Onset and resolution of neuroinflammation and inner blood-retinal barrier disruption in a mouse retinal ischemia-reperfusion injury model

Abstract

Purpose : The retina is considered a relatively immune-privileged tissue with an inner blood-retinal barrier (iBRB) including well-developed endothelial tight junctions (TJ) that restrict leukocyte trafficking and control the flux of plasma proteins and fluid. We used a mouse intraocular pressure (IOP)-induced ischemia-reperfusion (IR) model of neurodegeneration to examine the innate neuroinflammatory response to sterile retinal damage and the relationship to disruption of the iBRB.

Methods : Retinal IR injury was unilaterally produced by elevation of IOP for 90 min followed by natural reperfusion. Retinal edema and layer thinning was evaluated by spectral domain optical coherence tomography (SD-OCT). DNA fragmentation was used to follow retinal cell death. FITC-BSA leakage, NH2-biotin leakage, and immuno-fluorescence (IF) localization of TJ proteins were used to evaluate iBRB integrity. Leukostasis and infiltration of leukocytes was examined by IF and flow cytometry. Western blotting was used to examine expression of TJ proteins.

Results : IR injury caused edema at 4 h and day 1, followed by inner retinal layer thinning for 2 weeks, coincident with retinal cell death. Vascular permeability was elevated within 4 h and resolved by 3 weeks. No change in vessel density, endothelial cell number or vessel perfusion was observed. A marked leukostasis occurred at day 1, transitioned to leukocyte infiltration between days 1 and 4, and exhibited a logical progression with Ly6C^hi inflammatory monocytes and granulocytes predominating early and Ly6C^neg reparative patrolling monocytes predominating as resolution ensued. TJ changes began within hours, with increased occludin Ser490 phosphorylation followed by down-regulation of zonula occludins-1 (ZO-1) and claudin-5 expression at 1 and 2 days, commensurate with junction disruption and disorganization. By 4-weeks TJ protein levels and organization were restored or even increased.

Conclusions : Resolving inflammation and reestablishing integrity of the retinal vasculature are important therapeutic goals for treatment of several sight-threatening retinal diseases, including diabetic retinopathy. For the first time, we describe a close temporal correlation between onset and resolution of neurodegeneration, neuroinflammation, iBRB disruption and alterations of TJ proteins in the IR model.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.