Neuronal Apoptosis In Rats Research Articles

Effect of theanine pretreatment on DNA repair function in neurons during brain ischemia-reperfusion injury in rats

Objective To investigate the effect of theanine pretreatment on DNA repair function in neurons during brain ischemia-reperfusion(I/R)injury in rats. Methods One hundred and eight male Sprague-Dawley rats, weighing 290-310 g, aged 15 weeks, were randomly divided into 3 groups(n=36 each)using a random number table: sham operation group(S group), I/R group and theanine pretreatment group(T group). Global cerebral I/R was produced by 4-vessel occlusion method.Bilateral vertebral arteries were electrically cauterized, and bilateral common carotid arteries were clamped for 6 min.Theanine 1 g/kg was injected intravenously at 4 h before clamping bilateral common carotid arteries in T group, and the equal volume of normal saline was given in the other two groups.At 2, 6, 12, 24, 48 and 72 h of reperfusion, 6 rats were selected in each group and sacrificed, the brains were removed, and the hippocampus was isolated for determination of the number of viable neurons in the hippocampal CA1 region(with a light microscope), apoptosis in neurons in the hippocampal CA1 region(by TUNEL), and expression of DNA repair protein X-ray repair cross-complementing group 1(XRCC1)and Ku70(by immunohistochemistry). The apoptotic index was calculated. Results Compared with S group, the number of viable neurons was significantly decreased, and the apoptotic index was significantly increased at 6, 12, 24, 48 and 72 h of reperfusion, and the expression of XRCC1 and Ku70 was significantly down-regulated at 2, 6, 12, 24, 48 and 72 h of reperfusion in I/R group(P<0.01). Compared with I/R group, the number of viable neurons was significantly increased at 12, 24, 48 and 72 h of reperfusion, the apoptotic index was significantly decreased at 6, 12, 24, 48 and 72 h of reperfusion, and the expression of XRCC1 and Ku70 was significantly up-regulated at 2, 6, 12, 24, 48 and 72 h of reperfusion in T group(P<0.01). Conclusion The mechanism by which theanine pretreatment attenuates brain I/R injury is related to enhancement of DNA repair function and reduction of neuronal apoptosis in rats. Key words: Tea; Glutamates; Brain; Reperfusion injury; DNA Repair; Apoptosis; Neurons

Profile of retigabine-induced neuronal apoptosis in the developing rat brain.

Acute neonatal exposure to some, but not all, anticonvulsant drugs induces a profound increase in neuronal apoptosis in rats. Phenobarbital and phenytoin induce apoptosis at a therapeutically relevant dose range, lamotrigine and carbamazepine do so only at supratherapeutic doses or in polytherapy, and valproate does so even at subtherapeutic doses. Levetiracetam is devoid of pro-apoptotic effects. Retigabine, a new-generation drug, acts uniquely by enhancing the M-type potassium current. Because its safety profile in developing animals is unstudied, we sought to determine if retigabine would induce apoptosis. Postnatal day (P) 7 rat pups were treated with retigabine (5-30 mg/kg), vehicle (saline), or comparator drugs (phenobarbital, lamotrigine, levetiracetam, or carbamazepine). Cell death was assessed using amino-cupric-silver staining. A separate group of animals was treated repeatedly (three times over 24 h) with retigabine (15 mg/kg) or vehicle. To establish a pharmacokinetic profile for retigabine, we measured plasma and brain levels after drug treatment. Consistent with prior studies from our group and others, we found phenobarbital-induced cell death throughout thalamus, nucleus accumbens, and several neocortical areas. By contrast, levetiracetam, lamotrigine, and carbamazepine were found to have no appreciable apoptotic effect on the aforementioned structures. Acute (single) exposure to retigabine, even at doses of 30 mg/kg, was also without effect on apoptosis. However, repeated (three times) exposure to retigabine triggered apoptosis in a subset of brain areas. The half-life of retigabine in plasma was 2.5 h, with appreciable concentrations reached in the brain within 1 h of administration. These data demonstrate that retigabine, like many other anticonvulsant drugs, is capable of triggering neuronal apoptosis in the developing rat brain. Unlike other drugs, repeated dosing of retigabine was necessary to induce this effect. This may be due to its shorter half-life as compared to other drugs, such as phenobarbital.

