| BackgroudrSpinal cord injury(SCI) is a serious disease which do harm to human's health. Spinal cord injury combined with spine fracture is more and more common.The difficult functional restoration after SCI is a subject that have disturbed clinical doctors and researchers for many years, because of the poor ability of the nerve system to regenerate new axons.In traditional concept,not only the neuron in gray matter but also the tract in white matter can not regenerate.Whereas,nowadays we know that: neuroncan't regenerate; glial cell Can regenerate. Glial cell don't benefit the regenerateion of neurons or axons.A lot of gliosis will become the barrier of the axon regeneration;Axons can regenerate.Therefore axon's regeneration become the main direction of the SCI repairment.Two aspects are involved in the SCI repairment: the first one is inducing the nerve fiber to grow,that is,giving many kinds of nerve trophic factors to promote the nerve's growth, or promoting the expression of the nerve trophic factors ,providing bridges and pipelines for the axon's regeneration,providing the schwann cells and extracellular matrix which can support and guide the nerve growth;the second one is decreasing the inhibitory factors for axon's growthrfor example,reducing the formation of the cavum and scar tissue in broken end of the spinal cord and restraining the emergence of some inhibitory factors.At present,the transplantation of cells and tissues is the most important method that has a large probability to apply in clinical therapy.Cells transplantation can keep theregenerative axons away from the circumambient inhibitory environment.On the other hand,some cells can product some nerve nutrient substance,therefore can promote and guide axon's regeneration.these cells and tissues included peripheral nerve, embryonic brain or spinal cord tissue ,embryonic stem cells(ES cells), neural stem cells(NSCs), olfactory ensheathing cells(OECs), marrow stroma cells(MSCs), activated macrophages,and so on.Peripheral nerve graft is one of the earliest methods applied to the repairment of SCI.People have acknowledged its value since Cajal's time.Later people tried many methods to transplant the peripheral nerve,in which Cheng's experiment is most representative. Complete spinal cord gaps in adult rats were bridged with multiple intercostal nerve grafts that redirected specific pathways from white to gray matter. The grafted area was stabilized with fibrin glue containing acidic fibroblast growth factor . Hind limb function improved progressively during the first 6 months, as assessed by two scoring systems. The corticospinal tract regenerated through the grafted area to the lumbarenlargement, as did several bulbospinal pathways. Histological examination proved that there are regeneration of the motor neurons. Yet,more and more studies have proved that it has its limitations that using peripheral nerve by itself to repair the SCI.The regenerative axons often can not go through the distal interface between the grafts and the spinal cord.Macrophages are the main inflammatory cells that participate the organism's inflammatory reaction and tissue repair.They also play a important role in peripheral nerveous system(PNS).On the one hand, they are "street sweeper",on the other hand,they and the cytokine they secrete together participate the biochemical metabolism of the injuried nerve.The Walliam's degeneration occur at the injured part and the caudral part after the peripheral nerve injury.Axons and myelin sheath break into pieces. Afterwards lots of macrophages gather and are activated to phagocytose degenerative debris.Macrophages promote the regeneration through direct and indirect manner such as secreting cytokine. It is known that the concentration andactivation of the macrophages is an important reason for injuried peripheral nerve to regenerate. The CNS is "immune privilege organ" and has self-protective function of preventing the massive immune cells from infiltrating it in order to protect its own steady functions, and the inflammatory response in the CNS is much slighter than that in the PNS or the other tissue.There are different opinions on the effect of macrophages in SCI repair.But more and more study confirmed that macrophages can promote the regeneration of the axons.Then it can be concluded that if the peripheral nerve combined with activitied macrophage is transplanted to the injuried spinal cord,can we obtain a better result?Therefore,this study is designed to solve the problem.Objective: To explore the effect of SCI repair by single peripheral nerve graft, single activitied macrophages graft and peripheral nerve combined with activitied macrophages graft,and to make clear that whether there is difference between there methods.Methods: 1. Preparation of the activated macrophages:A BALB/C mouse of 30g was put to death by dislocation of the cervical joint, then its spinal cord was taken as antigen.another 5 mice were put to death and PBS was injected into the abdominal cavity in order to get macrophages. Activited macrophages were attained after the macrophages were incubated together with the spinal cord antigen.Then the density of the cells suspension was adjusted to 1 05/ml.2.The fabrication of the peripheral nerve:2 BALB/C mice were fixed and disinfected after anesthesia,then the bilateral sciatic nerves were removed and prepared to be grafted.3. Preparation of the model of the SCI: The spinal cord at T10 level was exposed after the spinous processes and lamina of vertebra were taken away carefully, then the left half spinal cord was resected using a needle tip under the microscope to get a gap of 3mm in the spinal cord.At last the hemi-resected model of the SCI was attained.4. 