Temporal Alterations in Cerebrovascular Glycocalyx and Cerebral Blood Flow after Exposure to a High-Intensity Blast in Rats.

The glycocalyx is a proteoglycan-glycoprotein structure lining the luminal surface of the vascular endothelium and is susceptible to damage due to blast overpressure (BOP) exposure. The glycocalyx is essential in maintaining the structural and functional integrity of the vasculature and regulation of cerebral blood flow (CBF). Assessment of alterations in the density of the glycocalyx; its components (heparan sulphate proteoglycan (HSPG/syndecan-2), heparan sulphate (HS), and chondroitin sulphate (CS)); CBF; and the effect of hypercapnia on CBF was conducted at 2-3 h, 1, 3, 14, and 28 days after a high-intensity (18.9 PSI/131 kPa peak pressure, 10.95 ms duration, and 70.26 PSI·ms/484.42 kPa·ms impulse) BOP exposure in rats. A significant reduction in the density of the glycocalyx was observed 2-3 h, 1-, and 3 days after the blast exposure. The glycocalyx recovered by 28 days after exposure and was associated with an increase in HS (14 and 28 days) and in HSPG/syndecan-2 and CS (28 days) in the frontal cortex. In separate experiments, we observed significant decreases in CBF and a diminished response to hypercapnia at all time points with some recovery at 3 days. Given the role of the glycocalyx in regulating physiological function of the cerebral vasculature, damage to the glycocalyx after BOP exposure may result in the onset of pathogenesis and progression of cerebrovascular dysfunction leading to neuropathology.

Exposure to Low-Intensity Blast Increases Clearance of Brain Amyloid Beta.

The long-term effects of exposure to blast overpressure are an important health concern in military personnel. Increase in amyloid beta (Aß) has been documented after non-blast traumatic brain injury (TBI) and may contribute to neuropathology and an increased risk for Alzheimer's disease. We have shown that Aß levels decrease following exposure to a low-intensity blast overpressure event. To further explore this observation, we examined the effects of a single 37 kPa (5.4 psi) blast exposure on brain Aß levels, production, and clearance mechanisms in the acute (24 h) and delayed (28 days) phases post-blast exposure in an experimental rat model. Aß and, notably, the highly neurotoxic detergent soluble Aß42 form, was reduced at 24 h but not 28 days after blast exposure. This reduction was not associated with changes in the levels of Aß oligomers, expression levels of amyloid precursor protein (APP), or increase in enzymes involved in the amyloidogenic cleavage of APP, the ß- and ϒ-secretases BACE1 and presenilin-1, respectively. The levels of ADAM17 α-secretase (also known as tumor necrosis factor α-converting enzyme) decreased, concomitant with the reduction in brain Aß. Additionally, significant increases in brain levels of the endothelial transporter, low-density related protein 1 (LRP1), and enhancement in co-localization of aquaporin-4 (AQP4) to perivascular astrocytic end-feet were observed 24 h after blast exposure. These findings suggest that exposure to low-intensity blast may enhance endothelial clearance of Aß by LRP1-mediated transcytosis and alter AQP4-aided glymphatic clearance. Collectively, the data demonstrate that low-intensity blast alters enzymatic, transvascular, and perivascular clearance of Aß.

The Neurovascular Unit as a Locus of Injury in Low-Level Blast-Induced Neurotrauma.

