OBJECTIVETo explore the neuroprotective effects of electroacupuncture (EA) on hypoxic-ischemic encephalopathy (HIE) and to further investigate the role of glial cell line-derived neurotrophic factor (GDNF) family receptor member RET (rearranged during transfection) and its key downstream phosphatidylinositol 3 kinase (PI-3K)/protein kinase B (Akt) pathway in the process.

METHODSA total of 220 seven-day-old SD rats (of either sex, from 22 broods) were randomly divided into two groups, one (30 rats) for sham-surgery group and the other (190 rats) for HIE model group. The HIE model was established using the left common carotid artery ligation method in combination with hypoxic treatment. The successfully established rats were randomly divided into five groups, including control model group, EA group, sham-EA group, antagonist group and antagonist plus electroacupuncture group, with 35 rats in each group. Baihui (GV 20), Dazhui (GV 14), Quchi (LI 11) and Yongquan (KI 1) acupoints were chosen for acupuncture. EA was performed at Baihui and Quchi for 10 min once a day for continuous 1, 3, 7 and 21 days, respectively. The rats were then killed after the operation and injured cerebral cortex was taken for the measurement of neurologic damage by hematoxylin-eosin (HE) staining and the degenerative changes of cortical ultrastructure by transmission electron microscopy. RET mRNA level and Akt protein level were detected by real-time reverse-transcription polymerase chain reaction (RT-PCR) and western blot analysis, respectively.

RESULTSEA could ameliorate neurologic damage of the first somatic sensory area (S1Tr) and alleviate the degenerative changes of ultrastructure of cortical neurons in rats subjected to HIE. And the longer acupuncture treatment lasted, the better its therapeutic effect would be. This was accompanied by gradually increased expression of GDNF family receptor RET at the mRNA level and its downstream signaling Akt at the protein level in the ischemic cortex.

CONCLUSIONEA has neuroprotective effects on HIE and could be a potential therapeutic strategy for HIE in the neonate. Activation of RET/Akt signaling pathway might be involved in this process.

Animals ; Blotting, Western ; Cerebral Cortex ; pathology ; ultrastructure ; Electroacupuncture ; Female ; Glial Cell Line-Derived Neurotrophic Factor ; genetics ; metabolism ; Hypoxia-Ischemia, Brain ; genetics ; pathology ; therapy ; Male ; Nerve Degeneration ; pathology ; Neurons ; pathology ; ultrastructure ; Neuroprotective Agents ; therapeutic use ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Proto-Oncogene Proteins c-ret ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction

OBJECTIVETo study the effects of Bushen Yisui Capsule (, BSYSC) on the oligodendrocyte lineage genes (Olig) 1 and Olig2 in C57BL/6 mice with experimental autoimmune encephalomyelitis (EAE) in order to explore the remyelination effect of BSYSC.

METHODSThe mice were randomly divided into normal control (NC), EAE model (EAE-M), prednisone acetate (PA, 6 mg/kg), BSYSC high-dose (3.02 g/kg) and BSYSC low-dose (1.51 g/kg) groups. The mice were induced by immunization with myelin oligodendrocyte glycoprotein (MOG) 35-55. The neurological function scores were assessed once daily. The pathological changes in mice brains were observed with hematoxylin-eosin (HE) staining and transmission electron microscope (TEM). The protein expressions of myelin basic protein (MBP), Olig1 and Olig2 in brains were measured by immunohistochemistry. The mRNA expressions of Olig1 and Olig 2 was also determined by quantitative real-time polymerase chain reaction.

RESULTSCompared with the EAE-M mice, (1) the neurological function scores were significantly decreased in BSYSC-treated mice on days 22 to 40 (P<0.01); (2) the inflammatory cells and demyelination in brains were reduced in BSYSC-treated EAE mice; (3) the protein expression of MBP was markedly increased in BSYSC-treated groups on day 18 and 40 respectively (P<0.05 or P<0.01); (4) the protein expression of Olig1 was increased in BSYSC (3.02 g/kg)-treated EAE mice on day 40 (P<0.01). Protein and mRNA expression of Olig2 was increased in BSYSC-treated EAE mice on day 18 and 40 (P<0.01).

