Saurav Bhowmick

Hibernating mammals exhibit an innate physiological ability to withstand dramatic fluctuations in blood flow that occurs during hibernation and arousal or experimental models of ischemia reperfusion without significant damage. These innate adaptations are of significance particularly to organs that are highly susceptible to energy deprivation, such as the brain and the heart. Among vertebrates, the arctic ground squirrel (AGS) is a species that tolerates ischemic/anoxic insult. During the process of entering hibernation, a state of prolonged torpor, the AGS undergoes a profound decrease in respiratory rate, heart rate, blood flow, cerebral perfusion, and body temperature (Tb). The reduced level of blood flow during torpor resembles an ischemic state, albeit without energy deficit. During the process of arousal or emergence from torpor, however, when Tb, respiratory rate, heart rate, and blood flow rapidly returns to pre-torpid levels, the rapid return of cerebral blood flow mimics a...

The long-term disabilities associated with spinal cord injury (SCI) are primarily due to the absence of robust neuronal regeneration and functional plasticity. The inability of the axon to regenerate after SCI is contributed by several intrinsic factors that trigger a cascade of molecular growth program and modulates axonal sprouting. Phosphatase and tensin homolog (PTEN) is one of the intrinsic factors contributing to growth failure after SCI, however, the underlying mechanism is not well known. Here, we developed a novel therapeutic approach for treating SCI by suppressing the action of PTEN in a mouse model of hemisection SCI. We have used a novel peptide, PTEN antagonistic peptide (PAP) to block the critical domains of PTEN to demonstrate its ability to potentially promote axon growth. PAP treatment not only enhanced regeneration of corticospinal axons into the caudal spinal cord but also promoted the regrowth of descending serotonergic axons in SCI mice. Furthermore, expression...

Recent studies from our group and others have demonstrated that oxidative stress, Ca2+ signaling, and neuroinflammation are major mechanisms contributing to post-traumatic neurodegeneration. The present study investigated the mechanisms of regulation of nuclear factor E2-related factor 2 (Nrf2) and its role in regulating antioxidant genes and oxidative stress-induced neuroinflammation and neurodegeneration following TBI. Nrf2 transcriptional system is the major regulator of endogenous defense mechanisms operating within the cells. Wild-type (Nrf2+/+) and Nrf2-deficient mice (Nrf2−/−) were subjected to 15 psi fluid percussion injury and demonstrated the regulatory role of Nrf2 in the expression antioxidant genes and oxidative stress, neuroinflammation, and cell death. Immunohistochemistry, q-RT-PCR, and western blotting techniques detected downregulation of Nrf2 and antioxidant proteins such as HO-1, GPx1, GSTm1, and NQO1 in mouse brain samples. Further, our study demonstrated that the downregulation of Nrf2 and antioxidant genes in TBI correlated with the induction of free radical-generating enzyme NADPH oxidase 1 and inducible nitric oxide synthase and their corresponding oxidative/nitrosative stress markers 4-hydroxynonenal and 3-nitrotyrosine. The decrease in Nrf2 with subsequent increase in oxidative stress markers led to the activation of MMP3/9, TGF-β1, and NF-kB that further led to neuroinflammation and apoptosis. The absence of Nrf2 function in mice resulted in exacerbated brain injury as shown by the increased oxidative stress markers, pro-inflammatory cytokines, and apoptosis markers at 24 h after TBI. In conclusion, this study could establish the significance of Nrf2 in transforming into a novel preventive approach against the pathophysiology of TBI. • Traumatic brain injury impairs Nrf2 signaling in mouse. • Nrf2-mediated activation of antioxidant genes are altered after TBI. • Impairment of Nrf2 signaling leads to oxidative stress. • TBI-induced downregulation of Nrf2 activates MMPs, TGF-β1, and NF-kB. • Nrf2 regulates neuroinflammation and apoptotic cell death in TB.