81 BALB/C mice were grouped in A^ B^ C% D groupsrandomly after their spinal cord were hemi-resected.20 mice of group A were dealed with activated macrophages graft; 20 of group B were dealed with peripheral nerve graft; 21 of group C were transplanted both two grafts; 20 of group D were contral group.Each of the 4 groups was respectively divided into 3 subgroups randomly. 7 of subgroup 1 were investigated by their restoration of the motor function and improvement of the somatosensory evoked potential (SEP). 7 of subgroup 2 were taken for histological examination. The spinal cords were fixed and cut into longitudinal frozen sections,and were immuneostained and stained with hematoxylin and eosin (HE).The the numbers of cells or neurofilaments were counted under microscope. 6 of subgroup 3 (7 of C3) were used as complement ones.Results: 1. The left hind limb of the mice in group A^ Bj and Ci began to move slightly after 2nd week. The BBB scale of them began to increased from 4th week,in which the scale of Cj mice increased more obviously.After 8th week the scale of animals increased rapidly,which can surpass 10 until 12th week. The BBB scale of group Di didnot increased obviously.There was a significant difference between group Ci and the other three groups , There was a significant difference between group A] > B] and D, ,too.(ONE WAY ANOVA,P<0.01).It was proved that the motor functional restoration of group Ajn B] and Ci was obvious.2. The SEP wave of group A^ B] and d appeared from 3rd week after injury,whose latent period was long and amplitude was low. Afterwards the latent period became shorter and the amplitude became higher gradually, which suggested the recovery of electrophysiological functions of spinal cord. The alteration became quickly after 8th week . The wave of group D] didn't appear until 4th week and its alteration was not significant. There was a significant difference between group Ci and the other three groups , There was a significant difference between group Aj> Bj and D, ,too.(ONE WAY ANOVA,P<0.01).3. The grafted macrophages existed in girdle-shape on the cavity wall of the injuried spinal cord,whose density decreased rapidly with away from the injuriedsite.Ferthermore,there were no grafted macrophages existed out of the injuried site.The number of the macrophages achieved maximum from 4th day to 7th day.The macrophages could survived for 4 weeks and decreased quickly after 4 weeks.In group B2 and C2 the macrophages located between the injuried spinal cord and the peripheral nerve,took part in the regeneration of the axons and existed in the cavity of the spinal cord and along the regenerated axons.There was a significant difference between the group A2^ C2 and group B2 > D2, (ONE WAY ANOVA,P<0.05) .There was no significant difference between the group A2 and C2,group B2 and D2,( ONE WAY ANOVA,P>0.05) .4. The number of the astrocyte in the injuried spinal cord increased in the early time after SCI,which became Stable in later time. There was a significant difference between the group D2 and the other three groups (ONE WAY ANOVA,P<0.05) .There was no significant difference between the group C2 and the other three groups, (ONE WAY ANOVA,P>0.05) .5. The number of the NF positive cells in the group A2> B2 and C2 began to increase from the 4th week.A part of myelin restored and some smaller cavum was formed.Till the 12th week,the grafted nerve fused with the spinal cord and a host of nerve fiber regenerated in group C2.Whereas in group D2 ,the axons was difficult to regenerated. There was a significant difference between the group C2 and the other three groups, the group D2 and the other three groups (ONE WAY ANOVA,P<0.05) .There was no significant difference between the group B2 and C2, (ONE WAY ANOVA,P>0.05) .Conclusions: 1. Both single peripheral nerve transplantation and single activated macrophages transplantation can promote the SCI repair to a certain extent,but their effect is finite.2. The peripheral nerve combined with activated macrophages transplantation can promote the SCI repair effectively.3. The BBB scale ^ immunocytochemistry histological examination and SEP together can evaluate the restoration of the SCI objectively.4. SEP is a sensitive and impartial test technique for SCI.Its change may be earlier than that of the BBB scale and the clinical situation.
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