Blast-induced neurotrauma has received much attention over the past decade. Vascular injury occurs early following blast exposure. Indeed, in animal models that approximate human mild traumatic brain injury or subclinical blast exposure, vascular pathology can occur in the presence of a normal neuropil, suggesting that the vasculature is particularly vulnerable. Brain endothelial cells and their supporting glial and neuronal elements constitute a neurovascular unit (NVU). Blast injury disrupts gliovascular and neurovascular connections in addition to damaging endothelial cells, basal laminae, smooth muscle cells, and pericytes as well as causing extracellular matrix reorganization. Perivascular pathology becomes associated with phospho-tau accumulation and chronic perivascular inflammation. Disruption of the NVU should impact activity-dependent regulation of cerebral blood flow, blood-brain barrier permeability, and glymphatic flow. Here, we review work in an animal model of low-level blast injury that we have been studying for over a decade. We review work supporting the NVU as a locus of low-level blast injury. We integrate our findings with those from other laboratories studying similar models that collectively suggest that damage to astrocytes and other perivascular cells as well as chronic immune activation play a role in the persistent neurobehavioral changes that follow blast injury.

Metabotropic Glutamate Receptor 2 Expression Is Chronically Elevated in Male Rats With Post-Traumatic Stress Disorder Related Behavioral Traits Following Repetitive Low-Level Blast Exposure.

Many military veterans who experienced blast-related traumatic brain injuries in the conflicts in Iraq and Afghanistan currently suffer from chronic cognitive and mental health problems that include depression and post-traumatic stress disorder (PTSD). Male rats exposed to repetitive low-level blast develop cognitive and PTSD-related behavioral traits that are present for more than 1 year after exposure. We previously reported that a group II metabotropic receptor (mGluR2/3) antagonist reversed blast-induced behavioral traits. In this report, we explored mGluR2/3 expression following blast exposure in male rats. Western blotting revealed that mGluR2 protein (but not mGluR3) was increased in all brain regions studied (anterior cortex, hippocampus, and amygdala) at 43 or 52 weeks after blast exposure but not at 2 weeks or 6 weeks. mGluR2 RNA was elevated at 52 weeks while mGluR3 was not. Immunohistochemical staining revealed no changes in the principally presynaptic localization of mGluR2 by blast exposure. Administering the mGluR2/3 antagonist LY341495 after behavioral traits had emerged rapidly reversed blast-induced effects on novel object recognition and cued fear responses 10 months following blast exposure. These studies support alterations in mGluR2 receptors as a key pathophysiological event following blast exposure and provide further support for group II metabotropic receptors as therapeutic targets in the neurobehavioral effects that follow blast injury.

Differential effects on TDP-43, piezo-2, tight-junction proteins in various brain regions following repetitive low-intensity blast overpressure.

Introduction: Mild traumatic brain injury (mTBI) caused by repetitive low-intensity blast overpressure (relBOP) in military personnel exposed to breaching and heavy weapons is often unrecognized and is understudied. Exposure to relBOP poses the risk of developing abnormal behavioral and psychological changes such as altered cognitive function, anxiety, and depression, all of which can severely compromise the quality of the life of the affected individual. Due to the structural and anatomical heterogeneity of the brain, understanding the potentially varied effects of relBOP in different regions of the brain could lend insights into the risks from exposures. Methods: In this study, using a rodent model of relBOP and western blotting for protein expression we showed the differential expression of various neuropathological proteins like TDP-43, tight junction proteins (claudin-5, occludin, and glial fibrillary acidic protein (GFAP)) and a mechanosensitive protein (piezo-2) in different regions of the brain at different intensities and frequency of blast. Results: Our key results include (i) significant increase in claudin-5 after 1x blast of 6.5 psi in all three regions and no definitive pattern with higher number of blasts, (ii) significant increase in piezo-2 at 1x followed by significant decrease after multiple blasts in the cortex, (iii) significant increase in piezo-2 with increasing number of blasts in frontal cortex and mixed pattern of expression in hippocampus and (iv) mixed pattern of TDP-3 and GFAP expression in all the regions of brain. Discussion: These results suggest that there are not definitive patterns of changes in these marker proteins with increase in intensity and/or frequency of blast exposure in any particular region; the changes in expression of these proteins are different among the regions. We also found that the orientation of blast exposure (e.g. front vs. side exposure) affects the altered expression of these proteins.

