Chloride channel blocker 4,4-diisothiocyanatostilbene-2,2'-disulfonic acid inhibits nitric oxide-induced apoptosis in cultured rat hippocampal neurons

Apoptosis in cultured rat hippocampal neurons was induced using the nitric oxide donor 3-morpholinosydnonimine, and cells were treated with the chloride channel blocker, 4,4- diisothiocyanatostilbene-2,2＇-disulfonic acid. Results showed that the survival rate of neurons was significantly increased after treatment with 4,4-diisothiocyanatostilbene-2,2＇-disulfonic acid, and the rate of apoptosis decreased. In addition, the expression of the apoptosis-related proteins poly（adenosine diphosphate-ribose）polymerase-1 and apoptosis-inducing factor were significantly reduced. Our experimental findings indicate that the chloride channel blocker 4,4- diisothiocyanatostilbene-2,2＇-disulfonic acid can antagonize apoptotic cell death of hippocampal neurons by inhibiting the expression of the apoptosis-related proteins poly（adenosine diphosphate-ribose）polymerase-1 and apoptosis-inducing factor.

The mechanism and relevant mediators associated with neuronal apoptosis and potential therapeutic targets in subarachnoid hemorrhage

Subarachnoid hemorrhage(SAH)is a dominant cause of death and disability wo rldwide.A sharp increase in intracranial pressure after SAH leads to a reduction in cerebral perfusion and insufficient blood supply for neuro ns,which subsequently promotes a series of pathophysiological responses leading to neuronal death.Many previous experimental studies have reported that excitotoxicity,mitochondrial death pathways,the release of free radicals,protein misfolding,apoptosis,nec rosis,autophagy,and inflammation are involved solely or in combination in this disorder.Among them,irreversible neuronal apoptosis plays a key role in both short-and long-term prognoses after SAH.Neuronal apoptosis occurs through multiple pathways including extrinsic,mitochondrial,endoplasmic reticulum,p53 and oxidative stress.Meanwhile,a large number of blood contents enter the subarachnoid space after SAH,and the secondary metabolites,including oxygenated hemoglo bin and heme,further aggravate the destruction of the blood-brain barrier and vasogenic and cytotoxic brain edema,causing early brain injury and delayed cerebral ischemia,and ultimately increasing neuronal apoptosis.Even there is no clear and effective therapeutic strategy for SAH thus far,but by understanding apoptosis,we might excavate new ideas and approaches,as targeting the upstream and downstream molecules of apoptosis-related pathways shows promise in the treatment of SAH.In this review,we summarize the existing evidence on molecules and related drugs or molecules involved in the apoptotic pathway after SAH,which provides a possible target or new strategy for the treatment of SAH.

Detecting the apoptosis of dopamine neurons with immunohistochemical staining and double-staining technique

BACKGROUND： It is proved that the onset of Parkinson disease companies with neuronal apoptosis of dopamine in substantia nigra of midbrain. Previous researches on neuronal apoptosis of dopamine were analyzed on their consecutive tissue sections with immunohistochemical single-labeling method, immunofluorescence and electron microscope, and there are significant differences.OBJECTIVE ： To observe the feasibility of neuronal apoptosis of dopamine with in situ end labeling and tyrosine-hydroxylase antibody immunohistochemical double-labeling staining technique.DESIGN ： Controlled study.SETTING： College of Pharmacology of Taishan Medical College; College of Management of Taishan Medical College. MATERIALS ： Wistar rats with 2 weeks old and of clean grade were provided by the Animal Center of Taishan Medical College. In situ end labeling kit （terminal deoxynucleotidyl transferase, mixed reactive solution of nucleotide, transfusion-POD）, monoclonal antibody of rat antibody against tyrosine hydroxylase （Boehriuser）. METHODS： The experiment was completed at the Pharmacological Laboratory of Taishan Medical College from February to December 2005. Tissue from midbrain of rats was taken out to make paraffin sections to observe the neuronal apoptosis of dopamine under microscope with in situ end labeling and tyrosine-hydroxylase antibody immunohistochemical double-labeling staining technique.MAIN OUTCOME MEASURES ： Neuronal apoptosis of dopamine with in situ end labeling and tyrosine-hydroxylase antibody immunohistochemical double-labeling staining technique. RESULTS： ① After double-labeling staining, two kinks of positive products were observed in neurons of dopamine which were suffered from apoptosis. One stained with tyrosine hydroxylase was hyacinthine, and the other stained with in situ end labeling was buffy. Cells of positive products stained with in situ end labeling shaped as strap and bend and was distributed in clustering. Cytoplasm was hyacinthine, staining was symmetrical, and cellular ecphyma was observed. Nucleus was stained vacantly which was coincidence with form of neurons of dopamine. ②Apoptosis showed strictly in cytoplasm and nucleus at the aspect of morphology. Cytoplasm stained with in situ end labeling was hardly to recognize because of the usage of double-labeling staining technique, but nucleus was still characterized by apoptosis. The behavior of positive products stained with in situ end labeling was described as following： nucleus was buffy; karyopycnosis was round and irregular; caryotin was integrated into clump which was distributed at the border of nucleus and shaped as demilune and anular; positive signals were limited in nucleus and coincidence with morphological changes of apoptosis. However, blue and positive products were observed in cytoplasm of neurons of dopamine which did not occur apoptosis, and the nucleus was not labeled. Therefore, processing apoptosis of neurons of dopamine could be recognized. CONCULSION： Double-labeling staining technique can be used to correctly reveal histological and morphological changes of neuronal apoptosis of dopamine during its onset and development.

