Imatinib preserves blood–brain barrier integrity following experimental subarachnoid hemorrhage in rats

Blood–brain barrier (BBB) disruption and consequent edema formation contribute to the development of early brain injury following subarachnoid hemorrhage (SAH). Various cerebrovascular insults result in increased platelet‐derived growth factor receptor (PDGFR)‐α stimulation, which has been linked to BBB breakdown and edema formation. This study examines whether imatinib, a PDGFR inhibitor, can preserve BBB integrity in a rat endovascular perforation SAH model. Imatinib (40 or 120 mg/kg) or a vehicle was administered intraperitoneally at 1 hr after SAH induction. BBB leakage, brain edema, and neurological deficits were evaluated. Total and phosphorylated protein expressions of PDGFR‐α, c‐Src, c‐Jun N‐terminal kinase (JNK), and c‐Jun were measured, and enzymatic activities of matrix metalloproteinase (MMP)?2 and MMP‐9 were determined in the injured brain. Imatinib treatment significantly ameliorated BBB leakage and edema formation 24 hr after SAH, which was paralleled by improved neurological functions. Decreased brain expressions of phosphorylated PDGFR‐α, c‐Src, JNK, and c‐Jun as well as reduced MMP‐9 activities were found in treated animals. PDGFR‐α inhibition preserved BBB integrity following experimental SAH; however, the protective mechanisms remain to be elucidated. Targeting PDGFR‐α signaling might be advantageous to ameliorate early brain injury following SAH. © 2014 Wiley Periodicals, Inc.     

Changes in the astrocytic aquaporin‐4 and inwardly rectifying potassium channel expression in the brain of the amyotrophic lateral sclerosis SOD1G93A rat model

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting upper and lower motor neurons. Dysfunction and death of motor neurons are closely related to the modified astrocytic environment. Astrocytic endfeet, lining the blood–brain barrier (BBB), are enriched in two proteins, aquaporin‐4 (AQP4) and inwardly rectifying potassium channel (Kir) 4.1. Both channels are important for the maintainance of a functional BBB astrocytic lining. In this study, expression levels of AQP4 and Kir4.1 were for the first time examined in the brainstem and cortex, along with the functional properties of Kir channels in cultured cortical astrocytes of the SOD1^G93A rat model of ALS. Western blot analysis showed increased expression of AQP4 and decreased expression of Kir4.1 in the brainstem and cortex of the ALS rat. In addition, higher immunoreactivity of AQP4 and reduced immunolabeling of Kir4.1 in facial and trigeminal nuclei as well as in the motor cortex were also observed. Particularly, the observed changes in the expression of both channels were retained in cultured astrocytes. Furthermore, whole‐cell patch‐clamp recordings from cultured ALS cortical astrocytes showed a significantly lower Kir current density. Importantly, the potassium uptake current in ALS astrocytes was significantly reduced at all extracellular potassium concentrations. Consequently, the Kir‐specific Cs⁺‐ and Ba²⁺‐sensitive currents were also decreased. The changes in the studied channels, notably at the upper CNS level, could underline the hampered ability of astrocytes to maintain water and potassium homeostasis, thus affecting the BBB, disturbing the neuronal microenvironment, and causing motoneuronal dysfunction and death. © 2012 Wiley Periodicals, Inc.     

Redistribution of the water channel protein aquaporin-4 and the K+ channel protein Kir4.1 differs in low- and high-grade human brain tumors

