Dandan Sun

Molecular Mechanisms of Ischemic Cerebral Edema: Role of Electroneutral Ion Transport

The brain achieves homeostasis of its intracellular and extracellular fluids by precisely regulating the transport of solute and water across its major cellular barriers: endothelia of the blood-brain barrier (BBB), choroid plexus epithelia, and neuroglial cell membranes. Cerebral edema, the pathological accumulation of fluid in the brains intracellular and extracellular spaces, is a major cause of morbidity and mortality following stroke and other forms of ischemic brain injury. Until recently, mechanisms of cerebral edema formation have been obscure; consequently, its treatment has been empiric and suboptimal. Here, we provide a paradigm for understanding ischemic cerebral edema, showing that its molecular pathogenesis is a complex yet step-wise process that results largely from impaired astrocytic cell volume regulation and permeability alterations in the cerebral microvasculature, both of which arise from pathological changes in the activities of specific ion channels and transporters. Recent data has implicated the bumetanide-sensitive NKCC1, an electroneutral cotransporter expressed in astrocytes and the BBB, in cerebral edema formation in several different rodent models of stroke. Pharmacological inhibition or genetic deficiency of NKCC1 decreases ischemia-induced cell swelling, BBB breakdown, cerebral edema, and neurotoxicity. Combination pharmacological strategies that include NKCC1 as a target might thus prove beneficial for the treatment of ischemic, and potentially other types of, cerebral edema.

Na+-Dependent Chloride Transporter (NKCC1)-Null Mice Exhibit Less Gray and White Matter Damage after Focal Cerebral Ischemia

We previously demonstrated that pharmacological inhibition of Na+−K+−Cl− cotransporter isoform 1 (NKCC1) is neuroprotective in in vivo and in vitro ischemic models. In this study, we investigated whether genetic ablation of NKCC1 provides neuroprotection after ischemia. Focal ischemia was induced by 2 hours occlusion of the left middle cerebral artery (MCAO) followed by 10 or 24 hours reperfusion. Two hours MCAO and ten or twenty-four hours reperfusion caused infarction (˜85 mm3) in NKCC1 wild-type (NKCC1+/+) mice. Infarction volume in NKCC1−/− mice was reduced by ˜30% to 46%. Heterozygous mutant (NKCC1+/–) mice showed ˜28% reduction in infarction (P>0.05). Two hours MCAO and twenty-four hours reperfusion led to a significant increase in brain edema in NKCC1+/+ mice. In contrast, NKCC1+/– and NKCC1−/− mice exhibited ˜50% less edema (P<0.05). Moreover, white matter damage was assessed by immunostaining of amyloid precursor protein (APP). An increase in APP was detected in NKCC1+/+ mice after 2 hours MCAO and 10 hours reperfusion. However, NKCC1−/− mice exhibited significantly less APP accumulation (P<0.05). Oxygen-glucose deprivation (OGD) induced ˜67% cell death and a fourfold increase in Na+ accumulation in cultured NKCC1+/+ cortical neurons. OGD-mediated cell death and Na+ influx were significantly reduced in NKCC1−/− neurons (P<0.05). In addition, inhibition of NKCC1 by bumetanide resulted in similar protection in NKCC1+/+ neurons and astrocytes (P<0.05). These results imply that stimulation of NKCC1 activity is important in ischemic neuronal damage.

Na-K-Cl Cotransporter-Mediated Intracellular Na+ Accumulation Affects Ca2+ Signaling in Astrocytes in an In Vitro Ischemic Model

