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

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Abstract and Figures

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.
| 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 Ca 2+ both in postsynaptic terminals and in astrocytes. In post-synaptic neurons, Ca 2+ elevation induces eCB release to the extracellular space, inhibiting neurotransmitter release from the presynaptic neuron. In astrocytes, eCB binding to their receptors mobilize Ca 2+ 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 Ca 2+ 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; CB 1 R: cannabinoid receptor 1: CB 2 R: cannabinoid receptor 2; CBs: cannabinoids; CSF: cerebrospinal fluid; eCB: endocannabinoids; ROS: reactive oxygen species.
… 
| 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 CB 1 R and CB 2 R. (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; CB 1 R: cannabinoid receptor 1: CB 2 R: cannabinoid receptor 2; CBs: cannabinoids; MBP: myelin binding protein; OL: oligodendrocyte; OPC: oligodendrocyte progenitor cell.
… 
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Cannabinoids as Glial Cell Modulators
in Ischemic Stroke: Implications for
Neuroprotection
Andrés Vicente-Acosta
1
,
2
, Maria Ceprian
3
, Pilar Sobrino
4
, Maria Ruth Pazos
5
* and
Frida Loría
5
*
1
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain,
2
Departamento de Biología Molecular, Universidad
Autónoma de Madrid, Madrid, Spain,
3
ERC Team, PGNM, INSERM U1315, CNRS UMR5261, University of Lyon 1, University of
Lyon, Lyon, France,
4
Departamento de Neurología, Hospital Universitario Fundación Alcorcón, Alcorcón, Spain,
5
Laboratorio de
Apoyo a la Investigación, Hospital Universitario Fundación Alcorcón, Alcorcón, Spain
Stroke is the second leading cause of death worldwide following coronary heart disease.
Despite signicant efforts to nd 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 ndings on the potential promising contribution of the endocannabinoid system
pharmacological manipulation in glial cells for the treatment of ischemic stroke.
Keywords: cannabinoids, neuroinammation, ischemic stroke, glia, drug target
INTRODUCTION
Stroke is a rapidly developing neurological pathology that involves the appearance of clinical
symptoms due to a global or focal disturbance of brain function, generally with vascular origin (Sacco
et al., 2013). It is the second leading cause of death worldwide after coronary and heart disease,
constituting 10% of total mortality, the rst cause of disability, and the second cause of dementia,
causing a signicant family, healthcare, and socioeconomic cost (Feigin, 2021). Moreover, data from
different studies indicate that stroke prevalence is increasing and will continue to rise, probably due
to an increment in life expectancy globally (Feigin, 2021). Stroke is a heterogeneous disease and
depending on the nature of the brain injury, two major types can be distinguished, hemorrhagic
Edited by:
Carmen Rodriguez Cueto,
Center for Biomedical Research on
Neurodegenerative Diseases
(CIBERNED), Spain
Reviewed by:
Alline C. Campos,
University of São Paulo, Brazil
Robert B. Laprairie,
University of Saskatchewan, Canada
*Correspondence:
Maria Ruth Pazos
ruth.pazos@salud.madrid.org
Frida Loría
frida.loria@salud.madrid.org
Specialty section:
This article was submitted to
Neuropharmacology,
a section of the journal
Frontiers in Pharmacology
Received: 02 March 2022
Accepted: 10 May 2022
Published: 01 June 2022
Citation:
Vicente-Acosta A, Ceprian M,
Sobrino P, Pazos MR and Loría F
(2022) Cannabinoids as Glial Cell
Modulators in Ischemic Stroke:
Implications for Neuroprotection.
Front. Pharmacol. 13:888222.
doi: 10.3389/fphar.2022.888222
Abbreviations: AEA, N-arachidonoylethanolamine or anandamide; BBB, blood-brain barrier; BCP, β-caryophyllene; CBD,
cannabidiol; CBDV, cannabidivarin; CBG, cannabigerol; CB, cannabinoid; CB
1
R, cannabinoid receptor type 1; CB
2
R, can-
nabinoid receptor type 2; CBF, cerebral blood ow; CNS, central nervous system; DAGL, diacylglycerol-lipase; eCB, endo-
cannabinoid; ECS, endocannabinoid system; GFAP, glial brillary acidic protein; HI, hypoxia-ischemia; IL-1β, interleukin-
1beta; intraperitoneal, i.p.; intravenous, i.v.; LPS, lipopolysaccharide.
Frontiers in Pharmacology | www.frontiersin.org June 2022 | Volume 13 | Article 8882221
REVIEW
published: 01 June 2022
doi: 10.3389/fphar.2022.888222
stroke (15% of cases) and ischemic stroke (85% of cases), which in
some cases could be transient ischemic attacks (van Asch et al.,
2010;Meschia and Brott, 2018). Hemorrhagic stroke is caused by
the rupture of a blood vessel in the brain and ischemic stroke, the
focus of this review, occurs when a cerebral artery is occluded by a
blood clot, causing a cerebral infarction (Pekny et al., 2019).
Thrombus formation can have different etiologies, such as
atherothrombosis, cardioembolism, small vessel occlusive
disease, rare cause (infection, neoplasia, myeloproliferative
syndrome, metabolic disorders, coagulation disorders) or even
cryptogenic stroke (Díez Tejedor et al., 2001;Meschia and Brott,
2018). As the brain and its proper functioning depend on an
adequate supply of oxygen and glucose, its cessation causes
neuronal death and glial activation that lead to functional
alterations not only in the affected area but also in related
brain areas (Pekny et al., 2019).
Due to the high prevalence of ischemic stroke and to a higher
opportunity for an effective therapeutic intervention than for
hemorrhagic, a great effort has generally been devoted to the
study of ischemic stroke. It has been observed that primary
prevention, which acts on modiable or potentially modiable
vascular risk factors, can considerably reduce the incidence of
ischemic strokes. Among the modiable risk factors, the most
important are arterial hypertension, tobacco use, diabetes
mellitus, hypercholesterolemia, obesity, physical inactivity, and
atrial brillation (Kuriakose and Xiao, 2020). However, due in
part to the estimated global increase in the prevalence of ischemic
stroke and the need to nd appropriate treatments to reduce
sequelae, it is essential to thoroughly study the molecular
mechanisms underlying the disease in order to nd new
treatments that will eventually lead to functional recovery of
patients.
Molecular Mechanisms of Ischemic Stroke
After the ischemic event, there is a central zone of irreversibly
damaged tissue known as core that receives less than 15% of
normal cerebral blood ow (CBF), a surrounding area of
damaged tissue that may recover its function known as
penumbra, receiving less than 40% of normal CBF, and the
peri-infarct region, which receives between 40 and 100% of
normal CBF (Meschia and Brott, 2018;Feske, 2021). In the
acute phase of cerebral ischemia, precisely due to the reduced
CBF, the affected tissue experiences cellular energy depletion,
with a dysfunction of the ATP-dependent ionic pumps producing
the intracellular accumulation of calcium (Ca
2+
). This in turn
induce the release and accumulation of excitotoxic amino acids
like glutamate in the extracellular space (Qureshi et al., 2003;Lai
et al., 2014). As a consequence of the intracellular Ca
2+
increase,
Ca
2+
-dependent enzymes are activated, resulting in
mitochondrial dysfunction and cell death, mainly by necrosis
(Fernández-Ruiz et al., 2015;Belov Kirdajova et al., 2020;
Kuriakose and Xiao, 2020). In this rst stage, microglial cells
act as early players and their activation leads to the production of
pro-inammatory mediators, such as tumour necrosis factor-
alpha (TNF-α), interleukin-1beta (IL-1β), and reactive oxygen
species (ROS) (Lambertsen et al., 2005;Clausen et al., 2008;Allen
and Bayraktutan, 2009;Chen et al., 2019). These factors recruit
other inammatory cell populations into the ischemic area,
mainly circulating monocytes, which interact with astrocytes
through the secretion of cytokines and chemokines, possibly
contributing to astrocyte activation (Kim and Cho, 2016;Frik
et al., 2018;Hersh and Yang, 2018;Rizzo et al., 2019). Once
astrocytes are activated, they shift their morphology and function
according to the biological context. Indeed, increasing evidence
sustains the critical role of these cells in the brains response to
stroke (Pekny et al., 2016), but their harmful or benecial
contribution to the ischemic pathway is currently under
intense debate (Liddelow et al., 2017). Astrocytes are also
critical for glial scar formation surrounding the infarct zone,
which may help limit immune cell inltration (Liddelow and
Barres, 2015,2017). Moreover, vasogenic edema, characterized by
extravasation and extracellular accumulation of uid into the
cerebral parenchyma caused by disruption of the blood-brain
barrier (BBB), takes place during the subacute phase (2472 after
the ischemic event) (Chen et al., 2019;Belov Kirdajova et al.,
2020). Regarding the role of oligodendrocytes in stroke, available
data indicate there is a substantial oligodendrocyte loss due to
excitotoxicity and oxidative stress in the ischemic core; however, a
signicant increase in this cell population takes place within the
penumbra (for a review see Jadhav et al., 2022). Finally, the
chronic phase can extend for weeks after the initial damage, being
probably caused by a delayed apoptotic neuronal death involving
several factors like an uncontrolled inammatory response, the
persistent presence of neurotoxic/neuroinhibitory factors, and
oxidative stress, among others (Allen and Bayraktutan, 2009;
Zhang et al., 2012;Belov Kirdajova et al., 2020).
Current Therapeutic Approaches to
Ischemic Stroke
In recent years, several effective treatments have been developed
for the treatment of ischemic stroke within the acute phase.
