BackgroundStudies have demonstrated that triptolide has good anti-inflammatory and immunosuppressive effects. However, the effect of triptolide on cerebral ischemia/reperfusion injury is still unclear.ObjectiveTo observe the effects of triptolide on neurologic function, infarct volume, water content of brain tissue, neutrophil number in microvascular wall and interleukin-1β(IL-1β) expression in rat models of local ischemia/reperfusion, and analyze the mechanism of triptolide for protecting brain.DesignRandomized controlled experiment.SettingDepartment of Pathology, Medical School of Ningbo University; Department of Forensic Medicine, Tongji Medical College of Huazhong University of Science and Technology.MaterialsSixty Wistar rats of either gender, aged 4 months old, weighing from 200 to 250 g, were provided by the Experimental Animal Center, Tongji Medical College, Huazhong University of Science and Technology. Triptolide was purchased from Fujian Institute for Medical Science (purity 99.98%; Batch No. 2000215). It was dissolved in 20 g/L propanediol, and filtered with 200-mesh filter for later use.MethodsThis experiment was carried out in the laboratory of Forensic Medicine, Tongji Medical College of Huazhong University of Science and Technology, Department of Pathology, Medical School of Ningbo University between January 2001 and September 2004. Sixty Wistar rats were randomized into 4 groups: sham-operation group, model group, low-dose triptolide group and high-dose triptolide group. Rats in each group, except for sham-operation group, were developed into rat models of cerebral ischemia/reperfusion according to the method of Longa et al. In the first 3 days of modeling, rats in the low- and high-dose triptolide groups were intraperitoneally injected with 0.2 and 0.4 mg/kg triptolide respectively, once a day, 3 days in total. At ischemia 1 hour and reperfusion 24 hours, infarct volume, neurologic deficit (five-point scale, higher scores indicated poor neurologic function), water content of brain tissue, neutrophil number in microvascular wall in the middle cerebral artery occlusive side of rats were detected, meanwhile, brain tissue injury degree and IL-1βimmunohistochemical staining changes in brain-derived nerve cells were observed under the optical microscope.Main outcome measuresNeurologic deficit, infarct volume percentage, water content of brain tissue, neutrophil number in microvascular wall and positive rate of IL-1β immunoreaction.ResultsSixty rats were all involved in the final analysis. Neurologic deficit scores of rats in the low- and high-dose triptolide groups were (1.96±0.14) and (1.75±0.16)points respectively, which were both significantly lower than those in model group [(2.58±0.11)points,P < 0.05,0.01]. Infarct volume percentages of rats in low- and high-dose triptolide groups were significantly lower than that in model group separately (P < 0.05, 0.01). Water content of brain tissue of rats in model group was significantly higher than that in the sham-operation group [(82.35±1.26)% vs. (76.65±1.17)%,P < 0.01]; Water content of brain tissue of rats in the low- and high-dose triptolide groups was respectively(80.15±1.43)%,(78.23± 1.15)%, which was significantly lower than that in the model group (P < 0.05, 0.01). Pathological changes of brain tissue of rats: Under the optical microscope, infarct focus was not found in the brain tissue of rats in the sham-operation group, while clear infarct focus could be found in the brain tissue of rats in the model group; Although infarct focus was found in the brain tissue of rats in the low- and high-dose triptolide groups, the whole infarct area was contracted as compared as that in the model group. Neutrophil number in microvascular wall of brain tissue of rats in the low- and high dose triptolide groups was 10.60±2.12,8.11±1.21 respectively, which was significantly less than that in model group(16.25±1.96,P < 0.05,0.01). Positive rate of IL-1βimmunoreaction in the brain tissue of rats in model group was significantly higher than that in the sham-operation group (P < 0.01); and positive rate of IL-1β immunoreaction in the brain tissue of rats in low- and high-dose triptolide groups was significantly lower than that in the model group (P < 0.05, 0.01), but higher than that in the sham-operation group, without significant difference (P > 0.05).ConclusionTriptolide protects against cerebral ischemia/reperfusion injury of rats that may be related with anti-inflammations. Triptolide inhibits IL-1β expression in brain tissue and reduces the attachment and aggregation of neutrophils in blood capillary, and further inhibits the infiltration of blood white cells, thus, it will lessen cerebral injury, contract cerebral infarct and improve cerebral function. 相似文献
BACKGROUND: Mechanical injury can cause the changes of polygene expression spectrum in rat cerebral cortical nerve cells, and then result in the changes of intracellular protein expression. At present, dielectrophoresis is combined with mass spectrum technique to detect the expression of different proteins in rat cortex after brain injury, but the protein chip technique requires further investigation.
OBJECTIVE: To analyze the differences of protein expression spectrum in rat cerebral cortex before and after closed traumatic brain injury using WCX-2 protein chip technique.
DESIGN: A randomized controlled animal experiment.
SETTING: Training Division of the Medical College of Chinese People's Armed Police Force.
MATERIALS: Seventy-two male SD rats of clean degree, 350–450 g, were provided by the Experimental Animal Center, Academy of Military Medical Sciences of Chinese PLA. Urea, trifluoroacetic acid, CHAPS and Tris (Sigma, USA); WCX-2 (Ciphergen, USA). Ultra-high speed hypothermia centrifuger (Bechman, USA); Rotary tissue microtome (Keuca, Germany); Biochip processor and PBSⅡ-C protein chip reader (Ciphergen, USA).
METHODS: The experiments were carried out in the Institute of Molecular Pathology, Central Laboratory, and Department of Pathology, Medical College of Chinese People's Armed Police Force from June 2005 to March 2006. ① Grouping and treatment: The experiments were completed in molecular pathological institute, central laboratory and pathological department. ① The rats were randomly divided into control group (n =12) and brain injury group (n =60). Marmarou's weight-dropping models were duplicated at different time points in the brain injury group. In the control group, the rats were only treated by incising the skin of head top, without fixing the stainless steel hitting backup plate at the vault of skull, and obtain brain cortex for pathological and protein chip research, and they were killed after 24 hours. The rats in the brain injury group were killed at 4, 8, 12, 24 and 48 hours after model establishment. ② Pathological observation: Longitudinal section was made on cerebral cortex, and sections of 5 μm were prepared, then stained with hematoxylin and eosin (HE). ③ Protein chip analysis: 100 mg cerebral cortex was collected from each rat, and the protein content in sample was detected with Bradford method, meanwhile, WCX-2 protein chip was used to analyze the protein spectrum. The data were automatically collected with Ciphergen proteinchip 3.0 software, and the results were analyzed using Biomarker Wizard software to compare the differences of protein spectrum in rat cortex between the groups.
MAIN OUTCOME MEASURES: Results of the pathological observation of cerebral cortex and the protein spectrum analysis.
RESULTS: ① Pathological changes of cerebral cortex: In the control group, no necrosis and edema was observed. In the brain injury group, injures of different severity occurred at different time points; After 4 hours, focal or scattered red nerve cells could be observed, the size of some cells was increased, cytoplasm was lightly stained, and only nuclear fragments were seen; After 8 hours, the necrotic nerve cells were increased, and the number of nerve cells was reduced, astrocytes (neuronophagia) could be seen in partial cytoplasm; there was small vascular dilatation, and endothelial cell proliferation; interstitial edema, regional rarefaction lightly stained. After 12–48 hours, the necrotic nerve cells were reduced, and astrocytes proliferated. ② Results of protein spectrum analysis: The WCX-2 experiment found that the expressions of 5 639, 3 212 and 7 536 u proteins in cerebral cortex changed after injury in the brain injury group. The peak intensity of 5 639 u protein in the brain injury group at 8 hours after injury was higher than that in the control group (P < 0.05); The peak intensity of 3 212 u protein in the brain injury group at 48 hours after injury was higher than that in the control group (P < 0.05); The peak intensity of 7 536 u protein at 24 hours after injury was higher than that in the control group (P < 0.05).
CONCLUSION: Brain injury can cause the changes of protein expression spectrum in cerebral cortex, it is suggested that brain injury can induce the expression of protein. 相似文献
AbstractObjectives: The present study investigated the effects of tetrahydrobiopterin (BH4) on cerebral infarction after transient focal ischemia in rats. Methods: Focal ischemia (1·5 hours) was created in male Sprague-Dawley rats (250-280 g) by middle cerebral artery occlusion. Some rats were treated with 20 mg/kg tetrahydrobiopterin by intraperitoneal injection 30 minutes before reperfusion. At 2, 6, and 12 hours of reperfusion, the brains were harvested for the nitric oxide synthase (NOS) activity and nitric oxide (NO) level assays. At 12 hours of reperfusion, the brains were harvested for infarct size measurement. Results: NOS activity and NO level were all augmented after reperfusion. BH4 treatment significantly further increased NOS activity and NO level. Cerebral infarct size was significantly bigger in BH4 treatment group compared to that in no treatment group. Conclusions: The data indicate that BH4 enhances cerebral infarction after transient focal ischemia in rats, through NOS and NO pathway. 相似文献
BACKGROUND: Batroxobin has been found to have protective effect on cerebral ischemia-reperfusion, and cardiopulmonary resuscitation (CPR) is the common cause of global brain ischemia-reperfusion.
