OBJECTIVE To observe the degeneration and regeneration of the Meissner’s corpuscles after implanted sensory nerve into the denervated monkey’s fingers under electron microscope. METHODS The two finger nerves of the monkey’s fingers were denervated. Afterwards, one finger nerve was cut off, and the other was reimplanted into the denervated finger. After 1, 3, 5, 8 and 12 months, the finger skin was cut off and observed under electron microscope. RESULTS The degenerative changes of nerve ending in Meissner’s corpuscles were observed after 1 month of denervation, and the basic structure of the corpuscles had no obvious changes. After 3 months, the axons of corpuscles were disappeared, and the volume of corpuscles was shrunk. The basic structure of nerves was disappeared, and the lemmocyte and neurolemma plate were changed after 5 months. The collagen fibrils in the corpuscles were gradually increased in 8 months, the endoneurial structure and interneurial matrix were completely disappeared and replaced by collagen fibrils in 12 months. After 3 months of nerve implantation, unmyelinated nerve fibers were appeared and grew into the corpuscles. A part of corpuscles innervated in 5 months. Most of corpuscles innervated and myelinated nerve fibers were observed in 8 months. And in 12 months, corpuscles innervated to normal level. CONCLUSION The implantative sensory nerve by means of reinnervating the original corpuscles and regenerating new corpuscles could innervate the degenerative Meissner’s corpuscles.
ObjectiveTo investigate the expression pattern and significance of Sonic Hedgehog (Shh) signaling pathway by observing whether the Shh signaling pathway components express in the adult rat after spinal cord injury (SCI).
MethodsSixty-four healthy male Sprague-Dawley rats were randomly divided into normal group (group A, 8 rats), sham group (group B, 8 rats), and SCI group (group C, 48 rats). In group A, the rats served as controls without any treatment; a decompressive laminectomy was performed on T7-9 levels without SCI in group B; and modified Allen's method was used to make SCI model in group C. Basso Beattie Bresnahan (BBB) scale was used to assess the hind limb motor function at 12 hours, 1 day, 3 days, 7 days, 14 days, and 21 days after SCI; the immunofluorescence staining, real-time PCR, and Western blot were performed to detect the mRNA and protein expression levels of Shh and Glioma-associated oncogene homolog-1 (Gli-1) in SCI zone.
ResultsThe BBB score slowly increased with time in group C, but the scores at each time point in group C were significantly lower than those in group A and group B (P<0.05). The results of immunofluorescence staining showed that Shh and Gli-1 rapidly increased after SCI in astrocytes. Real-time PCR and Western blot showed that the relative expression levels of Shh and Gli-1 mRNA and protein were gradually increased in group C and reached a maximum at 7 days. In addition, the relative expression levels of Shh and Gli-1 mRNA and protein in group C were significantly higher than those in group A and group B (P<0.05). On the other hand, compared with group A, the expression of Gli-1 protein was reduced in the cytoplasm but increased in nucleus in group C.
ConclusionAstrocytes synthesize and secrete Shh and Gli-1 signaling molecules after SCI, both Shh and Gli-1 significantly up-regulate and exhibit dynamic changes, which suggests Shh signaling pathway may be involved in nerve cell regeneration after SCI.
Objective To develop a technique that can directly demonstrate collateral sprouting of intact nerve fibers at endtoside neurorrhaphy site. Methods Five Wistar adult rats were used in this study. The common peroneal nerves at one side were sectioned at the level of knee joint, and their distal ends were sutured to the tibial nerves after removal of a 1 mm-diameter window in the epineurium. Three months after the operation, the nerve segments at neurorrhaphy site and the normal tibial nerves at the contralateral site were harvested. The specimens were fixed in 10% formaldehyde and postfixed in 1% osmium tetroxide, thenmacerated in glycerol. Single fiber was teased out in pure glycerol under an operative microscope, then transferred to a slide and observed under light microscope. The nerve segments at neurorrhaphy site and distal peroneal nerves were alsoharvested for histological evaluation. Results At the neurorrhaphy site, small nerve fibers sprouted from a donor nerve fiber near node’s of Ranvier. While such phenomena were not found in normal tibial nerve. From the longitudinal sectionof neurorrhaphy site, bundles of nerve fibers ranged from tibial nerve to peroneal nerve were observed. Lots of regenerative nerve fibers emerged in distal peroneal nerve. Conclusion The phenomena of collateral sprouting at end-to-side neurorrhaphy site can be demonstrated directly by nerve fiber micro-tease technique.