Besides, the coordination between the emergency teams,
neurologists, and hospital stroke units implementing what is
known as stroke code,allows a faster intervention, which
facilitates the patients admission to the stroke unit and
administration of treatments that help reduce mortality and
sequelae (Indredavik et al., 1991). Currently, the reperfusion
therapies available for the treatment of ischemic stroke in the
acute phase are intravenous (i.v.) thrombolysis and mechanical
thrombectomy (Kuriakose and Xiao, 2020;Feske, 2021); however,
due to the narrow therapeutic time window for effective
intervention, less than 5% of patients can benet from these
treatments (Fernández-Ruiz et al., 2015;Choi et al., 2019). There
are two types of thrombolytic treatments: i.v. injection of
recombinant tissue plasminogen activator and tenecteplase,
which are administered within the rst 4.5 h after stroke onset.
These treatments improve the patients clinical and functional
outcome, evaluated within 3 months (Alonso de Leciñana et al.,
2014). Another therapeutic option used when thrombolytic
treatment cannot be administered or has not been effective is
mechanical thrombectomy. There are several randomized studies
that demonstrate the efcacy of this procedure when applied
within 6 h of symptom onset (Powers et al., 2018).
Frontiers in Pharmacology | www.frontiersin.org June 2022 | Volume 13 | Article 8882222
Vicente-Acosta et al. Glial Endocannabinoid System in Stroke
With the aim of reducing sequelae and improving the
functional evolution of patients, neuroprotective treatments are
continuously under investigation (Miller et al., 2011;Kolb et al.,
2019). Still, despite having promising results in animal models
using neuroprotective drugs, these treatments have failed to
improve neurological outcomes after ischemic stroke in
clinical trials, probably because neuronal survival is not
enough to promote brain recovery (Gleichman and
Carmichael, 2014;Choi et al., 2019). For that reason, the study
of glial cells as novel therapeutic targets in stroke has gained
attention recently, not only because these cells have been
demonstrated to be essential for proper brain functioning but
also for their neuroprotective potential in different neurological
pathologies, including stroke (Gleichman and Carmichael, 2014;
Hersh and Yang, 2018;Jha et al., 2019).
The Endocannabinoid System
The endocannabinoid system (ECS) constitutes an intercellular
communication system that plays a fundamental role in the
regulation of multiple physiological processes such as synaptic
transmission, memory processes, nociception, inammation,
appetite, or thermoregulation, among others (Fernández-Ruiz
et al., 2015;Cristino, Bisogno and Di Marzo, 2020;Estrada and
Contreras, 2020). Consequently, the ECS and the elements that
constitute it (receptors, endogenous ligands, and synthesis and
breakdown enzymes) play a key role in neurotransmission, in the
endocrine and the immune system.
Cannabinoids (CBs) exert their effects mainly via cannabinoid
receptor 1 (CB
1
R) and cannabinoid receptor 2 (CB
2
R) (Matsuda
et al., 1990;Munro et al., 1993). CB
1
R is widely expressed in the
central nervous system (CNS), mostly in neurons but also in glial
cells, while CB
2
R is characteristic of the immune system, being
expressed as well by CNS cells like microglia, astrocytes and
oligodendrocytes (Munro et al., 1993;Howlett, 2002;Molina-
Holgado et al., 2002;Gulyas et al., 2004;Núñez et al., 2004;Benito
et al., 2007;Navarrete and Araque, 2008;Turcotte et al., 2016;
Fernández-Trapero et al., 2017). CBs also activate other receptors
such as orphan G protein-coupled receptors (GPCRs),
peroxisome proliferator-activated receptors (PPARs), or the
adenosine A
2A
receptor (A
2A
R) (Morales and Reggio, 2017;
Franco et al., 2019;Iannotti and Vitale, 2021).
The main endogenous ligands or endocannabinoids (eCBs)
are 2-arachidonoylglycerol (2-AG) (Mechoulam et al., 1995;
Sugiura et al., 1995) and N-arachidonoylethanolamine or
anandamide (AEA) (Devane et al., 1992). Both eCBs are
synthesized on demandfrom membrane lipid precursors. 2-
AG is the most abundant eCB and a full CB
1
R/CB
2
R agonist
(Sugiura et al., 1995;Stella et al., 1997). It is synthesized by the
enzyme diacylglycerol-lipase (DAGL) (Tanimura et al., 2010;
Shonesy et al., 2015) and metabolized to arachidonic acid and
glycerol by monoacylglycerol lipase (MAGL) (Dinh et al., 2002).
By contrast, AEA is a partial CB
1
R agonist and it does not bind to
CB
2
R(Silva et al., 2013;Zou and Kumar, 2018). AEA has a
complex synthesis mechanism involving the action of the enzyme
N-arachidonoyl phosphatidylethanolamine-phospholipase D
(NAPE-PLD) (Di Marzo et al., 1994;Blankman and Cravatt,
2013), meanwhile, its degradation is carried out by the enzyme
fatty acid amide hydrolase (FAAH), which metabolizes AEA to
arachidonic acid and ethanolamide (Zou and Kumar, 2018).
Role of the ECS in Ischemic Stroke
Similar to other neuropathologies, several studies have proved
that the ECS is altered in ischemic stroke, as it has been reviewed
elsewhere (Hillard, 2008;Fernández-Ruiz et al., 2015;Kolb et al.,
2019;Cristino et al., 2020). However, contradictory and
conicting results have been found and to date, the role of the
ECS in stroke has not been elucidated.
Endocannabinoid tone alterations have been reported in
clinical studies and plasma levels of AEA were signicantly
elevated in samples from acute stroke patients (Schäbitz et al.,
2002;Naccarato et al., 2010). Moreover, higher levels of 2-AG and
other ECS-lipid mediators, such as palmitoylethanolamide
(PEA), are positively correlated with neurological impairment
(Naccarato et al., 2010). Very recently, an increased expression of
CB
2
R and the microRNA miR-665, a potential CB
2
R regulator,
were found in circulating monocytes of patients with acute
ischemic stroke (Greco et al., 2021). These observations at the
peripheral level could be reecting disturbances at the central
level, in line with those observed in postmortem tissues (Caruso
et al., 2016), or suggesting its involvement in the modulation of
the peripheral immune response in stroke patients (Greco et al.,
2021).
The involvement of the ECS in the pathophysiology of stroke
is even more evident in animal models (Schäbitz et al., 2002;
Muthian et al., 2004;Zarruk et al., 2012;Sun, and Fang, 2013). In
the transient middle cerebral artery occlusion (tMCAO) model
using CB
1
R
/
mice, a greater lesion volume was observed than in
wild-type animals due to a decrease in CBF after reperfusion,
probably due to a direct effect of CB
1
R activation on
cerebrovascular smooth muscle cells (Hillard, 2000;
Parmentier-Batteur et al., 2002). However, the administration
of pharmacological treatments aimed at modulating CB
1
R
function show controversial results. On the one hand, several
works have shown that CB
1
R antagonism has neuroprotective
effects in animal models of stroke (Muthian et al., 2004;Zhang
et al., 2008;Schmidt et al., 2012;Knowles et al., 2016;Reichenbach
et al., 2016). For example, the treatment with CB
1
R antagonists
such as SR141716 (5 mg/kg) increases CBF in the affected brain
area, decreases the lesion volume in both the tMCAO and the
photothrombotic permanent MCAO (pMCAO) models, and
improves the neurological function after stroke (Zhang et al.,
2008;Reichenbach et al., 2016). In a rat model of global brain
ischemia, the treatment with the CB
1
R antagonist AM251
(2 mg/kg) also shows neuroprotective effects on areas of the
reward system, reducing neuronal death and improving
behavioral test performance (Knowles et al., 2016). On the
other hand, CB
1
R activation with the selective CB
1
R agonist
ACEA, both after intracerebral and intraperitoneal (i.p.)
administration (10 μM and 1 mg/kg, respectively), has also
shown neuroprotective effects in the endothelin-induced
MCAO (eMCAO) and pMCAO models, reducing neuronal
death and brain injury volume (Schmidt et al., 2012;Caltana
et al., 2015). Regarding the role of CB
2
R in ischemic stroke, there
seems to be a greater consensus, since the majority of studies
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Vicente-Acosta et al. Glial Endocannabinoid System in Stroke
report a neuroprotective effect i.e., a reduction of infarct volume
when CB
2
R agonists are administered in different animal models
(Schomacher et al., 2008;Zhang et al., 2008;Reichenbach et al.,
2016;Choi et al., 2018).
Although the role of the ECS in stroke may appear more
complex than in other neurological pathologies, CB-based
therapies begin to acquire special relevance for patients
suffering from ischemic stroke (Kolb et al., 2019;Cristino
et al., 2020). One of the main features of CB-based therapies is
that they are multi-target molecules able to regulate the three
main pathological mechanisms involved in neurodegenerative
diseases, especially in ischemic stroke: inammation,
excitotoxicity, and oxidative stress. These effects could be
mediated not only by CB
1
R and CB
2
R but other ECS-related
receptors may also be involved, such as PPARγor G-protein
receptor 55 (GPR55) (Fernández-Ruiz et al., 2015;Aymerich
et al., 2018). Although considerable progress has been made in the
study of the ECS in neurons, there is also extensive evidence
supporting the important modulatory role of the glial ECS for the
proper function of these cells and their interactions with other cell
types. However, the precise role of glial ECS remains a eld barely
explored but with great potential in stroke and other neurological
pathologies (Egaña-Huguet et al., 2021;Jadhav et al., 2022).
