High glucose-induced effects on Na+-K+-2Cl- cotransport and Na+/H+ exchange of blood-brain barrier endothelial cells: involvement of SGK1, PKCßII, and SPAK/OSR1.

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 (PKCßII) are involved in HG effects on BBB NKCC and NHE1. We found transient increases in phosphorylated SGK1 and PKCßII within the first hour of HG exposure, after 5-60 min for SGK1 and 5 min for PKCßII. However, no changes were observed in cerebral microvascular endothelial cell SGK1 or PKCßII abundance or phosphorylation (activity) after 24 or 48 h HG exposures. Further, we found that HG-induced increases in NKCC and NHE1 abundance were abolished by inhibition of SGK1 but not PKCßII, 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.

Exacerbated brain edema in a rat streptozotocin model of hyperglycemic ischemic stroke: Evidence for involvement of blood-brain barrier Na-K-Cl cotransport and Na/H exchange.

Cerebral edema is exacerbated in diabetic ischemic stroke through poorly understood mechanisms. We showed previously that blood-brain barrier (BBB) Na-K-Cl cotransport (NKCC) and Na/H exchange (NHE) are major contributors to edema formation in normoglycemic ischemic stroke. Here, we investigated whether hyperglycemia-exacerbated edema involves changes in BBB NKCC and NHE expression and/or activity and whether inhibition of NKCC or NHE effectively reduces edema and injury in a type I diabetic model of hyperglycemic stroke. Cerebral microvascular endothelial cell (CMEC) NKCC and NHE abundances and activities were determined by Western blot, radioisotopic flux and microspectrofluorometric methods. Cerebral edema and Na in rats subjected to middle cerebral artery occlusion (MCAO) were assessed by nuclear magnetic resonance methods. Hyperglycemia exposures of 1-7d significantly increased CMEC NKCC and NHE abundance and activity. Subsequent exposure to ischemic factors caused more robust increases in NKCC and NHE activities than in normoglycemic CMEC. MCAO-induced edema and brain Na uptake were greater in hyperglycemic rats. Intravenous bumetanide and HOE-642 significantly attenuated edema, brain Na uptake and ischemic injury. Our findings provide evidence that BBB NKCC and NHE contribute to increased edema in hyperglycemic stroke, suggesting that these Na transporters are promising therapeutic targets for reducing damage in diabetic stroke.

Histological and cognitive alterations in adult diabetic rats following an episode of juvenile diabetic ketoacidosis: Evidence of permanent cerebral injury.

Evidence suggests that diabetic ketoacidosis (DKA) may cause subtle cognitive alterations in children but the mechanisms are poorly understood. Acute DKA is associated with reactive astrogliosis and microglial activation in a rat model. Whether these inflammatory changes permanently alter brain histology is unknown. We aimed to determine whether DKA results in permanent alterations in brain histology and whether these changes are associated with cognitive deficits in a rat model. We induced diabetes in juvenile rats with streptozotocin at 4 weeks of age. We induced DKA in one group (n=21) at 5 weeks of age and compared this group to rats with diabetes without DKA episodes (n=13). Beginning at 7 weeks, rats underwent a series of cognitive tests to evaluate memory. At 15 weeks, rat brains were harvested and examined using immunohistochemistry (IHC). In tests of novel object recognition and social recognition, both groups performed similarly, however, the DKA group performed more poorly in object-place recognition tests, suggesting alterations in hippocampal function. IHC studies demonstrated increased glial fibrillary acidic protein staining intensity in the hippocampus of DKA rats suggesting astrogliosis, and decreased NeuN positive cell counts in the cortex suggesting neuron loss. These studies demonstrate that DKA results in permanent alterations in brain microstructure in a rat diabetes model. These structural changes are associated with deficits in hippocampal function.

Treatment with the KCa3.1 inhibitor TRAM-34 during diabetic ketoacidosis reduces inflammatory changes in the brain.

