Effect of intracranial hypertension on cerebral hemorrhage induced autonomic nerve imbalance

BACKGROUND: Cerebral hemorrhage can cause the imbalance of nerve function, whereas its mechanism and main impact factors are still not quite clear. OBJECTIVE: To explore the rules about the changes of intracranial pressure in brainstem hemorrhage and internal capsule hemorrhage, and analyze the role of intracranial hypertension in the changes of nerve function caused by cerebral hemorrhage. DESIGN: A self-controlled trial. SETTING: Department of Physiology, Tianjin Medical University. MATERIALS: Sixty-five healthy male Japanese white rabbits with long ears (1.5–1.8 kg) were supplied and fed by the Department of Animal Experiment of Tianjin Medical University. The RM6240B biological signal collecting and processing system was used. METHODS: The experiments were conducted in the Department of Physiology, Tianjin Medical University from August 2001 to May 2006. ① The rabbits were anesthetized, then fixed onto the brain stereotaxic apparatus, and afterwards fenestration on skull and intubation to lateral ventricle were performed.The dynamic changes of intracranial pressure were monitored continuously. Rabbits were infused with autologous arterial blood (0.3 mL) into midbrain corpora quadrigemina inferior colliculus to induce model of acute brainstem hemorrhage; models of internal capsule hemorrhage were established by infusing autologous arterial blood into internal capsule. ② The dynamic intracranial pressures under the above conditions were recorded continuously with the RM6240B biological signal collecting and processing system. ③ An animal model of persistent intracranial hypertension was established by infusion of physiologic saline into lateral ventricle. ④ The changes of the intensity of autonomic nerve discharge were analyzed, using the biological signal collecting and processing system before and after hemorrhage and under persistent intracranial hypertension. ⑤ Ten animal models of internal capsule hemorrhage and 10 of brainstem hemorrhage were selected respectively, then gross pathological samples were cut open, and the accuracy of hemorrhage models was affirmed. Histological sections in hemorrhage point and around this point were prepared for with hematoxylin and eosin staining, and the pathological changes were observed under light microscope. MAIN OUTCOME MEASURES: ① Changes of intracranial pressures before and after internal capsule hemorrhage and brainstem hemorrhage; ② Changes of the discharge intensity of cervical vagus nerve trunk in animal models of internal capsule hemorrhage, brainstem hemorrhage and persistent intracranial hypertension without hemorrhage; ③ Accuracy of location of internal capsule hemorrhage and brainstem hemorrhage confirmed by gross pathological samples and sections. RESULTS: Totally 65 rabbits were involved in the analysis of results. ① Dynamic state of intracranial pressure: Intracranial pressure increased obviously at 45 minutes after internal capsule hemorrhage and brainstem hemorrhage, the intracranial pressures were (1.31±0.30), (1.82±0.45) kPa, which were obviously higher than those before hemorrhage [(1.04±0.18), ( 1.05±0.19) kPa, P < 0.01]. ② Discharge of vagus nerve: Under intracranial hypertension, the discharge of cervical vagus nerve trunk was enhanced, and the discharge intensity of vagus nerve trunk was significantly different before and after persistent intracranial hypertension [(364.28±78.55), (1252.19±151.75) μV·s, P < 0.01]. The discharges of cervical vagus nerve trunk were significantly enhanced after internal capsule hemorrhage and brainstem hemorrhage (P < 0.01). ③ Validation of hemorrhage sites: The hemorrhage sites were internal capsule and brainstem on histopathological sections. CONCLUSION: Intracranial pressure may play an important role in the pathophysiological process of vagus nerve imbalance caused by cerebral hemorrhage.     

Spinal cord injury in patients with systemic lupus Erythematosus——Clinical manifestations, imaging characteristics and treatment

BACKGROUND： There are fewer reports on systemic lupus erythematosus （SLE） related myelitis, and definite and uniform therapeutic program is not available. OBJECTIVE： To observe the clinical manifestations, imaging characteristics, results of laboratory examination and treatment of SLE. DESIGN： A retrospective case analysis. SETTING： Department of Neurology, the Second Affiliated Hospital of Sun Yat-sen University. PARTICIPANTS： Totally 1 052 SLE inpatients were selected from the Second Affiliated Hospital of Sun Yat-sen University from January 1995 to May 2005, and they all accorded with the diagnostic standards for SLE set by American Rheumatism Association in 1982. 124 of them were diagnosed to have damage of central nervous system. Inclusive criteria： Patients with one of the focal physical signs, including mental and behavior disorders, headache, seizure and involvement of nervous system. Exclusive criteria： Patients with hypertensive encephalopathy, damage of nervous system due to uremia and infection of central nervous system. Spinal cord lesion occurred in 15 female cases of 23 - 51 years old. Informed consents were obtained from all the participants. METHODS： The physical signs, laboratory examinations, therapeutic program and prognosis were recorded in the 15 patients with symptoms of spinal cord lesions. All the patients underwent MRI scan of brain or lesioned segment of spinal cord, and 8 cases of them underwent lumbar puncture to determine intracranial pressure, routine and biochemical examinations were cerebrospinal fluid were performed. The disease activity of SLE in systems beyond central nervous system was evaluated with modified lupus activity criteria count （LACC）. MAIN OUTCOME MEASURES：① Incidence of SLE related myelitis, attack age distribution and its association with the activity of SLE; ② Comparisons of the clinical characteristics, cranial and spinal cord MRI manifestations, different therapeutic program and prognosis. RESULTS： All the 15 SLE patients were involved in the analysis of results. ① The incidence of SLE related myelitis was low （1%, 15/1 052）. ②SLE related myelitis occurred mostly when the SLE symptoms were active, and only a few occurred at the stable period. ③ Among the SLE patients, MRI displayed abnormal changes in 71% （10/14）, the typical changes appeared abnormal signals at corresponding spinal segments, manifested as prolonged T1 and T2 signals, thickened spinal segments. Lumbar segments were mostly involved. ④ Of the 9 patients treated with hormone impact, 7 cases （78%） had obvious improvements, and the effects were better in those treated with immunosuppressor combined with intravenous immunoglobulin of large dosage. CONCLUSION：① Myelitis is a rare complication of SLE.② MRI serves as a valuable supplementary approach in the diagnosis of SLE related myelitis without specificity. ③ Steroid pulse combined with immunosuppressor and intravenous immunoglobulin of large dosage is effective in the treatment.     

