Your browser does not support JavaScript!

Home    Search  

Results - Details

Search command : Author="Τσιλιμπάρης"  And Author="Μιλτιάδης"

Current Record: 4 of 102

Back to Results Previous page
Next page
Add to Basket
[Add to Basket]
Identifier 000451464
Title Investigation of the chronic administration of endogenous and synthetic cannabinoids on the function of cannabinoid receptor type-1 (CB1) in healthy retina and in an experimental model of retinopathy
Alternative Title Μελέτη της επίδρασης της χρόνιας χορήγησης ενδογενών και συνθετικών κανναβινοειδών στη λειτουργία του κανναβινοειδικού υποδοχέα τύπου-1 (CB1) στον υγιή αμφιβληστροειδή και σε πειραματικό μοντέλο αμφιβληστροειδοπάθειας
Author Παπαδογκωνάκη, Σοφία
Thesis advisor Θερμού, Κυριακή
Reviewer Τσιλιμπάρης, Μιλτιάδης
Χαραλαμπόπουλος, Ιωάννης
Γραβάνης, Αχιλλέας
Σιγανός, Χαράλαμπος
Σιδηροπούλου, Κυριακή
Καστελλάκης, Ανδρέας
Abstract ntroduction: The endocannabinoid system is considered as an important therapeutic target for new treatments of neurodegenerative diseases. The retina, as part of the CNS, comprises a full and functional endocannabinoid system that consists of the cannabinoid receptors (CB1 and CB2 receptors), endogenous agonists (endocannabinoids) and their metabolic enzymes. The endocannabinoids , anandamide (AEA) and 2-arachidonoyl-glycerol (2-AG), as well as synthetic cannabinoids, like R- (+)-Methanandamide (MethAEA), have been reported to provide neuroprotection to retinal neurons in different acute animal models of retinopathies. However, despite the neuroprotective actions of cannabinoids after acute administration, their therapeutic potential is limited due to the development of tolerance and dependence by repeated treatment that correlates with the downregulation of the CB1 receptor. Aims: Most of the studies suggesting agonist-induced desensitization and downregulation of CB1R have been performed in brain regions. In the present study, we focused on the investigation of the effect of endogenous and synthetic cannabinoids, and the inhibitors of the metabolic enzymes of ΑΕΑ (FAAH; ΑΜ5206), and 2-AG (MAGL/ABHD6; ΑΜ11920) on the expression of CB1 receptors in normal rat retina, after subchronic or chronic administration, as well as their potential neuroprotective properties in the experimental in vivo model of AMPA excitotoxicity. In addition, we examined the involvement of autophagy in the mechanisms involved in the downregulation of the CB1 receptor. Methods: We investigated the subchronic and chronic effects of cannabinoids in normal retina. Sprague-Dawley rats were administered intraperitoneally (i.p) with AEA, MetAEA, 2-AG or AM5206 and AM11920 daily for 4 or 14 days (subchronic or chronic administration, respectively). In order to assess the subchronic neuroprotective properties of these agents, the in vivo AMPA excitotoxicity model was employed, AMPA (8.4mM) was administered intravitreally and 24 hours later, rats were administered the above cannabinoids agents i.p daily for 4 days. We also employed the AMPA model of excitotoxicity in order to assess whether the administration of 2-AG by a less invasive route, via topical administration as eye drops (1%, twice/day for 2 days or once/day for 4 or 8 days) will protect the retina. Ph.D Thesis Sofia Papadogkonaki 8 The effect of the indirect elevation of endocannabinoid levels, by using inhibitors of the enzymes that metabolize AEA and 2-AG and subsequently lead to the elevation of their endogenous levels was also investigated. The FAAH inhibitor AM5206 or the dual MAGL/ABHD6 inhibitor (metabolic enzymes of AEA and 2-AG, respectively) were administered i.p for 4 days in control rats, to assess their subchronic effect on CB1R expression in normal rat retina, or following AMPA administration in order to study their neuroprotective effects. Immunohistochemical studies were performed employing antibodies against to CB1R and markers of retinal neurons to assess CB1R expression and retinal cell neuroprotection [nitric oxide synthetase, bNOS, marker of amacrine cells), calbindin (marker of amacrine and horizontal cells), caspase-3 (marker of apoptotic death) or Iba-1 (marker of microglia) to study the activation of microglia and the potential anti- inflammatory actions of the endocannabinoid 2-AG. Moreover, western blot analysis was performed to assess the protein levels of CB1R, and t he phosphorylation of its downstream signalling proteins, Akt or ERK1/2 kinases. The mechanism of CB1R’s downregulation was investigated in vivo using immunoprecipitation (IP) methodology in order to assess the potential interaction of the CB1R protein with the trafficking marker β-arrestin 2, or endosomal markers (Rab5, Rab7). In addition, we employed an ex vivo model, where retinas were incubated with 2-AG in combination with or without the autophagic inhibitor Bafilomycin A1 (BafA1), in order to examine the