Microstructure and glycosaminoglycan ratio of canine cornea after reconstructive transplantation with glycerin-preserved porcine amniotic membranes

Objective  Although amniotic membranes of canine, feline, and equine species have some advantages as corneal transplantation material in many canine ocular diseases, their softness, thinness, and low availability can pose problems. As an alternative, the more abundant porcine amniotic membranes may be used. This paper describes the use of glycerin-preserved porcine amniotic membranes in corneal transplantation in eight normal dogs.
Method  A 0.4-mm deep recipient bed in the axial cornea of the OS of all dogs was created using an 8-mm Barron radial vacuum trephine. The recipient bed was then filled with amnion, and the entire cornea was covered with another piece of the glycerin-preserved membrane. The ocular signs evaluated were corneal opacity and corneal vascularization. The dogs were euthanized on days 5, 10, 20, or 40 after surgery, and samples were collected to evaluate corneal thickness, parenchymal cell number, mean collagen fibril diameter, collagen fibril content and the glycosaminoglycan (GAG) ratio.
Results  Corneal opacity was observed immediately after surgery. Restoration of corneal transparency, regression of corneal vascularization, and visualization of the pupil and iris were noted on day 40.
Conclusions  The clinical observations were supported histologically by regained corneal thickness, parenchymal cell number, mean collagen fibril diameter, collagen fibril content, and GAG ratio, suggesting that this technique may be a novel method for the treatment of ocular surface disorders.     

Amniotic membrane transplantation for the reconstruction of the ocular surface in three cases

This paper describes the use of amniotic membrane in the reconstructive surgical repair of generalized keratomalacia, ankyloblepharon, and after fibrous histiocytoma removal in two dogs and a cat. Case 1 was an 11-year-old female Yorkshire terrier with severe bullous keratomalacia in the left eye (OS). A frozen canine amniotic membrane graft and a third eyelid flap were applied. At day 80 postoperatively, only a mild scar and corneal vascularization were present. Case 2 was a 4-year-old female Siamese cat with symblepharon of both eyes after rhinotracheitis. Resection of the conjunctiva and frozen canine amniotic membrane transplant were performed. One month later, there was a little corneal scarring and corneal vascularization. Case 3 was a 6-year-old female terrier with a scleral and corneal mass at the 11-12 o'clock position (OS). Resection of the mass and amniotic membrane transplantation were performed. The mass was a fibrous histiocytoma localized to the sclera and cornea. This eye healed with mild conjunctivalization and no pigmentation. Amniotic membrane transplantation can be used as a method of reconstruction of the ocular surface with good repair of the cornea and minimal scarring in small animals.     

A retrospective study of 30 cases of frozen lamellar corneal graft in dogs and cats

Frozen lamellar corneal grafts and nictitating membrane flaps were used in 18 dogs and 12 cats to repair deep corneal defects. In all dogs either melting corneal ulcers or descemetoceles were present. In the 12 cats, nine had either a melting corneal ulcer or descemetocele, two animals had acute bullous keratopathy, and one cat had corneal sequestrum. Initial vascularization with gradual clearing of the graft occurred during the first 45 days postoperatively. At 60 days postoperatively, all eyes were visual. Frequent postoperative complications included: focal dehiscence of the wound ( n  = 9); melting of part of the graft ( n  = 7); and pigmentation of the graft ( n  = 4). The frozen lamellar corneal graft was a very safe technique, and restored the tectonic and the optical function of the cornea. It provided the best results in corneas with nonperforating corneal defects. This technique provides poorer results when the cornea was perforated prior to surgery or during the surgical procedure.     

Canine amniotic membrane transplantation for corneal reconstruction after the excision of dermoids in dogs

Transplantation of canine amniotic membrane (AM) in conjunction with a third eyelid flap was performed after the removal of large dermoids by keratectomy and conjunctivectomy on 7 eyes of 7 dogs. Corneal epithelialization was completed within 2 weeks after the transplantation. Five eyes attained normal transparency of the cornea within 5 weeks. Slight pigmentation of the bulbar conjunctiva at the limbus was observed in 1 dog that had pre-existing pigmentary keratitis. Neovascularization and scarring of the cornea and impaired vision were not found in any dogs at 8 weeks after the transplantation. In conclusion, transplantation of canine AM can promote corneal healing after the excision of large dermoids in dogs.     

