ObjectiveTo evaluate the effect of using Schwann-like cells derived from human umbilical cord blood mesenchymal stem cells (hUCBMSCs) as the seed cells to repair large sciatic nerve defect in rats so as to provide the experimental evidence for clinical application of hUCBMSCs. MethodsFourty-five male Sprague Dawley (SD) rats in SPF grade, weighing 200-250 g, were selected. The hUCBMSCs were harvested and cultured from umbilical cord blood using lymphocyte separating and high molecular weight hydroxyethyl starch, and then was identified. The hUCBMSCs of 3rd generation were induced to Schwann-like cells, and then was identified by chemical derivatization combined with cytokine. The acellular nerve basal membrane conduit was prepared as scaffold material by the sciatic nerve of SD rats through repeated freezing, thawing, and washing. The tissue engineered nerve was prepared after 7 days of culturing Schwann-like cells (1×107 cells/mL) on the acellular nerve basal membrane conduit using the multi-point injection. The 15 mm sciatic nerve defect model was established in 30 male SD rats, which were randomly divided into 3 groups (10 rats each group). Defect was repaired with tissue engineered nerve in group A, with acellular nerve basal membrane conduit in group B, and with autologous sciatic nerve in group C. The nerve repair was evaluated through general observation, sciatic function index (SFI), nerve electrophysiology, weight of gastrocnemius muscle, and Masson staining after operation. ResultsThe hUCBMSCs showed higher expression of surface markers of mesenchymal stem cells, and Schwann-like cells showed positive expression of glia cell specific markers such as S100b, glial fibrillary acidic protein, and P75. At 8 weeks after operation, the acellular nerve basal membrane conduit had no necrosis and liquefaction, with mild adhesion, soft texture, and good continuity at nerve anastomosis site in group A; group B had similar appearance to group A; adhesion of group C was milder than that of groups A and B, with smooth anastomotic stoma and no enlargement, and the color was similar to that of normal nerve. SFI were gradually decreased, group C was significantly greater than groups A and B, group A was significantly greater than group B (P<0.05). The compound action potential could be detected in anastomotic site of 3 groups, group C was significantly greater than groups A and B, and group A was significantly greater than group B in amplitude and conduction velocity (P<0.05). Atrophy was observed in the gastrocnemius of 3 groups; wet weight's recovery rate of the gastrocnemius of group C was significantly greater than that of groups A and B, and group A was significantly greater than group B (P<0.05). Masson staining showed that large nerve fibers regeneration was found in group A, which had dense and neat arrangement with similar fiber diameter. The density and diameter of medullated fibers, thickness of myelinated axon, and axon diameter of group C were significantly greater than those of groups A and B, and group A was significantly greater than group B (P<0.05). ConclusionTissue engineered nerves from hUCBMSCs-derived Schwann-like cells can effectively repair large defects of the sciatic nerve. hUCBMSCs-derived Schwann-like cells can be used as a source of seed cells in nerve tissue engineering.
Objective To systematically evaluate effectiveness and safety of nerve block therapy for neck pain. Methods Databases including CENTRAL, PubMed, Ovid, ISI, EBSCO, CBM and CNKI were searched from the date of their establishment to November 2011, and relevant references were also retrieved manually to collect both domestic and abroad randomized controlled trials (RCTs) about nerve block therapy for neck pain. According to the inclusion and exclusion criteria, two researchers independently screened literature, extracted data, and assessed the quality of the included studies. Then the meta-analysis was conducted using RevMan 5.0 software. Results A total of 10 studies involving 625 participants were included. The results of qualitative analysis showed that: a) The short-term effectiveness of the nerve block therapy group was markedly superior to the placebo group, the cognitive therapy group and the transcutaneous electric nerve stimulation (TENS) group; and b) The short-term effectiveness of the combined nerve block therapy was markedly superior to the single nerve block therapy. The results of meta-analysis demonstrated that: a) There was no significant difference between the greater occipital nerve (GON) block group and the C2/3 nerve block group in neither short-term (SMD=−0.13, 95%CI −0.58 to 0.32, P=0.58) nor medium-term effectiveness (SMD=−0.01, 95%CI −0.46 to 0.44, P=0.98); and b): There was no significant difference between the injection with steroids group and the injection without steroids group in both short-term (SMD=0.16, 95%CI −0.13 to 0.44, P=0.28) and long-term effectiveness (SMD=0.27, 95%CI −0.02 to 0.55, P=0.07). Conclusion Current evidence shows nerve block therapy for neck pain is safe and especially good in short-term effectiveness. The combined nerve block therapy is probably more effective, but the effectiveness is not obviously improved by injection with or without steroids, and by different block methods. Due to the limitation of quality, quantity and total sample size of the included studies, this conclusion still needs to be proved by conducting more high quality and large scale studies.
