Adenosine Signalling and Regulation of Ca2+-permeable AMPA Receptors in Hypoxia

Stroke is one of major causes of death, and ischemic stroke attributes to approximately 87% of all strokes. Our lab has previously shown that during an ischemic insult, adenosine A1 receptor (A1R) activation in the rat hippocampus leads to AMPA receptor (AMPAR) downregulation and persistent synaptic inhibition. This persistent synaptic depression could contribute to neuronal damage, as neurons require constant excitatory inputs. Moreover, we observed an adenosine-induced post-hypoxia synaptic potentiation (APSP) in rat hippocampal CA1 field potential recordings. Hypoxia-induced cell death and APSP were significantly reduced when rat hippocampal slices were pretreated with either A1R or A2AR antagonist, indicating a potential interaction between A1Rs and A2ARs through yet unknown mechanisms. This study further explores the role of glutamate receptors in the generation of APSP through adenosine signalling. We hypothesize that hypoxia induces A1R-mediated, dynamin (a GTPase regulating endocytosis)-dependent internalization of A1Rs and GluA2/GluA1 AMPARs, which is then followed by an A2AR-mediated upregulation of these AMPARs, which is a prerequisite for full expression of APSP. Electrophysiology studies demonstrated that synaptic transmissions and APSPs in the rat hippocampal CA1 region are mostly mediated by AMPAergic mechanisms, instead of NMDA receptors, and that these required both A1R and A2AR signalling cross-talk. To determine whether Ca2+-permeable AMPARs (CP-AMPARs) underlie APSP, I performed experiments to test the effects of selective CP-AMPAR antagonists on APSP levels, including NASPM, IEM 1460 and Philanthotoxin-74. Application of the CP-AMPARs antagonists at the early phase of hypoxic stimulation blocked the generation of APSPs, whereas no attenuated effects were observed when applied after the expression of APSPs. In contrast, the clinically approved anti-seizure drug Perampanel, which is a non-competitive AMPAR antagonist, blocked the generation of APSP. Surprisingly, all CP-AMPAR antagonists tested were effective in preventing hypoxia-induced hippocampal neuronal damage during the early phase of hypoxic stimulation, but Perampanel was the only compound that prevented neuronal damage during normoxic brain slice reperfusion. Additional studies of leukocyte-specific protein1 (LSP1) knockout mice also revealed a potential contribution of LSP1 to altering synaptic plasticity during hypoxia-reperfusion injury models. In particular, LSP1 may regulate the levels of synaptic depression and CP-AMPARs during A1R stimulation in hypoxic conditions. Collectively, this study has provided further evidence for CP-AMPARs’ role in delayed hippocampal injury, which involves both A1Rs and A2ARs, and reveals GluA2-lacking AMPARs as a potential target for designing neuroprotective drugs in the late stage neurodegeneration related to hypoxia/ischemia

MODULATION OF THE ALPHA-7 NICOTINIC ACETYLCHOLINE RECEPTOR FOLLOWING EXPERIMENTAL RAT BRAIN INJURY IMPROVES CELLULAR AND BEHAVIORAL OUTCOMES

Traumatic brain injury (TBI) is a leading cause of death and long-term disability worldwide, and survivors are often left with cognitive deficits and significant problems with day to day tasks. To date, therapeutic pharmacological treatments of TBI remain elusive despite numerous clinical trials. An improved understanding of the molecular and cellular response to injury may help guide future treatment strategies. One promising marker for brain injury is the translocator protein (TSPO), which is normally expressed at a low level, but is highly expressed following brain damage and is associated with neuroinflammation. The isoquinoline carboxamide PK11195 binds selectively to the TSPO in many species, and has therefore become the most-studied TSPO ligand. To characterize the time-course of TSPO expression in the controlled cortical injury (CCI) model of TBI we subjected Sprague-Dawley rats to CCI and euthanatized them after 30 minutes, 12 hours, 1, 2, 4, or 6 days. Autoradiography with radiolabelled PK11195 was used to assess the time-course of TSPO binding following CCI. Autoradiographs were compared to adjacent tissue slices stained with the microglia/macrophage marker ED-1, with which a moderate positive correlation was discovered. PK11195 autoradiography was used as a tool with which to assess neuroinflammation following CCI and the administration of an α7 nAChR antagonist, methyllycaconitine (MLA). We hypothesized that blocking the calcium permeable α7 nAChR after brain injury would have a neuroprotective effect by attenuating excitotoxicity in the shortterm. Our study revealed clear dose-dependent tissue sparing in rats administered MLA after trauma and a modest improvement in functional outcome. The relatively modest recovery of function with MLA, which could be due to prolonged α7 nAChR blockade or downregulation lead us to explore the potential of α7 nAChR partial agonists in treating TBI. The α7 nAChR partial agonists tropisetron, ondansetron, and DMXB-A produced a moderate attenuation of cognitive deficits, but did not have a neuroprotective effect on tissue sparing. These studies show that following TBI, α7 nAChR modulation can have neuroprotective effects and attenuate cognitive deficits. Whether this modulation is best achieved through partial agonist treatment alone or a combination antagonist/agonist treatment remains to be determined

P2X7 receptor regulates leukocyte infiltrations in rat frontoparietal cortex following status epilepticus

