MCAO Rats Research Articles

Galectin-3 protects against ischemic stroke by promoting neuro-angiogenesis via apoptosis inhibition and Akt/Caspase regulation

Post-stroke neurological deficits and mortality are often associated with vascular disruption and neuronal apoptosis. Galectin-3 (Gal3) is a potent pro-survival and angiogenic factor. However, little is known about its protective role in the cerebral ischemia/reperfusion (I/R) injury. We have previously shown significant up-regulation of Gal3 in the post-stroke rat brain, and that blocking of Gal3 with neutralizing antibody decreases the cerebral blood vessel density. Our current study demonstrates that intracerebral local delivery of the Gal3 into rat brain at the time of reperfusion exerts neuroprotection. Ischemic lesion volume and neuronal cell death were significantly reduced as compared with the vehicle-treated MCAO rat brains. Gal3 increased vessel density and neuronal survival after I/R in rat brains. Importantly, Gal3-treated groups showed significant improvement in motor and sensory functional recovery. Gal3 increased neuronal cell viability under in vitro oxygen–glucose deprivation conditions in association with increased phosphorylated-Akt, decreased phosphorylated-ERK1/2, and reduced caspase-3 activity. Gene expression analysis showed down regulation of pro-apoptotic and inflammatory genes including Fas-ligand, and upregulation of pro-survival and pro-angiogenic genes including Bcl-2, PECAM, and occludin. These results indicate a key role for Gal3 in neuro-vascular protection and functional recovery following ischemic stroke through modulation of angiogenic and apoptotic pathways.

Overexpressed microRNA-539-5p inhibits inflammatory response of neurons to impede the progression of cerebral ischemic injury by histone deacetylase 1.

Several microRNAs (miRNAs or miRs) regulate cerebral ischemic injury outcomes; however, little is known about the role of miR-539-5p during cerebral ischemic injury or the postischemic state. Cerebral ischemic injury was modeled in vitro by exposing human cortical neurons to oxygen-glucose deprivation (OGD) and in vivo by occluding the middle cerebral artery (MCAO) in a rat model. The effects of miR-539-5p, histone deacetylase 1 (HDAC1), and early growth response 2 (EGR2) on cerebral ischemia were investigated using gain- and loss-of-function experiments. We identified changes in miR-539-5p, HDAC1, EGR2, and phosphorylated c-Jun NH2-terminal kinase (JNK). The interaction among miR-539-5p, HDAC1, and EGR2 was determined by dual luciferase reporter gene assay, chromatin immunoprecipitation, and coimmunoprecipitation. We also investigated the effects on cell viability and apoptosis and changes in inflammatory cytokine expression and spatial memory on MCAO rats. miR-539-5p and EGR2 were poorly expressed, while HDAC1 was highly expressed in OGD-treated HCN-2 cells. miR-539-5p targeted HDAC1, while HDAC1 prevented acetylation of EGR2 resulting in its downregulation and subsequent activation of the JNK pathway. Overexpression of miR-539-5p or EGR2 or silencing HDAC1 improved viability and reduced apoptosis of OGD-treated HCN-2 cells in vitro. Furthermore, overexpression of miR-539-5p improved spatial memory, while decreasing cell apoptosis and inflammation in MCAO rats. Collectively, these data suggest that miR-539-5p targets HDAC1 to upregulate EGR2, thus blocking the JNK signaling pathway, by which cerebral ischemic injury is alleviated.

Phoenixin 14 inhibits ischemia/reperfusion-induced cytotoxicity in microglia

The process of ischemia/reperfusion (IR) in ischemic stroke often leads to significant cell death and permanent neuronal damage. Safe and effective treatments are urgently needed to mitigate the damage caused by IR injury. The naturally occurring pleiotropic peptide phoenixin 14 (PNX-14) has recently come to light as a potential treatment for IR injury. In the present study, we examined the effects of PNX-14 on several key processes involved in ischemic injury, such as pro-inflammatory cytokine expression, oxidative stress, and the related cascade mediated through the toll-like receptor 4 (TLR4) pathway, using BV2 microglia exposed to oxygen-glucose deprivation and reoxygenation (OGD/R). Our results demonstrate an acute ability of PNX-14 to regulate the expression levels of proinflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). PNX-14 also prevented oxidative stress by reducing the generation of reactive oxygen species (ROS) and increasing the level of the antioxidant glutathione (GSH). Importantly, PNX-14 inhibited high-mobility group box 1 (HMGB1)/TLR4/myeloid differentiation primary response 88 (MyD88)/nuclear factor-κB (NF-κB) signaling pathway, by inhibiting the activation of TLR4 and preventing the nuclear translocation of p65 protein. We further confirmed the cerebroprotective effects of PNX-14 in an MCAO rat model, which resulted in reduced infarct volume and decreased microglia activation. Together, the results of this study implicate a possible protective role of PNX-14 against various aspects of IR injury in vitro.

