# Astrocyte CB1 receptors drive blood-brain barrier disruption in central nervous system inflammatory disease

Teresa Colomer, Ana Bernal-Chico, Ester Sánchez-Martín, Alvaro

Moreno-García, Andrés Mateo Baraibar, Aitziber Uribe-Irusta, Ander

Iriarte-Sarria, Sandra Beriain, Urszula Skupio, Charlotte

Gatuingt-Chasseriaud, et al.

► To cite this version:

Teresa Colomer, Ana Bernal-Chico, Ester Sánchez-Martín, Alvaro Moreno-García, Andrés Mateo Baraibar, et al.. Astrocyte CB1 receptors drive blood-brain barrier disruption in central nervous system inflammatory disease. Journal of Neuroinflammation, In press, 10.1186/s12974-026-03708-3. hal-05515805

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# Astrocyte CB₁ receptors drive blood-brain barrier disruption in central nervous system inflammatory disease

Received: 6 August 2025

Accepted: 17 January 2026

Published online: 29 January 2026

Cite this article as: Colomer T., Bernal-Chico A., Sánchez-Martín E. et al. Astrocyte CB₁ receptors drive blood-brain barrier disruption in central nervous system inflammatory disease. *J Neuroinflammation* (2026). https://doi.org/10.1186/s12974-026-03708-3

Teresa Colomer, Ana Bernal-Chico, Ester Sánchez-Martín, Alvaro Moreno-García, Andrés Mateo Baraibar, Aitziber Uribe-Irusta, Ander Iriarte-Sarria, Sandra Beriain, Urszula Skupio, Charlotte Gatuingt-Chasseriaud, Delphine Gonzales, Guillaume Laplagne, Román Serrat, Isabel Pidal-Ladrón Guevara, Carlos Matute, Diego Clemente, Vanja Tepavcevic, Ignacio Fernández-Moncada, Candice Chapouly, Giovanni Marsicano & Susana Mato

We are providing an unedited version of this manuscript to give early access to its findings. Before final publication, the manuscript will undergo further editing. Please note there may be errors present which affect the content, and all legal disclaimers apply.

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# Astrocyte CB₁ receptors drive blood-brain barrier disruption in central nervous system inflammatory disease

Teresa Colomer<fnref n="1" />,<fnref n="2" />,<fnref n="3" />†, Ana Bernal-Chico<fnref n="1" />,<fnref n="2" />,<fnref n="3" />†, Ester Sánchez-Martín<fnref n="1" />,<fnref n="2" />,<fnref n="3" />†, Alvaro Moreno-García<fnref n="1" />,<fnref n="2" />,<fnref n="3" />, Andrés Mateo Baraibar<fnref n="1" />,<fnref n="2" />,<fnref n="3" />, Aitziber Uribe-Irusta<fnref n="1" />,<fnref n="2" />, Ander Iriarte-Sarria<fnref n="1" />,<fnref n="2" />, Sandra Beriain<fnref n="4" />, Urszula Skupio<fnref n="4" />, Charlotte Gatuingt-Chasseriaud<fnref n="4" />, Delphine Gonzales<fnref n="4" />, Guillaume Laplagne<fnref n="4" />, Román Serrat<fnref n="4" />, Isabel Pidal-Ladrón de Guevara<fnref n="7" />,<fnref n="8" />,<fnref n="9" />, Carlos Matute<fnref n="1" />,<fnref n="3" />,<fnref n="9" />, Diego Clemente<fnref n="7" />,<fnref n="8" />,<fnref n="9" />, Vanja Tepavcevic<fnref n="2" />,<fnref n="9" />,<fnref n="10" />,<fnref n="11" />, Ignacio Fernández-Moncada<fnref n="4" />,<fnref n="5" />, Candice Chapouly<fnref n="6" />, Giovanni Marsicano<fnref n="4" />, Susana Mato<fnref n="1" />,<fnref n="2" />,<fnref n="3" />*

<footnote n="1">¹Department of Neurosciences, University of the Basque Country UPV/EHU, 48940 Leioa, Spain.</footnote>

<footnote n="2">²Achucarro Basque Center for Neuroscience, 48940 Leioa, Spain.</footnote>

<footnote n="3">³Neuroimmunology Group, Biobizkaia Health Research Institute, 48903 Barakaldo, Spain.</footnote>

<footnote n="4">⁴University of Bordeaux, INSERM, Neurocentre Magendie, U1215, F-33000 Bordeaux, France.</footnote>

<footnote n="5">⁵University of Bordeaux, INRAE, Bordeaux INP, NutriNeurO, UMR 1286, F-33000 Bordeaux, France.</footnote>

<footnote n="6">⁶Univ. Bordeaux, INSERM, BMC, U1034, F-33600 Pessac, France.</footnote>

<footnote n="7">⁷Neuroimmuno-Repair Group, Hospital Nacional de Parapléjicos (SESCAM), 45071 Toledo, Spain.</footnote>

<footnote n="8">⁸Castile-La Mancha Health Research Institute (IDISCAM), Spain.</footnote>

<footnote n="9">⁹Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), 28029 Madrid, Spain.</footnote>

<footnote n="10">¹⁰Laboratory of Comparative and Regenerative Neurobiology, Cavanilles Institute of Biodiversity and Evolutionary Biology, University of Valencia, 46980 Paterna, Spain.</footnote>

<footnote n="11">¹¹Department of Cell Biology, Functional Biology and Physical Anthropology, University of Valencia, 46100 Burjassot, Spain.</footnote>

<footnote>†These authors contributed equally</footnote>
*Correspondence: susana.mato@ehu.eus

**Abstract**

Reactive astrocytes shape central nervous system (CNS) inflammation and participate in myelin damage and repair mechanisms in multiple sclerosis (MS). Through the activation of cannabinoid CB₁ receptors (CB₁R) expressed by neurons and oligodendrocyte lineage cells, endocannabinoid signaling restricts neurodegeneration and promotes remyelination in preclinical MS models. However, despite accumulating evidence that supports cell-specific roles for CB₁R in brain physiology and pathology, the implications of astrocyte CB₁R signaling in MS initiation and progression remain uncertain. Using complementary *in vivo* disease models, here we investigated the effects of targeted genetic deletion of astrocyte CB₁R on the expression of MS-like pathology in mice. Interestingly, astrocyte-specific deletion of CB₁R reduced demyelinating neuropathology, attenuated astrocyte reactivity and improved clinical deficits during the time-course of experimental autoimmune encephalomyelitis (EAE). Mice with astrocyte CB₁R inactivation displayed unaltered oligodendrocyte populations both in EAE plaques and in lysolecithin-induced remyelinating spinal cord lesions, likely excluding that CB₁R expressed by astroglial cells modulate myelin repair processes. Conversely, inactivation of CB₁R in astrocytes restricted humoral and leukocyte parenchymal infiltration and reduced the expression of vascular effectors in EAE lesions. Finally, loss of blood-brain barrier (BBB) function induced by cortical microinjection of VEGF-A was less severe
in astrocyte CB₁R null mice. These results show that astrocyte CB₁R signaling constitutes a significant pro-inflammatory mechanism in experimental MS and bring to light a deleterious role for endocannabinoid-mediated modulation of astroglial cells with potential implications in the etiopathology and therapy of neuroinflammatory disorders.

**Keywords**

Multiple sclerosis; Astrocyte; CB₁ receptors; Experimental autoimmune encephalomyelitis; Blood-brain barrier; Remyelination

**Introduction**

Multiple sclerosis (MS) is an immune-mediated inflammatory disease of the central nervous system (CNS) and one of the most prevalent neurological disorders leading to chronic disability among young adults (1). The main pathological hallmark of MS is the formation of demyelinating lesions in the brain and spinal cord associated with neuroaxonal degeneration as primary substrate of the irreversible clinical deficits that characterize disease progression (2). Focal lesions in MS are thought to be caused by the bidirectional interaction between peripheral immune cells that infiltrate into the CNS parenchyma, including T cells, B cells and myeloid cells, and activated resident immune cells, mainly astrocytes and microglia (3). A subset of lesions in MS patients are characterized by a variable extent of remyelination suggested as a mechanism of neuroprotection and clinical recovery (4, 5). At present, treatment strategies for clinical exacerbation in MS include almost exclusively immunotherapeutic drugs that target
peripheral immune cells and their trafficking into the CNS. These pharmacological therapies lead to a substantial reduction in lesion formation and clinical relapse rate but do not prevent the progression of clinical disability.

Astrocytes support brain homeostasis and function through a plethora of mechanisms that include the modulation of synaptic transmission through the release of gliotransmitters, the metabolic assistance to neurons and oligodendrocytes, and the maintenance of the blood-brain barrier (BBB), among others. In recent years, complementary lines of evidence have shown that astrocytes adopt a wide spectrum of reactive states during neuroinflammation that confer these cells the potential to exacerbate damage or facilitate repair (6-8). Aberrant astrocyte activation critically contributes to inflammatory lesion formation and progression in MS through the release of molecules that promote the loss of BBB integrity and the recruitment of peripheral immune cells (8-10). However, astrocytes may also facilitate the differentiation of oligodendrocyte progenitor cells (OPCs) and support the survival of mature oligodendrocytes to ensure successful remyelination (11, 12). An improved understanding of astrocyte regulatory mechanisms in neuroinflammatory contexts may thus provide novel therapeutic targets that reduce MS pathology and clinical severity during acute and progressive phases of the disease.

Cannabinoid type-1 receptors (CB₁R) are among the most abundant G protein-coupled receptors in the mammalian brain and the primary molecular target of endogenous cannabinoids - anandamide and 2-
arachidonoylglycerol - and Δ⁹-tetrahydrocannabinol (THC), the main psychoactive component of the hemp plant *Cannabis sativa* (13). Endocannabinoids acting on CB₁R heterogeneously expressed in neuronal populations and glial cells modulate physiological brain functions through a wide variety of cellular processes and exhibit neuroprotective potential during CNS damage (14-16). Pharmacological and genetic studies conducted so far have demonstrated that cannabinoid agents that potentiate CB₁R-mediated signaling attenuate neurodegeneration and suppress neuroinflammation while promoting myelin repair in rodent models of MS (17-20). However, the clinical efficacy of cannabinoid-based medications in MS is limited, and therapeutic optimization requires a better understanding of endocannabinoid and CB₁R related networks in inflammatory demyelinating contexts (16, 21). On mechanistic grounds, there is consensus that the neuronal population of CB₁R provides neuroprotection from excitotoxic damage in rodent models of MS by limiting synaptic glutamate release (20, 22-24). Experimental *in vivo* evidence from conditional mutant mice has also recently grounded the hypothesis that CB₁R expressed by OPCs promote oligodendrocyte differentiation and favor remyelination in MS (25, 26). However, the implications of CB₁R in astrocytes during MS onset and progression have been largely neglected despite the fundamental contribution of these receptor populations as effectors of (endo)cannabinoid-mediated signaling in the brain (27, 28). Indeed, the combination of high-resolution electron microscopy, electrophysiology, calcium imaging
and cell-specific mutagenesis techniques has demonstrated, during the last decade, that astrocytes in the brain and spinal cord express functional CB₁R in perisynaptic, perivascular, and mitochondrial compartments (29-33). Accordingly, CB₁R activity has arisen as crucial modulator of astrocyte-derived gliotransmitter release and metabolic supply (27, 28, 33) with emerging implications in neuroinflammatory disorders (34).

In this study, we aimed at disentangling the roles of astrocyte CB₁R (aCB₁R) signaling in MS neuropathology using mice where this receptor population is selectively ablated in astroglial cells and a combination of rodent models that recapitulate autoimmune demyelination, BBB breakdown and myelin repair. Our results highlight that aCB₁R exacerbate neurological disability during autoimmune demyelination by fostering BBB permeability and recruitment of peripheral immune cells towards lesion sites. These observations identify a previously unexpected disease-promoting role of aCB₁R during CNS inflammatory lesion formation with potential implications in MS pathogenesis and therapy.

