| BackgroundsMany researches show that the normal architecture of muscle and the maintenance of its function depend on the contributions of some neurotrophic factors. When motor nerve is damaged, the greatest change of denervated skeletal muscle is the loss of such kind of factors. Meanwhile, skeletal muscle is disused, which finally results in the change of muscles in morphosis and physiological biochemistry. What displays in histomorphology is the cataplasia of end plate, the loss of the muscle cytoplasm and the diminutus of anteroposterior diameter. Now the re-establishment of motor nerve function after its injury centers on the recovery of the succession of nerve while ignores the protection of its target organs. Such factors as the lengthy reactivation process of nerve with re-cataplasia of nerve during this process, the baffling effect of injury region, the immobilization of limbs after its reparation, even if the nerve is promptly repaired, the process that muscles with denervation lasts for a long time, and the trophic action of nerve lost and gradual cataplasia of end plate bring great difficulty to the re-establishment of myoneural junction. In clinic, the features that muscle presents intestini degeneration, transverse striation abolishment, myatrophy, the death and disappearance of muscle are found. Then, the apraxia and atrophy of muscle have influence on the morphology of end plate, which will lead to an infernal circle between them. This is one of the most important factors nowadays which hinder the recovery of function after nerve repair. As a result, how to prevent the cataplasia of end plate and myatrophy becomes an urgent and tough problem nowadays which awaits to be solved urgently in clinic once motor nerve is damaged.However, there are few researches and treatments on how to obviate the cataplasia of end plate. The commonly used treatment in clinic is functional exercise, which can only avoid the disuse of muscle but can not offer neurotrophy. Particularly, in the morning of surgery recovery, the limbs are always fixed which limits the effect of such kind of treatment.Certain scholar once introduced that the excitability of denervated muscles can be replaced by electrical stimulation and a modality of electrical stimulation that using an experimental rat to imitate the excitability of normal end plate was established. And some achievements were obtained. However, the effect of electrical stimulation can not last for a long period and there were many drawbacks. There are also some achievements in the empirical study of drug on the treatment of this aspect in recent years. It is reported that the experiment on rats indicates that Dexamethasone through intraperitoneal injection can alleviate and lessen the consequence that non-mutilation nerve injury leads to the cataplasia of end plate. Clenbuterol by mouth can avoid the cataplasia of denervated end plate and muscular atrophy. Through a certain way, muscle satellite cell can generate such nutritive materials as insulin-like growth factor (IGF), which can imitate the trophic action of nerve to muscles. In addition, NGF and HGF can lessen the cataplasia of end plate. In 1996, initial researches indicated, which was presented by Margaret, that in theâ…¡stage, when nerves were repaired, once carrier of fibrin gel of acid fibroblast growth factor (aFGF) was imbedded into atrophic muscle belly, it could induce the regeneration of end plate but there was no obvious contribution to those whose nerves were not repaired. Such kind of research showed that the contribution of aFGF could be displayed on the condition that the nerve was repaired and whether it could protect denervated end plate was not still be proved. The contribution that aFGF did to the recovery of function was not indicated as well.AFGF can generate extensive biological effects such as enhancing cell multiplication and differentiation and promoting tissue repair. It is also a kind of morphogenetic factor whose target cell includes sarcoblast, collagenoblast, vascular endothelial cell. Angiogenesis can ameliorate microcirculation and can do great endotrophic action to many architectures. Collagenoblast can promote the formation of muscle cell's basal lamina, while basal lamina of skeletal muscle is one of the significant conditions in the formation of myoneural junction. As a result, aFGF plays an important role in promoting the differentiation of collagenoblast which does contribution to the