Neuroprotective effect of melatonin against ischemia/reperfusion-induced neuronal apoptosis in mouse cerebellum

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.     

Hyperglycemia induces protein nonenzymeatic glycosylation in brain neurons of diabetic rats at early stage

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.     

Application of biological dural graft made by meninges from porkers

BACKGROUND: Presently, over 40 kinds of dural grafts have been successively used in clinic. Among them, lyophilized human dura mater with good histocompatibility and less complications is applied most widely. But there are a few reports on cases of infected spongiform encephalopathy following application of lyodura. More ideal repair materials deserve to be further investigated. OBJECTIVE: To investigate the efficiency and safety of biological dural graft made by meninges from porkers to repair meningeal injury. DESIGN: A self-control observation. SETTING: Wuhan General Hospital of Guangzhou Military Area Command of Chinese PLA. MATERIALS: Sixteen New Zealand Rabbits, of either gender, weighing from 2 to 3 kg, of clean grade Ⅱ, with the age of 0.5–1 year, were involved in this experiment. The involved rabbits were provided by the Animal Experimental Center of the First Military Medical University of Chinese PLA. Biological surgical patch (dural graft) was developed by Guangdong Guanhao Biotechnological Co.,Ltd. It was processed by using meninges from porkers by tissue engineering technology. METHODS: This experiment was carried out in the Experimental Center of the 157 Hospital of Chinese PLA between December 2003 and June 2004. ① The experimental rabbits were anesthetized. Dura mater was exposed from two sides of postmedial line of coronal suture. A rectangular dura mater about 8 mm×8 mm in size was cut off. Then a biological surgical patch (dural graft) was sheared into insert with 8 mm diameter and sutured. The left dura mater was untouched and used as control. Scalp was sutured, and postoperative wound healing and recovery were observed. ②The anesthetized rabbits were sacrificed at postoperative 3, 14, 30 and 90 days, 4 rabbits once. The whole head was cut off, and its scalp was removed. Afterwards, the head was fixed by formalin. Tissues in operative site were obtained, performed routine paraffin embedding, sliced and conducted HE staining, finally, the sections were observed. White blood cells in venous blood were counted before operation and execution, separately. The obtained data were statistically analyzed. MAIN OUTCOME MEASURES: ① Wound healing and recovery following implantation of dural graft. ②The amount of white blood cells in venous blood from rabbits at each time point before operation and before execution. ③ Histological examination results of operative site. RESULTS: Sixteen experimental rabbits were involved in the final analysis. ① The experimental rabbits of each group had no local infection, effusion and abnormal appearance. They had good wound healing and were normal to access to food. ② There were no significant differences in amount of white blood cells in venous blood from experimental rabbits between at each time point after modeling and before operation (P > 0.05). ③Pathological observation of operative site : At postoperative 3 days, local acute inflammation repair reaction appeared; At postoperative 2 weeks, chronic inflammatory reaction appeared, endodermis in artificial dural graft formed, and artificial dural graft and host dura mater healed; In postoperative 1 month, wound began to chronically recover; In postoperative 3 months, host blood capillary began to form in artificial dural graft based on chronic repair. In all the control sides, fibroplasia was found, and a few neutrophils were found at postoperative 2 weeks. CONCLUSION: Biological surgical patch has high stability and good histocompatibility. It can provide dural epithelial epithelium, effectively prevent against the conglutination of scalp tissue and brain tissue, and avoid the leakage of cerebrospinal fluid.     

Clinical characteristics, neurophysiological grade and outcome of patients with carpal tunnel syndrome——A retrospective study