MICROGLIA
Microglia, discovered in 1919 by the Spanish physician and
histologist Pio del Rio-Hortega, constitute the rst line of
defense of the CNS (Mecha et al., 2016;Prinz, Jung and
Priller, 2019). Microglia are highly dynamic cells included in
the phagocytic-mononuclear cell lineage along with peripheral
and CNS-associated macrophages (CAMS), monocytes, and
dendritic cells (Ginhoux et al., 2010;Goldmann et al., 2016;
Mecha et al., 2016;Prinz et al., 2019). However, there is currently
an intense debate about its origin, since recent publications
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 activatio n 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 signicant 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-inammatory 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-inammatory
cytokines and ROS that contribute to exacerbate neuronal death, oligodendrocyte damage and astrocyte activation. The inammatory 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 CB
1
R and CB
2
R as well as other ECS-related receptors has been
upregulated in different in vitro and in vivo stroke models. However, its specic 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-
inammatory 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; CB
1
R: cannabinoid receptor 1: CB
2
R: cannabinoid receptor 2; CBs: cannabinoids;
eCBs: endocannabinoids; ROS: reactive oxygen species.
Frontiers in Pharmacology | www.frontiersin.org June 2022 | Volume 13 | Article 8882224
Vicente-Acosta et al. Glial Endocannabinoid System in Stroke
suggest that microglial cells originate in the yolk sac from myeloid
progenitors during embryonic development whereas peripheral
macrophages develop from hematopoietic stem cells (Prinz, Jung
and Priller, 2019). After embryogenesis, microglia maintain a
population of 520% of total glial cells in the mouse brain and
around 0.5%16.6% in the human brain by a process of self-
renewal (Mittelbronn et al., 2001;Ginhoux et al., 2010;Askew
et al., 2017). Long considered the macrophages of the brain,
microglia have multiple functions in physiological and
pathophysiological conditions. Very recently, it has been
shown that mouse and human microglia also exhibit regional
phenotypic heterogeneity (Böttcher et al., 2019), however,
whether this heterogeneity correlates with a regional-specic
function or if this is relevant for different pathologies, remains
to be investigated.
Under physiological conditions, microglial cells participate in
important functions like dendritic pruning, neural rewiring,
oligodendrocyte precursor cells (OPCs) differentiation, and
synaptic plasticity, among other cellular processes (Figure 1A)
(Nakajima et al., 2001;Wake et al., 2009;Matcovitch-Natan et al.,
2016;Filipello et al., 2018). In vivo imaging studies have
demonstrated that resting microglia show a small soma with
highly dynamic branching morphology, acting as sensors that
detect changes in the brain parenchyma. Following an acute
injury, microglia are activated, changing their morphology to
an amoeboid shape and modifying their branching pattern that
rapidly are directed towards the lesion site (Davalos et al., 2005;
Nimmerjahn et al., 2005). Changes also occur at the molecular
level, including epigenetic, transcriptomic, and proteomic
modications (Mecha et al., 2015;Prinz et al., 2019).
Moreover, once activated after a harmful event in the CNS,
microglia undergo the process of phenotypic polarization,
shifting toward one of two main opposite phenotypes: the
classically activated pro-inammatory state (M1-like) or the
alternative anti-inammatory protective state (M2-like). In
addition, similarly to the phenotypic classication of
macrophages, within the M2-like phenotype, different
microglial subtypes (M2a, M2b, and M2c) have been
associated with repairing, immunoregulatory, or deactivating
phenotype functions, respectively (Zawadzka et al., 2012;
Mecha et al., 2016;Kanazawa et al., 2017). However, because
most studies have been performed in cell culture, further in vivo
studies are needed to establish not only the function of the
different subtypes of the microglial M2-like phenotype, but
even their existence in the pathophysiological context, as
recently reviewed by Tanaka et al. (2020).
The phenotypic polarization of microglial cells to M1 upon
stimulation with bacterial-derivative molecules such as
lipopolysaccharide (LPS) or even interferon gamma (IFN-γ)
has been characterized in vitro. Under these conditions, M1
cells release a wide variety of pro-inammatory cytokines and
chemokines like TNF-α, IL-1α, IL-1β, IL-6, or IL-12 (Michelucci
et al., 2009;Zawadzka et al., 2012;Malek et al., 2015;Mecha et al.,
2015). Polarization to M1 also induces the expression of genes
such as iNOS, ROS production, and the activation of the
inammasome complex (Shi et al., 2012;Gong et al., 2018).
Stimulation with anti-inammatory cytokines, i.e. IL-4 or IL-10
promotes a polarization toward the M2-like phenotype
(Michelucci et al., 2009;Lively and Schlichter, 2013). Other
cytokines and certain chemokines, including IL-3, IL-21,
CCL2, and CXCL4, also induce M2 polarization. M2 cells in
turn release anti-inammatory cytokines such as IL-4, IL-10, IL-
13, or TFG-β(Michelucci et al., 2009;Mecha et al., 2015).
The role of the different microglial phenotypes has been the
subject of intense study in recent years and is becoming
increasingly relevant given the dual functions of these cells in
pathophysiological processes associated with acute and chronic
diseases, including ischemic stroke (Kanazawa et al., 2017;Qin
et al., 2019).
Microglial Function in Ischemic Stroke
The contribution of microglia to the neuroinammatory context
of ischemic stroke is controversial, as microglial cells could exert
both detrimental and benecial effects (for review consult Qin
et al., 2019). On the one hand, post-ischemic inammation has
been considered a negative factor that worsens patient outcome,
since activated microglia carry out striping processes that disrupt
synaptic connections resulting in the functional impairment of
neuronal circuits after ischemic damage (Wake et al., 2009). But
on the other hand, it seems to be a necessary process for the
clearance of cellular debris and dead cells through phagocytosis
and to trigger repairing processes that promote functional brain
recovery (Ma et al., 2017;Qin et al., 2019;Rajan et al., 2019).
Following an ischemic stroke, activated microglial cells change
their morphology and rapidly migrate to the focus of injury as
they are sensitive to uctuations in blood ow and respond to
BBB rupture and to cell death occurring in the acute phase of
stroke (Nimmerjahn et al., 2005;Masuda et al., 2011;Ju et al.,
2018). Besides, in response to damage, CNS resident microglia
continuously proliferate, contributing with new cells to the
resident microglial pool (Li et al., 2013). The extravasation
and migration of peripheral immune cells (Iadecola et al.,
2020), as well as the mobilization of pericytes close to the
injury, also increase the microglial pool site (Özen et al., 2014;
Roth et al., 2019). Despite monocyte extravasation in stroke has
recently gained a considerable amount of interest, the specic
contribution of individual cell types to the progression or repair
of ischemic damage is still under intense study (Urra et al., 2009;
Rajan et al., 2019). Overall, the microglial response to ischemic
stroke is very complex and follows a spatio-temporal pattern.
First, studies in animal models of both permanent and transient
ischemia, have demonstrated a dramatic increase in microglial
cells between 24 h and 7 days post-ischemia (Michalski et al.,
2012;Li et al., 2013;Cotrina et al., 2017;Rajan et al., 2019). This
peak in cell number in the ischemic core appears later in models
of photothrombotic ischemia, being a slightly more moderate
response (Li et al., 2013;Cotrina et al., 2017). Furthermore, in a
tMCAO model, M2-like microglia is greatly increased in the
ischemic zone, probably as an immediate response to neuronal
damage that tries to eliminate cellular debris and limiting the
extent of tissue damage (Figure 1B)(Hu et al., 2012). Soon after
microglial activation in the pMCAO model, phagocytic
microglial cells enclosing MAP2-positive neurons are observed
(Cotrina et al., 2017). However, this context changes during the
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Vicente-Acosta et al. Glial Endocannabinoid System in Stroke
chronic phase of stroke in which M1 microglial cells proliferate
and are recruited. M1 microglia release pro-inammatory
cytokines and ROS that contribute to exacerbating neuronal
death, BBB breakdown, and also have a reduced phagocytic
capacity that prevents tissue repair (Figure 1B)(Hu et al.,
2012;Chen et al., 2019). Phenotypic changes of microglia are
also region-specic, with amoeboid-shaped cells being located in
the core and penumbra of the lesion and less branched cells in the
peri-infarct zone (Li et al., 2013;Cotrina et al., 2017;Rajan et al.,
2019).
Activated microglia orchestrate the response to ischemic
damage by communicating not only with neurons but also
with non-neuronal cells and BBB structural components
(Mecha et al., 2016;Huang et al., 2020). In fact, the
interaction between activated microglia and astrocytes plays a
crucial role in the process of neuroinammation. The release of
cytokines and trophic factors by microglia promotes phenotypic
change in astrocytes, thus, M1 microglia releases, among other
factors TNF-α, IL-1βor C1q, favoring the neurotoxic reactivity
state of astrocytes (Liddelow et al., 2017). This communication is
bidirectional, such that astrocytes also inuence microglial
phenotypic changes in a neuroinammatory context by
secreting a wide range of chemokines (for review, Jha et al.,
2019;Liu et al., 2020). Besides, activated microglia also interact
with oligodendrocytes, with a vast amount of data suggesting a
deleterious effect of M1 microglia and the pro-inammatory
cytokines they release, on oligodendrocyte survival (Moore
et al., 2015;Fan et al., 2019). On the other hand, in multiple
sclerosis models, an increased differentiation of OPCs and,
therefore, activation of remyelination processes favored by
M2-like microglia has been described (Miron et al., 2013).
This oligodendrocyte-protective effect has also been observed
in an animal model of bilateral common carotid artery stenosis,
where the treatment with the immunomodulatory drug
Fingolimod promotes the polarization of microglia towards the
M2-like phenotype leading to increased survival of OPCs and
favoring myelin repair processes (Qin et al., 2017). In light of the
complex microglial response in stroke and the dual effect of the
phenotypes described, the search for new therapeutic options
with a modulating effect on cell polarization in stroke has
intensied in recent years (Qin et al., 2017;Liu et al., 2019;Lu
et al., 2021).
