Efficacious cellular therapy of descemetocele in a dog

Abstract

The cornea is comprised of 4 layers; the outermost layer is the epithelium, followed by the stroma, Descemet’s membrane, and endothelium. Corneal descemetocele is a serious consequence of progressive corneal ulceration, characterized by a herniation of the Descemet membrane through an overlying stromal defect. It requires urgent intervention due to the risk of perforation. Although there are several treatments available for this type of corneal ulcer, conservative approaches may be inadequate due to the typical severity of this injury. Surgical interventions, including conjunctival autograft transplantation and corneoscleral transposition, are commonly used. Mesenchymal stem cells (MSCs) have been used to effectively treat corneal ulcers, but there are limited reports regarding its use for descemetocele. A 7-year-old female shih tzu was diagnosed with descemetocele. In this dog, 2 × 10⁶ MSCs, provided by CellTech — Stem Cell Technologies, were injected bilaterally into the conjunctiva, with an additional 5 × 10⁵ MSCs applied topically to each eye. The ulcer achieved complete remission with an absence of corneal opacity after 75 d, supporting the claim that MSCs are an effective and safe option for the treatment of descemetocele.

Key clinical message:

The dog’s descemetocele healed completely after a single application of MSCs after 30 d, with scars and leukoma completely absent after 75 d. No surgical intervention was required.

The canine cornea is comprised of 4 layers; the outermost layer is the epithelium (0.08 mm), stroma (0.5 to 0.6 mm), Descemet’s membrane (4 times endothelium’s thickness), and endothelium (1 cell layer) (1).

A corneal ulcer, defined as an epithelial corneal rupture with stromal exposure, is considered the most prevalent ophthalmic disease in dogs, affecting approximately 21.7% of the canine population (2,3). There are numerous types of ulcers, classified as superficial (affecting only the epithelium) or deep (with loss of stroma). Although superficial ulcers are more painful, deep ulcers are a greater risk for complications (4).

Corneal descemetocele is a serious consequence of progressive corneal ulceration, characterized by a herniation of the Descemet membrane through an overlying stromal defect. It requires urgent intervention due to the risk of perforation (5). Typical clinical signs include stromal damage, corneal edema, and a black or white background, indicating exposure of the Descemet membrane (6). Diagnosis is based on an ophthalmic examination and fluorescein staining, which stains only the stroma and the margins of the ulcer while leaving the Descemet membrane unstained (7).

There are several treatments available for this type of corneal ulcer, although conservative approaches may be inadequate due to the severity of the injury (8). Currently, surgical intervention, including conjunctival autograft transplantation (60 d for recovery) and corneoscleral transposition (30 to 60 d for recovery) are common choices (9,10). In addition, stem cell therapy has been reported to be efficient in the treatment of several types of corneal ulcers (11,12). In that regard, descemetocele treatment recovery time was reduced to just 14 d with cellular therapy, circumventing the need for surgical intervention (13).

Stem cells are undifferentiated cells with the capacity to self-renew and differentiate (14). Mesenchymal stem cells (MSCs) are classified as adult stem cells because they are present after birth and are considered pluripotent as they can differentiate into lineages from all 3 germ layers (15). There have been many clinical uses for MSCs, as they release anti-inflammatory factors and modulate the immune system (15). More specifically, they can inhibit T-cell secretions and INF-2γ, reducing inflammation, activity levels of dendritic cells and cytokines, and antigenic presentation. Moreover, they protect ganglion cells and stimulate axon regeneration by secreting bioactive and neurotrophic factors (16,17).

In this case study, MSCs were used to treat descemetocele in a canine patient.

Case description

A 7-year-old female shih tzu was diagnosed with descemetocele. Initially, the antibiotics gentamicin and moxifloxacin were prescribed, as well as EDTA and Regencel (retinol, amino acids, methionine, and chloramphenicol; Itapira, São Paulo, Brazil). After 25 d, no clinical improvement was observed, prompting the owner to seek alternative approaches. At this point, 2 × 10⁶ allogeneic MSCs, derived from a canine ovary and provided by CellTech — Stem Cell Technologies following a previous protocol (18,19), were injected bilaterally into the patient’s conjunctiva, with an additional 5 × 10⁵ applied topically to each eye. Prior to these procedures, the dog’s owner had provided informed, written consent. The application day was defined as Day 0. The ulcer in the left eye was larger (Figure 1 A) than that in the right (Figure 1 G). After 72 h (Day 3), the ulcer in the right eye had healed and, in the left eye, there were reductions in corneal edema, hyperemia, and ulcer size (Figures 1 B, H). At Day 7, ulcer size and hyperemia were further reduced (Figures 1 C, I). This pattern of improvement continued through Day 10. At Day 30, complete ulcer remission was apparent, along with the absence of edema, hyperemia, or corneal rupture. The only alteration still observed was increased corneal opacity (Figures 1 E, K), which was no longer apparent at Day 75 (Figures 1 F, L).

