Experimental Allergic Encephalomyelitis in Connexin 43-Heterozygous Mice

Alterations in the expression of gap junction proteins (connexins) have previously been observed in experimental allergic encephalomyelitis (EAE). Demyelinating lesions have significantly decreased Cx43, while recovering lesions have greatly increased Cx43 and increased glial fibrillary acidic protein-expressing astrocytes. This suggests an important role for gap-junctional intercellular communication in astrocytes in the recovery from CNS inflammation. To study the effects of decreased Cx43 expression during acute disease (21 days post-immunization) and in recovering spinal cord tissue (55 days post-immunization) we induced EAE in Cx43 heterozygous and wild-type mice. Mice showed signs of disease by day 10, and signs of recovery by day 25. There were no clinical or pathological differences between heterozygous and wild-type mice in the acute disease stage, except that wild-type male mice had fewer clinical signs of disease. Male mice that were heterozygous for Cx43, and therefore had decreased expression of Cx43, had increased EAE disease severity. All demyelinating lesions had reduced numbers of reactive astrocytes and a significant decrease in Cx43 expression. In the 55-day study, all heterozygous and wild-type mice were clinically improved, showed decreased pathological signs, and showed increased laminin expression, indicative of CNS recovery. Furthermore, all heterozygous mice showed a striking increase in Cx43 expression during recovery, suggesting that the regulatory factors affecting Cx43 expression are still present in mice that have only one wild-type Cx43 allele.     

Gene expression alterations in connexin null mice extend beyond the gap junction

Connexin43 (Cx43) is the principal gap junction protein between astrocytes in the neonatal brain and also interconnects neural precursor cells during CNS development. In an attempt to understand global effects of expression of the Cx43 gap junction gene on development and function of the nervous system, we have compared gene expression patterns in cultured astrocytes and brains from wildtype mice with those in which Cx43 is deleted as well as in spinal cords of experimental autoimmune encepahlomyelitis (EAE) mice. One surprising result obtained from high densitity mouse cDNA studies was the large number of genes that were statistically altered in mice with decreased expression of Cx43. These altered genes encode proteins with a wide range of functions within cells, and thus deletion of normal gap junction expression appears to result in globally altered glial functions in addition to disruption of intercellular communication. Here we discuss those results in the context of the strategies and data analysis paradigms that we are using in such studies.     

A neuroprotective role for gap junctions

Glial-neuronal interactions have been implicated in both normal information processing and neuroprotection. One pathway of cellular interactions involves gap junctional intercellular communication (GJIC). In astrocytes, gap junctions are composed primarily of the channel protein, connexin43 (Cx43), and provide a substrate for formation of a functional syncytium implicated in the process of spatial buffering in the CNS. Thus gap junctional communication may be neuroprotective following a CNS insult that entails glutamate cytotoxicity (i.e. ischemia). We have shown that blocking gap junctions during a glutamate insult to co-cultures of astrocytes and neurons results in increased neuronal injury. To assess the effect of reduced Cx43 and GJIC on neuroprotection, we examined brain infarct volume in wild type and Cx43 heterozygote null mice following focal ischemia. Cx43 heterozygous null mice exhibited a significantly larger infarct volume compared to wild type. At the cellular level, a significant increase in TUNEL positive cells was observed in the penumbral region of the Cx43 heterozygote mice. These results suggest that augmentation of GJIC in astrocytes may contribute to neuroprotection following ischemic injury. These findings support the hypothesis that gap junctions play a neuroprotective role against glutamate cytotoxicity.     

A Neuroprotective Role for Gap Junctions

Glial-neuronal interactions have been implicated in both normal information processing and neuroprotection. One pathway of cellular interactions involves gap junctional intercellular communication (GJIC). In astrocytes, gap junctions are composed primarily of the channel protein, connexin43 (Cx43), and provide a substrate for formation of a functional syncytium implicated in the process of spatial buffering in the CNS. Thus gap junctional communication may be neuroprotective following a CNS insult that entails glutamate cytotoxicity (i.e. ischemia). We have shown that blocking gap junctions during a glutamate insult to co-cultures of astrocytes and neurons results in increased neuronal injury. To assess the effect of reduced Cx43 and GJIC on neuroprotection, we examined brain infarct volume in wild type and Cx43 heterozygote null mice following focal ischemia. Cx43 heterozygous null mice exhibited a significantly larger infarct volume compared to wild type. At the cellular level, a significant increase in TUNEL positive cells was observed in the penumbral region of the Cx43 heterozygote mice. These results suggest that augmentation of GJIC in astrocytes may contribute to neuroprotection following ischemic injury. These findings support the hypothesis that gap junctions play a neuroprotective role against glutamate cytotoxicity.     

