Effects Of Sevoflurane On The Activation Of Rat Spinal Cord Astrocytes And Its Possible Mechanisms

PartⅠ:The effect of sevoflurane on the activation of rat spinal cord astrocytes and pain behaviors in a rat model of hind paw plantar incision[Objective]:To observe the effects of sevoflurance on pain behaviors and spinal cord astrocytes activation in a rat model of hind paw plantar incision.[Methods]:80 adult healthy male SD rats were studied and divided at random into control and experimental groups(n=40).Rats underwent a unilateral hind paw plantar incision after placidity anaesthesia.8 rats were selected randomly for each time point and were tested before operation,6 hours,1 day,3 days and 5 days after operations. The rats were put to death immediately after cumulative pain score were calculated and the mechanical thresholds were measured by using von Frey filaments.The tissue of the forth to sixth lumbar spinal cord was obtained,and then the immunoreactivity of glial fibrillary acidic protein(GFAP) was observed by using immunohistofluorescence and the mRNA levels of GFAP were assayed by real-time fluorescence quantitative reverse transcription polymerase chain reaction(real-time RT-PCR) to assess the activation of astrocytes.[Results]:The cumulative pain score in both groups appeared highestly 6 hours after operation,then began to decrease gradually and came to normal 5 days after operation. The mechanical threshold decreased rapidly after operation in both groups,then increased gradually.It came to normal in sevoflurance group but was still lower in control group 5 days after operation.To control group,the GFAP immunoreactivity of ipsilateral spinal dorsal horn in the forth to sixth lumbar spinal cord began to enhance 6 hours after operation and reached peak 3 days after operation.By contrast,it enhanced slightly and with no peak in sevoflurance group.Real-time RT-PCR measurements suggested that in these two groups,the mRNA expression of GFAP in the forth to sixth lumbar spinal cord began to upregulate 6 hours after operation and kept on increasing until 5 days after operation.Nevertheless,no peak was observed in both groups.The mRNA expression of GFAP in sevoflurance group was lower than control group. [Conclusions]:The activation of spinal cord astrocytes may contribute to the occurrence and maintenance of mechanical hyperalgesia after hind paw incision.The mechanism that sevofluence may relieve mechanical hyperalgesia is probably through inhibiting the activation of spinal cord astrocytes. PartⅡ:The effect of sevoflurane on the activation of cultured rat spinal cord astrocytes and ERK signaling transduction pathway[Objective]:To observe the effect of sevoflurance on the activation of cultured rat spinal cord astrocytes and explore its possible effect on ERK signaling transduction pathway.[Methods]:Rat spinal cord astrocytes were cultured in vitro and devided into two groups:control group and sevoflurance group,with stimulation of 100μmol/L aminoglutaminic acid(Glu) for different time.The astrocytes activation was studied by ckk-8 alive cell proliferation,transmission electron microscope and GFAP expression that was detected by western blot.Then,the astrocytes were devided into six group:control group,PD98059 group,PD98059+Sevo group,Glu group, PD98059+Glu group and PD98059+Glu+Sevo group.Expression changes of GFAP and phosphorylation cAMP response element binding protein(pCREB) were detected by western blot.[Results]:The results of ckk-8 alive cell proliferation displayed that with stimulation of Glu(100μmol/L),absorbance ratios increased significantly after 20 min in both groups.After that,no differences were observed at each time point.But absorbance ratios of sevoflurance group were lower than those of control group.Observation on cellular morphology by transmission electron microscope displayed that,by contrast with sevofluence group,there were more euchromosomes in cell nuclei,plenty rough endoplasmic reticulum,Golgi's body,ribosome and mitochondria in the cytoplasm of cells in control group with stimulation of Glu at each time point.Many secretory vacuole and cytolysosome were also seen in control group.Detection by western blot displayed that GFAP expressions in sevofluence group were less than control group at each time point;GFAP and pCREB expressions in PD98059+Sevo group were less than both PD98059 group and control group;GFAP and pCREB expressions in PD98059+Glu+Sevo group were less than both PD98059+Glu group and control group.[Conclusions]:Sevoflurance can efficiently inhibit the activation of rat spinal cord astrocytes in vitro,stimulated by aminoglutaminic acid.Probably,its inhibitory effect on the activation of astrocytes was through the downstream of ERK in the ERK signaling transduction pathway——inhibiting the phosphorylation of CREB.