Puerarin reduces apoptosis in rat hippocampal neurons cultured in high glucose medium by modulating the p38 mitogen activated protein kinase and c-Jun N-terminal kinase signaling pathways

ObjectiveTo investigate the neuroprotective effect of puerarin on rat hippocampal neurons cultured in high glucose medium, and to examine the role of the p38 mitogen activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK) signaling pathways in this effect. MethodsPrimary cultures of hippocampal neurons were prepared from newborn Sprague Dawley rats. Neuron-specific enolase immunocytochemistry was used to identify neurons. The neurons were cultured with normal medium (control group) or with high-glucose medium (high-glucose group), and puerarin (puerarin group), a p38 MAPK inhibitor (SB239063; p38 MAPK inhibitor group) or a JNK inhibitor (SP600125; JNK inhibitor group) were added. After 72 h of treatment, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was performed to detect apoptosis, and western blotting was used to assess protein levels of p-p38, p38, p-JNK and JNK. ResultsIn the high-glucose group, the neuronal apoptosis rate and the p-p38/p38 and p-JNK/JNK ratios were higher than in the control group. The p38 MAPK and JNK inhibitors prevented this increase in the apoptosis rate. The apoptosis rates in the puerarin group, the p38 MAPK inhibitor group and the JNK inhibitor group were significantly decreased compared with the high-glucose group. Moreover, protein levels of p-p38 and p-JNK were significantly reduced, and the p-p38/p38 and p-JNK/JNK ratios were decreased in the puerarin group compared with the high-glucose group. In addition, compared with the high-glucose group, p-p38 levels and the p-p38/p38 ratio were reduced in the p38 MAPK inhibitor group, and p-JNK levels and the p-JNK/JNK ratio were decreased in the JNK inhibitor group. ConclusionPuerarin attenuates neuronal apoptosis induced by high glucose by reducing the phosphorylation of p38 and JNK.

Effect of exogenous hydrogen sulfide on neuronal apoptosis during focal cerebral ischemia-reperfusion in rats

Objective To investigate the effect of exogenous hydrogen sulfide (H2S) on neuronal apoptosis during focal cerebral ischemia-reperfusion (I/R) in rats. Methods One hundred eight male Sprague-Dawley rats, aged 6-8 weeks, weighing 250-270 g, were randomly divided into 3 groups (n = 36 each) using a random number table: control group (group C), I/R group and H2S group.Focal cerebral ischemia was induced by middle cerebral artery occlusion in anesthetized rats.A nylon thread with a rounded tip was inserted into the left internal carotid artery and advanced intracranially to block blood flow into the middle cerebral artery.Middle cerebral artery occlusion was maintained for 90 min followed by reperfusion.In group H2S, 0.25% NaSH (a donor of exogenous H2S) 10 mg/kg was injected intraperitoneally at the onset of reperfusion.The equal volume of normal saline was given in C and I/R groups.At 1, 3 and 7 days of reperfusion, neurological deficit was scored, and corner test was performed.Brains were removed for determination of myocardial infarct size, Bax-, Bcl-2- and caspase-3-positive cells, and cell apoptosis.The percentage of myocardial infarct size, rate of Bax-, Bcl-2-and caspase-3-positive cells and apoptosis rate were calculated. Results Compared with group C, the neurological deficit score was significantly decreased, and the corner score, percentage of myocardial infarct size, rate of Bax-, Bcl-2- and caspase-3-positive cells and apoptosis rate were increased at each time point of reperfusion in I/R and H2S groups (P<0.05). Compared with group I/R, the neurological deficit score and Bcl-2-positive cells were significantly increased, and the corner score, percentage of myocardial infarct size, rate of Bax- and caspase-3-positive cells and apoptosis rate were decreased at each time point of reperfusion in group H2S (P<0.05). Conclusion The mechanism by which exogenous H2S attenuates focal cerebral I/R is related to inhibition of neuronal apoptosis in rats. Key words: Hydrogen sulfide; Reperfusion injury; Brain; Apoptosis

Neuroserpin Protects Rat Neurons and Microglia-Mediated Inflammatory Response Against Oxygen-Glucose Deprivation- and Reoxygenation Treatments in an In Vitro Study

Background/Aims: Neuroserpin (NSP) is known for its neuroprotective role in cerebral ischemic animal models and patients. Our laboratory conducted a series of investigations on the neuroprotection of NSP in different cells in the brain. In the present study, we further observe the effects of NSP on neurons and microglia-mediated inflammatory response following oxygen-glucose deprivation (OGD), and explore possible mechanisms related to neuroprotection of OGD in the central nervous system (CNS). Methods: Neurons and microglia from neonatal rats were treated with OGD followed by reoxygenation (OGD/R). To confirm the effects of NSP, the neuronal survival, neuronal apoptosis, and lactate dehydrogenase (LDH) release were measured in cultured neurons. Furthermore, the levels of IL-1β and nitric oxide (NO) release were also detected in cultured microglia. The possible mechanisms for the neuroprotective effect of NSP were explored using Western blot analysis. Results: NSP administration can reverse abnormal variations in neurons and microglia-mediated inflammatory response induced by OGD/R processes. The neuronal survival rate, neuronal apoptosis rate, and LDH release were significantly improved by NSP administration in neurons. Simultaneously, the release of IL-1β and NO were significantly reduced by NSP in microglia. Western blot showed that the expression of ERK, P38, and JNK was upregulated in microglia by the OGD/R treatment, and these effects were significantly inhibited by NSP. Conclusion: These data verified the neuroprotective effects of NSP on neurons and microglia-mediated inflammatory response. Inhibition of the mitogen-activated protein kinase (MAPK) signaling pathways might play a potential role in NSP neuroprotection on microglia-mediated inflammatory response, which needs further verification.