Late chronic local inflammation, synaptic alterations, vascular remodeling and arteriovenous malformations in the brains of male rats exposed to repetitive low-level blast overpressures.

In the course of military operations in modern war theaters, blast exposures are associated with the development of a variety of mental health disorders associated with a post-traumatic stress disorder-related features, including anxiety, impulsivity, insomnia, suicidality, depression, and cognitive decline. Several lines of evidence indicate that acute and chronic cerebral vascular alterations are involved in the development of these blast-induced neuropsychiatric changes. In the present study, we investigated late occurring neuropathological events associated with cerebrovascular alterations in a rat model of repetitive low-level blast-exposures (3 × 74.5 kPa). The observed events included hippocampal hypoperfusion associated with late-onset inflammation, vascular extracellular matrix degeneration, synaptic structural changes and neuronal loss. We also demonstrate that arteriovenous malformations in exposed animals are a direct consequence of blast-induced tissue tears. Overall, our results further identify the cerebral vasculature as a main target for blast-induced damage and support the urgent need to develop early therapeutic approaches for the prevention of blast-induced late-onset neurovascular degenerative processes.

(2R,6R)-Hydroxynorketamine Treatment of Rats Exposed to Repetitive Low-Level Blast Injury.

Many military veterans who experienced blast-related traumatic brain injuries (TBIs) in the conflicts in Iraq and Afghanistan suffer from chronic cognitive and mental health problems, including post-traumatic stress disorder (PTSD). Male rats subjected to repetitive low-level blast exposure develop chronic cognitive and PTSD-related traits that develop in a delayed manner. Ketamine has received attention as a treatment for refractory depression and PTSD. (2R,6R)-hydroxynorketamine [(2R,6R)-HNK] is a ketamine metabolite that exerts rapid antidepressant actions. (2R,6R)-HNK has become of clinical interest because of its favorable side-effect profile, low abuse potential, and oral route of administration. We treated three cohorts of blast-exposed rats with (2R,6R)-HNK, beginning 7-11 months after blast exposure, a time when the behavioral phenotype is established. Each cohort consisted of groups (n = 10-13/group) as follows: 1) Sham-exposed treated with saline, 2) blast-exposed treated with saline, and 3) blast-exposed treated with a single dose of 20 mg/kg of (2R,6R)-HNK. (2R,6R)-HNK rescued blast-induced deficits in novel object recognition (NOR) and anxiety-related features in the elevated zero maze (EZM) in all three cohorts. Exaggerated acoustic startle was reversed in cohort 1, but not in cohort 3. (2R,6R)-HNK effects were still present in the EZM 12 days after administration in cohort 1 and 27 days after administration in NOR testing of cohorts 2 and 3. (2R,6R)-HNK may be beneficial for the neurobehavioral syndromes that follow blast exposure in military veterans. Additional studies will be needed to determine whether higher doses or more extended treatment regimens may be more effective.

Progressive Transcriptional Changes in the Amygdala Implicate Neuroinflammation in the Effects of Repetitive Low-Level Blast Exposure in Male Rats.

