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.
Objective To review research progress of the relation between glial cell line-derived neurotropic factor (GDNF) and motoneuron development and motoneuron disease. Methods The recent articles on GDNF and motonerons were extensively reviewed. The molecular structure, the mode of action and the route of administration of GDNF were investigated. Results GDNF plays extensive roles in the development anddisease of motoneuron. GDNF might regulate the development of the motonerons of the spinal cord to some extent and also save the injured motoneurons. Conclusion GDNF has a potential clinical value and inestimable futurein the treatment of motoneuron diseases.
OBJECTIVE: To purify and study Schwann cells cytoplasmic neurotrophic protein. METHODS: The dissociated SC taken from 300 newborn rats sciatic nerves were cultured, collected, ultrasonicated and ultraspeed centrifuged. The supernates were ultrafiltrated and concentrated by using ultrafiltration units with PM10, PM30, PM50 ultrafiltration membranes. The ultrafiltrated-concentrated solution with the protein molecular weight 10-30 ku, 30-50 ku and gt; 50 ku were collected respectively. The dissociated spinal cord motoneurons of 14 days embryonic rats were cultured with serum-free conditional medium and the additional SC cytoplasmic proteins were added into the medium. The results showed that the 10-30 ku and gt; 50 ku SC cytoplasmic proteins were able to maintain the survival of motoneurons for 24 hours. Then the 26 ku and 58 ku proteins were further extracted and purified from SC cytoplasm by high pressure liquid chromatography, and their neurobiological activities were studied. RESULTS: The 26 ku and 58 ku Schwann cell’s cytoplasmic proteins were able to maintain the survival of motoneurons cultured in the serum-free medium for 48 hours. The highest biological activity concentration is 20 ng per well. CONCLUSION: Schwann cells cytoplasm contains motoneuron neurotrophic proteins with molecular weight 26 ku and 58 ku.
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 study the method to inhibit perioperative internal mammary artery (IMA) spasm from the perspective of muscarinic receptor, and research the function of muscarinic cholinergic receptor subtypes of IMA. Methods IMA segments in vitro with intact endothelium were obtained from 30 patients who underwent coronary artery bypass grafting (CABG). According to muscarinic receptor antagonists of different concentrations， They were divided into control group (not using receptor antagonist), atropine group (nonselective M receptor antagonist), pirenzepine group (M1 receptor antagonist) and Methoctramine group(M2 receptor antagonist) by random number table. The effects of antagonists on vasodilatation were analyzed, Scott ratio was used to calculate affinity index (pD2) and Schild plot was used to count rivalry index (pA2). Results Acetylcholine (Ach)induced concentrationdependentrelaxation response of IMA segments with intact endothelium precontracted with potassium chloride (KCl). The pD2 was 6.92±0.05. The effects of atropine, pirenzepine and methoctramine on doseresponse curve induced by Ach with intact endothelium were all concentrationdependent. With the increase of the concentration of antagonists, the Achinduced doseresponse curves had a significant shift to right(Plt;0.05). Atropine, pirenzepine and Methoctramine competitively antagonized the reaction of vessel to Ach. The pA2 were 9.62±0.15,7.70±0.08 and 630±0.08, respectively. Conclusion The Achinduced relaxation response of IMA with intact endothelium is concentrationdependent. According to the affinity of different antagonist, IMA in Vitro Achinduced relaxation response is implemented by acting on nonneuronal muscarinic cholinergic M1 receptor subtype.
ObjectiveTo explore the correlation between the functional status of upper limb motor neurons and motor function in stroke patients, and provide guidance for rehabilitation assessment and functional prognosis.MethodsThe stroke patients who were hospitalized in Department of Rehabilitation Medicine of Zhongda Hospital of Southeast University between November 2020 and January 2021 were selected. Motor unit number estimation (MUNE) and F wave were examined to evaluate the functional status of motor neuron. The Fugl-Meyer Assessment (FMA) and Modified Ashworth Scale (MAS) were used to evaluate the upper limb motor function. The correlations of electrophysiological parameters with FMA score and MAS score were analyzed respectively.ResultsA total of 42 patients were enrolled, and 16 patients were complicated with carpal flexor spasm on the affected side. Among the 42 stroke patients, the MUNE of the abductor pollicis brevis on the affected side was lower than that on the unaffected side (t=−3.466, P=0.001), and the percentage of F waves with different shapes on the affected side was significantly lower than that on the unaffected side (Z=−5.583, P<0.001). Among the 16 stroke patients with carpal flexor spasm, the F wave amplitude was higher on the affected side than that on the unaffected side (t=2.764, P=0.014), while the F wave latency on the affected side was not statistically significant compared with the unaffected side (Z=−0.595, P=0.552). Among the 42 stroke patients, the affected/unaffected side ratio of the percentage of F waves with different shapes was positively correlated with FMA score (rs=0.377, P=0.014), while the correlation between the affected/unaffected side ratio of MUNE and FMA score was not statistically significant (rs=0.104, P=0.513). Among the 16 stroke patients with carpal flexor spasm, the affected/unaffected side ratio of the F wave amplitude was positively correlated with the MAS score of the carpi flexor muscle (rs=0.550, P=0.027).ConclusionStroke may result into the number of functional motor neurons of the upper limbs of the hemiplegic side decreased and the excitability of motor neurons increased simultaneously, and which were related to motor function and muscle tone.
