Safety of transglutaminase-induced corneal collagen cross-linking on the central cornea thickness and intraocular pressure in vivo

Subject and measures

Ethical approval was obtained from the Animal Care and Use Committee (ACUC) Faculty of Veterinary Medicine, Universitas Airlangga (No. 2.KEH.071.07.2022). All procedures were performed to ethical standards.

We calculated the sample size using the Lemeshow formula. Twenty-one normal New Zealand white rabbits were enrolled, and the right eye was treated. All the rabbits were divided into three groups, namely, transglutaminase-induced CXL group, epithelial-off CXL group, and transepithelial CXL group. There were seven rabbits in each group and we used random allocation. All rabbits were healthy males, between two and three months of age, and weighed between 3 and 4 kg. All rabbits were provided by the Faculty of Veterinary Medicine, Universitas Airlangga. The experiments were performed at Veterinary Hospital Universitas Airlangga.

The equipment utilized in this study included individual rabbit cages in an ideal room temperature (24°C), enough ventilation, and good ad libitum; the portions of food and drink were the same for all rabbits.

Cross-linking procedure

Anesthesia was induced via intramuscular injection of 0.1 mL/kgBW xylazine 2% (Xyla, Interchemie, Netherland) and 6–10 mg/kgBW ketamine (Ket-A-100^®, Agrovet Market s.a. Lima, Peru). The right eyes were treated, and the contralateral eyes served as untreated controls kept closed during the procedure. The corneal epithelium of the first group was removed using an 8 mm MacRae Photorefractive Keratectomy (PRK) well and alcohol 20% for 40 seconds, then the loose epithelium was removed with a hockey knife. After the epithelial-off procedure, transglutaminase solution 1 U/mL (Zedira, Germany) was dripped onto the cornea every two minutes for 30 minutes, for a total of 16 times. The cornea epithelial of the transepithelial group was not removed, then the riboflavin transepithelial (Peschke TE, Peschke Meditrade GmbH, Switzerland) was dripped onto the cornea every two minutes for 30 minutes. Subsequently, UVA exposure (365 ± 5 nm, 3 mW/cm², 8-mm diameter light spot) was performed using a UVA lamp CCL-Vario System (Peschke Trade GmbH, Switzerland) while continuously dripping the riboflavin onto the cornea every two minutes for 30 minutes. The cornea epithelial of the epithelial-off group was removed with the same procedure, then the riboflavin epithelial-off (Ribolink, Aurolab, India) was dripped two times onto the cornea. Subsequently, UVA exposure was performed using the same technique while continuing to drip the riboflavin onto the cornea every five minutes for 30 minutes.^12,13 Immediately following the procedure, an eye ointment containing chloramphenicol (Cendo, Indonesia) was used. The ointment was administered every eight hours for 14 days.

CCT and IOP evaluation procedure

Fourteen days after treatment, the CCT and IOP of the rabbits were evaluated using Corvis ST (Oculus Optikgeräte GmbH, Wetzlar, Germany). Before the procedure, the rabbits were first anesthetized using 0.15 mL/kg xylazine and 6–10 mg/kg ketamine intramuscularly. All examinations were conducted by the same experienced ophthalmology technician to remove potential interobserver variability. The heads of the rabbits were positioned in front of the instrument to ensure that the cornea was 11 mm away from the air nozzle. In the instance of improper imaging or alignment concerns, a retake was performed in order to meet quality standards. The air puff was released and the ultrafast camera captured 140 horizontal cross-sectional corneal images; subsequently, CCT and IOP were determined.

Statistical analysis

Comparative between data were tested using the one-way ANOVA followed with a post hoc test if the data were normally distributed. If the data were not normally distributed, they would be analyzed using the Kruskal–Wallis test and continued by using the Mann–Whitney test. The result is considered significant if the p-value is < 0.05. All statistical data were processed using SPSS 26.0 software (RRID:SCR_002865) (SPSS, Chicago. IL, USA).

All the protocols are published in protocols.io (https://doi.org/10.17504/protocols.io.261ge3eool47/v1).

Comparison of CCT using Corvis ST

We found no secondary infection in the rabbit's cornea during the observation period and 14 days after cross-linking (Figure 1). Our study demonstrated that the transglutaminase-induced CXL group had the highest value of CCT at 428 μm. The lowest value was from the UVA/RF epithelial-off group with CCT 321 μm. In comparison with the UVA/RF epithelial-off CXL group and the UVA/RF transepithelial CXL group, the CCT of the transglutaminase-induced CXL group displayed the highest mean score, whereas the UVA/RF epithelial-off CXL group had the lowest mean score (Table 1). Analysis using the one-way ANOVA test revealed no significant differences between any of the groups (p-value = 0.990).

Figure 1. Anterior segment of rabbit day 0; (A) Transglutaminase, (B) UVA/RF epithelial-off, (C) UVA/RF transepithelial. Anterior segment of rabbit day 14; (D) Transglutaminase, (E) UVA/RF epithelial-off, (F) UVA/RF transepithelial.