BACKGROUND: Carpal tunnel syndrome (CTS) is diagnosed mainly according to clinical symptoms, physical sign and neurodiagnostic laboratory examination. The therapeutic effect of conservative management and surgical operation in treating CTS need to be further observed and evaluated. OBJECTIVE: To analyze the clinical characteristics, neurophysiological grade and outcome in patients with CTS. DESIGN: Retrospective case-analysis. SETTING: Department of Neurology, First Affiliated Hospital, Medical College, Xi'an Jiaotong University. PARTICIPANTS: Totally 161 patients with suspected CTS from National Neuroscience Institute of Singapore referred to the Neurodiagnostic Laboratory for the confirmatory testing between January and September 2002. The involved patients, 137 male and 24 female, were aged 21–85 years. METHODS: ①The condition of diabetes mellitus complicated by abnormal thyroid function was observed. ② The effect on predominant hand, and paraesthesia were observed. ③Neuroelectrophysiological studies were performed and the results were graded into mild, moderate and severe CTS according to the American Association of Electrodiagnostic Medicine (AAEM) criteria. ④ Conservative management and surgical intervention were followed up 3 months later, and symptoms and physical sign basically disappeared, and function was basically recovered, which indicated that disease condition improved. MAIN OUTCOME MEASURES: ①Condition of CTS complicated by metabolic disease; ②Effects on predominant hand and paraesthesia; ③Electrophysiological grading; ④Prognosis. RESULTS: Totally 161 patients participated in the final analysis. ①Condition of CTS complicated by metabolic disease: Among 161 patients, 17.4% (28/161) were documented to have diabetes mellitus and 7(4.3%) had hypothyroidism. ②Effects on predominant hand and paraesthesia: Dominant hand involvement was present in 134 patients (83.2%) and more than 75% had onset of symptoms in the dominant hand. Sensory symptoms like numbness and paresthesias were the predominant symptoms, accounting for 89.1% (134/161), this discomfort was felt in all 5 digits of the hand in 47.6%, and lateral three and half digits in 21.4%. The noctural symptoms were present in 30.4% (49/161) patients. ③Electrophysiological typing: The most frequent abnormality was that of the prolonged mid-palm median and ulnar latency difference in 146(54.7%) hands; 103(38.6%) hands had prolonged median motor distal latency. Absent response from thenar muscle was present in 35 (13.1%) hands. Nerve conduction study showed bilateral CTS in 105 (65.2%) patients and unilateral CTS in 56 (34.8%) patients. Sixteen patients with bilateral CTS had symptoms in one hand only. Overall, 36.8% had mild, 49.2 % had moderate and 13.9 % had severe CTS, with median duration of symptoms of 6, 9 and 14 months, respectively. ④Delay in diagnosis: 37(22.9%) patients delayed in diagnosis from 1–4 months, 16(43.2%) were misdiagnosed as cervical spondylosis; 6(16.2%) were ignored due to their condition by busy work; 15(40.5%) were unware of their symptoms. ⑤Prognosis: Follow up data was available for only 72.7% (117/161) patients. Conservative management was conducted in 73.5% (86/117). Clinical symptoms were resolved or improved in 65.1% (56/86) patients with 17 mild CTS, 29 moderate CTS, and 10 severe CTS. 26.5% (31/117) patients underwent surgery for CTS release, and clinical symptoms were improved in 12(38.7%) with moderate CTS and 2 (6.5%) with severe CTS at 3 months of follow up. CONCLUSION: ①Sensory symptoms in CTS are more in severe and common in dominant hand. ②Conservative management showed resolution or improvement for mild and moderate CTS. Surgical intervention shows either resolution or improvement in clinical symptoms in moderate CTS. ③ The common reasons for delay in diagnosis were due to misdiagnosis as cervical spondylosis and lack of awareness of the condition. ④Assessment on severity of CTS by electrophysiological grade is of important significance for determining therapeutic mean.     

Correspondence of CT perfusion imaging to pathological manifestations in rabbit models of hyperacute cerebral infarction

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.     

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.     

Effects of tanshinone Ⅱ sodium sulfonate plus cinepazide maleate on the hemorrheologic indexes and blood lipids in patients with acute cerebral infarction

BACKGROUND: The severity of cerebral infarction is associated with the increase of blood viscosity caused by hyperfibrinogenemia and hyperlipidemia, etc. Thus it has become one of the target for treating cerebral infarction to decrease blood viscosity by integrated Chinese and western medicine. OBJECTIVE: To investigate the influence and clinical therapeutic effects of cinepazide maleate combined with tanshinone Ⅱ A sodium sulfonate on the hemorrheologic indexes and blood lipids of patients with acute cerebral infarction, and compare the results with those of simple cinepazide maleate treatment. DESIGN: A non-randomized case-controlled observation. SETTINGS: Hebei North University; the Second Affiliated Hospitals of Hebei North University; the Third Affiliated Hospitals of Hebei North University. PARTICIPANTS: Eighty-six inpatients with cerebral infarction were selected from the infirmary, the Second and Third Affiliated Hospitals of Hebei North University from September 2004 to October 2006. They were all diagnosed to have acute cerebral infarction by CT or MRI, and accorded with the diagnostic standards for acute cerebral infarction set by the Fourth National Academic Meeting for Cerebrovascular Disease in 1995. Meanwhile, 40 teachers and medical staff of voluntary physical examinees were selected as the control group. Informed contents were obtained from all the patients and their relatives. METHODS: The patients were divided into combined treatment group (n=43) and simple treatment group (n=43). In the combined treatment group, the patients were administrated with 160 mg cinepazide maleate injection (Beijing Four-ring Pharmaceutical, Co.,Ltd, No. H200220125; 80 mg/2 mL) added in 5% glucose, and 40 mg tanshinone Ⅱ sodium sulfonate (Shanghai No.1 Biochemical & Pharmaceutical Co.,Ltd., No. H31022558, 10 mg/2 mL) added in 250 mL normal saline. In the simple treatment group, the patients were only administrated with cinepazide maleate 320 mg added in 5% glucose or 250 mL normal saline. They were treated for 1 or 2 courses, once a day, and 14 days as a course. The patients were detected before treatment and at 14 and 28 days after treatment respectively. ① Determination of hemorrheologic indexes: Whole blood viscosity was determined with LBY-N6B automatic hemorrheologic meter; Plasma viscosity with LBY-F200B automatic plasma viscosity meter; Volume of fibrinogen was determined by the method of 12.5% sodium nitrate depositing biuret reaction. ② Determination of blood lipids: The serum levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) were determined. ③ Severity of neurological deficit: The total score of neurological deficit score (NDS) ranged from 0 to 45 points, 0–15 points was taken as mild, 16–30 points as moderate and 31–45 points as severe. ④ Evaluation of curative effects: Generally cured: NDS decreased by 91%–100%, and disabled severity of grade 0; Significantly improved: NDS decreased by 46%–90%, and disabled severity of grades 1–3; Improved: NDS decreased by 18%–45%; No change: NDS decreased by less than 18%; Aggravated: NDS increased by more than 18%. Generally cured and significant improved were taken as significant effect. ⑤ The adverse events and side effects after medication were observed. MAIN OUTCOME MEASURES: ① Results of hemorrheologic indexes and blood lipids; ② NDS results in the combined treatment group and simple treatment group; ③ Therapeutic effects and adverse events. RESULTS: All the 86 patients with cerebral infarction and 40 healthy controls were involved in the analysis of results. ① Results of hemorrheologic indexes and blood lipids: The hemorrheologic indexes and blood lipids before treatment were manifested as abnormalities to different extents in both the combined treatment group and simple treatment group; The hemorrheologic indexes after treatment were obviously improved in both groups. But the hemorrheologic indexes were improved more obviously in the combined treatment group as compared with those in the simple treatment group (P < 0.05); The levels of TC, TG and LDL-C after treatment in the combined treatment group were obviously lowered (P < 0.05), whereas those in the simple treatment group were not significantly changed (P > 0.05). ② NDS results: The NDS scores at 14 and 28 days after treatment in the combined treatment group [(6.23±2.34), (4.27±1.83) points] were obviously lower than those in the simple treatment group [(8.76±3.41), (6.65±2.49) points, P < 0.05]. ③ Therapeutic effects and side effects: The total significant effective rates in the combined treatment group and simple treatment group were 93% and 81% respectively. In the combined treatment group, 1 case suffered from palpitation, dizziness and agrypnia. In the simple treatment group, 1 case suffered from palpitation, dizziness and agrypnia, 1 case had itch of skin. All the above symptoms disappeared gradually after the transfusing speed was adjusted to be slower. No drug withdrawal occurred in the patients due to the adverse events. CONCLUSION: Cinepazide maleate combined with tanshinon can obviously improve the abnormalities of hemorrheologic indexes and blood lipids and nerve function in patients with acute cerebral infarction, and its curative effect is faster than that of simple cinepazide maleate treatment.     

