Role for periodontitis in the progression of lipid deposition in an animal model

Abstract

Epidemiologic studies have implicated periodontitis as a risk factor for the development of cardiovascular disease. However, no prospective studies investigating this potential relationship have been carried out. Age- and sex-matched New Zealand White rabbits were maintained on a diet consisting of 0.5% fat for 13 weeks to induce the accumulation of lipid deposits in the aorta as a model for atherogenesis. One-half of the animals received silk ligatures around their mandibular premolars followed by an application of a periodontal pathogen, Porphyromonas gingivalis, to induce periodontitis. Animals were sacrificed after 14 weeks. Periodontal disease severity was quantified radiographically, histologically, and by direct visualization of bone loss on defleshed skulls. Lipid deposition was evaluated by computer-assisted morphometry in the aortas en face after lipid deposits were stained with Sudan IV. Animals with experimentally induced periodontitis had more extensive accumulations of lipids in the aorta than did nonperiodontitis animals (P < 0.05), and there was a positive correlation between the severity of periodontal disease and the extent of lipid deposition (r(2) = 0.9501). The results provide direct evidence that periodontitis may be a risk factor and may contribute to the pathogenesis of atherosclerosis. The data support the concept that infections at remote locations can modulate atherosclerotic events distantly.

FIG. 1.

Digital standardized radiograph of test (left panel) and control (right panel) animal jaws. The radiographs were taken with standardized alignment in three directions. The percentage of the tooth outside the bone was calculated according to a modified technique of Bjorn et al. (3). The left panel shows clear bone loss in the second premolar area (arrows).

FIG. 2.

Histological assessment of bone resorption due to P. gingivalis application. Panel A shows cells from a control animal whose bone levels were not affected by the placement of the ligature plus vehicle. In contrast, panel B shows cells from an animal of the periodontitis group where resorption of the bone crest was observed in response to the placement of ligatures soaked with P. gingivalis. This animal developed severe aortic lipid deposition (hematoxylin and eosin staining; magnification, ×174).

FIG. 3.

Representative results of periodontal lesion quantification and en face analysis. (A) Test group. The images show an extensive loss of bone in the second premolar area (arrow) and extensive coverage by atherosclerotic plaque of the same animal's aorta. (B) Control group. The images show the crestal bone with no loss around the second premolar and the aorta of the same animal, where minimal plaque is visible. (C) Test group. The images show the crestal bone from an animal that failed to develop any periodontal lesion and the same animal's aorta, which shows minimal plaque development as well.

FIG. 4.

For PCR optimization, 10-fold serial dilutions of 3-day P. gingivalis cultures were amplified under the conditions described in the text. The conditions used in our study were able to detect P. gingivalis 16S rRNA genes from as few as 10¹ CFU obtained from a pure culture.