A study of optimal concentration range and time window of sevoflurane preconditioning for brain protection in MCAO rats

BackgroundSevoflurane preconditioning improves brain function in MCAO rats, and there are several methods for determining appropriate concentration and time windows for preconditioning. This study investigated the brain protective effects with a single sevoflurane preconditioning at different concentrations and different time windows on MCAO rats.MethodsAdult Sprague-dawley rats were randomly assigned to 14 groups. The rats in the sevoflurane preconditioning group inhaled 0.5 MAC, 1.0 MAC, and 1.3 MAC sevoflurane, respectively for 3 h, and then MCAO models were established at 6 h, 12 h, 24 h, and 48 h. MCAO and sham groups underwent no preconditioning with sevoflurane. The neurological severity score, cerebral infarct volume and brain water content of the rats were measured 24 h after reperfusion.ResultsAfter inhalation of 1.3 MAC sevoflurane for 3 h of preconditioning, the MCAO model was established after 24 h. This preconditioning improved the neurological severity score, reduce cerebral infarct volume and brain water content in MCAO rats. After inhalation of 1.0 MAC sevoflurane for 3 h of preconditioning, MCAO model established after 24 h reduced the cerebral infarct volume and brain water content of MCAO rats, but the neurological severity score showed no significant improvement, and no significant brain protective effects were observed at other concentrations and time windows.ConclusionsThese results suggested that after inhalation of 1.3 MAC sevoflurane for 3 h of preconditioning, MCAO model established after 24 h demonstrated significant brain protective effects in MCAO rats.

Method of Intra‐Arterial Administration in a transient MCAO Rat model to mimic the treatment of the human patient

Endovascular thrombectomy is routinely performed in patients undergoing a large vessel occlusion. This process not only allows for the removal of a blood clot but also for the selective delivery of potential therapeutics directly to the site of injury. The intra‐arterial (IA) route of drug administration in the mouse was developed to bridge the gap between animal stroke treatments and clinical stroke therapy. We have previously shown this delivery method targets the site of injury while blunting systemic effects that have proven problematic with intraperitoneal routes. Here, we adapted the IA method for use in rats, combining it with a clinically relevant large vessel occlusion stroke model (MCAO). Our goal was to characterize variances in the model in order to optimize potential therapeutic delivery methods to the ipsilaterally affected hemisphere. Male and female Sprague‐Dawley rats (4 months of age) were subjected to placement of micro‐angio tubing at the bifurcation of the common carotid artery (CCA), with delivery through the internal carotid artery (ICA). We then determined the optimal infusion rate and volume using injection of India ink and evaluation of vascular distribution within the brain and the liver. Importantly, we found the infusion rate and volume varied depending on sex and body weight. India ink was selectively delivered to the ipsilateral side of the brain in males (370–460 g) at 4 μl/min with a max volume of 25 μl and in females (250–300 g) at 2.5 μl/min with a max volume of 25 μl. Following these guidelines, no ink was observed in the liver of these animals, indicating reduced systemic circulation of administered compounds. We then performed a 5‐hour transient MCAO, on male and female Sprague‐Dawley rats (4 months of age), to mimic human stroke, since 5 hours is the average time from the clinical presentation of stroke to the thrombectomy procedure. A silicone coated monofilament was advanced till resistance was felt, occluding the MCA territory. At the 5 hour time point, we removed the monofilament and inserted the mico‐angio tubing at the same entry point on the bifurcation of the CCA. We performed variant injections using different volumes and rates, measuring delivery of dye (India Ink) and rate of induced subarachnoid hemorrhage. A rate of 4 ul/min with a max volume of 25 ul was optimal in males, and a rate of 2.5 ul/min with a max volume of 25 ul was optimal in females. The results showed that, even with a large vessel occlusion, and removal of the monofilament (recanalization), the IA injection using these sex‐specific rates and volumes resulted in appropriate limited dye delivery without ruptured subarachnoid hemorrhage. This IA method is ideally suited for combination with the MCAO stroke model and mirrors the human patient undergoing an endovascular thrombectomy. This model provides an investigative opportunity to test neuroprotective drugs and other pharmacotherapies.Support or Funding InformationUniversity of Kentucky Center for Advanced Translational Stroke Science, Surgery Core

Effect of electroacupuncture on neurological function and Wnt signaling pathway in ischemic brain tissue of cerebral infarction rats

