Cerebral Ischemia-reperfusion In Rats Research Articles

Automated label-free detection of injured neuron with deep learning by two-photon microscopy.

Stroke is a significant cause of morbidity and long-term disability globally. Detection of injured neuron is a prerequisite for defining the degree of focal ischemic brain injury, which can be used to guide further therapy. Here, we demonstrate the capability of two-photon microscopy (TPM) to label-freely identify injured neurons on unstained thin section and fresh tissue of rat cerebral ischemia-reperfusion model, revealing definite diagnostic features compared with conventional staining images. Moreover, a deep learning model based on convolutional neural network is developed to automatically detect the location of injured neurons on TPM images. We then apply deep learning-assisted TPM to evaluate the ischemic regions based on tissue edema, two-photon excited fluorescence signal intensity, as well as neuronal injury, presenting a novel manner for identifying the infarct core, peri-infarct area, and remote area. These results propose an automated and label-free method that could provide supplementary information to augment the diagnostic accuracy, as well as hold the potential to be used as an intravital diagnostic tool for evaluating the effectiveness of drug interventions and predicting potential therapeutics.

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.

Mechanisms Associated with Protective Effects of Ginkgo Biloba Leaf Extracton in Rat Cerebral Ischemia Reperfusion Injury

ABSTRACTCerebral infarction occurs as a consequence of cerebral ischemia-reperfusion injury (CIRI). Ginkgo biloba leaf extract (GbE) is composed predominantly of active ingredients such as flavonoids and terpene lactones and often used to treat cerebrovascular diseases. However, the mechanisms underlying the use of this herbal extract to treat cerebrovascular-mediated damage are not known. The aim of this study was to examine the effectiveness of administration GbE to ameliorate the observed consequences of CIRI. The following parameters were measured: (1) behavioral score (2) infarct area (3) the content of serum malondialdehyde (MDA) as well as activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and (4) interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α) expression levels in the infarcted brain tissue. Data demonstrated that treatment with GbE to CIRI rats resulted in significant reduction in cerebral-infarcted area associated with improvement in behavioral score. GbE was found to decrease serum MDA levels concomitant with elevated activity levels of SOD and GSH-PX. Immunohistochemistry and Western blot analysis showed that GbE significantly lowered the levels of IL-6 and TNF-α in the infarcted brain tissue. Data suggest that GbE may be therapeutically effective in improving behavioral score in CIRI rats through reduction of oxidative stress and anti-inflammation in the cerebral infarction region.

Influence of miR-34a on cerebral neuronal apoptosis in rats with cerebral ischemia reperfusion through the Notch1 signaling pathway.

To explore the influence and the underlying mechanism of micro-ribonucleic acid (miR)-34a on cerebral neuronal apoptosis in rats with cerebral ischemia-reperfusion (CIR). 60 adult male Wistar rats were randomly divided into 3 groups: Sham group, CIR group and miR-34a knockdown group. The rat model of CIR was established using the suture occlusion method. The expression level of miR-34a in lesion tissues in the three groups was determined via Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The pathological injury of brain tissues was detected via hematoxylin-eosin (HE) staining and the infarction region in each group was evaluated via triphenyl tetrazolium chloride (TTC) staining. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was performed to measure the cerebral neuronal apoptosis level. The level of Nissl's body in each group was detected via Nissl's staining. The expression level of the platelet-derived neurotrophic factor (PDNF) and Notch1/hypoxia-inducible factor-1α (HIF-1α) signaling pathway-related proteins in brain tissues were detected via immunohistochemistry and Western blotting, respectively. The expression level of miR-34a in brain tissues of the CIR group was significantly increased compared to that of the Sham group (p < 0.05). After the intervention with miR-34a, the infarction area of brain tissues was markedly reduced when comparing to the CIR group (p < 0.05). In addition, the results of HE staining and Nissl staining revealed that CIR treatment led to edema in cerebral neurons, disorderly arranged neurons, and remarkably decreased number of Nissl's bodies. However, miR-34a knockdown following CIR significantly alleviated the brain tissue injury and markedly increased the number of Nissl's bodies (p < 0.05). The results of TUNEL staining also indicated that miR-34a siRNA could remarkably reverse the cerebral neuronal apoptosis caused by CIR in rats (p < 0.05). According to the immunohistochemical staining results, the expression of PDNF in brain tissues declined in the CIR group, while enhanced in the miR-34a siRNA group (p < 0.05). Furthermore, the Western blotting results manifested that miR-34a siRNA could up-regulate the Notch1 and HIF-1α protein expressions in brain tissues of CIR rats. Our data demonstrated that the miR-34a knockdown could alleviate the brain tissue injury and neuronal apoptosis by activating the Notch1/HIF-1α signaling pathway CIR-treated rats.

