Morphological Study Of Neuroimmunomodulation In Chronic Inflammation In Digestive Mucosa

Neuropeptide is one kind of substances which possesses bioactivecharacter, can participate the nerve information transmitting in the way ofneurotransmitter and neuromodulator. In 1931 Von Euler and Gaddumfound substance P by chance. 40 years later, people dissociated substanceP from brain and bowel, and testified that they were the same substances.With the discovery of enkephalin, endorphin and dynorphinLater and theirmechanisms, especially with the development of protein chemical,radioimmunoassy, immnunohistochemistry and gene engineering,hundreds of neuropeptides were discovered by now. The moreneuropeptides were detected, the more functions were comprehended onphysiology and pathophysiology.The origin discoveries of neuropeptides include hypothalamus,anterior pituitary, brain and spinal cord, gastrointestinal tract, placenta,adrenal medulla and heart. However, most of neuropeptides which werefound in nervous system were also discoverable in periphery organs andtissues. Moreover, some peptides which were found in periphery existed innervous system. Now people have known that the gastrointestinalneuropeptides were secreted by endocrine cells which diffused distributedin gastrointestinal mucosa or nerve tissue cells. Any changes will irritate thereleasing hormone and peptides from gastrointestinal endocrine cells orgastrointestinal nerve tissue cells which will educe biological effect by theway of endocrine, paracrine, solinocrine and autocrine. The gastrointestinalneuron and fibres consist of enteric nervous system which dominatesgastrointestinal smooth muscle, gland, and vessels independent centralnervous system. Since half part of myenteric neuron and most ofsubmocous neuron containing at least one gastrointestinal peptide, andmore than one neuron perform the same activity, the present study planedto use multiple antibodies, and focus on SP, VIP and NPY to investigate thedistributions of SP, VIP and NPY containing nerve fibres in different organsand tissues in normal and different pathophysiological conditions in humanbeings. Moreover, chemical rat gastritis model was prepared for observingthe distribution of nerver fibres-containing different neuropeptides.That the biopsy sample from normal and H. pylori infected chronicgastritis were investigated by immunohistochemical andimmunocytochemical methods was the first part of the present study. Allneuropeptides-containing nerve fibres were similar presenting in themucosa among the glands with different density. Compared with the control,the number of VIP and NPY IR nerve fibres were enhanced in gastritisdistinctly and SP IR nerve fibres were increased slightly in gastritis. Theimmunocompetent cell show immunoreactivity to SP and NPY and thenumber of direct contact between the mast cell and IR nerve fibres wasincreased. Electron microscope testified that the varicosity of IR nerve fibrewhich possessed a large number of granulates and some of degranulatedmast cells. The mast cell was immunoreactivity to SP or NPY. Themorphous of mast cell in normal was round and round vesicles insidecytoplasma, however, it was irregular, degranulated and vacuole appearedin gastritis. Moreover, the degranulated mast cell connected to IR nervefibre. The gap between the IR nerve fibre and immunocompetent cell was20nm-200nm.The second part of present study consists of animal modelpreparation and ABC stain. Gastric inflammation was induced withiodoacetamide. During a 5-day period, gastritis rats received a dailyintragastric administration (0.1ml) of the iodoacetamide solution;in addition,iodoacetamide (0.1%) and sucrose (1%) were added to the drinking waterduring the same period. Controls received the drinking water withoutiodoacetamide. 5 days later all rats were sacrificed and gastric sampleswere got and the experimental procedures were performed in the sameway as humen beings gastritis. Macroscopic lesions were observed in thestomach of iodoacetamide-treated rats. The distribution ofneuropeptides-containing nerve fibres were not difference between thecontrol and the gastritis, which presented in the mucosa among the glandswith different density. Compared with the control, the number of VIP andNPY IR nerve fibres were enlarged in gastritis distinctly and SP IR nervefibres were increased slightly in gastritis. The immunocompetent cell showimmunoreactivity to SP and NPY and the number of direct contact betweenthe mast cell and IR nerve fibres was increased as