BACKGROUND: Diabetic ketoacidosis (DKA) causes brain injuries in children ranging from subtle to life-threatening. Previous studies suggest that DKA-related brain injury may involve both stimulation of Na-K-Cl cotransport and microglial activation. Other studies implicate the Na-K-Cl cotransporter and the Ca-activated K channel KCa3.1 in activation of microglia and ischemia-induced brain edema. In this study, we determined whether inhibiting cerebral Na-K-Cl cotransport or KCa3.1 could reduce microglial activation and decrease DKA-related inflammatory changes in the brain. METHODS: Using immunohistochemistry, we investigated cellular alterations in brain specimens from juvenile rats with DKA before, during and after insulin and saline treatment. We compared findings in rats treated with and without bumetanide (an inhibitor of Na-K-Cl cotransport) or the KCa3.1 inhibitor TRAM-34. RESULTS: Glial fibrillary acidic protein (GFAP) staining intensity was increased in the hippocampus during DKA, suggesting reactive astrogliosis. OX42 staining intensity was increased during DKA in the hippocampus, cortex and striatum, indicating microglial activation. Treatment with TRAM-34 decreased both OX42 and GFAP intensity suggesting a decreased inflammatory response to DKA. Treatment with bumetanide did not significantly alter OX42 or GFAP intensity. CONCLUSIONS: Inhibiting KCa3.1 activity with TRAM-34 during DKA treatment decreases microglial activation and reduces reactive astrogliosis, suggesting a decreased inflammatory response.

Diabetic ketoacidosis in juvenile rats is associated with reactive gliosis and activation of microglia in the hippocampus.

BACKGROUND: Type 1 diabetes may be associated with structural and functional alterations in the brain. The role of diabetic ketoacidosis (DKA) in causing these alterations has not been well explored. METHODS: We used immunohistochemical staining to investigate cellular alterations in brain specimens from juvenile rats with DKA before, during, and after treatment with insulin and saline, and compared these to samples from diabetic rats and normal controls. RESULTS: Glial fibrillary acidic protein (GFAP) staining intensity was increased in the hippocampus during DKA and increased further during insulin/saline treatment. Twenty-four and 72 h after treatment, hippocampal GFAP intensity declined but remained above control levels. There were no significant changes in GFAP intensity in the cortex or striatum. OX42 staining intensity was increased during untreated DKA and increased further during insulin/saline treatment in the hippocampus and cortex. NeuN staining intensity was decreased after DKA treatment in the striatum but not in other regions. CONCLUSIONS: DKA causes inflammatory changes in the brain including reactive gliosis and activation of microglia. These findings are present during untreated DKA, but intensify during insulin/saline treatment. The hippocampus was disproportionately affected, consistent with previous studies showing deficits in hippocampal functions in rats after DKA recovery and decreased memory capacity in children with a history of DKA.

Levels of S100B in brain and blood of rats with diabetic ketoacidosis.

Diabetic ketoacidosis (DKA) frequently causes subtle brain injuries in children. Rarely, these injuries can be severe and life threatening. The physiological processes leading to brain injury during DKA are poorly understood. S100B is a calcium-binding protein secreted by astrocytes. Elevated serum S100B levels are documented in several types of brain injuries. S100B may have either neuroprotective or neurotoxic effects, depending upon the concentration. We undertook the current studies to measure alterations in S100B production and secretion during DKA. We measured serum S100B concentrations in juvenile rats during and after DKA, and used immunohistochemistry to measure S100B expression in the hippocampus, cortex and striatum. Compared to levels in both normal and hyperglycemic control rats, serum S100B levels during DKA were significantly reduced. Serum S100B gradually rose after DKA, returning to levels of hyperglycemic controls by 72 h. S100B expression in the hippocampus was also significantly reduced 24h after DKA. There were no significant changes in S100B expression in other brain regions. Our findings contrast with those for other types of brain injuries in which both serum S100B levels and astrocyte S100B expression are typically elevated. These data suggest that serum S100B measurement cannot be used as an indicator of brain injury during DKA. Whether reduced S100B production or secretion is involved in the pathogenesis of DKA-related brain injury should be investigated.

Blood-brain barrier KCa3.1 channels: evidence for a role in brain Na uptake and edema in ischemic stroke.

