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Laboratory science

Effects of genipin corneal crosslinking in rabbit corneas

Avila, Marcel Y. MD, PhD*; Narvaez, Mauricio MD; Castañeda, Juan P. MD

Author Information
Journal of Cataract & Refractive Surgery: July 2016 - Volume 42 - Issue 7 - p 1073-1077
doi: 10.1016/j.jcrs.2016.04.025
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Abstract

Exogenous crosslinking has emerged as a new way to reduce corneal deformation and as a treatment for corneal ectasia and keratoconus.1 In the clinical setting, crosslinking involves the use of ultraviolet (UV) light and riboflavin; however, UV light induces changes in corneal cells that must be considered when treatment is proposed. Therefore, a methodology that avoids UV light is desirable.2,3

Genipin has emerged as a new crosslinking agent with excellent biocompatibility,4 low toxicity,5 and the ability to crosslink the cornea5 and sclera,6 as shown by in vitro and in vivo myopia model experiments.7 We also found lower toxicity to endothelial and stromal cells and improved stiffness of the cornea in comparison with UV and riboflavin crosslinking.6

We previously found an increase in corneal stiffness compared with accelerated UV crosslinking, similar to the Dresden UV protocol.8 To make this feasible in the clinical setting, a reduction in the delivery time of the crosslinker genipin would be desirable. Despite the low in vitro and in vivo toxicity of genipin–chitosan membranes in the anterior chamber in animal models,9 further studies are required to provide evidence of anterior segment effects and the effect of corneal flattening in animal models before clinical studies are considered.

In this study, the effects of genipin on the cornea were evaluated using an optimized genipin delivery system. The anterior segment and intraocular pressure (IOP) were measured to elucidate the hypothesis that genipin crosslinking induces a reduction in corneal curvature.

Materials and methods

Ex Vivo Experiments

Eight rabbits (aged 5 months) were obtained from a local abattoir and their eyes were used to assess penetration and cornea densitometry in vitro. Briefly, the fresh rabbit eyes were enucleated and treated with genipin at different concentrations (1.00%, 0.50%, and 0.25%, and vehicle; 4 eyes per group). Genipin was dissolved in dimethyl sulfoxide and then in phosphate-buffered saline pH 8.5 (Challenge Bioproducts Co., Ltd.) with a purpose-built vacuum device (the same used in the in vivo experiments) to increase the permeability of genipin and to avoid contact with other structures. A subset of experiments was performed in which the rabbit eyes were cannulated to obtain an IOP of 18 mm Hg. Then, suction was applied using the vacuum device and the IOP was measured with an applanation tonometer (Tono-Pen, Reichert). Treatment was performed for 5 minutes, after which the eyes were washed with saline and incubated at 30°C for 24 hours. The eyes were then evaluated using the Pentacam Scheimpflug pachymetry module (Oculus Optikgeräte GmbH) from the Scheimpflug images and corneal densitometry.

In Vivo Experiments

Twenty New Zealand albino rabbits (aged 5 months) were used. The rabbits were handled according to the Association for Research in Vision and Ophthalmology protocols for animal experimentation in ophthalmology. Protocols involved paired-eye design (1 eye treated with genipin and the contralateral eye with vehicle) to reduce the number of animals required and interanimal variability.

Under general anesthesia of ketamine (50 mg/kg), xylazine (10 mg/kg), and acepromazine (1 mg/kg), both eyes of each rabbit were deepithelialized and 1 eye was treated with genipin 0.25% in a custom vehicle for 5 minutes using a vacuum device designed to ensure contact between the cornea and the genipin and to prevent drops in the conjunctiva. Briefly, a plastic syringe (5 cm long) was connected to a 0.8 mm plastic cylinder, and a vacuum with 4 cm of negative pressure was applied for 5 minutes. This system was adapted from the principle of the vacuum device described by Myung et al.A; the contralateral eye was treated with the vehicle alone.