Objective To make individualized evidence-based treatment for patients with diabetic peripheral neuropathy. Methods Based on the clinical questions we raised, evidence was collected and critically assessed. Patients’ preferences was also taken into consideration in the decision-making treatment. Results 157 studies were retrieved and finally 15 randomized controlled trials, 14 systematic reviews and meta-analyses, and 1 clinical guidelines were considered eligible. The evidence indicated that the first step in management of patients with diabetic peripheral neuropathy should aim for stable and optimal glycemic control; there was no statistically significant difference between aldose reductase inhibitors and placebo in the treatment of diabetic polyneuropathy, the same to nerve growth factor; alpha-lipoic acid is superior to placebo in reducing symptoms of diabetic peripheral neuropathy; 5-hydroxytryptamine and norepinephrine uptake inhibitor, tricyclic antidepressants and anticonvulsants might alleviate the pain in patients with diabetic peripheral neuropathy; vitamin B and capsaicin cream are is effective and safe in the management of diabetic peripheral neuropathic pain. The individualized treatment plans were developed based on the available evidence. After 3 month of treatment, the blood sugar returned to normal and symptoms were alleviated. Conclusion The treatment efficacy in diabetic peripheral neuropathy has been improved by determining an individulized treatment plan according to evidence-based methods.
ObjectiveTo explore the mechanisms of perineural invasion (PNI) in pancreatic cancer so as to find a new treatment for pancreatic cancer. MethodsThe literatures on PNI, neurotropism, nerve-tumor microenvironment and nerve growth factor in pancreatic cancer were reviewed and the mechanisms of PNI were summarized. ResultsThe rich innervation of pancreatic tissue itself and the minute slits within perineural structure were the anatomic basis of PNI. Tumor cells expressed neural antigens were the pathological basis of PNI. Tumor-nerve microenvironment and nerve growth factor family and themselves receptors might play an important molecular role in PNI. However, tumor cells expressed neural antigens were not only closely related to the PNI, but also the interaction between tumor cells and nerves played an important role in PNI. ConclusionsThe detailed mechanisms of PNI are extremely complex and controversial up to today. However, it is possible to search a new therapeutic target in pancreatic cancer according to the mechanisms of PNI.
Objective To investigate the effects and significance of nerve growth factor (NGF) and its high affinity receptor of tyrosine kinase A (TrkA) expressions on proliferative connective tissue of bile duct in rats after bile duct ligation (BDL). Methods Forty-six female Sprague-Dawley rats were randomly divided into two groups: control group ( n =6) and BDL group ( n =40). The model of obstructive jaundice in rat was made by bile duct ligation, then duodenohepatic ligament was taken and treated with anti-NGF and anti-TrkA receptor antibody. Expressions of NGF and TrkA receptor in connective tissue of bile duct were investigated by immunohistochemistry, blood specimens were collected from left ventricle to detect serum total bilirubin (TB) and alanine aminotransferase (ALT). Results After BDL, TB level obviously elevated in the third day, and continued until the fourteenth day, then descended. By day 21 and 28, it returned to normal level. Compared with normal bile duct, due to bile stasis, an increased thickness of the bile duct wall was observed by microscope which correlated with the proliferation and differentiation of connective tissue cell. NGF and TrkA were expressed by the cell membrane and the cytoplasm of connective tissue cell and inflammatory infiltration cell after BDL. The trend between their expressions and bilirubin levels was similar. Conclusion NGF and its receptor TrkA regulate the proliferate and differentiation of connective cell in bile duct. They may play a key role in the formation of bile duct scar, which seems to be hardly reversed by relief of bile stasis in a short time.
【Abstract】Objective To evaluate the distribution of nerve growth factor receptor( P75 NGFR) in congenital choledochal cyst(CCC) and its clinical implication. Methods Specimens from 18 children with CCC and normal choledochal specimens from 9 controls were immuno-stained with P75 NGFR antibody. Results Extensive P75 NGFR staining was found in the nerve fibres of normal comnon bile duct，bly staining of ganglion cells were observed on the normal specimens. There was very little immunoreactive fibre in the CCC. Conclusion The abnormal distribution of P75 NGFR in the aganglionic choledochal suggests that abnormal P75 NGFR is related to the occurrance of the CCC.