Olfactory ensheathing cell transplantation in 106 patients with old spinal cord injury Differences in ages, sexes, disease courses, injured types and sites

BACKGROUND: It has been demonstrated that the transplantation of olfactory ensheathing cell (OEC) can promote the recovery of neurological function through ameliorating the local internal environment in spinal cord injury. OBJECTIVE: To evaluate the recent efficacy of OEC transplantation on old spinal cord injury. DESIGN: A self-controlled experiment. SETTING: Department of Neurosurgery, Taian Rongjun Hospital of Shandong Province. PARTICIPANTS: Totally 106 inpatients with old spinal cord injury were selected from the Department of Neurosurgery, Taian Rongjun Hospital of Shandong Province from June 2004 to December 2006, including 97 males and 9 females. Inclusive criteria: ① Complete data; ② Informed with the fact; ③ No further recover neurological function after drug therapy (neurotrophic factor, GM-1), traditional Chinese medicine, physiotherapy and rehabilitative exercises; ④ No obvious compression of the injured spinal cord displayed by MRI examination. METHODS: ① The olfactory bulb was obtained from embryo of induced labor in middle pregnancy above 4 months supplied voluntarily by pregnant women, and the survived cells after purification and culture for 1–2 weeks were collected. Dura mater was incised by posterior approach, then the cultured OEC suspension was transplanted to corresponding regions by means of multi-target injection using microscope. ② The patients were evaluated for twice with the standards suggested by American Spinal Injury Association (ASIA) at admission and 2–4 weeks postoperatively, in order to investigate the efficacy in different age groups, different sites and at different time points after the OEC transplantation. ③ Standards for evaluation: The International Standard for Neurological and Functional Classification of Spinal Cord Injury set by ASIA: The highest score of motor function was 100 points; The highest score of sensory function was 112 points for light touch and 112 for acupuncture sense. Frankel grading modified by ASIA in 1992: grades A, B, C, D and E (grade A for complete injury, and grade E for normal). ④ The data were statistically processed by Professor Cheng from the Department of Statistics, Taishan Medical College. MAIN OUTCOME MEASURES: Changes of motor and sensory functions postoperatively. RESULTS: All the 106 patients with old spinal cord injury were involved in the analysis of results. Grade A (n =72) at admission recovered to grade B in 31 cases and grade C in 7 cases after operation; Grade B (n =9) recovered to grade C in 2 cases; Grade C (n =15) recovered to grade D in 4 cases; Grade D (n =10) recovered to grade E in 1 cases, which referred to normal sensory and motor functions. The scores of motor, tactile sensation and pain sensation postoperatively were all obviously higher than those at admission (t =5.381, 7.036, 7.775, P < 0.05). The recovery of motor function had obvious difference among patients of different ages (F =5.235, P =0.001). There were no obvious differences in the recovery of motor and sensory functions among patients of different sexes, courses, injured types and sites. CONCLUSION: The neurological function can be ameliorated in a short time after OEC transplantation in patients with old spinal cord injury. The efficacy is generally the same in patients of different sexes, courses, injured types and sites, but there are differences in the effects on motor function of patients of different ages.     

Huayu capsule enhances limb-catching capability of rats with experimental open traumatic brain injury

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.     

Brain-derived neurotrophic factors increase the proliferation and differentiation of endogenous neural stem cells in mouse models of cerebral infarction