Successful treatment of an unusually large corneal epithelial inclusion cyst using equine amniotic membrane in a dog

A 10‐year‐old intact male Yorkshire Terrier was referred for investigation of a large raised and nonpainful corneal lesion oculus dexter. Clinical examination revealed a pale, translucent corneal mass, which occupied half of the corneal surface and measured 11 mm × 11 mm × 13 mm. The mass was removed by superficial keratectomy and equine amniotic membrane (AM) was transplanted into the large corneal defect to cover the wound and provide tectonic support for the remaining cornea. The mass was histologically confirmed as a corneal epithelial inclusion cyst. There was no evidence of recurrence or complication at the surgical site 100 days postoperatively. Corneal epithelial inclusion cysts are uncommon in dogs. Although superficial keratectomy is the recommended treatment for corneal inclusion cyst, the combination of superficial keratectomy and AM transplantation had to be considered as an alternative for repair of large corneal defects. This is the first case report of the combined application of AM and superficial keratectomy to successfully treat a corneal inclusion cyst in a dog.     

Amniotic membrane transplantation for the treatment of feline corneal sequestrum: pilot study

Objectives To describe and evaluate the use of equine amniotic membrane trans‐plantation after lamellar keratectomy for the treatment of corneal sequestrum in cats. Methods Six cats (seven eyes) of various breed and ages with corneal sequestra were treated surgically with lamellar keratectomy and amniotic membrane transplantation. All the sequestra and a small piece of the amniotic membranes used for each surgery were submitted for histopathologic examination. Results Five of the seven eyes showed minimal level of scarring in the cornea and good transparency. No recurrences of the sequestra have been noted during the follow‐up period (3–9 months). One eye had necrosis of the amniotic membrane 2 weeks after the surgery. The sequestrum of this eye showed a high level of bacterial contamination on histopathology. Three months later the same cat developed a descemetocele in the area where the necrotic amniotic membrane was rejected. A second eye developed a perforation under the amniotic membrane two weeks after the surgery. The sequestrum of this eye was deep and without vascularization. Conclusion Amniotic membrane transplantation after lamellar keratectomy was a valid procedure for surgical treatment of corneal sequestrum in cats. The procedure resulted in excellent cosmesis and functional vision in five of seven eyes; although case selection is important, particularly to exclude the very deep and non‐vascularized sequestra.     

Identification of putative orthologs of clinically relevant antimicrobial peptides in the equine ocular surface and amniotic membrane

Objectives

This study aimed to define the antimicrobial peptide (AMP) expression pattern of the equine ocular surface and amniotic membrane using a targeted qPCR approach and 3'Tag-sequencing. It will serve as a reference for future studies of ocular surface innate immunity and amniotic membrane therapies.

Procedures

A targeted qPCR approach was used to investigate the presence of orthologs for three of the most highly expressed beta-defensins (DEFB1, DEFB4B, and DEFB103A) of the human ocular surface and amniotic membrane in equine corneal epithelium, conjunctiva, and amniotic membrane. 3'Tag-sequencing was performed on RNA from one sample of corneal epithelium, conjunctiva, and amniotic membrane to further characterize their AMP expression.

Results

Equine corneal epithelium, conjunctiva, and amniotic membrane expressed DEFB1, DEFB4B, and DEFB103A. DEFB103A was expressed at the highest amounts in corneal epithelium, while DEFB4B was most highly expressed in conjunctiva and amniotic membrane. 3'Tag-sequencing from all three tissues confirmed these findings and identified expression of five additional beta-defensins, 11 alpha-defensins and two cathelicidins, with the alpha-defensins showing higher normalized read counts than the beta-defensins.

Conclusions

This study identified AMP expression in the equine cornea and conjunctiva, suggesting that they play a key role in the protection of the equine eye, similar to the human ocular surface. We also determined that equine amniotic membrane expresses a substantial number of AMPs suggesting it could potentiate an antimicrobial effect as a corneal graft material. Future studies will focus on defining the antimicrobial activity of these AMPs and determining their role in microbial keratitis.     

Use of porcine small intestinal submucosa for corneal reconstruction in dogs and cats: 106 cases

Objectives : To describe the efficacy of porcine small intestinal submucosa in corneal reconstructive surgery in dogs and cats through a large retrospective study. Methods : A retrospective evaluation of 106 cases of surgical reconstruction of the cornea with small intestinal submucosa seen between May 2005 and January 2010 was carried out. The corneal defect was filled by microsurgical grafting of porcine small intestinal submucosa. The biomaterial implant was deposited in one or several layers depending on the depth of the defect. The animals were examined 3, 6 and 12 weeks after surgery. Results : Vision was preserved in all eyes at three months post‐surgery. In 74 cases (69.8%) the corneal scar was either transparent or discrete, whilst in 32 cases (30.2%) a mild or marked scar was observed. Minor complications occurred in 9 cases (8.5%) with partial integration of the small intestinal submucosa and in 24 cases (22.6%) with faint or mild corneal pigmentation, without impairing vision. In cases followed over a period longer than three months, major complications occurred in five dogs resulting in vision impairment because of pronounced pigmentation. Clinical Significance : Corneal grafting of porcine small intestinal submucosa is an effective method for corneal reconstruction resulting in corneal transparency in most cases. It is an excellent alternative to conventional conjunctival grafts.     

