Venkata Siva Sai Sujith Sajja

Blast induces oxidative stress, inflammation, neuronal loss and subsequent short-term memory impairment in rats.

Molecular and cellular mechanisms of brain injury after exposure to blast overpressure (BOP) are not clearly known. The present study hypothesizes that pro-oxidative and pro-inflammatory pathways in the brain may be responsible for neuronal loss and behavioral deficits following BOP exposure. Male Sprague-Dawley rats were anesthetized and exposed to calibrated BOP of 129.23±3.01kPa while controls received only anesthesia. In situ dihydroethidium fluorescence staining revealed that BOP significantly increased the production of reactive oxygen species in the brain. In addition, real-time reverse transcriptase-polymerase chain reaction, immunofluorescence staining and enzyme-linked immunosorbent assay demonstrated a significant up-regulation of mRNA and protein expressions of pro-inflammatory mediators, such as interferon-γ and monocyte chemoattractant protein-1, in brains collected from BOP-exposed animals compared with the controls. Furthermore, immunoreactivity of neuronal nuclei in brains indicated that fewer neurons were present following BOP exposure. Moreover, novel object recognition paradigm showed a significant impairment in the short-term memory at 2weeks following BOP exposure. These results suggest that pro-oxidative and pro-inflammatory environments in the brain could play a potential role in BOP-induced neuronal loss and behavioral deficits. It may provide a foundation for defining a molecular and cellular basis of the pathophysiology of blast-induced neurotrauma (BINT). It will also contribute to the development of new therapeutic approaches selectively targeting these pathways, which have great potential in the diagnosis and therapy of BINT.

Blast-induced neurotrauma leads to neurochemical changes and neuronal degeneration in the rat hippocampus.

Blast‐induced neurotrauma is a major concern because of the complex expression of neuropsychiatric disorders after exposure. Disruptions in neuronal function, proximal in time to blast exposure, may eventually contribute to the late emergence of clinical deficits. Using magic angle spinning 1H MRS and a rodent model of blast‐induced neurotrauma, we found acute (24–48 h) decreases in succinate, glutathione, glutamate, phosphorylethanolamine and γ‐aminobutyric acid, no change in N‐acetylaspartate and increased glycerophosphorylcholine, alterations consistent with mitochondrial distress, altered neurochemical transmission and increased membrane turnover. Increased levels of the apoptotic markers Bax and caspase‐3 suggested active cell death, consistent with increased FluoroJade B staining in the hippocampus. Elevated levels of glial fibrillary acidic protein suggested ongoing inflammation without diffuse axonal injury measured by no change in β‐amyloid precursor protein. In conclusion, blast‐induced neurotrauma induces a metabolic cascade associated with neuronal loss in the hippocampus in the acute period following exposure. Copyright

Effects of blast-induced neurotrauma on the nucleus accumbens.

Blast‐induced neurotrauma (BINT) leads to deterioration at the cellular level, with adverse cognitive and behavioral outcomes. The nucleus accumbens (NAC) plays an important role in reward, addiction, aggression, and fear pathways. To identify the molecular changes and pathways affected at an acute stage in the NAC, this study focused on a time course analysis to determine the effects of blast on neurochemical and apoptotic pathways. By using a rodent model of BINT, acute damage to the NAC was assessed by proton magnetic resonance spectroscopy (1H‐MRS), high‐performance liquid chromatography, immunohistochemistry, and Western blotting. The results demonstrated ongoing neuroprotective effects from elevated levels of Bcl‐2, an antiapoptotic marker, at 24 hr and N‐acetyl aspartate glutamate at 48 hr following blast exposure. Selective loss of serotonin levels at 24 hr, increased levels of inflammation (elevated glycerophosphocholine at 48 and 72 hr), and increased levels of glial fibrillary acidic protein were also observed at 24 and 48 hr, leading to disruptive energy status. Furthermore, active cell death was indicated by the increased levels of the apoptotic marker Bax, decreased actin levels, and signs excitotoxicity (glutamate/creatine). In addition, increased evels of caspase‐3, an apoptotic marker, confirm active cell death in NAC. It is hypothesized that blast overpressure causes inflammation and neurochemical changes that trigger apoptosis in NAC. This cascade of events may lead to stress‐related behavioral outcomes and psychiatric sequelae.

