Resveratrol modulates SDF-1/CXCR4 signaling to inhibit hypoxia-ischemia-induced apoptosis of neurons from the brain of newborn rats

AIM:To investigate the effect of resveratrol (RSV) on apoptosis and stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) pathway in hypoxia-ischemia-induced neurons from the brain of newborn rats and its mechanism. METHODS:The cortex neurons from the brain of newborn rats were given oxygen-glucose deprivation (OGD) treatment to mimic neonatal hypoxic-ischemic encephalopathy (HIE). The cortex neurons were randomly divided into 4 groups:control group, HIE model group, HIE+RSV-low (10 μmol/L) group, and HIE+RSV-high (50 μmol/L) group. After OGD treatment for 2 h, the neurons were cultured with indicated dose of RSV for 24 h. The apoptosis was analyzed by flow cytometry. Western blot was used to determine the levels of apoptosis-related proteins, SDF-1 and CXCR4. Real-time PCR was used to detect the mRNA expression of SDF-1 and CXCR4. Additionally, to explore the effects of RSV on cell apoptosis and apoptosis-related proteins after the suppression of SDF-1/CXCR4 signaling, a CXCR4 antagonist AMD3100, and RSV were used to co-treat OGD-injured neurons for 24 h. RESULTS:RSV alleviated OGD-induced neuronal apoptosis, down-regulated cleaved caspase-3 and cytochrome C levels, and up-regulated the ratio of Bcl-2/Bax. Compared with the control group, OGD treatment increased the expression of SDF-1 and CXCR4 (P<0.05). Compared with the HIE model group, RSV further up-regulated the expression of SDF-1 and CXCR4 (P<0.05). AMD3100 reversed the effects of RSV on OGD-induced cell apoptosis. CONCLUSION:RSV suppresses hypoxia-ischemia-induced apoptosis of neurons from the brain of newborn rats via up-regulating SDF-1/CXCR4 signaling pathway.     

Protective effect and functional mechanism of curcumin on TNF-α induced neuronal damage in rat hippocampus

AIM: To observe the protective effect of curcumin on TNF-α induced neuronal damage in rat hippocampus and to explore the functional mechanism of curcumin. METHODS: The excitatory postsynaptic potential (EPSP) was recorded in CA1 pyramidal layer of rat hippocampal slices with in vitro brain slices recording techniques. High frequency stimulation was given on Schaffer branches to induce long-term potentiation (LTP). After treated with drugs, the initial slope of EPSP in each group was measured and calculated. RESULTS: Compared to control group, TNF-α and N-methyl-D-aspartate(NMDA) obviously inhibited the LTP in hippocampal slices of rat brain (P<0.05). Curcumin partly recovered the LTP, which was inhibited by TNF-α or NMDA, to near the control level (P>0.05). No effect of TNF-α, NMDA or curcumin on basal synaptic transmission in hippocampal slices was observed. CONCLUSION: Curcumin has protective effect on hippocampal neurons of rats. Curcumin can partly prevent the over-activation of NMDA receptor on neuronic membrane induced by TNF-α and maintain the long-term potentiation in neurons.     

Effect of Buyanghuanwu decoction on the expression of ERK2 and CaMKⅡβ in hippocampus tissue and on ability of learning and memory in vascular dementia rats