The cornea was then washed with saline solution, and a mixture of tobramycin and dexamethasone was applied to both eyes every 6 hours for 5 days until complete epithelialization occurred.

Measurements

The Scheimpflug system was used to measure corneal densitometry values. The Scheimpflug system quantifies the density of the cornea on a scale from 0 to 100 (arbitrary optical units). Peak densitometry values were recorded directly from the axis line appearing in the Scheimpflug image in the ex vivo eyes, and the depth was calculated as the percentage of denser areas in the center of the cornea.

Corneal thickness was measured using an ultrasound pachymeter (Pachette 3, DGH Technology, Inc.). Corneal curvature was measured using an autorefractor keratometer (KP 8000, Topcon Europe Medical B.V.). The IOP was measured using an applanation tonometer. Slitlamp evaluations were performed on 0, 1, 5, 15, 30, and 60 days, and changes in the cornea, lens, and anterior segment were evaluated. Changes in corneal transparency (haze) were graded from 0 (no haze) to 5 (severe haze that precluded visualization of the anterior chamber). These measurements were performed in both eyes on 0, 1, 5, 15, 30, and 60 days.

Statistical Analysis

In most instances, the statistical significance of comparisons was established with a paired Student t test. Corneal pachymetry, corneal keratometry (K), and IOP were compared using 1-way analysis of variance on ranks; then, statistically significant comparisons were established with the Kruskal-Wallis test using Sigmastat (Systat Software, Inc.). Differences were considered significant if the probability of null hypothesis (P) was less than 0.05.

Results

Ex Vivo Corneal Densitometry and Intraocular Pressure Changes

The mean Scheimpflug corneal densitometry was concentration dependent, with a maximum value with genipin 1.00% (mean 95 optical units ± 4.7 [SD]) followed by genipin 0.50% (mean 74 ± 12.3 optical units) and a minimum value with genipin 0.25% (mean 68.5 ± 9.8 optical units) in comparison with the control corneas. In the deep layers of the cornea, this effect was also concentration dependent, affecting 81% of the corneal layers with genipin 1.00%, 38.1% with genipin 0.50%, and 32.2% with genipin 0.25% (Figure 1). The IOP increased from a mean of 18.0 mm Hg to 33.0 ± 2.3 mm Hg with the vacuum device (n = 10).

Figure 1
Figure 1:
Effects of genipin in ex vivo corneas and effect on corneal densitometry.

In Vivo Study

Keratometry

The mean K values (20 eyes each group; all timepoints) were and 49.2 ± 0.8 D (genipin) and 49.7 ± 0.6 diopters (D) (control) at baseline, 47.1 ± 0.8 D (genipin) and 47.9 ± 0.3 D (control) at 30 days, and 44.8 ± 0.4 D (genipin) and 46.28 ± 0.5 D (control) at 60 days. The difference between the 2 groups was statistically significant at 60 days (t(7) = 2.655, P = .001; paired-samples t test). The mean reduction in the steepest values was 4.4 ± 0.5 D in the genipin group and 2.5 ± 2.0 D in the control group; the difference was statistically significant (P = .005) (Figure 2).

Figure 2
Figure 2:
Keratometric effects of genipin compared with the contralateral eye. The dotted blue line represents the genipin-treated corneas; the black line represents the control corneas.

Pachymetry

The mean preoperative pachymetry was 412 μm in the genipin-treated eyes and 417 μm in the control eyes at 0 day. At 60 days, the mean pachymetry was 415 μm in the genipin-treated eyes and 420 μm in the control eyes (Figure 3).

Figure 3
Figure 3:
Corneal thickness in treated eyes versus contralateral eyes (pre = pretreatment).

Slitlamp Evaluation

The endothelium was normal during the entire study period. Minimal corneal edema was observed 4 days after treatment, and complete epithelialization was observed at 5 days, with a slight blue coloration. No eye developed cataract during the study, and none had haze after 15 days. No eye presented with flare or cells in the anterior chamber (Figure 4).

