| BACKGROUNDIntercellular adhesion molecule-1(ICAM-1) is belonging the immunoglobulin gene superfamily.Through mediate adhesion between cells and cell,cells and extracellular matrix,ICAM-1 can participate in a series of physiological and pathological processes.ICAM-1,a single-chain transmembrane glycoprotein with a molecular weight of 80-110 KDa,consists of five Ig-like domains,a hydrophobic transmembrane domain and a short cytoplasmic C-terminal domain.And NF-κB, plays the leading role in the activation of ICAM-1.Trough the special interaction of protein-protein,NF-κB participate in the expression adjustment of ICAM-1 with other transcription factors and syn-activator activates together.Ligand of ICAM-1 includes lymphocyte function- associated antigen-1(LFA-1) and macrophage antigen-1(Mac-1).When LFA-1 is increased,resting affinity can be regulated.First,the affinity can be increase by an addition of the aggregation degree of integrin distribution on the membrane.And,integrins change configuration,so that its intrinsic affinity with ligand is increased.Under physiological conditions,ICAM-1 is expressed at a low level in endothelial cells and epithelial cells or constitutively on the surface of the cells,providing the underlying molecular basis for cell recognition, activation,proliferation,differentiation and motility,and thereby helping to stabilize the internal environment of the body.Moreover,ICAM-1 also plays a key role during pathological conditions,such as inflammatory reaction etc.For these reasons,a comprehensive and objective understanding of ICAM-1 is needed.Inflammatory cells leave the circulation at sites of local inflammation through a series of distinct sequential steps.The leukocyte-endothelinm interaction,the fundamental event of inflammation is orchestrated by the action of some cell adhesion molecules(CAMs) found both on leukocytes and endothelial cells.The initial adhesive event is the margination characterized by rolling of leukocytes along the luminal surface of the endothelium.Each member of the selectin family(E-, P-L-selectin) has been shown to support leukocyte rolling under flow conditions. Once the marginated leukocytes form a stationary adhesion with endothelial cells,it is stimulated by chemotactic factors to downregulate the selectin-based adhesion and upregulate adherence dependent on integrins.Subsequent stages of leukocyte immigration are mediated by the interactions between integrins and members of the immunoglobulin superfamily:intercellular cell adhesion molecules(ICAM-1, ICAM-2,and ICAM-3),vascular cell adhesion molecule(VCAM-1) and platelet cell adhesion molecule(PECAM-1).Thus,teukocytes rapidly immigrate into surrounding tissues.And according to literature review,ICAM-1 is believed to allow not only recruitment of naive lymphocytes and rapid accumulation of leukocytes at injured and infected sites,but also recirculation of lymphocytes stimulated by inflammatory agents.ICAM-1 is already playing the influential role in the cell adherence,it is also a player in the actual transmigration of leukocytes through the vessel wall (diapedesis).OBJECTIVESDental pulp is surrounded by dentin and isolated from other tissue,like an in vitro system,and it is almost always exposed to inflammatory agents entering through dentinal tubules in the case of dental caries and through the apical foramen in periodontitis.The pulp develops inflammation as a basic protective biologic defense mechanism in response to any type of injury.As a result,dental pulp could be a model for studying the roles of adhesion molecules on the vascular endothelium in transendothelial migration of leukocytes both with and without inflammation.Despite the extensive information on the tissue distribution of ICAM-1 in healthy and pathological conditions in other fields of medicine to date,there is limited scientific data concerning the expression of this molecule by the vascular endothelium of the oral tissues and particularly of the dental pulp.Therefore,the present study was undertaken to determine the expression of ICAM-1 on the vascular endothelium of healthy and inflamed dental pulp.Materials and methods1.Animals and reagentsWe used 60 female Sprague-Dawley rats(n=60,200-250 g body weight) for this study.The rats were divided equally into six groups included one control group and five experimental groups.The rats were purchased from the Center of Experimental Animals of Southern Medical University.ICAM-1 antibody was purchased from Wuhan Boster Biological Technology Co.,Ltd;SP-9001 HistostainTM-Plus Kits and DAB complex kit was supplied by Zhongshan Golden Bridge Biotechnology Corporation,Beijing,China.2.Sample preparationIn 50 female rats of five experimental groups,following anesthesia with 