The present case series aimed to determine if therapeutic or tectonic corneal grafts using cryopreserved donor tissue remnants are viable treatment options for refractory infectious keratitis or apparent/impending corneal perforation. Until recently, transplantation of fresh cornea tissue has been considered most appropriate in these cases if the donor cornea supply is sufficient. Sharma S. et al. reported that both functional and anatomical success were achieved for an average of 11 months in about 60% of patients with corneal ulceration, thinning, and perforation after corneal path graft21. However, cornea donor tissue is severely lacking in Asian countries22–24. From a non-medical point of view, Asian countries suffer from a significant shortage of organ donors due to deep-rooted ethnical and religious beliefs. This obstacle will be difficult to overcome for the foreseeable future5. Cornea tissues are now available from eye banks in countries with more abundant corneal donors, such as the United States. However, even in this case, there are significant limitations due to the high cost and limited availability of fresh tissue. In addition, the shortage of fresh corneal tissues worldwide is getting serious at a time of global crisis when the world is facing COVID-1925. Therefore, this strategy is insufficient to address the current shortage of donor tissue.
On the other hand, it is questionable whether fresh corneal tissue is the most appropriate for all transplants when the availability of fresh tissue is severely lacking. Therapeutic grafts for infectious lesions or tectonic grafts using fresh cornea for lesions existing in the corneal periphery pose additional problems such as reinfection or graft rejection. In addition, the use of immunosuppressants after transplantation, as would be necessary with the use of fresh tissue, can lead to uncontrollable systemic side effects and exacerbate existing physiological problems in elderly patients with poor medical condition, which is the patient cohort that most commonly requires tectonic or therapeutic keratoplasty for perforated corneas or refractory infectious keratitis. In these contexts, prompt intervention for infectious corneal ulcer and impending corneal perforation preclude the use of fresh donor tissue, which is not always available. Therefore, prior studies have evaluated the feasibility of conducting tectonic or therapeutic PKP or lamellar keratectomy (LK) using glycerol-preserved donor corneas13,26,27. Storage of corneal tissue in glycerol has advantages such as storage at room temperature and low probability of graft rejection due to acellularity caused by glycerol. However, when glycerol-preserved cornea is used in cases of infectious keratitis, reinfection may occur depending on the causative microorganism or secondary glaucoma may occur28.
More recently, tectonic keratoplasty using femtosecond laser intrastromal lenticules29 and therapeutic xenografts30 have been proposed as new therapeutic modalities. Although lenticule size could be sufficient to create tectonic grafts for keratoplasty, this modality is difficult to apply in cases with deep or large ulcerations due to limitations in the size and thickness of the lenticules31. In the case of xenografts, the probability of immune rejection is high, and a high degree of immunosuppression is therefore required. This modality would thus be contraindicated for the targeted patient population, in which poor medical condition is common, and immunosuppression would pose significant risk of reinfection in infectious keratitis32,33.
The long-term preservation method of donor tissue remnants is a major consideration in the use of preserved corneal tissue for keratoplasty. In previous reports, the advantages of glycerol storage have been reported. Glycerol storage at room temperature lowers the antigenicity of donor tissue by destroying endothelial cells through osmotic stress, and the tissue can be inexpensively stored over long periods of time9,34. However, as glycerol is a dehydrating agent with antimicrobial and antiprotease properties, it can damage corneal endothelial cells in the recipient eyes. When storing tissue remnants in glycerol, the storage media must be replaced with water before use, and caution is required because glycerol remains in the tissue and spreads to the ocular surface or anterior chamber, where it can cause serious damage to the intraocular tissues35,36. In addition, there are no data to support the safety of glycerol application to ocular tissues. On the other hand, Optisol-GS preserves the corneal epithelium at 4°C for more than 2 weeks, and has an antibiotic effect due to the use of gentamicin (100 µg/mL) and streptomycin (200 µg/mL) in this formula. Optisol-GS has been widely used for tissue storage and transplantation, and there are sufficient data to support its safety37,38. Since Optisol-GS is not a cryoprotectant, osmotic stress of cells and tissue freezing injury may occur if the remnant cornea is immersed in Optosol-GS for rapid freezing34. However in this process, the endothelial cells are definitely destroyed, so we thought it would be more helpful to prevent immune rejection. Therefore, we selected Optisol-GS as the preservative medium for cryopreservation of donor tissue remnants. Our results suggest that this approach has several benefits over previously used methods.