Chronic mental health problems are common among military veterans who sustained blast-related traumatic brain injuries. The reasons for this association remain unexplained. Male rats exposed to repetitive low-level blast overpressure (BOP) exposures exhibit chronic cognitive and post-traumatic stress disorder (PTSD)-related traits that develop in a delayed fashion. We examined blast-induced alterations on the transcriptome in four brain areas (anterior cortex, hippocampus, amygdala, and cerebellum) across the time frame over which the PTSD-related behavioral phenotype develops. When analyzed at 6 weeks or 12 months after blast exposure, relatively few differentially expressed genes (DEGs) were found. However, longitudinal analysis of amygdala, hippocampus, and anterior cortex between 6 weeks and 12 months revealed blast-specific DEG patterns. Six DEGs (hyaluronan and proteoglycan link protein 1 [Hapln1], glutamate metabotropic receptor 2 [Grm2], purinergic receptor P2y12 [P2ry12], C-C chemokine receptor type 5 [Ccr5], phenazine biosynthesis-like protein domain containing 1 [Pbld1], and cadherin related 23 [Cdh23]) were found altered in all three brain regions in blast-exposed animals. Pathway enrichment analysis using all DEGs or those uniquely changed revealed different transcription patterns in blast versus sham. In particular, the amygdala in blast-exposed animals had a unique set of enriched pathways related to stress responses, oxidative phosphorylation, and mitochondrial dysfunction. Upstream analysis implicated tumor necrosis factor (TNF)α signaling in blast-related effects in amygdala and anterior cortex. Eukaryotic initiating factor eIF4E (EIF4e), an upstream regulator of P2ry12 and Ccr5, was predicted to be activated in the amygdala. Quantitative polymerase chain reaction (qPCR) validated longitudinal changes in two TNFα regulated genes (cathepsin B [Ctsb], Hapln1), P2ry12, and Grm2. These studies have implications for understanding how blast injury damages the brain and implicates inflammation as a potential therapeutic target.

Transcranial Laser Therapy Does Not Improve Cognitive and Post-Traumatic Stress Disorder-Related Behavioral Traits in Rats Exposed to Repetitive Low-Level Blast Injury.

Many military veterans who experienced blast-related traumatic brain injuries (TBIs) in the conflicts in Iraq and Afghanistan suffer from chronic cognitive and mental health problems, including post-traumatic stress disorder (PTSD). Transcranial laser therapy (TLT) uses low-power lasers emitting light in the far- to near-infrared ranges. Beneficial effects of TLT have been reported in neurological and mental-health-related disorders in humans and animal models, including TBI. Rats exposed to repetitive low-level blast develop chronic cognitive and PTSD-related behavioral traits. We tested whether TLT treatment could reverse these traits. Rats received a 74.5-kPa blast or sham exposures delivered one per day for 3 consecutive days. Beginning at 34 weeks after blast exposure, the following groups of rats were treated with active or sham TLT: 1) Sham-exposed rats (n = 12) were treated with sham TLT; 2) blast-exposed rats (n = 13) were treated with sham TLT; and 3) blast-exposed rats (n = 14) were treated with active TLT. Rats received 5 min of TLT five times per week for 6 weeks (wavelength, 808 nm; power of irradiance, 240 mW). At the end of treatment, rats were tested in tasks found previously to be most informative (novel object recognition, novel object localization, contextual/cued fear conditioning, elevated zero maze, and light/dark emergence). TLT did not improve blast-related effects in any of these tests, and blast-exposed rats were worse after TLT in some anxiety-related measures. Based on these findings, TLT does not appear to be a promising treatment for the chronic cognitive and mental health problems that follow blast injury.

Low-level blast exposure induces chronic vascular remodeling, perivascular astrocytic degeneration and vascular-associated neuroinflammation.

Cerebral vascular injury as a consequence of blast-induced traumatic brain injury is primarily the result of blast wave-induced mechanical disruptions within the neurovascular unit. In rodent models of blast-induced traumatic brain injury, chronic vascular degenerative processes are associated with the development of an age-dependent post-traumatic stress disorder-like phenotype. To investigate the evolution of blast-induced chronic vascular degenerative changes, Long-Evans rats were blast-exposed (3 × 74.5 kPa) and their brains analyzed at different times post-exposure by X-ray microcomputed tomography, immunohistochemistry and electron microscopy. On microcomputed tomography scans, regional cerebral vascular attenuation or occlusion was observed as early as 48 h post-blast, and cerebral vascular disorganization was visible at 6 weeks and more accentuated at 13 months post-blast. Progression of the late-onset pathology was characterized by detachment of the endothelial and smooth muscle cellular elements from the neuropil due to degeneration and loss of arteriolar perivascular astrocytes. Development of this pathology was associated with vascular remodeling and neuroinflammation as increased levels of matrix metalloproteinases (MMP-2 and MMP-9), collagen type IV loss, and microglial activation were observed in the affected vasculature. Blast-induced chronic alterations within the neurovascular unit should affect cerebral blood circulation, glymphatic flow and intramural periarterial drainage, all of which may contribute to development of the blast-induced behavioral phenotype. Our results also identify astrocytic degeneration as a potential target for the development of therapies to treat blast-induced brain injury.