Inosine inhibits apoptosis and cytochrome C mRNA expression in rat neurons after cerebral ischemia/reperfusion

BACKGROUND: It has been demonstrated that adenosine can induce glial cell to release cytochrome C, enhance expression of apoptotic gene bax, inhibit anti-apoptotic gene bcl-2, and activate caspase-3 to apoptosis; Whereas inosine can inhibit neuronal apoptosis which is similar to bcl-2. OBJECTIVE: To observe the effects of inosine on neuronal apoptosis and expression of cytochrome C mRNA in rats after focal cerebral ischemia/reperfusion, and analyze the pathway of its neuroprotective effect. DESIGN: A randomised controlled animal trial. SETTINGS: Department of Neurology, Rongcheng Second People's Hospital; Department of Neurology, Affiliated Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. MATERIALS: Sixty-eight rats, weighing 230-280 g and clean grade, were used. TdT-mediated dUTP-biotin nick end labeling (TUNEL) and cytochrome C mRNA in situ hybridization kits and DAB staining kit were purchased from Wuhan Boster Biological Co., Ltd.; Inosine injection [200 mg (2 mL) each] from Qingdao First Pharmaceutical Factory. METHODS: The experiment was accomplished in the animal experimental center in Tongji Medical College of Huazhong University of Science and Technology from December 2003 to June 2005. ① Sixty-four rats were made into focal ischemia by middle cerebral artery occlusion (MCAO) with a nylon monofilament suture. The successfully induced rats were assigned to inosine group (n =32) and model group (n =32) at random. Rats in the inosine group were intraperitoneally administrated with inosine in dose of 100 mg/kg preoperatively, twice a day, 7 days in all. The rats in the control group were injected with the same dose of saline solution by the similar way preoperatively. Each group was randomized into ischemia /reperfusion 2, 6, 12, 24 hours, 2, 3, 7 and 14 days subgroups consisted of 4 rats. The other 4 rats were taken as the sham-operated group, the rats were given the same treatment except for not introduced the filament into the external carotid artery stump, and brain tissue was removed at 2 hours of reperfusion. ② In situ hybridization was performed to examine the expression of cytochrome C mRNA while TUNEL staining was made to characterize apoptosis. ③ The t test was used to compare the difference of measurement data. MAIN OUTCOME MEASURES: ① Neuronal apoptosis in the different regions of the ischemic brain tissue; ② Expression of cytochrome C mRNA in the different regions at different time points after MCAO. RESULTS: All the 68 rats were involved in the analysis of results. ① Neuronal apoptosis: A small number of TUNEL-positive cells were detected in the sham-operated brain and non-ischemic brain. The number of apoptotic cells in the ischemic cortex peaked at 24 hours of reperfusion [(72.00±1.98) cells] and that in the striatum peaked at 2 days [(94.75±3.57) cells], then decreased to the level of sham-operated group at 14 days. Inosine could reduce apoptotic cells from 12 hours to 7 days of reperfusion as compared with the model group (t =6.19-26.67, P < 0.01). ② Cytochrome C mRNA expression: There was weak expression of cytochrome C mRNA in both sham-operated brain and contralateral brain. Cytochrome C was detected at 2 hours of reperfusion in ischemic brain [(25.75±3.50), (39.75±2.49) cells], and strongly increased to a peak at 12 hours and 24 hours of reperfusion in cortex and striatum [(122.50±6.69), (119.25±5.12) cells], respectively. Furthermore, inosine could significantly decrease cytochrome C expression in cortex at 12 hours to 14 days of reperfusion after ischemic reperfusion and that in striatum at 12 hours to 3 days (t =8.67-43.26, P < 0.01). CONCLUSION: Inosine can exert a neuroprotective effect by inhibiting apoptosis and cytochrome C mRNA expression.

Effect of polygonatum polysaccharide on the hypoxia-induced apoptosis and necrosis in in vitro cultured cerebral cortical neurons from neonatal rats

