Corneal Resistance to Enzymatic Digestion After Rose Bengal and Combined Rose Bengal/Riboflavin Cross-Linking Is Oxygen Independent

Abstract

Purpose: To assess corneal resistance to enzymatic digestion after rose bengal (RB)/green light and RB/green light followed by riboflavin (RF)/ultraviolet A (UV-A) cross-linking (CXL), with or without oxygen.

Methods: Ex vivo porcine corneal buttons (n = 144) underwent CXL with RB/green or RB/green–RF/UV-A under atmospheric 21% oxygen conditions or in a nitrogen chamber with 0.1% oxygen (hypoxic conditions) to test 10- and 15-J/cm² fluences. After CXL, corneas were digested with 0.3% collagenase A, and mean digestion times (MDTs) were recorded.

Results: For the non-irradiated control group, the MDT was 19.75 ± 1.34 hours. Under atmospheric oxygen conditions, RB/green CXL yielded MDTs of 33.69 ± 1.4 and 34.38 ± 1.31 hours with fluences of 10 and 15 J/cm², respectively. RB/green + RF/UV-A showed MDTs of 39.56 ± 1.93 and 51.94 ± 4.2 hours for combined fluences of 10 + 10 J/cm² and 15 + 15 J/cm², respectively. Hypoxic RB/green MDTs were 33.88 ± 1.02 and 34.06 ± 1.57 hours, and RB/green + RF/UV-A MDTs were 39.62 ± 2.5 and 50.35 ± 1.59 hours for the same respective fluences. No significant differences were observed between the control groups and corresponding intervention groups (all P > 0.05).

Conclusions: CXL via RB/green and RB/green–RF/UV-A significantly enhanced corneal collagenase digestion resistance, irrespective of oxygen presence. These findings could help optimize infectious keratitis therapy CXL protocols.

Translational Relevance: Our findings aid the understanding of the molecular mechanisms underlying the therapeutic effect of CXL and may contribute to refining accelerated PACK-CXL protocols and other CXL treatment strategies.

Introduction

Infectious keratitis, when inappropriately treated, may lead to impaired vision or even blindness. Early intervention is vital to prevent irreversible damage, such as ulceration. Recent techniques, such as photoactivated chromophore for infectious keratitis–corneal cross-linking (PACK-CXL), also referred to as photodynamic therapy (PDT), enable treatment initiation before pathogen identification, as the pathogen-agnostic therapeutic effects can expedite the treatment process, saving precious time.^1–5 

PACK-CXL involves saturating the cornea with a chromophore, followed by irradiation with light at a specific wavelength that activates that chromophore. This results in the generation of reactive oxygen species (ROS), which enhance the bonding between collagen fibers and proteoglycans in the extracellular matrix.^6,7 Two primary antiseptic mechanisms have been identified: (1) the indirect inhibition of pathogen growth through cross-linking making corneal tissue denser, and (2) ROS-mediated damage to pathogen cellular membranes, RNA, and DNA. Recent studies indicate that PACK-CXL is an effective adjuvant treatment for advanced ulcerative infectious keratitis.^8–10 

Different chromophores and light source combinations can be employed in PACK-CXL. Studies indicate that the combinations of riboflavin (RF) and ultraviolet A (UV-A) light at 365 nm (RF/UV-A) and rose bengal (RB) and green light at 532 nm (RB/green) can both be effective in treating infectious keratitis.^11–16 Notably, riboflavin and RB have minimal overlap in their absorption spectra, and RF/UV-A and RB/green have different penetration depths. We have previously shown that both RF/UV-A and RB/green can significantly enhance corneal resistance to enzymatic digestion, and this resistance can be further improved when the chromophore/light combination procedures are performed sequentially.¹⁷ 

Oxygen plays a crucial role in RF/UV-A CXL for corneal ectasia and is consumed during the photochemical reaction between riboflavin and UV-A light, as it is necessary for generating ROS. Its availability in the stroma is rate limiting. Indeed, porcine corneas treated in low-oxygen environments show biomechanical properties similar to those of untreated controls.^8,18 However, in PACK-CXL, the goal is not to strengthen the cornea biomechanically but rather to kill pathogens and enhance the resistance of the cornea to digestion caused by pathogen-produced proteases. Our group recently showed that the pathogen-killing effect of RF/UV-A PACK-CXL is oxygen independent (Hafezi et al., submitted). In this study, we aimed to determine the role of oxygen in enhancing corneal resistance to digestion after PACK-CXL with RB/green, either alone or in combination with RF/UV-A. 

