Rat Model Of Cerebral Ischemia-reperfusion Injury Research Articles

Detection of miR-203 expression using a nanofluorescent probe to study the neuroprotective effect of sevoflurane in a neuroinflammatory injury rat model

Ischemic cerebrovascular disease has high disability and mortality rates that can result in related sequelae if not treated promptly and properly. Nanofluorescent probes are useful in the early diagnosis of the disease due to their high sensitivity and specificity, thus facilitating early therapeutic measures and observation of disease progression. Here, a novel nanostructure probe was designed to detect the expression of miR-203 in a cerebral ischemia-reperfusion injury (CIRI) model after sevoflurane treatment, and the effects of sevoflurane and neuroinflammatory injury were discussed. A CIRI rat model was established to study miR-203 expression, nerve injury, oxidative stress, and inflammatory factors in the brain tissue of rats treated with sevoflurane. The nanofluorescent probe showed high specificity and sensitivity for miR-203 detection. miR-203 was over-expressed in CIRI rats, which was decreased by sevoflurane treatment. In addition, sevoflurane treatment significantly improved neurological function, learning, and memory in CIRI rats; decreased malondialdehyde, lactate dehydrogenase, and superoxide dismutase levels; and inhibited the expression of tumor necrosis factor α and interleukins 1 and 6. Moreover, an inverse correlation was determined between miR-203 expression and the degree of neuroinflammation. This study demonstrated that miR-203 may be a useful diagnostic and prognostic index for neuroinflammatory injury.

Multi-parametric MRI assessment of melatonin regulating the polarization of microglia in rats after cerebral ischemia/reperfusion injury

ObjectivesMulti-parametric magnetic resonance imaging (MRI) can provide comprehensive and valuable information for precise diagnosis and treatment evaluation of a number of diseases. In this study, the neuroprotective effects of melatonin (Mel) on a rat model of cerebral ischemia/reperfusion injury (CIRI) were assessed by multi-parametric MRI combined with histopathological techniques for longitudinal monitoring of the lesion microenvironment. MethodsSixty Sprague Dawley (SD) rats were randomly divided into three groups: the Sham, CIRI and CIRI + Mel groups. At multiple time points after ischemia, MRI scanning was performed on a 7.0 Tesla MRI scanner. Multi-parametric MRI includes T2-weighted imaging (T2WI), diffusion weighted imaging (DWI), and chemical exchange saturation transfer (CEST)-MRI. CEST effects were calculated by the Lorentzian difference method, 3.5 ppm indicates amide protons of mobile proteins/peptide (Amide-CEST) and 2.0 ppm indicates amine protons (Guan-CEST). Multiple histopathological techniques were used to examine the histopathological changes and explore the therapeutic effects of Mel. ResultsT2WI and DWI-MRI could localize the infarct foci and areas in CIRI rats, which was further validated by staining, 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining, hematoxylin and eosin (H&E) staining, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling (TUNEL) staining. After Mel treatment, T2WI and DWI-MRI showed smaller infarct volume, and neurons displayed improved morphology with less apoptosis rates. Notably, Amide-CEST and Guan-CEST signal decreased as early as 2 h after CIRI (all P <0.001), reflecting the change of pH after ischemia. After Mel treatment, both Amide-CEST and Guan-CEST signal increased in ischemic cortex and striatum compared with control group (all P < 0.001). The immunofluorescence staining and western blotting analysis suggested the expression of M2 microglia increased after Mel treatment; While，after Mel treatment the inflammatory factor interleukin-1β (IL-1β) decreased compared with control CIRI rats. ConclusionsMulti-parametric MRI was shown to be an effective method to monitor the brain damage in a rat model of CIRI and assess the therapeutic effects of Mel treatment. Amide-CEST and Guan-CEST were especially sensitive to the changes in brain microenvironment during the early stage after CIRI. Furthermore, the neuroprotective effect of Mel treatment is associated with its promotion of the microglia polarized to M2 type in CIRI rats.

Lithium co-administration with rutin improves post-stroke neurological outcomes via suppressing Gsk-3β activity in a rat model.