BACKGROUND： It has been confirmed that brain-derived neurotrophic factor （BDNF） can promote the proliferation of neural stem cells （NSCs） and protect neuron-like cells in vitro. However, its effect on endogenous NSCs in vivo is still unclear. OBJECTIVE： To evaluate whether BDNF can induce the endogenous NSCs to proliferate and differentiate into the neurons in the mice model of cerebral infarction. DESIGN： A synchronal controlled observation. SETTINGS： Department of Neurology, Microbiology Division of the Department of Laboratory, Tianjin First Central Hospital; Howard Florey Institute, Medical College, the University of Melbourne. MATERIALS： Twenty-four pure breed C57BL/6J mice at the age of 10 weeks old （12 males and 12 females） were divided into saline control group and BDNF-treated group, 6 males and 6 females in each group. METHODS： The experiments were performed at the University of Melbourne from July 2004 to February 2005. ① The left middle cerebral artery （MCA） was ligated in both groups to establish models of cerebral infarction and the Matsushita measuring method was used to monitor the blood flow of the lesioned region supplied by MCA. 75% reduction of blood flow should be reached in the lesioned region. ② At 24 hours after infarction, mice in the BDNF-treated group were administrated with BDNF, which was slowly delivered using an ALZET osmium pump design. BDNF was dissolved in saline at the dosage of 500 mg/kg and injected into the pump, which could release the solution consistently in the following 28 days. The mice in the saline control group accepted the same volume of saline at 24 hours after infarction. ③ The Rotarod function test began at 1 week preoperatively, the time stayed on Rotarod was recorded. The mice were tested once a day till the end of the experiment. At 4 weeks post cerebral infarction, double labeling of Nestin and GFAP, BIH tubulin and CNPase immunostaining was performed to observe the differentiation directions of the re-expressed endogenous NSCs, and the percentages of the cells differentiated into astrocytes, neurons and oligodendrocytes were calculated. MAIN OUTCOME MEASURES： ① The differentiation directions of the re-expressed endogenous NSCs, and the percentage of the cells differentiated into astrocytes, neurons and oligodendrocytes.② Comparison of motor function between the two groups. RESULTS： All the 24 pure C57BL/6J mice were involved in the analysis of results. ①Positively expressed endogenous NSCs appeared in the mice of both groups, and they mainly distributed around the focus of lesion, as well as the contralateral side. The expressed cells in the BDNF-treated group were obviously more than those in the saline control group. ②Activations of endogenous NSCs： At 4 weeks after infarction, re-expressions of endogenous NSCs appeared in both groups. The number of the re-expressed cells in the BDNF-treated group was about 4.2 times higher than that in the saline control group. The percentage of the cells differentiated into neurons in the BDNF-treated group was significantly higher than that in the saline control group （36%, 15%）, the percentage of the cells differentiated into astrocytes was lower than that in the saline control group （54%, 77%）, whereas the percentage of the cells differentiated into oligodendrocytes was similar to that in the saline control group （10%, 8%）. ③ Results of motor functional test： Compared with before cerebral infarction, the mice in both groups manifested as obvious decrease in motor function at 1 week after infarction, whereas the recovery of motor function in the BDNF-treated group was significantly superior to that in the saline control group at 2, 3 and 4 weeks （P 〈 0.01）. CONCLUSION： BDNF can promote the proliferation of endogenous NSCs in the brain of mice with cerebral infarction, it can decrease the differentiation rate of astrocytes, and increase the differentiation rate of neurons. BDNF has small influence on the differentiation of endogenous NSCs into oligodendrocytes, which was not benefit for the recovery of neural axon. Endogenous NSCs may improve the motor function of mice through the above pathways.     

Mirror writing in elderly patients with Alzheimer disease and vascular dementia

BACKGROUND: The results showed that mirror writing (MW) was correlated with the development of written language, so that MW examination may be one of methods to examine the intelligence of elderly people. OBJECTIVE: To study the MW in elderly patients with Alzheimer disease (AD) and vascular dementia (VaD) and take appropriate scale for their evaluation. DESIGN: Taking the written portion of the Chinese Aphasia Examination Scale (1994) for assessment. SETTING: Department of Neurology, Neuropsychological Laboratory, Beijing Hospital. PARTICIPANTS: From March 1998 to January 2001, 33 patients with AD, 30 patients with VaD admitted into Department of Neurology, Beijing Hospital was enrolled into study. Criteria according to the Diagnostic and Statistical Manual of Mental Disorder, 4th edition (DSM-IV), published by the American Psychiatric Association was used to diagnose AD, while criteria according to the National Institute of Neurological Disorders and Stroke-Association Internationale pour la Recherche et l’ Enseignement en Neurosciences (NINDS-AIREN) and Alzheimer Disease Diagnostic and Treatment Center (ADDCT) were used for diagnosis of VaD. AD group contained 19 males and 14 females aged 60–83 years. Twenty-eight males and 2 females, aged 60–87 years made up the VaD group. The 63 healthy elderly subjects matched on age and education as controls were enrolled into study. The matched controls were categorized AD control (n =33) and VaD control (n =30). All patients and controls were understanding and agree with all items of assessment. METHODS: MW examination, Mini Mental State Examination (MMSE), Hachinski Ischemic Scale, the Global Deterioration Scale (GDS) were examined in all subjects. ① Use the written potion of the Chinese Aphasia examination Scale (1994), patient using MW for 91%–100% of dictation had complete MW, those using MW for 51%–90% of dictation had severe MW, those using MW for 11%–50% had moderate MW, those using MW for 1%–10% had mild MW. ② According to the MMSE, the patients were considered to have dementia if they were illiterate and had an MMSE score ≤ 17 score or educated time ≤ 6 years and MMSE ≤ 20 score or educated time > 6 years and MMSE ≤ 24 score. ③ Using the Hachinski Ischemic Scale to differentiate the AD and VaD, it included 6 items and total 9 scores. > 7 score was VaD, < 4 score was AD and 4–7 score was blended dementia. ④ Using the GDS to assess cognitive function: Standard criteria were divided in 7 degrees: 1 degree: no impairment of cognition and 7 degree: very severe impairment of cognition. MAIN OUTCOME MEASURES: The data of MW examination, evaluation of MMSE, Hachinski Ischemic Scale and the GDS of all assessed subjects. RESULTS: All 63 cases of AD and VaD and 63 healthy controls were entered for analysis. ①Results of MW examination: A total of 17 patients with AD were characterized as using MW, 3 with moderate MW and 14 patients with mild MW. In the corresponding control group, only 2 subjects were characterized as being mild MW. The VaD group has 23 patients with MW, 2 with moderate and 21 patients with mild MW. ② MMSE score: MMSE score of AD group was much lower than that of individuals in control group [(20.15±3.40), (29.73±0.40) score, P < 0.01], MMSE score of VaD group was much lower than that of individuals in control group [(19.33±2.75), (29.12±0.63) score, P < 0.01]. ③ Hachinski Ischemic Scale and GDS score: Rating according to the Hachinski Ischemic Scale was higher in VaD patients compared to AD patients [(9.61±1.69), (1.09±0.60) score, P < 0.01]. The GDS score did not significantly differ between the AD group and the VaD group. CONCLUSION: ① MW examination could be used as an indicator of intelligence in healthy elderly people and also could be used as one of methods to assess the intelligence in AD and VaD patients. ② Grade of severity of MW may indirectly reflect the degree of dementia.     