involvement of autophagy in the mechanism of CB1R downregulation. Results: This study showed that the cannabinoids AEA, MethAEA and 2-AG reduce CB1R expression in a dose dependent manner, after subchronic or chronic administration. The reduction of the CB1R’s expression by of MethAEA affected its downstream signaling (Akt and ERK1/2 phosphorylation). Subchronic treatment of AEA or 2-AG did not reduce Akt or ERK1/2 phosphorylation. Moreover, subchronic i.p administration of AEA, MethAEA or 2-AG induced downregulation of the CB1R in the model of AMPA excitotoxicity and failed to provide protection to bNOS expressing amacrine cells. However, 2-AG managed to attenuate AMPA- induced microglial activation, as it reduced the number of reactive microglial cells. We also assessed the effect of 2-AG, administered topically as eye drops for 2, 4 or 8 days. Our results showed that 2-AG (1%) did not affect CB1R’s expression at any time Ph.D Thesis Sofia Papadogkonaki 9 point and protected bNOS expressing amacrine cells after 2 or 4 days, but not after 8 days of treatment, but reduced the number of caspase-3 positive cells. Also 2-AG (1%) attenuated the AMPA induced increase in the number of reactive microglia after subchronic (4 days) administration. The elevation of the endogenous levels of AEA or 2-AG via the administration of FAAH or MAGL/ABHD6 inhibitor, respectively, does not induce downregulation of the CB1R at any of the doses studied (subchronically) in normal rat retina. In the model of AMPA excitotoxicity, AM5206 and AM11920 provided neuroprotection to bNOS expressing amacrine cells, after subchronic treatment (i.p) without affecting the expression of CB1R. Regarding the mechanism of CB1R downregulation in vivo, our findings suggest sthat the CB1R does not interact with β-arrestin 2 or Rab5 and Rab7 endosomal markers (early and late endosomes, respectively), however it is possible that the potential interaction could not be detected due to technical issues related with the use of tissue for IP studies. The data of our ex vivo studies showed that 2-AG induced downregulation of the CB1R. However, this effect was reversed when retinas were treated with 2-AG in combination with BafA1, suggesting that autophagy may be involved in the mechanism of CB1R downregulation. Conclusions: This study provides important information regarding agonist-induced CB1R downregulation in rat retina. Endogenous and synthetic cannabinoids may induce downregulation of the CB1R after subchronic or chronic i.p administration in normal rat retina, as well as in the experimental model of AMPA excitotoxicity, where they failed to protect amacrine cells. However, the topical eye drop administration of the endocannabinoid 2-AG provides neuroprotection to retina up to 8 days of treatment and does not affect the expression of CB1R. The inhibition of the metabolic enzymes of AEA and 2-AG, by FAAH and MAGL/ABHD6 inhibitors, respectively, led to the elevation of the endogenous levels of these cannabinoids and did not alter the levels of CB1R expression in normal retina or in the AMPA excitotoxicity model. Moreover, the subchronic i.p administration of these inhibitors was shown to exert neuroprotective actions to the retina, suggesting that the pharmacological inhibition of the endocannabinoids’ metabolism may be a more effective therapeutic approach compared to the exogenous systemic cannabinoid administration. The studies regarding the potential interaction of CB1R with trafficking markers did not provide satisfying results, however our ex vivo studies showed that inhibition of autophagy prevents the reduction of CB1R expression, implying that autophagy may play a role in the mechanism of downregulation of the receptor. Ph.D Thesis Sofia Papadogkonaki 10 In closing, this study provides novel information regarding the effect of repeated administration of endogenous and synthetic cannabinoids or inhibitors of the endocannabinoid metabolism in the expression of the CB1R in rat retina. Our results suggest that inhibitors of the metabolic enzymes of endocannabinoids may be effective in chronic treatment for retinal disease. Furthermore , we report new information regarding the trafficking of CB1R that may contribute to the elucidation of the still unknown mechanism via which the CB1R is downregulated in retina. The results obtained from this study will serve to evaluate the chronic use of cannabinoids as neuroprotectants in retinal disease
Language English
Subject Downregulation
Αμφιβληστροειδής χιτώνας
Μειορύθμιση
Issue date 2022-12-07
Collection   School/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work--Doctoral theses
Permanent Link https://elocus.lib.uoc.gr//dlib/5/9/c/metadata-dlib-1668065268-714649-7824.tkl Bookmark and Share
Views 664

Digital Documents
No preview available

No permission to view document.
It won't be available until: 2025-12-07