Rat Middle Cerebral Artery Research Articles

Electroacupuncture on Trigeminal Nerve-Innervated Acupoints Ameliorates Poststroke Cognitive Impairment in Rats with Middle Cerebral Artery Occlusion: Involvement of Neuroprotection and Synaptic Plasticity.

Poststroke cognitive impairment (PSCI) is a severe sequela of stroke. There are no effective therapeutic options for it. In this study, we evaluated whether electroacupuncture (EA) on the trigeminal nerve-innervated acupoints could alleviate PSCI and identified the mechanisms in an animal model. The male Sprague-Dawley rat middle cerebral artery occlusion (MCAO) model was used in our study. EA was conducted on the two scalp acupoints, EX-HN3 (Yintang) and GV20 (Baihui), innervated by the trigeminal nerve, for 14 sessions, daily. Morris water maze and novel object recognition were used to evaluate the animal's cognitive performance. Neuroprotection and synaptic plasticity biomarkers were analyzed in brain tissues. Ischemia-reperfusion (I/R) injury significantly impaired spatial and cognition memory, while EA obviously reversed cognitive deterioration to the control level in the two cognitive paradigms. Moreover, EA reversed the I/R injury-induced decrease of brain-derived neurotrophic factor, tyrosine kinase B, N-methyl-D-aspartic acid receptor 1, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor, γ-aminobutyric acid type A receptors, Ca2+/calmodulin-dependent protein kinase II, neuronal nuclei, and postsynaptic density protein 95 expression in the prefrontal cortex and hippocampus. These results suggest that EA on the trigeminal nerve-innervated acupoints is an effective therapy for PSCI, in association with mediating neuroprotection and synaptic plasticity in related brain regions in the MCAO rat model.

Neuroprotective effect of apigenin against cerebral ischemia/reperfusion injury.

ObjectiveThe therapeutic efficacy of apigenin in PC12 cells and rats remains uncertain. The aim of this study was to investigate the neuroprotective effects of apigenin against cerebral ischemia/reperfusion injury, both in vitro and in vivo.MethodsWe first treated PC12 cells with cobalt chloride (CoCl2) to create a model of oxidative stress injury. Cell viability was then determined using a multifunctional microplate reader. In addition, reactive oxygen species (ROS) levels, apoptosis, and mitochondrial membrane potentials (MMPs) were examined using high-content cytometer analysis. The efficacy of apigenin treatment was also analyzed in a rat middle cerebral artery occlusion (MCAO) model using TTC staining and neurological deficit scores.ResultsThe half-inhibitory concentration of CoCl2 was 1.2 mM. Pretreatment with 10 µg ⋅ mL−1 apigenin significantly enhanced cell viability, reduced ROS levels, alleviated apoptosis, and improved MMP in PC12 cells with CoCl2-induced injury in vitro. In addition, apigenin treatment in vivo significantly improved neurological deficit scores and reduced infarct areas in MCAO rats. These results suggest that the neuroprotective mechanisms of apigenin may be related to mitochondrial activation.ConclusionsApigenin had excellent neuroprotective effects for the treatment of cerebral ischemia/reperfusion injury in vitro and in vivo.

Neuroprotection by cattle encephalon glycoside and ignotin beyond the time window of thrombolysis in ischemic stroke.

Cattle encephalon glycoside and ignotin (CEGI) injection is known as a multi-target neuroprotective drug that contains numerous liposoluble molecules, such as polypeptides, monosialotetrahexosyl ganglioside (GM-1), free amino acids, hypoxanthine and carnosine. CEGI has been approved by the Chinese State Food and Drug Administration and widely used in the treatments of various diseases, such as stroke and Alzheimer's disease. However, the neuroprotective effects of CEGI beyond the time window of thrombolysis (within 4.5 hours) on acute ischemic stroke remain unclear. This study constructed a rat middle cerebral artery occlusion model by suture-occluded method to simulate ischemic stroke. The first daily dose was intraperitoneally injected at 8 hours post-surgery and the CEGI treatments continued for 14 days. Results of the modified five-point Bederson scale, beam balance test and rotameric test showed the neurological function of ischemic stroke rats treated with 4 mL/kg/d CEGI improved significantly, but the mortality within 14 days did not change significantly. Brain MRI and 2,3,5-triphenyltetrazolium chloride staining confirmed that the infarct size in the 4 mL/kg/d CEGI-treated rats was significantly reduced compared with ischemic insult only. The results of transmission electron microscopy and double immunofluorescence staining showed that the hippocampal neuronal necrosis in the ischemic penumbra decreased whereas the immunopositivity of new neuronal-specific protein doublecortin and the percentage of Ki67/doublecortin positive cells increased in CEGI-treated rats compared with untreated rats. Our results suggest that CEGI has an effective neuroprotective effect on ischemic stroke when administered after the time window of thrombolysis. The study was approved by the Animal Ethics Committee of The Third Military Medical University, China.

