Involvement of the Wnt signaling pathway and cell apoptosis in the rat hippocampus following cerebral ischemia/reperfusion injury

We investigated the role of the Wnt signaling pathway in cerebral ischemia/reperfusion injury by examining β-catenin and glycogen synthase kinase-3β protein expression in the rat hippocampal CA1 region following acute cerebral ischemia/reperfusion. Our results demonstrate that cell apoptosis increases in the CA1 region following ischemia/reperfusion. In addition, β-catenin and glycogen synthase kinase-3β protein expression gradually increases, peaking at 48 hours following reperfusion. Dickkopf-1 administration, after cerebral ischemia/reperfusion injury, results in decreased cell apoptosis, and β-catenin and glycogen synthase kinase-3β expression, in the CA1 region. This suggests that β-catenin and glycogen synthase kinase-3β, both components of the Wnt signaling pathway, participate in cell apoptosis following cerebral ischemia/reperfusion injury.

Effect and mechanism of adrenomedullin on apoptosis of renal tubular epithelial cell in rats induced by renal ischemia reperfusion injury

Objective To investigate the effect and mechanism of adrenomedullin ( AM ) on apoptosis of renal tubular epithelial cell in rats induced by renal ischemia reperfusion injury. Methods Thirty-two Wistar rats were randomly divided into 4 groups: control group,IRI group, empty plasmid group and AM group. One week after re-

Inhibition of cerebral ischemia/reperfusion injuryinduced apoptosis:nicotiflorin and JAK2/STAT3 pathway

Nicotiflorin is a flavonoid extracted from Carthamus tinctorius.Previous studies have shown its cerebral protective effect,but the mechanism is undefined.In this study,we aimed to determine whether nicotiflorin protects against cerebral ischemia/reperfusion injury-induced apoptosis through the JAK2/STAT3 pathway.The cerebral ischemia/reperfusion injury model was established by middle cerebral artery occlusion/reperfusion.Nicotiflorin（10 mg/kg） was administered by tail vein injection.Cell apoptosis in the ischemic cerebral cortex was examined by hematoxylin-eosin staining and terminal deoxynucleotidyl transferase d UTP nick end labeling assay.Bcl-2 and Bax expression levels in ischemic cerebral cortex were examined by immunohistochemial staining.Additionally,p-JAK2,p-STAT3,Bcl-2,Bax,and caspase-3 levels in ischemic cerebral cortex were examined by western blot assay.Nicotiflorin altered the shape and structure of injured neurons,decreased the number of apoptotic cells,down-regulates expression of p-JAK2,p-STAT3,caspase-3,and Bax,decreased Bax immunoredactivity,and increased Bcl-2 protein expression and immunoreactivity.These results suggest that nicotiflorin protects against cerebral ischemia/reperfusion injury-induced apoptosis via the JAK2/STAT3 pathway.

Neuronal apoptosis and interleukin 1-beta converting enzyme expression in the frontal cortex and hippocampus of mice with ischemia/reperfusion injury

BACKGROUND： Interleukin 1β-converting enzyme （ICE） gene expression can induce neuronal apoptosis. However, the dynamic changes in ICE gene expression and its effects on neuronal apoptosis under cerebral ischemia/reperfusion conditions remain unclear. OBJECTIVE： To observe neuronal apoptosis and changes in ICE gene expression in the frontal cortex and hippocampus following ischemia/reperfusion injury. DESIGN, TIME AND SETTING： A randomized, controlled animal study was conducted at the Laboratory of Experimental Animal Center, the Second Hospital of Jilin University and Central Laboratory, the Second Hospital of Jilin University, China, from November 2008 to September 2009. MATERIALS： The ICE gene primer sequence （TaKaPa Co., Dalian, China）, FACScan Flow cytometer （Becton Dickinson, Franklin Lakes, N J, USA）, and Perkin Elmer GeneAmp PCR system 2400 （Perkin Elmer, Waltham, MA, USA） were used in this study. METHODS： A total of 45 healthy, adult, male, Kunming mice were randomly assigned to normal control （n = 5）, sham surgery （n = 5）, and model （n = 35） groups. The mice in the model group were equally and randomly subdivided into seven subgroups according to various reperfusion time points （1 hour, 1,3, 7, 14, 28, and 42 days）. Animal models of ischemia/reperfusion injury were established by bilateral carotid artery ligation in the model group. The mice in the sham surgery group only received saline perfusion and surgery for carotid artery exposure. MAIN OUTCOME MEASURES： Neuronal apoptosis in the frontal cortex and hippocampus of mice was measured using flow cytometry. The time course of ICE mRNA levels in the frontal cortex and hippocampus were quantified using reverse transcription-polymerase chain reaction. RESULTS： Neuronal apoptosis in the frontal cortex and hippocampus peaked at 3 days following ischemia/reperfusion injury （P 〈 0.05）. ICE mRNA expression increased in the frontal cortex at 1 day following ischemia/reperfusion injury （P 〈 0.05）, decreased at 3 days, and then peaked at 14 days （P 〈 0.05）. ICE mRNA expression increased in the hippocampus at 3 days following ischemia/reperfusion injury （P 〈 0.05）, peaked at 7 days （P 〈 0.05）, and then decreased gradually to normal levels at 28 days. CONCLUSION： Neuronal apoptosis peaked at 3 days following ischemia/reperfusion injury, and both apoptosis and ICE mRNA levels remained high for 2 weeks after injury. Early apoptosis may result from increased ICE mRNA expression.