The blood-brain barrier (BBB) regulation is characterized by an interplay between endothelial cells, subendothelial basal laminae and astrocytic cells. Astroglial cells are highly polarized by the differentiation of perivascular membrane domains. These domains are characterized by the aggregation of, among other molecules, the water channel protein aquaporin-4 (AQP4), the dystrophin-dystroglycan complex, and the inwardly rectifying potassium channel protein Kir4.1. Normally, this ion channel plays an important role in spatial buffering of extracellular K⁺ in the central nervous system, which only can be performed due to the non-uniform distribution of Kir4.1 across the surface of the glial cell. In this study, we observed a mislocalization of Kir4.1 in various human brain tumors (low- and high-grade astrocytomas and oligodendrogliomas), suggesting that buffering capacity of glial cells may be compromised, leading to water influx (cytotoxic edema). Interestingly, whereas dystrophin remained regularly restricted at the endfeet membranes in all cases investigated, AQP4 was found to be redistributed only in high-grade astrocytomas, not in low-grade astrocytomas. If the mechanisms of redistribution of AQP4 and Kir4.1 are different in low- and high-grade gliomas, this may suggest that the mechanisms of clustering of AQP4 and Kir4.1 at the glial endfeet membrane domains are also different. The redistribution of AQP4 in glioblastoma cells is discussed as a reaction to the vasogenic edema, as induced by the breakdown of the BBB, to facilitate reabsorption of excess fluid.     

精氨酸加压素对星形胶质细胞水孔蛋白-4表达的调节及脑水肿形成影响的研究

目的研究精氨酸加压素(AVP)对星形胶质细胞水孔蛋白-4(AQP4)表达的调节,以及p38 MAPK信号通路在AQP4表达过程的作用,明确AVP及AQP4在脑水肿发生过程中的作用。方法大鼠大脑皮质分离星形胶质细胞,星形胶质细胞经分别用AVP、V1a受体(V1aR)拮抗剂和SB 203580进行处理,采用免疫组织化学技术及RT-PCR对AQP4 mRNA进行检测,Western blot检测p38 MAPK信号通路在AVP诱导AQP4表达中的活化程度。结果500nmol/L的AVP处理6h后,AQP4 mRNA表达开始升高(P<0.01),到12h达高峰(P<0.01),24h后仍维持在较高的水平(P<0.05)。加入p38 MAPK抑制剂SB 203580干预后,AQP4 mRNA表达水平与对照组比较差异不显著(P>0.05);AVP处理15min后p38 MAPK磷酸化水平开始增加,30min达高峰,持续到60min开始下降。V1aR拮抗剂处理后p38 MAPK磷酸化水平整个时间段均未出现明显变化。结论AVP通过激活V1aR引起p38MAPK信号通路活化从而诱导AQP4 mRNA高表达,从基因水平对AQP4进行调节,可能在脑水肿发生中,尤其是在星形胶质细胞水肿形成中起重要作用。V1aR拮抗剂及p38 MAPK抑制剂能抑制AQP4 mRNA的表达,避免星形胶质细胞肿胀。     

Aquaporin‐4 in manganese‐treated cultured astrocytes

Manganese in excess is neurotoxic and causes CNS injury resembling that of Parkinson's disease. In brain, astrocytes predominantly take up and accumulate manganese and are thus vulnerable to its toxicity. Manganese was shown to induce cell swelling in cultured astrocytes, and oxidative/nitrosative stress (ONS) mediates such swelling. As aquaporin‐4 (AQP4) is important in the mechanism of astrocyte swelling, we examined the effect of manganese on AQP4 protein levels in cultured astrocytes. Treatment of cultures with manganese increased AQP4 protein in the plasma membrane (PM), whereas total cellular AQP4 protein and mRNA levels were unchanged, suggesting that increased AQP4 levels is due to its increased stability and/or increased trafficking to the PM and not to its neosynthesis. AQP4 gene silencing by small interfering ribonucleic acid resulted in a marked reduction in astrocyte swelling by manganese. Antioxidants, as well as an inhibitor of nitric oxide synthase, diminished the increase in AQP4 protein expression, suggesting a role of ONS in the mechanism of AQP4 increase. As ONS is known to activate mitogen‐activated protein kinases (MAPKs) and MAPK activation has been implicated in astrocyte swelling, we examined the effect of manganese on the activation of MAPKs and found an increased phosphorylation of extracellular signal‐regulated kinase (ERK)1/2, C‐Jun amino‐terminal kinase (JNK)1/2/3, and p38‐MAPK. Inhibitors of ERK1/2 and p38‐MAPK (but not of JNK) blocked (40–60%) the manganese‐induced increase in AQP4 protein content and astrocyte swelling, suggesting the involvement of these kinases in the increased AQP4 content. Inhibition of oxidative stress or MAPKs may represent potential strategies for counteracting AQP4‐related neurological complications associated with manganese toxicity. © 2010 Wiley‐Liss, Inc.     

