Altered Expression Of Glutamic Acid Decarboxylase In Rat Small Intestine With Postoperative Ileus

BackgroundPostoperative ileus(POI),a frequent complication occurs after abdominal surgery,is considered as an intestinal motility disorder with immunocyte infiltration.Gamma-aminobutyric acid(GABA)is one of the most important inhibitory neurotransmitters in the central nervous system(CNS).GABA system is composed of GABA and its related enzymes,receptors and transporters.The immunocyte can also synthetize and secrete GABA.The activation of GABA receptors in gut can affect the intestinal motility through the cholinergic system.We speculate that the activation of GABA system by immunocyte plays an important role in the intestinal dysmotility of POI.Autofluorescence(AF)in tissue greatly interferes with the acquisition of immunofluorescence(IF)target signals and the judgment of experimental results.Lipofuscin and other lipid components in immune cells are important sources of AF,while Sudan black B(SBB)can absorb AF by combining lipid to form brown black granules.As one of the most used experimental animals,rats have a lot of immune cells in their intestines,but the distribution of AF in their intestines has not been clearly investigated.ObjectivesTo verify the expression and distribution of glutamate decarboxylase(GAD)and GABA receptors in jejunum,ileum and colon of normal rats.To observe the change of GABA secretion and GABARs expression in intestine of POI model rats,as well as the alteration of cholinergic system in the muscular layer,and eventually,explore the role of immunocyte and GABA system in the intestinal dysmotility of POI.To verify the expression of AF in rat intestine and evaluate the optimum concentration and staining time of SBB treatment to reduce the AF and improve the specificity of IF staining in rat intestine.Methods1.Animal model:Adult healthy male SD rats were anesthetized with 10%chloral hydrate.The small intestine was drawn out and wiped up and down with a wet cotton ball for five or six times in 10 min,and then returned to the abdominal cavity.2.Tissue harvest:24 hours after the operation,the intestine was harvest by abdominal surgery.Part of the fresh tissues were used for Western blot,tissues left were fixed with 4%paraformaldehyde.Frozen sections were made for morphological detection after dehydration with gradient sucrose.3.To evaluate the intestinal mobility by intestinal propulsion test.HE staining was used to observe the intestinal morphology of POI model rats.At the meaning time,the injury of intestinal mucosa was evaluated by pathological score,which followed by the Chiu's grade.4.The expression and distribution of GAD and GABA receptors in jejunum,ileum and colon of normal rats were detected by immunohistochemistry and immunofluorescence technology.5.The expression and distribution of GAD,GABAB receptor(GABAB),acetylcholine transferase(ChAT)in jejunum,ileum and colon of POI model rats were detected by immunohistochemistry and immunofluorescence technology.6.The alteration of GABAB receptor expression in jejunum and ileum were quantified by Western blot technology.7.After cryostat section,intestinal autofluorescence(AF)was investigated by immunofluorescence(IF)before and after Sudan black B(SBB)treatment.Results1.GAD was expressed in jejunum,ileum mucosa and muscular layer of normal rats.2.POI rat model was established successfully.3.The expression of GAD in intestinal muscular layer of POI rats was up-regulated.Either CD45 or CD68 was detected in most GAD-immunoactivity positive nurons in the muscular layer of the small intestine of POI rats.4.The expression of GABAB receptor in the muscular layer of small intestine of POI rats was up-regulated while the expression of ChAT in the myenteric plexus was down-regulated.5.AF was observed in both FITC-and TRITC-channel spectrum in all intestinal segments and not affected by serum blocking.0.3%SBB treatment for 10 min significantly reduce the AF and improved the specificity of IF staining in rat intestine.ConclusionsGAD positive cells were detected in the intestinal mucosa and muscular layer of normal rats.A large number of inflammatory cells infiltrate into the muscular layer of POI rats.These cells may synthesize GABA and activate GABA_B receptor expressed on cholinergic neurons in the myenteric plexus,inhibit the activity of cholinergic neurons and reduce the cholinergic signal,which maintain the contraction of intestine,ultimately,inhibit the intestinal motility.Our study provides a possible new mechanism for the dysmotility of POI intestine.In addition,GABA_B receptor inhibitors may be a new tool to alleviate or treat POI.AF was observed in both FITC-and TRITC-channel spectrum in all intestinal segments,which was not affected by serum blocking.The AF in rat intestinal frozen section was effectively reduced by 0.3%SBB treatment for 10 min with the target inflorescence intact.