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Blast-induced traumatic brain injury (bTBI) has been documented as a significant concern for both military and civilian populations in response to the increased use of improvised explosive devices. Identifying biomarkers that could aid in the proper diagnosis and assessment of both acute and chronic bTBI is in urgent need since little progress has been made towards this goal. Addressing this knowledge gap is especially important in military veterans who are receiving assessment and care often years after their last blast exposure. Neuron-specific phosphorylated neurofilament heavy chain protein (pNFH) has been successfully evaluated as a reliable biomarker of different neurological disorders, as well as brain trauma resulting from contact sports and acute stages of brain injury of different origin. In the present study, we have evaluated the utility of pNFH levels measured in the cerebrospinal fluid (CSF) as an acute and chronic biomarker of brain injury resulting from single and tightly coupled repeated blast exposures using experimental rats. The pNFH levels increased at 24 h, returned to normal levels at 1 month, but increased again at 6 months and 1 year post-blast exposures. No significant changes were observed between single and repeated blast-exposed groups. To determine whether the observed increase of pNFH in CSF corresponded with its levels in the brain, we performed fluorescence immunohistochemistry in different brain regions at the four time-points evaluated. We observed decreased pNFH levels in those brain areas at 24 h, 6 months, and 1 year. The results suggest that blast exposure causes axonal degeneration at acute and chronic stages resulting in the release of pNFH, the abundant neuronal cytoskeletal protein. Moreover, the changes in pNFH levels in the CSF negatively correlated with the neurobehavioral functions in the rats, reinforcing suggestions that CSF levels of pNFH can be a suitable biomarker of bTBI.

The interaction of explosion-induced blast waves with the head (i.e., a direct mechanism) or with the torso (i.e., an indirect mechanism) presumably causes traumatic brain injury. However, the understanding of the potential role of each mechanism in causing this injury is still limited. To address this knowledge gap, we characterized the changes in the brain tissue of rats resulting from the direct and indirect mechanisms at 24 h following blast exposure. To this end, we conducted separate blast-wave exposures on rats in a shock tube at an incident overpressure of 130 kPa, while using whole-body, head-only, and torso-only configurations to delineate each mechanism. Then, we performed histopathological (silver staining) and immunohistochemical (GFAP, Iba-1, and NeuN staining) analyses to evaluate brain-tissue changes resulting from each mechanism. Compared to controls, our results showed no significant changes in torso-only-exposed rats. In contrast, we observed significant changes in whole-body-exposed (GFAP and silver staining) and head-only-exposed rats (silver staining). In addition, our analyses showed that a head-only exposure causes changes similar to those observed for a whole-body exposure, provided the exposure conditions are similar. In conclusion, our results suggest that the direct mechanism is the major contributor to blast-induced changes in brain tissues.

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.

According to the US Defense and Veterans Brain Injury Center (DVBIC) and Centers for Disease Control and Prevention (CDC), mild traumatic brain injury (mTBI) is a common form of head injury. Medical imaging data provides clinical insight into tissue damage/injury and injury severity, and helps medical diagnosis. Computational modeling and simulation can predict the biomechanical characteristics of such injury, and are useful for development of protective equipment. Integration of techniques from computational biomechanics with medical data assessment modalities (e.g., magnetic resonance imaging or MRI) has not yet been used to predict injury, support early medical diagnosis, or assess effectiveness of personal protective equipment. This paper presents a methodology to map computational simulations with clinical data for interpreting blunt impact TBI utilizing two clinically different head injury case studies. MRI modalities, such as T1, T2, diffusion-weighted imaging (DWI) and apparent diffusion coefficient (ADC), were used for simulation comparisons. The two clinical cases have been reconstructed using finite element analysis to predict head biomechanics based on medical reports documented by a clinician. The findings are mapped to simulation results using image-based clinical analyses of head impact injuries, and modalities that could capture simulation results have been identified. In case 1, the MRI results showed lesions in the brain with skull indentation, while case 2 had lesions in both coup and contrecoup sides with no skull deformation. Simulation data analyses show that different biomechanical measures and thresholds are needed to explain different blunt impact injury modalities; specifically, strain rate threshold corresponds well with brain injury with skull indentation, while minimum pressure threshold corresponds well with coup–contrecoup injury; and DWI has been found to be the most appropriate modality for MRI data interpretation. As the findings from these two cases are substantiated with additional clinical studies, this methodology can be broadly applied as a tool to support injury assessment in head trauma events and to improve countermeasures (e.g., diagnostics and protective equipment design) to mitigate these injuries.

Exposure to the repeated low-level blast overpressure (BOP) periodically experienced by military personnel in operational and training environments can lead to deficits in behavior and cognition. While these low-intensity blasts do not cause overt changes acutely, repeated exposures may lead to cumulative effects in the brain that include acute inflammation, vascular disruption, and other molecular changes, which may eventually contribute to neurodegenerative processes. To identify these acute changes in the brain following repeated BOP, an advanced blast simulator was used to expose rats to 8.5 or 10 psi BOP once per day for 14 days. At 24 h after the final BOP, brain tissue was collected and analyzed for inflammatory markers, astrogliosis (GFAP), tight junction proteins (claudin-5 and occludin), and neurodegeneration-related proteins (Aβ40/42, pTau, TDP-43). After repeated exposure to 8.5 psi BOP, the change in cytokine profile was relatively modest compared to the changes observed following 10 psi BOP, which included a significant reduction in several inflammatory markers. Reduction in the tight junction protein occludin was observed in both groups when compared to controls, suggesting cerebrovascular disruption. While repeated exposure to 8.5 psi BOP led to a reduction in the Alzheimer’s disease (AD)-related proteins amyloid-β (Aβ)40 and Aβ42, these changes were not observed in the 10 psi group, which had a significant reduction in phosphorylated tau. Finally, repeated 10 psi BOP exposures led to an increase in GFAP, indicating alterations in astrocytes, and an increase in the mechanosensitive ion channel receptor protein, Piezo2, which may increase brain sensitivity to injury from pressure changes from BOP exposure. Overall, cumulative effects of repeated low-level BOP may increase the vulnerability to injury of the brain by disrupting neurovascular architecture, which may lead to downstream deleterious effects on behavior and cognition.