miR-21-3p alleviates neuronal apoptosis during cerebral ischemiareperfusion injury by targeting SMAD2

Cerebral ischemia is due to the formation of blood clots or embolisms in the brain arteries,which leads to local brain tissue necrosis and neural cell apoptosis.Recent studies have shown that microRNA(miRNA)plays an important regulatory role in the pathological process of ischemic injury.The aim of this study is to investigate the role and the mechanism of miR-21-3p and drosophila mothers against decapentaplegic 2(SMAD2)in cerebral ischemic reperfusion injured(CIRI)neural cells.The CIRI model was established by oxygen-glucose deprivation and recovery process for N2a cells.The cell viability and the apoptotic was evaluated by MTT assay and the Flow Cytometer,respectively.The expression of miR-21-3p and SMAD2 mRNA was detected by real-time fluorescence quantitative PCR(qRT-PCR),and the expression of SMAD2 and apoptotic-related proteins were detected by Western Blotting.Our results showed that miR-21-3p is down-regulated,and SMAD2 is up-regulated in CIRI.Overexpression of miR-21-3p inhibits the apoptosis of neural cells in CIRI.miR-21-3p targets SMAD2 and inhibits SMAD2 expression.Overexpression of SMAD2 eliminates the protective effect of over-expression of miR-21-3p on neural cells in CIRI.Token together,this study provides a theoretical basis for the mechanism of ischemic reperfusion injury in neural cells and a new molecular target for ischemic stroke therapy.

Protective effects of human umbilical cord mesenchymal stem cell vein transplantation against spinal cord ischemia/reperfusion injury in rats

BACKGROUND： The majority of studies addressing spinal cord ischemia/reperfusion injury （SCIRI） have focused on drugs, proteins, cytokines, and various surgical techniques. A recent study reports that human umbilical cord mesenchymal stem cell （hUCMSC） transplantation achieves good therapeutic effects, but the mechanisms underlying nerve protection remain poorly understood. OBJECTIVE： To observe survival of transplanted hUCMSCs in SCIRI rat models and the influence on motor function in the hind limbs, to determine interleukin-8 expression and cellular apoptosis in spinal cord tissues, and to verify the hypothesis that hUCMSC transplantation exhibits protective effects on SCIRI. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Laboratory of the Department of Orthopedics in the First Affiliated Hospital of Soochow University, China between January 2007 and December 2008. MATERIALS： hUCMSCs were harvested from umbilical cord blood of healthy pregnant women after parturition in the Obstetrical Department of the First Affiliated Hospital of Soochow University, China. Rabbit anti-human BrdU monoclonal antibody was provided by DAKO, USA. Terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） Kit and enzyme-linked immunosorbent assay （ELISA） Kit were purchased by Wuhan Boster, China. METHODS： A total of 72 healthy, Wistar, adult rats were randomly assigned to three groups： sham-surgery, model, and transplantation, with 24 rats in each group. SCIRI was induced in the model and transplantation groups via the abdominal aorta block method. The infrarenal abdominal aorta was not blocked in the sham-surgery group. Prior to abdominal aorta occlusion, 0.2 03 mL bromodeoxyuridine （BrdU）-Iabeled hUCMSCs suspension （cell concentration 5 × 10 3/uL） was injected through the great saphenous vein of the hind limb, and an equal volume of physiological saline was administered to the model and sham-surgery groups. MAIN OUTCOME MEASURES： Pathological observation of rat spinal cord tissues was performed by hematoxylin-eosin staining at 6, 24, and 48 hours post-surgery. Immunohistochemistry was applied to determine hUCMSCs survival in the spinal cord. The amount of cellular apoptosis and interleukin-8 expression in spinal cord tissues was assayed utilizing the TUNEL and ELISA methods, respectively. Motor function in the hind limbs was evaluated according to Jacob＇s score. RESULTS： Numerous BrdU-positive cells were observed in spinal cord tissues from the transplantation group. The number of apoptotic cells and interleukin-8 levels significantly decreased in the transplantation group （P 〈 0.05）, pathological injury was significantly ameliorated, and motor function scores significantly increased （P 〈 0.05） compared with the model group. CONCLUSION： Via vein transplantation, hUCMSCs were shown to reach and survive in the injury area. Results suggested that the transplanted hUCMSCs contributed to significantly improved pathological changes in the injured spinal cord, as well as motor function, following SCIRI. The protective mechanism correlated with inhibition of cellular apoptosis and reduced production of inflammatory mediators.

