Schunemann, R. | bioRxiv Science

bioRxiv2019

Retinal Plasticity: Functional Recovery after Bipolar Cell Loss in the Oxygen Induced Retinopathy Model

The development of non-invasive live ocular imaging and electrophysiological test systems for rodent eyes provides new tools for not only averaged analysis of the entire retina but also the ability to see, test, and compare different subregions of the same retina. These new capabilities provide the possibility for more detailed examinations of local structural and functional relationships within a single eye and the ability to also follow changes longitudinally over time. We have developed protocols based around the Micron-III/IV retinal imaging camera system for combining fluorescent imaging of the neural retinal micro-vasculature by FA (fluorescein angiography), imaging of all neural retinal layers by SD-OCT (Spectral-Domain Ocular Coherence Tomography), and focal "spot" light-targeted electroretinography (Focal-ERG) to relate the local neurovascular unit structure to the inner (photoreceptor) and outer-retinal electrical response to light stimulation. For demonstration purposes we have used the popular mouse oxygen induced retinopathy (OIR) model, which causes radial central patches of retinal neuron loss mostly in zones away from and between the primary retinal arteries and veins. In this model, the loss of central microvasculature is induced developmentally in mouse litters exposed to 75% oxygen from age P7 to P11. Return to room air on P12, causes several days of retinal ischemia during which neurons, mostly of the inner retina, perish. Bipolar and ganglion cell death ends as neovascular growth revascularizes the central retina. This model provides for non-uniform retinal damage as well as gradual progression and resolution over time. The OIR model was used to generate regions of inner retinal neuron loss in B6.Cg-TgThy1-YFP mice. Using image-guided focal-ERG, the dark-adapted mixed rod-cone light response was compared using stimulation of small circular (0.27 mm diameter) target areas located in the central retinas of the same eyes (OIR and control). The same areas of the same retinas were followed over three ages after revascularization (P21, P28 and P42). ConclusionsCombined FA and SD-OCT imaging can provide local geographic specific information on retinal structural changes and be used to select different retinal areas within the same eye for testing of local light response. This analysis strategy can be employed for studies with rodent disease models that do not uniformly impact the entire retinal area. Combining these techniques would also be useful for testing gene and cell replacement therapies in retinal degeneration models where typically a small zone of the retina is treated. Both treated and untreated retinal zones within the eye can be followed non-invasively over many weeks. SUMMARYMouse models utilized for retinal disease research including retinal vascular models can display nonuniform changes over the entire retina. Damage or loss of retinal layers and retinal neurons due to hypoxia can impact some retinal areas while leaving adjacent regions unaltered. Combining vascular imaging by fluoresceine angiography, vascular imaging and retinal layer imaging by SD-OCT, and focal-ERG provides us with new tools to examine retinal structure-function relationships within a single retina.

neuroscience↗

bioRxiv2019

The a-isoform of VEGFA165 is a Significantly Stronger Activator of Human Retinal Endothelial Cells compared to the b-isoform

PurposeThere are reports that a b-isoform of Vascular Endothelial Growth Factor-A-165 (VEGFA165b) is predominant in normal human vitreous, switching to the a-isoform (VEGFA165a) in the vitreous of some diseased eyes. While these isoforms appear to have a different ability to activate the VEGF-Receptor-2 (VEGFR2) in various endothelial cells, the nature of their ability to activate intracellular signalling pathways is not fully characterized, especially in retinal endothelial cells. We determined their activation potential for two key intracellular signalling pathways (MAPK, AKT) over complete dose-response curves and compared potential effects on the expression of several VEGFA165 target genes in primary human retinal microvascular endothelial cells (HRMECs). MethodsTo determine full dose-response curves for the activation of MAPK (ERK1/2), AKT and VEGFR2, direct in-cell western assays were developed using primary Human Retinal Microvascular Endothelial Cells (HRMECs). Potential 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. ResultsActivation dose-response analysis revealed much stronger activation of MAPK, AKT and VEGFR2 by the a-isoform at lower doses. MAPK activation in primary HRMECs displayed a sigmoidal dose-response to a range of VEGFA165a concentrations spanning 10-250 pM, which shifted higher into the 100-5,000 pM range with VEGFA165b. Similar maximum activation of MAPK was achieved by both isoforms at high concentration. Maximum activation of AKT by VEGFA165b was only half of the maximum activation from VEGFA165a. At a lower intermediate dose, where VEGFA165a activated intracellular signalling stronger than VEGFA165b, the changes to VEGFA target gene expression was generally greater with VEGFA165a. ConclusionsIn primary HRMECs, VEGFA165a could maximally activate MAPK and AKT at lower concentrations where VEGFA165b had relatively little effect. The timing for maximal activation of MAPK was similar for both isoforms, which is different than reprorted for non-retinal endothelial cells. While VEGFA165a and VEGFA165b are limited to the sequence of their six C-terminal six amino acids, this results in a large difference in their ablility to activate at least two key intracellular signalling pathways and potentially VEGF target gene expression in primary human retinal endothelial cells.

cell biology↗