The Study On The Mechanism Of Ischemia Reperfusion Injury In Acute Intussusception And Its Imaging In Vivo

Objective:The aim of this study was to establish an ischemia reperfusion injury animal model in intussusception,and apply immunohistochemical staining to explore whether TLR4 is involved in intestinal ischemia reperfusion injury,and utilize near infrared tracing in vivo and cell imaging experiments in vitro with TLR4 quantum dots probes to diagnose intestinal ischemia reperfusion injury from the level of molecular imaging,and to provide theoretical basis for the prevention and treatment of intestinal ischemia reperfusion injury.Methods:1.The tissue specimens of intestinal necrosis caused by intussusception were collected,and the expression of TLR4 and p38 MAPK in the necrotic intestinal tissue was observed by immunohistochemical staining.2.Of 62 BALB/c mice,36 mice were randomly selected to observe the pathological process,and 16 mice were used as control group and the remaining 10 mice were employed as ischemia reperfusion group.Then animal models of intussusception were established and Micro-CT was used to verify them.And the dynamic changes of intussusception were evaluated by H-E staining.The expression of TLR4 and p38 MAPK molecules was measured by immunohistochemistry in the intestinal ischemia reperfusion injury model.3.Eighteen nude mice were randomly allocated to 3 groups:control group,ischemia reperfusion group and ischemia reperfusion+TAK-242 group.The ischemia reperfusion injury model and hypoxia reoxygenation model of intestinal epithelial cells were established and utilized TLR4 quantum dots probes to explore the imaging effect of quantum dots probes in ischemia reperfusion injury model and hypoxia reoxygenation model of intestinal epithelial cells through experiments in vivo and in vitro.Results:1.For human specimens,the expression of TLR4 in necrotic intestinal tissues caused by intussusception increased significantly,and was distributed diffusely or granularly on thecell membrane and in the cytoplasm which was brown.P38 MAPK was highly expressed in the necrotic intestinal tissues,and its location was mainly in the cytoplasm.2.The intussusception model was successfully established in 46 mice.There was no significant vascular compromise at the beginning of intussusception and fifth minutes.After 15 minutes,vascular compromise was seen in the intussusception model.And vascular compromise or venous congestion were found after 30 minutes.As time went on,vascular compromise and intestinal obstruction aggravated.When intussusception was120 minutes,6 mice had intestinal necrosis and 2 cases had intestinal perforation.On Micro-CT,46 mice had masses.Of these masses,36 were doughnut shaped and 10 were pseudo-kidney shaped.On enhanced CT,small bowel obstruction was found at 30 minutes of intussusception.After 60 min,bowel walls of intussusception had thickened,and the small bowels had dilated as they filled with fluid.Histology confirmed 46 cases of intussusception.In the ?30 min group(5,15,and 30 min),there was no significant inflammatory change,suggesting that intussusception was acute and at an early stage,but in the >30 min group(60 min,120 min),there were some inflammatory changes and more necrotic areas were found in the intussusception model group.TLR4 mainly expressed on the cell membrane and in the cytoplasm of the intestinal epithelial cells.In the IR model of intussusception,the MQS score of TLR4 in the intestinal epithelial cells was5.189±2.483,while that in the control group was 1.186±1.171,and there was a significant difference between the two groups(P<0.05).P38 MAPK mainly expressed in the cytoplasm of the intestinal epithelial cells.In the IR model of intussusception,the MQS score of p38 MAPK in the intestinal epithelial cells was 8.712±0.920,while that in the control group was 2.109±0.109,and there was a significant difference between the two groups(P<0.05).3.In vivo experiments,there was obvious TLR4 fluorescence signal in abdominal bowel in IR group.In control group,there was no TLR4 fluorescence signal in abdominal bowel.In IR+TAK-242 group,there was no obvious TLR4 fluorescence signal in abdominal intestine.For cell experiments,TLR4 in intestinal epithelial cells in hypoxia reoxygenation+Anti-TLR4-QDs group showed intense red fluorescence signal,distributing diffusely or granularly on cell membrane and in the cytoplasm,and there was no red fluorescence signal in the nucleus.And TLR4 in the control group,normal intestinal epithelial cells+Anti-TLR4-QDs group and hypoxia reoxygenation+TLR4 preconditioning group all showed no obvious red fluorescence signal.Conclusion:1.TLR4 highly expressed in the necrotic intestinal tissue caused by intussusception,which distributed diffusely or granularly on the cell membrane and in the cytoplasm.And p38 MAPK also highly expressed in the necrotic intestinal tissue,and its location was mainly in the cytoplasm.2.In the mice intussusception models,intestinal ischemia had a tendency to deteriorate over time.Ischemia-reperfusion injury occurred during intussusception,and it was found that ischemia-reperfusion injury was associated with upregulation of TLR4 and p38 MAPK.3.TLR4 quantum dots probes could specifically combine with TLR4 molecules which highly expressed in the intestine with ischemia reperfusion injury and provided an effective method for the early imaging in vivo in the intestinal ischemia reperfusion injury.