Glia-mediated neuroinflammation is related to brain injury exacerbation after cerebral ischemia/reperfusion (I/R) injury. Astrocytic hemichannels or gap junctions, which were mainly formed by connexin-43, have been implicated in I/R damage. However, the exact roles of astrocytic hemichannels and gap junction in neuroinflammatory responses induced by I/R injury remain unknown. Primary cultured astrocytes were subjected to OGD/R injury, an in vitro model of I/R injury. Salvianolic acid B (SalB) or carbenoxolone (CBX) were applied for those astrocytes. Besides, Cx43 mimetic peptides Gap19 or Gap26 were also applied during OGD/R injury; Cx43 protein levels were determined by western blot and cytoimmunofluorescene staining, hemichannel activities by Ethidium bromide uptake and ATP concentration detection, and gap junction intercellular communication (GJIC) permeability by parachute assay. Further, astrocyte-conditioned medium (ACM) was collected and incubated with microglia. Meanwhile, ATP or apyrase were applied to explore the role of ATP during OGD/R injury. Microglial activation, M1/M2 phenotypes, and M1/M2-related cytokines were detected. Also, microglia-conditioned medium (MEM) was collected and incubated with astrocytes to further investigate its influence on astrocytic hemichannel activity and GJIC permeability. Lastly, effects of ACM and MCM on neuronal viability were detected by flow cytometry. We found that OGD/R induced abnormally opened hemichannels with increased ATP release and EtBr uptake but reduced GJIC permeability. WB tests showed decreased astrocytic plasma membrane’s Cx43, while showing an increase in cytoplasma. Treating OGD/R-injured microglia with ATP or OGD/R-ACM induced further microglial activation and secondary pro-inflammatory cytokine release, with the M1 phenotype predominating. Conversely, astrocytes incubated with OGD/R-MCM exhibited increased hemichannel opening but reduced GJIC coupling. Both SalB and CBX inhibited abnormal astrocytic hemichannel opening and ATP release and switched the activated microglial phenotype from M1 to M2, thus providing effective neuroprotection. Application of Gap19 or Gap26 showed similar results with CBX. We also found that OGD/R injury caused both plasma membrane p-Cx43(Ser265) and p-Src(Tyr416) significantly upregulated; application of SalB may be inhibiting Src kinase and attenuating Cx43 internalization. Meanwhile, CBX treatment induced obviously downregulation of p-Cx43(Ser368) and p-PKC(Ser729) protein levels in plasma membrane. We propose a vicious cycle exists between astrocytic hemichannel and microglial activation after OGD/R injury, which would aggravate neuroinflammatory responses and neuronal damage. Astrocytic Cx43, hemichannels, and GJIC play critical roles in OGD/R injury-induced neuroinflammatory responses; treatment differentially targeting astrocytic Cx43, hemichannels, and GJIC may provide novel avenues for therapeutics during cerebral I/R injury.

Stroke is the second leading cause of death worldwide and the most common cause of adult-acquired disability in many nations. Thus, attenuating the damage after ischemic injury and improving patient prognosis are of great importance. We have indicated that ischemic preconditioning (IP) can effectively reduce the damage of ischemia reperfusion and that inhibition of gap junctions may further reduce this damage. Although we confirmed that the function of gap junctions is closely associated with glutamate, we did not investigate the mechanism. In the present study, we aimed to clarify whether the blockade of cellular communication at gap junctions leads to significant reductions in the levels of glutamate released by astrocytes following cerebral ischemia. To explore this hypothesis, we utilized the specific blocking agent carbenoxolone (CBX) to inhibit the opening and internalization of connexin 43 channels in an in vitro model of oxygen-glucose deprivation/re-oxygenation (OGD/R), following IP. OGD/R resulted in extensive astrocytic glutamate release following upregulation of hemichannel activity, thus increasing reactive oxygen species (ROS) generation and subsequent cell death. However, we observed significant increases in neuronal survival in neuron-astrocyte co-cultures that were subjected to IP prior to OGD/R. Moreover, the addition of CBX enhanced the protective effects of IP during the re-oxygenation period following OGD, by means of blocking the release of glutamate, increasing the level of the excitatory amino acid transporter 1, and downregulating glutamine expression. Our results suggest that combined use of IP and CBX represents a novel therapeutic strategy to attenuate damage from cerebral ischemia with minimal adverse side effects.