OBJECTIVE: To observe the effect of Batroxobin on the morphological results of cerebral cortex and hippocampus in rabbit models of CPR, and the changes of serum concentration of tumor necrosis factor alpha (TNF-α) after CPR.
DESIGN: A randomized controlled observation.
SETTING: Laboratory of the Department of Burns, Changhai Hospital affiliated to the Second Military Medical University of Chinese PLA.
MATERIALS: Thirty healthy New Zealand rabbits of 2.5–3.0 kg, either male or female, were used. Kits for TNF-α determination were provided by LIFEKEY BioMeditech Company (USA).
METHODS: The experiments were carried out in the laboratory of Department of Burns, Changhai Hospital from February 2001 to January 2002. The 32 rabbits were randomly divided into sham-operated group (n=8), conventional resuscitation group (n=12) and Batroxobin-treated group (n=12). The animals in the conventional resuscitation group and Batroxobin-treated group were anesthetized, then induced into modified Pittsburg's model of mechanical ventricular fibrillation. Sham-operated group was discharged on the chest wall, which did not cause ventricular fibrillation. Conventional resuscitation group and Batroxobin-treated group were exposed to 6 minutes of cardiac arrest induced by ventricular fibrillation, then the resuscitation began. A dosage of 0.3 Bu/kg of Batroxobin was administered to the rabbits in the Batroxobin-treated group at the beginning of resuscitation. Blood sample was collected at 4 and 12 hours after CPR to determine the concentration of TNF-α in serum. After the second blood collection, brain tissue was taken out immediately, and the forms of nerve cells in cerebral cortex and hippocampal CA1 region were observed under light microscope.
MAIN OUTCOME MEASURES: ① TNF-αconcentration in serum at 4 and 12 hours after CPR; ② Forms of nerve cells in cerebral cortex and hippocampal CA1 region at 12 hours after CPR.
RESULTS: All the 31 New Zealand rabbits were involved in the analysis of results. ①TNF-α concentration in serum: At 4 hours after CPR, the TNF-α concentrations in serum in the conventional resuscitation group and Batroxobin-treated group [(5.947±2.366), (5.122±2.521) ng/L] were significantly higher than that in the sham-operated group [(2.604±1.623) ng/L, P < 0.05]. At 12 hours after CPR, the TNF-α concentration in serum in the conventional resuscitation group was (7.770±3.121) ng/L, it was significantly higher than that at 4 hours (P < 0.05), also significantly higher than that in the Batroxobin-treated group [(5.425±2.280) ng/L, P < 0.05]. ② Forms of nerve cells: In the sham-operated group, no abnormality was found in the hippocampal CA1 region and cerebral cortex. In the conventional resuscitation group, the pyramidal cells in hippocampal CA1 region were lined up in disorders, and edema, puff, vacuolization, nucleus concentration and anachromasis were also observed appeared; Edema of nerve cells, vacuole, pyknosis appeared in cerebral cortex; microthrombosis appeared in some blood capillaries. As compared with the conventional resuscitation group, cellular edema was relieved and pyknosis of nerve cells were obviously reduced, and no microthrombosis was found in hippocampal CA1 region and cerebral cortex in the Batroxobin-treated group.
CONCLUSION: Batroxobin have neuroprotective effect on CPR rabbits, and may inhibit the excessive increase of TNF-α concentration in serum. 相似文献
BACKGROUND: Lots of evidences have demonstrated that acute inflammatory reaction plays an important role in cerebral ischemia and cerebral ischemia/reperfusion injury. Tumor necrosis factor (TNF), as one of important inflammatory cytokines, also participates in the injury.
OBJECTIVE: To observe the changes in TNF-α expression and myeloperoxidase (MPO) activity of mouse models of local cerebral infarction induced by photochemical method, and analyze the correlation of TNF-α expression and MPO activity.
DESIGN: Randomized controlled experiment.
SETTING: Laboratory of Cerebral Microcirculation, Taishan Medical College.
MATERIALS: Sixty involved male adult Kunming mice were provided by the Experimental Animal Center of Shandong University. TNF-α primary antibody, kits for enzyme-linked immunosorbent assay(ELISA) and streptavidin-biotin complex immunohistochemical dyeing kit were purchased from Boster Company(Wuhan). MPO kit was purchased from Jiancheng Bioengineering Institute (Nanjing). Cold light source was developed by Hengfa Co.,Ltd.( LG-150, Xuzhou).
METHODS: This experiment was carried out in the Laboratory of Cerebral Microcirculation of Taishan Medical College between July 2004 and July 2005. The involved 60 Kunming mice were randomized into 3 groups: normal control group (n =6), sham-operation group (n =6) and model group (n =48). Mice in the model group were observed at 30 minutes, 1, 3, 6, 12, 24, 48 and 72 hours after illumination, separately, 6 mice at each time point. In the model group, mice models of local cerebral infarction were developed as follows: The mice were anesthetized to expose left skulls. Taking 2 mm left to sagittal suture and 2 mm posterior to coronal suture as center, a field with diameter of 3 mm for illumination was set. The optical fiber detecting head of cold light source was vertically close to exposed skull. The mice were injected with rose Bengal for 5 minutes, and then cold light source was open for 10 minutes. Illumination was omitted in the sham-operation group. Mice in the control group were not modeled. At postoperative 6 hours, TNF-α expression in infracted-side cortex was detected with immunohistochemical method and ELISA, and MPO activity in infracted-side cortex with chromatometry. MPO activity could reflect the infiltration degree of neutrophils in tissue. Stronger activity indicated severer infiltration. Single-factor analysis of variance was used for comparison among groups, q test for pairwise comparison and correlative analysis for detecting the inter-parameter correlation.
MAIN OUTCOME MEASURES: Changes in TNF-α expression and MPO activity of left cortex of mice in each group.
RESULTS: Sixty mice were involved in the final analysis. After cerebral infarction, TNF-α positive cells were neurons and glial cells mainly, distributing in and around the infarct region. TNF-α expression in cortex of mice of sham-operation group was （615.7±16.1） ng/L, and that of model group increased to （792.2±17.8） ng/L at 3 hours after illumination, and reached peak [（921.9±23.9） ng/L] at 6 hours after illumination, and decreased to （848.0±30.6） ng/L at 12 hours after illumination and recovered to the normal level [（625.3±14.3） ng/L] at 72 hours after illumination. MPO activity of sham-operation group was （7.151±0.433） nkat/g, and that of model group increased to （10.469±0.600） nkat/g at 3 hours after illumination, reached the peak [（15.486±0.650） nkat/g] at 12 hours after illumination, decreased to （11.052±0.617） nkat/g at 24 hours after illumination and recovered to the normal level [（7.418±0.617） nkat/g] at 72 hours after illumination. Change of MPO activity lagged behind that of TNF-α, and correlative analysis showed that the both were positively correlated（r =0.953, P < 0.01）.
CONCLUSION: In the acute stage of cerebral infarction of mice induced by photochemical method, TNF-α expression in infarcted-side cortex is closely related with infiltration of neutrophils. TNF-α induces inflammatory cells to intrude into ischemic brain tissue, and participates in the inflammatory reaction process at the early stage of cerebral ischemia. 相似文献
BACKGROUND: Some experiments have demonstrated that injecting orphanin FQ (OFQ) into lateral ventricle, which can obviously decrease the pain threshold. It is indicated that OFQ is an anti-opiate substance. However, whether OFQ has effects on sensory neuron ion channel in cerebral cortex needs to be further studied.
OBJECTIVE: To investigate the effects of OFQ, morphine or their combination on sodium channel current of somatosensory neurons in rat cerebral cortex.
DESIGN: Repeated measurement trial.
SETTING: Department of Physiology, Harbin Medical University.
MATERIALS: Fifty healthy Wistar rats, aged 12–16 days, of either gender, were provided by the Experimental Animal Center, Second Hospital Affiliated to Harbin Medical University. OFQ was purchased from Sigma-Aldrich Company, and morphine was provided by the Shenyang First Pharmaceutical Factory. PC2C patch clamp amplifier and LabmasterTL1were purchased from Yibo Life Science Instrument Co.,Ltd. of Huazhong University of Science and Techgnology.
