Vinpocetine Protects Against Cerebral Ischemia-Reperfusion Injury by Targeting Astrocytic Connexin43 via the PI3K/AKT Signaling Pathway

What is the EXACT METHOD your doctor is using to stop reperfusion injury? NOTHING? Then what research is your doctor working on to solve that problem?  You just might want to save those neurons that are dying during reperfusion. I'd suggest charging your doctor $1000 a dead neuron, that might concentrate the mind. Nothing else seems to get survivors closer to 100% recovery.

• reperfusion injury (26)

 

Vinpocetine Protects Against Cerebral Ischemia-Reperfusion Injury by Targeting Astrocytic Connexin43 via the PI3K/AKT Signaling Pathway

Mingming Zhao¹, Shuai Hou¹, Liangshu Feng¹, Pingping Shen¹, Di Nan¹, Yunhai Zhang^2,3, Famin Wang^2,3, Di Ma^1* and Jiachun Feng^1*

• ¹Department of Neurology and Neuroscience Center, The First Hospital of Jilin University, Changchun, China
• ²Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, China
• ³Jiangsu Key Laboratory of Medical Optics, Suzhou, China

Vinpocetine (Vinp) is known for its neuroprotective properties. However, the protective mechanism of Vinp against cerebral ischemia/reperfusion (I/R) injury should be further explored. This study was designed to investigate the neuroprotective effects of Vinp against oxygen-glucose deprivation/reoxygenation (OGD/R) injury in vitro and cerebral I/R injury in vivo and explore whether this mechanism would involve enhancement of astrocytic connexin 43 (Cx43) expression via the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway. In vitro, we detected astrocytic viability and extracellular nitric oxide by an assay kit, intracellular reactive oxygen species by a DCFH-DA probe, inflammation and apoptosis-related protein expression by immunofluorescence staining, and the astrocytic apoptosis rate by flow cytometry. In vivo, we measured the cerebral infarction volume, superoxide dismutase activity, malondialdehyde content, and the expression of inflammation and apoptosis-related proteins. The results indicated that Vinp ameliorated the detrimental outcome of I/R injury. Vinp attenuated astrocytic injury induced by OGD/R and reduced cerebral infarction volume and cerebral edema in rats with cerebral I/R injury. Moreover, Vinp reduced oxidative stress, inflammation, and apoptosis induced by cerebral I/R injury in brain tissues. Meanwhile, Vinp increased p-Cx43 and p-AKT expression, and the p-Cx43/Cx43 and p-AKT/AKT ratio, which was decreased by cerebral I/R injury. Coadministration of PI3K inhibitors LY294002 and BKM120 blunted the effects of Vinp. This study suggests that Vinp protects against cerebral I/R injury via Cx43 phosphorylation by activating the PI3K/AKT pathway.

Introduction

Ischemic stroke has high morbidity and mortality and seriously affects patient quality of life (Ribeiro et al., 2015). Timely recovery of blood and oxygen supply to the ischemic brain tissue is essential for ischemic penumbra survival. Thrombolytic therapy is the best treatment option for ischemic stroke (Sheth et al., 2015). However, reperfusion aggravates the damage and provokes dysfunction through a cascade of events such as calcium overload, excitotoxicity, oxidative stress, inflammatory responses, and apoptosis, which are collectively termed “ischemia-reperfusion injury” (I/R injury) (Dirnagl et al., 1999). Therefore, effectively blocking the cascade of cerebral I/R injury and exploring effective drugs for the treatment of ischemic stroke are very important.

Astrocytes are abundant in the central nervous system, and they play essential roles in maintaining brain function under physiologic conditions and in influencing neuronal survival under pathologic conditions, such as cerebral I/R injury and other brain insults (Garman, 2011; Falkowska et al., 2015; Verkhratsky et al., 2017). During ischemic stroke, astrocytes may be activated and produce and release reactive oxygen species (ROS), pro-inflammatory cytokines, and other factors that may negatively influence the survival of neurons in the penumbra (Swanson et al., 2004). Thus, preventing astrocytic inflammatory and apoptotic effects may be a promising strategy for neuroprotection in ischemic stroke (Cekanaviciute and Buckwalter, 2016; Choudhury and Ding, 2016; Liu and Chopp, 2016).