To explore the mechanism of electroacupuncture (EA) underlying improvement of cerebral infarction (CI) by investigating its influence on expression of cerebral Wnt7a, lymphoid enhancer factor-1 (LEF1), glycogen synthase kinase 3β(GSK-3β) and Dickkopf-1(DKK1) mRNA and proteins in CI rats. A total of 280 male Wistar rats were randomly divided into blank control (n＝10), sham-operation, model and EA groups，and 90 rats of the last 3 groups were further divided into 1, 3, 6, 9, 12 and 24 h, and 3, 7 and 12 d subgroups with 10 rats in each subgroup. The CI model was established by occlusion of the middle cerebral artery (MCAO). The sham-operation group received the same surgical operation but without thread embolus insertion. EA (2 Hz/15 Hz, 2 mA) was applied to "Shuigou" (GV26) for 20 min, once a day for 1, 3, 7 and 12 d, respectively. The neurological deficit was evaluated by using Neurological Severity Scores (NSS). The expression levels of Wnt7a，LEF1, GSK-3β and DKK1 mRNAs and proteins in the right ischemic brain tissues were detected by Quantative real-time PCR and Western blot, respectively. After MCAO, the NSS score was significantly increased in the model and EA groups relevant to the blank control and sham-operation groups (P<0.01) and gradually decreased with the prolongation of ischemia time. After EA, the NSS scores were notably decreased on day 3, 7 and 12 in the EA group compared with the model group (P<0.05, P<0.01). After modeling, the expression levels of Wnt7a and LEF1 mRNAs from 3 h to 12 d, Wnt7a and LEF1 proteins from 6 h to 12 d were considerably increased (P<0.01, P<0.05), while those of GSK-3β mRNA at 9, 12 and 24 h, GSK-3β protein at 24 h and 3 d, and DKK1 mRNA at 24 h and 3 d and DKK1 protein at 3 d were obviously decreased in the model group relevant to the sham-operation group (P<0.05, P<0.01). After the intervention, the expression levels of Wnt7a mRNA at 12 h to 3 d, Wnt7α protein from 24 h to 12 d, LEF1 mRNA from 24 h to 12 d, and LEF1 protein from 3 d to 12 d were further apparently up-regulated (P<0.01, P<0.05), while those of GSK-3β mRNA at 9 h, 3，7 and 12 d, and GSK-3β protein at 12 h, 7 d and 12 d, and DKK1 mRNA at 12 h, 24 h and 3 d, and DKK1 protein at 24 h to 12 d were obviously down-regulated in the EA group relevant to the model group (P<0.01, P<0.05). No significant difference was found between the blank and sham-operation groups in the NSS scores and expression levels of Wnt7a, LEF1, GSK-3β and DKK1 mRNAs and proteins at all the time points (P>0.05). EA of GV26 can significantly improve the neurological deficit symptoms in MCAO rats, which may be associated with its effects in up-regulating the expression of Wnt7a and LEF1 mRNAs and proteins, and in down-regulating the expression of GSK-3β and DKK1 mRNAs and proteins.

LncRNA ZFAS1 Improves Neuronal Injury and Inhibits Inflammation, Oxidative Stress, and Apoptosis by Sponging miR-582 and Upregulating NOS3 Expression in Cerebral Ischemia/Reperfusion Injury.

The interplay between lncRNAs and miRNAs was reported to be associated with cerebral ischemia reperfusion injury (CIRI). Besides, the lncRNA ZFAS1 was significantly downregulated in patients with ischemic stroke. However, the exact biological role of ZFAS1 and its mechanism in CIRI remain to be elucidated. The current study was designed to explore the protective effects of ZFAS1 on neuronal injury, inflammation, oxidative stress, and neuronal apoptosis in CIRI. ZFAS1 and miR-582-3p levels were assessed by RT-qPCR. Cerebral infarction was assessed by tetrazolium chloride (TTC) staining. Inflammation and oxidative stress were measured by ELISA and matched test kits. The apoptotic rate and apoptotic mediators in OGD/R-suffered PC12 cells were respectively detected by flow cytometry and western blot. NO production was measured by NO kit and western blot assay. The regulatory relationship between lncRNA ZFAS1 and miR-582-3p was analyzed using the luciferase reporter assay. ZFAS1 was downregulated in CIRI. ZFAS1 overexpression alleviated neurological function deficit and neuronal damage in a MCAO rat model. Besides, ZFAS1 upregulation or miR-582-3p downregulation could both inhibit the inflammation, oxidative stress, and apoptosis and enhance NO level in OGD/R-suffered PC12 cells. Reporter assay indicated that ZFAS1 served as a molecular sponge for miR-582-3p. In addition, ZFAS1 could negatively regulate miR-582-3p expression and release its effects in CIRI. Taken together, our study revealed that lncRNA ZFAS1 protected against neuronal damage and modulated the inflammation, oxidative stress, apoptosis, and NO level via regulating miR-582-3p in cerebral I/R injury. Therefore, lncRNA ZFAS1 might serve as a therapeutic application to suppress CIRI progression.

