Corneal Grafts in Pets: Cryopreserved vs Dehydrated

• This study compares using dehydrated and cryopreserved heterologous corneal grafts to manage full-thickness corneal defects in cats and dogs via penetrating keratoplasty.
• 132 animal eyes were examined, from 65 cats (81 eyes) and 42 dogs (51 eyes), all with full-thickness corneal defects.
• 57 eyes received cryopreserved grafts, and 75 eyes received dehydrated grafts; follow-up lasted 6 months.
• Optically clear corneas with mild scarring were achieved in 40.4% of cryopreserved cases and 42.7% of dehydrated cases.
• Moderate scarring was observed in 35.1% of cryopreserved and 37.3% of dehydrated cases.
• Severe scarring and corneal opacities with severe vision loss developed in 22.8% of cryopreserved and 18.7% of dehydrated cases.
• Overall, affected eyes were salvaged with visual function improved to varying degrees in 78.0% of cases.
• Transplant rejection led to enucleation in 1.75% of cryopreserved and 1.3% of dehydrated cases.
• No statistically significant differences were observed in clinical outcomes between cryopreserved and dehydrated corneal grafts.
• Dehydrated cornea is a viable alternative to cryopreserved grafts for managing full-thickness corneal defects.
• Keywords include cornea, corneal sequestrum, injury, keratectomy, and ulcer.

Methods

• Shilkin Center for Veterinary Ophthalmology conducted the clinical trial on heterologous corneal grafting from 2016 to 2019.
• Animals included 76 cats and 47 dogs that met criteria for penetrating keratoplasty.
• Inclusion criteria: deep/full-thickness corneal defects, dazzle reflex in the affected eye, and consensual pupillary light reflex in the contralateral eye.
• Exclusion criteria: age over 19 years for cats and 16 for dogs, severe systemic diseases, and iris prolapse older than 10 days.
• Animals were randomized into cryopreserved (28 cats, 19 dogs) and dehydrated corneal grafting groups (39 cats, 24 dogs).
• 110 animals (135 eyes) underwent penetrating keratoplasty.
• Follow-up and assessment occurred at 3, 10, 17, 24 days, and 2, 3, 6 months post-surgery.
• Outcomes were evaluated in 107 animals (65 cats and 42 dogs) after excluding those lost to follow-up.

Ethics & Examination

• The Bioethics Committee of Shilkin Center for Veterinary Ophthalmology approved this study.
• Owners provided informed consent to use their pets' data and photos.
• Penetrating keratoplasty was performed in 57 eyes with cryopreserved grafts (34 feline, 23 canine) and 75 eyes with dehydrated grafts (47 feline, 28 canine).
• Neuro-ophthalmic, slit-lamp biomicroscopy, rebound tonometry, indirect ophthalmoscopy, and fluorescein dye tests were done in the ophthalmic examination.

Graft Preparation

• Heterologous corneal grafts came from porcine eyes from a slaughterhouse, assessed for inflammation and neoplasms.
• Eyes were enucleated, disinfected with 0.2% povidone-iodine, frozen, and stored at -20°C, usable within 1-12 months.
• Before surgery, corneal tissue was partially thawed, dead epithelium was removed, and a trephine was used for graft diameter.
• The dehydrated corneal graft preparation used the same removal and disinfection as the cryopreserved
• After disinfection, the cornea was removed at the limbus and placed into a separate sterile container filled with silica gel.
• The containers were sealed and stored with a lid at room temperature where, culturing of the dehydrated material revealed no bacterial or fungal contamination at 4 months to 2 years after preservation.
• Immediately before surgery, the dehydrated cornea was rehydrated in 0.9% sodium chloride for 10 min
• Following rehydration, the cornea was placed in a donor chamber for ALTK, and a trephine was used to obtain a corneal graft

