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

PURPOSE: Lipopolysaccharide (LPS) may act as a key stimulatory agent in ocular surface diseases (OSDs) through TNF-alpha release. We used in vivo confocal microscopy (CM) and ex vivo flow cytometry, two new tools for assessing ocular inflammation induced by LPS.

METHODS

We investigated a model of acute inflammation in rabbits by subconjunctival injection of LPS and developed new evaluation techniques for animal models: CM, to observe inflammatory infiltrates, and conjunctival impression cytology (IC) specimens processed with in vitro CM and flow cytometry for assessing TNF-alpha and TNF receptor-1 (TNFR-1) expression. A neutralizing anti-TNF-alpha antibody was used to assess the role of TNF-alpha.

RESULTS

In vivo CM provided high-resolution images of inflammatory infiltrates and leukocyte rolling in blood vessels. It showed that the LPS group presented strong conjunctival inflammation, reaching its maximum level 4 h after injection. Flow cytometry and immunostaining in IC specimens showed an increased expression of TNF-alpha and TNFR-1 in the epithelium. Immunohistology confirmed these results and showed infiltration of vimentin+, CD4(+), and CD8(+) cells in the conjunctiva. TUNEL-positive cells were found 4 h after injection. Neutralizing anti-TNF-alpha significantly inhibited LPS-induced inflammation and apoptosis evaluated by in vivo CM; and inhibited LPS-induced TNF-alpha and TNFR-1 expression by ex vivo conjunctival IC specimens evaluated by flow cytometry.

CONCLUSIONS

IC specimens and new-generation in vivo CM were thus in good agreement with immunohistology and appeared to be reliable, effective, and nonharmful methods to investigate experimental models of OSDs. The two new tools applied here evaluate the animal models in vivo on the cellular lever. This study is consistent with the experimental research's strategy by reducing the number of experimental animals used.