well. Fewimmunocompetent cell exhibited IR in control. What electron microscopeobserved was similar with human gastritis that the mast cell wasimmunoreactivity to SP and NPY. Furthermore, varicosity of SP and NPY IRnerve fibre possessed a large number of granulates. Generally the distancebetween the IR nerve fibre and immunocompetent cells is 20nm-200nm.Moreover, some of mast cells degranulated in gastritis.The colonic biopsy samples from ulcerative colitis before and aftersalazosulfamide treatment for one month were detected byimmunohistochemical and immunocytochemical method. Light microscopeobserved that all the detected IR nerve fibre-containing differentneuropepetides were present all layers in human colon. Most of them weresituated in epithelium mucosa and very close to vessels in ulcerative colitisafter one month treatment. From the investigated neuropeptides, SP andVIP IR nerve fibres were most remarkable. The number of NPY IR nervefibre was moderate. IR neuron could be found in submucosa. Onlymoderate infiltration of inflammatory cells and increasing IR nerve fibreswere present in treatment .However, the numbers of IR nervefibres-containing different neuropeptides were reduced in the inflamedcolon from ulcerative colitis. The small and round lymphocytes and largerelliptic plasma cells were increased in ulcerative colitis before treatmentand present positive for SP, VIP and NPY. During the electron microscopicinvestigation of colitis the affected areas were characterized infiltration oflymphocytes, plasma cells, macrophages and degranulated mast cells.Most of the nerve fibres contained a large number of clear granulatedvesicles in control but only a few synapses and vesicles were detected incolonic enteric plexus from the ulcerative colitis. These varicosevesicle-containing nerve fibres were close to the epithelial lining, bloodvessel and lymphocytes. In most cases the IR nerve fibres were free ofSchwan cell. The distance between the membranes of IR nerve fibres andlymphocytes, plasma cell and mast cell was 20-200 nm. The direct contactwith immunocopetent cell was also observed.Changings of nerve fibres-containing different neuropeptides in thegallbladder mucosa with chronic cholecystitis accompaniedcholecyslithiasis and without inflammatory gallbladder were detected byABC method. Compared with the control, both the distribution and thenumber of nerve fibres-containing different neuropeptides were changedwith light microscope. The immunoreactive positive (IR) nervefibres-containing different neuropeptides could be found in all layers;moreover, SP and VIP IR neuron could be detected in muscle layer in thecontrol. However, NPY and VIP IR nerve fibres mainly located insubmucasa in case. The number of (IR) nerve fibres-containing SPdecreased obviously and NPY decreased, however, VIP increasedmarkedly in case. In the inflamed area the number of theimmunocompetent cells was also increased (being lymphocytes, plasmacells and mast cells) and some of them were also IR for SP and VIP. Closecontacts were detected between IR nerve fibres and the immunocytes inseveral cases. The electron microscope observed the varicosity of IR nervefibre which possessed a large number of granulates and some ofdegranulated mast cells. The mast cells were IR for SP and NPY and theIR nerve fibre adjoined immunocompetent cells.Meanwhile, the present study observed the changes of mast cell. Thesize and morphous were different in normal. Mast cells were ellipse orfusiform and granula stainable in cytoblast in control. In case, mast cellswere activated by inflammation or chemical stimulus and presentderegulation. VIP which was released from mast cell could dilate smallvessel, effect local circulation and inhibit the releasing of histamine or otherbioactive substances. SP which was also released from mast cell couldpromote neutrophil and eosinophile granulocyte to the inflamed area andendotheliocyte to secret adhesion molecule. SP could also irritate mast cellto release histamine. The gap between mast cell and immunoactivepositive nerve fibre was 20-200nm. The close in anatomy means theinteraction each other in function. The spatial relation provided basicmorphous between gastrointestinal neuropeptides and mast cell andtestified the two-ways regulation as well. Gastrointestinal neuropeptidescould act as a neuron-immnuno-modulation in digestive mucosainflammation.The present study investigated the distributions of nerve fibres-containing different neuropeptides in stomach, colon and gall bladder. SP,VIP and