<p>Abstract</p> <p>Background</p> <p>In the present study, we investigated the roles of P2X7 receptor in recruitment and infiltration of neutrophil during epileptogenesis in rat epilepsy models.</p> <p>Methods</p> <p>Status epilepticus (SE) was induced by pilocarpine in rats that were intracerebroventricularly infused with either saline, 2',3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP), adenosine 5'-triphosphate-2',3'-dialdehyde (OxATP), or IL-1Ra (interleukin 1 receptor antagonist) prior to SE induction. Thereafter, we performed immunohistochemical studies for myeloperoxidase (MPO), CD68, interleukin-1β (IL-1β), monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2).</p> <p>Results</p> <p>In saline-infused animals, neutrophils and monocytes were observed in frontoparietal cortex (FPC) at 1 day and 2 days after SE, respectively. In BzATP-infused animals, infiltrations of neutrophils and monocytes into the FPC were detected at 12 hr and 1 day after SE, respectively. In OxATP-infused animals, neutrophils and monocytes infiltrated into the FPC at 1 day and 2 days after SE, respectively. However, the numbers of both classes of leukocytes were significantly lower than those observed in the saline-infused group. In piriform cortex (PC), massive leukocyte infiltration was detected in layers III/IV of saline-infused animals at 1-4 days after induction of SE. BzATP or OxATP infusion did not affect neutrophil infiltration in the PC. In addition, P2X7 receptor-mediated MCP-1 (released from microglia)/MIP-2 (released from astrocytes) regulation was related to SE-induced leukocyte infiltration in an IL-1β-independent manner.</p> <p>Conclusions</p> <p>Our findings suggest that selective regulation of P2X7 receptor-mediated neutrophil infiltration may provide new therapeutic approaches to SE or epilepsy.</p

The Brain-Heart Connection: Establishment of a Novel Rodent Model of Focal Insular Ischemic Stroke to Examine the Pathophysiology of Stroke-Induced Heart Injury

The neurological influence of ischemic stroke in the generation of stroke-induced heart injury (SIHI) has been acknowledged for several years. However, pathophysiological mechanisms remain uncertain. Clinically, it is hypothesized that stroke involving the insular cortex (IC) initiates SIHI, since the IC controls autonomic regulation of cardiovascular function. Yet, given the high prevalence of shared risk factors between ischemic stroke and cardiovascular disorders, mechanistic conclusions from clinical studies are largely speculative. We therefore sought to establish a novel rodent model of focal insular ischemic stroke, used to evaluate chronic outcomes of SIHI. Focal ischemic stroke was induced into the right or left IC of male Wistar rats, through stereotaxic injection of endothelin-1. Control groups received an injection of ibotenic acid, phosphate-buffered saline or no injection. Before euthanasia, rats were assessed for autonomic deficits in sensorimotor gating. At 28 days post-injection, rats with left IC damage displayed trends of impaired sensorimotor gating; compared to rats with right IC damage and control groups. Pathologically, all injured groups exhibited a chronic increase in microglia activation, present at the IC and remote white/grey matter regions. Furthermore, these groups expressed left atrial cardiac fibrosis. When correlated, a positive association between microglia activation and cardiac fibrosis was observed. With this novel model, we have identified several downstream consequences of IC ischemic stroke within the brain and heart; affirming the focal contribution of IC damage to SIHI. Taken together, these results provide insight into potential mechanisms of post-stroke cardiac damage, serving as future therapeutic targets for SIHI

PRECLINICAL DEVELOPMENT OF PHYTOCANNABINOID- AND ENDOCANNABINOID- BASED PHARMACOTHERAPIES FOR THE TREATMENT OF ETHANOL-INDUCED NEURODEGENERATION

Excessive ethanol consumption, characteristic of alcohol use disorders (AUDs), is associated with widespread neurodegeneration and cognitive and behavioral impairments that may contribute to the chronic and relapsing nature of alcoholism. Therefore, identifying novel targets that can afford neuroprotection will undoubtedly aid current treatment strategies for AUDs. The cannabinoids have been shown to provide neuroprotection in a variety of preclinical models of neurodegeneration; however minimal data is available regarding the use of cannabinoid-based pharmacotherapies for treating ethanol-induced neurodegeneration. Therefore, the current dissertation examined the overarching hypothesis: the cannabinoids are a therapeutic strategy to afford neuroprotection in the context of ethanol-induced neurodegeneration. Importantly, this overarching hypothesis was approached with translational considerations in mind. Specifically, the use of many cannabinoids in the clinic is hindered due to multiple unfavorable pharmacokinetic/pharmacodynamic profiles, including high first pass metabolism and untoward psychoactivity. Therefore, the studies herein were designed to circumvent these PK/PD obstacles. The first set of studies examined whether transdermal delivery of the phytocannabinoid, cannabidiol (CBD), could attenuate binge ethanol induced neurodegeneration. Transdermal CBD afforded neuroprotection in the entorhinal cortex and neuroprotection was similar in magnitude as intraperitoneal administration. The second set of studies found that binge ethanol treatment transiently down-regulated the main CNS cannabinoid receptor, CB1R. Interestingly, these changes were not accompanied by alterations in one of the major endogenous ligands, anandamide (AEA), or other related n-acylethanolamides (NAEs). The latter finding is in contrast to other literature reports demonstrating that endocannabinoid content is substantially elevated in response to a CNS insult. Nevertheless, studies were carried out to determine if administration of the AEA and NAE catabolism inhibitor, URB597, could attenuate binge ethanol induced neurodegeneration. URB597 failed to produce neuroprotection in the entorhinal cortex and dentate gyrus of the hippocampus. However, additional studies found that URB597 failed to elevate AEA in the entorhinal cortex, and in general the biological activity of URB597 was impaired by ethanol exposure. Therefore, with further drug discovery/development efforts, it may be feasible to optimize such treatment strategies. In conclusion, the studies within the current dissertation demonstrated the feasibility of using some cannabinoid-based agents to prevent ethanol-induced neurodegeneration

Nicotinic Acetylcholine Receptor Signaling in Neuroprotection

Alzheimer’s disease; Neurodegenerative diseases; Nicotine; Transporter; Gli