AIM: To investigate the effect of Buyanghuanwu decoction, a Chinese medicine, on the ability of learning and memory in the rats with vascular dementia (VD) and on the protein expression of extracellular signal-regulated kinase 2(ERK2) and calcium/calmodulin-dependent protein kinase Ⅱβ(CaMKⅡβ) in hippocampus CA1 area.METHODS: The rats were divided into 4 groups: sham group, VD group, VD+Buyanghuanwu decoction group and VD+nimodipine group. The VD rat model was prepared by Pulsinelli's four-vessel occlusion. At 7th day, 14th day or 28th day after operation, the behaviors of the rats were tested by Morris water maze. The morphological changes of the neurons in hippocampus CA1 area were observed by HE staining 30 d after operation. Western blotting was used to observe the protein expression of ERK2 and CaMKⅡβ in the brain tissues of hippocampal CA1 area of the VD rats. RESULTS: Compared with sham group, the pathological changes such as irregular arrangement, coagulation necrosis and obvious deletion in the neurons of hippocampus CA1 area in VD group appeared significantly. The obstacle of learning and memory ability was observed and the protein expression of ERK2 and CaMKⅡβ in hippocampal CA1 area was significantly decreased (P<0.05). Compared with VD group, the neurons in hippocampal CA1 area of VD+Buyanghuanwu decoction group and VD+nimodipine group were in eumorphism, lined up in order, and the structure was close to that in sham group. The ability of learning and memory also significantly improved (P<0.05). The protein expression of ERK2 and CaMKⅡβ in hippocampal CA1 area significantly increased (P<0.05). CONCLUSION: Buyanghuanwu decoction promotes the protein expression of ERK2 and CaMKⅡβ in hippocampus CA1 area to protect the neurons from injury, builds up the synapses and promotes the ability of learning and memory in VD rats.     

PPARγ agonist pioglitazone attenuates cortical neuron lesion and gliosis in rat brain of post-traumatic injury

AIM: To investigate the neuroprotective effect of pioglitazone (Pio), a potent agonist of peroxisome proliferator-activated receptor gamma (PPARγ), on the traumatic brain injury (TBI) in rats. METHODS: SD rats were randomly divided into 4 groups: sham group, vehicle+TBI group, Pio+TBI group and Pio+T0070907+TBI group. TBI was induced by the method of controlled cortical impact (CCI) injury. Neutral red staining technique was used to determine the cortical lesion volume. NeuN, GFAP and OX-42 were measured by immunohistochemical technique to evaluate the morphology of neurons, activation and infiltration of astrocytes and microglia at the edge of cortical lesion. RESULTS: CCI injury in rat elicited activation and proliferation of the astrocytes and microglia. The glial scar wall formation at the edge of cortical lesion, which was accompanied by the loss of neurons, was observed. Pio significantly reduced the cortical lesion volume, the activation and infiltration of the astrocytes and microglia, and the loss of pyramidal neurons at the edge of cortical lesion. T0070907, an antagonist of PPARγ, reversed the effects of Pio. CONCLUSION: Pioglitazone exerts a neuroprotective efficacy, attenuates the loss of neurons and cortical lesion volume following CCI injury by inhibiting the activation and infiltration of astrocytes and microglia, especially glial scar formation.     

Mild hypothermia protects against injury of rat hippocampal neurons induced by oxygen-glucose deprivation

AIM：To study the protective effect of mild hypothermia (31~32 °C) on rat hippocampal neurons against oxygen-glucose deprivation (OGD)-induced injury and its possible mechanisms. METHODS：An OGD experimental model of rat hippocampal neurons in vitro was established to simulate cerebral ischemic-hypoxic injury. The rat hippocampal neurons were randomly divided into 4 groups：control group, mild hypothermia group, OGD group and mild hypothermia+OGD group. The cell morphology was observed under light and electron microscopes. The neuronal apoptosis was detected by flow cytometry. The activity of caspase-3 in the cytoplasm was measured by colorimetry. RESULTS：The neuronal injury was apparent after OGD, with a great increase in apoptotic rate (P<0.01). Compared with OGD group, the morphology of neuronal injury in mild hypothermia+OGD group was attenuated, and the neuronal apoptotic rate and the activity of caspase-3 in the cytoplasm decreased. The activity of caspase-3 in the cytoplasm increased after OGD, and was positively correlated with the neuronal apoptotic rate (r=0.823, P<0.05). The activity of caspase-3 in the cytoplasm also increased after mild hypothermia and OGD, and was also positively correlated with the neuronal apoptotic rate (r=0.841, P<0.05). CONCLUSION：OGD can increase caspase-3 activity in the neuronal cytoplasm and induce neuronal apoptosis. Restraint on caspase-3 activity in the neuronal cytoplasm may be the mechanism by which mild hypothermia protects against neuronal injury induced by OGD.     

