Hyperglycemia exacerbates edema formation and worsens neurological outcome in ischemic stroke. Edema formation in the early hours of stroke involves transport of ions and water across an intact blood-brain barrier (BBB), and swelling of astrocytes. We showed previously that high glucose (HG) exposures of 24 hours to 7 days increase abundance and activity of BBB Na+-K+-2Cl- cotransport (NKCC) and Na+/H+ exchange 1 (NHE1). Further, bumetanide and HOE-642 inhibition of these transporters significantly reduces edema and infarct following middle cerebral artery occlusion in hyperglycemic rats, suggesting that NKCC and NHE1 are effective therapeutic targets for reducing edema in hyperglycemic stroke. The mechanisms underlying hyperglycemia effects on BBB NKCC and NHE1 are not known. In the present study we investigated whether serum-glucocorticoid regulated kinase 1 (SGK1) and protein kinase C beta II (PKCβII) are involved in HG effects on BBB NKCC and NHE1. We found transient increases in phosphorylated SGK1 and PKCβII within the first hour of HG exposure, after 5-60 min for SGK1 and 5 min for PKCβII. However, no changes were observed in cerebral microvascular endothelial cell SGK1 or PKCβII abundance or phosphorylation (activity) after 24 or 48 hr HG exposures. Further, we found that HG-induced increases in NKCC and NHE1 abundance were abolished by inhibition of SGK1 but not PKCβII, whereas the increases in NKCC and NHE activity were abolished by inhibition of either kinase. Finally, we found evidence that STE20/SPS1-related proline/alanine-rich kinase and oxidative stress-responsive kinase-1 (SPAK/OSR1) participate in the HG-induced effects on BBB NKCC.

IntroductionCognitive decline is common in patients with type 1 diabetes and has been attributed to the effects of chronic hyperglycemia and severe hypoglycemia. Diabetic ketoacidosis (DKA) has only recently been suspected to be involved in causing cognitive decline. We hypothesized that DKA triggers both acute and chronic neuroinflammation, contributing to brain injury.Research methods and designWe measured concentrations of cytokines, chemokines and matrix metalloproteinases (MMP) in serum and brain tissue lysates in juvenile rats during and after DKA (during acute DKA, 24 hours and 7 days after DKA), and compared these to healthy controls and hyperglycemic controls. We also measured cytokine, chemokine and MMP concentrations in serum and brain tissue of adult rats (70 days) that had experienced DKA as juveniles and compared these measurements to those of adult diabetic rats without exposure to DKA.ResultsDuring acute DKA in the juvenile rats, serum concentrations of CCL3, tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and MMP-9 were significantly increased. Serum concentrations of IL-2 and IL-17A increased 7 days after DKA recovery. In brain tissue lysates, concentrations of CCL3, CCL5, interferon (IFN)-γ and MMP-9 were significantly elevated during acute DKA. In adult rats that had DKA as juveniles (28 days previously), serum concentrations of IL-1ß and brain concentrations of IL-10 and IL-12p70 were elevated in comparison to diabetic rats without prior DKA. Composite scores for highly correlated cytokines and chemokines (mean z-scores for IL-10, IL-1ß, TNF-α, IL-17A, IFN-γ, CXCL-1 and CCL5) were also significantly elevated in adult rats with prior DKA.ConclusionsThese data confirm that DKA causes acute systemic inflammation and neuroinflammation in a rat model. Importantly, the neuroinflammatory response triggered by DKA is long-lasting, suggesting the possibility that DKA-induced chronic neuroinflammation could contribute to long-term cognitive decline in individuals with diabetes.

Cerebral edema is exacerbated in hyperglycemic ischemic stroke through poorly understood mechanisms. We showed previously that hypoxia and other factors present during the early hours of ischemic stroke stimulate blood‐brain barrier (BBB) Na‐K‐Cl cotransport (NKCC1) and Na/H exchange (NHE1) activities, leading to increased secretion of Na, Cl and water from blood into brain across a still intact BBB and consequent edema formation. More recently we found that BBB endothelial cells exposed to high glucose (6 hr to 7 d) exhibit increased abundance of NKCC and NHE and greater stimulation of the transporter activities by ischemic factors compared to cells maintained in normoglycemic conditions. Further, we found that the more robust edema formation observed in hyperglycemic rats is significantly attenuated by bumetanide and HOE‐642, inhibitors of NKCC1 and NHE1 activities. The present study was conducted to investigate signaling events underlying these effects of hyperglycemia on BBB Na transporters and ischemia‐induced cerebral edema with the overall goal of identifying additional therapeutic targets for diabetic ischemic stroke. Compelling evidence suggests that intracellular Ca (Cai) signaling mechanisms are likely to be involved. Exposure to high glucose is known to alter Cai dynamics in several cell types, including non‐brain endothelial cells. In addition, stimulation of NKCC and/or NHE activity by increases in Cai has been demonstrated in a variety of cell types. In recent studies we found that exposing BBB endothelial cells to high glucose for 48 hours resulted in elevated resting levels of Cai and significantly increased the abundance of the Transient Receptor Potential V4 (TRPV4) channel. The present study was conducted to further evaluate the effects of hyperglycemia on BBB endothelial cell Cai and to investigate whether TRPV4 plays a role in hyperglycemia‐induced alteration of Cai dynamics in the cells. Bovine cerebral microvascular endothelial cells (CMEC) were exposed for 24 hr to hyperglycemic (HG, 30mM glucose), normoglycemic (NG, 5mM glucose) or osmotic control media (5mM glucose + 12.5 mM NaCl) then loaded with 5μM Cal‐520‐AM and imaged after exposure (1 hr) to normoxia or hypoxia (7% O2). Calcium levels were normalized to maximal non‐saturated calcium signal following the addition of 8μM ionomycin. We found that HG CMEC exhibited a 4‐fold increase in [Cai] when exposed to hypoxia compared to a 2‐fold in NG CMEC. This effect was abolished in the presence Ca‐free media or the TRPV4 inhibitor HC067047 (1μM). Western blot evaluation of CMEC lysates following exposure to HG media (6 to 48 hr) revealed significant increases in TRPV4 abundance after 24 and 48 hr but no effect of osmotic control medium (NG media with added 12.5 mM NaCl). Using conventional perforated patch‐clamp methods and a 1‐second voltage ramp (−100 mV to +100 mV) protocol, we also found that the TRPV4 agonist GSK1016790A activated an outwardly rectifying current in the cells that was blocked by the antagonist (HC067047; 1μM), indicating the presence of TRPV4 activity in the cells. Our findings indicate that hyperglycemia exposures cause more robust hypoxia‐induced increases in BBB endothelial cell [Cai] in a TRPV4‐dependent manner.Support or Funding InformationSupported by NINDS and the American Heart AssociationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.