Effect of levocarnitine on cerebral ischemia-reperfusion rats via activating Nrf2/ARE signaling pathway.

Levocarnitine plays a crucial role in the metabolism of organisms. The aim of this study was to explore the impact of Levocarnitine on cerebral ischemia-reperfusion (I/R) rats and the underlying mechanism. Cerebral I/R model was first successfully established. Two groups were set up, including drug group (I/R + Levocarnitine group) and control group (I/R group). The influences of Levocarnitine on brain injury and oxidative stress in cerebral I/R rats were evaluated. Furthermore, the impacts of Levocarnitine on the nuclear factor E2-related factor 2 (Nrf2)/antioxidant responsive element (ARE) signaling pathway and neuronal apoptosis in rats were detected. Compared with I/R group, I/R + Levocarnitine group exhibited markedly lowered neurological deficit score and cerebral infarct volume. However, superoxide dismutase (SOD) and notably decreased malondialdehyde (MDA) were significantly up-regulated in I/R + Levocarnitine group. This suggested that Levocarnitine could relieve cerebral nerve injury and oxidative stress in cerebral I/R rats. Additionally, in I/R + Levocarnitine group, the protein expressions of Nrf2, heme oxygenase-1 (HO-1), and B-cell lymphoma 2 (Bcl-2) were significantly up-regulated, whereas cleaved Caspase-3 (c-Caspase-3) was notably down-regulated. Furthermore, neuronal apoptosis in cerebral I/R rats was remarkably inhibited. Levocarnitine alleviates brain injury and neuronal apoptosis in cerebral I/R rats by activating the Nrf2/ARE signaling pathway.

MiR-183 inhibits microglia activation and expression of inflammatory factors in rats with cerebral ischemia reperfusion via NF-κB signaling pathway.

Ischemic stroke represents 87% of all strokes, and is the third leading cause of disability and mortality worldwide. The cause of ischemic stroke is the obstruction of blood flow through the artery that supplies oxygen-rich blood to the brain, with ischemia-reperfusion injury as its major cause. microRNAs (miRNA) are small non-coding RNAs, which serve important roles in the regulation of gene expression at the post-transcription level. The aim of the present study was to investigate the effect of miRNA-183 (miR-183) on microglia activation in rats with cerebral ischemia-reperfusion injury. To this end, a rat cerebral ischemia-reperfusion injury model was established. The results indicated that miR-183 expression was decreased by cerebral ischemia-reperfusion. In addition, treatment using miR-183 agomir significantly reduced the neurological function scores, percentage of cerebral infarction volume, and ionized calcium-binding adapter molecule-1 (IBA-1)-positive cells in the CA1 area of the hippocampus in rats subjected to cerebral ischemia-reperfusion injury, implicating a neuroprotective role for miR-183. MiR-183 agomir treatment also decreased the expression of pro-inflammatory-associated proteins interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α. Finally, the expression of the nuclear factor (NF)-κB p65 and IκBα was decreased and increased by miR-183 agomir treatment, respectively, indicating inhibition of the NF-κB signaling pathway. These observations suggest that miR-183 regulates the activation of microglia in rats with cerebral ischemia-reperfusion injury by inhibiting the NF-κB signaling pathway.