IFN-inducible protein 10/CXC chemokine ligand 10-independent induction of experimental autoimmune encephalomyelitis

In multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), autoaggressive T cells traffic into the CNS and induce disease. Infiltration of these pathogenic T cells into the CNS has been correlated with the expression of the chemokine IFN-inducible protein (IP)10/CXC chemokine ligand (CXCL)10, a chemoattractant for activated T cells, and its receptor CXCR3, in the CNS of both MS patients and mice with EAE. In the present study, we report that targeted deletion of IP-10 did not diminish the expression, severity, or histopathology of EAE induced by active immunization with 100 micro g of myelin oligodendrocyte glycoprotein peptide (MOG)p35-55. However, we found that IP-10-deficient mice had a lower threshold for expression of disease compared with wild-type littermates. EAE induced by immunization with 5 micro g of MOGp35-55 resulted in more severe disease characterized by a greater number of CNS lesions and infiltrating mononuclear cells in IP-10-deficient mice compared with wild-type controls. IP-10-deficient mice immunized with MOGp35-55 demonstrated increased levels of IFN-inducible T cell alpha-chemokine/CXCL11 mRNA in the CNS and decreased levels of monokine induced by IFN-gamma/CXCL9 mRNA in draining lymph nodes, suggesting differential compensation for loss of IP-10 in lymphoid vs parenchymal tissue compartments. EAE in IP-10-deficient mice induced by low-dose immunization was associated with enhanced Ag-specific Th1 responses in the draining lymph node, which corresponded with diminished lymph node TGF-beta1 expression. Our data demonstrated that IP-10 was not required for the trafficking of pathogenic T cells into the CNS in EAE but played an unexpected role in determining the threshold of disease susceptibility in the periphery.     

Contribution of Pannexin1 to Experimental Autoimmune Encephalomyelitis

Pannexin1 (Panx1) is a plasma membrane channel permeable to relatively large molecules, such as ATP. In the central nervous system (CNS) Panx1 is found in neurons and glia and in the immune system in macrophages and T-cells. We tested the hypothesis that Panx1-mediated ATP release contributes to expression of Experimental Autoimmune Encephalomyelitis (EAE), an animal model for multiple sclerosis, using wild-type (WT) and Panx1 knockout (KO) mice. Panx1 KO mice displayed a delayed onset of clinical signs of EAE and decreased mortality compared to WT mice, but developed as severe symptoms as the surviving WT mice. Spinal cord inflammatory lesions were also reduced in Panx1 KO EAE mice during acute disease. Additionally, pharmacologic inhibition of Panx1 channels with mefloquine (MFQ) reduced severity of acute and chronic EAE when administered before or after onset of clinical signs. ATP release and YoPro uptake were significantly increased in WT mice with EAE as compared to WT non-EAE and reduced in tissues of EAE Panx1 KO mice. Interestingly, we found that the P2X7 receptor was upregulated in the chronic phase of EAE in both WT and Panx1 KO spinal cords. Such increase in receptor expression is likely to counterbalance the decrease in ATP release recorded from Panx1 KO mice and thus contribute to the development of EAE symptoms in these mice. The present study shows that a Panx1 dependent mechanism (ATP release and/or inflammasome activation) contributes to disease progression, and that inhibition of Panx1 using pharmacology or gene disruption delays and attenuates clinical signs of EAE.     

Decreased connexin43 expression in the mouse heart potentiates pacing-induced remodeling of repolarizing currents

