Many extracellular matrix molecules are expressed in the embryonic nervous system and there is some evidence that they are important regulators of neural development. Of these molecules, laminin appears to be the most potent, affecting virtually all neurons of the peripheral and central nervous system. This study was undertaken to investigate the effects of laminin on the proliferation and differentiation of cultured neuroepithelial cells taken from fetal rat forebrains (embryonic day 17-19). The results are summarized as follows. 1) Neuroepithelial cells cultivated in epidermal growth factors containing serum-free medium subsequently differentiated into neurons, astrocytes, and oligodendrocytes. 2) Neuronal cells derived from neuroepithelial cells were immunoreactive for gamma-aminobutyric acid (GABA) or substance P, but were not for serotonin and tyrosine hydroxylase. 3) In western blot analysis, the phosphorylated neurofilament content in neuronal cells was higher in culture on laminin than in culture on poly-L-lysine (PLL). 4) The proliferation rate of GABAergic neurons was higher in culture on laminin than in culture on PLL. These results suggest that GABAergic and substance P-ergic neurons can be differentiated from neuroepithelial cells and that laminin promotes the differentiation of neuronal cells from neuroepithelial cells and the increased proliferation rate of GABAergic cells.
Animal ; Brain/drug effects* ; Brain/cytology ; Cell Aging/drug effects ; Cell Differentiation/drug effects ; Cell Division/drug effects ; Cells, Cultured ; Epidermal Growth Factor/pharmacology* ; Epithelial Cells/drug effects ; Epithelial Cells/cytology ; GABA/physiology ; Laminin/pharmacology* ; Neurons/physiology ; Neurons/drug effects* ; Neurons/cytology* ; Rats/embryology

Brain-derived neurotrophic factor (BDNF)is the richest neurophin in brain tissue and may act as an activity-dependent neuronal survival factor. In vitro, BDNF plays an important role in preventing cortical neurons from hypoxia-induced neurotoxicity. It could induce a variety of cellular responses such as cell growth, survival, differentiation, and anti-apoptosis mainly via activating mitogen-activated protein kinase (MAPK) and Ca2+/calmodulin-dependent kinase (CaMK) signaling pathways. And among these multiple signaling pathways there is growing evidence of complicated cross talk.
Animals ; Brain-Derived Neurotrophic Factor ; pharmacology ; physiology ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Calcium-Calmodulin-Dependent Protein Kinases ; drug effects ; physiology ; Cells, Cultured ; Humans ; Mitogen-Activated Protein Kinases ; drug effects ; physiology ; Neurons ; cytology ; drug effects ; enzymology ; Signal Transduction

OBJECTIVETo explore the mechanism of borneol in opening the blood-brain barrier (BBB).

METHODSBorneol contained serum was prepared and using Matin-Darby canine kidney epithelium (MDCKE) cell line as the in vitro BBB model to observe the effects of borneol on intercellular tight junction (ICTJ) and pinocytosis vesicles of BBB model.

RESULTSBorneol reduced the ICTJ and caused increase of the number and enlarged the diameter of vesicles. The ICTJ was opened firstly 4 hrs after borneol treatment, then the pinocytosis was affected 24 hrs later. The effects disappeared 24 hrs after removal of the borneol contained serum, indicating that the above-mentioned effects were reversible.

CONCLUSIONBorneol could obviously loosen the ICTJ in BBB, accelerate the transportation of substance through the intercellular passage, it also could increase the number and volume of pinocytosis vesicles in BBB cells, thus to accelerate the transportation of substance by way of cell pinocytosis.

