Purpose To investigate the characteristics of intraocular growth of mice embryonic stem cells (ESC) in nude mice. Methods The undifferentiated murine ESC in vitro were transplanted into the eyes of nude mice.Mophological and immunohistochemical examinations were implemented. Results Two to three days after transplantation,yellowish-white granules and masses were seen inside the anterior chamber and vitreous cavity and enlarged gradually.Morphological examination showed that there were undifferentiated cells and differentiated cells in anterior chamber and vitreous cavity.The morphology and alignment of some differentiated cells were similar to those of the retina of nude mice.The cells were highly positive in NSE staining. Conclusion The transplanted ESC could grow in the eyes of nude mice and differentiate into neurons and retina-like structure. (Chin J Ocul Fundus Dis，2000，16：213-284)
OBJECTIVE Following the delayed repair of peripheral nerve injury, the cell number of anterior horn of the spinal cord and its ultrastructural changes, motorneuron and its electrophysiological changes were investigated. METHODS In 16 rabbits the common peroneal nerves of both sides being transected one year later were divided into four groups randomly: the degeneration group and regeneration of 1, 3 and 5 months groups. Another 4 rabbits were used for control. All transected common peroneal nerves underwent epineural suture except for the degeneration group the electrophysiological examination was carried out at 1, 3 and 5 months postoperatively. Retrograde labelling of the anterior horn cells was demonstrated and the cells were observed under light and electronmicroscope. RESULTS 1. The number of labelled anterior horn cell in the spinal cord was 45% of the normal population after denervation for one year (P lt; 0.01). The number of labelled cells increased steadily from 48% to 57% and 68% of normal values at 1, 3 and 5 months following delayed nerve repair (P lt; 0.01). 2. The ultrastructure of the anterior horn cells of the recover gradually after repair. 3. With the progress of regeneration the latency become shortened, the conduction velocity was increased, the amplitude of action potential was increased. CONCLUSION Following delayed repair of injury of peripheral nerve, the morphology of anterior horn cells of spinal cord and electrophysiological display all revealed evidence of regeneration, thus the late repair of injury of peripheral nerve was valid.
In order to investigate the effect of nerve compression on neurons, the commonly used model of chronic nerve compression was produced in 48 SD rats. The rats were sacrificed in 1, 2, 3, 4, 5 and 6 months after compression, respectively. The number of neuron and ultrashruchure of alpha-motor neurons and ganglion cells of the corresponding spinal segment were examined. The results showed as following: After the sciatic nerve were crushed, the number of neuron and ultrastructure of alpha-motor neurons and ganglion cells might undergo ultrastructural changes, and even the death might occur. These changes might be aggravated as the time of crushing was prolonged and the compression force was increased. It was concluded that for nerve compression, decompression should be done as early as possible in order to avoid or minimize the ultructural changes of the neuron.
Transcranial magnetic stimulation (TMS) as a non-invasive neuroregulatory technique has been applied in the clinical treatment of neurological and psychiatric diseases. However, the stimulation effects and neural regulatory mechanisms of TMS with different frequencies and modes are not yet clear. This article explores the effects of different frequency repetitive transcranial magnetic stimulation (rTMS) and burst transcranial magnetic stimulation (bTMS) on memory function and neuronal excitability in mice from the perspective of neuroelectrophysiology. In this experiment, 42 Kunming mice aged 8 weeks were randomly divided into pseudo stimulation group and stimulation groups. The stimulation group included rTMS stimulation groups with different frequencies (1, 5, 10 Hz), and bTMS stimulation groups with different frequencies (1, 5, 10 Hz). Among them, the stimulation group received continuous stimulation for 14 days. After the stimulation, the mice underwent new object recognition and platform jumping experiment to test their memory ability. Subsequently, brain slice patch clamp experiment was conducted to analyze the excitability of granulosa cells in the dentate gyrus (DG) of mice. The results showed that compared with the pseudo stimulation group, high-frequency (5, 10 Hz) rTMS and bTMS could improve the memory ability and neuronal excitability of mice, while low-frequency (1 Hz) rTMS and bTMS have no significant effect. For the two stimulation modes at the same frequency, their effects on memory function and neuronal excitability of mice have no significant difference. The results of this study suggest that high-frequency TMS can improve memory function in mice by increasing the excitability of hippocampal DG granule neurons. This article provides experimental and theoretical basis for the mechanism research and clinical application of TMS in improving cognitive function.