formation of myoneural junction. Meanwhile many researches indicate that the principal cause of muscular atrophy is tunica muscularis suffers lipide peroxidation which also has effect on end plate. As a result, to enhance the formation of basal lamina will also enhance the replenishment of those impaired hymeno-structures. Other research indicates that aFGF can accommodate the affluence and effluence of Ca2+ in intra-cellular, which will enhance the transformation of xanthine oxidase from xanthine dehydrogenase and then eliminate free radical. Accordingly, those hymeno-structures such as end plate can be protected through the elimination of free radical. To hasten myoblastic differentiation will form muscle cell and differentiate myotube and thereby prevent muscle from myatrophy. AFGF can improve the blood circulation through the action of vascular endothelial cell.There is still no report shows that aFGF can prevent the cataplasia of end plate and myatrophy after motor nerve injury. This study was undertaken to probe the effect on the prevention of the cataplasia of denervated end plate and myatrophy by aFGF and its mechanism of action.Objectives1. To establish aFGF slow-releasing system which providing denervated end plate and muscle with substitution of neurotrophy.2. To establish the animal model of preventing cataplasia of denervated end plate and myatrophy with fibrin gel of aFGF.3. To approach the morphological and functional effect that how fibrin gel of aFGF prevents cataplasia of denervated end plate and myatrophy.4. To approach initially the mechanism of action that how fibrin gel of aFGF prevents cataplasia of denervated end plate and myatrophy.Materials and Methods1. The preparation of aFGF slow- releasing system: liquorâ… : cryodesiccant human fibrinogen and aprotinin were dissolved into aqua pro injectione directly with concentration as 60mg.ml-1 and 3000 KIU.ml-1 respectively; liquorâ…¡: 40mmol.L-1CaCL2 zymoplasm with concentration as 250 U.ml-1. Admixed aFGF 50μg with 0.5ml liquorâ… and add it with equal volumn liquorâ…¡by mixing thoroughly. Put this admixture into tooting and got a cylindrical, semitransparent ivory compound. Fibrin gel was prepared by admixed liquorâ… with equal volumn liquorâ…¡.2. Establishing and grouping animal models: 155 adult inbred line Sprague-Dawley rats were selected and abscised their right tibial muscle branch of the common peroneal nerves (the distance between abscising point and the point where nerve entering into muscle is 1cm) with complete anesthesia which was through 10% chloral hydrate through intraperitoneal injection. The models were randomly divided into five groups. Group NR+aFGFgel: those that epineuriums were sutured and carriers of Fibrin gel of aFGF (25μg aFGF per rat)were imbedded in tibial muscles. Group NR+gel: those that epineuriums were sutured and carriers of fibrin gel were imbedded in tibial muscles. Group aFGFgel: those that epineuriums were not sutured and carriers of fibrin gel of aFGF (25μg aFGF per rat)were imbedded in tibial muscles. Group NR: those that only epineurium were sutured. Group Blank: those that epineuriums were not sutured and no implant was imbedded in tibial muscles. Group NR+gel, Group NR and Group Bland were control groups. All rats were observed for 6 weeks postoperation.3. Morphological research: Observe the appearance of the muscle through morphological method with naked eyes and measure humid weight of tibial muscle. Staining with auric chloride was deployed to display end plate. Ultrathin sections were stained with uranium and lead and the ultrastructure of end plate was observed by transmission electron microscope. Calculate end plates and the percentage of cataplasia of denervated end plate in five groups and analyze it through statistical method. Observe the degradation of fibrin gel of aFGF and those possible adverse effects.4. Functional research: Karnovsky-Roots copper-ferrocyanide was employed to display AchE of end plate and observe the change of AchE in end plate of each group. The count of cataplasia of end plate: take randomly 30 end plates in each group and count the quantity of end plates which were with subdued activity through staining with AchE and the statistical analysis was conducted. Image analysis: 10 end plates of each staining section were intaken randomly through PC camera with 400X microscope. Image analyzer was employed to observe the staining area of AchE and gradation. And statistical analysis