Objective To investigate the effect of extract of ginkgo biloba leaves (EGb50) on the prol iferation of SCs cultured in vitro. Methods The SCs were isolated from 3-day-old SD rats’ sciatic nerves by the method of enzyme gradationdigestion (n=20) and the purified 2nd passage of SCs were divided into 2 groups: the experimental group, in which SCs were cultured in FBS-DMEM medium with EGb50 (terminal concentration: 50 μg/mL); the control group, in which SCs were cultured in the FBS-DMEM medium without EGb50. The absorbance (A) value was detected by the 2, 3-bis- (2-methoxy-4-nitro-5- sulfophenyl)-2H-tetrazol ium-5-carboxanil ide (XTT) method 1, 3, 5, 7 and 9 days after culture, then the growth curves was drawn. Cell cycle was detected by flow cytometry (FCM). Disintegration per minute (DPM) of SCs was detected by the method of 3H-thymine nucleoside (3H-TdR) 2 and 3 days after culture and nerve growth factor (NGF) synthesis in SCs culture media was detected by ELISA method. Results Most SCs were spindle-shaped with a purity above 90%. XTT detection showed that A value of SCs in the control group was gradually increased 3 days after culture, reached the peak 5 days after culture and gradually decreased from then; the A value in the experimental group experienced the similar changes, but it was higher than that in the control group at each time point (P lt; 0.01). 3H-TdR showed that the DPM of the experimental group was 1 961.78 ± 231.13 and 4 601.51 ± 605.08 at 2 and 3 days after culture, while for the control group, the A value was 1 347.15 ± 121.57 and 3 740.42 ± 158.73 at the same time point, indicating a significant difference between two groups (P lt; 0.01). FCM observation indicated that the SCs prol iferation index of the experimental group and the control group was 18.6% ± 3.2% and 9.7% ± 2.9%, indicating a significant difference between two groups (P lt; 0.01). ELISA observation showed that the NGF concentration in the experimental and the control group was (0.065 6 ± 0.003 9) ng/mL and (0.038 6 ± 0.003 6) ng/mL, indicating a significant difference (P lt; 0.01). Conclusion EGb50 is capable of enhancing the prol iferation of SCs cultured in vitro, which may be one of the important mechanisms to promote peripheral nerve regeneration.
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 explore the possibility to bridge peripheral nerve defects by xenogeneic acellular nerve basal lamina scaffolds. METHODS: Thirty SD rats were randomly divided into 5 groups; in each group, the left sciatic nerves were bridged respectively by predegenerated or fresh xenogeneic acellular nerve basal lamina scaffolds, autogenous nerve grafting, fresh xenogeneic nerve grafting or without bridging. Two kinds of acellular nerve basal lamina scaffolds, extracted by 3% Triton X-100 and 4% deoxycholate sodium from either fresh rabbit tibial nerves or predegenerated ones for 2 weeks, were transplanted to bridge 15 mm rat sciatic nerve gaps. Six months after the grafting, the recovery of function was evaluated by gait analysis, pinch test, morphological and morphometric analysis. RESULTS: The sciatic nerve function indexes (SFI) were -30.7% +/- 6.8% in rats treated with xenogeneic acellular nerve, -36.2% +/- 9.7% with xenogeneic predegenerated acellular nerve, and -33.9% +/- 11.3% with autograft respectively (P gt; 0.05). The number of regenerative myelinated axons, diameter of myelinated fibers and thickness of myelin sheath in acellular xenograft were satisfactory when compared with that in autograft. Regenerated microfascicles distributed in the center of degenerated and acellular nerve group. The regenerated nerve fibers had normal morphological and structural characters under transmission electron microscope. The number and diameter of myelinated fibers in degenerated accellular nerve group was similar to that of autograft group (P gt; 0.05). Whereas the thickness of myelin sheath in degenerated accellular nerve group was significantly less than that of autograft group (P lt; 0.05). CONCLUSION: The above results indicate that xenogeneic acellular nerve basal lamina scaffolds extracted by chemical procedure can be successfully used to repair nerve defects without any immunosuppressants.