BACKGROUND AND PURPOSE: KCa3.1, a calcium-activated potassium channel, regulates ion and fluid secretion in the lung and gastrointestinal tract. It is also expressed on vascular endothelium where it participates in blood pressure regulation. However, the expression and physiological role of KCa3.1 in blood-brain barrier (BBB) endothelium has not been investigated. BBB endothelial cells transport Na(+) and Cl(-) from the blood into the brain transcellularly through the co-operation of multiple cotransporters, exchangers, pumps, and channels. In the early stages of cerebral ischemia, when the BBB is intact, edema formation occurs by processes involving increased BBB transcellular Na(+) transport. This study evaluated whether KCa3.1 is expressed on and participates in BBB ion transport. METHODS: The expression of KCa3.1 on cultured cerebral microvascular endothelial cells, isolated microvessels, and brain sections was evaluated by Western blot and immunohistochemistry. Activity of KCa3.1 on cerebral microvascular endothelial cells was examined by K(+) flux assays and patch-clamp. Magnetic resonance spectroscopy and MRI were used to measure brain Na(+) uptake and edema formation in rats with focal ischemic stroke after TRAM-34 treatment. RESULTS: KCa3.1 current and channel protein were identified on bovine cerebral microvascular endothelial cells and freshly isolated rat microvessels. In situ KCa3.1 expression on BBB endothelium was confirmed in rat and human brain sections. TRAM-34 treatment significantly reduced Na(+) uptake, and cytotoxic edema in the ischemic brain. CONCLUSIONS: BBB endothelial cells exhibit KCa3.1 protein and activity and pharmacological blockade of KCa3.1 seems to provide an effective therapeutic approach for reducing cerebral edema formation in the first 3 hours of ischemic stroke.

Blood-brain barrier Na transporters in ischemic stroke.

Blood-brain barrier (BBB) endothelial cells form a barrier that is highly restrictive to passage of solutes between blood and brain. Many BBB transport mechanisms have been described that mediate transcellular movement of solutes across the barrier either into or out of the brain. One class of BBB transporters that is all too often overlooked is that of the ion transporters. The BBB has a rich array of ion transporters and channels that carry Na, K, Cl, HCO3, Ca, and other ions. Many of these are asymmetrically distributed between the luminal and abluminal membranes, giving BBB endothelial cells the ability to perform vectorial transport of ions across the barrier between blood and brain. In this manner, the BBB performs the important function of regulating the volume and composition of brain interstitial fluid. Through functional coupling of luminal and abluminal transporters and channels, the BBB carries Na, Cl, and other ions from blood into brain, producing up to 30% of brain interstitial fluid in healthy brain. During ischemic stroke cerebral edema forms by processes involving increased activity of BBB luminal Na transporters, resulting in "hypersecretion" of Na, Cl, and water into the brain interstitium. This review discusses the roles of luminal BBB Na transporters in edema formation in stroke, with an emphasis on Na-K-Cl cotransport and Na/H exchange. Evidence that these transporters provide effective therapeutic targets for reduction of edema in stroke is also discussed, as are recent findings regarding signaling pathways responsible for ischemia stimulation of the BBB Na transporters.

Ischemic factor-induced increases in cerebral microvascular endothelial cell Na/H exchange activity and abundance: evidence for involvement of ERK1/2 MAP kinase.

Brain edema forms rapidly in the early hours of ischemic stroke by increased secretion of Na, Cl, and water into the brain across an intact blood-brain barrier (BBB), together with swelling of astrocytes as they take up the ions and water crossing the BBB. Our previous studies provide evidence that luminal BBB Na-K-Cl cotransport (NKCC) and Na/H exchange (NHE) participate in ischemia-induced edema formation. NKCC1 and two NHE isoforms, NHE1 and NHE2, reside predominantly at the luminal BBB membrane. NKCC and NHE activities of cerebral microvascular endothelial cells (CMEC) are rapidly stimulated by the ischemic factors hypoxia, aglycemia, and AVP, and inhibition of NKCC and NHE activities by bumetanide and HOE642, respectively, reduces brain Na uptake and edema in the rat middle cerebral artery occlusion model of stroke. The present study was conducted to further explore BBB NHE responses to ischemia. We examined whether ischemic factor-stimulated NHE activity is sustained over several hours, when the majority of edema forms during stroke. We also examined whether ischemic factors alter NHE1 and/or NHE2 protein abundance. Finally, we conducted initial studies of ERK1/2 MAP kinase involvement in BBB NHE and NKCC responses to ischemic factors. We found that hypoxia, aglycemia, and AVP increase CMEC NHE activity through 5 h and that NHE1, but not NHE2, abundance is increased by 1- to 5-h exposures to these factors. Furthermore, we found that these factors rapidly increase BBB ERK1/2 activity and that ERK1/2 inhibition reduces or abolishes ischemic factor stimulation of NKCC and NHE activities.

Brain cell swelling during hypocapnia increases with hyperglycemia or ketosis.