Comparison of IOP using Corvis ST

The results of the research showed that the IOP was lowest in the UVA/RF transepithelial and the UVA/RF epithelial-off CXL groups with 5.50 mmHg. The highest IOP was in the transglutaminase-induced CXL group with 13.50 mmHg. The mean IOP in the transglutaminase-induced CXL group was highest among three groups (Table 2). Analysis using the Kruskal–Wallis test revealed no significant differences between any of the groups with p-value = 0.563. There were no signs of infection and inflammation in the three groups immediately after treatment and 14 days after treatment.

CCT after CXL

This study used two conventional CXL groups, namely, the UVA/RF epithelial-off group and the UVA/RF transepithelial group, compared with the transglutaminase-induced CXL group. In contrast to earlier research by Wu et al.¹², which solely employed UVA/RF epithelial-off techniques, transepithelial procedures were performed more often at the time of this investigation to prevent possible complications.

According to the results, there were no statistically significant differences in CCT between the groups (p-value = 0.990). The same result was obtained from the study that Wu et al. conducted in 2019, in which there was insignificant difference between the CCT of rabbits given CXL and CCT in the control group.^12,13 Although epithelial remodeling and stroma edema disappear a few days after treatment, CCT has been reported to be responsible for corneal thickness changes over a longer time; this theory may explain why there is currently no significant CCT thickening.¹⁴

The CCT decreases in the first few months after the UVA/RF epithelial-off CXL method, and then it will increase in the following 12 months. The CCT in UVA/RF transepithelial CXL tends to be more stable. The decrease in CCT after performing CXL with the epithelial-off method can be caused by several factors, including compaction of collagen fibers, dehydrated corneal stroma, and apoptosis of keratocytes caused by UVA rays. This could explain why, in this study, CXL using UVA/RF epithelial-off had the lowest CCT, because in this method a corneal epithelial debridement procedure was performed as well as UVA light irradiation. Factors that trigger an increase in CCT can be caused by repopulation of keratocytes, where this process requires a longer time, which is around six months.^14,15

Transglutaminase is an enzyme that catalyzes the formation of isopeptide bonds between proteins. This can occur through cross-linking of proteins due to γ-glutamyl lysine bonds or due to the unification of primary amino acids with peptide bond residues and glutamine. Recent studies have shown that mRNA, fibronectin, and transglutaminase were found to be more abundant in human corneal keratocytes treated with UVA and riboflavin. This induction by transglutaminase is proposed as a new mechanism for cross-linking. The CXL transglutaminase treatment did not use UVA irradiation, which could be the cause of the CCT in the transglutaminase group having the highest average.^13,15

IOP after CXL

The group with transglutaminase-induced CXL had the highest IOP among the groups, although there was no significant difference between groups (p-value = 0.563). The increase in IOP can be one of the factors that explain an increase in corneal rigidity after transglutaminase administration as CXL. A previous study by Kymionis et al.¹⁶ showed a mean rise of 2–3 mmHg after UVA/RF CXL due to increased corneal stiffness. Several studies stated that there were significant differences in IOP after CXL. These studies use different measuring instruments from those used in our study, namely Goldmann Applanation Tonometry and Tonopen.^16–18 In addition, these results can be interpreted as the safety of transglutaminase-induced CXL can be compared with UVA/RF CXL in influencing CCT and IOP, which is one of the factors in increasing corneal rigidity.^12,13

Cornea CXL is a procedure to create bonds that connect polymer chains. The CXL procedure can change the biomechanics of the human cornea by increasing corneal rigidity by up to 328.9%.¹⁹ Morphologically, the formation of cross-links increases the distance between molecules because the collagen polypeptide chains are separated.²⁰ In conventional CXL, two phases are employed. The corneal saturation step acts as a photosensitizer to promote cross-linking, while the UVA irradiation stage protects the underlying tissues from UVA-induced damage.²¹ Conventional CXL will result in cross-linking through two pathways, namely oxidative and glycosylation pathways.²² Activated riboflavin, in addition to activating oxidative pathways, can also support the occurrence of glycosylated cross-linking in proteoglycans and collagen fibers through a mechanism mediated by advanced glycation end products.^23,24

Recent studies have shown that mRNA, fibronectin, and transglutaminase are more abundant in human corneal keratocytes treated with UVA and riboflavin. Induction by transglutaminase is proposed as a new mechanism for cross-linking.^9,10 One of the possible advantages of CXL transglutaminase is that it prevents the loss of keratocytes and the discomfort usually experienced by patients treated with UVA and riboflavin.^12,13 Transglutaminase facilitates the formation of the ε-(γ-glutamyl) lysine isopeptide bond, which mediates the biochemical reaction between glutamic acid and lysine.²⁵ The cornea is mainly constructed of type I collagen and is rich in glutamic acid and lysine. Corneal collagen fibers could be cross-linked by transglutaminase, potentially resulting in more rigid mechanical properties.²⁶

Limitations

This study's limitations included the limited follow-up period and the absence of any measurements before the CXL procedure. Recolonization of stromal keratocytes and enhanced stromal fiber density are complete six months following CXL; hence, a six-month follow-up may be recommended.²⁷ This study's brief research period was a constraint; if the research had a longer period and pre-post CXL procedure examination, it might have produced the best results. Consequently, more research into the use of transglutaminase as a treatment for CXL and corneal ectasia is warranted.