Triglyceride-rich lipoprotein lipolysis products increase blood-brain barrier transfer coefficient and induce astrocyte lipid droplets and cell stress.

Elevation of blood triglycerides, primarily as triglyceride-rich lipoproteins (TGRL), has been linked to cerebrovascular inflammation, vascular dementia, and Alzheimer's disease (AD). Brain microvascular endothelial cells and astrocytes, two cell components of the neurovascular unit, participate in controlling blood-brain barrier (BBB) permeability and regulating neurovascular unit homeostasis. Our studies showed that infusion of high physiological concentrations of TGRL lipolysis products (TGRL + lipoprotein lipase) activate and injure brain endothelial cells and transiently increase the BBB transfer coefficient (Ki = permeability × surface area/volume) in vivo. However, little is known about how blood lipids affect astrocyte lipid accumulation and inflammation. To address this, we first demonstrated TGRL lipolysis products increased lipid droplet formation in cultured normal human astrocytes. We then evaluated the transcriptional pathways activated in astrocytes by TGRL lipolysis products and found upregulated stress and inflammatory-related genes including activating transcription factor 3 (ATF3), macrophage inflammatory protein-3α (MIP-3α), growth differentiation factor-15 (GDF15), and prostaglandin-endoperoxide synthase 2 (COX2). TGRL lipolysis products also activated the JNK/cJUN/ATF3 pathway, induced endoplasmic reticulum stress protein C/EBP homologous protein (CHOP), and the NF-κB pathway, while increasing secretion of MIP-3α, GDF15, and IL-8. Thus our results demonstrate TGRL lipolysis products increase the BBB transfer coefficient (Ki), induce astrocyte lipid droplet formation, activate cell stress pathways, and induce secretion of inflammatory cytokines. Our observations are consistent with evidence for lipid-induced neurovascular injury and inflammation, and we, therefore, speculate that lipid-induced astrocyte injury could play a role in cognitive decline.

MRI measurement of blood-brain barrier transport with a rapid acquisition refocused echo (RARE) method.

Dynamic Contrast Enhanced (DCE) MRI is increasingly being used to assess changes in capillary permeability. Most quantitative techniques used to measure capillary permeability are based on the Fick equation that requires measurement of signal reflecting both plasma and tissue concentrations of the solute being tested. To date, most Magnetic Resonance Imaging (MRI) methods for acquiring appropriate data quickly rely on gradient recalled echo (GRE) type acquisitions, which work well in clinical low field settings. However, acquiring this type of data on high field small animal preclinical MRIs is problematic due to geometrical distortions from susceptibility mismatch. This problem can be exacerbated when using small animal models to measure blood brain barrier (BBB) permeability, where precise sampling from the superior sagittal sinus (SSS) is commonly used to determine the plasma concentration of the contrast agent. Here we present results demonstrating that a standard saturation recovery rapid acquisition refocused echo (RARE) method is capable of acquiring T1 maps with good spatial and temporal resolution for Patlak analysis (Patlak, 1983) to assess changes in BBB Gd-DTPA permeability following middle cerebral artery occlusion with reperfusion in the rat. This method limits known problems with magnetic susceptibility mismatch and may thus allow greater accuracy in BBB permeability measurement in small animals.

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.

Acute mountain sickness prophylaxis: a high-altitude perspective.

Acute mountain sickness (AMS) is the most common form of altitude illness affecting athletes and adventurists who work or play at elevations greater than 10,000 ft above mean sea level. Considerable research has been conducted to mitigate risk for those who work or play in environmental extremes. This article describes the experiences of a group of U.S. Army Special Operations soldiers who tested recommended doses of acetazolamide prophylaxis for AMS during six expeditions to elevations between 19,000 and 23,000 ft. In addition, we briefly review the literature as it pertains to prophylaxis of AMS. In our experience, prophylaxis with the recommended doses of acetazolamide resulted in fewer AMS symptoms and seemed to confer a higher likelihood of successfully summiting each peak. We conclude that acetazolamide is an acceptable choice for AMS prevention along with a slow, controlled ascent and proper fitness, nutrition, clothing, and gear.

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.

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 17beta-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.

Bumetanide reduces cerebral edema formation in rats with diabetic ketoacidosis.

The mechanisms responsible for cerebral edema formation in diabetic ketoacidosis (DKA) are not well understood, although evidence suggests ischemia as a contributing factor. Previous studies have shown that the Na-K-Cl cotransporter of cerebral microvascular endothelial cells and astrocytes is a major participant in ischemia-induced cerebral edema in stroke. The present study was conducted to test the hypothesis that the Na-K-Cl cotransporter also contributes to cerebral edema in DKA. Sprague-Dawley rats were administered streptozotocin to induce DKA, and then cerebral edema was assessed by determination of apparent diffusion coefficients (ADC) with magnetic resonance diffusion-weighted imaging. Cerebral ADC values in DKA rats were significantly reduced in both cortex and striatum compared with non-DKA control rats, indicating the presence of cerebral edema. Intravenous administration of bumetanide to DKA rats abolished the drop in cortical ADC values, while having no significant effect in the striatum. Insulin and saline treatment had no effect when given after bumetanide but increased both cortical and striatal ADC values when given before bumetanide. Evidence is also presented here that acetoacetate and beta-hydroxybutyrate stimulate brain microvascular Na-K-Cl cotransporter activity. These findings suggest that the Na-K-Cl cotransporter contributes to brain edema in DKA.

