Repeated Blast Exposures Research Articles

Distinct patterns of expression of traumatic brain injury biomarkers after blast exposure: Role of compromised cell membrane integrity

Glial fibrillary acidic protein (GFAP), a protein enriched in astrocytes, and Tau, a protein abundant in neuronal microtubules, are being widely studied as biomarkers of brain injury, and persistent severity-dependent increases in brain and blood have been reported. Studies on the acute changes of these proteins after blast exposure are limited. Using a mouse model of closely-coupled repeated blast exposures, we have evaluated acute changes in the levels of GFAP and total Tau by Western blotting. Brain levels of GFAP and Tau proteins decreased significantly at 6h and increased considerably at 24h after repeated blast exposures. Plasma samples showed a similar initial decrease and later increase over this timeframe. This biphasic pattern points to possible absorption or sequestration of these proteins from plasma immediately after repeated blast exposures. Liver and spleen tissue showed significant increases in the levels of GFAP and Tau protein at 6 and 24h post-blast exposures whereas semi-quantitative RT-PCR analysis of liver showed no significant changes in the levels of GFAP or Tau mRNAs. These results suggest that blast exposure causes transient changes in cell membrane integrity in multiple organs leading to abnormal migration of proteins from the tissues to the plasma and vice versa. This transient changes in cell membrane permeability and subsequent bidirectional movement of molecules may contribute to the pathophysiology of TBI and polytrauma after blast exposure.

Extracellular cyclophilin A protects against blast-induced neuronal injury

Blast-induced traumatic brain injury (TBI) and subsequent neurobehavioral deficits are major disabilities suffered by the military and civilian population worldwide. Rigorous scientific research is underway to understand the mechanism of blast TBI and thereby develop effective therapies for protection and treatment. By using an in vitro shock tube model of blast TBI with SH-SY5Y human neuroblastoma cells, we have demonstrated that blast exposure leads to neurobiological changes in an overpressure and time dependent manner. Paradoxically, repeated blast exposures resulted in less neuronal injury compared to single blast exposure and suggested a potential neuroprotective mechanism involving released cyclophilin A (CPA). In the present study, we demonstrate accumulation of CPA in the culture medium after repeated blast exposures supporting the notion of extracellular CPA mediated neuroprotection. Post-exposure treatment of the cells with purified recombinant CPA caused significant protection against blast-induced neuronal injury. Furthermore, repeated blast exposure was associated with phosphorylation of the proteins ERK1/2 and Bad suggesting a potential mechanism of neuroprotection by extracellular CPA and may aid in the development of targeted therapies for protection against blast-induced TBI.

Contribution of systemic factors in the pathophysiology of repeated blast-induced neurotrauma

Blast-induced traumatic brain injury is complex and involves multiple factors including systemic pathophysiological factors in addition to direct brain injuries. We hypothesize that systemic activation of platelets/leukocytes plays a major role in the development and exacerbation of brain injury after blast exposure. A mouse model of repeated blast exposure that results in significant neuropathology, neurobehavioral changes and regional specific alterations in various biomolecules in the brain was used for the proposed study. Activation of platelets was evaluated by flow cytometry and serotonin content was analyzed by ELISA. Expression of myeloperoxidase was analyzed by Western blotting. Histopathology of the brain was used to assess blast-induced cerebral vasoconstriction. The data showed an increase in the activation of platelets at 4h after repeated blast exposures, indicating changes in platelet phenotype in blast neurotrauma. Platelet serotonin concentration showed a significant decrease at 4h after blast with a concurrent increase in the plasma serotonin levels, confirming the early onset of platelet activation after repeated blast exposures. Blood, plasma and brain myeloperoxidase enzyme activity and expression was increased in repeated blast exposed mice at multiple time points. Histopathological analysis of the brains of blast exposed mice showed constriction of blood vessels compared to the respective controls, a phenomenon similar to the reported cerebral vasoconstriction in blast affected victims. These results suggest that repeated blast exposure leads to acute activation of platelets/leukocytes which can augment the pathological effects of brain injury. Platelet/leukocyte targeted therapies can be evaluated as potential acute treatment strategies to mitigate blast-induced neurotrauma.

