Objective Peri pheral nerve injury is a common cl inical disease, to study the effects of the physical therapy on the regeneration of the injured sciatic nerve, and provide a reference for cl inical treatment. Methods Sixty-four female adult Wistar rats (weighing 252-365 g) were chosen and randomly divided into 4 groups (n=16): group A, group B, groupC, and group D. The experimental model of sciatic nerve defect was establ ished by crushing the right sciatic nerve in groups B, C, and D; group A served as the control group without crushing. At 2 days after injury, no treatment was given in group B, electrical stimulation in group C, and combined physical therapies (decimeter and infrared ray) in group D. At 0, 7, 14, and 30 days after treatment, the sciatic nerve function index (SFI) and the motor nerve conduction velocity (MNCV) were measured, and morphological and transmission electron microscopy (TEM) examinations were done; at 30 days after treatment, the morphological evaluation analysis of axons was performed. Results At 0 and 7 days after treatment, the SFI values of groups B, C, and D were significantly higher than that of group A (P lt; 0.05); at 14 and 30 days after treatment, the SFI value of group D decreased significantly, no significant difference was observed between group D and group A (P gt; 0.05) at 30 days; whereas the SFI values of groups B and C decreased, showing significant difference when compared with the value of group A (P lt; 0.05). At 0, 7, and 14 days after treatment, the MNCV values of groups B, C, and D were significantly lower than that of group A (P lt; 0.05), and there were significantly differences between group B and groups C, D (P lt; 0.05); at 14 days, the MNCV value of group D was significantly higher than that of group C (P lt; 0.05); and at 30 days, the MNCV values of groups B and C were significantly lower than that of group A (P lt; 0.05), but there was no significant difference between group D and group A (P gt; 0.05). At 0 and 7 days, only collagen and l i pid were observed by TEM; at 14 and 30 days, many Schwann cells and perineurial cells in regeneration axon were observed in groups B, C, and D, especially in group D. Automated image analysis of axons showed that there was no significant difference in the number of myelinated nerve fibers, axon diameter, and myelin sheath thickness between group D and group A (P gt; 0.05), and the number of myelinated nerve fibers and axon diameter of group D were significantly higher than those of groups B and C (P lt; 0.05). Conclusion Physical therapy can improve the regeneration of the injured sciatic nerve of rats.
Objective To investigate the effects of icariin and mixed prescri ption of icariin, radix hedysari polysaccharide, and l iquid extracted from earthworm on peri pheral nerve regeneration. Methods Twenty male SD rats weighing (200 ± 10) g were selected and randomized into four groups (n=5 per group): sham operated group (group A), model group (group B), icariin group (group C), and mixed l iquid group (group D). In group A, the left sciatic nerves of the rats were only exposed, and treated at fixed time from the following day with the NS (2 mL/d). In groups B, C, D, the models were made by clamping sciatic nerve and treated with NS, icariin and mixed l iquid, respectively (2 mL/d). The general state ofanimals was observed after the treatment daily. The nerve function index, motor nerve conductive velocity and the morphous and number of myel inated sciatic nerve fibers were measured at 21 days. Results Animals in various groups were all in good state. After 21 days, the weights of rats in groups A, B, C and D were (366.9 ± 14.0), (370.1 ± 16.3), (373.3 ± 19.6) and (374.0 ± 11.4) g, respectively, and there was no significant difference among these groups (P gt; 0.05). For sciatic function index, there was no significant difference between group A and group D (P gt; 0.05), between group B and group C (P gt; 0.05), while there was significant difference between group B and group D (P lt; 0.05). For tibial function index, there was significant difference between group A and groups B, C, D (Plt; 0.05), there was no significant difference between group B and groups C, D (Pgt; 0.05). For peroneal function index, there was no significant difference between group A and groups C, D (P gt; 0.05), between group B and groups C, D (P gt; 0.05). The sciatic motor nerve conductive velocities of group A, B, C and D were (45.0 ± 2.9), (8.0 ± 2.6), (13.4 ± 6.8), and (19.6 ± 9.3) m/s, respectively, there was no significant difference between group B and group C (P gt; 0.05), and there was significant difference between group A and groups B, C, D and between group B and group D (P lt; 0.05). The size of individual myel inated sciatic nerve fibers of regenerated nerves in groups B, C, and D was significantly smaller than that in group A. Comparing with group A, the number of myel inated sciatic nerve fibers in groups B, C, and D was 93.3% ± 35.6%, 90.6% ± 37.1%, and 115.4% ± 40.6%, respectively, but there was no significant difference among four groups (P gt; 0.05). Conclusion Icariin and mixed prescription are safe. The improving peripheral nerve regeneration effect of mixed prescription is more obvious than that of icariin, indicating the comprehensive study of modified formula radixhedysari is necessary to find the effective part or mixture of effective compounds with fixed percentage.