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-β-galactosidase (SA-β-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-β-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.

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.

At present, accurate and reliable biomarkers to ascertain the presence, severity, or prognosis of blast traumatic brain injury (bTBI) are lacking. There is an urgent need to establish accurate and reliable biomarkers capable of mbTBI detection. Currently, there are no studies that identify changes in miRNA and lipids at varied severities of bTBI. Various biological components such as lipids, circulating mRNA, and miRNA, could potentially be detected using advanced techniques such as next-generation sequencing and mass spectroscopy. Therefore, plasma analysis is an attractive approach with which to diagnose and treat brain injuries. Subacute changes in plasma microRNA (miRNA) and lipid composition for sphingolipids were evaluated in a murine model of mild-to-moderate bTBI using next-generation sequencing and mass spectroscopy respectively. Animals were exposed at 17, 17 × 3, and 20 psi blast intensities using a calibrated blast simulator. Plasma lipid profiling demonstrated decreased C18 fatty acid chains of sphingomyelins and increased ceramide levels when compared with controls. Plasma levels of brain-enriched miRNA, miR-127 were increased in all groups while let-7a, b, and g were reduced in the 17 × 3 and 20 psi groups, but let 7d was increased in the 17 psi group. The majority of the miRs and lipids are highly conserved across different species, making them attractive to explore and potentially employ as diagnostic markers. It is tempting to speculate that sphingolipids, miR-128, and the let-7 family could predict mTBI, while a combination of miR-484, miR-122, miR-148a, miR-130a, and miR-223 could be used to predict the overall status of injury following blast injury.

A history of mild traumatic brain injury (mTBI), particularly repeated mTBI (rmTBI), has been identified as a risk factor for late-onset neurodegenerative conditions. The mild and transient nature of early symptoms often impedes diagnosis in young adults who are disproportionately affected by mTBIs. A proportion of the affected population will incur long-term behavioral and cognitive consequences but the underlying pathomechanism is currently unknown. Diffusion tensor imaging (DTI) provides sensitive and quantitative assessment of TBI-induced structural changes, including white matter injury, and may be used to predict long-term outcome. We used DTI in an animal model of blast rmTBI (rmbTBI) to quantify blast-induced structural changes at 7 and 90 days post-injury, and their evolution between the two time points. Young adult male rats (~P65 at injury) were exposed to repeated mild blast overpressure, or anesthetized as shams, and their fixed brains were imaged using high-field (7 T) MRI. We found that whole brain volumes similarly increased in injured and sham rats from 7 to 90 days. However, we detected localized volume increases in blast-exposed animals 7 days post-injury, mainly ipsilateral to incident blast waves. Affected regions included gray matter of the frontal association, cingulate, and motor cortex, thalamus, substantia nigra, and raphe nuclei (median and dorsal), as well as white matter of the internal capsule and cerebral peduncle. Conversely, we measured volume reductions in these and other regions, including the hippocampus and cerebellum, at 90 days post-injury. DTI also detected both transient and persistent microstructural changes following injury, with some changes showing distinct ipsilateral versus contralateral side differences relative to blast impact. Early changes in fractional anisotropy (FA) were subtle, becoming more prominent at 90 days in the cerebral and inferior cerebellar peduncles, and cerebellar white matter. Widespread increases in radial diffusivity (RD) and axial diffusivity (primary eigenvalue or E1) at 7 days post-injury largely subsided by 90 days, although RD was more sensitive than E1 at detecting white matter changes. E1 effects in gray and white matter, which paralleled increases in apparent diffusion, were likely more indicative of dysregulated water homeostasis than pathologic structural changes. Importantly, we found evidence for a different developmental trajectory following rmbTBI, as indicated by significant injury x age interactions on volume. Our findings demonstrate that rmbTBI initiates dynamic pathobiological processes that may negatively alter the course of late-stage neurodevelopment and adversely affect long-term cognitive and behavioral outcomes., Highlights • Young adult rats exposed to mild blast show lasting microstructural brain changes. • The evolution of mTBI pathology was reflected by temporal changes in DTI measures. • Regional volume changes captured significant injury × age interactions. • DTI measures differentially captured injury effects in white and gray matter. • Significant interaction effects suggest an altered developmental trajectory.

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 overpressure exposure has been linked to transient, but measurably deteriorated performance and symptomatologies in law enforcement and military personnel. Overlapping sub-concussive symptomatology associated with the very low level blast overpressures (vLLB) but high sound pressure (

