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BACKGROUND: Bone marrow mesenchymal stem cells have the potential to differentiate into neuron-like cells, which have been listed as the preferred stem cells for the treatment of spinal cord injury. However, due to their low differentiation efficiency, it is particularly important to find a factor with high induction ability. Based on literature review and our previous studies, it is speculated that bone morphogenetic protein 7 (BMP-7) gene may play a vital role in promoting the differentiation of bone marrow mesenchymal stem cells into neuron-like cells. OBJECTIVE: To investigate the differentiation of rat bone marrow mesenchymal stem cells into neurons induced by BMP-7 lentivirus vector transfection. METHODS: Bone marrow mesenchymal stem cells of Sprague-Dawley rats were cultured by whole bone marrow adherence method, and then were transfected with LV-GFP when multiplicities of infection were 50, 25, 10, and 1. Green fluorescent protein expression was observed using fluorescence inversion microscope in each group at 3 days after transfection, to confirm the best multiplicity of infection. Passage 3 bone marrow mesenchymal stem cells were divided into blank control group (routine culture), LV-GFP group, and LV-BMP-7-GFP group, followed by transfection at the best multiplicity of infection. After 24, 48, 72, 96, and 120 hours of transfection, MTT assay was used to detect cell survival rate in each group. Immunocytochemical assay was used to detect the expression of nerve cell markers (neurofilament protein 200, synaptophysin-1) after 3 days of transfection. RESULTS AND CONCLUSION: (1) After 3 days of LV-GFP transfection, GFP-positive cells were observed under fluorescence microscopy when multiplicities of infection were 10, 25, and 50, whereas no GFP-positive cells were found when the multiplicity of infection was 1. The average fluorescence intensity was the highest when the multiplicity of infection was 10 (P < 0.05), indicating that multiplicity of infection=10 had the best infection effect. (2) Immunocytochemical results showed that the expression of neurofilament-200 and synaptophysin-1 was negative in the blank control group and LV-GFP group, but positive in the LV-BMP-7-GFP group. The cell body and axon in the LV-BMP-7-GFP group were dyed bright brown. In summary, lentivirus-mediated BMP-7 transfection can promote the differentiation of rat bone marrow mesenchymal stem cells into neuron-like cells.
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OBJECTIVE@#To observe the repair effect of bone marrow mesenchymal stem cells (BMSCs) combined with basic fibroblast growth factor (bFGF) on spinal cord injury in rats and explore its mechanism.@*METHODS@#SD rat BMSCs were obtained by serum culture technique. Eighty healthy 6-week-old male SD rats(weight about 240 g) were randomly divided into 4 groups with 20 each. The sham operation group underwent simple laminectomy without damaging spinal cord and was kept in the same condition as the other 3 groups. The other 3 groups underwent left T9 spinal cord hemisection to establish spinal cord injury model. After 9 days of modeling the local transplantation was performed. The Control group was implanted with gelatin sponge containing normal saline. The BMSCs transplantation group was implanted with gelatin sponge containing BMSCs. The bFGF+BMSCs transplantation group was implanted with gelatin sponge containing bFGF+BMSCs. After 4 and 8 weeks, the expression of NF-200 and GFAP in injured spinal cord tissue was analyzed by Western blotting and the recovery of hind limb function was evaluated by Basso Beattie Bresnahan(BBB) motor function score scale.@*RESULTS@#The BBB scores of BMSCs transplantation group and bFGF+BMSCs transplantation group were better than control group at 4 and 8 weeks after operation (<0.05) and there was significant difference between bFGF+BMSCs transplantation group and BMSCs transplantation group (<0.05). After 4 and 8 weeks postoperatively, NF-200 expression was minimal in control group and only a small amount was expressed in BMSCs transplantation group while in bFGF+BMSCs transplantation group NF-200 was highly expressed(<0.05). GFAP expression was high in control group, middle in BMSCs transplantation group and low in bFGF BMSCs transplantation group(<0.05). There was significant difference between bFGF+BMSCs transplantation group, BMSCs transplantation group and control group(<0.05).@*CONCLUSIONS@#The combined transplantation of BMSCs and bFGF can repair the spinal cord injury in rats. The mechanism may be related to the decrease of GFAP expression and the increase of NF-200 expression.