Acute remote ischemic preconditioning alleviates free radical injury and inflammatory response in cerebral ischemia/reperfusion rats

Remote ischemic preconditioning (IPreC) is an effective strategy to defend against cerebral ischemia/reperfusion (IR) injury; however, its mechanisms remain to be elucidated. The aim of the present study was to investigate the effect of IPreC on brain tissue following cerebral ischemia, as well as the underlying mechanisms. Adult male Sprague-Dawley rats were treated with IPreC for 72 h prior to the induction of transient cerebral ischemia and reperfusion. The results demonstrated that IPreC reduced the area of cerebral infarction in the IR rats by 2,3,5-triphenyl-tetrazolium chloride staining. In addition, cell apoptosis was markedly suppressed by IPreC with an increased expression of B-cell lymphoma 2 (Bcl-2)/Bcl-2-associatd X protein using Terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling assay and western blot analysis. IR induced a decrease in the level of superoxide dismutase, and IPreC significantly suppressed increased levels of malondialdehyde, lactate dehydrogenase and nitric oxide. The expression of CD11b and CD18 was markedly inhibited by IpreC unsing flow cytometry. Furthermore, IPreC markedly decreased the release of pro-inflammatory factors interleukin (IL)-6 and IL-1β, and enhanced the level of anti-inflammatory factors (IL-10 and IL-1 receptor antagonist) by ELISA assay. Finally, IPreC reduced the levels of transforming growth factor-β-activated kinase 1, phosphorylated-P65/P65, and tumor necrosis factor-α, indicating that the nuclear factor-κB pathway was involved in IPreC-mediated protection against cerebral ischemia. Taken together, the results suggested that IPreC decreased ischemic brain injury through alleviating free radical injury and the inflammatory response in cerebral IR rats.

Effects of selective brain hypothermia on expression of miR-484 during cerebral ischemia-reperfusion in rats

Objective To evaluate the effects of selective brain hypothermia on the expression of miR-484 during cerebral ischemia-reperfusion (I/R) in rats. Methods A total of 120 clean-grade healthy male Sprague-Dawley rats, aged 8-10 weeks, weighing 250-300 g, were divided into 4 groups (n=30 each) using a random number table method: sham operation group (group S), cerebral I/R group (group I/R), hypothermia group (group H) and normothermia group (group N). Blood vessels were only separated and ligated without blockade in group S. In the other three groups, cerebral I/R was induced by inserting a nylon thread with rounded tip into the right internal carotid artery, and the middle cerebral artery was occluded for 2 h followed by reperfusion.In group H, 4 ℃ normal saline was infused for 15 min at a rate of 80 ml·kg-1·h-1 through the internal carotid artery immediately after removing the nylon thread.In group N, 37 ℃ normal saline was infused in the same way.Neurological deficit scores were evaluated at 6, 24 and 48 h after reperfusion.Animals were then sacrificed, the cerebral cortex of the ischemic penumbra was removed for determination of nerve cell apoptosis (by TUNEL method), expression of mitochondrial fission protein 1 (Fis1) (by Western blot) and expression of miR-484 (quantitative real-time polymerase chain reaction). The apoptotic rate was calculated. Results Compared with group S, the neurological deficit score and apoptotic rate of nerve cells were significantly increased, and the expression of Fis1 was up-regulated at each time point in the other three groups, the expression of miR-484 was significantly down-regulated in I/R and N groups, and the expression of miR-484 was significantly up-regulated in group H (P<0.05 or 0.01). Compared with group I/R and group N, the neurological deficit score and apoptotic rate of nerve cells were significantly decreased, and the expression of Fis1 was down-regulated, and the expression of miR-484 was up-regulated at each time point in group H (P<0.05 or 0.01). Conclusion The mechanism by which selective cerebral hypothermia attenuates cerebral I/R injury is associated with up-regulating miR-484 expression and thus down-regulating Fis1 expression in rats. Key words: Hypothermia; Brain; Reperfusion injury; MicroRNAs

Immortalized Human Bone Marrow Derived Stromal Cells in Treatment of Transient Cerebral Ischemia in Rats.

