Transient Receptor Potential Melastatin 7 (TRPM7) is a ubiquitously expressed divalent cation channel implicated as a key regulator of neuronal cell death in stroke. Our research group has previously shown that TRPM7 dependent cytoskeletal regulation particularly via cofilin mediates neuronal death in oxygen glucose deprivation (in vitro stroke model). LIMK1 phosphorylation was also shown to decrease downstream of TRPM7 activation during anoxia. In the present study we investigated the effects of TRPM7 activation during anoxia, on three regulators of LIMK and cofilin; Rho-associated kinase 2 (ROCK2), P-21 activated kinase 3 (PAK3) and Slingshot family phosphatase 1 (SSH1). Our findings suggest that PAK3 activity is downregulated during OGD through TRPM7 independent mechanisms. However, SSH1 activity appears to be regulated downstream of TRPM7 in a manner that is consistent with LIMK and cofilin regulation. Overall, our work suggests that SSH1 is a new link between anoxia-induced TRPM7activity and cofilin hyperactivation.

Transient Receptor Potential Melastatin 7 (TRPM7) is a ubiquitously expressed divalent cation channel implicated as a key regulator of neuronal cell death in stroke. Our research group has previously shown that TRPM7 dependent cytoskeletal regulation particularly via cofilin mediates neuronal death in oxygen glucose deprivation (in vitro stroke model). LIMK1 phosphorylation was also shown to decrease downstream of TRPM7 activation during anoxia. In the present study we investigated the effects of TRPM7 activation during anoxia, on three regulators of LIMK and cofilin; Rho-associated kinase 2 (ROCK2), P-21 activated kinase 3 (PAK3) and Slingshot family phosphatase 1 (SSH1). Our findings suggest that PAK3 activity is downregulated during OGD through TRPM7 independent mechanisms. However, SSH1 activity appears to be regulated downstream of TRPM7 in a manner that is consistent with LIMK and cofilin regulation. Overall, our work suggests that SSH1 is a new link between anoxia-induced TRPM7activity and cofilin hyperactivation.

There is increasing evidence that inflammatory processes play a pivotal role in the pathophysiology of ischaemic brain injury. Cerebrovascular endothelial cells that form the blood-brain barrier are critical for maintaining brain homeostasis, however, during cerebral ischaemia they contribute to the post-ischaemic inflammatory responses. It is not yet fully understood how different cerebral cells interact during this inflammatory response. This study aimed to test the hypothesis that oxygen-glucose deprivation (OGD) induces the inflammatory activation of the cerebrovascular endothelium and glial cells in vitro and that intercommunication between these cells regulate their responses to OGD. Primary murine brain endothelial cells (MBECs) monocultures, murine mixed-glial monocultures and MBEC-glial co-cultures were exposed to OGD for up to 24 hours (h), then reperfused cultures were returned to normoxia for a further 24 hours. MBECs and glia remained viable over a 24 h OGD exposure and during reperfusion. OGD induced a time-dependent increase in MBEC glucose transporter 1 (GLUT-1) expression but a time-dependent decline in expression and secretion of monocyte chemoattractant protein-1 (MCP-1). A significant increase in keratinocyte-derived chemokine (KC) secretion by MBEC monocultures was observed during reperfusion after prolonged exposure (18-24 h) to OGD whereas, KC secretion by co-cultured MBECs was increased during reperfusion after short exposure (4 h) to OGD. Co-cultured MBECs displayed a significant increase in intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression in response to a short or prolonged exposure to OGD with 24 h of reperfusion. Neither OGD nor reperfusion had any effect on permeability of the MBEC monolayer. OGD induced a time-dependent increase in nuclear stabilisation of hypoxia inducible factor-1 alpha (HIF-1α) in glial cells which correlated to vascular endothelial growth factor (VEGF) secretion during OGD and subsequent reperfusion. Nuclear stabilisation of the nuclear factor kappa B (NFκB)p65 subunit by glial cells was dependent upon the duration of OGD. Reperfusion induced a significant increase in KC secretion by co-cultured glial cells after short exposure to OGD. Inflammatory activation of co-cultured MBECs and glia after 4 or 24 h OGD caused a significant increase in neutrophil transendothelial migration which correlated with MBEC expression of ICAM-1 and VCAM-1. A combination of these cell adhesion molecules with neutrophil integrins and soluble glial-derived mediators contributed to neutrophil transendothelial migration. These studies provide evidence that combined hypoxia and glucose withdrawal induces the activation of MBECs and glial cells in vitro. Cross-talk between these two cell types may further regulate their activation. As a result of this inflammatory activation, soluble MBEC and glial-derived mediators may contribute to neutrophil transendothelial migration through the regulation of MBEC cell adhesion molecule expression.