Objective Using nerve growth factor ( NGF) and anti-NGF microspheres injected directly into the asthmatic rat adrenal gland, to explore the possible role of anti-NGF microsphere treatment in asthma.Methods 32 male SD rats were randomly divided into a normal control group, an asthma group, a NGF microspheres group, and an anti-NGF microspheres group. The behavior of rats, lung function testing, light microscopy of lung biopsy, electron microscopy of adrenal medulla cell ultrastructure changes, NGF and phenylethanolamine N-methyltransferase ( PNMT) expressions in the adrenal gland were assayed by immunohistochemistry method, and serum NGF, cortisol, corticosterone, epinephrine and norepinephrine concentrations were detected by ELISA. Results Behavior in the asthma rats showed varying degrees of sneezing, runny nose, wheezing, scratching the head and face, irritability holes, incontinence, increased aggression and other acts, while in the anti-NGF rats showed relatively slighter symptoms. The rats in the asthma, anti-NGF and NGF groups showed significant airway hyperresponsiveness, while RL value reduced and Cdyn value increased in the anti-NGF group compared with the asthma group. HE staining of lung tissue revealed obvious bronchoconstriction, inflammatory cell infiltration around small vessels and alveolar spaces and in interstitum, bronchial epithelial cells desquamation in the asthma group. In anti-NGF group, tracheal epithelium was relatively complete, inflammatory exudation, bronchoconstriction and inflammatory cell infiltration were milder compared to the asthma group. Electron microscopy showed vacuolated changes of adrenal medulla cells, uneven distribution of chromaffin granules in the asthma group and the NGFgroup, and the quantity and concentration of chromaffin granules were significantly lower than normal. There were villous clubbing processes on the adrenal medulla cell membrane in the NGF group. While the anti-NGF group had no significant vacuolar changes in chromaffin granules and the concentration was close to normal. Image analysis showed that mean gray values of PNMT and NGF in the anti-NGF group were significantly different fromthe asthma group. The ELISA results showed that: ( 1) The average concentrations of epinephrine in each group were as follows, ie. the control group gt; anti-NGF group gt; asthma group gt; NGF group. ( 2) The average concentrations of norepinephrine in each group were as follows, ie. the NGF group gt; asthma group gt; anti-NGF group gt; control group. ( 3) There was no significant difference among the groups in the average concentration of cortisol. ( 4) The average concentrations of norepinephrine in each group were as follows, ie. , the control group gt; anti-NGF group gt; asthma group gt; NGF group. Conclusions Local embedding of anti-NGF microspheres can alleviate inflammatory infiltration in lung tissue and improve lung function of rat model with asthma. The mechanismmay be the anti-NGF antagonists the NGF receptor and reverse adrenal medulla cell transdifferentiation process primined by NGF.
Objective To investigate the technique of reduction by posterior approach for severe spondylolisthesis, and to discuss the method to prevent nerve stretch injury. Methods Between July 2007 and April 2011, 17 patients with severe spondylolisthesis underwent reduction, fixation, and fusion by posterior approach. There were 2 males and 15 females with a median age of 15 years (range, 8-67 years) and a median disease duration of 18 months (range, 5 months-16 years and 4 months). The level of spondylolisthesis was at L4 in 1 case and L5 in 16 cases; the spondylolisthesis was at degree III in 12 cases and degree IV in 5 cases according to Meyerding classification. There were 16 cases of developmental spondylolisthesis (high- dysplastic and low-dysplasia spondylolisthesis in 9 and 7 cases, respectively) and 1 case of traumatic spondylolisthesis; 16 cases of developmental spondylolisthesis at L5 level included 6 cases of type 4, 9 case of type 5, and 1 case of type 6 according to Spinal Deformity Study Group (SDSG) classification. All cases underwent posterior spinal decompression, Schanz screw fixation for the slipped vertebrae, the intervertebral and posterolateral fusion and reduction of the slipped vertebrae, and correction of the lumbosacral kyphosis. The reductive degree of slipped vertebrae was modulated according to the strain of exiting spinal root. The slip degree should be reduced within Meyerding degree II. The anteroposterior and lateral radiographs of whole spine were taken in a standardized standing position to observe the correction of displacement severity and lumbosacral angle. The nerve function and pain score of lower extremity were evaluated by neurological Frankel grade and visual analogue scale (VAS). Bony fusion was assessed by followed-up CT three-dimentional reconstruction. Results Exiting nerve root paralysis occurred in 1 case after operation, and released at 4 weeks after operation; no aggravation of nerve damage was observed in the other patients. The incisions primarily healed. All the patients were followed up 12-48 months (mean, 25 months). The slip percentage, the lumbosacral angle, and VAS score of lower extremity were improved from 72% ± 10%, (18.2 ± 3.5)°, and 7.0 ± 1.5 at preoperation to 12% ± 6%, ( — 7.3 ± 2.9)°, and 1.5 ± 1.3 at 12 months after operation respectively, all showing significant differences (P lt; 0.05). Osteosynthesis was seen at the bone grafting area by CT three-dimentional reconstruction at 12 months after operation. No breakage of screw and rod or reduction loss occurred. Conclusion It can obtain satisfactory clinical result to use spinal canal decompression by posterior approach, the Schanz screw fixation of the slipped vertebrae, the intervertebral and posterolateral fusion for severe spondylolisthesis. The risk of nerve stretch injury can be prevented by choosing the lowest height of intervertebral cage, modulating the reductive degree of slipped vertebrae according to the strain of exiting spinal root, and correcting lumbosacral kyphosis.