Cerebral ischemic stroke is one of the leading causes of adult disability worldwide. Reperfusion is the only therapeutic option with a lot of side effects. In the current study, we investigated the efficacy of rutin and lithium co-treatment in improving post-stroke neurological outcomes in a transient global cerebral ischemia-reperfusion injury rat model. Middle-aged male rats were subjected to transient global cerebral ischemia-reperfusion. NORT and Y-maze were used to assess the cognitive processes. Lipid peroxidation, protein carbonylation, and nitric oxide assays were performed to study oxidative stress. The excitotoxicity index was calculated by HPLC. Real time-PCR and western blotting were performed to study gene and protein expressions. The co-administration of rutin and lithium improved the overall survival, recognition memory, spatial working memory, and neurological score following cerebral ischemia-reperfusion in rats. Further, a marked decrease in malonaldehyde, protein carbonyls, and nitric oxide levels was observed following combined treatment. The mRNA expression of antioxidant (Hmox1 and Nqo1) and pro-inflammatory (Il2, Il6, and Il1β) markers were significantly attenuated in the rutin and lithium co-administrated group. The treatment inhibited the Gsk-3β and maintained a normal pool of the downstream β-catenin and Nrf2 proteins. The results revealed that co-administration of rutin and lithium had a neuroprotective potential, suggesting it to be a viable treatment to overcome post-stroke deaths and neurological complications.

Ro25-6981 alleviates neuronal damage and improves cognitive deficits by attenuating oxidative stress via the Nrf2/ARE pathway in ischemia/reperfusion rats

ObjectivesOxidative stress plays a crucial role in the initiation and progression of cerebral ischemia‒reperfusion injury (CIRI). Therefore, ameliorating oxidative damage is considered to be a beneficial strategy for the treatment of CIRI. NMDAR NR2B subunit antagonists have been reported to be beneficial for synaptic plasticity, neuropathic pain, epilepsy, and cerebral ischemia. However, it remains unclear whether the NR2B subunit antagonist Ro25-6981 has any effect on CIRI. MethodsIn this study, the Morris water maze test and passive avoidance test were used to detect spatial learning and memory. Neuronal loss was measured by Nissl staining. The expression of NSE was assayed by immunohistochemistry. The activities of MDA, 8-OHdG, SOD, GSH-Px, GST and CAT were detected by assay kits. Real-time PCR was used to detect the mRNA levels of hippocampal SOD, GSH-Px and HO-1. Western blotting was used to measure the activation of the Nrf2/ARE pathway by Ro25-6981. ResultsRo25-6981 ameliorated cognitive deficits and neuronal damage induced by ischemia‒reperfusion (I/R). Neuronal injury was decreased and the expression of NSE was increased in the CA1 regions of the hippocampus of I/R rats after Ro25-6981 treatment. Moreover, Ro25-6981 alleviated the levels of MDA and 8-OHdG by elevating the activities of SOD, GSH-Px, GST and CAT. Meanwhile, the mRNA levels of SOD, GSH-Px and HO-1 were increased in I/R rats after Ro25-6981 treatment. Furthermore, Ro25-6981 promoted the translocation of Nrf2 to the nucleus, promoting the expression of the Nrf2 downstream genes HO-1 and NQO1. ConclusionThe present study indicated that the improvement in the antioxidant properties of Ro25-6981 is mediated by the Nrf2/ARE pathway. This is the first study to demonstrate a favorable effect of Ro25-6981 on cognitive impairment in a CIRI rat model, rendering this NR2B subunit antagonist a promising agent for the treatment or prevention of CIRI.

Urinary Proteome Analysis of Global Cerebral Ischemia–Reperfusion Injury Rat Model via Data-Independent Acquisition and Parallel Reaction Monitoring Proteomics

Cerebral ischemia–reperfusion (I/R) injury is the leading cause of death in severe hypotension caused by cardiac arrest, drowning, and excessive blood loss. Urine can sensitively reflect pathophysiological changes in the brain even at an early stage. In this study, a rat model of global cerebral I/R injury was established via Pulsinelli’s four-vessel occlusion (4-VO) method. Overall, 164 urinary proteins significantly changed in the 4-VO rat urine samples compared to the control samples by data-independent acquisition (DIA) proteomics technique (1.5-fold change, p < 0.05). Gene Ontology annotation showed that the acute-phase response, the ERK1 and ERK2 cascade, endopeptidase activity, blood coagulation, and angiogenesis were overrepresented. After parallel reaction monitoring (PRM) validation, 15 differential proteins having human orthologs were verified as the potential urinary markers associated with cerebral I/R injury. Of these potential biomarkers, 8 proteins were reported to be closely associated with cerebral I/R injury. Nine differential proteins changed even when there were no clinical manifestations or histopathological cerebral damage, including FGG, COMP, TFF2, HG2A, KNG1, CATZ, PTGDS, PRVA, and HEPC. These 9 proteins are potential biomarkers for early screening of cerebral I/R injury to prevent the development of cerebral injury. KNG1, CATZ, PTGDS, PRVA, and HEPC showed an overall trend of upregulation or downregulation at 12 and 48 h after I/R injury, reflecting the progression of cerebral I/R injury. These 5 proteins may serve as potential biomarkers for prognostic evaluation of cerebral I/R injury. These findings provide important clues to inform the monitoring of cerebral I/R injury and further the current understanding of its molecular biological mechanisms.

