Effects Of Oxidative Stress On Rabbit Periodontitis When Exposed In Analogic High Altitude Anoxia Environment

Backgrounds and objectives:Periodontitis is one of the most common, chronic inflammory diseases, which threatenthe oral health of human. The epidemiological investigation shows that the prevalence andpathogenesis process of periodontitis is in a rising tendency accompanied with theincreasing of the altitude and the living time in highland.Stimulated by the pathogens as well as their productions, the immune cells wereinfiltrated and released the inflammatory mediators in local periodontal tissues; further themacrophages were activated to discharge the inflammatory exudates such as cytokinesTNF-α （Tumor necrosis factor-alpha）, PGE₂（Prostaglandin E₂）, IL-8（Interleukin-8）.Mediating by the numerous inflammatory cytokines, periodontitis eventually lead to thedestruction of the periodontal supporting tissues, including the degradation of collagenfibers and the bone resorption. Our preliminary animal experiments researches have alreadyindicated that the expressions of a few inflammatory mediators in plateau are notable higherthan in plain, but the pathogenesis of periodontitis in highland is still unclear. The factors ofstress, including hypoxia, cold, hard ultraviolet radiation and depriving of sleeping, areincreasing in highland environment, thus the various kinds of functions and metabolism oforganism can suffer from a series of changes under oxidative stress. Researches manifestthat hypoxia environment can change the redox signaling following by oxidative stress andthus affect the interactions of immune cells. Lacking of oxygen and inflammatory reactionin local tissues, as well as generally immune response, play important roles in thepathological processes of chronic periodontitis. These show that the oxidative stress causedby hypoxia environment maybe affect the pathogenesis of periodontitis in plateau. However,there is little known about the changes of oxidative stress in local periodontal tissues andthe effects of the adhesion molecules among cells in highland. Thus it is needed to furtherstudy. This study established the experimental animal models of chronic periodontitis in astable, analogic high altitude anoxia environment to observe the clinical modification andthe histological changes. The activity of superoxide dismutase （SOD） in blood serum andgingival tissues in all groups were measured and the expressions of soluble intercellularadhesion molecule-1（sICAM-1） and soluble vascular cell adhesion molecule-1（sVCAM-1）in blood serum and gingival tissues were detected by enzyme linked immunosorbent assay（ELISA）. And the relationships between these mediators and the periodontal clinical indiceswere analyzed too. Thus it is helpful for us to understand the roles of these mediators in thegenesis, progress and prognosis of periodontal diseases in highland, expecting to achieve anew thread for the prevention and cure of periodontitis in the special environment.Materials and Methods:1.The establishment of rabbit periodontitis animal modelsFourty male rabbits were randomly divided into four groups（ten each）: normoxiacontrol and experimental group, hypoxia control and experimental group. The periodontitismodels were established by being ligated the two central incisors of mandible and raised byperiodontitis diets referring to the methods of Keyes and Chen tielou. The anoxia groupswere raised in an established low pressure oxygen compartment of altitude5000meteres foreight weeks and the anoxic duration are23hours everyday. The normoxia groups wereraised in normal oxygen environment of average altitude400meteres.2.The evaluation of rabbit periodontitis animal modelsAfter eight weeks, the physiological index and the periodontal clinical indices indifferent groups were recorded, including attachment lost （AL）, plaque index （PLI）,gingival index （GI）. The X-rays were used to observe the changes of alveolar levels and theserum sampling were collected and saved. Then the rabbits were sacrificed and theperiodontium specimens were incised to undertake the section and carried out routinehematoxylin-eosin （HE） staining. HE staining and pathological assay were used to observethe general conditions and pathological changes of rabbit periodontal tissues. Allexperiments were conducted in accordance with the National of Health guidelines for thewelfare of experimental animals.3.The activity of enzyme detectionThe changes of the activity of SOD in serum and local gingival tissues