in vivo imaging of MMP-2 expression in the conjunctiva following implantation of Ilomastat or ethylcellulose tissue-tablet

Abstract

 

Purpose

 

Matrix metalloproteinases (MMP) and in particularly MMP-2 play a significant roles during wound healing; from inflammation and angiogenesis to tissue remodelling/scarring. A better understanding of MMP-2 expression during conjunctival healing would to predict in prognosis and the timing of dosing a selective therapeutic agent. Ilomastat is a broad spectrum MMP inhibitor which can be fabricated into a small tablet for subconjunctival implantation and has shown some degree of inflammatory response while retained the antiscarring activity. We have now used a novel MMP-2 inhibitor that was conjugated to fluorescein (FITC) to monitor conjunctival expression of MMP2 in the presence of a small implanted tablet. Two tablets were evaluated; (i) ethylcellulose which was used to act as a foreign body without any therapeutic activity and (ii) an ilomastat tablet of the same size. Imaging was conducted using HRA II fitted with adaptive lenses at various time points. Our aim was to determine if we could utilise an imaging strategy to monitor the expression of MMP-2 during conjunctival healing.

 

Methods

 

Two conjunctival pockets were created in the superior-nasal and superior-temporal of the left rabbit eye. A small implantable tablet fabricated from either ethylcellulose (2mm,1.94mg) or ilomastat (2mm,3.5mg) was implanted at the superior-nasal and superior-temporal respectively. The pockets were closed (Nylon10-0). The FITC labelled MMP-2 inhibitor (59.8μM, 50μL) was injected subconjunctivally near each tissue-tablet. HRA II was used to image the conjunctiva at days 0,1, 2, 3, 4, 5 and 9.

 

Results

 

Imaging of FITC-MMP-2 inhibitor at sufficient resolution was possible using HRA at various time points. The MMP2i-FITC displayed higher signal intensity around the ethylcellulose tablet at day 5 compared to ilomastat tablet (Figure1). At day 9, only the ethylcellulose tablet showed a signal.

 

Conclusions

 

MMP2i-FITC can be used for real time examination of MMP 2 protein expression. MMP2 around the ethylcellulose tablet appeared to be present longer than around the ilomastat tablet. This is consistent with suggesting the bioactivity of the released ilomastat. HRA with adaptive lenses can be used for real time study at molecular level in conjunctiva.

 

 

Figure 1. Day 5, post-surgery. small arrow=Ethylcellulose, long arrow = ilomastat A. Infrared Reflectance(IR) B. Fluorescein Angiography(FA)

 

Keywords: 765 wound healing • 474 conjunctiva • 514 enzymes/enzyme inhibitors