In this study, the mean age of patients who underwent graft was 69.4 years, and 26.1% of patients were over 80 years old. Therefore, the patients were elderly, and most had underlying medical conditions. Within the patient cohort, 11 patients (24.4%) had uncontrolled diabetes, and five (11.1%) had a history of stroke. One patient (2.2%) had hemiplegia, and one patient (2.2%) had intellectual disability. In general, high-dose steroids are needed to prevent graft rejection in corneal transplant39. However, in older patients, the use of immunosuppressants could aggravate cardiovascular risk, and cause malignancy and infections due to over-immunosuppression in long-term therapy40,41. Immunosuppressants generally used for solid organ transplantations, such as cyclosporine, tacrorimus, and glucocorticoids, could exacerbate high blood pressure or blood sugar in this vulnerable patient population42,43. In addition, the use of immunosuppressive drugs in patients with a history of stroke poses a significant infection risk44,45. Therefore, immunosuppressants should be used with significant caution in the elderly, patients with underlying diseases such as diabetes and hypertension, and patients who have suffered a stroke. In our case series, only topical antibiotics and steroid eye drops were used, and no systemic treatments were administered. Nevertheless, no graft rejection occurred in either tectonic or therapeutic grafts, suggesting that sufficient acellularity can be obtained only by deep freezing cornea donor tissue in Optisol-GS. Finally, anatomical success was achieved in 41 of 46 patients (89.1%). Anatomical integrity was maintained in 14 of 18 patients (77.8%) in the therapeutic keratoplasty group, and 27 of 28 patients (96.4%) in the tectonic keratoplasty group. Most of the therapeutic keratoplasty cases were elderly patients in poor general condition, with co-existing conditions such as cancers or uncontrolled diabetes. In the present study, because therapeutic keratoplasty was performed for refractory keratitis in which most of the patients did not respond to drugs for fungal keratitis, the success rate of approximately 80% is remarkable. Although reinfection occurred after the initial graft in five patients, the infection was controlled, and corneal integrity was successfully maintained after the patients underwent an additional therapeutic graft.
Particularly in corneal lesions located near the limbus, such as Mooren’s ulcer, Terrien’s marginal degeneration, or staphylococcal marginal keratitis, the incidence of graft rejection is high, ranging from 39.4–64% after keratolimbal allograft transplantation46,47. In the present study, nine patients with keratitis invading the marginal cornea or corneoscleral tissue were identified. All of the patients maintained successful anatomical integrity after grafting using cryopreserved cornea without rejection. In general, eccentric grafts have a higher risk of rejection than central position grafts because they are proximal to the vascularized limbus and are thus surrounded by host corneal tissue rich in Langerhans cells48. Therefore, for marginal lesions, it is more effective to use cryopreserved cornea than fresh cornea to reduce antigenicity.
In cases of infectious keratitis, the use of topical or systemic immunosuppressants after grafting could promote recurrence or exacerbation of the infection7,8. In addition, systemic broad-spectrum antimicrobial agents must be used for at least 2 weeks postoperatively until the corneal epithelium has healed49. If systemic immunosuppressants and antibiotics are used together, patients are at risk for severe nephrotoxicity and hepatotoxicity, especially in elderly cohorts50,51. Because the prevalence of infectious keratitis is relatively high in elderly and medically compromised patients, it would be inappropriate to use fresh cornea for therapeutic keratoplasty in these cases52,53. In fact, in the present study, the average age of patients who underwent therapeutic keratoplasty for infectious corneal ulcers was 70.9 years, and major medical conditions were present in most patients. Considering these factors, acellularized grafting of cryopreserved cornea is preferable for therapeutic keratoplasty, as the risk of systemic side effects due to immunosuppressant use is decreased.