BACKGROUND: Severe hypocapnia reduces cerebral blood flow (CBF) and is known to be a risk factor for diabetic ketoacidosis (DKA)-related cerebral edema and cerebral injury in children. Reductions in CBF resulting from hypocapnia alone, however, would not be expected to cause substantial cerebral injury. We hypothesized that either hyperglycemia or ketosis might alter the effects of hypocapnia on CBF and/or cerebral edema associated with CBF reduction. METHODS: We induced hypocapnia (pCO2 20 ± 3 mmHg) via mechanical ventilation in three groups of juvenile rats: 25 controls, 22 hyperglycemic rats (serum glucose 451 ± 78 mg/dL), and 15 ketotic rats (ß-hydroxy butyrate 3.0 ± 1.0 mmol/L). We used magnetic resonance imaging to measure CBF and apparent diffusion coefficient (ADC) values in these groups and in 17 ventilated rats with normal pCO2 (40 ± 3 mmHg). In a subset (n = 35), after 2 h of hypocapnia, pCO2 levels were normalized (40 ± 3 mmHg) and ADC and CBF measurements were repeated. RESULTS: Declines in CBF with hypocapnia occurred in all groups. Normalization of pCO2 after hypocapnia resulted in hyperemia in the striatum. These effects were not substantially altered by hyperglycemia or ketosis. Declines in ADC (suggesting brain cell swelling) during hypocapnia, however, were greater during both hyperglycemia and ketosis. CONCLUSIONS: We conclude that brain cell swelling associated with hypocapnia is increased by both hyperglycemia and ketosis, suggesting that these metabolic conditions may make the brain more vulnerable to injury during hypocapnia.

Intravenous HOE-642 reduces brain edema and Na uptake in the rat permanent middle cerebral artery occlusion model of stroke: evidence for participation of the blood-brain barrier Na/H exchanger.

Cerebral edema forms in the early hours of ischemic stroke by processes involving increased transport of Na and Cl from blood into brain across an intact blood-brain barrier (BBB). Our previous studies provided evidence that the BBB Na-K-Cl cotransporter is stimulated by the ischemic factors hypoxia, aglycemia, and arginine vasopressin (AVP), and that inhibition of the cotransporter by intravenous bumetanide greatly reduces edema and infarct in rats subjected to permanent middle cerebral artery occlusion (pMCAO). More recently, we showed that BBB Na/H exchanger activity is also stimulated by hypoxia, aglycemia, and AVP. The present study was conducted to further investigate the possibility that a BBB Na/H exchanger also participates in edema formation during ischemic stroke. Sprague-Dawley rats were subjected to pMCAO and then brain edema and Na content assessed by magnetic resonance imaging diffusion-weighed imaging and magnetic resonance spectroscopy Na spectroscopy, respectively, for up to 210 minutes. We found that intravenous administration of the specific Na/H exchange inhibitor HOE-642 significantly decreased brain Na uptake and reduced cerebral edema, brain swelling, and infarct volume. These findings support the hypothesis that edema formation and brain Na uptake during the early hours of cerebral ischemia involve BBB Na/H exchanger activity as well as Na-K-Cl cotransporter activity.

Effects of hyperglycemia and effects of ketosis on cerebral perfusion, cerebral water distribution, and cerebral metabolism.

Diabetic ketoacidosis (DKA) may cause brain injuries in children. The mechanisms responsible are difficult to elucidate because DKA involves multiple metabolic derangements. We aimed to determine the independent effects of hyperglycemia and ketosis on cerebral metabolism, blood flow, and water distribution. We used magnetic resonance spectroscopy to measure ratios of cerebral metabolites (ATP to inorganic phosphate [Pi], phosphocreatine [PCr] to Pi, N-acetyl aspartate [NAA] to creatine [Cr], and lactate to Cr) and diffusion-weighted imaging and perfusion-weighted imaging to assess cerebral water distribution (apparent diffusion coefficient [ADC] values) and cerebral blood flow (CBF) in three groups of juvenile rats (hyperglycemic, ketotic, and normal control). ATP-to-Pi ratio was reduced in both hyperglycemic and ketotic rats in comparison with controls. PCr-to-Pi ratio was reduced in the ketotic group, and there was a trend toward reduction in the hyperglycemic group. No significant differences were observed in NAA-to-Cr or lactate-to-Cr ratio. Cortical ADC was reduced in both groups (indicating brain cell swelling). Cortical CBF was also reduced in both groups. We conclude that both hyperglycemia and ketosis independently cause reductions in cerebral high-energy phosphates, CBF, and cortical ADC values. These effects may play a role in the pathophysiology of DKA-related brain injury.