on average results was conducted. Machine of evoked potential electromyogram was deployed to detect and stimulate ischiadic nerve through low frequency RNS and myoelectric wave amplitude of tibial muscle was recorded. Stimulate it repetitively for 9 times with the frequency from 3 to 5Hz pulsating current and observe whether there is decrement or not. Masculine was based on the fact that if the fifth myoelectric wave amplitude is lower 10% than that of the first one. Statistical analysis on the average decrement rate of the fifth myoelectric wave amplitude was conducted.5. Mechanism research: Observe the morphology of muscle satellite cell of tibial muscle around end plate in each group through transmission electron microscope. Observe randomly 20 ultrathin sections randomly and count the quantity of muscle satellite cells and statistical analysis about it was conducted. Observe the masculine expressing area of aFGF receptor (aFGFR) through immunohistochemiscal staining. Blood vessel masculine expressing of aFGFR was counted and average aFGFR gradation of capillary vessel was measured with image analyzer.6. Statistical analysis of the data was performed using the SPSS 13.0 software package(Department of Biostatistics, FIMMU). The measurement data were analyzed by one-way ANOVA and multiple comparison was performed by LSD. The numeration data were analyzed by Chi-square. The significant level of difference is 0.05.Results1. Morphological observation with naked eyes: group NR+aFGFgel: through own control, it could be observed that there was symmetry between bilateral tibial muscles and normal muscular tension while no manifest muscular atrophy in this group. There were similar performances in group NR+gel and NR: there was manifest muscular atrophy in the right tibial muscles and descending muscular tensions. Group aFGFgel: there was slight muscular atrophy in the right tibial muscle but not so manifest as that in group NR+gel and NR. There was no muscular tension. Group Blank: There was manifest muscular atrophy in the right tibial muscle and no muscular tension in this group. There was no abnormal reaction in which fibrin gel was emdedded in group NR+aFGFgel, group NR+gel and group aFGFgel.2. The humid weight of tibial muscle of group NR+aFGFgel was 1.26+0.10g, and that of group aFGFgel was 1.20±0.12 g. The humid weight of tibial muscle of group NR+aFGFgel and group aFGFgel was heavier than that of control groups. And statistical analysis on this showed predominant significance.3. Chromoscopy of end plate: majority end plates in group NR+aFGFgel were with integrated structure and normal morphology. There were similar performances in group NR+gel and group NR: the morphology of end plates in denervated muscle fiber was anomalistic. The branched ending of axon was decreasing and tangling as well. End plate was in the form of striga or punctiform. Group aFGFgel: no axon was observed, the morphology of majority end plates was basically normal, only the end plates region reduced. Group Blank: no axon was observed, the quantity of end plate decreased manifestly and the morphology of end plate was anomalistic.4. Electron microscope observation: there were great amount of end plates in group NR+aFGFgel and aFGFgel and their architecture was fundamentally normal. Few cataplasia of end plates were observed. The quantity of end plate in control groups decreased obviously and the architecture of end plates was cataplasia. The amount of end plates of group NR+aFGFgel was 12.00±2.24, and that of group aFGFgel was 11.40±1.14. The amount of end plates of group NR+aFGFgel and group aFGFgel was higher than that of control groups, and statistical analysis on this showed predominant significance. The cataplasia percentage of end plates of group NR+aFGFgel was 26.67% and that of group aFGFgel was 33.33%. The cataplasia percentage of end plates of group NR+aFGFgel and group aFGFgel was lower than that of control groups, and statistical analysis on this showed predominant significance.5. RNS detecting results: There was no obvious decrement of myoelectricity wave in group NR+aFGFgel when there was low frequency RNS. The decrement rate was lower than 10% and no masculine sample was observed. While there was obvious decrement of myoelectricity wave in group NR+gel and group NR when there was low frequency RNS. The masculine rate was 90% in these two groups. The decrement rate of group NR+aFGFgel was 5.70±3.13%, which was lower than that of