Hyperbaric oxygen therapy for cerebral blood flow and electroencephalogram in patients with acute cerebral infarction ——Choice for therapeutic occasion

BackgroundHyperbaric oxygen (HBO) therapy increases blood oxygen content, changes cerebral blood flow (CBF) and cerebral metabolism. Its therapeutic effects on cerebrovascular disease have been fully confirmed, but the occasion for HBO therapy is still unclear.ObjectiveTo observe the therapeutic effects of HBO therapy at different time on CBF and electroencephalogram (EEG) in patients with acute cerebral infarction (CI).DesignRandomized controlled trial.SettingDepartment of Neurology, Shidong Hospital, Yangpu District of Shanghai.ParticipantsNinety-six inpatients with acute CI, admitted to Department of Neurology, Shidong Hospital, Yangpu District of Shanghai from January 2001 to December 2006, were involved in this experiment. The involved participants met the diagnosis criteria of acute CI and confirmed by skull CT or MRI. They all were patients with moderate CI (16–30 points) according to neurologic deficit score formulated by Chinese Medical Association. Informed consents of detected items and therapeutic regimen were obtained from all the involved participants. They were randomized into two groups with 48 in each: early-stage treatment group and advanced-stage treatment group. Among the 48 patients in the early-stage treatment group, 21 male and 27 female, aged 53–68 years, 22 patients were found with basal ganglia infarction, 10 with brain lobe infarction, 16 with multiple infarction, 27 accompanied with hypertension and 2 accompanied with diabetes mellitus. Among the 48 patients in the advanced-stage treatment group, 23 male and 25 female, aged 52–71 years, 25 patients were found with basal ganglia infarction, 10 with brain lobe infarction, 12 with multiple infarction, 1 with brain stem infarction, 28 accompanied with hypertension and 1 accompanied with diabetes mellitus.MethodsAfter admission, patients of two groups received routine drug treatment.

Patients in the early-stage treatment group and advanced-stage treatment group began to receive HBO therapy within one week of CI and 4 weeks after CI, respectively. The total course of treatment both was 2 weeks. EEG examination was carried out before and after therapy, and CBF was determined with ¹³³Xe inhalation.

Assessment criteria of curative effects: Basically cured: neurologic symptoms and body signs disappeared, could work and do housework; Markedly effective: score of neurologic deficit was decreased by over 21 points, could manage himself/herself partially; Effective: score of neurologic deficit was decreased by 8 to 12 points; Non-effective: Score was increased or decreased less than 8 points, neurologic deficit was worsened, even died. Total effective rate = (number of cured+number of markedly effective+number of effective)/number of total cases×100%.

t test and Chi-square test were used for comparing the difference of measurement data and enumeration data respectively, and Ridit analysis was used for comparing the difference of clinical curative effects.Main outcome measures

Comparison of EEG and CBF of patients from two groups before and after treatment.

Comparison of post-treatment neurologic deficit of patients between two groups.ResultsAll the involved 96 patients with CI participated in the final analysis.

Clinical symptoms of patients from two groups after therapy were significantly improved as compared with those before therapy, and curative effects of early treatment group were better than those of advanced treatment group (U =1.99,P < 0.05).

After treatment, CBF in each region of brains, except for that in parietal lobe of patients in the advanced-stage treatment group, was significantly improved (P < 0.05–0.01); The improvement of CBF of patients in the early-stage treatment group was more obvious than that in the early-stage treatment group (P < 0.05–0.01).

The abnormal rate of EEF of patients from early-stage treatment group and advanced-stage treatment group before treatment was 94% and 96%, respectively. After treatment, improvement rate of EEG of patients in the early-stage treatment group was 95%, which was significantly different from that in the advanced-stage treatment group (82%,χ² =4.32,P < 0.05)ConclusionHBO therapy both at early and advanced stages of CI (within 1 week and 4 weeks after CI attack) can improve CBF and EEG of patients with early CI, especially.     

Changes of serum high sensitive C-reactive protein in patients with acute cerebral infarction