Repetitive Low-Level Blast Exposure Improves Behavioral Deficits and Chronically Lowers Aß42 in an Alzheimer Disease Transgenic Mouse Model.

Public awareness of traumatic brain injury (TBI) in the military increased recently because of the conflicts in Iraq and Afghanistan where blast injury was the most common mechanism of injury. Besides overt injuries, concerns also exist over the potential adverse consequences of subclinical blast exposures, which are common for many service members. A TBI is a risk factor for the later development of neurodegenerative diseases, including Alzheimer disease (AD)-like disorders. Studies of acute TBI in humans and animals have suggested that increased processing of the amyloid precursor protein (APP) toward the amyloid beta protein (Aß) may explain the epidemiological associations with AD. In a previous study, however, we found in both rat and mouse models of blast overpressure exposure that rather than increasing, rodent brain Aß42 levels were decreased after acute blast exposure. Here we subjected APP/presenilin 1 transgenic mice (APP/PS1 Tg) to an extended sequence of repetitive low-level blast exposures (34.5 kPa) administered three times per week over eight weeks. If initiated at 20 weeks of age, these repetitive exposures, which were designed to mimic human subclinical blast exposures, reduced anxiety and improved cognition as well as social interactions in APP/PS1 Tg mice, returning many behavioral parameters in APP/PS1 Tg mice to levels of non-transgenic wild type mice. Repetitive low-level blast exposure was less effective at improving behavioral deficits in APP/PS1 Tg mice when begun at 36 weeks of age. While amyloid plaque loads were unchanged, Aß 42 levels and Aß oligomers were reduced in the brain of mice exposed to repetitive low-level blast exposures initiated at 20 weeks of age, although levels did not directly correlate with behavioral parameters in individual animals. These results have implications for understanding the nature of blast effects on the brain and their relationship to human neurodegenerative diseases.

Blast-induced temporal alterations in blood-brain barrier properties in a rodent model.

The consequences of blast-induced traumatic brain injury (bTBI) on the blood-brain barrier (BBB) and components of the neurovascular unit are an area of active research. In this study we assessed the time course of BBB integrity in anesthetized rats exposed to a single blast overpressure of 130 kPa (18.9 PSI). BBB permeability was measured in vivo via intravital microscopy by imaging extravasation of fluorescently labeled tracers (40 kDa and 70 kDa molecular weight) through the pial microvasculature into brain parenchyma at 2-3 h, 1, 3, 14, or 28 days after the blast exposure. BBB structural changes were assessed by immunostaining and molecular assays. At 2-3 h and 1 day after blast exposure, significant increases in the extravasation of the 40 kDa but not the 70 kDa tracers were observed, along with differential reductions in the expression of tight junction proteins (occludin, claudin-5, zona occluden-1) and increase in the levels of the astrocytic water channel protein, AQP-4, and matrix metalloprotease, MMP-9. Nearly all of these measures were normalized by day 3 and maintained up to 28 days post exposure. These data demonstrate that blast-induced changes in BBB permeability are closely coupled to structural and functional components of the BBB.

Laterality and region-specific tau phosphorylation correlate with PTSD-related behavioral traits in rats exposed to repetitive low-level blast.