Effects of mitochondrial division inhibitor on neurological function and neuronal apoptosis in rats after cardiopulmonary resuscitation

To investigate the effects of mitochondrial division inhibitor 1 (mdivi-1) in rats after cardiopulmonary resuscitation (CPR) and its mechanism. Fifty Sprague-Dawley (SD) rats were randomly (random number table) divided into sham group (n = 8), cardiac arrest (CA) model group (n = 14), dimethyl sulfoxide post-treatment control group (DMSO group, n = 14), and mdivi-1 post-treatment group (mdivi-1 group, n = 14). Asphyxial CA was reproduced in animals, and they were resuscitated by CPR. In the mdivi-1 group or DMSO group, the animals were given mdivi-1 (1.2 mg/kg) or DMSO (0.1%) intravenously after restoration of spontaneous circulation ( ROSC ). The neurological functions were assessed using neurological deficit score (NDS) determined at 24, 48 and 72 hours after CPR. The brain tissues were harvested at 72 hours after CPR. The histopathologic changes were assessed by hematoxylin and eosin (HE) staining, and the normal neuron was counted. The neuronal apoptosis was assessed with terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, and the expressions of cytochrome C (Cyt-C) protein in mitochondria and cytoplasm from hippocampus were determined by Western Blot. NDS in all experiment groups was gradually increased after CPR, and they were significantly lower than those of the sham group at 24, 48, and 72 hours (51.5 ± 3.7 vs. 80.0 ± 0.0, 59.3 ± 3.6 vs. 80.0 ± 0.0, 66.7 ± 2.6 vs. 80.0 ± 0.0, all P < 0.05). The number of normal pyramidal neurons in the hippocampal CA1 region was markedly reduced (cells/HP: 4.4 ± 1.1 vs. 23.1 ± 4.0, P < 0.05), the apoptotic index was significantly increased [(86.9 ± 6.9)% vs. (3.4 ± 0.8)%, P < 0.05], the expressions of Cyt-C in mitochondria were significantly decreased (A value: 0.46 ± 0.18 vs. 1.00 ± 0.00, P < 0.05), and the expressions of Cyt-C in cytoplasm were significantly up-regulated (A value: 6.65 ± 0.21 vs. 1.00 ± 0.00, P < 0.05). Compared with the CA group, NDS at 24 hours and 48 hours in mdivi-1 group was slightly increased (55.2 ± 3.3 vs. 51.5 ± 3.7, 64.7 ± 2.4 vs. 59.3 ± 3.6, both P > 0.05), and it was significantly increased at 72 hours (74.5 ± 2.3 vs. 66.7 ± 2.6, P < 0.05), the number of normal pyramidal neurons in the hippocampal CA region was markedly increased (cells/HP: 16.2 ± 2.4 vs. 4.4 ± 1.1, P < 0.05), the apoptotic index was dramatically reduced [(42.3 ± 3.9 )% vs. (86.9 ± 6.9 )%, P < 0.05], the expressions of Cyt-C in mitochondria were significantly increased (A value: 0.83 ± 0.22 vs. 0.46 ± 0.18, P < 0.05), and the expressions of Cyt-C in cytoplasm were significantly decreased (A value: 3.84 ± 0.47 vs. 6.65 ± 0.21, P < 0.05). There was no statistically significant difference in above indexes between CA group and DMSO group. By inhibiting mitochondrial Cyt-C apoptotic pathway to reduce neuronal apoptosis in rats after CA-CPR, mdivi-1 can improve brain function after CPR.

Influence of acute ethanol intoxication on neuronal apoptosis and Bcl-2 protein expression after severe traumatic brain injury in rats

ObjectiveTo study the influence and mechanism of acute ethanol intoxication (AEI) on rat neuronal apoptosis after severe traumatic brain injury (TBI). MethodsNinety-six Sprague-Dawley rats were randomly divided into four groups: normal control, AEI-only, TBI-only and TBI+AEI (n=24 for each). Severe TBI model was developed according to Feeney’s method. Rats in TBI+AEI group were firstly subjected to AEI, and then suffered head trauma. In each group, animals were sacrificed at 6h, 24h, 72h, and 168h after TBI. The level of neuronal apoptosis and the expression of Bcl-2 protein were determined by TUNEL assay and immunohistochemical method, respectively. ResultsApoptotic cells mainly distributed in the cortex and white matter around the damaged area. Neuronal apoptosis significantly increased at 6h after trauma and peaked at 72h. Both the level of neuronal apoptosis and expression of Bcl-2 protein in TBI-only group and TBI+AEI group were higher than those in control group (P<0.05). Compared with TBI-only group, the two indexes were much higher in TBI+AEI group at all time points (P<0.05). ConclusionOur findings suggest that AEI can increase neuronal apoptosis after severe TBI.

