Application and Evaluation of Full-thickness Keratoplasty for the Management of Irreparable Corneal Affections in Dogs

Infected corneas of dogs can be treated medically or surgically depending on severity of corneal diseases (Gelatt and Samuelson, 1982). Usually better response is achieved through medical management at early stage with confirmatory diagnosis of corneal diseases but delayed or severely traumatised irreparable corneas demands surgical interventions to achieve visual or tectonic eyes (Hollingsworth, 2003). Moreover unsuccessful surgical management in corneal diseases can leads to panophthamitis followed by eye-globe enucleation (Maggs, 2018). There are many surgical techniques like pedicle grafting from bulbar conjunctiva, keratotomy, superficial keratectomy, use of cryogens and keratoplasties were described in previous literatures (Hakanson, 1987; Azoulay,T. 2014; Dorbandt et al., 2015; Lacerda et al., 2016; Rajasekaran, 2021; Qureshi et al., 2022) for verity of surgical affections of canine corneas with different success rates. For irreparable corneal affections, cornea transplant can be a good option and even after graft rejection, this procedure can save reaming surrounding cornea transparent and thereby eye remains visual (Hansen and Guandalini, 1999). However in diseases where complete cornea gets affected then tectonic outcomes are usually expected and many pet-owners prefers it over eye enucleation. The Term “Keratoplasty” consists surgical procedure which involved replacement of diseases corneal tissue with healthy cornea (Qureshi, 2020). Results of full-thickness keratoplasty (or penetrating keratoplaty) in animals were well documented in earlier studies (McEntyre, 1968; Mueller and Formston, 1969; Coster, 1981; Brooks et al., 2008; Lacerda et al., 2016) but promising results are still lacking. Corneal tissue after transplant shows comparatively less immune reaction and hence possesses higher success rates over other organ transplant in human ophthalmology (Coster, 1981; Völker Dieben et al., 1982; Wilson, 1990; Boisjoly et al., 1993; Panda et al., 2007). Therefore six clinical cases of dog affected with irreparable unilateral corneal affections such as bullous keratopathy, corneal sequestrum, full thickness corneal defect and corneal edema were selected, operated and evaluated for penetrating keratoplasty.

Total 6-clinical cases {bullous keratopathy (n=1), chemical burn (n=1), corneal sequestrum (n=1), full-thickness corneal defect (n=1) and in corneal oedema (n=2)} affected with irreparable severely damaged corneas were presented to Department of Veterinary Surgery and Radiology, GADVASU, Ludhiana. Detailed preoperativecase presentation findings are included in Table 1. All these cases were selected for full-thickness keratoplasty after comprehensive blood work and detailed ocular examinations. Fresh and frozen corneas were utilized for transplant in accordance to availability at the time of case presentation. Donor corneas along with 1-2 cm sclera rim were harvested from dogs died or euthanized due to systemic/traumatic diseases other than eye diseases. Immediate after harvesting, donor corneal tissue was placed in a commercially available ophthalmic antibiotic solution (“McCarey-Kaufmanmedium’’¹) and stored. For utilization of tissue as fresh graft (collected 48-hours prior to surgery), corneas were preserved refrigerated at 4°C. While for utilization of frozen grafts, the containers were frozen at -22°C and used as per need within 1 month of period. All the selected dogs were prescribed and advised to administer topical medicines to prevent panophthalmitis. Systemic antibiotic was also administered for 3-days prior to surgery. Eye medications which included topical lacrimomimetic (0.5%) carboxy methyl cellulose sodium², topical antibiotic eye drops (0.3%) gatifloxacin³, topical immunosuppressant (0.1%) cyclosporine⁴ from the day of reporting till the scheduled day of surgery. Where necessary, systemic antibiotics, non-steroidal anti-inflammatory medications and other supplements were given based on the findings of the pre-operative evaluation.To ensure ocular asepsis on the day of surgery, gatifloxacin eye drops were applied to the affected ocular surface at intervals of 15 to 20 minutes, beginning one hour before surgery and continuing until the procedure started. General anaesthesia was used for surgical interventions in present study. A combination of Butorphanol⁵ @ 0.2 mg/kg body weight, Atropine⁶ @ 0.02-0.04 mg/kg body weight and Acepromazine maleate⁷ @ 0.02-0.05 mg/kg body weight through intramuscular route were used in all dogs for premedication. For induction Propofol^®8 @ 4  mg/kg body weight was administered slowly by intravenous route. For Maintenance Isoflurane⁹ kept at 1-
 