Effects of acupuncture on Beclin-1 and Caspase-3 expression in hippocampal tissue of rats with cerebral ischemia-reperfusion injury: Observations at different time points ☆基于不同时间点观察针刺对脑缺血再灌注损伤大鼠海马组织Beclin-1及Caspase-3表达的影响

ObjectiveTo observe the changes in the neurological score, infarct size, and autophagy- and apoptosis-related indicators following the acupuncture treatment of rats with cerebral ischemia-reperfusion injury (CIRI) in order to investigate the effects of acupuncture on autophagy and apoptosis in the hippocampal tissue of CIRI rats MethodsIn this study, 170 Sprague-Dawley rats were randomly assigned to the normal group (n=34), sham group (n=34), and model establishment group (n=102). A CIRI rat model was established in the model establishment group. The rats of the model establishment group were then randomly and equally allocated to the middle cerebral artery occlusion/reperfusion group (MCAO/R group), acupuncture group (AC group), and edaravone group (ED group) (n=34 each). Each group was further subdivided into the 24-h and 72-h subgroups (n=17 each). The rats of the MCAO/R group were immobilized for 30 min and administered with an intraperitoneal injection of 0.9% sodium chloride solution. The rats of the AC group were immobilized, and they received acupuncture at the“Dà zhuī (大椎 GV 14) ”,“Shuĭ gōu (水沟 GV 26 ) ”, and “Băi huì (百会 GV 20) ” and an intraperitoneal injection of 0.9% sodium chloride solution, with the acupuncture needles left in place for 30 min. The rats of the ED group were immobilized for 30 min and administered with an intraperitoneal injection of edaravone (5 mg/kg). All interventions were performed once every 12 h. Investigations of the neurological score, infarct size using triphenyl tetrazolium chloride staining, hippocampal Caspase-3 protein expression by immunohistochemical assay, hippocampal Beclin-1 expression by Western blotting, autophagosomes in the hippocampus by transmission electron microscopy, and apoptotic rate in the brain tissue of the ischemic side using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay were performed both before and after the interventions. ResultsBefore intervention, the neurological score of the model establishment group was significantly lower than that of the normal and sham groups (P<0.01). After 24 h and 72 h, the neurological scores of the ED and AC groups were significantly increased (P<0.01), whereas the infarct size ratio were smaller (P<0.05 or P<0.01) and the apoptotic rates and Caspase-3 expression were lower (P<0.01) when compared with the MCAO/R group. The hippocampal Beclin-1 expression in the ED and AC groups was downregulated after 24 h of intervention (P<0.05) but upregulated after 72 h of intervention (P<0.05 and P<0.01, respectively). In the ED and AC groups, 72 h of intervention led to significant increases in the neurological score (P<0.01) and significant reductions in the apoptotic rate and Caspase-3 expression (P<0.05 or P<0.01), but no significant changes in the Beclin-1 expression (P>0.05) when compared with 24 h of intervention. Moreover, the AC group showed a significant decrease in the infarct size ratio (P<0.01). Electron microscopy revealed that after 24 h of intervention, only individual lysosome structures could be observed in the tissues of the AC and ED groups. After 72 h of intervention, co-existence of autophagosomes and lysosomes was observed in the ED group, whereas co-existence of lysosomes and autolysosomes was observed in the AC group. ConclusionAcupuncture significantly improved the neurological score and reduced the infarct size in the CIRI rats. The mechanisms of these effects could be attributed to the regulation of autophagy and inhibition of apoptosis in the hippocampal tissue as well as the duration of the intervention to a certain extent.

