Metabolic profile of porcine corneas after photodynamic cross‐linking treatment

Abstract

Editor, Corneal collagen cross-linking (CXL), using ultraviolet A (UVA) irradiation and photosensitizer riboflavin was introduced in 2003 by Wollensak et al. as an efficient treatment of progressive keratoconus. It halts the progression of corneal ectasia and leads to a slight reduction of keratectasia in about 50% of patients with keratoconus (Wollensak et al. 2003a; Wollensak 2010). We recently examined the influence of standard CXL on the metabolic profile of porcine corneas using the metabonomic approach based on magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. 24 porcine eyes were retrieved from a local abattoir within 24 hrs post-mortem. 12 eyes were cross-linked, another 12 eyes served as controls with epithelial debridement only. After complete epithelial debridement, 0.2 ml of a 0.1% riboflavin/20% dextran solution was applied every 3 min for 10 min. The corneas were then irradiated with UVA (370 nm) for 30 min using a double UVA diode with a focusing distance of 1 cm and a UVA irradiance of 3 mW/cm2 (total dose, 5.4 J/cm²). During irradiation, riboflavin/dextran solution was applied every 3 min. After treatment, central corneal buttons with 10 mm diameter were trephined and frozen at −80°C. Rectangular 4 × 4 mm, central samples were cut from the corneas and immersed in D2O in a zirconium HR-MAS rotor. HR-MAS 1H NMR spectroscopy, assignment of the metabolites in spectra and data analysis were performed as described previously (Kryczka et al. 2011). Peak areas were quantified using absolute integrals and were normalized by the mean wet weight of the control (14.4 mg). The significance of differences between individual means was tested by the Student’s t-test for independent samples (statistica for Windows software version 7.1; StatSoft, Tulsa, OK, USA). Twenty metabolites were assigned in HR-MAS 1H NMR spectra (Fig. 1A). Levels of formate, glutathione (GSH) and leucine concentration in the cross-linked corneas were significantly lower than in the controls, by 70% (p = 0.021), 28% (p = 0.048) and 26% (p = 0.043), respectively (Fig. 1B). Representative HR-MAS 1H NMR spectrum of a porcine cornea (panel A) and changes in metabolic profiles of porcine corneas exposed to cross-linking photodynamic therapy (panel B). (A) The p.p.m. values are assigned using TSP as the reference substance at 0 p.p.m. (B) The results shown are the Means ± SD, IU-institutional units: HR-MAS 1H NMR peak integrals divided by cornea wet weight, see Ref. Kryczka et al. (2011); n = 12 for every group of samples; *p < 0.05 versus control. Ac, acetate; Ala, alanine; Arg, arginine; Chol, choline; Creat, creatine; Cr, creatinine; For, formate; Glut, glutamate; GSH, glutathione; His, histidine; hypo-Tau, hypotaurine; Ileu, isoleucine; Lac, lactate; Leu, leucine; Lys, lysine; P-chol, phosphocholine; Tau, taurine; Try, tryptophan; TSP, sodium-3′-trimethylsilyl-propionate-2,2,3,3-d4; Tyr, tyrosine; Val, valine. Our results suggest that glutathione, which is one of the main nonenzymatic intracellular corneal antioxidants (Lassen et al. 2008), contributes significantly to the antioxidant defence system in the cornea during CXL and is therefore partially depleted after CXL. Similarly, formate that can act as an OH radical scavenger (Shiraishi et al. 2000) was also reduced. The scavenging action of antioxidants (Lassen et al. 2008) might play an important role in balancing the beneficial biomechanical and possibly negative cytotoxic side effects of CXL. The riboflavin/UVA system is known to decrease concentrations of free histidine and tyrosine in collagen solutions. Kato et al. (1994) proposed di-tyrosine formation as a probable explanation of those changes. In our study, only a slight tendency towards the reduction of these amino acids was observed (Fig. 1B). The less significant changes could be due to the low concentration of these amino acids in the corneal tissues. In conclusion, the results of this study have demonstrated a moderate impact of CXL on the antioxidant system of porcine corneas in the early post-treatment phase. No major alarming changes were found in the metabolic profiles of cross-linked corneas supporting the clinical view of CXL being a safe procedure. Further research is needed to elucidate the influence of CXL on the various antioxidant systems of the cornea (e.g. superoxide diamutase, catalase) and the long-term effects of corneal cross-linking on the metabolic profile. Modulation of the cross-linking and wound healing response by adding antioxidant eye drops like vitamin C protecting endothelial cells against apoptosis (Wollensak et al. 2003b) especially in thin corneas might become possible and should also be explored in the future.