Animals ; Blood-Brain Barrier ; drug effects ; physiology ; Bornanes ; pharmacology ; Cell Line ; Cell Membrane Permeability ; Epithelial Cells ; cytology ; Kidney ; cytology ; Male ; Models, Neurological ; Pinocytosis ; drug effects ; Rabbits ; Tight Junctions ; drug effects

The aim of the present study was to assess the ototoxicity of kanamycin sulfate (KM) in adult rats and its underlying mechanism. Forty male Sprague-Dawley rats (6-7 weeks old) were randomly divided into the experimental group and the control group. The animals in the experimental group were injected subcutaneously with KM (500 mg/kg per day) for two weeks, and the control group received equal volume of normal saline. To assess the ototoxicity of KM, the auditory brainstem response (ABR) was recorded to monitor the changes in hearing thresholds, and the density of spiral ganglion cells (SGCs) and morphology of cochlea were observed using surface preparations and frozen sections of cochlea. The results showed that the hearing threshold of rats in the experimental group was elevated by more than 60 dB across all the frequencies two weeks after the first administration of KM. And in the experimental group, the density of SGCs became lower, and organ of Corti suffered loss of hair cells. The loss of outer hair cells (OHCs) was more severe than that of inner hair cells (IHCs), correlated with the density decrease of SGCs. We conclude that the ototoxicity of KM in the adult rats was apparent and the underlying mechanism is associated with the loss of SGCs and hair cells.
Animals ; Cochlea ; drug effects ; pathology ; Evoked Potentials, Auditory, Brain Stem ; drug effects ; Hair Cells, Auditory, Outer ; cytology ; drug effects ; pathology ; Hearing Loss ; chemically induced ; physiopathology ; Kanamycin ; toxicity ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spiral Ganglion ; pathology ; physiology ; ultrastructure

Neural stem cells (NSCs) have mainly been applied to neurodegeneration in some medically intractable neurologic diseases. In this study, we established a novel NSC line and investigated the cytotoxic responses of NSCs to exogenous neurotoxicants, glutamates and reactive oxygen species (ROS). A multipotent NSC line, B2A1 cells, was established from long-term primary cultures of oligodendrocyte-enriched cells from an adult BALB/c mouse brain. B2A1 cells could be differentiated into neuronal, astrocytic and oligodendroglial lineages. The cells also expressed genotypic mRNA messages for both neural progenitor cells and differentiated neuronoglial cells. B2A1 cells treated with hydrogen peroxide and L-buthionine-(S,R)-sulfoximine underwent 30-40% cell death, while B2A1 cells treated with glutamate and kainate showed 25-35% cell death. Cytopathologic changes consisting of swollen cell bodies, loss of cytoplasmic processes, and nuclear chromatin disintegration, developed after exposure to both ROS and excitotoxic chemicals. These results suggest that B2A1 cells may be useful in the study of NSC biology and may constitute an effective neurotoxicity screening system for ROS and excitotoxic chemicals.
Animals ; Brain/*cytology ; Buthionine Sulfoximine/pharmacology ; Cell Differentiation ; Cell Line ; Cell Lineage ; Cytokines/pharmacology ; Enzyme Inhibitors/pharmacology ; Excitatory Amino Acid Agonists/pharmacology ; Glutamic Acid/pharmacology ; Humans ; Hydrogen Peroxide/pharmacology ; Intercellular Signaling Peptides and Proteins/pharmacology ; Kainic Acid/pharmacology ; Mice ; Mice, Inbred BALB C ; Multipotent Stem Cells/cytology/*drug effects/physiology ; Neuroglia/cytology/drug effects/physiology ; Neurons/cytology/*drug effects/physiology ; Neurotoxins/*pharmacology ; Oxidants/pharmacology ; Phenotype ; Reactive Oxygen Species/metabolism

OBJECTIVETo observe the effect of Rg1-induced NSCs in treatment of neonatal rat model with hypoxiaischemia.

METHODThe neonatal rat model of HIE was established and assessed by using TTC staining and behavioral observation, then Rg1-induced NSCs was transplanted into the neonatal rat of HIE by lateral ventricle injection. Water maze test and somatosensory evoked potential were detected to observe brain function and the immunohistochemistry was done to assess growth and differentiation about transplanted NSCs a month after transplanted.

RESULTThe transplantation of Rg1-induced NSCs could significantly shorten incubation period, swimming distance, exploration time of target quadrants of water maze test and incubation period and amplitude of somatosensory evoked potentials. Additionally, the concentrated expression appeared in the hippocampus and grew around the ischemic injury area in transplantation group.

CONCLUSIONTransplantation of Rg1-induced NSCs play a better role in the treatment of neonatal HIE rats.