Protective effects of brain-targeted dexmedetomidine nanomicelles on mitochondrial dysfunction in astrocytes of cerebral ischemia/reperfusion injury rats

Cerebral ischemia/reperfusion injury (CIRI) is closely related to mitochondrial dysfunction in astrocytes. Therefore, based on glucose transporter 1 (GLUT1), which is highly expressed in the brain tissue of rats with CIRI, we design a kind of brain-targeted dexmedetomidine (Man@Dex) nanomicelles. The results showed that Man@Dex not only had the advantages of small particle size, stability and non-toxicity, but also realized brain-targeted drug delivery. Primary astrocytes were cultured in vitro to construct CIRI cell model. It was found that Man@Dex could improve the activity of injured astrocytes. Man@Dex could exert antioxidant activity by inhibiting the reactive oxygen species (ROS) production of astrocytes, thus inhibiting the cytotoxicity induced by hypoxia and reoxygenation. Man@Dex could improve the ATP level and mitochondrial membrane potential (MMP) to protect mitochondrial function of damaged astrocytes. The CIRI rat model was constructed and confirmed by hematoxylin and eosin (HE), Triphenyl-2H-tetrazolium chloride (TTC) staining and nerve defect score. It indicated that Man@Dex could alleviate CIRI and improve MMP, which was beneficial to the recovery of brain injury in rats. This research provides a new theoretical basis and target for the development of brain-targeted nano-drugs of CIRI.

Electroacupuncture ameliorates ischemic injury in cerebral ischemia-reperfusion rats by regulating endogenous melatonin and inhibiting the activation of astrocytes

To observe the effect of electroacupuncture(EA)at "Baihui"(GV20) and "Shenting" (GV24) on the expression of melatonin synthesis rate-limiting enzyme-arylalkylamine N-acetyltransferase(AANAT)in pineal gland of rats with focal cerebral ischemia-reperfusion injury, so as to explore the mechanism of EA underlying improving ischemia-reperfusion injury. Forty-eight SD rats were randomly divided into sham operation, model, EA and non-acupoint groups, with 12 rats in each group. The focal cerebral ischemia-reperfusion injury rat model was established by occlusion of the middle cerebral artery. Rats of the EA group received EA at GV20 and GV24, while those in the non-acupoint group received EA at non-acupoints below the costal margins on both sides for 20 min, once daily for 7 days. The neurological deficit score (0 to 4 points) was given after successful modeling according to Longa's method. Morris water maze test was used to assess the cognitive function of rat. ELISA was used to detect the plasma melatonin content, and PCR and Western blot were used to detect the mRNA and protein expressions of AANAT in the pineal gland, separately. Immunofluorescence staining was used to detect the activation of astrocytes and neuronal injury in the hippocampus. After focal cerebral ischemia-reperfusion injury and compared with the sham operation group, the neurological deficit score, the escape latency, and the expression of GFAP were significantly increased (P<0.01)，while the times of platform quadrant crossing, the secretion of melatonin at 24:00，AANAT mRNA and protein expression levels and NeuN protein expression were significantly down-regulated (P<0.01). After EA at GV20 and GV24, the above-mentioned indexes all reversed in the EA group relative to the model group, and there were significant differences between the two groups(P<0.01). Compared with the model group, the changes of the abovementioned indexes in the non-acupoint group were not statistically significant (P>0.05). EA at GV20 and GV24 can alleviate neurological deficit and improve cognitive function in cerebral ischemia-reperfusion rats，which may be related to its effects in up-regulating endogenous melatonin levels, inhibiting the activation of astrocytes and protecting damaged neurons in the hippocampus.

Nose-to-brain delivery of borneol modified tanshinone IIA nanoparticles in prevention of cerebral ischemia/reperfusion injury

Targeted treatment of cerebral ischemia/reperfusion injury (CIRI) remains a problem due to the difficulty in drug delivery across the blood–brain barrier (BBB). In this study, we developed Bo-TSA-NP, a novel tanshinone IIA (TSA) loaded nanoparticles modified by borneol, which has long been proved with the ability to enhance other drugs’ transport across the BBB. The Bo-TSA-NP, with a particle size of about 160 nm, drug loading of 3.6%, showed sustained release and P-glycoprotein (P-gp) inhibition property. It demonstrated a significantly higher uptake by 16HBE cells in vitro through the clathrin/caveolae-mediated endocytosis and micropinocytosis. Following intranasal (IN) administration, Bo-TSA-NP significantly improved the preventive effect on a rat model of CIRI with improved neurological scores, decreased cerebral infarction areas and a reduced content of malondialdehyde (MDA) and increased activity of superoxide dismutase (SOD) in rat brain. In conclusion, these results indicate that Bo-TSA-NP is a promising nose-to-brain delivery system that can enhance the prevention effect of TSA on CIRI.