Modulation of cholinergic pathways and inflammatory mediators in blast-induced traumatic brain injury

Cholinergic activity has been recognized as a major regulatory component of stress responses after traumatic brain injury (TBI). Centrally acting acetylcholinesterase (AChE) inhibitors are also being considered as potential therapeutic candidates against TBI mediated cognitive impairments. We have evaluated the expression of molecules involved in cholinergic and inflammatory pathways in various regions of brain after repeated blast exposures in mice. Isoflurane anesthetized C57BL/6J mice were restrained and placed in a prone position transverse to the direction of the shockwaves and exposed to three 20.6psi blast overpressures with 1–30min intervals. Brains were collected at the 6h time point after the last blast exposure and subjected to cDNA microarray and microRNA analysis. cDNA microarray analysis showed significant changes in the expression of cholinergic (muscarinic and nicotinic) and gammaaminobutyric acid and glutamate receptors in the midbrain region along with significant changes in multiple genes involved in inflammatory pathways in various regions of the brain. MicroRNA analysis of cerebellum revealed differential expression of miR-132 and 183, which are linked to cholinergic anti-inflammatory signaling, after blast exposure. Changes in the expression of myeloperoxidase in the cerebellum were confirmed by Western blotting. These results indicate that early pathologic progression of blast TBI involves dysregulation of cholinergic and inflammatory pathways related genes. Acute changes in molecules involved in the modulation of cholinergic and inflammatory pathways after blast TBI can cause long-term central and peripheral pathophysiological changes.

Modulation of hearing related proteins in the brain and inner ear following repeated blast exposures

AbstractEmerging studies show that blast exposure causes traumatic brain injury (TBI) and auditory dysfunction without rupture of tympanic membrane, suggesting central auditory processing impairment after blast exposure. There is limited information on the mechanisms of blast-induced TBI and associated peripheral and central auditory processing impairments. We utilized a repetitive blast exposure mouse model to unravel the mechanisms of blast TBI and auditory impairment. C57BL/6J mice were exposed to three repeated blasts (20.6 psi) using a shock tube, and the cerebellum was subjected to proteomic analysis. The data showed that calretinin and parvalbumin, two major calcium buffering proteins, were significantly up-regulated after repeated blast exposures, and this was confirmed by Western blotting. Since these proteins are reportedly involved in auditory dysfunction, we examined the inner ear and found both calretinin and parvalbumin were up-regulated, suggesting that modulation of these proteins plays a role in blast-induced peripheral and central auditory processing impairments. Expression of cleaved caspase-3 was also up-regulated in both regions indicating ongoing cellular apoptosis, possibly due to altered calcium homeostasis. These results provide a molecular basis for changes in central and peripheral auditory processing involving abnormal calcium homeostasis resulting in hearing impairment after blast exposure.

Rapid Release of Tissue Enzymes into Blood after Blast Exposure: Potential Use as Biological Dosimeters

Explosive blast results in multiple organ injury and polytrauma, the intensity of which varies with the nature of the exposure, orientation, environment and individual resilience. Blast overpressure alone may not precisely indicate the level of body or brain injury after blast exposure. Assessment of the extent of body injury after blast exposure is important, since polytrauma and systemic factors significantly contribute to blast-induced traumatic brain injury. We evaluated the activity of plasma enzymes including aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH) and creatine kinase (CK) at different time points after blast exposure using a mouse model of single and repeated blast exposures to assess the severity of injury. Our data show that activities of all the enzymes in the plasma were significantly increased as early as 1 h after blast exposure. The elevated enzyme activity remained up to 6 h in an overpressure dose-dependent manner and returned close to normal levels at 24 h. Head-only blast exposure with body protection showed no increase in the enzyme activities suggesting that brain injury alone does not contribute to the systemic increase. In contrast to plasma increase, AST, ALT and LDH activity in the liver and CK in the skeletal muscle showed drastic decrease at 6 h after blast exposures. Histopathology showed mild necrosis at 6 h and severe necrosis at 24 h after blast exposures in liver and no changes in the skeletal muscle suggesting that the enzyme release from the tissue to plasma is probably triggered by transient cell membrane disruption from shockwave and not due to necrosis. Overpressure dependent transient release of tissue enzymes and elevation in the plasma after blast exposure suggest that elevated enzyme activities in the blood can be potentially used as a biological dosimeter to assess the severity of blast injury.