Na-K-Cl cotransporter isoform 1 (NKCC1) plays an important role in maintenance of intracellular Na+, K+, and Cl- levels in astrocytes. We propose that NKCC1 may contribute to perturbations of ionic homeostasis in astrocytes under ischemic conditions. After 3-8 hr of oxygen and glucose deprivation (OGD), NKCC1-mediated 86Rb influx was significantly increased in astrocytes from NKCC1 wild-type (NKCC1+/+) and heterozygous mutant (NKCC1+/-) mice. Phosphorylated NKCC1 protein was increased in NKCC1+/+ astrocytes at 2 hr of OGD. Two hours of OGD and 1 hr of reoxygenation (OGD/REOX) triggered an ∼3.6-fold increase in intracellular Na+ concentration ([Na+]i) in NKCC1+/+ astrocytes. Inhibition of NKCC1 activity by bumetanide or ablation of the NKCC1 gene significantly attenuated the rise in [Na+]i. Moreover, NKCC1+/+ astrocytes swelled by 10-30% during 20-60 min of OGD. Either genetic ablation of NKCC1 or inhibition of NKCC1 by bumetanide-attenuated OGD-mediated swelling. An NKCC1-mediated increase in [Na+]i may subsequently affect Ca2+ signaling through the Na+/Ca2+ exchanger (NCX). A rise in [Ca2+]i was detected after OGD/REOX in the presence of a sarcoplasmic-endoplasmic reticulum (ER) Ca2+-ATPase inhibitor thapsigargin. Moreover, OGD/REOX led to a significant increase in Ca2+ release from ER Ca2+ stores. Furthermore, KB-R7943 (2-[2-[4(4-nitrobenzyloxy)phenyl]ethyl]isothiourea mesylate), an inhibitor of reverse-mode operation of NCX, abolished the OGD/REOX-induced enhancement in filling of ER Ca2+ stores. OGD/REOX-mediated Ca2+ accumulation in ER Ca2+ stores was absent when NKCC1 activity was ablated or pharmacologically inhibited. These findings imply that stimulation of NKCC1 activity leads to Na+ accumulation after OGD/REOX and that subsequent reverse-mode operation of NCX contributes to increased Ca2+ accumulation by intracellular Ca2+ stores.

Inhibition of Na(+)-K(+)-Cl(-) cotransporter during focal cerebral ischemia decreases edema and neuronal damage.

Our previous study demonstrated that pharmacological inhibition of the Na(+)-K(+)-Cl(-) cotransporter isoform 1 (NKCC1) during ischemia and reperfusion attenuated neuronal damage and edema. In this study, we further investigated whether NKCC1 activity contributes to ischemic damage during either ischemia or reperfusion. Immunoblotting revealed that expression of NKCC1 protein was increased following 2-h focal ischemia in cerebral cortex. A sustained up-regulation of NKCC1 in cortex was detected at 4, 8, 12, and 24 h of reperfusion. An increase in the phosphorylated NKCC1 (NKCC1-p) was found at 4 and 8 h of reperfusion. In striatum, a significant increase in NKCC1 expression occurred between 4 and 24 h of reperfusion and no elevation of NKCC1-p signal was observed. Artificial cerebral spinal fluid (aCSF) or 100 microM bumetanide in aCSF were continuously microdialyzed into left cortices either 1 h prior to ischemia plus 2-h ischemia, or only during 24-h reperfusion. Infarction volume was significantly decreased in the pre-ischemic bumetanide-treated group (P<0.05) but not in the post-ischemic treatment group (P>0.05). In addition, pre-ischemic bumetanide treatment reduced the ipsilateral water content increase by 70% (P<0.05). Inhibition of NKCC1 did not attenuate poly (ADP-ribose) polymerase cleavage or the number of TUNEL-labeled apoptotic cells in ischemic brains. These results suggest that inhibition of NKCC1 attenuates cytotoxic edema and necrotic neuronal death during focal ischemia. Activation of NKCC1 activity plays a role in the early stage of ischemic damage.

Decreased Neuronal Death in Na+/H+ Exchanger Isoform 1-Null Mice after In Vitro and In Vivo Ischemia

Na+/H+ exchanger isoform 1 (NHE1) is a major acid extrusion mechanism after intracellular acidosis. We hypothesized that stimulation of NHE1 after cerebral ischemia contributes to the disruption of Na+ homeostasis and neuronal death. In the present study, expression of NHE1 was detected in cultured mouse cortical neurons. Three hours of oxygen and glucose deprivation (OGD) followed by 21 h of reoxygenation (REOX) led to 68 ± 10% cell death. Inhibition of NHE1 with the potent inhibitor cariporide (HOE 642) or genetic ablation of NHE1 reduced OGD-induced cell death by ∼40–50% (p < 0.05). In NHE1+/+ neurons, OGD caused a twofold increase in [Na+]i, and 60 min REOX triggered a sevenfold increase. Genetic ablation of NHE1 or HOE 642 treatment had no effects on the OGD-mediated initial Na+i rise but reduced the second phase of Na+i rise by ∼40–50%. In addition, 60 min REOX evoked a 1.5-fold increase in [Ca2+]i in NHE1+/+ neurons, which was abolished by inhibition of either NHE1 or reverse-mode operation of Na+/Ca2+ exchange. OGD/REOX-mediated mitochondrial Ca2+ accumulation and cytochrome c release were attenuated by inhibition of NHE1 activity. In an in vivo focal ischemic model, 2 h of left middle cerebral artery occlusion followed by 24 h of reperfusion induced 84.8 ± 8.0 mm3 infarction in NHE1+/+ mice. NHE1+/+ mice treated with HOE 642 or NHE1 heterozygous mice exhibited a ∼33% decrease in infarct size (p < 0.05). These results imply that NHE1 activity disrupts Na+ and Ca2+ homeostasis and contributes to ischemic neuronal damage.

