Blast Exposure Alters Synaptic Connectivity in the Mouse Auditory Cortex.

Blast exposure can cause auditory deficits that have a lasting, significant impact on patients. Although the effects of blast on auditory functions localized to the ear have been well documented, the impact of blast on central auditory processing is largely undefined. Understanding the structural and functional alterations in the central nervous system (CNS) associated with blast injuries is crucial for unraveling blast-induced pathophysiological pathways and advancing development of therapeutic interventions. In this study, we used electrophysiology in combination with optogenetics assay, proteomic analysis, and morphological evaluation to investigate the impairment of synaptic connectivity in the auditory cortex (AC) of mice following blast exposure. Our results show that the long-range functional connectivity between the medial geniculate nucleus (MGN) and AC was impaired in the acute phase of blast injury. We also identified impaired synaptic transmission and dendritic spine alterations within 7 days of blast exposure, which recovered at 28 days post-blast. Additionally, proteomic analysis identified a few differentially expressed proteins in the cortex that are involved in synaptic signaling and plasticity. These findings collectively suggest that blast-induced alterations in the sound signaling network in the auditory cortex may underlie hearing deficits in the acute and sub-acute phases after exposure to shockwaves. This study may shed light on the perturbations underlying blast-induced auditory dysfunction and provide insights into the potential therapeutic windows for improving auditory outcomes in blast-exposed individuals.

Assessment of auditory and vestibular damage in a mouse model after single and triple blast exposures.

The use of explosive devices in war and terrorism has increased exposure to concussive blasts among both military personnel and civilians, which can cause permanent hearing and balance deficits that adversely affect survivors' quality of life. Significant knowledge gaps on the underlying etiology of blast-induced hearing loss and balance disorders remain, especially with regard to the effect of blast exposure on the vestibular system, the impact of multiple blast exposures, and long-term recovery. To address this, we investigated the effects of blast exposure on the inner ear using a mouse model in conjunction with a high-fidelity blast simulator. Anesthetized animals were subjected to single or triple blast exposures, and physiological measurements and tissue were collected over the course of recovery for up to 180 days. Auditory brainstem responses (ABRs) indicated significantly elevated thresholds across multiple frequencies. Limited recovery was observed at low frequencies in single-blasted mice. Distortion Product Otoacoustic Emissions (DPOAEs) were initially absent in all blast-exposed mice, but low-amplitude DPOAEs could be detected at low frequencies in some single-blast mice by 30 days post-blast, and in some triple-blast mice at 180 days post-blast. All blast-exposed mice showed signs of Tympanic Membrane (TM) rupture immediately following exposure and loss of outer hair cells (OHCs) in the basal cochlear turn. In contrast, the number of Inner Hair Cells (IHCs) and spiral ganglion neurons was unchanged following blast-exposure. A significant reduction in IHC pre-synaptic puncta was observed in the upper turns of blast-exposed cochleae. Finally, we found no significant loss of utricular hair cells or changes in vestibular function as assessed by vestibular evoked potentials. Our results suggest that (1) blast exposure can cause severe, long-term hearing loss which may be partially due to slow TM healing or altered mechanical properties of healed TMs, (2) traumatic levels of sound can still reach the inner ear and cause basal OHC loss despite middle ear dysfunction caused by TM rupture, (3) blast exposure may result in synaptopathy in humans, and (4) balance deficits after blast exposure may be primarily due to traumatic brain injury, rather than damage to the peripheral vestibular system.

Blast Exposure Causes Long-Term Degeneration of Neuronal Cytoskeletal Elements in the Cochlear Nucleus: A Potential Mechanism for Chronic Auditory Dysfunctions.

Blast-induced auditory dysfunctions including tinnitus are the most prevalent disabilities in service members returning from recent combat operations. Most of the previous studies were focused on the effect of blast exposure on the peripheral auditory system and not much on the central auditory signal-processing regions in the brain. In the current study, we have exposed rats to single and tightly coupled repeated blasts and examined the degeneration of neuronal cytoskeletal elements using silver staining in the central auditory signal-processing regions in the brain at 24 h, 14 days, 1 month, 6 months, and 1 year. The brain regions evaluated include cochlear nucleus, lateral lemniscus, inferior colliculus, medial geniculate nucleus, and auditory cortex. The results obtained indicated that a significant increase in degeneration of neuronal cytoskeletal elements was observed only in the left and right cochlear nucleus. A significant increase in degeneration of neuronal cytoskeletal elements was observed in the cochlear nucleus at 24 h and persisted through 1 year, suggesting acute and chronic neuronal degeneration after blast exposure. No statistically significant differences were observed between single and repeated blasts. The localized degeneration of neuronal cytoskeletal elements in the cochlear nucleus suggests that the damage could be caused by transmission of blast shockwaves/noise through the ear canal and that use of suitable ear protection devices can protect against acute and chronic central auditory signal processing defects including tinnitus after blast exposure.