BACKGROUND: Serum high sensitive C-reactive protein (hs-CRP), which regards as a high sensitive mark of systemic inflammatory response syndrome, can provide a lot of valuable information for the treatment and prognosis of cerebrovascular disease. OBJECTIVE: To observe the differences of blood glucose, lipid, homocysteine and previous disease history among patients with acute cerebral infarction at various levels of hs-CRP and compare changes of hs-CRP of patients with various degrees of neurologic impairment. DESIGN: Contrast observation. SETTING: Department of Neurology, Shenzhou Hospital, Shenyang Medical College. PARTICIPANTS: A total of 102 patients with acute cerebral infarction were selected from Department of Neurology, Shenzhou Hospital of Shenyang Medical College from February 2005 to September 2006, including 55 males and 47 females aged from 55 to 86 years. All accepted patients met the diagnostic criteria of cerebral infarction established by the Fourth National Cerebrovascular Disease Academic Meeting and were diagnosed with CT or MRI examination. All patients provided the confirmed consent. Based on clinical criteria of neurologic impairment established by the Fourth National Cerebrovascular Disease Academic Meeting, patients were randomly divided into mild group (0–15 points, n =46), moderate group (16–30 points, n =38) and severe group (31–45 points, n =18). In addition, based on hs-CRP level within 72 hours, patients were divided into normal group (hs-CRP ≤ 3 mg/L, n =53) and increasing group (hs-CRP > 3 mg/L, n =49). METHODS: ① 2 mL venous blood was selected from hospitalized patients in the next morning to separate serum. Quantitative measurement of hs-CRP was dealt with Latex Enhnced Turbidimetric Immunoassay (LETIA). ② Fasting venous blood was colleted from hospitalized patients in the next morning to measure numeration of white blood cells, fibrinogen, blood glucose, total cholesterol (TC), triacylglycerol (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and homocysteine. ③ Measurement data were compared with t test or analysis of variance. MAIN OUTCOME MEASURES: ① Comparisons of serum biochemical indexes among patients with various levels of hs-CRP; ② comparisons of risk factors among patients with various levels of hs-CRP; ③ comparisons of levels of hs-CRP among patients with various degrees of clinical neurologic impairment. RESULTS: A total of 102 patients were involved in the final analysis. ① Plasma fibrinogen and numeration of leucocytes were more in the increasing group than those in the normal group (t =4.39, 3.54, P < 0.01); while, there were no significant differences of blood glucose, TC, TG, HDL-C, LDL-C and homocysteine between the two groups (P > 0.05). ② Percentage of patients with hypertension and diabetes mellitus (DM) was higher in the increasing group than the normal group (χ2=3.98, 4.23, P < 0.05); while, percentage of patients with smoking in the increasing group was not significantly different from that of patients in the normal group (P > 0.05). ③ Level of hs-CRP of patients with severe neurologic impairment was higher than that of patients with moderate neurologic impairment (t =2.273, P < 0.05); that of patients with moderate neurologic impairment was higher than that of patients with mild neurologic impairment (t =2.586, P < 0.05); that of patients with severe neurologic impairment was obviously higher than that of patients with mild neurologic impairment (t = 4.913, P < 0.01). CONCLUSION: ① With the increase of hs-CRP, plasma fibrinogen and numeration of leucocytes of patients with acute cerebral infarction is increased, especially, they are increased remarkably among patients who have history of diabetes mellitus and hypertension. ② Increase of level of hs-CRP can be regarded as one of marks to evaluate severity of acute stroke.     

Triptolide for cerebral ischemia/reperfusion injuryt

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.     

Protective effects of ginkgo biloba leaves extract on peroxide-induced oxidative stress damage in PC12 cells

BACKGROUND: Extracts of ginkgo biloba leaves (EGB) and its metabolites have been reported to enhance brain function and nerve behavior. It has also been hypothesized that they can protect neurons from oxidative stress. OBJECTIVE: To investigate protective effects of EGB on peroxide (H2O2)-induced oxidative stress damage in PC12 cells. DESIGN: Observational contrast study. SETTING: Department of Pathophysiology, Guangdong Pharmacological College. MATERIALS: EGB was provided by Xi'an Fujie Biotechnological Development Company; 1640 culture medium, methylthiazolyl tetrazolium (MTT), trypsin and dimathyl sulfoxide (DMSO) by Sigma Company; PC12 cell strain by Cell Center of Medical College of Zhongshan University; calf serum by Hangzhou Sijiqing Bioengineering Company; lactate dehydrogenase (LDH) kit by Nanjing Jiancheng Bioengineering Research Institute. METHODS: The experiment was carried out in Department of Cell Biology of Guangdong Pharmacological College from June to December 2005. ① Cell culture: PC12 cells were cultured in 1640 medium containing 200 g/L fetal calf serum. The cells were diluted to 1×107 L–1 and washed every two days. Those cells were used to experiment until they grew in logarithm on solid wall. ② Grouping and intervention: PC12 cells (1×108 L–1) were plated in 96-well plates with the density of 200 μL/hole and divided into three groups: normal control group (routinely adding media), H2O2 group (treating with media and H2O2 for 20 hours) and EGB group (adding media, 100 μmol/L EGB and 100 μmol/L H2O2). ③ MTT assay: PC12 cells (1× 108 L–1) were plated in 96-well plates and divided into three groups with 8 holes for each group. Under sterile condition, cells were added with 5 g/L MTT (100 μL) and cultured for 4 hours. And then, 200 μL DMSO fluid was added and shaken for 30 minutes until blue crystal products formed were dissolved soundly. ④ Experimental evaluation: Absorbance (A) at 630 nm was measured and LDH activity was measured at the same time. MAIN OUTCOME MEASURES: Results of MTT assay and LDH activity. RESULTS: ① Results of MTT assay: A value was lower in the H2O2 group than that in the normal control group (P < 0.01), while A value was higher in the EGB group than that in the H2O2 group (P < 0.01). ② LDH activity: LDH activity was higher in the H2O2 group than that in the normal control group (P < 0.01), while LDH activity was lower in the EGB group than that in the H2O2 group (P < 0.01). CONCLUSION: EGB can inhibit H2O2-induced oxidative stress damage in PC12 cells possibly by preventing damage to the cell membrane.     

Hyperbaric oxygen treatment for experimental intraocular hypertension in rats： An electroretinogram detection

