| Part I Effects of locally application of C/GP-NGF hydrogel in autologous vein conduit on the repair of facial nerve defect in a rat modelObjective:Recently, biodegradable nerve conduits have been widely used in research on the repair of peripheral nerve defects, but the results with these methods are still far from satisfactory. In this study, we investigated the effects of chitosan as a drug delivery vehicle and scaffold in the autologous vein conduit on the recovery of damaged facial nerve in a rat model.Materials and methods: Fresh chitosan-?-glycerophosphate (C/GP) hydrogel and chitosan-?-glycerophosphate-nerve growth factor (C/GP-NGF) hydrogel were made and observed in vitro. The microstructures of C/GP hydrogel and C/GP-NGF hydrogel were observed with scanning electron microscope. The NGF released from C/GP-NGF hydrogel was tested by ELISA kit at 12 h,1 d,3 d,5 d,7 d,9 d and 12 d, and the NGF release curve was fabricated. The animal experiment involved 75 adult Wistar rats (220 g-250 g) which were randomized divided into five groups (n=15 in each group). The right buccal branches of the animals' facial nerves were transected leaving a 5 mm defects in length. In group A (Auto group), the buccal branch was reconstructed by autologous nerves. In group B to E, the buccal branch was reconstructed with autologous veins, and fresh C/GP-NGF hydrogel (C/GP-NGF group), NGF solution (NGF group), blank C/GP hydrogel (C/GP group), or PBS (PBS group) was injected into the vein conduit. The left buccal branches were exposed without other treatment, which were used as control. At the 4th,8th, and 12th weeks after the operation, we analyzed the vibrissae movement grade and the compound muscle action potentials (CMAPs) of each rat to assess the function of regenerated nerves. At the 4th,8th, and 12th weeks after operation, the morphological changes of regenerated nerves were also observed by modified trichrome staining, and the matureness of regenerated nerves was evaluated by toluidine blue staining and transmission electron microscope. Additionally, the Tubulin and S-100 double immunofluorescent staining were performed to assess the regenerated nerves.Results:The in vitro results:The C/GP hydrogel was solution at room temperature, and turned into gel after coated under 37? for 30min. The C/GP hydrogel and C/GP-NGF hydrogel were porous showed by the scanning electron microscope. The NGF was sustainedly released from C/GP-NGF hydrogel for more than 12 days. The in vivo results:The right vibrissae movement of rats was completely lost after the operation. Better recovery of vibrissae movement was obtained in the Auto group and the C/GP-NGF group, and no significant differences were seen between these two groups (p> 0.05). The recovery rates of the vibrissae movement in the Auto group and C/GP-NGF group were significantly faster than that in the PBS group (p< 0.05). The recovery rates of the vibrissae movement in the NGF group and C/GP group had no significant differences compared to those in the PBS group (p> 0.05). At the 4th week after operation, CMAPs could only be recorded in the Auto and C/GP-NGF groups. However, at the 8th and 12th weeks after operation, the CMAPs could be recorded form the regenerated buccal branch in all groups. The amplitude of CMAPs and nerve conduit velocity in the C/GP-NGF group were similar to those in the Auto group, and significantly faster than those in the NGF group, C/GP group and PBS group. Histological results showed that more mature regenerated axons were achieved in the C/GP-NGF group than in the NGF group, C/GP group and PBS group. Besides, most of the chitosan hydrogel in the vein conduit has been biodegraded four weeks following operation. The toluidine blue staining showed that larger nerve fibers, thicker myelin sheath and higher density of fibers were observed in the C/GP-NGF group compared to those in the C/GP group, NGF group and PBS group (p< 0.05). The recovery of facial nerves in the NGF and C/GP groups was not superior to those in the PBS group. The regenerated fibers were positively stained by Tubulin and S-100. In addition, Tubulin-positive density was also significantly greater in the C/GP-NGF group than in the negative control groups (p< 0.05).Conclusion:NGF steadily released by the C/GP-NGF hydrogel in the vein conduit can improve the recovery of peripheral facial nerves. C/GP hydrogel was a good drug release vehicle, and