Intravenous injection of apelin-13 improves sensory-motor balance deficits caused by cerebral ischemic reperfusion injury in male wistar rats via restoration of nitric oxide

It has been reported that apelin-13 possesses neuroprotective effects against cerebral ischemia/reperfusion injury (IRI). Disabilities in sense, movement and balance are the major stroke complications which, result in a high rate of mortality. Here, effects of intravenous (IV) injection of apelin-13 on the severity of neural death, infarct volume, neurological defects and its association with nitric oxide (NO) were investigated. A rat model of cerebral IRI was created by middle cerebral artery occlusion (MCAO) for 60 min and restoration of blood flow for 23 h. Animals were randomly assigned into six groups: sham, ischemia (MCAO), vehicle (MCAO + PBS) and three treatment groups (MCAO + apelin-13 in 10, 20, 40 μg/kg doses, IV). All injections were carried out via tail vein injection 5 min before reperfusion. Neural loss and infarct volume were evaluated by Nissl and 2,3,5-triphenyltetrazolium chloride (TTC) staining, respectively. Neurological defects were scored by standard modified criteria. Serum NO was measured by colorimetric method. Apelin-13 in doses of 20 and 40 μg/kg significantly reduced neural death, infarct volume and disturbance of sensory-motor balance compared to control and vehicle groups (p < 0.05). Serum NO levels reduced in MCAO groups compared to sham. Apelin-13 restored serum NO levels at 20 μg/kg dose (p < 0.05). Our data showed beneficial effect of IV injection of apelin-13 on sensory-motor balance defects by reducing neural death and restoration of serum NO levels. The present study shows the validity of apelin-13 in treatment of ischemic stroke in different administration methods.

Lidocaine improves cerebral ischemia-reperfusion injury in rats through cAMP/PKA signaling pathway

Influence of lidocaine on rats with cerebral ischemia-reperfusion injury (CIRI) was studied to explore its mechanism of action. A total of 30 Sprague-Dawley rats were randomly divided into control group and model group, and the rat model of CIRI was prepared by the suture-occluded method in the model group. Then the rats in the model group were randomly assigned into the model group (n=10) and the lidocaine group (n=10). The neurological function score of rats was evaluated, and the levels of serum B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) in rats were determined using ELISA. TUNEL assay was performed to detect the neuronal apoptosis in the brain of rats. The messenger ribonucleic acid (mRNA) and protein expression levels of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) were measured via RT-PCR and western blotting, respectively. Compared with those in the control group, the rats in the model group had an elevated neurological function score, a raised level of Bcl-2, but a reduced level of Bax in the serum, an obviously increased rate of neuronal apoptosis in the brain and decreased mRNA and protein levels of cAMP and PKA in cerebral tissues. The rats in lidocaine group had a lower neurological function score, a lower level of Bcl-2, but a higher level of Bax in the serum, an evidently lower rate of neuronal apoptosis in the brain and higher mRNA and protein levels of cAMP and PKA in cerebral tissues than those in the model group. Lidocaine can improve the neurological function of rats with CIRI and inhibit neuronal apoptosis in the brain, and its mechanism of action may be related to the activation of the cAMP/PKA signaling pathway.

Remifentanil protects neurological function of rats with cerebral ischemia-reperfusion injury via NR2B/CaMKIIα signaling pathway.

The effects of remifentanil were studied on cerebral ischemia-reperfusion injury (IRI) in rats, and its regulatory effect was explored on the N-methyl D-aspartate receptor subtype 2B (NR2B)/calcium/calmodulin-dependent protein kinase type II subunit alpha (CaMKII) signaling pathway in cerebral tissues. A total of 40 Sprague-Dawley rats were randomly assigned into sham group, model group, low-dose group (remifentanil injected into the caudal veins at 2 μg/kg) and high-dose group (remifentanil injected into the caudal veins at 10 μg/kg). Then, in the model, low-dose and high-dose groups, the rat model of cerebral IRI was established through middle cerebral artery occlusion (namely, ischemia for 1 h and reperfusion for 2 h), while no thread was inserted into the rats in the sham group. Neurological function of rats in each group was evaluated, and the cerebral infarct size was measured using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Moreover, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was performed to determine the neuronal apoptosis level, and mitochondrial membrane potential was measured via JC-1 assay. Moreover, the reactive oxygen species (ROS) level in neurons and the malondialdehyde (MDA) content in cerebral tissues were determined using 2',7'-dichlorofluorescin diacetate (DCFH-DA) assay and enzyme-linked immunosorbent assay (ELISA), respectively. Finally, the expression levels of apoptosis-associated proteins and the NR2B/CaMKIIα signaling pathway-associated proteins in cerebral tissues were measured by Western blotting. Remifentanil preconditioning substantially decreased the neurological score of rats (p<0.01), cerebral infarct size (p<0.01), neuronal apoptosis level (p<0.01), ROS level in neurons (p<0.01), MDA content (p<0.01) and expression levels of cysteinyl aspartate specific proteinase-3 (Caspase-3), NR2B, phosphorylated CaMKIIα (p-CaMKIIα) and p-cAMP responsive element binding protein (p-CREB) (p<0.01), but it increased the mitochondrial membrane potential (p<0.01) and B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein (Bax) ratio (p<0.01). Remifentanil can repress the NR2B/ CaMKIIα signaling pathway in the neurons of rats with cerebral IRI to decrease the p-CREB expression, ROS level and MDA content in neurons, neuronal apoptosis level and cerebral infarct size, and increase the mitochondrial membrane potential, thereby protecting the neurological function.