CONCLUSIONThe effects of BSYSC on reducing demyelination and promoting remyelination might be associated with the increase of Olig1 and Olig2.

Animals ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; metabolism ; Brain ; drug effects ; pathology ; ultrastructure ; Bromodeoxyuridine ; metabolism ; Capsules ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Encephalomyelitis, Autoimmune, Experimental ; drug therapy ; genetics ; pathology ; physiopathology ; Female ; Fluorescent Antibody Technique ; Mice, Inbred C57BL ; Myelin-Oligodendrocyte Glycoprotein ; metabolism ; Nerve Tissue Proteins ; genetics ; metabolism ; Oligodendrocyte Transcription Factor 2 ; RNA, Messenger ; genetics ; metabolism

OBJECTIVETo establish red-green dual-color fluorescence glioma model in nude mice and to explore its practical values.

METHODSCM-DiI-stained rat glioma C6 cells (C6-CM- DiI cells) expressing red fluorescence were inoculated into the brain of athymic nude mice expressing green fluorescence protein (NC-C57BL/6J-EGFP). Then the whole-body dual-color fluorescence imaging was detected dynamically. Finally whole brains of the tumor-bearing mice were removed and 5 µm thick serial frozen slices were made. Light microscopy, fluorescence microscopy and confocal laser scanning microscopy were performed to observe the transplanted tumor tissue structure and fluorescent cells.

RESULTSTumor mass with red fluorescence increased gradually under continuous in-vivo fluorescence imaging monitoring. Under the fluorescence microscope, cells with red, green and yellow fluorescence were observed in the frozen sections of transplanted tumor tissue and the mutual structural relationship among them could be defined. The tumor cells migration, implantation and cell fusion between transplanted tumor cells and host cells could be observed. It could be distinguished according to the fluorescence, that blood vessels of tumor-origin displayed red fluorescence, blood vessels of host-origin displayed green fluorescence and mosaic blood vessels appeared yellow fluorescence. It was depicted that host innate astrocytes and oligodendrocytes in the microenvironment at the tumor periphery could be activated and dedifferentiated into nestin-positive cells.

CONCLUSIONSIn contrast to traditional animal model, the dual-color fluorescence imaging of nude mouse models of glioma possesses enormous advantages in investigating tumor mass in-vivo fluorescence imaging, tumor cells migration and metastasis, tumor angiogenesis and reactive activation of host innate cells in the microenvironment at tumor periphery, thus, has highly practical application value.

Animals ; Astrocytes ; metabolism ; Brain Neoplasms ; blood supply ; metabolism ; pathology ; ultrastructure ; Carbocyanines ; metabolism ; Cell Fusion ; Cell Line, Tumor ; Cell Movement ; Disease Models, Animal ; Fluorescent Dyes ; metabolism ; Glioma ; blood supply ; metabolism ; pathology ; ultrastructure ; Green Fluorescent Proteins ; metabolism ; Luminescent Proteins ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Nude ; Microscopy, Confocal ; Microscopy, Fluorescence ; Neoplasm Transplantation ; Neovascularization, Pathologic ; Nestin ; metabolism ; Oligodendroglia ; metabolism ; Rats

