The purpose of this study was to establish a method to record the dynamic process of vascular regeneration and remodeling in rat cerebral ischemic regions. An animal brain window model was established to continuously observe the changes of rat cortical vascular ischemia in vivo, and the model of cerebral ischemia was established by photochemical embolization. Optical coherence tomography (OCT) was performed to record the formation of vascular blockage and the injury and regeneration of small vessels during cerebral ischemia recovery. The results showed that 30 min of laser irradiation could completely block the cortical vessels in rats. Within 24-48 h after ischemia, the degree of brain injury was the greatest, and the number of blood vessels in the ischemic region reached the minimum. Then the blocked blood vessels began to be dredged, and the small blood vessels around the ischemic area began to regenerate. Small blood vessels in the superficial/deep layers of the cortex disappeared significantly after laser irradiation. During 10 d after ischemia, the blocked blood vessels were gradually dredged and recovered. On the 10th day after laser irradiation, a large number of neovascularization appeared in the superficial layer of cortex, but the deep vessels did not recover. These results indicate that the method established in this study can observe the changes of blood vessel in cerebral ischemic region continuously, which lays a foundation for further quantitative study on the dynamics of embolized blood vessels and peripheral capillaries during the recovery of cerebral ischemia.
Animals ; Brain ; blood supply ; Brain Ischemia ; Cerebral Cortex ; blood supply ; Rats ; Regeneration

Under global cerebral ischemia, the effect of different brain temperature on cerebral ischemic injury was studied. Male Sprague-Dawley rats were divided into normothermic (37-38°C) ischemia, mild hypothermic (31-32°C) ischemia, hyperthermic (41-42°C) ischemia and sham-operated groups. Global cerebral ischemia was established using the Pulsinelli four-vessel occlusion model and brain temperature was maintained at defined level for 60 min after 20-min ischemia. The expression of c-fos protein and the levels of malondialdehyde (MDA) and lactate in brain regions were detected by immunochemistry and spectrophotometrical methods, respectively. C-fos positive neurons were found in the hippocampus and cerebral cortex after cerebral ischemia reperfusion. Mild hypothermia increased the expression of c-fos protein in both areas, whereas hyperthermia decreased the expression of c-fos protein in the hippocampus at 24 h reperfusion, and the cerebral cortex at 48 h reperfusion when compared to normothermic conditions. In normothermic, mild hypothermic and hyperthermic ischemia groups, the levels of MDA and lactate in brain tissue were increased at 24, 48 and 72 h reperfusion following 20-min ischemia as compared with the sham-operated group (P<0.01). The levels of MDA and lactate in mild hypothermic group were significantly lower than those in normothermic group (P<0.01). It is suggested that brain temperature influences the translation of the immunoreactive protein product of c-fos after global cerebral ischemia, and MDA and lactate are also affected by hypothermia and hyperthermia.
Animals ; Body Temperature ; Brain ; blood supply ; metabolism ; physiopathology ; Brain Ischemia ; metabolism ; physiopathology ; Cerebral Cortex ; blood supply ; metabolism ; physiopathology ; Hippocampus ; blood supply ; metabolism ; physiopathology ; Immunochemistry ; Lactic Acid ; metabolism ; Male ; Malondialdehyde ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Proto-Oncogene Proteins c-fos ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; metabolism ; physiopathology ; Spectrophotometry ; Temperature ; Time Factors ; Tumor Suppressor Protein p53 ; metabolism