Pulmonary injury risk curves and behavioral changes from blast overpressure exposures of varying frequency and intensity in rats.

At present, there are no set guidelines establishing cumulative limits for blast exposure numbers and intensities in military personnel, in combat or training operations. The objective of the current study was to define lung injury, pathology, and associated behavioral changes from primary repeated blast lung injury under appropriate exposure conditions and combinations (i.e. blast overpressure (BOP) intensity and exposure frequency) using an advanced blast simulator. Male Sprague Dawley rats were exposed to BOP frontally and laterally at a pressure range of ~ 8.5-19 psi, for up to 30 daily exposures. The extent of lung injury was identified at 24 h following BOP by assessing the extent of surface hemorrhage/contusion, Hematoxylin and Eosin staining, and behavioral deficits with open field activity. Lung injury was mathematically modeled to define the military standard 1% lung injury threshold. Significant levels of histiocytosis and inflammation were observed in pressures ≥ 10 psi and orientation effects were observed at pressures ≥ 13 psi. Experimental data demonstrated ~ 8.5 psi is the threshold for hemorrhage/contusion, up to 30 exposures. Modeling the data predicted injury risk up to 50 exposures with intensity thresholds at 8 psi for front exposure and 6psi for side exposures, which needs to be validated further.

Blast-induced hearing impairment in rats is associated with structural and molecular changes of the inner ear.

Auditory dysfunction is the most prevalent injury associated with blast overpressure exposure (BOP) in Warfighters and civilians, yet little is known about the underlying pathophysiological mechanisms. To gain insights into these injuries, an advanced blast simulator was used to expose rats to BOP and assessments were made to identify structural and molecular changes in the middle/inner ears utilizing otoscopy, RNA sequencing (RNA-seq), and histopathological analysis. Deficits persisting up to 1 month after blast exposure were observed in the distortion product otoacoustic emissions (DPOAEs) and the auditory brainstem responses (ABRs) across the entire range of tested frequencies (4-40 kHz). During the recovery phase at sub-acute time points, low frequency (e.g. 4-8 kHz) hearing improved relatively earlier than for high frequency (e.g. 32-40 kHz). Perforation of tympanic membranes and middle ear hemorrhage were observed at 1 and 7 days, and were restored by day 28 post-blast. A total of 1,158 differentially expressed genes (DEGs) were significantly altered in the cochlea on day 1 (40% up-regulated and 60% down-regulated), whereas only 49 DEGs were identified on day 28 (63% up-regulated and 37% down-regulated). Seven common DEGs were identified at both days 1 and 28 following blast, and are associated with inner ear mechanotransduction, cytoskeletal reorganization, myelin development and axon survival. Further studies on altered gene expression in the blast-injured rat cochlea may provide insights into new therapeutic targets and approaches to prevent or treat similar cases of blast-induced auditory damage in human subjects.

Blast Exposure Leads to Accelerated Cellular Senescence in the Rat Brain.

Blast-induced traumatic brain injury (bTBI) is one of the major causes of persistent disabilities in Service Members, and a history of bTBI has been identified as a primary risk factor for developing age-associated neurodegenerative diseases. Clinical observations of several military blast casualties have revealed a rapid age-related loss of white matter integrity in the brain. In the present study, we have tested the effect of single and tightly coupled repeated blasts on cellular senescence in the rat brain. Isoflurane-anesthetized rats were exposed to either a single or 2 closely coupled blasts in an advanced blast simulator. Rats were euthanized and brains were collected at 24 h, 1 month and 1 year post-blast to determine senescence-associated-ß-galactosidase (SA-ß-gal) activity in the cells using senescence marker stain. Single and repeated blast exposures resulted in significantly increased senescence marker staining in several neuroanatomical structures, including cortex, auditory cortex, dorsal lateral thalamic nucleus, geniculate nucleus, superior colliculus, ventral thalamic nucleus and hippocampus. In general, the increases in SA-ß-gal activity were more pronounced at 1 month than at 24 h or 1 year post-blast and were also greater after repeated than single blast exposures. Real-time quantitative RT-PCR analysis revealed decreased levels of mRNA for senescence marker protein-30 (SMP-30) and increased mRNA levels for p21 (cyclin dependent kinase inhibitor 1A, CDKN1A), two other related protein markers of cellular senescence. The increased senescence observed in some of these affected brain structures may be implicated in several long-term sequelae after exposure to blast, including memory disruptions and impairments in movement, auditory and ocular functions.

