Time representation of mitochondrial morphology and function after acute spinal cord injury

Changes in mitochondrial morphology and function play an important role in secondary damage after acute spinal cord injury. We recorded the time representation of mitochondrial morphology and function in rats with acute spinal cord injury. Results showed that mitochondria had an irregular shape, and increased in size. Mitochondrial cristae were disordered and mitochondrial membrane rupture was visible at 2–24 hours after injury. Fusion protein mitofusin 1 expression gradually increased, peaked at 8 hours after injury, and then decreased to its lowest level at 24 hours. Expression of dynamin-related protein 1, amitochondrial fission protein, showed the opposite kinetics. At 2–24 hours after acute spinal cord injury, malondialdehyde content, cytochrome c levels and caspase-3 expression were increased, but glutathione content, adenosine triphosphate content, Na+-K+-ATPase activity and mitochondrial membrane potential were gradually reduced. Furthermore, mitochondrial morphology altered during the acute stage of spinal cord injury. Fusion was important within the first 8 hours, but fission played a key role at 24 hours. Oxidative stress was inhibited, biological productivity was diminished, and mitochondrial membrane potential and permeability were reduced in the acute stage of injury. In summary, mitochondrial apoptosis is activated when the time of spinal cord injury is prolonged.     

Mitochondrial division inhibitor 1 protects cortical neurons from excitotoxicity:a mechanistic pathway

Mitochondrial division inhibitor 1(Mdivi-1) is a selective cell-permeable inhibitor of dynamin-related protein-1(Drp1) and mitochondrial division.To investigate the effect of Mdivi-1 on cells treated with glutamate,cerebral cortex neurons isolated from neonatal rats were treated with 10 m M glutamate for 24 hours.Normal cultured cells and dimethyl sulfoxide-cultured cells were considered as controls.Apoptotic cells were detected by flow cytometry.Changes in mitochondrial morphology were examined by electron microscopy.Drp1,Bax,and casp ase-3 expression was evaluated by western blot assays and immunocytochemistry.Mitochondrial membrane potential was detected using the JC-1 probe.Twenty-four hours after 10 m M glutamate treatment,Drp1,Bax and caspase-3 expression was upregulated,Drp1 and Bax were translocated to mitochondria,mitochondrial membrane potential was decreased and the rate of apoptosis was increased.These effects were inhibited by treatment with 50 μM Mdivi-1 for 2 hours.This finding indicates that Mdivi-1 is a candidate neuroprotective drug that can potentially mitigate against neuronal injury caused by glutamate-induced excitotoxicity.     

Oxymatrine reduces neuroinflammation in rat brain A signaling pathway

Cerebral neuroinflammation models were established by injecting 10 μg lipopolysaccharide into the hippocampus of male Sprague-Dawley rats. The rats were treated with an intraperitoneal injection of 120, 90, or 60 mg/kg oxymatrine daily for three days prior to the lipopolysaccharide injection. Twenty-four hours after model induction, the hippocampus was analyzed by real-time quantitative PCR, and the cerebral cortex was analyzed by enzyme-linked immunosorbent assay and western blot assay. The results of the enzyme-linked immunosorbent assay and the real-time quantitative PCR showed that the secretion and mRNA expression of the pro-inflammatory cytokines interleukin-1β and tumor necrosis factor-α were significantly decreased in the hippocampus and cerebral cortex of model rats treated with oxymatrine. Western blot assay and real-time quantitative PCR analysis indicated that toll-like receptor 4 mRNA and protein expression were significantly decreased in the groups receiving different doses of oxymatrine. Additionally, 120 and 90 mg/kg oxymatrine were shown to reduce protein levels of nuclear factor-κB p65 in the nucleus and of phosphorylated IκBα in the cytoplasm of brain cells, as detected by western blot assay. Experimental findings indicate that oxymatrine may inhibit neuroinflammation in rat brain via downregulating the expression of molecules in the toll-like receptor 4/nuclear factor-κB signaling pathway.     