Cerebral ischemic stroke may cause a number of adverse neurological complications or behavioral disorders. Moreover, cerebral ischemic stroke is a prevalent disease with limited treatment strategies and may increase chances of developing neurodegenerative diseases, e.g., Alzheimer's disease, in the future. Therefore, alternative strategies are highly needed for improving the therapy for ischemic stroke. Human bone marrow stroma stem cells, also known as mesenchymal stem cells (hMSC), have a demonstrative role in treating transient cerebral ischemia, yet whether immortalized hMSC may have a better effect than hMSC is unknown. Here, we addressed this question. A rat cerebral ischemia reperfusion (IR) model was used to compare the effects of hMSC and an immortalized hMSC by human telomerase reverse transcriptase (hMSC-TERT). We found that both hMSC and hMSC-TERT similarly attenuated the elevation of serum neuron specific enolase levels after IR. Transplantation with hMSC alleviated cerebral infarction, alleviated brain edema, improved neural function, and reduced brain cell apoptosis, in a significantly better degree than transplantation with hMSC. Thus, these data suggest that immortalized hMSC may have an advantage over hMSC in treatment of transient cerebral ischemia.

Involvement of hippocampal miR-664-3p in electroacupuncture induced improvement of learning-memory ability of cerebral ischemia-reperfusion rats

To explore the effect of electroacupuncture (EA) on learning-memory ability and its correlation with miR-664-3p levels in the hippocampus in cerebral ischemia-reperfusion injury (CI/RI) rats. A total of 27 male SD rats were randomly divided into sham operation, CI/RI model and EA groups (n＝9 in each group). The CI/RI model was established by occlusion of the middle cerebral artery (MACO) according to the modified Longa methods. EA was applied to "Baihui" (GV20) and "Shenting" (GV24) for 20 min, once a day for 7 days. The Morris water maze tests (place navigation tasks and spatial probe trials) were used to evaluate the rats' learning-memory ability. After EA intervention, the hippocampal tissue was collected to measure the mRNA and miRNA expression profiles by using high-throughput next generation sequencing. Bioinformatics analyses including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to screen the most abundant differentially-expressed miRNAs and mRNAs, particularly the target genes of miR-664-3p. Their biological functional categories including the key biochemical metabolic pathways and signaling pathways involved in CI and EA effect in the left hippocampus tissue were analyzed. The average escape latency of place navigation tests was significantly longer in the CI/RI model group than in the sham operation group (P<0.01), and obviously shorter in the EA group than in the model group (P<0.01); and the times of escape-platform quadrant crossing of the spatial probe trials were considerably fewer in the CI/RI model group than in the sham operation group (P<0.05), and markedly more in the CI/RI EA group than in the model group (P<0.05). Combined analysis of miRNA and mRNA showed that the differentially-expressed target genes of miR-664-3p were significantly up-regulated after CI/RI relevant to the sham operation rats (P<0.05) and notably down-regulated in the EA group relevant to the CI/RI model group (P<0.05). The GO analysis displayed that the target genes of miR-664-3p were significantly enriched in the category of cell transportation activity, ion binding，and transmembrane transporter activity, the production of precursor metabolites and energy, transportation, signal transduction，localization, etc. KEGG analysis showed that the target genes of miR-664-3p in this study were mainly enriched in the thyroid hormone pathway, Hippo signaling pathway, MAPK signal transduction pathway，cAMP signaling pathway, neurotrophic factor signal transduction pathway, etc. EA of GV20 and GV24 can improve the learning-memory ability of CI/RI rats, which is closely related to its effect in down-regulating the levels of hippocampal target genes of miR-664-3p involving cellular transmembrane transportation，cell to environment interaction，cell-cell communication, etc.

Sodium Dichloroacetate Stimulates Angiogenesis by Improving Endothelial Precursor Cell Function in an AKT/GSK-3β/Nrf2 Dependent Pathway in Vascular Dementia Rats.

Sodium dichloroacetate (DCA) is a mitochondrial pyruvate dehydrogenase kinase inhibitor, and has been shown to display vasoprotective effects in chronic ischemic stroke. The purpose of this study was to evaluate the therapeutic effect of DCA on vascular dementia (VD) and endothelial progenitor cell (EPC)-mediated angiogenesis. After cerebral ischemia-reperfusion in rats, DCA was administered continuously for 21 days; following which, histological analysis, and cognitive functional tests were conducted. Rat bone marrow-derived EPCs were isolated, their function and quantity were measured, and the effects of long-term administration of DCA on EPCs in a rat model of VD was studied. We found that long-term DCA administration improved cognitive function in VD rats, reduced brain infarct size and brain atrophy, increased VEGF and bFGF levels in vivo, promoted angiogenesis in damaged areas, and significantly improved EPC function in VD rats. Compared with the VD group, AKT, Nrf2, eNOS expression, and intracellular NO levels were elevated in EPCs of DCA-treated VD rats. In addition, GSK3β and intracellular ROS levels were decreased. Simultaneously, it was found that DCA directly acted on EPCs, and improved EPC functional behavior. Taken together, these findings suggested that long-term DCA administration improved cognitive function in a rat model of VD, and did so in part, by improving EPC function. Observations suggest that prolonged DCA administration might be beneficial in treating VD.

