Ocular Application of the Kinin B1 Receptor Antagonist LF22-0542 Inhibits Retinal Inflammation and Oxidative Stress in Streptozotocin-Diabetic Rats

Purpose: Kinin B1 receptor (B1R) is upregulated in retina of Streptozotocin (STZ)-diabetic rats and contributes to vasodilation of retinal microvessels and breakdown of the blood-retinal barrier. Systemic treatment with B 1R antagonists reversed the increased retinal plasma extravasation in STZ rats. The present study aims at determining whether ocular application of a water soluble B1R antagonist could reverse diabetes-induced retinal inflammation and oxidative stress. Methods: Wistar rats were made diabetic with STZ (65 mg/kg, i.p.) and 7 days later, they received one eye drop application of LF22-0542 (1 % in saline) twice a day for a 7 day-period. The impact was determined on retinal vascular permeability (Evans blue exudation), leukostasis (leukocyte infiltration using Fluorescein-isothiocyanate (FITC)-coupled Concanavalin A lectin), retinal mRNA levels (by qRT-PCR) of inflammatory (B1R, iNOS, COX-2, ICAM-1, VEGF-A, VEGF receptor type 2, IL-1b and HIF-1a) and anti-inflammatory (B2R, eNOS) markers and retinal level of superoxide anion (dihydroethidium staining). Results: Retinal plasma extravasation, leukostasis and mRNA levels of B 1R, iNOS, COX-2, VEGF receptor type 2, IL-1b and HIF-1a were significantly increased in diabetic retinae compared to control rats. All these abnormalities were reversed to control values in diabetic rats treated with LF22-0542. B1R antagonist also significantly inhibited the increased production of superoxide anion in diabetic retinae. Conclusion: B1R displays a pathological role in the early stage of diabetes by increasing oxidative stress and proinflammator

Effect of diabetes and LF22-0542 on glycemia and body weight.

<p>Values are mean ± s.e.m.</p>***<p>P<0.001, significantly different from control group.</p

Effect of LF22-0542 on retinal oxidative stress in STZ-diabetic rats.

<p>(A) Representative pictures of superoxide anion production stained with dihydroethidine on retinal section from a control rat, a control rat treated with LF22-0542, a STZ-diabetic rat and a STZ-diabetic rat treated with LF22-0542. Scale bar is 75 µm. (B) Fluorescence intensity of superoxide anion was quantify by the evaluation of mean pixel energy ratio of DHE staining versus TO-PRO-3 in the retinal ganglion cells layer (RGC), the inner nuclear layer (INL) and the outer nuclear layer (ONL). Data are mean ± s.e.m. of values obtained from 3 rats in each group. Statistical comparison with control (*) or STZ (+) rats is indicated by **P<0.01, ⁺P<0.05, ⁺⁺⁺P<0.001.</p

Effect of LF22-0542 on retinal leukostasis in STZ-diabetic rats.

<p>(A) Representative pictures of adherent leucocytes in retinal vessels of a control rat, a control rat treated with LF22-0542, a STZ-diabetic rat and a STZ-diabetic rat treated with LF22-0542. Scale bar is 50 µm. (B) Number of adherent leukocytes per retina. Data are mean ± s.e.m. of values obtained from 5 to 7 rats in each group. Statistical comparison with control (*) or STZ (+) rats is indicated by *⁺P<0.05.</p

Effect of LF22-0542 on the expression of retinal inflammatory mediators in STZ-diabetic rats.

<p>mRNA levels of B₁R, B₂R, eNOS, iNOS, ICAM-1, IL-1β, COX-2, HIF-1α,VEGF-R₂ and VEGF-A. Data are mean ± s.e.m. of values obtained from 7–8 rats in each group. Statistical comparison with control rats (*) is indicated by *P<0.05, **P<0.01.</p

Effect of LF22-0542 on retinal vascular permeability in STZ-diabetic rats.

<p>Data are mean ± s.e.m. of values obtained from 9 to 11 rats. Statistical comparison with control (*) or STZ (+) rats is indicated by *⁺P<0.05.</p

Primers list.

<p>Primers list.</p

Chemical structure of LF22-0542.

<p>Chemical structure of LF22-0542.</p

Tau accumulation in the retina promotes early neuronal dysfunction and precedes brain pathology in a mouse model of Alzheimer’s disease

Abstract Background Tau is an axon-enriched protein that binds to and stabilizes microtubules, and hence plays a crucial role in neuronal function. In Alzheimer’s disease (AD), pathological tau accumulation correlates with cognitive decline. Substantial visual deficits are found in individuals affected by AD including a preferential loss of retinal ganglion cells (RGCs), the neurons that convey visual information from the retina to the brain. At present, however, the mechanisms that underlie vision changes in these patients are poorly understood. Here, we asked whether tau plays a role in early retinal pathology and neuronal dysfunction in AD. Methods Alterations in tau protein and gene expression, phosphorylation, and localization were investigated by western blots, qPCR, and immunohistochemistry in the retina and visual pathways of triple transgenic mice (3xTg) harboring mutations in the genes encoding presenilin 1 (PS1M146 V), amyloid precursor protein (APPSwe), and tau (MAPTP301L). Anterograde axonal transport was assessed by intraocular injection of the cholera toxin beta subunit followed by quantification of tracer accumulation in the contralateral superior colliculus. RGC survival was analyzed on whole-mounted retinas using cell-specific markers. Reduction of tau expression was achieved following intravitreal injection of targeted siRNA. Results Our data demonstrate an age-related increase in endogenous retinal tau characterized by epitope-specific hypo- and hyper-phosphorylation in 3xTg mice. Retinal tau accumulation was observed as early as three months of age, prior to the reported onset of behavioral deficits, and preceded tau aggregation in the brain. Intriguingly, tau build up occurred in RGC soma and dendrites, while tau in RGC axons in the optic nerve was depleted. Tau phosphorylation changes and missorting correlated with substantial defects in anterograde axonal transport that preceded RGC death. Importantly, targeted siRNA-mediated knockdown of endogenous tau improved anterograde transport along RGC axons. Conclusions Our study reveals profound tau pathology in the visual system leading to early retinal neuron damage in a mouse model of AD. Importantly, we show that tau accumulation promotes anterograde axonal transport impairment in vivo, and identify this response as an early feature of neuronal dysfunction that precedes cell death in the AD retina. These findings provide the first proof-of-concept that a global strategy to reduce tau accumulation is beneficial to improve axonal transport and mitigate functional deficits in AD and tauopathies