# Materials and methods

## Mice

All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council Committee, 2011) and the European Communities Council Directive of 22 September 2010 (2010/63/EU74). Experiments were approved by the local ethical committees of the University of the Basque Country
(approval numbers 2017140, 2020005 and 2022245) and the University of Bordeaux (approval numbers A33063098 and 16901). Inducible mutant mice of a C57BL/6N background lacking CB₁R in cells expressing the astrocytic marker glial fibrillary acidic protein GFAP (aCB₁-KO) and aCB₁-WT littermates were bred at the Neurocentre Magendie (Bordeaux, France). Cages were enriched and mice were maintained under standard conditions (food and water *ad libitum*; 12 h-12 h light-dark cycle). Experiments were performed during dark cycle (light off at 8:00 h a.m.). *In vivo* models were induced in female mice based on epidemiological evidence that MS affects 2-4 times more women than men (35). The number of mice in each experimental group was similar. No statistical methods were used to predetermine sample size.

aCB₁-KO mice were generated by crossing mice carrying LoxP sites flanking the coding region of the *Cnr1* gene (CB₁f/f) (36) with GFAP-CreERT2 mice (37) using a three-step backcrossing procedure to obtain CB₁f/f;GFAP-CreERT2 and CB₁f/f littermates, called aCB₁-KO and aCB₁-WT respectively. Deletion of the *Cnr1* gene was obtained in adult aCB₁-KO mice (6-12 weeks of age) by daily intraperitoneal (i.p.) injections of tamoxifen (1 mg dissolved at 10 mg/mL in 90% sesame oil, 10% ethanol) for 8 days (29, 38) and corroborated using a PCR-based strategy with genomic DNA (Figure S1). Tamoxifen-treated aCB₁-WT were used as controls. Mutant mice were used for experiments 3-4 weeks after the last tamoxifen injection.

## EAE model
Mice were immunized in the flank by subcutaneous (s.c.) injection of 200 µg MOG35-55 peptide (MEVGWYRSPFSRVVHLYRNGK) (Peptide Synthesis Core Facilities of the Pompeu Fabra University, Spain) in incomplete Freund's adjuvant supplemented with 1.2 mg *Mycobacterium tuberculosis* H37Ra (Difco Laboratories). Pertussis toxin (500 ng; Calbiochem) was injected i.p. on the day of immunization and again at 2 days post-immunization (dpi). Body weight and motor symptoms were recorded daily and scored from 0 to 8 as follows: 0, no detectable changes in muscle tone and motor behavior; 1, flaccid tail; 2, paralyzed tail; 3, impairment or loss of muscle tone in hindlimbs; 4, hindlimb hemiparalysis; 5, complete hindlimb paralysis; 6, complete hindlimb paralysis and loss of muscle tone in forelimbs; 7, tetraplegia; and 8, moribund.

## Demyelinating lesion induction

Demyelinating lesions were induced by stereotaxic injection of 1% lysophosphatidylcholine (LPC; 0.5 µL; Sigma-Aldrich) diluted in sterile saline solution (0.9% NaCl) into the spinal cord (39). Mice were anesthetized by i.p. injection of a ketamine (100 mg/Kg; Fatro) and xylazine (20 mg/Kg; Calier) cocktail. Two longitudinal incisions into the *longissimus dorsi* at each side of the vertebral column were performed and the muscle tissue covering the column was removed. Animals were placed into a stereotaxic frame and the intervertebral space of the 13th thoracic vertebra was exposed by removing the connective tissue. An incision into dura mater was performed using a 30-gauge needle and LPC was injected into the white matter of the *dorsal funiculus* at a rate
of 0.5 µL/min via a Hamilton syringe attached to a glass micropipette using a stereotaxic micromanipulator. The lesion site was marked with sterile charcoal. Following LPC injection, the muscle sheaths were closed using 3/0 Monocryl and the wound was sutured with 4/0 silk. Buprenorphine (0.1 mg/Kg; Dechra) was subcutaneously administered as postoperative analgesic treatment. Mice were euthanized and processed for immunohistochemistry 14 days after the surgery.

## VEGF-A microinjection

Mice were anesthetized using a local injection of lidocaine (20 mg/mL) under the skull skin and a mix of air and isoflurane (3% for induction and 1.5% for support). Animals were placed into a stereotaxic apparatus and mouse VEGF-A₁₆₅ (60 ng in 3 µL of 0.9% NaCl) or vehicle were delivered into the cerebral cortex at y = 1 mm caudal to Bregma, x = 2 mm, z = 1.5 mm as previously described (40). Mice received a subcutaneous injection of buprenorphine (0.1 mg/Kg; Vetergesic) 30 minutes before surgery and again 8 hours post-surgery to assure constant analgesia. Animals were sacrificed 48 h after VEGF-A₁₆₅ injection and processed for immunohistochemistry.

## Surgery for AAV administration and fiber implantation

Mice were anesthetized with isoflurane and placed on a heating-pad to keep the body temperature at 37°C. Eye dehydration was prevented by topical application of ophthalmic gel and analgesia was achieved by s.c. injection of buprenorphine (0.05 mg/Kg; Buprecare). The skin above the skull was shaved with a razor and disinfected with modified ethanol 70% and betadine before an incision was made. Mice were
placed into a stereotaxic frame and injected with an AAV encoding the
genetically encoded calcium indicator GCaMP6f under the GFAP
promoter (AAV-9/2-GFAP-hHBbI/E-GCaMP6f-bGHp(A)) (ETH Zürich) to
carry out fiber photometry imaging of calcium activity in astrocytes.

Virus titers were between 10¹⁰-10¹² genomic copies per mL.

Stereotaxic injections were targeted to the mouse somatosensory
cortex according to the following coordinates (from bregma): anterior-
posterior -1.5; medial-lateral ± 2.5; dorsal-ventral -1.5. Viral particles
(400-500 nL) were injected at a maximum rate of 100 nL/min using a
glass pipette attached to a Nanojet III (Drummond, Broomall, USA).

Following virus delivery, the syringe was left in place for 10 min before
being slowly withdrawn from the brain. The optical fiber (400 µm
diameter) was placed 250 µm above the injection site during the same
surgical session. Mice were weighed daily and individuals that failed to
return to their pre-surgery body weight were excluded from
subsequent experiments. All animals were treated with tamoxifen 1
week after the surgery.

Fiber photometry imaging

Freely moving mice were imaged after 3 days of handling habituation.

The day of recording each mouse was placed in a rectangular chamber
and its behavior recorded using a camera placed above the chamber.

Baseline recordings of spontaneous astrocyte activity were made for
15 min every 1-2 days starting 3 days before MOG administration. The
calcium signal evoked by sensory stimulation of the tail was assessed
at the end of the baseline period.
Cortical astrocyte GCaMP6f was imaged *in vivo* using 470 and 405 nm LEDs. The emitted fluorescence is proportional to the calcium concentration for stimulation at 470 nm (41, 42). The isosbestic 405 nm stimulation (UV light) was used in alternation with the blue light (470 nm) for analysis purposes as the fluorescence emitted after this stimulation is not calcium-dependent (43). The GCaMP6f fluorescence from the astrocytes was collected with a sCMOS camera through an optic fiber divided in 2 sections: a short fiber implanted in the brain of the mouse and a long fiber (modified patchcord), both connected through a ferrule-ferrule (1.25 mm) connection. MATLAB program (Matlabworks) was used to synchronize each image recording made by the camera, and the GCaMP6s light excitation made by the LEDs (470 and 405 nm). The two wavelengths of 470 and 405 nm at a power of 0.1 mW were alternated at a frequency of 20 Hz each (40 Hz alternated light stimulations).

To calculate fluorescence due specifically to calcium fluctuations and to remove bleaching and movement artifacts, the isosbestic 405 nm signal was subtracted from the 470 nm calcium signal. Specifically, normalized fluorescence changes ($\Delta F/F_0$) were calculated by subtracting the mean fluorescence (2 min sliding window average) from the fluorescence recorded by the fiber at each time point and dividing this value by the mean fluorescence ($F-F_{mean})/F_{mean}$) using a customized Matlab software. Subsequently, the calcium independent isosbestic signal was subtracted from the raw signal emitted after the 470 nm excitation to eliminate unspecific fluorescence. The result is
the global calcium signal ($\Delta F/F(\%) = \Delta F_{Ca} - \Delta F_{iso}$), that was used as an estimate of tonic activity of the astrocytes. Calcium transients were detected on the filtered trace using a threshold to identify them (2 median absolute deviation - MAD - of the entire trace). Amplitude was determined as the MAD of each studied period (44).

## Flow cytometry analysis of splenic populations

Fresh spleens were collected from aCB₁-KO and aCB₁-WT mice anesthetized with isoflurane and mechanically processed to obtain a single cell suspension, which was passed through a 40-µm filter (BD Biosciences), and washed with cold supplemented RPMI medium (Gibco, #1640) containing 2 mM L-Glutamine (Thermo Fisher), 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). Erythrocytes were lysed with 1 mL of ACK buffer (8.29 g/L NH₄Cl, 1 g/L KHCO₃, 1 mM EDTA in distilled H₂O; pH 7.4) and the reaction was stopped with phosphate buffer saline (PBS). Splenocytes were recovered by centrifugation at 210 x g for 5 min, and 2 x 10⁶ cells were resuspended in 50 µL of staining buffer (PBS supplemented with 10% FBS, 25 mM HEPES buffer, 2% P/S). The Fc cell receptors were blocked with an anti-CD16/CD32 antibody (10 µg/mL; BD Biosciences; #553142) for 10 min at 4°C. Cells were then labelled for 30 min at 4°C in the dark with fluorochrome-conjugated antibodies targeting lymphoid (CD3e, CD4, CD8a, CD25, CD69 and CD19) and myeloid (CD11b, Ly6C, Ly6G, CD11c) markers at 0.025-0.2 µg/10⁶ cells (Table S1). Cells were rinsed with PBS, centrifuged at 210 x g for 5 min, resuspended in PBS and analyzed in a FACS Canto II cytometer (BD
Biosciences). Data were processed using FlowJo 10.9.0 software (Tree Star Inc.). Fluorescence Minus One (FMO) controls were used to set gates and determine positive expression for each marker.

## Genotyping of the *Cnr1* allele

Genotyping of the *Cnr1* locus was performed following previously described procedures (36) with some modifications. Genomic DNA was extracted from forebrain and spinal cord lysates using NucleoMag B-beads (Macherey-Nagel). DNA concentration and purity were assessed using a DeNovix spectrophotometer and a Qubit fluorometer (Thermo Fisher). To assess the presence of Cre-mediated recombination, the *Cnr1* locus was amplified using primers P50 (5'-GCTGTCTCTGGTCCTCTTAAA-3') and P53 (5'-CTCCTGTATGCCATAGCTCTT-3') (**Figure S1a**). This reaction produced a ~2900 bp amplicon for the unrecombined allele and a ~689 bp product corresponding to the excised DNA fragment following Cre-mediated recombination. The presence of the floxed allele was evaluated in a separate reaction using primers P50 and P51 (5'-GGTGTCACCTCTGAAAACAGA-3'), which resulted in a ~595 bp product for the floxed allele and a ~475 bp product for the wild-type allele (**Figure S1a**). To determine the presence of Cre recombinase, samples were amplified with primers 5'-CGGCATGGTGCAAGTTGAATA-3' and 5'-GCGATCGCTATTTTCCATGAG-3', generating a band with 300 bp. PCR products were resolved by capillary electrophoresis and visualized using LabChip® GX (Revvity).

## Quantitative RT-PCR
Mice were anesthetized with ketamine/xylazine (80/10 mg/Kg, i.p; Imalgene®/Rompun®) and transcardially perfused with cold PBS for 30-60 s with an average of an average of 5 mL buffer per mouse in order to remove circulating blood cells. The lumbar spinal cord was dissected in lysis buffer containing 1% β-mercaptoethanol for optimal template preservation. Total RNA was purified with on-column DNAse treatment using RNeasy Plus Mini kit (Qiagen; 74104) following manufacturer's instructions. RNA was eluted with 14-35 µL of RNAse-free deionized water and stored at -80°C until analysis. Synthesis of cDNA, pre-amplification and amplification steps were performed at the Genome Analysis Platform of the UPV/EHU following quality control of RNA samples with an Agilent 2100 Bioanalyzer (Agilent Technologies). Pre-amplified cDNA samples were measured with no reverse transcriptase and no template controls in the BioMark HD Real-Time PCR System using 48.48 Dynamic Arrays of integrated fluidic circuits (Fluidigm Corporation). We used commercial primers from IDT Integrated DNA Technologies or Fluidigm Corporation (Table S2). Data pre-processing and analysis were completed using Fluidigm Melting Curve Analysis Software and Real-time PCR Analysis Software (Fluidigm Corporation) to determine valid PCR reactions. *Gapdh*, *Hprt* and *Ppia* were included as candidate reference genes for normalization purposes. Data were corrected for differences in input RNA using the geometric mean of reference genes selected according to results from the normalization algorithms *geNorm* (https://genorm.cmgg.be/) and *Normfinder*
(https://moma.dk/normfinder-software). Relative expression values
were calculated with the 2⁻⁰·⁰⁵ΔΔCt method.

