The Expression Of Angiotensin 1-7 And Mas Receptor In Spinal Cord Of Rat With Gastrocnemius Incision

Part one:Objection: To explore the changes of pain threshold and the expression of Ang1-7 and Mas receptor at spinal level on rat with gastrocnemius incision.Method:1 Models and groups: Sixteen male Sprague-Dawley rats were divided into Sham group and gastrocnemius incision group randomly. Before and 30 minutes after surgery,postoperatively 1st, 2th, 4th, 6th, 12 th, 24 th, 48 th hour thermal withdrawal latency and mechanical withdrawal threshold were measured on plantar hind paw.2 Enzyme-linked immunosorbent assay(ELISA): Rats were executed and the section of spinal cord was collected when the measurement of pain threshold was completed.The spinal concentration of Ang1-7 was detected by ELISA.3 Immunohistochemistry(IHC): The section of spinal cord was collected and sliced after heart perfusion postoperatively 2th day. We Observed the expression of Mas receptor in the tissues of each group by immunohistochemitry. Image Pro Plus 6.0 was applied to calculate the value of AOD(average optical density) on Mas receptor immunoreactive cells in four randomly selected visions without overlapping fields.Result:1 Mechanical withdrawal threshold and thermal withdrawal latency in two groups:There was no statistical significance of mechanical withdrawal threshold and thermal withdrawal latency between two groups before surgery(P>0.05). TWL and MWT values decreased 30 minutes after surgery, hyperalgesia related to surgical pain reached their peak on the 2th hour, continued to the 48 th hour postoperatively. Compared to the Sham group, there were significant difference of TWL and MWT values decreasing on each time point(P<0.05).2 ELISA result shown that spinal concentration of Ang1-7 was significantly lower in gastrocnemius incision rats compared with normal ones.3 Immunohistochemical result shown that there were more Mas R positive cells in surgical group than those in Sham group. Significant difference was found between two groups according to the value of AOD(P<0.05).Conclusion: Acute postoperative pain can downregulate the expression of Ang1-7, but upregulate the expression of Mas receptor in spinal cord of rat.Part two:Objection: To observe the effect of Ang1-7 administered i.t. at various doses on rats.Method: Rats with successful Intrathecal catheterization were divided randomly into four groups. Intrathecal(i.t.) injected saline and different concentration of Ang1-7 to investigate the rats’ behavior changes and measure pain threshold. Rats were executed and the section of spinal cord was collected when the measurement of pain threshold was completed. We Observed the morphology of neurons and glial cells by HE staining to identify necrosis of tissues.Result: 15 minutes after i.t. injection of Ang1-7, MWT and TWL of rats increased dose-related compared with saline group(P<0.05). The morphology of neurons and glial cells were normal. Morphological changes such as karyopycnosis and nuclear fragmentation of cell necrosis were hardly observed in spinal cord by HE staining.Conclusion: Administered Ang1-7 into the subarachnoid cavity was safe and effective.Intrathecal injection of Ang 1-7 can increase the pain threshold of the rats by its analgesic effect.Part three:Objection: To examine the analgesic effect on gastrocnemius incision rats after Ang1-7 administered i.t.Method: Gastrocnemius incision rats were randomly divided into six groups,morphine or Ang1-7(3.0 pmol) was co-administered i.t. with or without different concentrations of A779(0.3 pmol, 3.0 pmol, 30.0 pmol) immediately after gastrocnemius incision surgery. 15 minutes after the i.t. injection, the rats were placed in the transparent cage to record accumulated time response to pain. Rats were executed and the section of spinal cord was collected when the measurement of pain threshold was completed. We Observed the morphology of neurons and glial cells by HE staining to identify necrosis of tissue.Result: Compared with the saline group, MWT and TWL increased significantly after morphine or Ang1-7 administered i.t(P <0.05). There were significant difference between morphine group and Ang1-7 group according to the MWT and TWL values(P<0.05). MWT and TWL increasing were dose-related counteracted when Ang1-7co-administered with different concentrations of A779(P>0.05). The morphology of neurons and glial cells were normal. Morphological changes such as karyopycnosis and nuclear fragmentation of cell necrosis were hardly observed in spinal cord by HE staining.Conclusion: A-779 was safe enough to administer into the subarachnoid space.Ang1-7 probability participate in the transmission of acute postoperative pain in spinal cord level by specifically binding with its Mas receptor.