Excessive microglial activation and subsequent neuroinflammation lead to neuronal cell death, which are involved in the pathogenesis and progression of several neurodegenerative diseases such as Parkinson's disease. The objective of this study was to determine the involvement of chlorpyrifos (CPF) in the activation of microglia and production of inflammatory factors in response to CPF stimulation and the influence on the viability of dopaminergic (DA) neurons. We detected the change of BV-2 cells morphology and expression of inducible nitric oxide (iNOS), cyclooxygenase-2 (COX-2) mRNA and protein level upon CPF stimulation (0, 1, 3, 6, 12, 24 h) in BV-2 (mouse brain microglia) cells by reverse transcription polymerase chain reaction (RT-PCR) or Western blot. We randomly assigned BV-2 cells into CPF, menstruum dimethysulfoxide (DMSO) and normal saline (NS) groups. We stimulated The BV-2 cells in the CPF group with CPF, and we stimulated the two control groups with DMSO or NS for 12 hours, respectively. We then collected the used culture media from the culture dishes and centrifuged it to remove the detached cells. Then, we used the supernatants as microglial conditioned media. We treated SH-SY5Y neurons with various groups of microglial conditioned media for 24 hours. We observed the effect of conditioned media collected from BV-2 cell on the viability of dopaminergic cell lines SH-SY5Y using MTT assay. We found that inflammatory factors iNOS, COX-2 mRNA and protein levels were up-regulated upon CPF stimulation. Conditioned media from BV-2 upon CPF stimulation is toxic to SH-SY5Y. It might be concluded that the exposure to CPF may induce dopaminergic neuronal damage by the activation of inflammatory response, and a mechanism may be involved in Parkinson's disease pathogenesis.
To investigate the mechanism of cAMP/Ca2+ signaling pathway inducing bone marrow mesenchymal stem cells to differentiate into neuronal cells, we cultured the bone marrow mesenchymal stem cells D1 cells in the present study. D1 cells were divided into two groups: control group and salidroside inducing groups. Control group was cultured with complete culture solution D/F12, while salidroside inducing groups were induced with 100 mg·L–1 salidroside for different time periods (24, 48 and 72 hours). PCR-array assay was used to detect expression of 84 calcium related mRNA, and significantly different genes were chosen to analyse. As a result, there were 4 significantly upregulated mRNAs inclu-ding DNA damage-inducible transcript 3 (Ddit3), heat shock protein 5 (Hspa5), protein phosphatase 1 regulatory subunit (Ppp1r15a) and prostaglandin-endoperoxide synthase 2 (Ptgs-2), and 4 significantly downregulated mRNAs including glucagon (Gcg), interleukin 2 (Il2), tumor necrosis factor (Tnf) and somatostatin (Sst) in the cAMP/Ca2+ signaling pathway. They probably had an effect on the process of salidroside induced D1 cells differentiating into neuronal cells.
The purpose of this study was to identify specific microRNAs (miRNAs) during differentiation and maturation of interneurons and to predict their possible functions by analyzing the expression of miRNAs during in vitro differentiation of the rat interneuron precursor cell line GE6. In the experiment, the interneuron precursor cell line GE6 was cultured under three different conditions, i.e. the first was that had not added growth factors and the normal differentiation cultured for 4 days (Ge6_4d); the second was that cultured with bone morphogenetic protein-2 (BMP2) for 4 days (Ge6_bmp2); and the third was that cultured with sonic hedgehog (SHH) for 4 days (Ge6_shh). In addition, another group of undifferentiated GE6 (Ge6_u) was applied as a control. We found in this study that the expression levels of a large number of miRNAs changed significantly during GE6 differentiation. The expression levels of miR-710, miR-290-5p and miR-3473 increased in the GE6 cells with secreted factor BMP2. These miRNAs may play important regulatory roles during interneuron differentiation.
This study aims to construct the recombinant lentivirus vector containing specific small interfering RNA (siRNA) targeting rat CREB binding protein(CBP)gene and to identify its function of inhibiting the expressions of acetylated histone in primarily cultured hippocampal neurons. Firstly, we constructed four kinds of recombinant lentivirus siCBP. And then we used them to infect the primarily cultured hippocampal neurons, and performed real-time PCR, western blot respectively to detect the expressions of CBP. Afterwards, the most effective lentivirus siCBP was used to infect the primarily cultured hippocampal neurons, and then the HAT activity and protein expressions of acetylated histone Ac-H3, Ac-H4 of the neurons were examined. By using PCR, endonuclease cutting and gene sequencing, we confirmed that the target genes were correctly cloned in lentivirus vector. Besides, CBP mRNA and protein expressions in neurons were found to be with varying degrees of decreases after infections of the four kinds of lentivirus siCBP. Furthermore, the representative and most effective lentivirus GR806 could effectively inhibit the HAT activity and the protein expressions of Ac-H3, Ac-H4 in neurons. It provides the experimental basis for the subsequent application of siCBP to clarify the effects and corresponding molecular mechanism of the CBP-dependent histone acetylation on learning and memory function in hippocampus.