Western blot

Anesthetized mice were transcardially perfused with cold PBS and the
lumbar spinal cords were homogenized (1:20 w/v) in ice-cold RIPA
buffer (Thermo Fisher; #89900) containing a protease inhibitor cocktail
(Thermo Fisher; #87786) using a Potter homogenizer with a loosely
fitting Teflon pestle. Samples were incubated in ice for 30 min and
subjected to centrifugation (12000 x g at 4°C for 8 min) to remove
insoluble material. Solubilized proteins were quantified in the
supernatants using the BioRad Protein Assay Kit (Protein Assay
Reagents; 5000-114-13-15). Protein samples (4 µg) were loaded into
polyacrylamide Criterion TGX Precast (BioRad) gels before
electrophoretic transfer onto Nitrocellulose membranes
(Amersham™ Protran® Western blotting membranes, pore size
0.2 µm). Membranes were blocked for 1 h in Tris-buffered saline (TBS;
50 mM Tris, 200 mM NaCl; pH 7.4) with 0.05% Tween-20, 5% BSA.
Subsequently, membranes were incubated overnight at 4°C with
primary antibodies raised against myelin basic protein (MBP; 1:1000;
BioLegend; #808401), Claudin 4 (CLN-4; 1:500; Thermo Fisher; #32-
9400), Cadherin 5 (CDH-5; 1:500; R&D Systems; #AF1002),
Intercellular Adhesion Molecule 1 (ICAM-1; 1:500; R&D Systems;
#AF796), Podocalyxin (PODXL; 1:500; R&D Systems; #AF1556),
Vascular cell adhesion protein 1 (VCAM-1; 1:1000; Abcam;
#ab134047), zona occludens (ZO-1; 1:1000; Invitrogen; #02200) and
α-Tubulin (1:5000; Abcam; #ab7291) in blocking solution. Membranes were incubated for 1 h at RT with horseradish peroxidase-conjugated secondary antibodies (1:5000; Cell Signaling Technology) and developed with NZY Standard ECL Western Blotting Substrate (NZYtech). Volumetric analysis of relevant immunoreactive bands was carried out after acquisition on a ChemiDoc XRS System (Bio-Rad) controlled by The Quantity One software v 4.6.3 (BioRad).

**Histology and fluorescence immunohistochemistry**

*Mice sacrifice and tissue processing*

EAE mice were deeply anesthetized by intraperitoneal injection of Dolethal (200 mg/Kg) and perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) (25 mM NaH₂PO₄·H₂O, 75 mM Na₂HPO₄; pH 7.4) for 10 min. The spinal cords were extracted and post-fixed in the same fixative solution for 24 h at 4°C. Alternatively, anesthetized mice were transcardially perfused with cold PBS and the brains extracted and stored at -80°C. Mice injected with LPC were perfused with 2% PFA in 0.1 M PB for 15 min and spinal cords post-fixed for 30 min. Spinal cords were dissected into 1 or 4-5 longitudinal 2-mm-thick blocks containing either the demyelinating LPC lesion or equivalent lumbar spinal cord portions from EAE mice, respectively. Tissue blocks were then placed in 15% sucrose-7% gelatin in PBS, frozen in isopentane for 2 min at -65°C and stored at -80°C. Coronal 10-12 µm-thick spinal cord and forebrain sections were cut into Superfrost glass slides (Thermo Fisher, #11976299) using a CM3050 S cryostat (Leica Biosystems) and stored at -20°C.
Mice injected with VEGF-A₁₆₅ received a subcutaneous injection of buprenorphine (0.1 mg/kg) (Vetergesic) 30 min before surgery. Mice were then profoundly anesthetized by i.p. injection of a mix of ketamine (100 mg/kg; Imalgene) and xylazine (20 mg/kg; Rompun), and transcardially perfused with PBS for 5 min and then with 10% formalin (Merck; #252549) for 12 min. The brains were post-fixed in 10% formalin for 3 h and incubated in 30% sucrose overnight. Forebrain tissues were embedded on Tissue-Tek O.C.T. Compound (Sakura, #4583) and stored at -80°C. Coronal 12 µm-thick sections containing the marked lesion were cryostat sectioned into Superfrost glass slides and stored at -20°C until further processing.

*Luxol fast blue myelin staining*

Spinal cord sections were incubated overnight at 37-42°C with 0.1% luxol fast blue (LFB) (Sigma-Aldrich; 1328-51-4) diluted in 95% ethanol and 0.5% glacial acetic acid and subsequently differentiated with a 0.01% lithium carbonate solution. Tissues were dehydrated by immersion in increasing concentrations of ethanol, processed with xylene and mounted with DPX mounting medium.

*Hematoxylin and eosin staining*

Spinal cord sections were rinsed in distilled water, incubated in hematoxylin solution (Epredia; #6765008) for 2 min and rinsed for 3 min in 0-3% clorhidric acid. Tissues were subsequently counterstained by incubation in eosin solution (Epredia; #6766008) for 25 min and rinsed in tap water for 5 min. Sections were dehydrated by immersion
in increasing concentrations of ethanol, processed with xylene and
mounted with DPX.

*Immunolabelling of spinal cord and cortical tissue*

Spinal cord sections were air-dried for 30 min at RT and rehydrated in TBS (20 mM Tris, 1.4 M NaCl; pH 7.6) for 30 min. Antigen retrieval was performed for OLIG2 immunostaining by adding low-pH R-Universal retrieval buffer (Aptum Biologics; #AP0530-500) and heating the slices in a microwave for 45 s at maximum temperature. For MBP immunolabelling, slices were permeabilized in absolute ethanol for 15 min at -20°C followed by extensive washing in TBS. Tissue sections were incubated for 1 h at RT in blocking solution containing 5-10% normal goat serum (NGS) (Vector Labs; S-1000) or donkey serum (NDS) (Interchim; #UP77719A) and 0.2% Triton X-100 in TBS. The blocking solution was supplemented with 3% Fab fragment (Jackson ImmunoResearch) when using primary antibodies made in mouse. Slides were incubated overnight at 4°C with primary antibodies (Table S3) diluted in TBS containing 5% NGS and 0.1% Triton X-100, washed in TBS (3 x 10 min) and incubated for 1 h at RT with Alexa Fluor secondary antibodies made in goat or donkey and Hoechst (4 µg/mL) (Sigma-Aldrich; B2261) in antibody solution. Tissue sections were washed with TBS (3 x 10 min) and mounted with ProLong Gold Antifade (Thermo Fisher; #P36930) or Fluoromount-G (Thermo Fisher; #00-4959-52) mounting media for microscopy analysis.

Cryostat sections from EAE mice containing the somatosensory
cortex were air-dried for 30 min at RT, fixed in 4 % PFA for 15 min and
washed in TBS (3 x 10 min). Tissue slides were incubated for 1 h at RT
in a blocking-permeabilization solution containing 5% NGS and 0.2%
Triton X-100 in TBS and subsequently incubated for 12-48 h at 4°C with
primary antibodies (Table S3) diluted in blocking solution. Following
extensive washing, primary antibodies were detected by incubation
with appropriate Alexa Fluor secondary antibodies for 2 h at RT.
Hoechst was used for chromatin staining. Sections were then washed
in TBS (3 x 10 min) and mounted in ProLong mounting medium using
coverslips for microscopy analysis.

Tissue sections from VEGF-A₁₆₅-injected mice were tempered for 20 min and then rehydrated in PBS for 20 min. Antigen retrieval was performed by incubation in a Tris-EDTA solution (10 mM Tris HCl, 1 mM EDTA; pH 8) for 30 min at 100°C. Slices were then washed in PBS (3 x 5 min) and incubated for 1 h at RT in a blocking solution containing 10% NDS and 0.3% Triton X-100 in PBS. Sections were subsequently incubated overnight at 4°C with the corresponding primary antibodies (Table S3) prepared in 5% NGS and 0.1% Triton X-100 in PBS. Tissue sections were washed in PBS (3 x 5 min) and incubated with Alexa Fluor secondary antibodies for 1 h at RT. Following extensive washing tissues were mounted on glass coverslips using Fluoromount-G with DAPI mounting medium (Thermofisher; #00-4959-52) for microscopy analysis. All the immunohistochemical experiments included tissue samples run in parallel without primary antibodies as internal controls.

Image acquisition and analysis
Quantitative analysis of demyelination and inflammatory lesion number in EAE mice was performed in 4-10 tissue sections per mouse imaged using a 3D Histech Panoramic MIDI II slide scanner and the CaseViewer and CaseConverter softwares (3DHistech). For the immunohistochemical characterization of brain and spinal cord tissues, optical images from sections processed in parallel were acquired using 20X and 40X lens on a Leica TCS STED CW SP8 super-resolution microscope or a Zeiss Axioplan 2 pseudoconfocal microscope coupled to an Axiocam MRc5 digital camera. Image acquisition was carried out using fluorescence intensity settings at which the control sections without primary antibody gave no signal.

Immunohistochemical characterization of demyelinating lesions in the EAE model was performed by examining 4 objective pictures taken from 2 non-consecutive spinal cord sections per mouse separated by ~2 mm. Regions of interest (ROIs) corresponding to the demyelinated plaque and the adjacent periplaque were established based on MBP immunohistochemistry and on the density of the cell nuclei. The plaque of demyelinated lesions was characterized by the total lack of MBP immunostaining and a high nuclear density, while the periplaque was determined as the area corresponding to a 100 µm perimeter measured from the lesion edge to the adjacent area, and characterized by weak or less dense MBP immunostaining. Immunolabelling of LPC injected spinal cords was evaluated in 2 tissue sections containing the central part of the demyelinating lesion. The somatosensory cortex of EAE mice was examined for inflammatory lesion load and astrocyte
reactivity and immunohistochemical characterization performed in 2-4 images collected from 2 non-consecutive coronal sections per mouse separated by 150-200 µm. Cortical lesions induced by VEGF-A$_{165}$ were evaluated in 3-4 images collected from the lesion area.

Image analysis was performed using Fiji Image J (45). Immunopositive cells were counted in a selected ROI using a cell counter plugin and data expressed as mean cell number per square millimeter (mm²) of tissue area. Analysis of immunostained areas was performed in 16-bit gray scale transformed pictures. Fluorescence signals were considered positive if they were above a defined intensity threshold and normalized to total selected ROI area. For colocalization analysis, pixels positive for GFAP or aquaporin-4 and C3, ICAM-1, VCAM-1 or VEGF-A immunoreactivity were counted in projections of Z series stacks with the same number of images taken at a spacing of 0.8 µm by a blinded observer.

## Data collection and statistical analyses

No statistical methods were used to pre-determine sample sizes but they are similar to those reported in previous publications. Experimenters were always blinded to mice genotype but not to treatments. Statistical analyses were performed using GraphPad Prism 10 for Windows (GraphPad Software Inc). Summary results are presented as the mean of independent data points ± SEM. Individual datasets were initially tested for normal distribution with the Shapiro-Wilk test and differences between groups were determined by two-tailed unpaired Student *t* test, Mann-Whitney test or Wilcoxon
matched-pairs signed rank test. Differences in EAE disease progression over time were assessed with the Wilcoxon matched-pairs signed-rank test. Calcium responses in the EAE model were analyzed using two-way ANOVA followed by Šídák's test for multiple comparisons. Differences were considered to be significant when *p* < 0.05.

## Results

### Astrocyte CB₁ receptors exacerbate clinical deficits and myelin pathology in EAE

To study the role of aCB₁R in MS we analyzed the phenotype of conditional mutant mice lacking CB₁R in GFAP positive cells (29, 32) in the EAE model of autoimmune demyelination. Upon EAE induction, mice lacking CB₁R specifically in astrocytes (aCB₁-KO) displayed similar disease onset but significantly decreased clinical scores during the acute phase of the disease (Figure 1a) as well as at the experimental end-point (score at 22 dpi = 4.250 ± 0.240 in aCB₁-WT versus 2.750 ± 0.377 in aCB₁-KO; *n* = 18-19 mice; *p* = 0.0073; Mann-Whitney test).

Histological evaluation of spinal cords at 22 dpi revealed that the number of inflammatory lesions (Figure S2a), predominantly found in white matter areas close to the tissue edge, and the proportion of demyelinated white matter (Figure 1b), were reduced in aCB₁-KO mice as compared to aCB₁-WT controls. Consistent with a preserved neurological function, the levels of non-phosphorylated neurofilaments (SMI-32) and amyloid precursor protein (APP) within spinal cord lesions were significantly reduced in aCB₁-KO mice, indicative of preserved neuroaxonal integrity (Figure 1c). We next interrogated the protective
phenotype of aCB₁-KO mice at chronic EAE disease stages. Astrocyte-
specific CB₁R null mice displayed a sustained reduction in disability
scores during EAE progression to a more chronic clinical plateau
(Figure S3a, left panel). Comparative analysis at 35 dpi evidenced a
non-significant attenuation of motor symptomatology in aCB₁-KO mice
(Figure S3a, right panel) that was associated with improved spinal
cord myelin pathology in terms of demyelinating lesion numbers
(Figure S3b) and proportion of demyelinated white matter area
(Figure S3c-d). Inflammatory spinal cord lesions from aCB₁-KO mice
at the chronic stage also showed reductions in the extent of
neuroaxonal degeneration that were encompassed by an attenuated
presence of microglia/macrophages and reactive astrocytes, as
determined by double immunolabelling for MBP and SMI32, Iba1 or
GFAP (Figure S3c, e). Thus, aCB₁R exacerbate autoimmune
inflammation and associated clinical symptomatology during EAE
progression.