This study describes the neuropathologic features of normal canine brain ablated with non-thermal irreversible electroporation (N-TIRE). The parietal cerebral cortices of four dogs were treated with N-TIRE using a dose-escalation protocol with an additional dog receiving sham treatment. Animals were allowed to recover following N-TIRE ablation and the effects of treatment were monitored with clinical and magnetic resonance imaging examinations. Brains were subjected to histopathologic and ultrastructural assessment along with Bcl-2, caspase-3, and caspase-9 immunohistochemical staining following sacrifice 72 h post-treatment. Adverse clinical effects of N-TIRE were only observed in the dog treated at the upper energy tier. MRI and neuropathologic examinations indicated that N-TIRE ablation resulted in focal regions of severe cytoarchitectural and blood-brain-barrier disruption. Lesion size correlated to the intensity of the applied electrical field. N-TIRE-induced lesions were characterized by parenchymal necrosis and hemorrhage; however, large blood vessels were preserved. A transition zone containing parenchymal edema, perivascular inflammatory cuffs, and reactive gliosis was interspersed between the necrotic focus and normal neuropil. Apoptotic labeling indices were not different between the N-TIRE-treated and control brains. This study identified N-TIRE pulse parameters that can be used to safely create circumscribed foci of brain necrosis while selectively preserving major vascular structures.
Animals ; Brain/metabolism/*pathology/surgery/ultrastructure ; Caspase 3/metabolism ; Caspase 9/metabolism ; Dogs ; Electroporation/veterinary ; Magnetic Resonance Imaging/methods ; Microscopy, Electron, Transmission ; Necrosis/metabolism/pathology ; Neurosurgical Procedures/*adverse effects

OBJECTIVETo investigate the therapeutic mechanism of baicalin on rat brain glioma.

METHODSDeep brain glioma models were established by injection of glioma cell line C6 cells into the brain of Wistar rats. The rats at 7 days after modeling were randomly divided into tumor control group (0.9% NaCl solution 30 mg×kg(-1)×d(-1) gavage)and experimental groups. The experimental rats was divided into 3 groups: low dose group (50 mg×kg(-1)×d(-1)), middle dose group (100 mg×kg(-1)×d(-1)) and high dose group (200 mg×kg(-1)×d(-1)), given the baicalin by gavage. Pathological and electron microscopic changes were observed. The expressions of p53 and Bcl-2 were determined by immunohistochemistry, and the changes of MRI, the average survival time and body weight of the rats in each group after treatments were analyzed.

RESULTSCompared with the control group, the tumor diameter and volume of high dose group rats before sacrifice were significantly reduced (P < 0.01), and the survival time was significantly prolonged (P < 0.01). Immunohistochemistry revealed strong positive expression rate of mutant p53 (84.47 ± 3.74)% and moderately positive rate (47.28 ± 2.38)% in the control group, significantly higher than that in the negative group (12.91 ± 1.07)% (P < 0.01). The positive rate of mutant p53 of the high dose group was (46.42 ± 2.19)%, significantly lower than that of the control group (84.47 ± 3.74)% (P < 0.01). The expression rate of Bcl-2 in the control group was strongly positive (86.51 ± 4.17)% and moderate positive (48.19 ± 2.11)%, significantly higher than that of the negative group (10.36 ± 1.43)% (P < 0.01). Electron microscopy revealed that baicalin caused damages of the cell nuclei and organelles in the gliomas.

CONCLUSIONSBaicalin has significant inhibitory effect on glioma in vivo, and its mechanism may be related to cell apoptosis induced by down-regulated expression of mutant p53, but not related with Bcl-2 expression.

Animals ; Antineoplastic Agents, Phytogenic ; administration & dosage ; pharmacology ; Apoptosis ; drug effects ; Brain Neoplasms ; metabolism ; pathology ; ultrastructure ; Cell Nucleus ; drug effects ; Dose-Response Relationship, Drug ; Down-Regulation ; Flavonoids ; administration & dosage ; pharmacology ; Glioma ; metabolism ; pathology ; ultrastructure ; Magnetic Resonance Imaging ; Male ; Microscopy, Electron, Transmission ; Neoplasm Transplantation ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Tumor Burden ; drug effects ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo explore the effects of taurine on the ultrastructure and P2X7 receptor protein expression in brain following traumatic brain injury (TBI) in rats.

METHODSForty male SD rats, were divided randomly into four groups that were sham-operated group, TBI group, TBI plus low-dose taurine group and TBI plus high-dose taurine group. The TBI model was established by Marmarou's method, the expression of P2X7 receptor protein in parietal cortex and hippocampus was detected by the immunohistochemical method, the ultrastructure of parietal cortex were observed by transmission electron microscope.