Objective To prepare nerve growth factor (NGF)-insulin composite gel and observe the effects of NGF-insulin composite gel on deep second degree scald wound healing in diabetic rats. Methods Carbomer 980, NGF (4 000 U), and insulin (800 U) were used to prepare the insulin gel, NGF gel, and NGF-insulin composite gel. The character of NGF-insulin composite gel was observed, and the in vitro drug release was tested. Seventy-five SPF Wistar male rats, weighing 200-250 g, were divided into 5 groups randomly, 15 rats each group: normal control group (group A), diabetes control group (group B), insulin gel treatment group (group C), NGF gel treatment group (group D), and NGF-insulin composite gel treatment group (group E). The type 1 diabetes rat model was established by intraperitoneal injection of Streptozotocin (55 mg/kg) in groups B, C, D, and E, while the rats in group A were injected with the same dose of citric acid and calcium citrate buffer. After modeling success, deep second degree scald wound on the back was made with constant temperature water bath box. Wounds were treated with carbomer blank gel in groups A and B, with insulin composite gel in group C, with NGF gel in group D, and with NGF-insulin composite gel in group E, once a day. At 3, 7, 11, 15, and 21 days after injury, the scald wound healing was observed and healing rate was calculated; the full-thickness skin specimens were harvested from 3 rats of each group for histological and immuohistochemical staining observation. Results The NGF-insulin composite gel was clear and transparent, and had good moisture retention capacity and adhesion; it was easy to apply and clean up. The drug release in vitro lasted more than 24 hours and maintained for 30 days. No rat died during the experiment. At 3 days after injury, wound area did not reduce in all groups; at 7, 11, 15, and 21 days, group E had the highest wound healing rate, and group B had the lowest; significant differences were found between group E and group B and when compared with the other groups (P lt; 0.05). HE staining showed that group E surpassed other groups in the growth of granulation tissue and collagen fiber. Immunohistochemical results showed that the CD34 and proliferating cell nuclear antigen (PCNA) expressed at 3 days, and the number of positive cells increased gradually with time; the microvessel density and PCNA expression were highest in group E and were lowest in group B, showing significant differences when compared with the other groups and between group E and group B (P lt; 0.05). Conclusion NGF-insulin composite gel can improve deep second degree scald wound healing in diabetic rats.
Objective To obtain highly purified and large amount of Schwann cells (SCs) by improved primary culture method, to investigate the biocompatibility of small intestinal submucosa (SIS) and SCs, and to make SIS load nerve growth factor (NGF) through co-culture with SCs. Methods Sciatic nerves were isolated from 2-3 days old Sprague Dawley rats and digested with collagenase II and trypsin. SCs were purified by differential adhesion method for 20 minutes and treated with G418 for 48 hours. Then the fibroblasts were further removed by reducing fetal bovine serum to 2.5% in H-DMEM. MTT assay was used to test the proliferation of SCs and the growth curve of SCs was drawn. The purity of SCs was calculated by immunofluorescence staining for S-100. SIS and SCs at passage 3 were co-cultured in vitro. And then the adhesion, proliferation, and differentiation of SCs were investigated by optical microscope and scanning electron microscope (SEM). The NGF content by SCs was also evaluated at 1, 2, 3, 4, 5, and 7 days by ELISA. SCs were removed from SIS by repeated freeze thawing after 3, 5, 7, 10, 13, and 15 days of co-culture. The NGF content in modified SIS was tested by ELISA. Results The purity of SCs was more than 98%. MTT assay showed that the SCs entered the logarithmic growth phase on the 3rd day, and reached the plateau phase on the 7th day. SCs well adhered to the surface of SIS by HE staining and SEM; SCs were fusiform in shape with obvious prominence and the protein granules secreted on cellular surface were also observed. Furthermore, ELISA measurement revealed that, co-culture with SIS, SCs secreted NGF prosperously without significant difference when compared with the control group (P gt; 0.05). The NGF content increased with increasing time. The concentration of NGF released from SIS which were cultured with SCs for 10 days was (414.29 ± 20.87) pg/cm2, while in simple SIS was (4.92 ± 2.06) pg/cm2, showing significant difference (P lt; 0.05). Conclusion A large number of highly purified SCs can be obtained by digestion with collagenase II and trypsin in combination with 20-minute differential adhesion and selection by G418. SIS possesses good biocompatibility with SCs, providing the basis for further study in vivo to fabricate the artificial nerve conduit.