Sustained scleral stiffening in rats after a single genipin treatment

Abstract

Scleral stiffening has been proposed as a therapy for glaucoma and myopia. Previous in vivo studies have evaluated the efficacy of scleral stiffening after multiple treatments with a natural collagen crosslinker, genipin. However, multiple injections limit clinical translatability. Here, we examined whether scleral stiffening was maintained after four weeks following a single genipin treatment. Eyes from brown Norway rats were treated in vivo with a single 15 mM genipin retrobulbar injection, sham retrobulbar injection, or were left naive. Eyes were enucleated either 1 day or four weeks post-injection and underwent whole globe inflation testing. We assessed first principal Lagrange strain of the posterior sclera using digital image correlation as a proxy for scleral stiffness. Four weeks post-injection, genipin treatment resulted in a 58% reduction in scleral strain as compared to controls ( p = 0.005). We conclude that a single in vivo injection of genipin effectively stiffened rat sclera for at least four weeks which motivates further functional studies and possible clinical translation of genipin-induced scleral stiffening.

Assessment of Visual and Retinal Function Following In Vivo Genipin-Induced Scleral Crosslinking

“…Previous work from our laboratory demonstrated successful stiffening of the posterior rat sclera for 4 weeks after a single retrobulbar injection of genipin 21 as a possible therapy to mitigate axial elongation in myopia or optic neuropathy in glaucoma. In the present study, we have used the same genipin injection procedure to evaluate potential adverse effects of genipin-induced scleral crosslinking at the molecular and functional levels in the eye.…”

“…Additionally, a lower dose of genipin (volume or concentration, or a combination of the two) could be considered to potentially mitigate the slight loss of RGC axons that we observed, although we note that the dose and concentration that we used was selected because it was the lowest dose that achieved a desired level of scleral stiffening. 21 …”

“…All retrobulbar injections were performed using a sterile 31G insulin syringe needle (BD 300 µl Insulin Syringe Ultra-Fine needle; Becton, Dickinson and Company, Franklin Lakes, NJ) inserted into the inferior quadrant. A genipin concentration of 15 mM was previously used in our in vivo stiffening study, 21 and successfully stiffened the posterior rat sclera for 4 weeks. Rats received a topical antibiotic (Certi-sporyn, Kansas City, MO) to avoid infection and antisedan (1 mg/kg) to reverse anesthesia.…”

“…We have previously shown the efficacy of genipin (15 mM) to induce sustained scleral stiffening for up to 4 weeks after retrobulbar injection in Brown Norway rat eyes. 21 However, it is important to ensure that long-term scleral stiffening is safe and has negligible adverse effects on visual function. Here, we aim to evaluate the potential use of genipin-induced collagen crosslinking in the sclera on visual and retinal function over 4 weeks.…”

Smarce1 and Tensin 4 Are Putative Modulators of Corneoscleral Stiffness

“…We suggest this is, however, actually a benefit: measurement of ocular compliance, unlike alternative methods [e.g., strip testing (Phillips and McBrien, 1995 ; Elsheikh and Anderson, 2005 ; Lari et al, 2012 ; Hatami-Marbini and Rahimi, 2014 ), compression (Battaglioli and Kamm, 1984 ; Mortazavi et al, 2009 ; Boazak et al, 2019 )] evaluates the eye in its natural geometrical configuration. Further, ocular compliance measurements are faster and easier to carry out in small murine eyes as compared to alternatives (Myers et al, 2010 ; Hannon et al, 2019 ), and they do not require fixation of the eye to a mounting block, which can potentially introduce non-physiological boundary conditions. A third possible limitation was the use of eye mass to calculate eye volume, which in turn was used to calculate ϕ norm .…”