Bone marrow mesenchymal stem cells induce M2 microglia polarization through PDGF-AA/MANF signaling.

BACKGROUNDBone marrow mesenchymal stem cells (BMSCs) are capable of shifting the microglia/macrophages phenotype from M1 to M2, contributing to BMSCs-induced brain repair. However, the regulatory mechanism of BMSCs on microglia/macrophages after ischemic stroke is unclear. Recent evidence suggests that mesencephalic astrocyte–derived neurotrophic factor (MANF) and platelet-derived growth factor-AA (PDGF-AA)/MANF signaling regulate M1/M2 macrophage polarization.AIMTo investigate whether and how MANF or PDGF-AA/MANF signaling influences BMSCs-mediated M2 polarization.METHODSWe identified the secretion of MANF by BMSCs and developed transgenic BMSCs using a targeting small interfering RNA for knockdown of MANF expression. Using a rat middle cerebral artery occlusion (MCAO) model transplanted by BMSCs and BMSCs–microglia Transwell coculture system, the effect of BMSCs-induced downregulation of MANF expression on the phenotype of microglia/macrophages was tested by Western blot, quantitative reverse transcription-polymerase chain reaction, and immunofluorescence. Additionally, microglia were transfected with mimics of miR-30a*, which influenced expression of X-box binding protein (XBP) 1, a key transcription factor that synergized with activating transcription factor 6 (ATF6) to govern MANF expression. We examined the levels of miR-30a*, ATF6, XBP1, and MANF after PDGF-AA treatment in the activated microglia.RESULTSInhibition of MANF attenuated BMSCs-induced functional recovery and decreased M2 marker production, but increased M1 marker expression in vivo or in vitro. Furthermore, PDGF-AA treatment decreased miR-30a* expression, had no influence on the levels of ATF6, but enhanced expression of both XBP1 and MANF.CONCLUSIONBMSCs-mediated MANF paracrine signaling, in particular the PDGF-AA/miR-30a*/XBP1/MANF pathway, synergistically mediates BMSCs-induced M2 polarization.

Calpain-mediated cleavage of Fbxw7 during excitotoxicity

Neuronal cell death induced by ischemic injury has been attributed to glutamate receptor-mediated excitotoxicity, which is known to be accompanied by Ca2+ overload in the cytoplasm with concomitant activation of calcium-dependent mechanisms. More specifically, the overactivation of calpains, calcium-dependent cysteine proteases, have been associated with neuronal cell death following glutamate treatment. Previously, we observed decreased expression levels of F-box/WD repeat domain-containing protein 7 (Fbxw7) after the hyperactivation of cyclin-dependent kinase 5 (Cdk5) in cortical neurons challenged with glutamate. As determined using in vitro calpain cleavage assays, we demonstrated that the cleavage of Fbxw7 was mediated by activated calpain and attenuated in the presence of the calpain inhibitor, calpeptin. Using the rat middle cerebral artery occlusion model, we confirmed that Fbxw7 was indeed cleaved by activated calpain in the ipsilateral cortex. Based on our data, we hypothesize that the negative regulation of Fbxw7 by calpain may contribute to neuronal cell death and that the preservation of Fbxw7 by the inhibition of calpain, Cdk5, or both composes a novel protective mechanism following excitotoxicity.

TREM-1-targeting LP17 attenuates cerebral ischemia-induced neuronal injury by inhibiting oxidative stress and pyroptosis

Stroke ranks as the second leading cause of disability and death globally. Trigger receptors expressed on myeloid cells (TREM) −1 are responsible for the activation of the innate immune response and also play a critical role in inflammation. In this study, we reported the contribution of TREM-1 after ischemic damage in a rat middle cerebral artery occlusion (MCAO) model. This study also demonstrated that TREM-1 expression was upregulated following cerebral infarction in rats. TREM-1 inhibition was determined using its selective inhibitor, LP17, which indicated a neuroprotective effect on cerebral infarction damage. The findings revealed that inhibition of TREM-1 by administering LP17 improved cerebral damage and decreased ischemic areas and brain water contents. Moreover, LP17 decreased MCAO-induced microglial activation and neurodegeneration, evidenced by a reduction in the expression of microglial Iba-1 and FJ-B positive cells, and reversed neuronal loss. Besides, the contribution of LP17 to ischemic neuronal damage may be associated with a decrease in the production of pro-inflammatory cytokines, and enhanced production of anti-inflammatory cytokine IL-10. Both in vivo and in vitro studies showed that inhibiting TREM-1 attenuated ROS accumulation, lipid per-oxidation (LPO) contents such as malondialdehyde (MDA) and enhanced the superoxide dismutase (SOD) activity after ischemia. Inhibiting TREM-1 alleviated inflammation and pyroptosis found in MCAO rats. This was achieved through the inhibition of the levels of NLRP3, caspase-1, ASC (an apoptosis-associated speck-like protein containing a CARD) and gasdermin D. These results confirmed that inhibiting TREM-1 protects against ischemia-induced neuronal damage and alleviates microglial mediated neuro-inflammation by reducing oxidative stress and pyroptosis. Therefore, blocking TREM-1 expression provides an effective intervention for improving ischemic stroke.

