The sodium-potassium-chloride cotransporter (NKCC), sodium-hydrogen exchanger (NHE), and sodium bicarbonate transporter (NBC) have been shown to be involved in increased sodium ion transport across the blood-brain barrier (BBB) during ischemic stroke and subsequent cerebral edema formation due to osmotically obligate secretion of water across the BBB. A clinically relevant observation is that stroke patients who are also hyperglycemic, whether from type I or type II diabetes or another cause, exhibit worsened edema, infarct, and neurological outcome compared to normoglycemic stroke patients. Recent work in the lab has shown that hyperglycemia increases both the abundance and activity of NKCC and NHE in vitro. In the present studies, I focused on the hypothesis that hyperglycemia primes the BBB sodium ion transport system to react more robustly to the onset of ischemia, leading to increased sodium ion secretion into the brain followed by increased edema formation. To address the involvement of NKCC and NHE during hyperglycemic ischemic stroke, I analyzed NMR data collected from rats made hyperglycemic by administration of streptozotocin and then subjected to permanent middle cerebral artery occlusion (pMCAO). The rats were treated intravenously with bumetanide (NKCC inhibitor), HOE-642 (NHE inhibitor), or vehicle. I found that both inhibitors significantly reduced edema and infarct in the hyperglycemic rats compared to the control, confirming their relevance in edema formation under hyperglycemic conditions as had been previously observed in normoglycemic rats. In addition, in my analysis of NMR data from normoglycemic rats administered intravenous S0859 (NBC inhibitor) and subjected to pMCAO, I found that the inhibitor significantly reduced edema and infarct. This demonstrates the additional involvement of NBC during edema formation and is to be followed up by experiments with hyperglycemic rats. I also studied the effect of chronic and intermittent exposures of 10mM and 20mM glucose on NKCC abundance in cultured bovine cerebral microvascular endothelial cells (BCMECs), supplementing recent studies by other lab members using 30mM glucose. My initial results showed no significant changes in NKCC abundance when exposed to 10mM and 20mM glucose, but more work will need to be completed to explain differences found between my observations and those of others. Finally, I worked towards optimizing calcium imaging techniques for use in studying the role of the TRPV4 channel in hyperglycemia-induced changes in intracellular calcium levels of BCMECs in response to hypoxia. The results of my work have demonstrated a connection between hyperglycemia and increased sodium ion transport across the BBB and edema formation, in agreement with the hypothesis that hyperglycemia primes the BBB to react more robustly to ischemia. My optimizations to calcium imaging experiments will also allow future studies to delve further into the mechanisms by which hyperglycemia exerts its effects on BBB ion transport in the context of ischemic stroke.
|Advisor:||O'Donnell, Martha E|
|Commitee:||Ferns, Michael, Sack, Jon|
|School:||University of California, Davis|
|Department:||Molecular, Cellular and Integrative Physiology|
|School Location:||United States -- California|
|Source:||MAI 81/8(E), Masters Abstracts International|
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