Role of p53 and apoptosis in cerebral vasospasm after experimental subarachnoid hemorrhage.

Our previous studies indicate that apoptosis in endothelial cells of major cerebral arteries contributes to cerebral vasospasm after subarachnoid hemorrhage (SAH). This study examined the pathologic roles of tumor suppressor p-53 in cerebral vasospasm using an established dog double-hemorrhage model. Twenty mongrel dogs were divided into four groups: (1) control, (2) SAH, (3) SAH+DMSO (vehicle), and (4) SAH+pifithrin-alpha (PFT) (p53 inhibitor). The p53 inhibitor (200 nmol/L) was injected into the cisterna magna daily from Day 0 through Day 3. Angiogram was performed on Day 0 and Day 7. Western blot, cell proliferation assay, histology, and TUNEL staining were conducted on the basilar arteries collected on Day 7 after SAH. The arterial diameter on Day 7 was 42%+/-4%, 40%+/-5%, and 59%+/-4% for SAH, SAH+DMSO, and SAH+PFT, respectively. In addition, positive staining of TUNEL and increased protein expression of p53, Bax, and PCNA in the basilar artery were observed on Day 7. PFT suppressed apoptosis in endothelial cells and proliferation in smooth muscle cells, and attenuated angiographic vasospasm. In conclusion, p53 may be a key factor in endothelial apoptosis and smooth muscle proliferation after SAH. Inhibition of p53 may potentially reduce or even prevent cerebral vasospasm.     

Aquaporin-4 independent Kir4.1 K+ channel function in brain glial cells

Functional interaction of glial water channel aquaporin-4 (AQP4) and inwardly rectifying K+ channel Kir4.1 has been suggested from their apparent colocalization and biochemical interaction, and from the slowed glial cell K+ uptake in AQP4-deficient brain. Here, we report multiple lines of evidence against functionally significant AQP4-Kir4.1 interactions. Whole-cell patch-clamp of freshly isolated glial cells from brains of wild-type and AQP4 null mice showed no significant differences in membrane potential, barium-sensitive Kir4.1 K+ current or current-voltage curves. Single-channel patch-clamp showed no differences in Kir4.1 unitary conductance, voltage-dependent open probability or current-voltage relationship. Also, Kir4.1 protein expression and distribution were similar in wild-type and AQP4 null mouse brain and in the freshly isolated glial cells. Functional inhibition of Kir4.1 by barium or RNAi knock-down in primary glial cell cultures from mouse brain did not significantly alter AQP4 water permeability, as assayed by calcein fluorescence quenching following osmotic challenge. These studies provide direct evidence against functionally significant AQP4-Kir4.1 interactions in mouse glial cells, indicating the need to identify new mechanism(s) to account for altered seizure dynamics and extracellular space K+ buffering in AQP4 deficiency.     

Loss of astrocyte polarization upon transient focal brain ischemia as a possible mechanism to counteract early edema formation

Brain edema is the main cause of death from brain infarction. The polarized expression of the water channel protein aquaporin‐4 (AQP4) on astroglial endfeet surrounding brain microvessels suggests a role in brain water balance. Loss of astrocyte foot process anchoring to the basement membrane (BM) accompanied by the loss of polarized localization of AQP4 to astrocytic endfeet has been shown to be associated with vasogenic/extracellular edema in neuroinflammation. Here, we asked if loss of astrocyte polarity is also observed in cytotoxic/intracellular edema following focal brain ischemia after transient middle cerebral artery occlusion (tMCAO). Upon mild focal brain ischemia, we observed diminished immunostaining for the BM components laminin α4, laminin α2, and the proteoglycan agrin, in the core of the lesion, but not in BMs in the surrounding penumbra. Staining for the astrocyte endfoot anchorage protein β‐dystroglycan (DG) was dramatically reduced in both the lesion core and the penumbra, and AQP4 and Kir4.1 showed a loss of polarized localization to astrocytic endfeet. Interestingly, we observed that mice deficient for agrin expression in the brain lack polarized localization of β‐DG and AQP4 at astrocytic endfeet and do not develop early cytotoxic/intracellular edema following tMCAO. Taken together, these data indicate that the binding of DG to agrin embedded in the subjacent BM promotes polarized localization of AQP4 to astrocyte endfeet. Reduced DG protein levels and redistribution of AQP4 as observed upon tMCAO might therefore counteract early edema formation and reflect a beneficial mechanism operating in the brain to minimize damage upon ischemia. © 2012 Wiley Periodicals, Inc.     