Repeated mild blast-induced traumatic brain injury (rmbTBI), caused by recurrent exposure to low levels of explosive blast, is a significant concern for military health systems. However, the pathobiology of rmbTBI is currently poorly understood. Animal models are important tools to identify the molecular changes of rmbTBI, but comparisons across different models can present their own challenges. In this study, we compared two well-established rodent models of mbTBI, the “KI model” and the “USU/WRAIR model.” These two models create different pulse forms, in terms of peak pressure and duration. Following single and double exposures to mild levels of blast, we used in situ hybridization (ISH) to assess changes in mRNA levels of tyrosine hydroxylase (TH), tryptophan hydroxylase (TPH2), and galanin in the locus coeruleus (LC) and dorsal raphe nucleus (DRN). These systems and their transmitters are known to mediate responses to stress and anxiety. We found increased mRNA levels of TH, TPH2 and galanin in the LC and DRN of single-exposed rats relative to sham rats in the KI but not the USU/WRAIR model. Sham mRNA values measured in the USU/WRAIR model were substantially higher than their KI counterparts. Double exposure caused similarly significant increases in mRNA values in the KI model but not the USU/WRAIR model, except TPH2 and galanin levels in the DRN. We detected no cumulative effect of injury in either model at the used inter-injury interval (30 min), and there were no detectable neuropathological changes in any experimental group at 1 day post-injury. The apparent lack of early response to injury as compared to sham in the USU/WRAIR model is likely caused by stressors (e.g., transportation and noise), associated with the experimental execution, that were absent in the KI model. This study is the first to directly compare two established rodent models of rmbTBI, and to highlight the challenges of comparing findings from different animal models. Additional studies are needed to understand the role of stress, dissect the effects of psychological and physical injuries and to identify the window of increased cerebral vulnerability, i.e., the inter-injury interval that results in a cumulative effect following repeated blast exposure.

Despite the large number of promising neuroprotective agents identified in experimental traumatic brain injury (TBI) studies, none has yet shown meaningful improvements in long-term outcome in clinical trials. To develop recommendations and guidelines for pre-clinical testing of pharmacological or biological therapies for TBI, the Moody Project for Translational Traumatic Brain Injury Research hosted a symposium attended by investigators with extensive experience in pre-clinical TBI testing. The symposium participants discussed issues related to pre-clinical TBI testing including experimental models, therapy and outcome selection, study design, data analysis, and dissemination. Consensus recommendations included the creation of a manual of standard operating procedures with sufficiently detailed descriptions of modeling and outcome measurement procedures to permit replication. The importance of the selection of clinically relevant outcome variables, especially related to behavior testing, was noted. Considering the heterogeneous nature of human TBI, evidence of therapeutic efficacy in multiple, diverse (e.g., diffuse vs. focused) rodent models and a species with a gyrencephalic brain prior to clinical testing was encouraged. Basing drug doses, times, and routes of administration on pharmacokinetic and pharmacodynamic data in the test species was recommended. Symposium participants agreed that the publication of negative results would reduce costly and unnecessary duplication of unsuccessful experiments. Although some of the recommendations are more relevant to multi-center, multi-investigator collaborations, most are applicable to pre-clinical therapy testing in general. The goal of these consensus guidelines is to increase the likelihood that therapies that improve outcomes in pre-clinical studies will also improve outcomes in TBI patients.

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.

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.

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.

Use of improvised explosive devices has significantly increased the incidence of traumatic brain injury (TBI) and associated neuropsychiatric deficits in the recent wars in Iraq and Afghanistan. Acute deleterious effects of single and repeated blast exposure can lead to long-term neurobiological effects and neuropsychiatric deficits. Using in vitro and in vivo shock tube models of blast-induced TBI, we studied changes in mitochondrial energy metabolism after blast exposure. Single and repeated blast exposures in vitro resulted in significant decreases in neuronal adenosine triphosphate (ATP) levels at 6 h post-blast that returned towards normal levels by 24 h. Similar changes in ATP also were observed in the cerebral cortices of mice subjected to single and repeated blast exposures. In neurons, mitochondrial glutamate oxaloacetate transaminase (GOT2) plays a critical role in metabolism and energy production. Proteomic analysis of brain cortices showed a significant decrease in GOT2 levels 6 h after repeated blast exposures, which was further confirmed by Western blotting. Western blot analysis of GOT2 and pyruvate dehydrogenase in the cortex showed direct correlation only between GOT2 and ATP levels. Activity of GOT2 in the isolated cortical mitochondria also showed significant decrease at 6 h supporting the results of proteomic and Western blot analyses. Knowing the significant role of GOT2 in the neuronal mitochondrial energy metabolism, it is quite likely that the down regulation of GOT2 after blast exposure is playing a significant role in mitochondrial dysfunction after blast exposure.

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 6 h and increased considerably at 24 h 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 24 h 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.

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.

Blast has been the leading cause of injury, particularly traumatic brain injury and visual system injury, in combat operations in Iraq and Afghanistan. We determined the effect of shock tube-generated primary blast on retinal electrophysiology and on retinal and brain optic tract histopathology in a rat model. The amplitude of a- and b-waves on the electroretinogram (ERG) for both right and left eyes were measured prior to a battlefield simulation Friedlander-type blast wave and on 1, 7, and 14 days thereafter. Histopathologic findings of the right and left retina and the right and left optic tracts (2.8 mm postoptic chiasm) were evaluated 14 days after the blast. For two experiments in which the right eye was oriented to the blast, the amplitude of ERG a- and b-waves at 7 days post blast on the right side but not on the left side was diminished compared to that of sham animals (P = 0.005–0.01) Histopathologic injury scores at 14 days post blast for the right retina but not the left retina were higher than for sham animals (P = 0.01), and histopathologic injury scores at 14 days for both optic tracts were markedly higher than for shams (P

Mild traumatic brain injury, caused by the exposure to single or repeated blast overpressure, is a principal concern due to its pathological complexity and neurobehavioral similarities with posttraumatic stress disorder. In this study, we exposed rats to a single or multiple (five total; administered on consecutive days) mild blasts, assessed their behavior at 1 and 16 days postinjury) and performed histological and protein analyses of brains and plasma at an early (2 h) and a late (22 days) termination time point. One day postinjury, multiple-injured (MI) rats showed the least general locomotion and the most depression- and anxiety-related behaviors among the experimental groups; there were no such differences at 16 days. However, at the later time point, both injured groups displayed elevated levels of select protein biomarkers. Histology showed significantly increased numbers of TUNEL+ (terminal-deoxy-transferase-mediated dUTP nick-end labeling)-positive cells in the dorsal and ventral hippocampus (DHC and VHC) of both injured groups as early as 2 h after injury. At 22 days, the increase was limited to the VHC of MI animals. Our findings suggest that the exposure to mild blast overpressure triggers early hippocampal cell death as well as neuronal, glial, and vascular damage that likely contribute to significant, albeit transient increases in depression- and anxiety-related behaviors. However, the severity of the observed pathological changes in MI rats failed to support the hypothesized cumulative effect of repeated injury. We infer that at this blast frequency, a potential conditioning phenomenon counteracts with and reduces the extent of subsequent damage in MI rats.

