Form deprivation is an effective technique to establish the myopia model of the guinea pig eye [7]. Significant amounts of myopia, axial elongation and thinning of the scleral fibril diameter can be found in the guinea pig eyes after 21-day FD [14]. In the present study, the average axial length of the FD eyes in Group B (11.99 mm) was 109 % of the untreated control eyes in Group A (10.95 mm); −6 D of myopia (from 3.50 to −2.51 D) was developed in the guinea pig eyes at the end of this FD process. Although changes induced by FD were observed throughout the whole eye after FD, the elongation of the vitreous chamber and the thinning of the sclera were the dominant responses [7, 14]. These ocular changes were similar to the myopic process in human and monkey eyes [19, 20].
Cross-linking plays an important role for the regulation of ocular elongation and myopia development [21]. Intraperitoneal injections of cross-linking blocker (such as β-aminoproprionitrile and D-penicillamine) have been found to increase the FD myopia of tree shrew eyes [21]. As a physical CXL technique, scleral CXL using ultraviolet A (UVA) and riboflavin has been proven to strengthen the scleral biomechanical rigidity without any obvious side-effects [22]. Compared with the clearly defined target area of physical CXL technique, sub-Tenon injections with chemical CXL reagents have several advantages, such as minimally invasive procedure (via small incision), large treatment area (may include the entire sclera), and ease of manipulation without the need for any specialized UV device nor UV irradiation [16].
The amount of axial myopia was found to correlate with the mechanical properties of the sclera [23]. As a natural CXL reagent, genipin has remarkable efficacy [24], stability [25] and low cytotoxicity [10], and has been used for improving the mechanical property of scleral tissue [12]. In previous ex vivo studies [13, 26], it has been found that the stiffness of porcine sclera increases 280–820 % after a 30-min incubation with 1 % genipin. In the present study, the scleral Young modulus in the eyes with FD and genipin CXL (Group C, 7.23 MPa) was similar to that in the untreated control eyes (Group A, 4.52 MPa; P = 0.0873), and was 3 times stronger than those eyes with FD only (Group B, 2.17 MPa; P = 0.0024). On the other hand, sub-Tenon injections using 0.50 % genipin eliminated the eye elongation and stopped the myopia development induced by the FD in guinea pig eyes. At the end of this study, the amount of axial length and myopia were statistically less than the FD eyes in Group B (P < 0.01), and were statistically equal to the value of the untreated eyes in Group A (P > 0.05).
Furthermore, the development of FD myopia in mammalian eyes is associated with the ultrastructural performances of scleral fibrils [27]. It has been proven that smaller diameter collagen fibrils provide tissues with lower tensile strength [28]. In this study, the FD induced the axial elongation and myopic growth in guinea pig eyes, which was accompanied with a decrease in scleral fibril diameter; in turn, the genipin CXL eliminated the FD-induced axial elongation and prevented the myopic development accompanied with the thickening of scleral collagen fibrils. However, unlike the adenosine receptor antagonists (such as 7-methylxanthine) [14], the thickening of the target sclera was not present in the current and in some previous studies [17]. This is because CXL is a process of bonding the present existing collagen fibrils, but not a process of collagen regeneration. In the present study, both the increase in fibril diameter and the reduction of fibril density were observed under the transmission electron microscope. Previous studies also found the damages of fibroblasts and the thinning of corneal or scleral tissues after CXL procedures [29]. Long-term efficacy of the scleral CXL using genipin for halting the process of FD myopia should be observed in the future.
In the present study, mild reversible side effects were observed in the peripheral cornea and sclera adjacent to the injection sites, whereas the retina and choroid were not affected. The ocular barriers were believed to play important roles in protecting the retina and choroid from the histological damages of the sub-Tenon injection and genipin toxicity [16]. Yet, it was observed that genipin toxicity is dose-dependent but not time-dependent [13]. The promising genipin concentration and treatment time in tissue-engineering practices are 0.5 mM (equivalent to 0.0113 %) and 30 min, respectively [13]. This is much lower than the present dose used in our study. Thus, more attention should be concentrated on the visual function of cross-linked eyes in the future to examine the possible cytotoxic effects of this technique.
In addition, some limitations of the current study should be noted. The limited number of eyes, as well as the time-frame, for instance, cannot elaborate information about the long-term efficacy and safety of the current CXL technique. Additionally, the changes in intraocular pressure and optic nerve were not investigated in the current study.