Objective To investigate the effect of tetramethylpyrazine (TMP) with a certain concentration added to vitrification solution on peripheral nerve allografts regeneration. Methods Forty-eight healthy clean SD male rats were selected as donors, and 96 healthy clean Wistar male rats as recipients, all rats being 3 months old and weighing 200-250 g. The sciatic nerves segments of 15 mm were removed from the donors, then randomly divided into 4 groups according to vitrificationsolution containing TMP. No TMP was used in group A as the control group; 100 mg/L, 200 mg/L and 400 mg/L TMP were used in group B, group C and group D, respectively. Then them were cryo-preserved at — 196 ℃ for 3 weeks. Nerve defect of 10 mm in length was made in the sciatic nerves of recipients. After rewarming, the allografts were transplanted to the corresponding rats. The gross appearance, the morphological and electrophysiological changes, the image analysis of axons and motor end-plate were detected at 4, 8, 12 and 16 weeks. Results All rates survived to the end of the experiment. The adhesion and edema of allografts in group A and group B were obvious 4 weeks after operation; then adhesion and edema was obvious in group A and were improved in the other groups 8 weeks after operation. Adhesion was observed in groups A and B; no adhesion was observed in groups C and D at 12 weeks. The number of regeneration nerve, the latent, the ampl itude, the nerve conduction velocity, the medullary sheath/μm2, the medullary sheath density/μm2 and the image analysis of axons and motor end-plate in groups A and B were significantly lower than those in groups C and D (P lt; 0.01); and there were no significant differences between groups C and D (P gt; 0.05). The observation of transmission electron microscope showed that medullated nerve fibers and myel in sheath of groups C and D were thicker than groups A and B, layers of groups C and D were clear. Conclusion The vitrification solution with 200 mg/L tetramethylpyrazine has protective effect on regeneration of peripheral nerve allografts．
Objective To study the biological activities ofthe nerve regeneration conditioned fluid (NRCF), especially to further separateand identify the protein bands of the relative molecular mass of (232-440)×103. Methods The silicone nerve regeneration chambers were implanted between the cut ends of the sciatic nerve in 6 New Zealand white rabbits (weight, 1.8-2.5 kg). The proteins in NRCF were separated by the native-polycrylamide gel electrophoresis (Native-PAGE), the protein bands of the relative molecular mass of (232-440)×103 were analyzed by the Shotgun technique, liquid chromatography, and mass spectrometry. Results The Native-PAGE result showed that there was 1 protein band of the relative molecular mass over 669×103, (232-440)×103 and (140-232)×103,respectively, and 6 bands of the relative molecular mass of (67-140)×103.Besides, 54 proteins were identified with at least 2 distinct peptides in 1 protein band of the relative molecular mass of (232-440)×103, including 4 unnamed protein products, mainly at the isoelectric points of 5.5-8.0 and of the relative molecular mass of (10-40)×103. Based on their functions in the protein database, allthe identified proteins in this study were classified into the following 5 groups: conjugated protein (43%), transport protein (30%), enzyme (6%), signal transducer (4%), and molecular function-unknown protein (17%). At the subcellular localization of the identified proteins, there was mainly a secreted protein (63%), and the remaining proteins were localized in the membrane and cytoplasm. Conclusion Native-PAGE and the Shotgun technique can effectively separate and identify proteins from NRCF, and can identify the components of the protein band of the relative molecular mass of (232-440)×103 and provide basicinformation on the unnamed protein products in NRCF.
