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. 2016 Nov 18;11(11):e0166827.
doi: 10.1371/journal.pone.0166827. eCollection 2016.

Modulation of Type-1 and Type-2 Cannabinoid Receptors by Saffron in a Rat Model of Retinal Neurodegeneration

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Free PMC article

Modulation of Type-1 and Type-2 Cannabinoid Receptors by Saffron in a Rat Model of Retinal Neurodegeneration

Rita Maccarone et al. PLoS One. .
Free PMC article

Abstract

Experimental studies demonstrated that saffron (Crocus sativus) given as a dietary supplement counteracts the effects of bright continuous light (BCL) exposure in the albino rat retina, preserving both morphology and function and probably acting as a regulator of programmed cell death [1]. The purpose of this study was to ascertain whether the neuroprotective effect of saffron on rat retina exposed to BCL is associated with a modulation of the endocannabinoid system (ECS). To this aim, we used eight experimental groups of Sprague-Dawley rats, of which six were exposed to BCL for 24 hours. Following retinal function evaluation, retinas were quickly removed for biochemical and morphological analyses. Rats were either saffron-prefed or intravitreally injected with selective type-1 (CB1) or type-2 (CB2) cannabinoid receptor antagonists before BCL. Prefeeding and intravitreally injections were combined in two experimental groups before BCL. BCL exposure led to enhanced gene and protein expression of retinal CB1 and CB2 without affecting the other ECS elements. This effect of BCL on CB1 and CB2 was reversed by saffron treatment. Selective CB1 and CB2 antagonists reduced photoreceptor death, preserved morphology and visual function of retina, and mitigated the outer nuclear layer (ONL) damage due to BCL. Of interest, CB2-dependent neuroprotection was more pronounced than that conferred by CB1. These data suggest that BCL modulates only distinct ECS elements like CB1 and CB2, and that saffron and cannabinoid receptors could share the same mechanism in order to afford retinal protection.

Conflict of interest statement

Competing Interests: This investigation was partially supported by the commercial companies Essse Caffè S.p.A. and Hortus Novus s.r.l. We confirm that this does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Effect of BCL on ECS mRNA expression.
qRT-PCR analysis of main components of ECS (NAPE-PLD, DAGL, FAAH, MAGL, CB1, CB2 and TRPV1) in the retinas from untreated controls and rats exposed to bright continuous light (LD). Data were expressed as means ± SEM (n = 6), and were analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc analysis. ***p<0.001 vs control.
Fig 2
Fig 2. Effect of BCL on ECS protein expression.
(A) Representative Western blot of main components of ECS (NAPE-PLD, DAGL, FAAH, MAGL, CB1, CB2 and TRPV1) in the retinas from controls and rats exposed to bright continuous light (LD). Panel B shows densitometry of ECS components expression normalised for its own control. Data were expressed as means ± SEM (n = 6), and were analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc analysis.*p<0.05 vs control.
Fig 3
Fig 3. CB1 and CB2 mRNA levels following saffron treatment and BCL exposure.
qRT-PCR analysis of CB1 and CB2 in the retinas from untreated controls, rats exposed to bright continuous light alone (LD), or in combination with saffron (saffron + LD). Data were expressed as means ± SEM (n = 6), and were analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc analysis. ***p<0.001, ****p<0.0001 vs control, ###p<0.001, ####p<0.0001 vs LD.
Fig 4
Fig 4. CB1 and CB2 protein levels following saffron treatment and BCL exposure.
(A) Representative Western blot of CB1 and CB2 in the retinas from controls, rats exposed to bright continuous light alone (LD), or in combination with saffron (saffron + LD). Panel B shows densitometry of CB1 and CB2 expression normalised for its own control. Data were expressed as means ± SEM (n = 6), and were analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc analysis.*p<0.05 vs control, ##p<0.01 vs LD.
Fig 5
Fig 5. CB1 immunolabelling following saffron treatment and BCL exposure.
Localization of CB1 in retinal sections. Images are representative of the same retinal region in the four tested groups: control, saffron, LD, saffron+LD. Scale bar: 50 μm. ONL (outer nuclear layer), OPL (outer plexiform layer), INL (inner nuclear layer), IPL (inner plexiform layer), GCL (ganglion cell layer).
Fig 6
Fig 6. CB2 immunolabelling following saffron treatment and BCL exposure.
Localization of CB2 in retinal sections. Images are representative of the same retinal region in the four tested groups: control, saffron, LD, saffron+LD. Scale bar 50 μm.
Fig 7
Fig 7. Quantitative analisys of TUNEL+ cells.
Fig 7A: representative images acquired by confocal microscopy in the same region (“hot spot”) showing the apoptotic cells after exposure to bright light in the six experimental conditions examined. The quantitative analysis of the dying cells in outer nuclear layer (ONL) is reports in the graph (panel B). Dying photoreceptors are counted from superior to inferior edge in all experimental conditions normalized respect to LD group. Data were expressed as means ± SEM (n = 6), and were analyzed by one-way analysis of variance (ANOVA) followed by Tukey-test post hoc analysis. ***p<0.001 all experimental groups vs LD, *p<0,05 SR1+LD vs saffron+LD and vs saffron+SR1+LD.
Fig 8
Fig 8. fERG b-wave amplitude (μV) as a function of flash luminance (cd/m2).
In both panels A and B are reported the same data for control, LD and saffron+LD. Panel A: CB1 receptors antagonist [SR141716A (SR1)] with or without saffron, panel B: CB2 receptor antagonist [SR144528 (SR2)], with or without saffron. Data were expressed as means ± SEM (n = 6). # p<0.05 SR1+LD vs saffron+LD; ££p<0,01 SR2+LD vs saffron+LD; §§ p<0.01 and §§§p<0.001 LD vs saffron+LD, SR1+LD, saffron+SR1+LD, SR2+LD, saffron+SR2+LD; ***p<0.001 control vs all experimental groups.
Fig 9
Fig 9. Bisbenzimide labelling and quantitative analysis of hot spot extension.
Panel A: Representative images labelled with a nuclear staining (bisbenzimide) in a dorsal position one millimeter from optic disc (“hot spot”) in LD, saffron+LD, SR1+LD, saffron+SR1+LD, SR2+LD, saffron+SR2+LD, one week after light damage (LD). Scale bar, 50 μm. Panel B: Ratio of hot spot/superior retinal length. Data were expressed as means ± SEM (n = 6). *** p<0.001 vs LD.

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Grant support

This investigation was supported by Ministero dell’Istruzione, dell’Università e della Ricerca (PRIN 2010-2011 grant) to MM, to SB and by Mr. Francesco Segafredo, Essse Caffè S.p.A. The company Hortus Novus s.r.l. provided the saffron used in this study. These supporters had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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