P2X4 receptor in the dorsal horn partially contributes to brain‐derived neurotrophic factor oversecretion and toll‐like receptor‐4 receptor activation associated with bone cancer pain

Previous studies have suggested that the microglial P2X7 purinoceptor is involved in the release of tumor necrosis factor‐α (TNFα) following activation of toll‐like receptor‐4 (TLR4), which is associated with nociceptive behavior. In addition, this progress is evoked by the activation of the P2X4 purinoceptor (P2X4R). Although P2X4R is also localized within spinal microglia in the dorsal horn, little is known about its role in cancer‐induced bone pain (CIBP), which is in some ways unique. With the present rat model of CIBP, we demonstrate a critical role of the microglial P2X4R in the enhanced nociceptive transmission, which is associated with TLR4 activation and secretion of brain‐derived neurotrophic factor (BDNF) and TNFα in the dorsal horn. We assessed mechanical threshold and spontaneous pain of CIBP rats. Moreover, P2X4R small interfering RNA (siRNA) was administered intrathecally, and real‐time PCR, Western blots, immunofluorescence histochemistry, and ELISA were used to detect the expression of P2X4R, TLR4, OX‐42, phosphorylated‐p38 MAPK (p‐p38), BDNF, and TNFα. Compared with controls, intrathecal injection of P2X4R siRNA could prevent nociceptive behavior induced by ATP plus lipopolysaccharide and CIBP and reduce the expression of P2X4R, TLR4, p‐p38, BDNF, and TNFα. In addition, the increase of BDNF protein in rat microglial cells depended on P2X4 receptor signaling, which is partially associated with TLR4 activation. The ability of microglial P2X4R to activate TLR4 in spinal cord leading to behavioral hypersensitivity and oversecretion of BDNF could provide an opportunity for the prevention and treatment of CIBP. © 2014 Wiley Periodicals, Inc.     

Atorvastatin Pretreatment Attenuates Ischemic Brain Edema by Suppressing Aquaporin 4

Background: Cerebral edema, a serious complication of acute cerebral infarction, has a crucial impact on morbidity and mortality in the early stage of cerebral infarction. And aquaporin 4 (AQP4), a bidirectional water transporting protein, plays a pivotal role in edema formation. At experimental model, it has proven that atorvastatin could exert pleiotropic neuroprotection on acute cerebral infarction independent of its cholesterol-lowering action. It was a common protective manifestation that atorvastatin can reduce the infarct volume and cerebral edema. However, little is known about atorvastatin improving ischemic brain edema by regulating AQP4 expression. This study intended to investigate the neuroprotection effects of atorvastatin pretreatment in rats with cerebral ischemia and further explore the potential relationship between atorvastatin and AQP4 expression. Methods: Fifty-one adult male Sprague Dawley rats were randomly divided into 3 groups: sham, middle cerebral artery occlusion (MCAO), and atorvastatin pretreatment (Ator) group. For Ator group, 20 mg/kg of atorvastatin injectable suspension was administered once for 7days by gavage before operation, whereas the others were administered the same volume of saline matching. Except for sham group, MCAO and Ator groups were subjected to permanent MCAO by modified intraluminal suture method. Infarct volume, neurological deficit, brain water content (BWC), immunohistochemistry, western blot, and polymerase chain reaction (PCR) were measured at 24 hours after MCAO. Results: Compared with sham group, the mNSS, infarct volume, and BWC of ischemic hemisphere were significantly increased (P < 0.001) in MCAO group. Positive cells and protein levels of p-p38MAPK and AQP4 in peri-infarction were significantly increased (P < 0.01). The mRNA levels of p38MAPK and AQP4 were also prominently upregulated (P < 0.01). Interestingly, preadministration of atorvastatin dramatically decreased infarct volume and the BWC of ischemic hemisphere compared with MCAO group (P < 0.05). The overexpressions of p-p38MAPK and AQP4 in peri-infarction were significantly decreased (P < 0.05) and their mRNA levels were downregulated by atorvastatin pretreatment (P < 0.05). Neurological deficits were also dramatically improved (P < 0.001). Conclusion: To the best of our knowledge, this is the first study that demonstrates an effect of atorvastatin on expression of AQP4, and we propose that decreased AQP4 expression through a p38MAPK-suppression pathway may be the mechanism of atorvastatin alleviating ischemic cerebral edema.     