control groups by statistical analysis.6. AchE activity of end plate: end plates in group NR+aFGFgel and aFGFgel were stained thickly, which indicates that the acitivity of AchE was strong. The architecture of end plates was normal. Ache staining area of the end plates in group NR+aFGFgel was 1693.98±168.161μm2, and the gradation was 24.81±1.44. AchE staining area of the end plates in group aFGFgel was 1520.60±303.00μm2, and the gradation was 24.93±1.54. The acitivity of AchE was stronger than that of control groups by statistical analysis. The rate of cataplasia of end plate in group NR+aFGFgel was 20.42% and that in group aFGFgel was 24.17%,which was lower than that of control groups by statistical analysis.7. Observation of MSC: the volume and the amount of MSC in group NR+aFGFgel and group aFGFgel increased. The amount of MSC in group NR+aFGFgel was 11.00±3.00 and that in group aFGFgel was 10.40±1.14, which was higher than that of control groups by statistical analysis.8. Immunohistochemical observation: masculine expressing area of aFGFR mainly existed in capillary vessel wall. Observation showed that high density of expressing area of aFGFR was in group NR+aFGFgel and group aFGFgel. Meanwhile there were intensive capillary vessels in these two groups. Observation also showed that low density of masculine expressing area of aFGFR in capillary vessel was in control groups. And there were rare capillary vessels in control groups. The masculine percentage of aFGFR expression of capillary vessels of group NR+aFGFgel was 87.50%, that of group aFGFgel was 85.00%, which was higher than that of control groups by statistical analysis. The aFGFR gradation of capillary wall was 25.92±1.55 in group NR+aFGFgel, that was 26.85±1.23 in group aFGFgel, which indicated that the expressing amount of aFGFR in capillary wall was higher than that of control groups by statistical analysis.Conclusions1. Experiment confirmed that fibrin gel of aFGF could be deployed as the drug slow-releasing system which providing denervated end plate and muscle with substitution of neurotrophy and there was no abnormal reaction.2. Combining nerve repair and the protection for end plate with fibrin gel of aFGF could manifestly prevent end plate from cataplasia and avoid myatrophy in rats.3. Even if the nerves were not repaired, fibrin gel of aFGF could still protect end plate effectively and avoid myatrophy.4. RNS detection for conduction of neuromuscular junction indicated that when there was motor nerve injury, combining nerve repair and the protection for end plate with fibrin gel of aFGF could effectively improve the conduction of neuromuscular junction.5. Observation of the activity of AchE through enzymohistochemical method indicated that when there was motor nerve injury, combining nerve repair and the protection for end plate with fibrin gel of aFGF could effectively recover the activity of AchE of end plate and enhance normal metabolism of neurotransmitter and transmission of nerve impulse.6. Those groups with fibrin gel of aFGF were with much more muscle satellite cells than those control groups without fibrin gel of aFGF. It indicated that the function of preventing end plate from cataplasia and avoiding myatrophy by fibrin gel of aFGF correlated with the proliferation of muscle satellite cell.7. For those groups with fibrin gel of aFGF, there were more blood vessels with abundant transportation of blood around end plates. While for those without fibrin gel of aFGF, there were less blood vessels and bad microcirculation. This indicated that aFGF could enhance the formation of blood vessel manifestly and improve microcirculation.8. For those groups with fibrin gel of aFGF, the percentage of masculine expressing of aFGFR in capillary vessel and the amount of expressing of aFGFR were much higher than that of those groups without fibrin gel of aFGF. This further indicated that fibrin gel of aFGF prevented end plate from cataplasia and avoided myatrophy correlated to its action to capillary vessel. Through improving the function of microcirculation around end plate, the neural trophic action of denervated muscles was redeemed.9. Through observing the morphous of muscle satellite cells and the microcirculation around end plate, this experiment deemed that one effective way to prevent devervated end plate from cataplasia and myatrophy was to provide end plate and muscles with substituting neurotrophy.
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