Le rôle du récepteur B1 des kinines dans le développement de la rétinopathie diabétique

La rétinopathie diabétique est associée à plusieurs changements pathologiques du lit vasculaire rétinien, incluant l’ouverture de la barrière hémato-rétinienne, l’inflammation vasculaire et la modification du débit sanguin. Récemment, il a été proposé que le récepteur B1 des kinines, qui est surexprimé dans la rétine diabétique, puisse être impliqué dans le développement de ces altérations vasculaires. Ainsi, cette thèse présente les effets de traitements pharmacologiques avec des antagonistes du récepteur B1 sur la perfusion rétinienne, la perméabilité vasculaire, l’infiltration des leucocytes (leucostasie), l’expression de médiateurs de l’inflammation et la production d’anion superoxyde dans la rétine du rat rendu diabétique avec la streptozotocine (STZ). Les résultats obtenus montrent que l’application oculaire (10 µl d’une solution à 1%, deux fois par jour pendant 7 jours) de LF22-0542, un antagoniste hydrosoluble du récepteur B1, bloque significativement l’hyperperméabilité vasculaire, la leucostasie, le stress oxydatif et l’expression génique de médiateurs de l’inflammation (B1R, iNOS, COX-2, VEGF-R2, IL-1β et HIF-1α) dans la rétine chez le rat à 2 semaines de diabète. L’administration orale (3 mg/kg) d’un antagoniste non-peptidique et sélectif pour le récepteur B1, le SSR240612, entraîne une diminution du débit sanguin rétinien 4 jours après l’induction du diabète mais n’a aucun effet sur la réduction de la perfusion rétinienne à 6 semaines. Le récepteur B1 joue donc un rôle protecteur au tout début du diabète en assurant le maintien d’un débit sanguin normal dans la rétine; un effet qui n’est toutefois pas maintenu pendant la progression du diabète. Ces données présentent ainsi la dualité du récepteur B1 avec des effets à la fois protecteurs et délétères. Elles suggèrent aussi un rôle important pour le récepteur B1 dans l’inflammation rétinienne et le développement des altérations vasculaires. Le récepteur B1 pourrait donc représenter une nouvelle cible thérapeutique pour le traitement de la rétinopathie diabétique.Diabetic retinopathy is associated with retinal vascular changes, including blood retinal barrier breakdown, vascular inflammation and blood flow alterations. It has been proposed that kinin B1 receptor, which is upregulated in the diabetic retina, could be involved in the development of these pathological features of diabetic retinopathy. In a rat model of diabetes induced by Streptozotocin (STZ), the effects of kinin B1 receptor antagonists on retinal perfusion, vascular permeability, leukostasis, gene expression of inflammatory mediators and production of superoxide anion in the retina were evaluated. The results show that in 2-week diabetic rats, topical ocular application of the water soluble kinin B1 receptor antagonist LF22-0542 (10 µl of 1% solution, twice per day) for a 7-day period reverses vascular hyperpermeability, leukostasis, oxidative stress and gene expression of inflammatory mediators (B1R, iNOS, COX-2, VEGF-R2, IL-1β and HIF-1α) in the retina. Single oral administration (3 mg/kg) of SSR240612, a selective non-peptide B1 receptor antagonist, induces a decrease of retinal blood flow in 4-day diabetic rats but has no effect on retinal blood flow reduction present at 6 weeks of diabetes. Therefore, B1 receptor has a protective role in early diabetes by preserving a normal blood flow in the retina. These data suggest that B1 receptor exerts protective and adverse effects in the diabetic retina. They also support a key role for B1 receptor in retinal inflammation and the development of vascular alterations. B1 receptor could therefore represent a promising therapeutic target for the treatment of diabetic retinopathy

Estimating moss growth in arctic conditions: a comparison of three methods

ABSTRACT. Except for Sphagnum mosses of peatland habitats, reliable methods to assess moss productivity in arctic or boreal biomes give usually highly variable results. Therefore, ecosystem processes are poorly understood in these biomes where mosses are an important component of the system. The aim of this study was to compare three methods to estimate moss growth in polygon patterned fens: cranked wires, natural markers and artificial white marks (an alternative to the spray method). Precision of estimates was significantly higher when natural markers were used (coefficients of variation, CV, between 17 and 27%), compared to cranked wires (CV537%) or white marks (CV556%). Natural markers also provided estimates for growth of moss stems 32 to 113% higher than the other methods. Although cranked wires were calibrated shortly after snowmelt, some moss growth is still missed and consequently moss growth is underestimated. Accuracy of cranked wires was poor, mainly caused by frost heaving or permafrost activities that can affect wire position. Thus, this method should be avoided in arctic ecosystems. Even if white marks were painted on moss stems at the end of the growing season prior to the sampling year, lower estimates of moss growth were still found. We suspect some interference with moss growth processes during the marking process, at least when used with brown mosses. The natural marker method, which provides increment for an entire growing season, appears to be the most accurate method of the three. Additionally, it is also the easiest and the least time consuming method to use. Its main drawback is that relatively few species have natural growth marks and these species may not always be present among the targeted species under study. Also, measurements of stem growth on the same sample did not differ between observers, even if the second measurement was done 12 years later. In conclusion, when species with natural markers are present, this method should be used to assess moss growth. For arctic/sub-arctic studies where such species are lacking, the artificial white marks method should be refined further

Modulation of retinal blood flow by kinin B 1 receptor in Streptozotocin-diabetic rats

a b s t r a c t The vasoactive kinin B 1 receptor (B 1 R) is overexpressed in the retina of diabetic rats in response to hyperglycemia and oxidative stress. The aim of the present study was to determine whether B 1 R could contribute to the early retinal blood flow changes occurring in diabetes. Male Wistar rats were rendered diabetic with a single i.p. injection of Streptozotocin (STZ) and studied 4 days or 6 weeks after diabetes induction. The presence of B 1 R in the retina was confirmed by Western blot. The impact of oral administration of the B 1 R selective antagonist SSR240612 (10 mg/kg) was measured on alteration of retinal perfusion in awake diabetic rats by quantitative autoradiography. Data showed that B 1 R was upregulated in the STZ-diabetic retina at 4 days and 6 weeks. Retinal blood flow was not altered in 4-day diabetic rats compared with age-matched controls but was significantly decreased following SSR240612 treatment. In 6-week diabetic rats, retinal blood flow was markedly reduced compared to control rats and SSR240612 did not further decrease the blood flow. These results suggest that B 1 R is upregulated in STZ-diabetic retina and has a protective compensatory role on retinal microcirculation at 4 days but not at 6 weeks following diabetes induction

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 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 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 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

Primers list.

<p>Primers list.</p