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Browsing by Author "Saxena, Saahil"
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Item 459 Caspase-1 mediated inflammatory response - a critical player in concussive mild traumatic brain injury (mTBI) associated long term pain(Cambridge University Press, 2023-04-24) Nguyen, Tyler; Talley, Sarah; Nguyen, Natalie; Cochran, Ashlyn G.; Al-Juboori, Mohammed; Smith, Jared A.; Saxena, Saahil; Campbell, Edward M.; Obukhov, Alexander G.; White, Fletcher A.; Anesthesia, School of MedicineOBJECTIVES/GOALS: Patients who have experienced conjunctive mild traumatic brain injuries (mTBIs) suffer from a number of comorbidities, including chronic pain. Despite extensive studies investigating the underlying mechanisms of mTBI-associated chronic pain, the role of inflammation after mTBI and its contribution to long-term pain are still poorly understood. METHODS/STUDY POPULATION: Given the shifting dynamics of inflammation, it is important to understand the spatial-longitudinal changes and their effects on TBI-related pain. Utilizing a recently developed transgenic caspase-1 luciferase reporter mouse, we characterized the bioluminescence signal evident in both in vivo and ex vivo tissues following repetitive closed head mTBIs. This allowed us to reveal the spatiotemporal dynamics of caspase-1 activation in individual animals over time. Furthermore, we utilize various proteomic and behavioral assays to evaluate the role of caspase-1 mediated inflammation in the development and progression of injury-associated chronic pain. Lastly, by blocking inflammasome caspase-1 activation with a specific inhibitor, we assess its clinical potential as the next therapeutic approach to pain. RESULTS/ANTICIPATED RESULTS: We established that there were significant increases in bioluminescent signals upon protease cleavage in the brain, thorax, abdomen, and paws in vivo, which lasted for at least one week after each injury. Enhanced inflammation was also observed in ex vivo brain slice preparations following injury events that lasted for at least 3 days. Concurrent with the in vivo detection of the bioluminescent signal were persistent decreases in mouse hind paw withdrawal thresholds that lasted for more than two months postinjury. Using MCC950, a potent small molecule inhibitor of NLRP3 inflammasome-caspase 1 activity, we observed reductions in both caspase-1 bioluminescent signals in vivo and caspase-1 p45 expression by immunoblotting and an increase in hind paw withdrawal thresholds. DISCUSSION/SIGNIFICANCE: Overall, these findings suggest that neuroinflammation in the brain following repeated mTBIs is coincidental with a chronic nociplastic pain state, and repeated mTBI-associated events can be ameliorated by a highly specific small molecule inhibitor of NLRP3 inflammasome activation.Item Repeated closed-head mild traumatic brain injury-induced inflammation is associated with nociceptive sensitization(BMC, 2023-08-27) Nguyen, Tyler; Nguyen, Natalie; Cochran, Ashlyn G.; Smith, Jared A.; Al‑Juboori, Mohammed; Brumett, Andrew; Saxena, Saahil; Talley, Sarah; Campbell, Edward M.; Obukhov, Alexander G.; White, Fletcher A.; Anesthesia, School of MedicineBackground: Individuals who have experienced mild traumatic brain injuries (mTBIs) suffer from several comorbidities, including chronic pain. Despite extensive studies investigating the underlying mechanisms of mTBI-associated chronic pain, the role of inflammation in long-term pain after mTBIs is not fully elucidated. Given the shifting dynamics of inflammation, it is important to understand the spatial-longitudinal changes in inflammatory processes following mTBIs and their effects on TBI-related pain. Methods: We utilized a recently developed transgenic caspase-1 luciferase reporter mouse model to monitor caspase-1 activation through a thinned skull window in the in vivo setting following three closed-head mTBI events. Organotypic coronal brain slice cultures and acutely dissociated dorsal root ganglion (DRG) cells provided tissue-relevant context of inflammation signal. Mechanical allodynia was assessed by mechanical withdrawal threshold to von Frey and thermal hyperalgesia withdrawal latency to radiant heat. Mouse grimace scale (MGS) was used to detect spontaneous or non-evoked pain. In some experiments, mice were prophylactically treated with MCC950, a potent small molecule inhibitor of NLRP3 inflammasome assembly to inhibit injury-induced inflammatory signaling. Bioluminescence spatiotemporal dynamics were quantified in the head and hind paws, and caspase-1 activation was confirmed by immunoblot. Immunofluorescence staining was used to monitor the progression of astrogliosis and microglial activation in ex vivo brain tissue following repetitive closed-head mTBIs. Results: Mice with repetitive closed-head mTBIs exhibited significant increases of the bioluminescence signals within the brain and paws in vivo for at least one week after each injury. Consistently, immunoblotting and immunofluorescence experiments confirmed that mTBIs led to caspase-1 activation, astrogliosis, and microgliosis. Persistent changes in MGS and hind paw withdrawal thresholds, indicative of pain states, were observed post-injury in the same mTBI animals in vivo. We also observed enhanced inflammatory responses in ex vivo brain slice preparations and DRG for at least 3 days following mTBIs. In vivo treatment with MCC950 significantly reduced caspase-1 activation-associated bioluminescent signals in vivo and decreased stimulus-evoked and non-stimulus evoked nociception. Conclusions: Our findings suggest that the inflammatory states in the brain and peripheral nervous system following repeated mTBIs are coincidental with the development of nociceptive sensitization, and that these events can be significantly reduced by inhibition of NLRP3 inflammasome activation.