| A series of degeneration reaction will happen about pericaryo-n,axon,medullary and effector after injury of peripheral nerve. Walleriandegeneration distal end of anon and retrogr degeneration of subterminal axonwill happen within several hours after completely mutilation of axon. Thecataplasia death of neutron cell and atrophy of skeletal muscle were unavoidablyhappen before nerval relegation. so the key point of therapy is to protect neuroncell,prevent atrophy of skeletal muscle and promote regeneration of peripheralnerve. people have find that neurotrophic factors have such effect with thedevelopment of molecular biology, among these insulin-like factor-1(IGF-1) isinvestigative object for everyone 10 years recently. but main fields of researchnow are central nervous system, the study reports about effects of peripheralnerve were not much.On the basis of the experimental studies on IGF-1 at home and abroad, weknew well the biological characteristics of IGF-1 through looking up the relatedliteratures. IGF-1 is a biological multiactivity polypeptide composed of 70 aminoacids, which has the effect on endocrine, autocrine and paracrine and central andperipheral nerves, and has the neurotrophic and neuroregulatory effects. IGF-1,after bonding to the IGF-1 receptor on the surface of the neuron in the injuredpart, can improve the survival of neurons. Previous studies found that IGF-1 hasthe neurotrophic and protective effect on various central and peripheral nervecells, capable of affecting the growth, survival and differentiation of nerve cells;IGF-1 can promote regeneration of the injured facial nerve. It has been reportedthat IGF-1 has been used for clinical treatment of diabetic pathological changesof peripheral nerve. Viral vector brain-derived neurotrophic factor (BDNF),ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor(GDNF) and others injected into the part near the injured sciatic nerve can beexpressed in the Schwann cells and reduce the death of motor neurons. So wespeculated that in vivo transfection of the IGF-1 gene may be feasible and itcould protect motor neurons, inhibit the atrophy of the skeletal muscle andpromote the regeneration of the sciatic nerve through axoplasmic transport.Based on the small gap theory of nerve regeneration, an appropriate gap canuse the trophism and chemotaxis of nerve regeneration to solve the phenomenonof malpositioned nerve growth, giving more accurate connection. In thisexperiment, a 3-mm-long nerve regeneration chamber was caused by sciaticnerve crush injury in the rat and the liposome mediated hIGF-1 plasmid wasinjected into the nerve regeneration chamber via the epineurium. According toliteratures, the plasmid generally stays in the body not more than 28 d, so theimmunohistochemical method was used to detect the expression of IGF-1 in thespinal cord motoneuron and gastrocnemius at different times (2d, 7d, 14d and28d) in this experiment to understand whether IGF-1 could be ingested by theinjured nerve axon and transported to the spinal cord motoneuron andgastrocnemius through axoplasm and the time law of expression;and understandmotor neuron degeneration and changes in the tigroid body in the cytoplasmthrough special staining of the spinal cord. 28d after transfection, the number ofapoptosed motor neurons was observed to understand whether the motor neuronsdied or apoptosed by the immunohistochemical method. 56 d after transfection,histological and ultrastructural observations of the spinal cord tissue and theregenerated nerve fiber of the sciatic nerve were made to understand the role ofhIGF-1 in promoting peripheral nerve regeneration and protecting the motorneuron;and histological and electrophysiological observations were made tounderstand the role of IGF-1 in inhibiting the atrophy of skeletal muscles. Nerverecovery was observed through the determination of the sciatic functional index(SFI). The experimental results were analyzed and the possible actionmechanism of IGF-1 behind the promotion of peripheral nerve regeneration,inhibition of the atrophy of skeletal muscles and protection of the motor neuronwas examined.After transfection of IGF-1 gene, the footprints of the rats at different timeswere measured and the sciatic functional indexes at different times werecalculated. The results showed that SFI in the IGF-1 treated group recoveredbetter than that in the model group and the blank control group, indicating thathIGF-1 has the effect of promoting the recovery of the sciatic nerve function.The results from the detection of hIGF-1 expression in the spinal cord andgastrocnemius at different time points confirmed that IGF-1 can be ingested bythe injured sciatic nerve axon and reached the tissues of the spinal cord andgastrocnemius via current and countercurrent of axoplasm and brought into play.We speculated that the hIGF-1 plasmid was ingested through some kinds of cellmembrane transport especially pinocytosis possibly because injury increased thepermeability of the nerve cell membrane.The results of the determination of hIGF-1 protein in the spinal cord motorneuron and gastrocnemius by the immunohistochemical method at different timepoint (2d, 7d, 14d and 28d) showed that small amount of expression of hIGF-1started at 2d and then the expression of hIGF-1 in the spinal cord andgastrocnemius continued to increase;the expression between 7d and 14d wasmost obvious. The peak expression of the spinal cord motor neuron appeared at7d while that of gastrocnemius at 14d. There was no significance of difference(P>0.05)between the gray-scale values of the rats in the three groups at the twotime points, 7 d and 14 d;but there was significance of difference(P<0.01)between the gray values at 7 d and 14 d and the gray values at 2d and 28 d;theexpression of hIGF-1 gradually decreased after 14 d, and there was still smallamount of expression of hIGF-1 in the gastrocnemius at 28 d while almost noexpression of hIGF-1 could be detected in the spinal cord motor neuron. Atvarious time points, the gene expression in the hIGF-1 treated group wasobviously higher than that in the other two groups and it showed a very obvioussignificance of difference(P<0.01)in the measured gray values compared withthe other two groups. We found from Marsland and LFB double staining of thespinal cord motor neuron at different time points(2d, 7d, 14d and 28d)that 2dafter transfection, the degeneration of motor neurons