The Influence Of Seawater Immersion On The Pain Behavior Of SMIR(Skin/Muscle Incision And Retraction) Model In Rats And Its Mechanism

Objective1. To observe the influence of seawater immersion on the pain behavior of SMIR (skin/muscle incision and retraction) model in rats and explore the relationship between the internal mechanism and neural morphological changes.2. To study Relevance of the pain behavior change of rats with SMIR (skin/muscle incision and retraction) model after seawater immersion and the activation of TRPV1, microglia on DRG(dorsal root ganglion) and intumescentia lumbalis cornu dorsale medullae spinalis, and explore the immanent mechanism.3. The effect of different flushing fluid on the postoperative pain behavior and incision healing time of SMIR model rats after seawater immersionMethods1. To observation of pain behavior changes of SMIR model rats after seawater immersion for different time:24 healthy male SD rats were randomly divided into four groups:group SMIR (only SMIR model), SI5 group (local wound after the establishment of SMIR model, to be immersed in seawater for 5 minutes), SI20 group (after the establishment of SMIR model. Local wound to seawater immersion for 30 minutes), SI60 group (after the establishment of SMIR model, the local wound seawater immersed for 60 minutes). After Id,3d,5d,7d,14d, 21d and 35d,50%Paw mechanical withdrawal threshold (PMWT) and Paw thermal withdrawal latency (PTWL) were tested.2. To observation the morphological changes of nerve systems of SMIR rats after seawater immersion:48 healthy male SD rats were choosen based on pain threshold after screening, then randomly divided into three groups:model control group (MC) and physiological saline immersion group (NS group), seawater immersion group(SW). after SMIR models were set up, the incision of rats in saline immersion group in the rat were immersed in saline for 60 minutes. Rats in seawater immersion group were immersed in seawater immersion for 60 minutes. No special processing were done on model group model. On Id,3d,5d, 7d,14d and 21d 35d after the operations,50% Paw mechanical withdrawal threshold (PMWT) and Paw thermal withdrawal latency (PTWL) were tested. The ipsilateral saphenous nerves were taken out to operate HE dyeing and the ultrastructure of neuronso of ipsilateral L3 and L4 dorsal root ganglion were observe through electron microscopy techniques. We recorded general situation and healing time.3. The relationship between changes of expression of the dorsal root ganglia TRPVl and microglial activation and pain behavior:respectively after 3d,35d randomly 4 rats were selected from MC group, NS group, SW group, the expression of TRPVl in dorsal root ganglia and the lumbar enlargement of the spinal dorsal horn were tested by immunohistochemical assay, as well as the level of activation with the use of immunofluorescence staining labeled OX-42 determination of dorsal root ganglion and the lumbar enlargement of spinal dorsal horn microglia.4 to observe the effects of different washing liquid on healing time of rats with seawater immersion after SMIR and the wound pain behavior:healthy male SD 48 rats, the pain threshold after screening were randomly divided into seawater immersion group (SW group) and seawater immersion+distilled water group (SW+DW group) and seawater immersion+low permeability saline group (SW+HS group) and seawater immersion+saline group (SW+NSgroup), after the establishment of SMIR model, in addition to seawater immersion group, the other groups were treated with distilled water, saline, saline and low permeability. After operation on postoperative Id,3d,5d,7d,14d,21d and 35d were measured in 50%rats of the mechanical withdrawal threshold (PMWT) and heat pain threshold (PTWL). The general situation of rats were observed and recorded during the experiment, rats wound healing time.Results1. SI5 group after the dl of the operation side of the PMWT values were significantly reduced compared with SMIR group (P＜0.05). Compared with SMIR group, SI20 group, postoperative 1D,3D,5D side PMWT, the corresponding value of 7D decreased (P＜0.05). PMWT based SMIR group, SI5 group and SI20 group were on postoperative 35d recovery of postoperative side level. SI60 group after surgery side PMWT 1D to 35d the value of each time point were compared with SMIR group and SI5 group decreased (P<0.05); and compared with SI20, SI60 group after 1D,14d,21 d,35d, PMWT on the ipsilateral side decreased (P＜0.05), and 35d after the operation can not be restored to the preoperative MWT value. SMIR group, SI5 group, SI20 group compared with before modeling and there is no significant difference between the PMWT values. SI60 group after 1D to 14d each time point value and the side of the PMWT SMIR group, SI5 group, SI20 group and DO group were significantly decreased (P＜0.05),21d returned to baseline after operation. SMIR group, SI5 group and SI20 group at each time point and contralateral side was no significant difference between PTWL values; SI60 group after 1D to 14d at each time point postoperatively PTWL than SMIR group, SI5 group, SI20 group and before making the model slightly decreased (P＜0.05); and the 1D,3D,5D 7d, the PTWL value is SMIR side after operation group, SI5 group, SI20 group and before modeling (P＜0.05) decreased slightly.2. Compared with NS group and SW group, the corresponding ipsilateral PMWT value of SW group from postoperative Id to 35d were significantly lower (P ＜0.05), and even in 35 d can’t return to preoperative and postoperative base PMWT value. The contralateral PMWT value SW group from the first day to 21 days after operation were significantly decreased (P＜0.05) than the basic PMWT level. On d35 it returned to normal leve.The corresponding ipsilateral PTWL value of SW group for dl to dl4 after operations were lower significantly than the MC group and NS group, the difference was statistically significant, while the contralateral PTWL of SW group were slightly decreased than the values of basic level,NS group and MC group.There were no difference in the PWTL values of all the time points between MC group and NS group.3. The saphenous nerves epineurium of the MC group and NS group in 3d、 35d after operations were smooth and complete, closely connected with nerve fibers, and the nerve fibers are close. The saphenous nerve epineuriums of SW group at d3 after the operation were significant drape, and nerve fiber gap widened, nerve fiber attenuate arranged loosely, loss of part of the nerve fibers, fiber density uneven. The saphenous nerve epineuriums of SW group at d35 were implicit slightly fold, nerve fibers looked like a loose wave with uniform density there were little swollen mitochondria and cytolysosome in dorsal root ganglion neurons of MC group and NS group d3 and d35 d after operation, and mitochondrial cristae were clearly visible. At 3d a large number of bubble like mitochondria and cytolysosome or autophagy vesicles were seen in the DRG neurons with mitochondrial cristae disappeared, and at 35d a little swelling mitochondria and cytolysosome were found with mitochondrial cristae vague. At 3d snd 35d postoperatively, the numbers of abnormal mitochondria and lysosome in dorsal root ganglion neurons of SW group were significantly higher than the MC group, NS group (P＜0.05). compared to the third day, the number of abnormal mitochondria and lysosome in the three groups at d35 postoperatively were significantly decreased (P ＜0.05).4. the integrated optical density of TRPVl on ipsilateral DRG and intumescentia lumbalis cornu dorsale medullae spinalis were significantly greater than the MC group and NS group (P＜0.05). Compare the postoperative day 3, the integrated optical density of TRPVl of three groups at d35 decreased (P＜0.05). After d3 days and d35, the mean density of the microglia specific protein marker OX-42 on the dorsal root ganglion and lumbar intumescence spinal cord dorsal horn of SW group were significantly greater than the that in MC group and NS group (P＜0.05).5. Compared with the basic mechanical pain threshold, the ipsilateral PMWT values of four groups of rats from postoperative day 1 to 35 days of operation decreased (P＜0.05). compared with SW group, SW+DW, SW+HS group, ipsilateral corresponding PMWT values of the SW+NS group from 1 to 35 days after surgery were increased (P＜0.05).While compared With the SW+DW, SW+IIS group, the ipsilateral corresponding PMWT of SW+NS group from 1 to 35 days after surgery at each time point were increased (P＜.05). Compared with base PMWT value, SW group, the contralateral PMWT value of SW+DW, SW+HS group, the SW+ NS group from postoperative day 1 to 7 days reduced to some extent (P＜0.05), compared with SW group, the contralateral PMWT value of SW+DW group of postoperative 1 day and 7 days is significantly higher (P＜0.05), the corresponding point of PMWT values of SW+NS group from 1 to 7 days were lower than that of SW group (P＜0.05);There were no difference between the SW+ DW and SW+HS group in contralateral PMWT at postoperative each time point, the contralateral PMWT of SW+NS grou at postoperative day 1,3,5 was significantly higher than the SW+DW and SW+HS group (P＜0.05). The Ipsilateral corresponding PTWL values of SW+DW, SW+HS group, the SW+NS group at each point from the first day to 14 days after surgery was higher than SW group were higher (P＜0.05). Postoperative day 1 to 7 days, the SW +NS group ipsilateral corresponding PTWL value was higher than the SW+high DW, SW+HS group (P＜0.05). There were no difference between the basic level and postoperatively each point of SW+NS group in contralateral PTWL value, the contralateral PTWL value of SW+NS group at dl d3 d5 postoperatively is significantly higher than the SW group, the SW+DW, SW+HS group (P＜0.05). no difference were found in healing time of SW group, the SW+DW, SW+HS group and the healing time of SW+NS group was about 2 days in advance than SW group, the SW+DW, SW+HS group.ConclusionSeawater immersion for a short time (5 minutes,20 minutes)will reduce psilateral mechanical pain threshold level of the acute phase(after three days)of SMIR rats. Seawater immersion (60 minutes) could aggravate the pain of the SMIR model rats with the durative hyperalgesia in ipsilateral paw and mirror pain in contralateral side. Seawater immersion can lead to peripheral nerve injury in rats, the changes of the ultrastructure of the dorsal root ganglion neurons may be involved in worse pain and nerve pathological pain produced by the process. Spinal dorsal horn and dorsal root ganglion TRPV1 excessive expression make sustain microglia activation pathway may be the internal mechanism of the constant pain; Seawater immersion may delay SMIR model rat incision healing, immediate flushing not only can alleviate postoperative pain worse situation, among them with physiological saline flushing effect is better, and also to have certain effect in incision healing.