2% with 100% oxygen was utilized. The patient’s head was elevated and positioned to the surgeon’s preference by placing padding material beneath the patient’s head. Hair was clipped (not shaved) from the surrounding area (periorbital area and eyelids) and a 5% povidone iodine solution was applied. Before the procedure started, the dorsal and ventral conjunctival fornixes were also flushed with povidone iodine solution (1:50 ratio). To cover the eye during surgery, sterile surgical drapes or commercially available eye drapes with an operating window in the middle were used. The eyelids were held in place with a Lieberman speculum and the surgeon operated while seated in front of the patient. Both fresh (n=3) and frozen (n=3) homologous corneas collected from donor dogs were applied to replace the recipient corneas affected with irreparable corneal affections (opaque/weakened/infected) that do not permit vision. All efforts were made specially to eliminate ocular bacterial infections, if any. For surgery eyes were prepared similarly mentioned in earlier techniques of present study. Two different sized sterilized corneal trephines were used for trephining of donor and receipt corneas, respectively. The corneal lesions to be removed were positioned centrally within the minimum sized corneal trephine. In recipient eye, holding the corneal trephine perpendicular to the corneal lesions, it was twisted both clockwise and counter-clock wise until approximately 80% of the corneal stroma was incised. A stab was made using 2.8 mm keratotome in circumscribed incision made through corneal trephine and ultimately it allowed entry into the anterior chamber. Diseased cornea was removed with the help of keratoplasty scissors. Anterior chamber was collapsed immediate after incisions where larger corneal defects were presented and thereby larger recipient’s corneal tissue were removed. Donor cornea was first placed over Teflon block with endothelial side up (identified through attached scleral-rim) and donor corneal button was harvested with 0.5 mm larger sized corneal trephines. Corneal button was grabbed carefully (to prevent endothelial damage) using Lim’s forceps and placed over the recipient’s bed where diseased corneal was already trephined. Patson spatula (double ended) was used to secure the corneal button in position on recipient’s bed during application of cardinal sutures. The  corneal  graft was  sutured  in  each  quadrant  with  four  8-0  simple interrupted Vicryl sutures. Suture needle was carefully penetrated upto 2/3^rd of stroma in both donor and recipient’s corneal tissue thereby penetration of endothelium was prevented. Once the donor button was stabilized with cardinal sutures, additional sutures were placed in simple interrupted manner. Partial temporary tarsorraphy was done and removed after 2-weeks.All animals were administered with systemic antibiotics (Inj. Cefotaxime @ 25 mg/kg body weight, b.i.d.for 5 days) and NSAIDs (Inj, Meloxicam @ 0.2 mg/kg body weight, o.d. for 3 days) intramuscularly to prevent further infections and reduce post-operative inflammation.Topical lacrimomimetic eye drops (Carboxy  methyl  cellulose  sodium) was instilled 4-times a day for 1 month post-operatively. Topical antibiotic eye drops (Gatifloxacin) was instilled on every 2 hour basis during the 1^st post-operative week which was tapered to every 4 hour basis, 8 hour basis and 12 hour basis in the successive post-operative weeks on the basis of fluorescein dye test (FDT). Topical cycloplegic eye drops (1%Tropicamide) instillation was started for 1^st-week after surgery (one-drop in a day) to relieve uveal-spasm. Topical steroid independent of FDT were started from 2^nd week of surgery till recovery (to stop initiation of graft reaction) and topicalimmunosuppressant drug (0.1% Cyclosporine, 2-drops t.i.d.) was started from the day of surgery till eye become comfortable. Owners were advised to place dogs in dark room forfirst month post-operatively.Corneal clarity was graded from 0 (crystal clear), 1 (clear), 2 (hazy), 3 (very hazy) and 4 (opaque) whereas corneal pigmentation score was calculated according to Charbiwala (2019). This extent of corneal pigmentation was assessedusing particular grading system in accordance to Charbiwala, (2019). Total area of deposition of brown to blackish pigment either melanin or interms of melanocytes over the ocular surface (mainly cornea) was referred as degree/extent of corneal pigmentation. In all the casesof ocular surface pigmentation, corneo-scleral limbus was used as periphery (rim) andthen ocular surface within circle was divided into total  12 sectors‘. It is the graded as ½ (Single line of pigment in a sector), 1 (Single sector affected with pigmentation), 1+1 (Pigment extending upto the resting pupil edge), 2+1 (Visual axis is affected {pigment extending beyond the resting pupil edge}) and 1 point perclock hour (Pigment extending just beyond the limbus).