10% chloral hydrate,the roof of the pulp chamber of the right first and second maxillary molar teeth was removed using a 005 round bur in a high-speed handpiece.The pulp chambers were left open to the oral environment.10 female rats were control group and were untreated in all animals.After experimental pulpal exposure,the rats were killed at various time points: 1h,1d,3d,5d and 7d;each n=10.The teeth and the part of maxillary bone were rapidly removed to determine quantitative changes in pulpitis induced ICAM-1 activity.3.Observation under light microscopeThe specimens were fixed in 4%paraformaldehyde for 48 hours,and further trimmed and transferred into 15%EDTA for decalcification for 15 days and then embedded by routine paraffin.Slices were continuously sectioned parallel to the long axis of tooth with a thickness of 5μm.One paraffin section of each sample was selected and stained with HE,and then the dental pulp histological and morphological changes were observed.4.Immunohistochemistry examinationImmunohistochemical SP methods were employed in this study.After being dewaxed and reacted with 3%H2O2 for 10 min,the slices were put in citrated saline buffer liquid(pH 6.0±0.1) and heated in the microwave stove adjusted to grade"3"at 92～98℃for 10min.And then the slides were pre-incubated with 10%goat serum for 15 min at room temperature.Then immune-stained were began with adding monoclonal antibodies to ICAM-1,for 12 hours at 4℃in refrigerator.The slides were sequentially incubated with biotinconjugated secondary antibodies followed by horse radish peroxidase-conjugated streptoavidin.The first step lasted 15 min and the second step lasted 10 min at 37℃in thermostat with 5min PBS-buffered saline washes three times each step.The reactions were revealed using DAB substrate, slides were then washed in running tap water.Contrast with hematoxylin and mounted in balsam,observed with light microscopy.Blank staining was performed similarly with elimination of the primary antibody.5.The method used to quantify labelingEssentially,computer-assisted image analysis software(Image-Pro Plus,Version 5.0;Media Cybernetics,Silver Spring,MD,USA) was used to create a digital image from the microscopic image.The OD value of ICAM-1-labelled tissue was then determined within each field of analysis.6.Statistical analysisAll the data were shown as mean±S.D.Statistical analysis of data was performed by applying one - way analysis of variance(ANOVA) with SPSS V.13.0. The q-test was conducted after the test for homogeneity of variance and analysis of variance,and the correct t test was employed in the two-two comparison.In all statistical comparisons,P＜0.05 or P＜0.01 was considered significantly difference statistically.RESULTS1.HE stain:In the normal control group,pulp tissue have a small amount of blood vessels and a great quantity of fibroblasts,and at the edging of the medullary cavity there are a layer of columnar odontoblast cells.The typical pulpitis was observed in experimental groups.The dental pulp inflammation may appear in 1 hour group,and aggravates gradually along with the time development.Leucocyte appears in early stage of pulpitis,but lymphocyte appears latterly from the third day.Blood vessel hyperplasia and dilatation and vascular engorgement are also main manifestation of pulpitis.2.Immunohistochemistry examination results: Very less positive expressions of ICAM-1 were found in microvascular endothelial cells of the dental pulp regions in thenormal control group,but in the five experimental groups,the expression was higher than those in the normal control group,especially at the first and the third and and fifth day.The seventh day was gradually reduction.3.Statistical analysis results:There were showed significant difference,indicating that the ICAM-1 positive cell expression in microvascular endothelial cells increased sharply in the dental pulp of the pulpitis.CONCLUSIONSThis study showed that the method to establish a rat pulpitis induced by exposure of dental pulp was feasible and ICAM-1 had a significant increase in the experiment pulpitis compared with normal dental pulp tissue.ICAM-1 may play a role in the process of dental pulpitis.ICAM-1 guides the stable adhesion between leukocytes and vascular endothelial cells,which enables leukocytes to be recruited and anchored on the inflamed tissue. But,for recruitment of lymphocytes,ICAM-1 played the very minor role.There must have different regulatory mechanism for recruitment of lymphocytes in the process of dental pulpitis.Through the animal experiment,we can observe the entire dynamic process of pulpitis from start at the molecular level,further clarify the mechanism of ICAM-1 involve in the development of pulpitis.
|
PDF Full Text Request