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Objective To explore the effects of moxibustion and electroacupuncture at points on the Du meridian on the expression of calcitonin gene-related peptide ( CGRP ) in rats with spinal cord injury ( SCI ).Methods Twenty Sprague-Dawley rats were divided into a normal control group, a SCI group, a moxibustion group and an electroacupuncture group. Rats in the latter three groups were subjected to spinal cord transection under a microscope. Moxibustion and electroacupuncture were administered to rats in those groups at points on the Du meridian daily for 3 days beginning on the 7th day after the operation, Immunofluorescence staining was used to observe any changes in the CGRP-positive area of the spinal cord's dorsal horn. Western blotting was used to detect changes in the content of CGRP in the spine. Results The CGRP-positive stained area of spinal cord's dorsal horn was significantly larger in the moxibustion and electroacupuncture groups than in the SCI group. CGRP content was also significantly higher. Any differences between the moxibustion and electroacupuncture groups were not significant. The CGRP-positive area and its content in the normal control group were not significantly different from those in the moxibustion and electroacupuncture groups. Conclusions Either moxibustion or electroacupuncture at points on the Du meridian can promote the expression of CGRP in rats after SCI. There is no significant difference between their effects.
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OBJECTIVE: To explore the effects of Rhodiola rosea on the body weight and the intake of sucrose and water in depressive rats induced by chronic mild stress.dz METHODS: A total of 70 male SD rats were divided into seven groups, including normal control group (treated with 0.5% sodium carboxymethycellulose), untreated group, negative control group (treated with 0.5% sodium carboxymethycellulose), positive control group (treated with fluoxetine), low-, medium- and high-dose Rhodiola rosea group (treated with 1.5, 3, 6 g/kg Rhodiola rosea respectively). Except for rats in normal control group, the other sixty rats endured chronic stress for 4 weeks to establish the depression model. After that, rats were administered Rhodiola rosea for 3 weeks. During the whole experiment, the body weight, and sucrose intake, tap water intake of all rats were examined once a week. RESULTS: After the termination of the stress regime, compared with the normal control group, the body weight and 1% sucrose intake in depressive rats were decreased. After 3-week Rhodiola rosea treatment, the body weight and 1% sucrose intake increased in rats of the low-dose Rhodiola rosea group and recovered to the level of the normal control group. CONCLUSION: Low-dose Rhodiola rosea can increase the body weight and sucrose intake of depressive rats, making them recover to normal status.
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OBJECTIVE: To explore the effects of Valerian on the level of 5-hydroxytryptamine (5-HT), cell proliferation and neuron number in cerebral hippocampus of rats with depression induced by chronic mild stress. METHODS: Seventy rats were divided into 7 groups: normal control, untreated, negative control, positive control, and low-, medium- and high-dose Valerian-treated groups. There were 10 rats in each group. Except for the normal control group, depression was induced in rats by chronic mild stress. The depressive rats in the other six groups were intragastrically administered with sodium carboxymethycellulose, fluoxetine, and low, medium and high-dose Valerian, respectively for 3 weeks. After the treatment, the proliferating cells in the hippocampus were labeled by injecting bromodeoxyuridine (BrdU) in 7 groups. The content of 5-hydroxytryptamine (5-HT) in the hippocampus was detected by high-performance liquid chromatography (HPLC), and the number of hippocampal neurons was counted by morphometry. RESULTS: Compared with the normal control group, the levels of 5-HT in the hippocampus in the low- and medium-dose Valerian-treated groups were increased and recovered to normal level. After the administration of low-dose Valerian for 3 weeks, the number of BrdU positive cells and neurons in the hippocampus of the depressive rats were recovered to the normal status. CONCLUSION: Minidose Valerian may promote the level of 5-HT and cell proliferation in the hippocampus of the depressive rats, and may play a role in saving injured neurons of the hippocampus.