BACKGROUND： Cardiocerebrovascular diseases induced cerebral circulation insufficiency and senile vascular dementia can result in ischemic/hypoxic apoptosis of central neurons, which we should pay more attention to and prevent and treat as early as possible. Traditional Chinese medicine possesses the unique advantage in this field. Polygonatum, a Chinese herb for invigorating qi, may play a role against the hypoxic apoptosis of brain neurons. OBJECTIVE ： To observe the protective effect of polygonatum polysaccharide on hypoxia-induced apoptosis and necrosis in cerebral cortical neurons cultured in vitro. DESIGN： A comparative experiment.SETTING： Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine. MATERIALS： The experiment was carried out in the Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine from November 2003 to April 2005. Totally 218 Wistar rats （male or female） of clean degree within 24 hours after birth were purchased from the animal center of Jiangxi Medical College （certification number was 021-97-03）. METHODS：① Preparation of cerebral cortical neurons of rats： The cerebral cortical tissues were isolated from the Wistar rats within 24 hours after birth, and prepared to single cell suspension, and the cerebral cortical neurons of neonatal rats were in vitro cultured in serum free medium with Neurobasal plus B27 Supplement. ② Observation on the non-toxic dosage of polygonatum polysaccharide on neurons： After the neurons were cultured for 4 days, polygonatum polysaccharide of different dosages （1-20 g/L） was added for continuous culture for 48 hours, the toxicity and non-toxic dosage of polygonatum polysaccharide on neurons were observed and detected with trypan blue staining. ③Grouping： After hypoxia/reoxygenation, the cultured neurons were divided into normal control group, positive apoptotic group and polygonatum polysaccharide group. In the normal control group, the neurons were cultured at 37℃ in CO2 with the volume fraction of 0.05 under saturated humidity for 6 days. In the apoptotic positive group, the neurons were cultured with hypoxia for 12 hours after 4-day culture, and followed by reoxygenation for 48 hours. In the polygonatum polysaccharide group, polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to some neurons at 10 hours before the hypoxia culture, and then the neurons were cultured with hypoxia for 12 hours, followed by reoxygenation for 48 hours; polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to the other neurons at 12 hours after hypoxia followed by reoxygenation for 48 hours.④ The Hoechst33342 fluorescence staining, Annexin V/PI flow cytometer, appearance of DNA agarose gel electrophoresis gradient strap and immunohistochemical staining were used to observe the expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax, and observe the effect of polygonatum polysaccharide against the hypoxic apoptosis of cerebral cortical neurons of neonatal rats. MAIN OUTCOME MEASURES： ① Toxicity and non-toxic dosage of polygonatum polysaccharide on neurons;② Apoptotic rate of neurons detected with Hoechst33342 fluorescence staining;③ Early apoptotic rate and necrotic rate of neurons detected with Annexin V/PI flow cytometer; ④DNA agarose gel electrophoresis ladder-like strap appeared or not;⑤ Expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax. RESULTS：① Polygonatum polysaccharide within 6 g/L had no cytotoxicity on the normal cultured cerebral cortical neurons （P 〉 0.05）. ②The apoptotic rates of neurons detected with Hoechst33342 fluorescence staining had significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 10 hours before the hypoxia culture [（13.00±4.52）%,（12.72±2.15）%, （11.80±1.18）%,（38.03±1.05）%, P 〈 0.01], and had no significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 12 hours after the hypoxia culture （36.77±1.45）%, （36.60±1.61）%, （36.37±2.02）%, （38.03±1.05）%, P 〉 0.05].③ Annexin V/PI flow cytometer detected that the anti-necrotic effect was enhanced with the increased concentration of polygonatum polysaccharide within 0.5-1.5 g/L （P 〈 0.01）. Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia could significantly decrease the apoptotic rate of neurons induced by hypoxia/reoxygenation （P 〈 0.01）. ④ No DNA agarose gel electrophoresis ladder-like strap appeared in the groups with polygonatum polysaccharide of 0.5-1.5 g/L added at 10 hours before hypoxia;⑤ After Polygonatum polysaccharide of 0.5-1.5 g/L was added before hypoxia, the expression of Bcl-2 protein of hypoxic neurons was increased （P 〈 0.01）, and those of Bax protein and Caspase-3 protein were reduced （P 〈 0.01）, and the ratio of Bcl-2/Bax was increased （P 〈 0.01）. CONCLUSION： Polygonatum polysaccharide within 6 g/L has no cytotoxicity on the normal cultured cerebral cortical neurons. Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia plays a role agains necrosis of neurons induced by hypoxia. Polygonatum polysaccharide of 0.5-1.5 g/L can significantly reduce the apoptosis of neurons induced by hypoxia through up-regulating the expression of Bcl-2 protein, down-regulating the expressions of Bax protein and Caspase-3 protein, and increasing the ratio of Bcl-2/Bax.

Duodenal-jejunal bypass improves hypothalamic oxidative stress and inflammation in diabetic rats via glucagon-like peptide 1-mediated Nrf2/HO-1 signaling

BACKGROUND Type 2 diabetes mellitus(T2DM)is often accompanied by impaired glucose utilization in the brain,leading to oxidative stress,neuronal cell injury and inflammation.Previous studies have shown that duodenal jejunal bypass(DJB)surgery significantly improves brain glucose metabolism in T2DM rats,the role and the metabolism of DJB in improving brain oxidative stress and inflammation condition in T2DM rats remain unclear.AIM To investigate the role and metabolism of DJB in improving hypothalamic oxidative stress and inflammation condition in T2DM rats.METHODS A T2DM rat model was induced via a high-glucose and high-fat diet,combined with a low-dose streptozotocin injection.T2DM rats were divided into DJB operation and Sham operation groups.DJB surgical intervention was carried out on T2DM rats.The differential expression of hypothalamic proteins was analyzed using quantitative proteomics analysis.Proteins related to oxidative stress,inflammation,and neuronal injury in the hypothalamus of T2DM rats were analyzed by flow cytometry,quantitative real-time PCR,Western blotting,and immunofluorescence.RESULTS Quantitative proteomics analysis showed significant differences in proteins related to oxidative stress,inflammation,and neuronal injury in the hypothalamus of rats with T2DM-DJB after DJB surgery,compared to the T2DM-Sham groups of rats.Oxidative stress-related proteins(glucagon-like peptide 1 receptor,Nrf2,and HO-1)were significantly increased(P<0.05)in the hypothalamus of rats with T2DM after DJB surgery.DJB surgery significantly reduced(P<0.05)hypothalamic inflammation in T2DM rats by inhibiting the activation of NF-κB and decreasing the expression of interleukin(IL)-1βand IL-6.DJB surgery significantly reduced(P<0.05)the expression of factors related to neuronal injury(glial fibrillary acidic protein and Caspase-3)in the hypothalamus of T2DM rats and upregulated(P<0.05)the expression of neuroprotective factors(C-fos,Ki67,Bcl-2,and BDNF),thereby reducing hypothalamic injury in T2DM rats.CONCLUSION DJB surgery improve oxidative stress and inflammation in the hypothalamus of T2DM rats and reduce neuronal cell injury by activating the glucagon-like peptide 1 receptor-mediated Nrf2/HO-1 signaling pathway.

