Objective To research the protective effects of different allogeneic cells injected into denervated muscles on ventricornual motor neuron. Methods Thirty-six adult female SD rats, weighting 120-150 g, were individed into four groups randomly and each group had nine. Left ischiadic nerves of all the SD rats, which were cut down on germfree conditions,were operated by primary suture of epineurium. Different cells were injected into the triceps muscles of calf in each group after operation with once a week for 4 weeks:1 ml Schwann cells (1×106/ml) in group A, 1 ml mixed cells ofSchwann cells and myoblast cells (1∶1,1×106/ml) in group B, 1 ml extract from the mixed cells of Schwann cells, myoblast cells and endotheliocytes (1∶1∶1,1×106/ml)in group C,and 1 ml culture medium without FCS as control group(group D). The observation of enzymohistochemistry and C-Jun expression in the ventricornual motor neuron was made after three months of operation. Results After 3 months of operation, the expressions of C-Jun in groups A, B and C were superiorto that in group D; the number of neuron was more than that of group D. The expressions of C-Jun in the ventricornual motor neuron were as follows: 128.591±0.766 in group A, 116.729±0.778 in group B, 100.071±2.017 in group C and 144.648±2.083 in group D; showing statistically significant difference between groupsA, B, C and D(P＜0.01). Enzymohistochemistry showed the well outlined and wellstacked cell body of neuron in groups A, B and C, and illdefined boundary of cytoplasm and nucleus. There was statistically significant defference in enzyme activity of the ventricornual motor neuron between groups(P＜0.01). Conclusion All of the Schwann cells,mixed cells of Schwann cells with myoblast cells,and the extract from Schwann cells, myoblast cells and endotheliocytes can protect the ventricornual motor neuron. And the protectiveeffect of the extract from Schwann cells, myoblast cells and endotheliocytes is superior to that of Schwann cells and mixed cells.
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.
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.
OBJECTIVE: To study the effects of Schwann cell cytoplasmic derived neurotrophic proteins (SDNF) on the regeneration of peripheral nerve in vivo. METHODS: Ninety adult SD rats were chosen as the experimental model of degenerated muscle graft with vascular implantation bridging the 10 mm length of right sciatic nerve. They were divided randomly into three groups, 30 SD rats in each groups. 25 microliters of 26 ku SDNF (50 micrograms/ml, group A), 58 ku SDNF (50 micrograms/ml, group B) and normal saline(group C) were injected respectively into the proximal, middle and distal part of the degenerated muscle grafts at operation, 7 and 14 days postoperatively. The motorial function recovery assessment was carried out every 15 days with the sciatic nerve function index(SFI) after 15 days to 6 months of operation. Histological and electrophysiological examination of regenerating nerve were made at 1, 3 and 6 months postoperatively. RESULTS: There were significant statistic differences between the both of experimental groups(group A and B) and control group(group C) in the respects of the histological, electrophysiological examination and SFI(P lt; 0.01). CONCLUSION: The 26 ku SDNF and 58 ku SNDF can improve the regeneration of the injured peripheral nerve in vivo.
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.
ObjectiveTo investigate the effect of serum on the differentiation of neural stem cells.MethodsThe neural stem cells were isolated from the embryonic hippocampus tissues of Sprague Dawley rats at 14 day of pregnancy. After culturing and passaging, the 3rd generation cells were identified by immunocytochemical staining. Then, the cells were divided into 3 groups according to the concentrations of fetal bovine serum (FBS) used in the differentiation cell culture medium: 5% (group A), 1% (group B), 0 (group C), respectively. The other components of the culture media in 3 groups were the same. Cell viability was determined by using the Live/Dead cell staining at 8 days; the expressions of glial cell marker [glial fibrillary acidic protein (GFAP)] and neuronal marker (β-Ⅲ Tubulin) were determined and analyzed by immunocytochemical staining and real-time fluorescent PCR at 4 and 8 days of culture.ResultsBased on cell morphology and immunocytochemical staining, neural stem cells were identified. Cells were growing well with no death in all groups. With decreasing FBS concentration, the expression of GFAP was significantly decreased on both protein and mRNA level, whereas the expression of β-Ⅲ Tubulin was evidently increased. The staining of each group at 8 days was more obvious than that at 4 days. There were significant differences in mRNA expressions of GFAP and β-Ⅲ Tubulin at 4 and 8 days between groups (P<0.05).ConclusionSerum can promote the differentiation of neural stem cells into glial cells. At the same time, it inhibits the differentiation of neural stem cells into neurons, the lower the serum concentration, the smaller the effect.