Effect of ulinastatin on expression of metabotropic glutamate receptor II during cerebral ischemia-reperfusion in rats

Objective To evaluate the effect of ulinastatin on the expression of metabotropic glutamate receptor Ⅱ (mGluRⅡ) during cerebral ischemia-reperfusion (I/R) in rats. Methods Forty-eight male Sprague-Dawley rats, aged 6-8 weeks, weighing 230-280 g, were divided into 3 groups (n=16 each) by a random number table method: sham operation group (S group), cerebral I/R group (I/R group) and ulinastatin group (U group). The model of cerebral I/R injury was established by occluding the right middle cerebral artery using a nylon thread with a rounded tip inserted into internal carotid artery and advanced cranially until resistance was met.Middle cerebral artery occlusion was maintained for 2 h followed by 24-h reperfusion.Ulinastatin 100 000 U/kg was injected via the tail vein immediately after onset of reperfusion in group U. The neurological deficit score (NDS) was assessed after 24 h of reperfusion.The rats were then sacrificed, and brain tissues were obtained for determination of brain infarction (by TTC staining), expression of IκB-α in cerebral ischemic penumbra (by Western blot) and expression of mGluR Ⅱ in cerebral ischemic penumbra (by immunofluorescent staining). The percentage of cerebral infarct volume was calculated. Results Compared with S group, the NDS and percentage of cerebral infarct volume were significantly increased, the expression of mGluRⅡ in cerebral ischemic penumbra was up-regulated, and the expression of IκB-α in cerebral ischemic penumbra was down-regulated in I/R and U groups (P<0.05). Compared with I/R group, the NDS and percentage of cerebral infarct volume were significantly decreased, the expression of mGluRⅡ in cerebral ischemic penumbra was down-regulated, and the expression of IκB-α in cerebral ischemic penumbra was up-regulated in U group (P<0.05). Conclusion The mechanism by which ulinastatin mitigates cerebral I/R injury may be related to inhibiting the expression of mGluR Ⅱ in cerebral ischemic penumbra of rats. Key words: Trypsin inhibitor; Reperfusion injury; Brain; Receptor, glutamate

Effects of isoflurane postconditioning on angiogenesis during cerebral ischemia-reperfusion in rats and the role of Shh signaling pathway

Objective To evaluate the effects of isoflurane postconditioning on angiogenesis during cerebral ischemia-reperfusion (I/R) in rats and the role of Shh signaling pathway. Methods Thirty-two clean-grade healthy male Sprague-Dawley rats, aged 6-8 weeks, weighing 220-280 g, were divided into 4 groups (n=8 each) by a random number table method: sham operation group (Sham group), I/R group, isoflurane postconditioning group (ISO group), and isoflurane postconditioning plus Shh signaling pathway specific inhibitor cyclopamine group (ISO + CYC group). Cerebral ischemia was produced by inserting a 3-0 nylon thread with a rounded tip into the internal jugular vein. The nylon thread was threaded cranially until resistance was met. Occlusion was maintained for 1.5 h followed by 24 h reperfusion. Neurological deficit was scored at 24 h of reperfusion. Rats were then sacrificed, and brains were removed for determination of cerebral infarct volume (by TTC) and expression of glioma-associated oncogene homolog 1 (Gli1), vascular endothelial growth factor (VEGF) and transmembrane phosphoglycoprotein protein (CD34) in cerebral cortex (by Western blot) and for examination of the pathological changes (by Nissl staining). Results Compared with Sham group, the neurological deficit score and cerebral infarct volume were significantly increased, and the expression of Gli1, VEGF and CD34 in the cerebral cortex was up-regulated in I/R and ISO groups (P 0.05). Compared with ISO group, the neurological deficit score and cerebral infarct volume were significantly increased, and the expression of Gli1, VEGF and CD34 in the cerebral cortex was down-regulated in ISO+ CYC group (P<0.05). Conclusion The mechanism by which isoflurane post-conditioning attenuates cerebral I/R injury is related to activating Shh signaling pathway and promoting angiogenesis in rats. Key words: Isoflurane; Ischemic postconditioning; Reperfusion injury; Brain; Hedgehog proteins; Angiogenesis

