Does brimonidine tartarate have any effect on viability and/or proliferation of limbal mesenchymal limbal stem cells?

Abstract

Purpose : Mesenchymal stem cell transplantation is still one of the hot topics within translational medicine. The limbus, rim of the corneoscleral junction of human eye, is highly rich in terms of stem cells. Limbal mesenchymal stem cells (LMSCs) have been used in many ocular diseases. Brimonidine tartrate (BT) is selective alpha-2 adrenergic agonist that its neuroprotective effect had been proven. BT drops are preferred in glaucoma patients especially for neuroprotection. Our purpose is to evaluate the effect of BT on viability and/or proliferation of LMSCs in vitro. Methods : LMSCs were isolated from corneoscleral rim. The cells in third passage were characterized and differentiated. For MTT assay, cells were seeded in the density of 3x103 in 96 well plate and pretreatment with BT in 0.1, 1, 5,10 and 200 μM concentrations. After 48 hours, MTT solution was added to each wells and plate was read in 570nm absorbance. For scratch assay, cells were seeded in the density of 6x104 in 12 well plate and pretreatment with BT in pervious mentioned concentrations. For Annexin V/PI cell viability test, cells were seeded in the density of 2x105 in 6 well plate and pretreatment with BT in pervious mentioned concentrations. After 48 hours, cells were washed and stained with Annexin V The cells were analysed by flowcytometry. Results : Our experiments showed that pre-treated LMSCs with 10 μM BT increased cell viability significantly compared to untreated cells in MTT assay (p<0.05). In scratch assay pre-treated LMSCs with 5 μM and 10 μM BT displayed statistically significant rapid migration at 20th hours regarding to other untreated cells (p<0.05). According to cell viability results, pre-treated LMSCs with 10 μM BT decreased Annexin V expression compared to untreated group. And it is statistically significant (p<0.05). Conclusions : Since limbal mesenchymal stem cells and optic nerve share the same embryological origin (ectodermal), it is not surp rising that in our study BT enhanced the viability of limbal mesenchymal stem cells and improved proliferation. From this point of view, we hypothesized that intravitreally injection of brimonidine tartrate loaded limbal mesenchymal stem cells may not only protect axon loss, but also induce axon regeneration in traumatic optic nerve injuries. This preliminary study supported our hypothesis and we are in the process of planning future studies about this topic.