Inhibiting the Src/STAT3 signaling pathway contributes to the anti-melanoma mechanisms of dioscin.

Late stage melanoma is associated with a high mortality rate. Signal transducer and activator of transcription 3 (STAT3) is currently a target for melanoma treatment as it is constitutively activated with high frequency in melanoma. Dioscin is a natural steroid saponin that is present in several medical herbs. A previous study demonstrated that dioscin inhibits STAT3 signaling in a cerebral ischemia-reperfusion injury rat model. Furthermore, dioscin has been reported to exert anti-melanoma effects in B16 melanoma cells and a B16 allograft mouse model. The present study investigated whether inhibition of STAT3 signaling is involved in the anti-melanoma effects of dioscin. The results of the present study demonstrated that dioscin significantly decreased viability, induced apoptosis and suppressed migration of human A375 melanoma cells and murine B16F10 melanoma cells. Furthermore, dioscin inhibited the phosphorylation of STAT3 and Src (an upstream kinase of STAT3), and downregulated mRNA levels of STAT3-targeted genes, including B-cell lymphoma-2, cyclin D1 and matrix metalloproteinase-2. In addition, overexpression of STAT3 decreased the anti-proliferative effects of dioscin. Overall, the results of the present study indicate that inhibiting the Src/STAT3 signaling pathway contributes to the anti-melanoma molecular mechanisms of dioscin. These results provide further pharmacological groundwork for developing dioscin as a novel anti-melanoma agent.

Involvement of apoptosis in the protective effects of Dracocephalum moldavaica in cerebral ischemia reperfusion rat model

ABSTRACTAn extract of Dracocephalum moldevica (DML) was found to exert protective effects on cerebral ischemia-reperfusion injury (CIRI); however, the mechanisms underlying the observed actions of this plant-derived mixture remain to be determined. Thus, the aim of this study was to examine the influence of DML on CIRI rat model induced by middle cerebral artery occlusion (MCAO). The following parameters were measured: (1) viable neurons in the infarcted area using Nissl staining; and (2) immunohistochemistry and Western blot were employed to determine protein expression levels of p53, bcl-2 associated X protein (bax) and B-cell lymphoma-2 (bcl-2), three biomarkers of apoptosis. MCAO significantly decreased the number of viable cortical pyramidal neurons in the infarcted area, while treatment with DML extract significantly elevated the number of viable neurons. MCAO was found to significantly elevate in gene expression levels of p53 and protein expression levels bax accompanied by diminished protein expression levels of bcl-2. Prior administration of DML extract produced marked reduction in gene expression levels of p53 and protein expression levels bax but increased in protein expression levels of bcl-2. Data suggested apoptosis was initiated in MCAO and that DML was effective in treating CIRI via an anti-apoptotic action as evidenced by inhibition of gene expression levels of p53 and protein expression levels of bax with concomitant elevation in protein expression levels of bcl-2. Our findings suggest that extract of DML may prove beneficial in treatment of cerebrovascular disorders.