Antibodies Against Lysophosphatidic Acid Protect Against Blast-Induced Ocular Injuries.

Exposure to blast overpressure waves is implicated as the major cause of ocular injuries and resultant visual dysfunction in veterans involved in recent combat operations. No effective therapeutic strategies have been developed so far for blast-induced ocular dysfunction. Lysophosphatidic acid (LPA) is a bioactive phospholipid generated by activated platelets, astrocytes, choroidal plexus cells, and microglia and is reported to play major roles in stimulating inflammatory processes. The levels of LPA in the cerebrospinal fluid have been reported to increase acutely in patients with traumatic brain injury (TBI) as well as in a controlled cortical impact (CCI) TBI model in mice. In the present study, we have evaluated the efficacy of a single intravenous administration of a monoclonal LPA antibody (25 mg/kg) given at 1 h post-blast for protection against injuries to the retina and associated ocular dysfunctions. Our results show that a single 19 psi blast exposure significantly increased the levels of several species of LPA in blood plasma at 1 and 4 h post-blast. The anti-LPA antibody treatment significantly decreased glial cell activation and preserved neuronal cell morphology in the retina on day 8 after blast exposure. Optokinetic measurements indicated that anti-LPA antibody treatment significantly improved visual acuity in both eyes on days 2 and 6 post-blast exposure. Anti-LPA antibody treatment significantly increased rod photoreceptor and bipolar neuronal cell signaling in both eyes on day 7 post-blast exposure. These results suggest that blast exposure triggers release of LPAs, which play a major role promoting blast-induced ocular injuries, and that a single early administration of anti-LPA antibodies provides significant protection.

Long-Term Effects of Blast Exposure: A Functional Study in Rats Using an Advanced Blast Simulator.

Anecdotal observations of blast victims indicate that significant neuropathological and neurobehavioral defects may develop at later stages of life. To pre-clinically model this phenomenon, we have examined neurobehavioral changes in rats up to 1 year after exposure to single and tightly coupled repeated blasts using an advanced blast simulator. Neurobehavioral changes were monitored at acute, sub-acute, and chronic time-points using Morris water maze test of spatial learning and memory, novel object recognition test of short-term memory, open field exploratory activity as a test of anxiety/depression, a rotating pole test for vestibulomotor function, and a rotarod balance test for motor coordination. Single and repeated blasts resulted in significant functional deficits at both acute and chronic time-points. In most functional tests, rats exposed to repeated blasts performed more poorly than rats exposed to single blast. Interestingly, several functional deficits post-blast were most pronounced at 6 months and beyond. Significant neuromotor impairments occurred at early stages after blast exposure and the severity increased with repeated exposures. The novel object recognition testing revealed short-term memory deficits at 6 and 12 months post-blast. The water maze test revealed impairments at acute and chronic stages after blast exposure. The most substantial changes in the blast-exposed rats were observed with the center time and margin time legacies in the open field exploration test at 6, 9, and 12 months post-blast. Notably, these two outcome measures were minimally altered acutely, recovered during sub-acute stages, and were markedly affected during the chronic stages after blast exposures and may implicate development of chronic anxiety and depressive-like behaviors.

Defective methionine metabolism in the brain after repeated blast exposures might contribute to increased oxidative stress.

