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H. Liang, A. Labbé, F. Brignole–Baudouin, C. Martin, J.–M. Warnet, C. Baudouin; Lipopolysaccharide Induced–Rabbit Conjunctival Inflammation and Apoptosis: In vivo Confocal Microscopy and Histological Approaches . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4932.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies suggested that lipopolysaccharide (LPS) is a major inducer of inflammation during ocular surface bacterial infection. The purpose of this study was to investigate in rabbits a model of LPS–induced conjunctival inflammation using in vivo confocal microscopy, immunohistology and impression cytology (IC) specimens.
Acute conjunctivitis was induced in rabbit eyes by subconjunctival injection of LPS and results were compared to those found in balanced salt solution (BSS)–injected rabbits. The new–generation HRT II/RCM in vivo confocal microscope was used to observe conjunctival inflammatory patterns in the epithelium and substantia propria. IC specimens from rabbit conjunctiva were analyzed using immunohistochemistry, confocal microscopy and flow cytometry. Cryosections of conjunctiva were used for immunostaining and were observed under a confocal microscope for assessing inflammatory cells and tumor necrosis factor alpha (TNF–α)–related markers. Apoptotic cell death in the conjunctiva was determined by TUNEL assay.
The LPS–injected group presented an acute conjunctivitis with redness, swelling and purulent secretion, maximal 4 hours after injection. The HRT II in vivo confocal microscope provided high–resolution images of inflammatory cell infiltration and leukocyte rolling in blood vessels. Flow cytometry analysis and immunostaining of IC specimens showed an increased expression of TNF–α and TNF receptor 1 (TNFR–1) in the epithelium. Immunostaining of cryosections also showed strong expression of TNF–α, TNFR–1, infiltration of vimentin–positive cells, CD4+ and CD8+ lymphocytes in the conjunctiva. Examination of conjunctival sections showed TUNEL–positive apoptotic cells in the conjunctiva as early as 4 hours after injection.
LPS induced inflammation and apoptosis with important expression of TNF–α and TNFR–1 in the conjunctival epithelium and substantia propria. IC specimens and HRT II in vivo confocal microscopy were in good agreement with immunohistology, and appeared as being reliable, effective and non–harmful methods to evaluate experimental models of ocular surface diseases.
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