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OBJECTIVE: To investigate the effects of Ganoderma spores on mitochondria-related molecular substances in hippocampus of young rats birthed by rats with gestational hypertension. METHODS: Nitric oxide synthase (NOS) inhibitor Nw-nitro-L-arginine methylester (L-NAME) was intraperitoneally injected into pregnant rats to induce gestational hypertension, and Ganoderma spores were administered orally. The effects of Ganoderma spores on levels of mitochondria-related molecular substances in hippocampus of young rats birthed by the rats with gestational hypertension were evaluated with immunoradiometric assay of cAMP, RT-PCR analysis of related genes, and detection of enzyme activity. RESULTS: In hippocampus of the new-born rats birthed by rats with gestational hypertension, the cAMP level, mitochondrial DNA (mtDNA) level and adenosine triphosphatase (ATPase) activity were decreased, and the expression level of peroxisome proliferator activated receptor gamma coactivator 1 alpha (pgc1 alpha) was unchanged compared to the normal control group. The cAMP level, mtDNA level, ATPase activity and pgc1 alpha expression level in hippocampus of 30-day post-natal rats were lower than those of the rats in normal control group. After oral administration of Ganoderma spores, the cAMP and mtDNA levels in hippocampus of the new-born rats and 30-day post-natal rats recovered almost to the levels of normal control rats, and the ATPase activity and pgc1 alpha expression level were also increased significantly. CONCLUSION: Ganoderma spores may regulate the levels of mitochondria-related molecular substances in hippocampus of young rats birthed by rats with gestational hypertension.
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OBJECTIVE: To explore whether pre-administration of Ganoderma lucidum spore (GASP) can reduce incidence of neural tube defects (NTDs) induced by all-trans retinoic acid (ATRA) in pregnant mice. METHODS: Twenty pregnant mice were randomly divided into four groups: normal control group, solvent-treated group, ATRA-induced group, and GASP-treated plus ATRA-induced group. GASP solution, which was prepared with solvent (sodium carboxymethyl cellulose), was fed to the pregnant mice in the GASP-treated plus ATRA-induced group twice a day from embryo (E) 0 d to E10.5 d. The same dose of solvent was given to the pregnant mice in the solvent-treated group. At E7.75 d, ATRA (50 mg/kg) was given to the pregnant mice in both ATRA-induced group and GASP-treated plus ATRA-induced group for single time. Embryos were sampled from pregnant mice at E10.5 d. Then the incidence rate of NTDs in mouse embryo was calculated and the crown-rump length of mouse embryo was measured. The positive rate of nestin expression and the distribution of cell cycle of embryonic neural tube neuroepithelial cells were detected by histochemical staining technique and flow cytometry respectively. Reverse transcription-polymerase chain reaction method was used to detect the gene expressions of cyclin-dependent protein kinase 2 (Cdk2) and Cdk4 mRNAs. RESULTS: The incidence rates of NTDs in mouse embryos in the ATRA-induced group and the GASP-treated plus ATRA-induced group were 79.41% and 21.67% respectively, while the crown-rump length of mouse embryos in these two groups were (3.62+/-1.27) mm and (5.84+/-0.92) mm respectively. The positive rate of nestin expression in embryonic neural tube neuroepithelial cells of mouse embryo at E10.5 d in the ATRA-induced group was 32.44%, while that in the GASP-treated plus ATRA-induced group was 77.65%. The cell cycle of embryonic neural tube neuroepithelial cells was obviously arrested at G(0)/G(1) phase in the ATRA-induced group as compared with that in the GASP-treated plus ATRA-induced group. The Cdk4 mRNA was transcripted at a high level in embryonic neural tube in the GASP-treated plus ATRA-induced group, but the Cdk2 mRNA was not detected in this group. CONCLUSION: Pre-administration of GASP can reduce the incidence of NTDs induced by ATRA in pregnant mice.
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OBJECTIVE: To detect some proteins associated with the effect of ganoderma lucidium spores (GASP) on promoting the survival and axon regeneration of injured spinal motor neurons in rats. METHODS: The rats were divided into normal control group, untreated group and GASP-treated group, and the rats in the last two groups received ventral root avulsion. GASP preparation was fed to the rats in the GASP-treated group for 14 days. The gray matter tissues of the lumbar spinal were sampled from rats in each group after 14 days following ventral root avulsion, and the extracted proteins from these tissues were detected by using 2-dimensional electrophoresis. Matrix assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS) was utilized to identify the differentially expressed proteins among these three groups. RESULTS: There were six kinds of proteins differentially expressed among the three groups, which were collapsin response mediator protein 2 (CRMP-2), F-actin capping protein beta subunit (FCP-beta), isocitrate dehydrogenase [NAD] subunit beta (IDH-beta), ATPase, glutamate oxaloacetate transaminase-1 (GOT1) and M2 pyruvate kinase (M2-PK). The expression levels of CRMP-2, IDH-beta, ATPase and GOT1 were higher in the GASP-treated group than those in the untreated group, while the expression levels of FCP-beta and M2-PK were lower than those in the untreated group. CONCLUSION: GASP maybe promotes the survival and axon regeneration of injured spinal motor neurons in rats by virtue of up- or down-regulating the expression levels of the proteins mentioned above.