Glibenclamide Improves Survival and Neurologic Outcome After Cardiac Arrest in Rats.

Glibenclamide confers neuroprotection in animal models as well as in retrospective clinical studies. This study determines whether glibenclamide improves outcome after cardiac arrest in rats. Prospective randomized laboratory study. University research laboratory. Male Sprague-Dawley rats (n = 126). Rats successfully resuscitated from 8-minute asphyxial cardiac arrest were randomized to glibenclamide or vehicle group. Rats in the glibenclamide group were intraperitoneally administered glibenclamide with a loading dose of 10 μg/kg at 10 minutes and a maintenance dose of 1.2 μg at 6, 12, 18, and 24 hours after return of spontaneous circulation, whereas rats in the vehicle group received equivalent volume of vehicle solution. Survival was recorded every day, and neurologic deficit scores were assessed at 24, 48, and 72 hours and 7 days after return of spontaneous circulation (n = 22 in each group). Results showed that glibenclamide treatment increased 7-day survival rate, reduced neurologic deficit scores, and prevented neuronal loss in the hippocampal cornu ammonis 1 region. To investigate the neuroprotective effects of glibenclamide in acute phase, we observed neuronal injury at 24 hours after return of spontaneous circulation and found that glibenclamide significantly decreased the rate of neuronal necrosis and apoptosis. In addition, glibenclamide reduced the messenger RNA expression of tumor necrosis factor-α and monocyte chemoattractant protein-1 in the cortex after return of spontaneous circulation. Furthermore, the sulfonylurea receptor 1 and transient receptor potential M4 heteromers, the putative therapeutic targets of glibenclamide, were up-regulated after cardiac arrest and cardiopulmonary resuscitation, indicating that they might be involved in neuroprotective effect of glibenclamide. Glibenclamide treatment substantially improved survival and neurologic outcome throughout a 7-day period after return of spontaneous circulation. The salutary effects of glibenclamide were associated with suppression of neuronal necrosis and apoptosis, as well as inflammation in the brain.

Role of mitochondrial permeability transition pore in sevoflurane-induced inhibition of apoptosis induced by oxygen-glucose deprivation and restoration in rat neurons

Objective To evaluate the role of mitochondrial permeability transition pore (mPTP) in sevoflurane-induced inhibition of apoptosis induced by oxygen-glucose deprivation and restoration (OGD/R) in rat neurons. Methods Cortical neurons isolated from neonatal Sprague-Dawley rats born within 24 h, were cultured primarily and seeded in 6-well plates (2 ml/well) at a density of 1×106 cells/ml.The neurons were randomly divided into 5 groups (n=72 each) using a random number table: control group (group C), OGD/R group (group O), mPTP opener group (group A), mPTP opener + sevoflurane group (group AS), and sevoflurane group (group Sev). The cells were cultured in normal culture medium in group C, and the cells were subjected to OGD for 90 min followed by restoration of O2-glucose supply for 24 h in the other groups.In A and AS groups, mPTP opener atractyloside 20 μmol/L was added immediately after OGD.In Sev and AS groups, the cells were post-conditioned with 2% sevoflurane for 1 h immediately after OGD.The neurons were collected at 24 h of OGD in O and A groups, or at 23 h of OGD in Sev and AS groups.Annexin Ⅴ-FITC/PI double staining was performed to count apoptotic neurons, apoptotic rate was calculated, and cell survival rate was measured using MTT assay.The mitochondrial membrane potential (MMP) was measured by using JC-1 fluorescence probe as indicator.The opening of mPTP was determined through assessing the changes of mitochondrial optical density (ΔOD540 of mPTP). The expression of pro-apoptotic factors Bid, Bim, Puma protein and mRNA was detected by semi-quantitative reverse transcription polymerase chain reaction and Western blot. Results Compared with group C, apoptotic rate was significantly increased, cell survival rate and MMP were decreased, the opening of mPTP was increased, and the expression of Bid, Bim, Puma protein and mRNA was up-regulated in the other groups.Compared with group O, apoptotic rate was significantly decreased, cell survival rate and MMP were increased, the opening of mPTP was decreased, and the expression of Bid, Bim, Puma protein and mRNA was down-regulated in Sev group.Compared with group Sev, apoptotic rate was significantly increased, cell survival rate and MMP were decreased, the opening of mPTP was increased, and the expression of Bid, Bim, Puma protein and mRNA was up-regulated in A and As groups.Compared with group A, apoptotic rate was significantly decreased, cell survival rate and MMP were increased, the opening of mPTP was decreased, and the expression of Bid, Bim, Puma protein and mRNA was down-regulated in group AS. Conclusion Sevoflurane mitigates OGD/R-induced apoptosis in rat neurons through inhibiting mPTP opening. Key words: Mitochondrial membrane transport proteins; Anesthetics, inhalation; Reperfusion injury; Neurons; Apoptosis