Neuroprotective Influence of miR-301a Inhibition in Experimental Cerebral Ischemia/Reperfusion Rat Models Through Targeting NDRG2.

The objective of this study is to find out the potential influence of miR-301a in an experimental cerebral ischemia-reperfusion (I/R) rat model through targeting NDRG2. Rats with cerebral I/R injury were constructed and classified into model, miR-301a inhibitor, miR-301a mimic, NC (negative control), siNDRG2, NDRG2, and miR-301a inhibitor + si-NDRG2 groups, as well as another sham group. Cerebral infarct volume and cell apoptosis were observed by TTC staining and TUNEL staining. The targeting relationship between miR-301a and NDRG2 was verified by luciferase assay. ELISA, qRT-PCR, and Western blot were used to detect the expressions of related molecules. Compared with sham group, rats in the model group had elevated neurological function score and infarct volume; meanwhile, the cell apoptosis rate and inflammatory response were also increased with enhanced expression of miR-301a and NDRG2 (all P < 0.05). These changes were worsened in the miR-301a mimic and si-NDRG2 groups. Conversely, those rats in the miR-301a inhibitor and NDRG2 groups presented increased NDRG2, and at the same time, other above concerning factors also exhibited opposite tendencies (all P < 0.05). Dual-luciferase reporter gene assay confirmed that NDRG2 was a target gene of miR-301a, and si-NDRG2 could reverse the neuroprotective effect of miR-301a inhibitor in rats with cerebral I/R injury. Inhibiting miR-301a has a neuroprotective effect on rats with cerebral I/R injury to ameliorate cell apoptosis and inflammatory response through possibly targeting NDRG2.

Cdh1 overexpression improves emotion and cognitive-related behaviors via regulating hippocampal neuroplasticity in global cerebral ischemia rats

Post-stroke survivors exhibited cognitive deficits and performed emotional impairment. However, the effect of global cerebral ischemia on standard behavioral measures of emotionality and underlying mechanism remain largely unknown. Our previous work identified that down-regulation of Cdh1 contributed to ischemic neuronal death in rat, thus we hypothesized that Cdh1 exerts a role in emotionality after cerebral ischemia, and we investigated the effect of Cdh1 overexpression on neurogenic behaviors and possible mechanisms in transient global cerebral ischemia reperfusion (tGCI/R) rats. A series of behavioral tests were used to evaluate emotion and cognitive related behaviors, and molecular biological techniques were employed to investigate hippocampal neuroplasticity. The results showed that tGCI/R rats displayed anxiety- and depression-like behaviors and a certain degree of cognitive impairment, and these abnormal behaviors accompanied with a loss of hippocampal synapses and dendritic spines, disruption of dendrite arborization and decline in the level of GAP-43, synaptophysin, synapsin and PSD-95. However, Cdh1 overexpression improved negative emotionality, ameliorated cognitive deficits, rescued hippocampal synapses loss, prevented dendritic network disorganization, and increased the level of synaptic-associated proteins after tGCI/R. Taken together, these findings suggest that Cdh1 overexpression exerts a neuroprotective effect by regulating hippocampal neuroplasticity thus improving negative emotionality and cognitive deficits after tGCI/R.

Retigabine protects the blood-brain barrier by regulating tight junctions between cerebral vascular endothelial cells in cerebral ischemia-reperfusion rats.

