Ultrastructure Study On Peritoneal Lymphatic Stomata And Its Intraperitoneal Endotoxin Translocation

Objective:Lymphatic stomata is peritoneal mesothelial cellswith cytoplasmic processes extending cytoplasmic projections of adjacent cellsconnected together,and it is the lymphatic capillaries in the skin surface of theperitoneal’s opening.As a constant structure of the human body,the lymphaticstomata morphology is concerned by many researchers,many of theexperiments have been confirmed on relationship between peritoneal lymphaticstomata and drainage of ascites,it is also closely related to intra-abdominaltumor metastasis,but there is little information about the transfer relationshipbetween the lymphatic stomata and abdominal infections substancesendotoxin.This experiment,we use electron microscopyto observe theultrastructure of peritoneal lymphatic stomata way,and through intraperitonealinjection of tracer labeled exogenous endotoxin,we continuous observedendotoxin transport in the path of the peritoneal lymphatic stomata and thecharacteristics of it.This can provide evidence for the further research on thefunction of peritoneal lymphatic stomata.Methods:1.Get rat’s diaphragmperitoneal tissue according to the literature.Observe the peritoneal lymphaticstomata’s distribution,size and morphology under the scanning electronmicroscope(SEM).Observe the lymphatic stomata’s structure under thetransmission electron microscope(TEM).Use Elescope computer imageprocessing software to analyze the lymphatic stomata’s SEM images.UesSPSS19.0software to analysis the data to get rat’s peritoneal lymphatic stomata average diameter (μm) and density (/0.01mm2).2.Intraperitoneal inject in ratswith FITC labeled endotoxin,and then in5min,30min,1h,2h,3h,take rat’speritoneal,cisterna chyli,portal vein,then frozen-section them.Observe FITClabeled endotoxin on the peritoneum,cisterna chyli,portal vein under afluorescence microscope.Use IMAGE-PRO PLUS6.0image processingsoftware analyzes the fluorescence intensity of FITC on the peritoneum,cisternachyli,portal vein.Compares differences between them. The fluorescenceintensity of fluorescence analysis of portal vein blood and the cisternachyli liquid FITC spectrophotometer, using SPSS19.0software to get the datafor statistical analysis, and comparison of difference.3.Intraperitoneal inject inrats with nano-gold-labeled endotoxin,respectively at5min,30min,1h,2h,3htake the rat’s peritoneal,cisterna chili,portal vein.Observe the transportation ofnano-gold-labeled endotoxin under scanning electron microscopy andtransmission electron microscopy. Use Elescope computer image processingsoftware analyzes the density(/0.01mm2)of the nano-gold-labeled endotoxinon the lymphatic stomata,cisterna chili,portal vein under scanning electronmicroscopy.Use software SPSS19.0to analyze the resulting data. Compare thedifference between the transportation of nano-gold-labeled endotoxin inlymphatic stomata way and portal vein way.Results:1.Under the scanningelectron microscope,we can observe the peritoneal lymphatic stomata arelocated in the voids between the cube peritoneal mesothelial cells.It is extendedby cubic mesothelial cells between the protuberance and adjacent cell fingerlike cytoplasmic processes which are connected mutually.Lymphatic stomata is round or ellipse.There is a subcutaneous connective tissue fibers in the bottomof lymphatic stomata.Under the transmission electron microscope,we find theperitoneal lymphatic stomata cubic mesothelial cells clearly,largenuclei,prominent nucleoli.Peritoneal lymphatic stomata is the opening oflymphatic capillaries in the peritoneum,and down through a small tube andperitoneal lymphatic drainage unit (LDU),lymphatic lacunae,and it is connectedwith lymphatic capillaries at last.2.Under a fluorescence microscope,5minFITC-labeled endotoxin appears on the peritoneum.The amount of endotoxinon the peritoneal reaches peak in30min,decreased when1h (compared to30min,P>0.05),2h decreased significantly (compared to30min,P <0.05).Eachtime point, the change of cisterna chyli as follows:in5min,there has been asmall FITC-labeled endotoxin in cisterna chyli,30min gradually increased,thecisterna chyli’s endotoxin reaches peak in1h,2h declined(compared with1h,P>0.05),3h decreased significantly(compared with1h,P<0.05).The case of theportal vein:in5min,the portal vein is difficult to observe FITC-labeledendotoxin.As time progresses,the portal vein’s endotoxin reach the peak in2h,3hdecline(and2h comparison,P<0.05).In the experiment5min,30min,1h,2h,3h,the portalvein’s endotoxin are lower than endotoxin on the cisterna chyli (P<0.05).Fluorescence spectrophotometer to analyze changes in the fluorescenceintensity of portal vein blood and the cisterna chyli liquid FITC and the changesof portal vein and celiac wall FITC had the same trend.3.Under scanningelectron microscopy,in5min,nano-gold-labeled endotoxin apears in the vicinityof the peritoneal lymphatic stomata aggregation.30min endotoxin reaches the peak,decreases in1h (compared with30min, P>0.05),2h decreased significantly(compared with30min,P<0.05),3h continued to decline.The change of cisternachyli as follows:in5min,a small amount of nano-gold-labeled endotoxinappears in the cisterna chyli.Endotoxin of cisterna chyli reach the peak in1h,decreased in2h(compared with1h,P>0.05),3h decreased significantly(compared with1h,P<0.05).The case of the portal vein:in5min,nano-gold-labeledendotoxin in portal vein is difficult to observe.As time progresses,endotoxin in portalvein reaches the peak in2h,3h decreases (compared with2h,P<0.05).In the experimentof5min,30min,1h,2h,3h,endotoxin of portal vein is lower than that of the peritoneallymphatic stomata and the cisterna chyli(P<0.05). Under the transmission electronmicroscope,we find nano-gold-labeled endotoxin in peritoneal lymphatic stomata, andunder the subperitoneal tubular,lymphatic drainage units and lymphaticcapillaries.Conclusion:Through this study,we get the preliminaryconclusions:1.Peritoneal lymphocinesia bases on the structure of peritoneallymphatic stomata,it is existed in rats.2.In our experimental observation timepoints,the way of peritoneal lymphocinesia has the function of transportingexogenous endotoxin.3.Peritoneal lymphocinesia for endotoxin transshipmentis through the peritoneal lymphatic stomata,via the peritoneum small tube underperitoneum,lymphatic drainage unit into the lymphatic capillary,at last into thecisterna chili.4.Peritoneal lymphocinesia transports on peritoneal exogenousendotoxin is earlier than the portal approach, and transport intensity ishigher than it, suggesting that the peritoneal lymphatic stomata pathwayplays an important role in the translocation.