Martha E. O'Donnell

Engaging neuroscience to advance translational research in brain barrier biology

The delivery of many potentially therapeutic and diagnostic compounds to specific areas of the brain is restricted by brain barriers, of which the most well known are the blood–brain barrier (BBB) and the blood–cerebrospinal fluid (CSF) barrier. Recent studies have shown numerous additional roles of these barriers, including an involvement in neurodevelopment, in the control of cerebral blood flow, and — when barrier integrity is impaired — in the pathology of many common CNS disorders such as Alzheimers disease, Parkinsons disease and stroke.

Bumetanide inhibition of the blood-brain barrier Na-K-Cl cotransporter reduces edema formation in the rat middle cerebral artery occlusion model of stroke.

Increased transport of Na+ across an intact blood-brain barrier (BBB) participates in edema formation during the early hours of cerebral ischemia. In previous studies, the authors showed that the BBB Na-K-Cl cotransporter is stimulated by factors present during ischemia, suggesting that the cotransporter may contribute to the increased brain Na+ uptake in edema. The present study was conducted to determine (1) whether the Na-K-Cl cotransporter is located in the luminal membrane of the BBB, and (2) whether inhibition of the BBB cotransporter reduces brain edema formation. Perfusion-fixed rat brains were examined for cotransporter distribution by immunoelectron microscopy. Cerebral edema was evaluated in rats subjected to permanent middle cerebral artery occlusion (MCAO) by magnetic resonance diffusion-weighted imaging and calculation of apparent diffusion coefficients (ADC). The immunoelectron microscopy studies revealed a predominant (80%) luminal membrane distribution of the cotransporter. Magnetic resonance imaging studies showed ADC ratios (ipsilateral MCAO/contralateral control) ranging from 0.577 to 0.637 in cortex and striatum, indicating substantial edema formation. Intravenous bumetanide (7.6–30.4 mg/kg) given immediately before occlusion attenuated the decrease in ADC ratios for both cortex and striatum (by 40–67%), indicating reduced edema formation. Bumetanide also reduced infarct size, determined by TTC staining. These findings suggest that a luminal BBB Na-K-Cl cotransporter contributes to edema formation during cerebral ischemia.

Estradiol Reduces Activity of the Blood–Brain Barrier Na–K–Cl Cotransporter and Decreases Edema Formation in Permanent Middle Cerebral Artery Occlusion

Estrogen has been shown to protect against stroke-induced brain damage, yet the mechanism is unknown. During the early hours of stroke, cerebral edema forms as increased transport of Na and Cl from blood into brain occurs across an intact blood–brain barrier (BBB). We showed previously that a luminal BBB Na–K–Cl cotransporter is stimulated by hypoxia and arginine vasopressin (AVP), factors present during cerebral ischemia, and that inhibition of the cotransporter by intravenous bumetanide greatly reduces edema in rats subjected to permanent middle cerebral artery occlusion (MCAO). The present study was conducted to determine whether estrogen protects in stroke at least in part by reducing activity of the BBB cotransporter, thereby decreasing edema formation. Ovariectomized rats were subjected to 210 mins of permanent MCAO after 7-day or 30-min pretreatment with 17 β-estradiol and then brain swelling and 2,3,5-triphenyltetrazolium chloride staining were assessed as measures of brain edema and lesion volume, respectively. Diffusion-weighed imaging was used to monitor permanent MCAO-induced decreases in apparent diffusion coefficient (ADC) values, an index of changes in brain water distribution and mobility. Na–K–Cl cotransporter activity of cerebral microvascular endothelial cells (CMECs) was assessed as bumetanide-sensitive K influx and cotransporter abundance by Western blot analysis after estradiol treatment. Estradiol significantly decreased brain swelling and lesion volume and attenuated the decrease in ADC values during permanent MCAO. Estradiol also abolished CMEC cotransporter stimulation by chemical hypoxia or AVP and decreased cotransporter abundance. These findings support the hypothesis that estrogen attenuates stimulation of BBB Na–K–Cl cotransporter activity, reducing edema formation during stroke.

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.

Cerebral microvascular endothelial cell Na/H exchange: evidence for the presence of NHE1 and NHE2 isoforms and regulation by arginine vasopressin

Blood-brain barrier (BBB) Na transporters are essential for brain water and electrolyte homeostasis. However, they also contribute to edema formation during the early hours of ischemic stroke by increased transport of Na from blood into brain across an intact BBB. We previously showed that a luminal BBB Na-K-Cl cotransporter is stimulated by hypoxia, aglycemia, and AVP and that inhibition of the cotransporter by intravenous bumetanide significantly reduces edema and infarct in the rat middle cerebral artery occlusion (MCAO) model of stroke. More recently, we found evidence that intravenous cariporide (HOE-642), a highly potent Na/H exchange inhibitor, also reduces brain edema after MCAO. The present study was conducted to investigate which Na/H exchange protein isoforms are present in BBB endothelial cells and to evaluate the effects of ischemic factors on BBB Na/H exchange activity. Western blot analysis of bovine cerebral microvascular endothelial cells (CMEC) and immunoelectron microscopy of perfusion-fixed rat brain revealed that Na/H exchanger isoforms 1 and 2 (NHE1 and NHE2) are present in BBB endothelial cells. Using microspectrofluorometry and the pH-sensitive dye BCECF, we found that hypoxia (2% O(2), 30 min), aglycemia (30 min), and AVP (1-200 nM, 5 min) significantly increased CMEC Na/H exchange activity, assessed as Na-dependent, HOE-642-sensitive H(+) flux. We found that AVP stimulation of CMEC Na/H exchange activity is dependent on intracellular Ca concentration and is blocked by V(1), but not V(2), vasopressin receptor antagonists. Our findings support the hypothesis that a BBB Na/H exchanger, possibly NHE1 and/or NHE2, is stimulated during ischemia to participate in cerebral edema formation.