Effects of normobaric oxygen on acute ischemic area monitored by diffusion-weighted imaging

To investigate the protective effects of normobaric oxygen (NBO) therapy on acute cerebral ischemia in middle cerebral artery occlusion (MACO) rats using diffusion-weighted imaging (DWI). A total of twenty adult male Sprague–Dawley rats (280–300 g) of right MCAO model were used in this study. All these MCAO rats were divided into NBO and control group with 10 rats per group. All rats were scanned with DWI sequence of MRI at MCAO 0.5 h, 2 h, 6 h, 12 h, and 24 h. The relative cerebral infarction volume (rCIV) and relative cerebral infarction volume growth rate (rCIVGR) were calculated at all these MCAO time points respectively. The actual ADC value and relative ADC (rADC) of the core (ROI-a), marginal (ROI-b) and peripheral areas (ROI-c) of acute infarction were measured and calculated respectively. The ADC value, rADC, rCIV and rCIVGR at different MCAO time points were compared between NBO and control group. No significant difference were found in rCIV and rCIVGR of all these MCAO time points between NBO and control group (p > 0.05), while significant difference was found in ADC value and rADC of ROI-a at MCAO 0.5 h between two group (**p < 0.01), as well as in the ADC value and rADC of ROI-b from MCAO 2–24 h between two group (**p < 0.01). The ADC value and rADC of ROI-c in NBO group were significantly higher than that of control group at MCAO 2 h, 6 h, 12 h and 24 h (**p < 0.01). NBO treatment could protect IP of acute infarct areas and slow down the progress of IP when acute cerebral ischemia happens. The application of DWI technology helps to reveal the protective mechanism of NBO treatment on acute cerebral ischemia, and provides an important theoretical basis and an exploration way for future clinical studies on acute cerebral ischemia.

Iron Sucrose as MRI Contrast Agent in Ischemic Stroke Model

Despite the growing concern about the safety of gadolinium-based contrast agents (GBCAs), they are still the most commonly used. Ferumoxytol, as an off-label alternative MRI contrast agent, cannot be administered by a rapid bolus for dynamic susceptibility contrast perfusion-weighted imaging (DSC-PWI). To assess the feasibility of iron sucrose (IS) as a contrast agent for MR angiography (MRA) and DSC-PWI. Prospective animal model. Thirty-six normal rats (16 for MRA, 20 for biocompability tests) and 36 occlusion of the middle cerebral artery (MCAO) model rats. 3.0T; head and neck angiography, using a fast spoiled gradient-recalled-echo (FSPGR) sequence and DSC-MRI using echo planar imaging(EPI) sequence. MRA was performed on normal rats to examine the signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) of different doses of IS. DSC-PWI was performed on MCAO rats at 0, 24, 48, and 72 hours postreperfusion to investigate the lesion detectability of IS. Arterial spin labeling (ASL) and DSC-PWI enhanced by GBCAs were conducted on MCAO rats as controls. Kruskal-Wallis test was used to compare qualitative assessment. One-way analysis of variance (ANOVA) was used to compare the parametric data. Pearson's r values were evaluated between relative cerebral blood flow(rCBF)-ASL, rCBF-DSCIS , and rCBF obtained from DSC-PWI enhanced by GBCA. The mean SNR and CNR of the common carotid artery at doses of 10 mg Fe/kg of IS were comparable with the standard dose of GBCAs (SNR: 68.04 ± 12.55 vs. 67.72 ± 14.66; CNR: 23.78 ± 7.21vs. 21.63 ± 6.83). In MCAO rat models, rCBF and relative cerebral blood volume (rCBV) of ipsilateral striatum declined (0.72 ± 0.14, 0.86 ± 0.11) with prolonged relative mean transit time (rMTT) and relative time-to-peak (rTTP) (1.27 ± 0.24, 1.07 ± 0.03) following occlusion. Hyperperfusion was observed in all rats at 48 and 72 hours postreperfusion, in 4/6 rats at 24 hours postreperfusion for IS-mediated DSC-PWI. IS may be an effective contrast agent for both MRA and DSC-PWI in ischemic stroke models. 1 TECHNICAL EFFICACY STAGE: 1 J. Magn. Reson. Imaging 2020;52:836-849.

