Cultured primary hippocampal neurons are ideal tool for investigating the subcellular localization and trafficking of neuronal proteins. The aim of the present study was to establish a method to co-culture hippocampal neurons and cortical astrocytes, which would guarantee well conditions of neurons. Newborn Sprague-Dawley (SD) rats were sacrificed by decapitation. The cortex of cerebrum was cut into pieces, and the cortical tissue was digested with trypsin. The liquid suspension of single cells was planted onto a 25 cm² culture flask. On the fourth day of culture, the tissue cells except astrocytes were removed by intensive agitation of culture flask. Purified astrocytes were allowed to grow continuously until they reached most area of flask. At this time point, we replaced the culture media with neuronal cell media containing cytarabine, and planted the primary culture of rat hippocampal neurons onto the feed layer of cortical astrocytes. The microscopic observation results showed that, the astrocytes evenly grew without obvious boundaries between each other, and exhibited good purity. The co-cultured hippocampal neurons were in good condition, developed intertwined network of axons and dendrites, lived for a long time, and could tolerate gene transfection. Above all, this method is relatively simple from a technical point of view, yet provides healthy and reliable neuronal culture.
Animals ; Astrocytes ; cytology ; Cells, Cultured ; Cerebral Cortex ; cytology ; Coculture Techniques ; Culture Media ; Hippocampus ; cytology ; Neurons ; cytology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the expression of metabotropic glutamate receptor 5 (mGluR5) and its cellular distribution in the frontal cortex, ventricular zone (VZ) and subventricular zone (SVZ) in human fetuses.

METHODSAccording to the gestational age, the collected fetuses were divided into 4 groups, namely 9-11 weeks, 14-16 weeks, 22-24 weeks and 32-36 weeks. Brain tissue blocks including the frontal lobe or VZ/SVZ were prepared into slices, and the expression pattern and cellular distribution of mGluR5 in the frontal cortex and VZ/SVZ were observed by immunohistochemistry or immunofluorescence.

RESULTSmGluR5 immunoreactivity was present in the cell membrane in the frontal cortex, VZ and SVZ from the 9th to 36th weeks and the immunoreactivity in the marginal zone (MZ) and cortical plate (CP) was markedly stronger than that in VZ and SVZ. The cells expressing mGluR5 included neural stem/progenitor cells in the VZ and SVZ, immature neurons in the VZ and MZ, and numerous mature neurons in the CP.

CONCLUSIONmGluR5 is expressed by a variety of cells such as neural stem cells in the frontal cortex, VZ and SVZ in human fetus, suggesting a role of mGluR5 in the development of human cerebral cortex.

Cerebral Cortex ; cytology ; Cerebral Ventricles ; cytology ; metabolism ; Fetus ; cytology ; metabolism ; Frontal Lobe ; cytology ; metabolism ; Humans ; Neural Stem Cells ; cytology ; metabolism ; Receptor, Metabotropic Glutamate 5 ; Receptors, Metabotropic Glutamate ; metabolism

The methods of purification, expanding, marking, conservation, induced differentiation and identification of neural stem cells (NSCs) in vitro were explored for further research and treatment of tethered cord syndrome in children and other neural system diseases. The cells derived from the cerebral cortex of frontal lobe in 14.5 d rat embryos were maintained in serum-free DMEM/F12 medium containing 20 ng/ml bFGF, 20 ng/ml EGF and B27 supplement. The NSCs of suspending single-cell-colon were isolated and passaged, were purified by limited dilution, and were expanded numerously with sub-colon in consecutive generations. Then, Nestin antigen expression was detected by immunohistochemistry techniques. The cells of the purified and expanded NSCs were frozen, recovered and incubated in BrdU, and the NSCs were induced to differentiate in serum or feeder layer. These revived NSCs from frozen cells could express Nestin antigen and could be induced in serum or feeder layer to differentiate into neurons and glias expressing tubulin-III and GFAP respectively. It is good and simple for purification and proliferation of NSCs numerously by the limited dilution and consecutive generations suspending single-cell-colon of NSCs from the cerebral cortex of frontal lobe in rat embryos. The NSCs could be induced on feeder layer to differentiate into neural cells numerously. BrdU can mark and trace NSCs for the research and treatment of the animal model of neural system diseases. By good command of the technlogies for the purification proliferation, and induced differentiation of NSCs in vitro, it is possible to find a new way for further research of the biologic specificity and the treatment of the disease in nervous system.
Animals ; Cell Differentiation ; Cell Separation ; Cells, Cultured ; Cerebral Cortex ; cytology ; Embryo, Mammalian ; Female ; Frontal Lobe ; cytology ; Male ; Neurons ; cytology ; Rats ; Rats, Wistar ; Stem Cells ; cytology