Gap junction redistribution and reduced expression, a phenomenon termed gap junction remodeling (GJR), is often seen in diseased hearts and may predispose toward arrhythmias. We have recently shown that short-term pacing in the mouse is associated with changes in connexin43 (Cx43) expression and localization but not with increased inducibility into sustained arrhythmias. We hypothesized that short-term pacing, if imposed on murine hearts with decreased Cx43 abundance, could serve as a model for evaluating the electrophysiological effects of GJR. We paced wild-type (normal Cx43 abundance) and heterozygous Cx43 knockout (Cx43+/-; 66% mean reduction in Cx43) mice for 6 h at 10-15% above their average sinus rate. We investigated the electrophysiological effects of pacing on the whole animal using programmed electrical stimulation and in isolated ventricular myocytes with patch-clamp studies. Cx43+/- myocytes had significantly shorter action potential durations (APD) and increased steady-state (Iss) and inward rectifier (I(K1)) potassium currents compared with those of wild-type littermate cells. In Cx43+/- hearts, pacing resulted in a significant prolongation of ventricular effective refractory period and APD and significant diminution of Iss compared with unpaced Cx43+/- hearts. However, these changes were not seen in paced wild-type mice. These data suggest that Cx43 abundance plays a critical role in regulating currents involved in myocardial repolarization and their response to pacing. Our study may aid in understanding how dyssynchronous activation of diseased, Cx43-deficient myocardial tissue can lead to electrophysiological changes, which may contribute to the worsened prognosis often associated with pacing in the failing heart.     

CD24 on the resident cells of the central nervous system enhances experimental autoimmune encephalomyelitis

CD24 is a cell surface glycoprotein that is expressed on both immune cells and cells of the CNS. We have previously shown that CD24 is required for the induction of experimental autoimmune encephalomyelitis (EAE), an experimental model for the human disease multiple sclerosis (MS). The development of EAE requires CD24 expression on both T cells and non-T host cells in the CNS. To understand the role of CD24 on the resident cells in the CNS during EAE development, we created CD24 bone marrow chimeras and transgenic mice in which CD24 expression was under the control of a glial fibrillary acidic protein promotor (AstroCD24TG mice). We showed that mice lacking CD24 expression on the CNS resident cells developed a mild form of EAE; in contrast, mice with overexpression of CD24 in the CNS developed severe EAE. Compared with nontransgenic mice, the CNS of AstroCD24TG mice had higher expression of cytokine genes such as IL-17 and demyelination-associated marker P8; the CNS of AstroCD24TG mice accumulated higher numbers of Th17 and total CD4+ T cells, whereas CD4+ T cells underwent more proliferation during EAE development. Expression of CD24 in CD24-deficient astrocytes also enhanced their costimulatory activity to myelin oligodendrocyte glycoprotein-specific, TCR-transgenic 2D2 T cells. Thus, CD24 on the resident cells in the CNS enhances EAE development via costimulation of encephalitogenic T cells. Because CD24 is increased drastically on resident cells in the CNS during EAE, our data have important implications for CD24-targeted therapy of MS.     

Gamma delta T cells regulate the extent and duration of inflammation in the central nervous system by a Fas ligand-dependent mechanism

Gamma delta T cells have been shown to regulate immune responses associated with inflammation, but the mechanism of this regulation is largely unknown. Using the experimental autoimmune encephalomyelitis (EAE) model of the human CNS autoimmune disease multiple sclerosis, we demonstrate that gamma delta T cells are important regulators of CNS inflammation. This was shown using gamma delta T cell-deficient mice that were unable to recover from EAE. The chronic disease was accompanied by a prolonged presence of both macrophages and lymphocytes in the CNS. This extended inflammatory response was due to alterations in both cell proliferation and death. In mice lacking gamma delta T cells, proliferation of encephalitogenic T cells was 3-fold higher, and caspase activity, indicating apoptosis, was 2-fold lower compared with those in control mice recovering from EAE. gamma delta T cell-deficient mice reconstituted with wild-type gamma delta T cells recovered from EAE and resolved inflammation in the CNS, whereas mice reconstituted with Fas ligand-dysfunctional gamma delta T cells did not. Thus, gamma delta T cells regulate both inflammation in the CNS and disease recovery via Fas/Fas ligand-induced apoptosis of encephalitogenic T cells, and a quick resolution of inflammation in the CNS is essential to prevent permanent damage to the CNS resulting in chronic disease.     