Cognitive dysfunction associated with diabetic ketoacidosis in rats.

BACKGROUND: Type 1 diabetes mellitus in children may be associated with neurocognitive deficits of unclear cause. A recent retrospective study in children suggested possible associations between diabetic ketoacidosis (DKA) and decreased memory. The current investigation was undertaken to determine whether cognitive deficits could be detected after a single episode of DKA in an animal model. METHODS: Streptozotocin was used to induce diabetes in juvenile rats, and rats were then treated with subcutaneous insulin injections. In one group, insulin was subsequently withdrawn to allow development of DKA, which was then treated with insulin and saline. After recovery from DKA, subcutaneous insulin injections were re-started. In the diabetes control group, rats continued to receive subcutaneous insulin and underwent sham procedures identical to the DKA group. One week after recovery, cognitive function was tested using the Morris Water Maze, a procedure that requires rats to locate a hidden platform in a water pool using visual cues. During a 10 day period, mean time to locate the platform (latency) during 4 trials per day was recorded. RESULTS: Comparison of latency curves demonstrated longer mean latency times on days 7 and 8 in the DKA group indicating delayed learning compared to diabetic controls. CONCLUSIONS: These data demonstrate that a single DKA episode results in measurable deficits in learning in rats, consistent with findings that DKA may contribute to neurocognitive deficits in children with type 1 diabetes.

Ischemia-induced stimulation of cerebral microvascular endothelial cell Na-K-Cl cotransport involves p38 and JNK MAP kinases.

Previous studies have provided evidence that, in the early hours of ischemic stroke, a luminal membrane blood-brain barrier (BBB) Na-K-Cl cotransporter (NKCC) participates in ischemia-induced cerebral edema formation. Inhibition of BBB NKCC activity by intravenous bumetanide significantly reduces edema and infarct in the rat permanent middle cerebral artery occlusion model of ischemic stroke. We demonstrated previously that the BBB cotransporter is stimulated by hypoxia, aglycemia, and AVP, factors present during cerebral ischemia. However, the underlying mechanisms have not been known. Ischemic conditions have been shown to activate p38 and JNK MAP kinases (MAPKs) in brain, and the p38 and JNK inhibitors SB-239063 and SP-600125, respectively, have been found to reduce brain damage following middle cerebral artery occlusion and subarachnoid hemorrhage, respectively. The present study was conducted to determine whether one or both of these MAPKs participates in ischemic factor stimulation of BBB NKCC activity. Cultured cerebral microvascular endothelial cell NKCC activity was evaluated as bumetanide-sensitive (86)Rb influx. Activities of p38 and JNK were assessed by Western blot and immunofluorescence methods using antibodies that detect total vs. phosphorylated (activated) p38 or JNK. We report that p38 and JNK are present in cultured cerebral microvascular endothelial cells and in BBB endothelial cells in situ and that hypoxia (7% O(2) and 2% O(2)), aglycemia, AVP, and O(2)-glucose deprivation (5- to 120-min exposures) all rapidly activate p38 and JNK in the cells. We also provide evidence that SB-239063 and SP-600125 reduce or abolish ischemic factor stimulation of BBB NKCC activity. These findings support the hypothesis that ischemic factor stimulation of the BBB NKCC involves activation of p38 and JNK MAPKs.

The KCa3.1 blocker TRAM-34 reduces infarction and neurological deficit in a rat model of ischemia/reperfusion stroke.

Microglia and brain infiltrating macrophages significantly contribute to the secondary inflammatory damage in the wake of ischemic stroke. Here, we investigated whether inhibition of KCa3.1 (IKCa1/KCNN4), a calcium-activated K(+) channel that is involved in microglia and macrophage activation and expression of which increases on microglia in the infarcted area, has beneficial effects in a rat model of ischemic stroke. Using an HPLC/MS assay, we first confirmed that our small molecule KCa3.1 blocker TRAM-34 effectively penetrates into the brain and achieves micromolar plasma and brain concentrations after intraperitoneal injection. Then, we subjected male Wistar rats to 90 minutes of middle cerebral artery occlusion (MCAO) and administered either vehicle or TRAM-34 (10 or 40 mg/kg intraperitoneally twice daily) for 7 days starting 12 hours after reperfusion. Both compound doses reduced infarct area by ≈ 50% as determined by hematoxylin & eosin staining on day 7 and the higher dose also significantly improved neurological deficit. We further observed a significant reduction in ED1(+)-activated microglia and TUNEL-positive neurons as well as increases in NeuN(+) neurons in the infarcted hemisphere. Our findings suggest that KCa3.1 blockade constitutes an attractive approach for the treatment of ischemic stroke because it is still effective when initiated 12 hours after the insult.