Objective To investigate the effect of olfactory ensheathing cell culture medium (OECCM) on the growth of spinal cord neurons and its protective effect on the injured neurons by H2O2, and to disscuss the probable protective mechanisms of olfactory ensheathing cells (OECs). Methods The primary olfactory ensheathing cells (OECs) were isolated from olfactory bulb of adult SD rat, and OECCM were prepared. The morphology of OECs was observed by inverted phase contrast microscope, identified by rabbit-antiratlow-affinity nerve growth factor p75 (NGFRp75), and its purity were calculated.Primary spinal cord neurons were cultured from 15 to 17 days pregnant SD rats, and injury model of neurons were prepared by H2O2. OECCM and control culture medium were added into the normal spinal neurons (groups A, B). OECCM and control culture medium were added into the injured spinal neurons by H2O2 (groups C, D). In groups A and C, 200 μL of control culture medium was used; in groups B and D, 100 μL of control culture medium and 100 μL of OECCM were used. Then the growth index such as average diameter of neuron body, the number and length of neuron axons were measured. The viabil ities of normal and injured neurons were assessed by MTT. Results OECs showed bipolar or tripolar after 6-9 days of culture. Primary spinal cord neurons were round and bigger, and neuron axons grew significantly and showed bipolar after 5-7 days of culture. The immunocytochemisty of OECs by NGFRp75 showed that membrane were stained. The degree of purity was more than 90%. Primary spinal cord neurons grew well after 6-9 days of culture, and compared with group A, neurons of group B grew b, whose cell density and diameter were bigger. The average diameter of neuron body, the number and length of neuron axons were (33.38 ± 6.80) D/μm, (1.67 ± 0.80), and (91.19 ± 62.64) L/μm in group A, and (37.39 ± 7.28) D/μm, (1.76 ± 0.82), and (121.33 ± 81.13) L/μm in group B; showing statistically significant differences (P lt; 0.05). The absorbency (A) value of neurons was 0.402 0 ± 0.586 9 in group A and 0.466 0 ± 0.479 0 in group B; showing statistically significant difference (P lt; 0.01). After 2 hours of injury by H2O2, the cell density of spinal cord neurons decreased, and neuron axons shortened. The A value of injured neurons was 0.149 0 ± 0.030 0 in group C and 0.184 0 ± 0.052 0 in group D, showing statistically significant difference (P lt; 0.01). Conclusion The results above suggest that OECCM could improve the growth of spinal cord neurons and protectthe injured neurons. The neurotrophic factors that OECs secrete play an important role in the treatment of spinal cord injury.
This study aims to investigate the effect ofγ-Secretase Inhibitor DAPT, (N-[N-(3, 5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester), on the differentiation of neural precursor cells and the production of neurons in the neural precursor cell line GE6. GE6 was cultured in medium with 4μmol/L DAPT added as the experimental group and the untreated medium separately as the control group. After 4 days of differentiation, we carried out the following experiments. We used immuno-fluorescent staining to observe the ratio of Tuj1, GFAP and O4 positive cells. We also used qRT-PCR to detect the effect of the DAPT on Tuj1 and GFAP mRNA transcription in the GE6. The results of immuno-fluorescent staining indicated that the Tuj1 ratio of experimental group was higher compared to that of the control group, but the GFAP and O4 ratio of experimental group was lower than that of the control group. The differences were statistically significant (P < 0.05). The result of qRT-PCR was in accordance with immunofluorescent staining results. It was well concluded that DAPT could promote the neurogenic differentiation of neural precursor cell line rather than leading to gliogenic differentiation. More neurons could be obtained for transplantation with the addition of DAPT.