Prazosin blocks apoptosis of endothelial progenitor cells through downregulating the Akt/NF-κB signaling pathway in a rat cerebral infarction model.

Endothelial progenitor cells (EPCs) can enhance the recanalization of thrombosis during the progression of cerebral infarction. Prazosin plays a therapeutic role in expanding the peripheral vasculature and regulating infarction cardiosclerosis by inhibiting phosphoinositide signaling. However, the possible mechanisms underlying the therapeutic effects of prazosin have not been fully explored. The purpose of the present study was to analyze the anti-apoptotic effects of prazosin on EPCs in a rat cerebral infarction model. The results showed that prazosin treatment decreased apoptosis of EPCs. Prazosin treatment decreased the serum expression levels of the inflammatory factors, interleukin-1β and tumor necrosis factor-α in rats with cerebral infarctions as well as in EPCs in vitro. In addition, prazosin reduced the expression levels of Akt, NF-κB, phosphorylated (p)-Akt and p-NF-κB in EPCs and the middle cerebral artery of rats with cerebral infarction. These findings demonstrated that prazosin inhibited EPC apoptosis in the cerebral infarction rats through targeting the Akt/NF-κB signaling pathway. In conclusion, these results indicated that prazosin has a preventive effect on cerebral infarction by inhibiting EPC apoptosis and by inhibiting the inflammatory response in vitro and in vivo through regulating the Akt/NF-κB signaling pathway.

D1 receptor‐mediated endogenous tPA upregulation contributes to blood‐brain barrier injury after acute ischaemic stroke

Blood‐brain barrier (BBB) integrity injury within the thrombolytic time window is becoming a critical target to reduce haemorrhage transformation (HT). We have previously reported that BBB damage was initially damaged in non‐infarcted striatum after acute ischaemia stroke. However, the underlying mechanism is not clear. Since acute ischaemic stroke could induce a significant increase of dopamine release in striatum, in current study, our aim is to investigate the role of dopamine receptor signal pathway in BBB integrity injury after acute ischaemia using rat middle cerebral artery occlusion model. Our data showed that 2‐h ischaemia induced a significant increase of endogenous tissue plasminogen activator (tPA) in BBB injury area and intra‐striatum infusion of tPA inhibitor neuroserpin, significantly alleviated 2‐h ischaemia‐induced BBB injury. In addition, intra‐striatum infusion of D1 receptor antagonist SCH23390 significantly decreased ischaemia‐induced upregulation of endogenous tPA, accompanied by decrease of BBB injury and occludin degradation. More important, inhibition of hypoxia‐inducible factor‐1 alpha with inhibitor YC‐1 significantly decreased 2‐h ischaemia‐induced endogenous tPA upregulation and BBB injury. Taken together, our data demonstrate that acute ischaemia disrupted BBB through activation of endogenous tPA via HIF‐1α upregulation, thus representing a new therapeutic target for protecting BBB after acute ischaemic stroke.

LncRNA MEG8 Attenuates Cerebral Ischemia After Ischemic Stroke Through Targeting miR-130a-5p/VEGFA Signaling.