Phenytoin inhibited changes in nitric oxide of rat hippocampus induced by stress

AIM: To investigate the changes in nNOS and iNOS expression of hippocampal CA3 pyramidal neurons and NO₂^-/NO₃^- level of hippocampal homogenate of rats induced by stress, and to explore the effect of phenytoin on them. METHODS: Rats were subjected to forced-swimming stress, phenytoin was administered(ip) at 30 min before stress. Using the immunohistochemistry and the computerized image technique, the expression levels of nNOS and iNOS of rat hippocampal CA3 pyramidal neurons were assayed quantitatively, and the NO₂^-/NO₃^- level of hippocampal homogenate was also measured using nitric acid deoxidize enzyme method. RESULTS: The nNOS average grey degree of hippocampal CA3 pyramidal neurons was significantly lower in stress group (155.42±3.77)than that in control group(164.54±4.62)and in stress plus phenytoin group(164.27±2.55)(P<0.01); The iNOS relative sectional area proportion of hippocampal CA3 pyramidal neuron was significantly larger in stress group(5.87%±2.90%) than that in control group (0.90％±0.89%) and in strers plus phenytoin groups (0.90%±0.88%)(P<0.01); The NO₂^-/NO₃^- level of hippocampal homogenate was significantly higher in stress group(42.75 umol/L±14.49 umol/L)than that in control group(21.23 umol/L±6.99 umol/L)and in stress plus phenytoin group(18.40 umol/L±8.11 umol/L)(P<0.01). CONCLUSION: It is suggested that the stress could induce nNOS and iNOS expression in CA3 pyramidal neurons and excessive production of NO in hippocampus of rats, which could be inhibited by phenytoin.     

Effects of heroin at different concentrations on electrophysiological properties of rat hippocampal CA1 pyramidal neurons

AIM: To investigate changes of electrophysiological properties of rat hippocampal CA1 pyramidal neurons in perfusion with heroin at different concentrations. METHODS: The intracellular recordings were made from CA1 pyramidal neurons of isolated hippocampal slices in perfusion with 0.03-0.3 mmol·L-1 heroin. Compared with control parametes before herion perfusion, the electrophysiological data obtained from identical neurons at 15 min after acute perfusion of the different concentration heroin were analyzed. RESULTS: With the increase of the heroin concentrations in perfusion, the absolute values of neuron resting potential and threshold potential decreased (P<0.05). In 0.3 mmol·L-1 heroin perfusion group，the amplitude of action potential decreased （P<0.05）, 10%-90% risetime and 10%-90% decaytime of spike potential elongated （P<0.05， P<0.01）, and I-V relation curve indicated membran slope resistance increased in 0.1-0.3 mmol·L^-1 perfusion group （P<0.05，P<0.01）. I-F relation curve showed that neuron average firing frequency recored at 15 min after 0.03 mmol·L^-1 heroin infusion was similar to the control frequency when intracellular stimulus intensity was 0.1-1.8 nA, however, firing frequency kept the higher level （P<0.05，P<0.01） in 1.9-2.0 nA vs the control frequence. It was evident that EPSPs of pyramidal neurons were inhibited in perfusion of three concentration heroin, especially in 0.3 mmol·L^-1. CONCLUSION: These results indicate acute heroin perfusion has a significantly influence on electrical activities of hippocampus CA1 pyramidal neurons.     