Microglia and the ECS
Under physiological conditions, both in animals and humans,
microglia hardly express CB
1
R and CB
2
R(Benito et al., 2007;
López et al., 2018;Egaña-Huguet et al., 2021). However,
numerous studies show that the expression pattern of both
receptors are altered in microglial cells in neuropathological
conditions, e.g., Alzheimers disease (Benito et al., 2003;López
et al., 2018), multiple sclerosis (Benito et al., 2007), Downs
syndrome (Núñez et al., 2008), spinocerebellar ataxia
(Rodríguez-Cueto et al., 2014), immunodeciency virus
infection (Benito, 2005) or Huntingtons disease (Palazuelos
et al., 2009).
In general, in an in vivo neuroinammatory context, an
increase in CB
2
R levels is associated mostly with the presence
of microglia around neuropathological hallmarks, e.g., protein
aggregates (Benito et al., 2007;Núñez et al., 2008;López et al.,
2018). However, in vitro studies show the complexity of the
microglial response since microglial activation and polarization
seem to vary depending on the stimulus used, the manipulation of
the cell culture, or even the intrinsic heterogeneity of these cells
(Pietr et al., 2009;Mecha et al., 2016;Gosselin et al., 2017). A few
years ago, Mecha and others demonstrated changes in the
different constituents of the ECS when microglia polarization
proceeds in vitro. The classical activation of rodent microglia with
LPS induces a downregulation not only of CB
1
R and CB
2
R but
also of the eCB synthesis and degradation enzymes (Malek et al.,
2015;Mecha et al., 2015). By contrast, alternative activation,
which polarizes microglia towards the M2-like phenotype,
upregulates the expression of CB
2
R and the eCB synthesis
enzymes. Consequently, M2 microglia are able to produce and
release 2-AG and AEA in greater quantities than in the resting
state (Walter et al., 2003;Mecha et al., 2015). However, the use of
other stimuli, such as IFNɣ, does not seem to inuence the
expression of CB
2
R(Carlisle et al., 2002;Maresz et al., 2005).
Finally, activation of CB
2
R regulates pivotal functions of
microglia, such as their migration capacity (Walter et al., 2003;
Guida et al., 2017), phagocytosis (Tolón et al., 2009), and cytokine
release (Malek et al., 2015)(Figure 1A).
Regarding CB
1
R, it has been recently demonstrated that the
human microglial cell line N9 expresses this receptor in the
resting state. Although no changes in CB
1
R expression levels
are detected after stimulation with LPS and IFNɣ, proximity
ligation assays show that CB
1
R-CB
2
R heterodimers are formed
following the inammatory stimulus (Navarro and Borroto-
Escuela, 2018). Another study has shown that despite low
CB
1
R expression, the treatment with the selective CB
1
R
antagonist SR141716A (1 μM) induces the polarization of BV-
2 microglia towards the M1 phenotype. Moreover, the use of this
antagonist prevents the anti-inammatory effects of the non-
selective cannabinoid agonist, WIN55,212-2 (1 μM) (Lou et al.,
2018). All these data could suggest the involvement of CB
1
Rin
microglial polarization and function, raising the possibility of its
pharmacological manipulation to modulate the inammatory
response in neurological diseases.
Microglia also express other non-CB receptors through which
certain CBs can exert their effects. This is the case of PPARs,
which seem to play a relevant role in microglial polarization (Ji
et al., 2018). In particular, PPARαcan be activated in vitro by the
eCB-like compound PEA, leading to an increase in CB
2
R
expression and 2-AG production (Guida et al., 2017), and the
migration capacity of these cells (Franklin et al., 2003;Guida et al.,
2017). The orphan receptor GPR55 is also expressed in microglia
and is attracting special attention in different neuroinammatory
pathologies (Marichal-Cancino, 2017;Saliba et al., 2018;Burgaz
et al., 2021). This receptor appears to follow a similar expression
pattern to CB
2
R when microglia are stimulated with LPS.
However, differences have been observed between the use of
cell lines and primary microglial cultures. These differences are
probably due to an intrinsic heterogeneity in the microglia cell
lines used, or even to the possibility that in primary cultures,
microglia are in a primed state due to the handling necessary for
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Vicente-Acosta et al. Glial Endocannabinoid System in Stroke
the development of in vitro assays (Pietr et al., 2009;Saliba et al.,
2018). GPR55 blockage has anti-inammatory effects by reducing
prostaglandin production by microglia following LPS stimulation
(Saliba et al., 2018). Finally, GPR55 activation by 2-AG or the
synthetic cannabinoid abnormal-cannabidiol (abn-CBD),
promotes BV-2 cell activation and migration (Franklin and
Stella, 2003;Walter et al., 2003;Ryberg et al., 2007).
Microglial ECS Pharmacological
Modulation in Stroke
Although the molecular mechanisms involved in CB-based
neuroprotection are still not known in detail, we do know that
microglia and microglial ECS play a relevant role in stroke. Due to
the role of CB
2
R on microglial migration and polarization and
also on extravasation of peripheral immune cells to the CNS, the
study of this receptor has received a great interest in stroke
(Zhang et al., 2008;Hosoya et al., 2017;Greco et al., 2021). Several
reports have shown an increased expression of CB
2
R in the
ischemic penumbra in tMCAO, eMCAO, and also in both
adult and neonatal hypoxia-ischemia (HI) murine models
(Ashton et al., 2007;Zhang et al., 2008;Fernández-López
et al., 2012;Schmidt et al., 2012). This upregulation occurred
in macrophage-like cells that could be resident microglia or
inltrated peripheral monocytes (Ashton et al., 2007;Schmidt
et al., 2012). A more recent study, using a model of
photothrombotic ischemia combined with positron emission
tomography and histological techniques, describes an early
increase in CB
2
R expression in the peri-infarct area that
colocalizes with some microglial cells showing amoeboid
morphology. They even detect CB
2
R in branched microglia of
the contralateral hemisphere that could be in a primed state,
highlighting the role of this receptor in the different states of
microglial activation (Hosoya et al., 2017). Pharmacological CB
2
R
activation using selective agonists leads to a reduction in lesion
volume and cognitive improvement in different animal models of
stroke (Ashton et al., 2007;Zhang et al., 2008;Schmidt et al., 2012;
Ronca et al., 2015). In addition, improved regional
microcirculation in the affected area, decreased leukocyte
rolling and extravasation, and preserved BBB integrity (Zhang
et al., 2007,2008). The involvement of microglial CB
2
R in BBB
preservation has also been studied in animal models of
intracerebral hemorrhage and traumatic brain injury. In these
studies, selective CB
2
R activation reduces the release of pro-
inammatory cytokines by microglia and upregulates the
expression of molecules necessary for the maintenance of tight
junctions such as zo-1 or claudin-5, which are essential for BBB
integrity (Figure 1C)(Amenta et al., 2012;Li et al., 2018).
Recently, the protective and neuroinammatory-modulating
potential of β-caryophyllene (BCP), a terpene derived from
Cannabis sativa, which acts as a CB
2
R agonist, has been
demonstrated. In a model of photothrombotic ischemia, the
treatment with BCP alone or in combination with cannabidiol
(CBD), the main non-psychoactive constituent of Cannabis
sativa, reduced the infarct area in a dose-dependent manner,
and modulated both the number and morphology of microglial
cells (Yokubaitis et al., 2021).
CB
1
R expression is also altered in stroke patients and in
animal models (Zhang et al., 2008;Schmidt et al., 2012;
Caltana et al., 2015;Caruso et al., 2016). A study performed in
postmortem samples from patients revealed increased CB
1
R
immunohistochemical labeling in the ischemic area. This
pattern was associated both with neuronal and non-neuronal
cells suggesting a role of CB
1
R in the glial neuroinammatory
response following acute ischemic damage (Caruso et al., 2016).
However, the use of CB
1
R agonists and antagonists in different
animal models of stroke have shown controversial results as
previously explained. These results could be explained by the
diversity of animal models used that may affect differently the
receptors abundance after ischemic injury, to CB
1
R
desensitization effects depending on the ligand used and the
dose, or even by the different roles played by this receptor
depending on the cell type where it is expressed. To date, little
is known about the specic role of CB
1
R in microglia in the
context of ischemic stroke. In the tMCAO model, an early and
modest increase in CB
1
R expression has been described in
microglia after ischemic damage in the ipsilateral hemisphere
(Schmidt et al., 2012). Moreover, CB
1
R activation by 1 mg/kg i.p.
administration of ACEA in a pMCAO model, not only reduces
lesion volume, but also reduces glial reactivity, by decreasing the
number of lectin-positive cells. Notably, it also reduced the
number of microglial cells with amoeboid morphology in
favor of cells with a more branched morphology, both in the
short and long term (Caltana et al., 2015). These data, together
with those previously mentioned (Lou et al., 2018;Navarro and
Borroto-Escuela, 2018), indicate that the study of this receptor
and its function in relation to the phenotypic polarization of
microglia should be further explored.
In recent years, CBD has gained special importance in the
context of ischemic brain injury (Hayakawa et al., 2010;
Fernández-Ruiz et al., 2015;Mori et al., 2017,2021;Martínez-
Orgado et al., 2021;Yokubaitis et al., 2021;Khaksar et al., 2022).
CBD is a multitarget molecule with a complex pharmacology.
Although it initially showed a low afnity for CB receptors, it has
subsequently been shown that it can act as an antagonist of CB
1
R
and CB
2
R at low concentrations (Pertwee, 2008;Navarro and
Reyes-Resina, 2018). Noteworthy, CBD also has an afnity for
other ECS-related receptors such as GPR55, 5-HT
1A
, TRPA1,
TRPV1-4 or PPARɣ(Pertwee, 2008;Britch et al., 2021). In the
different animal models of stroke used, it has been shown that
CBD treatment improves the motor decits observed after
ischemic damage and reduces the area of injury (Hayakawa
et al., 2004;Schiavon et al., 2014;Ceprián et al., 2017;Mori
et al., 2017). In adult animals, CBD facilitates neuroplasticity after
tMCAO by decreasing glial reactivity, reducing both the number
of reactive microglia and astrocytes in the hippocampus, and
favoring the release of neurotrophic factors, such as brain-derived
neurotrophic factor (Schiavon et al., 2014;Mori et al., 2017).
