Therapeutic Penetrating Keratoplasty Using Full - Thickness Gamma - Irradiated Sterile

Purpose: To report the ultrastructural and biological features of a gamma-irradiated sterile cornea based on its successful use in a therapeutic penetrating keratoplasty for a case of severe fungal keratitis. Methods: A commercially available acellular, sterile cornea was examined by electron microscopy and immunohistochemical staining after in-vivo explantation in a 50 year-old diabetic male who presented with a culture positive Aspergillus keratitis that did not respond to maximal medical therapy. A penetrating keratoplasty was performed for tectonic and therapeutic purposes using 1 Cornea and External Disease Service, Medstar Washington Hospital Center, Washington, DC 2 Departments of Ophthalmology and Pathology, Medstar Georgetown University Medical Center, Washington DC 3 Department of Pathology, Medstar Georgetown University Medical Center, Washington DC 4 Department of Pathology, Medstar Georgetown University Medical Center, Washington DC 5 Ocular Surface Disease and Dry Eye Clinic, Cornea and External Disease Service, The Wilmer Eye Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA and decreased vision. He had no history of contact lens wear or previous eye problems. On presentation his visual acuity was handmotions. Slit-lamp examination of the left eye demonstrated a central mid-stromal ring infiltrate, with an overlying epithelial defect measuring 1.5 mm in diameter as well as several satellite infiltrates. The left eye was promptly cultured and the patient was started on fortified vancomycin (25mg/ ml), tobramycin (15mg/ml) and voriconazole 1% every hour due to the high suspicion of a fungal or polymicrobial infection. The initial corneal culture was negative for any microorganisms. However, a subsequent culture grew Aspergillus flavus. Despite aggressive medical therapy, including oral itraconazole, hourly topical voriconazole 1%, and topical fortified antibiotics, the patient’s condition deteriorated over the next few weeks. He developed a hemorrhagic hypopyon, iris neovascularization, and corneal thinning with impending perforation or microperforation. The hypopyon increased in size and became organized, raising the suspicion of intraocular invasion (figure 1). An emergency surgical approach was selected and the use of a sterile acellular cornea versus a fresh donor cornea was discussed with the patient. Owing to sterility and lack of antigenicity it was recommended that the transplantation be performed using VisionGraft. The patient provided an informed consent. A therapeutic penetrating keratoplasty was performed using an 8.75 mm VisionGraft cornea, oversizing the graft by 0.25mm. The donor was sutured in place with interrupted 10-0 nylon sutures. Aqueous fluid sample was sent for culture and an intracameral injection of 0.2 ml of 100 mcg/100 microliter voriconazole was given at the end of the procedure. In the immediate postoperative period, the patient developed a hyphema due to intraoperative bleeding from the iris. The intraocular pressure increased to 40 mmHg. Despite maximal medical therapy the patient had to be taken back to the operating room for an anterior chamber wash-out. Following the anterior chamber wash-out the intraocular pressure remained normal for the rest of the follow-up. The topical antifungals were continued for three weeks after the keratoplasty. The VisionGraft integrated very well with the recipient bed. The graft-host junction healed well, with normal epithelialization. However, after about two weeks a central epithelial defect that slowly gained the appearance of a sterile neurotrophic defect was noted. This central defect persisted despite aggressive use of lubricants and a bandage contact lens (figure 2). Although the graft remained clear, a dense, white cataract developed and necessitated a triple procedure with cataract extraction, intraocular lens implantation, and a full thickness penetrating corneal transplantation using fresh donor tissue for visual rehabilitation. The fresh donor tissue healed as expected with the host epithelium covering the entire donor within 10 days. The last ocular examination was performed nine months after the second keratoplasty and demonstrated a clear corneal graft with a visual acuity of 20/200 limited by diabetic macular edema. Results: Light microscopy of sections stained with hematoxylin and eosin showed corneal tissue with complete absence of the endothelium. The epithelium was present in the periphery of some sections, but largely absent over the rest (figure 3). The stroma was predominantly devoid of keratocytes. However, in the periphery of the graft, a few stromal keratocytes were visible with irregular nuclei. The lamellae of the stroma appeared regular, but lacked normal artifactitious clefting throughout most of the sections. Bowman’s and Descemet’s membranes appeared normal, but no endothelial cells were present. Keratocytes were largely absent, except for some irregular cells in the periphery of the graft. No inflammatory cells were present. Immunohistochemical staining with neurofilament for axonal nerve fibers, and S-100 for Schwann cells, were both negative. Electron microscopy showed the maintenance of the typical hexagonal arrangement of the corneal stromal collagen fibers arranged in lamellae. There was evidence of cross-linking Figure 4