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

BACKGROUND: The high concentration of glutamate release is the main cause for neuronal cell death. The relationship between glutamate level and apoptosis during ischemia/reperfusion injury is still unclear. OBJECTIVE: To observe the neuronal apoptosis at 24 and 72 hours following cerebral ischemia/reperfusion in rats, and analyze the possible influencing factors. DESIGN: A randomized controlled animal experiment. SETTING: School of Medicine, Southern Yangtze University. MATERIALS: Totally 30 male adult Sprague Dawley (SD) rats of clean grade, weighing 240–290 g, were obtained from Shanghai Experimental Animal Center, Chinese Academy of Sciences. The rats were randomly divided into sham-operated group (n=10) and model group (n=20). Each group was observed at 24 and 72 hours after ischemia/reperfusion, 5 rats at each time point in the sham-operated group, whereas 12 at 24 hours and 8 at 72 hours in the model group. Kits for determining apoptosis and Bcl-2 were bought from Wuhan Boster Biological Technology, Co., Ltd.; Kit for calcineurin from Nanjing Jiancheng Bioengineering Institute. METHODS: The experiment was carried out in the Functional Scientific Research Room of Southern Yangtze University from June to October in 2006. ① Right middle cerebral artery was occluded by inserting a thread through internal carotid artery (ICA). The surgical process for the sham-operated rats was the same as that in the model group except a nylon suture inserted the ICA. According to Longa five-degree standard, the neurological deficit evaluation of rats was evaluated after surgery, and grades 1–3 were taken as successful model establishment. The blood was recirculated by withdrawing the nylon filament under anesthesia at 2 hours after ischemia in successful rat models. ②After reperfusion, the brain tissue was quickly removed at 24 or 72 hours and the slices were obtained from optic chiasma to funnel manubrium. The changes of the number of apoptotic cells were observed using the terminal deoxynucleotidyl transferase mediated dUTP-biotin nick-end labeling method. The expressions of Bcl-2 protein were determined with immunohistochemical staining. The activity of calcineurin was determined by the inorganic phosphorus method. The content of excitatory amino acid was detected by high performance liquid chromatography. MAIN OUTCOME MEASURES: ① Glutanate content in brain tissue; ② Conditions of apoptosis; ③ Calcineurin activity in brain tissue; ④ Bcl-2 expression in brain tissue. RESULTS: Totally 30 SD rats were used, 5 died and the other 25 were involved in the analysis of results. ① Changes of apoptosis: There were 0–3 apoptotic cells in the sham-operated group. In the model group, the numbers of apoptotic cells were obviously increased at 24 and 72 hours of reperfusion (P < 0.01), and it was markedly reduced at 72 hours as compared with 24 hours (P < 0.01). ② Changes of glutanate content: The glutamate contents at 24 and 72 hours of reperfusion in the model group were obviously higher than those in the sham-operated group (P < 0.01); In the model group, it was obviously increased at 24 hours as compared with 72 hours (P < 0.01). ③ Changes of Bcl-2 protein: In the model group, the Bcl-2 protein expression had no obvious changes at 24 hours of reperfusion, and it was obviously enhanced at 72 hours, which was obviously different from that in the sham-operated group and that at 24 hours (P < 0.01). ④ Changes of calcinerin activity: In the model group, the activity of calcineurin in brain tissue had no obvious changes at 24 hours of reperfusion; The activity of calcineurin at 72 hours was obviously higher than that in the sham-operated group and that at 24 hours (P < 0.01). CONCLUSION: The brain cyto-apoptosis action at different time points following reperfusion incompletely depends on the glutamate levels, while it depends on the interaction of some apoptosis related factors, such as amino acid, calcineurin, and Bcl-2, etc.     

Edaravone combined with Schwann cell transplantation may repair spinal cord injury in rats

Edaravone has been shown to delay neuronal apoptosis, thereby improving nerve function and the microenvironment after spinal cord injury. Edaravone can provide a favorable environment for the treatment of spinal cord injury using Schwann cell transplantation. This study used rat models of complete spinal cord transection at T9. Six hours later, Schwann cells were transplanted in the head and tail ends of the injury site. Simultaneously, edaravone was injected through the caudal vein. Eight weeks later, the PKH-26-labeled Schwann cells had survived and migrated to the center of the spinal cord injury region in rats after combined treatment with edaravone and Schwann cells. Moreover, the number of PKH-26-labeled Schwann cells in the rat spinal cord was more than that in rats undergoing Schwann cell transplantation alone or rats without any treatment. Horseradish peroxidase retrograde tracing revealed that the number of horseradish peroxidase-positive nerve fibers was greater in rats treated with edaravone combined with Schwann cells than in rats with Schwann cell transplantation alone. The results demonstrated that lower extremity motor function and neurophysiological function were better in rats treated with edaravone and Schwann cells than in rats with Schwann cell transplantation only. These data confirmed that Schwann cell transplantation combined with edaravone injection promoted the regeneration of nerve fibers of rats with spinal cord injury and improved neurological function.     