Objective To evaluate the feasibil ity of direct anastomosis in the rat model of the brachial plexus extravertebral foramen nerve root division of C5-7. Methods Forty-eight SD rats (male or female) aging 4-6 months and weighing 250-300 g were selected to make the model of extravertebral foramen nerve root division of C5-7. The left C5-7 nerve roots, as the experimental sides, were separated to the brachial plexus nerve trunk and the transected roots were sutured to theproximal stump immediately after cutting off the brachial plexus extravertebral foramen nerve root division. The right C5-7nerve roots, as the control sides, received no operation. The general condition of rats after operation was observed. The gross observation, the histological observation and BDA nerve tracing technology were adopted to observe the wet weight of musculus biceps brachii, the cross section of biceps brachii and the spinal cord and distal nerve trunk at 3 weeks, 3 months and 6 months after operation. Results All rats survived well after operation. Claudication and unfold claw reflex were observed in the experimental sides, and the unfold claw reflex disappeared 3 months later. Comparatively, the control sides were normal. Nerve adhesion aggravated gradually and the neural stems were shriveled within 6 months after operation in the experimental sides. Comparatively, the control sides were normal. The wet weight of biceps brachii in the experimental sides and the control sides at 3 weeks, 3 months and 6 months after operation was (0.28 ± 0.12), (1.37 ± 0.33), (0.58 ± 0.10), (1.36 ± 0.35), (1.39 ± 0.31), (1.37 ± 0.38) g, respectively, indicating significant differences between two sides at 3 weeks and 3 months (P lt; 0.05), but no significant difference at 6 months (P gt; 0.05). The modified Marsland and the LFB staining of spinal cord and superior trunk of brachial plexus showed that the number of neurons, cell nuclear and Nissl body decreased and cell bodies changed from swell ing to shrinkage, dyeing nerve fibers increased, neural axone was thin and myel in sheath was sl ightly stained at each time point in experimental side. The number of motor neurons in cornu anterius medullae spinal is in the experimental side was 84.5% ± 3.2%, 74.4% ± 4.5%, 73.7% ± 3.8% of that in the control side at each time point, respectively. HE staining of biceps brachii detected thatthe muscle denaturation was very serious at 3 months after operation and then recovered. Neural tracing used BDA showed that the closer to the proximal of nerve trunk, the more obviously stained it was of myel in sheath and the more massive of axon at 6 months after operation. And there was almost no myel in and axon stained in musculocutaneous nerve. Conclusion In the rat model of brachial plexus extravertebral foramen nerve root division, the motoneuron in cornu anterius medullae spinal is necrosis rate reaches 20%-30%, and most of the residual neurons are pathologic. The regenerated fibers manifest as insufficient dynamic power and incomplete development, making no sense for the recovery of end organ function. Therefore, the exact mechanism of the recovery of biceps brachial muscle demands further study.
Objective To evaluate the diagnostic value of serum neuron specific enolase (NSE) in patients with small cell lung cancer. Methods We searched MEDLINE, EMBASE, The Cochrane Library and other databases (1966 to March 2007) to collect studies which evaluated the diagnostic value of NSE in patients with small cell lung cancer. The heterogeneity of included studies was tested by the Cochrane Collaboration’s software RevMan 4.2. The Summary Receiver Operating Characteristic (SROC) curve and meta-analyses were performed by MetaDisc. Results Fifteen studies involving 4221 patients (672 SCLC and 3549 NSCLC patients, all diagnosed by the gold standard) were included. Meta-analyses showed that the heterogeneity among studies was high (P=0.000 2, I2=66.1%), the pooled sensitivity was 0.67 (95%CI 0.64 to 0.71) and the pooled specificity was 0.91 (95%CI 0.90 to 0.92). Subgroup analyses indicated that 4 of the studies which used the reagent supplied by The Academy of Military Medical Sciences (P=0.33, I2=13.4%, AUC= 0.9672, SE=0.0393) and another 4 which used the reagent supplied by Roche (P=0.23, I2=29.9%, AUC=0.8311, SE=0.0836) had no heterogeneity. Conclusion NSE could be regarded as one of the reference tests in patients with small cell lung cancer, but more high quality trials are required.
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.