Role of p38MAPK in S1P receptor‐mediated differentiation of human oligodendrocyte progenitors

FTY720 is a sphingosine 1‐phosphate receptor (S1PR) modulator used as a daily therapy to reduce disease activity in the relapsing form of multiple sclerosis (MS). FTY720 readily accesses the CNS. Previous studies have shown that phosphorylated FTY720 (FTY720‐p) enhances oligodendrocyte progenitor cell (OPC) survival, differentiation, and remyelination following experimentally induced demyelination in rodents. To elucidate the underlying mechanism, human fetal OPCs alone or in co‐culture with rat dorsal root ganglia neurons (DRGN) were treated daily with FTY720‐p, a condition that desensitizes cellular responses to S1P, the natural ligand of S1PR. In co‐cultures, FTY720‐p and S1P given daily or every three days increased the number of O1/MBP double positive cells and axonal ensheathment. In cultures composed of PDGFRα‐antibody selected cells alone, daily application of FTY720‐p also increased the number of O4/GC double positive cells. At an early time point (day 2), FTY720‐p activated ERK1/2, CREB and p38MAPK in O4‐positive cells, as well as in β‐III Tubulin positive neurons and GFAP positive astrocytes. In later cultures (day 6), FTY720‐p activated p38MAPK in O4 positive cells, p38MAPK and ERK1/2 in neurons, and p38MAPK, ERK1/2 and CREB in astrocytes. A MEK inhibitor (U0126) prevented the differentiation of OPCs into O4‐positive cells, while a p38MAPK inhibitor (PD169316) blocked progression into O4‐positive and into GC‐positive stages of differentiation. Our results demonstrate that FTY720‐p, under conditions that model daily clinical use, can act directly on OPCs to impact differentiation, and also indirectly via neurons and astrocytes by activating ERK1/2 and p38MAPK. GLIA 2014;62:1361–1375     

p53 induction contributes to excitotoxic neuronal death in rat striatum through apoptotic and autophagic mechanisms

The present study sought to investigate mechanisms by which p53 induction contributes to excitotoxic neuronal injury. Rats were intrastriatally administered the N‐methyl‐d ‐aspartate (NMDA) receptor agonist quinolinic acid (QA), the changes in the expression of p53 and its target genes involved in apoptosis and autophagy, including p53‐upregulated modulator of apoptosis (PUMA), Bax, Bcl‐2, damage‐regulated autophagy modulator (DRAM) and other autophagic proteins including microtubule‐associated protein 1 light chain 3 (LC3) and beclin 1 were assessed. The contribution of p53‐mediated autophagy activation to apoptotic death of striatal neurons was assessed with co‐administration of the nuclear factor‐kappaB (NF‐κB) inhibitor SN50, the p53 inhibitor Pifithrin‐alpha (PFT‐α) or the autophagy inhibitor 3‐methyladenine (3‐MA). The increased formation of autophagosomes and secondary lysosomes were observed with transmission electron microscope after excitotoxin exposure. QA induced increases in the expression of p53, PUMA, Bax and a decrease in Bcl‐2. These changes were significantly attenuated by pre‐treatment with SN50, PFT‐α or 3‐MA. SN50, PFT‐α or 3‐MA also reversed QA‐induced upregulation of DRAM, the ratio of LC3‐II/LC3‐I and beclin 1 protein levels in the striatum. QA‐induced internucleosomal DNA fragmentation and loss of striatal neurons were robustly inhibited by SN50, PFT‐α or 3‐MA. These results suggest that overstimulation of NMDA receptors can induce NF‐κB‐dependent expression of p53. p53 participates in excitotoxic neuronal death probably through both apoptotic and autophagic mechanisms.     