Na+-K+-Cl- cotransporter in rat focal cerebral ischemia

In cultured neurons, the authors previously demonstrated that the Na+-K+-Cl− cotransporter is significantly stimulated by elevated extracellular potassium and glutamate, which are important factors in cerebral ischemic damage. These findings led the authors to hypothesize that stimulation of the cotransporter after ischemia might result in Na+, K+, and Cl− influx, and might contribute to neuron damage. In the current study, the authors investigated such a role of the Na+-K+-Cl− cotransporter in focal cerebral ischemia. Cerebral ischemia was induced by 2-hour occlusion of the left middle cerebral artery (MCA) and 24-hour reperfusion in male spontaneously hypertensive rats (SHRs). Immunocytochemical staining and immunoblotting revealed an up-regulation of expression of the cotransporter protein in neurons in cortex at 24 hours of reperfusion. Artificial cerebral spinal fluid (aCSF) or 100 μmol/L bumetanide (a cotransporter inhibitor) in aCSF were continuously microdialyzed through a microdialysis probe into left cortices throughout 2-hour MCA occlusion and 24-hour reperfusion. Compared with the aCSF-treated group, infarction volume was significantly reduced in the bumetanide-treated group (25%, P < 0.05). In addition, brain water content in the bumetanide-treated brains was decreased by 70% (P < 0.05). These results strongly suggest that the Na+-K+-Cl− cotransporter may play an important role in cerebral ischemic neuronal damage.

The role of cation-dependent chloride transporters in neuropathic pain following spinal cord injury

BackgroundAltered Cl- homeostasis and GABAergic function are associated with nociceptive input hypersensitivity. This study investigated the role of two major intracellular Cl- regulatory proteins, Na+-K+-Cl- cotransporter 1 (NKCC1) and K+-Cl- cotransporter 2 (KCC2), in neuropathic pain following spinal cord injury (SCI).ResultsSprague-Dawley rats underwent a contusive SCI at T9 using the MASCIS impactor. The rats developed hyperalgesia between days 21 and 42 post-SCI. Thermal hyperalgesia (TH) was determined by a decrease in hindpaw thermal withdrawal latency time (WLT) between days 21 and 42 post-SCI. Rats with TH were then treated with either vehicle (saline containing 0.25% NaOH) or NKCC1 inhibitor bumetanide (BU, 30 mg/kg, i.p.) in vehicle. TH was then re-measured at 1 h post-injection. Administration of BU significantly increased the mean WLT in rats (p < 0.05). The group administered with the vehicle alone showed no anti-hyperalgesic effects. Moreover, an increase in NKCC1 protein expression occurred in the lesion epicenter of the spinal cord during day 2–14 post-SCI and peaked on day 14 post-SCI (p < 0.05). Concurrently, a down-regulation of KCC2 protein was detected during day 2–14 post-SCI. The rats with TH exhibited a sustained loss of KCC2 protein during post-SCI days 21–42. No significant changes of these proteins were detected in the rostral region of the spinal cord.ConclusionTaken together, expression of NKCC1 and KCC2 proteins was differentially altered following SCI. The anti-hyperalgesic effect of NKCC1 inhibition suggests that normal or elevated NKCC1 function and loss of KCC2 function play a role in the development and maintenance of SCI-induced neuropathic pain.