The effect and mechanism of combination of total paeony glycosides and total ligustici phenolic acids against focal cerebral ischemia

The root of Paeonia lactiflora Pall. (Chishao, CS) and Ligusticum chuanxiong Hort. (Chuanxiong, CX) were widely used as a drug pair in Chinese Medicine, and the combination of CS and CX showed a more significant inhibition on neuronal apoptosis in our previous study. In the present study, total paeony glycosides (TPGs) from CS and total ligustici phenolic acids (TLPAs) from CX were combined to evaluate the synergistic effects against focal cerebral ischemia both in vitro and in vivo. The combination of TPGs and TLPAs at 7:3 had the best anti-oxidative stress and anti-inflammatory effect on OGD-induced HUVEC. Additionally, the infarction area proportion and neuron apoptosis of rats by TPGs:TLPAs (7:3) was significantly lower than their alone in MCAO rats. Moreover, TPGs: TLPAs of 7:3 showed a more significant effect on decreasing the expression of MMP-2 and MMP-9, and increasing the protein expression or mRNA level of TIMP-1 than other combinations. The optimal ratio of TPGs and TLPAs at 7:3 could bring more remarkable protective effects against focal cerebral ischemia in MCAO rats by alleviating oxidative stress, inflammatory and neuronal apoptosis to protect the blood-brain barrier. Overall, the present study provided benefical evidence for clinical application of CS and CX as a “drug pair”.

Brozopine Inhibits 15-LOX-2 Metabolism Pathway After Transient Focal Cerebral Ischemia in Rats and OGD/R-Induced Hypoxia Injury in PC12 Cells.

The goal of this study was to elucidate the mechanisms of protection of Sodium (±)-5-bromo-2-(α-hydroxypentyl) benzoate (trade name: Brozopine, BZP) against cerebral ischemia in vivo and in vitro. To explore the protective effect of BZP on focal cerebral ischemia-reperfusion injury, we evaluated the effects of various doses of BZP on neurobehavioral score, cerebral infarction volume, cerebral swelling in MCAO rats (ischemia for 2 h, reperfusion for 24 h). In addition, the effects of various doses of BZP on OGD/R-induced-PC12 cells injury (hypoglycemic medium containing 30 mmol Na2S2O4 for 2 h, reoxygenation for 24 h) were evaluated. Four in vivo and in vitro groups were evaluated to characterize targets of BZP: Control group, Model group, BZP group (10 mg/kg)/BZP group (30 μmol/L), C8E4 group (10 mg/kg)/C8E4 group (30 μmol/L). An ELISA kit was used to determine the levels of 15-HETE (a 15-LOX-2 metabolite) in vivo and in vitro. Rat nuclear factor κB subunit p65 (NF-κB p65), tumor necrosis factor (TNF-α), interleukin-6 (IL-6), and intercellular adhesion molecule-1 (ICAM-1) were also quantified in vivo and in vitro. The results showed that BZP improved focal cerebral ischemia-reperfusion injury in rats and PC12 cells treated with Na2S2O4 in dose/concentration-dependent manners through inhibition of production of 15-HETE and expression of NF-κB, IL-6, TNF-α, and ICAM-1. In conclusion, BZP exerted protective effects against cerebral ischemia via inhibition of 15-LOX-2 activity.

Hydroxysafflor Yellow A Confers Neuroprotection from Focal Cerebral Ischemia by Modulating the Crosstalk Between JAK2/STAT3 and SOCS3 Signaling Pathways.

Natural bioactive compounds have increasingly proved to be promising in evidence- or target-directed treatment or modification of a spectrum of diseases including cerebral ischemic stroke. Hydroxysafflor yellow A (HSYA), a major active component of the safflower plant, has drawn more interests in recent year for its multiple pharmacological actions in the treatment of cerebrovascular and cardiovascular diseases. Although the Janus kinase signaling, such as JAK2/STAT3 pathway, has been implicated in the modulation of the disease, the inhibition or activation of the pathway that contributed to the neuronal prevention from ischemic damages remains controversial. In this study, a series of experiments were performed to examine the dose- and therapeutic time window-related pharmacological efficacies of HSYA with emphasis on the HSYA-modulated interaction of JAK2/STAT3 and SOCS3 signaling in the MCAO rats. We found that HSYA treatment significantly rescued the neurological and functional deficits in a dose-dependent manner in the MCAO rats within 3h after ischemia. HSYA treatment with a dosage of 8mg/kg or higher markedly downregulated the expression of the JAK2-mediated signaling that was activated in response to ischemic insult, while it also promoted the expression of SOCS3 coordinately. In the subsequent experiments with the use of the JAK2 inhibitor WP1066, we found that the treatment of WP1066 alone or combination of WP1066/HSYA all exhibited inhibitory effects on JAK2-mediated signaling, while there was no influence on the SOCS3 activity of corresponding efficacious data in the MCAO rats, suggesting that excessive activation of JAK2/STAT3 might be necessary for HSYA to provoke SOCS3-negative feedback signaling. Taking together, our study demonstrates that HSYA might modulate the crosstalk between JAK2/STAT3 and SOCS3 signaling pathways that eventually contributed to its therapeutic roles against cerebral ischemic stroke.