Objective To explore the facilitative effects of different allogenic cells injected into the denervated muscles on the nerve regeneration, the protection of the myoceptor degeneration, and the promotion for rehabilitation of the muscular function. Methods Schwann cells, myoblast cells, and renal endothelial cells were prepared from 400 SD rats aged 7 days and weighing 20.0±2.3 g. Thirty-six adult female SD rats weighing 120-150 g were randomly divided into 4 groups(n=9). Under the asepsis condition, the left ischiadic nerves of all the SD rats were cut off, and the primary suture of the epineurium was performed. After operation, the different corresponding cells were injected into the triceps muscles of the rat calf in each group once per week for 4 times in all. One ml of Schwann cells (1×106/ml) was injected into the rats in Group A; 1 ml of the mixed cells of Schwann cells and myoblast cells (1×106/ml) was injected into the rats in Group B; 1 ml of the extract from the mixed cells of Schwann cells, myoblast cells, and renal endothelial cells (1×106/ml) was injected into the rats in Group C; 1 ml of the culture medium without any serum was injected into the rats in Group D as a control. After operation, observation was made for the general condition of the rats; 3 months after operation, enzymohistochemistry and the CJun expression were performedin the ventricornual motor neuron. At the proximal and the distal ends of the nerve suture, the density of neurilemma cells in the unit area and the area size of the regenerated nerve fibers were observed and measured. Results The affected limbs of the rats in Groups A, B and C improved 13 months after operation. The ulcers and swelling at the ankles gradually relieved and the rats could move normally 3 months after operation. However, the affected limbsof the rats in Group D still had ulcers and swelling, with an obvious contracture of the toes and a difficult movement. Three months after operation, the number of the target muscle myoceptor, the number of the Actin positive cells, the activity of the various enzymes in the denervated muscles, and the histological changes of the regenerated nerves were better in Group C than in Groups A and B (P＜0.01); and they were all better in Groups A, B and C than in Group D（Plt;0.01）. Conclusion Schwann cells, the mixture of Schwann cells and myoblast cells, and the extract from the mixture of Schwann cells, myoblast cells and renal endothelial cells can all promote neurotization and rehabilitation of the muscular function, and protect against the myoceptor degeneration. However, the effect of the extract is superior to that of Schwann cells or the mixed cells.
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.
OBJECTIVE: To explore the mechanism of tissue specificity of neurotropism in peripheral nerve regeneration, we investigated the biological characteristics of the nerve regeneration conditioned fluids(NRCF) on motoneuron of SD rats cultured in vitro. METHODS: Silicon chambers were sutured respectively to the distal stumps of motorial branch of femoral nerve and saphenous nerve to collect NRCF, namely MD-NRCF and SD-NRCF. The rats cortex motoneuron were divided into 4 groups and cocultured with MD-NRCF, SD-NRCF, b-FGF and serum-free medium respectively. The cultured cells were photoed under phase-contrast microscope, their longest neurites and cell-body areas were measured by cell image processing computer system. MTT automated colorimetric microassay was also adopted to quantify the activation of cultured motoneurons in each group. RESULTS: Cells of MD-NRCF group had longer neurites than those of the other three groups, and their activation was also superior to those of the other groups. CONCLUSION: The results suggest that MD-NRCF has more significantly neurite-promoting and neurobiological effects on motoneuron than SD-NRCF and b-FGF.
To observe the effect of percutaneous electrical stimulation on peripheral nerve regeneration, a model was created on the sciatic nerves of 56 rats from either sectioned and followed by direct anastomosis or clamping of the nerve. The indices, such as conducting velocity of nerve, maximal induced action potential of muscle, growth speed of nerve, rateof axon crossing anastomosis site, number of muscular fiber on transverse area and weight of muscle by autocontrol were compared. In this study, 36 rats were divided into two groups, 24 rats in Group 1 and 12 rats in Group 2. In Gourp 1, both sciatic nerves were sectioned and was anastomozed 4 weeks later. One side of the nerve was stimulated with percutaneous electric current, the other side was served as control. In Group 2, both sides of nerves were clamped and the electical stimulationwas carried out on one side. The parameters of the electric current were 2～5HZ, 0.4m/s, 24～48V. The electrophysiological and histomorphological features were observed 1 to 6 weeks after operation. The results showed that in the stimulatedside, the indices were all superior to that of the control side. This suggestedthat electrical stimulation could promote peripheral nerve regeneration.