Hydrogen sulfide inhibits beta-amyloid peptide-induced apoptosis in PC12 cells and the underlying mechanisms*★

BACKGROUND: Studies have demonstrated that hydrogen sulfide (H2S) levels are 55% lower in brains of Alzheimer’s disease (AD) patients than in age-matched normal individuals, which suggests that H2S might be involved in some aspects of AD pathogenesis. OBJECTIVE: To observe the protective mechanisms of varied concentrations of H2S against β-amyloid-peptide (Aβ) induced apoptosis in pheochromoytoma (PC12) cells, and to analyze the pathway of action. DESIGN, TIME AND SETTING: A controlled, observational, in vitro experiment was performed at Neurophysiology Laboratory in Zhongshan Medical School, Sun Yat-sen University between July 2006 and May 2007. MATERIALS: PC12 cells were provided by the Animal Experimental Center of Medical School of Sun Yat-sen University. Glybenclamide, rhodamine123, and dihydrorhodamine123 were purchased from Sigma (USA). METHODS: PC12 cells were incubated at 37 ℃ in a 5% CO2-enriched incubator with RPMI-1640 medium, supplemented with 5% horse-serum and 10% fetal bovine serum. Cells in logarithmic growth curves received different treatment: The PC12 cells were maintains at 37 ℃ with the original medium, then incubated in Aβ25-35, sodium hydrosulfide (NaHS), glybenclamide, NaHS+ Aβ25-35, or pretreated with glybenclamide 30 minutes prior to administration of and Aβ25-35, respectively. MAIN OUTCOME MEASURES: (1) The survival rate of PC12 cells was detected by MTT assay and Hoechst staining. (2) The apoptosis rate of PC12 cells was detected utilizing flow cytometry with propidium iodide staining, and morphological changes of apoptotic cells were observed. (3) The mitochondrial membrane potential was detected by Rhodamine123-combined flow cytometry. (4) The intracellular reactive oxygen species content was detected by dihydrorhodamine123-combined flow cytometry. RESULTS: Aβ25-35 induced significantly decreased viability and increased percentage of apoptosis in PC12 cells, as well as dissipated mitochondrial membrane potential expression and an overproduction of reactive oxygen species. When PC12 cells were co-treated with NaHS and Aβ25-35, the decreased cell viability induced by 20 μmol/L Aβ25-35 was concentration-dependently blocked by NaHS (50, 100, and 200 μmol/L). NaHS (100 μmol/L) obviously reduced the percentage of apoptotic PC12 cells induced by 20 μmol/L Aβ25-35. In addition, 100 μmol/L NaHS inhibited mitochondrial membrane potential dissipation and reactive oxygen species overproduction. When the ATP-sensitive K channel (KATP) inhibitor, glybenclamide, was administered 30 minutes prior to NaHS and Aβ25-35 treatment, the NaHS-dependent cellular protection was partly blocked. This resulted in reduced PC12 cell viability and increased the percentage of apoptosis, as well as significantly blocked mitochondrial membrane potential preservation and inhibited reactive oxygen species overproduction due to NaHS treatment. CONCLUSION: NaHS protected PC12 cells against Aβ25-35-induced damage. NaHS-dependent cellular protection was associated with mitochondrial membrane potential preservation and inhibition of reactive oxygen species overproduction. The KATP channel inhibitor, glybenclamide, significantly blocked the cellular protective effects of NaHS, indicating that KATP channel activation plays an important role in NaHS-induced protection of PC12 cells to Aβ25-35-induced damage. Key Words: apoptosis; β-amyloid peptide; cytoprotection; hydrogen sulfide; mitochondrial membrane potential; reactive oxygen species     

8-hydroxy-2-(di-n-propylamino)tetralin intervenes with neural cell apoptosis following diffuse axonal injury

Previous studies have reported a neuroprotective effect of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) against traumatic brain injury. In accordance with the Marmarou method, rat models of diffuse axonal injury were established. 8-OH-DPAT was intraperitoneally injected into model rats 8-OH-DPAT treated rats maintained at constant temperature served as normal temperature controls TUNEL results revealed that neural cell swelling, brain tissue necrosis and cell apoptosis occurred around the injured tissue. Moreover, the number of Bax-, Bcl-2- and caspase-3-positive cells increased at 6 hours after diffuse axonal injury, and peaked at 24 hours. However, brain injury was attenuated, the number of apoptotic cells reduced, Bax and caspase-3 expression decreased, and Bcl-2 expression increased at 6, 12, 24, 72 and 168 hours after diffuse axonal injury in normal temperature control and in 8-OH-DPAT-intervention rats. The difference was most significant at 24 hours. All indices in 8-OH-DPAT-intervention rats were better than those in the constant temperature group. These results suggest that 8-OH-DPAT inhibits Bax and caspase-3 expression, increases Bcl-2 expression, and reduces neural cell apoptosis, resulting in neuroprotection against diffuse axonal injury. This effect is associated with a decrease in brain temperature.     

