In vivo confocal microscopy and ex vivo flow cytometry:new tools for assessing ocular inflammation applied to rabbitlipopolysaccharide-induced conjunctivitis

[摘要] Purpose: Lipopolysaccharide (LPS) may act as a keystimulatory agent in ocular surface diseases (OSDs) through TNF-αrelease. We used in vivo confocal microscopy (CM) and ex vivo flowcytometry, two new tools for assessing ocular inflammation induced byLPS.Methods: We investigated a model of acute inflammation inrabbits by subconjunctival injection of LPS and developed new evaluationtechniques for animal models: CM, to observe inflammatory infiltrates,and conjunctival impression cytology (IC) specimens processed with invitro CM and flow cytometry for assessing TNF-α and TNF receptor-1(TNFR-1) expression. A neutralizing anti-TNF-α antibody was usedto assess the role of TNF-α.Results: In vivo CM provided high-resolution images ofinflammatory infiltrates and leukocyte rolling in blood vessels. Itshowed that the LPS group presented strong conjunctival inflammation,reaching its maximum level 4 h after injection. Flow cytometry andimmunostaining in IC specimens showed an increased expression ofTNF-α and TNFR-1 in the epithelium. Immunohistology confirmedthese results and showed infiltration of vimentin+, CD4+, andCD8+ cells in the conjunctiva. TUNEL-positive cells were found 4 hafter injection. Neutralizing anti-TNF-α significantly inhibitedLPS-induced inflammation and apoptosis evaluated by in vivo CM; andinhibited LPS-induced TNF-α and TNFR-1 expression by ex vivoconjunctival IC specimens evaluated by flow cytometry.Conclusions: IC specimens and new-generation in vivo CM werethus in good agreement with immunohistology and appeared to be reliable,effective, and nonharmful methods to investigate experimental models ofOSDs. The two new tools applied here evaluate the animal models in vivoon the cellular lever. This study is consistent with the experimentalresearch's strategy by reducing the number of experimental animalsused.

[学科分类] 生物化学/生物物理