20 21 Purpose: Bipolar cells can perish during inner retinal ischemia resulting from disruption of the 22 inner retinal microvasculature. Can surviving inner retinal neurons recover and integrate into the 23 functional network after ischemic damage? Using the mouse oxygen induced retinopathy (OIR) 24 model, we applied focal-ERG to determine if B-wave recovery occurs after ischemic bipolar cell 25 death after revascularization. 26 Methods: The OIR model was used to generate regions of inner retinal neuron loss in B6.Cg-27 Tg Thy1-YFP mice. Using image-guided focal-ERG, the dark-adapted mixed rod-cone light response 28 was compared using stimulation of small circular (0.27 mm diameter) target areas located in the 29 central retinas of the same eyes (OIR and control). The same areas of the same retinas were 30 followed over three ages after revascularization (P21, P28 and P42). 31 Results: There was a minimal effect on the photoreceptor-derived A-wave amplitude in the 32 central damaged regions. B-wave amplitudes were almost absent in these damaged central 33 regions at age P21, and progressively recovered through ages P28 to P42. The magnitude of the 34 recovered B-wave amplitude by P42 remained less than the normal control retina (room air), but 35 did show oscillatory potentials. 36 Conclusions: Recovery of retinal functionality, a neural plasticity, was detected in the central 37 damaged regions of the mouse OIR model after the initial loss of bipolar cells and 38 revascularization of these zones. These results suggest that any treatments that would enhance 39 the overall survival of inner retinal neurons result in a significant improvement in neural function 40 after any surviving neurons establish connectivity. 41 42

Keywords: blood-retinal barrier, VEGFA165b, MAPK, AKT, Endothelial blood-retinal barrier, diabetic retinopathy, image analysis, optical coherence tomography, retinal cell culture, retinal vasculature 2 ABSTRACT Purpose: Studies show that the b-isoform of Vascular Endothelial Growth Factor-A-165 (VEGFA 165 b) is predominant in normal human vitreous, switching to the a-isoform (VEGFA 165 a) in the vitreous of eyes with active diabetic retinopathy or ROP. The potential of this isoformswitching to impact the retinal vasculature is not clear, particularly in primary human retinal endothelial cells, which are important targets of VEGFA. We do not know how these two isoforms compare in their ability to activate key intracellular signalling pathways (MAPK, AKT) or alter VEGFA-target gene expression in primary human endothelial cells from the neural retina. Methods: Effects of saturating amounts of both VEGFA 165 isoforms (a/b) on the rat retinal vasculature were compared using intravitreal injection, fluorescein-angiography and Optical Coherence Tomography to monitor primary vein dilation and retinal edema. Full dose-response curves for the activation of MAPK (ERK1/2), AKT and VEGFR2 were determined using direct in-cell western assays of primary Human Retinal Microvascular Endothelial Cells (HRMECs). Differences in dose-response effects on gene expression markers related to endothelial cell / leukocyte adhesion (ICAM1, VCAM1 and SELE ) and tight-junctions (CLDN5 and OCLN ) were tested by quantitative-PCR. Results: In rats, dilation of primary retinal veins and edema could be induced within 24 hours by intravitreal injection of a saturating dose of either isoform. In HRMECs, activation dose-response analysis revealed much stronger activation of MAPK, AKT and VEGFR2 by the a-isoform at lower doses. While similar maximum activation of VEGFR2 and MAPK could be achieved by both isoforms at higher doses, maximum activation of AKT by the b-isoform was only half that observed for the a-isoform. At the level of gene expression, VEGFA 165 a was also more effective 3 at increasing expression of ICAM1, VCAM1 and SELE and decreasing expression of CLDN5 and OCLN at intermediate and high doses in primary HRMECs. Conclusions: VEGFA 165 a maximally activated MAPK and AKT in HRMECs at lower concentrations where VEGFA 165 b had little effect. The timing for maximal activation of MAPK was similar for both isoforms in HRMECs, which is different from non-retinal endothelial cells. While the dose-responses for VEGFR2 and MAPK activation had similar maximums with both isoforms, there were large differences between the isoforms in their effects on endothelial cell gene expression even at a high dose. The shifts of VEGFA 165 expression from mostly b-isoform to mostly a-isoform, as reported in some human retinal vascular diseases, could potentially impact the activation of intracellular signalling and VEGFA target gene expression in endothelial cells of the human neural retina. Receptor-2 (VEGFR2). A seminal analysis of the vitreous fluids of pati...

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