METHODS: This experiment was carried out in the Department of Physiology (provincial laboratory), Harbin Medical University between January 2005 and May 2006. Cortical neurons were acutely isolated from rats, and prepared into cell suspension following culture. ①Sodium channel current of somatosensory neurons in rat cerebral cortex was recorded before and after administration by whole-cell Patch clamp technique after 50 nmol/L OFQ being added to extracellular fluid. ②The amplitude of sodium channel current of somatosensory neurons in rat cerebral cortex was recorded before and after administration by the same method after 20 μmol/L morphine being added to extracellular fluid, and then the change of sodium channel current was recorded after 50 nmol/L OFQ being added.
MAIN OUTCOME MEASURES: The amplitude of sodium channel current of somatosensory neurons in rat cerebral cortex following the administration of OFQ, morphine separately or their combination..
RESULTS: ①The amplitude of sodium channel current of somatosensory neurons in rat cerebral cortex was significantly lower after administration of 50 nmol/L OFQ than before at the clampe of the voltage of –30 mV (P < 0.05). ②The amplitude of sodium channel current of somatosensory neurons in rat cerebral cortex was significantly lower after administration of 20 μmol/L morphine than before at the clampe of the voltage of –30 mV (P < 0.05). The sodium channel current recovered to –（2 345.24±174.18）pA after 50 nmol/L OFQ was administrated. There were significant differences in the amplitude of Na+ channel current between two interventions (P < 0.05).
CONCLUSION: Morphine and OFQ can respectively reduce the amplitude of sodium channel current of somatosensory neurons in rat cerebral cortex, and OFQ can reverse the effect of morphine partly. It is indicated that OFQ can produce antiopioid activity in the central nervous system by influencing sodium channel current. 相似文献
BackgroundThe enhanced expression of c-Fos protein in nerve cells after hypoxia is the marker for converting extracellular hypoxia information to intracellular changes at hypoxia, and it is suspected that the increase of c-Fos protein can lead to the synthesis and excretion of related neurotrophic factor and nerve growth factor. However, it is still unclear what functional changes of nerve cells are induced by the increase of c-Fos protein at hypoxia, and whether it is good for the survival of damaged neurons.ObjectiveTo observe the expression of c-Fos in the cerebral neurons from embryos of rats with hypoxia in uterus, and investigate the pathway for the protective effect of Angelica sinensis injection on the cerebral neurons from rat embryos under hypoxia.DesignA completely randomized controlled study.SettingDepartment of Histology and Embryology, Luzhou Medical College.MaterialsTwelve female Wistar rats in oestrum and 1 male adult Wistar rat with body mass of 220 to 250 g were selected. Rabbit-anti-rat neuro-specific enolase (NSE) and rabbit-anti-rat c-Fos were purchased from Wuhan Boster Biological Technology Co., Ltd.; Double-staining kit was bought from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. Angelica sinensis injection was produced by the Department of Pharmacy, the Second Affiliated Hospital of Hubei Medical University.MethodsThe experiments were completed in the experimental animal center and the Department of Histology and Embryology of Luzhou Medical College from December 2004 to December 2005. Twelve adult female Wistar rats in oestrum and 1 male Wistar rat were housed in one rearing cage. The appearance of vaginal embolus at 8:00 in the next morning was recorded as 0 day of pregnancy and the rats were recorded for 15 days, and they were divided randomly into three groups, control group (n =4), hypoxia group (n =4) and Angelica group (n =4). The pregnant rats in the hypoxia group were firstly injected with saline (8 mL/kg), then put into 2 L wide-mouthed bottle containing 100 g sodalime, and then the lid of the bottle was closed tightly to induce hypotonic hypoxia for 1 hour followed by 1-hour re-oxygenation. The pregnant rats were killed under anesthesia, and then fetuses were taken out by rapid cesarean. Part of the brain tissues were exposed and then fixed in formaldehyde (40 g/L). The pregnant rats in the Angelica group were treated the same as those in the hypoxia group except that saline was replaced by 250 g/L Angelica sinensis injection which was injected via caudal vein (8 mL/kg). The rats in the control group were injected with saline (8 mL/kg) slowly via caudal vein, but not put into the wide-mouthed bottle for hypoxia, and then the brain tissues were removed and fixed as those in the hypoxia group after 1 hour. Twenty embryos from rats were chosen randomly in each group and then routinely embedded in paraffin. Paraffin sections of 4 μm thick were prepared through the anterior fontanelle of head of the fetal rats. The sections were immunohistologically stained with c-Fos/NSE. The one-way analysis of variance (ANOVA) was used to compare the differences of measurement data among the groups, and the q test was applied in the two-two comparison.Main outcome measuresThe numbers of c-Fos and c-Fos/NSE positive neurons in cerebrum from rat embryos were observed.Results Numbers of NSE positive neurons in cerebrum of rat embryos in the control group, hypoxia group and Angelica group were (84.3±9.0), (90.2±12.5) and (86.7±9.7) cells/high power field (P > 0.05). The number of c-Fos/NSE positive neurons was more in the hypoxia group than in the control group and Angelica group [(38.4±5.28), (11.35±2.67), (20.65±4.07) cells/high power field, q =29.17, 19.14, P < 0.05].ConclusionHypoxia can stimulate the expression of c-Fos in cerebral neurons from rat embryos. Angelica sinensis injection could reducing the damage of hypoxia to neurons and play a neuroprotective role by decreasing the expression of c-Fos protein in hypoxic neurons. 相似文献
BackgroundSome researches demonstrate that high-sensitivity C-reactive protein may be a risk factor to cause carotid atherosclerosis in patients with cerebral infarction. Inflammatory reaction may participate in formation of carotid atherosclerosis in patients with acute cerebral infarction.ObjectiveTo investigate the correlation between levels of serum high-sensitivity C-reactive protein and carotid atherosclerosis in patients with acute cerebral infarction accompanied with carotid atherosclerosis.DesignContrast observation between two groups.SettingDepartment of Neurology, Zhenzhou Hospital, Shenyang Medical College.ParticipantsA total of 102 patients with acute cerebral infarction regarded as cerebral infarction group were selected from Department of Neurology, Shenzhou Hospital Affiliated to Shenyang Medical College from February 2005 to September 2006. There were 55 males and 47 females and their ages ranged from 55 to 86 years. All patients met the variously diagnostic points of cerebral infarction established by the Fourth National Cerebrovascular Disease Academic Meeting and were finally diagnosed with CT or MRI examination. Illness course was in an acute phase. A total of 96 healthy subjects were regarded as control group, including 51 males and 45 females aged from 48 to 78 years. All accepted subjects provided the confirmed consent.Methods Patients in the cerebral infarction group received carotid ultrasound Doppler examination and serum high-sensitivity C-reactive protein detection within 72 hours after onset. IMMAGE immune biochemical system and latex reinforcement particle-enhanced nephelometric immunoassay (PENIA) were used for quantitative detection of serum high-sensitivity C-reactive protein. Healthy subjects in the control group received the same detection. SEQUOIA512 color Doppler ultrasound (Siemens Company, USA) was used to detect carotid artery of all subjects so as to observe intima media thickness of artery and formation of artery atherosclerostic plaques. If artery atherosclerostic plaques were formed, their properties and amounts were determined based on the characteristics of light-echo signals. Evaluating criteria: Intima media thickness of artery was the vertical dimension from crossed face between lumen and tunica intima to crossed face between tunica media and tunica adventitia. Intima media thickness ≤ 0.9 mm was regarded as normal; 0.9 mm < intima media thickness ≤ 1.2 mm was regarded as thickening; when local eminence thickening was processed towards to lumen, the intima media thickness was more than 1.2 mm and plaque of tunica intima was formed at the same time. Properties of plaque were classified into 4 types: steady low-echo lipid malacoplakia, equal-echo fiber plaque, strong-echo or sound-imaging calcification hard plaque and unsteady-echo ulcer mixed plaque. Fiber plaque and calcification hard plaque were steady but malacoplakia and mixed plaque were unsteady.Main outcome measuresThickness of tunica media, characteristics of plaque and level of serum high-sensitivity C-reactive protein in carotid artery in two groups.ResultsAll 102 patients with cerebral infarction and 96 healthy subjects were involved in the final analysis. Comparisons of level of high-sensitivity C-reactive protein: Level of high-sensitivity C-reactive protein in normal tunica media was higher in the cerebral infarction group [(4.66±1.55) mg/L] than the control group [(3.49±1.24) mg/L, t =2.541, P < 0.05]. In addition, level of high-sensitivity C-reactive protein in patients with thickening tunica media and plaque was not significantly different between the cerebral infarction group and the control group (P > 0.05). Correlation between various degrees of vascular lesion and level of high-sensitivity C-reactive protein in the cerebral infarction group: Level of high-sensitivity C-reactive protein was statistically significantly higher in patients with thickening tunica media [(8.16±2.42) mg/L] than patients with normal tunica media [(4.66±1.55) mg/L, t =4.132, P < 0.01]. In addition, level of high-sensitivity C-reactive protein was statistically significantly higher in patients with carotid plaque [(12.08±3.85) mg/L] than patients with normal tunica media (t =5.994, P < 0.01) and thickening tunica media (t =4.197, P < 0.01). Levels of high-sensitivity C-reactive protein in patients with various kinds of carotid plaque: Level of high-sensitivity C-reactive protein was statistically significantly higher in patients with unsteady carotid plaque [(13.54±2.62) mg/L] than patients with steady carotid plaque [(8.61±3.71) mg/L, t =2.002, P < 0.05]. That was to say level of serum high-sensitivity C-reactive protein in patients who suffered acute cerebral infarction combined with carotid atherosclerosis especially carotid plaque was higher than that in those patients who did not have carotid lesions. This suggested that serum high-sensitivity C-reactive protein had a certain correlation with onset of carotid atherosclerosis in patients with acute cerebral infarction.ConclusionSerum high-sensitivity C-reactive protein certainly correlates with onset of carotid atherosclerosis in patients with acute cerebral infarction, while inflammatory reaction may participate in formation of carotid atherosclerosis in patients with acute cerebral infarction. 相似文献
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. 相似文献
BackgroundAt present, inhalation of nitrogen monoxidum (NO) or other angiotenic is widely used to cure hypoxic pulmonary artery hypertension. In addition, recent researches demonstrate that postganglionic fiber of stellate ganglion can regulate contents of blood vessel endothelium-calcitonin gene-related peptide (BVE-CGRP) and nitricoxide synthase (NOS) in lung tissue. Therefore, stellate ganglion which is blocked with the local anesthetic may cause therapeutic effects on hypoxic pulmonary artery hypertension.ObjectiveTo observe the effects of stellate block on calcitonin gene-related peptide (CGRP) of vasodilation factors, prostacyclin, endothelin-1 of vasoconstriction factors, thromboxan, blood vessel endothelium-nitricoxide synthase (BVE-NOS) and mean arterial pressure of lung tissue in rabbits with hypoxic pulmonary artery hypertension.DesignRandomly controlled animal study.SettingNeurological Institute of Taihe Hospital Affiliated to Yunyang Medical College.MaterialsA total of 24 adult Japanese rabbits of both genders and weighing 2.3–2.6 kg were provided by Animal Experimental Center of Hubei Academy of Medical Science. SP kit was provided by Beijing Zhongshan Biotechnology Co., Ltd.; moreover, kits of endothelin-1, CGRP, prostacyclin and thromboxan were provided by Radioimmunity Institute, Scientific and Technological Developing Center, General Hospital of Chinese PLA, and color image analytical system (Leica-Q500IW) was made in Germany.MethodsThe experiment was carried out in the Neurological Institute of Taihe Hospital affiliated to Yunyang Medical College from February to December 2002. Rabbits were performed with aseptic manipulation to exposure left stellate ganglion and then it was put in epidural catheter for 1 week. In addition, one end of epidural catheter was fixed near by stellate ganglion and the other end was fixed through dorsal neck. All rabbits were randomly divided into 4 groups, including normal control group, stellate block group, hypoxia group and hypoxia + stellate block group, with 6 in each group. Rabbits in the normal control group were perfused with saline through epidural catheter with 0.5 mL once for three times per day and 3 successive days in total; in addition, rabbits in the stellate block group were perfused with 2.5 g/L bupivacaine through epidural catheter with 0.5 mL once for three times per day and 3 successive days in total. Rabbits in the hypoxia group were used to establish hypoxic pulmonary artery hypertension models. That was to say, the experimental rabbits were put in hypoxic box (containing sodalime and calcium chloride to absorb CO2 and water) and given various flows of oxygen and nitrogen through the two lateral wells simultaneously. And then, oxygen was monitored with oxygen-concentration monitoring device to control the concentration in (10±2)% for 8 hours per day and 2 successive weeks in total. Rabbits in the hypoxia + stellate block group were used to establish hypoxia models as the same as those in the hypoxia group. Two weeks later, 2.5 g/L bupivacaine was pushed into epidural catheter with 0.5 mL once for three times per day and 3 successive days in total. Breast was directly opened to measure mean pulmonary artery pressure. 6 mL blood was collected through pulmonary arterial duct to measure levels of plasma CGRP, prostacyclin, endothelin-1 and thromboxane with radio-immunity technique; meanwhile, immunohistochemical staining was used to observe the changes of BVE-NOS content of the experimental rabbits in all groups.Main outcome measuresChanges of CGRP, prostacyclin, endothelin-1 and thromboxane and BVE-NOS.ResultsA total of 24 experimental rabbits were involved in the final analysis. As compared with those in the normal control group, hypoxic pulmonary artery hypertension of the experimental rabbits was higher in the hypoxia group and hypoxia + stellate block group after hypoxia [(3.84±0.30), (3.16±0.45), (2.60±0.27) kPa, P < 0.05, 0.01]; CGRP was lower [(68.20±8.78), (108.24±14.35), (130.25±22.70) ng/L, P < 0.05, 0.01]; prostacyclin was lower [(94.45±10.68), (98.77±12.31), (155.27±20.67) ng/L, P < 0.01]; endothelin-1 was higher [(184.74±29.66), (115.27±13.62), (98.20±11.52), ng/L, P < 0.05, 0.01]; thromboxan was higher [(226.27±30.46), (207.67±27.32), (124.25±16.89) ng/L, P < 0.01]. As compared with that in hypoxia group, hypoxic pulmonary artery hypertension was decreased in hypoxia + stellate block group (P < 0.05), CGRP was increased (P < 0.01), and endothelin-1 was decreased remarkably (P < 0.05). Level of BVE-NOS of the experimental rabbits was higher in stellate block group, hypoxia group and hypoxia + stellate block group than that in the normal control group [(0.25±0.06), (0.27±0.07), (0.46±0.12), (0.14±0.03), P < 0.05], and NOS level was higher in the hypoxia + stellate block group than that in hypoxia group (P < 0.05).ConclusionMean arterial pressure is decreased in rabbits with hypoxic pulmonary artery hypertension after stellate block and level of endothelin-1 is also decreased; however, levels of CGRP and NOS are increased respectively. 相似文献
BACKGROUND: Delayed neuronal death after total cerebral ischemia may accompany with apoptosis, but acupuncture may play a certain role in protecting nerve through inhibiting ischemic neuronal apoptosis.
OBJECTIVE: To observe the effect of acupuncture on neuronal apoptosis in rats after cerebral ischemia and analyze its cerebral protective mechanism.
DESIGN: Contrast observation among groups.
SETTING: Heilongjiang University of Traditional Chinese Medicine.
MATERIALS: A total of 30 male healthy Wistar rats of general grade and weighing (250±20) g were randomly divided into three groups, including sham operation group, cerebral ischemia group and acupuncture group with 10 rats in each group. Apoptosis in situ kit was provided by Baolingman Company, Germany.
METHODS: The experiment was carried out in the Laboratory Center, Heilongjiang University of Traditional Chinese Medicine from May to November 2004. ① Rats in the cerebral ischemia group and the acupuncture group were used to establish total cerebral ischemic models with four vessels occlusion; in addition, models in the sham operation group were established with the same method as mentioned above. However, four vessels of rats in the sham operation were exposured and cerebral ischemia did not occur. Rats in the acupuncture group were given acupuncture treatment after operation. Needle of 40 mm in length was used to acupuncture bilateral Zusanli (St 36) and Quchi (LI 11) with the depth of 3 mm, and then bilateral acupoints were connected with KWD-808II omnipotenc impulse electro-therapeutic apparatus (frequency: 1 Hz; thin waves; voltage: 2 V) once a day for totally 30 minutes. Meanwhile, needle of 25 mm in length was used to acupuncture Baihui (Du 20) with the depth of 3 mm, and then the needle was twirled once every 5 minutes for 30 minutes in total. The course was 7 days. ② Neuronal injuries in hippocampal CA1 area after cerebral ischemia were observed with Nissl body staining method at 7 days after treatment; neuronal apoptosis was observed with TUNEL staining; manifestations of neuronal apoptosis in cerebral cortex and hippocampal CA1 area were observed with electron microscope.
MAIN OUTCOME MEASURES: Neuronal injuries in hippocampal CA1 area after cerebral ischemia; neuronal apoptosis in cerebral cortex and hippocampal CA1 area after cerebral ischemia; morphological changes under electron microscope.
