blast levels necessary to cause blast brain injury. Anesthetized male New Zealand White rabbits were exposed to varying levels of shock tube blast exposure focused on the head, while their thoraces were protected. The specimens were euthanized and evaluated when the blast resulted in respiratory arrest that was non-responsive to resuscitation or at 4?h post-exposure. Injury was evaluated by gross examination and histological evaluation. The fatality data from brain injury were then analyzed using Fisher's exact test to determine a brain fatality risk function. Greater blast intensity was associated with post-blast apnea and the need for mechanical ventilation. Gross examination revealed multifocal subdural hemorrhages, most often near the brainstem, at more intense levels of exposure. Histological evaluation revealed subdural and subarachnoid hemorrhages in the non-responsive respiratory-arrested specimens. A fatality risk function from blast exposure to the head was determined for the rabbit specimens with an LD(50) at a peak overpressure of 750?kPa. Scaling techniques were used to predict injury risk at other blast overpressure/duration combinations. The fatality risk function showed that the blast level needed to cause fatality from an overpressure wave exposure to the head was greater than the peak overpressure needed to cause fatality from pulmonary injury. This risk function can be used to guide future research for blast brain injury by providing a realistic fatality risk to guide the design of protection or to evaluate injury.

Pulmonary injury risk assessment for long-duration blasts: a meta-analysis.

BACKGROUND: Long-duration blasts are an increasing threat with the expanded use of thermobaric and other novel explosives. Other potential long-duration threats include large explosions from improvised explosive devices, weapons caches, and other explosives including nuclear explosives. However, there are very few long-duration pulmonary blast injury assessments, and use of short-duration exposure injury metrics is inappropriate as the injury mechanism for long-duration exposures is likely different from that of short-duration exposures. METHODS: This study develops an injury model for long-duration (>10 milliseconds positive overpressure phase) blasts with sharp rising overpressures. For this study, data on more than 2,730 large animal experiments were collected from more than 55 experimental studies on blast. From this dataset, nearly 850 large animal experiments were selected with positive phase overpressure durations of 10 milliseconds or more. Various models were evaluated to determine the best fit of injury risk as a function of pressure and duration. A linear logistic regression was performed on the experimental data for threshold injury and lethality in terms of pressure and duration. The effects of mass, pressure, and duration scaling were all evaluated, and two goodness-of-fit indicators were used to assess the different models. RESULTS AND CONCLUSIONS: New injury risk assessment curves were determined for both incident and reflected pressure conditions for reflecting surface and free-field exposures. Position dependent injury risk curves were also determined. The resulting curves are an improvement to existing assessments, because they use actual data to demonstrate theoretical assumptions on the injury risk.

Thoracic and lumbar spinal impact tolerance.

INTRODUCTION: Thoracolumbar injuries resulting from motor vehicle accidents, falls, and assaults have a high risk of morbidity and mortality. However, there are no biomechanically based standards that address this problem. METHODS: This study used four cadaveric porcine specimens as a model for direct spinal impact injuries to humans to determine an appropriate injury tolerance value. The anthropometric parameters of these specimens are compared with values found in a large human cadaveric dataset. Each specimen was subjected to five impacts on the dorsal surface of the lower thorax and abdomen. RESULTS: The injuries ranged from mild spinous process fractures to endplate fractures with anterior longitudinal ligament (ALL) transactions with a maximum AIS=3. The average peak reaction force for the thoracic failure tests was 4720+/-1340 N, and the average peak reaction force for the lumbar failure tests was 4650+/-1590 N. DISCUSSION: When scaled to human values using anthropometric parameters determined in this study, the force at which there is a 50% risk of injury is 10,200+/-3900 N. This value favorably compares to that found in the existing literature on isolated vertebral segments. SUMMARY: After demonstrating that the porcine model can be used as a spinal impact model for the human, the resulting injury risk value can be used in determining new standards for human injury risk or in guiding the design of safety equipment for the back.

Development of injury criteria for pelvic fracture in frontal crashes.

OBJECTIVE: This article assesses the position-dependent injury tolerance of the hip in the frontal direction based on testing of eight postmortem human subjects. METHODS: For each subject, the left and right hemipelvis complex was axially loaded using a previously developed test configuration. Six positions were defined from a seated femur neutral condition, combining flexed, neutral, and extended femur positions with abducted, neutral, and adducted positions. RESULTS: Axial injury tolerances based on peak force were found to be 6,850 +/- 840 N in the extended, neutral position and 4,080 +/- 830 N in the flexed, neutral position. From the flexed neutral orientation, the peak axial force increased 18% for 20 degrees abduction and decreased 6% for 20 degrees adduction. From the extended, neutral orientation, the peak axial force decreased 4% for 20 degrees abduction and decreased 3% for 20 degrees adduction. However, as there is evidence that increases in loading may occur after the initiation of fracture, the magnitude of the peak force is likely related to the extent of injury, not to the initial tolerance. Using the axial femur force at the initiation of fracture (assessed with acoustic crack sensors) as a potentially more relevant indicator of injury may lower the existing injury criteria. This fracture initiation force varied by position from 3,010 +/- 560 N in the flexed, neutral position to 5,470 N in the extended, abducted position. Further, there was a large position-dependent variation in the ratio of fracture initiation force to the peak axial force. The initiation of fracture was 83% of the peak axial force in the extended, abducted position, but the ratio was 34% in the extended, adducted position. CONCLUSIONS: This may have significant implications for the development of pelvic injury criteria by automobile designers attempting to mitigate pelvis injuries.

A methodology for assessing blast protection in explosive ordnance disposal bomb suits.

To reduce human casualties associated with explosive ordnance disposal, a wide range of protective wear has been designed to shield against the blast effects of improvised explosive devices and munitions. In this study, 4 commercially available bomb suits, representing a range of materials and armor masses, were evaluated against 0.227 and 0.567 kg of spherical C-4 explosives to determine the level of protection offered to the head, neck, and thorax. A Hybrid III dummy, an instrumented human surrogate [1], was tested with and without protection from the 4 commercially available bomb suits. 20 tests with the dummy torso mounted to simulate a kneeling position were performed to confirm repeatability and robustness of the dummies, as well as to evaluate the 4 suits. Correlations between injury risk assessments based on past human or animal injury model data and various parameters such as bomb suit mass, projected area, and dummy coverage area were drawn.