Studies on evaluation of autologous concentrated growth factor membrane for the treatment of corneal ulcer in dogs

Introduction

Cornea acts as a barrier to shield the eye from the outside world (Kaushal et al., 2024) [1] . Numerous factors such as trauma, preocular tear film shortages or defects, irritants, abnormalities of the eyelids or lashes, immune-mediated or allergic inflammation, foreign bodies or the inability to blink, could cause corneal ulcers. Breed-specific predispositions based on eyelid and craniofacial conformation has also been linked to an increased incidence of corneal ulcers (Packer et al., 2015) [2] . There are four types of corneal ulcers: superficial keratitis, deep corneal keratitis, descemetocele keratitis and perforation keratitis (Kim et al., 2009) [3] . Appropriate treatment of corneal ulcer is required for preventing the development of more complicated ulcer forms, which could result in increased ocular pain, corneal perforation and blindness (James-Jenks et al., 2023) [4] . Many treatments have been tried in corneal lesions. Some of these methods included corneal tissue sealants, conjunctival grafts, third eyelid flap, tarsorrhaphy and keratotomy, applications of soft contact lens, amniotic membrane application, small intestinal submucosa application, renal capsule application, pericardial application, stem cell applications and platelet-rich plasma application (Melek et al., 2022) [6] . Concentrated Growth Factor (CGF) is a new generation of platelet concentrate products, which exhibits an interesting clinical and biotechnological application potential (Borsani et al., 2015) [5] . This study was undertaken to evaluate the efficacy of concentrated growth factor membrane for the treatment of corneal ulcer in dogs.

Materials and Methods

The study on evaluation of autologous concentrated growth factor membrane for healing of corneal ulcer was conducted in clinical cases of dogs presented to the Department of Veterinary Surgery and Radiology, Veterinary College, Hebbal, Bengaluru. Six dogs with Corneal affections like deep corneal ulcer, melting stromal ulcer, descemetocele and staphyloma which were found fit for surgery were chosen for the study irrespective of age, breed and sex. A detailed and thorough clinical and ophthalmic examination was carried out in all the six cases under present study. Evaluation of haematological parameters (total erythrocyte count (millions/cmm), haemoglobin (g/dL), total leukocyte count (thousands/cmm), differential leukocyte count (%) and platelet count (thousands/μL)) and biochemical parameters (serum creatinine (mg/dL), blood urea nitrogen (mg/dL) and serum alanine aminotransferase (U/L)) were done before and after treatment with CGFM. Under general anaesthesia, following aseptic precautions and sufficient wound debridement, corneal grafting was done with CGFM. All the corneas were protected using tarsorrhaphy which was retained up to seven days.

Ophthalmic examination

A detailed ophthalmic examination was carried out to assess the condition of the eye. The tear production was assessed using STT-1 (figure 1) and the extent of ulceration was assessed by means of fluorescein dye test (figure 2). The Semi quantitative preclinical ocular toxicity scoring (SPOTS) system of grading for monitoring changes in corneal opacity and vascularisation as described by Eaton et al. (2017) [8] was also done before and after CGFM graft.

Anaesthetic protocol

All the dogs were premedicated with atropine sulphate (Atropine Sulphate® injection IP, (0.6 mg/ml), Harson Laboratories, Akota, Baroda -20) at the dose rate of 0.045 mg/kg body weight intramuscularly. Sedation was achieved with administration of xylazine hydrochloride (Xylaxin® injection USP, (20 mg/ml), Indian Immunologicals Ltd., Hyderabad -33) at the dose rate of 1.0 mg/kg body weight intramuscularly. General anaesthesia was induced and maintained with thiopentone sodium (Thiosol® injection (500 mg), Neon laboratories Ltd, Mumbai) at the dose rate of 12.5mg/kg intravenously given to effect.

Preparation of autologous concentrated growth factor membrane (CGFM)

According to the Sacco's (2006) protocol for preparation of the autologous Concentrated Growth Factor (CGF) membrane, a 7ml of fresh blood was collected in 15mL anticoagulant-free tube with a scalp vein (21-23G), under sedation prior to surgery. The tubes were immediately centrifuged in a centrifuge (Eppendorf Centrifuge 5804/5804 R, Eppendorf SE, Hamburg, Germany) with a special program of centrifugation; acceleration x 30", 2,700 rpm x 2', 2,400 rpm x 4', 2,700 rpm x 4', 3,000 rpm x 3' and deceleration x 36" and stop. Following centrifugation red blood cells aggregated at the tube's bottom, concentrated growth factor gel in the center, and platelet-poor plasma (PPP) at the tube's top. The gel in the middle part of the sample and the red blood cells underneath were gently removed from the tube and the red blood cells below were cut out with sterile scissors. The clots were gently pressed between sterile gauze to obtain the CGF membrane (Figure 3). The CGFM graft was applied over the lesion and secured using polyglactin 910(VICRYL *TM , Johnson and Johnson Pvt.Ltd, Maharashtra-36) size 6-0. Simple interrupted sutures were placed to secure the graft to bulbar conjunctiva. Peri operatively, continuous irrigation with sterile isotonic normal saline was done to rehydrate the graft and to prevent desiccation of cornea. A temporary tarsorrhaphy was performed in all the 6 cases using 3-0 nylon (TRULON®, Healthium Medtech Ltd, Bangalore-58) to protect the cornea and the graft in all the dogs (Figure 4).