The cortexes were obtained from new-born rats and dissociated to single cells by triturating. The cells were cultured in neural stem cell (NSC) culture medium (DMEM supplemented with bFGF, EGF and B27) and formed primary neurospheres after 7 days. Single cells dissociated from neurosphere were cultured in 96-well plates and formed single-cell cloning neurosphere 7 days later. The primary and single-cell cloning neurospheres were both positive for the immunofluorescent staining of nestin and were identified as NSC. It was proved that NSC can be expanded in vitro and provide seed cells for neural tissue engineering.
Animals, Newborn ; Cell Separation ; Cells, Cultured ; Cerebral Cortex/*cytology ; Culture Media ; Neurons/*cytology ; Rats, Sprague-Dawley ; Stem Cells/*cytology ; Tissue Engineering

The cortexes were obtained from new-born rats and dissociated to single cells by triturating. The cells were cultured in neural stem cell (NSC) culture medium (DMEM supplemented with bFGF, EGF and B27) and formed primary neurospheres after 7 days. Single cells dissociated from neurosphere were cultured in 96-well plates and formed single-cell cloning neurosphere 7 days later. The primary and single-cell cloning neurospheres were both positive for the immunofluorescent staining of nestin and were identified as NSC. It was proved that NSC can be expanded in vitro and provide seed cells for neural tissue engineering.
Animals ; Animals, Newborn ; Cell Separation ; Cells, Cultured ; Cerebral Cortex ; cytology ; Culture Media ; Neurons ; cytology ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; cytology ; Tissue Engineering

This study is to establish a simple and practical co-culture method of cortical neurons and astrocytes of rats. The cortex of the new-born SD rats was digested by 0.125% pancreatic enzyme, and the differential adherence was applied to obtain the mixed cell suspension of neurons and astrocytes. A low concentration of cytarabine was used to inhibit the astrocytes in a moderate way to get neuronal and astrocyte co-culture. The morphological characteristics of the cells in different times were observed under the inverted microscope. The cells began to adhere the wall 2 h after the inoculation. Neurons and astrocytes grew in a good condition under the inverted microscope 9 days after the inoculation. The results of the immunofluorescence staining and Rosenfeld's staining indicated that the co-culture of neurons and astrocytes was successful and the ratio of neurons and astrocytes was close to 1:1. A new neurons and astrocytes co-culture method, which is simple and convenient, was successfully established. It will be an efficient method for the related researches about neuronal and astrocyte co-culture in vitro.
Animals ; Animals, Newborn ; Astrocytes ; cytology ; Cells, Cultured ; Cerebral Cortex ; cytology ; Coculture Techniques ; methods ; Female ; Male ; Neurons ; cytology ; Primary Cell Culture ; methods ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo evaluate the efficiency of a modified culture method for rat cerebral cortical astrocytes in vitro.

METHODSThe astrocytes derived from the cerebral cortex of 3-day-old Sprague-Dawley rats were first purified as described previously, then the cells were replanted at a low density. The culture flask was changed after 1 hour and substratum was replaced after 24 hours. Cells were syncretized to a monolayer, followed by cell passage. After three passages the cells were cultured in DMEM medium containing 10% fetal serum for a long period. The derivation of the cells was identified by immunofluorescent staining with anti-GFAP polyclonal antibodies.