The role of the MHC class II transactivator in class II expression and antigen presentation by astrocytes and in susceptibility to central nervous system autoimmune disease

The role of the MHC class II transactivator (CIITA) in Ag presentation by astrocytes and susceptibility to experimental autoimmune encephalomyelitis (EAE) was examined using CIITA-deficient mice and newly created transgenic mice that used the glial fibrillary acidic protein promoter to target CIITA expression in astrocytes. CIITA was required for class II expression on astrocytes. Like class II-deficient mice, CIITA-deficient mice were resistant to EAE by immunization with CNS autoantigen, although T cells from immunized CIITA-deficient, but not class II-deficient, mice proliferated and secreted Th1 cytokines. CIITA-deficient splenic APC presented encephalitogenic peptide to purified wild-type encephalitogenic CD4(+) T cells, indicating that CIITA-independent mechanisms can be used for class II-restricted Ag presentation in lymphoid tissue. CIITA-deficient mice were also resistant to EAE by adoptive transfer of encephalitogenic class II-restricted CD4(+) Th1 cells, indicating that CIITA-dependent class II expression was required for CNS Ag presentation. Despite constitutive CIITA-driven class II expression on astrocytes in vivo, glial fibrillary acidic protein-CIITA transgenic mice were no more susceptible to EAE than controls. CIITA-transfected astrocytes presented peptide Ag, but in contrast to IFN-gamma-activated astrocytes, they could not process and present native Ag. CIITA-transfected astrocytes did not express cathepsin S without IFN-gamma activation, indicating that CIITA does not regulate other elements that may be required for Ag processing by astrocytes. Although our results demonstrate that CIITA-directed class II expression is required for EAE induction, CIITA-directed class II expression by astrocytes does not appear to increase EAE susceptibility. These results do not support the role of astrocytes as APC for class II-restricted Ag presentation during the induction phase of EAE.     

C57BL/6小鼠性别差异对髓鞘少突胶质糖蛋白诱导的实验性自身脑脊髓炎疾病的影响

多发性硬化是人类常见的中枢神经系统自身免疫性炎症致脱髓鞘疾病．流行病学研究发现,女性患者多于男性,其平均发病时间早于男性．实验性自身免疫性脑脊髓炎(EAE)与多发性硬化症有相似的临床症状和病理特征,是被广泛应用于人类疾病研究的动物模型．本实验利用髓鞘少突胶质糖蛋白MOG_33-35免疫C57BL/6小鼠建立EAE模型,观察29天．通过疾病评分发现雌雄小鼠在发病率、起病时间上均无明显差别,但雄鼠的发病症状明显比雌鼠严重．在其病理切片HE染色中观察到雄性小鼠中枢浸润的炎性细胞多于雌性小鼠,并且在LFB染色中同样观察到雄鼠脱髓鞘区域明显增大．对其发病高峰期中枢浸润细胞的染色分析时,可以发现雄性小鼠中浸润的CD4 T细胞及其亚群T_H-1和T_H-17细胞均有明显增加．这些都表明MOG_33-35免疫C57BL/6小鼠建立的EAE模型存在着性别差异的影响,这一发现为今后建立多发性硬化症的动物模型中动物性别的选择提供了一定的参考依据．     

Relapsing murine experimental allergic encephalomyelitis induced by myelin basic protein

Experimental allergic encephalomyelitis (EAE), an experimental autoimmune disease of the central nervous system (CNS), is readily induced in many mammalian species by immunization with CNS tissue or myelin basic protein (MBP) purified from the CNS. EAE has been frequently used as a model for multiple sclerosis (MS). However, EAE generally presents as an acute monophasic disease in the adult animal after immunization with MBP. After recovery, the animal is resistant to rechallenge with encephalitogen (1). Two exceptions to these observations have been reported. McFarlin et al. (2) reported that a variable number of Lewis rats showed signs of a single, mild relapse about a week after recovery from MBP-induced acute EAE. Panitch and Ciccone (3) have reported induction of recurrent EAE in rats immunized with human MBP. Chronic, relapsing EAE has been induced in the mouse; however, an apparent requirement for CNS tissue had been noted (4, 5). Recently, during the course of a series of experiments on the induction of EAE in SJL/J, PL/J, and (SJL/J X PL/J)F1 (SPL F1) mice, it was observed that the F1 mice frequently had paralytic relapses after recovery from MBP-induced symptoms. Experiments were initiated to examine this phenomenon, and the findings are presented below.     