Subject(s)
Animals , Male , Rats , Bone Marrow Cells , Fibroblast Growth Factor 2 , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Rats, Sprague-Dawley , Spinal Cord , Spinal Cord InjuriesABSTRACT
Objective To investigate the effects of quercetin on glial scar formation and axonal regeneration after spinal cord injury (SCI) and its association with the p38 mitogen activated protein kinase (MAPK) signal pathway.Methods 128 female Sprague-Dawley (SD) rats were randomly divided into a control group (SCI + saline),an intervention group (SCI + quercetin + anisomycin),a treatment group (SCI + quercetin) and a sham-operation group (n =32).Basso Beattie Bresnahan (BBB) assessment and footprint analysis of the hind limb were performed on days 1,3,7,14,21 and 28 postoperation in each group.The expression levels of p38MAPK,phosphorylation p38MAPK,glial fibrillary acidic protein (GFAP) and neurofilament protein-200 (NF-200) were detected by Western blot.The numbers of GFAP and NF-200 positive staining cells in the injured spinal cord in each group were detected by immunohistochemistry.Results The BBB scores in the treatment group were significantly higher than in the intervention and control groups at each time point after SCI except on day 3 postoperation (P ＜ 0.05).The expression levels of phosphorylation p38MAPK protein in each SCI group were significantly higher than in the sham-operation group on days 3 and 7 postoperation (P ＜ 0.05).The expression levels of phosphorylation p38MAPK protein in the treatment group were significantly lower than in the control and intervention groups on days 3,7 and 14 postoperation (P ＜ 0.05),but there was no significant difference on day 28 postoperation among all the groups (P ＞ 0.05).The numbers of NF-200 and GFAP positive staining cells were significantly greater than in the sham-operation group at each time point postoperation (P ＜ 0.05);the NF-200 positive staining cells in the treatment group were significantly increased in comparison with the control and intervention groups (P ＜ 0.05);the GFAP positive staining cells in the treatment group were significandy fewer than in the control and intervention groups on days 7,14 and 28 postoperation (P ＜ 0.05).Conclusions Quercetin may have protective effects against acute SCI by decreasing glial scar formation,increasing axonal regeneration and promoting recovery of locomotor and nerve function in rats.The effects may be correlated with inhibition of the p38MAPK signal pathway.
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Objective To investigate the neurofilament protein (NF) and synaptophysin(SYN) distribution in the developing stage of human embryonic spinal cord and their clinical significance . Methods Totally 16 of human embryos, aged from 2 to 4 months were used in this study .Immunohistochemical staining was uesd to detect the expression and distribution of NF and SYN in the spinal cord .Results At the second month stage , NF expression was weakly positive in only the marginal layer on both sides of the spinal cord .At 3-4 months, NF expression was positive or strong positive in the spinal cord center crack before and after the middle of partition and cortex .In the ependymal layer of the lateral part of the expression of NF positive nerve fibers were seen in the gray matter .From 2 to 4 month-age, all visible SYN positive expressions distributed in the spinal cord .With the increase of gestational age , SYN positive expression in the spinal cord staining intensity value was increased first , and then decreased .The number of positive values increased month by month . Conclusion NF and SYN both take part in the regulation of human embryonic spinal cord tissue growth .