Interestingly, neuroprotective effects of CBD have also been
observed in a neonatal HI stroke model, both in the short and
long term. Besides, there is an improvement in the performance
of motor tests despite the fact that no decrease in lesion volume is
observed. In the same study, administration of 5 mg/kg of CBD,
also decreased glial reactivity, decreasing the number of
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Vicente-Acosta et al. Glial Endocannabinoid System in Stroke
microglial cells in the ipsilateral hemisphere (Ceprián et al.,
2017). These results are similar to those obtained in neonatal
models of HI in piglets and in rodents. In those studies, 1 mg/kg
of CBD, shows neuroprotective effects by decreasing neuronal
death and anti-inammatory effects by modulating cytokine
release and decreasing the number of reactive astrocytes and
microglia after HI injury (Figure 1C)(Lafuente et al., 2011,2016;
Pazos et al., 2012,2013;Mohammed et al., 2017;Barata et al.,
2019). CBD appears to modulate microglial polarization by
promoting a less amoeboid and more branching phenotype
(Mohammed et al., 2017;Barata et al., 2019). Several pieces of
evidence demonstrate the multitarget effect of CBD in stroke and/
or HI models involving the CB
1
R, CB
2
R, GPR55, 5-HT
1A
, and
PPARɣreceptors (Mishima et al., 2005;Castillo et al., 2010;Pazos
et al., 2013;Mori et al., 2021). Since microglia express most of
these receptors, this strengthens the idea of the important role
played by the ECS in the polarization, cell renewal and migration
of microglia particularly in the context of stroke. However, there
are still many unknowns about the precise role of the ECS in
microglial polarization and function and the molecular
mechanisms involved in those processes, which must be
addressed to nd new CB-based therapies for stroke treatment.
ASTROCYTES
Astrocytes are one of the most numerous cell populations in the
CNS, where they exert many crucial homeostatic functions that
allow the development and proper function of this system and the
brain cells (Sofroniew and Vinters, 2010;Clarke and Barres, 2013;
Verkhratsky and Nedergaard, 2018). These functions include the
regulation of extracellular concentrations of ions and
neurotransmitters, the formation and elimination of synapses,
cytokine and neurotrophin secretion, CBF and metabolism
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 Ca
2+
both in post-
synaptic terminals and in astrocytes. In post-synaptic neurons, Ca
2+
elevation induces eCB release to the extracellular space, inhibiting neurotransmitter release
from the presynaptic neuron. In astrocytes, eCB binding to their receptors mobilize Ca
2+
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 Ca
2+
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 signicant
morphological and functional changes like hypertrophy, hyperplasia and increased GFAP levels. To increase neuronal survival, astrocytes release neurotrophic factors
and anti-inammatory cytokines. Nevertheless, they also release pro-inammatory cytokines that negatively affec t neuronal survival. Moreover, at the neurovascular unit,
astrocytes upregulate the expression of surface receptors and enzymes that are strongly associated with inammatory responses that actively contribute to BBB
disruption and leukocyte inltration into the CNS, becoming a source of inammation 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; CB
1
R: cannabinoid receptor 1: CB
2
R: cannabinoid
receptor 2; CBs: cannabinoids; CSF: cerebrospinal uid; eCB: endocannabinoids; ROS: reactive oxygen species.
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Vicente-Acosta et al. Glial Endocannabinoid System in Stroke
regulation, among others (Figure 2A)(Sofroniew, 2020).
Classically, astroglial cells have been classied in two different
groups according to their location and morphology with
protoplasmic astrocytes located mainly in gray matter, and
brous astrocytes predominantly found in white matter (Miller
and Raff, 1984). However, over the last years, increasing evidence
has changed this conception and now it is acknowledged that
astrocytes are highly heterogeneous, exhibiting important
morphological and physiological differences among brain
regions and signicant differences in gene expression and
protein content (Molofsky et al., 2012;Höft et al., 2014;John
Lin et al., 2017;Miller, 2018). Thus, astrocyte functions vary
depending on the neural populations they are associated with
and/or the biological environment surrounding them. Moreover,
astrocyte heterogeneity is species-dependent, with higher
morphological and possibly functional complexity in the
human brain compared to rodents (Kettenmann and
Verkhratsky, 2008). In the healthy brain, astroglial cells
provide structural support for neurons, actively participating
in the regulation of neuronal growth and synapse formation,
maturation, maintenance, and pruning (Sofroniew and Vinters,
2010;Clarke and Barres, 2013). They also play an active role in
synaptic transmission, by being part of the tri-partite synapse,
they support neuronal signaling, neurotransmitter uptake
regulation, gliotransmitter and calcium release, modulating in
this way synaptic plasticity and learning (Pereira and Furlan,
2010;Sofroniew and Vinters, 2010;Verkhratsky et al., 2012). One
important function of astrocytes is their involvement in the
maintenance and functionality of the BBB, particularly via
astrocyte endfeet, together with endothelial cells and pericytes
(Alvarez et al., 2013;Siqueira et al., 2018). Astrocytes are
responsible for the selective diffusion of molecules through the
BBB, allowing ion diffusion and regulating the entry of small
molecules and water to the CNS. At the same time, these cells
regulate the supply of oxygen and nutrients to neurons by taking
up glucose, lactate, or ketone bodies from the bloodstream and
transferring them to neurons as a source of energy, and/or by
releasing trophic factors that are essential for neuronal survival
(Rouach et al., 2008;Alvarez et al., 2013;Dezonne et al., 2013;
Sotelo-Hitschfeld et al., 2015;Benarroch, 2016). In addition, these
cells directly regulate BBB function by releasing molecules i.e.
sonic hedgehog, nitric oxide, and vascular endothelial growth
factor, which are involved in tight junction development,
vasodilation, and angiogenesis (Nian et al., 2020).
In homeostatic conditions, astrocytes are in a quiescent or
resting state and become reactive in response to different stimuli
or insults to the CNS like infections, trauma, neurodegenerative
diseases, and stroke (Moulson et al., 2021). Astrocyte reactivity is
in the rst instance a physiological response that involves
phenotypic and molecular changes aimed at restoring
homeostasis and neurological function through diverse
mechanisms (Sofroniew, 2020). However, in pathological
conditions, these cells have biphasic functions, being benecial
or detrimental through cell-autonomous or non-cell-
autonomous mechanisms, depending on the biological context.
For example, if the initial insult is not resolved and becomes
chronic, astrocytes can contribute to exacerbating the damage
either by losing/gaining functions (Sofroniew, 2020). Recently, it
was demonstrated the existence of at least two different types of
reactive astrocytes (Zamanian et al., 2012;Liddelow et al., 2017).
Under neuroinammatory conditions, astrocytes polarize toward
an A1-neurotoxic reactivity state, expressing different pro-
inammatory proteins and possibly other toxic molecules that
induce synapse loss and neuronal death, whilst A2-
neuroprotective reactive astrocytes are induced after an
ischemic insult and promote neuronal survival (Liddelow
et al., 2017;Guttenplan et al., 2021). Nevertheless, late
discoveries on regional and local heterogeneity of astrocytes
are shedding light on their complex developmental,
morphological, molecular, physiological, and functional
diverseness, modifying this dual classication of astrocytes (for
a review on this topic see (Pestana et al., 2020). Newer hypotheses
sustain the existence of mixed populations (subtypes) of astroglial
cells that coexist in the resting state with a continuum in the
intensity of reactivity states (Miller, 2018;Khakh and Deneen,
2019;Pestana et al., 2020;Sofroniew, 2020). Thus, the existence of
different astrocyte subtypes in the resting state could explain
different responses to the same insult, resulting in a variety of
reactive states, an idea supported by data from various
neurodegenerative disease models (Clarke et al., 2018;Yun
et al., 2018;Smith et al., 2020). As the knowledge of astrocyte
biology is constantly evolving due to the development and
availability of new tools/experimental approaches, like single-
cell transcriptomics, which provides valuable data on astrocyte
heterogeneity and reactivity (Moulson et al., 2021), we could
expect more rened and possibly unied concepts in the
forthcoming years, as recently discussed by Escartin et al. (2021).
Astrocyte Function in Ischemic Stroke
Similar to what occurs with other CNS cells, astrocytes undergo
signicant morphological, molecular, and functional
modications after an ischemic event (Figure 2B). These
changes are very dynamic and rely not only on astrocytes but
also on interactions and intercommunication with other CNS
cells, notably neurons, microglia, and oligodendrocytes. In
pathological circumstances like stroke, injured neurons and
other cells communicate with astrocytes by releasing cytokines
and other molecules, triggering astrocyte activation and causing
profound changes in the synthesis and expression of other
molecules (Sofroniew, 2009;Scarisbrick et al., 2012). After the
stroke, there is a massive response of astrocytes, called reactive
astrogliosis, but the timeline of astroglial activation is slower than
in neurons or in microglia (Revuelta et al., 2019). Reactive
astrogliosis is characterized by an increased expression of the
glial brillary acidic protein (GFAP) and changes in cell
morphology like hyperplasia and hypertrophy (Sofroniew,
2009;Scarisbrick et al., 2012;Kozela et al., 2017). Moreover,
activated astrocytes release pro-inammatory cytokines, like IL-
1β, IL-6, and TNF-, modulating the immune response and
actively participating in the inammatory process initiated
after an ischemic event (Zamanian et al., 2012). Reactive
astrocytes also synthesize and release some anti-inammatory
cytokines and neurotrophic factors that protect neurons, enhance
neuronal synapses and plasticity, and improve functional
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Vicente-Acosta et al. Glial Endocannabinoid System in Stroke
outcomes after the stroke (Swanson et al., 2004;Li et al., 2008;
Cekanaviciute et al., 2014). Noteworthy, astrocyte response after
an ischemic event will signicantly depend on the astrocyte
subtype and possibly on the brain region affected. On the one
hand, proliferative reactive astrocytes will increase their number
and form limitant borders surrounding the infarcted area,
constituting in conjunction with other cells a physical barrier
around the necrotic tissue in the brain. These limitant borders
allow setting boundaries to the damaged area, releasing molecules
that promote neuronal growth and survival, and avoiding the
spreading of neuroinammation (Swanson, Ying and Kauppinen,
2004;Li et al., 2008;Huang et al., 2014;Sofroniew, 2020).
