An Experimental Study Of Serum DAO,MDA Activity And The Histological Changes Of Intestine After Acute Intestinal Obstruction In Rats

Objective:Intestinal obstruction is one of the frequent s-urgical abdomen.Intestinal obstruction has complicated pathophysiology.The study of systematic pathophysiology is promoting recent years,including in intestinal peristalsis increasing,enteric cavity pneumatosis and fluidify,intestinal ischemia-hypoxia,infecting,poisoning and so on.And it can alsocause the alteration of intestinal anatomy and the function.Finaldiagnosis rate have great degree to increase and the mortality to decrease,along with the diagnosis and treatment levelraised continuously.But how to diagnose the intestinal obstruction especially the pedo-intestinal obstruction in time andaccurately and to draw up a corresponding treatment measure still has different opinions on the clinic.so thistopic needfurther research.The succession of patient's conditions is closely related with damaged degree on the intestine wall.It is crucial important that the accurate judgment of damaged degree onobstructive intestine is for treatment and prognosis in intestinal obstruction.This experiment plans to examine the change of Dianime oxidase(DAO) and Malondialdehyde(MDA) a ctivity in serumand to investigate the pathomorphology change of intestinedamaged through enzymohistochemistry and transmission electron microscope(TEM) after acute intestinalobstruction inrats,and to discuss their interactive relationships.This experimental result can provide a certain help for the diagnosis and the selection of the surgical management of the pedo-intestinal obstruction on the clinic.Materials and methods:Hebei Medical University Animal Center offered forty-nine healthy immature Wistar rats.They were divided randomly into seven groups(A,B,C,D,E,F,G).Every group had 7 rats.Group A was control group,and the rest was experimental groups.Abrosia overnight before experiment and free drinking. They were anesthetized with 3-percent pentobarbital (30mg/kg,intraperitoneally).When anesthesia took effect,rats were Stationary in the stage.And belly-thrix was sheared off,then disinfected.Go into pneumoperitoneum in median inferior belly. From Group B toGroup G,the small intestines were all ligatured with thick suture apart from ileocecal junction 3cm so as not to make intestinal contents pass through.And not ligature mesenterium vessels,then close abdominal incisal opening.The experimental steps of Group A were same besides the intestines werenot ligatured.To Group A-G,a piece of small intestine(about 1.0cm)were got apart from ileocecal junction 4cm at 0h,3h,6h,12h,24h,48h,72h after operation and divided equally into two pieces,then rinsed with 4℃Sodium Chl oride.One was embedded in OCT and intoserial frigorific sections of 5um cut.These sections respectively were carriedout acidphosphatase(ACP) staining,alkaline phosphatase(AKP) staining and nicotinamide adenine dinucleotide tetrazolium oxidoreductase(NADH-TR) staining,then observed with light microscope.Another section was cut into 1mm3 tissue pieces and fixed in 4℃Glutaral of 4 percentage,then madeinto ultrathin sections.TEM observed them and photographed,then recorded.Meanwhile,4ml blood was collected with decapitation and placed into aseptic test tube.Blood-serum was abstracted from and carried on quantitative assay of DAO and MDA activity.All data were reported as mean±standard deviation andanalysised with t-test.P<0.05 was considered statistically significant.Results: 1 The general state of rats:The response of Group A was sensitive,and the temperament vivid,the smallintestines had not abnormal.The immature rats were graduallyirascible and irritable along with intestinal obstruction lasting.The subterminal intestines were extending and congesting,flexibility droopping after 24 hours.The mind of immature rats were dispirited,one of seven rats died, the dark purpleguts extended extremely and came necrotic partly,the flexibility vanished after 48 hours.The mind of immature ratsweredull,four of seven rats died,and most part of subterminal intestines came necrotic after 72 hours. 2 DAO activity in serum:The average value in GroupA 0.7393±0.2978 U/ml.The value of the rest group washigher than GroupA,especially for GroupDGroupEGroupF,thedifferences were significant(P<0.05).Although for GroupB GroupCGroupG,all had already go up, the differences with GroupAwere no significant.3 MDA activity in serum:The average value in GroupA 3.46±0.66umol/1.The value of the rest group was higher than GroupA,especially for GroupDGroupEGroupF GroupG,the differences were significant(P<0.05).Although for GroupB GroupC,both had already go up,the differences with Group A were no significant.4 Pathologic observations of the intestine:4.1 The results of ACP staining:ACP is the notate enzymeof lysosome.ACP staining shows red formless deposition.For Group A,ACP locates in lysosome of kytoplasm;For GroupB,red is uniform,and red zones are mainly continuous; ForGroupC,intestinal villus are partly damaged,and redzones are not continuous;For GroupD,the structuresof intestinal villus are not complete and edematous,and ACP overflows and presents massive;For Group E,massive