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Objective To study the effects of repetitive transcranial magnetic stimulation (rTMS) on rats'locomotor function after hemisectional spinal cord injury (SCI). Methods A model of hemisectional SCI was established at the T10 segment in 12 rats,which were then randomly divided into an SCI-rTMS group and an SCI-control group.Another 6 rats served as normal controls.The SCI-rTMS group received threshold rTMS daily for 4 weeks,while the SCI-control group was given sham rTMS.The normal control group was left without any treatment.BassoBeatti-Bresnahan (BBB) locomotor scores were recorded weekly.Motor evoked potential (MEP) was detected at the 28th day after the operation.Neurofilament-200 (NF-200) was detected with immunofluorescent staining of the spinal cord lesions. Results The BBB scores in the SCI-rTMS group improved more than those in the SCI-control group.MEP of the fight hindlimb was detected in the rTMS group,but not in the control group.Expression of NF-200 marker increased significantly more than in the rTMS group. Conclusions rTMS can improve locomotor function of rats after spinal cord hemisectioning,which may result from increased expression of NF-200 and the regeneration of axons.
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Phosphatidylinositol phosphates (PtdInsPs) are ubiquitous membrane phospholipids that play diverse roles in cell growth and differentiation. To clarify the regulation mechanism acting on neurofilament light chain (NF-L) self assembly, we examined the effects of various PtdInsPs on this process. We found that PtdInsPs, including PI(4,5)P2, directly bind to the positively charged Arg54 of murine NF-L, and this binding promotes NF-L self assembly in vitro. Mutant NF-L (R53A/R54A) proteins lacking binding affinity to PtdInsPs did not have the same effect, but the mutant NF-L proteins showed greater self assembly than the wild-type in the absence of any PtdInsP. These results collectively suggest that Arg54 plays a pivotal role in NF-L self assembly by binding with PtdInsPs.
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Animals , Mice , Fluorescent Antibody Technique , Mutation/genetics , Neurofilament Proteins/genetics , Phosphatidylinositol Phosphates/metabolism , Phospholipase C gamma/metabolism , Protein MultimerizationABSTRACT
@#Objective To explore the effects of early acupuncture on the expression of neurofilament protein (NFP) in the brain and the neurobehavior of the neonatal rats with brain injury. Methods The pregnant Wistar rats were randomly divided into two groups: lipopolysaccharide (LPS) group and control group, which were consecutively injected intraperitoneally with LPS or saline. The full-term newborn rats from LPS group were randomly divided into acupuncture group and model group. Acupuncture group was acupunctured from postnatal 8 d. They were evaluated with hanging test, and the expression of NFP was detected with immunohistochemical stain 21 d after birth. Results Both the scores of hanging test and the expression of NFP were the highest in the control group, the lowest in the model group and medium in the acupuncture group (P<0.01). Conclusion Acupuncture may increase the expression of NFP and improve locomotor function of the neonatal rats with brain injury caused by intrauterine infection.
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Objective To explore the effect of early enriched environment intervention on expression of neurofilament protein (NFP) in brain and the neurobehavior of filial rats with brain injury.Methods The pregnant Wistar rats of lipopolysaccharide (LPS) group were consecutively injected intraperitoneally with LPS on the 17th and 18th day of gestation while the control group only received an injection of same dose of 9 g/L saline.All premature rats,whose gestational age was less than 22 days,were removed from both groups;While the full-term newborn rats were chosen.After delivering,the uterus and placenta were taken out immediately to examine the infection situation by HE staining.Twenty-four hours after born,the brains of the newborn rats were taken out to observed the white matter damage by HE staining.LPS group was randomly divided into 2 groups:intervention group and non-intervention group.The intervention