Blast-induced traumatic brain injury (bTBI) is one of the major disabilities in Service Members returning from recent military operations. The neurobiological underpinnings of bTBI, which are associated with acute and chronic neuropathological and neurobehavioral deficits, are uncertain. Increased oxidative stress in the brain is reported to play a significant role promoting neuronal damage associated with both brain injury and neurodegenerative disorders. In this study, brains of rats exposed to repeated blasts in a shock tube underwent untargeted profiling of primary metabolism by automatic linear exchange/cold injection GC-TOF mass spectrometry and revealed acute and sub-acute disruptions in the metabolism of the essential amino acid methionine and associated antioxidants. Methionine sulfoxide, the oxidized metabolite of methionine, showed a sustained increase in the brain after blast exposure which was associated with a significant decrease in cysteine, the amino acid derived from methionine. Glutathione, the antioxidant synthesized from cysteine, also concomitantly decreased as did the antioxidant ascorbic acid. Reductions in ascorbic acid were accompanied by increased levels of its oxidized metabolite, dehydroascorbic acid and other metabolites such as threonic acid, isothreonic acid, glycolic acid and oxalic acid. Fluorometric analysis of the brains showed acute and sub-acute increase in total reactive oxygen species. In view of the fundamental importance of glutathione in the brain as an antioxidant, including its role in the reduction of dehydroascorbic acid to ascorbic acid, the disruptions in methionine metabolism elicited by blast exposure might prominently contribute to neuronal injury by promoting increased and sustained oxidative stress.

Cerebrospinal Fluid Chemokine (C-C Motif) Ligand 2 Is an Early-Response Biomarker for Blast-Overpressure-Wave-Induced Neurotrauma in Rats.

Chemokines and their receptors are of great interest within the milieu of immune responses elicited in the central nervous system in response to trauma. Chemokine (C-C motif)) ligand 2 (CCL2), which is also known as monocyte chemotactic protein-1, has been implicated in the pathogenesis of traumatic brain injury (TBI), brain ischemia, Alzheimer's disease, and other neurodegenerative diseases. In this study, we investigated the time course of CCL2 accumulation in cerebrospinal fluid (CSF) after exposures to single and repeated blast overpressures of varied intensities along with the neuropathological changes and motor deficits resulting from these blast conditions. Significantly increased concentrations of CCL2 in CSF were evident by 1 h of blast exposure and persisted over 24 h with peak levels measured at 6 h post-injury. The increased levels of CCL2 in CSF corresponded with both the number and intensities of blast overpressure and were also commensurate with the extent of neuromotor impairment and neuropathological abnormalities resulting from these exposures. CCL2 levels in CSF and plasma were tightly correlated with levels of CCL2 messenger RNA in cerebellum, the brain region most consistently neuropathologically disrupted by blast. In view of the roles of CCL2 that have been implicated in multiple neurodegenerative disorders, it is likely that the sustained high levels of CCL2 and the increased expression of its main receptor, CCR2, in the brain after blast may similarly contribute to neurodegenerative processes after blast exposure. In addition, the markedly elevated concentration of CCL2 in CSF might be a candidate early-response biomarker for diagnosis and prognosis of blast-induced TBI.

Acute decrease in alkaline phosphatase after brain injury: A potential mechanism for tauopathy.

Dephosphorylation of phosphorylated Tau (pTau) protein, which is essential for the preservation of neuronal microtubule assemblies and for protection against trauma-induced tauopathy and chronic traumatic encephalopathy (CTE), is primarily achieved in brain by tissue non-specific alkaline phosphatase (TNAP). Paired helical filaments (PHFs) and Tau isolated from Alzheimer's disease (AD) patients' brains have been shown to form microtubule assemblies with tubulin only after treatment with TNAP or protein phosphatase-2A, 2B and -1, suggesting that Tau protein in the PHFs of neurons in AD brain is hyperphosphorylated, which prevents microtubule assembly. Using blast or weight drop models of traumatic brain injury (TBI) in rats, we observed pTau accumulation in the brain as early as 6h post-injury and further accumulation which varied regionally by 24h post-injury. The pTau accumulation was accompanied by reduced TNAP expression and activity in these brain regions and a significantly decreased plasma total alkaline phosphatase activity after the weight drop. These results reveal that both blast- and impact acceleration-induced head injuries cause an acute decrease in the level/activity of TNAP in the brain, which potentially contributes to trauma-induced accumulation of pTau and the resultant tauopathy. The regional changes in the level/activity of TNAP or accumulation of pTau after these injuries did not correlate with the accumulation of amyloid precursor protein, suggesting that the basic mechanism underlying tauopathy in TBI might be distinct from that associated with AD.