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 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.

The overlapping pathologies and functional outcomes of blast-induced TBI (bTBI) and stress-related neurobehavioral disorders like post-traumatic stress disorder (PTSD) are significant military health issues. Soldiers are exposed to multiple stressors with or without suffering bTBI, making diagnosis and treatment as well as experimental modeling of bTBI a challenge. In this study we compared anxiety levels of Naïve rats to ones that were exposed to each of the following conditions daily for 4 consecutive days: C I: transportation alone; C II: transportation and anesthesia; C III: transportation, anesthesia, and blast sounds; Injured: all three variables plus mild blast overpressure. Following behavioral testing we analyzed sera and select brain regions for protein markers and cellular changes. C I, C II, and C III animals exhibited increased anxiety, but serum corticosterone levels were only significantly elevated in C III and Injured rats. C III and Injured animals also had elevated interferon-γ (IFN-γ) and interleukin-6 (IL-6) levels in the amygdala (AD) and ventral hippocampus (VHC). Glial fibrillary acidic protein (GFAP) levels were only significantly elevated in the VHC, prefrontal cortex (PFC), and AD of Injured animals; they showed an apparent increase in ionized calcium-binding adapter molecule (Iba1) and GFAP immunoreactivity, as well as increased numbers of TUNEL-positive cells in the VHC. Our findings demonstrate that experimental conditions, particularly the exposure to blast acoustics, can increase anxiety and trigger specific behavioral and molecular changes without injury. These findings should be taken into consideration when designing bTBI studies, to better understand the role of stressors in the development of post-traumatic symptoms, and to establish a differential diagnosis for PTSD and bTBI.

It is likely that the mild TBI and cognitive impairments observed among many of the troops returning from OIF and OEF result from repeated exposures to blast overpressure. Although the clinical symptoms of concussion are typically transient, there is both a cumulative risk for persistent damage due to repeated concussions, and a post-concussion period of greatest vulnerability to a second impact. Specific risk assessments and guidelines should be established for exposure to blast overpressure. We are using a preclinical model of blast overpressure in rats to investigate the cumulative effects of multiple blast exposures on neurologic status, neurobehavioral function, and brain histopathological endpoints. Repeated exposures to blast overpressure with varied inter-blast intervals are used to characterize and define the temporal window of brain vulnerability to repeated blast overpressure. We anticipate that these data will provide a critical first step in establishing rational risk guidelines and developing mitigation strategies.

The high incidence of mild TBI in Warfighters exposed to blast has triggered interest in injury mitigation and increased TBI resilience as well as concerns over safe return-to-duty and minimization of long-term and delayed TBI-related debilitations in returning veterans. These objectives require the utilization of high fidelity animal models to investigate the underlying neurobiological mechanisms of injury as a rational basis for defining risks and establishing effective countermeasures. The etiology of blast TBI (bTBI) is largely undefined, and several mechanisms, likely interactive, have been proposed. Using a well-validated blast model, we are exploring blast-induced acceleration of the head as one of the primary components of bTBI. Having successfully worked with a range of inanimate objects of varied areal densities to define relations of blast flow conditions to acceleration and displacement, we have begun examination of the effects of systematically varied conditions on anesthetized rats to isolate and distinguish the contributions of blast-induced head acceleration and displacement from other biomechanical components and effects of the shockwave.

Halothane-anesthetized male rats were subjected to either moderately severe parasagittal fluid percussion-induced traumatic brain injury (TBI) or sham injury, and for 30 min immediately after injury hypoxia was induced in half the rats from each group by substituting a 13% O 2 source to deliver halothane for continued anesthesia. At 60min post-TBI, Northern blot analysis showed a significant increase in c-fos mRNA levels, by 60-100% above sham control levels in the frontal cortex, cerebellum and hippocampus. Although hypoxia in sham-injured rats did not by itself alter c-fos mRNA levels, it did significantly potentiate the TBI-induced changes in c-fos mRNA in all three brain regions. These findings show that hypoxia is an important factor influencing genomic responses to TBI.

In an adult rat model of blast wave exposure, we examined the efficacy of four drugs known to be polyunsaturated fatty acid derived lipid mediators of inflammation, i.e., lipoxin A4 (LXA4), protectin DX (PDX), resolvin D1 (RVD1), and resolvin E1 (RVE1), to alleviate neuronal cell damage to the eyes (retinas) and brain visual centers. Rats were placed in a compressed air driven shock tube and exposed, right side on, once to a 20 psi (260 Hz) blast over pressure wave. The drugs were immediately administered to the blasted rats by intravenous injection (25 g/kg), and then given every other day out to 14 days. Injury outcome measures were electroretinography (ERG), visual discrimination behavioral testing, and histopathology (n = 8, 9, 9, and 9, respectively). Blasted rats were assessed at baseline and then out to 14 days post-exposure. ERG based light signaling responses of their eyes showed a drug efficacy order of RVE1 LXA4 PDX RVD1. Visual discrimination testing of their ability to press a lever with a cue light, yielded a drug efficacy order of RVD1 RVE1 PDX LXA4. Histopathology of their retinas and associated brain optic tracts for neuronal cell degeneration gave a drug efficacy order of RVD1 LXA4 PDX RVE1 and LXA4 RVD1 RVE1 PDX, respectively. For all outcome measures, these drugs at best produced modest injury improvements for the blasted retina and brain, suggesting they have a rather limited potential as visual system therapeutics.