Expression of Na+-K+-Cl− cotransporter in rat brain during development and its localization in mature astrocytes

Na(+)-K(+)-Cl(-) cotransporter has been proposed to play an important role in the regulation of intracellular Cl(-) concentration in neurons during development. In this study, the expression pattern of the cotransporter in different regions of rat brain was examined at birth (P0), postnatal days 7 (P7), P14, P21, and adult by Western blotting analysis. In cortex, thalamus, cerebellum and striatum, the cotransporter expression level was low at P0 and significantly increased at P14 (P<0.05). The expression peaked at P21 and was maintained at the same level in adulthood. However, in hippocampus, a peak level of the cotransporter expression was detected in adult brain. The immunocytochemistry study of adult rat brain revealed that an intense staining of the Na(+)-K(+)-Cl(-) cotransporter protein was observed in dendritic processes of CA1-CA3 hippocampal pyramidal neurons. In contrast, abundant immuno-reactive signals of the cotransporter were found in somata of thalamic nucleus. Immunofluorescence double staining demonstrates that the Na(+)-K(+)-Cl(-) cotransporter was expressed in astrocytes within cortex, corpus callosum, hippocampus and cerebellum. In addition, co-localization of the cotransporter and glial fibrillary acidic protein (GFAP), or with aquaporin 4, was found in perivascular astrocytes of cortical cortex and white matter. The results indicate that a time-dependent expression of the Na(+)-K(+)-Cl(-) cotransporter protein occurs not only in cortex but also in hippocampus, striatum, thalamus and cerebellum. In addition, the cotransporter is expressed in astrocytes and perivascular astrocytes of adult rat brain.

Calcium dysregulation induces apoptosis-inducing factor release: cross-talk between PARP-1- and calpain-signaling pathways.

Recent discoveries show that caspase-independent cell death pathways are a pervasive mechanism in neurodegenerative diseases, and apoptosis-inducing factor (AIF) is an important effector of this mode of neuronal death. There are currently two known mechanisms underlying AIF release following excitotoxic stress, PARP-1 and calpain. To test whether there is an interaction between PARP-1 and calpain in triggering AIF release, we used the NMDA toxicity model in rat primary cortical neurons. Exposure to NMDA resulted in AIF truncation and nuclear translocation, and shRNA-mediated knockdown of AIF resulted in neuroprotection. Both calpain and PARP-1 are involved with AIF processing as AIF truncation, nuclear translocation and neuronal death were attenuated by calpain inhibition using adeno-associated virus-mediated overexpression of the endogenous calpain inhibitor, calpastatin, or treatment with the PARP-1 inhibitor 3-ABA. Activation of PARP-1 is necessary for calpain activation as PARP-1 inhibition blocked mitochondrial calpain activation. Finally, NMDA toxicity induces mitochondrial Ca(2+) dysregulation in a PARP-1 dependent manner. Thus, PARP-1 and mitochondrial calpain activation are linked via PARP-1-induced alterations in mitochondrial Ca(2+) homeostasis. Collectively, these findings link the two seemingly independent mechanisms triggering AIF-induced neuronal death.

Ischemia-Induced Changes in Cerebral Mitochondrial Free Fatty Acids, Phospholipids, and Respiration in the Rat

Abstract: Changes in the free fatty acid pool size and fatty acyl chain composition of mitochondrial membrane phospholipids and their relation to disruption of mitochondrial function were examined in rat brains after 30 min of cerebral ischemia (Pulsinelli‐Brierley model) and 60 min of normoxic reoxygenation. During ischemia, significant hydrolysis of polyunsaturated molecular species from diacyl phosphatidylcholine, particularly fatty acyl 20:4 (arachidonic acid; 20% decrease) and 22:6 (docosahexaenoic acid; 15% decrease), was observed. Thirty minutes of ischemia caused a 16% loss of 18:2 (linoleic acid) from phosphatidylethanolamine. Recirculation for 60 min did not return the polyunsaturated fatty acid content of phospholipids to normal. Total content of free fatty acids increased during ischemia, particularly 18:2 and 22:6, which exhibited the most dramatic rise. The free fatty acid pool size continued to increase during 60 min of recirculation. The respiratory control ratio decreased significantly during 30 min of ischemia with no apparent recovery following 60 min of reoxygenation. The degree of free radical‐mediated lipid peroxidation in mitochondria was significantly increased during ischemia and reperfusion. It was concluded that (a) 30 min of cerebral ischemia caused differential degradation in each of the phospholipid classes and preferential hydrolysis of the polyunsaturated molecular species and (b) 60 min of normoxic reperfusion failed to promote reacylation of the mitochondrial phospholipids and restoration of normal respiration.