ObjectiveTo investigate the expression regulation of inflammation cytokines interleukin 4 (IL-4), IL-6, IL-13, and tumor necrosis factor α (TNF-α) in rats with sciatic nerve defect following olfactory ensheathing cell (OEC) transplantation. MethodsThe primary OEC for cell culture and identification was dissociated from the olfactory bulb of the green fluorescent protein-Sprague Dawley (GFP-SD) rat. One hundred SD rats were randomly divided into 2 groups, and the right sciatic nerve defect (10 mm in length) model was made, then repaired with poly (lactic acid-co-glycolic acid) (PLGA). The mixture of equivalent cultured GFP-OEC and extracellular matrix (ECM) was injected into both ends of PLGA nerve conduit in the experimental group (n=55), and the mixture of DMEM and ECM in the control group (n=45). The general situation of rats was observed after operation. At 6 hours, 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, and 6 weeks, the inflammatory cytokines were detected by Western blot. At 2, 4, and 6 weeks, the survival of GFP-OEC was observed in the experimental group. At 9 weeks, HE staining was used to observe the morphology of nerve tissue, and the sensory and motor function and the electrophysiological index were detected. ResultsThe cultured primary cells were GFP-OECs by immunofluorescence staining. Compared with the control group, the experimental group showed significantly increased expression level of IL-4 at 2-6 weeks (P < 0.05), significantly decreased expression level of IL-6 and TNF-α at 3 days and 1 week (P < 0.05) and significantly increased expression level of IL-13 at 1 day and 3-6 weeks (P < 0.05) by Western blot detection. At 2, 4, and 6 weeks, the surviving GFP-OEC of regenerative nerve end was observed in the experimental group under the fluorescence microscope. At 9 weeks, regenerative nerve tissue was loose, and cell morphology was irregular in the experimental group, while the regenerative nerve tissue had vesicular voids and the cell number decreased significantly in the control group. At 9 weeks, the functional recovery of sciatic nerve in the experimental group was better than that of the control group, showing significant difference in the lateral foot retraction time, sciatic nerve function index, muscle action potential latency, and the amplitude of compound muscle action potential (P < 0.05). ConclusionOEC can promote the anti-inflammation cytokines expression of IL-4 and IL-13 and inhibit the pro-inflammatory cytokines expression of IL-6 and TNF-α, which can improve the local inflammatory microenvironment of sciatic nerve and effectively promote the structure and function recovery of sciatic nerve.
Objective To investigate the expression change of endogenous Spastin after sciatic nerve injury in rats, and to discuss the role and significance in the peripheral nerve regeneration. Methods Thirty-six adult male Sprague Dawley rats weighing 180–220 g were randomly divided into the experimental group (n=30) and the control group (n=6). Sciatic nerve compression damage model was established in the experimental group, and the sciatic nerve was only exposed in the control group. The L4-6 spinal cord tissue was obtained to detect Spastin mRNA and protein levels by real-time fluorescence quantitative PCR and Western blot at 1, 3, 7, 14, and 28 days after operation in the experimental group (n=6) and at 7 days in the control group. Meanwhile, the sciatic nerve at 5 mm distal to the injured site was obtained to observe the ultrastructure of the distal axon by transmission electron microscope (TEM). Results The expression trends of Spastin gene and Spastin protein in L4-6 spinal cord tissue of 2 groups were basically identical. In the experimental group, the expressions of Spastin gene and protein decreased at the beginning, and then increased; the expressions reduced to the minimum at 7 days after operation, and came back to the initial level at 28 days. The expression levels of Spastin mRNA and protein at 3, 7, and 14 days were significantly lower in the experimental group than the control group (P<0.05), but no significant difference was noted between 2 groups at 1 and 28 days (P>0.05). The expression levels of Spastin mRNA and protein at 3, 7, and 14 days were significantly lower than those at 1 and 28 days in the experimental group (P<0.05), but no significant difference was noted between at 1 day and 28 days (P>0.05). At 1, 3, and 7 days after operation, the myelin damage was observed by TEM; at 14 days, there were regenerating Schwann cells; at 28 days, a large number of myelinated nerve fibers were seen, which were closed to normal form. Conclusion In the process of sciatic nerve regeneration after injury, a complex succession of changes take place in the expression of endogenous Spastin protein in rats, indicating that Spastin protein plays an important role in the process.