Intracisternal administration of SB203580, a p38 mitogen-activated protein kinase inhibitor,attenuates cerebral vasospasm via inhibition of tumor-necrosis factor-α

Tumor-necrosis factor-α (TNF-α) is critical to the development of cerebral vasospasm after subarachnoid hemorrhage (SAH). Hence, therapeutic strategies targeting TNF-α can attenuate cerebral vasospasm. This study investigated the effects of SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor, on TNF-α concentration in the cerebral arteries and the cerebrospinal fluid (CSF) after SAH and on subsequent cerebral vasospasm. Twenty-three rabbits were divided into four groups: (i) control (without SAH), (ii) SAH (SAH only), (iii) dimethylsulfoxide (DMSO, vehicle), and (iv) SB203580. The severity of vasospasm and the immunoreactivities of TNF-α and phosphorylated p38 MAPK in the brain vessels were determined in all animals, and the concentrations of TNF-α in the CSF were also assessed. Severe vasospasm was observed in the rabbits from the SAH and DMSO groups. SB203580 reversed vasospasm after SAH. Lower immunoreactivities of TNF-α and phosphorylated p38 MAPK were found in the basilar artery in the SB203580 group than in the DMSO group. The concentration of TNF-α in the CSF increased after SAH, but treatment with SB203080 after SAH suppressed this increase. Our data show that SB203580 reversed cerebral vasospasm by inhibiting the phosphorylation of p38 MAPK in the basilar artery and by suppressing the increase in TNF-α in the basilar artery and CSF after SAH. SB203580 could therefore potentially be used for the treatment of cerebral vasospasm after SAH.     

Kir4.1和AQP4在大鼠局灶性脑缺血再灌注损伤中的作用

目的 观察大鼠局灶性脑缺血再灌注模型Kir4.1和AQP4的变化.探讨其在再灌注损伤中的作用. 方法 雄性Wistar大鼠50只.按照随机数字表法分成假手术组、缺血2 h再灌注3 h组、12 h组、24 h组和72 h组.每组10只.应用"线栓法"实现大鼠右侧大脑中动脉闭塞,2 h后拔出线栓进行再灌注,并在相应时间点处死大鼠.利用免疫组化和实时定量PCR(RT-PCR)法观察Kir4.1和AQP4蛋白表达及mRNA水平的变化. 结果与假手术组相比,缺血再灌注组大鼠梗死灶周围区皮质Kir4.1、AQP4的蛋白表达和mRNA水平明显升高.于再灌注后3 h开始升高,24 h达到高峰,72 h开始下降.假手术组、缺血再灌注3 h、12 h、24 h和72 h组Kir4.1 mRNA水平分别为0.34±0.02、0.47±0.06、0.61±0.08、0.83±0.10、0.68±0.09.AQP4的mRNA水平分别为0.49±0.05、0.66±0.09、0.91±0.09、1.12±0.11、0.94±0.08.Kir4.1与AQP4的mRNA水平呈正相关(r=0.780,P=0.000). 结论 Kir4.1和AQP4相互作用,共同参与了脑缺血再灌注损伤的形成.     