The blood-brain barrier (BBB) constitutes a neurovascular unit formed by microvascular endothelial cells, pericytes, and astrocytes. Brain pericytes are important regulators of BBB integrity, permeability, and blood flow. Pericyte loss has been implicated in injury; however, how the crosstalk among pericytes, endothelial cells, and astrocytes ultimately leads to BBB dysfunction in traumatic brain injury (TBI) remains elusive. In this study, we demonstrate the importance of pericyte-endothelium interaction in maintaining the BBB function. TBI causes the platelet-derived growth factor-B (PDGF-B)/PDGF receptor-β signaling impairment that results in loss of interaction with endothelium and leads to neurovascular dysfunction. Using in vivo mild (7 psi) and moderate (15 psi) fluid percussion injury (FPI) in mice, we demonstrate the expression of various pericyte markers including PDGFR-β, NG2 and CD13 that were significantly reduced with a subsequent reduction in the expression of various integrins; adherent junction protein, N-cadherin; gap junction protein, connexin-43; and tight junction proteins such as occludin, claudin-5, ZO-1, and JAM-a. Impairment of pericyte-endothelium interaction increases the BBB permeability to water that is marked by a significant increase in aquaporin4 expression in injured animals. Similarly, pericyte-endothelium integrity impairment in FPI animals greatly increases the permeability of small-molecular-weight sodium fluorescein and high-molecular-weight-tracer Evans blue across the BBB. In addition, the injury-inflicted animals show significantly higher levels of S100β and NSE in the blood samples compared with controls. In conclusion, our data provide an insight that brain trauma causes an early impairment of pericyte-endothelium integrity and results in BBB dysregulation that initiates pathological consequences associated with TBI.

Cerebral ischemia-reperfusion (I/R) injury initiates a cascade of events, generating nitric oxide (NO) and superoxide(O) to form peroxynitrite (ONOO), a potent oxidant. Arctic ground squirrels (AGS; Urocitellus parryii) show high tolerance to I/R injury. However, the underlying mechanism remains elusive. We hypothesize that tolerance to I/R modeled in an acute hippocampal slice preparation in AGS is modulated by reduced oxidative and nitrative stress. Hippocampal slices (400µm) from rat and AGS were subjected to oxygen glucose deprivation (OGD) using a novel microperfusion technique. Slices were exposed to NO, Odonors with and without OGD; pretreatment with inhibitors of NO, Oand ONOOfollowed by OGD. Perfusates collected every 15min were analyzed for LDH release, a marker of cell death. 3-nitrotyrosine (3NT) and 4-hydroxynonenal (4HNE) were measured to assess oxidative and nitrative stress. Results show that NO/Oalone is not sufficient to cause ischemic-like cell death, but with OGD...

Traumatic brain injury (TBI) can result in persistent sensorimotor and cognitive deficits, which occur through a cascade of deleterious pathophysiological events over time. In this study, we investigated the hypothesis that neurodegeneration caused by TBI leads to impairments in sensorimotor function. TBI induces the activation of the caspase-3 enzyme, which triggers cell apoptosis in an in vivo model of fluid percussion injury (FPI). We analyzed caspase-3 mediated apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and poly (ADP-ribose) polymerase (PARP) and annexin V western blotting. We correlated the neurodegeneration with sensorimotor deficits by conducting the animal behavioral tests including grid walk, balance beam, the inverted screen test, and the climb test. Our study demonstrated that the excess cell death or neurodegeneration correlated with the neuronal dysfunction and sensorimotor impairments associated with TBI.