The PI3K/AKT signaling pathway regulates a wide range of cellular functions, including cellular differentiation, proliferation, inflammation, and apoptosis (Cantley, 2002). Studies have shown that phosphorylation of AKT (Ser473) reduces neuronal apoptosis caused by cerebral I/R injury (Fukunaga and Kawano, 2003; Zhao et al., 2006), and LY294002-mediated inhibition of the PI3K/AKT pathway blocked the cardioprotective effect of atorvastatin against I/R injury in cardiocytes by downregulating Connexin 43 (Cx43) (Bian et al., 2015). Moreover, activated AKT can phosphorylate the C-terminal Ser373 residue of Cx43 (Solan and Lampe, 2014). Since Cx43 is the most commonly expressed gap junction protein in astrocytes (Orellana et al., 2011), and increased Cx43 expression can reduce neuronal damage after cerebral I/R (Nakase et al., 2003), we speculate that Cx43 is involved in the PI3K/AKT pathway’s protective effects against cerebral I/R injury.

Vinpocetine (Vinp) is a semi-synthetic alkaloid derivative isolated from the leaves of Phyllostachys pubescens. Its anti-inflammatory and anti-platelet aggregation effects on improving cerebral blood flow, brain metabolism, and cognition have been confirmed by various studies (Zhang et al., 2018; Zhang et al., 2018). Vinp has been widely used in the treatment of stroke, cerebral arteriosclerosis, and chronic cerebral insufficiency, and it exhibits unique advantages in the treatment of dementia and epilepsy. A previous study showed that Vinp similarly decreased the inflammatory response by inhibiting NF-κB and TNF-α expression after cerebral I/R injury (Wang et al., 2014); however, its specific mechanism remains unknown. Cerebral I/R injury can activate both astrocytes and microglia, which may produce inflammatory cytokines and other toxic mediators (Kim et al., 2016; Duris et al., 2018). Microglial TLR4/MyD88/NF-κB has been shown to be one of the mechanisms by which Vinp protects against cerebral I/R injury (Wu et al., 2017). However, so far, no study has focused on whether Vinp’s protective effects against cerebral I/R injury is related to astrocytes. Hence, we hypothesized that Vinp may affect astrocytic Cx43 via the PI3K/AKT pathway and thereby provide neuroprotection.

In this study, we explored the neuroprotective roles of vinpocetine against oxygen-glucose deprivation/reoxygenation (OGD/R) injury in vitro and cerebral I/R injury in vivo and explore whether this mechanism would involve enhancement of astrocytic connexin 43 (Cx43) expression via the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway.

Vasculogenesis in Experimental Stroke After Human Cerebral Endothelial Cell Transplantation

Sounds interesting.  What will your doctor do to initiate human clinical trials?
http://stroke.ahajournals.org/content/44/12/3473.abstract?etoc

1. Hiroto Ishikawa, MD, PhD;
2. Naoki Tajiri, PT, PhD;
3. Kazutaka Shinozuka, PhD;
4. Julie Vasconcellos, BS;
5. Yuji Kaneko, PhD;
6. Hong J. Lee, PhD;
7. Osamu Mimura, MD, PhD;
8. Mari Dezawa, MD, PhD;
9. Seung U. Kim, MD, PhD;
10. Cesar V. Borlongan, PhD

+ Author Affiliations

1. From the Department of Neurosurgery and Brain Repair, University of South Florida College of Medicine, Tampa, FL (H.I., N.T., K.S., J.V., Y.K., C.V.B.); Department of Ophthalmology, Hyogo College of Medicine, Nishinomiya, Japan (H.I., O.M.); Medical Research Institute, Chung-Ang University College of Medicine, Seoul, Korea (H.J.L.); Department of Stem Cell Biology and Histology and Department of Anatomy and Anthropology, Tohoku University Graduate School of Medicine, Sendai, Japan (M.D.); and Department of Neurology, University of British Columbia, Vancouver, British Columbia, Canada (S.U.K.).