RESULTSCompared with sham-operated group, the positive expression cells of P2X7 receptor protein in parietal cortex and hippocampus of TBI group were significantly increased (P < 0.01). Compared with TBI group, the positive expression cells of P2X7 receptor protein in parietal cortex and hippocampus of TBI plus low-dose taurine group and TBI plus high-dose taurine group were significantly decreased (P <0.01 or P <0.05). Compared with TBI plus low-dose taurine group, the positive expression cells of P2X7 receptor protein in parietal cortex and hippocampus of TBI plus high-dose taurine group were significantly decreased (P < 0.05 or P < 0.01). The pathological damage of parietal cortex in the TBI plus high-dose taurine group was obviously lightened.

CONCLUSIONTaurine exerts the neuroprotective effect on TBI in rats, the protective mechanism might be associated with down-regulating the expression of P2X7 receptor protein in parietal cortex and hippocampus.

Animals ; Brain ; metabolism ; ultrastructure ; Brain Injuries ; metabolism ; pathology ; Male ; Rats ; Rats, Sprague-Dawley ; Receptors, Purinergic P2X7 ; metabolism ; Taurine ; pharmacology

OBJECTIVETo observe the effects of electroacupuncture (EA) on the expressions of Nogo-A and the ultrastructure in the cerebral cortex at different time points after the cerebral ischemia-reperfusion in rats.

METHODSOne hundred and thirty male Sprague Dawley (SD) rats were randomly divided into the EA group (n = 30), the sham-EA group (n = 30), the model group (n = 30), the sham-operation group (n = 30), and the blank group (n = 10). The modified ZeaLonga method was used to prepare the left middle cerebral artery occlusion (MCAO) model in the first three groups. After the operation Baihui (DU20) and Dazhui (DU14) were daily needled in the EA group. One inch beside Baihui (DU20) and Dazhui (DU14) were daily needled in the sham-EA group. Rats in the model group were only treated with MCAO ischemia/reperfusion. Rats in the sham-operation group only received surgical wound. No treatment was given to rats in the blank group. The ultrastructures of ischemic cells and the intervention of the Nogo-A expressions were observed using the immunohistochemical staining and the transmission electron microscope 1, 7, and 28 days after EA.

RESULTS(1) In the EA group, the damage of ultrastructures of neurons, gliocytes, and blood brain barrier in the ischemic region was alleviated when compared with that of the sham-EA group and the model group. (2) On the 1st, 7th and 28th day after the cerebral ischemia-reperfusion, the expressions of Nogo-A in the ischemic cortex in the EA group was lower when compared with those in the sham-EA group and the model group at the corresponding time points, showing significant difference (P < 0.05). But there was no statistical difference between the sham-EA group and the model group at the same time point (P > 0.05).

CONCLUSIONThe mechanism of EA for protecting cerebral ischemia/reperfusion might be closely associated with alleviating the damage on the ultrastructures of brain cells, and down-regulating the expressions of Nogo-A.

Acupuncture Points ; Animals ; Brain Ischemia ; metabolism ; pathology ; therapy ; Cerebral Cortex ; metabolism ; ultrastructure ; Electroacupuncture ; Male ; Myelin Proteins ; metabolism ; Nogo Proteins ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; metabolism ; pathology ; therapy

OBJECTIVETo observe the effects of hyperbaric oxygen (HBO) on synaptic ultrastructure and the synaptophysin expression (p38) in hippocampal CA3 after hypoxia-ischemic brain damage (HIBD) in neonatal rats.

METHODSThe rat model of HIBD was made by the method of Bjelke and divided randomly into two groups (n = 10)--HIBD group and HBO-treated HIBD group. Another 20 rats underwent sham-operation and were also divided randomly into HBO-treated control group and the control group. After 24 h of the operation, the rats of the HBO-treated groups received HBO (2ATA, 1 h/d) for 14 days. When rats were 4 weeks old, the learning-memory ability of rats in every group was evaluated through water-maze test. Their hippocampal ultrastructure was observed with electron microscope and the p38 expression was detected immunohistochemically.

