Objective To investigate the effect on differentiation of denervated skeletal muscle-derived stem cells (MDSCs) induced by TGF-β1 in vitro.Methods MDSCs were obtained from the rat denervated skeletal muscle by preplate technique,with TGF-β1 adding on medium.Cultured cells were divided into two groups.A,control group; B,10 ng/ml TGF-β1 group.Cell growth was observed with phase contrast microscope.lmmunocytochemistry,quantitative RT-PCR and Western blot was used to detect the expression of Sca-1,COL-Ⅰ,COL-Ⅲ,α-SMA and vimentin in denervated MDSCs.Results The synthesis of COL-Ⅰ,COL-Ⅲ,α-SMA and vimentin by denervated MDSCs was extremely low at protein level in vitro,while Sca-1 level was really high.Belong to the treatment with TGF-β1,COL-Ⅰ,COL-Ⅲ,oα-SMA and vimentin in the denervated MDSCs had strong expression,but Sca-1 in which had a weak expression.Under the stimulation of TGF-β1,COL-Ⅰ expression reached peak at the 2nd day (12.5591 ± 0.3389),which was about 3 times as control group.COL-Ⅲ reached highest value at the 5th day (0.8956 ± 0.0438),which was about 23 times as control group.α-SMA topped out to 18 times at the 5th day (1.1090 ± 0.0018).Vimentin expression rose by 8.5 times and peaked at the 5th day (0.1794 ± 0.0019).The expression of Sca-1 began to decline at the 2nd day,with a remarkable reduction at the 5th day (0.0636 ± 0.0015).Conclusion TGF-β1 could induce differentiation of the denervated MDSCs to myofibroblasts in vitro,and promote the synthesis and excretion of extracellular matrix.

BACKGROUND: Auto-neural transplantation is used widely on peripheral neurological defect, but it also has some difficulties. So some scholars try to use xenoma-neural transplantation; however, it is hard todeal with immunological rejection.OBJECTIVE: To study the effect of transforming growth factor-β1 (TGFβ1) used in local area on neural regeneration after transplantation of fresh nerve allograft.DESIGN: Randomized controlled study.SETTING: Hand Surgery Department of Union Hospital Affiliated to Tongji Medical College of Huazhong University of Science and TechnologY.MATERIALS: The experiment was conducted in the Union Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology between August 2001 and October 2002. Totally 60healthy adult Wistar rats from different confinements were randomly divided into three groups including experimental group, blank group and control group with 20 in each group.METHODS: TGF-β1 plasmid was prepared for using. Establishment of animal model: Sciatic nerve at the 0.5 cm deep of piriformis muscle of rats in the two groups was cut with disinfectant razor into chip regularly about 2.0 cm. The excisional nerve segment was exchanged to transplant plerosis neurological defect. TGF-31 was injected into the local muscles and bisection of nerve in the experimental group, and equal volume of saline was injected into rats in the blank group and the control group. In addition, rats in the experimental group and the blank group were not treated with any drugs, but cyclosporine A (15 mg/kg) was used to feed rats in the control group. Ten rats from each group were taken for section and staining at the 6th and the 12th week: ① Glees-luxot fast blue staining method; ② myelin sheath fast blue staining method. Axonal amount: Fields were randomly taken from the middle staining samples 12 weeks later and 1.0 mm2 interaxis-cylinder was counted under light microscope of 400 times. Comparisons among groups were analyzed with i test.MAIN OUTCOME MEASURES: Morphological observation and axonal amount of transplanted area in each group.RESULTS: Quantitative analysis of the experimental animals: Totally 60rats entered the final analysis without any loss. ① Infiltration of monocytes was observed widely in various areas of graft in the blank group;meanwhile, desiccation of myelin sheath and plenty of vacuolations were also observed, especially at the sixth week. The whole graft was infiltrated by monocyte with severe rejection. Few axis-cylinders were regenerated in the transplanted segment. At the 12th week, graft was slender, plenty of scar tissues were proliferated, edema was observed obviously, few Schwann cells and regenerated axis-cylinders were observed, and lots of regenerated axis-cylinders did not pass the whole graft. A few infiltrative monocytes were observed, and edema was observed obviously, but new vessel was formed in transplanted nerve, and regenerated axis-cylinders passed the whole graft in the experimental group and the control group.Lots of Schwann cells were observed at the 6th week; meanwhile, regenerated axis-cylinders passed the whole graft at the 12th week, a quantitative myelinization was formed, Schwann cells proliferated obviously, and edema between axis-cylinder was relieved. Numbers of peripherally regener ated axis-cylinder of nerve and remyelination in each ransplanted area were more than those in the central area, and edema between peripheral axis-cylinder was milder than that in the central area in the experimental group. ② Twelve weeks after operation, 5 rats in each group were selected to observe their fields, which were taken randomly from neural graft,under the microscope of 400 times to count 1.0 mm2 inter-axis-cylinders.Number of axis-cylinder was higher in the experimental group and the control group than that in the blank group, and the differences were significant [(78.3±4.6), (76.1±4.2) , (15.0±3.5) ,t=3.056, t=2.948, P ＜ 0.01];however, number in the experimental group was similar to that in the control group, and differences were not significant [(78.3±4.6), (76.1±4.2),t=1.982 P ＞ 0.05].CONCLUSION: TGF-β1 used in local area plays an immunosuppressive action locally, decreases host immunological rejection, increases the number of axis-cylinder, and accelerates growth of nerve.

