Abstract
Purpose :
Melatonin is involved in many biological functions in different tissues, including eye tissues. Two main receptors, MT1 and MT2 have been described in the cornea of different species and a role suggested in corneal wound healing. Our aim was to study the presence of MT1 and MT2 receptors and the aralkylamine N-acetyltransferase (AANAT) enzyme in the porcine ocular surface, and to assess the role of melatonin in an ex vivo porcine corneal wound healing model.
Methods :
Porcine eyeballs were proccessed for immunodetection (n=4) in paraffin-embedded sections and protein analysis in tissue extracts by Western blot (n=4) of MT1 and MT2 receptors and AANAT. For the ex vivo wound healing model, porcine eyeballs (n=24) were wounded in central cornea. Anterior surfaces were subsequently isolated. Tissue preparations were then exposed to 250 µl of melatonin (5, 30, 60, 90 or 120 µg/ml) for 4h. Wound size at T0 and after 24, 48, 72 and 96h was measured in images from fluorescein-stained wounds. Control (unexposed) and melatonin-exposed corneal tissues were processed for morphology analysis. The wounded area/total corneal surface ratio was calculated using ImageJ software. Results were given as % of wound healing (wounded areax100/total corneal area) relative to the baseline (wounded area 24h after treatment=100%). Data (mean±SEM) were analized by the Student’s t test.
Results :
MT2 was present in conjunctival and corneal tissues, while MT1 was less expressed and preferentially localized in conjunctival tissues. AANAT was detected in corneal epithelium and conjunctival epithelium to a lesser extent. Western blotting confirmed the differential location observed for both receptors and AANAT in ocular surface tissues. Control wounds progresively healed until almost complete closure after 96h. Wounds exposed to 60 µg/ml and 90 µg/ml melatonin healed 26.3% and 20.6% faster than control wounds at 48h, respectively. At 72h the fastest healing rate was showed by 90 µg/ml melatonin exposed wounds. Surprisingly, wounds exposed to 120 µg/ml melatonin healed 11.8% slower than control wounds at 96h.
Conclusions :
We demonstrated the presence of MT1, MT2 and AANAT in the porcine ocular surface. Also, melatonin can act as a wound healing modulator in an ex vivo corneal wound healing model depending on its concentration. This porcine model can help clarify the role of melatonin in corneal physiology.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.