Objective To explore the histochemical staining for distinguishing and local izing nerve fibers and fascicles at histological level in three-dimensional reconstruction of peri pheral nerves. Methods The right median nerve was harvested from one fresh cadaver and embedded in OCT compound. The sample was serially horizontally sl iced with 6 μm thickness. All sections were stained with Karnovsky-Roots method (group A, n=30) firstly and then stained with toluidine blue (group B, =28) and Ponceau 2R (group C, n=21) in proper sequence. The results of each step were taken photos (× 100). After successfully stitching, the two-dimensional panorama images were compared, including texture feature, the number and aver gray level of area showing acetylchol inesterase (AchE) activity, and result of auto microscopic medical image segmentation. Results In groups A, B, and C, the number of AchE-positive area was (21.63 ± 4.06)× 102, (20.64 ± 3.51)× 102, and (20.54 ± 5.71)× 102, respectively, showing no significant difference among 3 groups (F=0.64, P=0.54); the mean gray level was (1.41 ± 0.06)× 102, (1.10 ± 0.05)× 102, and (1.14 ± 0.07)× 102, respectively, showing significant differences between group A and groups B and C (P lt; 0.001). In the image of group A, only AchE-positive area was stained; in the image of group B, myelin sheath was obscure; and in the image of group C, axons and myelin sheath could be indentified, the character of nerve fibers could be distinguished clearly and accurately, and the image segmentation of fascicles could be achieved easier than other 2 images. Conclusion The image of Karnovsky-Roots-toluidine blue-Ponceau 2R staining has no effect on the AchE-positive area in the image of Karnovsky-Roots staining and shows better texture feature. This improved histochemical process may provide ideal image for the three-dimensional reconstruction of peri pheral nerves.
Objective To investigate the effects of chitosan/polyvinyl alcohol (PVA) nerve conduits for repairing radial nerve defect in Macaques. Methods Twelve adult Macaques weighing 3.26-5.35 kg were made the models of radial nerve defect (2 cm in length) and were randomly divided into 3 groups according to nerve grafting, with 4 Macaques in each group. Chitosan/PVA nerve conduit, non-graft, and autografts were implanted in the defects in groups A, B, and C, respectively. And the right radial nerves were used as normal control. At 8 months postoperatively, the general observation,electrophysiological methods, and histological examination were performed. Results At 8 months postoperatively, theregenerated nerve bridged the radial nerve defect in group A, but no obvious adhesion was observed between the tube and the peripheral tissue. The regenerated nerve had not bridged the sciatic nerve defect in group B. The adhesions between the implanted nerve and the peri pheral tissue were significant in group C. Compound muscle action potentials (CMAP) were detected in group A and group C, and no CMAP in group B. Peak ampl itude showed a significantly higher value in normal control than in groups A and C (P lt; 0.05), but there was no significant difference between groups A and C (P gt; 0.05). Nerve conduction velocity and latency were better in normal control than in groups A and C, and in group C than in group A, all showing significant differences (Plt; 0.05). The density of myl inated fibers in groups A and C was significantly lower than that in normal control (P lt; 0.05), but there was no significant difference between groups A and C (P gt; 0.05). The diameter and the myel in sheath thickness of the myl inated fibers in normal control were significantly higher than those in groups A and C, and in group C than in group A, all showing significant differences (P lt; 0.05). Conclusion The chitosan/PVA nerve conduits can promote the peripheral nerve regeneration, and may promise alternative to nerve autograft for repairing peripheral nerve defects.