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.

The role of the blood-brain barrier Na-K-2Cl cotransporter in stroke.

Studies from this and other laboratories have shown that the Na-K-2Cl cotransporter is present in BBB endothelial cells is stimulated by factors present during cerebral ischemia. Further, our in situ studies have shown that the cotransporter resides predominantly in the luminal BBB membrane. This is consistent with the hypothesis that a luminal cotransporter works with abluminal Na/K ATPase to secrete NaCl into the brain, and during stroke, BBB cotransporter activity is increased such that the barrier hypersecretes NaCl and water into the brain, facilitating cytotoxic edema formation. Our in vivo MCAO stroke studies provide further support for a role of the BBB cotransporter in cerebral ede-ma formation. Collectively, these findings suggest that the BBB Na-K-2Cl cotransporter does indeed substantially contribute to cerebral edema formation in stroke.

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.

Cerebral metabolic alterations in rats with diabetic ketoacidosis: effects of treatment with insulin and intravenous fluids and effects of bumetanide.

OBJECTIVE: Cerebral edema is a life-threatening complication of diabetic ketoacidosis (DKA) in children. Recent data suggest that cerebral hypoperfusion and activation of cerebral ion transporters may be involved, but data describing cerebral metabolic alterations during DKA are lacking. RESEARCH DESIGN AND METHODS: We evaluated 50 juvenile rats with DKA and 21 normal control rats using proton and phosphorus magnetic resonance spectroscopy (MRS). MRS measured cerebral intracellular pH and ratios of metabolites including ATP/inorganic phosphate (Pi), phosphocreatine (PCr)/Pi, N-acetyl aspartate (NAA)/creatine (Cr), and lactate/Cr before and during DKA treatment. We determined the effects of treatment with insulin and intravenous saline with or without bumetanide, an inhibitor of Na-K-2Cl cotransport, using ANCOVA with a 2 x 2 factorial study design. RESULTS: Cerebral intracellular pH was decreased during DKA compared with control (mean +/- SE difference -0.13 +/- 0.03; P < 0.001), and lactate/Cr was elevated (0.09 +/- 0.02; P < 0.001). DKA rats had lower ATP/Pi and NAA/Cr (-0.32 +/- 0.10, P = 0.003, and -0.14 +/- 0.04, P < 0.001, respectively) compared with controls, but PCr/Pi was not significantly decreased. During 2-h treatment with insulin/saline, ATP/Pi, PCr/Pi, and NAA/Cr declined significantly despite an increase in intracellular pH. Bumetanide treatment increased ATP/Pi and PCr/Pi and ameliorated the declines in these values with insulin/saline treatment. CONCLUSIONS: These data demonstrate that cerebral metabolism is significantly compromised during DKA and that further deterioration occurs during early DKA treatment--consistent with possible effects of cerebral hypoperfusion and reperfusion injury. Treatment with bumetanide may help diminish the adverse effects of initial treatment with insulin/saline.

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 pCO(2). 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.

Na+/Ca2+-exchanger-mediated Mn2+-enhanced (1)H2O MRI in hypoxic, perfused rat myocardium.

Paramagnetic Mn2+ has emerged in the search for non-invasive magnetic resonance imaging (MRI) techniques to monitor Ca2+ in diagnostic and prognostic cardiovascular disease tests because it both alters MRI contrast and behaves as a Ca2+ 'surrogate' in vivo. However, the reliance on macroscopically averaged measurements to infer microscopic processes constitutes a major limitation of MRI. This investigation circumvents this limitation and contributes an MRI-based myocardial Ca2+-transporter assay, which probes the Na+/Ca2+-exchanger involvement in Mn2+ (and presumably Ca2+) transport by virtue of its response to pharmacological inhibition. In the model employed herein, ex vivo arrested rat hearts underwent normoxia and then hypoxia while a constant (hyperkalemic) perfusion minimized flow (and uncontrolled Ca2+-channel) contributions to Mn2+-enhanced MRI measurements. The results (i) demonstrate that Mn2+ (and presumably Ca2+) accumulates via Na+/Ca2+-exchanger-mediated transport during hyperkalemic hypoxia and further, (ii) implicate hypo-perfusion (rather than the diminished participation of an isolated sarcolemmal Ca2+-transporter) as the mechanism that underlies the reported reductions of Mn2+ accumulation (relative to healthy myocardium) subsequent to myocardial insults in MRI studies. Although myriad studies have employed Mn2+-enhanced MRI in myocardial investigations, this appears to be the first attempt to assay the Na+/Ca2+-exchanger with MRI under highly circumscribed conditions. MRI-based Ca2+)transporter assays, such as the Na+/Ca2+-exchanger assay utilized here, will inevitably impact disciplines in the medical sciences and beyond.