Surgical Procedure

• Animals received intravenous cefazolin (25 mg/kg) and subcutaneous meloxicam (0.2 mg/kg), intramuscular tiletamine (2-4 mg/kg), and medetomidine (2 mg/kg).
• Propofol (2–4 mg/kg) induced general anesthesia, maintained with 1.5%-2% isoflurane in 100% oxygen.
• Rocuronium bromide was used for neuromuscular block if necessary.
• The surgical site was prepared with 1% povidone-iodine.
• Corneal defect sizes were measured with a Castroviejo caliper.
• Full-thickness surgical intervention on non-perforated corneas was performed with a trephine incision.
• Paracentesis with a 1.2 mm paracentesis knife was performed followed by viscoelastic introduction to prevent IOP drop and fibrin exudation
• Complete removal of the corneal defect was achieved by using a crescent blade knife.
• If corneal integrity was compromised, necrotic tissue was removed with a scarificator and microscissors, then prolapsed iris was cleaned of fibrin
• Synechiae were broken down using mechanical movement and by means of introducing a viscoelastic solution
• The corneal graft, prepared in advance, then was placed into the corneal bed
• Single interrupted sutures using 9-0 polyglactin or nylon were used to suture.
• The graft was fixed with four cardinal sutures then secured to the recipient cornea using simple interrupted sutures placed 1 mm from each other.
• Phenylephrine 0.1% and tPA were injected intracamerally to prevent postoperative synechiae.
• Mean surgery time: 30 ± 10 min.
• Postoperative topical medications: tropicamide (2–3 times daily for 5–7 days), tobramycin or ofloxacin (4–6 times daily for 2–4 weeks), and sodium hyaluronate (4–6 times daily for 2–4 weeks).
• Systemic treatment: peroral doxycycline or cefixime (once daily for 7–12 days), and robenacoxib (once daily for 3–5 days).
• Nylon sutures were removed at 3–4 weeks post-surgery.
• Follow-up and outcome assessment was performed at 3, 10, 17, 24 days, and 2, 3, 6 months after surgery.
• Absolute values and means are shown, using the x² test for statistical significance.
• Clinical outcome differences were assessed using relative risks (RR) and 95% confidence intervals (CI).

Results

• Penetrating keratoplasty was done in 65 cats and 42 dogs with deep corneal lesions
• Dog breeds: French Bulldog, Pug, Yorkshire terrier, Bulldog, Chihuahua, Shih Tzu, crossbreed dogs.
• Cat breeds: crossbreed, Sphynx, Persian, and Abyssinian, with mean ages of 6.14 years in dogs and 6.80 in cats.
• All animals underwent slit-lamp biomicroscopy, rebound tonometry, neuro-ophthalmic testing, at set postoperative times.
• Mydriasis was induced to assess for synechia, and visual function was assessed postoperatively.

Postoperative Complications

• Suture erosion, graft melting, synechiae, ocular hypertension, recurrent sequestration, and transplant rejection were observed in the 24 days after surgery.
• There were no statistically significant differences in postoperative complication rates between cryopreserved and dehydrated grafting, and all complications were promptly managed.
• Suture erosions led to new suture placement, recurrent sequestration required repeat surgery, and graft malacia was treated with cross-linking and/or topical serum and ethylenediaminetetraacetic acid.
• Elevated IOP was successfully managed with topical hypotensive agents.
• Transplant rejection resulted in enucleation in one animal per group, with eyes sent for histopathologic examination.
• Granulation was observed but not classified as transplant rejection.
• Corneal scarring and transparency were assessed at 6 months based on: mild scarring, moderate scarring, and severe scarring.
• Optically clear cornea with mild scarring was achieved in 40.4% of cases; moderate scarring in 35.1%; severe scarring in 22.8% after cryopreserved grafting.

Corneal Scarring Values

• Optically clear cornea with mild scarring was achieved in 42.7% of cases; moderate scarring in 37.3%, and severe scarring in 18.7% after dehydrated corneal grafting.
• Successful penetrating keratoplasty was defined as salvaged globes and preserved central vision, while enucleation was considered failure.
• The affected eyes were salvaged and visual function improvement was achieved in 78.0% of cases overall.
• Transplant rejection warranted enucleation in 1.75% and 1.3% cases after cryopreserved and dehydrated corneal grafting

Discussion

• Common corneal disorders (ulcer, injury, feline corneal sequestrum) are managed with keratoplasty, keratoprosthesis, adhesives, synthetics, and biomaterials.
• Veterinary medicine also uses corneoscleral transposition, conjunctival grafts, amniotic membrane, renal capsule, peritoneum, and cartilage
• Grafts usually come from eyes of euthanized animals which are suitable for donation to the eye bank.
• Cryopreserved corneal grafting covers up to 90% of keratoplasty surgeries in dogs.
• The most common postoperative complication is transplant rejection and corneal opacities which can cause wound dehiscence and aqueous humor leakage.
• Large numbers of patients and lack of homologous corneas make heterologous corneal grafting the most satisfactory solution
• Preserved material is challenging to keep completely transparent after keratoplasty
• Surgery time (surgical site preparation to placement of the last corneal suture) didn't exceed 40 minutes
• The majority of researchers link graft rejection to vascularization of the recipient cornea and antigenic activity of the graft
• True transplant rejection was only observed in two cases which indicates blood vessel growth and granulation of the recipient cornea, the graft itself wasn’t affected by vascularization