RESULTS: Among 30 Wistar rats, 24 rats were involved in the final analysis. ① Expression of positive neurons in cerebral cortex and hippocampal CA1 area with Nissl body staining: Neuronal defect was obvious in cerebral cortex and hippocampal CA1 area in the cerebral ischemia group as compared with that in the sham operation group (P < 0.05), and neuronal defect was decreased in hippocampal CA1 area in the cerebral ischemia group as compared with that in the acupuncture group (P < 0.05). ② Expression of positive neurons in cerebral cortex and hippocampal CA1 area with TUNEL staining: Positive neurons with TUNEL staining were not observed in the sham operation group, but positive neurons were increased in the cerebral ischemia group as compared with those in the acupuncture group (P <0.05). ③ Observational results of electron microscope: Neuronal apoptosis was not found in the sham operation group; neuronal apoptosis was rarely found in the acupuncture group; neuronal apoptosis was typical in the cerebral ischemia group.
CONCLUSION: Delayed neuronal death after total cerebral ischemia may accompany with apoptosis, but acupuncture may play a certain role in protecting nerve through inhibiting ischemic neuronal apoptosis. 相似文献
BACKGROUND: The direct current electrical field can effectively promote the regeneration of the spinal cord; moreover, methylprednisolone (MP) can relieve secondary edema after spinal cord injury. Tetrandrine (Tet) is an effective component of hanfangji and can protect the effect of spinal cord and axis-cylinder. Whether direct current electrical field combining with MP or Tet has synergic or strengthening effect on treating complete spinal cord injury or not should be studied further.
OBJECTIVE:To study the effect of direct current electrical field assisted by MP and Tet on treating spinal cord injury.
DESIGN: Randomized controlled animal study.
SETTING: People's Hospital of Hainan Province.
MATERIALS: A total of 45 healthy hybrid dogs, of both genders, weighing 10–12 kg, aged 1.5–2 years, were provided by Animal Center of Hainan Province. Somatosensory evoked potential meter (DANTEC Company), IBAS-2.0 imaging analysis meter (Germany), and self-made electronic stimulator.
METHODS: The experiment was carried out in Hainan People's Hospital from May 2001 to June 2004. All experimental dogs were randomly divided into 4 groups: control group (n =9), electrostimulating group (n =12), MP + electrostimulating group (n =12) and Tet + electrostimulating group (n =12). ① After anesthesia, Allen WD method was used to induce complete spinal cord injury. The metal bar, which was 10 cm in height fell freely and vertically hit the spinal cord to provide a complete spinal cord injury. Dogs in control group and electrostimulating group were implanted electrical stimulators 6 hours after spinal cord injury (no electricity in control group); dogs in MP + electrostimulating group were injected 30 mg/kg MP for 15 minutes at 2 hours after spinal cord injury and electrical stimulators implanted at 6 hours after injury; dogs in Tet + electrostimulating group were intravenously injected with 7.5 mg/kg Tet at 2 hours after spinal cord injury and electrical stimulators implanted at 6 hours after injury; and then, 7.5 mg/kg Tet injected at days 2 and 3 after injury. ② Specimens were taken from control group from three dogs of every month; from the injured segments of spinal cords at 1 month, 2 months and 3 months; and from electrostimulating group, MP + electrostimulating group and Tet + electrostimulating group of 4 dogs for histological examinations. ③ Detection of neurological function: Neurological function was evaluated with the functional 10 grading system. The scores ranged from 0 to 10 (0: complete paraplegia; 10: normality). ④ Detection of cortical somatosensory evoked potential (CSEP): According to the scheme formulated by the International Electroencephalographical Association, the patterns of the fundamental waves were P1–N1–P1 waves. The latency of the P1 wave and the amplitude of P1–N1 waves were mainly observed individually at 1, 2 and 3 months after the injury. ⑤ Histological detection: All spinal cord specimens of the injuried segment were harvested at 1, 2 and 3 months after injury. They were stained with hematoxylin and Nissl staining methods, and then were observed under an optical microscope, and the neurons were counted. The sectional areas of the neurons and the density of the Nissl bodies were measured by a system image pattern analysis (IBAS-2.0, Germany).
MAIN OUTCOME MEASURES: The neurological function, cortical somatosensory evoked potential, neuronal amount, sectional area of neurons and Nissl body density at 1 to 3 months after injury.
RESULTS: All 45 experimental dogs were involved in the final analysis. ① Detection of neurological function: One month later, the dogs in MP + electrostimulating group could walk, but the dogs in electrostimulating group and Tet + electrostimulating group could stand. Two months after injury, the dogs in MP + electrostimulating group almost recovered to normal, but the dogs in electrostimulating group could walk and those in Tet + electrostimulating group could run. Those in control group had no parent recovery. ② Detection of P1 latency and P1–N1 amplitude: Changes of P1 latency in control group were long and P1–N1 amplitude was very low at 1 month later. Compared to electrostimulating group, MP + electrostimulating group and Tet + electrostimulating group, there were significant differences (P < 0.05). P1 latency was manifestly shortened and amplitude were raised in electrostimulating group, MP + electrostimulating group and Tet + electrostimulating group. Those in MP + electrostimulating group and Tet + electrostimulating group were superior to those in electrostimulating group and there were significant differences (P < 0.05). ③ Sectional areas of neurons and Nissl body density: At 1–3 months after injury, sectional areas of neurons were larger in electrostimulating group [(170.14±7.45), (209.60±14.80), (312.47±12.63) μm2], MP + electrostimulating group [(282.18±15.25), (418.18±16.27), (515.25±15.10) μm2] and Tet + electrostimulating group [(231.81±7.38), (322.67±8.45), (386.82±10.42) μm2] than control group[(98.12±4.93), (113.50±6.74), (122.59±8.03) μm2, P < 0.05]; especially, sectional area was the largest in MP + electrostimulating group. At 1–3 months after injury, Nissl body density was more in electrostimulating group (170.14±7.45, 209.60±14.80, 312.47±12.63), MP + electrostimulating group (282.18±15.25, 418.18±16.27, 515.25±15.10) and Tet + electrostimulating group (231.81±7.38, 322.67±8.45, 386.82±10.42) than control group (98.12±4.93, 113.50±6.74, 122.59±8.03, P < 0.05); especially, Nissl body density was the most in MP + electrostimulating group.
CONCLUSION: The direct current electrical field can effectively promote spinal cord regeneration. The combination of direct current electrical field with large dose MP or Tet has synergistic effects for treating spinal cord injury. The curative effects of direct current electrical field with large dose MP are much better than those with Tet. 相似文献
BACKGROUND: It is hard to cure the open traumatic brain injury (TBI), especially for the brain functional recovery after brain injury. In this regard, traditional Chinese medicine (TCM) has a wide prospect.
OBJECTIVE: To observe the effect of Huayu capsule on limb-catching capability of rat models of open TBI, and investigate its possible mechanism.
DESIGN: Randomized and controlled study.
SETTING: Grade 3 Pharmacological Laboratory of TCM, State Administration of TCM, Chengdu University of TCM.
MATERIALS: This study was performed from October 2005 to January 2006. Fifty Sprague-Dawley rats of either gender, aged 3 months old, weighing from 190 to 220 g, were involved in this study. Huayu capsule was made and supplied by the Department of TCM Processing of Chengdu University of TCM, Lot No. 050121; Xuefuzhuyu oral liquid was manufactured by Jilin Aodong Yanbian Pharmaceutical Industry Co.,Ltd., Lot No. 050406.
METHODS: Open right parietal lobe TBI rat models were made as described in references. The involved rat models were randomized into 5 groups according to gender and body mass: model group, high-, middle-, low-dose Huayu capsule groups and Xuefuzhuyu oral liquid group, with 10 rats in each. Rats in the model group were administrated with distilled water of 5 mL/kg; Rats in the high-, middle- and low-dose Huayu capsule groups were administrated with 1.030, 0.515, 0.258 g/kg raw herbs; Rats in the Xuefuzhuyu oral liquid group were administrated with Xuefuzhuyu oral liquid of 5 mL/kg, intragastrically once a day for 7 days successively for all after recovering consciousness from anesthetization. ① One hour after administration on the 6th day, rats in each group were placed on a 100 cm fine straight iron wire paralleling to the ground and 20 cm above the operational table. The time of the rats keeping on the wire was counted and it indicated the nerve-muscle catching capability. The longer the remained time, the better the nerve-muscle catching capability.② Twenty-four hours after the administration on the 7th day, the samples of the whole brain were carefully taken out and stained by toluidine blue for observing the morphology of cells in the injured brain tissue. ③ The nerve cells in 4 visual fields from 4 directions (upper, lower, left, right) of injured area of brain tissue were counted. The amount was the total number of the four visual fields. The nerve cells in the injured brain tissue were measured by the same way. ④ On the basis of nerve cell counting, the content of Nissl's body in the corresponding nerve cells was measured.
MAIN OUTCOME MEASURES: ①Nerve-muscle catching capability. ② Histopathomorphological examination of the injured areas in brain. ③Measurement of nerve cells and macrophages in the injured areas of brain. ④Measurement of content of Nissl's body in the injured areas of brain.