CB₁ receptor deletion prevents astrocyte dysfunction during
EAE

To study the role of CB₁R in the phenotypic transformation of astroglial
cells during EAE we assessed the expression of molecules related to
the acquisition of astrocyte pathogenic properties (46-48) in spinal cord
tissue. Astrocyte-specific CB₁R null mice showed reduced expression
levels of several genes associated with the conversion of these cells to
disease-promoting phenotypes at acute EAE disease as determined by
real-time qPCR analysis of spinal cord lysates (Figure 1d). We next
examined inflammatory spinal cord lesions for the presence of
complement component 3 (C3) as marker of pathogenic astrocytes in
MS and EAE (47-49). Double immunofluorescence staining of GFAP and
C3 indicated reduced expression of both proteins in demyelinating
spinal cord lesions from aCB₁-KO mice that were encompassed by lower
numbers of Iba1⁺ microglia/macrophages (Figure S2b-c).
Colocalization analysis of GFAP and C3 evidenced reductions in the
expression levels of C3 within astrocytic profiles (Figure 1e). These
results may suggest that aCB₁R facilitate the astrocyte transformation
into pathogenic phenotypes during EAE.

We next sought for possible differences between genotypes regarding astrocyte functional properties. Reactive astrocytes in the somatosensory cortex display impaired calcium responses that correlate to the severity of clinical symptomatology during acute EAE (34, 49). Thus, we reasoned that astrocyte-specific CB₁R deletion might attenuate glial reactivity and preserve astrocyte network function in this brain area. Fiber photometry analysis of cortical astrocytes during EAE time-course (Figure 1f) showed reductions in the amplitude of sensory-evoked calcium signals in freely behaving aCB₁-WT mice at acute disease as compared to non-immunized mice (Figure 1g, left panel). This result resembles our recent observations on astrocyte calcium deregulation in this rodent model of MS using non-transgenic mice (49) and adds to the growing body of evidence showing that attenuation of aCB₁R function encompasses the pathogenic activation of these cells during acute autoimmune inflammation (34, 46).
Comparison of astrocyte calcium signals recorded from non-immunized aCB₁-KO and aCB₁-WT mice did not evidence differences between genotypes (Figure S4) showing that aCB₁R deletion does not modulate calcium responses under our experimental paradigm in control conditions. However, astrocyte calcium signals evoked by sensory stimulation in the brain cortex of aCB₁-KO mice at acute EAE disease were not significantly reduced as compared to those recorded in the control aCB₁-KO group (Figure 1g, right panel). Together, these combined observations suggest an attenuation of disease-associated astrocyte hypo-responsiveness at the calcium signaling level in mice lacking aCB₁R. Consistently, cortical GFAP levels were reduced in the aCB₁-KO group when compared to aCB₁-WT mice without reaching statistical significance (p = 0.0806; unpaired t test) while C3 immunostaining was significantly downregulated (Figure S2d). Colocalization analysis showed astrocyte-specific reductions in C3 expression within deep cortical layers of aCB₁-KO mice at acute disease (Figure 1h). Furthermore, immunofluorescence staining revealed that the presence of cortical inflammatory lesions, defined by the presence of intraparenchymal CD3+ T cells, was also significantly reduced in aCB₁-KO animals (Figure 1i). Thus, aCB₁R deletion attenuates cortical inflammation and astrocyte network dysfunction during EAE.

Mice with astrocytic CB₁ receptor inactivation display intact oligodendrocyte populations in the EAE and LPC models

Oligodendrocyte differentiation prevents axonal loss and attenuates clinical symptomatology in the EAE model (50-52), thus pinpointing to
functional remyelination as potential mechanism of disease
attenuation during autoimmune inflammation. Neurotoxic astrocytes
release factors that promote oligodendrocyte apoptosis and delay
lineage progression, leading to reduced remyelination and subsequent
neuronal death (8, 48). Based on these evidences, we hypothesized
that the protective phenotype of aCB₁-KO mice in terms of astrocyte
reactivity and demyelination extent may be related to the engagement
of repair mechanisms during disease time-course. To determine
whether aCB₁R impede oligodendrocyte differentiation-promoting
effects of astroglial cells as mechanism of clinical exacerbation, we
immunostained spinal cord sections from aCB₁-WT and aCB₁-KO mice
for the oligodendrocyte lineage marker OLIG2 in combination with CC1
and NG2 to identify myelinating oligodendrocytes and OPCs,
respectively. Astrocyte-specific CB₁R mutants at acute EAE disease
displayed unaltered numbers of OLIG2⁺ oligodendrocyte lineage cells,
CC1⁺/OLIG2⁺ oligodendrocytes and NG2⁺/OLIG2⁺ OPCs as compared to
littermate controls, both in the demyelinating lesions and in the
surrounding perilesion areas (Figure 2a-d). The percentages of
CC1⁺/OLIG2⁺ mature oligodendrocytes and NG2⁺/OLIG2⁺ OPCs in the
plaques and periplaques were also similar between aCB₁-WT and aCB₁-
KO mice (Figure 2e). Thus, aCB₁R do not modulate oligodendrocyte
populations at acute EAE disease. Consistently, gene expression
analysis of oligodendrocyte/myelin genes (Olig2, Pdgfra, Mbp, Mog)
and factors that promote oligodendrocyte differentiation and
(re)myelination (Bdnf, Cntf, Ifg1, Ntf3, Pdgfa, Tgfb1) did not show
significant differences between genotypes (Figure 2f). Collectively, these results suggest that aCB₁R do not hinder myelin repair as mechanism of clinical deterioration during EAE.

Mechanistic studies of remyelination in EAE mice are challenging as autoimmune inflammation produces concomitant demyelination, axonal damage and myelin repair (53). To gain further insights on the role of aCB₁R during remyelination *in vivo* we used a toxin-induced model in which demyelination of focal lesions generated by localized injection of LPC in spinal cord white matter is followed by spontaneous remyelination (54-56). OPCs are recruited into the demyelinated lesion between 3-7 days post-lesion (dpl) and differentiate to mature oligodendrocytes during the second week post-lesion, thus providing a defined time window to study changes in the rate of remyelination. We analyzed oligodendrocyte populations in LPC lesions from aCB₁-WT and aCB₁-KO mice at 14 dpl corresponding to the peak of endogenous OPC differentiation during the remyelination phase (Figure S5a). The demyelination extent of LPC lesions, assessed by co-immunostaining against MBP, was similar between genotypes (Figure S5b). Quantification of OLIG2+ oligodendrocyte lineage cells and double-labelled CC1+/OLIG2+ mature and CC1-/OLIG2+ immature populations in LPC lesions revealed no variations between aCB₁-KO and aCB₁-WT mice (Figure S5c-e). Consistently, the numbers of PDGFRα+ precursor cells in LPC lesions were similar in aCB₁-KO and aCB₁-WT mice (Figure S5f). We also investigated potential changes in oligodendrocytes expressing brain-enriched myelin-associated protein 1 (BCAS1), which
have been identified as a population of immature cells actively involved
in (re)myelination (57, 58). Immunohistochemistry for BCAS1+
revealed significant numbers of active oligodendrocytes in the lesion
area of both aCB₁-KO and aCB₁-WT mice that remained unaltered
between genotypes (Figure S5g). Thus, aCB₁R do not modulate
spontaneous OPC differentiation following toxic oligodendrocyte loss.
We next assessed LPC lesions from aCB₁-KO and aCB₁-WT mice for the
presence of inflammatory cells. GFAP expression was not significantly
different between groups, suggesting that LPC injections activate
astrogliosis to a similar extent in aCB₁-KO and aCB₁-WT mice (Figure
S5h). The presence of Iba1⁺ microglia/macrophages (Figure S5i) and
CD45 infiltrating inflammatory cells (Figure S5j) was also similar
between genotypes. These combined results suggest that aCB₁R do not
interfere with myelin repair processes in inflammatory and toxin
induced demyelinating contexts and point to alternative mechanisms
for clinical exacerbation during autoimmune inflammation.

## Reduced humoral and cellular infiltration in EAE lesions from astrocyte-specific CB₁ receptor null mice

Perivascular astrocyte processes are enriched in CB₁R that modulate
BBB permeability during stress-induced inflammation (59-62). Thus, we
wondered whether changes in the BBB properties contribute to the
protective phenotype of astrocyte-specific CB₁R null mice during EAE.
To address this question, we initially measured parenchymal entry of
humoral factors and immune cells as readouts of BBB opening at acute
EAE disease (40, 63, 64). The areas immunopositive for the serum
proteins fibrinogen and IgG as markers of humoral factor extravasation
were markedly reduced in spinal cord lesions from aCB₁-KO mice
(Figure 3a). Moreover, spinal cord tissues from aCB₁-KO mice
contained significantly lower numbers of CD45⁺ leukocytes (Figure
3b) and reduced densities of CD3⁺, B220⁺ and Ly6G⁺ leukocyte
subsets (Figure 3c) within EAE lesions or perilesion areas, as
measured using histopathology. Of note, the comparative analysis of
splenic lymphoid and myeloid cell populations revealed no detectable
differences between non-immunized aCB₁-WT and aCB₁-KO mice in any
of the evaluated cellular subsets (Figure S6). This result suggest that
astrocyte-specific CB₁R mutants do not display an impaired ability to
develop autoimmune responses, consistent with the lack of differences
between genotypes in terms of symptom onset. Collectively, these
observations highlight that aCB₁-KO mice display preserved BBB
function during autoimmune inflammation.

Astrocytes promote the formation of demyelinating lesions in EAE
and MS by mechanisms that lead to the repression of endothelial tight
junction (TJ) proteins and drive BBB permeability (63, 64).
Concomitantly, astroglial cells activated in response to
neuroinflammation upregulate claudin 1 (CLN-1) and claudin 4 (CLN-4)
to form protective astrocytic TJ complexes that control humoral and
cellular transit (65). Expression of Cldn1 and Cldn4 in spinal cord tissue
was not significantly modulated in the aCB₁-KO group as compared to
aCB₁-WT mice (Figure 3d). However, western blot analysis of spinal
cord lysates showed reduced expression levels of the main astrocytic
TJ protein CLN-4 (65) (Figure 3e), which sits well with the attenuated inflammatory phenotype of aCB₁-KO mice (Figure 3b-c). To investigate whether aCB₁R modulate the endothelial BBB during autoimmune inflammation we analyzed the expression of vascular endothelial cell markers and TJ associated proteins in spinal cord tissues from EAE mice. Astrocyte-specific CB₁R null mice displayed unaltered levels of the vascular endothelial membrane proteins podocalyxin (PODXL) and laminin, as measured using immunoblotting and confirmed by immunohistochemistry of EAE lesions (Figure S7a-b). These findings indicate that aCB₁-KO mice do not display abnormal angiogenesis at acute EAE disease as compared to wild-type animals. The expression of the endothelial TJ components cadherin 5 (CDH-5), zonula occludens (ZO-1) and claudin 5 (CLN-5) was also similar between aCB₁-WT and aCB₁-KO mice (Figure S7c-d). Thus, aCB₁R null mice display microvascular endothelial cells similar to wild-type controls at established EAE disease despite the attenuated humoral and leukocyte infiltration in lesion and perilesion sites.

Mice with astrocyte CB₁ inactivation show reduced expression of immune and vascular effector molecules in EAE lesions

Reactive astrocytes contribute to the pathogenesis of CNS inflammatory lesions through the release of intercellular molecules that enable circulating inflammation to reach the brain and spinal cord parenchyma. Astrocyte effector molecules that mediate autoimmune inflammation include the chemokines CCL2, CCL5 and CXCL2 as recruiters of perivascular leukocytes (8, 10), adhesion molecules such
as ICAM-1 and VCAM-1 aberrantly expressed by astroglial cells (66-69), and angiogenic factors, mainly VEGF-A, that signal to the vascular endothelium and promote permeability (40, 63, 64). To gain further insights on the mechanistic implications of aCB₁R during autoimmune inflammation we addressed the expression of immune and vascular effector molecules at acute EAE disease. The expression levels of *Cc/2*, *Cc/5* and *Cxc/2* determined by RT-qPCR in spinal cord lysates of EAE mice were highly heterogeneous and not significantly modulated in the aCB₁-KO group (**Figure 4a**). Conversely, gene expression analysis highlighted significantly lower levels of *Icam1* and *Vcam1* that were confirmed by immunoblotting (**Figure 4a-b**) and confocal imaging (**Figure S8**) of spinal cord tissue. Moreover, double immunostaining for GFAP or aquaporin-4 with ICAM-1 or VCAM-1 showed reduced expression of both adhesion molecules in astrocytic profiles within EAE lesions and perilesion areas of aCB₁-KO mice (**Figure 4c-d**). Of note, VEGF-A expression was drastically downregulated in spinal cord tissue (**Figure S8**) and GFAP and aquaporin-4 immunopositive astrocyte profiles (**Figure 4c-d**) of aCB₁-KO mice as compared to the aCB₁-WT group. Thus, the inhibition of humoral and lymphocyte infiltration in white matter lesions of mice with aCB₁R deletion is accompanied by a restricted astrocyte production of adhesion molecules and VEGF-A during autoimmune inflammation.