Pretreatment with Dingnaofang reduces vascular endothelial cell apoptosis induced by cerebral ischemia/perfusion injury

BACKGROUND： Vascular endothelial cell apoptosis participates in cerebral ischemia/reperfusion injury, and the method of Qi-supplementation and blood-activation has remarkable neuroprotective effects against cerebral ischemia/reperfusion injury. OBJECTIVE： To explore the molecular mechanism that Dingnaofang （Chinese herbs for supplementing Qi and activating blood circulation） inhibits vascular endothelial cell apoptosis induced by cerebral ischemia/reperfusion injury. DESIGN, TIME AND SETFING： A randomized controlled animal experiment was performed at the Department of Central Laboratory, Third Xiangya Hospital, Central South University, China, between October 2007 and December 2008. MATERIALS： Dingnaofang consisted of Huangqi （Milkvetch Root; Radix Astragah）, Chuanxiong （Szechwan Lovage Rhizome; Rhizoma Chuanxiong）, Yinxingye （ginkgo leaf; Fofium Ginkgo）, Dilong （earthworm; Pheretima）, Danggui （Chinese Angelica; Radix Angelicae Sinensis）, Tianqi （Radix Notoginseng）, and Gancao （Radix Glycyrrhizae; Radix Glycytthizae）, with a proportion of 5：2：2： 1： 1： 1： 1. METHODS： A total of 130 Sprague Dawley rats were randomly divided into sham-surgery （n = 10）, cerebral ischemia/reperfusion （n = 40）, cerebral ischemia pretreatment （n = 40） and Dingnaofang pretreatment groups （n = 40）. Middle cerebral artery occlusion was used to induce cerebral ischemic injury. The bilateral common carotid artery in the cerebral ischemia pretreatment group was blocked for 10 minutes on days 7, 3 and 1 prior to ischemia/reperfusion injury, while rats in Dingnaofang pretreatment group were intragastrically administrated with 4 g Dingnaofang 1 week prior to cerebral ischemia once per day, for 7 successive days. MAIN OUTCOME MEASURES： Apoptosis ratios in vascular endothelial cells were measured using Hoechst 33258 staining and flow cytometry; apoptosis was detected by monitoring DNA gradient bands and the activation of caspase-3, 8, 9 and Bid using Western blot. RESULTS： Following cerebral ischemiaJreperfusion injury, the number of apoptotic vascular endothelial cells in the middle cerebral artery significantly increased （P 〈 0.01）; however, cerebral ischemia pretreatment and Dingnaofang pretreatment groups significantly reduced apoptosis induced by cerebral ischemia/reperfusion injury （P 〈 0.01）. In particular, DNA gradient bands were not observed following Dingnaofang pretreatment. At 24 hours after cerebral ischemia/reperfusion injury, cleaved fragments of caspase-3, 8 and 9 were detected at 11 kD （P 11）, 20 kD （P 20） and 10 kD （P 10）, respectively, following Western blot. Bid was also cleaved into its truncated form （tBid; 15 kD）. Gray scale analysis indicated that P 11, P 20, P 10 and tBid band values in the Dingnaofang pretreatment group were significantly less than in the cerebral ischemia/reperfusion and cerebral ischemia pretreatment groups （P 〈 0.01 or P 〈 0.05）. CONCLUSION： Dingnaofang inhibits vascular endothelial cell apoptosis induced by cerebral ischemia/reperfusion injury via inhibition of apoptotic signal transduction pathways.