Sevoflurane prevents miR-181a-induced cerebral ischemia/reperfusion injury

BackgroundSevoflurane (sevo) has been reported to be an effective neuroprotective agent in cerebral ischemia/reperfusion injury (CIRI). However, the precise molecular mechanism underlying sevo preconditioning in CIRI remains largely unknown. MethodsA middle cerebral artery occlusion (MCAO) rat model and primary cortical neurons after oxygen-glucose deprivation and reoxygenation (OGDR) were used as the in vivo and in vitro models of CIRI. The expression profiles of miR-181a and X chromosome-linked inhibitor-of-apoptosis protein (XIAP) in the cerebral cortex of rats and in cortical neurons were examined by qRT-PCR and Western blot, respectively. The infarct volumes were measured by TTC staining and neurological deficits in rats was determined by Zea-Longa scoring criteria. The cell viability, lactate dehydrogenase (LDH) release and apoptotic rate were detected in cortical neurons by MTT assay, LDH analysis and flow cytometry. Western blot analysis was performed to assess the expression of apoptosis-related protein. Luciferase reporter assay was used to confirm the interaction between miR-181a and XIAP. ResultsmiR-181a was upregulated and XIAP was downregulated in rats after MCAO. Sevo preconditioning attenuated miR-181a expression and promoted XIAP level in a rat model of CIRI. Sevo preconditioning ameliorated anti-miR-181a-mediated protective effects on cerebral ischemia in rat model of CIRI, presented as the decrease of infarct volume, neurological deficit and apoptosis. Moreover, sevo pretreatment abated miR-181a-induced cellular injury in primary cortical neurons after OGD, embodied by the increase of cell viability, the reduction of LDH release and the decline of apoptosis. Furthermore, miR-181a suppressed XIAP expression by binding to its 3′UTR in cortical neurons, and sevo-mediated increase on XIAP expression was counteracted by miR-181 overexpression in OGDR-treated neurons. ConclusionSevo preconditioning protected against CIRI in vitro and in vivo possibly by inhibiting miR-181a and facilitating XIAP.

MAPK pathway mediates the anti-oxidative effect of chicoric acid against cerebral ischemia-reperfusion injury in vivo.

The aim of the present study was to investigate the protective effect of chicoric acid on oxidative stress and inflammation in rats with cerebral ischemia-reperfusion injury. A cerebral ischemia-reperfusion injury rat model was created via transient middle cerebral artery occlusion (MCAO) and rats were treated with various doses of chicoric acid (0, 1, 10 and 100 mg/kg). Neurological deficits and infarct volume were used to estimate the protective effects of chicoric acid treatment. Levels of reactive oxygen species (ROS), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, nitric oxide (NO) and prostaglandin E2 (PGE2) were assessed. Western blot analysis was also used to measure the expression of cyclooxygenase (COX)-2, p38-mitogen activated protein kinase (MAPK), c-Jun, phosphorylated protein kinase B (p-AKT) and AKT. Chicoric acid exposure was observed to reduce neurological deficits and infarct volume in rats with cerebral ischemia-reperfusion injury. In addition, ROS production and inflammation were significantly suppressed following treatment with chicoric acid. Chicoric acid was demonstrated to significantly inhibit the upregulation of NO and PGE2 levels in rats following MCAO. Furthermore, chicoric acid significantly suppressed the MCAO-induced promotion of COX-2, p38-MAPK and c-Jun protein expression and enhanced the inhibition of p-AKT/AKT. These results suggest that chicoric acid has a protective effect, preventing oxidative stress and inflammation in rats with cerebral ischemia-reperfusion injury via the p38-MAPK, c-Jun and AKT signaling pathways.

Effect of electroacupuncture at Neiguan (PC 6) and Baihui (GV 20) on CHOP and caspase-12 gene expressions in rats after ischemia-reperfusion injury

To investigate the effects of electroacupuncture (EA) at Neiguan (PC 6) and Baihui (GV 20) by observing the changes of CCAAT/enhancer-binding protein(C/EBP) homologous protein (CHOP) and caspase-12 gene expressions in rats after cerebral ischemia-reperfusion injury (IRI), and explore whether the apoptosis pathway of endoplasmic reticulum stress (ERS) is involved in the protective mechanisms of EA. Sixty rats were randomly assigned to five groups (12 in each group): a normal control group (group A), a sham-operation group (group B), an operation group (group C), an Edaravone group (group D) and an EA group (group E). The cerebral IRI rat model was induced by middle cerebral artery occlusion (MCAO) using intraluminal monofilament. 2,3,5-triphenyl tetrazolium chloride (TTC) staining was adopted in the measurement of cerebral infarction volume. Real-time polymerase chain reaction (RT-PCR) was used to determine the mRNA expressions of CHOP and caspase-12. Compared with group A and group B, the volume of cerebral infarction and mRNA expressions of CHOP and caspase-12 in group C, group D and group E were increased, with statistical significances (P 0.05). EA at Neiguan (PC 6) and Baihui (GV 20) can effectively suppress the volume of cerebral infarction. Furthermore, the underlying mechanism of EA at Neiguan (PC 6) and Baihui (GV 20) is possibly related to the down-regulation of CHOP and caspase-12 mRNA expressions, so as to decrease cell apoptosis.