were assayed by the principle of xanthine oxidase. The correlations were evaluated among the activityand the periodontal clinical indices.4.Adhesion molecules quantificationThe concentrations of sICAM-1and sVCAM-1in serum and local gingival tissueswere detected by ELISA. The correlations were also analyzed among the contents and theperiodontal clinical indices.Results:1.The physical indexCompared with the normoxia groups, the weights of the anoxia groups grew retardedand gained slightly, showing a descending tendency especially in the later stage of theexperiment. At eight weeks, the weights of the normoxia experimental group and the anoxiaexperimental group are higher than the respectively control group （P<0.01）. Compared theanoxia experimental group to the normoxia experimental group and compared the anoxiacontrol group with the normoxia control group, the deviations have statistic significances（P<0.01）.2.The clinical indices and the pathological changesThe periodontal tissues in anoxia experimental group have severity inflammation,including obviously red swelling and retraction of gingiva, deep periodontal pockets,positive bleeding of probing, active hemorrhagic tendency and abundant chymus andbacterial plaques in the gingival crest and the dental face. The sections of the anoxiaexperimental group were infiltrated with massive inflammatory cells and the alveolar boneswere suffered from great loss. The X-rays show the dramatically widened periodontalmembrane space and the decreasing height of alveolar bone. Compared with other groups,the periodontal clinical indices of the anoxia experimental group, such as AL, PLI and GI,have statistic significances （P<0.01）.3.The activity of enzyme detectionThe activities of SOD in anoxia experimental group were distinguished lower than theother groups （P<0.05）. The activities of SOD in serum and gingival tissues presenteddramatically negative correlations with periodontal clinical indices （AL, PLI, GI, P<0.01）.And the activities in serum were positively correlated with that in the gingival tissues（P<0.01, r value is0.846）. 4.Adhesion molecules quantificationThe concentrations of sICAM-1and sVCAM-1in serum and gingival tissues of theanoxia experimental group are higher than all the other groups （P<0.01）. Theconcentrations of sICAM-1and sVCAM-1in serum and gingival tissues exhibitedsignificantly positive correlations with periodontal clinical indices （PLI, P<0.05; GI, AL,P<0.01）. And the levels in serum were positively correlated with in the gingival tissues（P<0.01, r values were respectively0.997and0.988）.Conclusion:1. Our experiments have successfully built up the rabbit periodontitis animal modelwhen exposed in analogic high altitude anoxia environment. The periodontal clinicalsituations and pathological changes of the anoxia experimental group represent the typicallypathological changes of periodontitis, including retraction of gingiva, hyperplasia ofjunctional epithelium towards the root of teeth, deep periodontal pockets, infiltration withsignificant inflammatory cells in epithelium and connective tissues, the serious resorptionof alveolar ridge. In our model, hypoxia and high altitude environment aggravated thepathological process of periodontitis.2．At a simulated altitude of hypoxia environment in our model, the activity of SODdramatically decreased, while the levels of sICAM-1and sVCAM-1significantly increased,indicating the high altitude and hypoxia environment can increase the oxidative stress byreducing the anti-oxidant ability and influence the expression of adhesion molecules inlocal and systemic periodontal tissues.3. The concentrations of sICAM-1and sVCAM-1exhibited strikingly positivecorrelations with periodontal clinical indices, while the activity of SOD negativelycorrelated with the indices, indicating that in our simulated altitude hypoxia environment,the increasing of sICAM-1and sVCAM-1and the decreasing of the activity of SOD canaffect the balance of reactive oxygen species（ROS） and the adhesion of inflammatory cells,thus accelerating the pathogenesis process of periodontitis and bone resorption.4. The pathogenesis of periodontitis companied with the immunologic reaction locallyand generally. Our researches show that the notable correlations exist in the levels ofinflammatory mediators between the serum and gingival tissues, proclaiming that thehypoxia environment not only can sharpen and accelerate the inflammatory reaction in local tissues, but also can contribute to the inflammatory state generally.