**Diminished inflammatory neuropathology of astrocyte CB₁ receptor null mice in the VEGF-A model of BBB breakdown**
Collectively, the above findings suggest that aCB₁R facilitate BBB permeability to promote inflammatory lesion formation during autoimmune inflammation. To further investigate potential links between aCB₁R and BBB opening *in vivo*, we addressed the phenotype of astrocyte-specific CB₁R null mice in the VEGF-A injection model, which triggers a rapid, localized breakdown of BBB structure and function mirroring pathological leakage during autoimmune inflammation (40, 63, 64). We stereotaxically delivered mouse VEGF-A₁₆₅ into the left cerebral cortex of aCB₁-KO mice and littermate aCB₁-WT animals and measured changes in barrier function at 2 days post-injection (**Figure 5a**). Compatible with published data (40, 64), we observed BBB breakdown as measured by fibrinogen and IgG immunoreactivity in VEGF-A₁₆₅-injected areas (**Figure 5b**). The endothelial TJ markers CDH-5 and ZO-1 appeared patchy and discontinuous in both aCB₁-WT and aCB₁-KO mice (**Figure 5d**) and these changes were accompanied by parenchymal accumulation of CD45⁺ cells (**Figure 5b**). Importantly, BBB disruption, as measured by serum protein and immune cell extravasation, was attenuated in aCB₁-KO mice (**Figure 5b**) despite unaltered expression levels of the endothelial cell marker PODXL (**Figure 5c**), and TJ proteins CDH-5 and ZO-1 (**Figure 5d**). Reminiscent of our observations in the EAE lesions, aCB₁R null mice showed reduced astrocyte reactivity (**Figure 5e**), and lower levels of the astrocyte TJ protein CLN-4, but not of CLN-1, in areas of BBB disruption (**Figure 5f**). These studies show that aCB₁R engage mechanisms downstream of VEGF-A that promote BBB leakiness and
allow for the infiltration of peripheral immune cells during inflammatory
lesion formation.

Discussion

Studies conducted during the past decades have demonstrated that
CB₁R signaling restricts clinical disability in MS (16, 21). Potential
mechanisms for (endo)cannabinoid-mediated protection mediated by
CB₁R in MS patients include neuroprotective and remyelination
promoting effects, as defined by analyzing the phenotype of
constitutive CB₁R knockout mice (22) and transgenic mice lacking CB₁R
on neurons (20) and OPCs (25) in preclinical disease models.
Nevertheless, the specific roles of CB₁R expressed by astrocytes in MS
have been scarcely investigated despite the critical involvement of
these cells in disease initiation and progression (8-10). This study is the
first to our knowledge that highlights the significance of CB₁R
endocannabinoid signaling mediated by astrocytes in the generation of
clinical deficits during CNS inflammatory disease. Upon characterizing
the phenotype of mice lacking aCB₁R in models of acute and chronic
CNS damage, we depict an unprecedented deleterious role of CB₁R in
astroglial cells during inflammatory lesion formation linked to the
modulation of BBB permeability. On mechanistic grounds, we show that
aCB₁R deletion restricts humoral and cellular leakage associated with
BBB disruption, at least in part, by engaging cellular processes
downstream VEGF-A signaling. These observations uncover a novel
mechanism underlying astrocyte-related control of the BBB in
neuroinflammation and shed light on the roles of endocannabinoids
and CB₁R in the pathogenesis of MS.

In this study, we show protective effects of aCB₁R inactivation during
the time-course of EAE at the clinical and neuropathological level.
Attenuation of neurological disability occurred in concert with changes
in astrocyte reactivity and expression of molecules related to the
acquisition of disease promoting functions in MS patients and
preclinical disease models (46, 48). These observations are consistent
with a scenario in which aCB₁R signaling facilitates the emergence of
neurotoxic phenotypes that impede astrocyte-oligodendrocyte
interactions underlying myelin repair as potential mechanism of
disease exacerbation (8). Studies specifically designed to investigate
the involvement of cell autonomous astrocyte responses mediated by
CB₁R in remyelination are currently lacking. Thus, we addressed this
possibility by histologically targeting oligodendrocyte populations in
lesion and perilesion areas of aCB₁-KO mice at acute EAE disease. This
analysis did not evidence significant modulatory effects encompassing
the attenuation of inflammation and myelin pathology that resulted
from deletion of CB₁R in astrocytes. Consistently, analysis of
remyelinating spinal cord lesions induced by microinjection of LPC did
not highlight oligodendrocyte differentiation promoting effects in
astrocyte-specific CB₁R null mice. Although the possibility that
endocannabinoids target CB₁R in astroglial cells to modulate
remyelination in vivo cannot be fully disregarded in the absence of a
detailed myelin ultrastructure analysis during the time-course of
myelin repair, these results suggest that aCB₁R do not impede remyelination as potential mechanism of clinical exacerbation in demyelinating disease.

Our present data support the hypothesis that astrocyte responses to CB₁R signaling modulate the formation of CNS lesions as principal mechanism of disease exacerbation. This possibility sits well with the fundamental roles of astroglial cells in regulating the BBB breakdown during CNS inflammation (8, 10) and with reports of enriched CB₁R expression in perivascular astrocytic profiles (59, 61, 62). Indeed, the clinical benefits of astrocyte-specific CB₁R inactivation during EAE are observed during the onset of neurological symptomatology and occur in concert with reduced inflammatory lesion load at established disease, both in cortical and spinal cord tissue. These observations prompted us to investigate the phenotype of aCB₁-KO mice in terms of BBB disruption during EAE. Here, as expected if CB₁R in astrocytes would promote BBB permeability defects allowing circulating inflammation to enter the CNS parenchyma, aCB₁-KO mice exhibited lower levels of humoral mediators and infiltrating leukocytes in inflammatory demyelinating EAE lesions and surrounding perilesion areas. Notably, these changes were associated with reduced expression levels of the adhesion molecules ICAM-1 and VCAM-1 by reactive astrocytes in EAE lesions. These findings may explain, at least in part, the restricted presence of infiltrating immune cells observed in mice with astrocyte-specific CB₁R deletion, according to the established roles of both adhesion molecules in mediating leukocyte
movement across the BBB during CNS inflammatory disease (66-69). Reactive astrocytes in EAE lesions from aCB₁-KO mice also exhibited lower levels of the angiogenic and pro-permeability factor VEGF-A, identified as a key driver of astrocyte-mediated BBB disruption, leukocyte infiltration and neuropathology during CNS inflammation (63, 64). Reductions in astrocyte VEGF-A expression might limit leukocyte entry and contribute to reducing lesion load in astrocyte-specific CB₁R null mice. Concomitantly, lower levels of adhesion molecules or other potential astrocyte mediators may further restrict BBB permeability as evolving inflammation drives astrocyte VEGF-A expression (70). Using the focal, directly induced VEGF-A model in transgenic mice allowed us to selectively examine the role of CB₁R-related astrocyte responses on BBB breakdown and lymphocyte entry downstream this vascular effect molecule. Critically, mice with astrocyte-specific CB₁R deletion showed milder humoral and cellular extravasation induced by VEGF-A, thus mirroring the restricted inflammatory phenotype observed in EAE lesions. Altogether, these findings suggest that CB₁R signaling in astrocytes facilitates the formation of CNS inflammatory lesions by targeting effector mechanisms downstream VEGF-A that promote BBB disruption.

Notably, the above changes occurred in concert with reduced levels of the astrocyte TJ protein CLN-4, whose induction has been associated with the emergence of a protective astrocyte barrier that limits the access of peripheral inflammation to the CNS parenchyma (65). These seemingly contradictory findings can be explained on the basis that the
evolving inflammation in lesion sites, which we find to be significantly
reduced in mice lacking aCB₁R, is a driving force for the induction of
junctional adhesion proteins at the glia limitants (65). A striking
observation in this study, however, is that attenuated parenchymal
leakage of circulating inflammation in lesions from aCB₁-KO mice was
not encompassed by significant endothelium abnormalities, as
expected according to reports that BBB breakdown results from
disruptions of endothelial junctional proteins such as CLN-5 (40, 63,
64). It should be borne in mind, however, that our present findings do
not exclude the possibility that astrocyte-specific CB₁R null mice exhibit
preserved endothelial barrier functions at initial stages of BBB
dysfunction in the EAE and VEGF-A models, as compared to non-
transgenic animals. Disruption of the BBB precedes immune cell
infiltration during CNS lesion formation (40, 71) and it seems plausible
that early-onset differences in endothelial permeability between
genotypes are masked at later states by the evolving inflammatory
neuropathology. Future research targeting the time-course of CNS
lesion formation *in vivo* combined with clinically relevant *in vitro*
models of BBB dysfunction that combine pharmacological approaches
and gene knockdown strategies may uncover the mechanistic basis of
astrocyte signaling to endothelial cells under the control of
endocannabinoids and aCB₁R that lead to clinical exacerbation during
demyelinating disease. In this context, the paucity of studies that
investigate the signaling events engaged by endocannabinoids at the
BBB permeability level (60, 72, 73) contrasts the overwhelming
evidence supporting that these lipid mediators and their exogenous counterparts dampen CNS inflammation in multiple disease paradigms (14, 16). Result from the Theiler's murine encephalomyelitis virus (TMEV) infection model supported a protective role for CB₁R expressed by endothelial cells in attenuating leukocyte transmigration (74). More recently, perivascular aCB₁R have been postulated to promote stress resilience in mice through the preservation of BBB functions (60). In this landmark study, viral-mediated astrocyte overexpression of CB₁R mitigated inflammatory responses and morphological changes in the chronic social defeat stress (CSDS) mouse model of depression. These observations are in apparent contrast with our present findings that aCB₁R deletion preserves BBB function and restrains circulating inflammation to enter the brain parenchyma. It should be noted, however, that stress-induced inflammation and autoimmune demyelination are intrinsically different disease contexts that involve dissimilar etiopathological mechanisms. Furthermore, the protective phenotype of aCB₁R at the vascular level reported by Dudek and collaborators was ascribed to astrocyte populations within the nucleus accumbens shell in male mice resilient to CSDS. Conversely, this study, that we performed in female mice, suggests more generalized mechanisms underlying the control of BBB permeability by aCB₁R during CNS lesion formation, as aCB₁-KO mice showed attenuated spinal cord white matter and cortical grey matter inflammatory pathology in complementary disease models. Remarkably, CSDS and viral-induced downregulation of endothelial *C/n5* increase aCB₁R and
endocannabinoid levels *in vivo* while acute inflammatory challenges
with IL-6 upregulate the expression levels of *Cnr1* in culture systems
(60). These recent observations suggest that BBB disruption facilitates
aCB₁R signaling through inflammation-related mechanisms. In this
scenario, it seems plausible that distinct inflammatory environments
engage endocannabinoid signaling through specific astrocytic pools of
CB₁R located at plasmatic membranes and/or mitochondrial
compartments, leading to context-specific modulation of BBB
functions. Unfortunately, astrocyte-specific adaptations in CB₁R
function and signaling in the EAE model have not been investigated
until recently. Gene expression analysis of endocannabinoid signaling
genes in astrocytes purified during the time-course of EAE highlighted
reduced *Cnr1* transcript levels both at acute (14-17 dpi) and chronic
(28-31 dpi) stages that were absent at presymptomatic disease (7-8
dpi) (46). More recently, *ex vivo* and *in vivo* analysis of astrocyte
calcium dynamics in mice at acute EAE demonstrated reductions in
CB₁R coupling to the modulation of intracellular calcium responses
that involve deficits in the intracellular pathways operated by G
proteins (34). In this study, impairments in aCB₁R-mediated calcium
signaling translated into gliotransmission defects leading to
exacerbated synaptic excitation, with still unknown consequences in
terms of cortical pathology. Given that aCB₁R signaling is
downregulated at acute EAE, we assume that the pathogenic
activation of the astrocytic receptor pools in this rodent model
autoimmune demyelination takes place during the initial phase of the
disease, consistent with an early role in the formation of inflammatory
lesions. Of note, astrocyte activation during EAE involves early-onset
reductions in most endocannabinoid hydrolysis genes that can be
detected at presymptomatic stages (46). These findings provide a
plausible cell autonomous mechanism for the early activation of
aCB₁R in this model of autoimmune inflammation potentially leading
to receptor downregulation at later stages. Further, locally produced
endocannabinoids might also target astrocytic CB₂R according to the
reported expression of this protein in these cells within MS
inflammatory lesions (75). However, these hypotheses remain to be
further explored using high-resolution anatomical techniques and cell-
type specific endocannabinoid probes (76) applied to the analysis of
astrocytes during EAE. Mechanistic considerations notwithstanding,
our study adds to the growing body of evidence that supports essential
roles for the astrocytic populations of CB₁R in regulating the astrocyte-
endothelial interface in neuroinflammatory disorders.