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Objective To explore a fast,simplified and economical method for Schwann cells(SCs) cultured and purified in vitro.In this way,a stable and reliable cell sources can be provided in order to study SCs transplanted in vivo. Methods SCs cultures were prepared from the sciatic and brachial nerves of 3 to 5-day-old SD neonatal rats with double enzyme digestion method to acquire dissociated cells and one enzyme digestion method to acquire incomplete digested tissues. Results With the same number of neonatal rats,one enzyme digestion(hemi-explant)method acquired at least two times more SCs than double enzyme digestion(single cell) method did and saved at least 40 minutes.96% of S-100 positive SCs cultured were shown by immunohistochemical staining.Conclusion When the one enzyme digestion method(hemi-explant) is used to culture SCs,sufficient SCs with qualified purity can be acquired in a short time.
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Objective To detect the expression and the biological activity of a recombinant adenovirus expression vector carrying human neurotrophin-3 (NT-3) receptor TrkC gene (Adeno-TrkC) in neural stem cells. Methods The expression of TrkC mRNA in 293 cells infected with Adeno-TrkC was detected by RT-PCR, and the expression of TrkC protein in neural stem cells infected by Adeno-TrkC was detected with immunocytochemistry and Western blotting. The effects of human neurotrophin_3 (NT-3) on the neural stem cells infected by Adeno-TrkC differentiating into neuron-like cells and astrocyte-like cells in vitro were observed. Results The transcription of TrkC mRNA and the expression of TrkC protein was detected in 293 cells and neural stem cells infected by Adeno-TrkC. This kind of TrkC was able to make more neural stem cells differenting into neuron-like cells in vitro with its ligand NT-3 and the percentage of neuron-like cell’s differentiation was 55
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Objective To explore the effects of morphine on the terminal of primary afferent fiber distributing in spinal lamina Ⅱ after the sciatic nerve crush. Methods The positive reactive areas of fluoride-resistant acid phosphatase(FRAP) at spinal lamina Ⅱ were measured by the FRAP histochemistry and microcomputer image analysis techniques, after sciatic nerve was injured 15 days and 30 days both in morphine and control groups of rats. Results The positive reactive areas of FRAP at spinal lamina Ⅱ were depleted on different degree in two groups of rat after sciatic nerve was injured. The positive reactive areas of FRAP were greater 40% on injured sciatic nerve in 30 days than in 15 days in control group. In morphine group, the positive reactive areas of FRAP were larger 22% on injured sciatic nerve in 30 days than in 15 days; simulaneously also were bigger 19% than on injured sciatic nerve 30 days of control group.Conclusion The positive reactive areas of FRAP at spinal lamina Ⅱ show recovering enlargement as the surviving time lengthens in both groups of rats injured sciatic nerve.Morphine may enhance the positive reactive area of FRAP at spinal lamina Ⅱ in rat of sciatic nerve crush.
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Objective To explore the effects of neurotrophin-3 genetically modified Schwann cells(NT-3-SCs) and neural stem cells(NSCs) combinative transplantation to promote the neurons'survival and axonal regeneration of the spinal cord transected rat. Methods After the transected spinal cord injury(SCI) model was established in SD rats,NT-3-SCs or LacZ report gene modified Scwhann cells(LacZ-SCs) were combinative transplanted with NSCs into the transected site.60 days later,fluorogold(FG) was injected into the caudal spinal cord fo the transected site.7days after FG injected,the sacrifice,cryosection and morphology serious studies were performed to observe the FG labeled neurons in the rostral spinal cord(RSC) of transected site,red nuclei(RN) and the sensorimotor cortex(SMC);The survival neurons in the L_1 Clark's nuclei(CN),RN and SMC were couut,and regenerated axons within or near the transected spinal cord observed. Results From more to less,the order of groups of the survival neurons in the CN,RN and SMC was NT-3-SCS & NSCs group,LacZ-SCs & NSCs group,experimental control group.In the NT-3-SCs & NSCs group and LacZ-SCs & NSCs group,there were 5-HT,CGRP and SP positive axons within or near the transected spinal cord.And some FGlabeled cells were found in RSC,RN and SMC. Conclusion Combinative grafting NSCs and NT-3-SCs could promote the neurons'survival and axonal regeneration of the spinal cord injured rat.
