Live Conjunctiva-Associated Lymphoid Tissue Analysis in Rabbit under Inflammatory Stimuli Using In Vivo Confocal Microscopy

Abstract

Conjunctiva-associated lymphoid tissue (CALT) plays an important role in ocular surface immunity. No study until now has been able to show its in vivo aspects, and few have demonstrated its reactions after pathologic patterns. The authors investigated in rabbit eyes the cell reactions occurring in the organized CALT during conjunctival inflammation models. Using in vivo confocal microscopy (IVCM), the authors analyzed, for the first time in vivo inflammatory cell infiltration and circulation inside the lymph vessels in rabbit CALT after inflammatory stimuli (LPS, TNFalpha, LPS+/-anti-TNF). Cresyl violet staining was performed to observe the morphology of CALT, and immunohistology was performed in whole mount conjunctiva and cryosections for detecting CD45(+) lymphocytes. Human CALT in vivo aspects were also explored in a patient with vernal keratoconjunctivitis (VKC). The conjunctivitis model induced by LPS was characterized by inflammatory cell infiltration in the dome and intrafollicular layers of CALT and cell circulation inside the lymph vessels. TNF alone induced moderate inflammatory infiltration in CALT. However anti-TNF antibodies could significantly decrease LPS/TNFalpha-induced inflammation. CD45(+) lymphocytes were strongly expressed in the CALT after injection of LPS or TNFalpha at 4 hours and decreased with injection of anti-TNFalpha. The authors also showed the presence of the CALT pattern in VKC. The authors showed for the first time the in vivo aspects of normal and pathologic cell reactions in rabbit CALT after inflammatory stimuli. IVCM-CALT could be a pertinent tool in the future for the comprehension of ocular surface defense mechanisms, and inflammatory cell analysis in CALT could constitute a new criterion for evaluating ocular surface inflammation.