The study on the relationship between hippocampus neuronal apoptosis and hippocampus synaptic plasticity in rats exposed to aluminum

To investigate the effect of aluminum exposure on neuronal apoptosis of rats hippocampus and the correlation of and synaptic plasticity. There were 40 SPF grade SD rats which were randomly divided into four groups: the control group, the low dose group, the medium dose group and the high dose group, 10 rats in each group. The rats were daily gavaged with aluminum lactate for 30 days. The hippocampal fEPSPs in rat was measured by electrophysiological grapher and the neuronal apoptosis in hippocampus was detected by Flow cytometer. In addition, the relative expression of gene which includes caspase-3, 8, 9 was measured by Real-time PCR. Compared to the control group, the average of fEPSPs which after HFS 10, 20, 30, 40, 50, 60 min was decreased at different time point in the low dose group, the medium dose group and the high dose group (P < 0.05). Compared with the control group, the rate of apoptosis was significantly increased in the medium dose group and the high dose group (P < 0.05). Compared to the control group, the relative expression of caspase-3 in the medium dose group and the high dose group was significantly increased in Real-time PCR (P < 0.05), and the relative expression of caspase-8 in the high dose group was significantly increased (P < 0.05). Aluminum exposure may induced neuronal apoptosis in rats, and then affect hippocampal synaptic plasticity.

Effect of chronic aluminum exposure on neuron apoptosis and expression of P53 phosphorylation in rats

To explore the role of P53 phosphorylation in neuron apoptosis of rats by chronic aluminum exposure. A total of male 40 SD rats were divided randomly into 4 groups (n = 10/dose), the exposed groups were fed with normal diet with different concentration of AlCl3 · 6H2O for 6 months respectively. The dosage of low, middle and high groups were 10.73, 107.33, 1073.33 mg/kg in sequence. The control group received normal diet. The neuron apoptosis was measured by method of Tunel. The expressions of P53 and pP53-ser15 protein in the cortex were detected by Western-blot. Tunel staining showed that the low, middle and high group rats had increased apoptosis rate than control group (P < 0.01). Western-blot test demonstrated that the expression of P53 protein in the cortex of high group rats were significantly higher than the control and low groups (P < 0.05). The expression of pP53-ser15 protein in the cortex of middle and high group rats were also higher than the control and low groups (P < 0.05). Chronic aluminum exposure can lead to over expression of P53 and pP53-ser15 protein in cerebral cortex, which maybe one of the most important mechanisms of neuron apoptosis induced by AlCl3.

Hypoxiс postconditioning is an effective method of protection from severe hypoxia induced lipid peroxidation and neuronal apoptosis in rats

Postconditioning (PostC) is an exposure of the damaged organism to extreme factors of the mild intensity to mobilize endogenous protective mechanisms. In our laboratory method of PostC using three daily trials of mild hypobaric hypoxia (MHH) was developed. It has been found that such method of the PostC effectively prevents degeneration of the hippocampal and neocortical neurons in rats, subjected to severe hypoxia (SH). Present study has been aimed at examination of the impact of oxidative processes in the development of the neuroprotection acquired in the course of hypoxic PostC during first three days of reoxygenation after SH in rats. The levels of thiobarbituric acid reactive substances (TBARS) and Schiff bases (SB) were used as markers of lipid peroxidation. In addition, the intensity of the apoptotic DNA fragmentation has been studied. During the three days after the SH a sustained increase of SB in the rat hippocampus was observed (700-1000% of the control value). After the first PostC episode the SB levels decreased to 150% of the baseline. Subsequently this parameter did not differ significantly from the control values. TBARS showed accumulation on the first day following the SH but afterwards its levels dropped to 40% of control and did not recover then to normal values. In the PostC animals, the levels of TBARS after each of three PostC episodes did not differ from the control values. These facts indicate that the PostC MHH balances the activity of pro- and antioxidant systems in vulnerable brain regions and promotes the effective utilization of components damaged by peroxidation. Fragments ladder typical for cells undergone apoptosis was obtained by the electrophoretic separation of the total DNA, extracted from a rat brain after one, two and three days after the SH. In the PostC group, the DNA fragmentation was revealed only after the first PostC episode, demonstrating antiapoptotic action PostC MHH.

