Experimental Study Of Repairment Of Rat Spinal Cord Injuries Using Activated Macrophages Transplantation

Background: Spinal cord injury (SCI) leads to an irreversible loss of motor and sensory functions below the level of injury due to the poor ability of the cell bodies to regenerate new axons and inhibitory acting for the myelin or the formation of the glial scar and cavum. In the other way, the functions of the spinal cord will be damaged furthermore for the secondary damage in spinal cord after primary traumatic injury. Now there are few effective therapeutic tools to promote the regeneration of the axons in clinic, excluding the decompression and fixation of the vertebral canal to prohibit the further damage towards the spinal cord. For many years, investigators have been devoting themselves to explore the failure cause of the axons regeneration and search for the therapy methods.At present, the failure cause of recovery of the spinal cord functions due to these factors: i. Inferior regeneration reactive of the axons.ii.The hostility of the microenvironment to fiber regeneration in the central nervous system (CNS). iii. Prohibitting the regeneration of the axons because of the glial scar formation. To aim directly at those factors, a few methods have been detected for the partial recovery of the spinal cord functions after SCI.The major methods includ: decreasing the acute injury of nerve; using trophic factors; transplantation of cells and tissues; alternation of the hostility of the microenvironment to fiber regeneration.Intensive studies have indicated that axons in the CNS of mammals have poorer regenerative ability compared with that of peripheral nervous system (PNS).Wallerian degeneration will happen in the distal tip of the peripheral nerves after injury, including destruction of axons, necrosis of Schwann cell and infiltration of local inflammatory cell, among them the major cell are macrophages. Activated macrophages that infiltrate the PNS could clear the degenerated axons and myelin debris, and secrete cytokines and growth factors to promote the regeneration of the axons. The CNS belongs to immune privilege organ and has self-protective action of preventing the massive immunologic cells from infiltrating it in order to protect the resistant functions, and the inflammatory response in the CNS is slighter than that in the PNS or the other tissue. So some one suggested that macrophages transplantation is one of the effective methods for the spinal cord regrowth and might be an effective treatment for the SCI.In the present study, there are two different viewpoints about the action of the macrophage transplantation for the repairment in the CNS.One point is that macrophage transplantation does harm to the regrowth of axons. Macrophage transplantation could enhance the permeability across the blood-spine barrier. Macrophages not only release inflammatory neurotoxin and cytokines, but also augment the degeneration and necrosis, which leads to the secondary damages to the spinal cord. Another point is that macrophages transplantation has beneficial effects for the axons regrowth. Macrophages could scavenge debris and inhibitors of axons regeneration, which is sphingomyelin. Macrophages also secrete and stimulate trophic substance of the axons regrowth,such as nerve growth factor(NGF),IL-1, IL-6 etc.Furthermore, they could modulate substrates and proteases, which are needed for the regeneration of the axons.It is still obscure what action the macrophages play during the SCI and which requirement is beneficial or inhibitory for the regeneration of the axons. In this investigation, the correlation between the timing and effects of the macrophages transplantation for the SCI was studied.Objective: To investigate the restoration of the motor functions, improvement of the electrophysiological recordings , alteration of the pathomorphology and the relation to each other in different time of post-injury after the activated macrophageswere transplanted. In order to provide the theory data for the repair of the SCI using macrophages transplatation, the correlation between the timing of macrophages transplantation and the restoration of the spinal cord functions ought to be decided.Methods: 1. Incubating > purifying and counting of the activated macrophages: A BALB/C rat was sacrificed by dislocation of the cervical joint, which body weight was 30g, then it's spinal cord was taken as antigen. Another rats was sacrificed and PBS was injected into the abdominal cavity in order to get macrophages. Activated macrophages were attained after the macrophages were incubated together with the spinal cord antigen. Then the density of cells suspension was adjusted to 10 /ml.2.The fabrication 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 2 mm in the spinal cord .At last the semi-resected model of the SCI was attained.3. 62 BALB/C rats were grouped in Ax B, C groups randomly after their spinal cords were semi-resected. 5 (al of activated macrophages were injected into the gap of the spinal cord in the left of the Group A rats when the spinal cord had been semi-resected for an hour. 5 fal of activated macrophages were also injected into the gap of the spinal cord in the left of the Group B rats when the spinal cord had been semi-resected for eight hours. 8 rats were taken respectively in An B> C groups randomly and their restoration of the motor functions and improvement of the somatosensory evoked potential (SEP) were investigated following every week after injury. The motor functions improvement was evaluated by Basso, Beattie and Bresnahan locomotor rating scale(BBB scale).Another 8 rats were taken respectively in Ax B, C groups randomly and at various survival times such as 1,4 days and 1,2,4, 8,12,16 weeks, spinal cords were fixed and cut into transverse frozen sections, and were immuneostained and stained with hematoxylin and eosin (HE) .Then the numbers of cells or neurofilaments were counted under microscope.Results: l.The BBB scale of Group A rats increased gradually from 4th week to 12' week, which score exceeded 4 rarely, and increased quickly after 12th week,...