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.

Mild traumatic brain injury (mTBI) represents a significant challenge for the civilian and military health care systems due to its high prevalence and overall complexity. Our earlier works showed evidence of neuroinflammation, a late onset of neurobehavioral changes, and lasting memory impairment in a rat model of mild blast-induced TBI (mbTBI). The aim of our present study was to determine whether acute treatment with the non-steroidal anti-inflammatory drug minocycline (Minocin(®)) can mitigate the neurobehavioral abnormalities associated with mbTBI, Furthermore, we aimed to assess the effects of the treatment on select inflammatory, vascular, neuronal, and glial markers in sera and in brain regions associated with anxiety and memory (amygdala, prefrontal cortex, ventral, and dorsal hippocampus) following the termination (51 days post-injury) of the experiment. Four hours after a single exposure to mild blast overpressure or sham conditions, we treated animals with a daily dose of minocycline (50 mg/kg) or physiological saline (vehicle) for four consecutive days. At 8 and 45 days post-injury, we tested animals for locomotion, anxiety, and spatial memory. Injured animals exhibited significantly impaired memory and increased anxiety especially at the later testing time point. Conversely, injured and minocycline treated rats' performance was practically identical to control (sham) animals in the open field, elevated plus maze, and Barnes maze. Protein analyses of sera and brain regions showed significantly elevated levels of all of the measured biomarkers (except VEGF) in injured and untreated rats. Importantly, minocycline treatment normalized serum and tissue levels of the majority of the selected inflammatory, vascular, neuronal, and glial markers. In summary, acute minocycline treatment appears to prevent the development of neurobehavioral abnormalities likely through mitigating the molecular pathologies of the injury in an experimental model of mbTBI.

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.

Lumbar subarachnoid injection of dynorphin A causes an ischemia-induced neuronal degeneration and persistent hindlimb paralysis. The protective effects of a variety of competitive and non-competitive N-methyl-D-aspartate (NMDA) receptor antagonists indicate that activation of the NMDA receptor complex is essential for dynorphin A-induced spinal cord injury. 1-Aminocyclopropanecarboxylic acid (ACPC) is a high affinity, partial agonist at strychnine-insensitive glycine receptors associated with the NMDA receptor complex. Pretreatment of rats with ACPC (100 and 200 mg/kg, i.p., 30 min prior to dynorphin A) significantly eliminated the persistent hindlimb motor deficits and neuropathological changes produced by 20 nmol of this peptide. The neuroprotective effects of ACPC (100 mg/kg, i.p.) were abolished by parenteral administration of glycine (800 mg/kg, 30 min prior to ACPC), consistent with other in vivo and in vitro studies indicating that the pharmacological actions of ACPC are effected through strychnine-insensitive glycine receptors. When given instead as six daily injections (200 mg/kg, i.p.) followed by an injection-free day, ACPC also significantly improved neurological recovery following dynorphin-A injection. These results support earlier indications that: (1) activation of the NMDA receptor complex plays a critical role in mediating dynorphin A-induced rat spinal cord injury; (2) ACPC provides an effective means of antagonizing excitotoxic phenomena; and (3) chronic administration of ACPC can elicit a persistent change in the NMDA receptor complex.

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.

De novo hippocampal neurogenesis contributes to functional recovery following traumatic brain injury (TBI). Enriched environment (EEN) can improve the outcome of TBI by positively affecting neurogenesis. Blast induced traumatic brain injury (bTBI) characterized by memory impairment and increased anxiety levels, is a leading cause of chronic disability among soldiers. Using a rodent model of bTBI we asked: (a) whether long-term exposure to EEN after injury can ameliorate behavioral abnormalities and (b) what the effects of EEN are at the molecular and cellular levels and on de novo neurogenesis. We found that housing injured animals in EEN resulted in significantly improved spatial memory while animals in normal housing (NH) showed persistent memory impairment. VEGF and Tau protein but not Interleukin-6 (IL-6) levels were normalized in the dorsal hippocampus (DHC) of EEN rats while all three markers remained elevated in NH rats. Interestingly, after peaking at 6 weeks post-injury, anxiety returned to normal levels at 2 months independent of housing conditions. Housing animals in EEN had no significant effect on VEGF and Tau protein levels in the ventral hippocampus (VHC) and the amygdala (AD). We also found that EEN reduced IL-6 and IFNγ levels in the VHC; these markers remained elevated following NH. We observed an increase in GFAP and DCX immunoreactivities in the VHC of NH animals at 2 months post-injury. Conversely, injured animals housed in EEN showed no increase in GFAP or DCX immunoreactivity in their VHC. In summary, long-term exposure of injured animals to EEN appears to play a positive role in the restoration of memory functions but not on anxiety, which returned to normal levels after a significant period of time. Cellular and molecular changes in response to EEN appear to be a part of neurogenesis-independent as well as dependent recovery processes triggered by bTBI.

Psychological stress and traumatic brain injury (TBI) can both result in lasting neurobehavioral abnormalities. Post-traumatic stress disorder and blast induced TBI (bTBI) have become the most significant health issues in current military conflicts. Importantly, military bTBI virtually never occurs without stress. In this experiment, we assessed anxiety and spatial memory of rats at different time points after repeated exposure to stress alone or in combination with a single mild blast. At 2 months after injury or sham we analyzed the serum, prefrontal cortex (PFC), and hippocampus (HC) of all animals by proteomics and immunohistochemistry. Stressed sham animals showed an early increase in anxiety but no memory impairment at any measured time point. They had elevated levels of serum corticosterone (CORT) and hippocampal IL-6 but no other cellular or protein changes. Stressed injured animals had increased anxiety that returned to normal at 2 months and significant spatial memory impairment that lasted up to 2 months. They had elevated serum levels of CORT, CK-BB, NF-H, NSE, GFAP, and VEGF. Moreover, all of the measured protein markers were elevated in the HC and the PFC; rats had an increased number of TUNEL-positive cells in the HC and elevated GFAP and Iba1 immunoreactivity in the HC and the PFC. Our findings suggest that exposure to repeated stress alone causes a transient increase in anxiety and no significant memory impairment or cellular and molecular changes. In contrast, repeated stress and blast results in lasting behavioral, molecular, and cellular abnormalities characterized by memory impairment, neuronal and glial cell loss, inflammation, and gliosis. These findings may have implications in the development of diagnostic and therapeutic measures for conditions caused by stress or a combination of stress and bTBI.