Role of HIF-1α in reduction of apoptosis in cortical neurons of rats by sevoflurane preconditioning: the relationship with Bid, Bim and Puma

Objective To evaluate the role of hypoxia inducible factor-1α(HIF-1α)in reduction of apoptosis in cortical neurons of rats by sevoflurane preconditioning. Methods Primary cortical neurons obtained from neonatal Sprague-Dawley rats were seeded in 6-well plates(2 ml/well), and randomly divided into 4 groups(n=15 each)using a random number table: control group(C group), anoxia-reoxygenation(A/R)group, sevoflurane preconditioning group(SP group)and HIF-1α inhibitor 2-methoxyestradiol group(H group). The neurons were subjected to O2-glucose deprivation for 90 min followed by restoration of O2-glucose supply for 24 h. In group SP, the neurons were exposed to 2.0% sevoflurane for 2 h followed by 5 min washout for 3 times, and then sevoflurane preconditioning was performed immediately.In group H, sevoflurane preconditioning was performed at 72 h of incubation with 5 μmol/L 2-methoxyestradiol.The apoptosis in neurons was assessed using Annexin Ⅴ-FITC/PI assay, and apoptosis rate was calculated.The expression of Bid, Bim, Puma and activated caspase-3 in neurons was detected by Western blot. Results Compared with group C, apoptosis rate was significantly increased, and the expression of Bid, Bim, Puma and activated caspase-3 was up-regulated in group A/R.Compared with group A/R, apoptosis rate was significantly decreased, and the expression of Bid, Bim, Puma and activated caspase-3 was down-regulated in group SP.Compared with group SP, apoptosis rate was significantly increased, and the expression of Bid, Bim, Puma and activated caspase-3 was up-regulated in group H. Conclusion HIF-1α mediates reduction of apoptosis in rat neurons by sevoflurane preconditioning, and down-regulated expression of Bid, Bim, and Puma is involved in the mechanism. Key words: Hypoxia-inducible factor 1, alpha subunit; Apoptosis regulatory proteins; Anesthetics, inhalation; Ischemic preconditioning; Cell hypoxia; Oxygen; Neurons; Apoptosis

Effect of intermittent hypoxia on neuronal apoptosis and inducible nitric oxide synthase expression in rat hippocampus

Objective To investigate the effect of intermittent hypoxia on neuronal apoptosis and inducible nitric oxide synthase (iNOS) expression in rat hippocampus, in order to explore the potential mechanism of hippocampal neuronal apoptosis induced by intermittent hypoxia. Methods Twenty-four Wistar male rats (280-350 g) were randomly divided into three groups: the normal control, intermittent normoxia and intermittent hypoxia group (n=8 each). The animal model of intermittent hypoxia was established by an automated nitrogen/oxygen profile system. The three groups were respectively exposed to continuous normoxia (21%O2), cyclical normoxia (21%O2), and cyclical hypoxia [alternating between normoxia (21%O2) and hypoxia (5%O2), every 120 seconds] throughout the eight hours of light time(8∶00-16∶00). The rats were dissected and the hippocampus was removed at the end of the designated duration of exposures for six weeks. TdT-mediated dUTP nick end labeling (TUNEL) was used to detect the apoptotic rate of the hippocampus region in each group. Reverse transcription-PCR, immunohistochemistry (IHC) and Western blotting were used to examine mRNA and protein expressions of iNOS in the hippocampus tissue. Results The apoptotic rate of hippocampal neurons was higher in intermittent hypoxia group than in intermittent normoxia group 〔(28.236±0.081)% vs. (9.341±0.026)%, P<0.05〕. The mRNA and protein expressions of iNOS were higher in intermittent hypoxia group than in intermittent normoxia group (3.394±1.344 vs. 0.125±0.040, 7.793±0.052 vs. 1.356±0.039, both P<0.05). Conclusions Intermittent hypoxia can induce hippocampal neuronal apoptosis in rats, accompanied by the upregulation of iNOS gene transcription and protein expression, which indicates that iNOS may involve in the process of hippocampal neuronal apoptosis induced by intermittent hypoxia. Key words: Sleep apnea, Obstructive; Apoptosis; Nitric oxide synthase