In the present study, we examined the effect of oxygen glucose deprivation (OGD) post-conditioning (PostC) on neural cell apoptosis in OGD-PostC model and the protective effect on primary cortical neurons against OGD injury in vitro. Four-h OGD was induced by OGD by using a specialized and humidified chamber. To initiate OGD, culture medium was replaced with de-oxygenated and glucose-free extracellular solution-Locke's medium. After OGD treatment for 4 h, cells were then allowed to recover for 6 h or 20 h. Then lactate dehydrogenase (LDH) release assay, Western blotting and flow cytometry were used to detect cell death, protein levels and apoptotic cells, respectively. For the PostC treatment, three cycles of 15-min OGD, followed by 15 min normal cultivation, were applied immediately after injurious 4-h OGD. Cells were then allowed to recover for 6 h or 20 h, and cell death was assessed by LDH release assay. Apoptotic cells were flow cytometrically evaluated after 4-h OGD, followed by re-oxygenation for 20 h (O4/R20). In addition, Western blotting was used to examine the expression of heat-shock protein 70 (HSP70), Bcl-2 and Bax. The ratio of Bcl-2 expression was (0.44±0.08)% and (0.76±0.10)%, and that of Bax expression was (0.51±0.05)% and (0.39±0.04)%, and that of HSP70 was (0.42±0.031)% and (0.72±0.045)% respectively in OGD group and PostC group. After O4/R6, the rate of neuron death in PostC group and OGD groups was (28.96±3.03)% and (37.02±4.47)%, respectively. Therefore, the PostC treatment could up-regulate the expression of HSP70 and Bcl-2, but down-regulate Bax expression. As compared with OGD group, OGD-induced neuron death and apoptosis were significantly decreased in PostC group (P<0.05). These findings suggest that PostC inhibited OGD-induced neuron death. This neuro-protective effect is likely achieved by anti-apoptotic mechanisms and is associated with over-expression of HSP70.
Animals ; Apoptosis ; drug effects ; Blotting, Western ; Cell Hypoxia ; Cell Survival ; drug effects ; Cells, Cultured ; Cerebral Cortex ; blood supply ; cytology ; embryology ; Flow Cytometry ; Glucose ; pharmacology ; HSP70 Heat-Shock Proteins ; metabolism ; Ischemic Postconditioning ; methods ; Neurons ; cytology ; drug effects ; metabolism ; Oxygen ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; prevention & control ; bcl-2-Associated X Protein ; metabolism

BACKGROUNDCerebral ischemia-reperfusion injury is the main reason for the loss of neurons in the ischemic cerebrovascular disease. Therefore, to deeply understand its pathogenesis and find a new target is the key issue to be solved. This research aimed to investigate the neuroprotective effects of salvianolic acid B (SalB) against oxygen-glucose deprivation/reperfusion (OGD/RP) damage in primary rat cortical neurons.

METHODSThe primary cultures of neonatal Wister rats were randomly divided into the control group, the OGD/RP group and the SalB-treatment group (10 mg/L). The cell model was established by depriving of oxygen and glucose for 3 hours and reperfusion for 3 hours and 24 hours, respectively. The neuron viability was determined by MTT assay. The level of cellular reactive oxygen species (ROS) was detected by fluorescent labeling method and spin trapping technique respectively. The activities of neuronal Mn-superoxide dismutase (Mn-SOD), catalase (CAT) and glutathione peroxidase (GSH-PX) were assayed by chromatometry. The mitochondria membrane potential (ΔΨ(m)) was quantitatively analyzed by flow cytometry. The release rate of cytochrome c was detected by Western blotting. The neuronal ultrastructure was observed by transmission electron microscopy. Statistical significance was evaluated by analysis of variance (ANOVA) followed by Student-Newman-Keuls test.

RESULTSOGD/RP increased the level of cellular ROS, but decreased the cell viability and the activities of Mn-SOD, CAT and GSH-PX; SalB treatment significantly reduced the level of ROS (P < 0.05); and enhanced the cell viability (P < 0.05) and the activities of these antioxidases (P < 0.05). Additionally, OGD/RP induced the fluorescence value of ΔΨ(m) to diminish and the release rate of cytochrome c to rise notably; SalB markedly elevated the level of ΔΨ(m) (P < 0.01) and depressed the release rate of cytochrome c (P < 0.05); it also ameliorated the neuronal morphological injury.

CONCLUSIONThe neuroprotection of SalB may be attributed to the elimination of ROS and the inhibition of apoptosis.