Neuroprotective role of astrocytic gap junctions in ischemic stroke

The role of astrocytic gap junctions in ischemia remains controversial. Several studies support that astrocytic gap junctions play a role in the spread of hypoxic injury, while other reports have demonstrated that blocking astrocytic gap junctions increases neuronal death. Using a stroke model on animals in which the astrocytic gap junction protein connexin43 (Cx43) was compromised, we explored the neuroprotective role of astrocytic gap junctions. A focal brain stroke was performed on heterozygous Cx43 null [Cx43(+/-)] mice, wild type [Cx43(+/+)] mice, astrocyte-directed Cx43 deficient [Cx43(fl/ fl)/hGFAP-cre] mice (here designated as Cre(+) mice), and their corresponding controls [Cx43(fl/fl)] (here designated as Cre(-) mice). Four days following stroke, ischemic lesions were measured for size and analyzed immunohistochemically. Stroke volume was significantly larger in Cx43(+/-) and Cre(+) mice compared to Cx43(+/+) and Cre(-) mice, respectively. Apoptosis as detected by TUNEL labeling and caspase-3 immunostaining was amplified in Cx43(+/-) and Cre(+) mice compared to their control groups. Furthermore, increased inflammation as characterized by the immunohistochemical staining of the microglial marker CD11b was observed in the Cre(+) mice penumbra. Astrocytic gap junctions may reduce apoptosis and inflammation in the penumbra following ischemic insult, suggesting that coupled astrocytes fulfill a neuroprotective role under ischemic stroke conditions.     

Neuroprotective Role of Astrocytic Gap Junctions in Ischemic Stroke

The role of astrocytic gap junctions in ischemia remains controversial. Several studies support that astrocytic gap junctions play a role in the spread of hypoxic injury, while other reports have demonstrated that blocking astrocytic gap junctions increases neuronal death. Using a stroke model on animals in which the astrocytic gap junction protein connexin43 (Cx43) was compromised, we explored the neuroprotective role of astrocytic gap junctions. A focal brain stroke was performed on heterozygous Cx43 null [Cx43(+/?)] mice, wild type [Cx43(+/+)] mice, astrocyte-directed Cx43 deficient [Cx43^{fl/ fl}/hGFAP-cre] mice (here designated as Cre(+) mice), and their corresponding controls [Cx43^fl/fl] (here designated as Cre(?) mice). Four days following stroke, ischemic lesions were measured for size and analyzed immunohistochemically. Stroke volume was significantly larger in Cx43(+/?) and Cre(+) mice compared to Cx43(+/+) and Cre(?) mice, respectively. Apoptosis as detected by TUNEL labeling and caspase-3 immunostaining was amplified in Cx43(+/?) and Cre(+) mice compared to their control groups. Furthermore, increased inflammation as characterized by the immunohistochemical staining of the microglial marker CD11b was observed in the Cre(+) mice penumbra. Astrocytic gap junctions may reduce apoptosis and inflammation in the penumbra following ischemic insult, suggesting that coupled astrocytes fulfill a neuroprotective role under ischemic stroke conditions.     

Dual benefit of reduced Cx43 on atherosclerosis in LDL receptor-deficient mice

Paracrine cell-to-cell interactions are crucial events during atherogenesis, however, little is known on the role of gap junctional communication during this process. We recently demonstrated increased expression of Cx43 in intimal smooth muscle cells and in a subset of endothelial cells covering the shoulder of atherosclerotic plaques. The purpose of this study was to examine the role of Cx43 in the development of atherosclerosis in vivo. Atherosclerosis-susceptible LDL receptor-deficient (LDLR(-/-)) mice were intercrossed with mice heterozygous for Cx43 (Cx43(+/-) mice). Male mice with normal (Cx43(+/+)LDLR(-/-)) or reduced (Cx43(+/-)LDLR(-/-)) Cx43 level of 10 weeks old were fed a cholesterol-rich diet (1.25%) for 14 weeks. Both groups of mice showed similar increases in serum lipids and body weight. Interestingly, the progression of atherosclerosis was reduced by 50% (P < 0.01) in the thoraco-abdominal aorta and in the aortic roots of Cx43(+/-)LDLR(-/-) mice compared with Cx43(+/+)LDLR(-/-) littermate controls. In addition, atheroma in Cx43(+/-)LDLR(-/-) mice contained fewer inflammatory cells and exhibited thicker fibrous caps with more collagen and smooth muscle cells, important features associated, in human, with stable atherosclerotic lesions. Thus, reducing Cx43 expression in mice provides beneficial effects on both the progression and composition of the atherosclerotic lesions.     