Synthesis and biological evaluation of 1,2,4-triazole derivatives as potential neuroprotectant against ischemic brain injury

A series of 1,2,4-triazole derivatives 1–14 was synthesized to investigate their neuroprotective effects and mechanisms of action. Compounds 5–11 noticeably protected PC12 cells from the cytotoxicity of H2O2 or sodium nitroprusside (SNP). Compound 11 was the most effective derivative. Compound 11 chelated Fe (II) iron, scavenged reactive oxygen species (ROS), and restored the mitochondrial membrane potential (MMP). Moreover, it enhanced the activity of the antioxidant defense system by increasing the serum level of superoxide dismutase (SOD) and promoting the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). Compound 11 caused certain improvements in behavior, the cerebral infarction area, and serum levels of biochemical indicators (TNF-α, IL-1β, SOD and MDA) in a rat MCAO model. The lethal dose (LD50) of compound 11 in mice receiving intraperitoneal injections was greater than 400 mg/kg. Meanwhile, pharmacokinetic experiments revealed high bioavailability of this compound after both oral and intravenous administration (F = 60.76%, CL = 0.014 mg/kg/h) and a longer half-life (4.26 and 5.11 h after oral and intravenous administration, respectively). Based on these findings, compound 11 may be a promising neuroprotectant for the treatment of ischemic stroke.

HIF‑1α attenuates neuronal apoptosis by upregulating EPO expression following cerebral ischemia‑reperfusion injury in a rat MCAO model.

Hypoxia-inducible factor-1α (HIF-1α) is a key transcriptional factor in response to hypoxia and is involved in ischemic stroke. In the present study, the potential for HIF-1α to inhibit neuronal apoptosis through upregulating erythropoietin (EPO) was investigated in a transient middle cerebral artery occlusion (tMCAO) rat stroke model. For this purpose, a recombinant adenovirus expressing HIF-1α was engineered (Ad-HIF-1α). Control adenovirus (Ad group), Ad-HIF-1α (Ad-HIF-1α group) or Ad-HIF-1α in addition to erythropoietin mimetic peptide-9 (EMP9), an EPO-receptor (-R) antagonist (Ad-HIF-1α+EMP9 group), were used for an intracranial injection into rat ischemic penumbra 1 h following MCAO. All rats demonstrated functional improvement following tMCAO, while the improvement rate was faster in rats treated by Ad-HIF-1α compared with all other groups. The EPO-R inhibitor partially reversed the benefits of Ad-HIF-1α. Apoptosis induced by tMCAO was significantly inhibited by Ad-HIF-1α (P<0.05). The expression of HIF-1α, evaluated by immunohistochemistry either in neurons or astrocytes, was upregulated by Ad-HIF-1α. Both EPO mRNA and protein expression were increased by Ad-HIF-1α, however, there was no significant change of EPO-R either on an mRNA level or protein level. Furthermore, EMP9 did not change the EPO expression which was upregulated by Ad-HIF-1α. Activated caspase 3 in neurons was suppressed by Ad-HIF-1α. Activated caspase 3 downregulated by HIF-1α was partially blocked by EMP9. Altogether, the present data demonstrated that HIF-1α attenuates neuronal apoptosis partially through upregulating EPO following cerebral ischemia in rat. Thus, upregulating HIF-1α subsequent to a stroke may be a potential treatment for ischemic stroke.

Metabolomic Profiling of the Synergistic Effects of Ginsenoside Rg1 in Combination with Neural Stem Cell Transplantation in Ischemic Stroke Rats.