Urolithin A alleviates blood-brain barrier disruption and attenuates neuronal apoptosis following traumatic brain injury in mice

Urolithin A(UA)is a natural metabolite produced from polyphenolics in foods such as pomegranates,berries,and nuts.UA is neuroprotective against Parkinson’s disease,Alzheimer’s disease,and cerebral hemorrhage.However,its effect against traumatic brain injury remains unknown.In this study,we established adult C57BL/6J mouse models of traumatic brain injury by controlled cortical impact and then intraperitoneally administered UA.We found that UA greatly reduced brain edema;increased the expression of tight junction proteins in injured cortex;increased the immunopositivity of two neuronal autophagy markers,microtubule-associated protein 1A/B light chain 3A/B(LC3)and p62;downregulated protein kinase B(Akt)and mammalian target of rapamycin(mTOR),two regulators of the phosphatidylinositol 3-kinase(PI3K)/Akt/mTOR signaling pathway;decreased the phosphorylation levels of inhibitor of NFκB(IκB)kinase alpha(IKKα)and nuclear factor kappa B(NFκB),two regulators of the neuroinflammation-related Akt/IKK/NFκB signaling pathway;reduced blood-brain barrier permeability and neuronal apoptosis in injured cortex;and improved mouse neurological function.These findings suggest that UA may be a candidate drug for the treatment of traumatic brain injury,and its neuroprotective effects may be mediated by inhibition of the PI3K/Akt/mTOR and Akt/IKK/NFκB signaling pathways,thus reducing neuroinflammation and enhancing autophagy.

Postischemic Housing Environment on Cerebral Metabolism and Neuron Apoptosis after Focal Cerebral Ischemia in Rats

The purpose of this study was to evaluate the roles of different housing environments in neurological function, cerebral metabolism, cerebral infarction and neuron apoptosis after focal cerebral ischemia. Twenty-eight Sprague-Dawley rats were divided into control group （CG） and cerebral ischemia group, and the latter was further divided into subgroups of different housing conditions： standard environment （SE） subgroup, individual living environment （IE） subgroup, and enriched environment （EE） subgroup. Focal cerebral ischemia was induced by the middle cerebral artery occlusion （MCAO）. Beam walking test was used to quantify the changes of overall motor function. Cerebral infarction and cerebral metabolism were studied by in vivo magnetic resonance imaging and 1H-magnetic resonance spectra, respectively. Neuron necrosis and apoptosis were detected by hematoxylin-eosin and TUNEL staining methods, respectively. The results showed that performance on the beam-walk test was improved in EE subgroup when compared to SE subgroup and IE subgroup. Cerebral infarct volume in IE subgroup was significantly larger than that in SE subgroup （P〈0.05） and EE subgroup （P〈0.05） on day 14 after MCAO. NAA/Cr and Cho/Cr ratios were lower in MCAO groups under different housing conditions as compared to those in CG （P〈0.05）. NAA/Cr ratio was lower in IE subgroup （P〈0.05） and higher in EE subgroup （P〈0.05） than that in SE subgroup. NAA/ Cr ratio in EE was significantly higher than that in IE subgroup （P〈0.05）. Cho/Cr ratio was decreased in MCAO groups as compared to that in CG （P〈0.05）. A significant decrease in normal neurons in cerebral cortex was observed in MCAO groups as compared to CG （P〈0.05）. The amount of normal neurons was less in IE subgroup （P〈0.05）, and more in EE subgroup （P〈0.05） than that in SE subgroup after MCAO. The amotmt of normal neurons in EE subgroup was significantly more than that in IE subgroup after MCAO （P〈0.05）. The ratio of TUNEL-positive neurons in EE was significantly lower than that in SE subgroup （P〈0.05） and IE subgroup （P〈0.05）. Correlation analysis showed that the beam walking test was negatively correlated with NAA/Cr ratio （P〈0.05）. Cerebral infarct volume was negatively correlated with both NAA/Cr ratio （P〈0.01） and Cho/Cr ratio （P〈0.01）. The amount of normal cortical neurons was positively correlated with both NAA/Cr ratio （P〈0.0I） and Cho/Cr ratio （P〈0.05）. The TUNEL-positive neurons showed a negative correlation with both NAA/Cr ratio （P〈0.01） and Cho/Cr ratio （P〈0.01）. This study goes further to show that EE may improve neurological functional deficit and cerebral metabolism, decrease cerebral infarct volume, neuron necrosis and apoptosis, while IE may aggravate brain damage after MCAO.