Tumor necrosis factor‐α potentiates long‐term potentiation in the rat dentate gyrus after acute hypoxia

An inadequate supply of oxygen in the brain may lead to an inflammatory response through neuronal and glial cells that can result in neuronal damage. Tumor necrosis factor‐α (TNF‐α) is a proinflammatory cytokine that is released during acute hypoxia and can have neurotoxic or neuroprotective effects in the brain. Both TNF‐α and interleukin‐1β (IL‐1β) have been shown by a number of research groups to alter synaptic scaling and also to inhibit long‐term potentiation (LTP) in the hippocampus when induced by specific high‐frequency stimulation (HFS) protocols. This study examines the effects of TNF‐α on synaptic transmission and plasticity in hippocampal slices after acute hypoxia using two HFS protocols. Field excitatory postsynaptic potentials were elicited in the medial perforant pathway of the dentate gyrus. Exogenous TNF‐α (5 ng/ml) attenuated LTP induced by theta burst stimulation but had no effect on LTP induced by a more prolonged HFS. Pretreatment with lipopolysaccharide (100 ng/ml) or TNF‐α but not IL‐1β (4 ng/ml) prior to a 30‐min hypoxic insult resulted in a significant enhancement of LTP post hypoxia when induced by the HFS. Anti‐TNF, 3,6′‐dithiothalidomide (a TNF‐α synthesis inhibitor), and SB203580 (a p38 MAPK inhibitor) significantly reduced this effect. These results indicate an important modulatory role for elevated TNF‐α levels on LTP in the hippocampus after an acute hypoxic event. © 2015 Wiley Periodicals, Inc.     

Distinct detergent-resistant membrane microdomains (lipid rafts) respectively harvest K(+) and water transport systems in brain astroglia

The detergent-resistant microdomains (DRM) of cell membranes scaffold different molecules and regulate cell functions by orchestrating various signaling pathways including G-proteins and tyrosine kinase. Here we report a novel role for DRM in astroglial cells. K(+)-buffering inwardly rectifying Kir4.1 channels and the water channel AQP4 are expressed in astrocytes and they may be functionally coupled to maintain ionic and osmotic homeostasis in the brain. Kir4.1 and AQP4 channel proteins were abundant in noncaveloar DRM in the brain and also in HEK293T cells when exogenously expressed. In HEK293T cells, depletion of membrane cholesterol by methyl-beta-cyclodextrin (MbetaCD) resulted in loss of Kir4.1 association with DRM and its channel activity but did not affect either the distribution or the function of AQP4. Immunolabeling showed that Kir4.1 and AQP4 were occasionally distributed in close proximity but in distinct compartments of the astroglial cell membrane. Astroglial noncaveolar DRM may therefore be made up of at least two distinct compartments, MbetaCD-sensitive and MbetaCD-resistant microdomains, which control localization and function of, respectively, Kir and AQP4 channels on the cell membrane.     

水通道蛋白4对一氧化碳中毒后迟发性脑病大鼠神经损伤的影响

目的 探讨水通道蛋白4(Aquaporin 4,AQP4)对一氧化碳(Carbon monoxide,CO)中毒后迟发性脑病(Delayed encephalopathy,DEACMP)大鼠神经损伤的影响。方法 将210只雄性SD大鼠随机分为空白对照(Blank control,BC)组、CO中毒(CO)组、钠-钾-氯共转运体(Na⁺-K⁺-Cl^- cotransporter,NKCC)抑制剂处理(布美他尼)组、p38-丝裂原活化蛋白激酶(Mitogen activated protein kinase,MAPK)抑制剂处理(MAPK)组和AQP4特异性抑制剂处理(AQP4抑制剂)组,每组各42只; 根据造模后不同时间点将每组大鼠进一步分为染毒3、6、12、24、48、72 h和7 d后共7个亚组,每亚组各6只; 取大鼠脑前额叶皮质组织,计算脑皮质含水量,采用HE法观察脑皮质形态; 采用免疫组化链霉菌抗生物素蛋白-过氧化物酶(Streptavidin-perosidase,SP)染色法测定大鼠脑皮质AQP4,p38 MAPK,NKCC1、胶质纤维酸性蛋白(Glial fibrillary acidic protein,GFAP)和S100钙结合蛋白B(S100 calcium binding protein B,S100B)蛋白表达水平。结果 与BC组比较,CO组大鼠在染毒3、6、12、24、48、72 h后尾静脉COHB水平和脑皮质含水量显著升高,脑皮质AQP4,p38 MAPK,NKCC1,GFAP和S100B蛋白表达水平显著升高,染毒7 d后恢复正常(P<0.05); 与CO组比较,布美他尼组、MAPK组和AQP4抑制剂组大鼠脑皮质含水量显著降低,脑皮质AQP4,p38 MAPK,NKCC1,GFAP和S100B蛋白表达水平显著降低,且AQP4抑制剂组变化更明显(P<0.05)。结论 p38-MAPK/NKCC信号通路可能参与调控CO中毒DEACMP大鼠脑皮质AQP4表达,抑制AQP4表达可有效减轻大鼠脑水肿并改善预后,有望成为预防和治疗DEACMP的新靶点。     