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AIM: To explore the effects of neurotrophin-3 (NT-3)-genetically modified Schwann cells (NT-3-SCs) on differentiation of neural stem cells (NSCs) into the neuron-like cells. METHODS: The NSCs were co-cultured with NT-3-SCs. Report gene LacZ genetically modified Schwann cells (LacZ-SCs) and normal SCs respectively in vitro. 7 d later, the differentiation of NSCs was studied by immunohistochemistry, and the percentage of neuron-like cells was calculated. RESULTS: NSCs differentiated to the GFAP-positive cells (glial-like cells) and NF-positive cells (neuron-like cells) in vitro. Compared to the normal SCs, NT-3-SCs more efficiently promoted NSCs to differentiate into the neuron-like cells. The effect of LacZ-SCs was as the same to the normal SCs. CONCLUSION: NT-3-SCs promote NSCs to differentiate into the neuron-like cells. [
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Nerve growth factor (NGF) can promote the outgrowth of neurites of the target ganglia. In order to further explore the relationship between this effect and the synthesis of RNA and DNA in the neurons, an autoradiography of 3~H-uridine and 3~H-thymidine was used. Superior cervical ganglia (SCG) from newborn rats were cultivated by Maximow's double coverslip method. All cultures were divided to one group of cultures a crude preparation of NGF was added to the medium and another group without NGF served as control. Before tissue culture was stopped, the. covership cultures were transferred to thelabeling-medium and incubated, and then they were fixed, and cut into serial sections and subjected to autoradiographie processes. The results show that the percentage and the level of grains of neurons labeled by 3~H-uridine in the NGF group are higher than that of control. Moreover, before the growth rate of neurites reaches a peak, the level of grains of neurons labeled by 3~H-uridine in the NGF group is obviously increased. The evidence suggests that NGF can promote the synthesis of RNA in neurons of SCG, which has a direct bearing on the quick outgrowth of neurites. In the experiments with 3~H-thymidine incorporation, that the NGF may promote the synthesis of DNA in some neurons of the third day SCG in vitro was also observed.
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Objective To explore the effects of DU meridian electroacupuncture on the survival,differentiation and immigration of neural stem cells(NSCs) transplanted in the injured site of rat spinal cord transection. Methods Twenty adult SD rats were divided into NSCs transplanted 14d group(NSCsl4d group),DU meridian electroacupuncture plus NSCs transplanted 14d group(EA+NSCs14d group),NSCs transplanted 30d group(NSCs30d group) and DU meridian electroacupuncture plus NSCs transplanted 30d group(EA+NSCs30d group).T10 spinal cord segments of all animals were completely transected.The rats of EA+NSCs14d group and EA+NSCs30d group were treated with DU electroacupuncture at 5 days after operation.The sacrifice times of the rats were respectively at 14 days and 30 days after operation.The spinal cords were taken for observing the survival,differentiation and immigration of neural stem cells transplanted in the injured site of rat spinal cord transection. Results 1.The surviving number of transplanted NSCs in EA+NSCs14d group or EA+NSCs30d group was more than that of NSCsl4d group or NSCs30d group,and the surviving number of transplanted NSCs in EA+NSCs30d group and NSCs30d group was less than that in EA+NSCs14d group and NSCs14d group.2.Some transplanted NSCs at injured site of transected completely spinal cord and neighboring tissue showed microtubule association protein 2(MAP2) positive staining in EA+NSCs30d group or NSCs30d group.3.Many transplanted NSCs at injured site of transected completely spinal cord and neighboring tissue could be observed to show glial fibrillary acidic protein(GFAP) positive staining in EA+NSCs 30d group or NSCs 30d group.4.The immigrating distance of transplanted NSCs toward caudal tissue of injured site of spinal cord was longer in EA+NSCs 14d group and EA+NSCs 30d group than that in NSCs 14d group and NSCs 30d group.Conclusion DU meridian electroacupuncture may promote the survival of neural stem cells transplanted in injured site of rat spinal cord transected completely,and these cells can differentiate into MAP2 or GFAP possive cells.DU meridian electoacupuncture may affect the immigrating direction of neural stem cells transplanted at injured site of spinal cord toward host spinal cord tissue.