To study the characteristics of acute rejection after limb allotransplantation, 29 male Sprague-Dawley rats were randomly divided into 2 groups, with 15 rats in control group and 14 rats in experimental group. Each rat in control group underwent limb replantation. Each rat in experimental group received limb transplantation from Wistar rat. No immunosuppressive drugs were used after operation. The circulation of the transplanted limb, time and signs of rejection, histopathological changes in the tissues of the limb graft when rejected and survival time of limb grafts were evaluated. In the control group, no signs of rejection were observed, the circulation of each replanted limb was normal, it could survive for a longer time. The experimental group showed clinical signs of rejection (sub dermal edema and erythema) after a mean time of 3.36+/-1.15 days, and the mean survival time of the allografts was only 7+/-0.78 days. Histopathological examination showed most violent rejection reaction in skin. It is concluded that with Wistar-to-SD limb transplantation without use of immunosuppression, rejection of the grafts would occur after a mean time of 3.36+/-1.15 days; the earliest signs of rejection were edema and erythema of the skin, skin being the most representative component of limb graft rejection.
Acute Disease ; Extremities/*transplantation ; *Graft Rejection ; Graft Survival ; Random Allocation ; Rats, Sprague-Dawley ; Rats, Wistar ; Skin/immunology ; Transplantation, Homologous

Protective effect of interleukin-1beta (IL-1beta) on motor neurons was studied after peripheral nerve injury. Twenty Wistar rats were divided into 2 groups randomly. The right sciatic nerve of each rat was resected. After silicon tubulization of sciatic nerve in rat, 15 microl 1 ng/ml IL-1beta and PBS solution were injected into the silicon capsule respectively. Enzyme histochemistry was performed to show acetyle cholesterase (AchE) and nitric oxide staining (NOS) activity of spinal alpha motor neurons in spinal segments 2 weeks later. Neurons were counted and the diameter and cross sectional (c/s) area of neurons were analyzed by using computer image analysis system. The results showed that as compared with the normal side, both enzyme activities significantly changed in motor neurons in PBS group. The diameter and c/s area of both neurons changed significantly too (P < 0.01). These results suggest that exogenous IL-1beta protects alpha-motor neurons from degeneration and necrosis after peripheral nerve injury.
Interleukin-1/*pharmacology ; Motor Neurons/*pathology ; Neuroprotective Agents/*pharmacology ; Random Allocation ; Rats, Wistar ; Sciatic Nerve/*injuries ; Spinal Cord/pathology