Primary blast survival and injury risk assessment for repeated blast exposures

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. 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. 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. 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.

Preliminary studies on differential expression of auditory functional genes in the brain after repeated blast exposures

The mechanisms of central auditory processing involved in auditory/vestibular injuries and subsequent tinnitus and hearing loss in Active Duty servicemembers exposed to blast are not currently known. We analyzed the expression of hearing-related genes in different regions of the brain 6 h after repeated blast exposures in mice. Preliminary data showed that the expression of the deafness-related genes otoferlin and otoancorin was significantly changed in the hippocampus after blast exposures. Differential expression of cadherin and protocadherin genes, which are involved in hearing impairment, was observed in the hippocampus, cerebellum, frontal cortex, and midbrain after repeated blasts. A series of calcium-signaling genes that are known to be involved in auditory signal processing were also found to be significantly altered after repeated blast exposures. The hippocampus and midbrain showed significant increase in the gene expression of hearing loss-related antioxidant enzymes. Histopathology of the auditory cortex showed more significant injury in the inner layer compared to the outer layer. In summary, mice exposed to repeated blasts showed injury to the auditory cortex and significant alterations in multiple genes in the brain known to be involved in age- or noise-induced hearing impairment.

Sensory and communication disorders in traumatic brain injury

Patients with traumatic brain injury (TBI) often present with a constellation of symptoms that may interact, persist, and exacerbate if untreated [1-7]. Recently, clinicians have noted that auditory, vestibular, and visual symptoms were frequently reported by combat returnees during their clinical evaluation and treatment [8-12]. These sensory issues are likely to negatively affect the ability of individuals with TBI to process cognitive information and perform daily tasks such as communication and ambulation. While researchers are still investigating the effects of sensory and communication disorders on rehabilitation outcome, preliminary data have shown that dual sensory impairment may adversely influence functional outcome in patients with TBI [10]. Founded by Congress in 1992, the Defense and Veterans Brain Injury Center (DVBIC) is the TBI operational component of the Defense Centers of Excellence for Psychological Health and Traumatic Brain Injury (www.dvbic.org) [13]. With its headquarters in the nation's capital region, DVBIC has been a bridging force between the U.S. Military Health System and Veterans Health Administration, as well as academic and private rehabilitation facilities, all of which share the common goal of providing the best care to servicemembers and Veterans with TBI. As a part of its mission, DVBIC has continued to support educational initiatives and research programs so that clinicians can learn and implement the highest quality of evidence-based practice. This special issue on sensory and communication disorders in TBI represents another endeavor to highlight the latest findings by clinicians and researchers who serve patients with TBI. It contains a wide spectrum of high-quality articles, encompassing innovative research findings, insightful clinical observations, focused reviews, and consensus expert opinions. The following topics are discussed: 1. Multisensory impairment reported by Veterans with and without TBI. 2. Sensorintegrative dysfunction underlying vestibular disorders after TBI. 3. Audiological issues and hearing loss among Veterans with mild TBI. 4. Central auditory processing by individuals exposed to high-intensity blasts. 5. Telehealth tinnitus management for persons with and without TBI. 6. Blast exposure and dual sensory impairment. 7. Implications of blast exposure for central auditory function. 8. Visual symptomatology and referral patterns for Operation Iraqi Freedom/Operation Enduring Freedom (OIF/OEF) Veterans with TBI. 9. Vergence in mild TBI. 10. Visual attention in OIF/OEF Veterans. 11. Relationship of screen-based symptoms for mild TBI and mental health problems. 12. Mild TBI and pain in OIF/OEF Veterans. 13. Preliminary framework for a familiar auditory sensory training task during coma recovery. 14. Preliminary studies on differential expression of auditory functional genes in the brain after repeated blast exposures. Throughout its preparation process, this special issue has generated enthusiastic responses from a wide range of subject matter experts who have worked diligently with the editorial staff and volunteers to bring this project to fruition. A key focus has been to increase health professionals' awareness of the diverse sensory and communication disorders that may result from a TBI so that a team-oriented, patient-centered rehabilitation plan can be formulated and implemented expediently, thereby enhancing the likelihood of an optimal outcome [14]. The issue also includes a synopsis prepared by expert speech-language pathologists who work in the military, Department of Veterans Affairs, and civilian systems that summarizes their experience and identifies recommendations for the management of cognitive-communication problems after combat-related mild TBI. As we receive more articles on the expressive communication deficits related to TBI, another special issue may be warranted. …