As the greatest medical and socioeconomic problem in developing countries, stroke is the second or third leading cause of death in China and worldwide. In adult organisms suffering from stroke, transplanted stem cells have the ability to repair damaged tissues by regenerating autologous cells, while ginsenoside Rg1 could promote proliferation and differentiation of stem cells. Although obvious antistroke effects of ginsenoside Rg1 and transplanted stem cells have been verified in publications, the mechanism exploration remains challenging. Our study was carried out to investigate the synergistic effects of ginsenoside Rg1 and neural stem cell (NSC) transplantation on MCAO rats with a 1H NMR-based nontargeted metabolomics method to identify potential biomarkers and protein targets and discover the potential mechanism. NSCs transplantation after MCAO combined with ginsenoside Rg1 administration could significantly improve the cerebral infarct and neurological deficits. The treatment significantly intervened the levels of ten metabolites, and perturbed energy metabolism, amino acids metabolism, and lipids metabolism. And 11 enzymes were identified and verified as the targets of NSCs transplantation and ginsenoside Rg1 administration on MCAO rats. Our findings helped to improve the antistroke mechanism of NSCs transplantation and ginsenoside Rg1 and supply a theory basis for the combined research of stem cells and Chinese medicine in the future.

Downregulated miRNA-324-5p aggravates neuronal injury induced by oxygen-glucose deprivation via modulating RAN.

Differentially expressed miRNAs in the GEO profile of ischemic stroke were analyzed to clarify the specific role of microRNA-324-5p (miRNA-324-5p) in ischemic stroke and the potential mechanism. After screening out miRNA-324-5p, its level in peripheral blood of stroke patients and in vitro oxygen-glucose deprivation (OGD)-induced primary rat neurons was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Regulatory effects of miRNA-324-5p on viability, and apoptosis of OGD-induced neurons were evaluated by CCK-8 and Annexin V fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining, respectively. Glucose uptake and caspase-3 activity in OGD-induced neurons transfected with miRNA-324-5p mimics or inhibitor were also examined. The binding of miRNA-324-5p to its target gene RAN was analyzed by dual-luciferase reporter gene assay and western blot analysis. By analyzing the data of GSE46266 profile, miRNA-324-5p expression was shown markedly lower in MCAO rats relative to controls. Identically, we also observed the downregulated miRNA-324-5p in peripheral blood of stroke patients and in vitro OGD-induced primary neurons. Overexpression of miRNA-324-5p accelerated viability, induced apoptosis and strengthened glucose uptake ability of OGD-induced neurons. Knockdown of miRNA-324-5p, conversely, obtained the opposite results. Furthermore, we confirmed the binding of miRNA-324-5p to RAN, the target gene that was negatively regulated by miRNA-324-5p. Importantly, RAN overexpression partially reversed the regulatory effect of miRNA-324-5p on viability and glucose uptake of OGD-induced neurons. miRNA-324-5p is downregulated after ischemic stroke, which aggravates the disease condition by inhibiting neuronal proliferation and glucose uptake via upregulating RAN.

The role of PI3K-mediated AMPA receptor changes in post-conditioning of propofol in brain protection

BackgroundWe aimed to study the role of amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) glutamate receptor 2 (GluR2) subunit trafficking, and activity changes in short-term neuroprotection provided by propofol post-conditioning. We also aimed to determine the role of phosphoinositide-3-kinase (PI3K) in the regulation of these processes.MethodsRats underwent 1 h of focal cerebral ischemia followed by 23 h of reperfusion were randomly divided into 6 groups (n = 36 per group): sham- operation (S), ischemia–reperfusion (IR), propofol (P group, propofol 20 mg/kg/h at the onset of reperfusion for 2 h after 60 min of occlusion), and LY294002 (PI3K non-selective antagonist) + sham (L + S, LY294002 of 1.5 mg/kg was infused 30 min before sham operation), LY294002+ ischemia–reperfusion (L + IR, LY294002 of 1.5 mg/kg was infused 30 min before middle cerebral artery occlusion), LY294002 + IR + propofol (L + P, LY294002 of 1.5 mg/kg was infused 30 min before middle cerebral artery occlusion and propofol 20 mg/kg/h at the onset of reperfusion for 2 h after 60 min of occlusion).ResultsCompared with group IR, rats in group P had significant lower neurologic defect scores and infarct volume. Additionally, consistent with enhanced expression of PI3K-AMPAR GluR2 subunit complex substances in ipsilateral hippocampus, GluR2 subunits showed increased levels in both the plasma and postsynaptic membranes of neurons, while pGluR2 expression was reduced in group P. Furthermore, LY294002, the PI3K non-selective antagonist, blocked those effects.ConclusionThese observations demonstrated that propofol post-conditioning revealed acute neuroprotective role against transient MCAO in rats. The short-term neuroprotective effect was contributed by enhanced GluR2 subunits trafficking to membrane and postsynaptic membranes of neurons, as well as down-regulated the expression of pGluR2 in damaged hippocampus. Finally, the above-mentioned protective mechanism might be contributed by increased combination of PI3K to AMPAR GluR2 subunit, thus maintained the expression and activation of AMPAR GluR2 in the ipsilateral hippocampus.