Animals ; Cell Differentiation ; drug effects ; Evoked Potentials ; Female ; Ginsenosides ; pharmacology ; Hippocampus ; pathology ; physiopathology ; Hypoxia-Ischemia, Brain ; pathology ; physiopathology ; therapy ; Male ; Maze Learning ; Neural Stem Cells ; cytology ; drug effects ; transplantation ; Rats ; Rats, Sprague-Dawley ; Recovery of Function ; physiology

OBJECTIVEThis study aimed to investigate the expression pattern and function of Nuclear receptor subfamily 2 group E member 1 (Nr2e1) in retinoic acid (RA)-induced brain abnormality.

METHODSThe mouse model of brain abnormality was established by administering 28 mg/kg RA, and neural stem cells (NSCs) were isolated from the mouse embryo and cultured in vitro. Nr2e1 expression was detected by whole mount in situ hybridization, RT-PCR, and Western blotting. Nr2e1 function was determined by transducing Nr2e1 shRNA into NSCs, and the effect on the sonic hedgehog (Shh) signaling pathway was assessed in the cells. In addition, the regulation of Nr2e1 expression by RA was also determined in vitro.

RESULTSNr2e1 expression was significantly downregulated in the brain and NSCs of RA-treated mouse embryos, and knockdown of Nr2e1 affected the proliferation of NSCs in vitro. In addition, a similar expression pattern of Nr2e1 and RA receptor (RAR) α was observed after treatment of NSCs with different concentrations of RA.

CONCLUSIONOur study demonstrated that Nr2e1 could be regulated by RA, which would aid a better understanding of the mechanism underlying RA-induced brain abnormality.

Animals ; Brain ; cytology ; embryology ; Cell Proliferation ; Down-Regulation ; Gene Expression Regulation ; Gene Expression Regulation, Developmental ; drug effects ; Mice ; Mice, Inbred C57BL ; Neural Stem Cells ; drug effects ; physiology ; Receptors, Cytoplasmic and Nuclear ; genetics ; metabolism ; Tretinoin ; pharmacology

In the present study, we tested whether maintenance of adequate cerebral perfusion pressure (CPP) by pharmacologically preventing systemic hypotension with dopamine infusion would prevent cerebral ischemia and attenuate energy depletion and neuronal injury even though intracranial pressure remains elevated in a newborn piglet meningitis model. Cerebral blood flow, measured at the end of the experiment using fluorescent microspheres, was significantly increased by dopamine infusion. The decreased cerebral cortical cell membrane Na+, K+-ATPase activity and increased lipid peroxidation products, indicative of meningitis-induced brain damage, were significantly attenuated by dopamine infusion. Dopamine also significantly attenuated the meningitis-induced reduction in both brain ATP and phosphocreatine levels and the increase in brain lactate level. In summary, maintenance of adequate CPP with dopamine prevented cerebral ischemia, reduced cerebral energy depletion, and attenuated brain injury in neonatal bacterial meningitis.
Animals ; Animals, Newborn ; Brain/cytology/drug effects/*physiology ; Cell Membrane/*metabolism ; Cerebrovascular Circulation/*drug effects ; Dopamine/metabolism/*pharmacology ; Fluorescent Dyes/metabolism ; Hemodynamic Processes ; Meningitis, Escherichia coli/*metabolism ; Microspheres ; Random Allocation ; Regional Blood Flow ; Support, Non-U.S. Gov't ; Swine

OBJECTIVETo study the influence and mechanism of acute ethanol intoxication (AEI) on rat neuronal apoptosis after severe traumatic brain injury (TBI).

METHODSNinety-six Sprague-Dawley rats were randomly divided into four groups: normal control, AEI-only, TBI-only and TBI+AEI (n equal to 24 for each). Severe TBI model was developed according to Feeney's method. Rats in TBI+AEI group were firstly subjected to AEI, and then suffered head trauma. In each group, animals were sacrificed at 6 h, 24 h, 72 h, and 168 h after TBI. The level of neuronal apoptosis and the expression of Bcl-2 protein were determined by TUNEL assay and immunohistochemical method, respectively.