Protective effects of auricularia auricular polysaccharide on chronic cerebral ischemia injury in rats

AIM: To investigate the effects of auricularia auricular polysaccharide (AAP) on chronic cerebral ischemia injury in rats. METHODS: The chronic cerebral ischemia mode1 was made by permanent middle cerebral artery occlusion (MCAO) on the right side. AAP at different doses (50 mg/kg and 100 mg/kg) was intragastrically administered at the onset of ischemia and in the following days after operation, once a day for 4 weeks. After 4 weeks of MCAO, Morris water maze test was introduced to examine the learning and memory functions. Nissl staining was performed to detect the survival neurons in hippocampal slices. Level of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in brain tissue were measured. RESULTS: Rats treated with AAP showed a shorter escaping latency in spacial navigation test because the AAP treated rats spent less time to find the platform in spatial probe test. More survival neurons in hippocampal slices were observed from AAP treated rats. Also, the MDA level in brain tissue was reduced and SOD activity in brain tissue was increased in the AAP treated rats with MCAO. CONCLUSION: AAP protects rats from chronic brain ischemic injury, in which its anti-oxidative effect might be involved.     

A rat model of repeated seizures induced by pentylenetetrazol at different maturational stages and the role of NF-κB in the pathogenesis of epilepsy

AIM: To observe histopathologic changes and NF-κB expression in hippocampus in neonatal and matural rats after repeated seizures, and to explore the role of NF-κB in the pathogenesis of epilepsy in premature brain of rats. METHODS: Neonatal rats and mature rats were divided into 2 experimental groups at 10 days and 60 days after birth (P10 and P60). Convulsions were induced by repeated injection of pentylenetetrazol (PTZ) intraperitoneally for first 5 days. The animals in control group were injected with NS at the same volume in the same conditions. The neurons in CA1, CA3, dentate granule (DG), as well as in hilar were counted by thionin staining, in order to observe the profile of the necrosis and apoptosis. NF-κB expression was examined by immunohistochemistry assay. Timm's method of silver sulfide staining was adopted to observe the mossy fiber sprouting. RESULTS: (1) In immature rats (10 days old), neurons in CA1, CA3 and hilar demonstrated no differences from controls, whereas adult rats (P60) had a significant decrease in number of neurons in CA1 and CA3 (8.22±1.88, 5.62±1.68 vs 6.31±1.50, 3.62±1.40). In adult rats, neurons in dentate granule showed no differences with controls, whereas immature rats with daily seizures had a significant increase (23.25±3.06 vs 16.25±1.58). (2) There was prominent sprouting in the CA3 stratum pyramidal layer in all experimental rats after 5 daily seizures, regardless of the age. However, the degree of sprouting was significantly different between the two experimental groups (3.25±1.03 vs 1.50±0.92, P<0.05). (3) NF-κB was highly expressed in CA3, CA1 and DG after 24 hours by PTZ-kindling, whereas it was little expressed in control group. NF-κB expression was higher in P10 experimental rats than that in P60 rats. CONCLUSION: No cell loss was observed in hippocampus in neonatal rats after recurrent kindling. The high expression of NF-κB may be one of the important molecular mechanisms underlying the special resistance of the neurons in premature brain to the epileptic cerebral lesions.     

Adenosine A2A receptor antagonist SCH58261 attenuates hypoxic-ischemic brain damage in a fetal rabbit model