NPY stained well and SP, VIP and NPY nerve fibres andimmunoconpetent cells which positive to SP, VIP or SP, NPY could beobserved in gastritis and control.The distribution of SP nerve fibres in gastritis and control was similar.SP immunoactive positive neuron and fibres presented in submucosa and/or muscle layer in inflamed and normal tissue. Inflammation produced SPreleased from damaged tissue. The vasopermeability increased, bloodplasma extravasated and regional edema caused by SP. SP could increasethe gastrointestinal contraction which contract arteriole and cause thereduction of blood flow, as result , the mucosa were damaged. Moreover,the proinflammatory factors secretion enhanced by SP aggravatedinflammation. The releasing of histamine, prostaglandin, bradykinin and HTfrom degranulated mast cell could produce neuron-inflammation and pain.The problem is immunoactive positive nerve fibre-containing SP increasedin chronic gastritis and colitis after treatment but decreased in cholecystitis.The reason may be the central nerve system participate the SP secreting.In the future study will find out the real reason.Neuron and fibre-containg VIP were abundant in digestive system.The number was increased in all cases. Moreover the rat model andhuman beings gastritis were the same phenomenon has demonstrated thatrespond of gastric mucosa to stimulate. VIP could modulate the generation,differentiation and activation of immunocyte and inhibit the secretion andcomposition of proinflammatory cytokine result in decreasing ofinflammation. VIP enhanced the peroxidase activity result in inhibitingdegranulation of eosinophile granulocyte. The increasing of VIP aftertreatment reflected the healing.Immunoactive positive nerve fibre-containing NPY stained well andDAB well. Although the increasing in chemical gastritis and decreasing incholecystitis without statistical significance, the tendency was similar to SP.In the future study will investigate the contribution of NPY.Conclusions: The releasing of neuropeptide from gastric mucosainduced by H pylori infection would activate mast cell and otherinflammatory cells which caused inflammation and enlarged inflammatoryprocess. Meanwhile, lymphocyte may secret and release neuropeptide.There was direct bidirectional communication between immune system andnervous system. The neuropeptide which releases from sensory nerveterminal participated in the gastric protecting regulation.Iodoacetamide could induce non-infectious gastritis. Degranulatemast cell could be detected both in H.pylori infected gastritis andiodoacetamide-induced rat gastritis. Moreover degranulate mast cellcontacting with immunoreactive nerve fibre certificated that the gastricinflammation was related to immune response which proofed thatneuropeptide participate in neuron-immuno-modulation in gastric mucosa.The changing of number and distribution of nerve fibre-containingdifferent nerve fibres in ulcerative colitis mucosa may be the result ofmucosa inflammation and impaired. The direction contact between theimmunocompetent cell and nerve fibre-containing neuropeptide providesthe evidence for immunoreactive response and interaction.During the formation of cholecystitis a large number ofgastrointestinal neuropeptids participated in the process. VIP, which coulddilate gall bladder, increasing and SP, which could contract gall bladder,decreasing unbalanced gall bladder contraction and relaxation. Result indecreased contraction, loosening and distention in gall bladder.The achievements of present study as follows:The study observed the changing of nerve fibres-containing differentneuropeptides in common inflammation in digestive system includinggastritis, colitis and cholecystitis, the relationship between nerve fibre andimmunocompetent cell and mast cell degranulating which confirmed theneuropepides played an important role on inflammatoryneuro-immuno-modulation in digestive system.The present study successfully prepared animal gastritis induced byiodoacetamid, which certified the contribution of neuropeptide inneuro-immuno-modulation induced by chemical irritation.This clinic significance of present study is that will benefit thealternative treatment of gastrointestinal inflammation. The balance of pro-and anti-infalmmation will determine the successful control of inflammtion.The pro-inflammatory and anti-inflammatory neuropeptide are ofimportance in inflammation. The therapeutic efficacy of uncreative colitissuggested that anti-inflammatory neuropeptide increasing conduce toinflammation.