The Effects Of Progesterone On The Expression Of AQP4 And GFAP In Spinal Cord Of Rats After Acute Spinal Cord Injury

The edema followed spinal cord injury(SCI) is a major factor effecting the function recovery. Recently, many studies demonstrate that progesterone(PG) and some of its metabolites can significantly reduce cerebral edema and enhance functional recovery from TBI and stroke in several animal modele. Now,it has been accepted that AQP4,which mainly express at astrocyte in CNS, has a dominating role in regulating the edema in CNS. The mechanisms of progesterone modulating spinal edema have not been detected.Objective1. To observe the changes of spinal cord water content after injury and therapied with PG.2. To observe the change of AQP4 and GFAP in distribute and quantity after spinal cord injury.3. To observe the change of AQP4 and GFAP in distribute and quantity in injuried spinal cord after therapied with PG. Investigate the posibility that PG modulate the epression of AQP4 and GFAP in spinal cord.Materials and MethodsA total of 144 adult male Sprague-Dawley (SD) rats served as subjects, Weight range:250-300g. the rats SCI model was made based on previous documents, with some modification. The cords at T6 was injured by bilateral compression for 1 min using forceps that were 1mm wide with a 1mm spacer. Rapidly remove the forceps,when find the tails and double low limbs began to swing or spasm, which evidence the spinal cord has been injuried.Based on previous studies determining optimal dose response, all progesterone-treated animals received 16mg/kg progesterone in Soybean Oil (vehicle).The animals received the first dose of either Progesterone or an equal volume of Soybeal Oil at 0.5h after the injury,intraperitoneal injection,subsequent injections were given at 24h and 48h after injury,hypodermic injection.The experimental animals were divided randomly into three groups:Sham+ vehicle (SV), Lesion+vehicle (LV), Lesion+progesterone (LP). Every group wase divided into 6 subgroups, according different time points of tissue specimens, i.e. Oh, 6h,12h,24h,48 h,72h.There are 9 animals in each subgroup.3 rats of them, spinal cords were removed from T4-T8 vertebral body levels after perfused with 4% paraformaldehydein 0.1 M phosphate buffer via the heart. postfixed for overnight at 4℃, cryoprotected in 30% sucrose in phosphate buffer 3-7 days, and embedded in OCT compound, prepare for Double immunofluorescence staining.6 rats of them, spinal cords were removed from T4-T5 vertebral body levels, weighed, heated at 80℃for 48 h and re-weighed. Percent water content was calculated as [wet weight-dry weight)/wet weight×100]. Spinal cords were removed from T7-T8 vertebral body levels, freezed rapidly in-70℃, prepare for Western blotting.Result1. Spinal cord water content:the water content in SV group is lowest, LP group is higher(P<0.05), LV group is the highest(P<0.01).2. Results with HE staining:the lesion areas in LV group and LP group contains hematoma zone, peri-hematoma zone and normal tissue zone. There are a lot of eryrocytes in hematoma zone, necrotic and degenerated nerve cell; surrounding the hematoma are a small quantity swelling nerve cell, inflammatory lymphocyte. The edema zone surround the hematoma, whith noticeable at 72h with foam cell emerging. The edema necrosis and inflammatory reaction in LP group are all relieved compared with LV group.3. Double immunofluorescence staining:AQP4 was expressed in gray and white matter, co-localization of GFAP labeled astrocytes (green) with AQP4 labeling (red) in the white matter and grey matter of the normal spinal cord. AQP4 was abundantly expressed around blood vessels, in the glia limitans externa facing CSF in subdural spaces fluid (CSF).,thus supporting the hypothesis that AQP4 mediates water transport between the spinal cord parenchyma and blood or CSF.After SCI, there were some GFAP positive cells in the injured regions of the spinal cord that did not express AQP4. This figure shows apparently in injured groups at 72h. It might be that these GFAP-positive cells, which did not express AQP4 after SCI, are astrocytes that migrated into the lesion site.4. AQP4 and GFAP expression in electrophoresis and Western blotting:Compared with SV groups, the AQP4 expression levels in LV groups are increased significantly at 12h,24h,48h and 72h after the injury (P<0.01); the LP groups are moderately decreased at 12h,24h,48h and 72h (P<0.05). Compared with LV groups, the AQP4 expression levels in LP groups are decreased significantly at 12h, 24h,48h and 72h after the injury (P<0.01).Compared with SV groups, the GFAP expression levels in LV and LP groups are all decreased significantly at 6h,12h,24h,48h and 72h after the injury (P<0.01), lowest at 12h, restore to step-up at 24h. Compared with LV groups, the GFAP expression levels in the LP groups are no obviously diference (P>0.05) at 6h,12h and 24h after injury; but significantly increased at 48h and 72h (P<0.05).Conclusion1. The progesterone can reduce the water content in injuried spinal cord in acute course, extenuate spinal cord edema.2. In normal spinal cords, AQP4 and GFAP are co-localization in normal spinal cords. AQP4 was expressed in astrocytes, concentrate on the foot process involved in the construction of the BBB.In injuried spinal cords, the distribution of AQP4 and GFAP are discrepancy. There are no AQP4 expression in peri-lesion areas.3. Progesterone encourage reactive astrocyte accumulated around the lesion of spinal cord。4. Progesterone reduced the AQP4 expression levels in spinal cord damage zone.