Corticotropin-releasing hormone contributes to the peripheral inflammatory response in experimental autoimmune encephalomyelitis

Peripheral corticotropin-releasing hormone (CRH) is thought to have proinflammatory effects. We used the model of experimental autoimmune encephalomyelitis (EAE) to study the role of CRH in an immune-mediated disease. We showed that CRH-deficient mice are resistant to EAE, with a decrease in clinical score as well as decreased cellular infiltration in the CNS. Furthermore, Ag-specific responses of primed T cells as well as anti-CD3/anti-CD28 TCR costimulation were decreased in crh(-/-) mice with decreased production of Th1 cytokines and increased production of Th2 cytokines. Wild-type mice treated in vivo with a CRH antagonist showed a decrease in IFN-gamma production by primed T cells in vitro. This effect of CRH is independent of its ability to increase corticosterone production, because adrenalectomized wild-type mice had similar disease course and severity as control mice. We found that IkappaBalpha phosphorylation induced by TCR cross-linking was decreased in crh(-/-) T cells. We conclude that peripheral CRH exerts a proinflammatory effect in EAE with a selective increase in Th1-type responses. These findings have implications for the treatment of Th1-mediated diseases such as multiple sclerosis.     

Experimental autoimmune encephalomyelitis on the SJL mouse: effect of gamma delta T cell depletion on chemokine and chemokine receptor expression in the central nervous system

Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease of the central nervous system (CNS) that is a model for multiple sclerosis. Previously, we showed that depletion of gamma delta T cells significantly reduced clinical and pathological signs of disease, which was associated with reduced expression of IL-1 beta, IL-6, TNF-alpha, and lymphotoxin at disease onset and a more persistent reduction in IFN-gamma. In this study, we analyzed the effect of gamma delta T cell depletion on chemokine and chemokine receptor expression. In the CNS of control EAE mice, mRNAs for RANTES, eotaxin, macrophage-inflammatory protein (MIP)-1 alpha, MIP-1 beta, MIP-2, inducible protein-10, and monocyte chemoattractant protein-1 were detected at disease onset, increased as disease progressed, and fell as clinical signs improved. In gamma delta T cell-depleted animals, all of the chemokine mRNAs were reduced at disease onset; but at the height of disease, expression was variable and showed no differences from control animals. mRNA levels then fell in parallel with control EAE mice. ELISA data confirmed reduced expression of MIP-1 alpha and monocyte chemoattractant protein-1 at disease onset in gamma delta T cell-depleted mice, and total T cell numbers were also reduced. In normal CNS mRNAs for CCR1, CCR3, and CCR5 were observed, and these were elevated in EAE animals. mRNAs for CCR2 were also detected in the CNS of affected mice. Depletion of gamma delta T cells reduced expression of CCR1 and CCR5 at disease onset only. We conclude that gamma delta T cells contribute to the development of EAE by promoting an inflammatory environment that serves to accelerate the inflammatory process in the CNS.     

The Role of Connexin 43 and Hemichannels Correlated with the Astrocytic Death Following Ischemia/Reperfusion Insult

The aim of this study was to investigate the role of connexin 43 (Cx43) and its hemichannel (HC1) in the death of astrocytes following ischemia/reperfusion (IR) or oxygen–glucose deprivation/reoxygenation (OGDR) insult. Wistar rats had their bilateral common carotid artery clamped for 1.5 h followed by 0, 4, and 24 h of reperfusion (n = 8 for each time point), respectively. All rats were sacrificed and Cx43, HC1, and caspase 3 (Casp3) in cerebral ischemic tissues were examined by immunohistochemistry and western blotting. Astrocytes cell line, astrocytes transduced with a retroviral empty vector (Psup astrocyte), or a Cx43-specific shRNA construct (shRNA astrocytes) were treated with OGDR insult for various periods. The viability of astrocytes was assessed by MTT assay. The expression of Cx43, HC1, and Casp3 was detected with western blotting. The results showed that the expression of Cx43, HC1, and Casp3 in rats’ brain, astrocytes, and Psup astrocytes was significantly increased after 4 h of IR/OGDR and recovered on 24 h of the insult. Cell viability decreased after 4 h of the insult whereas the cell viability increased on 24 h after the insult. In contrast, the expression of Cx43, HC1, Casp3, and cell viability had no statistical differences in the null Cx43 gene—shRNA transfected astrocytes after the treatment of OGDR. The results suggest that Cx43 and HC1 are likely to play the pivotal roles in the mediation of the astrocytic death.