Military veterans who experience blast-related traumatic brain injuries often suffer from chronic cognitive and neurobehavioral syndromes. Reports of abnormal tau processing following blast injury have raised concerns that some cases may have a neurodegenerative basis. Rats exposed to repetitive low-level blast exhibit chronic neurobehavioral traits and accumulate tau phosphorylated at threonine 181 (Thr181). Using data previously reported in separate studies we tested the hypothesis that region-specific patterns of Thr181 phosphorylation correlate with behavioral measures also previously determined and reported in the same animals. Elevated p-tau Thr181 in anterior neocortical regions and right hippocampus correlated with anxiety as well as fear learning and novel object localization. There were no correlations with levels in amygdala or posterior neocortical regions. Particularly striking were asymmetrical effects on the right and left hippocampus. No systematic variation in head orientation toward the blast wave seems to explain the laterality. Levels did not correlate with behavioral measures of hyperarousal. Results were specific to Thr181 in that no correlations were observed for three other phospho-acceptor sites (threonine 231, serine 396, and serine 404). No consistent correlations were linked with total tau. These correlations are significant in suggesting that p-tau accumulation in anterior neocortical regions and the hippocampus may lead to disinhibited amygdala function without p-tau elevation in the amygdala itself. They also suggest an association linking blast injury with tauopathy, which has implications for understanding the relationship of chronic blast-related neurobehavioral syndromes in humans to neurodegenerative diseases.

Repeated Low-Level Blast Acutely Alters Brain Cytokines, Neurovascular Proteins, Mechanotransduction, and Neurodegenerative Markers in a Rat Model.

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 (Aß40/42, 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-ß (Aß)40 and Aß42, 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.

Progressive Cognitive and Post-Traumatic Stress Disorder-Related Behavioral Traits in Rats Exposed to Repetitive Low-Level Blast.

Many military veterans who experienced blast-related traumatic brain injuries (TBI) in the conflicts in Iraq and Afghanistan currently have chronic cognitive and mental health problems including post-traumatic stress disorder (PTSD). Besides static symptoms, new symptoms may emerge or existing symptoms may worsen. TBI is also a risk factor for later development of neurodegenerative diseases. In rats exposed to repetitive low-level blast overpressure (BOP), robust and enduring cognitive and PTSD-related behavioral traits develop that are present for at least one year after blast exposure. Here we determined the time-course of the appearance of these traits by testing rats in the immediate post-blast period. Three cohorts of rats examined within the first eight weeks exhibited no behavioral phenotype or, in one cohort, features of anxiety. None showed the altered cued fear responses or impaired novel object recognition characteristic of the fully developed phenotype. Two cohorts retested 36 to 42 weeks after blast exposure exhibited the expanded behavioral phenotype including anxiety as well as altered cued fear learning and impaired novel object recognition. Combined with previous work, the chronic behavioral phenotype has been observed in six cohorts of blast-exposed rats studied at 3-4 months or longer after blast injury, and the three cohorts studied here document the progressive nature of the cognitive/behavioral phenotype. These studies suggest the existence of a latent, delayed emerging and progressive blast-induced cognitive and behavioral phenotype. The delayed onset has implications for the evolution of post-blast neurobehavioral syndromes in military veterans and its modeling in experimental animals.

Repeated Low-Level Blast Overpressure Leads to Endovascular Disruption and Alterations in TDP-43 and Piezo2 in a Rat Model of Blast TBI.