Acute brain cell swelling (cytotoxic edema) can occur in the first minutes of stroke, presumably as a result of brain cells taking up water. In extreme hypo-osmotic situations such as excessive water-loading by patients, uptake by brain cells can expand the brain, causing seizures. But is ischemic brain cell swelling the same as hypo-osmotic swelling? Water can passively diffuse across the plasma membrane. However the presence of water channels termed aquaporins (AQP) facilitates passive water diffusion by 10-100 times. Unlike astrocytes, there is no evidence of water channels on neuronal plasma membrane. However, there is still much debate about which cells (neurons or astrocytes) swell during over-hydration or during stroke and if neurons and astrocytes can volume-regulate during osmotic stress. The purpose of this study was to examine and compare the volume responses of PyNs and magnocellular neuroendocrine cells (MNCs) to acute osmotic challenge and to OGD. We examined MNCs because they are intrinsically osmosensitive to small changes (2-3 mOsm) of plasma osmolality. We also examined if the same neurons behave similarly in brain slices or when dissociated and if they respond differently to acute osmotic stress and stroke-like stress. Our results indicate that during acute osmotic stress (±40 mOsm) half of dissociated PyNs and MNCs tended to show appropriate responses. MNCs in brain slices showed similar responses to when they were dissociated, while brain slice PyNs were less responsive than when dissociated. Exposure to OGD resulted in obvious differences between the two types of in vitro preparations. Dissociated PyNs and MNCs showed no consistency in their volume responses to 10 minutes of OGD. Dissociated neurons swelled, shrunk or were unchanged in about equal numbers. In contrast, brain slice PyNs underwent profound swelling whereas, brain slice MNCs showed minor volume decreases. We conclude that about half of our dissociated neurons were too variable and unpredictable in their osmotic volume responses to be useful for osmotic studies. They also were too resistant to stroke-like stress to be good models for ischemia. Brain slice neurons were similar in their osmotic responses to dissociated neurons but proved to have consistent and predictable responses to stroke-like stress. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2011-02-07 17:55:08.333

Exposure to ethanol (ETOH) during fetal development results in a range of cognitive/behavioral deficits. There are differences in sensitivity to the effects of ETOH that could be explained by other factors, such as hypoxia. Similar mechanisms of damage underlie both ETOH, more specifically ETOH withdrawal, and hypoxia. Based on this overlap, it was hypothesized that sub threshold levels of these insults may interact to produce increased damage in sensitive brain regions. This study used a rodent organotypic hippocampal slice culture model to investigate the interaction of hypoxia and ETOH withdrawal and to determine possible developmental differences in the sensitivity to these insults. The combination of ETOH and hypoxia produced greater damage in the CA1 and CA3 hippocampal regions, as measured by propidium iodide uptake. Differences in outcome were noted between on postnatal (PND) 2 and PND 8 tissue. ETOH alone caused damage as measured by the neuronal marker NeuN, suggesting the ETOH/hypoxia interaction involves different cell types and that caution should be taken when determining appropriate levels of exposure. This data could explain why some offspring appear more sensitive to ETOH and/or hypoxic challenges during early life.

The role of gap junctions in modulating the dynamics of axonal dysfunction in spinal cord white matter injury remains uncertain; hence, I examined the functional role and changes in expression of gap junctions following CNS injury. I hypothesized that inhibition of gap junctions improves axonal conduction during oxygen and glucose deprivation (OGD) in vitro. Carbenoxolone and octanol, gap junction blockers, did not change CAP amplitude in non-injured tissue, yet they significantly reduced the extent of its decline during OGD. No difference in mRNA expression of connexins 32, 36 was found. However, during OGD in the presence of gap junction blockers, expression of connexins 30, 43 was downregulated. Immunohistochemistry confirmed the presence of connexins in spinal cord slices: connexins 30, 43 overlapping with GFAP, connexin 32 with MBP and connexin 36 with CC1. Thus, blocking gap junctions enhances axonal conduction during OGD and promotes dynamic changes in connexin mRNA expression.

The role of gap junctions in modulating the dynamics of axonal dysfunction in spinal cord white matter injury remains uncertain; hence, I examined the functional role and changes in expression of gap junctions following CNS injury. I hypothesized that inhibition of gap junctions improves axonal conduction during oxygen and glucose deprivation (OGD) in vitro. Carbenoxolone and octanol, gap junction blockers, did not change CAP amplitude in non-injured tissue, yet they significantly reduced the extent of its decline during OGD. No difference in mRNA expression of connexins 32, 36 was found. However, during OGD in the presence of gap junction blockers, expression of connexins 30, 43 was downregulated. Immunohistochemistry confirmed the presence of connexins in spinal cord slices: connexins 30, 43 overlapping with GFAP, connexin 32 with MBP and connexin 36 with CC1. Thus, blocking gap junctions enhances axonal conduction during OGD and promotes dynamic changes in connexin mRNA expression.