In order to investigate the biological function of transforming growth factor-β1 (TGF-β1) during fibrosis in denervated skeletal muscle, we recruited sciatic nerve injury model of SD rats in which denervated gastrocnemius was isolated for analysis. At different time points after operation, denervated muscle was examined by several methods. Masson trichrome staining showed morphological changes of denervated skeletal muscle. Quantitative RT-PCR detected the rapid increase of TGF-β1 expression at mRNA level after nerve injury. It was found that a peak of TGF-β1 mRNA expression appeared one week post-operation. The expression of collagen I (COL I) mRNA was up-regulated in the nerve injury model as well, and reached highest level two weeks post-injury. Immunoblot revealed similar expression pattern of TGF-β1 and COL I in denervated muscles at protein level. In addition, we found that the area of the gastrocnemius muscle fiber was decreased gradually along with increased interstitital fibrosis. Interestingly, this pathological change could be prevented, at least partly, by local injection of TGF-β1 antibodies, which could be contributed to the reduced production of COL I by inhibiting function of TGF-β1. Taken together, in this study, we demonstrated that the expression of TGF-β1 was increased significantly in denervated skeletal muscle, which might play a crucial role during muscle fibrosis after nerve transection.

BACKGROUND: In repair of nerve defect with allogenic nerve graft, to reduce immune rejection is one of the key problems. At present, the main approach is to reduce antigenicity of grafted nerve segment and apply generally immune inhibitor.OBJECTIVE: To observe the effects of freeze/thaw treatment and local application of transforming growth factor beta 1 (TGF-β1) plasmid on frozen nerve allograft.DESIGN: Randomized controlled animal experiment was designed.SETTING: Department of Hand Surgery, Union Hospital, Tongji Medical College Affiliated to Huazhong University of Science and Technology.MATERIALS: The experiment was performed in Tongji Medical College of Huazhong University of Science and Technology from January 2003 to December 2004, in which 40 Wistar healthy and adult rats were employed,from different delivery and were randomized into experimental group and control, 20 rats in each one.METHODS: Transforming growth factor-β1 (TGF-β1) plasmid and frozen allogenic sciatic nerve were prepared. In experimental group and control,sciatic nerve was cut off 2.0 cm in length, in the foramen 0.5 cm beneath piriformis. The nerve defect was repaired with pre-frozen allogenic nerve 2.0 cm in length. In experimental group, TGF-β1 plasmid was injected in local muscle and two broken ends of nerve. In the control group, physiological saline of equal volume was injected. In the 6th and 12th weeks, the samples were collected from 10 rats in each group for sectioning, staining,axonal counting and statistical analysis.RESULTS: No any animal was died in experiment and all of animals entered result analysis. In the 6th weeks, in the control group, mild edema appeared among axons on the grafted segment of nerve and in the experimental group, there was no edema among axons and the regenerated nerve numbers were close to the normal. In the 12th week, in the experimental group, the entire grafted nerve segment was basically filled up by the regenerated axons;myelinated nerve fiber was arranged in order and both axons and myelins were developed well. The regenerated axonal count in experimental group was more significantly than the control, indicating extremely significant difference [(98.6±4.8), (75.8±5.1) counts/μm2, t=2.962, P ＜ 0.01].CONCLUSION: Freeze/thaw treatment can decrease antigenicity of allogenic nerve, which provides the possibility of repair of nerve defect. Local application of TGF-β1 plasmid can provide immune inhibition locally and reduce immune rejection in the host.

BACKGROUND: At present, the repair by means of suture is still commonly used to repair the peripheral nerve injury and rupture, while the adhesion of the fibrin glue repairing peripheral nerve injury has been considered as a new topic of study.OBJECTIVE: To study the countertraction intensity of peripheral nerve and its dynamic changes after repaired with the adhesion of fibrin glue.DESIGN: A randomized controlled experimental study.SETTING and MATERIALS: The study was completed in the Laboratory of Biodynamics, Department of Orthopaedics, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology. The healthy adult male Wistar rats weighing 250- 300 g were selected for the experiment.INTERVENTIONS: Totally 96 Wistar rats were completely randomized into the suture group and the adhesion group. Their sciatic nerves were cut, and the incisions were well lined. The fibrin glue was adopted in the adhesion group, while 11 -0 suture was adopted in the suture group. On the very day and 3, 7, 14, 21, 28 days after the operation, 8 rats were respectively taken each from the suture group and the adhesion group. The free sciatic nerves of them were detected immediately by the biodynamic test.MAIN OUTCOME MEASURES: The peak load and the power consumption were measured when the nerves ruptured and the nerve stress-strain curve was described.RESULTS: In normal countertraction intensity curve of the nerve, the elastic peculiarity can be manifested. Between the suture group and the adhesion group, there were no notable significances of the maximal countertraction intensity and power consumption on the very day and 14, 21, 28 days after the operation( P ＞ 0.05). While 3 days after the operation, the maximal countertraction intensity of the two groups was(1.35± 0. 27),( 1.97 ± 023) N/mm2 respectively, the power consumption was (0. 028 ± 0.007), (0.040 ± 0.003) J/mm2 respectively. Seven days after the operation, the maximal countertraction intensity was( 1.93 ± 0.26), (2.74± 0.30) N/mm2 respectively, the power consumption was(0.047±0.009), (0.063±0.007) J/mm2 respectively. The differences both had the notable significance ( P ＜ 0.05).CONCLUSION: The fibrin glue has enough countertraction intensity and can gratify the need of such nerve repairs.