might serve as a scaffold in the vein conduit. However, C/GP hydrogel or NGF alone in autologous vein was insufficient to enhance the regeneration of facial nerves.Part II The injury of marginal mandibular branch unexpectedly promotes the repair of buccal branch of facial nerve in a rat modelOBJECTIVE:Peripheral facial paralysis is a common disease in clinic. So far, the functional recovery of facial nerve injury is still far from satisfied, especially those with facial nerve defects. The buccal branch of facial nerve in rat is commonly used for facial nerve reconstruction research. In order to evaluate the recovery of facial nerve function by the vibrissae movement of rat, the marginal mandibular branch was also damaged in many studies. However, whether the injury of the marginal mandibular branch has any effect on the recovery of the buccal branch has not been reported. The aim of this study was to investigate whether the injury of the marginal mandibular branch affects the repair of facial nerve buccal branch in a rat model.STUDY DESIGN:This study involved 36 adult Wistar rats (220g-250g), and randomized divided into two groups. In group A, the buccal branch of the facial nerve was transected and reconstructed with vein conduit, the marginal mandibular branch was simultaneously damaged; in group B, the buccal branch of the facial nerve was transected and reconstructed with vein conduit, particularly, the marginal mandibular branch was intact. At the 4th,8th and 12th weeks after operation, the vibrissae movement of rat was assessed. At the 8th and 12th weeks after operation, electrophysiological tests were performed to assess the functional recovery of the buccal branch. The morphological changes of injured buccal branches were analyzed by modified trichrome staining, and the matureness of regenerated nerves was evaluated by toluidine blue staining. Additionally, the Tubulin and S-100 double immunofluorescent staining were performed to assess the regenerated nerves. At the 12th week after operation, the levator labii superioris muscle was harvest, and Tubulin and alpha-bungarotoxin were used to mark the neural muscle junctions (NMJs). Additionally, the DiI and DiD were used to trace the motoneurons of the buccal branch and marginal mandibular branch in the facial nerve nucleus.RESULTS:In group A, vibrissae movement of rats was eliminated after the operation, and it gradually recovered since the third or fourth week after repair. At the 12th week after operation, the average vibrissae movement grade of rats in group A was 2.66± 0.50. However, in group B, the right vibrissae movement of rat was similar to the health side after operation. Compound muscle action potentials (CMAPs) were recorded from regenerated buccal branches in group A since the 8th week following operation, but no CMAPs could be recorded in group B at each time point. The amplitude and latency of CMAP in group A were worse than those measured in intact nerves, and there was significantly difference (p< 0.05). More mature regenerated fibers were observed on the sections in group A than those in group B displayed by modified trichrome staining. Furthermore, larger fibers, thicker myelin sheath, and greater number of regenerated fibers were observed on the sections of regenerated nerves in group A than in group B (p< 0.05). The myelinated fibers in the sections were specially stained by Tubulin and S-100. The density of positive Tubulin spots in group A was significantly higher than in group B. The NMJs immunofluorescent staining showed that new NMJs were regenerated in the levator labii superioris muscles in group A, and the formation of the NMJs were normal. However, abnormal NMJs were formed in the levator labii superioris muscles in group B. The retrograde tracing results showed that there were motoneurons in all groups stained by DiI or DiD. In group A, the number of motoneurons stained by DiI was significantly increased (p< 0.05), while, the number of motoneurons stained by DiD was significantly decreased (p< 0.05). However, in group B, the number of motoneurons stained by DiD was significantly increased (p< 0.05), and the number of motoneurons stained by DiI was similar to those in the control group (p> 0.05).CONCLUSIONS:Our results indicate that the injury of the marginal mandibular branch improved the recovery of buccal branch in a rat model. |
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