Amniotic membrane transplantation for corneal surface reconstruction after excision of corneolimbal squamous cell carcinomas in nine horses

OBJECTIVE: The purpose of this study was to evaluate the usefulness and effectiveness of permanent amniotic membrane transplantation as an adjunctive treatment to superficial keratectomy alone or combined with strontium-90 irradiation for treatment of equine corneolimbal squamous cell carcinoma (SCC) to decrease corneal scarring and recurrence rate. STUDY: The retrospective case study included 11 horses (n = 12 eyes) diagnosed and treated for ocular SCC that involved the limbus and cornea. Nine of those horses (n = 9 eyes) were treated between 2002 and 2006, with superficial lamellar keratectomy alone or combined with strontium-90 irradiation and followed by placement of a permanent amniotic membrane graft in the surgical defect. The level of scarring (i.e. the clarity of the cornea) resulting with the use of amniotic membrane was subjectively compared to cases where a permanent bulbar conjunctival graft was performed following keratectomy combined with strontium-90 irradiation or cryotherapy (n = 3 eyes). Recurrence was defined as the postoperative and postirradiation regrowth of SCC in the same site and globe. RESULTS: The nine horses that received an amniotic membrane graft after keratectomy alone or combined with irradiation showed a minimal level of scarring in a cornea that regained a greater transparency in comparison to the horses that were treated with a bulbar conjunctival graft. All of the horses that received an amniotic membrane graft had 226 +/- 218 days of follow-up without tumor recurrence (mean +/- SD), ranging from 21 days to 778 days. CONCLUSIONS: The combination of superficial keratectomy alone or associated with beta-irradiation and permanent amniotic membrane transplantation is an effective treatment of corneal or corneolimbal SCC in horses. The placement of an amniotic membrane material represents an alternative surgical procedure to bulbar conjunctival grafts, especially if there is a lack of bulbar conjunctiva tissue available after tumor resection or if a particularly large corneal resection is necessary. The amniotic membrane is incorporated into the corneal defect and seems to create noticeably much less scarring than a corneal defect covered by bulbar conjunctiva.     

The use of porcine small intestinal submucosa for the repair of full-thickness corneal defects in dogs, cats and horses

OBJECTIVE: To evaluate the efficacy of using a porcine small intestinal submucosa (SIS) graft covered by a conjunctival flap for the surgical repair of full-thickness corneal wounds in dogs, cats and horses. PROCEDURE: All records dating from August 1999 to February 2003 from Purdue University Veterinary Teaching Hospital of patients that had undergone ophthalmic surgical procedures and received a SIS corneal graft for a full-thickness lesion were reviewed. Fifteen cases were identified including six dogs, two cats and seven horses. Requirements for inclusion in this study were that SIS was used as a corneal graft in a full-thickness corneal defect and that the graft was completely covered with a conjunctival flap. RESULTS: Of the 15 cases, one canine patient had received SIS following removal of an epibulbar melanocytoma. The remaining five canine patients had undergone this surgical procedure for the repair of corneal perforation. The two feline patients had been presented for corneal perforation following chronic ulceration. One equine patient had been presented for a deep melting ulcer, three for stromal corneal abscesses, and three for corneal perforations. Complications encountered postoperatively included aqueous leakage, conjunctival flap dehiscence, synechia, cataract and fibrin in the anterior chamber. Fourteen out of 15 patients were visual at the final re-evaluation. CONCLUSION: SIS is an inexpensive, easy-to-handle biomaterial that appears to be suitable for the repair of full-thickness corneal wounds in dogs, cats and horses. Results of our study support the conclusion that this relatively new product is an effective alternative to traditional implantation materials utilized in veterinary ophthalmology.     

Repair of a full thickness corneoscleral defect in a German shepherd dog using porcine small intestinal submucosa

Porcine small intestinal submucosa (SIS) was used, in conjunction with a conjunctival graft, to repair a full thickness corneoscleral defect resulting from the excision of a limbal melanoma in a German shepherd dog. The SIS was found to provide adequate mechanical support and to act as a suitable physical barrier in place of the excised cornea and sclera. Corneal vascularisation was present distant to the graft by two weeks postoperatively but this was effectively controlled with topical cyclosporin. By six weeks postoperatively, the graft had become incorporated into the cornea and sclera, and the associated corneal neovascularisation had resolved. From this initial case, porcine SIS would appear to be a suitable material for the repair of corneoscleral defects in dogs.     