To investigate the effect of retigabine on the blood-brain barrier permeability in rats with cerebral ischemia-reperfusion and its mechanism. A total of 90 Sprague-Dawley (SD) rats were selected to prepare a rat model of focal cerebral ischemia-reperfusion. The blood flow changes were detected using a laser Doppler flow meter, the percentage of the cerebral infarction volume was measured by means of the triphenyl tetrazolium chloride (TTC) staining, the effect of retigabine on the permeability of the blood-brain barrier after cerebral ischemia-reperfusion was examined via Evans blue (EB) staining, and the state of tight junctions between endothelial cells was determined via the transmission electron microscopy (TEM) technique. Immunohistochemistry was used to detect the effects of retigabine on the distribution and expressions of tight junction-associated proteins in the cerebral ischemia-reperfusion blood-brain barrier. Western blotting was adopted to examine the changes in the expressions of related proteins in cerebral ischemia-reperfusion tissues. At 48 h and 96 h after cerebral ischemia-reperfusion, retigabine notably reduced the cerebral infarction volume of rats, and the tight junctions between microvascular endothelial cells in the ischemic area opened up, the permeability of the blood-brain barrier was remarkably increased, and the permeability of the blood-brain barrier was significantly reduced under the action of retigabine. The expressions of claudin-5, occludin, and ZO-1 in the blood-brain barrier of the ischemic brain tissue significantly declined, and retigabine notably increased the expressions of three proteins and their distributions along the microvessels. At 3 h, 24 h, 48 h, and 96 h after cerebral ischemia-reperfusion, the expressions of the MMP-2 protein and MMP-9 protein in the ischemic brain tissue were evidently increased, which were inhibited by retigabine. Moreover, the expressions of the PKCδ protein in the ischemic brain tissue were markedly increased, which were significantly inhibited by retigabine. The regulatory roles of retigabine in the distribution and expressions of claudin-5, occludin, and ZO-1 may be associated with the inhibition of the expressions of the MMP-2, MMP-9, and PKCδ proteins.

Effect of Sargentodoxa cuneata total phenolic acids on focal cerebral ischemia reperfusion injury rats model.

ObjectiveExplore the possible protective effect of Sargentodoxa cuneata total phenolic acids on cerebral ischemia reperfusion injury rats.MethodsFocal cerebral ischemia reperfusion rats model were established by linear thrombus. Nimodipine group, Naoluotong group, the high, middle and low dose of Sargentodoxa cuneata total phenolic acids groups were given related drugs via intragastric administration before operation for seven days, once a day. At the same time sham operation group, and ischemia reperfusion group were given the same volume of physiological saline. One hour after the last administration, establish focal cerebral ischemia- reperfusion model in rats by thread method, and the thread was taken out after 2 h ischemia to achieve cerebral ischemia reperfusion injury in rats. After reperfusion for 24 h, the rats were given neurologic deficit score. The brain tissue was taken to measure the levels of IL-6, IL-1β, TNF-α, Bcl-2, Bax, Casp-3 and ICAM-1; HE staining observed histopathological changes in the hippocampus and cortical areas of the brain; Immunohistochemistry was used to observe the expression of NGF and NF-KBp65.ResultFocal cerebral ischemia reperfusion rats model was copyed successed. Compared with model group, each dose group of Sargentodoxa cuneata total phenolic acids could decreased the neurologic deficit score (P < 0.05 or P < 0.01), decreased the levels of IL-6, IL-1β, ICAM-1, TNF-α, Bax and Caspase-3 in brain tissue (P < 0.05 or P < 0.01), increased the levels of IL-10, Bcl-2, NGF in brain tissue (P < 0.05 or P < 0.01), decreased the express of NF-KBp65 in brain (P < 0.05 or P < 0.01).ConclusionSargentodoxa cuneata total phenolic acids can improve focal cerebral ischemia reperfusion injury rats tissue inflammation, apoptosis pathway, increase nutrition factor to protect the neurons, reduce the apoptosis of nerve cells, activate brain cells self-protect, improve the histopathological changes in the hippocampus and cortical areas of the brain, reduce cerebral ischemia reperfusion injury.