BACKGROUND: Hyperbaric oxygen (HBO) is used for treating glaucoma, and affirmative curative effect has been obtained. HBO can sensitively reflect the obviously heightened b wave of electroretinogram (ERG) of injured tissue. OBJECTIVE: To observe the effect of HBO treatment on retinal function of rats with acute experimental intraocular hypertension with ERG. DESIGN: Randomized controlled experiment. SETTING: Department of Ophthalmology, Third Xiangya Hospital, Central South University; Department of Hyperbaric Oxygen, Xiangya Hospital, Central South University; Department of Anatomy, Xiangya Hospital, Central South University. MATERIALS: Eighteen adult healthy Wistar rats, of either gender, weighing from 150 to 250 g, were provided by the Animal Room of Central South University. Type YLCO. 5/ⅠA baby hyperbaric oxygen chamber, type LMS-2A two-channel physiological recorder, type BG-1 retina exposure system, Jiangwan type Ⅰstereotaxis instrument. METHODS: This experiment was carried out in the Central South University between March and September 2006. Eighteen healthy Wistar rats were made into models of acute experimental intraocular hypertension. Then, they were divided into two groups: model group and HBO treatment group, with 9 in each group. Following 7 days of HBO treatment, the rats in HBO treatment group were placed in Type YLCO. 5/ⅠA baby hyperbaric oxygen chamber, which was pressurized with pure oxygen( volume fraction 0.825±0.025).The treatment pressure was 0.2 MPa. The rats in HBO treatment group daily inhaled HBO for 80 minutes within 7 days; Rats in the model group were untouched. The performance of eyes was observed under the status of intraocular hypertension. ERG was recorded before, during and 7 days after modeling, meanwhile, the recovery rate of b wave from ERG was calculated. Recovery rate of b wave from ERG=(amplitude of b wave 7 days after modeling/ amplitude of b wave before modeling)×100%. MAIN OUTCOME MEASURES: ① Performance of eyes under the status of intraocular hypertension. ② Recovery rate of b wave of ERG. RESULTS: All the 18 rats were involved in the final analysis. ① Performance of eyes under the status of intraocular hypertension.: When intraocular pressure increased until b wave of ERG disappeared, two eyes of rats with corneal opacity, dilated pupils, pale iris and stiffened eyeballs were found. ② Recovery rate of b wave of ERG in the HBO treatment group was significantly higher than that in the model group [(60.04± 19.33)% vs. (41.85±13.20)%,t =3.298,P < 0.01]. CONCLUSION: HBO treatment can obviously promote the recovery of retinal function following acute intraocular hypertension.     

Expression of angiogenic factors in cerebral arteriovenous malformations

BACKGROUND： In the process of vascularization, vascular endothelial growth factor （VEGF）, angiopoietin-2 and Tie2 are involved in the migration, differentiation and proliferation of vascular endothelial cells, and stimulate the rapid angiogenesis; Tiel and angiopoietin-1 play important roles in facilitating the formation of vascular lumen and maintaining the integrity of vascular wall. Thus the distributions and expressions may be associated with the occurrence of cerebral arteriovenous malformation. OBJECTIVE： To observe the biological effects of angiogenic factors in the occurrence and development of cerebral arteriovenous malformation. DESIGN： An observational comparative experiment. SETTINGS： Department of Neurosurgery, General Hospital of Shenyang Military Area Command of Chinese PLA; Department of Neurosurgery, General Hospital of Tianjin Medical University. PARTICIPANTS： Fresh samples of complete cerebral arteriovenous malformations resected in 47 patients were collected from the Department of Neurosurgery, General Hospital of Tianjin Medical University from August 1999 to May 2001, including 22 males and 25 females, the mean age was 34.5 years. Informed consents were obtained from all the patients or their relatives. The initial symptom was hemorrhage in 28 cases. All the patients were classified according to the clinical imaging data and Spetzler-Martin grading standard, including 11 cases of grade Ⅰ, 17 cases of grade Ⅱ, 11 cases of grade Ⅲ, and 8 cases of grade Ⅳ - Ⅴ. Normal brain tissues resected by decompression due to trauma were taken from 8 patients as controls, including 5 males and 3 females, aging 12 - 65 years. METHODS： ① The expressions of VEGF, Tie receptors, angiopoietin-1, angiopoietin-2, proto-oncogene c-myc and proliferating cell nuclear antigen（PCNA） in the samples of cerebral arteriovenous malformation were detected with immunohistochemical method. Under light microscope, the positively stained rat-anti-human factor Ⅷ-related antigens （specific marker of vascular endothelial cells） were counted, then the immuno-positive cells of the other antibodies in the visual field of neighboring section which was in ＂mirror＂ relation were counted, and the percentage of the latter to the former was considered as the labeling index of positive cells. The immunostaining intensity was classified negative （ - ）： no positive cells; positive （＋）： number of positive cells 〈 20%; moderately positive （＋＋）： number of positive cells 20% - 50%; strongly positive （＋＋＋）： number of positive cells 〉 50%. ② The differences of the enumeration data were compared with chi-squam test, and the correlation were analyzed with the linear correlation analysis. MAIN OUTCOME MEASURES： Expressions and distributions of VEGF, Tie 1 and Tie2 receptors, angiopoietin-1, angiopoietin-2, PCNA and c-myc in the samples of cerebral arteriovenons malformation and normal brain tissue. RESULTS： ① Expressions of angiogenic factors in the control group and cerebral arteriovenons malformation groups of each grade： The positive rates of VEGF, Tie2, angiopoietin-2, c-myc and PCNA expressions in the control group were significantly different from those in the cerebral arteriovenous malformation groups of each grade （ x^2=21.09 - 34.23, P 〈 0.05）, whereas the positive rates of Tiel and angiopoietin-1 expressions were close （ x^2=3.43 - 3.869, P 〉 0.05）. ② Expressions of angiogenic factors in hemorrhage group and non-hemorrhage group： The expressions of VEGF, angiopoietin-2 and PCNA in the hemorrhage group were significantly lower than those in the non-hemorrhage group （ x^2= 16.22 - 26.56, P 〈 0.05）. There ware no obvious differences in the expressions of Tiel and angiopoietin-1 expressions between the hemorrhage group and non-hemorrhage group （ x^2=3.22 - 3.78, P 〉 0.05）.The VEGF was positively correlated with the expressions of c-myc and PCNA （r = 0.728, 0.916, P 〈 0.05）. CONCLUSION： ①The expressions of angiogenic factors and related receptors may be involved in the process of cerebral arteriovenous malformation, and had important correlation the its clinical grading. ② Angiogenic factors may induce the expression of endothelial cell c-myc in cerebral arteriovenous malformation, and then interfere the cell proliferation and apoptosis.     

Distinct proteins in cortex of rats with closed traumatic brain injury detected by a WCX-2 protein chip

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.