group was treated with neonatal handling and enriched environment from postnatal 8 days,while no management was performed in control group and non-intervention group.Ethological examination was tested with hanging test,and immunohistochemical technique was used to detect the expression of NFP,when the neonatal rats were 21 days old.Results There were a large amount of neutrophilic granulocyte in the uterus and placenta in LPS-treated group; In the 1-day-old rats in LPS group,brain tissue pathology test showed diffuse white matter lucencies.The scores of hanging test in control group was the highest,the ones in non-intervention group was the lowest among the 3 groups,and there was significant difference between them (Pa
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Phosphoinositide-specific phospholipase C-gamma1 (PLC-gamma1) has two pleckstrin homology (PH) domains: an amino-terminal domain (PH1) and a split PH domain (PH2). Here, we show that overlay assay of bovine brain tubulin pool with glutathione-S-transferase (GST)-PLC-gamma1 PH domain fusion proteins, followed by matrix-assisted laser-desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), identified 68-kDa neurofilament light chain (NF-L) as a binding protein of amino-terminal PH domain of PLC-gamma1. NF-L is known as a component of neuronal intermediate filaments, which are responsible for supporting the structure of myelinated axons in neuron. PLC-gamma1 and NF-L colocalized in the neurite in PC12 cells upon nerve growth factor stimulation. In vitro binding assay and immunoprecipitation analysis also showed a specific interaction of both proteins in differentiated PC12 cells. The phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P2] hydrolyzing activity of PLC-gamma1 was slightly decreased in the presence of purified NF-L in vitro, suggesting that NF-L inhibits PLC-gamma1. Our results suggest that PLC-gamma1-associated NF-L sequesters the phospholipid from the PH domain of PLC-gamma1.
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Rats , Animals , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Protein Interaction Mapping , Protein Biosynthesis/drug effects , Protein Binding/drug effects , Phosphoproteins/chemistry , Phospholipase C gamma/antagonists & inhibitors , Phosphatidylinositol 4,5-Diphosphate/metabolism , Peptides/chemistry , PC12 Cells , Neurofilament Proteins/chemistry , Nerve Growth Factor/pharmacology , Molecular Weight , Molecular Sequence Data , Microtubules/metabolism , Microscopy, Fluorescence , Isoenzymes/metabolism , Glutathione Transferase/metabolism , Blotting, Far-Western , Blood Proteins/chemistry , Binding Sites , Amino Acid SequenceABSTRACT
BACKGROUND: Charcot-Marie-Tooth (CMT) disease is the most common form of inherited motor and sensory neuropathy. Neurofilament light chain polypeptide (NEFL) is one of the most abundant cytoskeletal components of the neuron. The NEFL gene encoding the neurofilament light chain plays an important role in the axonal structure that includes an extensive fibrous network in the cytoplasm of the neuron. Mutations in the NEFL gene are also present in CMT2E, CMT type 1 and Dejerine-Sottas syndrome. However, there have been no reports to investigate the NEFL genes in Korean CMT patients. Therefore, we investigated to find the clinical characteristics in patients with the NEFL gene mutation. METHODS: We examined mutations of the NEFL gene in 125 Korean CMT families. Mutations were confirmed by the sequencing of both strands. Nerve conduction studies were carried out on CMT patients having each mutation. RESULTS: Three pathogenic mutations were found in 3 families, and 2 polymorphisms in 2 families. Two mutations (Leu334Pro, Pro22Arg) were determined too novel, and those were not detected in 105 healthy controls. A de novo missense mutation was found in a CMT family with the NEFL mutation. The frequency of the NEFL mutation was 2.4%, which was similar in Europeans, and lower than those found in Japanese. Pro22Arg and Glu397Lys mutations showed demyelinating neuropathy and Leu334pro mutation showed axonal neuropathy. CONCLUSIONS: We found NEFL mutations in patients with sporadic or dominantly inherited CMT. NEFL mutations should be considered in the evaluation of CMT or related neuropathies with various clinical features.