The etiology of blast-induced traumatic brain injury (TBI) is largely undefined. Along with reducing mortality, in preliminary experiments Kevlar vests significantly protected against BOP-induced neuropathological changes in rats. We postulate that: 1) much of the blast-induced fiber degeneration in brain results from pressure surges transmitted through the vasculature that elicit a series of intracranial disruptions, and 2) Kevlar vests are neuroprotective by uncoupling this pressure transmission following exposure to blast. Using a compression driven shock tube, we compare external, systemic (e.g. vascular arterial and venous), and central (e.g. intracranial pressure) BOP-induced pressure changes, and assess the impact of Kevlar vests on these changes. We seek to: 1) determine if measured pressure changes are blast severity-dependent and correspond with outcome measures, and 2) assess the impact of Kevlar vests on measured BOP-induced pressure changes and outcome measures and establish whether a protective vest encasing the thorax ameliorates blast-induced brain injury, pointing to a significant contribution of the effects of blast on the thorax to brain injury. These studies will provide critical insights into the etiology of blast-induced brain injury, and will advance the development of mitigation strategies.

beta-Funaltrexamine (beta-FNA) is an alkylating derivative of naltrexone. In addition to acting as an irreversible inhibitor of mu-receptor-mediated physiological effects, intracerebroventricular (i.c.v.) administration of beta-FNA to rat attenuates the ability of selective delta receptor antagonists and naloxone to reverse delta receptor-mediated effects. Moreover, recent work demonstrated that i.c.v. administration of beta-FNA alters the conformation of the opioid receptor complex, as inferred by a decrease in the Bmax of the lower affinity [3H][D-ala2,D-leu5]enkephalin binding site. Consistent with the decreased potency of naloxone as an inhibitor of delta receptor mediated effects, beta-FNA doubled the naloxone IC50 for displacing [3H][D-ala2,D-leu5]enkephalin from its lower affinity binding site. These data collectively support the hypothesis that the opioid receptor complex postulated to mediate mu-delta interactions in vivo is identical to the opioid receptor complex as defined by vitro ligand binding studies. A direct prediction of this hypothesis is that beta-FNA should increase the Kd of antagonists for the mu binding site (mu cx) of the receptor complex. The data reported in this paper demonstrate that beta-FNA doubled the IC50 of the potent narcotic antagonist, 6-desoxy-6 beta-fluoronaltrexone (cycloFOXY) for displacing [3H][D-ala2,D-leu5]enkephalin from its lower affinity binding site, and doubled the Kd of [3H]cycloFOXY for its mu binding site, providing additional data that the mu binding site labeled by [3H]cycloFOXY is the mu binding site of the opioid receptor complex. beta-FNA also altered the kappa binding site labeled by [3H]cycloFOXY, and when administered intrathecally to mice, beta-FNA produced a longlasting antinociception in the acetic acid writhing test.

Studies conducted after the development of the rapid filtration assay for opiate receptors, and before the recognition of multiple opioid receptors, failed to detect changes in opioid receptors induced by chronic morphine. Recent experiments conducted in our laboratories were designed to examine the hypothesis that only one of several opioid receptor types might be altered by chronic morphine. Using binding surface analysis and irreversible ligands to increase the "resolving power" of the ligand binding assay, the results indicated that chronic morphine increased both the Bmax and Kd of the opioid receptor complex, labeled with either [3H][D-Ala2,D-Leu5]enkephalin, [3H][D-Ala2-MePhe4,Gly-ol5]enkephalin or [3H]6-desoxy-6 beta-fluoronaltreone. In the present study rats were pretreated with drugs known to attenuate the development of tolerance and dependence [the irreversible mu-receptor antagonist, beta-funaltrexamine (beta-FNA), and the inhibitor of tryptophan hydroxylase, para-chlorophenylalanine], prior to subcutaneous implantation of morphine pellets. The results demonstrated that 1) unlike chronic naltrexone, beta-FNA failed to upregulate opioid receptors and 2) both beta-funaltrexamine and PCPA pretreatment attenuated the chronic morphine-induced increase in the Bmax, but not the Kd, of the opioid receptor complex. These results provide evidence that naltrex-one-induced upregulation of the opioid receptor complex might occur indirectly as a consequence of interactions at beta-funaltrexamine-insensitive opioid receptors and that morphine-induced upregulation (increased Bmax) of the opioid receptor complex is a relevant in vitro marker related to the development of tolerance and dependence. These data collectively support the hypothesis that endogenous antiopiate peptides play an important role in the development of tolerance and dependence to morphine.

