Swetha Ravichandran, B.S. Optometry

M. Optometry Student, Manipal College of Health Professions, Manipal University, India



Corneal collagen cross-linking (CXL) is a procedure that was first explained by Wollensak et al in 2003 (1). Currently, it is the most common successful treatment used for halting the progression of Keratoconus (KC) and thereby strengthening bonds in collagen (Refer Figure 1). The traditional CXL procedure uses Riboflavin and Ultraviolet-A (UVA) and the minimum corneal thickness (CT) required is 400µm (1). But, some studies suggest the use of hypo-osmolar riboflavin solution which makes the CT reach 400µm in an otherwise thin cornea (Less than 300µm). However, in these cases, the increase in biomechanical resistance was not sufficient to arrest the progression of KC (2). CXL using Rose Bengal and Green light (RGX) is found to be penetrating only 100µm of the corneal stroma (3). This report addresses the advantages of RGX and its effect on the cornea.



Figure 1: Mechanism of Corneal Collagen cross-linking. (Image Courtesy: https://keratoconusgb.com/2012/11/20/what-is-cxl-collagen-cross-linking-a-guide/)

RGX effects on the cornea:

Corneal Stiffening: The first important effect of RGX on the cornea is – it makes the stroma stiff by about 11-fold in ≈12 minutes whereas, in the case of Riboflavin-UVA (UVX), stiffening is found to be about 6.25-fold (1, 3, 4).

Corneal Toxicity: RGX is found to be non-toxic to the keratocytes. On the contrary, UVX was found to be a significant cause of keratocyte apoptosis (3).

Biomechanical Changes: Although the stiffening properties were comparatively more with RGX, the biomechanical response which involved deformation was smaller in the case of UVX than of RGX (4). This was captured using the Air-puff system and images are shown in Figure 2. However, another study reported there was no significant difference in the stiffness post RGX (5).

Wound Healing: Re-epithelialization and pachymetry were similar post RGX and UVX. Based on the amount of stroma being treated, UVX resulted in cell death in the anterior and middle stroma. Similarly in RGX, cell death was pertaining only to the anterior third of the stroma (6).

Keratolysis: The time taken for dissolution was studied to understand the resistance to enzymatic degradation. The collagenolytic activity being the most important cause leading to ectasia was significantly reduced. But, corneas treated with either RGX or UVX was shown to degrade significantly slower than untreated corneas (7).

Role in Keratitis: In addition to the above-mentioned effects of RGX on the cornea, it also arrests the growth of methicillin-resistant Staphylococcus aureus and fungal cultures (8,9).

Efficacy and Safety: The green light used in RGX was not found to have any detrimental effect on the retina or iris. The duration of RGX being lesser than that of UVX, absence of keratocyte apoptosis, and attachment of rose bengal to collagen makes RGX an effective and safe method of CXL (10).



Figure 2: Corneal profile before and after spatial deformation in virgin eyes, after application of photosensitizer and after CXL treatments. (A) RGX; (B) UVX. (Image extracted from Bekesi et al. 2016)



 Although many in-vitro studies and experiments on rabbit cornea seem to be very promising proving many advantages, there are still requirements for more studies to demonstrate this theory on human cornea. With more studies depicting successful results on the human cornea, the procedure can be incorporated into regular practice for the treatment of corneal ectasia and keratitis.



  1. Wollensak, G., Spoerl, E., & Seiler, T. (2003). Riboflavin/ultraviolet-A–induced collagen crosslinking for the treatment of keratoconus. American journal of ophthalmology, 135(5), 620-627.
  2. Hafezi, F. (2011). Limitation of collagen cross-linking with hypoosmolar riboflavin solution: failure in an extremely thin cornea. Cornea, 30(8), 917-919.
  3. Cherfan, D., Verter, E. E., Melki, S., Gisel, T. E., Doyle, F. J., Scarcelli, G., & Kochevar, I. E. (2013). Collagen cross-linking using rose bengal and green light to increase corneal stiffness. Investigative ophthalmology & visual science, 54(5), 3426-3433.
  4. Bekesi, N., Kochevar, I. E., & Marcos, S. (2016). Corneal biomechanical response following collagen cross-linking with Rose Bengal–green light and Riboflavin-UVA. Investigative Ophthalmology & Visual Science, 57(3), 992-1001.
  5. Singh, M., Li, J., Han, Z., Vantipalli, S., Liu, C. H., Wu, C., … & Larin, K. V. (2016). Evaluating the effects of riboflavin/UV-A and rose-bengal/green light cross-linking of the rabbit cornea by noncontact optical coherence elastography. Investigative ophthalmology & visual science, 57(9), OCT112-OCT120.
  6. Lorenzo-Martín, E., Gallego-Muñoz, P., Ibares-Frías, L., Marcos, S., Pérez-Merino, P., Fernández, I., & Martínez-García, M. C. (2018). Rose Bengal and Green Light Versus Riboflavin–UVA Cross-Linking: Corneal Wound Repair Response. Investigative Ophthalmology & Visual Science, 59(12), 4821-4830.
  7. Fadlallah, A., Zhu, H., Arafat, S., Kochevar, I., Melki, S., & Ciolino, J. B. (2016). Corneal resistance to keratolysis after collagen crosslinking with rose bengal and green light. Investigative Ophthalmology & Visual Science, 57(15), 6610-6614.
  8. Halili, F., Arboleda, A., Durkee, H., Taneja, M., Miller, D., Alawa, K. A. & Parel, J. M. (2016). Rose bengal–and riboflavin-mediated photodynamic therapy to inhibit methicillin-resistant Staphylococcus aureus keratitis isolates. American Journal of Ophthalmology, 166, 194-202.
  9. Arboleda, A., Miller, D., Cabot, F., Taneja, M., Aguilar, M. C., Alawa, K., … & Parel, J. M. (2014). Assessment of rose bengal versus riboflavin photodynamic therapy for inhibition of fungal keratitis isolates. American journal of ophthalmology, 158(1), 64-70.
  10. Zhu, H., Alt, C., Webb, R. H., Melki, S., & Kochevar, I. E. (2016). Corneal crosslinking with rose bengal and green light: efficacy and safety evaluation. Cornea, 35(9), 1234-1241.