Abstract |
Introduction: Diabetic Retinopathy (DR) is one of the major complications of diabetes
mellitus. It is characterized by microvascular disruption, the presence of inflammation and
progressive degeneration of neuronal cells, which collectively lead to attenuation of optical
acuity and eventually bnindness. The major event that leads to the manifestation of DR is the
disruption of the “neurovascular unit” (NVU) homeostasis, a term used to describe the
functional interaction and interdependency among neural, glial cells and blood vessels in the
retina. Chronic hyperglycemia reduces expression of protective neurotrophic factors and
favors an increase in the expression of pro-inflammatory mediators, such as cytokines and
vascular endothelial growth factor (VEGF). This imbalance contributes to the progressive loss
of endothelial cells, the tight junctions of which form the inner blood retina barrier (iBRB).
As a result, there is an increase in vascular permeability which allows cytotoxic factors enter
the retina and induce the development of inflammation and oxidative stress. This is also
accompanied by the progressive degeneration of neural cells, through the activation of
apoptotic pathways. Furthermore, due to the drastic changes in retina physiology at the early
stages of DR, glial cells become activated and prarticipate and mediate pro-inflammatory
responses. The earlier tenet that DR is only a microvascular complication has been revised,
with studies examining the critical contribution of neurodegeneration in the pathophysiology
of DR. A growing body of evidence suggests that oxidative stress, neurodegeneration and
inflammation are present at the early stage of the disease, even before the development of thw
microvascular complications. Current treatments for DR (laser, intravitreal injection of anti-
VEGF agents and corticosteroids) are applied during the advance stage of the disease, where
patient’s vision is already compromised. These treatments mainly focus on the treatment of
vasculopathy and inflammation. Therapeutic intervention at the early stage of DR is only
limited in the tight control of risk factors for the development of the disease, such as blood
glucose and blood pressure. Finding and implementing new treatments with neuroprotective
and anti-inflammatory properties at the early stage of DR is imperative, since this could be a
good strategy for intercepting the development of the disease towards the vision threatening
advance stage.
The endocannabinoid system (ECS) has a neuromodulatory role in the central nervous system
(CNS), as it controls the activation of voltage gated ion channels, the presynaptic release of
glutamate and the activation of pro-survival cell signaling pathways. In retina CB1 receptors
are expressed in various neuronal cell populations, like photoreceptors, amacrine and
ganglion cells. CB2 receptors are mainly located in glial cells, where they participate in the
regulation of inflammatory responses. Numerous studies have reported the neuroprotective
properties of the ECS in the CNS. In the retina the neuroprotective properties, mediated by
CB1 receptor activation, are also well described in various models of retinopathies. However
extensive data indicate that in diabetic complications, other than DR, CB1 receptor activation
promotes oxidative stress and inflammation, while the use of CB1R antagonists reduces cell
death, inflammation and vascular damage. The protective properties, mediated by inhibition
of CB1 receptor activity, have also been confirmed in neurodegenerative conditions in the
ocular system. In contrast to CB1 receptor activation, CB2 receptor activation displays
neuroprotective effects, since it reduces inflammation, oxidative stress and cell death. The
protective properties of CB2 agonists have also been evaluated in diabetic complications,
such as diabetic nephropathy and also in neurodegenerative diseases in brain and retina.
Numerous studies have confirmed the neuroprotective properties of cannabinoid receptors agonists in various retinopathies, including excitotoxicity models. Apart from direct
cannabinoid agonists, inhibition of the metabolic enzymes of endocannabinoids seems to be
an effective strategy for indirectly controlling signaling through cannabinoid receptors.
Although numerous reports in the brain have confirmed the neuroprotective action of the
inhibitors of the endocannabinoids metabolism, the data in the retina are limited.
Aim of the study: The goal of the present thesis was the study of the neuroprotective
properties of the ECS in two in vivo animal model of retinopathy: A. In the first part, an in
vivo two week, streptozotocin (STZ) induced model of DR, which resembles the phenotype
observed at the early stages of the disease, was employed for the study of the neuroprotective,
anti-inflammatory and vasculoprotective actions of the eyedrop administration of a)
SR141716 (CB1 antagonist), b) AM1710 (CB2 agonist) and c) their combination
(SR141716+AM1710). B. In the second part we employed an in vivo model of AMPA
induced retinal excitotoxicity, in order to study the neuroprotective and anti-inflammatory
actions of two novel inhibitors of 2-AG hydrolytic enzymes: a) AM12100 [alpha/beta-
Hydrolase domain containing 6 (ABHD6) inhibitor] and b) AM11920 [dual alpha/beta-
Hydrolase domain containing 6/ monoacylglycerol lipase (ABHD6/MAGL) inhibitor].