The purpose of these experiments was to determine whether secondary hypoxia exacerbates the metabolic consequences of fluid percussion injury (FPI). In Experiment I, rats were trained to press a lever for their entire daily ration of food at any time during a 12-h light/dark cycle and run in an activity wheel. After food intake and body weight stabilized, rats were surgically prepared, assigned to one of four groups [FPI+Hypoxia (IH), FPI+Normoxia (IN), Sham Injury+Hypoxia (SH), Sham Injury+Normoxia (SN)] and, after recovery from surgery, anesthetized with halothane delivered by a 21% O2 source. Immediately after injury or sham injury, the O2 source was switched to 13% for rats in Groups IH and SH for 30 min. Post-traumatic hypoxemia exacerbated the ensuing FPI-induced reductions of food intake and body weight, but did not change FPI-induced reduction in wheel running. In Experiment II, rats were assigned to one of three groups (SH, IN, or IH) and subjected to sham injury and 13% O2 or FPI and either 13 or 21% O2. Immediately after 30 min of hypoxia or normoxia, rats were confined to metabolism cages that were used to quantify rates of oxygen consumption (VO2), carbon dioxide production (VCO2), and heat production (H). Post-traumatic hypoxia exacerbated the FPI-induced increases in VO2, VCO2, and H. The results of Experiments I and II provide convergent confirmation that secondary hypoxemia exacerbates the FPI-induced hypermetabolic state in rats and therefore might significantly exacerbate the brain injury-induced disruptions of energy metabolism in humans.

EEP is a tripeptide structurally similar to thyrotropin releasing hormone (TRH) and, like TRH, it is found in the mammalian brain. TRH has been found to increase in brain regions after seizures and to be neuroprotective. EEP has also been shown to increase in brain regions following seizure activity. We therefore sought to determine whether the similarities between these two peptides might be extended to include neuroprotection. Both TRH and EEP were found to be neuroprotective in vitro against an excitotoxic insult. Interestingly, the two tripeptides appeared to have different mechanisms of action. Even though EEP was as much as four times more neuroprotective than TRH, its ability to reduce glutamate-stimulated increases in intraneuronal Ca 2+ was about half that of TRH.

Laser-Doppler flowmetry (LDF) was used to record subcortical cerebral blood flow in hippocampus and striatum immediately following parasaggital fluid percussion brain injuries of mild to moderate severity (2.58 +/- 0.09 atm, 10-11 msec duration) in spontaneously breathing anesthetized rats. At 5 min postinjury, mean blood flow decreased bilaterally by 20-30% in both brain structures, and remained significantly reduced during the remainder of the 60 min postinjury recording interval. Blood flow did not change in the sham-injured rats. Subsequent beam-walk, beam-balance, and rope-hang assessments revealed significant neurological impairments in the injured rats but not in the sham controls. The magnitude of the blood flow changes and the severity of the ensuing neurological impairment were significantly correlated. Histopathological assessments revealed hemorrhagic contusions within ipsilateral cortical regions, occasional neuronal necrosis within underlying thalamus and CA3 and CA4 sectors of the hippocampus, and neuronal cell loss in the hilus of the dentate gyrus. In a second series of experiments, radiolabeled microspheres were used to validate the LDF blood flow measurements. The microsphere measurements revealed that the preinjury baseline and postinjury right hippocampal blood flow changes were not significantly altered by the intrahippocampal presence of an LDF probe, verifying that the LDF probe was not by itself an unacceptably disruptive influence on local cerebrovascular reactivity. Moreover, when right hippocampal blood flow was simultaneously evaluated in injured rats by both techniques, the relative blood flow changes were significantly correlated. These results indicate that laser-Doppler flowmetry provides a potentially useful means to appreciate acute regional cerebrovascular changes relative to other measures of outcome after brain trauma.

Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (NPFF), an endogenous mammalian antiopioid peptide, has been shown by other laboratories to attenuate the acute antinociceptive effects of morphine, the development of morphine tolerance, and naloxone-induced withdrawal in morphine-dependent rats. The present study determined the effect of chronic NPFF on mu opioid receptors and mRNA for the endogenous opioids dynorphin and enkephalin. Rats received ICV infusions of either saline or NPFF (5 μg/h) for 13 days via Alzet 2002 osmotic minipumps. Homogenate binding studies, which used whole brain membranes, demonstrated that NPFF decreased the B max of mu binding sites (labeled by [ 3 H][ d -Ala 2 -MePhe 4 ,Gly-ol 5 ]enkephalin) from 262 ± 12 to 192 ± 12 fmolmg protein, and increased the K d from 1.1 to 2.3 n M . Quantitative receptor autoradiography and in situ hybridization experiments were conducted with sections collected at the level of the striatum. The density of mu opioid binding sites labeled by [ 3 H][ d -Ala 2 -MePhe 4 ,Gly-ol 5 ]enkephalin was decreased in all brain areas measured except the corpus callosum, and there was no change in dynorphin mRNA or enkephalin mRNA in the caudate, the nucleus accumbens, or the ventral pallidum. Rats chronically administered ICV morphine sulfate (20 μg/h) for 14 days developed tolerance to morphine and a low degree of dependence, as measured by naloxone-precipitated withdrawal. Chronic administration of NPFF concurrently with morphine sulfate did not significantly alter naloxone-induced withdrawal signs or the development of morphine tolerance. Viewed collectively with previous findings that chronic ICV infusion of anti-NPFF IgG upregulates mu receptors, these data provide additional evidence that the density of CNS mu receptors is tonically regulated by NPFF in the extracellular fluid. The action of NPFF to decrease mu receptors is consistent with an antiopioid role for this peptide; however, the fact that NPFF (administered into the lateral ventricle) did not appreciably alter expression of morphine tolerance and dependence contrasts with previous findings and reinforces the view that this effect is most reliably seen after third ventricle administration.