Materials and Methods

Specimen Acquisition and Preparation

Freshly enucleated porcine eyes were obtained from a local abattoir and used within 6 hours. After the epithelium was removed using a hockey blade (FEATHER, pfm medical, Cologne, Germany), the corneas were excised circumferentially, leaving a 3-mm corneoscleral rim. The corneal buttons were then immersed in a 400-mOsmol/L phosphate-buffered saline solution for 10 minutes to standardize hydration. Following this, the central 8-mm region of the cornea was trephined with a biopsy punch (SMI AG, St. Vith, Belgium) to obtain corneal buttons. These buttons were soaked in either a 0.1% riboflavin solution (Ribo-Ker; EMAGine AG, Zug, Switzerland) or a 0.1% RB solution (Bengalrosa; Bichsel AG, Interlaken, Switzerland) for 10 minutes. To prepare the RB solution, 0.3207 g of RB powder was combined with 0.0107 g of trometamol, and the pH was adjusted to 7.5. Then, 320 g of balanced salt solution and 0.66403 g of sodium chloride were added, resulting in an osmolality of 350 to 400 mOsmol/L, yielding a 0.1% RB solution. 

According to the group settings, after the buttons were rinsed, CXL was performed either in ambient atmospheric conditions (21% oxygen) or in a nitrogen-rich, oxygen-depleted environment (0.1% oxygen). Corneas treated with riboflavin were cross-linked using 365-nm UV-A light (C-eye device; EMAGine AG), whereas those treated with RB received 522-nm green light from a custom-built experimental device provided by CSO Italia (Scandicci, Italy). Like the UV light source, this device also used LEDs. This research followed the tenets of the Declaration of Helsinki. 

Study Group Allocation and CXL Protocols

We randomly assigned 144 porcine eyes to one of three study groups (A, B, or C), with each group containing 64 corneas: 

• • Group A (control): This group served as a baseline control to demonstrate the general effects of CXL. These corneas (n = 16) did not undergo chromophore soaking or irradiation.
• • Group B (RB/green): All corneas underwent 10 minutes of RB soaking. Groups B1 and B2 corneas (n = 16 each) were irradiated in ambient room atmosphere (oxygen concentration 21%) with 365-nm UV-A light at either 10 J/cm² (15 mW/cm²; 11 minutes, 7 seconds) or 15 J/cm² (15 mW/cm²; 16 minutes, 40 seconds), respectively. In groups B3 and B4 (n = 16 each), corneas were treated in the absence of oxygen (nitrogen-rich environment, oxygen concentration of 0.1%), using either 10 J/cm² (15 mW/cm²; 11 minutes, 7 seconds) or 15 J/cm² (15 mW/cm²; 16 minutes, 40 seconds).
• • Group C (same-session RF/UV-A and RB/green): All corneas were soaked in riboflavin for 10 minutes, followed by 10 minutes of RB soaking. In subgroups C1 and C2, the treatment was carried out in ambient atmospheric conditions. C1 corneas received UV-A irradiation with a total fluence of 10 J/cm² at 18 mW/cm² for 9 minutes, 15 seconds. This was immediately followed by green light irradiation with a fluence of 10 J/cm² at 15 mW/cm² for 11 minutes, 7 seconds. C2 corneas were treated with UV-A irradiation at a higher fluence of 15 J/cm² at 30 mW/cm² for 8 minutes, 20 seconds. This was followed by green light at 15 J/cm² for 16 minutes, 40 seconds. Subgroup C3 followed the same treatment protocol as C1, and C4 followed the protocol for C2, but both C3 and C4 were irradiated in a low-oxygen environment.

Low-Oxygen Environment

The low-oxygen environment was generated using a hermetically sealed custom-built polymer-based chamber (45 × 45 × 33 cm). The chamber was floated with nitrogen (purity >99.9%; PanGas AG, Dagmersellen, Switzerland) that was linked to a humidification bottle of distilled water (Schott AG, Mainz, Germany) (Fig. 1). To prevent overpressure, a tube enabling outflow was integrated. Also, an oxygen sensor indicating oxygen percentage and temperature was mounted in the chamber (Greisinger, GHM Messtechnik GmbH, Regenstauf, Germany). The oxygen percentage remained stable at 0.1% during all experiments. 

Figure 1.

 

Experimental setup for the low-oxygen/nitrogen-enriched environment. The main components include a hermetically sealed polymer chamber (center), a humidification bottle containing distilled water (left) to maintain appropriate humidity levels, and an oxygen sensor (right) for real-time oxygen percentage and temperature monitoring during the PACK-CXL procedure.

Enzymatic Digestion of Corneal Buttons and Assessment

Each 24-well plate (Merck AG, Darmstadt, Germany) was filled with 2.0 mL of a 0.3% collagenase A solution (Roche, Basel, Switzerland). After completing the designated corneal cross-linking protocol, the corneal buttons were placed into the wells and incubated at 37°C in a thermoshaker set to 200 revolutions per minute. Visual inspection and photographic documentation were conducted hourly. The time taken for complete enzymatic digestion of each cornea was recorded. Digestion was considered complete when the corneas had fully fragmented into a dust-like layer. 