Recent evidence linking repeated low-level blast overpressure exposure in operational and training environments with neurocognitive decline, neuroinflammation, and neurodegenerative processes has prompted concern over the cumulative deleterious effects of repeated blast exposure on the brains of service members. Repetitive exposure to low-level primary blast may cause symptoms (subclinical) similar to those seen in mild traumatic brain injury (TBI), with progressive vascular and cellular changes, which could contribute to neurodegeneration. At the cellular level, the mechanical force associated with blast exposure can cause cellular perturbations in the brain, leading to secondary injury. To examine the cumulative effects of repetitive blast on the brain, an advanced blast simulator (ABS) was used to closely mimic "free-field" blast. Rats were exposed to 1-4 daily blasts (one blast per day, separated by 24 h) at 13, 16, or 19 psi peak incident pressures with a positive duration of 4-5 ms, either in a transverse or longitudinal orientation. Blood-brain barrier (BBB) markers (vascular endothelial growth factor (VEGF), occludin, and claudin-5), transactive response DNA binding protein (TDP-43), and the mechanosensitive channel Piezo2 were measured following blast exposure. Changes in expression of VEGF, occludin, and claudin-5 after repeated blast exposure indicate alterations in the BBB, which has been shown to be disrupted following TBI. TDP-43 is very tightly regulated in the brain and altered expression of TDP-43 is found in clinically-diagnosed TBI patients. TDP-43 levels were differentially affected by the number and magnitude of blast exposures, decreasing after 2 exposures, but increasing following a greater number of exposures at various intensities. Lastly, Piezo2 has been shown to be dysregulated following blast exposure and was here observed to increase after multiple blasts of moderate magnitude, indicating that blast may cause a change in sensitivity to mechanical stimuli in the brain and may contribute to cellular injury. These findings reveal that cumulative effects of repeated exposures to blast can lead to pathophysiological changes in the brain, demonstrating a possible link between blast injury and neurodegenerative disease, which is an important first step in understanding how to prevent these diseases in soldiers exposed to blast.

N-acetylcysteine Amide Ameliorates Blast-Induced Changes in Blood-Brain Barrier Integrity in Rats.

Traumatic brain injury resulting from exposure to blast overpressure (BOP) is associated with neuropathology including impairment of the blood-brain barrier (BBB). This study examined the effects of repeated exposure to primary BOP and post-blast treatment with an antioxidant, N-acetylcysteine amide (NACA) on the integrity of BBB. Anesthetized rats were exposed to three 110 kPa BOPs separated by 0.5 h. BBB integrity was examined in vivo via a cranial window allowing imaging of pial microcirculation by intravital microscopy. Tetramethylrhodamine isothiocyanate Dextran (TRITC-Dextran, mw = 40 kDa or 150 kDa) was injected intravenously 2.5 h after the first BOP exposure and the leakage of TRITC-Dextran from pial microvessels into the brain parenchyma was assessed. The animals were randomized into 6 groups (n = 5/group): four groups received 40 kDa TRITC-Dextran (BOP-40, sham-40, BOP-40 NACA, and sham-40 NACA), and two groups received 150 kDa TRITC-Dextran (BOP-150 and sham-150). NACA treated groups were administered NACA 2 h after the first BOP exposure. The rate of TRITC-Dextran leakage was significantly higher in BOP-40 than in sham-40 group. NACA treatment significantly reduced TRITC-Dextran leakage in BOP-40 NACA group and sham-40 NACA group presented the least amount of leakage. The rate of leakage in BOP-150 and sham-150 groups was comparable to sham-40 NACA and thus these groups were not assessed for the effects of NACA. Collectively, these data suggest that BBB integrity is compromised following BOP exposure and that NACA treatment at a single dose may significantly protect against blast-induced BBB breakdown.

Exposure to Blast Overpressure Impairs Cerebral Microvascular Responses and Alters Vascular and Astrocytic Structure.

Exposure to blast overpressure may result in cerebrovascular impairment, including cerebral vasospasm. The mechanisms contributing to this vascular response are unclear. The aim of this study was to evaluate the relationship between blast and functional alterations of the cerebral microcirculation and to investigate potential underlying changes in vascular microstructure. Cerebrovascular responses were assessed in sham- and blast-exposed male rats at multiple time points from 2 h through 28 days after a single 130-kPa (18.9-psi) exposure. Pial microcirculation was assessed through a cranial window created in the parietal bone of anesthetized rats. Pial arteriolar reactivity was evaluated in vivo using hypercapnia, barium chloride, and serotonin. We found that exposure to blast leads to impairment of arteriolar reactivity >24 h after blast exposure, suggesting delayed injury mechanisms that are not simply attributed to direct mechanical deformation. Observed vascular impairment included a reduction in hypercapnia-induced vasodilation, increase in barium-induced constriction, and reversal of the serotonin effect from constriction to dilation. A reduction in vascular smooth muscle contractile proteins consistent with vascular wall proliferation was observed, as well as delayed reduction in nitric oxide synthase and increase in endothelin-1 B receptors, mainly in astrocytes. Collectively, the data show that exposure to blast results in delayed and prolonged alterations in cerebrovascular reactivity that are associated with changes in the microarchitecture of the vessel wall and astrocytes. These changes may contribute to long-term pathologies involving dysfunction of the neurovascular unit, including cerebral vasospasm.