Animals ; Apoptosis ; drug effects ; Benzofurans ; pharmacology ; Catalase ; metabolism ; Cells, Cultured ; Cerebral Cortex ; blood supply ; Cytochromes c ; secretion ; Glutathione Peroxidase ; metabolism ; Hypoxia-Ischemia, Brain ; drug therapy ; Membrane Potential, Mitochondrial ; drug effects ; Neuroprotective Agents ; pharmacology ; Rats ; Rats, Wistar ; Reactive Oxygen Species ; metabolism ; Reperfusion Injury ; prevention & control ; Superoxide Dismutase ; metabolism

OBJECTIVETo study the effects of the Weinaokang (WNK), the active compounds extracted from Ginkgo, Ginseng, and saffron, on ischemia/reperfusion (I/R)-induced vascular injury to cerebral microvessels after global cerebral ischemia.

METHODSMale C57BL/6J mice were randomly divided into 5 groups (10 animals/group): the sham group (0.5% CMC-Na, 20 mL/kg), the I/R model group (0.5% CMCNa, 20 mL/kg), the I/R+Crocin control group (20 mg/kg), the I/R+high dose WNK group (20 mg/kg), and the I/R+low dose WNK group (10 mg/kg). Bilateral common carotid artery occlusion (BCCAO, 20 min) in mice, followed by 24 h reperfusion, was built. The generation of nitric oxide (NO), the activity of nitric oxide synthase (NOS), the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), and the expression of matrix metalloproteinases-9 (MMP-9) and G protein-coupled receptor kinase 2 (GRK2) in cortical microvascular homogenates were evaluated. The ultrastructural morphology of cortical microvascular endothelial cells (CMEC) was observed.

RESULTSThe transient global cerebral ischemia (20 min), followed by 24 h of reperfusion, significantly promoted the generation of NO and the activity of NOS. The reperfusion led to serious edema with mitochondrial injuries in the cortical CMEC, as well as enhanced membrane GRK2 expression and reduced cytosol GRK2 expression. Furthermore, enhanced phosphorylation of ERK1/2 and decreased expression of MMP-9 were detected in cortical microvessels after I/R (20 min/24 h). As well as the positive control Crocin (20 mg/kg, 21days), pre-treatment with WNK (20, 10 mg/kg, 21 days) markedly inhibited nitrative injury and modulated the ultrastructure of CMEC. Furthermore, WNK inhibited GRK2 translocation from cytosol to the membrane (at 20 mg/kg) and reduced ERK1/2 phosphorylation and MMP-9 expression in cortical microvessels.

CONCLUSIONWNK and its active compounds (Crocin) are effective to suppress I/R-induced vascular injury to cerebral microvessels after global cerebral ischemia with the target on GRK2 pathways.

Animals ; Brain Ischemia ; drug therapy ; metabolism ; Cerebral Cortex ; blood supply ; drug effects ; metabolism ; ultrastructure ; Drug Evaluation, Preclinical ; Drugs, Chinese Herbal ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; G-Protein-Coupled Receptor Kinase 2 ; metabolism ; Male ; Matrix Metalloproteinase 9 ; metabolism ; Mice ; Mice, Inbred C57BL ; Microvessels ; drug effects ; metabolism ; pathology ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; metabolism ; Phosphorylation ; Reperfusion Injury ; drug therapy ; metabolism ; Signal Transduction ; drug effects ; Tissue Distribution ; drug effects