MEG8 is involved in ischemia stroke, however, its role in ischemia stroke remains unknown. The current research aimed to investigate the effects and mechanisms of MEG8 in ischemic stroke. Mouse brain microvascular endothelial cells (BMECs) were treated by oxygen-glucose deprivation (OGD). Then, the expressions of MEG8 and miR-130a-5p were detected by quantitative reverse transcription-polymerase chain reaction (q-PCR). Cell counting kit-8 (CCK-8), wound-healing, tube formation, Western blot, and q-PCR assays were performed to detect the effects of MEG8 and miR-130a-5p on cell viability, migration, and angiogenesis and VEGFA expression. Bioinformatics, dual-luciferase reporter assay, and RNA immunoprecipitation analysis were carried out to investigate the targeting relationship between MEG8 and miR-130a-5p, and between miR-130a-5p and VEGFA. Then, rat middle cerebral artery occlusion (MCAO) model and MEG8 overexpression MCAO model were established, and neurological deficit and infarct volume of the model rats were evaluated. Finally, Western blot and q-PCR were carried out to detect the expressions of MEG8, miR-130a-5p, and VEGFA. MEG8 was upregulated and miR-130a-5p was downregulated in OGD-treated BMECs. MiR-130a-5p was found to be a target of MEG8, and VEGFA was predicted to be a potential target of miR-130a-5p. Downregulation of MEG8 inhibited the cell viability, migration, and angiogenesis and the expression of VEGFA via negatively regulating miR-130a-5p of BMECs treated by OGD/non-OGD. In addition, MEG8 reduced cerebral ischemia, neurological score and miR-130a-5p expression, and increased VEGFA expression of MCAO rat. Our findings proved that MEG8 regulates angiogenesis and attenuates cerebral ischemia after ischemic stroke via miR-130a-5p/VEGFA signaling.

Vascular-Cognitive Impairment following High-Thoracic Spinal Cord Injury Is Associated with Structural and Functional Maladaptations in Cerebrovasculature.

Individuals living with chronic spinal cord injury (SCI) often exhibit impairments in cognitive function, which impede their rehabilitation and transition into the community. Although a number of clinical studies have demonstrated the impact of impaired cardiovascular control on cognitive impairment, the mechanistic understanding of this deleterious relationship is still lacking. The present study investigates whether chronic disruption of cardiovascular control following experimental SCI results in cerebrovascular decline and vascular cognitive impairment. Fourteen weeks following a high thoracic SCI (at the third thoracic segment), rats were subjected to a battery of in vivo and in vitro physiological assessments, cognitive-behavioral tests, and immunohistochemical approaches to investigate changes in cerebrovascular structure and function in the middle cerebral artery (MCA). We show that in the MCA of rats with SCI, there is a 55% (SCI vs. control: 13.4 ± 1.9% vs. 29.63 ± 2.8%, respectively) reduction in the maximal vasodilator response to carbachol, which is associated with reduced expression of endothelial marker cluster of differentiation 31 (CD31) and transient receptor potential cation channel 4 (TRPV 4) channels. Compared with controls, MCAs in rats with SCI were found to have 50% (SCI vs. control: 1.5 ± 0.2 vs. 1 ± 0.1 a.u., respectively) more collagen 1 in the media of vascular wall and 37% (SCI vs. control: 30.5 ± 2.9% vs. 42.0 ± 4.0%, respectively) less distensibility at physiological intraluminal pressure. Further, the cerebral blood flow (CBF) in the hippocampus was reduced by 32% in the SCI group (SCI vs. control: 44.3 ± 4.5 mL/100 g/min vs. 65.0 ± 7.2 mL/100 g/min, respectively) in association with impairment of short-term memory based on a novel object recognition test. There were no changes in the sympathetic innervation of the vasculature and passive structure in the SCI group. Chronic experimental SCI is associated with structural alterations and endothelial dysfunction in cerebral arteries that likely contribute to significantly reduced CBF and vascular cognitive impairment.

Hydrogen Protons Modulate Perivascular Axo-axonal Interactions in the Middle Cerebral Artery of Rats.

Previous studies have demonstrated that nicotine can induce relaxation of the middle cerebral artery (MCA). However, whether this relaxation is associated with the activity of sensory calcitonin gene-related peptide (CGRP) nerves and whether this is modulated by hydrogen protons (H), facilitating the release of CGRP from sensory CGRPergic nerve terminals in the MCA, remains unclear. In this study, we examined the role of H in the modulation of neurogenic vasomotor responses in the rat-isolated endothelium-denuded MCA. Wire myography was used to measure vasoreactivity and indicated that nicotine-induced relaxation was sensitive to tetrodotoxin and lidocaine and drastically reduced levels of guanethidine (an adrenergic neuronal blocker), N-nitro-L-arginine (L-NNA), CGRP8-37, vasoactive intestinal polypeptide (VIP)6-28, capsaicin, capsazepine (a transient receptor potential vanilloid-1 inhibitor), and tetraethylammonium. However, this nicotine-induced relaxation was not sensitive to propranolol. Lowering the pH of the buffer solution with HCl caused pH-dependent vasorelaxation and deceased intracellular pH in the MCA rings, which was sensitive to L-NNA, CGRP8-37, VIP6-28, capsazepine, 4-aminopyridine (a voltage-gated potassium channel antagonist), and paxilline (a large conductance Ca-activated K channel antagonist). However, HCl-induced relaxation was not inhibited by glibenclamide (an ATP-sensitive K channel blocker). These results suggested that electrical and chemical activation of cerebral perivascular adrenergic nerves led to the release of H, which then facilitated the release of NO, VIP, and CGRP, resulting in vasorelaxation. Lowering the pH of the buffer solution caused potassium channels of vascular smooth muscle cells and perivascular nerves to open. In conclusion, our results demonstrated that H may act as a modulator on MCA perivascular nerves and/or smooth muscles.