Ultra-early treatment of bone marrow-derived mesenchymal stem cells for focal cerebral ischemia/ reperfusion injury

The time point at which bone marrow-derived mesenchymal stem cells（BMSCs）can be used in transplantation for the treatment of ischemic brain injury remains unclear.In the present study,BMSCs were transplanted to the ischemic site 90 minutes post-ischemia.The results demonstrated that the transplanted BMSCs improved neurological function,reduced infarct volume,increased survivin expression,decreased caspase-3 expression and reduced apoptosis.This suggests that BMSCs transplanted at an ultra-early stage ameliorated brain ischemia by increasing survivin expression,decreasing caspase-3 expression and reducing apoptosis at the ischemia/reperfusion injury site.

Electroacupuncture reduces apoptotic index and inhibits p38 mitogen-activated protein kinase signaling pathway in the hippocampus of rats with cerebral ischemia/reperfusion injury

Electroacupuncture attenuates cerebral hypoxia and neuronal apoptosis induced by cerebral ischemia/reperfusion injury.To further identify the involved mechanisms,we assumed that electroacupuncture used to treat cerebral ischemia/reperfusion injury was associated with the p38 mitogen-activated protein kinase（MAPK） signaling pathway.We established rat models of cerebral ischemia/reperfusion injury using the modified Zea-Longa＇s method.At 30 minutes before model establishment,p38 MAPK blocker SB20358 was injected into the left lateral ventricles.At 1.5 hours after model establishment,electroacupuncture was administered at acupoints of Chize（LU5）,Hegu（LI4）,Zusanli（ST36）,and Sanyinjiao（SP6） for 20 minutes in the affected side.Results showed that the combination of EA and SB20358 injection significantly decreased neurologic impairment scores,but no significant differences were determined among different interventional groups.Hematoxylin-eosin staining also showed reduced brain tissue injuries.Compared with the SB20358 group,the cells were regularly arranged,the structures were complete,and the number of viable neurons was higher in the SB20358 ＋ electroacupuncture group.Terminal deoxynucleotidyl transferase（Td T）-mediated d UTP nick-end labeling assay showed a decreased apoptotic index in each group,with a significant decrease in the SB20358 ＋ electroacupuncture group.Immunohistochemistry revealed reduced phosphorylated p38 expression at 3 days in the electroacupuncture group and SB20358 ＋ electroacupuncture group compared with the ischemia/reperfusion group.There was no significant difference in phosphorylated p38 expression between the ischemia/reperfusion group and SB20358 group.These findings confirmed that the electroacupuncture effects on mitigating cerebral ischemia/reperfusion injury are possibly associated with the p38 MAPK signaling pathway.A time period of 3 days could promote the repair of ischemic cerebral nerves.

Hypoxic preconditioning reduces NLRP3 inflammasome expression and protects against cerebral ischemia/reperfusion injury

Hypoxic preconditioning can protect against cerebral ischemia/reperfusion injury. However, the underlying mechanisms that mediate this effect are not completely clear. In this study, mice were pretreated with continuous, intermittent hypoxic preconditioning;1 hour later, cerebral ischemia/reperfusion models were generated by middle cerebral artery occlusion and reperfusion. Compared with control mice, mice with cerebral ischemia/reperfusion injury showed increased Bederson neurological function scores, significantly increased cerebral infarction volume, obvious pathological damage to the hippocampus, significantly increased apoptosis;upregulated interleukin-1β, interleukin-6, and interleukin-8 levels in brain tissue;and increased expression levels of NOD-like receptor family pyrin domain containing 3(NLRP3), NLRP inflammasome-related protein caspase-1, and gasdermin D. However, hypoxic preconditioning significantly inhibited the above phenomena. Taken together, these data suggest that hypoxic preconditioning mitigates cerebral ischemia/reperfusion injury in mice by reducing NLRP3 inflammasome expression. This study was approved by the Medical Ethics Committee of the Fourth Hospital of Baotou, China(approval No. DWLL2019001) in November 2019.