The aim of this study was to develop a nonlinear mixed-effects model for the increase in cerebral oximetry (rSO2) during the rapid introduction of desflurane, and to determine the effect of hypocapnia and N2O on the model. Twelve American Society of Anesthesiologist physical status class 1 and 2 subjects were allocated randomly into an Air and N2O group. After inducing anesthesia, desflurane was then increased abruptly from 4.0 to 12.0%. The PET(CO2), PET(DESF) and rSO2 were recorded at 12 predetermined periods for the following 10 min. The maximum increase in rSO2 reached +24-25% during normocapnia. The increase in rSO2 could be fitted to a four parameter logistic equation as a function of the logarithm of PET(DESF). Hypocapnia reduced the maximum response of rSO2, shifted the EC50 to the right, and increased the slope in the Air group. N2O shifted the EC50 to the right, and reduced the slope leaving the maximum rSO2 unchanged. The N2O-effects disappeared during hypocapnia. The cerebrovascular reactivity of rSO2 to CO2 is still preserved during the rapid introduction of desflurane. N2O slows the response of rSO2. Hypocapnia overwhelms all the effects of N2O.
Adult ; Anesthetics, Inhalation/*pharmacology ; *Cerebral Cortex/blood supply/drug effects/physiology ; Cerebrovascular Circulation/*drug effects/physiology ; Female ; Hemodynamics ; Humans ; Hypocapnia/*metabolism ; Isoflurane/*analogs & derivatives/pharmacology ; Male ; Middle Aged ; Models, Theoretical ; Nitrous Oxide/*metabolism ; *Oximetry ; Random Allocation ; Regional Blood Flow/drug effects

OBJECTIVETo develop a simple method for assessment of RNA integrity in laser capture microdissection (LCM) samples.

METHODSThe total RNA were isolated from the LCM samples and the sections before and after microdissection and examined by agarose gel electrophoresis. Real-time PCR was employed to assess the RNA from LCM samples, and the quantity of RNA was theoretically estimated according to the average total RNA product in mammalian cells (10 ng/1000 cells).

RESULTSWhen the total RNA from the sections before and after microdissection was intact, the RNA from LCM samples also had good quality, and the 28S and 18S rRNAs were visualized by ethidium bromide staining. Real-time PCR also showed good RNA quality in the LCM samples.

CONCLUSIONA simple method for quantitative and qualitative assessment of the RNA from LCM samples is established, which can also be applied to assessment of DNA or proteins in LCM samples.

Animals ; Capillaries ; pathology ; Cerebral Cortex ; blood supply ; pathology ; Lasers ; Male ; Microdissection ; methods ; Neurons ; pathology ; RNA ; analysis ; isolation & purification ; Rats ; Rats, Sprague-Dawley

AIMTo observe the expressional alterations of colony stimulating factor-1 receptor (CSF-1R) after ischemic injury of cerebral cortex, and study the function of colony stimulating factor-1 (CSF-1)/CSF-1R signal during the process of ischemic injury and repair of central nervous system (CNS).

METHODSWe examined the distribution and expression of CSF-1R in normal brain tissues and ischemic brain tissues by immunohistology and Western blot analysis.

RESULTSThe expression of CSF-1R in neurons could be up-regulated by ischemic injury in CNS.

CONCLUSIONCSF-1/CSF-1R might take part in the process of ischemic injury and repair.

Animals ; Brain Ischemia ; pathology ; physiopathology ; Cerebral Cortex ; blood supply ; Female ; Macrophage Colony-Stimulating Factor ; physiology ; Male ; Mice ; Mice, Inbred BALB C ; Neurons ; metabolism ; Random Allocation ; Receptor, Macrophage Colony-Stimulating Factor ; genetics ; metabolism ; physiology ; Reperfusion Injury ; metabolism ; physiopathology

OBJECTIVETo investigate the protective effects of adenovirus-mediated vascular endothelial growth factor (Ad-VEGF)165 gene transfer against hypoxic-ischemic brain damage (HIBD) in neonatal rats.

METHODSAd-VEGF recombinant adenovirus was constructed by bacterial homologous recombination technology. Seven-day-old Sprague-Dawley rats were randomly assigned to 4 groups: sham-operated (n=20), HIBD (n=25), buffer-treated (n=20), and Ad-VEGF-treated (n=25). The HIBD model was prepared by permanent occlusion of left common carotid artery, followed by exposure to 8% oxygen for 2 hrs. In the Ad-VEGF-treated and the Buffer-treated groups, 2 microL recombinant adenovirus suspension or buffer was injected into the left sensorimotor cortex of the rat brain 3 days after HIBD. Seven days after transplantation, VEGF165 mRNA expression was detected using RT-PCR. Neuronal apoptosis was detected by the terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nickel end labeling (TUNEL). CD34 and VEGF protein were detected using immunohistochemistry. Microvascular density in the cerebral cortex was measured based on CD34 positive cells. A radial arm maze test was performed from 30 postnatal days to evaluate long-term learning and memory functions. At 35 postnatal days, the rats were sacrificed for cerebral histological examinations by hematoxylin and eosin.

