Characterization of Rabbit Corneas Subjected to Stromal Stiffening by the Açaí Extract (Euterpe oleracea).

PURPOSE: In this study, we characterized rabbit corneas subjected to corneal cross-linking (CXL) with açaí extract compared with a riboflavin photo-stimulated procedure. MATERIALS AND METHODS: The corneas of the slaughterhouse rabbits were divided into three groups: control, consisting of untreated corneal samples; riboflavin/UVA, where corneas were treated with 0.1% riboflavin photo-stimulated at 365 nm as the standard protocol; and açaí, where the samples were subjected to 4% açaí extract for 0.5-2 h. After the CXL procedure, corneas of the three groups were characterized by analyzing their elastic modulus and thermal denaturation profile. RESULTS: The elastic modulus at 3% strain showed an approximately threefold increase in the riboflavin/UVA group and 10.5 times in the corneas treated with 4% açaí extract for 2 h, compared with the control group (p < 0.01). The denaturation temperature values of the two groups of crosslinked corneas increased significantly (p < 0.05) and were more pronounced in the açaí group. CONCLUSIONS: The açaí extract was effective in promoting CXL in rabbit corneas as characterized by the different techniques.

Characterization of angiotensin I-converting enzyme from anterior gills of the mangrove crab Ucides cordatus.

Angiotensin I-converting enzyme (ACE) is a well-known metallopeptidase that is found in vertebrates, invertebrates and bacteria. We isolated from the anterior gill of the crab Ucides cordatus an isoform of ACE, here named crab-ACE, which presented catalytic properties closely resembling to those of mammalian ACE. The enzyme was purified on Sepharose-lisinopril affinity chromatography to apparent homogeneity and a band of about 72 kDa could be visualized after silver staining and Western blotting. Assays performed with fluorescence resonance energy transfer (FRET) selective ACE substrates Abz-FRK(Dnp)P-OH, Abz-SDK(Dnp)P-OH and Abz-LFK(Dnp)-OH, allowed us to verify that crab-ACE has hydrolytic profile very similar to that of the ACE C-domain. In addition, we observed that crab-ACE can hydrolyze the ACE substrates, angiotensin I and bradykinin. The enzyme was strongly inhibited by the specific ACE inhibitor lisinopril (Ki of 1.26 nM). However, in contrast to other ACE isoforms, crab-ACE presented a very particular optimum pH, being the substrate Abz-FRK(Dnp)-P-OH hydrolyzed efficiently at pH 9.5. Other interesting characteristic of crab-ACE was that the maximum hydrolytic activity was reached at around 45°C. The description of an ACE isoform in Ucides cordatus is challenging and may contribute to a better understanding of the biochemical function of this enzyme in invertebrates.

Corneal absorption of a new riboflavin-nanostructured system for transepithelial collagen cross-linking.

Corneal collagen cross-linking (CXL) has been described as a promising therapy for keratoconus. According to standard CXL protocol, epithelium should be debrided before treatment to allow penetration of riboflavin into the corneal stroma. However, removal of the epithelium can increase procedure risks. In this study we aim to evaluate stromal penetration of a biocompatible riboflavin-based nanoemulsion system (riboflavin-5-phosphate and riboflavin-base) in rabbit corneas with intact epithelium. Two riboflavin nanoemulsions were developed. Transmittance and absorption coefficient were measured on corneas with intact epithelia after 30, 60, 120, 180, and 240 minutes following exposure to either the nanoemulsions or standard 0.1% or 1% riboflavin-dextran solutions. For the nanoemulsions, the epithelium was removed after measurements to assure that the riboflavin had passed through the hydrophobic epithelium and retained within the stroma. Results were compared to de-epithelialized corneas exposed to 0.1% riboflavin solution and to the same riboflavin nanoemulsions for 30 minutes (standard protocol). Mean transmittance and absorption measured in epithelialized corneas receiving the standard 0.1% riboflavin solution did not reach the levels found on the debrided corneas using the standard technique. Neither increasing the time of exposure nor the concentration of the riboflavin solution from 0.1% to 1% improved riboflavin penetration through the epithelium. When using riboflavin-5-phosphate nanoemulsion for 240 minutes, we found no difference between the mean absorption coefficients to the standard cross-linking protocol (p = 0.54). Riboflavin nanoemulsion was able to penetrate the corneal epithelium, achieving, after 240 minutes, greater stromal concentration when compared to debrided corneas with the standard protocol (p = 0.002). The riboflavin-5-phosphate nanoemulsion diffused better into the stroma than the riboflavin-base nanoemulsion.