Regional specific alterations in brain acetylcholinesterase activity after repeated blast exposures in mice

Acetylcholinesterase (AChE) which catalyzes the hydrolysis of the neurotransmitter acetylcholine has been recognized as one of the major regulators of stress responses after traumatic brain injury (TBI). Repeated blast exposure induces TBI (blast TBI) with a variable neuropathology at different brain regions. Since AChE inhibitors are being used as a line of treatment for TBI, we sought to determine the time course of AChE activity in the blood and different brain regions after repeated blast exposures using modified Ellman assay. Our data showed that repeated blast exposures significantly reduced AChE activity in the whole-blood and erythrocytes by 3–6 h, while plasma AChE activity was significantly increased by 3 h post-blast. In the brain, significant increase in AChE activity was observed at 6 h in the frontal cortex, while hind cortex and hippocampus showed a significant decrease at 6 h post-blast, which returned to normal levels by 7 days. AChE activity in the cerebellum and mid brain showed a decrease at 6 h, followed by significant increase at 3 days and that was decreased significantly at 14 days post-blast. Medulla region showed decreased AChE activity at 24 h post-blast, which was significantly increased at 14 days. These results suggest that there are brain regional and time-related changes in AChE activity after tightly coupled repeated blast exposures in mice. In summary, acute and chronic regional specific changes in the AChE activity after repeated blast exposures warrant systematic evaluation of the possibility of AChE inhibitor therapeutics against blast TBI.

Tightly Coupled Repetitive Blast-Induced Traumatic Brain Injury: Development and Characterization in Mice

A mouse model of repeated blast exposure was developed using a compressed air-driven shock tube, to study the increase in severity of traumatic brain injury (bTBI) after multiple blast exposures. Isoflurane anesthetized C57BL/6J mice were exposed to 13.9, 20.6, and 25 psi single blast overpressure (BOP1) and allowed to recover for 5 days. BOP1 at 20.6 psi showed a mortality rate of 2% and this pressure was used for three repeated blast exposures (BOP3) with 1 and 30 min intervals. Overall mortality rate in BOP3 was increased to 20%. After blast exposure, righting reflex time and body-weight loss were significantly higher in BOP3 animals compared to BOP1 animals. At 4 h, brain edema was significantly increased in BOP3 animals compared to sham controls. Reactive oxygen species in the cortex were increased significantly in BOP1 and BOP3 animals. Neuropathological analysis of the cerebellum and cerebral cortex showed dense silver precipitates in BOP3 animals, indicating the presence of diffuse axonal injury. Fluoro-Jade B staining showed increased intensity in the cortex of BOP3 animals indicating neurodegeneration. Rota Rod behavioral test showed a significant decrease in performance at 10 rpm following BOP1 or BOP3 at 2 h post-blast, which gradually recovered during the 5 days. At 20 rpm, the latency to fall was significantly decreased in both BOP1 and BOP3 animals and it did not recover in the majority of the animals through 5 days of testing. These data suggest that repeated blast exposures lead to increased impairment severity in multiple neurological parameters of TBI in mice.