However, these densely packed reactive astrocytes are also
considered a source of pro-inammatory molecules, ROS, and
neurotoxicity that ultimately inhibit axonal regeneration (Gris
et al., 2007). On the other hand, nonproliferative reactive
astrocytes acquire diverse reactivity states. In contrast to
microglial cells, which are very mobile, these astrocytes do not
migrate from the penumbra to the ischemic core, instead, they
polarize their processes to be able to exert their phagocytic
abilities (Huang et al., 2014), having as well the ability to
change their gene expression pattern and functions depending
on their particular context. If the acute initial insult is not resolved
and becomes chronic, nonproliferative reactive astrocytes can
contribute to exacerbating the damage either by losing/gaining
functions, as mentioned lines above (Sofroniew, 2020).
Another major event that occurs after stroke is BBB integrity
disruption (Fernández-López et al., 2012;Arba et al., 2017),
which favors ROS generation, the inltration of inammatory
cells like leukocytes, and the production of proteolytic enzymes
that ultimately exacerbate brain edema and neuroinammation
(Figure 2B)(Fernández-López et al., 2012;Arba et al., 2017). The
BBB is constituted by endothelial cells, pericytes, and astrocytes.
Some evidence indicates that changes in astrocytic proteins
involved in BBB maintenance like metalloproteinase-2 and the
toll-like receptor 4/metalloproteinase-9 (TLR4/MMP9) signaling
pathway are upregulated after stroke, contributing to the
disruption of astrocyte-endothelial junctions, consequently
altering BBB permeability (Liu et al., 2017;Rosciszewski et al.,
2018).
There are apparently contradictory roles of reactive astrocytes
after stroke regarding the extent of their putative toxic or protective
effects that could be difcult to measure, but several studies using
GFAP
/
mice in models of stroke and acute trauma are shedding
somelightonthismatter(Nawashiro et al., 2000;Wilhelmsson et al.,
2004;Li et al., 2008). For example, GFAP
/
mice showed an
impaired physiological response to ischemia in the pMCAO with
transient carotid artery occlusion (CAO) model (Nawashiro et al.,
2000), and GFAP
/
Vimentin
/
mice had a higher infarct area and
decreased glutamate transport by astrocytes than wild type mice after
MCA transection (Li et al., 2008). Besides, GFAP
/
Vimentin
/
mice subjected to an acute entorhinal cortex lesion model showed an
attenuated astrocyte reactivity response, evidenced by fewer
processes and dysregulation of endothelin B receptors, which
allowed synaptic recovery in the hippocampus, changes that were
associated with improved post-traumatic regeneration
(Wilhelmsson et al., 2004).
Recently, Rakers and co-authors explored in more detail
astrocyte reactivity in mice subjected to the tMCAO stroke
model and found an upregulation of the canonical markers of
reactive astrogliosis, the so-called pan-reactive transcripts, and a
prominent increase of A2-reactivity specic transcripts (Rakers
et al., 2019). Their observations suggest that these A2-like reactive
astrocytes protect neurons and promote neuroregeneration after
stroke. Moreover, they also observed signicant changes in the
expression of genes related to extracellular matrix composition,
cell migration, cell-cell adhesion, and glial scar formation, further
indicating that A2-astrocytes may help contain and restrict
neuroinammation and support neuronal survival (Rakers
et al., 2019). Nevertheless, among the upregulated genes they
found were several genes related to neuroinammation, the
complement cascade, apoptosis, and leukocyte transendothelial
migration. Thus, they observed the coexistence of genes with
potentially neurotoxic and neuroprotective functions in
astrocytes from brain homogenates of mice subjected to
tMCAO. Whether this phenomenon is due to the presence of
astrocytes with a spectrum of different phenotypes that vary from
neuroprotective to neurotoxic, or due to the activation of
neuroprotective and neurotoxic signaling pathways within
individual astrocytes remains to be determined.
Astrocytes and the ECS
The presence of CB
1
R, CB
2
R, and other CB-like receptors has
been demonstrated in astrocytes (Pazos et al., 2005;Sheng et al.,
2005;Navarrete and Araque, 2008;Stella, 2010;Yang et al., 2019;
Cristino et al., 2020). Besides, these glial cells are able to produce
and release the endogenous ligands 2-AG and AEA and also
express the intracellular degradation enzymes FAAH and MAGL
(Stella et al., 1997;Walter et al., 2002;Vázquez et al., 2015;
Grabner et al., 2016). CB
1
R activation in astrocytes not only
controls their metabolic functions and signaling but also regulates
synaptic transmission, through the tripartite synapse
(Gorzkiewicz and Szemraj, 2018). When the electrical impulse
causes neurotransmitter release from a presynaptic neuron,
depolarization of the postsynaptic neuron occurs, leading to
eCB release into the synaptic cleft and their binding to
receptors located both in neurons and astrocytes (Stempel
et al., 2016). While eCB binding to CB
1
R inhibits
neurotransmitter release in presynaptic neurons, a process
known as retrograde signaling (Stempel et al., 2016), it
increases intracellular Ca
2+
levels in neighboring astrocytes
(Navarrete and Araque, 2010;Covelo and Araque, 2016). This
Ca
2+
increase stimulates glutamate release from astrocytes, which
in turn causes a synaptic potentiation through mGluR1 receptors
located in the presynaptic neuron. As the intracellular Ca
2+
signal
extends within astrocytes, it stimulates glutamate release in distal
astrocyte regions, modulating in this way the synaptic
transmission of many lateral synapses to the original source of
eCBs (Figure 2A)(Navarrete and Araque, 2010;Covelo and
Araque, 2016). In addition, CB
1
R activation in astrocytes also
contributes to the regulation of CBF and the energy supply to
neurons by increasing the glucose oxidation rate and ketogenesis
(Shivachar et al., 1996;Bermudez-Silva et al., 2010;Stella, 2010;
Jimenez-Blasco et al., 2020). Notably, most perivascular
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Vicente-Acosta et al. Glial Endocannabinoid System in Stroke
astrocytes express CB
1
R, highlighting their importance for CBF
and metabolism (Rodriguez et al., 2001). On the other hand,
despite CB
2
R expression in astrocytes is limited under
physiological conditions, data show a signicant upregulation
of this receptor and the endocannabinoid tone in general upon
different insults. Moreover, it also changes in neuroinammatory
conditions, suggesting an important role of the astroglial ECS in
processes associated with brain damage and/or recovery
(Shohami et al., 2011;Fernández-Ruiz et al., 2015;Cassano
et al., 2017). In this sense, the study of different CBs has
attributed them anti-oxidant and anti-inammatory effects in
experimental models of several pathologies (Cristino et al., 2020).
In astrocytes, CBs regulate astrocyte activation and astrocyte-
mediated neurotoxicity by reducing the release of inammatory
mediators and increasing prosurvival factors (Fernández-Ruiz
et al., 2015;Estrada and Contreras, 2020). In different
experimental settings, ECS modulation in astrocytes reduces
TNF-αand IL-1βlevels, which are upregulated following
various inammatory challenges (Grabner et al., 2016;Labra
et al., 2018;Rodríguez-Cueto et al., 2018;Jia et al., 2020),
suggesting that modulation of these cells with CBs could be
contributing to neuroprotection through non-cell-autonomous
mechanisms.
Astrocyte ECS Pharmacological
Modulation in Stroke
After an ischemic event, astrocytes are more resilient than
neurons, being important for the post-acute phase because
they preserve their viability and remain metabolically active
both at the infarct core and penumbra regions (Thoren et al.,
2005;Gürer et al., 2009). Considering the critical functions of
these cells in the CNS, astrocytes are gaining notoriety as possible
therapeutic targets for different neurological conditions including
hypoxia and/or brain ischemia. At the same time, given their
potent anti-oxidant and immunomodulatory effects, numerous
studies have focused on the neuroprotective effects of CBs, mainly
CBD, for stroke therapy (England et al., 2015). However, limited
evidence is available concerning the mechanisms by which CBs
modulate astrocyte function and astrocyte-mediated effects in the
context of ischemic stroke. Here, we summarize the ndings
regarding the effects of CBs on astrocyte activation in the
tMCAO, pMCAO and related models in adult animals, the HI
model in neonate animals, and the oxygen and glucose
deprivation/re-oxygenation (OGD/R) in vitro model of stroke.