AIM: To study the effect of adenosine A_2A receptor antagonist SCH58261 on hypoxic-ischemic brain damage (HIBD) in a mature fetal rabbit model.METHODS: Pregnant New Zealand white rabbits at gestational day 29 were selected and were randomly divided into sham-operated group, hypoxic-ischemic group, SCH58261 0.04 mg/kg group, SCH58261 0.12 mg/kg group and DMSO group. The intrauterine rabbit HIBD model was established. All pregnant rabbits were subjected to cesarean section 24 h after the sham operation or experimental procedure to induce hypoxic-ischemic injury in the fetus. The survival neonatal rabbits were kept in a neonatal incubator at 35℃. The general conditions of the newborn rabbits were recorded. The degree of neurobehavioral damage in the newborn rabbits was estimated by a neurobehavioral scoring protocol. The concentration of SCH58261 in the serum of pregnant rabbits, the serum of neonatal rabbits and the brain tissues of neonatal rabbits was measured by mass spectrometry. The mRNA expression of Bcl-2/Bax and protein levels of p-P38 mitogen-activated protein kinase (MAPK) in the cortex, hippocampus and striatum area in the brain of the neonatal rabbits were determined by real-time PCR and Western blot. RESULTS: SCH58261 was detected in the serum and brain tissues of the newborn rabbits. The SCH58261 concentration was approximately 40 μg/L in the brain tissue of the newborn rabbits. The mRNA expression of Bcl-2 in the cortex, hippocampus and striatum of brain tissues in SCH58261 0.04 mg/kg group and SCH58261 0.12 mg/kg group was higher, and the mRNA expression of Bax was lower than those in HI group (P<0.05). The protein level of p-P38 MAPK in the cortex, hippocampus and striatum of brain tissues was reduced in SCH58261 0.04 mg/kg group and SCH58261 0.12 mg/kg group compared with HI group (P<0.05). The protein level of p-P38 MAPK in SCH58261 0.12 mg/kg group was a little lower than that in SCH58261 0.04 mg/kg group (P<0.05). CONCLUSION: Adenosine A_2A receptor antagonist SCH58261 attenuates hypoxia-ischemia induced neonatal brain injury by blocking adenosine A_2A receptor, subsequently inhibiting p-P38 MAPK phosphorylation to reduce neuronal apoptosis.     

Expression of p-p38 MAPK in partial cerebral tissues after hypoxic-ischemic brain damage in neonatal A2AR knockout mice

AIM： To study the expression of p-p38 MAPK in partial cerebral tissues after hypoxic-ischemic brain damage (HIBD) in the neonatal adenosine A2A receptor knockout (A2AR-/-) mice. METHODS：Base on the modified Rice method, the model of HIBD was established. The total 64 C57/BL6 neonatal mice (7 days old) of A2AR-/-(KO) and corresponding wild type (A2AR+/+, WT) were randomized into sham-operated group and model group. The mice in model group were divided into 3 subgroups： 1 d after HIBD, 3 d after HIBD and 7 d after HIBD (n=8 for each group). The cortex and hippocampal CA1 region were used as the study areas. The neuronal apoptosis was detected using TUNEL assay combined with Nissl staining. The expression of p-p38 MAPK and activated caspase-3 was determined by the method of immunohistochemistry. The KO mice and WT mice were also taken from sham-operated group (SKO and SWT, n=10) and model group (MKO and MWT, n=30) 1 d after HIBD to assess the early neurological behavior. RESULTS：The apoptotic neurons, activated caspase-3 and p-p38 MAPK increased after HIBD and peaked at 1 d after HIBD in the cortex and the hippocampal CA1 region. The apoptotic neurons and the expression of activated caspase-3 in KO mice were significantly higher than those in WT mice at the same time point after HIBD. The expression level of p-p38 MAPK in KO mice were significantly higher than that in WT mice at 1 d and 3 d after HIBD. The expression of activated caspase-3 was positively correlated with the expression of p-p38 MAPK in neonatal mice after HIBD (in the cortex：r=0.957, P<0.01; in the hippocampal CA1 region： r=0.939, P<0.01). CONCLUSION：p-p38 MAPK might be involved in the aggravated neuron apoptosis and brain damage induced by A2AR knockout after neonatal HIBO.     

Grafting neural stem cells improve the impaired cognitive deficits and spatial recognition after ischemic-hypoxic brain damage in neonatal rats