Statistical Analysis

Statistical analysis was performed by using RStudio 2022.12.0 (R Foundation for Statistical Computing, Vienna, Austria), Prism 9.5.1 (GraphPad, Boston, MA), and Excel 16.70 (Microsoft, Redmond, WA). The descriptive statistics are presented as mean ± SD. Analysis of variance was used to analyze the differences between the groups, with a value of P < 0.05 indicating statistical significance. 

Results

This study involved a total of 144 ex vivo porcine corneas. The mean digestion time (MDT) for group A was 19.75 ± 1.34 hours, which was significantly lower than in all intervention groups (all P < 0.05). For the RB/green-treated groups (B1 to B4) (Fig. 2), the MDTs were 33.69 ± 1.4, 34.38 ± 1.31, 33.88 ± 1.02, and 34.06 ± 1.57 hours, respectively, with no significant difference in digestion resistance between these groups. In the combination treatment groups (C1 to C4) (Fig. 2), the MDTs were 39.56 ± 1.93, 51.94 ± 4.2, 39.62 ± 2.5, and 50.35 ± 1.59 hours. Corneas in groups C2 and C4 demonstrated greater digestion resistance compared to those in groups C1 and C3 (all P < 0.05). No significant differences were observed between groups C1 and C3 or between groups C2 and C4 (P = 0.38). 

Figure 2.

 

Mean enzymatic digestion times for the control corneas (group A), for the RB/green-treated corneas (groups B1 to B4), and for the combined RF/UV-A and RB/green-treated corneas (groups C1 to C4).

Discussion

Corneal cross-linking can be used to treat several different corneal pathologies. Its principal use is for the treatment of corneal ectasias; by cross-linking molecules within the stroma, CXL can increase the biomechanical strength of the cornea.⁶ The combination of RF/UV-A and RB/green light protocols provides key advantages due to their varying tissue penetration depths, with riboflavin penetrating deeper and RB affecting more superficial layers. Additionally, these chromophores utilize different wavelengths with minimal overlap in their absorption spectra, allowing them to complement rather than compete with each other in cross-linking. When applied sequentially, their effects are additive.¹⁷ This dual approach is particularly beneficial for treating infectious keratitis, offering physicians an enhanced therapeutic option, especially in cases of therapy-resistant infections. 

Moreover, photoactivating riboflavin with UV light to generate ROS has other effects beyond biomechanical strengthening. The binding of stromal molecules (principally collagen and proteoglycans) hides protease binding sites through steric hindrance, whereas ROS exert a direct pathogen-killing effect. This occurs through a combination of binding to pathogen nucleic acids (inhibiting replication) and directly damaging pathogen cell membranes, leading to cell lysis.¹⁹ Both mechanisms make PACK-CXL an interesting option for the treatment of infectious keratitis.^2,10 

Oxygen plays several important roles in CXL. Its presence in the stroma is crucial for the biomechanical stiffening effect of CXL, and its concentration is rate limiting, as it is consumed during the photochemical reaction between UV light and riboflavin.²⁰ This limitation has posed a challenge for the development of accelerated CXL protocols, as increasing the speed of UV fluence application (e.g., 5.4 J/cm²) results in a reduced stiffening effect, because oxygen is not replenished from the atmosphere as quickly as it is consumed in the stroma. However, the presence or absence of oxygen in the cornea does not seem to impact the pathogen-killing effect of PACK-CXL protocols.^21–24 This allows PACK-CXL protocols to be accelerated in ways that CXL protocols for ectasia cannot. 

This study suggests that the effect of CXL on increasing corneal resistance to enzymatic digestion is also oxygen independent. Regardless of whether the chromophore and light wavelength combination is RF/UV-A, RB/green, or a sequential use of both in a single session, the presence or absence of oxygen does not seem to influence the enhancement in enzymatic digestion resistance conferred by these CXL protocols. These findings are consistent with recent studies on thicker animal corneas.^25,26 

Our study has some limitations. We conducted PACK-CXL ex vivo in transparent tissue. The appropriate fluence required for an opaque cornea, as seen in infectious keratitis, remains unknown and should be explored in future studies. Our findings emphasize that corneal resistance to enzymatic digestion after PACK-CXL, whether with RB/green light alone or in combination with RF/UV-A, is oxygen independent. These results enhance our understanding of the molecular mechanisms underlying the therapeutic effects of CXL and may contribute to the refinement of accelerated PACK-CXL protocols and other CXL treatment approaches. 

Acknowledgments

Supported by the Light for Sight Foundation. 

Disclosure: M.E. Aydemir, None; N.L. Hafezi, EMAGine AG (E); N.-J. Lu, None; E.A. Torres-Netto, None; M. Hillen, None; C. Koppen, None; F. Hafezi, Apparatus for the treatment and/or prevention of corneal diseases (P), EMAGine AG (I)