In adult animals subjected to transient or permanent ischemia,
the most consistent outcome in astroglial cells is increased
astrogliosis, i.e., high GFAP immunoreactivity in CNS areas
such as the motor cortex, the striatum, the hippocampus, or
the spinal cord. Astrocytes with longer and wider projections, and
other parameters that suggest a functional impairment of these
cells are also observed (Figure 2B)(Hayakawa et al., 2008,2009;
Schiavon et al., 2014;Caltana et al., 2015;Kossatz et al., 2016;
Ceprián et al., 2017;Jing et al., 2020). The inhibition of stroke-
induced reactive gliosis was also observed in the pMCAO mouse
model at 7 and 28 days after administering 1 mg/kg of ACEA
(Caltana et al., 2015). In that study, CB
1
R expression was
downregulated in ischemic conditions, which could be
contributing to increase inammation, neuronal degeneration,
and astroglial reactivity, suggesting that upregulation of the eCB
tone with ACEA could help revert these deleterious effects
(Caltana et al., 2015). On the other hand, GFAP staining was
signicantly elevated in different brain areas of both adult wild
type and CB
2
R
/
mice after HI (Kossatz et al., 2016), and in rats
subjected to HI in the spinal cord (Jing et al., 2020). In the study
by Jing et al. (2020), i.p. pretreatment of rats with 1 mg/kg of the
CB
2
R selective agonist JWH-133 1 h before ischemia not only
inhibited astrocyte reactivity, determined by GFAP
immunostaining but also reduced perivascular expression of
TLR4/MMP9. Notably, TLR4 upregulation in astrocytes has
been associated with a pro-inammatory reactivity phenotype
in astrocytes and with BBB disruption in the cortical
devascularization brain ischemia model (Rosciszewski et al.,
2018). The TLR4/MMP9-mediated reduction of astrocyte
reactivity after ECS activation via CB
2
R is of special interest
for stroke, as it has been suggested elsewhere that attenuation of
the inammatory process could be neuroprotective after tMCAO
in rats (Piao et al., 2003). Although it remains to be determined
whether the limitation of inammation in those experimental
conditions is mediated by astrocytes. In summary, in addition to
having neuroprotective effects, the administration of different CB
compounds like CBD, ACEA, and JWH-133 at various doses,
duration of administration, and delivery methods prevented the
increase in GFAP immunoreactivity, limiting astrocyte activation
(Hayakawa et al., 2008,2009;Schiavon et al., 2014;Caltana et al.,
2015;Ceprián et al., 2017). In the majority of the aforementioned
studies, the reduction of astroglial activation was observed with
the administration of CBs that act through different receptors.
For instance, while ACEA and JWH-133 are CB
1
R and CB
2
R
agonists, respectively, it has been demonstrated that CBD
preferentially binds to other receptors. Nevertheless, the
precise molecular mechanism(s) by which the activation of the
ECS is able to limit astrogliosis after stroke is not known yet and
remains to be addressed experimentally. Moreover, there is scarce
direct evidence showing that the modulation of astrocyte activity
with CBs increases neuronal survival after stroke. Even so, a
recent study in mice has shown that the increase in the eCB tone
through the inhibition of FAAH and MAGL with the compound
JZL195 (20 mg/kg, i.p.), induces long-term depression (LTD) at
CA3-CA1 synapses in the hippocampus, and confers astrocyte-
mediated neuroprotection after stroke (Wang et al., 2019). In that
study, JZL195-induced LTD was used as a preconditioning
insult to determine its potential neuroprotective effect against
subsequent ischemia. Noteworthy, it was observed that
preconditioning before tMCAO increased the number of
surviving neurons through a mechanism dependent on a
sequential activation of astroglial CB
1
R, and not neuronal
CB
1
R, and postsynaptic glutamate receptors (Wang et al.,
2019).
Over the years, the neuroprotective effects of CBD have been
clearly demonstrated in experimental models of HI in rodents
and notably in newborn pigs (Kicman and Toczek, 2020), but
only a limited number of studies have characterized/evaluated
astroglial reactivity as a neurological outcome. Evidence of GFAP
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Vicente-Acosta et al. Glial Endocannabinoid System in Stroke
immunoreactivity reduction after CBD treatment (5 mg/kg) has
been reported both in newborn rats after tMCAO (Ceprián et al.,
2017), and in newborn mice (1 mg/kg) after HI (Mohammed
et al., 2017). In addition to decreasing perilesional gliosis volume
in rats, CBD treatment limited astrocyte dysfunction, evidenced
by the recovery of the ex vivo H
+
-MRS myoinositol/creatinine
ratio, which had diminished after tMCAO (Ceprián et al., 2017).
However, another work found that the number of activated
astrocytes and IL-1βexpression levels were downregulated in
TRPV1
/
neonatal mice following HI, indicating that TRPV1 is
modulating astrocyte reactivity. These results might suggest that
in vivo, the neuroprotective effects of CBD may not involve
TRPV1 binding, at least in astrocytes (Yang et al., 2019).On
the other hand, in newborn pigs, some data indicate that CBD
modulates astrogliosis after HI-induced brain damage. In the
short term, i.v. administration of CBD (1 mg/kg) after acute HI
promotes an increase in the number of astrocytes in the peri-
infarct area (Pazos et al., 2013). However, by using the same
animal model but conducting histological analyses 72 h after the
induction of HI, CBD treatment (0.1 mg/kg) preserved the
number, size, and morphology of GFAP-positive astrocytes in
newborn pigs (Lafuente et al., 2011). Apart from analyzing GFAP
reactivity by immunohistochemistry, some studies have detected
high levels of the protein S100β, a possible biomarker of astrocyte
damage, in the cerebrospinal uid (CSF) of piglets after HI
(Lafuente et al., 2011;Garberg et al., 2016,2017). Noteworthy,
in only one of those studies CBD administration decreased S100β
levels (Lafuente et al., 2011), but this result might be explained by
the lack of neuroprotective effects of i.v. administration of
1 mg/kg and 50 mg/kg of CBD observed in the works by
Garber and co-authors (Garberg et al., 2016,2017). Future
research will clarify the ability of CBD to revert or not the
increase in S100βlevels observed after HI.
The most widely used experimental paradigm to investigate
in vitro the effects of ischemia is OGD/R. Given the relevance of
the BBB and the neurovascular unit in this pathology, the status of
the ECS, as well as the effects of CBs have been studied in BBB
models. In normoxia, the ECS has a modulatory role in BBB
permeability in co-cultures of endothelial cells and astrocytes. In
specic, AEA (10 µM) and OEA (10 µM) decrease BBB
permeability through CB
2
R, TRPV1, CGRP, and PPARα
receptors (Hind et al., 2015). Besides, in astrocyte
monocultures, CBD diminishes IL-6 and vascular cell adhesion
molecule-1 and increases lactate dehydrogenase release (LDH)
values when administered at high concentrations (10 µM) (Hind
et al., 2016). In these same in vitro BBB models, the eCB-like
molecules OEA, PEA, and virodhamine (all 10 µM), as well as
CBD (100 nM and 10 µM) attenuated the increase in BBB
permeability induced by OGD/R (Hind et al., 2015;Hind
et al., 2016). In a similar way, it was recently demonstrated
that cannabidivarin (CBDV) and cannabigerol (CBG), two
phytocannabinoids, are protective against OGD/R in human
endothelial cells, astrocytes, and pericytes, the different cells
that form the BBB (Stone et al., 2021). In astrocyte
monocultures, CBG (10 nM3 µM) and CBDV (30 nM, 1 and
3 µM) diminished IL-6 levels after OGD/R. And 1 and 3 µM of
CBG and 10 nM, 1 and 3 µM of CBDV, reduced OGD/R-induced
levels of LDH release, but the mechanisms by which these two
compounds provide protection need to be further investigated
(Stone et al., 2021). Despite the evidence indicating an active role
of the ECS in regulating astrocyte metabolism in vitro, there is a
generalized lack of evidence regarding the newest ndings on the
role of astrocytes as neuroprotectors or neurotoxic in this and
other in vitro models of stroke or in ex vivo experiments. Overall,
the evidence available so far indicates that the modulation of
astrocyte function/reactivity with CBs could be used as a possible
therapeutic approach to limit/arrest neurotoxic processes or
promote recovery mechanisms in ischemic stroke (Figure 2C).
OLIGODENDROCYTES
Oligodendrocytes are the myelinating cells of the CNS. The
myelin layers, composed mostly of water and lipids but also
proteins, enwrap the axon and form a multilamellar compacted
myelin sheath, protecting and isolating the axon (Morell and
Quarles, RH, 1999). Myelin electrically isolates axons, allowing
the saltatory impulse propagation and speeding the impulse
transmission (Nave and Trapp, 2008). Besides this structural
function, oligodendrocytes play a key role in the metabolic
support of axons by producing lactate that is then transported
to axons (Figure 3A)(Fünfschilling et al., 2012;Jha and
Morrison, 2020). OPCs are widely distributed throughout the
adult rat brain and participate in the modulation of the BBB and
in angiogenesis (Dawson et al., 2003;Maki et al., 2015;Maki,
2017). Thus, oligodendrocytes are vital for brain circuit activity
and neuron support, and their death and later remyelination
failure have deleterious consequences in stroke outcome.
Oligodendrocytes in Ischemic Stroke
Although the majority of studies on stroke focus on gray matter
damage, the relevance of white matter injury has rapidly grown
over the last years. Noteworthy, white matter injury occupies
approximately half of the infarct area after a stroke (Ho et al.,
2005), and myelinating disturbances resulting from stroke
directly correlate with a poorer cognitive and motor outcome
(Wang et al., 2016).
Oligodendrocytes are particularly susceptible to stroke due to
their sensitivity to excitotoxicity and oxidative stress. In these
cells, the expression of AMPA and NMDA receptors is
developmentally regulated and correlates with their maturation
from OPCs to mature myelinating oligodendrocytes (Káradóttir
et al., 2005;Salter and Fern, 2005;Spitzer et al., 2019). The
activation of AMPA and NMDA receptors in oligodendrocytes
induces the retraction of their processes and causes
oligodendrocyte cell death in the OGD model, effects that are
prevented by blocking both receptors (Salter and Fern, 2005).
Oligodendrocytes are also sensitive to the increase in the
excitatory neurotransmitter ATP that takes place in stroke,
through the P2X7 receptor (Domercq et al., 2009).