The exact cascade of events upstream and downstream aCB₁R
signaling that modulate BBB permeability during CNS lesion formation
remains a matter of future research in the context of autoimmune
inflammation, which should be addressed in clinically relevant *in vivo*
models that enable reliable, high-throughput assessment of astrocytic
function at early time points of the inflammatory pathology. According
to the existing literature, early-onset activation of aCB₁R may facilitate
BBB dysfunction by attenuating astrocyte reactivity (8, 10). This
possibility is supported by a series of *in vivo* ablation studies showing
that early inhibition of reactive astrogliosis facilitates circulating
inflammation to reach the CNS parenchyma and exacerbates clinical
disease in the EAE model of MS (77-79). Some tentative additional
support for this hypothesis comes from prior research showing that
pharmacological blockade of endocannabinoid hydrolysis inhibits
astrocyte reactivity while attenuating disease neuropathology and
clinical severity in the EAE and TMEV models of neuroinflammation (80,
81). However, these *in vivo* studies did not target CB₁R as potential
underling mechanism and research specifically designed to address the
role of CB₁R in regulating the functions of reactive astrocytes is still
scarce. In this context, it is worth mentioning that the protective
phenotype of astrocyte-specific CB₁R null mice in the EAE model that
we report here is consistent with recent *in vivo* observations that aCB₁-
KO mice display significantly less severe neuronal death following
induction of global ischemia (82).

To summarize, we propose that early, endogenous activation of CB₁R
in astrocytes drives the pathogenicity of CNS inflammatory lesions by
mechanistic interactions that promote BBB permeability and
recruitment of immune cells. This study challenges the traditional
neuroprotective role of endocannabinoids in MS and adds to the
accumulating evidence that points to relevant roles of
endocannabinoid signaling via aCB₁R in the neurovascular adaptations
that shape neuroinflammation.

Limitations of the study
The present study has some limitations to consider. First, like most
studies addressing astrocyte-mediated functions of CB₁R in complex
systems, the loss of function approach is based on the expression of
Cre recombinase under the control of the human GFAP promoter. Of
note, the pattern of recombination efficiency in the tamoxifen GFAP-
CreERT2 reporter mouse line, which reflects the transgenic expression
level of the human GFAP promoter, varies between brain regions in a
similar manner as the intrinsic reporter (37). Indeed, mouse
astrocytes express heterogeneous levels of GFAP, as defined by
single-cell transcriptional profiling (83-85). According to these
evidences, astrocyte populations in aCB₁-KO mice are heterogeneous
in terms of Cre-LoxP recombination and deletion of the *Cnr1* gene,
which limits the biological interpretation of our results. This technical
pitfall, together with the fact that astrocytes express very low levels of
CB₁R as compared to presynaptic elements (31, 86), limits the reliable
detection of *Cnr1* gene expression variations between aCB₁-KO and
aCB₁-WT mice using astrocyte preparations purified based on the
expression of cell-surface markers (87). However, the aCB₁-KO mouse
strain used in this study has been well characterized in terms of aCB₁R
downregulation throughout the CNS using high resolution electron
microscopy and functional approaches (29, 31, 32, 38, 86, 88).
Further, aCB₁-KO mice present a plethora of behavioral phenotypes
that include memory impairments (38), defects in social transmission
of stress (89) and protection from stroke (82), as well as resistance to
the amnesic (29) and anti-tremor (32) effects of cannabinoids. In the
context of these previous evidences, our data state the principle that
CB₁R expressed by astrocytes can exacerbate CNS inflammation.

Second, the low perfusion volume prior to dissection of spinal cord tissue for gene expression analysis may potentially limit the confidence in the results presented. In this study, EAE mice for RT-PCR analysis were perfused with an average of 5 mL of perfusion solution, which makes 3-4 times total blood volume in animals weighing 15-20 g. This approach differs from standardized procedures - typically using 10-20 mL of perfusate - in studies aiming to remove intravascular blood cells prior to CNS analysis. However, to the best of our knowledge there are no direct comparative studies of perfusion volume for capillary cell removal in the EAE model that take into account the remarkable loss of body weight during the acute phase of the disease, and most methodological choices are guided by general CNS perfusion practices.

With regard to the gene expression results presented in **Figures 1d** and **4a**, it should be borne in mind that variability is an intrinsic feature of the EAE model. Both aCB₁-KO and aCB₁-WT mice presented marked interindividual differences in terms of disease severity and dispersion in the expression of inflammatory genes within each group is not surprising. Therefore, although we cannot fully exclude the presence of residual blood cells in CNS tissue we are confident that our gene expression results in EAE mice are not limited by technical pitfalls related to inadequate perfusion.

**Data availability**
The datasets generated during the current study are available from the
corresponding authors on reasonable request.

Abbreviations

aCB₁R: Astrocyte cannabinoid type-1 receptor

BBB: Blood-brain barrier

CB₁R: Cannabinoid type-1 receptor

CDH-5: Cadherin 5

cDNA: Complementary DNA

CLN-1: Claudin 1

CLN-4: Claudin 4

CLN-5: Claudin 5

CNS: Central nervous system

FBS: Fetal bovine serum (FBS)

GFAP: Glial fibrillary acidic protein

ICAM-1: Intercellular adhesion molecule 1

LED: Light-emitting diode

LFB: Luxol fast blue

LPC: lysophosphatidylcholine

MBP: Myelin basic protein

MOG: Myelin oligodendrocyte glycoprotein

MS: Multiple sclerosis

NDS: Normal donkey serum

NGS: Normal goat serum

OPC: Oligodendrocyte precursor cell

PBS: Phosphate buffered saline
PCR: Polymerase chain reaction

PODXL: Podocalyxin

RIPA: Radioimmunoprecipitation assay

RNA: Ribonucleic acid

RT: Room temperature

TBS: Tris buffer saline

THC: Δ⁹-tetrahydrocannabinol

VCAM-1: Vascular cell adhesion protein 1

VEGF-A: Vascular endothelial growth factor A

ZO-1: Zonula occludens

## Acknowledgements

We thank all members of the S. Mato´s and G. Marsicano's laboratories for useful discussions and advice. We also wish to thank the staff from the Neurocentre Magendie Genotyping Facility, the Genome Analysis Platform of the University of the Basque Country, and the Achucarro Basque Center of Neuroscience Imaging, Cell Analytics and Molecular Biology Facilities, as well as the Animal Facilities of the University of the Basque Country and Neurocentre Magendie for mouse care.

## Funding

This work was funded by Carlos III Health Institute (PI21/00629, to S.M. and PI24/00447, to D.C. were cofounded by the European Union; CB22/05/00016, to D.C.), Basque Government (PIBA_2023_1_0046; 2023111031 and IT1473-22, to S.M.; CannaMetHD, to S.M. and G.M.; IT1203-19, to C.M.), ARSEP Foundation (ARSEP-1310 to S.M. and G.M.; ARSEP-1317 and SEP-10 to V.T.), the European Research Council
(MiCaBra, ERC-2017-AdG-786467, to G.M.), INSERM (to G.M.), Fondation pour la Recherche Medicale (FRM, DRM20101220445 to G.M.), Region Aquitaine (CanBrain, AAP2022A-2021-16763610 and -17219710 to G.M.), French State/Agence Nationale de la Recherche (HippObese, ANR-23-ce14-0004-03; ERA-Net Neuron CanShank, ANR-21-NEU2-0001-04; CaMeLS, ANR-23-CE16-0022-01, to G.M.), La Caixa Research Health 2023 (PsychoCannabis, HR23-00793, to G.M.), the Spanish Ministry of Science and Innovation (SAF2015-74332-JIN and PID2023-152688OB-100 to V.T.; PID2019-109724RB-100 to C.M.), Consellería de Educación, Universidades y Empleo-Generalitat Valenciana (CIDEXG/2023/23 to V.T.), BIOEF-EITB-Maratoia (BIO23/EM/008 to C.M.) and Walk on Project Foundation. T.C., E.S.-M. A.M.-G., A.M.B. and I.P.-L. held predoctoral fellowships from the Basque Government (T.C., A.M.-G. and A.M.B.), the University of the Basque Country (E.S.-M.) and the Spanish Ministry of Science, Innovation and Universities (I.P.-L.; FPU22/00693). The cartoon in Figure 1a was created with BioRender.com.

## Author contributions

T.C., A.B.-C., E.S.-M., A.U. and A.I-S. performed and analyzed histological, immunoblot and gene expression experiments and reviewed the manuscript. A.M.-G. and R.S. performed fiber photometry analysis. S.B., U.S. and I.F-M. were involved in mouse breeding and performed pharmacological treatments. C. G.-C.C., D.G. and G.L. performed and supervised mouse genotyping experiments. I.P.-L. and D.C. conceptualized and performed flow cytometry analysis. V.T.
conceptualized LPC experiments, performed surgeries and was involved in data analysis and interpretation. C.C. performed cortical surgeries for VEGF-A₁₆₅ injection and provided guidance for analysis and interpretation. C.M. performed EAE experiments. A.M.B. was involved in the analysis of calcium imaging experiments. G.M. and S.M. conceptualized and supervised the study. A.M.B., T.C., S.B. and S.M. produced the figures and wrote the paper. All authors approved the final manuscript.

**Competing interests**

The authors declare no competing interests.

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80. Feliú A, Bonilla Del Río I, Carrillo-Salinas FJ, Hernández-Torres G, Mestre L, Puente N, et al. 2-Arachidonoylglycerol reduces proteoglycans and enhances remyelination in a progressive model of demyelination. *J Neurosci.* 2017;37(35):8385-98.

81. Guadalupi L, Mandolesi G, Vanni V, Balletta S, Caioli S, Pavlovic A, et al. Pharmacological blockade of 2-AG degradation ameliorates clinical, neuroinflammatory and synaptic alterations in
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82. Wang F, Han J, Higashimori H, Wang J, Liu J, Tong L, et al. Long-term depression induced by endogenous cannabinoids produces neuroprotection via astroglial CB₁R after stroke in rodents. *J Cereb Blood Flow Metab*. 2019;39(6):1122-37.

83. Zeisel A, Muñoz-Manchado AB, Codeluppi S, Lönnerberg P, La Manno G, Juréus A, et al. Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq. *Science*. 2015;347(6226):1138-42.

84. Bayraktar OA, Bartels T, Holmqvist S, Kleshchevnikov V, Martirosyan A, Polioudakis D, et al. Astrocyte layers in the mammalian cerebral cortex revealed by a single-cell in situ transcriptomic map. *Nat Neurosci*. 2020;23(4):500-9.

85. Ben Haim L, Rowitch DH. Functional diversity of astrocytes in neural circuit regulation. *Nat Rev Neurosci*. 2017;18(1):31-41.

86. Bosier B, Bellocchio L, Metna-Laurent M, Soria-Gomez E, Matias I, Hebert-Chatelain E, et al. Astroglial CB₁ cannabinoid receptors regulate leptin signaling in mouse brain astrocytes. *Mol Metab*. 2013;2(4):393-404.

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<figure><img src="image_2.png" /></figure>
<figure><img src="image_3.png" /><figcaption>**Figure 1.** Astrocyte CB₁R exacerbate EAE pathology and clinical deficits. (a) Time-course of EAE progression in aCB₁-KO mice. Data are representative of 2 independent EAE experiments pooled together (n = 18-19 mice; ***p < 0.0001; Wilcoxon matched-pairs signed rank test</figcaption></figure>
for the comparison of score curves from the onset of EAE symptoms at 8 dpi to 22 dpi). (b-c) Representative images and/or quantitative analysis of MBP, SMI32 and APP immunostaining in spinal cord sections at 22 days post-immunization (dpi) (n = 6 mice). The dashed line delimits the demyelinating lesion (L) in c. Scale bar = 200 µm (b) and 25 µm (c). (d) Relative expression of neurotoxic astrocyte genes in spinal cord tissue (n = 9-10 mice). Right panel: Comparative analysis of neurotoxic astrocyte gene expression profile between genotypes (n = 9 genes) (p = 0.0039; Wilcoxon matched-pairs signed rank test). The expression of inflammatory genes in aCB₁-KO mice was normalized to the mean expression of each gene in the control group. (e) Representative confocal z-stack projections and quantification of GFAP and complement component 3 (C3) colocalization in spinal cord tissue (n = 6 mice). Scale bar = 100 µm. (f-i) Attenuated cortical astrocyte pathology and functional deficits in aCB₁-KO mice during EAE. (f) *In vivo* time-course analysis of astrocytic calcium activity for 3 consecutive days before EAE induction and at 12, 14, 17, 19 and 21 dpi. (g) Representative traces show calcium responses of cortical astrocytes evoked by tail-holding at -3 (top) and 19 (bottom) dpi. Graphs depict the amplitude of cortical astrocyte calcium responses recorded during the EAE time course compared to values from non-immunized animals recorded in parallel (n = 8-12 mice). (h) Confocal micrographs and quantification of C3 immunoreactivity in GFAP immunopositive astrocyte profiles within somatosensory cortex layer V-VI at acute EAE disease (n = 8-9 mice). Scale bar = 25 µm. (i) Confocal micrographs
and quantification of CD3 immunopositive T inflammatory lesions (arrowheads) in cortical tissue (n = 8-9 mice). Scale bar = 40 µm. **b-e** and **h-i**, *p* < 0.05, **p** < 0.01 and ***p** < 0.001, unpaired t-test or Mann-Whitney test. **g**, *p* < 0.05 and **p** < 0.01, two-way ANOVA followed by Šídák's test for multiple comparisons. Error bars express SEM.