Stroke is one of the leading causes of morbidity and mortality worldwide, and it is increasing in prevalence. The limited therapeutic window and potential severe side effects prevent the widespread clinical application of the venous injection of thrombolytic tissue plasminogen activator and thrombectomy, which are regarded as the only approved treatments for acute ischemic stroke. Triggered by various types of mild stressors or stimuli, ischemic preconditioning (IPreC) induces adaptive endogenous tolerance to ischemia/reperfusion (I/R) injury by activating a multitude cascade of biomolecules, for example, proteins, enzymes, receptors, transcription factors, and others, which eventually lead to transcriptional regulation and epigenetic and genomic reprogramming. During the past 30 years, IPreC has been widely studied to confirm its neuroprotection against subsequent I/R injury, mainly including local ischemic preconditioning (LIPreC), remote ischemic preconditioning (RIPreC), and cross preconditioning. Although LIPreC has a strong neuroprotective effect, the clinical application of IPreC for subsequent cerebral ischemia is difficult. There are two main reasons for the above result: Cerebral ischemia is unpredictable, and LIPreC is also capable of inducing unexpected injury with only minor differences to durations or intensity. RIPreC and pharmacological preconditioning, an easy-to-use and non-invasive therapy, can be performed in a variety of clinical settings and appear to be more suitable for the clinical management of ischemic stroke. Hoping to advance our understanding of IPreC, this review mainly focuses on recent advances in IPreC in stroke management, its challenges, and the potential study directions.

: The role of gut microbiota in health and diseases has been receiving increased attention recently. Emerging evidence from previous studies on gut-microbiota-brain axis highlighted the importance of gut microbiota in neurological disorders. Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the central nervous system (CNS) resulting from T-cell-driven, myelin-directed autoimmunity. The dysbiosis of gut microbiota in MS patients has been reported in published research studies, indicating that gut microbiota plays an important role in the pathogenesis of MS. Gut microbiota have also been reported to influence the initiation of disease and severity of experimental autoimmune encephalomyelitis, which is the animal model of MS. However, the underlying mechanisms of gut microbiota involvement in the pathogenesis of MS remain unclear. Therefore, in this review, we summerized the potential mechanisms for gut microbiota involvement in the pathogenesis of MS, including increasing the permeability of the intestinal barrier, initiating an autoimmune response, disrupting the blood-brain barrier integrity, and contributing to chronic inflammation. The possibility for gut microbiota as a target for MS therapy has also been discussed. This review provides new insight into understanding the role of gut microbiota in neurological and inflammatory diseases.

Background: Leukoaraiosis (LA), widely accepted as a feature of cerebral small vessel disease, significantly increases the incidence of stroke, dementia, and death. Cerebral small artery disease has been considered as one of the main causes of LA. However, since the term “venous collagenosis” (VC) was proposed in an atrophy research in 1995, there have been pathological and neuroimaging studies proving the association between the venous system and LA in aging, Alz­heimer’s disease (AD), and Parkinson’s disease. Summary: Autopsy studies confirmed that thickening of the lumen wall in venules, which results from the deposition of collagen I and III, leading to vessel stenosis or occlusion, is closely associated with LA. Susceptibility-weighted imaging research revealed a controversial association of deep medullary veins and LA in vivo, regarding which there are no standard criteria currently. Nevertheless, retinal venous changes had been reported to increase the risk of LA development, providing a novel way for in vivo evaluation. As for the internal jugular vein, jugular venous reflux could double the LA score in aging and modulate circulation of cerebral spinal fluids. Key Messages: Disruption of the venous system was notably associated with LA in aging, AD, and Parkinson’s disease post-mortem and in in vivo models. The venous pathological changes may induce cerebral hypoperfusion, drainage system disruption, and vasogenic oedema in the veins around the periventricular white matter. The clarification of VC in LA may provide an early prevention and early treatment strategy for LA patients.

Connexin-43 (Cx43) is the most abundant gap junction protein in the nervous system. It enables cell communication and has important physiological roles including ion transport and substrate exchange, all of which have been implicated in cerebral ischemia injury. Our previous in vitro and in vivo studies have demonstrated that Cx43 is internalized and degraded during ischemia stress. However, the significance of ischemia-induced degradation of Cx43 remains unclear. Herein, we demonstrated that Cx43 degradation during ischemia injury is mediated by selective autophagy; additionally, we identified two related autophagy receptors-OPTN and NDP52. Cx43 degradation during ischemia requires its phosphorylation and ubiquitination, which are mediated by PKC, Src kinases, and ubiquitin kinase PINK1. Using point mutagenesis, we identified three phosphorylation sites underlying Cx43 autophagy degradation under ischemic stress. Cx43 degradation inhibition promoted the transition of astrocytes from a pro-inflammatory to an anti-inflammatory status, based on the levels of IL-10 and TNF in ischemia. Knockdown or accelerated degradation of Cx43 protected astrocytes from apoptosis under ischemic stress. These findings elucidate the underlying mechanism of astrocytic Cx43 autophagic degradation during ischemia. The study has identified potentially novel therapeutic strategies against ischemic stroke and evidence of crosstalk between autophagic degradation of Cx43, astrocytic apoptosis, and neuroinflammation.