RESULTS: All the 50 rats were involved in the final analysis. ①The catching time of rats in the high- and middle-dose Huayu capsule groups as well as Xuefuzhuyu oral liquid group was extended to （23.6±10.12）,（18.6±8.17） and (22.6±9.43) s, respectively, which was significantly higher than that in the model group [ (12.1±4.15) s, P < 0.05–0.01]. ② The injured areas of brain tissue of rats in the Huayu capsule-treated groups and Xuefuzhuyu oral liquid group were decreased to different extents. The nerve cells adjacent to brain injured area were increased. ③ The number of macrophages around the brain injured area of rats in the high- and middle-dose Huayu capsule groups as well as Xuefuzhuyu oral liquid group was 63.9±7.99, 59.7±7.41 and 62.9±7.37, respectively, which was significantly larger than that in the model group（49.2±8.00,P < 0.01）. The number of nerve cells adjacent to brain injured area of rats in the high-, middle-, and low-dose Huayu capsule groups as well as Xuefuzhuyu oral liquid group was 86.2±25.93, 93.5±31.79, 92.1±14.54 and 125.2±34.25, respectively, which was significantly larger than that in the model group（62.5±16.98,P < 0.05–0.01）. ④ The total area, the total and integral absorbance , the average gray degree of Nissl's body in the cytoplasm of nerve cells of rats in the Huayu capsule-treated groups and Xuefuzhuyu oral liquid group were all significantly increased (P < 0.05–0.01).
CONCLUSION: Huayu capsule at different doses can promote the limb-catching capability of rat models of open TBI to different extent. This promoting effect may be related to increasing macrophages in the injured area, lessening the apoptosis of nerve cells and increasing the content of Nissl's body in the nerve cells. 相似文献
BACKGROUND: Protein nonenzymatic glycosylation is supposed to be one of mechanisms for chronic complications development in diabetes mellitus, and therefore, might play an important role in the neuronal degeneration.
OBJECTIVE: To study the protein nonenzymatic glycosylation in brain neurons of diabetic rats, and to analyze the pathway of neuronal degeneration at the early stage of hyperglymecia.
DESIGN: Randomized controlled animal experiment.
SETTING: Department of Endocrinology, First hospital Affiliated to General Hospital of Chinese PLA and Beijing Laboratory for Brain Aging, Xuanwu Hospital Affiliated to Capital Medical University.
MATERIALS: Thirty-five male Wistar rats (grade Ⅱ), aged 3 months old, and 11 male purebred Kunming mice (grade Ⅲ) without special pathogen, aged 3 months old, were provided by the Animal Room of Capital Medical University.
METHODS: This experiment was carried out in the Beijing Laboratory for Brain Aging, Xuanwu Hospital Affiliated to Capital Medical University in 1998. The rats in the diabetic model group were intraperitoneally injected into 10 g/L STZ according to 60 mg/kg to establish rat models of diabetes mellitus. The blood glucose and body mass of rats in each group were determined respectively at 1, 2 and 3 months after modeling. The antibodies of advanced glycosylation end products (AGEs) of bovine serum albumin (anti-BSA) were self-prepared: ①The antigen of AGEs-BSA was prepared.②Eleven male Kuming mice (grade Ⅱ) of 3 months old without special pathogen were selected to inoculate AGEs-BSA. ③ The animals were immunized. ④Primary purification and detection of poly-antibodies of AGEs: the AGEs were performed immunohistochemical examination at 1 month after diabetic modeling by ELISA method.
MAIN OUTCOME MEASURES: ① Detection results of blood glucose and body mass of rats in two groups at different time points. ② Determination of polyclonal antibody titer of AGEs-BSA. ③ Changes in immunohistochemical image of AGEs in brain tissue of rats in two groups.
RESULTS: Thirteen rats in the diabetic model group and fifteen rats in the normal model group entered the stage of final analysis. ①Changes of blood glucose and body mass: At 1, 2 and 3 months after modeling, the blood glucose of rats in the diabetic model group were respectively(28.8±2.8),(23.1±5.5),(25.4±5.1) mmol/L, which were significantly higher than those in the normal control group [(6.2±0.9),(6.1±0.8),(6.1±0.7) mmol/L,P < 0.01]; At 1, 2 and 3 months after modeling, the body mass of rats in the diabetic model group were respectively (250.1±52.2),(263.8±50.0),(261.5±42.6) g, which were significantly lower than those in the normal control group [(422.6±36.2),(462.6±39.0),(485.0±28.8) g,P < 0.01].②Determination of antibody titer of immune serum: The mice were treated by AGEs-BSA of different concentrations twice. After that, the titer of AGEs -BSA was determined, and the results of which indicated that a higher absorbance existed at 1∶1 000. ③Determination of antigen concentration: The final titer of antibody in the abdominal dropsy was determined, and the results of which suggested that there was a much higher absorbance in the AGEs-BSA at the concentration of 5–50 mg/L. ④Determination of antibody titer in abdominal dropsy: The antibody titer in abdominal dropsy was detected by ELISA method with antigen at 20 mg/L, which indicated that the maximum absorbance (1.265±0.039) existed at 1∶4 000, and very larger absorbance (0.982±0.067) at 1∶20 000. The polyclonal antibody of AGEs-BSA was successfully prepared. ⑤Immunohistochemical detection results: The immunohistochemical staining of AGEs showed there were positive neurons in the first month in the diabetic model group, whereas it was not significant in the normal control group. The positive substances were found mainly in the cytoplasm.
CONCLUSION: Hyperglycemia at the early stage of diabetes mellitus (1 month after modeling) can lead to protein nonenzymeatic glycosylation in brain neurons, and no obvious reactions mentioned above are found in the normal control group. It suggests that the degenerative changes of tissue structure of central nervous system are related with protein nonenzymeatic glycosylation caused by hyperglycemia. 相似文献
BACKGROUND: The expressions of P2X3 receptor in dorsal root ganglia (DRG) after different peripheral nerve injuries are diverse. It indicates the different roles of P2X3 in different models-caused neuropathologic pains.
OBJECTIVE: To observe the expressions of P2X3 in corresponding DRG after sciatic nerve ligation in rats.
DESIGN: Controlled observation experiment.
SETTING: Department of Morphology, Hunan Traditional Chinese Medical College; Department of Human Anatomy and Neurobiology, Xiangya Medical College, Central South University.
MATERIALS: Thirty-five healthy adult SD rats of clean grade an d either gender, weighing （200±20）g, were involved. According to the random digits table, the involved rats were randomized into 3 groups: normal group (n =5), sham-operated group (n =5) and experimental group (n =25). The experimental group were subdivided into 3,7,14,21,28 days groups according to different surviving time after operation, 5 rats at each time point. Polyclonal rabbit anti-P2X3 antibody (ABCAM company); biotinylated goat anti-rabbit IgG (Zhongshanjingqiao Biotechnical Co., Ltd., Beijing); Motic fluorescence microscope (Motic, Germany).
METHODS: The experiments were carried out in the Department of Human Anatomy and Neurobiology, Xiangya Medical College, Central South University from June to December 2006. ① Rats of experimental group were created into models by ligation of right sciatic nerve according to the method of Seltzer et al. Left sciatic nerve was used as self-control. As for rats in the sham-operated group, ligation of sciatic nerve was omitted, but other procedures were the same as those in the experimental group. Rats of normal group were untouched. ② Rats of the normal group and sham-operated group survived for 14 days separately, and those of experimental group survived for corresponding time. After being deeply anesthetized by intraperitoneal injection of over-dose sodium pentobarbital, the rats of experimental group were transcardially perfused. L4–6 corresponding DRG connected to sciatic nerve were taken for preparing transverse sections serially. ③P2X3 expression in L4–6 DRG was detected by immunohistochemistry, immunofluorescence and image analysis techniques.
MAIN OUTCOME MEASURES: P2X3 expression in L4–6 DRG of rats in each group.