Application of 18F-FDG microPET/CT in the screening of cerebral ischemia reperfusion models

Objective To investigate the feasibility of 18F-fluorodeoxyglucose (FDG) microPET/CT in the screening of cerebral ischemia reperfusion (CIR) models. Methods The suture-occluded method was used to establish CIR rat models with reversible middle cerebral artery embolism. After that only awake rats whose Zea-Longa scores were 1-4 were selected for the following experiments, and 18 male SD rats were selected. Garcia scale with 18 points was used to evaluate the neurological function of rats at 2 and 24 h post-operation. At the same time points, 18F-FDG was injected into caudal vein after anesthesia and microPET/CT scan was conducted at 40 min post-injection. Visual and semi-quantitative analyses were adopted to analyze the images. The autopsy and HE staining were performed on accidentally dead rats. The other alive rats were sacrificed after microPET/CT scan at 24 h post-operation, and their brain tissues were taken out quickly to detect the infarction by triphenyl tetrazolium chloride (TTC) staining. The pathological results were taken as the gold criteria. Fisher exact test was used to compare the difference of accuracy for diagnosing CIR models between neurological function score (NFS) and microPET/CT. Results According to the pathology, there were 11 CIR models, 4 with subarachnoid hemorrhage (SAH), 3 with SAH and cerebral hemorrhage. Between 8-12 h post-operation, 4 rats died accidentally. At 2 h post-operation, the diagnostic accuracies of NFS and microPET/CT were 11/18 and 15/18 (P 0.05). Conclusion 18F-FDG microPET/CT is better than NFS in screening CIR models in early stage. Key words: Brain ischemia; Reperfusion; Positron-emission tomography; Tomography, X-ray computed; Deoxyglucose; Rats

The protective mechanism underlying total flavones of Dracocephalum (TFD) effects on rat cerebral ischemia reperfusion injury

ABSTRACTPreviously, total flavones of Dracocephalum (TFD), derived from Dracocephalum, were found to exert protective effects in cerebral ischemia reperfusion injury (CIRI) in middle cerebral artery occlusion (MCAO) rat model. However, the mechanisms underlying these observed effects of TFD on MCAO-induced rats still remain to be determined. Therefore, the aim of this study was to examine whether TFD alleviated MCAO through mechanisms involving anti-inflammatory and anti-apoptotic using MCAO rats. The following parameters were measured: (1) percentage (%) area of brain infarction; (2) serum levels of inflammatory cytokines, including tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) and (3) expression protein levels of caspase-3 and AMP-activated protein kinase (AMPK). Results showed that MCAO significantly increased the % area of brain infarction, while TFD administration in these animals markedly reduced % area of brain infarction. A significant elevation on serum levels of TNF-α and IL-6 was noted with MCAO which was markedly reduced by TFD. In addition, MCAO produced a significant rise in protein expression levels of caspase-3 and AMPK. In contrast, TFD markedly lowered protein expression levels of caspase-3 and AMPK. Data suggest that the protective effects of TFD in MCAO model animals may involve inhibition of inflammatory mediator release associated with apoptosis through down regulation of AMPK signaling pathway.

The protective mechanism underlying total flavones of Dracocephalum (TFD) effects on rat cerebral ischemia reperfusion injury

ABSTRACTPreviously, total flavones of Dracocephalum (TFD), derived from Dracocephalum, were found to exert protective effects in cerebral ischemia reperfusion injury (CIRI) in middle cerebral artery occlusion (MCAO) rat model. However, the mechanisms underlying these observed effects of TFD on MCAO-induced rats still remain to be determined. Therefore, the aim of this study was to examine whether TFD alleviated MCAO through mechanisms involving anti-inflammatory and anti-apoptotic using MCAO rats. The following parameters were measured: (1) percentage (%) area of brain infarction; (2) serum levels of inflammatory cytokines, including tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) and (3) expression protein levels of caspase-3 and AMP-activated protein kinase (AMPK). Results showed that MCAO significantly increased the % area of brain infarction, while TFD administration in these animals markedly reduced % area of brain infarction. A significant elevation on serum levels of TNF-α and IL-6 was noted with MCAO which was markedly reduced by TFD. In addition, MCAO produced a significant rise in protein expression levels of caspase-3 and AMPK. In contrast, TFD markedly lowered protein expression levels of caspase-3 and AMPK. Data suggest that the protective effects of TFD in MCAO model animals may involve inhibition of inflammatory mediator release associated with apoptosis through down regulation of AMPK signaling pathway.