Hypoxia effects on cell volume and ion uptake of cerebral microvascular endothelial cells

Abstract

Increased transport of Na across an intact blood-brain barrier (BBB) contributes to cerebral edema formation in ischemic stroke. Our previous studies have shown that ischemic factors stimulate activity of a luminal BBB Na-K-Cl cotransporter, and we have hypothesized that during ischemia, the cotransporter together with the abluminal Na/K pump mediates increased transport of Na from blood into the brain. However, it is possible that elevated Na-K-Cl cotransporter activity could also cause cell swelling if it outpaces ion efflux pathways. The present study was conducted to evaluate the effects of hypoxia on intracellular volume of BBB cells. Cerebral microvascular endothelial cell (CMEC) monolayers were exposed to varying levels of hypoxia for 1 to 5 h in an O(2)-controlled glove box, and cell volume was assessed using 3-O-methyl-D-[(3)H]glucose and [(14)C]sucrose as markers of total and extracellular water space, respectively. Cells exposed to either 7.5%, 3%, or 1% O(2) showed gradual increases in volume (compared with 19% O(2) normoxic controls) that became significant after 3 or more hours. By ion chromatography methods, we also found that a 30-min exposure to 7.5% O(2) caused an increase in bumetanide-sensitive net Na uptake by the cells without increasing cell Na content. CMEC Na content was significantly increased, however, following 3 or more hours of exposure to 7.5% O(2). These findings are consistent with the hypothesis that during cerebral ischemia, the BBB Na-K-Cl cotransporter is stimulated to mediate transendothelial uptake of Na into the brain and that increased cotransporter activity also contributes to gradual swelling of the cells.

Effects of estradiol on ischemic factor-induced astrocyte swelling and AQP4 protein abundance

“…Furthermore, we have shown that CMEC exposed to moderate-to-severe hypoxia do not swell or exhibit significant increases in Na content until 3 or more hours after the onset of hypoxia, a finding consistent with increased NKCC and NHE activities contributing to BBB secretion of Na from blood into brain during the early hours of stroke rather than simple swelling of the endothelial cells (12,21). However, CMEC swelling observed after 5-h exposure to hypoxia is abolished in the presence of bumetanide and HOE-642, suggesting that both NKCC and NHE participate in the gradual swelling of the cells that occurs with hypoxia (12). There is evidence that the rapid astrocyte swelling that occurs during cerebral ischemia also involves NKCC and NHE activities.…”

Cerebral Blood Flow and Cerebral Edema in Rats With Diabetic Ketoacidosis

“…Rats with DKA manifest a reduction of apparent diffusion coefficient (ADC) values for brain water, indicating cytotoxic edema, and administration of the Na-K-Cl cotransport inhibitor bumetanide, which has been shown to reduce both astrocyte and brain microvascular endothelial cell swelling under hypoxic and ischemic conditions (13)(14)(15)(16)(17), returns ADC values to normal (18). These findings are similar to those in experimental models of stroke (18 -20).…”

In vivo effects of bumetanide at brain concentrations incompatible with NKCC1 inhibition on newborn DGC structure and spontaneous EEG seizures following hypoxia-induced neonatal seizures

“…Under ischemic conditions, the luminally located NKCC1 in BBB was functionally upregulated and played an important role in ionic edema and swelling of the brain parenchyma (Kahle et al, 2009;Wallace et al, 2011). In addition, there were evidences showing that hypoxia alone could stimulate the endothelial NKCC1 activity in BBB (Kawai et al, 1996;Brillault et al, 2008). Therefore, the neuroprotective effects mediated by BBB-located NKCC1 may contribute to the suppressing seizure activity of bumetanide.…”