Effects of L-Tetrahydropalmatine on Neuron Apoptosis during Acute Cerebral Ischemia-Reperfusion of Rats

To investigate the effects of L-Tetrahydropalmatine (L-THP ) on neuron apoptosis during acute cerebral ischemia-reperfusion of rats and explore the effects of heat shock protein (HSP) on neuron apoptosis, Wistar rats were randomly divided into 3 groups: normal group, ischemia- reperfusion group and treatment group. The condition of neuron apoptosis, the survival state of neuron, pathological changes under an electron microscope and the number of HSP70 positive cells were measured in all groups. Results showed that the apoptosis neuron number was increased obviously at the 24th h during reperfusion and was further increased at the 48th h, the 72th h. While the number of survival neurons was decreased gradually with the prolongation of reperfusion time. Treatment with L-THP could decrease the apoptosis neuron number but increase the survival neuron number and the HSP70 positive cell number. Our study suggested that L-THP could decrease apoptosis and necrosis of neuron, up-regulate the expression of HSP70 and protect the cerebral ischemic injury.

MicroRNA-101a-3p mimic ameliorates spinal cord ischemia/reperfusion injury

miR-101a-3p is expressed in a variety of organs and tissues and plays a regulatory role in many diseases,but its role in spinal cord ischemia/reperfusion injury remains unclear.In this study,we established a rat model of spinal cord ischemia/reperfusion injury by clamping the aortic arch for 14 minutes followed by reperfusion for 24 hours.Results showed that miR-101a-3p expression in L4-L6 spinal cord was greatly decreased,whereas MYCN expression was greatly increased.Dual-luciferase reporter assay results showed that miR-101a-3p targeted MYCN.MYCN immunoreactivity,which was primarily colocalized with neurons in L4-L6 spinal tissue,greatly increased after spinal cord ischemia/reperfusion injury.However,intrathecal injection of an miR-101a-3p mimic within 24 hours before injury decreased MYCN,p53,caspase-9 and interleukin-1βexpression,reduced p53 immunoreactivity,reduced the number of MYCN/NeuN-positive cells and the number of necrotic cells in L4-L6 spinal tissue,and increased Tarlov scores.These findings suggest that the miR-101a-3p mimic improved spinal ischemia/reperfusion injury-induced nerve cell apoptosis and inflammation by inhibiting MYCN and the p53 signaling pathway.Therefore,miR-101a-3p mimic therapy may be a potential treatment option for spinal ischemia/reperfusion injury.

The novel chalcone analog L2H17 protects retinal ganglion cells from oxidative stress-induced apoptosis

Chalcone is a plant metabolite widely found in fruits,vegetables,spices and tea,and has anti-tumor,anti-inflammation,immunomodulation,antibacterial and anti-oxidation activities,as well as many other pharmacological and biological effects.Our team has shown that its analogs have antioxidant activity,and oxidative stress is a pathological hallmark of retinal ischemia/reperfusion injury that can lead to retinal damage and visual loss.This investigation aims to identify a chalcone that protects retinal ganglion cells in vitro from the effects of oxidative stress and examine its mechanism.Rat retinal ganglion cell-5 cells were pretreated with chalcones and then exposed to tert-butyl hydroperoxide that causes oxidative damage.Controls received dimethyl sulfoxide only or tert-butyl hydroperoxide in dimethyl sulfoxide.Only（E）-3,4-dihydroxy-2′-methylether ketone（L2 H17）,of the five chalcone analogs,markedly increased the survival rate of oxidatively injured RGC-5 cells.Thus,subsequent experiments only analyzed the results of the L2 H17 intervention.Cell viability and apoptosis were measured.Intracellular superoxide dismutase and reactive oxygen species levels were used to assess induced oxidative stress.The mechanism of action by L2 H17 was explored by measuring the ER stress/UPR pathway and the expression and localization of Nrf2.All results demonstrated that L2 H17 could reduce the apoptosis of oxidatively injured cells,inhibit caspase-3 activity,increase Bcl-2 expression,decrease Bad expression,increase the activity of superoxide dismutase,inhibit the production of reactive oxygen species,increase Nrf2 immunoreactivity,and reduce the activating transcription factor 4,phospho-eukaryotic initiation factor 2 and CHOP expression.L2 H17 protects retinal ganglion cells induced by oxidative stress by regulating Nrf2,which indicates that it has the potential to become a drug for retinal ischemia/reperfusion.