The effects of piracetam on heroin-induced CPP and neuronal apoptosis in rats

BackgroundPiracetam is a positive allosteric modulator of the AMPA receptor that has been used in the treatment of cognitive disorders for decades. Recent surveys and drug analyses have demonstrated that a heroin mixture adulterated with piracetam has spread rapidly in heroin addicts in China, but its addictive properties and the damage it causes to the central neural system are currently unknown. MethodsThe effect of piracetam on the reward properties of heroin was assessed by conditioned place preference (CPP). Electron microscopy and radioimmunoassay were used to compare the effects of heroin mixed with equivalent piracetam (HP) and heroin alone on neuronal apoptosis and the levels of beta-endorphin (β-EP) in different brain subregions within the corticolimbic system, respectively. ResultsPiracetam significantly enhanced heroin-induced CPP expression while piracetam itself didn’t induce CPP. Morphological observations showed that HP-treated rats had less neuronal apoptosis than heroin-treated group. Interestingly, HP normalized the levels of β-EP in the medial prefrontal cortex (mPFC) and core of the nucleus accumbens (AcbC) subregions, in where heroin-treated rats showed decreased levels of β-EP. ConclusionsThese results indicate that piracetam potentiate the heroin-induced CPP and protect neurons from heroin-induced apoptosis. The protective role of HP might be related to the restoration of β-EP levels by piracetam. Our findings may provide a potential interpretation for the growing trend of HP abuse in addicts in China.

Is there an association between histopathological changes in the lower ureter and renal functions? Evaluation of patients who underwent ureteroneocystostomy for ureterovesical obstruction or vesicoureteral reflux

We aimed to assess the relationship between the histological changes of the ureterovesical junction (UVJ) and renal functions. Therefore, we evaluated histological changes of the lower ureter and renal scintigraphy findings of patients for whom ureteroneocystostomy was performed because of vesicoureteral reflux (VUR) or ureterovesical junction obstruction (UVO). UVJ specimens were obtained from 18 children. We investigated the changes in neuronal innervation, muscular morphology, extracellular matrix, and apoptosis rate with renal scintigraphy findings. Seven UVO and 11 VUR patients were treated. Alpha-actin expression in smooth muscle cells was found to be lower (P < 0.001) while neuronal defect was more prominent in the UVO group (P = 0.002). The renal functions decreased as the smooth muscle structural defect increased in the VUR group (P < 0.05). Neuronal tissue and muscle tissue were more defective in the UVO group. The decrease in neuronal fibers and muscle cells explains the pathogenesis of the obstructive group, but no difference was observed regarding the accumulation of collagen type 3 and cellular apoptosis between the VUR and UVO groups. In the VUR group, renal functions decreased while the smooth muscle defect at the distal end of the ureter increased.

Cloning, expression, and purification of cold inducible RNA-binding protein and its neuroprotective mechanism of action

Background: Cold-inducible RNA-binding protein (CIRP) is induced in response to hypothermia, where it exerts neuroprotective effects. Our preliminary studies revealed that it inhibits H2O2-induced apoptosis in rat neurons. In the current study, we report effective expression and purification approaches for the synthesis of CIRP, and assess its potential protective effects against oxidative stress. Methods: CIRP-encoding was expressed using the prokaryotic expression system pGEX-4T-1, and SP-Sepharose and Sephacryl S-200 columns were used to purify rCIRP. To mimic ischemia/reperfusion injury-associated oxidative stress, neuro2a cells (N2a) were pre-treated with rCIRP for 2h, followed by hydrogen peroxide (H2O2 60μmol/ml) for 24h. Cell viability was then quantified using an MTT assay. In addition, western blotting was performed to measure the cell cycle related signal transduction pathways. Results: N2a cells exhibited decreased viability following H2O2 treatment, whereas rCIRP significantly improved viability following H2O2 treatment. CIRP also accelerated cell cycle progression from S to G2/M phase in cultured mouse neuroblastoma cells. In addition, CIRP increased levels of p-ERK and p-Akt, and also re-activated the cell cycle-related protein cyclin D1 and c-Myc. These results suggest that CIRP activated the Akt and ERK signal transduction pathways in N2a cells. Conclusions: Our findings suggest that CIRP could exert protective effects against oxidative stress, and that it might be a novel neuroprotective agent.

Phenolic alkaloids from Menispermum dauricum inhibits apoptosis of cerebral ischemia in rats