Natural neuroprotective alkaloids from Stephania japonica (Thunb.) Miers

Modulating inflammatory responses after stroke can prevent brain injury and, therefore, improve neurological outcome. Stephania japonica (Thunb.) Miers is a Chinese folk medicine with the function of dispelling the “wind and blockage” in the human body according to the Chinese medicine theory, in which the symptoms of stroke are caused by the “wind and blockage” in the body. In this paper, we for the first time linked S. japonica to stroke by clarifying fifteen alkaloidal constituents including five undescribed (1–5) ones and screening out six hasubanan type alkaloids (1–4, 7, 15) that elicited stronger anti-neuroinflammatory activities than the positive drug. Moreover, the total alkaloid fraction (ASJ) with previously undescribed 3 as the main component was subject to the in vivo evaluation of the protective effect in the MCAO-induced brain injury. The results showed that ASJ exhibited potent protective effect against brain injury in the MCAO rat model. The results reported in this paper suggested that the hasubanan alkaloids from S. japonica would be an important molecular source for discovering novel therapeutic agents for neuroinflammation-related diseases, such as stroke diseases.

Delayed treatment of \u03b15 GABAA receptor inverse agonist improves functional recovery by enhancing neurogenesis after cerebral ischemia-reperfusion injury in rat MCAO model

Development of effective therapeutics and treatment strategy to promote recovery after cerebral ischemia-reperfusion injury necessitates further understandings of the complex pathophysiology of ischemic stroke. Given that α5-GABAAR inhibition has been shown to be involved in functional recovery after stroke, the present study was designed to evaluate the effects of treatment timing of α5 GABAAR inhibition on post-middle cerebral artery occlusion (MCAO) functional recovery. To this end, we examined the effects of L655,708 (α5 GABAAR inverse agonist) treatment at 3 or 7 days post-ischemia on apoptosis and neurogenesis in the peri-infarct region, brain infarction size, as well as modified neurological severity score (mNSS) and rotarod test time in rats. Consistent with previous reports, we found that when the treatment of L655,708 was initiated at post-MCAO day 3, it did not alter the functional recovery in rats. However, when the treatment of L655,708 was initiated at post-MCAO day 7, it demonstrated beneficial effects on functional recovery in rats. Interestingly, this phenomenon was not associated with altered brain infarction size nor with changes in brain cell apoptosis. However, we found that delayed treatment of L655,708 at post-MCAO day 7 significantly increased neurogenesis in peri-infarct zone in rats. These results suggested that removing α5 GABAAR-mediated tonic inhibition after cerebral ischemia-reperfusion injury may be an effective therapeutic strategy for promoting functional recovery from stroke.

Conditioned medium obtained from mesenchymal stem cells attenuates focal cerebral ischemia reperfusion injury through activation of ERK1/ERK2-BDNF signaling pathway

Previous studies have shown that conditioned medium (CM) obtained from mesenchymal stem cells (MSCs) might exert neuroprotective effects against focal cerebral ischemia reperfusion (I/R) injury. This study was conducted to investigate if CM obtained from MSCs gives rise to neuroprotection by targeting neurogenesis. To induce focal cerebral ischemia in rats, middle cerebral artery (MCA) was occluded for 1 h and the amniotic mesenchymal stem cells-conditioned medium (AMSC-CM) at the dose of 0.5 μl was administered 30 min after reperfusion by stereotactic intracerebral infusion. The animals were randomly divided into three groups: sham operated animals received all procedures except occlusion of MCA (sham, n = 12), I/R group only received occlusion of MCA (MCAO, n = 17), treatment group received MCAO + 0.5 μl of AMSC-CM (MCAO + AMSC-CM, n = 17). The expression of Phospho-ERK1/ERK2, BDNF, VEGF and NGF were determined using immunohistochemical assay. Neuronal loss and DNA fragmentation were evaluated by Nissl and TUNEL assay, respectively.Our results demonstrated that the expression of Phospho-ERK1/ERK2 and BDNF, VEGF and NGF significantly decreased in MCAO rats and was reversed by AMSC-CM. Likewise, AMSC-CM markedly reduced neuronal loss and DNA fragmentation at 24 h after reperfusion.In sum, our study showed that AMSC-CM administration at the onset of reperfusion led to neuroprotection by activating neuronal ERK1/ERK2-BDNF signaling pathway, neurogenesis, angiogenesis as well as suppression of apoptosis.