| The transplantation of chromaffin cell into the spinal subarachnoid space can produce pain attenuation. However, the human adrenal medullary chromaffin cell is scarcely obtained, the source lack restricts its wide use in clinical therapy. Researchers turn to find animal cells to replace human cells for xenotransplantation. The function of Bovine chromaffin cell (BCC) is similar to human cell, its source of BCC is abundant, and the cells can be separated easily, so the BCC is selected. Researchers have recently studied the BCC transplantation into the rat, but only described the changes of pain behaviors. The concentrations of the releasing factors from the BCC implanted in the subarachnoid space has rarely been reported.The spearated BCC were implanted into the subarachnoid space of rat. Both the antinocicepive effects in the animal pain model and the lasting time of antinociception were evaluated. The actions of rats with BCC transplantion followed by the injection with naloxone or phentolamine in the formalin test were observed. The concentration of the leucine enkephalin and catecholamine in the rats cerebral spinal fluid (CSF) was examined. The mechanism of the pain suppression after BCCimplanted was explored.MethodsThe collagenase was used to isolate the BCC from tissue, BCC cells were purified by density gradient sedimentation centrifugation and twice inoculations. The purity of the cells was indentified with dopamine beta hydroxylase (D & H) antibody immunocytochemical technology. The surviving rate of the cells was indentified with the trypan blue staining exclusion test.80 Sprague-Dawley rats were randomly allocated into transplantation group and control group. The rats of transplantation group were injected into the spinal subarachnoid with 20 u 1 BCC suspensions , including 4X107 cells. The rats of control group were implanted with equal amount of Dulbecco' s Modff ied Eagle' s Medium (DMEM) supplemented with 10% fetal calf serum. Each group was divided into 4 subgroups (2-week subgroup, 4-week subgroup, 6-week subgroup, 8-week subgroup), and the CSF of each subgroup rats was collected in the end.The thermal pain threshold: The tail-flicking test was used to detect the response to the acute heat stimulus. The longest time of enduring pain was limited in 15 seconds, and the basal thermal pain threshold was expressed with the mean value before the transplantation. The formula of the antinociceptive effects of heat: antinociceptive effects of heat = (the heat pain threshold tested - the basal threshold) / (The longest time of enduring pain - the basal threshold). Both groups were examined once a week during the 8 weeks.Formalin test: 2 weeks after the operations, the right hind paws of rats were injected with 50 u 1 5% formalin, then the pain behaviors were recorded in a period of the first minute every 5 minutes during the following one hour, the mean weighingscores of the pain behaviors in one minute were calculated. The mean weighing scores = p t /60. the p value was the scores of the pain behaviors, and the t value was the lasting time of the behaviors. Blocking test: 3 weeks after the operations, the rats of transplantation group were divided into 3 subgroups , 10 rats in each subgroup. Before injecting formalin, the rats of each subgroup were injected with naloxone (2mg/kgX phentolamine (lOmg/kg) or 0.9% saline water .respectively. Then the influence of the drugs was observed.CSF of the rats was collected and stored in -60. The concentrations of leucine enkephalin were assaied with RIA and those of catecholamine were assaied with High Performance Liquid Chromatography (HPLC). ResuIts:Acute heat stimulating responses: The antinociceptive effect values of rats of the control group were almost zero while those of the transplantation group were 0.6368 at the first weekend, 0.6735 at the second weekend, respectively. The antinociceptive effect values gradually declined in the next several weeks, and fell to 0.456 at the 8th weekend. The antinociceptive effect va... |
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