The Possible Damaged Mechanism and the Preventive Effect of Monosialotetrahexosylganglioside in a Rat Model of Cerebral Ischemia-Reperfusion Injury

The pathogenesis of cerebral ischemia-reperfusion injury (CIRI) is not completely clear and therapies are limited now. Therefore, our study aimed to investigate the possible pathogenesis and preventive approach of CIRI through analyzing changes of aspartate (Asp), glutamate (Glu), mitochondrial calcium (MCa), calmodulin (CaM), and malondialdehyde (MDA) contents and ultramicropathology in hippocampus and cerebral cortex of ischemic susceptible injured regions and the effect of monosialotetrahexosylganglioside (GM1) in the rat model of CIRI. Contents of Asp, Glu, MCa, CaM, and MDA in hippocampus and cerebral cortex tissues were measured by a high-performance liquid chromatography, atomic absorption spectrophotometer, and ordinary spectrophotometer, respectively, changes of ultramicrostructure in neurons of the hippocampus CA1 region and frontal cerebral cortex were observed by a transmission electron microscope. Contents of Asp and Glu in hippocampus and cerebral cortex tissues of CIRI groups significantly decreased and contents of MCa, CaM, and MDA significantly increased than those in control groups, and the ultramicrostructure in neurons of the hippocampus CA1 region and frontal cerebral cortex revealed a significant damaged change, and GM1 significantly ameliorated changes of Asp, Glu, MCa, CaM, and MDA contents in hippocampus and ultrastructural changes in neurons of the hippocampus CA1 region and frontal cerebral cortex. Our findings further support that the abnormal release and/or reuptake of excitatory amino acid neurotransmitters, the disordered calcium homeostasis and the excessive production and/or reduced elimination of reactive oxygen species contribute to the pathogenesis of CIRI, and GM1 can partially prevent these pathogenesises to exert the protective effect on CIRI.

Effects of acupuncture at Baihui (DU20) and Zusanli (ST36) on the expression of heat shock protein 70 and tumor necrosis factor α in the peripheral serum of cerebral ischemia-reperfusion-injured rats

To explore the effects of acupuncture on the peripheral serum expression of heat shock protein 70 (HSP70) and tumor necrosis factor α (TNF-α) in rats with cerebral ischemia-reperfusion injury (CIRI). In total, 152 Sprague-Dawley (SD) rats were randomly divided into an operated group and a non-operated group according to a random digits table. The operated group included a sham-operated group, a model group and an acupuncture group, whereas the non-operated group consisted of a normal group. Except for the normal group, each group was further divided into 12, 24, 48, 72, 96, and 144 h time points according to different reperfusion times. Eight rats were assigned in each operated group and in the normal group. The rat model of CIRI was established by the thread occlusion method in the model and acupuncture groups. The acupuncture group was treated with electroacupuncture at Baihui (DU20) and Zusanli (ST36) for the required time after successful operation. Blood was sampled to detect the HSP70 and TNF-α content by enzyme linked immunosorbent assay. The expression of HSP70 protein in the peripheral serum of the experimental groups was higher than that in the normal control group. The peak time in both the model and the sham-operated groups was 12 h, and the peak time in the acupuncture group was 24 h. The expression in the acupuncture group declined to a lower level at 72 h and was lower than that in the model and sham-operated groups (P<0.05). The peak time for the expression of TNF-α protein in the peripheral serum of both the model and the acupuncture groups was 24 h, but the expression in the acupuncture group was lower than the model group. Additionally, the expression of TNF-α in all experimental groups was higher than the normal group (P<0.05). Acupuncture at DU20 and ST36 in rats attenuated CIRI, which was associated with a reduction in the expression of HSP70 and TNF-α. These results provide clues to acupuncture's neuroprotective properties. Acupuncture at DU20 and ST36 in rats after CIRI can adjust the expression of HSP70 and TNF-α in the peripheral serum, which might be one of the mechanisms of acupuncture's attenuation of CIRI.

Effects of Tongluo-Jiunao Injection on Cerebral Ischemia–Reperfusion Injury Rats

Abstract The aim of this study was to investigate the protective effects of Tongluo-Jiunao (TLJN) injection on brain tissues of focal cerebral ischemia-reperfusion injury rats. Cerebral ischemia-reperfusion injury rat model was established by middle cerebral artery occlusion. All animal models were divided into the following four groups: sham, model, TLJN, and edaravone groups. The brain infarct volume, concentration of IGF-1 in brain tissues and serum were detected at 24 h, 3 d, and 7 d after cerebral ischemia-reperfusion injury. The results showed that compared to the model group, the brain infarct volume of TLJN group was significantly reduced, and the expression of IGF-1 of cerebral cortex was upregulated in TLJN group. It was concluded that TLJN has significant protective effects on brain tissues in focal cerebral ischemia-reperfusion injury rat, and the IGF-1 may play an important role in the protective effects of brain tissues.