RESULTSA variety of morphologically distinct astrocytes with many long processes and small cell bodies were obtained. Finally an astrocytic network occurred through cellular process connections. The immunofluorescent staining demonstrated the percentage of GFAP-positive cells was above 98%.

CONCLUSIONSThe modified culture method for astrocytes from rat cerebral tissue is reliable, with a high purity. The cultured astrocytes have a similar morphological development to those in vivo.

Animals ; Astrocytes ; physiology ; Cell Culture Techniques ; Cerebral Cortex ; cytology ; Female ; Glial Fibrillary Acidic Protein ; analysis ; Male ; Rats ; Rats, Sprague-Dawley

Animals ; Cerebral Cortex ; cytology ; physiology ; Delayed Rectifier Potassium Channels ; physiology ; Neurons ; physiology ; Patch-Clamp Techniques ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effect of propofol on the expression of thrombospondin-1 (THBS-1) mRNA and protein in purified newborn rat cortical astrocytes in vitro.

METHODSAstrocytes were isolated from newborn rat cortex and grown in culture before exposure to propofol at 3, 10, 30, 100 or 300 µmol/L for 6 h, 12, or 24 h. The mRNA level of THBS-1 was detected by RT-PCR, and the protein level of THBS-1 was detected by immunofluorescence cytochemistry and Western blotting.

RESULTSPropofol exposure caused significantly upregulated THBS-1 level in cultured astrocytes (P<0.05) to a level about 1.3 times higher than that in control cells. The mRNA and protein levels of THBS-1 in cultured rat cortical astrocytes were upregulated by exposures to 10, 30 and 100 µmol/L propofol (P<0.01). High expression of THBS-1 mRNA and protein was detected in the cells with exposures for different durations (P<0.05), especially in the 12 h group (P<0.01).

CONCLUSIONPropofol at clinically relevant concentrations can modulate the level of THBS-1 secreted by astrocytes of rat cerebral cortex in vitro.

Animals ; Astrocytes ; drug effects ; metabolism ; Cells, Cultured ; Cerebral Cortex ; cytology ; Propofol ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Thrombospondin 1 ; metabolism

OBJECTIVETo establish a method for organotypic slice culture of neonatal rat cortex in a modified condition and investigate the effect of spatial signals on neural stem cell (NSC) differentiation.

METHODSThe brain slices (200 µm in thickness) of neonatal SD rats (3 to 5 days old) were prepared and cultured in modified serum-free DMEM/F12 medium at 37 degrees celsius; with 95% O(2) and 5% CO(2). The organotypic slice cultures were observed regularly. NSCs isolated from the cortex of rat embryos (14-15 embryonic days) were cultured in serum-free DMEM/F12 supplemented with B27 and N2, and the passage 3 NSCs were labeled by CM-DiI before transplanted onto the organotypic slices cultured for 2 weeks. The survival of transplanted NSCs was assessed, and the cell differentiation was identified by immunofluorescence staining.

RESULTSThe organotypic slice cultures were well maintained for at least 4 weeks in the modified medium. The thickness of the organotypic slices reduced from 200 µm to 130 µm after 2-week culture in vitro due to the migration of the cells on the edge of the slices. CM-DiI-labeled NSCs survived well and differentiated into GFAP(+) glia and β-tubullin III(+) neurons.

CONCLUSIONNeonatal rat organotypic brain slice can be successfully cultured in a modified condition to serve as a model for studying NSC differentiation induced by spatial signals.

Animals ; Animals, Newborn ; Cell Differentiation ; physiology ; Cerebral Cortex ; cytology ; Coculture Techniques ; methods ; Fetus ; Neural Stem Cells ; cytology ; transplantation ; Organ Culture Techniques ; methods ; Rats