AIM: To investigate whether grafting neural stem cells (NSCs) improves the impaired cognitive deficits and spatial recognition after ischemic-hypoxic brain damage (HIBD) in neonatal rats. METHODS: Non-immunosuppressed 7-day-old SD rats were used as research subject and randomly divided into 3 groups: (1) sham group (n=10); (2) HIBD group (n=11); (3) transplant group (n=13). (2) and (3) were anesthetized and subjected to a hypoxic/ischemic injury obtained by combination of left carotid ligation and exposure to 8% oxygen for 2 h. At 3 days post injury, hypoxic-ischemic brain damaged animals were re-anesthetized and randomized to receive stereotactic injection of NSCs prelabeling with BrdU or control media into the hippocampus in the ipsilateral hemisphere. Cognitive (i.e., learning) deficits were assessed at 2 to 4 weeks after transplantation. At the end of the behavioral tests, the animals were killed and evaluated for NSC survival and histopathological analysis. RESULTS: Transplant group showed significantly improved cognitive function in selected tests as compared with HIBD group during the 4-week observation period. They took less time than HIBD group in finding the 3 arms baited with water and had a decreased number of working and reference memory errors in radial maze acquisition tests. Histological analysis showed that transplanted NSCs attenuated CA1 cell loss after HIBD, and NSCs survived for as long as 4 weeks after transplantation and were detected in the hippocampus. CONCLUSION: These data suggest that transplanted NSCs attenuate brain damage and cognitive dysfunction after hypoxic-ischemic brain damage. This approach warrants continued investigation in light of potential therapeutic uses.     

《果品品质研究》

AIM: To investigate the effects of diazoxide, an ATP-sensitive K⁺ channel opener on the μ-calpain activation, c-Fos and c-Jun expression in neonatal hypoxic-ischemic rat brain. METHODS: The animal model of hypoxic-ischemic brain injury (HIBI) was made in the 7-day-old SD rats. Diazoxide was injected into the left lateral ventricle prior or post hypoxic-ischemia (HI) insults. Western blot was applied to detect the integrated density (ID) of the nuclear c-Fos and c-Jun at 4h, and the cleavage of cytosolic μ-calpain at 24 h after HI insults. RESULTS: Low c-Fos and c-Jun expressions from cortical and hippocampal samples were observed in the two diazoxide groups, and significant differences in their expressions were found by comparison with the HI controls (P<0.05 respectively). Furthermore, the administration of diazoxide prior or post HI insults inhibited the cleavage of μ-calpain. CONCLUSION: Diazoxide down-regulated the expression of c-Fos and c-Jun, and inhibited the activation of μ-calpain may contribute to the neuroprotection from as well as therapeutical effects on HIBI.     

Neuronal apoptosis and the chance of apoptosis-related proteins in ovariectomied rats with the effect of App17-mer peptide

AIM: To observe the expression of apoptosis-related proteins in hippocampal neurons of ovariectomized (OVX) rats and explore the neuroprotective mechanism of the App17-mer peptide. METHODS: Female Wistar rats were randomly divided into three groups. Bilaterally ovariectomized rats with injection of App 17P peptide (3.5 μg in 0.1 mL/per rat, three times a week) formed the experimental group (17P+ OVX group). Anti-AIF, Bcl-2 and Bax antibodies were applied in the immunohistochemistry experiment. TUNEL was employed to detect apoptosis. RESULTS: The number of apoptotic neurons was clearly higher in hippocampal and cortex in OVX group than that in OVX+17P group. Immunohistochemistry demonstrated the increased expression of AIF, Bax in hippocampal neurons of OVX group. OVX group showed a significantly reduced expression of Bcl-2 in hippocampal neurons. Hippocampal tissue from OVX group showed the increased expression of AIF,Bax ,and showed diminished expression of Bcl-2 , treating with App17-mer peptide normalized the expression of these proteins. CONCLUSIONS: The expression of apoptosis-related proteins were abnormal in the OVX rats. App17-mer peptide normalized these changes ; Estrogen deficiency induced neuronal apoptosis. App17-mer peptide diminished apoptosis.     