Objective To investigate the neuroprotective effects of recombinant adeno-associated virus (rAAV) expressing vascular endothel ial growth factor (VEGF) on traumatic spinal cord injury (SCI) of rat and its mechanisms. Methods The 144 male Sprague Dawley rats were randomly divided into 4 groups, and each group contained 36 rats. The rats in sham group (group A) received dorsal laminectomy without SCI and microinjection, the rats in model control group (group B), rAAV-green fluorescent protein (GFP) group (group C), and rAAV-hVEGF165-GFP group (group D) received dorsallaminectomy with SCI and injection of 20 μL sal ine, rAAV-GFP viruses, or rAAV-hVEGF165-GFP viruses, respectively. At 3 and 7 days after operation, Basso-Beattie-Bresnahan (BBB) score was used to evaluate the neurologic function. At 7 days after operation, Nissl’s body staining was used to evaluate the histopathological changes; apoptosis was confirmed by transmission electron microscope examination and TUNEL staining; the expression of aquaporin 4 (AQP-4) was detected by Western blot assay. At 1, 3, 5, and 7 days, ELISA assay was used to detect the VEGF165 protein expression. Results According to BBB scores, the neurologic function in group D was significantly better than those in groups B and C at 3 and 7 days after operation (P lt; 0.05). Nissl’s body staining showed that tissue damage in group D was significantly milder than those in groups B and C at 7 days after operation (P lt; 0.05). ELISA results showed that VEGF165 protein expression was slowly-released in low dose in group D, and the expression in group D was significantly higher than that in groups A, B, and C at 3, 5, and 7 days after operation (P lt; 0.05). The results of transmission electron microscope and TUNEL staining showed that apoptosis rate of spinal cord neurons in group D was significantly lower than that in groups B and C at 7 days after operation (P lt; 0.05). The results of Western blot showed that AQP-4 expression in group D was significantly decreased when compared with that in groups B and C at 7 days after operation (P lt; 0.05). Conclusion TherAAV expressing VEGF has neuroprotective effects by inhibiting apoptosis of spinal cord neurons and relieving spinal cord edema.
Objective To investigate the influence of nerve growth factor (NGF) on neuroal regeneration of somatovisceral heterogenic reinnervation using a rat phrenic-to-vagus anastomosis model. Methods Forty male SD rats, aging 3 months and weighing 200 g, were selected and randomly divided into 3 groups. In group A (n=10, control group), phrenic and vagusnerves were exposed and no neurorraphy was performed. In group B (n=15) and group C (n=15), both nerves were transected and proximal stump of phrenic nevers were microsurgically anastomosed to the distal stump of vagus nerves. Postoperatively, group C was intraperitoneally injected with NGF (20 μg/kg·d), while groups A and B were given matching sal ine solution. Twelve weeks later, cardiac function was examined under electrical stimulation of the regenerated nerve. Light and electron microscopies were used to examine the heterogenic regenerated nerve, and the passing rate of axon and thickness of myel in sheath were calculated. Results Under electrical never stimulation in groups A, B, and C, the decreases of blood pressure were (20.12 ± 2.57), (10.63 ± 2.44), and (14.18 ± 2.93) mmHg (1 mmHg=0.133 kPa), respectively; and the decreases of heart rate were (66.77 ± 9.96), (33.44 ± 11.82), and (43.27 ± 11.02)/minutes, respectively. In group B, the decrease ampl itudes of blood pressure and heart rate were 52.83% and50.08% of group A, respectively. Blood pressure and heart rate in group C also decreased dramatically; the decrease ampl itudes of blood pressure and heart rate in group C were 70.48% and 64.80% of group A. There were significant differences in the decrease ampl itudes of blood pressure and heart rate (P lt; 0.05) between group B and group C. Morphological observation showed that heterogenic nerve fibers had the structure of matured myel in sheath and their axons could regenerate into the vagus nerve. In group B and group C, the passing rates of axon were 66.83% ± 4.46% and 81.63% ± 3.56%, respectively; and the thicknesses of myel in sheath were (0.25 ± 0.10) μm and (0.46 ± 0.08) μm, respectively; showing significant differences (P lt; 0.05) between group B and group C. Conclusion Heterogenic nerve is primarily a somatic motor nerve; NGF can promote the axons of heterogenic nerve to regenerate into the parasympathetic nerve.