Objective To observe the histomorphology and the biocompatibil ity of acellular nerve prepared by different methods, to provide the experimental evidence for the selection of preparation of acellular nerve scaffold. Methods Forty-eight adult Sprague Dawley rats, male or female, weighing 180-220 g, were selected. The sciatic nerves were obtained from 30 rats and were divided into groups A, B, and C (each group had 20 nerves). The acellular sciatic nerves were prepared by the chemical methods of Dumont (group A), Sondell (group B), and Haase (group C). The effect to remove cells was estimated by the degree of decellularization, degree of demyel ination, and intergrity of nerve fiber tube. The histocompatibil ity was observed by subcutaneous implant test in another 18 rats. Three points were selected along both sides of centre l ine on the back of rats, and the points were randomly divided into groups A1, B1, and C1; the acellular nerve of groups A, B, and C were implanted in the corresponding groups A1, B1, and C1. At 1, 2, and 4 weeks after operation, the rats were sacrificed to perform the general observation and histological observation. Results The histomorphology: apart of cells and the dissolved scraps of axon could be seen in acellular never in the group A, and part of Schwann cell basilar membrane was broken. In group B, the cells in the acellular never were not removed completely, the Schwann cell basilar membrane formed bigger irregular hollows, part of the Schwann cell basilar membrane was broken obviously. But in the group C, the cells were completely removed, the Schwann cell basilar membrane remained intactly. Group C was better than group A and group B in the degree of decellularization, degree of demyel ination, integrity of nerve fiber tube and total score, showing significant differences (P lt; 0.05). The subcutaneous implant test: there were neutrophils and lymphocytes around the acellular nerve in 3 groups at 1 week after implant. A few of lymphocytes were observed around the acellular nerve in 3 groups at 2 weeks after implant. The inflammation was less in groups A1, B1, and C1 at 4 weeks after implant, part of the cells grew into the acellular nerve and arranged along the Schwann cell basilar membrane. The reaction indexes of the inflammational cells in group A1 and group B1 were higher than that in group C1 at 1, 2, and 4 weeks after implant, showing significant differences (P lt; 0.01), but there was no significant difference between group A1 and group B1 (P gt; 0.05). Conclusion The acellular sciatic nerves prepared by Haase method has better acellular effect and the histocompatibil ity than those by the methods of Dumont and Sondell.
Objective To investigate the promotion effect of neurotropic reinnervation with chemically extracted acellular nerve allograft. Methods The sciatic nerves of 5 healthy adult SD rats, regardless of gender and weighing 270-300 g, were collected to prepare chemically extracted acellular nerve allograft. Eighteen healthy adult Wistar rats, regardless of genderand weighing 300-320 g, were made the model of left sciatic nerve defect (10 mm) and randomly divided into 2 groups: autograft (control group, n=9) and allograft group (experimental group, n=9). The defects were bridged by acellular nerve allograft in experimental group and by autograft by turning over the proximal and distal ends of the nerve in control group. At 3 months after transplantation, dorsal root ganglion (DRG) resection operation was performed in 6 rats of 2 groups. At 3 weeks after operation, the sural nerves were harvested to calculate the misdirection rate of nerve fibers with pathological staining and compute-assisted image analysis. Cholinesterase staining and carbonic anhydrase staining were performed in the sural nerve of 3 rats that did not undergo DRG resection at 3 months. Results The results of chol inesterase staining and carbonic anhydrase staining showed that experimental group had less brown granules and more black granules than control group. After DRG resection, count of myelinated nerve fiber were 4 257 ± 285 in the experimental group and 4 494 ± 310 in the control group significant (P lt; 0.05). The misdirection rate was 2.27% ± 0.28% and 7.65% ± 0.68% in the experimental group and in the control group respectively, showing significant difference (P lt; 0.05). Conclusion Chemically extracted acellular nerve allograft can not only act as a scaffold in the period of nerve defects repair, but markedly enhance the effects of neurotropic reinnervation.
