Choriorétinopathie de type birdshot (analyse des taches caractéristiques de la maladie à 5 ans)

PARIS7-Xavier Bichat (751182101) / SudocSudocFranceF

Cytokine/chemokine concentrations in aqueous humor.

<p>Cytokine/chemokine concentrations in aqueous humor were measured by multiplex ELISA 6 or 24 hours after LPS injection associated to vehicle (Veh) or aldosterone (Aldo) or spironolactone (Spiro) intravitreal injection. <b>A:</b> At 6 hours, TNF-α, IFN-γ, IL-2, MCP-1, and MIP-1α concentrations were significantly reduced in inflamed eyes with injection of aldosterone, compared to vehicle-injected inflamed eyes; spironolactone enhanced IL-6 concentrations in rats with EIU. Data are mean ± SEM; n = 5 rats per condition. <b>B:</b> At 24 hours, Aldosterone significantly reduced TNF-α, IFN-γ and MIP-1α concentrations; spironolactone increased TNF-α, IFN-γ, IL-6, IL-2 and MCP-1 concentrations. Data are mean ± SEM; n = 8 rats per condition. *p<0.05.</p

Real-time PCR primers.

<p>Real-time PCR primers.</p

Aldosterone reduces activation of microglia/macrophages in EIU. A:

<p>Immunofluorescence detection of IBA-1 microglia/macrophages on sections of inflamed eyes injected with vehicle, aldosterone or spironolactone. Twenty-four hours after LPS injection, in vehicle and spironolactone-injected eyes, many microglial cells had a round amoeboid shape attesting for their activation in the iris/ciliary body. By contrast, in aldosterone-injected eyes, more microglial cells had a resting dendritic shape with long branchings (white arrowheads). Merge images of IBA-1 (green) and DAPI (blue); scale bar: ciliary body: 40 µm; 25 µm for insert; iris: 60 µm. <b>B:</b> Quantification of activated IBA-1 microglia/macrophages on sections of inflamed eyes that were injected with vehicle, aldosterone or spironolactone. Twenty-four hours after LPS injection, less number of round activated IBA-1 positive cells was found in eyes sections with injection of aldosterone (Aldo) vs vehicle (Veh) or spironolactone (Spiro). Data are expressed as means ± SEM of 2 sections/rat; n = 4 rats per condition; *p<0.05, **p<0.01. <b>C:</b> Immunofluorescence of iNOS on sections of inflamed eyes injected with vehicle, aldosterone or spironolactone. Twenty-four hours after LPS injection, few round IBA-1 positive macrophages/microglia expressed iNOS in vehicle and spironolactone-injected eyes (colocalization: yellow, arrow). By contrast, iNOS signal was reduced in IBA-1 positive macrophages/microglia in aldosterone-treated eyes. Merge image of IBA-1 (green), iNOS (red) and DAPI (blue); scale bar: 10 µm; n = 4 rats per condition. <b>D:</b> iNOS mRNA expression (real-time PCR) in cells infiltrating the aqueous humor in EIU. In EIU, inflammatory cells (neutrophils and macrophages) accumulate in aqueous humor. In cells collected in aqueous humor from EIU eyes, iNOS mRNA expression was significantly reduced by aldosterone (Aldo) IVT compared to vehicle (Veh) IVT. This effect was prevented by the co-administration of aldosterone with the MR antagonist RU26752. The 18S was used as housekeeping gene. Results are relative changes compared to vehicle-injected eyes. Data are means ± SEM; n = 4–5 rats per condition; *p<0.05.</p

MR, GR, 11β-HSD1, 11β-HSD2 expression in iris/ciliary body and corticosterone concentration in aqueous humor during EIU. A–B:

<p>Time-course of MR (A) and GR (B) mRNA expression (real-time PCR). After LPS injection, down-regulation of GR was observed only at 24 hours, whilst MR expression was down-regulated already at 3 hours. The 18S was used as housekeeping gene. Results in EIU are relative to those without LPS injection. Data are means ± SEM; n = 6 rats per conditions; **p<0.01, ***p<0.001. <b>C:</b> Immunohistochemistry of MR and GR in iris sections. In normal iris rat, MR and GR are localized in the nuclei of the iris epithelial cells (arrow), endothelial cells (arrowhead) and probably in other resident cells of the iris stroma. Twenty-four hours after LPS injection, MR and GR expression signals were reduced in uveitis eyes (star: vessels). Negative controls are signals yielded in the absence of primary antibody. Scale bar = 40 µm; 20 µm for insert. <b>D–F:</b> Time-course of 11β-HSD2 (D), 11β-HSD1 (F) expression (real-time PCR) and corticosterone concentration in aqueous humor (E). LPS injection induced an early (3 hours) down-regulation of 11β-HSD2 transcripts and a late up-regulation of 11β-HSD1 transcripts at 24 hours compared to rats without LPS injection. The 18S was used as housekeeping gene. Results in EIU are relative to those without LPS injection. Corticosterone concentration in aqueous humor increases at 3 and 6 hours after LPS injection. Data are means ± SEM; n = 6 rats per conditions; *p<0.05, **p<0.01, ***p<0.001.</p