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Humans , Asian People , Axons , Charcot-Marie-Tooth Disease , Cytoplasm , Hereditary Sensory and Motor Neuropathy , Mutation, Missense , Neural Conduction , NeuronsABSTRACT
In the olfactory bulb, normal and transected olfactory axons are able to enter, regenerate, and reestablish lost synaptic contacts with their targets, throughout the lifetime of the organism. It was expected that studies of olfactory bulb ensheathing glia will provide important advances for the field of neural regeneration. Purpose of this study is to analyze morphologically the effects of olfactory bulb transplants into the cord after complete transection. Forty Sprague-Dawley rats were used in this study. Spinal cord of the rats were transected after laminectomy followed by insertion of chopped olfactory bulb tissues immediately and 3 weeks after the operation. In this study, transplants of olfactory bulb were successfully used to promote functional and structural recovery after complete spinal cord transection. The area of damaged spinal cord was greatly diminished after olfactory bulb transplantation. Nearly normal anterior horn cells were observed immediately distal to the transected region. Tyrosine hydroxylase immunoreactive descending fibers were observed in the distal region beyond transected area.
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Animals , Rats , Anterior Horn Cells , Axons , Immunohistochemistry , Laminectomy , Neuroglia , Olfactory Bulb , Rats, Sprague-Dawley , Regeneration , Spinal Cord Injuries , Spinal Cord , Tyrosine 3-MonooxygenaseABSTRACT
Objective To investigate the effects of transcranial magnetic stimulation(TMS)on the recovery of neurological function and the expression of neurofilament protein(NFP)-200 in rats with cerebral infarction.Methods The string-fastening method was used to establish focal ischemia-reperfusion models in 60 Sprague-Dawley rats and the rats were divided into the TMS group and the sham TMS group(composed of 30 rats each).Then each group were divided into the treated 1 d,3 d,7 d,14 d and 21 d subgroups(composed of 6 rats each).TMS group rats were administered with 2 sessions of TMS(200 pulses total)daily,keeping on corresponding days in different groups.The recovery of neurological function and the optical density of NFP-200 by immunohistochemistry in the infarct peripheral zone in two groups were detected after therapy.Results At the 14 d and 21 d after treatment,neurological deficit scores of TMS group(2.67?0.82,1.50?0.55) were significantly lower than those of the sham TMS group(3.67?0.52,3.17?0.75)(P
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Objective: To study the difference between the facial nerve injury by gun-shot and that by blast. Methods: 40 adult dogs were divided into 2 groups, with 20 dogs in each. In the blast group, the impact wave was simulated by detonator blast in the distance of 10 cm over the dog's masseter. At the same time,a steel ball was projected to the dog's face by a rifle to imitate segments in blast. In the gun-shot group, only the steel ball was used. Then the facial nerve was taken at the time of immediateness, 6 hours, 1 day, 3 days and 7 days following the injury for HE or immunocytochemical staining. And the facial nerve conduction velocity was examined 6 hours, 3 days and 7 days after injury.Results: The facial nerve edema was found in both groups just after injury. There was scattering hemorrhage and little hematoma below the spineurium in the gun-shot wound, as well as the diffusing bleeding and larger hematoma in blast wound. In the blast group, the severe edema could be found by light microscope in the whole extracranial facial nerve. Axonal fragments, infiltrating imflammatory cells and few neurofilament protein NF positive axons were observed. In the gun-shot group, only scattering damage was found within 2 cm from the wound track.Conclusion: The facial nerve injury by gun-shot bacomes less severe with the increase of distance from the wound track, but the blast injury is wider and more severe.
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We report a case of neuroblastoma with multiple skin metastases as a chief complaint in a 2-month-old girl. the skin lesions were rnultiple, pea-sized, bluish, nontender, moable subcutaneous nodules on abdomen, back and scalp. Histopathology showed small round or poly gonal tumor cells which have deeply stained, basophilic, hyperchromatic nuclei with some mitoses. Th.se tumor cells showed clumping tendency which is one of early menifestations of rosette formation. Immunohistochemically positive reaction was demonstrated by anti-NSE(neuron specific enolase) antilody but negative reaction by anti-NFP (neurofilament proteiin ) antibody. She has been succesfully treated with combined chemotherapy for 10 months without relapse.