The use of preserved equine renal capsule to repair lamellar corneal lesions in normal dogs

The purpose of this study was to evaluate the use of equine renal capsule preserved in 98% glycerine to repair lamellar corneal lesions in normal dogs. For this purpose, 12 dogs, divided into six groups ( n  = 2), were used to evaluate the 1st to 7th day, 15th day and 30th to 60th postoperative day. In order to perform the histologic study, the clinical procedures were analyzed, while the recipient's corneas were collected. The photophobia and blepharospasm also were more intense in the 1st to 7th postoperative day, and regressed in the 15th postoperative day. Therefore, the edema and the vascular events were both more frequent in the intermediary phases and regressed in the late periods. On the other hand, the morphological evaluation demonstrated an inflamatory exudate, also in the intermediary and late periods. These results suggested that the equine renal preserved capsule could be a useful alternative tissue to repair lamellar corneal lesions in dogs.     

Corneal diagnostic procedures

The cornea is the anterior, transparent portion of the fibrous tunic of the eye. It is continuous with the sclera at a transition called the limbus. In healthy conditions, the transparency of the cornea is maintained by the smooth, nonkeratinized, squamous epithelium, which is further enhanced by the precorneal tear film, the lack of corneal vascularization or pigmentation, the size and regular arrangement of the collagen fibrils that make up the corneal stroma, and the relative dehydration of the cornea (which is maintained by the endothelium and epithelium). The cornea can respond to adverse stimuli through vascularization, pigmentation, fibrosis, accumulation of cellular or noncellular infiltrate, and/or edema. Because of these limited responses, routine diagnostic procedures are critical in the diagnosis and treatment of corneal disorders. This article discusses tests of the precorneal tear film, corneal staining procedures, culture and sensitivity, cytology, and a few other procedures that are performed less commonly or require specialized instrumentation.     

Keratitis due to microfilariae in dogs: a newly recognized disease

Parasitic agents have been associated with keratitis, but a diagnosis of parasitic keratitis has not been commonly made in domestic animals. The purpose of this study was to describe the clinical and histopathological findings in seven dogs with chronic keratitis caused by microfilariae diagnosed in Brazil. All dogs presented with superficial corneal opacities of varying degrees affecting the perilimbal and central regions of the cornea, with other opaque areas appearing as crystalline deposits and corneal vascularization. The lesions were bilateral and were associated with mild‐to‐moderate conjunctival hyperemia. There was no history of blepharospasm or pruritus, and no subjects presented with epithelial erosions. Corneal biopsy revealed free microfilariae in the corneal stroma, with varying degrees of inflammation and collagen fiber destruction. The microfilariae were also found in skin lesions by skin snip technique. No adult worms were found in these dogs, and no dogs were on heartworm preventative before diagnosis. Monthly doses of oral ivermectin improved ocular and dermal lesions. One dog showed complete remission with the treatment. The species of the microfilariae was not identified.     

Equine corneal surgery and transplantation.

Corneal disease is common in equine ophthalmology and requires vigilant monitoring and appropriate therapy to optimize the outcome.Many equine corneal diseases, particularly those that progress rapidly, may benefit from surgical intervention. These include descemetoceles, deep corneal lacerations and ulcers, corneal perforation/iris prolapse, ulcerative keratitis, corneal stromal abscesses, and corneoscleral neoplasia. Indications for corneal transplantation include optical, tectonic, therapeutic, and cosmetic purposes. Corneal transplantation is most often implemented in equine patients for tectonic and therapeutic reasons when a cornea is compromised by corneal stromal abscess, iris prolapse, or neoplasia.This article provides an outline of when to consider surgical intervention for corneal disease, the procedures available and expected outcomes, and how appropriate early surgical intervention can dramatically improve the end result.     