Studies on the survival of central nervous system of hypoxia-tolerant species under challenges of reduced energy availability have characterised adaptive mechanisms of brain at the cell and tissue level that lead to reduced excitability and protection. However, evidence of hypoxic suppression of retinal activity in these species has not been followed up with mechanistic studies. Microspectrofluorometric monitoring of intracellular free Ca2+ concentration ([Ca2+]i) is useful for identifying cellular mechanisms that may lead to adaptive strategies, as unregulated increases in [Ca2+]i cause toxicity. Horizontal cells (HCs) are second order retinal neurons that receive tonic excitatory input from photoreceptors, and possess voltage-gated Ca2+ conductances and other channels that can facilitate toxic increases in [Ca2+]i under conditions of reduced energy availability (modeled as oxygen-glucose deprivation, OGD). It was demonstrated that isolated HCs of the hypoxia-tolerant goldfish display spontaneous, transient [Ca2+]i activity (SA) which decreased in amplitude and area under the curve following OGD or glucose removal (20 min) without recovery. SA was shown to be dependent on extracellular Ca2+ influx through voltage-gated Ca2+ channels, though mechanisms of SA generation and regulation has yet to be determined. Additionally, glutamate-elicited peak increases in [Ca2+]i were reduced after 20 min of OGD. The removal of O2 during OGD insult seemed to be protective as an increase in baseline [Ca2+]i was seen during and following glucose removal under normoxic conditions. The mechanisms mediating these decreases in spontaneous and elicited [Ca2+]i activity are currently unknown, though candidate pathways are discussed. This thesis contributes a hint towards how HCs may tolerate conditions of low energy availability, which may also inform investigations on their role in situ during these insults.

Hyperthermia damages both developing and adult brains, especially when it occurs after ischemia or stroke. Work presented in this dissertation used in vitro models of these stresses to investigate mechanisms underlying damage to immature neurons and neural precursors cultured from embryonic rat brain. Studies described in Chapter 2 investigated the effects of a brief, intense hyperthermic stress (30-45 min at 43ºC). This stress produced a selective depletion of nestin-immunoreactive neural precursor cells, and reduced proliferation, as evidenced by reduced BrdU incorporation into young Tuj1-immunoreactive neurons. The stress activated caspase 3, and produced multiple signs of nuclear damage as well as early and persisting mitochondrial depolarization. Cycloheximide, an inhibitor of protein synthesis, reduced cell death. All these findings suggest an apoptotic death process. Studies described in Chapter 3 used a combination of oxygen-glucose deprivation (OGD, 2 h) followed by mild 41ºC hyperthermia for 90 min (T). The combined OGDT stress reduced both survival in monolayer cultures and colony-forming ability in neurospheres. Cell death occurred gradually over 2 days, and was accompanied by caspase activation that began within 6 h post-stress. Post-stress application of cycloheximide or a general caspase inhibitor (especially qVD-OPH) reduced cell death, but specific inhibitors of caspases 2, 3, 8 or 9 were ineffective. OGDT led to upregulation of the pro-apoptotic protein Bim as well as redistribution of Bax from cytoplasm to mitochondria within 6 h. Persisting mitochondrial depolarization began within 3 h following the combined OGDT stress, but not following individual OGD or T stresses alone. These findings suggest that OGD sensitizes neural precursor cells to hyperthermia-induced damage, and that the combined OGDT stress kills neural precursors via apoptotic mechanisms that include activation of mitochondrial death pathways. Results of these studies suggest that immature neurons and neural precursors are especially vulnerable to hyperthermia-induced damage via apoptotic mechanisms. Pan-caspase inhibitors may be a promising therapeutic strategy to preserve viability of these cells following stroke with hyperthermia.

Adenosine (ADO) is an endogenous neuroprotectant. Under ischemic conditions ADO levels rise in the brain up to 100-fold. ADO in the brain is dependent on the movement across cell membranes by equilibrative nucleoside transporters (ENT) or produced from membrane bound ecto-5’ nucleotidase (eN). We used transgenic neurons with neuronal specific expression of human ENT1 (hENT1) and eN knockout (CD73 KO) astrocytes. The aim of this research was to determine the role of ENT1 and eN in ADO release from ischemic-like conditions in primary cultured neurons, astrocytes or co-cultures. Neurons primarily release intracellular ADO via ENTs; this effect was blocked by transporter inhibitor, dipyridamole (DPR). Astrocytes primarily convert ADO extracellularly from eN; this effect was with eN inhibitor α, β-methylene ADP (AOPCP). Combined neuron and KO astrocytes produced less ADO, extracellular ADO was inhibited by DPR but not AOPCP. Overall these results suggest that eN is prominent in the formation of ADO but other enzymes or pathways contribute to rising ADO levels in ischemic conditions.