<figure><img src="image_4.png" /></figure>
<figure><img src="image_5.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>e</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>f</figcaption></figure>

<figure><img src="image_9.png" /></figure>

**Figure 2.** Astrocyte CB₁R do not modulate oligodendrocyte populations in EAE inflammatory lesions. (a) Representative images of spinal cord lesions from aCB₁-KO and aCB₁-WT mice at acute EAE immunostained for OLIG2, CC1 and NG2 as markers of oligodendrocyte lineage cells, mature oligodendrocytes and oligodendrocyte precursor cells, respectively, show low cell densities in the lesion (L) and enrichment in the perilesion area (PL, dashed lines). Scale bar = 40 µm. (b-d) Quantitative analyses show similar densities of oligodendrocyte populations in the plaque and periplaque of aCB₁-KO and aCB₁-WT mice (n = 6 mice). (e) Percentages of oligodendrocytes (CC1⁺) and OPCs
(NG2+) in the OLIG2+ population indicate equal proportions of mature cells in the lesion and perilesion of aCB₁-KO and control littermates. (f) Gene expression analysis of oligodendrocyte/myelin markers and pro-myelinating molecules in spinal cord tissue from aCB₁-KO and aCB₁-WT mice at acute disease (n = 9-10 mice). Error bars express SEM.

<figure><img src="image_10.png" /></figure>

**Figure 3.** Restricted humoral and immune cell infiltration in EAE inflammatory lesions of aCB₁-KO mice. (a) Representative confocal z-stack projections of spinal cord lesions from aCB₁-KO and control aCB₁-WT mice at acute EAE (25 dpi) immunostained for fibrinogen (Fbn) and
IgG. Morphometry of parenchymal immunoreactivity shows reduced protein extravasation, measured as immunopositive area normalized to demyelinating lesion area, in aCB₁-KO mice compared to the aCB₁-WT group (n = 6 mice). Scale bar = 25 µm. (b) Representative micrographs and quantification of CD45⁺ infiltrating inflammatory cells in demyelinating lesions of aCB₁-KO and aCB₁-WT mice (n = 6 mice).

Scale bar = 25 µm. (c) Confocal images and quantitative analysis of CD3⁺ T cells, B220⁺ B cells and Ly6G⁺ neutrophils in lesion (L) and perilesion (PL, dashed lines) spinal cord tissue from aCB₁-KO mice as compared to aCB₁-WT animals at acute EAE disease (n = 6 mice). Scale bar = 50 µm. (d) Gene expression analysis of the astrocyte TJ proteins CLN-1 and CLN-4 in spinal cord tissue from aCB₁-KO and aCB₁-WT mice at 22 dpi (n = 9-10 mice). (e) Immunoblots and densitometry analysis of CLN-4 expression in spinal cord lysates from aCB₁-KO at 25 dpi compared to the aCB₁-WT group (n = 6-7 mice). \*p < 0.05, \*\*p < 0.01 and \*\*\*p < 0.001, unpaired t-test or Mann-Whitney test. Error bars express SEM.
<figure><img src="image_11.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_14.png" /></figure>
**Figure 4.** Astrocyte-specific CB₁R mutants show reduced expression of immune and vascular effector molecules in EAE lesions. (a) Relative gene expression of permeability effector molecules in spinal cord tissue from aCB₁-KO mice as compared to control aCB₁-WT animals at acute disease EAE (n = 9-10 mice). (b) Immunoblotting for the adhesion molecules ICAM-1 and VCAM-1 shows reduced expression levels in aCB₁-KO spinal cords (n = 6-7 mice). (c-d) Confocal z-stack projections of spinal cord sections double immunostained for (c) GFAP or (d) aquaporin-4 (Aqp-4) and ICAM-1, VCAM-1 or VEGF-A. Colocalization analysis highlights attenuated expression of permeability molecules in astroglial profiles within lesion (L) and/or perilesion (PL, dashed lines) areas from aCB₁-KO animals (n = 5-6 mice). Scale bar = 50 µm. *p* < 0.05, **p** < 0.01 and ***p** < 0.001, unpaired t-test or Mann-Whitney test. Error bars express SEM.
ARTICLE IN PRESS
<figure><img src="image_15.png" /><figcaption>**Figure 5.** Astrocyte CB₁R deletion reduces cortical BBB leakage, astrocytic tight-junction deregulation and leukocyte infiltration by VEGF-A. (a) Experimental design for the analysis of the BBB breakdown following cortical microinjection of murine VEGF-A₁₆₅ in aCB₁-KO and aCB₁-WT mice. Cerebral cortices from animals injected in 2 independent experiments were processed for histopathology at 2 days post-injection (dpi). (b) Representative cortical tissue sections immunostained for fibrinogen, IgG and CD45 and quantitative analysis</figcaption></figure>
show reduced extravasation of serum proteins, measured as
immunopositive area normalized to total lesion area, and attenuated
leukocyte infiltration in the aCB₁-KO group (n = 9 mice). Scale bars =
100 µm (fibrinogen, IgG) and 50 µm (CD45). (c, d) Confocal
micrographs and morphometry of (c) PODXL and (d) endothelial TJ
proteins CDH-5 and ZO-1 in aCB₁-KO and aCB₁-WT mice. Scale bar =
50 µm. (e, f) Attenuated astrocyte reactivity measured by
immunostaining for GFAP (e) and levels of astrocyte TJ proteins CLN-4
and CLN-4 (f) following microinjection of VEGF-A₁₆₅ in aCB₁-KO and
aCB₁-WT mice. Scale bar = 50 µm. \*p < 0.05 and \*\*p < 0.01, unpaired
t-test or Mann-Whitney test. Error bars express SEM.

<figure><img src="image_16.png" /></figure>
a

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

b

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c

<figure><img src="image_21.png" /></figure>

**Supplementary Figure 1.** (a) PCR-based strategy for the analysis of recombination in aCB₁-WT and aCB₁-KO mice. The diagram represents the wild-type, floxed, and Cre-recombined *Cnr1* alleles, indicating the position of the primers used for genotyping and PCR amplicon sizes for each reaction. (b) Detection of the excised fragment as a result of Cre-mediated recombination in forebrain and spinal cord lysates. Probe hybridization, amplification and electrophoresis resulted in a 689 bp product corresponding to the excision in forebrain and spinal cord tissues from tamoxifen-treated aCB₁-KO (CB₁<sup>f/f</sup>;GFAP-CreERT<sub>2</sub>) mice that was absent in samples from aCB₁-WT (CB₁<sup>f/f</sup>) mice. The ~2900 bp amplicon for the unrecombined allele was enriched in lysates from aCB₁-WT mice. DNA samples with germinal deletion (GD) of the *Cnr1* gene were used as control for recombination. (c) Genotyping of the
floxed allele rendered a ~595 bp product in aCB₁-WT and aCB₁-KO samples and a ~475 bp product in samples from wild-type (WT) mice. The presence of Cre recombinase generated a specific band with 300 bp in aCB₁-KO samples. DNA samples from Cre⁺ and Cre⁻ mice were run as positive and negative controls.

<figure><img src="image_22.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_25.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /></figure>

**Supplementary Figure 2.** Restricted inflammatory neuropathology in aCB₁-KO mice at acute EAE disease. (a) Representative images of spinal cord lesions (*) stained with H&E. Scale bar = 200 µm. The bar graph depicts the quantitative analysis of inflammatory and demyelinating lesions identified in lumbar spinal cord sections from aCB₁-KO and aCB₁-WT mice at acute EAE using H&E staining and MBP/Hoechst immunofluorescence, respectively (n = 6 mice). (b, c) Confocal micrographs and quantification of (b) GFAP and C3, and (c)
Iba1 immunostaining in spinal cord demyelinating lesions from aCB₁-KO and aCB₁-WT mice normalized to white matter tissue area (n = 6 mice). Scale bar = 100 µm. (d) Representative images and analysis of GFAP and C3 expression in somatosensory cortex layers V-VI from in aCB₁-KO and aCB₁-WT mice (n = 8-9 mice). Scale bar = 25 µm. *p* < 0.05 and **p** < 0.01, unpaired *t*-test or Mann-Whitney test. Error bars express SEM.

<figure><img src="image_28.png" /></figure>

**Supplementary Figure 3.** Reduced clinical severity and spinal cord pathology of chronic EAE in astrocyte-specific CB₁R null mice. (a) Clinical scores of aCB₁-KO and aCB₁-WT mice during chronic EAE. Comparison of motor scores from symptom onset to 35 dpi revealed significantly attenuated neurological disability in aCB₁-KO mice. Data are representative of 2 independent EAE experiments pooled together
(n = 8-12 mice; ***p < 0.0001; Wilcoxon matched-pairs signed rank
test for the comparison of score curves from the onset of EAE
symptoms at 6 dpi to 35 dpi). (b) Representative images of luxol fast
blue (LFB) myelin staining and quantification of demyelinating lesions
(*) in spinal cord sections from aCB₁-KO and aCB₁-WT mice at 35 dpi (n
= 8-9 mice). Scale bar = 50 µm. (c) Confocal micrographs depict spinal
cord sections from aCB₁-KO and aCB₁-WT mice double immunostained
for MBP and SMI32, GFAP or Iba1. (d) Quantification of

MBP immunostaining shows a reduced proportion of demyelinated
tissue normalized to white matter area in aCB₁-KO mice. (e) Attenuated
neuroaxonal damage (left panel) and astrocyte reactivity (middle
panel), and lower numbers of microglia/macrophages (right panel)
within inflammatory lesions from aCB₁-KO mice as quantified by
measuring SMI32 or GFAP immunopositive areas and Iba1+ cells (n =
7-9 mice). Scale bar = 100 µm. *p* < 0.05, **p* < 0.01 and ***p* < 0.001,
unpaired *t*-test or Mann-Whitney test. Error bars express SEM.
<figure><img src="image_29.png" /></figure>

**Supplementary figure 4.** Comparative analysis of cortical astrocyte calcium responses in control aCB₁-WT and aCB₁-KO mice. The graph depicts the amplitude of sensory-evoked astrocyte calcium signals recorded from non-immunized animals (n = 8-12 mice) during 5 non-consecutive days that correspond to 12, 14, 17, 19 and 21 dpi in EAE mice tested in parallel. Analysis using two-way ANOVA followed by Šídák's test for multiple comparisons did not render significant differences between aCB₁-WT and aCB₁-KO mice.
ARTICLE IN PRESS
a

<figure><img src="image_30.png" /></figure>

b

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c

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d

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f

<figure><img src="image_34.png" /></figure>

e

<figure><img src="image_35.png" /></figure>

g

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h

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i

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j

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<figure><img src="image_40.png" /></figure>