Post-operative care

Post-operatively an Elizabethan collar was advised in all cases till complete healing to prevent self-mutilation and injury to the eye. Ceftriaxone (Intacef, Intas Pharmaceuticals Ltd., Ahmedabad-54) was given parenterally at the dose rate of 20 mg/kg body weight, in all the cases on the day of surgery. Use of moxifloxacin eye drop (Ophthocare-M, Corise Healthcare Pvt Ltd., Thane-01) at the dose rate of one drop twice a day topically was advised in cases which retained fluorescein stain after the seventh post-operative day. Cephalexin (Cephavet ®, Sava Healthcare Ltd., Gujarat-35) at the dose rate of 20 mg/kg body weight was given orally twice a day for five days and meloxicam at the dose rate of 0.2 mg/kg body weight was given for three days (Melonex, Intas Pharmaceuticals Ltd., Ahmedabad). The tarsorrhaphy sutures were removed on seventh day post-grafting.

Results and Discussion

In almost all the cases presence of corneal edema and scar tissue negatively affected the visual function scoring. Those animals which had an extensive corneal injury took longer time to regain visual functions. It was assessed by menace response, PLR and cotton ball test. By day 21 st post grafting, complete vision function was noticed in all the six cases under present study. This was in agreement with the findings of Melek et al. (2022) [6] who used CGFM graft for treating corneal alkaline burns in Rabbits.

The reflex and basal aqueous tear production was measured by STT-1in the present study. The higher readings in schirmer tear test pre-operatively as compared to post-operative readings could be due to the excessive lacrimation due to pain and ocular irritation which gradually reduced as the ulcer healed. This was in accordance with the findings of Williams and Burg (2017) [7] . The Semi quantitative preclinical ocular toxicity scoring (SPOTS) system of grading for monitoring changes in corneal opacity and vascularisation was done before the surgery, on the day of surgery (0 th day) and postsurgery on 7 th , 14 th , 21 st and 28 th days as described by Eaton et al. (2017) [8] . A minimal corneal opacity (severity and area) with score one was present in all the dogs except in one dog at the end of the study period. This was attributed to the findings of Riestra et al. (2016) [9] who reported in her study that blood products can modulate TGF-β activity thereby reducing tissue fibrosis and promoting cell regeneration.

In the present study all the six dogs showed positive fluorescein dye test on affected eye on the day of presentation which could be due to the fluorescein sodium stain taken up by exposed corneal stroma (Singh et al., 2004) [10] . A minimal fluorescein staining (area and severity) with score one was present in three dogs by 7 th post-operative day. By 14 th postoperative day all the six cases showed negative fluorescein staining. This was similar to the findings of Ajin (2023) [11] Who did autologous PRFM graft for deep corneal ulcer in dogs and was attributed to the epithelialisation of the cornea due to the grafted membrane. In the present study corneal neovascularisation increased during the healing period up to 14 th day post grafting. By 21 st day post grafting, corneal vascularisation was absent in all the cases except two. By day 28 it was almost nil in all the six cases. This finding was similar to the observations of Mishra et al. (2021) [12] who reported that anti-inflammatory nature of platelets and its growth factors promoted rapid healing with earlier and complete withdrawal of neovascularization.

Various haematological parameters estimated under the present study were within the normal range. The values of serum creatinine, BUN and alanine transaminase were also within the normal range and there were no significant difference observed between the pre and post-operative values.This was in accordance with the findings of Kim et al.

(2009) [3] , Vidhyasree (2022) [13] . In all the six cases under the study, a membrane of adequate size to cover the lesion was obtained by following Sacco's. The only complication observed after treatment with CGFM graft was corneal opacity. A minimal corneal opacity was present in all cases except one at the end of observation period. This was similar to the findings of Melek et al. (2022) [6] and Ajin (2023) [11] who used autologous grafts for corneal ulcer treatment. Bernardi et al. (2017) [14] stated that tissue regeneration was one of the current therapies proposed. Due to the autologous nature of these concentrates, all biocompatibility concerns were avoided. High quantity of growth factors, such as Platelet Derived Growth Factor (PDGF), Transforming Growth Factor-B1 (TGF), TGF-B2, Fibroblast Growth Factor (FGF), Vascular Endothelial Growth Factor (VEGF) and Insulin like Growth Factor (IGF) were derived from platelets. They were reported to promote cellular proliferation, matrix remodeling and neo-angiogenesis (Figure 5, 6, 7, 8, 9 and 10).

Conclusion

The autologous CGFM prepared as per Sacco's protocol provided a good tectonic and chemotactic support which in turn resulted in early epithelialisation and healing with minimal scarring which consequently lead to restoration of complete vision function in all the cases under present study. Timely diagnosis and appropriate treatment with canine autologous CGFM were effective in successful surgical management of deep corneal ulcers, melting ulcer, descemetocele and staphyloma in dogs.

References

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