Expression of nerve growth factor precursor, mature nerve growth factor and their receptors during cerebral ischemia-reperfusion injury

We investigated nerve growth factor precursor （proNGF） and mature NGF expression in ischemic and non-ischemic cortices after cerebral ischemia-reperfusion injury. In both ischemic and non-ischemic cortices, proNGF was found to be present in the extracellular space and cytoplasm. In addition, mature NGF was expressed in extracellular space, but with a very low signal. In ischemic cortex only, proNGF was significantly decreased, reaching a minimal level at 1 day. Mature NGF was increased at 4 hours, then reached a minimal level at 3 days. The p75 neurotrophin receptor （p75NTR） was significantly decreased after ischemia, and increased at 3 days after ischemia. These results confirmed that proNGF was the predominant form of NGF during the pathological process of cerebral ischemia-repeffusion injury. In addition, our findings suggest that ischemic injury may influence the conversion of proNGF to mature NGF, and that proNGF/p75NTR may be involved in reperfusion injury.

Silencing Huwe1 reduces apoptosis of cortical neurons exposed to oxygen-glucose deprivation and reperfusion

HECT, UBA and WWE domain-containing 1(Huwe1), an E3 ubiquitin ligase involved in the ubiquitin-proteasome system, is widely expressed in brain tissue. Huwe1 is involved in the turnover of numerous substrates, including p53, Mcl-1, Cdc6 and N-myc, thereby playing a critical role in apoptosis and neurogenesis. However, the role of Huwe1 in brain ischemia and reperfusion injury remains unclear. Therefore, in this study, we investigated the role of Huwe1 in an in vitro model of ischemia and reperfusion injury. At 3 days in vitro, primary cortical neurons were transduced with a control or shRNA-Huwe1 lentiviral vector to silence expression of Huwe1. At 7 days in vitro, the cells were exposed to oxygen-glucose deprivation for 3 hours and reperfusion for 24 hours. To examine the role of the c-Jun N-terminal kinase(JNK)/p38 pathway, cortical neurons were pretreated with a JNK inhibitor(SP600125) or a p38 MAPK inhibitor(SB203508) for 30 minutes at 7 days in vitro, followed by ischemia and reperfusion. Neuronal apoptosis was assessed by TUNEL assay. Protein expression levels of JNK and p38 MAPK and of apoptosis-related proteins(p53, Gadd45 a, cleaved caspase-3, Bax and Bcl-2) were measured by western blot assay. Immunofluorescence labeling for cleaved caspase-3 was performed. We observed a significant increase in neuronal apoptosis and Huwe1 expression after ischemia and reperfusion. Treatment with the shRNA-Huwe1 lentiviral vector markedly decreased Huwe1 levels, and significantly decreased the number of TUNEL-positive cells after ischemia and reperfusion. The silencing vector also downregulated the pro-apoptotic proteins Bax and cleaved caspase-3, and upregulated the anti-apoptotic proteins Gadd45 a and Bcl-2. Silencing Huwe1 also significantly reduced p-JNK levels and increased p-p38 levels. Our findings show that downregulating Huwe1 affects the JNK and p38 MAPK signaling pathways as well as the expression of apoptosis-related genes to provide neuroprotection during ischemia and reperfusion. All animal experiments and procedures were approved by the Animal Ethics Committee of Sichuan University, China in January 2018(approval No. 2018013).

Changes in neuronal apoptosis and apoptosis modulatory factors in cerebral ischemia/reperfusion