Cerebral Blood Flow and Cerebral Edema in Rats With Diabetic Ketoacidosis

OBJECTIVE— Cerebral edema (CE) is a potentially life-threatening complication of diabetic ketoacidosis (DKA) in children. Osmotic fluctuations during DKA treatment have been considered responsible, but recent data instead suggest that cerebral hypoperfusion may be involved and that activation of cerebral ion transporters may occur. Diminished cerebral blood flow (CBF) during DKA, however, has not been previously demonstrated. We investigated CBF and edema formation in a rat model of DKA and determined the effects of bumetanide, an inhibitor of Na-K-Cl cotransport. RESEARCH DESIGN AND METHODS— Juvenile rats with streptozotocin-induced DKA were treated with intravenous saline and insulin, similar to human treatment protocols. CBF was determined by magnetic resonance (MR) perfusion–weighted imaging before and during treatment, and CE was assessed by determining apparent diffusion coefficients (ADCs) using MR diffusion–weighted imaging. RESULTS— CBF was significantly reduced in DKA and was responsive to alterations in pCO2. ADC values were reduced, consistent with cell swelling. The reduction in ADCs correlated with dehydration, as reflected in blood urea nitrogen concentrations. Bumetanide caused a rapid rise in ADCs of DKA rats without significantly changing CBF, while saline/insulin caused a rapid rise in CBF and a gradual rise in ADCs. DKA rats treated with bumetanide plus saline/insulin showed a trend toward more rapid rise in cortical ADCs and a larger rise in striatal CBF than those observed with saline/insulin alone. CONCLUSIONS— These data demonstrate that CE in DKA is accompanied by cerebral hypoperfusion before treatment and suggest that blocking Na-K-Cl cotransport may reduce cerebral cell swelling.

Intracellular Cl regulates Na-K-Cl cotransport activity in human trabecular meshwork cells

The trabecular meshwork (TM) of the eye plays a central role in modulating intraocular pressure by regulating aqueous humor outflow, although the mechanisms are largely unknown. We and others have shown previously that aqueous humor outflow facility is modulated by conditions that alter TM cell volume. We have also shown that the Na-K-Cl cotransport system is a primary regulator of TM cell volume and that its activity appears to be coordinated with net efflux pathways to maintain steady-state volume. However, the cellular mechanisms that regulate cotransport activity and cell volume in TM cells have yet to be elucidated. The present study was conducted to investigate the hypothesis that intracellular Cl concentration ([Cl]i) acts to regulate TM cell Na-K-Cl cotransport activity, as has been shown previously for some other cell types. We demonstrate here that the human TM cell Na-K-Cl cotransporter is highly sensitive to changes in [Cl]i. Our findings reveal a marked stimulation of Na-K-Cl cotransport activity, assessed as ouabain-insensitive, bumetanide-sensitive K influx, in TM cells following preincubation of cells with Cl-free medium as a means of reducing [Cl]i. In contrast, preincubation of cells with media containing elevated K concentrations as a means of increasing [Cl]i results in inhibition of Na-K-Cl cotransport activity. The effects of reducing [Cl]i, as well as elevating [Cl]i, on Na-K-Cl cotransport activity are concentration dependent. Furthermore, the stimulatory effect of reduced [Cl]i is additive with cell-shrinkage-induced stimulation of the cotransporter. Our studies also show that TM cell Na-K-Cl cotransport activity is altered by a variety of Cl channel modulators, presumably through changes in [Cl]i. These findings support the hypothesis that regulation of Na-K-Cl cotransport activity, and thus cell volume, by [Cl]i may participate in modulating outflow facility across the TM.

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.

Effect of cell shrinkage on permeability of cultured bovine aortic endothelia and frog mesenteric capillaries

1 We have tested the hypothesis that a reduction in endothelial cell volume increases microvessel permeability and that the degree of endothelial cell attachment to their basement membranes determines the magnitude of permeability changes caused by a reduction in endothelial cell volume. 2 A decrease in endothelial cell volume was imposed on both intact microvessels and cultured endothelial monolayers by raising osmolarity by 100 mosmol l−1 3 We found that hypertonic solutions did not increase the hydraulic permeability (Lp) of individually perfused venular microvessels in frog mesentery when the perfusate contained albumin. Hypertonic solutions did increase Lp, however, after we perfused the microvessels with the peptide Gly‐Arg‐Gly‐Asp‐Thr‐Pro (GEGDTP; 0.3 mmol l−1), to disrupt integrin‐dependent endothelial cell (EC) attachment to the extracellular matrix (ECM). 4 After albumin was removed from the perfusate, hypertonic solutions increased Lp of microvessels and the permeability of endothelial monolayers to α‐lactalbumin. 5 Our findings indicate that endothelial cell integrin‐ECM binding plays a role in transducing changes in cell volume and/or shape into changes in permeability. We hypothesize that removal of albumin from the vascular perfusate may compromise EC–ECM interactions via an integrin‐dependent mechanism.

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.