Effects of cold cardioplegia on pH, Na, and Ca in newborn rabbit hearts.

Many studies suggest myocardial ischemia-reperfusion (I/R) injury results largely from cytosolic proton (H(i))-stimulated increases in cytosolic Na (Na(i)), which cause Na/Ca exchange-mediated increases in cytosolic Ca concentration ([Ca]i). Because cold, crystalloid cardioplegia (CCC) limits [H]i, we tested the hypothesis that in newborn hearts, CCC diminishes H(i), Na(i), and Ca(i) accumulation during I/R to limit injury. NMR measured intracellular pH (pH(i)), Na(i), [Ca]i, and ATP in isolated Langendorff-perfused newborn rabbit hearts. The control ischemia protocol was 30 min for baseline perfusion, 40 min for global ischemia, and 40 min for reperfusion, all at 37 degrees C. CCC protocols were the same, except that ice-cold CCC was infused for 5 min before ischemia and heart temperature was lowered to 12 degrees C during ischemia. Normal potassium CCC solution (NKCCC) was identical to the control perfusate, except for temperature; the high potassium (HKCCC) was identical to NKCCC, except that an additional 11 mmol/l KCl was substituted isosmotically for NaCl. NKCCC and HKCCC were not significantly different for any measurement. The following were different (P < 0.05). End-ischemia pH(i) was higher in the CCC than in the control group. Similarly, CCC limited increases in Na(i) during I/R. End-ischemia Na(i) values (in meq/kg dry wt) were 115 +/- 16 in the control group, 49 +/- 13 in the NKCCC group, and 37 +/- 12 in the HKCCC group. CCC also improved [Ca]i recovery during reperfusion. After 40 min of reperfusion, [Ca](i) values (in nmol/l) were 302 +/- 50 in the control group, 145 +/- 13 in the NKCCC group, and 182 +/- 19 in the HKCCC group. CCC limited ATP depletion during ischemia and improved recovery of ATP and left ventricular developed pressure and decreased creatine kinase release during reperfusion. Surprisingly, CCC did not significantly limit [Ca]i during ischemia. The latter is explained as the result of Ca release from intracellular buffers on cooling.

Acute effects of 17beta-estradiol on myocardial pH, Na+, and Ca2+ and ischemia-reperfusion injury.

Evidence suggests that 1) ischemia-reperfusion injury is due largely to cytosolic Ca(2+) accumulation resulting from functional coupling of Na(+)/Ca(2+) exchange (NCE) with stimulated Na(+)/H(+) exchange (NHE1) and 2) 17beta-estradiol (E2) stimulates release of NO, which inhibits NHE1. Thus we tested the hypothesis that acute E2 limits myocardial Na(+) and therefore Ca(2+) accumulation, thereby limiting ischemia-reperfusion injury. NMR was used to measure cytosolic pH (pH(i)), Na(+) (Na(i)(+)), and calcium concentration ([Ca(2+)](i)) in Krebs-Henseleit (KH)-perfused hearts from ovariectomized rats (OVX). Left ventricular developed pressure (LVDP) and lactate dehydrogenase (LDH) release were also measured. Control ischemia-reperfusion was 20 min of baseline perfusion, 40 min of global ischemia, and 40 min of reperfusion. The E2 protocol was identical, except that 1 nM E2 was included in the perfusate before ischemia and during reperfusion. E2 significantly limited the changes in pH(i), Na(i)(+), and [Ca(2+)](i) during ischemia (P < 0.05). In control OVX vs. OVX+E2, pH(i) fell from 6.93 +/- 0.03 to 5.98 +/- 0.04 vs. 6.96 +/- 0.04 to 6.68 +/- 0.07; Na(i)(+) rose from 25 +/- 6 to 109 +/- 14 meq/kg dry wt vs. 25 +/- 1 to 76 +/- 3; [Ca(2+)](i) changed from 365 +/- 69 to 1,248 +/- 180 nM vs. 293 +/- 66 to 202 +/- 64 nM. E2 also improved recovery of LVDP and diminished release of LDH during reperfusion. Effects of E2 were diminished by 1 microM N(omega)-nitro-L-arginine methyl ester. Thus the data are consistent with the hypothesis. However, E2 limitation of increases in [Ca(2+)](i) is greater than can be accounted for by the thermodynamic effect of reduced Na(i)(+) accumulation on NCE.