Changes of dendritic morphology and spine density in hippocampal CA1 pyramidal cells of chronic cerebral ischemic rats

AIM: To investigate the changes of dendritic morphology and spine density in hippocampal CA1 pyramidal cells of the chronic cerebral ischemic rats. METHODS: The model of chronic cerebral ischemia was established by permanent occlusion of the bilateral common carotid arteries (2VO) in rats. Two weeks, 4 weeks or 8 weeks later, the behavior of the rats in each group was evaluated through the Morris water maze to select the successful modeling, and the brains were collected for processing Golgi staining. The changes in dendritic branch and length, and spine density in hippocampal CA1 pyramidal cells were observed under optical microscope. RESULTS: Compared with sham-operated group, dendritic branch and length in model group was significantly reduced in 4-week group and 8-week group (P<0.01), and spine density in model group were significantly reduced in 2-week, 4-week and 8-week groups (P<0.01). With prolonged ischemia, dendritic branch and length, and spine density in model group were all significantly reduced (P<0.05). CONCLUSION: Chronic cerebral ischemia leads to traumatic changes in dendrites and spines in hippocampal CA1 pyramidal cells, which constitutes the pathophysiological basis in the progressive cognitive dysfunction.     

Protective effect of ethyl pyruvate on brain tissues in neonatal rats with hypoxic-ischemic brain damage

AIM：To study the effects of ethyl pyruvate (EP) on brain tissues in neonatal rats with hypoxic-ischemic brain damage (HIBD) and its underlying mechanisms. METHODS：A total of 165 seven-day-old Sprague-Dawley (SD) rats were randomly divided into 3 groups：sham operation group (n=43), HIBD group (n=61) and HIBD+EP group (n=61). The rats in HIBD+EP group were intraperitoneally injected with EP (50 mg/kg) 30 min before operation, and once a day after surgery. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in brain homogenate, water content of brain and apoptotic cells in cortex were detected 3 days later. Ischemic and non-ischemic brain tissues were weighed to assess the extent of brain atrophy 14 days later. RESULTS：Higher level of SOD ［(125.78±18.35)×103 U/(g protein)］ and lower level of MDA ［(4.42±1.04) μmol/(g protein)］ in HIBD+EP group than that in HIBD group ［(97.84±15.50)×103 U/(g protein) and (6.02±0.89) μmol/(g protein), respectively］ was observed (P<0.05)．In addition, the water content of ischemic hemisphere was significantly higher than that of non-ischemic one in HIBD group (P<0.05), and was indistinguishable from that of non-ischemic one after EP treatment (P>0.05), indicating the protective effect of EP against brain edema. The apoptotic cells in cortex and hippocampus in HIBD+EP group ［(96.63±10.08)/field and (41.91±9.96)/field, respectively］ were obviously decreased compared with HIBD group ［(111.54±1.64)/field and (51.73±1.77)/field, respectively］, but still higher than those in sham operation group (P<0.05). The atrophy ratio of ischemic hemisphere in HIBD+EP group was (13.25±5.19)%, significantly lower than that in HIBD group ［(20.32±5.10)%, P<0.05］. CONCLUSION：Ethyl pyruvate is neuroprotective against HIBD in neonatal rats via increasing SOD level, decreasing MDA level, attenuating brain edema, decreasing apoptotic cells in cortex and alleviating atrophy of hypoxic-ischemic hemisphere.     

Effects of maternal limb ischemic preconditioning on fetal hippocampal neuron apoptosis induced by intrauterine distress-reoxygenation in rats

AIM: To evaluate the influence of maternal limb ischemic preconditioning (LIP) on the apoptosis of fetal hippocampal neurons induced by intrauterine distress-reoxygenation in rats. METHODS: Intrauterine ischemia was induced by clamping the uterine and uterine branch of the ovarian blood vessels with aneurysm clamps for a period of 15 min followed by removal of the clamps to permit reperfusion. Sprague-Dawley (SD) rats (n=12) were randomly divided into 4 groups on the 19th pregnant day: sham (S) group, LIP group, fetal distress (FD) group and LIP+FD group. The cesarean birth occurred on embryonic day 21 to obtain 12 fetal rats alive in each group. The fetal rats were decapitated and the pyramidal cells in CA1 hippocampus were observed under light microscope. The neuronal apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining and the apoptotic rate was calculated. The expression of Bcl-2 and Bax were detected by immunohistochemical method and Western blotting. RESULTS: The rates of neuronal apoptosis in FD group and LIP+FD group were significantly higher than that in S group (P<0.05), while no significant difference was observed between S group and LIP group (P>0.05). The expression of Bcl-2 and Bax in FD group and LIP+FD group was significantly higher than that in S group (P<0.05), while no significant difference was observed between S group and LIP group (P>0.05). The ratio of Bcl-2/Bax was lower in FD group than that in S group. Compared with FD group, the rate of neuronal apoptosis was significantly lower (P<0.05), while the ratio of Bcl-2/Bax was significantly higher (P<0.05) in LIP+FD group. CONCLUSION: Maternal limb ischemic preconditioning attenuates the apoptosis of fetal hippocampal neurons induced by intrauterine distress-reoxygenation in rats, which may be associated with the up-regulation of Bcl-2 expression.     