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Objective To investigate whether the skin-derived neural stem cells inducted by the culture could survive,differentiate and migrate in the lesioned site of rat spinal cord.Methods The skin of new born rat trans-gene of green fluorescence protein(GFP) was applied to be dissociated into cells,cultured and inducted to proliferate in vitro.Skin-derived neural stem cells were identified by immunocytochemistry staining.Then,skin-derived neural stem cells inducted by the culture were transplanted into the lesioned site of rat spinal cord hemisection.Thirty days and sixty days after the operation,the survival,migration and differentiation of transplanted cells were observed by immunocytochemistry staining. Results Ten days at cultured skin-dissociated cells,many cell-spheres had been formed by the proliferation of suspensive growing cells.These cell-spheres showed nestin positive staining of immunocytochemistry.It suggestes that the cell-spheres are neurospheres.In vivo,many transplanted skin-derived neural stem cells with GFP were observed in the lesioned area of spinal cord.Some transplanted cells migrated into host spinal cord tissue far away from the lesioned area.Some transplanted surviving cells showed nestin,MAP2 and GFAP positive staining of immunocytochemistry separately.Conclusion Skin-derived neural stem cells inducted by the culture may survive,migrate and differentiate into neuron-like cells and astrocyte-like cells in injured spinal cord of rat.
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The superior cervical ganglia (SCG) were dissected from neonatal rats.Dissoci-ated cell cultures were grown in Eagle's MEM supplemented with 20% calf serumwhich contain nerve growth factor (NGF).The cultures were divided into two groups:laser irradiation group and control.The former was exposed to lower dose of Helium-Neon laser (power:4 milliwatt)5 minutes every two hours.Two groups cultured for 20,22,24 and 28 hoursrespectively.Then the length of neurites of neurons were measured.At 22 hours,RNA and DNA synthesis of neurons labeled by ~3H-uridine and ~3H-thymidine wereobserved.The results showed that lower dose of Helium-Neon laser irradiation not onlypromotes growth of neurite but also enhances RNA synthesis of neurons in culture.However DNA synthesis in neurons does not promote in this experiment.
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Objective To explore whether ganoderma spores could help the neural epithelial cell of embryonic neural tube stagnated by retinoic acid in G-0/G-1 phase reenter the cell cycle,keep on proliferating and differentiating,and could reduce the occurrence of neural tube defects(NTDs). Methods When E7.75d,mice of the control group and the ganoderma spores group were given intragastrically onetime retinoic acid all-trans.Then the ganoderma spores solution was given intragastrically to the ganoderma spores group.At E10.5d,The embryos of the two groups were taken out and the ratio of NTDs was counted respectively Immunofluorescence histochemistry and flow cytometry were applied to examine the expression of nestin,and DNA quantity staining and RT-PCR to detect the mRNA transcriptions of Cdk2 and Cdk4. Results The radio of NTDs in the ganoderma spores group was lower than that in the control group remarkably,but the level of nestin was higher.With the neural tube cell cycles compared the cell radio of G-0/G-1 phase in the control group was higher than that in the ganoderma spores group,while the ratio in the S phase in the control group,it was lower than that of the normal embryos in the ganoderma spores group.The neural epithelial cell of embryonic neural tube in the ganoderma spores group could transcript Cdk4 mRNA normally,with a low transcription rate in the control group.Conclusion Ganoderma spores can reduce the occurrence of embryonic NTDs induced by retinoic acid in pregnant mice.