Objective Inducing human amniotic membrane mesenchymal stem cells (hAMSCs) to Schwann cells-like cells (SCs-like cells) in vitro, and to evaluate the efficacy of transplantation of hAMSCs and SCs-like cells on nerves regeneration of the rat flaps. Methods hAMSCs were isolated from placenta via two-step digestion and cultured by using trypsin and collagenase, then identified them by flow cytometry assay and immunofluorescence staining. The 3rd generation of hAMSCs cultured for 6 days were induced to SCs-like cells in vitro; at 19 days after induction, the levels of S-100, p75, and glial fibrillary acidic protein (GFAP) were detected by immunofluorescence staining, Western blot, and real-time fluorescence quantitative PCR (qPCR). The levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) were measured by ELISA in the supernatant of the 3rd generation of hAMSCs cultured for 6 days and the hAMSCs induced within 19 days. In addition, 75 female Sprague Dawley rats were taken to establish the rat denervated perforator flap model of the abdominal wall, and were divided into 3 groups (n=25). The 3rd generation of hAMSCs (1×106 cells) in the proliferation period of culturing for 6 days, the SCs-like cells (1×106 cells), and equal volume PBS were injected subcutaneously in the skin flap of the rat in groups A, B, and C, respectively. At 2, 5, 7, 9, and 14 days after transplantation, 5 rats in each group were killed to harvest the flap frozen sections and observe the positive expression of neurofilament heavy polypeptide antibody (NF-01) by immunofluorescence staining. Results The cells were identified as hAMSCs by flow cytometry assay and immunofluorescence staining. The results of immunofluorescence staining, Western blot, qPCR showed that the percentage of positive cells, protein expression, and gene relative expression of S-100, p75, and GFAP in SCs-like cells group were significantly higher than those in hAMSCs group (P<0.05). The results of ELISA demonstrated that the expression of BDNF and NGF was significantly decreased after added induced liquid 1, and the level of BDNF and NGF increased gradually with the induction of liquids 2 and 3, and the concentration of BDNF and NGF was significantly higher than that of hAMSCs group (P<0.05). Immunofluorescence staining showed that the number of regenerated nerve fibers in group B was higher than that in groups A and C after 5-14 days of transplantation. Conclusion The hAMSCs can be induced into SCs-like cells with the proper chemical factor regulation in vitro, and a large number of promoting nerve growth factor were released during the process of differentiation, and nerve regeneration in flaps being transplanted the SCs-like cells was better than that in flaps being transplanted the hAMSCs, which through a large number of BDNF and NGF were released.
ObjectiveTo investigate the effect of human adipose-derived stem cells (hADSCs) on pressure ulcers in mouse.MethodsThe subcutaneous adipose tissue from voluntary donation was harvested. Then the hADSCs were isolated and cultured by mechanical isolation combined with typeⅠcollagenase digestion. The 3rd generation cells were identified by osteogenic, adipogenic, chondrogenic differentiations and flow cytometry. The platelet rich plasma (PRP) from peripheral blood donated by healthy volunteers was prepared by centrifugation. The pressure ulcer model was established in 45 C57BL/6 mice by two magnets pressurized the back skin, and randomly divided into 3 groups (n=15). The wounds were injected with 100 μL of hADSCs (1×106 cells) transfected with a green fluorescent protein (GFP)-carrying virus, 100 μL human PRP, and 100 μL PBS in hADSCs group, PRP group, and control group, respectively. The wound healing was observed after injection. The wound healing rate was calculated on the 5th, 9th, and 13th days. On the 5th, 11th, and 21st day, the specimens were stained with HE staing, Masson staining, and CD31 and S100 immunohistochemical staining to observe the vascular and nerve regeneration of the wound. In hADSCs group, fluorescence tracer method was used to observe the colonization and survival of the cells on the 11th day.ResultsThe cultured cells were identified as hADSCs by induced differentiation and flow cytometry. The platelet counting was significantly higher in PRP group than in normal peripheral blood group (t=5.781, P=0.029). General observation showed that the wound healing in hADSCs group was superior to those in PRP group and control group after injection. On the 5th, 9th, and 13th days, the wound healing rate in hADSCs group was significantly higher than those in PRP group and control group (P<0.05). Histological observation showed that compared with PRP group and control group, inflammatory cell infiltration and inflammatory reaction were significantly reduced in hADSCs group, collagen deposition was significantly increased, and skin appendage regeneration was seen on the 21st day; at each time point, the expression of collagen was significantly higher in hADSCs group than in PRP group and control group (P<0.05). Immunohistochemical staining showed that the number of neovascularization and the percentage of S100-positive cells in hADSCs group were significantly better than those in PRP group and control group on the 5th, 9th, and 13th days (P<0.05). Fluorescent tracer method showed that the hADSCs could colonize the wound and survive during 11 days after injection.ConclusionLocal transplantation of hADSCs can accelerate healing of pressure ulcer wounds in mice and improve healing quality by promoting revascularization and nerve regeneration.