Figure 4
Figure 4:
Clinical photography of (A) control eye, (B) genipin-treated cornea 10 days after treatment, (C) genipin-treated cornea 30 days after treatment, and (D) genipin-treated cornea 60 days after treatment.

Intraocular Pressure

The mean pretreatment IOP was 10.0 ± 0.5 mm Hg in the genipin group and 9.8 ± 0.4 mm Hg in the control group. There was no statistically significant change in IOP in posterior controls, with mean values of 9.5 ± 0.6 mm Hg (genipin) versus 9.8 ± 0.5 mm Hg (control).

Discussion

The study results show an excellent safety profile in genipin-treated corneas and structures of the anterior chamber. Minimal blue staining of the cornea was observed; however, it did not compromise corneal transparency, and this slight coloration reflects crosslinking. The ex vivo experiments showed a concentration-dependent increase in corneal density and a change in the penetration of genipin as a function of the concentration. Based on this, a concentration of 0.50% was used for the in vivo experiments. A change in the corneal density has been described in UV crosslinking using Pentacam Scheimpflug pachymetry, with an increase of 75% from the baseline.10 A similar effect was observed in these experiments.

The use of the adapted vacuum device led to an increase in the permeability of genipin, reducing the time of exposure and making it feasible for the clinical setting as well as increasing the permeability of the corneal stroma, as shown in the Scheimpflug pachymetry modules. These results indicate that this could be a faster crosslinking system than actual systems and that it could be considered for clinical use. Also, a reasonable increase in IOP was observed with the vacuum, similar to the change in IOP obtained during corneal trephination with manual suction.11

The changes in corneal curvature were significant in this study, with a mean flattening of the corneas of 4.4 ± 0.5 D; this was more evident in steeper corneas. A flattening and reduction in maximum K values was evident in clinical studies, showing the effects of UV corneal collagen crosslinking. Those studies include the French National Reference Center for Keratoconus12 and a study in Italy,13 which showed a flattening in the maximum K value from 1.0 D to 2.0 D. Our observation of flattening of 4.4 D in treated rabbit eyes indicates an efficient procedure to stabilize corneas and improve corneal shape. Genipin acts by increasing the fibril diameter and creating intermolecular and intramolecular crosslinking, thus increasing the resistance in the cornea, in the sclera, and in models of myopia without sacrificing corneal transparency and related structures, as found in this study.

Although the absence of changes related to toxicity in corneal cells has been reported in previous studies, this study shows the long-term effects on these structures, as well as on the lens, with no changes in IOP.

One might expect this low toxicity because genipin is a natural crosslinking agent with a lower toxicity than UV light and riboflavin in corneas in ex vivo models and in acellular implanted corneas.14 Moreover, the injection of genipin did not lead to toxicity in the retina in guinea pigs in vivo when used for the control of eye growth in myopia models.7 Membranes implanted in rabbit anterior chambers containing genipin have excellent biocompatibility and great potential in ocular therapeutics, tissue repair, and pharmacology.9

In conclusion, genipin induced corneal flattening in rabbit eyes without toxic effects and might have potential for the management of corneal ectasia and keratoconus.

What Was Known

  • Genipin induces crosslinking in several tissues in the eye, including the cornea and sclera; however, penetration requires time and is not well standardized.

What This Paper Adds

  • Genipin induced corneal flattening in rabbit eyes without toxicity.

References

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Other Cited Material

A. Myung D, Zaler G, Abbate A, DiGiore D, Eaton D, Manche EE, “Vacuum-Mediated Transepithelial Delivery of Riboflavin to the Cornea,” presented at the annual meeting of the Association for Research in Vision and Ophthalmology, Denver, Colorado, USA, May 2015. Abstract available at: http://iovs.arvojournals.org/article.aspx?articleid=2332843. Accessed May 22, 2016
© 2016 by Lippincott Williams & Wilkins, Inc.