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Title:	Repeated low-level blast induces chronic neuroinflammation and neurobehavioral changes in rat models
Author:	Ravula, Arun Reddy
View Online:	njit-etd2022-078 (xvii, 125 pages ~ 3.2 MB pdf)
Department:	Federated Biological Sciences Department of NJIT and Rutgers-Newark
Degree:	Doctor of Philosophy
Program:	Biology
Document Type:	Dissertation
Advisory Committee:	Chandra, N. (Committee chair) Nadim, Farzan (Committee member) Pfister, Bryan J. (Committee member) Citron, Bruce A. (Committee member) Tran, Tracy (Committee member)
Date:	2022-05
Keywords:	Behavioral studies Inflammasome Neuroinflammation Oxidative stress Repeated low-level blast Traumatic brain injury
Availability:	Unrestricted
Abstract:	Blast-induced neurotrauma (bTBI) is a signature medical concern for military personnel when they are exposed to explosions in active combat zones. However, soldiers as well as law enforcement personnel are also repeatedly exposed to low-level blasts during training sessions with heavy weaponries as part of combat readiness. Service personnel who sustain brain injuries from repeated low-level blasts (rLLBs) do not display overt pathological symptoms immediately but rather develop cognitive impairments, attention deficits, anxiety, and sleep disturbances over time. An improved rat model of rLLB was developed in this thesis by applying controlled low-level blast pressures (10 psi) repeated five times to model the true mechanism of injuries sustained by service members. Neither the etiology of rLLB nor the consequences of repeated exposure to this low-level blast are well understood. Thus, this study examined rLLB-induced acute and chronic pathological and behavioral consequences in our rat model. The first aim investigated anxiety, motor, and memory impairments at acute (1-3 days) and chronic (>25 days) time points following rLLB using elevated plus maze (EPM), novel object recognition (NOR), and Rotarod tasks. Rationales for choosing these behavioral tasks were based on the literature; for example, EPM has been widely used in bTBI to assess anxiety-like symptoms, and the NOR test has shown consistent memory impairments at different BOPs whereas Morris water maze (MWM) has not. Finally, compared to other motor assessments, Rotarod has sensitivity for detecting motor coordination impairments in low-level blast. Results indicated that animals exposed to rLLB significantly displayed acute and chronic anxiety-like symptoms, motor, and short-term memory impairments compared to control (unexposed) and single low-level blast rats. The second aim explored the molecular mechanisms involved in neurobehavioral changes, including superoxide-producing NADPH oxidase (NOX1), microglial activation, and reactive astrocytosis as likely contributing factors. Of the many pathological mechanisms present following brain injury, chronic neuroinflammation has been observed for up to 17 years post-TBI and has neuroinflammation been observed even months after bTBI. Chronic persistent neuroinflammation can induce neurotoxicity. Microglia are the innate immune cells of the central nervous system and have both beneficial and detrimental effects based on their activation period. Chronic microglial activation causes continued release of free radicals, cytokines (IL-1β, TNF-α), chemokines, and other signaling molecules, resulting in neurobehavioral and pathological changes. Microglial-mediated behavioral deficits can be caused by proinflammatory cytokine IL-1β. Studies have shown that excessive IL-1β levels have an influence on anxiety, motor, and short-term recognition memory impairments in clinical and preclinical research. A molecular mechanism that is involved in IL-1β release is the inflammasome complex, particularly NLRP3. NOX1-mediated oxidative stress and NLRP3 -mediated IL-1β release have not been investigated in rLLB. This aim explored the role of the NLRP3 inflammasome complex in regulating caspase-1 activation and subsequent release of the proinflammatory cytokines IL-1β in chronic neuroinflammation. Using immunofluorescence, this study examined NOX1 and NLRP3 expression, microglial activation, and astrocytosis using specific primary antibodies such as NOX1, NLRP3, Iba-1, and GFAP, respectively in the hippocampus due to its high susceptibility for blast wave compared with any other brain region. Results indicated that there was an increase NOX1 and NLRP3 protein expression, microglial activation, and reactive astrocytosis following rLLB The third aim investigated the therapeutic effects of MCC950, a specific NLRP3 inhibitor, on neurobehavioral and neuropathological abnormalities following rLLB. This study mainly examines MCC950's protective role against rLLB-induced chronic microglial activation and inflammation, especially NLRP3-IL-1β-mediated neurobehavioral changes. This study performed single immunofluorescence to assess microglial activation and double immunofluorescence followed by colocalization analysis to evaluate NLRP3+ microglia in the hippocampus and perirhinal cortex following rLLB. Treatment with MCC950 prevented short-term recognition memory impairments and mitigated NLRP3 and cleaved caspase-1 expression and IL-1β release. Furthermore, inhibition of the NLRP3 inflammasome by administration of MCC950 displayed an improvement in behavioral and pathological changes caused by rLLB, validating the original hypothesis. Therefore, targeting microglial activation by inhibiting the NLRP3 inflammasome activation may have a therapeutic potential to counteract rLLB-induced chronic neurobehavioral changes.