NR 3/2013 ART.81


Azabu University, 1-17-71, Huchinobe, Chuou-ku, Sagamihara-shi, Kanagawa 252-5201, Japan


Hist. Vet. Inc., 22-9-F2, Sakae-cho, Kanagawa-ku, Yokohama-shi, Kanagawa 221-0052, Japan

Yasuhiro NAKANO

Hist. Vet. Inc., 22-9-F2, Sakae-cho, Kanagawa-ku, Yokohama-shi, Kanagawa 221-0052, Japan

Tomone ZAMA

Bayside Animal Clinic, 22-9-F1, Sakae-cho, Kanagawa-ku, Yokohama-shi, Kanagawa 221-0052, Japan


Bayside Animal Clinic, 22-9-F1, Sakae-cho, Kanagawa-ku, Yokohama-shi, Kanagawa 221-0052, Japan


Hist. Vet. Inc., 22-9-F2, Sakae-cho, Kanagawa-ku, Yokohama-shi, Kanagawa 221-0052, Japan
Bayside Animal Clinic, 22-9-F1, Sakae-cho, Kanagawa-ku, Yokohama-shi, Kanagawa 221-0052, Japan

A 6-year-old male Maltese with nodular episclerokeratitis received a glycerin-preserved homologous corneal graft after removal of a nodule. Three weeks before the initial visit, the patient was treated by instillations of corticosteroids and antibiotics, but showed no improvement. Upon the initial visit, a raised pink nodule on the medial limbus of the right eye was observed. The CBC and blood chemistry were normal, except for a high alkaline phosphatase level (1389 IU/L). After the nodule was removed, a glycerin-preserved stroma was used as a tectonic graft to repair the corneoscleral defect, as continuous corticosteroid therapy was not recommended because of a high risk of liver failure. Although no recurrence had been confirmed at least 59 days after surgery, additional follow-up is recommended.


Canine nodular granulomatous episclerokeratitis (NGE) presents as subcutaneous nodules within the episclera and sclera, and varies in progression. (Bellhorn i Henkind) (Paulsen, Lavach i Snyder) Focal firm, painless, smooth, pink, 0.5 cm to 1.0 cm diameter nodules are usually visible upon examination in the bulbar conjunctiva. The nodules are immune-mediated and inflammatory. (Grahn i Sandmeyer) (Breaux, Sandmeyer i Grahn). The most successful medical treatment for nodular fasciitis is immunosuppressive drugs; however, in many cases, the NGE still recurs and infiltrates the eye, which then requires removal. (Gwin, Gelatt i Peiffer) (Breaux, Sandmeyer i Grahn). This report discusses the use of a glycerin-preserved homologous corneal graft to repair a corneoscleral defect after nodule removal.

Case Report

A 6-year-old male Maltese, weighing 7 kg, was referred to our clinic for a raised pink mass on the medial limbus of the right eye (Figure 1).

Figure 1. A raised pink mass in the limbus of the right eye at the initial visit.

Three weeks before referral to our clinic, the dog had undergone a physical examination, during which the mass in the right eye was detected. Corticosteroids and antibiotics were administered into the eye, but the mass did not change.

At the initial medical examination in our clinic, a 6-mm diameter granulomatous-like nodule was present on the medial limbus of the right eye. The lesion had expanded into the corneal stroma, and was edematous. Conjunctival hyperemia was present in both eyes. The left eye was and slightly painful and had photophobia, as well as had a corneal erosion, as determined by a fluoresceine-staining test. Schirmer tear test values were 24 mm/min in the right eye and 14 mm/min in the left eye. The intraocular pressure was 14 mmHg in the right eye and 12 mmHg in the left eye. The menace, dazzle, and pupillary light reflex tests indicated normal vision in both eyes. The complete blood count (CBC) and blood biochemistry values were normal, except for a high serum alkaline phosphatase level (1389 IU/L). The dog was diagnosed with nodular granulomatous episclerokeratitis in the right eye and a corneal erosion in the left eye.

At the initial visit, we formulated eye drops of both ofloxacillin and acetylcysteine to be administered 3 times a day in both eyes. Additionally, we prescribed prednisolone eye ointment to be administered twice a day in the right eye. Eight days later, the size of the mass was slightly diminished, but neovascularization and pigmentation were observed near the mass. However, the corneal erosion of the left eye had disappeared.

Thirty-two days after the initial visit, the mass in the right eye increased to be 7 mm in diameter. At this point, we excised the nodule under general anesthesia with a myoblock injection of rocuronium bromide, by using a disposable cautery (Figure 2, 1-8).