RESULTSApoptotic cells mainly distributed in the cortex and white matter around the damaged area. Neuronal apoptosis significantly increased at 6 h after trauma and peaked at 72 h. Both the level of neuronal apoptosis and expression of Bcl-2 protein in TBI-only group and TBI+AEI group were higher than those in control group (P less than 0.05). Compared with TBI-only group, the two indexes were much higher in TBI+AEI group at all time points (P less than 0.05).

CONCLUSIONOur findings suggest that AEI can increase neuronal apoptosis after severe TBI.

Animals ; Apoptosis ; drug effects ; Brain Injuries ; Cerebral Cortex ; cytology ; Disease Models, Animal ; Ethanol ; poisoning ; Immunohistochemistry ; In Situ Nick-End Labeling ; Male ; Neurons ; physiology ; Prosencephalon ; cytology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley

Hepatocyte growth factor (HGF) pretreatment could protect multiple cell types from apoptosis induced by various damages including oxidative stress. The present study was designed to investigate the protective effect of HGF on rat cortical neurons against apoptosis induced by hydrogen peroxide (H2O2) in culture, and then to explore whether HGF could influence the mitochondrial pathway of apoptosis. Primary rat cortical neurons were isolated from Sprague-Dawley rats and cultured in serum free medium containing 2% B27 and Neurobasal-A. To mimic the oxidative stress damage, cortical neurons were exposed to 100 mumol/L H2O2 for 4 h. To explore the effects of HGF on the neurons subjected to H2O2 injury, cells were pretreated with HGF 15, 30, 60 ng/mL for 24 h, respectively, and then exposed to 100 mumol/L H2O2 for 4 h. The cell viability was measured by MTT colorimetric assay and cell injury was evaluated by lactate dehydrogenase (LDH) leakage rate. Apoptotic cells were detected by Hoechst 33258 staining and Annexin V-FITC/PI double labeled flow cytometry. The caspase-3 activity was assessed by colorimetry. The alteration of transmembrane potential of mitochondria was determined by confocal laser scanning microscopy. The expression of cytochrome C protein was measured by Western blot analysis. The results showed that H2O2 treatment significantly decreased the cell viability, increased LDH leakage rate and the percentage of apoptotic cells. Pretreatment of HGF at different concentrations (15-60 ng/mL) could remarkably increase the cell viability of neurons. Compared with that of H2O2 group (53.4%+/-7.4%), the cell viabilities of neurons treated with 15, 30, and 60 ng/mL HGF significantly increased to (69.3+/-6.4)%, (77.5+/-6.1)% and (82.9+/-9.3)% (P<0.05), respectively. HGF preincubation also evidently decreased the LDH leakage rate in cortical neurons damaged by H2O2. The results of Hoechst staining revealed that HGF pretreatment could significantly reduce the apoptotic rate of neurons. The apoptotic rate of H2O2 group was (62.8+/-7.1)%, while that of HGF groups decreased significantly to (34.8+/-8.4)%, (23.5+/-3.2)% and (18.6+/-4.5)% (P<0.05), respectively. The data from caspase-3 activity assay indicated that HGF preconditioning could also remarkably decrease the caspase-3 activity of neurons. In addition, in the presence of various concentrations of HGF, the decrease of transmembrane potential of mitochondria in neurons caused by H2O2 injury could be reversed. Moreover, as detected by Western blot analysis, HGF downregulated the expression of cytochrome C protein in neurons. These results suggest that HGF has a protective effect on rat cortical neurons against apoptosis induced by H2O2, which might be related to the inhibition of the mitochondrial apoptotic pathway and the suppression of the caspase-3 activity.
Animals ; Apoptosis ; Brain ; cytology ; Caspase 3 ; metabolism ; Cell Survival ; Cells, Cultured ; Cytochromes c ; metabolism ; Hepatocyte Growth Factor ; pharmacology ; Hydrogen Peroxide ; pharmacology ; Mitochondria ; physiology ; Neurons ; cytology ; drug effects ; Oxidative Stress ; Rats ; Rats, Sprague-Dawley