Stroke was a debilitating disease for which limited therapeutic approaches are available currently. Thus, there was an urgent need for developing novel therapies for stroke. Cell apoptosis played an important role in embryonic development, maturity of the immune system, and other respects. Cells received apoptotic signals, activated the cells’ protein, started the gene regulation mechanisms, and caused the suicide death. Recently many research showed that neuron apoptosis took part in ischemic cell damage and was the important form of neurons loss caused by ischemia. After ischemia, necrosis or apoptosis was observed in cells because of inflammation, calcium overload, oxygen free radical, damage of mitochondria and DNA. Phenolic alkaloids from Menispermum dauricum (PAMD) have gained growing appreciation for its beneficial biological effects through its anti-inflammatory property. There was a close relationship between apoptosis and inflammatory reaction during cerebral ischemia. In this study we investigated the protective effect of PAMD on apoptosis and the potential molecular mechanisms in cerebral ischemia rats. Cerebral ischemia in male Sprague–Dawley rats was induced by middle cerebral artery occlusion (MCAO) and PAMD was pre-administered intragastrically once daily for five consecutive days before surgery. The effect of the PAMD on the ethology and pathomorphology of the rats with MCAO were observed before and after treatment. Neurological deficit scores and morphological characteristics were measured after cerebral ischemia. The expression of caspase-3 was determined by immunohistochemistry at 24, 48, and 72 h after cerebral ischemia. RT-qPCR was used to analyze the levels of caspase-3 mRNA of middle and high dosage groups at 72 h after cerebral ischemia. Neurons apoptosis rate was measured by flow cytometry. The result showed that, compared with MCAO groups, PAMD noticeably improved neurological deficit at 24, 48, and 72 h after ischemic insult. Consistent with these results, our data also indicated that PAMD significantly downregulated the expression of caspase-3 protein and caspase-3 mRNA (P < 0.01, P < 0.05), decreased the neuron apoptosis rate (P < 0.05). The results provided a neuroprotective effect of PAMD on cerebral ischemia and this effect was exerted by inducing the bcl-2 expression and suppressing the bax upregulation, then inhibiting the expression of caspase-3 protein and caspase-3 mRNA, and terminating the cascade reaction of caspases.

Progesterone alleviates hypoxic-ischemic brain injury via the Akt/GSK-3β signaling pathway.

This aim of this study was to investigate whether progesterone (PROG) alleviates the neuronal apoptosis in neonatal rats with hypoxic-ischemic (HI) brain damage through the phosphatidylinositol 3-kinase (PI3K)/Akt/glycogen synthase kinase-3β (GSK-3β) signaling pathway. A total of 96 newborn Wistar rats aged 7 days were randomly divided into four groups: sham surgery, HI, drug prevention (PROG) and Akt inhibitor groups. HI animal models were established by a conventional method. All animals were sacrificed 24 h after hypoxia. Immunohistochemistry was used to detect the distribution and expression of phosphorylated Akt (p-Akt) and the GSK-3β proteins in the brain, and western blot analysis was used to determine the p-Akt and GSK-3β protein contents. An enzyme-linked immunosorbent assay was also used to determine the GSK-3β content of the brain tissue, and flow cytometry was used to evaluate the apoptosis rate of neural cells. The expression of p-Akt protein was reduced in the brain tissues of the HI group, whereas GSK-3β expression was increased. In addition, the GSK-3β content of the brain and the neuronal apoptosis rate were significantly increased. PROG pre-treatment increased p-Akt expression, decreased GSK-3β expression and GSK-3β content, and also reduced neuronal apoptosis. Following administration of the Akt inhibitor wortmannin, p-Akt expression decreased, GSK-3β expression increased, and the GSK-3β content and neuronal apoptosis rate significantly increased (P<0.05). In conclusion, PROG activates the PI3K/Akt/GSK-3β pathway to promote Akt activation, enhance p-Akt expression and inhibit GSK-3β expression, thereby inhibiting neuronal apoptosis, alleviating HI brain injury and inducing a cerebroprotective effect.

Fungal encephalitis in human autopsy cases is associated with extensive neuronal damage but only minimal repair

The present study aimed at examining neuronal injury and repair in post mortem brain sections of humans who died from fungal central nervous system infections. Histological and immunohistochemical abnormalities in 15 autopsy cases with fungal central nervous system infections from 1990 to 2008 were compared with findings in 10 age- und sex-matched control cases that died from acute non-neurological causes. The fungal pathogens were identified by culture or polymerase chain reaction and morphology in post mortem tissue. Seven patients with fungal encephalitis had either an organ transplantation or a malignant haematological disorder; five out of 15 did not have a classical predisposing illness but suffered from severe septic infections as the principal cause of immunosuppression, and three from alcoholism. Fungal organisms detected were Aspergillus spp. and other moulds, Candida spp. and black yeast-like fungi including Cladosporium spp. Histological analyses identified microglial activation, astrocytosis and axonal injury in the white matter without additional demyelination as characteristic features of this infectious disease. An increased rate of hippocampal neuronal apoptosis was detected in fungal encephalitis, while the number of recently generated TUC-4 and calretinin-expressing neurones in the dentate gyrus did not differ between patients and controls. Unlike in other infectious diseases of the nervous system where a coexistence of damage and repair was observed, fungal encephalitis is characterized by strong damage and minimal neuronal regeneration.