Surgical repair of deep melting ulcers with porcine small intestinal submucosa (SIS) graft in dogs and cats

PURPOSE: To evaluate the efficacy of using a porcine small intestinal submucosa (SIS) graft for the surgical repair of deep melting ulcers in dogs and cats. METHODS: Two cats and five dogs presented with deep and large melting ulcers of the cornea. In each case, the necrotic and collagenolytic tissue of the cornea was removed by keratectomy. A SIS graft, 1 mm greater than the corneal defect, was rehydrated in sterile saline and sutured to the edges of the ulcer with a simple interrupted pattern of 9/0 polyglactin 910. A nictitating membrane flap was utilized in two cats and four dogs for 2 weeks. All cases were treated postoperatively with topical and systemic antibiotics, a systemic anti-inflammatory drug and topical atropine. All animals were re-evaluated 15 days, 4 weeks, 35-45 days, 2-3 months and 6 months postsurgery. RESULTS: At 15 days postsurgery, a superficial intense corneal neovascularization surrounded the SIS graft. No ocular discomfort was present and fluorescein staining was negative in all cases. At 4 weeks the SIS graft was thick and opaque in all cases, although in one cat the SIS graft had partially detached. Between 35 and 45 days, SIS graft integration was evident in all eyes, and corneal neovascularization had decreased progressively. All eyes healed without complications and retained corneal transparency. This occurred even in the presence of corneal perforation in two cases: one prior to and one during surgery. CONCLUSION: Results of our study suggest the SIS graft may be an effective alternative surgical treatment to the traditional conjunctival grafts commonly used to repair melting ulcers in dogs and cats. The advantages of using a SIS graft include good corneal transparency, preservation of corneal integrity and maintenance of vision.     

Reconstruction of corneal epithelium with cryopreserved corneal limbal stem cells in a rabbit model

The integrity and transparency of the cornea plays a key role in preserving vision. This paper reports a procedure to create an artificial sheet of corneal epithelium from cryopreserved limbal stem cells (LSCs) and to use this for corneal transplantation. Corneal LSCs were isolated from biopsy specimens of rabbit limbal lamellar and cryopreserved in liquid nitrogen at 2–4 passages. The cells were grown in culture medium for 12–14 days on top of a cell-free human amniotic membrane framed on a nitrocellulose sheet. The corneal epithelium generated was transplanted into the right eyes of 14 LSC deficient (LSCD) rabbits (seven experimental animals, seven controls) with corneal damage. The seven LSCD rabbits in the experimental group were transplanted with a corneal epithelial sheet generated from the cryopreserved corneal LSCs. Four LSCD rabbits were used as the vehicle control and were transplanted with a cell-free amniotic membrane, and the remaining three LSCD rabbits were negative controls without transplantation. Over a 2-month recovery period, 2/7 animals in the experimental group recovered completely, four recovered partially and one did not respond. In the control groups, three negative controls and three vehicle controls lost their vision completely, and one of the vehicle controls partially recovered transparency of the cornea Following treatment, corneal transparency of the experimental rabbits was significantly improved compared to controls (P < 0.05). The results indicated that cryopreserved corneal LSCs can repair damaged rabbit cornea, suggesting a possible new clinical approach to reconstruction of corneal epithelium.     

Histological differences in full-thickness vs. lamellar corneal pig-to-rabbit xenotransplantation

To evaluate the differences in graft survival and histopathological characteristics between full-thickness and lamellar orthotopic corneal xenotransplantation in a pig-to-rabbit model, we orthotopically transplanted a full-thickness or the anterior half of a pig's cornea onto the OD of 16 rabbits. As a result, the median survival were 16.83 and 29.07 days for the full-thickness and lamellar xenografts, respectively ( P  = 0.0005). Histologically, the full-thickness corneal xenografts had massive infiltration by eosinophils, whereas the lamellar xenografts showed predominantly mononuclear infiltrates ( P  < 0.05). Given these preliminary findings, lamellar corneal xenografts in rabbits survived longer than the full-thickness xenografts and each type of graft demonstrated different rejection mechanisms.     

羊膜对山羊角膜缘上皮细胞培养影响的研究

比较完整羊膜和去上皮羊膜、不同冻存时间的羊膜对山羊角膜缘上皮细胞体外培养的影响,筛选出羊膜最适处理方法。将山羊角膜缘组织块分别接种于完整羊膜、去上皮羊膜、新鲜去上皮羊膜、冻存30 d去上皮羊膜、冻存90 d去上皮羊膜、冻存180 d去上皮羊膜上,比较在完整羊膜和去上皮羊膜及不同冻存时间羊膜上角膜缘上皮细胞的生长特性和组织结构。试验结果显示,在去上皮羊膜上角膜缘组织块贴壁和细胞增殖能力较完整羊膜强,角膜缘上皮细胞在2组羊膜上均能形成多层结构,但完整羊膜上形成的组织表层角质化较严重;在冻存30 d羊膜上角膜缘组织块贴壁和细胞增殖能力较新鲜羊膜、冻存90 d羊膜、冻存180 d羊膜强,角膜缘上皮细胞在各组羊膜上均形成多层结构,其结构无明显差异。结果表明,冻存30 d去上皮羊膜是构建组织工程人工角膜最适的羊膜载体材料。