red further increases;For Group F,the pathological changes aggravate,thecells are edematous, middle is smooth muscle,and the red zones arenot inform;For Group G,solution is not inform,and the muscular layers disappear.4.2 The results of AKP staining:AKP is the notate enzyme of cytolemma.AKP staining shows dark blue.For Group A,AKP locates in basilar part of absorptive cells'kytoplasm of intestinal villus and approaches cell nucleus;ForGroup B,the basement membrane is not complete;For Group C,the basement membrane further is damaged;For Group D,theintestinal villus are obviously edematous,and the soomth muscle is partly absent; For Group E,the intestinal villus aremore edematous and partly depauperate,the soomth muscledestroyed,the gap under the mucosa widen,and the cells of intestinal villus come necrotic and swell so that AKP mostly exists in the mesenchyma of the intestinal villus;For Group F,AKP has completely released into the mesenchyma and do not exists in the middle intestinal villus;For Group G,AKP has completely separated from the basement membrane and gland of the intestinal villus.4.3 The results of NATH-TR staining:NADH-TR is the notate enzyme of mitochondria and shows amethyst.ForGroup A,NADH-TR staining locates in kytoplasm,especially inthe cell's edge;For Group B,the intestinal villus areedematous and broaden,and enzyme has already appeared inthe mesenchyma;For Group C,NATH-TR presents massive,and the epithelium has not already be complete;For GroupD,the intestinal villus are more edematous,even not continual;For Group E,NADH-TR staining come thin,and mitochondria reduce;For Group F,NADH-TR staining come thinner,and the intestinal villus damaged takes place unreversible changes;For Gro up G,NADH-TR staining come thinnest.4.4 Observations of the ultrastructure:For Group A,theepithelium of intestinal mucosa are constituted of monolayercolumnar cell,the microvilli are complete,tidy and not engorge,compact junctions of cell are complete,the organelle have not obviously be damaged,and the goblet cells secrete highly,the membrane of secretory vesicles are distinct and complete.For Group B, the microvilli are complete,and the junctionof cells has slightest change;For Group C,the microvilli of the absorptive cells are basically normal and tidy,thecavity of intermediate junction partly enlarges and fuses,andthe mitochondria change slightly.the goblet cells have slightest change.For Group D,the microvilli of the absorptive cells arebasically normal,the cavity of intermediate junction enlargesslightly,and the mitochondria and the rough endoplasmic reticulum change slightly.The organelle of the goblet cells have slighter change. For Group E, the microvilli of the absorptive cells decrease,the mitochondria are edematous,the cristae decrease slightly,the membrane is complete,and the rough endoplasmic reticulum partly degranulate.Around the capillary of stroma obviously edematous involve fibrocytes.For Group F, the microvilli of the absorptive cells further decrease,the mitochondria are moreedematous,the cristae further decrease,the cristae and the membrane partly fuse and disappear,and the rough endoplasmic reticulum expand and degranulate.The goblet cells havemore secretory granules.For G roup G,the pathological changes are most serious.the microvilli of the absorptive cells are rare,compact junctionsof cellspartly fuse,and the desmosome is normal.the mitochondria are more edematous so that the cristae and the membrane mostly disappear.The ribosome of the rough endoplasmic reticulum partly fuse evendisappear.The pathological changes of the goblet cells are also more serious than the above.Conclusion:This experiment indicates that the changes of serum DAO,MDA activity are relevant to pathological change of intestines damaged.Along with time prolonging,the damaged degree of obstructive intestines aggravates,the cell is damaged,then the enzyme releases.Meanwhile concentration of serum MDA,DAO increases gradually.concentration of serum MDA,DAO do not increase obviously after 24hours,and concentration of serum DAO starts to decrease after 48 hours.These may correlate with them that most part of subterminal intestines were being damaged severely and intestinal mucosa was necrotic subtotally,then taking place unreversible changes.So the damaged degree of the intestinecan be calculated according to serum DAO,MDA activity.We should perform a surgical operation to relieve obstruction before theintestine takes place unreversible changes.So,acute completepedo-intestinal obstruction also should turn toperform a surgical operation in time when patient's conditions do not better,even though not worsen,though the non-surgical operation treat between 24 hours and 48 hours.Ther efore,to determine the change of DAO and MDA activity in serum havea certain help,especially to monitor continually,for the diagnosis and the selection of the surgical management of the pedo-intestinal obstruction on the clinic.But this result come from animal experiment,so it need still further research thatto determine the change of serum DAO,MDA is used as important index of the diagnosis and the selection of the surgical management of the pedo-intestinal obstruction on the clinic.