Figure 2. Photographs showing a repair of the limbal lesion using a glycerin-preservedhomologous corneal graft.
(1) Exposure of the limbal lesion after a canthotomy. (2) Incision of the sclera with a disposable cautery.
(3) The mass was removed. (4) A full-thickness corneoscleral defect.
(5) A glycerin-preserved graft was prepared with a crescent knife. (6) The graft was placed on the defect.
(7)The graft was sutured at the defect. (8) The conjunctival flap was placed on a part of the graft.

Then, we decided to perform a grafting procedure in order to repair the corneoscleral defect due to the nodule removal.

After receiving approval from our university’s animal research committee, we obtained a donor cornea with a 3-mm rim of adjoining sclera, which was aseptically preserved in pure sterile glycerin in the freezer (-30°C) for 2.5 years. (King i Townsend) (Chen, Lin i Zhang). The preserved cornea was placed into sterile saline for 3 h, and a layer of epithelium was removed by wiping thoroughly with gauze. The corneal graft was prepared by removing the endothelium,so the cornea was two-thirds its thickness and had an 11-mm-diameter.The cornea and sclera were sutured with 9-0 nylon (Mani, Inc., Japan) and 6-0 polyglactin 910 suture(Ethicon, Inc., USA. The conjunctival flap was covered with a part of the graft to create the limbus.

After the operation, the dog was hospitalized for a week, during which he received intravenous drip infusions of 20% D-mannitol (2 mL/kg, twice daily), flunixinmeglumine (0.5 mg/kg, once daily), and ampicillin (20 mg/kg, 3 times daily). Steroid and antibiotic instillations were performed 3 times a day, and 3% acetylcysteine eye drops were administered 6 days after surgery. An Elizabethan collar was placed postoperatively to prevent self-trauma to the surgical site. Two weeks after surgery, the dog was orally administered bromelain (1750 units/kg, twice daily), acetazolamide (4.4 mg/kg, once daily), and lysozyme hydrochloride (30 mg/head, twice daily). The left eye corneal erosion disappeared 2 weeks later.

Fifty-nine days after surgery, the graft was engrafted, and conjunctival hyperemia and corneal neovascularization were not observed (Figure 3).

Figure 3. The right eye 59 days after surgery.

At that time, the fluorescein-staining test was negative, and the dog’s vision was normal.

Histologic Findings

A mass that was not covered in a surrounding membrane around was observed (Figure 4, 1,2).

Figure 4. Photomicrographs of section of the removed tissue showing features
of a nodular granulomatous episclerokeratitis, using a hematoxylin-eosin stain.
(1) A low magnification photograph showing a large and invasive infiltrate.
The epithelium was intact. (2) The infiltrate border was relatively wide.
(3) A photograph showing spindle-shaped cell.

The mass was composed of numerous fibroblast-like spindle cells that were arranged in various manners, including unstructured, irregularly bundled, wavy, and cross-shaped (Figure 4, 3). These cells had irregularly or oval-spindle-shaped nuclei and a small amount of cytoplasm. A moderate number of lymphocytes, fewer plasma cells, and macrophages were admixed in the mass.


The condition of NGE has been referred by several other terms, such as fibrous histiocytoma, proliferative keratoconjunctivitis, nodular episclerokeratitis, pseudotumor, collie granuloma, and nodular fasciitis (Paulsen, Lavach i Snyder) (Grahn i Sandmeyer). The improvement of NGE is usually expected with medical therapy involving corticosteroids or immune-modulators, such as cyclosporine, cyclophosphamide, and azathioprine; however, NGE often requires lifelong topical or systemic immunosuppression. (Bellhorn i Henkind) (Gwin, Gelatt i Peiffer) (Paulsen, Lavach i Snyder) (Grahn i Sandmeyer) (Breaux, Sandmeyer i Grahn). In this case report, we did not consider the dog’s NGE to be controlled with continuous corticosteroid therapy because of the high serum alkaline phosphatase levels; hence, we decide to removed the mass.