Figure 1.

A — Left eye on the day of application (D0). B — Left eye 72 h after application (D3), reduction in corneal edema, hyperemia, and ulcer size. C — Left eye 7 d after application (D7), ulcer size and hyperemia further reduced. D — Left eye 10 d after application (D10), partial remission of ulcer, normalization of ophthalmic standards. E — Left eye 30 d after application (D30), complete remission of ulcer, persistent corneal opacity. F — Left eye 75 d after application (D75), complete resolution of the condition, corneal opacity is absent, and relapse is not observed. G — Right eye at the day of application (D0). H — Right eye 72 h after application (D3), reduction in corneal edema, hyperemia, and ulcer size. I — Right eye 7 d after application (D7), ulcer size and hyperemia further reduced. J — Right eye 10 d after application (D10), partial remission of ulcer, normalization of ophthalmic standards. K — Right eye 30 d after application (D30), complete remission of ulcer, persistent corneal opacity. L — Right eye 75 d after application (D75), complete resolution of the condition; corneal opacity is absent, and relapse is not observed.

Discussion

In the present case report, MSCs were used to treat descemetocele, with an observed recovery time of 3 d in the right eye and 30 d in the left eye, attributed to the difference in injury size. The MSCs were applied by 2 distinct routes: topical and subconjunctival. In a study of corneal lesions induced by a manual motorized brush in mice, 5 × 10⁵ autologous mesenchymal stem cells were administered by subconjunctival, topical, intravenous, or intraperitoneal routes. At the end of the 4-day evaluation period, subconjunctival and intravenous routes were the most effective (18). In another study involving an 18-year-old poodle diagnosed with recurrent corneal ulcer, 3 × 10⁶ mesenchymal stem cells were applied topically on the first day and repeated after 48 d; there was complete ulcer remission after 69 d, albeit with the presence of leukoma (12). In the present study, each eye received 2 × 10⁶ MSCs via a subconjunctival route and 5 × 10⁵ were administered topically. As the lesion in this patient was deeper than those in the mouse and poodle studies, we speculated that recovery may be delayed when cells are applied topically compared to other routes of administration.

Another study analyzed 26 dogs diagnosed with various types of corneal ulcers, previously treated with antibiotics and artificial tears for 15 d. In those dogs, 2.5 × 10⁵ mesenchymal stem cells were applied via the subconjunctival route, followed by 11 additional instillations of the same number of cells once per hour for a total of 11 h. As a result of this therapy, 84% of the animals achieved closure of corneal perforation in 14 d (13). This finding further supported the inference that topical application may only be sufficient when combined with subconjunctival administration, and not in isolation.

These therapeutic outcomes of MSCs are attributed to secretion of bioactive factors with anti-apoptotic, anti-fibrotic, and tropic effects (15). In the present study, the dog achieved complete remission of the corneal ulcer 3 d after application of mesenchymal stem cells in the right eye (Figure 1 H), where the lesion was smaller, and 30 d after application in the left eye, where the lesion was larger (Figure 1 E). This indicates that the size of the lesion influences recovery time and that, likely, the larger the lesion, the greater the dose of stem cells needed to expediently achieve the therapeutic objective. Another important aspect is corneal regeneration. In most cases, after corneal trauma, leukoma is observed, which presents as a dense, opaque, white area of cornea (19). The healthy cornea should be clear to refract light into the eye and form the image on the retina (20–22). As such, development of a therapeutic technique that removes leukoma is of high importance to the patient’s vision and consequently to their quality of life. In the present experiment, 75 d post-application, leukoma was no longer present, indicating complete regeneration of corneal tissue. Leukoma typically persists after other therapeutic approaches (20–22) or even after topical application of stem cells (12).

In conclusion, the findings of this study provided evidence regarding the efficacy of mesenchymal stem cells in promoting corneal regeneration. The dose and route of application used in this study achieved lesion closure without complications over a shorter interval, less invasively, and with less stress compared to what would be expected for conventional treatments. CVJ