ObjectiveTo investigate expression of TGF-β1,CTGF and collagen deposition in skeletal muscle during chronic entrapment of peripheral nerve. MethodsFifty rats were separated into two groups,control group and experimental group. At different time points after operation, the right gastrocnemius of 5rats from each group were collected for further analysis such as HE, Masson stain, immunohistochemical staining,RT-PCR and Western-blot. Results It was observed that axon degeneration occurred during chronic nerve entrapment,and which was in line with reports from other groups.Moreover,it had been demonstrated that after nerve entrapment,skeletal muscles may form fibrosis and degeneration consequently.Within this pathological procedure,expression of TGF-β1. CTGF and deposition of collagenⅠ changed rapidly when compared with control group.ConclusionOverall,these results indicated that these factors may be important during skeletal muscle degeneration after chronic nerve entrapment.

Protective effect of interleukin-1beta (IL-1beta) on motor neurons was studied after peripheral nerve injury. Twenty Wistar rats were divided into 2 groups randomly. The right sciatic nerve of each rat was resected. After silicon tubulization of sciatic nerve in rat, 15 microl 1 ng/ml IL-1beta and PBS solution were injected into the silicon capsule respectively. Enzyme histochemistry was performed to show acetyle cholesterase (AchE) and nitric oxide staining (NOS) activity of spinal alpha motor neurons in spinal segments 2 weeks later. Neurons were counted and the diameter and cross sectional (c/s) area of neurons were analyzed by using computer image analysis system. The results showed that as compared with the normal side, both enzyme activities significantly changed in motor neurons in PBS group. The diameter and c/s area of both neurons changed significantly too (P < 0.01). These results suggest that exogenous IL-1beta protects alpha-motor neurons from degeneration and necrosis after peripheral nerve injury.
Animals ; Female ; Interleukin-1 ; pharmacology ; Male ; Motor Neurons ; pathology ; Neuroprotective Agents ; pharmacology ; Random Allocation ; Rats ; Rats, Wistar ; Sciatic Nerve ; injuries ; Spinal Cord ; pathology

To study the characteristics of acute rejection after limb allotransplantation, 29 male Sprague-Dawley rats were randomly divided into 2 groups, with 15 rats in control group and 14 rats in experimental group. Each rat in control group underwent limb replantation. Each rat in experimental group received limb transplantation from Wistar rat. No immunosuppressive drugs were used after operation. The circulation of the transplanted limb, time and signs of rejection, histopathological changes in the tissues of the limb graft when rejected and survival time of limb grafts were evaluated. In the control group, no signs of rejection were observed, the circulation of each replanted limb was normal, it could survive for a longer time. The experimental group showed clinical signs of rejection (sub dermal edema and erythema) after a mean time of 3.36+/-1.15 days, and the mean survival time of the allografts was only 7+/-0.78 days. Histopathological examination showed most violent rejection reaction in skin. It is concluded that with Wistar-to-SD limb transplantation without use of immunosuppression, rejection of the grafts would occur after a mean time of 3.36+/-1.15 days; the earliest signs of rejection were edema and erythema of the skin, skin being the most representative component of limb graft rejection.
Acute Disease ; Animals ; Extremities ; transplantation ; Graft Rejection ; Graft Survival ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar ; Skin ; immunology ; Transplantation, Homologous