Yanling Wei

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.

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 20.6 psi blast overpressures with 1-30 min 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.

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.

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.

A combination of [+] and [−]-Huperzine A improves protection against soman toxicity compared to [+]-Huperzine A in guinea pigs

The neuropathologic mechanisms after exposure to lethal doses of nerve agent are complex and involve multiple biochemical pathways. Effective treatment requires drugs that can simultaneously protect by reversible binding to the acetylcholinesterase (AChE) and blocking cascades of seizure related brain damage, inflammation, neuronal degeneration as well as promoting induction of neuroregeneration. [-]-Huperzine A ([-]-Hup A), is a naturally occurring potent reversible AChE inhibitor that penetrates the blood-brain barrier. It also has several neuroprotective effects including modification of beta-amyloid peptide, reduction of oxidative stress, anti-inflammatory, anti-apoptotic and induction and regulation of nerve growth factor. Toxicities at higher doses restrict the neuroporotective ability of [-]-Hup A for treatment. The synthetic stereoisomer, [+]-Hup A, is less toxic due to poor AChE inhibition and is suitable for both pre-/post-exposure treatments of nerve agent toxicity. [+]-Hup A block the N-methyl-D-aspartate (NMDA)-induced seizure in rats, reduce excitatory amino acid induced neurotoxicity and also prevent soman induced toxicity with minimum performance decrement. Unique combinations of two stereo-isomers of Hup A may provide an excellent pre/post-treatment drug for the nerve agent induced seizure/status epilepticus. We investigated a combination of [+]-Hup A with a small dose of [-]-Hup A ([+] and [-]-Hup A) against soman toxicity. Our data showed that pretreatment with a combination [+] and [-]-Hup A significantly increased the survival rate and reduced behavioral abnormalities after exposure to 1.2 × LD(50) soman compared to [+]-Hup A in guinea pigs. In addition, [+] and [-]-Hup A pretreatment inhibited the development of high power of EEG better than [+]-Hup A pretreatment alone. These data suggest that a combination of [+] and [-]-Hup A offers better protection than [+]-Hup A and serves as a potent medical countermeasure against lethal dose nerve agent toxicity in guinea pigs.

Repeated blast exposures cause brain DNA fragmentation in mice.

The pathophysiology of blast-induced traumatic brain injury (TBI) and subsequent behavioral deficits are not well understood. Unraveling the mechanisms of injury is critical to derive effective countermeasures against this form of neurotrauma. Preservation of the integrity of cellular DNA is crucial for the function and survival of cells. We evaluated the effect of repeated blast exposures on the integrity of brain DNA and tested the utility of cell-free DNA (CFD) in plasma as a biomarker for the diagnosis and prognosis of blast-induced polytrauma. The results revealed time-dependent breakdown in cellular DNA in different brain regions, with the maximum damage at 24 h post-blast exposures. CFD levels in plasma showed a significant transient increase, which was largely independent of the timing and severity of brain DNA damage; maximum levels were recorded at 2 h after repeated blast exposure and returned to baseline at 24 h. A positive correlation was observed between the righting reflex time and CFD level in plasma at 2 h after blast exposure. Brain DNA damage subsequent to repeated blast was associated with decreased mitochondrial membrane potential, increased release of cytochrome C, and up-regulation of caspase-3, all of which are indicative of cellular apoptosis. Shock-wave-induced DNA damage and initiation of mitochondrial-driven cellular apoptosis in the brain after repeated blast exposures indicate that therapeutic strategies directed toward inhibition of DNA damage or instigation of DNA repair may be effective countermeasures.

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 3-6h, while plasma AChE activity was significantly increased by 3h post-blast. In the brain, significant increase in AChE activity was observed at 6h in the frontal cortex, while hind cortex and hippocampus showed a significant decrease at 6h post-blast, which returned to normal levels by 7 days. AChE activity in the cerebellum and mid brain showed a decrease at 6h, followed by significant increase at 3 days and that was decreased significantly at 14 days post-blast. Medulla region showed decreased AChE activity at 24h 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.

Cytoskeletal protein α–II spectrin degradation in the brain of repeated blast exposed mice

Repeated blast exposures commonly induce traumatic brain injury (TBI) characterized by diffuse axonal injury (DAI). We hypothesized that degradation of cytoskeletal proteins in the brain can lead to DAI, and evaluated α-II spectrin degradation in the pathophysiology of blast-induced TBI using the tightly-coupled three repetitive blast exposure mice model with a 1-30 min window in between exposures. Degradation of α-II spectrin and the expression profiles of caspase-3 and calpain-2, the major enzymes involved in the degradation were analyzed in the frontal cortex and cerebellum using Western blotting with specific antibodies. DAI at different brain regions was evaluated by neuropathology with silver staining. Repeated blast exposures resulted in significant increases in the α-II spectrin degradation products in the frontal cortex and cerebellum compared to sham controls. Expression of active caspase-3, which degrades α-II spectrin, showed significant increase in the frontal cortex after blast exposure at all the time points studied, while cerebellum showed an acute increase which was normalized over time. The expression of another α-II spectrin degrading enzyme, calpain-2, showed a rapid increase in the frontal cortex after blast exposure and it was significantly higher in the cerebellum at later time points. Neuropathological analysis showed significant levels of DAI at the frontal cortex and cerebellum at multiple time points after repeated blast injury. In summary, repeated blast exposure results in specific degradation of α-II spectrin in the brain along with differential expression of caspase-3/calpain-2 suggesting cytoskeletal breakdown as a possible contributor of DAI after repeated blast exposure.

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

Abstract Emerging 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...

Neuroprotective effects of imidazenil against chemical warfare nerve agent soman toxicity in guinea pigs.

The chemical warfare nerve agent, soman irreversibly inhibits acetylcholinesterase (AChE) leading to hypercholinergy and seizures which trigger glutamate toxicity and status epilepticus ultimately resulting in neuropathology and neurobehavioral deficits. The standard emergency treatment comprising of anticholinergic, AChE reactivator and anticonvulsant does not completely protect against soman toxicity. We have evaluated imidazenil, a new anticonvulsant imidazo benzodiazepine with high affinity and intrinsic efficacy at α5-, α2-, and α3- but low intrinsic efficacy at α1-containing GABA(A) receptors and is devoid of cardiorespiratory depression, sedative/hypnoitc and amnestic actions and does not elicit tolerance and dependence liabilities unlike diazepam, for protection against soman toxicity. Guinea pigs implanted with bipotential radiotelemetry probes for recording EEG and ECG were administered with 26 μg/kg pyridostigmine bromide 30 min prior to 2× LD(50) soman exposure and 1 min later treated with a combination of 2mg/kg atropine sulfate and 25mg/kg 2-pralidoxime and various doses of imidazenil. Intramuscular administration of imidazenil, dose-dependently protected against 2× LD(50) of soman toxicity up to 1mg/kg. Further increase in the dose of imidazenil to 2.5mg/kg was less effective than 1mg/kg probably due to non-specific actions at sites other than GABA(A) receptors. Compared to vehicle group, 1mg/kg imidazenil treatment showed optimal increase in survival rate, reduction in behavioral manifestations and high power of EEG spectrum as well as neuronal necrosis. These data suggest that imidazenil is an effective anticonvulsant for medical countermeasure against soman-induced toxicity.