appeared: transformedmotor neuron cell body, decreased, smaller Nissl granules, with eccentric nuclei,but the degeneration of motor neurons in the IGF-1 treated group was less thanthat in the model group and blank control group;after that the degree of thedegeneration of motor neurons gradually increased. The number of the motorneurons in the anterior horn of the spinal cord decreased at 7d and 14d, obviousmotor neuron cell degeneration and necrosis occurred and neuronophagia wasfound, most obvious at 14 d, more obvious in the model group and blank controlgroup, and relatively less neuron necrosis was found in the IGF-1 treated group;there were more normal motor neurons under an optical microscope;the numberof the motor neurons was slightly increased at 28 d and degenerative necrosis ofthe motor neuron cell were coexisted with repair. Degenerative necrosis occurredin some neuron cells while repair occurred in other cells. More neuroglial cellswere found around the injured motor neuron, or setallitosis. Compared with theother two groups, the hIGF-1 treated group showed more and earlier repairs, andthe Nissl bodies in the cell body were found in some neurons.7d,14d and 21d after transfection, the number of apoptosed spinal neuronswas detected by the TUNEL method and the results showed that the number ofapoptosed neurons in time of 7d was most serious in three groups and the numberof apoptosed cells in the hIGF-1 treated group was obviously less than that in theother two groups. The apoptosed cells were not only the motor neurons of thespinal cord anterior horn but also the neuroglial cells, so we speculated that themotor neurons degenerate and die after peripheral nerve injury mainly in theform of cell apoptosis.56d after transfection, the results of the determination of the maximumwave amplitude and latency of the motor nerve conduction velocity (MNCV) ofthe regenerated sciatic nerve of the rat and the compound muscle action potential(CMAP) showed that nerve regeneration in the hIGF-1 treated group was betterthan that in the model group and blank control group, indicating that it was betterin both the quantity and quality of the regenerated nerve fiber in the hIGF-1treated group. After electrophysiological examination, the paraffin sections of theregenerated nerve fiber on the experimental side were taken to conduct Marslandand LFB double staining. The results suggested that the number of theregenerated nerve fibers and the diameter of the axon and medullary sheath in thehIGF-1 treated group were obviously larger than those in the other two groups,with obvious significance of difference (P<0.01). The measuring results alsoshowed that the muscle wet weight and the cross-section area of thegastrocnemius in the hIGF-1 treated group were larger than those in the othertwo groups(P<0.01). All these indicated that hIGF-1 has the effect of promotingregeneration of the peripheral nerve and inhibiting muscular atrophy The resultsof the ultrastructural observation of the spinal cord and the regenerated nervefiber 56d after hIGF-1 transfection demonstrated that the structure of theneuropik in the spinal cord was basically normal in the hIGF-1 treated group,with relatively more and mature regenerated sciatic nerve fibers: thicker nervefibers and thicker and uniform medullary sheath;in the model group larger gapsbetween the nerve processes were found in the neuropik and compact mattersformed in the chondriosome in the axon, the medullary sheath of the myelinatednerve fiber was loose, the low-ripeness regenerated sciatic nerve fibers werethinner, the degree of maturation was lower and the medullary sheaths were lessthick and very different. In the blank control group, vacuoles were found in theneuropik with obvious local cavitation, the chondriosomes swelled and the cristadisappeared, the neurofilaments in the axon of the myelinated nerve fiber wereless and cavitated, and the regenerated sciatic nerve fibers were small andimmature and the medullary sheath was thin and irregular. The results of theultrastructural observation of the spinal cord and regenerated nerve fiberconfirmed the nutritional effect of hIGF-1 on the central and peripheral nerve.Based on the above results, the paper thinks that IGF-1 as a member of theneurotrophic factor family may play its role in the repair and reconstruction ofthe nerve function after injury in the following four respects: ⑴ after nerveinjury, the permeability of the neurolemma increases and through transport of theneurolemma especially pinocytosis, IGF-1 is ingested by the axon and reachesthe spinal cord and target muscle for proliferation through axoplasmic transport.⑵ protecting the spinal cord anterior horn motor neuron and inhibiting itsapoptosis. ⑶ inhibiting atrophy of the skeletal muscles. ⑷ inducing thegrowth of the axon and promoting the regeneration of the peripheral nerve.The experiment, on the basis of reviewing the existing literatures andexperiments both at home and abroad, summarized the pathological changemechanism of the degradation and death mechanism of the spinal cord motorneuron and the atrophy of the skeletal muscles and the biological characteristicsof IGF-1, and obtained the theoretical basis for IGF-1 promoting nerveregeneration and inhibiting atrophy of the skeletal muscles. Using the plasmid asthe vector, the experiment in animals confirmed that liposome mediated IGF-1plasmid can be ingested by the nerve axon in the injured part, and throughaxoplasmic transport reach the spinal cord and target muscle, having the effect ofprotecting the motor neuron cell, slowing the atrophy of the skeletal muscles andpromoting the regeneration of peripheral nerves. And it was found in theexperiment that IGF-1 had the largest effect in the spinal cord and skeletalmuscle between 7d and 14d after transfection. A preliminary study wasconducted on the pathological mechanism of the neurotrophic and myotrophiceffects and treatment at molecular biological levels, and preliminary observationsof the pathological change in the spinal cord motor neuron after peripheral nerveinjury at cellular levels. The experiment, which was simple and used highly safe,low production cost plasmid vectors that can be given many times, will provide anew idea and way for the clinical application of IGF-1 to the nerve functionrecovery and reconstruction after peripheral nerve injury.
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