Brain oxygenation with a non-vasoactive perfluorocarbon emulsion in a rat model of traumatic brain injury.

OBJECTIVE: The aim of this study was to assess, in two experiments, the safety and efficacy of the PFC emulsion Oxycyte as an oxygen therapeutic for TBI to test the hypothesis that early administration of this oxygen-carrying fluid post-TBI would improve brain tissue oxygenation (Pbt O2 ). METHODS: The first experiment assessed the effects of Oxycyte on cerebral vasoactivity in healthy, uninjured rats using intravital microscopy. The second experiment investigated the effect of Oxycyte on cerebral Pbt O2 using the PQM in TBI model. Animals in the Oxycyte group received a single injection of Oxycyte (6 mL/kg) shortly after TBI, while NON animals received no treatment. RESULTS: Oxycyte did not cause vasoconstriction in small- (<50 µm) or medium- (50-100 µm) sized pial arterioles nor did it cause a significant change in blood pressure. Treatment with Oxycyte while breathing 100% O2 did not improve Pbt O2 . However, in rats ventilated with ~40% O2 , Pbt O2 improved to near pre-TBI values within 105 minutes after Oxycyte injection. CONCLUSIONS: Although Oxycyte did not cause cerebral vasoconstriction, its use at the dose tested while breathing 100% O2 did not improve Pbt O2 following TBI. However, Oxycyte treatment while breathing a lower enriched oxygen concentration may improve Pbt O2 after TBI.

Cerebral Vasoactivity and Oxygenation with Oxygen Carrier M101 in Rats.

The severity of traumatic brain injury (TBI) may be reduced if oxygen can be rapidly provided to the injured brain. This study evaluated if the oxygen-carrier M101 causes vasoconstricton of pial vasculature in healthy rats (Experiment 1) and if M101 improves brain tissue oxygen (PbtO2) in rats with controlled cortical impact (CCI)-TBI (Experiment 2). M101 (12.5 mL/kg intravenous [IV] over 2 h) caused a mild (9 mm Hg) increase in the mean arterial blood pressure (MAP) of healthy rats without constriction of cerebral pial arterioles. M101 (12 mL/kg IV over 1 h) caused a modest (27 mm Hg) increase in MAP (peak, 123 ± 5 mm Hg [mean ± standard error of the mean]) of CCI-TBI rats and restored PbtO2 to near pre-injury levels. In both M101 and untreated control (NON) groups, PbtO2 was ∼30 ± 2 mm Hg pre-injury and decreased (p ≤ 0.05) to ∼16 ± 2 mm Hg 15 min after CCI. In NON, PbtO2 remained ∼50% of baseline but M101 administration resulted in a sustained increase in PbtO2 (peak, 25 ± 5 mm Hg), which was not significantly different from pre-injury until the end of the study, when it decreased again below pre-injury (but was still higher than NON). Histopathology showed no differences between groups. In conclusion, M101 increased systemic blood pressures without concurrent cerebral pial vasoconstriction (in healthy rats) and restored PbtO2 to 86% of pre-injury for at least 80 min when given soon after CCI-TBI. M101 should be evaluated in a clinically-relevant large animal model for pre-hospital treatment of TBI.