Indications of corneal transplant for irreparable corneal affections in small animals were already mentioned in earlier literature (Townsend et al., 2008; Gelatt et al., 2013; Maggs, 2018). In present study, fresh (n=3, C1 to C3) and frozen grafts (n=3, C4 to C6) were applied in equal distribution and independent of type of corneal affection. Size of affected cornea was also calculated in four-cases (mean was 6.87±1.22 mm, median was 6.2 mm, ranges from 4.9 to 10.2 mm) whereas in two cases (C2 and C4) it was not possible because of complete cornea involvement. The mean surgical time was recorded as 73.33±14.25 minutes. All the grafts were stabilised with the help of absorbable sutures (size- 8-0, polyglactin 910). In research of Lacerda et al., (2016) on penetrating keratoplasty, similar to present study, polyglactin 910 (9-0 in their study, but 8-0 in present study) absorbable suture material was applied to stabilize the graft but they followed cardinal suture pattern (12-6-9-3 O’clock positions). Townsend et al., (2008) transplanted cornea for treatment of feline sequestrum and used non-absorbable polyamide (9-0) sutures. McEntyre (1968) applied 7-0 silk sutures in canine cornea transplant. They placed simple interrupted pattern in few millimetre gap (1-2 mm). In present study, the mean value of number of sutures used during FTKP to affix the donor cornea with recipients’ bed was recorded as 8.83±1.01, with median of 9 ranges from 6 to 12 (in numbers). In a study (McEntyre, 1968) on experimental keratoplasty on dogs, range of total number of interrupted sutures was 8 to 16 along with continuous suture application. In his study, wound leakage and out-riding of donor edges were reported even after use of total 16 simple interrupted sutures. However such intra-operative complications might remained undiagnosed in present study hence not documented. Mueller and Formston (1969) explained that a more satisfactory method of preparing donor full-thickness graft was to trephine the transplant tissue from endothelial side of donor corneal button (corneal trephine should be firmly placed over the endothelium and then disk is punched out) which was similarly followed in present study. Procedure of cornea collection was also similarly followed in accordance to their study and they described that cornea with a rim of sclera should be dissected from intact globe; the ciliary body adherent to the scleral rim should be removed together with iris and lens. Many hurdles and complications like intra-operative iatrogenic damage to lens and vitreous (C5), faulty entry into anterior chamber (C1, C2 and C5), intra-operative iris protrusion (C5), difficulty to identify the exact epithelial and endothelial surface of donor corneal button after harvesting and thus the button was not utilized ahead (C4), oedematous/swellon donor corneal button (C4 to C6) and difficulty in placement of suture knot due to oedematous margins of recipients’ cornea and donor corneal button (C4 to C6) were recorded as Intra-operative complications. All these complications were similarly reported in research of Qureshi (2020) on canine keratoplasties. The mean value of donor graft size (in diameter) utilized in FTKP was recorded as 9.58 ± 0.49 mm. Same value is recorded as size of trephines for donor button retrieval. The mean value of recipient’s bed size was 9.08 ± 0.49 mm. In research of Lacerda et al., (2016), graft size ranged from 3 to 15 mm (median 7.5 mm). Donor graft must be larger than the recipients’ bed which ranged from 0.1 mm to 0.5 mm in previous literature (McEntyre, 1968; Mueller and Formston, 1969; Gelatt et al., 2013; Lacerda et al., 2016; Qureshi 2020) because graft-shrinkage during post-operative period. McEntyre (1968) utilized 7.0 mm corneal trephine for a depth of 0.4 mm for incision in recipient’s cornea. He mentioned that to minimize damage and save maximum of endothelial cells, recipient’s button was excised with a bevelled incision.
       