Hippocampal vulnerability and subacute response following varied blast magnitudes

Clinical outcomes from blast neurotrauma are associated with higher order cognitive functions such as memory, problem solving skills and attention. Current literature is limited to a single overpressure exposure or repeated exposures at the same level of overpressure and is focused on the acute response (<3 days). In an attempt to expand the understanding of neuropathological and molecular changes of the subacute response (7 days post injury), we used an established rodent model of blast neurotrauma. Three pressure magnitudes (low, moderate and high) were used to evaluate molecular injury thresholds. Immunohistochemical analysis demonstrated increased cleaved caspase-3 levels and loss of neuronal population (NeuN+) within the hippocampus of all pressure groups. On the contrary, selective activation of microglia was observed in the low blast group. In addition, increased astrocytes (GFAP), membrane signal transduction protein (Map2k1) and calcium regulator mechanosensitive protein (Piezo 2) were observed in the moderate blast group. Results from gene expression analysis suggested ongoing neuroprotection, as brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF) and Mn and CuZn superoxide dismutases (SOD) all increased in the low and moderate blast groups. Ongoing neuroprotection was further supported by increased SOD levels observed in the moderate group using immunohistochemistry. The gene expression level of glutamate aspartate transporter (GLAST) was upregulated in the low, but downregulated in the high blast group, while no changes were found in the moderate group. Overall, the data shown here provides evidence of a diverse neuroprotective and glial response to various levels of blast exposure. This mechanistic role of neuroprotection is vital in understanding ongoing cellular stress, both at the gene and protein levels, in order to develop interventional studies for the prognosis of injury.

Blast neurotrauma impairs working memory and disrupts prefrontal myo-inositol levels in rats

Working memory, which is dependent on higher-order executive function in the prefrontal cortex, is often disrupted in patients exposed to blast overpressure. In this study, we evaluated working memory and medial prefrontal neurochemical status in a rat model of blast neurotrauma. Adult male Sprague-Dawley rats were anesthetized with 3% isoflurane and exposed to calibrated blast overpressure (17 psi, 117 kPa) while sham animals received only anesthesia. Early neurochemical effects in the prefrontal cortex included a significant decrease in betaine (trimethylglycine) and an increase in GABA at 24 h, and significant increases in glycerophosphorylcholine, phosphorylethanolamine, as well as glutamate/creatine and lactate/creatine ratios at 48 h. Seven days after blast, only myo-inositol levels were altered showing a 15% increase. Compared to controls, short-term memory in the novel object recognition task was significantly impaired in animals exposed to blast overpressure. Working memory in control animals was negatively correlated with myo-inositol levels (r=-.759, p<0.05), an association that was absent in blast exposed animals. Increased myo-inositol may represent tardive glial scarring in the prefrontal cortex, a notion supported by GFAP changes in this region after blast overexposure as well as clinical reports of increased myo-inositol in disorders of memory.

Enduring deficits in memory and neuronal pathology after blast-induced traumatic brain injury.

Few preclinical studies have assessed the long-term neuropathology and behavioral deficits after sustaining blast-induced neurotrauma (BINT). Previous studies have shown extensive astrogliosis and cell death at acute stages (<7 days) but the temporal response at a chronic stage has yet to be ascertained. Here, we used behavioral assays, immmunohistochemistry and neurochemistry in limbic areas such as the amygdala (Amy), Hippocampus (Hipp), nucleus accumbens (Nac), and prefrontal cortex (PFC), to determine the long-term effects of a single blast exposure. Behavioral results identified elevated avoidance behavior and decreased short-term memory at either one or three months after a single blast event. At three months after BINT, markers for neurodegeneration (FJB) and microglia activation (Iba-1) increased while index of mature neurons (NeuN) significantly decreased in all brain regions examined. Gliosis (GFAP) increased in all regions except the Nac but only PFC was positive for apoptosis (caspase-3). At three months, tau was selectively elevated in the PFC and Hipp whereas α-synuclein transiently increased in the Hipp at one month after blast exposure. The composite neurochemical measure, myo-inositol+glycine/creatine, was consistently increased in each brain region three months following blast. Overall, a single blast event resulted in enduring long-term effects on behavior and neuropathological sequelae.

Subacute Oxidative Stress and Glial Reactivity in the Amygdala are Associated with Increased Anxiety Following Blast Neurotrauma.