Characterization of culture from smooth muscle cells isolated from rat middle cerebral arteries

Although human brain represents only 2% of the body mass, it uses around 20 % of the organism energy. Due to the brain’s limited energy storage, the oxygen and glucose necessary to support brain functions depends on the correct blood supply. The main components of the arteries are smooth muscle cells, which are considered the main regulators of vascular tone and blood flow distribution. The information currently available on the functioning of the cerebral arteries and their cell constituents is extremely scarce. Thus, the aim of this work was to develop an in vitro model of smooth muscle cells derived from rat middle cerebral artery. Explants were collected from rat middle cerebral artery and adhered to collagen-coated culture dishes. Immunocytochemical analysis showed that the cells present in the culture expressed α-actin, a protein characteristic of the contractile phenotype of these cells. In addition, these cells did not express the endothelial marker, vWF. To evaluate the functionality of these cells the response to contractile agents, serotonin and noradrenaline, and to relaxing agent, sodium nitroprusside was determine by Planar Cell Surface Area analysis. Together the data obtained show that the cell culture obtained through the procedure described resulted in cells presenting the markers characteristic of smooth muscle cells and maintaining the usual contractile response, indicating that the cells obtained through this may be used as a model for characterization and study of functional behavior of the middle cerebral artery, as well as interaction studies between vascular and neuronal system.

Fe Porphyrin-Based SOD Mimic and Redox-Active Compound, (OH)FeTnHex-2-PyP4+, in a Rodent Ischemic Stroke (MCAO) Model: Efficacy and Pharmacokinetics as Compared to Its Mn Analogue, (H2O)MnTnHex-2-PyP5+.

Mn(III) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin, (H2O)MnTnHex-2-PyP5+ (MnHex) carrying long hexyl chains, is a lipophilic mimic of superoxide dismutase (SOD) and a redox-active drug candidate. MnHex crosses the blood–brain barrier, and improved neurologic outcome and decreased infarct size and inflammation in a rat middle cerebral artery occlusion (MCAO) ischemic stroke model. Yet, the dose and the therapeutic efficacy of Mn porphyrin were limited by an adverse effect of arterial hypotension. An equally lipophilic Fe analog, (OH)FeTnHex-2-PyP4+ (FeHex), is as redox-active and potent SOD mimic in vitro. With different coordination geometry of the metal site, FeHex has one hydroxo (OH) ligand (instead of water) bound to the Fe center in the axial position. It has ~2 orders of magnitude higher efficacy than MnHex in an SOD-deficient E. coli model of oxidative stress. In vivo, it does not cause arterial hypotension and is less toxic to mice. We thus evaluated FeHex versus MnHex in a rodent MCAO model. We first performed short- and long-term pharmacokinetics (PK) of both porphyrins in the plasma, brain, and liver of rats and mice. Given that damage to the brain during stroke occurs very rapidly, fast delivery of a sufficient dose of drug is important. Therefore, we aimed to demonstrate if, and how fast after reperfusion, Fe porphyrin reaches the brain relative to the Mn analog. A markedly different plasma half-life was found with FeHex (~23 h) than with MnHex (~1.4 h), which resulted in a more than 2-fold higher plasma exposure (AUC) in a 7-day twice-daily treatment of rats. The increased plasma half-life is explained by the much lower liver retention of FeHex than typically found in Mn analogs. In the brain, a 3-day mouse PK study showed similar levels of MnHex and FeHex. The same result was obtained in a 7-day rat PK study, despite the higher plasma exposure of FeHex. Importantly, in a short-term PK study with treatment starting 2 h post MCAO, both Fe- and Mn- analogs distributed at a higher level to the injured brain hemisphere, with a more pronounced effect observed with FeHex. While a 3-day mouse MCAO study suggested the efficacy of Fe porphyrin, in a 7-day rat MCAO study, Mn-, but not Fe porphyrin, was efficacious. The observed lack of FeHex efficacy was discussed in terms of significant differences in the chemistry of Fe vs. the Mn center of metalloporphyrin; relative to MnHex, FeHex has the propensity for axial coordination, which in vivo would preclude the reactivity of the Fe center towards small reactive species.

