Subconjunctival use of allogeneic mesenchymal stem cells to treat chronic superficial keratitis in German shepherd dogs: Pilot study

Abstract

Background: Chronic superficial keratitis (CSK) is an ocular condition in dogs characterized by corneal opacification leading to visual function impairment. Control of this chronic condition requires the use of topical immunomodulators or corticosteroids daily. Regenerative medicine has shown promising results in several fields of medicine.

Aim: The aim of this study was to evaluate the clinical effect of allogeneic mesenchymal stem cells (MSCs) of adipose tissue applied via subconjunctival in dogs with CSK.

Methods: A series of cases of eight dogs diagnosed with CSK were divided into two groups, four dogs each; the conventional treatment group received prednisolone 1% as topical eye drops and the experimental group (EG) received allogeneic MSCs transplantation. The dogs had not previously been treated for CSK. Systemic and ophthalmologic examinations were performed to exclude other abnormalities. An administered amount of MSC (1 × 10⁶ cells each time) was injected via subconjunctival in the peri-limbal region at 0 and 30 days. The animals were followed for 110 days for clinical evaluation, and, at the same time, the images of the corneal abnormalities were obtained and analyzed in the ImageJ software. The statistical analysis was performed in the GrandPrism 7.0 software.

Results: Initial and final images revealed that areas with neovascularization, inflammatory infiltrate, and opacity regressed in most eyes in both groups (7/8 eyes in each group) at the end of the 110 days, p = 0.0391 and p = 0.0078 respectively, but this response was minor in the EG comparing to conventional group (CG) (p = 0.026). No local or systemic side effects were observed.

Conclusions: Despite the small melioration, MSCs treatment suggests clinical improvement in patients with CSK after 110 days without any local or systemic side effects. However, the improvement achieved was significantly less than the observed within CG. Further studies still are needed to evaluate the use and benefits of stem cells as an adjunct treatment for CSK.

Keywords: Autoimmune diseases; Chronic superficial keratitis; Cornea; Mesenchymal stem cells; Regenerative medicine.

Fig. 1.. Participants of the EG. A and B present the right eye of participant #1 on days 0 and 110, respectively. C and D present the right eye of participant #2 on days 0 and 110, respectively. In A, the neovascularization blocked anterior chamber visibility along with poor visual ability behavior evidenced by the negative result in the menace response test (arrow). In B, neovascularization declined to the point where only a small number of blood vessels were visible (small arrows), the periphery of the cornea became more transparent (large arrows), and visual behavior was clinically normal. In C, the disease stage was just the lateral portion of the cornea affected. As a model, the color lines represent the measurements analyzed: yellow represents the total area of the cornea, blue is the affected area, and green is the pigmented area. In D, at the end of the follow-up, the cornea had no blood vessels with the cornea becoming clear (arrow) but the pigmentation clinically occupied the same area as the initial blood vessels.

Fig. 2.. Participants of the CG. A and B present the right eye of participant #6 on days 0 and 110, respectively. C and D present the left eye of participant #7 on days 0 and 110, respectively. In A, the disease is at the initial stage with only two lower quadrants affected (arrows). In B, the neovascularized area had regressed without becoming pigmented (large arrow) and the initial pigmented area had established peripherally (small arrows). In C, the disease was in a more advanced stage with a chronic pattern due to the presence of extensive visible pigmentation associated with neovascularization. In D, the cornea showed the dorsal area cleaned (arrow) and the pigmented area had regressed in the central area allowing visual menace response and visual capability.

Fig. 3.. A. Comparison between groups on day 0, with no statistical difference between their values (p = 0.28). Columns represent mean and standard deviation, n = 8. B. Comparison between groups on day 30, with statistical difference (*) among their values (p = 0.04). Columns represent mean and standard deviation, n = 8. C. Comparison between groups on day 110, with statistical difference (*) among their values (p = 0.03). Columns represent mean and standard deviation, n = 8.

Fig. 4.. Experimental group: A. Comparison between follow-up days: affected areas. There was no statistical difference between day 0 and day 30 (p = 0.25) and with statistical difference (*) between day 0 and day 110 (p = 0.008). Columns represent the mean and standard deviation, n = 8. B. Comparison between follow-up days: pigmented areas. Comparison between follow-up days. No statistical difference between day 0, 30 and day 110 (p = 0.25). Columns represent the mean and standard deviation, n = 8.

Fig. 5.. Conventional Treatment Group: A. Comparison between follow-up days: affected areas. With statistical difference (*) between day 0 and day 30 (p = 0.03) and with statistical difference (**) between day 0 and day 110 (p = 0.04). Columns represent the mean and standard deviation, n = 8. B. Comparison between follow-up days: pigmented areas. Comparison between follow-up days. No statistical difference between day 0 and day 110 (p = 0.4). Columns represent the mean and standard deviation, n = 8.