Surgical therapy of NGE includes local or complete excision, which requires a repair of full-thickness corneoscleral defects in some cases (Bellhorn i Henkind) (Gwin, Gelatt i Peiffer) (Paulsen, Lavach i Snyder) (Grahn i Sandmeyer). In this case,the defect was restored with a tectonic graft, using a glycerin-preserved homologous corneal stroma. Fresh full-thickness corneal grafting might be accompanied by a rejection reaction, especially an acute endothelial rejection.For this reason, deep anterior lamellar keratoplastyhas been replacing full-thickness corneal grafting for disorders affecting the corneal stromal layers, while eliminating the risk of an endothelial rejection, if this procedure were performed in humans (Watson, Tuft i Dart) (Al-Torbak, Malak i Teichmann). Anterior lamellar keratoplasty (ALK) is a surgical procedure for replacing the epithelium and stroma, with an occurrence of stromal rejection in 1-2% of cases (Watson, Tuft i Dart). In ALK, the cellular components of the fresh cornea tissues are sources of major histocompatibility complex antigens and minor H antigens (Yamagami, Ebihara i Usui) (Kamiya, Hori i Kagaya). However, the antigenicity of the tissue can be lowered with a glycerin preservation, as compared to that of fresh cornea tissues, and the immunoreactivity was located mainly on the corneal epithelium, rather than on the stroma (Li, Shi i Zhang). Therefore, glycerin-preserved homologous corneal stroma is presumed to be suitable for tectonic grafts. Given, the above-mentioned advantages of and the applications for glycerol-preserved corneal tissue, we believe this procedure could be beneficial in humans.

In veterinary medicine, we have applied glycerol-preserved corneal tissues for corneal and corneoscleral diseases in dogs (Kanemaki, Kudo i Shimomura) (Kanai, Kanemaki i Matsuo) (Unpublished data). Although no recurrence of limbal melanoma in dogs had been confirmed for 1 more years after the same tectonic grafting procedures were performed for a full-thickness corneoscleral defects (Kanai, Kanemaki i Matsuo), we believe additional follow-up of this patient is necessary to ensure the patient’s condition does not recur 1 year after the operation.


  1. Al-Torbak, A, i inni. „Presumed stromal graft rejection after deep anterior lamellar keratoplasty.” Cornea. 2005: 24:241–243.
  2. Bellhorn, RW i P. Henkind. „Ocular nodular fasciitis in a dog.” J Am Vet Med Assoc. 1967: 15:150(2):212-213.
  3. Breaux, CB, LS Sandmeyer i BH. Grahn. „Immunohistochemical investigation of canine episcleritis.” Vet Ophthalmol. 2007: 10(3):168-172.
  4. Chen, W, i inni. „Comparison of fresh corneal tissue versus glycerin-cryopreserved corneal tissue in deep anterior lamellar keratoplasty.” Invest Ophthalmol Vis Sci. 2010: 51(2):775-781.
  5. Grahn, BH i LS. Sandmeyer. „Canine episcleritis, nodular episclerokeratitis, scleritis, and necrotic scleritis.” Vet Clin North Am Small AnimPract. 2008: 38(2):291-308.
  6. Gwin, RM, NK Gelatt i RL. Peiffer. „Ophthalmic nodular fasciitis in the dog.” J Am Vet Med Assoc. 1977: 170(6): 611-614.
  7. Kamiya, K, i inni. „Preservation of donor cornea prevents corneal allograft rejection by inhibiting induction of alloimmunity.” Exp Eye Res. 2000: 70(6):737-743.
  8. Kanai, K, i inni. „Repair of full-thickness corneoscleral defects using glycerin-preserved corneal grafts in five dogs with limbal melanoma.” J Jpn Vet Med Assoc. 2004: 57(1):647-650.
  9. Kanemaki, N, i inni. „Lamellar keratoplasty using glycerin-preserved porcine cornea in dogs.” Proceedings of World Veterinary Congress 1995: FCS11.2.3, 642
  10. King, JH Jr i WM. Townsend. „The prolonged storage of donor corneas by glycerine dehydration.” Trans Am Ophthalmol Soc. 1984: 82:106-110.
  11. Li, J, i inni. „Comparison of different methods of glycerol preservation for deep anterior lamellar keratoplasty eligible corneas.” Invest Ophthalmol Vis Sci. 2012: 53(9):5675-5685
  12. Paulsen, ME, i inni. „ Nodular granulomatous episclerokeratitis in dogs: 19 cases (1973 – 1985).” J Am Vet Med Assoc. 1987: 190(12):1581-1587.
  13. „Unpublished data.” -.
  14. Watson, SL, SJ Tuft i JK. Dart. „Patterns of rejection after deep lamellar keratoplasty.” Ophthalmology. 2006: 113:556–560.
  15. Yamagami, S, i inni. „Bone marrow-derived cells in normal human corneal stroma.” Arch Ophthalmol. 2006: 124(1):62-69.