Mitochondrial protective and anti-apoptotic effects of Rhodiola crenulata extract on hippocampal neurons in a rat model of Alzheimer's disease

In our previous study, we found that the edible alcohol extract of the root of the medicinal plant Rhodiola crenulata（RCE） improved spatial cognition in a rat model of Alzheimer＇s disease. Another study from our laboratory showed that RCE enhanced neural cell proliferation in the dentate gyrus of the hippocampus and prevented damage to hippocampal neurons in a rat model of chronic stress-induced depression. However, the mechanisms underlying the neuroprotective effects of RCE are unclear. In the present study, we investigated the anti-apoptotic effect of RCE and its neuroprotective mechanism of action in a rat model of Alzheimer＇s disease established by intracerebroventricular injection of streptozotocin. The rats were pre-administered RCE at doses of 1.5, 3.0 or 6.0 g/kg for 21 days before model establishment. ATP and cytochrome c oxidase levels were significantly decreased in rats with Alzheimer＇s disease. Furthermore, neuronal injury was obvious in the hippocampus, with the presence of a large number of apoptotic neurons. In comparison, in rats given RCE pretreatment, ATP and cytochrome c oxidase levels were markedly increased, the number of apoptotic neurons was reduced, and mitochondrial injury was mitigated. The 3.0 g/kg dose of RCE had the optimal effect. These findings suggest that pretreatment with RCE prevents mitochondrial dysfunction and protects hippocampal neurons from apoptosis in rats with Alzheimer＇s disease.

Extracts of Bauhinia Championii Alleviate Acute Neuronal Injury After Ischemic Reperfusion by Improving Endoplasmic Reticulum Stress-Mediated Neuronal Apoptosis

Objective Cerebral ischemia/reperfusion(I/R)is a potential factor for lethal injury,and currently lacks effective remedies.Bauhinia championii extracts(BCEs)have been reported to exhibit anti-oxidative and anti-hypoxia properties.The current work aimed to study whether BCE could alleviate neuronal injury caused by I/R.Methods To investigate the protective effects of BCE,oxygen-glucose deprivation/reperfusion(OGD/R)was applied to the HT22 cell line in vitro and to a cerebral I/R mouse model in vivo.Results Under OGD/R,the survival of HT22 cells was significantly prolonged after treatment with BCE.In vivo,BCE significantly reduced the infarct area and decreased neuronal apoptosis caused by I/R.It was further found that OGD/R could trigger endoplasmic reticulum(ER)stress and induce ER stress-mediated neuronal apoptosis in vivo and in vitro,while BCE could effectively alleviate ER stress and neuronal apoptosis.Conclusion These results suggested that BCE exhibits neuroprotective effects by reducing ER stress-mediated apoptosis after cerebral I/R injury.BCE may therefore be an effective therapeutic regimen against cerebral I/R damage.

Relevance and therapeutic potential of Cyp A targeting to block apoptosis inducing factor-mediated neuronal cell death

Programmed cell death （PCD） signaling pathways are import- ant contributors to acute neurological insults such as hypox- ic-ischemic brain damage, traumatic brain injury, stroke etc. The pathogenesis of all these diseases is closely linked with ab- erration of apoptotic cell death pathways. Mitochondria play a crucial role during PCD, acting as both sensors of death signals, and as initiators of biochemical path- ways, which cause cell death （Bras et al., 2005）. Cytochrome c was the firstly identified apoptogenic factor released from mitochondria into the cytosol, where it induces apoptosome formation through the activation of caspases. Other proteins, such as apoptosis inducing factor （AIF）, have been subsequently identified as mitochondrial released factors. AIF contributes to apoptotic nuclear DNA damage （Bras et al., 2005）. in a caspase-independent way

Pro-urokinase promotes angiogenesis but does not reduce neuronal apoptosis in infarcted cerebral tissue

Ischemic stroke is most commonly caused by vascular occlusion due to thrombosis or arterial embolism. Recently, thrombolysis has been used with increasing frequency for the treatment of acute ischemic stroke. Among the drugs used for thrombolysis, only recombinant tissue plasminogen activator is widely accepted internationally （Albers et al., 2008）. In China, urokinase has been widely used for thrombolysis after acute ischemic stroke. Pro-uro- kinase is the precursor of urokinase. Compared with urokinase, pro-uroki- nase has greater ability to dissolve thrombus and is safer to use.