ABSTRACT Behavioral symptoms, such as anxiety, are widely reported after blast overpressure (BOP) exposure. Amygdalar vulnerability to increasing magnitudes of BOP has not been investigated, and single exposures to blast have been limited to acute (<72 h) assessment. Rats were exposed to a single low, moderate, or high BOP (10, 14, or 24 psi) with an advanced blast simulator to test the susceptibility of the amygdala. Anxiety-like behavior was observed in the low- and moderate-pressure groups when subjected to the light/dark box assessment 7 days after the blast but not in high-pressure group. Immunohistochemistry was performed to measure apoptosis (cleaved caspase-3), neuronal loss (NeuN), reactive astrocytes (glial fibrillary acidic protein), microglia (Iba-1), and oxidative stress (CuZn superoxide dismutase). Slower progression of injury cascades was associated with a significant increase in anxiety, apoptosis, and astrogliosis in the low pressure group compared with others. A significant increase of CuZn superoxide dismutase in the low pressure group could be associated with neuroprotection from cell death caused by oxidative stress because neuronal loss was significant in the moderate- and high- but not the low-pressure group. Overall, this study demonstrated that overpressure as low as 10 psi can induce subacute anxiety, in addition to neuropathologic changes in the amygdala.

Evaluation of Impact-Induced Traumatic Brain Injury in the Göttingen Minipig Using Two Input Modes

Objectives: Two novel injury devices were used to characterize impact-induced traumatic brain injury (TBI). One imparts pure translation, and the other produces combined translation and rotation. The objective of this study was to evaluate the neuropathology associated with two injury devices using proton magnetic resonance spectroscopy (1H-MRS) to quantify metabolic changes and immunohistochemistry (IHC) to evaluate axonal damage in the corpus callosum. Methods: Young adult female Göttingen minipigs were exposed to impact-induced TBI with either the translation-input injury device or the combined-input injury device (n = 11/group). Sham animals were treated identically except for the injury event (n = 3). The minipigs underwent 1H-MRS scans prior to injury (baseline), approximately 1 h after injury, and 24 h post injury, at which point the brains were extracted for IHC. Metabolites of interest include glutamate (Glu), glutamine (Gln), N-acetylaspartate (NAA), N-acetylaspartylglutamate (NAAG), and γ-aminobutyric acid (GABA). Repeated measures analysis of variance with a least significant difference post hoc test were used to compare the three time points. IHC was performed on paraffin-embedded sections of the corpus callosum with light and heavy neurofilament antibodies. Stained pixel percentages were compared between shams and 24-h survival animals. Results: For the translation-input group (27.5–70.1 g), 16 significant metabolite differences were found. Three of these include a significant increase in Gln, both 1 h and 24 h postinjury, and an increase in GABA 24 h after injury. For the combined-input group (40.1–95.9 g; 1,014.5–3,814.9 rad/s2; 7.2–10.8 rad/s), 20 significant metabolite differences were found. Three of these include a significant increase in Glu, an increase in the ratio Glu/Gln, and an increase in the ratio Glu/NAAG 24 h after injury. The IHC analysis revealed significant increases in light and heavy neurofilament for both groups 24 h after injury. Conclusions: Only five metabolite differences were similar between the input modes, most of which are related to inflammation or myelin disruption. The observed metabolite differences indicate important dissimilarities. For the translation-input group, an increase in Gln and GABA suggests a response in the GABA shunt system. For the combined-input group, an increase in Glu, Glu/Gln, and Glu/NAAG suggests glutamate excitotoxicity. Importantly, both of these input modes lead to similar light and heavy neurofilament damage, which indicates axonal disruption. Identifying neuropathological changes that are unique to different injury mechanisms is critical in defining the complexity of TBI and can lead to improved prevention strategies and the development of effective drug therapies.

Chronic Hormonal Imbalance and Adipose Redistribution Is Associated with Hypothalamic Neuropathology following Blast Exposure.

Endocrine disorders have been shown to be a consequence of blast traumatic brain injury in soldiers returning from military conflicts. Hormone deficiency and adrenocorticotropic hormone (ACTH) dysfunction can lead to symptoms such as fatigue, anxiety, irritability, insomnia, sexual dysfunction, and decreased quality of life. Given these changes following blast exposure, the current study focused on investigating chronic pathology within the hypothalamus following blast, in addition to systemic effects. An established rodent model of blast neurotrauma was used to induce mild blast-induced neurotrauma. Adipose tissue, blood, and brain samples were collected at one and three months following a single blast exposure. Adipose tissue and blood were evaluated for changes in ACTH, adiponectin, C-reactive protein, glial fibrillary acidic protein, interleukin (IL)-1β, and leptin. The hypothalamus was evaluated for injury using immunohistochemical techniques. The results demonstrated that the weight of the blast animals was significantly less, compared with the sham group. The slower rate of increase in their weight was associated with changes in ACTH, IL-1β, and leptin levels. Further, histological analysis indicated elevated levels of cleaved caspase-3 positive cells within the hypothalamus. The data suggest that long-term outcomes of brain injury occurring from blast exposure include dysfunction of the hypothalamus, which leads to compromised hormonal function, elevated biological stress-related hormones, and subsequent adipose tissue remodeling.