Effects of maternal limb ischemic preconditioning on structural and functional changes of mitochondria in fetal hippocampal neurons induced by intrauterine distress-reoxygenation in rats

AIM: To investigate the effects of maternal limb ischemic preconditioning (LIP) on the mitochondrial structures and functions of the hippocampal neurons induced by reoxygenation in the intrauterine distress fetal rats. METHODS: Pregnant rats (n=40) were randomly divided into 4 groups: sham (S) group, LIP group, fetal distress (FD) group and LIP+FD group. Intrauterine ischemia model was established through the experimental design. The ultrastructure of the mitochondria in CA1 area of the hippocampus was observed. The mitochondrial membrane potential and reactive oxygen species (ROS) were measured. The content of ATP and MDA in the hippocampus tissue was detected. The activity of Mn-SOD was observed. RESULTS: Compared with sham group, the ultrastructure of mitochondria in CA1 area of the hippocampus was damaged in FD group and LIP+FD group. The mitochondrial membrane potential, the content of ATP and the activity of Mn-SOD were decreased. However, the content of ROS and MDA was increased. Compared with FD group, the ultrastructure of mitochondria in CA1 area of the hippocampus was intact in LIP+FD group. Furthermore, the reduced mitochondrial membrane potential and ATP content were inhibited. The activity of Mn-SOD was increased, but the content of ROS and MDA was decreased in LIP+FD group. CONCLUSION: Limb ischemia preconditioning inhibits the damage the mitochondria of fetal hippocampal neurons induced by reoxygenation in the intrauterine distress fetal rats.     

JNK pathway promotes apoptosis of rat hippocampal neurons after cerebral ischemia and reperfusion

AIM:To investigate the effect of c-Jun N-terminal kinase(JNK) pathway on the apoptosis of hippocampal neurons after cerebral ischemia-reperfusion(IR) in SD rats. METHODS:Ninety rats were randomly divided into 5 groups:sham group, cerebral IR group,cerebral IR+JNK inhibitor(SP600125) group,cerebral IR+JNK agonist(anisomycin) group and cerebral IR+vehicle group. The brain samples were collected 24 h after reperfusion. The protein level of caspase-3 in hippocampal neurons was measured by immunohistochemical and Western blotting techniques. The mRNA expression of caspase-3 in the hippocampus was determined by real-time fluorescence quantitative PCR. The apoptosis of hippocampal neurons was detected by TUNEL staining. RESULTS:Compared with sham group, the expression of caspase-3 at mRNA and protein levels in cerebral IR group increased obviously(P<0.05). Compared with cerebral IR group, the expression of caspase-3 at mRNA and protein levels in cerebral IR+JNK inhibitor group decreased obviously(P<0.05), and those in cerebral group increased obviously(P<0.05). However, the expression of caspase-3 at mRNA and protein levels in cerebral IR+vehicle group had no obvious change(P>0.05).The apoptosis of hippocampal neurons in each group was consistent with the changes of caspase-3 at mRNA and protein levels. CONCLUSION:Activation of JNK pathway enhances caspase-3 expression in rat hippocampal neurons after cerebral IR,thus promoting the apoptosis of the neurons.