Исследование механизма нейропротективного действия пептидного препарата Семакс в мозге крыс в условиях ишемии-реперфузии

Синтетический пептид АКТГ(4-7)PGP ускоряет регресс неврологических нарушений при ишемическом инсульте, однако молекулярные механизмы его действия полностью не известны. На модели полуторачасовой окклюзии средней мозговой артерии крыс был проведен анализ влияния пептида на экспрессию ряда генов и белков, вовлеченных в сигнальные пути, приводящие к воспалению и гибели клеток в условиях ишемии-реперфузии. Показано, что пептид через 24 ч от начала окклюзии снижает повышенный при ишемии уровень экспрессии в ишемизированной ткани мозга крыс мРНК провоспалительных цитокинов и хемокинов IL-1α, IL-1β, IL-6, TNF-α, Cxcl2 и Ccl3. Пептид снижал повышенные при ишемии уровни экспрессии белков металлопротеиназы ММР-9, транскрипционного фактора c-Fos, активных JNK киназ и предотвращал ишемическое снижение уровня активного транскрипционного фактора CREB. The synthetic peptide ACTH (4-7) PGP accelerates the regression of neurological disorders in ischemic stroke, but the molecular mechanisms of its action are not completely known. On the model of an hour and a half occlusion of the rat middle cerebral artery, an analysis was made of the effect of the peptide on the expression of a number of genes and proteins involved in signaling pathways leading to inflammation and cell death under conditions of ischemia-reperfusion. It was shown that the peptide 24 hours after the onset of occlusion reduces the level of expression of mRNA of pro-inflammatory cytokines and chemokines IL-1α, IL-1β, IL-6, TNF-α, Cxcl2 and Ccl3 in ischemic brain tissue. The peptide decreased the levels of expression of proteins metalloproteinase MMP-9, transcription factor c-Fos, active JNK kinases wich were increased under ischemia, and prevented ischemic decrease in the level of active transcription factor CREB.

Altered COVID-19 receptor ACE2 expression in a higher risk group for cerebrovascular disease and ischemic stroke

Coronavirus disease 2019 (COVID-19) is a worldwide pandemic. It has a high transmission rate among humans, and is a threat to global public health. However, there are no effective prophylactics or therapeutics available. It is necessary to identify vulnerable and susceptible groups for adequate protection and care against this disease. Recent studies have reported that COVID-19 has angiotensin-converting enzyme 2 (ACE2) as a functional receptor, which may lead to the development of severe cerebrovascular diseases (CVD), including strokes, in patients with risk factors for CVD such as diabetes and smoking. Thus, the World Health Organization (WHO) advised caution against COVID-19 for smokers and patients with underlying clinical symptoms, including cardiovascular diseases. Here, we observed ACE2 expression in the brain of rat middle cerebral artery occlusion (MCAO) model and evaluated the effects of cigarette smoke extract (CSE) and diabetes on ACE2 expression in vessels. We showed that the levels of ACE2 expression was increased in the cortex penumbra after ischemic injuries. CSE treatment significantly elevated ACE2 expression in human brain vessels. We found that ACE2 expression was upregulated in primary cultured human blood vessels with diabetes compared to healthy controls. This study demonstrates that ACE2 expression is increased in ischemic brains and vessels exposed to diabetes or smoking, makes them vulnerable to COVID-19 infection.

Circ-camk4 involved in cerebral ischemia/reperfusion induced neuronal injury

Stroke and subsequent cerebral ischemia/reperfusion (I/R) injury is a frequently occurring disease that can have serious consequences in the absence of timely intervention. Circular RNAs (circRNAs) in association with microRNAs (miRNAs) and RNA-binding proteins (RBPs) can influence gene expression. However, whether circRNAs have a role in cerebral I/R injury pathogenesis, especially soon after onset, is unclear. In this study, we used the SD rat middle cerebral artery occlusion (MCAO) model of stroke to examine the role of circRNAs in cerebral I/R injury. We used high-throughput sequencing (HTS) to compare the expression levels of circRNAs in cerebral cortex tissue from MCAO rats during the occlusion-reperfusion latency period 3 hours after I/R injury with those in control cerebral cortices. Our sequencing results revealed that expression levels of 44 circRNAs were significantly altered after I/R, with 16 and 28 circRNAs showing significant up- and down-regulation, respectively, relative to levels in control cortex. We extended these results in vitro in primary cultured neuron cells exposed to oxygen-glucose deprivation/reperfusion (OGD/R) using qRT-PCR to show that levels of circ-camk4 were increased in OGD/R neurons relative to control neurons. Bioinformatics analyses predicted that several miRNAs could be associated with circ-camk4 and this prediction was confirmed in a RNA pull-down assay. KEGG analysis to predict pathways that involve circ-camk4 included the glutamatergic synapse pathway, MAPK signaling pathway, and apoptosis signaling pathways, all of which are known to be involved in brain injury after I/R. Our results also demonstrate that levels of the human homolog to circ-camk4 (hsa-circ-camk4) are elevated in SH-SY5Y cells exposed to OGD/R treatment. Overexpression of hsa-circ-camk4 in SH-SY5Y cells significantly increased the rate of cell death after OGD/R, suggesting that circ-camk4 may play a key role in progression of cerebral I/R injury.