Angiotensin II (Ang II) is a mediator of oxidative stress via activation/induction of reactive oxygen and nitrogen species-generating enzymes, NADPH oxidase (NOX) and inducible nitric oxide synthase (iNOS). We investigated the hypothesis that overproduction of Ang II during traumatic brain injury (TBI) induces the activation of the oxidative stress, which triggers neuroinflammation and cell apoptosis in a cell culture model of neuronal stretch injury. We first established that stretch injury causes a rapid increase in the level of Ang II, which causes the release of pro-inflammatory cytokines, IL-1β and TNF-α, via the induction of oxidative stress. Since angiotensin-converting enzyme (ACE) mediates the production of Ang II via the conversion of Ang I into Ang II, we analyzed the expression of ACE by western blotting. Further, we analyzed caspase-3-mediated apoptosis by TUNEL staining and annexin V western blotting. Angiotensin type I (AT1) receptor antagonist losartan attenuated Ang...

Ischemic reperfusion (I/R) injury is associated with a complex and multifactorial cascade of events involving excitotoxicity, acidotoxicity, and ionic imbalance. While it is known that acidosis occurs concomitantly with glutamate-mediated excitotoxicity during brain ischemia, it remains elusive how acidosis-mediated acidotoxicity interacts with glutamate-mediated excitotoxicity. Here, we investigated the effect of acidosis on glutamate-mediated excitotoxicity in acute hippocampal slices. We tested the hypothesis that mild acidosis protects against I/R injury via modulation of NMDAR, but produces injury via activation of acid sensing ion channels (ASIC1a). Using a novel microperfusion approach, we monitored time course of injury in acutely prepared, adult hippocampal slices. We varied the duration of insult to delay the return to preinsult conditions to determine if injury was caused by the primary insult or by the modeled reperfusion phase. We also manipulated pH in presence and abs...

Cerebral ischemia/reperfusion (I/R) triggers a cascade of uncontrolled cellular processes that perturb cell homeostasis. The arctic ground squirrel (AGS), a seasonal hibernator resists brain damage following cerebral I/R caused by cardiac arrest and resuscitation. However, it remains unclear if tolerance to I/R injury in AGS depends on the hibernation season. Moreover, it is also not clear if events such as depletion of ATP, acidosis and glutamate efflux that are associated with anoxic depolarization are attenuated in AGS. Here, we employ a novel microperfusion technique to test the hypothesis that tolerance to I/R injury modeled in an acute hippocampal slice preparation in AGS is independent of the hibernation season. Acute hippocampal slices were harvested from summer euthermic AGS, hibernating AGS and interbout euthermic AGS. Slices were subjected to oxygen glucose deprivation (OGD), an in vitro model of I/R injury to determine cell death marked by lactate dehydrogenase (LDH) rel...

Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases that play a critical role in signal transduction and are activated by phosphorylation in response to a variety of pathophysiology stimuli. While MAP kinase signaling has a significant role in the pathophysiology of several neurodegenerative diseases, the precise function of activation of MAP kinase in traumatic brain injury (TBI) is unknown. Therefore, it is important to study the role of MAP kinase signaling in TBI-associated neurological ailments. In this study, using an in vitro stretch injury model in rat embryo neuronal cultures and the in vivo fluid percussion injury (FPI) model in rats, we explored the role of MAP kinase signaling in the mechanisms of cell death in TBI. Our study demonstrated that the stretch injury in vitro and FPI in vivo upregulated the phosphorylation of MAP kinase proteins ERK1/2 and JNK, but not p38. Using ERK1/2 inhibitor U0126, JNK inhibitor SP600125, and p38 inhibitor SB203580, we validated the role of MAP kinase proteins in the activation of NF-kB and caspase-3. By immunofluorescence and western blotting, further, we demonstrated the role of ERK1/2 and JNK phosphorylation in neurodegeneration by analyzing cell death proteins annexin V and Poly-ADP-Ribose-Polymerase p85. Interestingly, combined use of ERK1/2 and JNK inhibitors further attenuated the cell death in stretch-injured neurons. In conclusion, this study could establish the significance of MAP kinase signaling in the pathophysiology of TBI and may have significant implications for developing therapeutic strategies using ERK1/2 and JNK inhibitors for TBI-associated neurological complications.