Objective To investigate the appropriate concentration of tripterygium wilfordii and immunological rejection of rats’ sciatic nerve allograft with the tripterygium wilfordii’s pretreatment so as to explore tripterygium wilfordii’ s suppression. Methods Sixty SD rats (male, weighing 270-290 g), as sciatic nerve allograft acceptor were randomized into5 groups (groups A, B, C, D and E, n=12). To repair the sciatic nerve defect of SD rats, the Wistar rats’ sciatic nerve allografts about 15 mm long were used with 24 hours’ soak of different concentrations of tripterygium wilfordii (group A: 200 mg/L, group B: 400 mg/L, group C: 800 mg/L). The control groups (group D: the fresh sciatic nerve allograft from donors; group E: the fresh sciatic nerve allograft from themselves) were establ ished. At different time points after operation, the morphological examinations (the observation of histology, l ight microscope, electron microscope), the detection of myelin basic protein’s (MBP) content and the analyses of CD4+ and CD8+ T cells on the allografts in the acute phase were performed Results There was no significant difference in morphology among groups A, B and C, the adhesions between allografts and connective tissue were milder than that of group D, and the allografts’ morphous and the inflammatory cell infiltration were better than that of group D. The degeneration of myel in sheath was observed at different levels and there was no significant difference between group B and group E (P gt; 0.05). There was a significant difference in immunological rejection between groups A, B, C and group D (P lt; 0.05). Conclusion Tripterygium wilfordii can effectively suppress the acute immunological rejection in the early stage after operation, and protect the myel in sheath to a certain extent.
Objective To study the outcomes of nerve defect repair with the tissue engineered nerve, which is composed of the complex of SCs, 30% ECM gel, bFGF-PLGA sustained release microspheres, PLGA microfilaments and permeable poly (D, L-lacitic acid) (PDLLA) catheters. Methods SCs were cultured and purified from the sciatic nerves of 1-day-old neonatal SD rats. The 1st passage cells were compounded with bFGF-PLGA sustained release microspheres andECM gel, and then were injected into permeable PDLLA catheters with PLGA microfilaments inside. In this way, the tissueengineered nerve was constructed. Sixty SD rats were included. The model of 15-mm sciatic nerve defects was made, and then the rats were randomly divided into 5 groups, with 12 rats in each. In group A, autograft was adopted. In group B, the blank PDLLA catheters with PBS inside were used. In group C, PDLLA catheters, with PLGA microfilaments and 30% ECM gel inside, were used. In group D, PDLLA catheters, with PLGA microfilaments, SCs and 30% ECM gel inside, were used. In group E, the tissue engineered nerve was appl ied. After the operation, observation was made for general conditions of the rats. The sciatic function index (SFI) analysis was performed at 12, 16, 20 and 24 weeks after the operation, respectively. Eelectrophysiological detection and histological observation were performed at 12 and 24 weeks after the operation, respectively. Results All rats survived to the end of the experiment. At 12 and 16 weeks after the operation, group E was significantly different from group B in SFI (P lt; 0.05). At 20 and 24 weeks after the operation, group E was significantly different from groups B and C in SFI (P lt; 0.05). At 12 weeks after the operation, electrophysiological detection showed nerve conduct velocity (NCV) of group E was bigger than that of groups B and C (P lt; 0.05), and compound ampl itude (AMP) as well as action potential area (AREA） of group E were bigger than those of groups B, C and D (P lt; 0.05). At 24 weeks after the operation, NCV, AMP and AREA of group E were bigger than those of groups B and C (Plt; 0.05). At 12 weeks after the operation, histological observation showed the area of regenerated nerves and the number of myel inated fibers in group E were significantly differents from those in groups A, B and C (Plt; 0.05). The density and diameter of myel inated fibers in group E were smaller than those in group A (Plt; 0.05), but bigger than those in groups B, C and D (P lt; 0.05). At 24 weeks after the operation, the area of regenerative nerves in group E is bigger than those in group B (P lt; 0.05); the number of myel inated fibers in group E was significantly different from those in groups A, B, C (P lt; 0.05); and the density and diameter of myel inated fibers in group E were bigger than those in groups B and C (Plt; 0.05). Conclusion The tissue engineered nerve with the complex of SCs, ECM gel, bFGF-PLGA sustained release microspheres, PLGA microfilaments and permeables PDLLA catheters promote nerve regeneration and has similar effect to autograft in repair of nerve defects.