1. Correspondence to Cesar V. Borlongan, PhD, Department of Neurosurgery and Brain Repair, University of South Florida, 12901 Bruce B. Downs Blvd MDC78, Tampa, FL 33612. E-mail cborlong@health.usf.edu

Abstract

Background and Purpose—Despite the reported functional recovery in transplanted stroke models and patients, the mechanism of action underlying stem cell therapy remains not well understood. Here, we examined the role of stem cell–mediated vascular repair in stroke.

Methods—Adult rats were exposed to transient occlusion of the middle cerebral artery and 3 hours later randomly stereotaxically transplantated with 100K, 200K, or 400K human cerebral endothelial cell 6 viable cells or vehicle. Animals underwent neurological examination and motor test up to day 7 after transplantation then euthanized for immunostaining against neuronal, vascular, and specific human antigens. A parallel in vitro study cocultured rat primary neuronal cells with human cerebral endothelial cell 6 under oxygen-glucose deprivation and treated with vascular endothelial growth factor (VEGF) and anti-VEGF.

Results—Stroke animals that received vehicle infusion displayed typical occlusion of the middle cerebral artery–induced behavioral impairments that were dose-dependently reduced in transplanted stroke animals at days 3 and 7 after transplantation and accompanied by increased expression of host neuronal and vascular markers adjacent to the transplanted cells. Some transplanted cells showed a microvascular phenotype and juxtaposed to the host vasculature. Infarct volume in transplanted stroke animals was significantly smaller than vehicle-infused stroke animals. Moreover, rat neurons cocultured with human cerebral endothelial cell 6 or treated with VEGF exhibited significantly less oxygen-glucose deprivation–induced cell death that was blocked by anti-VEGF treatment.

Conclusions—We found attenuation of behavioral and histological deficits coupled with robust vasculogenesis and neurogenesis in endothelial cell–transplanted stroke animals, suggesting that targeting vascular repair sets in motion a regenerative process in experimental stroke possibly via the VEGF pathway.

Microglial P2Y12 Deficiency/Inhibition Protects against Brain Ischemia

I'm sure your doctor can explain this(snort, snort).
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0070927

Abstract

Objective

Microglia are among the first immune cells to respond to ischemic insults. Triggering of this inflammatory response may involve the microglial purinergic GPCR, P2Y₁₂, activation via extracellular release of nucleotides from injured cells. It is also the inhibitory target of the widely used antiplatelet drug, clopidogrel. Thus, inhibiting this GPCR in microglia should inhibit microglial mediated neurotoxicity following ischemic brain injury.

Methods

Experimental cerebral ischemia was induced, in vitro with oxygen-glucose deprivation (OGD), or in vivo via bilateral common carotid artery occlusion (BCCAO). Genetic knock-down in vitro via siRNA, or in vivo P2Y₁₂ transgenic mice (P2Y₁₂−/− or P2Y₁₂+/−), or in vivo treatment with clopidogrel, were used to manipulate the receptor. Neuron death, microglial activation, and microglial migration were assessed.

Results

The addition of microglia to neuron-astrocyte cultures increases neurotoxicity following OGD, which is mitigated by microglial P2Y₁₂ deficiency (P<0.05). Wildtype microglia form clusters around these neurons following injury, which is also prevented in P2Y₁₂ deficient microglia (P<0.01). P2Y₁₂ knock-out microglia migrated less than WT controls in response to OGD-conditioned neuronal supernatant. P2Y₁₂ (+/−) or clopidogrel treated mice subjected to global cerebral ischemia suffered less neuronal injury (P<0.01, P<0.001) compared to wild-type littermates or placebo treated controls. There were also fewer microglia surrounding areas of injury, and less activation of the pro-inflammatory transcription factor, nuclear factor Kappa B (NFkB).

Interpretation

P2Y₁₂ participates in ischemia related inflammation by mediating microglial migration and potentiation of neurotoxicity. These data also suggest an additional anti-inflammatory, neuroprotective benefit of clopidogrel.