RESULTS: Thirty-five SD rats were involved in the final analysis. ① P2X3 expression in DRG: In normal DRG of rats, there were abundant P2X3 immuno-positive small- and medium-sized primary sensory neurons, especially the small ones, which mostly received the input from C fibers. There were only a few large neurons expressing P2X3. The immuno-positive products mostly were located in the cytoplasm and processes. The expression of P2X3 had a slight but significant decrease in ipsilateral L4–6 DRG 3 days after sciatic nerve ligation, and a decreasing tendency was observed with the elongation of time. At 28 days, the expression had not returned to base line, and still maintained at a low level. ② P2X3 immuno-positive gray scale in DRG: P2X3 immuno-positive gray scale in ipsilateral side L4–6 DRG was 117.74±2.38,129.12±4.86,133.56±3.79,148.75±6.90 and 150.49±5.15, respectively at 3,7,14, 21 and 28 days after sciatic nerve ligation, which was significantly higher than that in the normal group and sham-operated group （105.11±3.52,104.22±5.41,F =78.861,P < 0.05）, also significantly higher than that in the contralateral side （105.53±5.85,108.54±3.70,104.07±4.16,106.55±2.02,106.29±5.19,t =3.48–13.95,P < 0.05）; There were no significant differences when comparing sham-operated group or contralateral side at each time point with normal group (P > 0.05)
CONCLUSION: P2X3 is significantly down regulated in L4–6 DRG after sciatic nerve ligation. It may exert certain effects in neuropathic pain. 相似文献
BACKGROUND: Some experiments have demonstrated that melatonin (N-aceyl-5-methoxytryptamine, Mel) has antioxidation. However, whether it has neuroprotective effect in the ischemia/reperfusion injury of central nervous system is unclear.
OBJECTIVE: To observe the protective effect of Mel on ischemia/reperfusion-induced cerebellar neuronal apoptosis of rats, and the action mechanism.
DESIGN: Controlled observation experiment.
SETTING: Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology.
MATERIALS: Eight Sprague-Dawley rats aged 7–8 days and weighing 10–12 g were provided by Medical Experimental Animal Center, Tongji Medical College,Huazhong University of Science and Technology. Anti-cytochrome C monoclonal antibody was purchased from R & D Company; 7-dichlorodihydrofluorescein diacetate(DCFH-DA), rhodamine 123 and Mel were purchased from Sigma Company (USA). Lactate dehydrogenase (LDH) kit was purchased from Nanjing Jiancheng Bioengineering Institute.
METHODS: This experiment was carried out in the laboratory for Department of Biochemistry and Molecule Biology, Tongji Medical College between October 2002 and March 2004. Cerebellar neurons of rats were cultured in vitro. After oxygen-glucose deprivation (OGD) for 90 minutes, 1×10–4,1×10–6, 1×10–9 mol/L Mel was added, respectively, namely high-, middle-, and low-concentration Mel groups. Cells, which were cultured by OGD, served as model group, and control group, in which OGD intervention was omitted, was set. ①Cytochrome C level of mitochondrial cells in each group was detected by ELISA method. ②LDH activity in the cell culture fluid was measured, and cell membrane permeability change was analyzed. The cells in the Mel group with the lowest LDH activity served as Mel treatment group, i.e. cells were cultured with OGD, and then Mel was added; Meanwhile, Mel prevention group was set, i.e. Mel was added before OGD. Intervention was not changed in the model group and control group. ③ DNA level was analyzed and cell apoptosis was observed by agarose gel electrophoresis(AGE). ④Mitochondrial transmembrane potential of cells, and apoptotic way in each group were analyzed by confocal laser scanning microscopy.
MAIN OUTCOME MEASURES: ①Mitochondrial cytochrome C level of cerebellar nerve cells. ②LDH activity of cerebellar nerve cells. ③ DNA AGE results. ④Mitochondrial transmembrane potential change.
RESULTS: ①Mitochondrial cytochrome C level of cerebellar nerve cells: cytochrome C was obviously released at 6 hours of OGD-reperfusion. Mel inhibited the release of cytochrome C in dose-dependent manner. ②LDH activity of cerebellar nerve cells: LDH activity (A value) was significantly lower in the high- and middle-concentration Mel groups than in the model group (P < 0.05). LDH activity (A value) in the low-concentration Mel group was 0.415 0±0.012 9, indicating that Mel could decrease LDH activity of OGD-treated cell supernatant and promote membrane stablization in dose-dependent manner. ③AGE results of DNA: 1×10–9 mol/L was considered as the best concentration of melatonin. Cell DNA was extracted for AGE. Results presented typical ladder shape, indicating apoptosis appeared, while apoptosis was lessened in the Mel treatment group and Mel prevention group.④Mitochondrial transmembrane potential change: Experimental results showed that green fluorescein was evenly distributed in cerebellar granule cells cultured normally, and the axons of neurons were very clear. The body of neurons was condensed and the axons disappeared after cerebellar granule cells undergoing OGD injury. Mel could completely reverse the effect of OGD.
CONCLUSION: Mel can enhance cerebellar neuronal membrane stabilization of rats in dose-dependent manner, and suppress OGD-induced apoptosis of cerebellar granule cells by preventing against mitochondrial apoptosis. 相似文献
BackgroundCould the infarction be diagnosed quickly and accurately at the acute stage by CT perfusion imaging (CTPI) technology? Whether the images of CTPI will correspond with the pathological changes or not? All the questions need to be solved by experimental and clinical studies.ObjectiveTo reveal the rules of perfusion map changes and guide the early diagnosis of hyperacute cerebral infarction by analyzing the correlation of CTPI with pathological manifestations for hyperacute cerebral infarction.DesignA randomized controlled animal experiment.SettingExperimental Center of Medical Radiology, Longgang Central Hospital of Shenzhen City.MaterialsForty-two adult New Zealand rabbits of (2.6±0.5) kg, either male or female, were randomly divided into experimental group (n =36) and control group (n =6). Six rabbits in the experimental group were observed after ischemia for 0.5, 1, 2, 3, 4 and 6 hours respectively, and 1 rabbit in the control group was observed at each corresponding time point.MethodsThe experiments were carried out in the Experimental Center of Medical Radiology, Longgang Central Hospital of Shenzhen City from March 2003 to July 2004. Rabbit models of cerebral infarction were established by modified O'Brein method. The rabbits in the experimental group were scanned at 0.5, 1, 2, 3, 4 and 6 hours after ischemia respectively. The dynamic CT scan slice was 13 mm from the anterior edge of the frontal cortex, and six fake color functional images were obtained, including cerebral blood flow map (CBF map), cerebral blood volume map (CBV map), peak to enhancement map (PE map), flow without vessels map, time to peak map (TP map), time to start map (TS map). The manifestations and changes of the functional maps in different interval were observed. Bilateral symmetric ranges of interest (ROI) were drawn separately on the CBF map, CBV map, TP map and TS map. The blood flow parameters of focal and contralateral cerebral tissues could be obtained to calculate relative cerebral blood flow (rCBF, rCBF=focal CBF/contralateral CBF), relative cerebral blood volume (rCBV, rCBV= focal CBV/contralateral CBV), a relative time to peak (rTP, rTP= focal TP–contralateral TP), a relative time to start (rTS, rTS= focal TP–contralateral TP). The perfusion maps were input into AutoCAD software. The percents of ischemic cores and peri-ischemic areas accounting for contralateral cerebral hemisphere were calculated. The animals were anesthetized and killed, then the cerebellum and low brain stem were taken out. The brain tissues were cut on coronal plane at 14 mm from the anterior edge of the frontal cortex, a 2-mm piece anterior to the incision, and a 3-mm piece posterior to the incision. The anterior piece was fixed, stained and observed. A 1-mm slice was cut from the front of the posterior piece tissues as electron microscope sample, the remnant was fixed and then taken out, and the location and size of stained “white” areas were observed as the reference for electron microscope sample. The correlation between CTPI and pathological manifestations was observed.Main outcome measures Laws of time and spatial changes of ischemic areas; Pathological changes of the ischemic tissues; Correspondency between CTPI and pathological manifestations.Results Laws of time and spatial changes of ischemic areas: Relative ischemic-core areas were consistent in each perfusion map, increased incessantly along with the ischemic times. Relative peri-ischemic areas were inconsistent in each perfusion map, on CBF map from 1 to 6 hours after ischemia, the area of ischemic core increased from (1.503±0.523)% to (7.125±1.054)%, the ascending trend occurred. But the peri-ischemic areas showed a descending trend on CBF map, the areas decreased from (8.960±0.719)% to (5.445±0.884)% from 0.5 to 6 hours; The relative areas were the largest one on TP maps, the average value was (32.796±3.029)% at 0.5 hour after ischemia happening (60.540±1.683)% at 6 hours. The trend of ischemic areas was increased. No obvious change was observed on TS maps. Pathological changes of the ischemic tissues: Under light microscope, there was no obvious change at 0.5–2 hours after ischemia, edema at 3 hours, karyopycnosis at 4 hours and eosinophilous changes at 6 hours; Under electron microscope, there was edema in ischemic cores within 4 hours after ischemia, whereas karyopycnosis or structure vanished after 4 hours; Edema was observed in peri-ischemic areas. Correlation between CTPI and pathological manifestations: On CTPI maps, the ischemic core was blue on CBF and CBV maps, black on TP and TS maps. Along with the ischemic times, the rCBF and rCBV decreased, whereas the rTP and rTS prolonged. Hemodynamic parameters were not significantly different within 2 hours of ischemia and 2 hours after ischemia. The rTP and rTS became 0 after 1 and 2 hours respectively. On CTPI maps the peri-ischemic area was red on CBF and CBV maps, red and yellow on TS maps, red on TP maps. Along with the ischemic times, the rCBF decreased, and the lowest level was always at about 20%, whereas the rTP and rTS prolonged.Conclusion CTPI manifestations corresponded well with pathological findings, and it is a sensitive, stable and reliable technique to diagnose hyperacute cerebral infarction. TP map was more sensitive than CBF map and TS map in exhibiting the peri-ischemic areas, thus TP maps could be a good choice for observing peri-ischemic areas. 相似文献