Oligodendrocytes are the brain cells with the highest
concentration of iron, which is used to synthesize myelin
(Reinert et al., 2019). This makes them extremely sensitive to
variations in oxidative stress, as nicely reviewed elsewhere
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Vicente-Acosta et al. Glial Endocannabinoid System in Stroke
(Bresgen and Eckl, 2015). Furthermore, mature oligodendrocytes
and, especially OPCs, are characterized by having limited
antioxidant defenses (Fragoso et al., 2004;Spaas et al., 2021).
This vulnerability is particularly strong in earlier stages of
oligodendrocyte maturation, which may affect the stroke-
induced oligoreparative response (Fragoso et al., 2004).
Conrming this high oligodendrocyte sensitivity to stroke,
oligodendrocyte cell death can be identied in vivo as early as
30 min after stroke (Pantoni et al., 1996). Meanwhile, alterations
in the morphology of oligodendrocytes that survive have been
described 24 h after insult (Mages et al., 2019). On the other hand,
stroke induces a strong proliferative response of OPCs, which
migrate to the affected area and mature into myelinating
oligodendrocytes (Figure 3B)(Zhang et al., 2011;Bonfanti
et al., 2017). This proliferative response seems to be age-
dependent (Dingman et al., 2018). However, the proportion of
the newly formed oligodendrocytes that reach a mature stage after
stroke is surprisingly low (Bonfanti et al., 2017;Dingman et al.,
2018). The mechanisms of this developmental impairment are
not yet clear, although excitotoxicity, inammation, and
oxidative stress seem to play a key role (Figure 3B).
Oligodendrocytes and the ECS
The ECS modulates oligodendrocyte maturation at every step:
from the proliferation of OPCs, to their migration and
maturation until the nal step of myelination (Gomez et al.,
2010;Fernández-López et al., 2012;Sanchez-Rodriguez et al.,
2018;Tomas-Roig et al., 2020). In these cells, CB receptors are
found along white matter tracts, and the rst experiments
activating CB
1
R showed that it promotes myelin basic protein
(MBP) expression in the rat subcortical white matter
(Herkenham et al., 1991;Arévalo-Martín et al., 2007).
Particularly important for oligodendrocyte development is the
constitutive production of 2-AG (Gomez et al., 2010,2015;
Sanchez-Rodriguez et al., 2018). The expression of the 2-AG
synthesis enzymes, DAGLand DAGLβ, is higher in OPCs than
in mature oligodendrocytes, whereas the degradation enzyme
MAGL is upregulated in mature oligodendrocytes The effect of 2-
AG in these cells is mediated by CB
1
R/CB
2
R(Figure 3A)(Gomez
et al., 2010). The administration of different antagonists of these
receptors reduces oligodendrocyte proliferation and migration. It
also impairs oligodendrocyte maturation, revealed by a reduced
arborization of immature oligodendrocytes, and myelin
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 bindi ng to CB
1
R and CB
2
R. (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 decient antioxidant system, and 3) inammat ion, 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 decits 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; CB
1
R: cannabinoid receptor 1: CB
2
R: cannabinoid receptor 2; CBs: cannabinoids; MBP:
myelin binding protein; OL: oligodendrocyte; OPC: oligodendrocyte progenitor cell.
Frontiers in Pharmacology | www.frontiersin.org June 2022 | Volume 13 | Article 88822213
Vicente-Acosta et al. Glial Endocannabinoid System in Stroke
production in vitro, with lower expression levels of MBP and
myelin-associated glycoprotein (Gomez et al., 2011,2015;
Sanchez-Rodriguez et al., 2018). Actually, CB
1
R
/
animals are
characterized by having less cell proliferation, evidenced by
BrdU
+
cells, in the subventricular zone (SVZ) and in the
dentate gyrus of rats (Jin et al., 2004). Although the molecular
pathways associated with these effects are not well characterized,
the PI3K/mTOR pathway has been proved to be involved in the
proliferative effect of 2-AG in oligodendrocytes, and the ERK/
MAPK signaling pathway has been associated with
oligodendrocyte maturation (Gomez et al., 2010,2011).
Interestingly, the pharmacological modulation of the ECS has
direct effects on oligodendrocyte maturation and migration, and
increased survival of OPCs has been observed in models of white
matter injury. The in vitro administration of the MAGL inhibitor
JZL-184 at 1 mg/kg, which increases 2-AG levels, accelerates
oligodendrocyte differentiation, and increases the percentage
of migrating cells (Gomez et al., 2010;Sanchez-Rodriguez
et al., 2018). Indeed, it has been observed that the direct
administration of 2-AG promotes oligodendrocyte migration
(Sanchez-Rodriguez et al., 2018). The therapeutic effects of 2-
AG have also been described in pathologies like spinal cord
injury. For instance, the administration of 5 mg/kg of 2-AG
30 min after a moderate contusive SCI in rats reduced white
matter injury and promoted oligodendrocyte survival, even
28 days after injury (Arevalo-Martin, Garcia-Ovejero and
Molina-Holgado, 2010). Furthermore, the inhibition of 2-AG
degradation with the MAGL inhibitor UCM03025 (5 mg/kg)
improved motor impairment and recovered MBP expression
in a multiple sclerosis model, with higher BrdU
+
/Olig2
+
cells,
i.e., new OPCs, in the spinal cord of affected mice that were
treated with the compound (Feliú et al., 2017).
CB
1
R/CB
2
R selective agonists or even non-selective agonists,
such as WIN55,212-2, also promote OPCs proliferation,
oligodendrocyte maturation, with cells showing a more
complex morphology, and myelinization, by increasing MBP
production (Arévalo-Martín et al., 2007;Gomez et al., 2011,
2015;Tomas-Roig et al., 2020). The daily administration of
0.5 mg/kg of WIN55,212-2 prevented demyelination and
promoted remyelination, increasing the number of myelinated
axons, in a cuprizone model of demyelination in mice (Tomas-
Roig et al., 2016;Tomas-Roig et al., 2020). However, the CB dose
should be thoroughly tested, as the daily administration of
1 mg/kg potentiated axonal demyelination, probably due to a
downregulation of CB
1
R(Tomas-Roig et al., 2016;Tomas-Roig
et al., 2020).
CB
2
R activation has also been shown to be oligoprotective
in vitro with the CB
2
R agonist BCP. This compound reduced
LPS-induced oligodendrocyte death by decreasing oxidative
stress and TNF-α(Askari and Shaee-Nick, 2019). Actually,
administration of tetrahydrocannabinol (THC), the main
psychoactive compound in Cannabis sativa,for5daysat
3 mg/kg in the cuprizone mouse model, reduced myelin loss
andimprovedmotorimpairment(Aguado et al., 2021). In this
study, electron microscopy analysis showed lower g-ratios in
the THC-treated group versus control, indicating that THC
effect is on remyelination (Aguado et al., 2021). CBs can also
promote oligodendrocyte survival by CB
1
RandCB
2
R
independent mechanisms. For example, 1 μMCBDwasable
to prevent oligodendrocyte death induced by inammatory
and oxidative stress stimuli through the reduction of
endoplasmic reticulum stress in primary cell cultures
(Mecha et al., 2012).
In summary, the evidence suggests that, due to its role in
promoting oligodendrocyte lineage survival and remyelination,
the ECS is a promising therapeutic target for functional recovery
after demyelinating pathologies, including stroke.
Oligodendrocyte ECS Pharmacological
Modulation in Stroke
Despite the aforementioned data on the possible therapeutic
effects of the ECS modulation in oligodendrocytes, very few
works have explored either the ECS system itself or the
oligoprotective potential of CBs during or after an ischemic
event. In agreement with the above-mentioned results, the
administration of WIN55,212-2, at the high concentration of
9 mg/kg, increased the proliferation rate of OPCs in the ipsilateral
SVZ of adult rats 24 h after pMCAO (Sun and Fang, 2013).
Moreover, WIN55,212-2 was also able to increase the number of
NG2
+
-OPCs within the stroke penumbra and reduce the NG2
+
/
caspase-3
+
cells during 14 days post-damage in a pMCAO model,
an effect that could be related to the increased expression of CB
1
R
in that area (Sun and Fang, 2013). Interestingly, this increased
proliferation/protection in OPCs seemed to translate into new
mature myelinating oligodendrocytes, an effect that was partially
mediated by CB
1
R(Figure 3C). In addition, the amelioration of
MBP loss was also prevented via CB
1
R activation and that was
associated with an increase in the number of myelinated axons
and lower g-ratio values (Sun and Fang, 2013;Sun and Fang,
2013). In a rodent model of neonatal HI, 1 mg/kg WIN55,212-2
also promoted oligodendrocyte proliferation in the SVZ up to
14 days after the insult. This increase positively correlated with
the presence of new APC
+
/BrdU
+
mature oligodendrocytes in the
injured dorsal striatum observed 28 days after the damage. In
addition, an upregulation of CB
2
R expression was observed in the
SVZ in the short term; however, it is not known whether
WIN55,212-2 modulates the expression of this receptor in the
SVZ (Fernández-López et al., 2010). This CB-mediated
protection of oligodendrocytes and myelin has also been
observed with the administration of CBD, a compound with
excellent antioxidant and anti-inammatory properties (Atalay
et al., 2019). Notably, the administration of the low dose of
1 mg/kg CBD in a neonatal model of HI was oligoprotective in the
ipsilateral cortex and corpus callosum. Similar to what happens in
humans, the hypomyelination induced by the insult was directly
related to the motor and cognitive impairment outcomes.
Interestingly, CBD treatment reduced insult-induced
oligodendrocyte impairment and preserved myelin
(Figure 3C)(Ceprián et al., 2019).
We can conclude from these studies that the modulation of the
oligodendroglial ECS is a promising eld for the treatment of
myelin disturbances associated with stroke and its motor/
cognitive sequelae. Although more experimental evidence is
Frontiers in Pharmacology | www.frontiersin.org June 2022 | Volume 13 | Article 88822214
Vicente-Acosta et al. Glial Endocannabinoid System in Stroke