Cannabinoids as Glial Cell Modulators in Ischemic Stroke: Implications for Neuroprotection

Abstract

Stroke is the second leading cause of death worldwide following coronary heart disease. Despite significant efforts to find effective treatments to reduce neurological damage, many patients suffer from sequelae that impair their quality of life. For this reason, the search for new therapeutic options for the treatment of these patients is a priority. Glial cells, including microglia, astrocytes and oligodendrocytes, participate in crucial processes that allow the correct functioning of the neural tissue, being actively involved in the pathophysiological mechanisms of ischemic stroke. Although the exact mechanisms by which glial cells contribute in the pathophysiological context of stroke are not yet completely understood, they have emerged as potentially therapeutic targets to improve brain recovery. The endocannabinoid system has interesting immunomodulatory and protective effects in glial cells, and the pharmacological modulation of this signaling pathway has revealed potential neuroprotective effects in different neurological diseases. Therefore, here we recapitulate current findings on the potential promising contribution of the endocannabinoid system pharmacological manipulation in glial cells for the treatment of ischemic stroke.

Keywords: cannabinoids; drug target; glia; ischemic stroke; neuroinflammation.

FIGURE 1

Microglial ECS function. (A). In healthy conditions, microglia participate in pivotal functions for proper neuronal functioning such as dendritic pruning, neural rewiring, secretion of trophic factors and synaptic plasticity. The main function of resting microglia is to monitor the brain parenchyma through their widely branched morphology and to quickly detect any type of cellular alteration or damage. Cannabinoid receptor activation regulates the phenotypic polarization of microglia, migration, cytokine production and phagocytic capacity of these cells. (B). Microglial response to ischemic stroke follows a spatio-temporal pattern. Initially, in the acute phase, there is a significant increase in the number of M2-like microglial cells in the ischemic area. The M2-like phenotype is considered protective since it acquires phagocytic capacity that allows it to eliminate the dead cells debris. In addition, they release neurotrophic factors and anti-inflammatory cytokines in an attempt to limit neuronal damage. However, during the chronic phase of stroke M1-like cells proliferate and are recruited to the injury area. M1 microglia release pro-inflammatory cytokines and ROS that contribute to exacerbate neuronal death, oligodendrocyte damage and astrocyte activation. The inflammatory response also contributes to BBB rupture and the release of chemoattractant factors from peripheral immune cells. The M1 phenotype is characterized by the acquisition of an amoeboid morphology, without branching and losing its phagocytic capacity thus preventing tissue repair. The expression of CB₁R and CB₂R as well as other ECS-related receptors has been upregulated in different in vitro and in vivo stroke models. However, its specific role in microglia is still unknown. (C). The protective effects observed after treatment with CBs in different stroke models include modulation of microgliosis. Reduction in the number of microglia cells was not only observed, but also induced polarization towards the M2-like phenotype. The M2-like microglia contributes to tissue repair, as it has phagocytic capacity and releases trophic factors. In addition, it releases anti-inflammatory cytokines limiting brain damage and preserving the BBB, thus decreasing peripheral immune cell extravasation. Green arrows and boxes: eCB-mediated effects; blue arrows and boxes: CB-mediated effects. BBB: brain-blood barrier; CB₁R: cannabinoid receptor 1: CB₂R: cannabinoid receptor 2; CBs: cannabinoids; eCBs: endocannabinoids; ROS: reactive oxygen species.

FIGURE 2

The ECS functioning in astrocytes. (A). In physiological conditions, astrocytes regulate processes that are crucial for neuronal functioning, e.g., providing metabolic and trophic support for neurons, controlling CBF, BBB permeability, and regulating synapse maintenance and plasticity, among others. Astrocytes participate in synapse plasticity through the tripartite synapse. When neurotransmitter is released by presynaptic neurons, it increases intracellular Ca²⁺ both in post-synaptic terminals and in astrocytes. In post-synaptic neurons, Ca²⁺ elevation induces eCB release to the extracellular space, inhibiting neurotransmitter release from the presynaptic neuron. In astrocytes, eCB binding to their receptors mobilize Ca²⁺ from internal stores, triggering gliotransmitter release, i.e., glutamate, which in turn promotes mGluR1-mediated neurotransmitter release from the presynaptic neuron, a phenomenon called synaptic potentiation. Besides, as Ca²⁺ spreads intracellularly in the astrocyte, it is able to release glutamate at distal points, stimulating synapsis that are at a certain distance from the synapse that was initially stimulated. This results in the so-called lateral synaptic potentiation. (B). During the acute/subacute phase after an ischemic event, astrocytes undergo significant morphological and functional changes like hypertrophy, hyperplasia and increased GFAP levels. To increase neuronal survival, astrocytes release neurotrophic factors and anti-inflammatory cytokines. Nevertheless, they also release pro-inflammatory cytokines that negatively affect neuronal survival. Moreover, at the neurovascular unit, astrocytes upregulate the expression of surface receptors and enzymes that are strongly associated with inflammatory responses that actively contribute to BBB disruption and leukocyte infiltration into the CNS, becoming a source of inflammation if this process becomes chronic. Over time, reactive glial cells rearrange, creating a barrier composed of densely packed cells that separate the ischemic core from the penumbra. (C). Main molecular changes induced in astrocytes by CBs after stroke. CB modulation of astrocyte reactivity includes reduced GFAP immunoreactivity and release of catalytic enzymes, leading to attenuation of BBB disruption. These molecules also increase neuronal survival after ischemia; however, it remains unclear whether the neuroprotection exerted by CBs is mediated by astrocytes. Green arrows and boxes: eCB-mediated effects; blue arrows and boxes: CB-mediated effects. BBB: brain-blood barrier; CB₁R: cannabinoid receptor 1: CB₂R: cannabinoid receptor 2; CBs: cannabinoids; CSF: cerebrospinal fluid; eCB: endocannabinoids; ROS: reactive oxygen species.

FIGURE 3

The ECS functioning in oligodendrocytes. (A). Under physiological conditions, oligodendrocytes play a key role in the metabolic support of axons, myelin sheath synthesis and BBB regulation, among others. In the CNS, the endocannabinoid system, particularly 2-AG, is involved in oligodendrocyte proliferation, maturation, migration and myelination of oligodendrocytes. 2-AG is produced in an autocrine manner and exerts its effects through its binding to CB₁R and CB₂R. (B). An ischemic event induces mature oligodendrocytes cell death due to the high sensitivity of these cells to: 1) glutamate and ATP receptor-induced excitotoxicity, 2) oxidative stress, i.e., high iron content and deficient antioxidant system, and 3) inflammation, through release of cytokines like TNF-α. In an attempt to repair the damage caused by stoke there is a strong oligoproliferative response. However, the maturation of these new oligodendrocytes is impaired, and they do not reach the mature myelinating oligodendrocyte stage, perpetuating myelinating deficits that contribute to motor and sensitive impairment observed after stroke. (C). Although the evidence of the oligoprotective potential of CBs after stroke is scarce, they seem to reduce the myelination impairment by 1) reducing oligodendrocyte cell death and 2) promoting oligodendrocyte proliferation and maturation into myelinating oligodendrocyte after the insult. Green arrows and boxes: eCB-mediated effects; blue arrows and boxes: CB-mediated effects. 2-AG: 2-Arachidonoylglycerol; BBB: blood-brain barrier; CB₁R: cannabinoid receptor 1: CB₂R: cannabinoid receptor 2; CBs: cannabinoids; MBP: myelin binding protein; OL: oligodendrocyte; OPC: oligodendrocyte progenitor cell.