BackgroundThe findings about the alterations in cerebrospinal fluid beta-amyloid protein (Aβ) and apolipoprotein E (ApoE) after subarachnoid hemorrhage indicate that they have significant correlation with prognosis of patients.ObjectiveTo observe the alterations in cerebrospinal fluid Aβ and ApoE after subarachnoid hemorrhage (SAH).DesignContrast observation.SettingDepartment of Neurosurgery, the First Hospital of Lanzhou University.ParticipantsA total of 25 SAH patients including 16 males and 9 females aged from 13 to 72 years were selected form Department of Neurosurgery, the First Affiliated Hospital of Lanzhou University from October 2003 to February 2004. The Hunt-Hess grade ranged from I to IV, and patients admitted hospital in 24 hours after invasion, affirmed by the brain CT scan and lumbar vertebra puncture, no other severe complications and important organs' functional defect and severe infection, no hematological system disease.MethodsAll admitted patients were collected CSF by lumbar vertebra puncture in 24 hours. The cerebrospinal fluid (CSF) of control group came from the admitted 15 patients of our hospital that have no nervous system disease. Aβ content was detected by enzyme linked immunosorbent assay (ELISA), the kit was provided by the Central Laboratory of the First Hospital of Lanzhou University; ApoE concentration was detected by monoclone enzyme linked immunosorbent assay (ELISA), the kit was provided by the Immunotechnique Research Institute of the Fourth Military Medical University. S100B concentration was detected by enzyme linked immunosorbent assay double antibody sandwich method, the kit was provided by the Physiological Research Room of the Fourth Military Medical University. The data were indicated on Mean±SD and were analyzed by SPSS 10.0 statistical package. All data were handled through test of significance variance analysis, and groups were compared through independent sampler t test. The concentration was handled through Pearson correlation analysis between Aβ and ApoE. The relationship between Aβ, ApoE concentration with pathogenetic condition and prognosis of the patients was handled through Spearman ranking correlation analysis.Main outcome measures The concentration of ApoE, Aβ and S100B after SAH in contrast to the control group in CSF by different Hunt-Hess and Glasgow Outcome Scale (GOS) grades; The level of correlation between ApoE and Aβ; Correlation between ApoE and Aβin pathogenetic condition and prognosis of the patients.ResultsAll 25 SAH patients and 15 controls were involved in the final analysis. The concentration of ApoE, Aβand S100B in CSF: The concentration of ApoE decreased after SAH in contrast to the control group [(0.46±0.007), (0.85±0.11) μg/L, P < 0.01], the concentration of ApoE decreased after SAH in contrast to the control group [(5.36±1.19), (8.41±1.60) μg/L, P < 0.01], and the concentration of S100B increased after SAH in contrast to the control group [(18.60±7.31), (6.56±1.02) pg/L, P < 0.01]. The concentration of ApoE, Aβ and S100B in CSF after SAH on different Hunt-Hess and GOS grades: The concentration of Aβin Hunt-Hess I–III grade was higher than Hunt-Hess IV, V grade [(6.63±1.25), (3.35±1.02) μg/L, P < 0.01], and the concentration of ApoE in Hunt-Hess I–III grade was higher than Hunt-Hess IV, V grade [(0.56±0.07), (0.38±0.04) μg/L, P < 0.05], the concentration of S100B in Hunt-Hess I–III grade was lower than Hunt-Hess IV–V grade [(16.32±5.58), (22.85±8.10) pg/L, P < 0.01]; the concentration of Aβ in GOSIV–III grade was lower than GOS IV, V grade [(3.76±1.04), (5.89±1.20) μg/L, P < 0.01], and the concentration of ApoE in GOS I–III grade was lower than GOS IV, V grade [(0.32±0.02), (0.58±0.07) μg/L, P < 0.01], and the concentration of S100B in GOS I–III grade was higher than GOS IV, V grade [(25.36±9.70), (14.33±6.69) pg/L, P < 0.01]. The results of Pearson correlation analysis and Spearman ranking correlation analysis: There was significantly positive correlation between CSF Aβconcentration and clinical outcome (r =0.65, P < 0.01), and the decrease in CSF Aβconcentration correlated significant with that of ApoE (r =0.85, P < 0.01).ConclusionThere is a significant decrease in both Aβ and ApoE in the CSF after SAH, and there is significant correlation between CSF Aβand ApoE concentration with clinical outcome, the interactions between these proteins may have important effects on SAH, ApoE and Aβas surrogate markers for the outcome of patients with SAH. 相似文献
BACKGROUND: Studies on febrile convulsion (FC)-caused brain injury are disputed in many aspects. How FC cause nervous system injury in the developmental period and what are the characteristics of these pathological injury are unknown. The current studies have demonstrated that heme oxygenase-1 (HO-1) exerts effects on brain injury mainly by catalyzing hemoglobin to produce degradation products, and HO-1 not only has neuroprotective effects, but also has neurotoxic effects during the FC-caused brain injury. Study on the effect of zinc protoporphyrin (ZnPP) on brain injury is still in the stage of animal experiment.
OBJECTIVE: To observe the effects of ZnPP on carbon monoxide (CO)/HO-1 system of rats subjected to FC, and to analyze the action pathway of ZnPP in brain protective effect.
DESIGN: A randomized controlled animal experiment.
SETTING: Department of Pediatrics, First Hospital Affiliated to Jiamusi University.
MATERIALS: Sixty-five Wistar rats, of either gender, were involved in this study. They were randomized into normal control group( n =14, 37 ℃ water bath) and febrile treatment group (n =51, 44.5 ℃ hot water bath). Febrile treatment group was sub-divided into febrile non-convulsion group (FNC group, n =16) and FC group (n =35). FC group was further sub-divided into simple convulsion group (n =20) and ZnPP treatment group (n =15). HO-1 mRNA in situ hybridization kit was provided by Boster Bioengineering Co.,Ltd. ZnPP(dark brown powder) was the product of Jingmei Bioengineering Company.
METHODS: This study was carried out in the postgraduate laboratory of Jiamusi University between January 2004 and January 2007. Rats in the febrile treatment group were placed in the 44.5 ℃ hot water bath box. If rats did not convulse in the water within 5 minutes, they were taken out, namely FNC group (n = 16), and those, which were convulsed within 5 minutes, were taken out immediately when they presented such a phenomenon, namely FC group (n =35). Convulsion induction was conducted once every other day, totally 10 times. Rats were euthanized for analysis at 24 hours after the last induction. Rats in the control group were placed in the 37 ℃ water. Rats in the ZnPP treatment group were intraperitoneally injected with ZnPP at 45 μmol/kg before FC attack. Rats in the simple convulsion group were only induced to be convulsed but not administrated.
MAIN OUTCOME MEASURES: CO level in the brain tissue homogenate and plasma of rats in each group was detected with a spectrophotometer. HO-1 mRNA expression in the hippocampal CA1 region, CA3 region and dentate gyrus of rats was observed by in situ hybridization technique.
RESULTS: Sixty-five Wistar rats were involved in the study. Two rats died respectively due to drowning and convulsion in the FC group. One rat died due to convulsion drowning in the ZnPP treatment group. ① Plasma CO concentration of control group and ZnPP treatment group was significantly lower than that of the FC group (P < 0.01), and was significantly higher in the ZnPP treatment group than in the FNC group (P < 0.05). ② CO level in the brain tissue homogenate was significantly lower in the control group and ZnPP treatment group than in the FC group (P < 0.01), and was very significantly higher in the ZnPP treatment group than in the control group (P < 0.01). ③ HO-1 mRNA expressions in the neuron of hippocampal CA1 region, CA3 region and dentate gyrus of the control group were the lowerest, and those in the FC group were the highest. HO-1 mRNA expression in the neuron of dentate gyrus in the FC group was significantly higher than that in the ZnPP treatment group (P < 0.01), and those in the FNC group and control group was significantly lower than that in the ZnPP treatment group (P < 0.01).
CONCLUSION: FC can cause brain injury. Over-expression of HO-1 mRNA and the increase of CO are involved in the patho-physiological process of FC. ZnPP can inhibit HO-1mRNA activity and decrease CO level, which is one of pathways for protecting brain. 相似文献