Ischemia-induced stimulation of Na-K-Cl cotransport in cerebral microvascular endothelial cells involves AMP kinase.

Increased blood-brain barrier (BBB) Na-K-Cl cotransporter activity appears to contribute to cerebral edema formation during ischemic stroke. We have shown previously that inhibition of BBB Na-K-Cl cotransporter activity reduces edema and infarct in the rat middle cerebral artery occlusion (MCAO) model of ischemic stroke. We have also shown that the BBB cotransporter is stimulated by the ischemic factors hypoxia, aglycemia, and arginine vasopressin (AVP), although the mechanisms responsible are not well understood. AMP-activated protein kinase (AMPK), a key mediator of cell responses to stress, can be activated by a variety of stresses, including ischemia, hypoxia, and aglycemia. Previous studies have shown that the AMPK inhibitor Compound C significantly reduces infarct in mouse MCAO. The present study was conducted to evaluate the possibility that AMPK participates in ischemic factor-induced stimulation of the BBB Na-K-Cl cotransporter. Cerebral microvascular endothelial cells (CMEC) were assessed for Na-K-Cl cotransporter activity as bumetanide-sensitive (86)Rb influx. AMPK activity was assessed by Western blot analysis and immunofluorescence methods using antibodies that detect total versus phosphorylated (activated) AMPK. We found that hypoxia (7% and 2% O(2)), aglycemia, AVP, and oxygen-glucose deprivation (5- to 120-min exposures) increase activation of AMPK. We also found that Compound C inhibition of AMPK reduces hypoxia-, aglycemia-, and AVP-induced stimulation of CMEC Na-K-Cl cotransporter activity. Confocal immunofluorescence of perfusion-fixed rat brain slices revealed the presence of AMPK, both total and phosphorylated kinase, in BBB in situ of both control and ischemic brain. These findings suggest that ischemic factor stimulation of the BBB Na-K-Cl cotransporter involves activation of AMPK.

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

In the early hours of ischemic stroke, cerebral edema forms as Na, Cl, and water are secreted across the blood-brain barrier (BBB) and astrocytes swell. We have shown previously that ischemic factors, including hypoxia, aglycemia, and arginine vasopressin (AVP), stimulate BBB Na-K-Cl cotransporter (NKCC) and Na/H exchanger (NHE) activities and that inhibiting NKCC and/or NHE by intravenous bumetanide and/or HOE-642 reduces edema and infarct in a rat model of ischemic stroke. Estradiol also reduces edema and infarct in this model and abolishes ischemic factor stimulation of BBB NKCC and NHE. There is evidence that NKCC and NHE also participate in ischemia-induced swelling of astrocytes. However, little is known about estradiol effects on astrocyte cell volume. In this study, we evaluated the effects of AVP (100 nM), hypoxia (7.5% O(2)), aglycemia, hypoxia (2%)/aglycemia [oxygen glucose deprivation (OGD)], and estradiol (1-100 nM) on astrocyte cell volume using 3-O-methyl-d-[(3)H]glucose equilibration methods. We found that AVP, hypoxia, aglycemia, and OGD (30 min to 5 h) each significantly increased astrocyte cell volume, and that estradiol (30-180 min) abolished swelling induced by AVP or hypoxia, but not by aglycemia or OGD. Bumetanide and/or HOE-642 also abolished swelling induced by AVP but not aglycemia. Abundance of aquaporin-4, known to participate in ischemia-induced astrocyte swelling, was significantly reduced following 7-day but not 2- or 3-h estradiol exposures. Our findings suggest that hypoxia, aglycemia, and AVP each contribute to ischemia-induced astrocyte swelling, and that the edema-attenuating effects of estradiol include reduction of hypoxia- and AVP-induced astrocyte swelling and also reduction of aquaporin-4 abundance.