Interventional effect of magnesium sulfate on nitric oxide synthase activity after acute craniocerebral injury

BACKGROUND: Abnormal changes in magnesium ion are closely related to cerebral injury. At present, some evidence indicates that magnesium reagent can improve nerve function and prognosis of patients with cerebral injury. OBJECTIVE: To observe the effect of magnesium sulfate on changes in nitric oxide synthase (NOS) activity in brain tissue of rats with acute craniocerebral injury. DESIGN: Completely randomized grouping design and randomly controlled study. SETTING: Laboratory of Neurosurgery, the Third Hospital of Chinese PLA. MATERIALS: Fifty-four male SD rats of clean grade and weighing 220–250 g were randomly divided into normal control group (n =6), cerebral injury group (n =24) and magnesium sulfate group (n =24). Especially, rats in cerebral injury group and magnesium sulfate group were equally divided into four subgroups and observed at 0.5, 2, 6 and 24 hours after model establishment. A solution of 125 g/L of magnesium sulfate was provided by the Seventh Pharmaceutical Factory of Wuxi and the NOS assay kit by Nanjing Jiancheng Bioengineering Institute. METHODS: The experiment was carried out in the Institute of Neurosurgery, the Third Hospital of Chinese PLA from August 2000 to August 2002. ① Rats in the cerebral injury group and magnesium sulfate group were anesthetized to establish cerebral injury models based on modified Feeney technique; magnesium sulfate group were intraperitoneally injected 600 mg/kg magnesium sulfate (125 g/L), but rats in the normal control group remained untreated. ② At 0.5, 2, 6 and 24 hours after cerebral injury, rats in cerebral injury group and magnesium sulfate group were decapitated and brains were dissected. Cerebral cortex of rats in cerebral injury group was selected for NOS assay; in addition, at 0.5 hour after cerebral injury, a portion of the parietal lobe was selected from the brains of rats in the normal control group. Brain samples were homogenized, the homogenated centrifuged and the supernatants were used to measure NOS activity with NOS kit. ③ Differences among the three groups were compared with t test. MAIN OUTCOME MEASURES: NOS activity in cerebral cortex of rats in each group. RESULTS: A total of 54 SD rats were involved in the final analysis. At 0.5 hour after cerebral injury, NOS activity in cerebral cortex was (42.45±13.46) nmol/L in cerebral injury group and (41.17±12.53) nmol/L in magnesium sulfate group, respectively, which was higher than that in normal control group [(39.45±11.84) nmol/L, P < 0.05]. At 2 hours after cerebral injury, NOS activities were (66.48±21.43) and (63.24±19.18) nmol/L, respectively, while at 6 hours after cerebral injury, NOS activities were (62.45±24.18) and (51.97±20.46) nmol/L, respectively, which were higher than those in normal control group (P < 0.01). At 24 hours after cerebral injury, NOS activity returned to basal level. Moreover, NOS activity was significantly lower in the magnesium sulfate group than that in the cerebral injury group at 2 and 6 hours after cerebral injury (P < 0.05, 0.01). CONCLUSION: NOS activity is increased in injured brain tissue of rats with craniocerebral injury, and treatment with magnesium sulfate provides some degrees of protection possibly through inhibition of NOS activity after cerebral injury.     

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.     

Antenatal taurine reduces cerebral cell apoptosis in fetal rats with intrauterine growth restriction*

From pregnancy to parturition, Sprague-Dawley rats were daily administered a low protein diet to establish a model of intrauterine growth restriction. From the 12 th day of pregnancy, 300 mg/kg taurine was daily added to food until spontaneous delivery occurred. Brain tissues from normal neonatal rats at 6 hours after delivery, neonatal rats with intrauterine growth restriction, and neo-natal rats with intrauterine growth restriction undergoing taurine supplement were obtained for fur-ther experiments. The terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick-end labeling assay revealed that the number of apoptotic cells in the brain tissue of neonatal rats with intrauterine growth restriction significantly increased. Taurine supplement in pregnant rats reduced cell apoptosis in brain tissue from neonatal rats with intrauterine growth restriction. Immu-nohistochemical staining revealed that taurine supplement increased glial cell line-derived neuro-trophic factor expression and decreased caspase-3 expression in the cerebral cortex of intrauterine growth-restricted fetal rats. These results indicate that taurine supplement reduces cell apoptosis through the glial cell line-derived neurotrophic factor-caspase-3 signaling pathway, resulting in a protective effect on the intrauterine growth-restricted fetal rat brain.