Retinal ischemia (RI), despite relative resistance of the retina to the ischemic insult, frequently results in visual impairment and blindness. One of risk factors leading to ischemic diseases of retina, except systemic hypertension or atherosclerosis, is increased intraocular pressure (IOP). Thus, IOP model is useful in studying both molecular mechanisms present during RI, as well as methods of reduction of its progression. Decreased conductivity between retinal cells may contribute to the lessened injury following RI. Serotonin (5-HT) appeared to be effective in reduction of IOP and thus attenuate retinal ganglion cell (RGC) death. Selective serotonin reuptake inhibitors (SSRI), increasing 5-HT levels in synapses, are widely used in psychiatry. They have shown many beneficial effects as antioxidative, anti-inflammatory, antiapoptotic and neuroprotective. They affect also level of neurotransmitters, including brain-derived neurotrophic factor (BDNF) that is known for its neuroprotective effects. Escitalopram is a SSRI showing a more pronounced inhibition of 5-HT uptake, resulting in higher extracellular serotonin levels.

Since SSRIs have not been examined yet during RI, we decided to evaluate influence of escitalopram on morphologic and functional changes in mouse retina ischemia-reperfusion model. For this purpose, in first step we will pre-condition mice with oral administration of 1 mg Escitalopram every other day for 12 weeks before further procedures. Then, transient RI will be performed by 45-min elevation of IOP. This induces apoptotic process that tends to progress via secondary neuronal degeneration. In a pilot study, we found that Escitalopram reduces RGC loss after RI. To check the role of BDNF in the neuroprotection of RGCs mediated by Escitalopram, we will apply genetic model of BDNF knockout. Expected alleviation of beneficial effect of Escitalopram would be a proof that its activity involves BDNF. In our pilot study, we reported that Escitalopram reduced amplitude of oscillatory potentials (OP) and prevented further reduction of these potentials during RI. This reduction of conductivity may be cause by (i) decreased expression of synaptic proteins or (ii) by reduced conductivity of synapses themselves. To find the role of gap junctions in Escitalopram mediated neuroprotection, we will use intravitreal delivery of AAV-cx36 construct in order to increase cx36 content in retinal neurons. We expect that increased expression of cx36 will limit Escitalopram-mediated neuroprotection. The retinal activity will be estimated by electroretinography (ERG) and visual evoked potentials (VEP) measured before SSRI treatment starts, before and 2 weeks after induction of RI, and after intravitreal treatments. Collected specimens – retinas, optic nerves, and superior colliculi – will be subjected to morphologic and biochemical analysis. Fractionated Western Blots will be used for analysis of Cx36 (neuronal connexin) as well as BDNF and serotonin. Retinas labeled with DAPI (for all nuclei), RBPMS antibody (for RGCs) and glycine antibody (for amacrine cells) will be subjected to stereology to evaluate density of RGCs in reference to total density of cells. To assess intensity of apoptosis, TUNEL assay will be applied.

According to our knowledge, this is the first project examining the possible neuroprotective features of SSRI towards RGC in retinal ischemia model in relation to BDNF expression and antioxidant systems. We hope to gain – through better understanding of potential molecular mechanisms associated with retinal ischemia – a new knowledge about pathomechanism of this disease which might result in possible translational impact in the future.