RESULTSCompared with the control group [(10.6 +/- 3.4) times], the water-maze learning ability of the rats in HIBD group [(15.5 +/- 4.9) times] was significantly decreased (P < 0.01), while the learning-memory ability of the HBO-treated HIBD group [(11.3 +/- 2.6) times] was significantly improved. There was no significant difference in the water-maze test between the HBO-treated HIBD group and the control group (P > 0.05). Compared with the control group, the ultrastructure of pyramidal neuron of hippocampal CA3 was distorted in HIBD group under the electron microscope. Compared with that in HBO-treated HIBD group (0.77 +/- 0.17, 0.67 +/- 0.16, 0.46 +/- 0.13, 0.86 +/- 0.14) and the control group (0.82 +/- 0.16, 0.70 +/- 0.16, 0.53 +/- 0.15, 0.91 +/- 0.17), the corrected optical densities (COD) of immunoreactive products of the hippocampal CA3 p38 were significantly decreased in HIBD group (0.41 +/- 0.19, 0.21 +/- 0.11, 0.08 +/- 0.03, 0.38 +/- 0.16) (P < 0.01). There was no significant difference in either ultrastructure or immunohistochemically reactive COD of p38 between the HBO-treated HIBD group and the control group (P > 0.05).

CONCLUSIONUnderlying the induction of synaptic plasticity and reducing the ultrastructural damage may be involved in the mechanism of HBO in the brain rehabilitation in perinatal brain damage with hypoxia-ischemia.

Animals ; Animals, Newborn ; Female ; Hippocampus ; metabolism ; pathology ; Hyperbaric Oxygenation ; Hypoxia-Ischemia, Brain ; metabolism ; pathology ; therapy ; Pregnancy ; Rats ; Rats, Sprague-Dawley ; Synapses ; metabolism ; ultrastructure ; Synaptophysin ; 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

OBJECTIVETo investigate the effect of physical training on cerebral structure and spatial learning and memory in neonatal rats submitted to hypoxic-ischemic brain damage (HIBD).

METHODSForty-eight 7-day-old Sprague-Dawley rats were randomly divided into three groups: a group that was subjected to left carotid ligation followed by 2 hrs hypoxic stress (HIBD); a group that received physical training 2 weeks after the HIBD event; a control group that was subjected to a sham-operation without ligation and hypoxic stress. Following four weeks physical training, motor function test and water maze tasks were performed. Bilateral brain weight, cerebral morphology and left hippocampal ultrastructrue of the animals were examined. The expression levels of phosphor calmodulin-dependent protein kinase II (CaMKII) and brain derived neurotrophic factor (BDNF) were determined by immunohistochemistry.

RESULTSCompared with the control group, the motor function and the spatial learning and memory ability in the non-trained HIBD group were significantly decreased, whereas there was no significant difference between the trained-HIBD and the control groups. The left hemisphere weight and neurons in the left hippocampal CA1 zone of both HIBD groups decreased and the reduction was more significant in non-trained HIBD group. The ultrastructure of the left hippocampus was remarkably abnormal in the non-trained HIBD group, while no obvious abnormality was observed in the trained HIBD and the control groups. Phosphor-CaMKII and BDNF expression in the left hippocampus in the trained HIBD group increased significantly compared with that in the non-trained HIBD group.

CONCLUSIONSPhysical training can restrain brain damage and ameliorate spatial learning and memory impairments in rats with HIBD.

Animals ; Animals, Newborn ; Brain-Derived Neurotrophic Factor ; metabolism ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; metabolism ; Hypothalamus ; metabolism ; ultrastructure ; Hypoxia-Ischemia, Brain ; pathology ; physiopathology ; psychology ; therapy ; Maze Learning ; Memory ; Phosphorylation ; Physical Conditioning, Animal ; Rats ; Rats, Sprague-Dawley