Neuroprotective Effects of Emodin against Ischemia/Reperfusion Injury through Activating ERK-1/2 Signaling Pathway.

Background: Stroke is one of the leading causes of death and disability worldwide and places a heavy burden on the economy in our society. Current treatments, such as the use of thrombolytic agents, are often limited by a narrow therapeutic time window. However, the regeneration of the brain after damage is still active days, even weeks, after stroke occurs, which might provide a second window for treatment. Emodin, a traditional Chinese medicinal herb widely used to treat acute hepatitis, has been reported to possess antioxidative capabilities and protective effects against myocardial ischemia/reperfusion injury. However, the underlying mechanisms and neuroprotective functions of Emodin in a rat middle cerebral artery occlusion (MCAO) model of ischemic stroke remain unknown. This study investigates neuroprotective effects of Emodin in ischemia both in vitro and in vivo. Methods: PC12 cells were exposed to oxygen-glucose deprivation to simulate hypoxic injury, and the involved signaling pathways and results of Emodin treatment were evaluated. The therapeutic effects of Emodin in ischemia animals were further investigated. Results: Emodin reduced infarct volume and cell death following focal cerebral ischemia injury. Emodin treatment restored PC12 cell viability and reduced reactive oxygen species (ROS) production and glutamate release under conditions of ischemia/hypoxia. Emodin increased Bcl-2 and glutamate transporter-1 (GLT-l) expression but suppressed activated-caspase 3 levels through activating the extracellular signal-regulated kinase (ERK)-1/2 signaling pathway. Conclusion: Emodin induced Bcl-2 and GLT-1 expression to inhibit neuronal apoptosis and ROS generation while reducing glutamate toxicity via the ERK-1/2 signaling pathway. Furthermore, Emodin alleviated nerve cell injury following ischemia/reperfusion in a rat MCAO model. Emodin has neuroprotective effects against ischemia/reperfusion injury both in vitro and in vivo, which may be through activating the ERK-1/2 signaling pathway.

Traumatic Brain Injury Impairs Flow‐Dependent Constrictions of Isolated Rat Middle Cerebral Arteries, Primarily by Interfering with the Flow Sensing Mechanisms

BackgroundTraumatic brain injury (TBI) is a major health problem worldwide due to its high mortality (35–40%), and morbidity (headache, brain edema, cognitive, behavioral, and communicative disabilities). Previously we have shown that mechanotransductions of two hemodynamic factors, pressure and flow are importantly involved in the autoregulation of cerebral blood flow, which may become inefficient after TBI. Indeed, previously we have shown that TBI diminishes pressure‐induced myogenic constriction of middle cerebral arteries (MCA). In the present study we hypothesized that TBI effects also flow‐induced constrictor response of MCA.MethodsTBI was induced in anaesthetized rat by Marmarou’s weight drop model, then MCA was isolated and transferred into pressure‐flow chamber. Inflow and outflow pressures were controlled and measured. The internal diameter was continuously measured by a videomicroscopy equipped with a microangiometer and recorded digitally by PowerLab system (AD Instruments). Data are mean±SEM.ResultsThe active (242,2±6,1 μm) and passive (277,9±8,8) diameters (p<0.05) of isolated MCAs of control rats (n=39) indicate that a substantial myogenic tone developed in response to pressure (80 mmHg: Δ−35,7 ±2,7 μm, p<0.05). Arachidonic acid (AA) ‐ after initial dilations (+6±3 and +9±2,5 μm) ‐ elicited dose dependent constrictions (−9±2,1 and −20±3,2 μm) of MCA of control rats. Step increases in flow elicited gradual constrictions of MCA of control rats (from max.: 229,3±6,6 to 191±6 μm, Δ−38,3 ±0,6 μm, p<0.05), which was eliminated after TBI (from 227,3±14,1 to 227,4±13,3 μm). U46619, a thromboxane A2 (TXA2) / prostaglandin H2 (PGH2) receptor (TP receptor) agonist elicited similar constrictions of MCA of control and TBI rats (max.: Δ−66,1 ±0,7 μm vs. Δ−49 ±4 μm) rats, which were eliminated by SQ 29,548 a TP receptor antagonist. CYP‐450 metabolite 20‐HETE elicited similar constrictions of MCA in control (Δ−18 ±0,9 μm and TBI rats (Δ−15 ±0,1 μm) which were eliminated by HET0016, an inhibitor of CYP‐450. Paxilline (an inhibitor of potassium channel blocker) inhibited flow‐induced constrictions of MCA of control (max.: Δ−37,5 ±3 μm vs. max.: Δ−2,8 ±1 μm, p<0.05), whereas did not affect the diameters of MCA after TBI (max.: Δ−0,1 ±1,6 μm vs. Δ−2,5 ±3 μm).ConclusionsThese findings suggest that 1) flow‐induced constrictor response of MCA depends on AA‐metabolites acting on TP receptors, 2) constrictor responses to TP agonists remain intact after TBI, 3) thus TBI interferes primarily with the flow sensing mechanisms of the vascular wall, which 4) likely leads to diminished autoregulation of cerebral blood flow.Support or Funding InformationScientific Excellence Program 2019., at the University of Physical Education, Innovation and Technology Ministry, Hungary TUDFO/51757/2019‐ITM and Higher Education Institutional Excellence Program at Semmelweis University and the National Bionika Program ED_17‐1‐2017‐0009, Hungary, and the University of Pecs within the framework of the 5th. thematic programme, National Research Development and Innovation Office NKFI‐FK123798.