**Supplementary figure 5.** Astrocyte-encoded CB<sub>1</sub>R do not modulate oligodendrocyte populations in toxin induced remyelinating lesions. (a) Analysis of aCB<sub>1</sub>-KO and aCB<sub>1</sub>-WT mice in the LPC model of toxic demyelination. LPC lesions were analyzed at 14 dpl corresponding to
the peak of oligodendrocyte differentiation and the onset of
remyelination. Data are representative of 2 independent LPC
experiments pooled together. (b) Immunohistochemistry for MBP
depicts demyelinated lesions (dashed lines) in the *dorsal funiculus* of
LPC-injected mice. Scale bar = 50 µm. Quantitative analysis of lesion
area shows no differences between genotypes (n = 4-7 mice). (c, d)
Quantification of CC1+/OLIG2+ oligodendrocytes and CC1-/OLIG2+
immature oligodendroglia in LPC lesions indicates equal cell
populations in aCB₁-KO and aCB₁-WT animals (n = 4-7 mice). Figure e
shows representative micrographs of LPC lesions triple labelled for
MBP, OLIG2 and CC1. Scale bar = 40 µm. (f, g) Confocal images of LPC
lesions double stained for MBP and (f) PDGFRα or (g) BCAS1. Analysis
of PDGFRα+ OPCs and BCAS1+ myelinating oligodendrocytes in lesions
from aCB₁-KO and aCB₁-WT mice shows no variations between
genotypes (n = 3-7 mice). Scale bars = 40 µm. (h, j) Representative
microphotographs and quantitative analysis of (h) astrocyte reactivity,
(i) microglia/macrophage numbers and (j) infiltrating inflammatory
cells in LPC lesions from aCB₁-KO and aCB₁-WT mice double labelled for
MBP and GFAP, Iba1 or CD45, respectively. Scale bars = 40 µm. Error
bars express SEM.
<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /><figcaption>**Supplementary figure 6.** Comparative analysis of splenic lymphoid and myeloid populations in naive aCB₁-WT and aCB₁-KO mice. (a) Flow</figcaption></figure>
cytometry gating strategy for immune cell analysis presents representative dot-plots from an aCB₁-WT mouse. (b) The total number of splenocytes was similar between genotypes, as were the proportions of (c) global T cells (CD3⁺), T helper cells (CD3⁺ CD4⁺), cytotoxic T cells (CD3⁺ CD8⁺), B cells (CD3⁻ CD19⁺) and (d) activated T cells (early stage, CD3⁺ CD4⁺/CD8⁺ CD69⁺; late stage CD3⁺ CD4⁺/CD8⁺ CD25⁺). Likewise, the percentages of (e) global myeloid cells (CD11b⁺), (f) inflammatory monocytes (iMos; CD11b⁺ Ly6Chi Ly6G⁻/low), patrolling monocytes (pMos; CD11b⁺ Ly6Cint Ly6G⁻), neutrophils (CD11b⁺ Ly6Cint Ly6Ghi), and classical dendritic cells (cDCs; CD11b⁺ CD11c⁺ Ly6C⁻) showed no differences between aCB₁-WT and aCB₁-KO mice (n = 4-5 mice). Error bars express SEM.

<figure><img src="image_43.png" /></figure>
a

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b

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c

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d

<figure><img src="image_47.png" /></figure>

**Supplementary figure 7.** Analysis of the vascular endothelium in EAE lesions from mice lacking astrocyte-encoded CB₁R. (a, b) Immunoblotting and/or morphometry analysis of the endothelial cell markers PODXL and laminin in spinal cord tissue from aCB₁-KO mice and control aCB₁-WT mice at acute EAE disease (n = 6 mice). Scale
bars = 50 µm (PODXL) and 100 µm (laminin). (c, d) Expression of endothelial TJ proteins CDH-5, ZO-1 and CLN-5 in spinal cord tissue from aCB₁-KO and aCB₁-WT mice was determined by (c) immunoblotting and/or (d) immunofluorescence (n = 6 mice). Scale bar = 25 µm.

a

<figure><img src="image_48.png" /></figure>

ICAM-1 Dapi

VCAM-1 Dapi

VEGF-A Dapi

b

<figure><img src="image_49.png" /></figure>

aCB₁-WT

aCB₁-KO

**Supplementary figure 8.** Reduced expression of vascular effector molecules in EAE lesions from astrocyte-specific CB<sub>1</sub>R null mice. (a, b) Representative confocal micrographs and morphometry show lower expression levels of the adhesion molecules ICAM-1, VCAM-1 and VEGF-A within lesion (L) and perilesion (PL) areas from aCB<sub>1</sub>-KO mice (n = 6 mice). Scale bar = 100 µm. \*p < 0.05 and \*\*p < 0.01, unpaired t-test or Mann-Whitney test. Error bars express SEM.
<table><caption>Supplementary Table 2. Primers for RT-qPCR analysis</caption><thead><tr><td>Gene</td><td>Forward primer</td><td>Reverse primer</td></tr></thead><tbody><tr><td>B2m</td><td>ACTGACCGGCCTGTATGCTA</td><td>ATGTTCGGCTTCCCATTCTCC</td></tr><tr><td>Bdnf</td><td>TCCAAAGGCCAACTGAAGCA</td><td>CTGCAGCCTTCCTTGGTGTA</td></tr><tr><td>C1ra</td><td>CCTTCCACCCCATAGAACAG</td><td>CCTTCAACTTACAGTGCCTGA</td></tr><tr><td>C3</td><td>AGGGAGTGTTTGTGCTGAAC</td><td>GCCAATGTCTGCCTTCTCTAC</td></tr><tr><td>Ccl2</td><td>GTAGTTTTTGTCACCAAGCTC</td><td>CAGAAGTGCTTGAGGTGGTT</td></tr><tr><td>Ccl5</td><td>GCTCCAATCTTGCAGTCGT</td><td>CCTCTATCCTAGCTCATCTCCA</td></tr><tr><td>Cldn1</td><td>ACAGCATGGTATGGAAACAGA</td><td>AGGAGCAGGAAAGTAGGACA</td></tr><tr><td>Cldn4</td><td>CACTCAGCCTACACGTTACTC</td><td>CACTCAGCACACCATGACTT</td></tr><tr><td>Cntf</td><td>GTGAAGACAGAAGCAAACCAG</td><td>AGATAGAGCGGCTACAGA GG</td></tr><tr><td>Cxcl10</td><td>ATTTTCTGCCTCATCCTGCT</td><td>TGATTTCAAGCTTCCCTATGGC</td></tr><tr><td>Fkbp5</td><td>GACACCAAAGAAAAGCTGACG</td><td>ACTATCTTCCTGTACTGA ATC</td></tr><tr><td>Gapdh</td><td>AGACGGCCGCATCTTCTT</td><td>TTCACACCGACCTTCACCAT</td></tr><tr><td>Gbp2</td><td>GCAGAATTCACCTCATACATCTTG</td><td>GATGGCACCAACATAGGTCTG</td></tr><tr><td>Ggta1</td><td>TCCTGTTGATGCTGATTGTCTC</td><td>GTCCTTCTGCCATCTGTT CTC</td></tr><tr><td>H2-</td><td>ACAGTCCCGACCCAGAGTAG</td><td>CCACGTAGCCGACAATGATGA</td></tr><tr><td>Hprt</td><td>CAGTACAGCCCCAAAATGGTTA</td><td>AGTCTGGCCTGTATCCAACA</td></tr><tr><td>Icam1</td><td>CTGTGCTTTGAGAACTGTGG</td><td>GGTCCTTGCCCTACTTGCTG</td></tr><tr><td>Igf1</td><td>ATGCTCTTCAGTTCGTGTGT</td><td>AGTACATCTCCAGTCTCC TCA G</td></tr><tr><td>ligp1</td><td>CTCATGTGAAGAGCCTGTAGC</td><td>CTGACCCATGACTTCAAGCA</td></tr><tr><td>Mbp</td><td>CCCTCACAGCGATCCAAGTA</td><td>CTCTGTGCCTTGGGAGGAA</td></tr><tr><td>Mog</td><td>CACTTGTGCCTACGATCCTC</td><td>AGTCCGATGGAGATTCTCTACT</td></tr><tr><td>Ntf3</td><td>AGTCCACCTTTCTCTTCATGTC</td><td>ACATCACCTTGTTCACCTGTA</td></tr><tr><td>Olig2</td><td>AGGGATGATCTAAGCTCTCGAA</td><td>ATTACAGACCGAGCCAAC AC</td></tr><tr><td>Pdgfa</td><td>ATTAACCATGTGCCCGAGA</td><td>GTATCTCGTAAATGACCGTCC</td></tr><tr><td>Pdgfra</td><td>TCACAGCCACCTTCATTACAG</td><td>GTTGCCTTACGACTCCAGATG</td></tr><tr><td>Ppia</td><td>AGGGTTCCTCCTTTCACAGAA</td><td>TGCCGCCAGTGCCATTA</td></tr><tr><td>Psmb8</td><td>GCTGCTTTCCAACATGATGC</td><td>CCGAGTCCCATTGTCATC TAC</td></tr></tbody></table>
<table><caption>**Supplementary Table 3. Primary antibodies for immunohistochemistry**</caption><tr><td>Serpin</td><td>CTACCAAGATGGCTAAGACCAA</td><td>GATGCTCTCCAAGTTGCT CT</td></tr><tr><td>Tgfb1</td><td>GCTGCGCTTGCAGAGATTAA</td><td>GTAACGCCAGGAATTGTTGCTA</td></tr><tr><td>Vcam1</td><td>GCAAAGGACACTGGAAAAGAG</td><td>TGTGCAGTTGACAGTGACA</td></tr><tr><td>Vegfa</td><td>AGAAAGACAGAACAAAGCCAGA</td><td>TCGTTTAACTCAAGCTGCCT</td></tr></table>

<table><thead><tr><td>Antibody</td><td>Host</td><td>Dilution</td><td>Reference</td></tr></thead><tbody><tr><td>Aquaporin-4</td><td>Guinea pig</td><td>1:1000</td><td>Synaptic Systems (#429004)</td></tr><tr><td>APP</td><td>Mouse</td><td>1:200</td><td>Merck (#MAB348)</td></tr><tr><td>B220</td><td>Rat</td><td>1:200</td><td>BD Biosciences (#557390)</td></tr><tr><td>BCAS1</td><td>Rabbit</td><td>1:500</td><td>Synaptic Systems (#445003)</td></tr><tr><td>C3</td><td>Rabbit</td><td>1:1000</td><td>Dako (#A0063)</td></tr><tr><td>CDH-5</td><td>Goat</td><td>1:100</td><td>R&D Systems (#AF1002)</td></tr><tr><td>CC1</td><td>Mouse</td><td>1:200</td><td>Calbiochem (#OP80)</td></tr><tr><td>CCL2</td><td>Mouse</td><td>1:300</td><td>Santa Cruz (#sc-32771)</td></tr><tr><td>CD3</td><td>Rat</td><td>1:50</td><td>Bio-Rad (#MCA1477)</td></tr><tr><td>CD45</td><td>Rat</td><td>1:100</td><td>BD Pharmigen, (#550539)</td></tr><tr><td>CD68</td><td>Rat</td><td>1:100</td><td>Bio-Rad (#MCA1957GA)</td></tr><tr><td>CLN-1</td><td>Mouse</td><td>1:100</td><td>ThermoFisher (#37-4900)</td></tr><tr><td>CLN-4</td><td>Mouse</td><td>1:100</td><td>ThermoFisher (#32-9400)</td></tr><tr><td>CLN-5</td><td>Mouse</td><td>1:100</td><td>Invitrogen (#35-2500)</td></tr><tr><td>Fibrinogen</td><td>Sheep</td><td>1:200</td><td>US Biological (#F4199)</td></tr><tr><td>GFAP</td><td>Rabbit</td><td>1:2000</td><td>Dako (#(#Z0334)</td></tr><tr><td>GFAP</td><td>Chicken</td><td>1:1000</td><td>Abcam Ab4674)</td></tr><tr><td>Iba1</td><td>Rabbit</td><td>1:500</td><td>Wako (#019-19741)</td></tr><tr><td>ICAM-1</td><td>Goat</td><td>1:500</td><td>R&D Systems (#AF796)</td></tr><tr><td>Laminin</td><td>Rabbit</td><td>1:500</td><td>Merck (#L9393)</td></tr><tr><td>Ly6G</td><td>Rat</td><td>1:100</td><td>BioLegend (#127601)</td></tr><tr><td>MBP</td><td>Chicken</td><td>1:200</td><td>Merck (#ab9348)</td></tr><tr><td>MBP</td><td>Mouse</td><td>1:1000</td><td>BioLegend (#808402)</td></tr><tr><td>NG2</td><td>Rabbit</td><td>1:200</td><td>Merck (#AB5320)</td></tr></tbody></table>
<table><tr><td>Olig2</td><td>Mouse</td><td>1:500</td><td>Merck (#MABN50)</td></tr><tr><td>PDGFRα</td><td>Goat</td><td>1:250</td><td>R&D Systems (#AF1062)</td></tr><tr><td>PODXL</td><td>Goat</td><td>1:500</td><td>R&D Systems (#AF1556)</td></tr><tr><td>SMI32</td><td>Mouse</td><td>1:200</td><td>BioLegend (#801702)</td></tr><tr><td>VCAM-1</td><td>Rabbit</td><td>1:500</td><td>Abcam (#ab134047)</td></tr><tr><td>VEGF-A</td><td>Rabbit</td><td>1:500</td><td>Santa Cruz (#sc-507)</td></tr><tr><td></td><td>Mouse</td><td>1:500</td><td>Santa Cruz (#sc-7269)</td></tr><tr><td>ZO-1</td><td>Rabbit</td><td>1:100</td><td>Invitrogen (#402200)</td></tr></table>

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