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Objective The extract of spinal cord tissue in morphine spared root rat was isolated and purified to look for some neurotrophic active substances. Methods Neurotrophic active substances were isolated and analysed by Sephacryl S 200 HR gel chromatography、high performance liquid chromatography(HPLC)and tissue culture,ect. Results The extract of spinal cord tissue of spared root rat could enhance the neurite growth of chick embryonal dorsal root ganglion(DRG) in vitro.There was also same effect in the extract of spinal cord tissue of morphine\|treated rat.But there was no significant difference in the extract of spinal cord tissue promoting the neurite growth between spared root rat and morphine treated rat.The extract of spinal cord tissue of morphine spared root rat had evident neurotrophic active role.The peak Ⅱ eluate and peak Ⅳ eluate obtained from the spinal cord tissue extract of morphine spared root rat through Sephacryl S 200 HR gel chromatography could promote the neurite growth of DRG.According to the analysis of SDS PAGE,the peak Ⅱ eluate showed one main protein zone with a molecular weight of 65kD and the protein composition of peak Ⅳ eluate was more complicated.The peak Ⅳ eluate of gel chromatography was then furhter isolated by HPLC.It was observed.That the peak A eluate of HPLC could promote the neurite growth of DRG. It was showed by SDS PAGE that the peak A eluate presented two main protein zones with molecular weight of 30kD and 18 kD.Conclusion\ The molecular weight of neurotrophic active substances,which were isolated from the extract of spinal cord tissue of morphine spared root rat,might be 65kD,30kD and 18kD proteins.\;[
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Objective To investigate the intervening effects of Ganoderma spore on the decrease of cell proliferation and neuronal survival in the hippocampus of fetal and postnatal rats induced with gestational hypertension. Methods Fourty SD pregnant rats were divided into four groups including the control group,Nw-nitro-L-arginine methylester(L-NAME)+distilled water(DW) group,L-NAME+L-Arginine group and L-NAME+Ganoderma spore(GS) group.The hippocampal tissue of the brain was detected by immunohistochemistry,Western blotting,RT-PCR,flow cytometry and electron microscopy. Results After the application of L-NAME,the expressions of hypoxia inducing factor-1?(HIF-1?) and vascular endothelial growth factor(VEGF) were increased at the hippocampus of E21 brain and continued up to P30 brain.The microvessel density of E21 hippocampus was increased and the structural abnormalities of blood capillary at P30 hippocampus were showed.The cell proliferation was decreased at E21 hippocampus and so was the neuronal number at P30 hippocampus.With the administration of Ganoderma spore,HIF-1? and VEGF were down-regulated at E21 hippocampus and were not detected at P30 hippocampus.The microvessel density of E21 hippocampus reached a normal level and the blood capillary ultrastructure of P30 hippocampus was restored.The cell proliferation of E21 hippocampus and neuronal number of P30 hippocampus were recuperatively increased.Conclusion Ganoderma spore may prevent the decrease of cell proliferation and neuronal survival in the hippocampus of fetal and postnatal rats induced with gestational hypertension.
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10kD fraction from operated side of two groups of animal was tending towards increasing, especially the approximate 67kD protein content from operated side of morphine group of animal.\;
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Objective Whether Schwann cells could promote the survival and differentiation of neural stem cells was explored in vitro. Methods Neural stem cells were dissociated and cloned from the hippocampal tissue of newborn rats.Schwann cells were also dissociated and purified simultaneously from the sciatic nerves and brachial plexus nerves of newborn rats.Then Schwann cells and neural stem cells were Co cultured.The expression of nestin of the neural stem cells and the expression of neurofilament(NF) or glial fibrillary acidic protein(GFAP)of the differentiated cells were detected by immunohistochemistry.The morphological changes of neural stem cells were examined with scanning electronic microscope. Results Compared with control group,the number of surviving neural stem cells and differentiated neuron like cells was significantly increased in Co cultured (Schwann cells add neural stem cells)groups.The primary processes of neuron like cells in Co cultured groups were obviously longer than that in control groups.The irregular convex or concave of the body of neural stem cells became plain and smooth in Co cultured group.Co cultured Schwann cells and neural stem cells have grown to touch together in following manner:1.Body touch body;2.Body touch process;3.Process touch process.Conclusion\ Schwann cells can promote the survival and differentiation of co cultured neural stem cells in vitro.