Alzheimer’s disease (AD) is a chronic central neurodegenerative disease. The pathological features of AD are the extracellular deposition of senile plaques formed by amyloid-β oligomers (AβOs) and the intracellular accumulation of neurofibrillary tangles formed by hyperphosphorylated tau protein. In this paper, an in vitro pathological model of AD based on neuronal network chip and its real-time dynamic analysis were presented. The hippocampal neuronal network was cultured on the microelectrode array (MEA) chip and induced by AβOs as an AD model in vitro to simultaneously record two firing patterns from the interneurons and pyramidal neurons. The spatial firing patterns mapping and cross-correlation between channels were performed to validate the degeneration of neuronal network connectivity. This biosensor enabled the detection of the AβOs toxicity responses, and the identification of connectivity and interactions between neuronal networks, which can be a novel technique in the research of AD pathological model in vitro.
Objective To explore the method that can inducethe mesenchymal stem cells (MSCs) to differentiate into the neuronlike cells in vitro．Methods The neuron-like cells were isolated froman SD rat (age, 3 months; weight, 200 g). They underwent a primary culture; theinduced liquid supernatant was collected, and was identified by the cell immunohistochemistry. The C3H1OT1/2 cells were cultured, as an MSCs model, and they were induced into differentiation by β-mercaptoethanol (Group A) and by the liquid supernatant of the neuron-like primary cells (Group B), respectively. The cells were cultured without any induction were used as a control (Group C). Immunohistochemistrywas used to identify the type of the cells． Results The result of the immunochemistry showed that the cells undergoing the primary culture expressed the neurofilament protein (NF) and the neuronspecific enolase (NSE), and they were neuron-like cells. β-mercaptoethanol could induce the C3H1OT1/2 cells toexpress NF and NSE at 2 h, and the expression intensity increased at 5 h. The liquid supernatant of the primarily-cultured neuron-like cells could induce theC3H1OT1/2 cells to express NF and NSE at 1 d, but the expression intensity induced by the liquid supernatant was weaker than that induced by β-mercaptoethanol. The positivity rate and the intensity expression of NSE were higher than those of NF. Conclusion MSCs can differentiate into the neuron-like cells by β-mercaptoethanol and the microenvironment humoral factor, which can pave the way for a further study of the differentiation of MSCs and the effectof the differentiation on the brain trauma repair.
OBJECTIVE To study the protective effects of Schwann cell derived neurotrophic factor (SDNF) on motoneurons of spinal anterior horn from spinal root avulsion induced cell death. METHODS Twenty SD rats were made the animal model of C6.7 spinal root avulsion induced motoneuron degeneration, and SDNF was applied at the lesion site of spinal cord once a week. After three weeks, the C6.7 spinal region was dissected out for motoneuron count, morphological analysis and nitric oxide synthase (NOS) enzyme histochemistry. RESULTS 68.6% motoneurons of spinal anterior horn death were occurred after 3 weeks following surgery, the size of survivors was significantly atrophy and NOS positive neurons increased. However, in animals which received SDNF treatment, the death of motoneurons was significantly decreased, the atrophy of surviving motoneurons was prevented, and expression of NOS was inhibited. CONCLUSION SDNF can prevent the death of motoneurons following spinal root avulsion. Nitric oxide may play a role in these injury induced motoneuron death.
OBJECTIVE: To research the protective effect of Schwann cell and extracellular matrix (ECM) gel on neurons in dorsal root ganglion. METHODS: 1. Schwann cells were seeded into 30% ECM at 1 x 10(8)/ml and then implanted into PLA hollow fiber conduits to repair 10 mm length defects of rat sciatic nerve, and histological observation was taken at 8 and 12 weeks after operation. 2. To observe the survival of Schwann cells, Schwann cells labeled BrdU were seeded into 30% ECM at 1 x 10(8)/ml and then implanted into PLA hollow fiber conduits to repair 10 mm length defects of rat sciatic nerve. Histological observation and immunohistochemical method stained with BrdU were done at 3 and 6 weeks after operation. RESULTS: 1. When seeded into ECM gel and transplanted into rats, most of the Schwann cells survived to 3 weeks and a part of them survived up to 6 weeks. 2. The survival neuron ratios of Schwann cells with ECM gel group and ECM gel group were 83.5% and 81.3% respectively, and significantly higher than that of saline group (72.9%, P lt; 0.05). CONCLUSION: When seeded into ECM gel and transplanted into rats, most of the Schwann cells survive and protect 83.5% neurons in dorsal root ganglion from retrograde death.