BACKGROUND： The high concentration of glutamate release is the main cause for neuronal cell death. The relationship between glutamate level and apoptosis during ischemia/reperfusion injury is still unclear. OBJECTIVE： To observe the neuronal apoptosis at 24 and 72 hours following cerebral ischemia/reperfusion in rats, and analyze the possible influencing factors. DESIGN： A randomized controlled animal experiment. SETTING： School of Medicine, Southern Yangtze University. MATERIALS： Totally 30 male adult Sprague Dawley （SD） rats of clean grade, weighing 240 - 290 g, were obtained from Shanghai Experimental Animal Center, Chinese Academy of Sciences. The rats were randomly divided into sham-operated group （n=10） and model group （n=20）. Each group was observed at 24 and 72 hours after ischemia/reperfusion, 5 rats at each time point in the sham-operated group, whereas 12 at 24 hours and 8 at 72 hours in the model group. Kits for determining apoptosis and Bcl-2 were bought from Wuhan Boster Biological Technology, Co., Ltd.; Kit for calcineurin from Nanjing Jiancheng Bioengineering Institute. METHODS： The experiment was carried out in the Functional Scientific Research Room of Southern Yangtze University from June to October in 2006.①Right middle cerebral artery was occluded by inserting a thread through internal carotid artery （ICA）. The surgical process for the sham-operated rats was the same as that in the model group except a nylon suture inserted the ICA. According to Longa five-degree standard, the neurological deficit evaluation of rats was evaluated after surgery, and grades 1 - 3 were taken as successful model establishment. The blood was recirculated by withdrawing the nylon filament under anesthesia at 2 hours after ischemia in successful rat models. ②After reperfusion, the brain tissue was quickly removed at 24 or 72 hours and the slices were obtained from optic chiasma to funnel manubrium. The changes of the number of apoptotic cells were observed using the terminal deoxynucleotidyl transferase mediated dUTP-biotin nick-end labeling method. The expressions of Bcl-2 protein were determined with immunohistochemical staining. The activity of calcineurin was determined by the inorganic phosphorus method. The content of excitatory amino acid was detected by high performance liquid chromatography. MAIN OUTCOME MEASURES： ①Glutanate content in brain tissue; ② Conditions of apoptosis; ③Calcineurin activity in brain tissue; ④ Bcl-2 expression in brain tissue. RESULTS： Totally 30 SD rats were used, 5 died and the other 25 were involved in the analysis of results. ① Changes of apoptosis： There were 0 - 3 apoptotic cells in the sham-operated group. In the model group, the numbers of apoptotic cells were obviously increased at 24 and 72 hours of reperfusion （P 〈 0.01 ）, and it was markedly reduced at 72 hours as compared with 24 hours （P 〈 0.0l）. ② Changes of glutanate content： The glutamate contents at 24 and 72 hours of repeffusion in the model group were obviously higher than those in the sham-operated group （P 〈 0.01）; In the model group, it was obviously increased at 24 hours as compared with 72 hours （P 〈0.01）. ③ Changes of Bcl-2 protein： In the model group, the Bcl-2 protein expression had no obvious changes at 24 hours of reperfusion, and it was obviously enhanced at 72 hours, which was obviously different from that in the sham-operated group and that at 24 hours （P 〈 0.0l）. ④Changes of calcinerin activity： In the model group, the activity of calcineurin in brain tissue had no obvious changes at 24 hours of reperfusion; The activity of calcineurin at 72 hours was obviously higher than that in the sham-operated group and that at 24 hours （P 〈 0.01）. CONCLUSION： The brain cyto-apoptosis action at different time points following reperfusion incompletely depends on the glutamate levels, while it depends on the interaction of some apoptosis related factors, such as amino acid, calcineurin, and Bcl-2, etc.

Gender difference in the neuroprotective effect of rat bone marrow mesenchymal cells against hypoxiainduced apoptosis of retinal ganglion cells

Bone marrow mesenchymal stem cells can reduce retinal ganglion cell death and effectively prevent vision loss. Previously, we found that during differentiation, female rhesus monkey bone marrow mesenchymal stem cells acquire a higher neurogenic potential compared with male rhesus monkey bone marrow mesenchymal stem cells. This suggests that female bone marrow mesenchymal stem cells have a stronger neuroprotective effect than male bone marrow mesenchymal stem cells. Here, we first isolated and cultured bone marrow mesenchymal stem cells from female and male rats by density gradient centrifugation. Retinal tissue from newborn rats was prepared by enzymatic digestion to obtain primary retinal ganglion cells. Using the transwell system, retinal ganglion cells were co-cultured with bone marrow mesenchymal stem cells under hypoxia. Cell apoptosis was detected by flow cytometry and caspase-3 activity assay. We found a marked increase in apoptotic rate and caspase-3 activity of retinal ganglion cells after 24 hours of hypoxia compared with normoxia. Moreover, apoptotic rate and caspase-3 activity of retinal ganglion cells significantly decreased with both female and male bone marrow mesenchymal stem cell co-culture under hypoxia compared with culture alone, with more significant effects from female bone marrow mesenchymal stem cells. Our results indicate that bone marrow mesenchymal stem cells exert a neuroprotective effect against hypoxia-induced apoptosis of retinal ganglion cells, and also that female cells have greater neuroprotective ability compared with male cells.

Does closure of acid-sensing ion channels reduce ischemia/reperfusion injury in the rat brain?