The interactive effects of mu and kappa opioid receptor agonists on respiratory function were investigated following their i.c.v. injection into conscious rats. The highly selective mu receptor agonist [D-Ala2,N-Methyl-Phe4,Gly-ol] enkephalin (DAMGO; 1.2-10 nmol) and the relatively selective mu agonist morphine (20 and 30 nmol) significantly decreased arterial pH and PO2, and increased arterial PCO2 and blood pressure. Morphine and a low dose of DAMGO (1.2 nmol) also significantly elevated respiratory rate. Heart rate was decreased by DAMGO and, depending upon dose, was either decreased (20 nmol) or increased (30 nmol) by morphine. The selective kappa opioid agonist U-50,488H (200 nmol i.c.v.), which by itself had no significant effect on either respiration or cardiovascular function, dose-dependently antagonized the acidotic, hypoxemic and hypercapnic effects of both DAMGO (2.5 nmol) and morphine (30 nmol). Furthermore, these mu antagonistic properties of U-50,488H were blocked completely after pretreatment with 25 nmol of the highly selective kappa opioid antagonist nor-binaltorphimine. These results indicate that the antagonism of mu opioid respiratory depressant effects by U-50,488H is kappa opioid receptor mediated.

The short-term cardiovascular effects of dynorphin A (1-13), as well as its effects upon morphine bradycardia were investigated. In unanesthetized, unrestrained rats, intracerebroventricular (ICV) dynorphin A (1-13) injections (10-20 micrograms) produced a dose-related pressor effect, whereas intravenous (IV) dynorphin A (1-13) (1.0 mg/kg) produced a depressor effect; these responses persisted less than five min. Heart rate was not significantly altered by these doses or routes of administration. Dynorphin A (1-13) also produced behavioral effects in the unanesthetized animals, such as wet dog shakes in response to IV administration and wet dog shakes accompanied by barrel rolling in response to ICV administration. To evaluate the effects of dynorphin A (1-13) pretreatment on the bradycardic response to IV morphine, rats were pretreated with 10 micrograms dynorphin A (1-13) ICV four, six or eight hours prior to challenge with morphine sulfate (0.1 mg/kg IV). Four hour pretreatment with dynorphin A (1-13) (tested at 14:00 hr) resulted in a potention of morphine bradycardia, with six hours pretreatment (tested at 16:00 hr) no effect was observed, and eight hours following dynorphin A (1-13) pretreatment (tested at 18:00 hr) morphine bradycardia was attenuated. Additionally, the bradycardic response to IV morphine alone became more exaggerated as rats approached their nocturnal activity cycle. These data further establish that dynorphin A (1-13) exerts a potent, long lasting modulatory effect on morphine bradycardia and emphasize the importance of circadian variables in altering the magnitude of cardiovascular responses to opioid agonists.

The biochemical and pharmacological properties of an endogenous anticonvulsant substance(s) found in rat cerebrospinal fluid (CSF) following seizures are described. CSF taken from donor rats following a single maximal electroshock (MES) seizure caused significant elevations in seizure thresholds in naive recipient rats when intracerebroventricularly injected 15 min prior to exposure to the volatile convulsant flurothyl. Anticonvulsant activity was antagonized by pre-injection in recipients of high doses of naloxone or the selective delta-opioid receptor antagonist ICI 174,864. The anticonvulsant activity was also lost when the CSF was exposed to heat (90 degrees C) or immobilized trypsin. Although unaffected by the peptidase inhibitors thiorphan and bestatin, the anticonvulsant activity was significantly potentiated by a combination of aprotinin and bacitracin. Ultrafiltration of CSF revealed that the anticonvulsant activity passed through membranes with a 10,000 molecular weight cut-off, but was retained by membranes with a 5000 molecular weight cut-off. CSF removed from rats following MES had significantly increased concentrations of beta-endorphin-like, but not dynorphin A, Leu- or Met-enkephalin-like immunoreactivities relative to CSF from sham-treated rats. However, significant increases in Met-enkephalin-like immunoreactivity were measured following exposure of the CSF to the proteolytic enzymes trypsin and carboxypeptidase B, suggesting the seizure-induced presence of a higher molecular weight form of Met-enkephalin not recognized immunologically prior to enzyme exposure. These data reconfirm the anticonvulsant actions of postseizure CSF, and indicate that these effects require mediation through delta-opioid receptors in the recipient rat. These data additionally argue against these effects being mediated by Met-enkephalin, Leu-enkephalin or dynorphin A in the CSF, and suggest instead that anticonvulsant effects are attributable to a heat- and trypsin-sensitive opioid peptide(s) with a molecular weight approximately in the range of 5000-10,000 Da.

Dynorphin A-(1-17) acts through non-opioid mechanisms to produce dose-related neurological deficits following injection into the lumbar spinal subarachnoid space in rats. Hindlimb motor function was examined following subarachnoid injection of dynorphin A fragments and other opioid peptides derived from prodynorphin to establish: (1) which portion(s) of the dynorphin A molecule cause hindlimb motor dysfunction, and (2) whether these paralytic actions are shared by other opioids (dynorphin B, alpha-neo-endorphin, and beta-neo-endorphin) derived from the same promolecule. To minimize the influence of enzymatic inactivation on relative bioactivities, peptides were coinjected with a combination of peptidase inhibitors previously shown to enhance the actions of dynorphin A fragments in vitro. Dynorphin A-(1-17) and -(2-17) produced dose-related neurological deficits with equal potencies and durations. Although without effect when injected alone, dynorphin A-(1-8), -(1-7) and -(3-8) caused transient motor dysfunction when co-injected with peptidase inhibitors. In contrast, dynorphin A-(1-6), -(1-5) and -(6-17) did not disrupt hindlimb motor function with or without peptidase inhibition. Dynorphin B, alpha-neo-endorphin and beta-neo-endorphin also caused hindlimb dysfunction which was potentiated by peptidase inhibition. These deficits appeared to result from non-opioid actions of these three peptides, since they were not blocked by the opioid antagonist naloxone. Thus, the paralytic effects of dynorphin A: (1) result from non-opioid actions involving the 3-7 or 3-8 positions of the molecule, and (2) are shared by other prodynorphin-derived opioid peptides.