Sex differences in the structure and function of rat middle cerebral arteries.

Epidemiological studies demonstrate that there are sex differences in the incidence, prevalence, and outcomes of cerebrovascular disease (CVD). The present study compared the structure and composition of the middle cerebral artery (MCA), neurovascular coupling, and cerebrovascular function and cognition in young Sprague-Dawley (SD) rats. Wall thickness and the inner diameter of the MCA were smaller in females than males. Female MCA exhibited less vascular smooth muscle cells (VSMCs), diminished contractile capability, and more collagen in the media, and a thicker internal elastic lamina with fewer fenestrae compared with males. Female MCA had elevated myogenic tone, lower distensibility, and higher wall stress. The stress/strain curves shifted to the left in female vessels compared with males. The MCA of females failed to constrict compared with a decrease of 15.5 ± 1.9% in males when perfusion pressure was increased from 40 to 180 mmHg. Cerebral blood flow (CBF) rose by 57.4 ± 4.4 and 30.1 ± 3.1% in females and males, respectively, when perfusion pressure increased from 100 to 180 mmHg. The removal of endothelia did not alter the myogenic response in both sexes. Functional hyperemia responses to whisker-barrel stimulation and cognition examined with an eight-arm water maze were similar in both sexes. These results demonstrate that there are intrinsic structural differences in the MCA between sexes, which are associated with diminished myogenic response and CBF autoregulation in females. The structural differences do not alter neurovascular coupling and cognition at a young age; however, they might play a role in the development of CVD after menopause.NEW & NOTEWORTHY Using perfusion fixation of the middle cerebral artery (MCA) in calcium-free solution at physiological pressure and systematically randomly sampling the sections prepared from the same M2 segments of MCA, we found that there are structural differences that are associated with altered cerebral blood flow (CBF) autoregulation but not neurovascular coupling and cognition in young, healthy Sprague-Dawley (SD) rats. Understanding the intrinsic differences in cerebrovascular structure and function in males and females is essential to develop new pharmaceutical treatments for cerebrovascular disease (CVD).

Accelerated cerebral vascular injury in diabetes is associated with vascular smooth muscle cell dysfunction.

Individuals with diabetes are more susceptible to cerebral vascular aging. However, the underlying mechanisms are not well elucidated. The present study examined whether the myogenic response of the middle cerebral artery (MCA) is impaired in diabetic rats due to high glucose (HG)-induced cerebral vascular smooth muscle cell (CVSMC) dysfunction, and whether this is associated with ATP depletion and changes in mitochondrial dynamics and membrane potential. The diameters of the MCA of diabetic rats increased to 135.3 ± 11.3% when perfusion pressure was increased from 40 to 180mmHg, while it fell to 85.1 ± 3.1% in non-diabetic controls. The production of ROS and mitochondrial-derived superoxide were enhanced in cerebral arteries of diabetic rats. Levels of mitochondrial superoxide were significantly elevated in HG-treated primary CVSMCs, which was associated with decreased ATP production, mitochondrial respiration, and membrane potential. The expression of OPA1 was reduced, and MFF was elevated in HG-treated CVSMCs in association with fragmented mitochondria. Moreover, HG-treated CVSMCs displayed lower contractile and proliferation capabilities. These results demonstrate that imbalanced mitochondrial dynamics (increased fission and decreased fusion) and membrane depolarization contribute to ATP depletion in HG-treated CVSMCs, which promotes CVSMC dysfunction and may play an essential role in exacerbating the impaired myogenic response in the cerebral circulation in diabetes and accelerating vascular aging.