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Identifier 000397350
Title Μελέτη της νευροπροστατευτικής δράσης των κανναβινοειδών στην αμφιβληστροειδική ισχαιμία
Alternative Title Study of the neuroprotective action of cannabinoids in retinal ischemia
Author Κοκονά, Δέσποινα
Thesis advisor Θερμού, Κυριακή
Reviewer Πλαϊτάκης, Α.
Παπαχατζής, Δ.
Τσιλιμπάρης, Μ.
Καστελλάκης, Α.
Λιαπάκης, Γ.
Χαραλαμπόπουλος, Ι.
Abstract Retinal ischemia leads to neovascularization and excitotoxicity (cell death) both of which can lead to the development of very severe retinopathies, such as diabetic retinopathy and age related macular degeneration (Lipton and Rosenberg, 1994; Campochiaro, 2000; Osborne et al., 2004; Grant et al., 2005), that often result in poor visual acuity and blindness. Surgical and new pharmacological approaches are available to target the neovascular component of ischemia induced retinopathies but not the neurodegenerative component. In order to preserve vision both components must be treated. Therefore, it is mandatory to establish new pharmacological targets for the treatment of the neurodegenerative component. The main aim of the present study was to investigate whether the endocannabinoid system may serve as an efficacious therapeutic target for the treatment of neurodegenerative retinal disease. Specifically, the neuroprotective actions of the endocannabinoids AEA (Narachidonoylethanolamine, anandamide) and 2-AG (2-arachidonoylglycerol) and synthetic cannabinoids MethAEA (Methanandamide) and HU-210 were studied, as well as the neuroprotective actions of the inhibitors of the enzymes responsible for the metabolism of the endocannabinoids. In addition, the involvement of the cannabinoid CB1 and CB2 receptors and the TRPV1 vallinoid receptor in the neuroprotective actions of endo- and synthetic cannabinoids, as well as the downstream signaling pathways leading to their putative neuroprotective effects were also investigated. We employed the in vivo AMPA model of retinal excitotoxicity, previously developed in our laboratory (Kiagiadaki and Thermos, 2008) and we investigated the neuroprotective actions of the synthetic HU-210 and MethAEA and the endogenous cannabinoids AEA and 2- AG. Cannabinoids were intravitreally injected with AMPA (42nmoles/eye) and 24 hours post injection, the animals were euthanized and their retinas were prepared for immunohistochemical studies or western blot analysis. AMPA caused a reduction of bNOS (brain nitric oxide synthase) and ChAT (choline acetyltransferase) immunoreactive neurons compared to the control tissues and the cannabinoids afforded neuroprotection against AMPA excitotoxicity, increasing the number of bNOS and ChAT immunoreactive cells compared to the AMPA treated tissues. The toxic effects of AMPA and the neuroprotective actions of cannabinoids were also substantiated by the TUNEL assay. Additional studies revealed that the cell death observed in the presence of AMPA does not involve activation of the apoptosis mediator caspase-3. To investigate the involvement of CB1 and CB2 receptors in the neuroprotection, we performed a series of studies, namely the intravitreal co-injection of cannabinoid receptor antagonists with AMPA and cannabinoids, radioligand binding studies, study of the neuroprotective actions of the CB2 selective agonist JWH015, RT-PCR and studies in CB1-/- andCB2-/- mice, which revealed that the CB1 but not the CB2 receptor is involved in the neuroprotective actions of cannabinoids. We also performed pharmacological studies to assess the involvement of the TRPV1 vallinoid receptor in the cannabinoids’ neuroprotective actions. The data obtained from these studies revealed that the TRPV1 receptor, is not involved in the neuroprotective actions of cannabinoids in the vivo retinal model of AMPA excitotoxicity. Having assessed the neuroprotective properties of the cannabinoids in the AMPA model of excitotoxicity in the retina and the involvement of the CB1 cannabinoid receptor in their actions, we subsequently investigated the signaling pathways that lead to the neuroprotection. We performed western blot analysis against phosphorylated and total forms of the PI3K/Akt and MEK/ERK1/2 kinases, functional studies using the PI3K/Akt inhibitor wortmannin and neuroprotection studies in Akt2-/- mice. The data obtained suggested the involvement of PI3K/Akt and/or MEK/ERK1/2 signaling pathways in the neuroprotection afforded by the cannabinoids. Specifically, the PI3K/Akt signaling pathway seems to be involved in the Abstract 124 neuroprotective actions of ΑΕΑ, 2-AG and HU-210, while the MEK/ERK1/2 pathway is involved in the neuroprotective actions of ΑΕΑ and 2-AG, but not those of HU-210. Besides the direct activation of cannabinoid receptors with specific agonists, cannabinergic signaling can also be modulated through inhibition of endocannabinoid metabolism. Therefore, we examined the putative neuroprotective actions of the inhibitors of the two metabolic enzymes of AEA and 2-AG (Fatty Acid Amide Hydrolase, FAAH and Monoacylglycerol lipase, MGL for AEA and 2-AG, respectively), to substantiate whether these inhibitors could provide a new therapeutic target for the treatment of retinal disease. We performed experiments employing a FAAH inhibitor (AM6642), which inhibits the degradation of AEA and a dual FAAH/MGL inhibitor (AM9928), which inhibits the degradation of both AEA and 2-AG. These compounds afforded neuroprotection when co-injected with AMPA, with AM9928 affording greater neuroprotection than AM6642.The neuroprotective actions of the two inhibitors were reversed in the presence of the CB1 receptor antagonist AM251. Subsequently, we co-injected the above mentioned enzyme inhibitors with AMPA plus endogenous and synthetic cannabinoids to examine if the neuroprotective effects could be increased due to the elevation of endocannabinoid levels in the synapse and the exogenously added agents. Both the FAAH and the FAAH/MGL inhibitors failed to afford neuroprotection in the presence of exogenously applied cannabinoids. These results suggest that blockade of endocannabinoids’ degradation protects the retina from the AMPA insults. However, the coadministration of the enzyme inhibitors and exogenously administered endocannabinoids did not have an additive effect, probably due to desensitization and downregulation of CB1 receptor (Schlosburget al., 2010). This tenet must be investigated further. MethAEA, a metabolically stable analogue of AEA that is metabolized by pathways independent of FAAH, did not alter the neuroprotective action of AM6642 (Abadji et al., 1994). The second synthetic cannabinoid employed (HU-210) reversed the neuroprotective actions of both AM6642 and AM9928. Since there are no reports in the literature regarding the metabolic pathways of HU-210, one cannot make any hypotheses regarding HU-210’s actions. The data obtained from the present study suggest that endogenous and synthetic cannabinoids protect the retina from AMPA excitotoxicity, via activation of the CB1, but not CB2 or TRPV1 receptors, with the involvement of the PI3K/Akt and/or MEK/ERK1/2 signaling pathways. Inhibition of the enzymes that metabolize the endocannabinoids also afforded neuroprotection, yet the co-administration of exogenous endocannabinoids leads to its attenuation. In conclusion, the present study revealed an important role for the endocannabinoid system in the physiology and pathophysiology of the retina. This system could provide an important therapeutic target for the development of more efficacious therapeutics for the treatment of retinopathies whose pathophysiology involves excitotoxic insults.
Language Greek
Subject Amacrine cells
Βραχύινα κύτταρα
Issue date 2015-12-18
Collection   Faculty/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work--Doctoral theses
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