RESULTSThe expression of VEGF165 mRNA increased in the Ad-VEGF-treated group more than in the untreated HIBD and the buffer-treated groups (p<0.05). The number of apoptotic neurons was less in the Ad-VEGF-treated group compared with that in the untreated HIBD and the buffer-treated groups (p<0.05). Microvascular density and VEGF positive cells increased in the Ad-VEGF-treated group compared with that in the untreated HIBD and the buffer-treated groups (p<0.05). In the radial arm maze test, the Ad-VEGF-treated group had more improved achievements than the HIBD and the buffer groups (p<0.05). Neuronal degeneration and necrosis were lessened in the Ad-VEGF-treated group compared with the HIBD and the buffer groups.

CONCLUSIONSAd-VEGF gene transfer can increase the expression of VEGF mRNA and VEGF protein, decrease neuronal apoptosis, and increase angiopoiesis in the brain. This attenuates brain damage and improves long-term learning and memory functions in neonatal rats after HIBD.

Adenoviridae ; genetics ; Animals ; Animals, Newborn ; Cerebral Cortex ; blood supply ; chemistry ; Female ; Genetic Therapy ; Hypoxia-Ischemia, Brain ; therapy ; In Situ Nick-End Labeling ; Male ; Neuroprotective Agents ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Vascular Endothelial Growth Factor A ; analysis ; genetics

OBJECTIVETo study the influence of conditioned medium of rat brain microvascular endothelial cells on mitochondrial function of cortical neurons and the protective effect of Tongluo Jiunao Injection (TJI) on it.

METHODSFour kinds of conditioned endothelial cell (EC) cultured medium were prepared, i.e. the N-CM medium prepared by EC cultured in the normal conditioned medium without any treatment; the NT-CM prepared by EC cultured in N-CM and treated with TJI 1 microl/ml for 10 h; the I-CM prepared by EC cultured in the non-glucose kreb medium under hypoxia condition; and the IT-CM by EC pre-treatce with TJI 1 microl/ml for 4 h and cultured as that of I-CM. The levels of neuronic mitochondrial activity, membrane potential (MMP) and cytochrome C (Cyt C) were determined before and after the glucose-oxygen deprived model neurons of brain cortex being cultured with different kinds of conditioned EC cultured medium for assessing the effects of these media on mitochondria of injured neuron.

RESULTSAs compared with those of the normal neuron, the mitochondrial activity and MMP of all injured neurons decreased and Cyt C level increased significantly. But comparison of these indexes among neurons cultured with different conditioned EC culture media showed that the greatest extent abnormality revealed in the N-CM cultured neurons, which even greater than that in the model neuron; while that was less in the N-CM cutured neuron than in model neuron; as for those cultured in the NT-CM and IT-CM, i.e. the TJI treated cuture medium, the abnormal changes were reduced significantly when compared with those cultured in medium untreated with TJI (N-CM and I-CM), respectively (all P < 0.05).

CONCLUSIONThe paracrine secretion of the brain microvascular endothelial cells has evident regulatory effect on survival of the injured neurons, which might possibly be related to its protective effect on neuron mitochondrial function, and TJI could enhance the protective effect.

Animals ; Capillaries ; cytology ; Cells, Cultured ; Cerebral Cortex ; blood supply ; cytology ; Culture Media, Conditioned ; pharmacology ; Cytochromes c ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Endothelial Cells ; cytology ; drug effects ; ultrastructure ; Male ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria ; drug effects ; metabolism ; physiology ; Neurons ; cytology ; drug effects ; ultrastructure ; Rats