High mobility group box protein‐1 promotes cerebral edema after traumatic brain injury via activation of toll‐like receptor 4

Traumatic brain injury (TBI) is a major cause of mortality and morbidity worldwide. Cerebral edema, a life‐threatening medical complication, contributes to elevated intracranial pressure (ICP) and a poor clinical prognosis after TBI. Unfortunately, treatment options to reduce post‐traumatic edema remain suboptimal, due in part, to a dearth of viable therapeutic targets. Herein, we tested the hypothesis that cerebral innate immune responses contribute to edema development after TBI. Our results demonstrate that high‐mobility group box protein 1 (HMGB1) was released from necrotic neurons via a NR2B‐mediated mechanism. HMGB1 was clinically associated with elevated ICP in patients and functionally promoted cerebral edema after TBI in mice. The detrimental effects of HMGB1 were mediated, at least in part, via activation of microglial toll‐like receptor 4 (TLR4) and the subsequent expression of the astrocytic water channel, aquaporin‐4 (AQP4). Genetic or pharmacological (VGX‐1027) TLR4 inhibition attenuated the neuroinflammatory response and limited post‐traumatic edema with a delayed, clinically implementable therapeutic window. Human and rodent tissue culture studies further defined the cellular mechanisms demonstrating neuronal HMGB1 initiates the microglial release of interleukin‐6 (IL‐6) in a TLR4 dependent mechanism. In turn, microglial IL‐6 increased the astrocytic expression of AQP4. Taken together, these data implicate microglia as key mediators of post‐traumatic brain edema and suggest HMGB1‐TLR4 signaling promotes neurovascular dysfunction after TBI. GLIA 2013;62:26–38     

Laminin-induced aggregation of the inwardly rectifying potassium channel, Kir4.1, and the water-permeable channel, AQP4, via a dystroglycan-containing complex in astrocytes

Dystroglycan (DG) is part of a multiprotein complex that links the extracellular matrix to the actin cytoskeleton of muscle fibers and that is involved in aggregating acetylcholine receptors at the neuromuscular junction. This complex is also expressed in regions of the central nervous system where it is localized to both neuronal and glial cells. DG and the inwardly rectifying potassium channels, Kir4.1, are concentrated at the interface of astroglia and small blood vessels. These channels are involved in siphoning potassium released into the extracellular space after neuronal excitation. This raises the possibility that DG may be involved in targeting Kir4.1 channels to specific domains of astroglia. To address this question, we used mixed hippocampal cultures to investigate the distribution of DG, syntrophin, dystrobrevin, and Kir4.1 channels, as well as aquaporin-permeable water channels, AQP4. These proteins exhibit a similar distribution pattern and form aggregates in astrocytes cultured on laminin. Both DG and syntrophin colocalize with Kir4.1 channel aggregates in astrocytes. Similarly, DG colocalizes with AQP4 channel aggregates. Quantitative studies show a significant increase of Kir4.1 and AQP4 channel aggregates in astrocytes cultured in the presence of laminin when compared with those in the absence of laminin. These findings show that laminin has a role in Kir4.1 and AQP4 channel aggregation and suggest that this may be mediated via a dystroglycan-containing complex. This study reveals a novel functional role for DG in brain including K+ buffering and water homeostasis.