Methods: Male and female rats weighting 250-300 g were employed for all the experiments.
For the DR study, diabetes was induced by a single intraperitoneal (i.p) injection of STZ (70
mg/kg). Animals with blood glucose levels >250 mg/dL were considered diabetic.
Cannabinoids were administered daily as eyedrops for 14 days at a dosage of 10 mg/ml,
starting two days post STZ injection. The experimental design was as following: 1.
Control+vehicle, 2. Diabetic+vehicle, 3. Diabetic+AM1710, 4. Diabetic+SR141716 and 5.
Diabtic+AM1710+SR141716. Twenty-four hours after the last eyedrop, animals were
euthanized ant their retinas were collected. Imunnohistochemical studies with antibodies
against NFL (neurofilament, ganglion cell axon marker), cleaved caspase 3 (apoptotic cell
death marker) and bNOS (brain nitric oxide synthase, amacrine cell marker) were conducted
for the estimation of neuroprotection. An antibody against NT (nitrotyrosine,
nitrative/oxidative stress marker) was used for the study of the oxidative damage.
Furthermore a histological stain with Eosin/Hematoxylin was employed in order to assess
retina thickness. For the study of the anti-inflammatory actions of the cannabinoid treatment
we used antibodies against GFAP (Glial fibrillary acidic protein, microglia marker) and Iba1
(Ionized calcium-binding adaptor molecule 1, microglia marker) and ELISA for TNFα
(Tumor necrosis factor α). Finally, for the estimation of the vasculoprotective actions of
cannabinoids we performed a colocalization study between CD-31 (cluster of differentiation
31, endothelial cell marker), ELISA for VEGF (Vascular endothelial growth factor A) and
Evans Blue (EB) assay (assessment of vascular leakage). For the AMPA excitotoxicity
experiments animals received intravitreal injections of AMPA (42 nmol/ eye), either alone or
in combination with AM12100 (10-5-10-3 M) or AM11920 (10-6-10-4 M). Twenty-four hours
after the injections, retinas were collected after euthanization of the animals. Cannabinoid-
mediated neuroprotection was assessed by an immunohistochemical study with an antibody
against bNOS, while for the anti-inflammatory actions antibodies againt GFAP and Iba1 were
employed.
Results: Topical administration of cannabinoids via eyedrops displayed various protective
astions in the DR model. The diabetes induced reduction in NFL-immunoreactivity (IR)
thickness and intensity was blocked by AM1710, SR141716 and their combination. AM1710
and SR141716 also blocked the diabetes induced reduction in total retinal thickness and [16]
especially in the inner nuclear layer (INL). Only AM1710 inhibited apoptotic cell death in the
INL. AM1710 and the dual treatment (SR141716+AM1710) protected bNOS+ cells in the
INL. All of the three cannabinoid treatmens effectively attenuated nitrative damage in the
diabetic rat retina. AM1710 was the only cannabinoid treatment that displayed significant
anti-inflammatory actions, since it reduced microglia and microglia activation, as well as the
levels of TNF-α. Single administration of AM1710 and SR141716 attenuated vascular
leakage and the levels of VEGF in the retina. In the AMPA excitotoxixity model, the
manipulation of endogenous 2-AG levels by the use of inhibitors of its hydrolytic enzymes
displayed protective properties, too. AM12100 (ABHD6 inhibitor) and AM11920 (dual
ABHD6/MAGL inhibitor) provided protection against AMPA in a dose dependent manner,
with the greatest efficacy observed after administration of the dual inhibitor. Both inhibitors
also mediated significant anti-inflammatory actions, by attenuating macroglia activation, but
only the dual inhibitor, AM11920, reduced the number of reactive microglial cells in the
retina.
Conclusions: The employment of CB2 agonists and CB1 antgonists at the early stages of DR,
may be a beneficial therapeutic strategy for attenuating the early pathophysiological
symptoms and intercepting disease progression towards the vision threatening, advanced
stages. The manipulation of endogenous cannabinoid levels with the use of inhibitors of 2-AG
metabolism seems to be an alternative method for attenuating neurodegeneration and
inflammation induced by excitotoxicity, which in fact possesses several advantages over the
use of direct cannabinoid agonists. Collectively, data from the present study suggest that ECS
represents a promising target for the treatment of retinopathies. However, more studies are
needed in order to study the exact mechanism via which cannabinoids exert their protective
properties and eliminate any limitations resulting from the use of cannabinoids as therapeutic
agents
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