Protective effects of (-)-epigallocatechin-3-gallate on D-galactose-induced neuronal apoptosis in mice

BACKGROUND： The neuroprotective effects of （-）-epigallocatechin-3-gallate （EGCG）, the main polyphenolic constituent of green tea, have been widely reported. However, the action mechanisms, in particular in D-galactose-induced aging mice, remain poorly understood. OBJECTIVE： The present study investigated the protective effects of EGCG on D-galactose-induced hippocampus neuronal apoptosis in aging mice, as well as the relationship with expression of p751CD, JNK2, and p53 proteins. DESIGN, TIME AND SETTING： A randomized, controlled, molecular biological, animal experiment was performed at the Laboratory of Pharmacology, Pharmaceutical College of China Medical University, China, from September 2006 to July 2008. MATERIALS： D-galactose and EGCG （Sigma, USA）, as well as terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling （TUNEL） In Situ Cell Apoptosis Detection Kit （Promega, USA）, were used in this study. METHODS： A total of 64 mice were equally and randomly divided into D-galactose model, low-dose EGCG, high-dose EGCG, and control groups. Mice in the D-galactose model, low-dose EGCG, and high-dose EGCG groups were subcutaneously injected with 3% D-galactose （150 mg/kg）, daily for 6 weeks, to establish a mouse model of aging. Mice in the control group were treated with saline （5 mL/kg）. At 3 weeks following injection, mice in the low-dose EGCG and high-dose EGCG groups were orally administered EGCG at a dose of 2 mg/kg and 6 mg/kg, respectively, once a day, for 4 consecutive days. Mice in the control and D-galactose model groups received distilled water （5 mL/kg）. MAIN OUTCOME MEASURES： Memory function was evaluated using a step-through passive avoidance test. Neuronal apoptosis in the mouse hippocampus was detected using TUNEL staining. Expression levels of the intracellular domain of the p75 neurotrophin receptor （p75NTR）-p751CD, JNK2, and p53 proteins in the hippocampus were determined using Western blot analysis. RESULTS： The aging mouse model was induced by subcutaneous injection of D-galactose, which resulted in obvious memory impairment, increased apoptotic index, and increased protein expression levels of p751CD, JNK2, and p53 in the hippocampus, compared with control mice （P 〈 0.01）. Oral EGCG administration （2 or 6 mg/kg） for 4 weeks significantly improved levels of memory deficit in the aging mice and reduced apoptotic indices and protein expression levels of p751CD, JNK2, and p53 in the mouse hippocampus （P 〈 0.01）. CONCLUSION： Results from this study demonstrated increased protein expression levels of p751CD, JNK2, and p53, as well as increased hippocampal neuronal apoptosis in a D-galactose-induced mouse model of aging. EGCG provided protective effects against D-galactose-induced neuronal apoptosis in the hippocampus by reducing protein expression levels of p751CD, JNK2, and p53 proteins in the hippocampus of aging mice.

Exploring the mechanism of neuronal apoptosis and brain developmental damage in the hippocampus of hypoxicischemic neonatal rats based on BDNF/TrkB/CREB pathway

Objective:Based on the BDNF/TrkB/CREB pathway,to explore the mechanism of neuronal apoptosis and brain developmental injury in the hippocampus of hypoxic-ischemic neonatal rats.Methods:Wistar young rats were ligated on one side of the common carotid artery and placed in an 8%oxygen and 92%nitrogen hypoxia box for 2 h to prepare hypoxic-ischemic brain injury models.Healthy rats were used as the control group.Control group and model group were selected,with 10 rats in each group,and the learning and memory ability was tested by Y-maze;2,3,5-triphenyltetrazolium chloride(TTC)staining was used to detect brain tissue damage;Western blot was performed to determine the expression of brain-derived neurotrophic factor(BDNF),tyrosine protein kinase B(TrKB)and cAMP-response element binding protein(CREB)in hippocampal tissue.Another 15 mice in the control group and 60 mice in the model group were divided into negative control group(NC),BDNF overexpression group(LV-BDNF),TrkB overexpression group(LV-TrkB),and CREB overexpression group(LV-CREB),blank vector,BDNF,TrkB,CREB adenovirus overexpression vector was injected into the tail vein.Y-maze test for learning and memory ability;TTC staining method to detect brain tissue damage;neuronal apoptosis in the hippocampus were detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling;Western blot to detect the level of neuronal apoptosis in the hippocampus.Apoptosis-related protein B-cell lymphoma-2(Bcl-2),BCL2associated X protein(Bcl-2 Assaciated X,Bax)and nuclear factor kappaB(NFκB)expression.Results:The learning and memory ability of the young mice in the model group was significantly reduced,the brain infarct volume was significantly increased,the expressions of BDNF and TrkB proteins in the hippocampus were significantly increased,and the expression of CREB proteins was significantly decreased;After overexpression of BDNF and TrkB CREB,in the LVBDNF,LVTrkB,and LVCREB group,the learning and memory ability of young mice were significantly improved,the brain infarct volume were significantly reduced,the hippocampal neuronal apoptosis were significantly reduced,The protein expression of Bax and NFκB were significantly decreased and the protein expression of Bcl2 were significantly enhanced.Conclusion:The expression of BDNF/TrkB/CREB is abnormal in HIBI model young mice.Overexpression of BDNF/TrkB/CREB can improve the learning and memory ability of young mice,repair brain tissue damage,and inhibit neuronal apoptosis.Therefore,the mechanism of HIBI may be related to BDNF/TrkB/CREB pathways.