Previous researchers (McEntyre, 1968; Mueller and Formston, 1969; Coster, 1981; Gelatt et al., 2013; Lacerda et al., 2016) stated that irreversible oedema, graft reaction (invasion of blood vessels towards donor tissue) and graft-opacity/fibrosis were considered as gross clinical signs of graft rejections after FTKP. The main reason behind all these clinical signs was considered as endothelial destruction which is usually not easy to identify. Similar clinical signs were also reported in present study in post-operative periods. Association of graft failure with numbers of invaded vessels in postoperative period in per cornea quadrant (away from limbus at least 2mm) was considered as risk factors of graft rejection in earlier studies ((McEntyre 1968). Similarly in present study grafts were invaded by vessels within a week of surgery.
       
Total 4-dogs of present study were survived whereas 2-dogs died in later period due to other systemic causes. The mean time of disappearance of corneal sutures or mean value of time taken to dissolve absorbable sutures was recorded as 37.6±3.26 days. The surgical Procedure and long term follow-up of penetrating keratoplasty in dog affected with Bullous keratopathy (C1) is shown in Fig 1.


               
Postoperatively, overall corneal clarity score 2, 3 and 4 was reported in total 1-case (C3; upto 1-month, afterwards dog died), 2-cases (C1 and C6) and 3-cases (C2, C4 and C6) respectively (Fig 3). Up to 1-months total 3-cases (C1, C2 and C6) showed positive menace reflex. 2-cases died and excluded from follow-up statistics (Table 2). In total 2-cases (C1 and C6) were reported with sluggish menace reflexes. Therefore, postoperatively visual outcome was 50% (2/4) and tectonic outcome was 100% (4/4) in present study. Post-operatively, mean value of corneal pigmentation score (for 4-cases, C1, C2, C5 and C6) was 9.00±1.58 (Fig 2). Pre-operatively visual outcome was recorded as 33.33% (2/6) which was remained same in post-operative period up to 6-months of follow-up. Moreover topical steroid was advised for long term application to reduce the scar/opacity but prolonged outcome was not available.There was only one report (Lacerda et al., 2016) in which rate of graft-failure/rejection has been mentioned as 56% which was almost similar to medical practice (Wilson and Kaufman, 1990; Panda et al., 2007) but many reports of medical science possess comparatively less rejection rates than veterinary practice (McEntyre, 1968; Mueller and Formston, 1969; Gelatt et al., 2013; Lacerda et al., 2016; Qureshi, 2020).

Good tectonic outcomes were achieved with use of full-thickness keratoplasty to manage irreparable corneas in dogs. Out of 6-eyes, 2-eyes at last stages of diseases were saved and existing visual capacity was preserved. Severe graft reaction, corneal opacity, graft-melanosis and graft rejections were noticed and recorded as complications of this study.

The financial support was provided by ICAR under DIMSCA-project and GADVASU, Ludhiana for completion of present research.

No conflict of interest.