Effects of intravitreal injection of aldosterone or spironolactone on clinical EIU.

<p>Clinical scores of the severity of EIU were determined 24 hours after induction of EIU. Steroids were injected IVT in each eye, simultaneously to footpad LPS injection; 24 hours later, both eyes were evaluated, and scores of each eye were gathered as the clinical score of the animal. <b>A:</b> Intravitreal injection (IVT) of aldosterone (aldo; 1, 20 or 200 nM) or spironolactone (spiro; 10 µm): aldosterone (20–200 nM) induced a significant reduction of the clinical severity of EIU, compared to vehicle (Veh) IVT. Spironolactone did not modify the severity of EIU. Data are mean ± SEM; n = 8 rats per condition. <b>B:</b> Intravitreal injection of aldosterone (200 nM) alone or associated with the MR antagonist RU26752 (20 µM) or with the GR antagonist RU38486 (20 µM): benefit of aldosterone injection was confirmed, which was blunted in the presence of RU26752. There was no statistical difference between aldosterone+RU38486 IVT and vehicle injection or aldosterone IVT. Data are mean ± SEM; n = 10 rats per condition; *p<0.05, **p<0.01, ***p<0.001.</p

MR and GR mRNA expression after intravitreal injection of aldosterone or spironolactone in the eye of LPS-injected rats.

<p>EIU was associated to down-regulation of MR and GR (Fig. 1). <b>A–B:</b> MR expression in iris/ciliary body (real-time PCR): In the inflamed eye, intravitreal injection of aldosterone (Aldo) increased MR transcripts at 6 hours (A) and 24 hours (B) in EIU vs vehicle-injected eyes (Veh). Intravitreal injection of spironolactone (Spiro) decreased MR transcripts compared to vehicle-injected rats at 24 hours (B) and had no effect at 6 hours (A). <b>C–D:</b> GR expression in iris/ciliary body (real-time PCR): In the inflamed eye, intravitreal injection of aldosterone increased GR transcripts at 6 hours (C) and 24 hours vs vehicle-injected eyes (D). GR expression was not statistically modified by spironolactone. The 18S was used as housekeeping gene. Results are relative values of aldo/spiro relative to vehicle injection. Data are means ± SEM; n = 5–7 rats per condition; *p<0.05.</p

The aldosterone-mineralocorticoid receptor pathway exerts anti-inflammatory effects in endotoxin-induced uveitis.

We have previously shown that the eye is a mineralocorticoid-sensitive organ and we now question the role of mineralocorticoid receptor (MR) in ocular inflammation. The endotoxin-induced uveitis (EIU), a rat model of human intraocular inflammation, was induced by systemic administration of lipopolysaccharide (LPS). Evaluations were made 6 and 24 hours after intraocular injection of aldosterone (simultaneous to LPS injection). Three hours after onset of EIU, the MR and the glucocorticoid metabolizing enzyme 11-beta hydroxysteroid dehydrogenase type 2 (11β-HSD2) expression were down-regulated in iris/ciliary body and the corticosterone concentration was increased in aqueous humor, altering the normal MR/glucocorticoid receptor (GR) balance. At 24 hours, the GR expression was also decreased. In EIU, aldosterone reduced the intensity of clinical inflammation in a dose-dependent manner. The clinical benefit of aldosterone was abrogated in the presence of the MR antagonist (RU26752) and only partially with the GR antagonist (RU38486). Aldosterone reduced the release of inflammatory mediators (6 and 24 hours: TNF-α, IFN-γ, MIP-1α) in aqueous humor and the number of activated microglia/macrophages. Aldosterone partly prevented the uveitis-induced MR down-regulation. These results suggest that MR expression and activation in iris/ciliary body could protect the ocular structures against damages induced by EIU