Role of alpha-synuclein in neuronal apoptosis induced by rotenone

BACKGROUND： Aggregation of α-synuclein is the major component of Lewy bodies, which are the pathological hallmarks of Parkinson disease （PD）. Although the mechanism of this protein aggregates is unclear, previous study showed that environmental toxins such as rotenone could induce the expression and aggregation of α-synuclein. OBJECTIVE： To observe the role of α-synuclein in PD.DESIGN ： A randomized controlled trial.SETTING ： Beijing Institute for Neuroscience, Capital University of Medical Sciences.MATERIALS ： This study was performed from July 2005 to January 2006 at the Beijing Institute for Neuroscience, Capital University of Medical Sciences. Human dopaminergic neuroblastoma SH-SY5Y cells were provided by Beijing Institute for Neuroscience, Capital University of Medical Sciences. METHODS： Human dopaminergic neuroblastoma SH-SY5Y cells were treated to make α-synuclein over express. Rotenone was added into the medium of cultured both native SH-SY5Y cells and α-synuclein-overexpression SH-SY5Y cells. Lactate dehydrogenase （LDH） assay was used to detect with the cell viability. Flow cytometry and electrophoresis were adopted to measure the cell apoptosis. MAIN OUTCOME MEASURES ： Cell viability, DNA fragmentation, and the number of cell apoptosis.RESULTS： After being treated with rotenone, LDH activity of α-synuclein overexpressed SH-SY5Y cells was （76.625±6.34） μkat/L, which was significantly lower than that of control group （P 〈 0.05）. As compared with normal SH-SY5Y cell, α-synuclein over-expressed SH-SY5Y cells had less DNA fragments and apoptotic cells, α-synuclein might play a role in cell apoptosis induced by rotenone, which was also confirmed by using of antioxidant reagent. CONCLUSION： α-synuclein may partially protect against cell apoptosis induced by rotenone in SH-SY5Y cells.

Effect of ketamine on aquaporin-4 expression and neuronal apoptosis in brain tissues following brain injury in rats

BACKGROUND： Aquaporin-4 （AQP-4） is closely related to the formation of brain edema. Neuronal apoptosis plays an important part in the conversion of swelled neuron following traumatic brain injury. At present, the studies on the protective effect of ketamine on brain have involved in its effect on aquaporin-4 expression and neuronal apoptosis in the brain tissues following brain injury in rats. OBJECTIVE： To observe the effect of ketamine on AQP-4 expression and neuronal apoptosis in the brain tissue following rat brain injury, and analyze the time-dependence of ketamine in the treatment of brain injury.DESIGN： Randomized grouping design, controlled animal tria SETTING ： Department of Anesthesiology, the Medical School Hospital of Qingdao University MATERIALS： Totally 150 rats of clean grade, aged 3 months, were involved and randomized into control group and ketamine-treated group, with 75 rats in each. Each group was divided into 5 subgroups separately at 6,12, 24, 48 and 72 hours after injury, with 15 rats at each time point. Main instruments and reagents： homemade beat machine, ketamine hydrochloride （Hengrui Pharmaceutical Factory, Jiangsu）, rabbit anti-rat AQP-4 polyclonal antibody, SABC immunohistochemical reagent kit and TUNEL reagent kit （Boster Co.,Ltd., Wuhan）. METHODS： This trial was carried out in the Institute of Cerebrovascular Disease, Medical College of Qingdao University during March 2005 to February 2006. A weight-dropping rat model of brain injury was created with Feeney method. The rats in the ketamine-treated group were intraperitoneally administered with 50 g/L ketamine （120 mg/kg） one hour after injury, but ketamine was replaced by normal saline in the control group. In each subgroup, the water content of cerebral hemisphere was measured in 5 rats chosen randomly. The left 10 rats in each subgroup were transcardiacally perfused with ketamine, then the brain tissue was made into paraffin sections and stained by haematoxylin and eosin. Neuronal morphology was observed. AQP-4 expression and neuronal apoptosis were measured with immunohistochemical method and TUNEL method respectively. MAIN OUTCOME MEASURES： Water content in brain tissue, neuronal morphology, the number of AQP-4 positive neurons and TUNEL positive neurons in rats of two groups at each time point after injury. RESULTS： Totally 150 rats entered the stage of result analysis. （1） Water content of brain tissue： The water content of brain tissue at each time point after injury in the ketamine-treated group was lower than that in the control group. There were very significant differences in water content at 12 and 24 hours after injury respectively between ketamine-treated group and control group [（77.34±2.35）% vs. （82.31 ±1.48）%; （78.01 ±2.21 ）% vs. （83.86±2.37）%, t=-4.001 6,4.036 7, both P 〈 0.01]. （2） Neuronal morphology： Pathological changes in traumatic region and peripheral region of injury in the ketamine-treated group were significantly lessened, and necrotic and apoptotic cells in the ketamine-treated group were also significantly reduced as compared with control group. （3） AQP-4 expression： AQP-4 positive neurons at each time point in the ketamine-treated group were significantly less than those in the control group. There were very significant differences in AQP-4 expression at 12 and 24 hours after injury between ketamine-treated group and control group [（34.17±4.74） /visual field vs. （43.42±5.65） /visual field;（40.83±3.17） /visual field vs. （58.88±6.23） /visual field,t=3.966 3,8.165 7, both P〈 0.01]. （4） Neuronal apoptosis： TUNEL positive neurons at each time point in the ketamine-treated group were less than those in the control group. There were very significant differences in the neuronal apoptosis at 12 and 24 hours after injury between ketamine-treated group and control group [（26.25±3.04） /visual field vs. （32.75±4.39） /visual field; （29.33± 4.02） /visual field vs. （39.83±5.61） /visual field,t=-3.849 3,5.169 2,both P 〈 0.01]. CONCLUSION： Ketamine can reduce brain edema, AQP-4 expression and neuronal apoptosis following brain injury in rats, and has obvious therapeutic effect on brain injury, especially at 12 and 24 hours after injury.