General anesthesia must be used for elderly corneal transplantation patients with dementia or poor cooperation, and for patients with intellectual disabilities. The probability of postoperative neurological and cardiac complications is increased when elderly persons are placed under general anesthesia54. In addition, general anesthesia use and incidence of dementia or cognitive imparement are positively correlated55–57. The risk of postoperative delirium is also high in elderly patients over 70 years of age or in patients with underlying intellecture disabilities58 which can lead to long-term cognitive imparement59. In these patients, performing therapeutic or tectonic keratoplasty as a preliminary step for future optical keratoplasty is not desirable due to the risk of systemic and neurologic complications related to anesthesia. Therefore, a method of obtaining therapeutic effects with a single operation, rather than repetitive operations, is clinically desirable. The therapeutic goal in emergency corneal transplantation is to maintain anatomical structure, eliminate infection, and, if possible, improve visual acuity. In the present study, patients who underwent tectonic keratoplasty exhibited significantly improved UCVA postoperatively relative to preoperative UCVA. In the therapeutic keratoplasty cohort, although there was no improvement in visual acuity, anatomical success was attained in 14 of 18 patients (77.8%). Because the main purpose of therapeutic keratoplasty is eradication of infected corneal tissue49,53, it could be reasonable to perform primary corneal transplantation using cryopreserved cornea tissue. Using this method, we can expect to preserve ocular integrity and improve visual acuity in non-infectious keratopathy, and to eliminate infected tissue in infectious keratitis, especially in elderly patients and/or patients with poor general condition.
There are several limitations to this study that should be acknowledged to avoid its overinterpretation. This study is a retrospective analysis, which is subject to selection bias. Further, the study relied on electronic medical records, such that the follow-up period was variable between patients. Patients with no final follow-up and patients under follow-up were included in analyses. Among patients with follow-up loss, a significant number of patients died due to underlying medical conditions early in the follow-up period. Patient 1 died from cerebral infarction, and Patient 31 died from subarachnoid hemorrhage during follow-up. Finally, only 19 of 46 patients were followed up for more than 1 year, which is insufficient to determine long-term surgical success. Although the study has the advantage of a relatively large number of cases with 55 cases in 46 patients, the information collection period was relatively long at 11 years. The overall surgical procedure was the same, but there could have been changes in minor details over time. Next, it was not confirmed whether the antimicrobial effect of Optisol-GS remained after the corneal tissue was thawed before surgery, or whether infection by other bacteria or fungi occurred in the media. To prevent contamination, before the step of cryopreserving remnant donor cornea, fresh donor cornea was excised rapidly, such that contamination would not occur. Subsequently, the remnant tissue was immediately and aseptically immersed in Optisol-GS and cryopreserved. However, a prior study suggested that the antimicrobial effect of Optisol-GS is diminished at low temperatures37. If the storage period is prolonged, the antimicrobial effect could decrease or infection of the media could occur, affecting postoperative outcomes. Finally, there is currently no legislation in Korea restricting the transplantation of the same organ to multiple recipients. Therefore, this procedure may not be possible in countries where it is prohibited to transplant the same cornea more than once. Further prospective and long-term follow-up study of a larger number of patients is needed to validate our findings.
In conclusion, therapeutic or tectonic keratoplasty using cornea cryopreserved in Optisol-GS is potentially an inexpensive, safe, and effective surgical option that can be expected to maintain anatomical integrity in cases of corneal perforation and infectious keratitis. Additionally, improved visual acuity can be expected in patients who undergo tectonic keratoplasty using cryopreserved cornea. In elderly patients or patients with poor general condition, who are highly likely to undergo the above procedures, use of cryopreserved corneal tissue can be considered a superior method to keratoplasty using fresh donor cornea. This method can reduce side effects by reducing the use of immunosuppressants, and successful treatment can be obtained with a single procedure without further surgeries in most cases.