Acidosis is a common characteristic of brain damage. Because studies have shown that permeable Ca2＋-acid-sensing ion channels can mediate the toxic effects of calcium ions, they have become new targets against pain and various intracranial diseases. However, the mechanism associated with expression of these channels remains unclear. This study sought to observe the expression characteristics of permeable Ca2＋-acid-sensing ion channels during different reperfusion inflows in rats after cerebral ischemia. The rat models were randomly divided into three groups： adaptive ischemia/reperfusion group, one-time ischemia/reperfusion group, and severe cerebral ischemic injury group. Western blot assays and immunofluorescence staining results exhibited that when compared with the one-time ischemia/reperfusion group, acid-sensing ion channel 3 and Bcl-x/I expression decreased in the adaptive ischemia/reperfusion group. Calmodulin expression was lowest in the adaptive ischemia/reperfusion group. Following adaptive reperfusion, common carotid artery flow was close to normal, and the pH value improved. Results verified that adaptive reperfusion following cerebral ischemia can suppress acid-sensing ion channel 3 expression, significantly reduce Ca2＋ influx, inhibit calcium overload, and diminish Ca2＋ toxicity. The effects of adaptive ischemia/reperfusion on suppressing cell apoptosis and relieving brain damage were better than that of one-time ischemia/reperfusion.

达格列净预处理对糖尿病大鼠心肌缺血再灌注损伤的影响

目的:研究达格列净预处理对糖尿病大鼠心肌缺血—再灌注(I-R)损伤的影响,并对其作用机制进行探讨。方法:选择糖尿病大鼠作为样本,对模型进行构建,本次实验共使用雄性SD大鼠24只,把其平均分为三组(n=8),第1组为假手术组,第2组为达格列净组,第3组为I-R组。假手术组样本在给予其开胸处理之后不进行结扎操作,无其他药物处理。达格列净组在结扎左前降支(LAD)前连续口服达格列净,给药30d(1次/d,1mg/kg),第31天结扎LAD 30min,再灌注2h。I-R组结扎LAD 30min,再灌注2h,无其他药物处理。连续监测肢体Ⅱ导联心电图,观察心率及ST段改变,再灌注2h末抽血查血清磷酸肌酸激酶同工酶(CK-MB)与乳酸脱氢酶(LDH),用伊文斯蓝(Evans blue)及三苯氯化四氮唑(TTC)双重染色方法测定心肌梗死及缺血面积,采用TUNEL法测心肌细胞凋亡指数(AI)。结果:与I-R组相比,达格列净组可减少CK-MB与LDH的释放(P<0.05),减少心梗面积与细胞AI(P<0.05);与假手术组相比,达格列净组与I-R组CK-MB与LDH的释放明显增加(P<0.05),心梗面积与细胞AI也明显增加(P<0.05)。结论:达格列净预处理可减少心梗面积与细胞AI,减少CK-MB与LDH的释放,起到对I-R心肌的保护作用。

香叶醇通过调控Nrf2/HO-1途径调节氧化应激减轻大鼠脑缺血/再灌注损伤

目的研究香叶醇对脑缺血/再灌注(cerebral ischemia reperfusion injury,CIRI)后Nrf2/HO-1信号通路的调控作用。方法随机将雄性SD大鼠分成假手术组(sham)、假手术+200 mg·kg^(-1)香叶醇组、缺血/再灌注(I/R)组、I/R+50 mg·kg^(-1)香叶醇组、I/R+100 mg·kg^(-1)香叶醇组、I/R+200 mg·kg^(-1)香叶醇组、依达拉奉组,I/R+brusatol(Nrf2抑制剂)组、I/R+200 mg·kg^(-1)香叶醇+brusatol组。大鼠在MCAO术前5 d连续腹腔注射香叶醇,术后再次腹腔注射香叶醇。假手术组和假手术+200 mg·kg^(-1)香叶醇组只分离血管不插栓线,其余组别使用线栓法建立大鼠CIRI损伤模型。通过改良神经功能评分表评定大鼠神经功能受损情况;TTC脑片染色测定大鼠脑梗死体积;HE染色观察大鼠缺血侧皮层受损情况;TUNEL凋亡染色测定大鼠皮层细胞凋亡情况;测定氧化应激相关参数;免疫组织化学染色法检测Nrf2和HO-1蛋白的表达;Western blot检测受损侧皮层组织中目的蛋白表达水平。结果与模型组相比,香叶醇给药组能明显改善大鼠神经损伤症状、减少脑梗死体积和改善大鼠脑受损皮层的损伤情况;同时减少细胞凋亡的发生;并且香叶醇干预组能明显增加大鼠右侧皮质Nrf2/HO-1蛋白的表达,降低氧化应激水平。结论香叶醇通过激活Nrf2/HO-1信号途径,改善缺血/再灌注所致的大鼠缺血性脑损伤。