Dantrolene enhances the protective effect of hypothermia on cerebral cortex neurons

Therapeutic hypothermia is the most promising non-pharmacological neuroprotective strategy against ischemic injury. However, shivering is the most common adverse reaction. Many studies have shown that dantrolene is neuroprotective in in vitro and in vivo ischemic injury models. In addition to its neuroprotective effect, dantrolene neutralizes the adverse reaction of hypothermia. Dantrolene may be an effective adjunctive therapy to enhance the neuroprotection of hypothermia in treating ischemic stroke. Cortical neurons isolated from rat fetuses were exposed to 90 minutes of oxygen-glucose deprivation followed by reoxygenation. Neurons were treated with 40 μM dantrolene, hypothermia（at 33°C）, or the combination of both for 12 hours. Results revealed that the combination of dantrolene and hypothermia increased neuronal survival and the mitochondrial membrane potential, and reduced intracellular active oxygen cytoplasmic histone-associated DNA fragmentation, and apoptosis. Furthermore, improvements in cell morphology were observed. The combined treatment enhanced these responses compared with either treatment alone. These findings indicate that dantrolene may be used as an effective adjunctive therapy to enhance the neuroprotective effects of hypothermia in ischemic stroke.

The neuroprotective effects of oxygen therapy in Alzheimer’s disease:a narrative review

Alzheimer’s disease(AD)is a degenerative neurological disease that primarily affects the elderly.Drug therapy is the main strategy for AD treatment,but current treatments suffer from poor efficacy and a number of side effects.Non-drug therapy is attracting more attention and may be a better strategy for treatment of AD.Hypoxia is one of the important factors that contribute to the pathogenesis of AD.Multiple cellular processes synergistically promote hypoxia,including aging,hypertension,diabetes,hypoxia/obstructive sleep apnea,obesity,and traumatic brain injury.Increasing evidence has shown that hypoxia may affect multiple pathological aspects of AD,such as amyloid-beta metabolism,tau phosphorylation,autophagy,neuroinflammation,oxidative stress,endoplasmic reticulum stress,and mitochondrial and synaptic dysfunction.Treatments targeting hypoxia may delay or mitigate the progression of AD.Numerous studies have shown that oxygen therapy could improve the risk factors and clinical symptoms of AD.Increasing evidence also suggests that oxygen therapy may improve many pathological aspects of AD including amyloid-beta metabolism,tau phosphorylation,neuroinflammation,neuronal apoptosis,oxidative stress,neurotrophic factors,mitochondrial function,cerebral blood volume,and protein synthesis.In this review,we summarized the effects of oxygen therapy on AD pathogenesis and the mechanisms underlying these alterations.We expect that this review can benefit future clinical applications and therapy strategies on oxygen therapy for AD.

MicroRNAs:protective regulators for neuron growth and development

MicroRNAs(miRNAs)play an important regulatory role in neuronal growth and development.Different mi RNAs target different genes to protect neurons in different ways,such as by avoiding apoptosis,preventing degeneration mediated by conditional mediators,preventing neuronal loss,weakening certain neurotoxic mechanisms,avoiding damage to neurons,and reducing inflammatory damage to them.The high expression of mi RNAs in the brain has significantly facilitated their development as protective targets for therapy,including neuroprotection and neuronal recovery.mi RNA is indispensable to the growth and development of neurons,and in turn,is beneficial for the development of the brain and checking the progression of various diseases of the nervous system.It can thus be used as an important therapeutic target for models of various diseases.This review provides an introduction to the protective effects of mi RNA on neurons in case of different diseases or damage models,and then provides reference values and reflections on the relevant treatments for the benefit of future research in the area.

Electroacupuncture preconditioning attenuates ischemic brain injury by activation of the adenosine monophosphate-activated protein kinase signaling pathway

Electroacupuncture has therapeutic effects on ischemic brain injury, but its mechanism is still poorly understood. In this study, mice were stimulated by electroacupuncture at the Baihui（GV20） acupoint for 30 minutes at 1 m A and 2/15 Hz for 5 consecutive days. A cerebral ischemia model was established by ligating the bilateral common carotid artery for 15 minutes. At 72 hours after injury, neuronal injury in the mouse hippocampus had lessened, and the number of terminal deoxynucleotide transferase-mediated d UTP nick-end labeling-positive cells reduced after electroacupuncture treatment. Moreover, expression of adenosine monophosphate-activated protein kinase α（AMPKα） and phosphorylated AMPKα was up-regulated. Intraperitoneal injection of the AMPK antagonist, compound C, suppressed this phenomenon. Our findings suggest that electroacupuncture preconditioning alleviates ischemic brain injury via AMPK activation.