Up-regulation of CB 2 receptors in reactive astrocytes in canine degenerative myelopathy, a disease model of amyotrophic lateral sclerosis

Abstract

Targeting the CB2 receptor afforded neuroprotection in SOD1(G93A) mutant mice, a model of amyotrophic lateral sclerosis (ALS). The neuroprotective effects of CB2 receptors were facilitated by their up-regulation in the spinal cord in SOD1(G93A) mutant mice. Herein, we have investigated whether a similar CB2 receptor up-regulation, as well as parallel changes in other endocannabinoid elements, are evident in the spinal cord of dogs with degenerative myelopathy (DM), caused from mutations in the superoxide dismutase 1 gene (SOD1). We used well-characterized post-mortem spinal cords from unaffected and DM-affected dogs. Tissues were used first to confirm the loss of motor neurons using Nissl staining, which was accompanied by glial reactivity (elevated GFAP and Iba-1 immunoreactivity). Next, we investigated possible differences in the expression of endocannabinoid genes measured by qPCR between DM-affected and control dogs. We found no changes in the CB1 receptor (also found with CB1 receptor immunostaining) as well as in NAPE-PLD, DAGL, FAAH and MAGL enzymes. In contrast, CB2 receptor levels were significantly elevated in DM-affected dogs determined by qPCR and Western-blotting, results reconfirmed in the grey matter using CB2 receptor immunostaining. Using double-labelling immunofluorescence, CB2 receptor immunolabelling co-localized with GFAP but not Iba-1, indicating up-regulation of CB2 receptors on astrocytes in DM-affected dogs. In summary, our results demonstrated a marked up-regulation of CB2 receptors occurring in the spinal cord in canine DM, which was concentrated in activated astrocytes. Such receptors may be used as a potential target to enhance the neuroprotective effects exerted by these glial cells.

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Up-regulation of CB2 receptors in reactive astrocytes in canine degenerative
myelopathy, a disease model of amyotrophic lateral sclerosis
María Fernández-Trapero1-4#, Francisco Espejo-Porras1-3#, Carmen Rodríguez-
Cueto1-3, Joan R Coates5, Carmen Pérez-Díaz4, Eva de Lago1-3* and Javier
Fernández-Ruiz1-3*
1Departamento de Bioquímica y Biología Molecular, Instituto Universitario de
Investigación en Neuroquímica, Facultad de Medicina, Universidad Complutense,
Madrid, Spain; 2Centro de Investigación Biomédica en Red de Enfermedades
Neurodegenerativas (CIBERNED), Madrid, Spain; 3Instituto Ramón y Cajal de
Investigación Sanitaria (IRYCIS), Madrid, Spain; 4Departamento de Medicina y
Cirugía Animal, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain;
5Department of Veterinary Medicine and Surgery, College of Veterinary Medicine,
University of Missouri, Columbia, MO, USA.
#Both authors shared the first position in this study
*Both authors shared the senior authorship of this study
Key words: Cannabinoids, endocannabinoid signaling, CB2 receptors, canine
degenerative myelopathy, amyotrophic lateral sclerosis, SOD1,
activated astrocytes.
Correspondence: Eva de Lago (e-mail: elagofem@med.ucm.es)
Javier Fernández-Ruiz (e-mail: jjfr@med.ucm.es)
Department of Biochemistry and Molecular Biology, Faculty of
Medicine, Complutense University, 28040-Madrid, Spain
Phone number: 34-913941450; Fax number: 34-913941691
Summary statement
CB2 receptors become up-regulated in activated astrocytes recruited at the damaged
spinal cord in dogs with degenerative myelopathy, a canine model of amyotrophic
lateral sclerosis
Disease Models & Mechanisms • DMM • Advance article
http://dmm.biologists.org/lookup/doi/10.1242/dmm.028373Access the most recent version at
DMM Advance Online Articles. Posted 9 January 2017 as doi: 10.1242/dmm.028373

Abstract
Targeting the CB2 receptor afforded neuroprotection in SOD1G93A mutant mice, a
model of amyotrophic lateral sclerosis (ALS). The neuroprotective effects of CB2
receptors were facilitated by their up-regulation in the spinal cord in SOD1G93A mutant
mice. Herein, we have investigated whether a similar CB2 receptor up-regulation, as
well as parallel changes in other endocannabinoid elements, are evident in the spinal
cord of dogs with degenerative myelopathy (DM), caused from mutations in the
superoxide dismutase 1 gene (SOD1). We used well-characterized post-mortem
spinal cords from unaffected and DM-affected dogs. Tissues were used first to
confirm the loss of motor neurons using Nissl staining, which was accompanied by
glial reactivity (elevated GFAP and Iba-1 immunoreactivity). Next, we investigated
possible differences in the expression of endocannabinoid genes measured by qPCR
between DM-affected and control dogs. We found no changes in the CB1 receptor
(also found with CB1 receptor immunostaining) as well as in NAPE-PLD, DAGL,
FAAH and MAGL enzymes. In contrast, CB2 receptor levels were significantly
elevated in DM-affected dogs determined by qPCR and Western-blotting, results
reconfirmed in the grey matter using CB2 receptor immunostaining. Using double-
labelling immunofluorescence, CB2 receptor immunolabelling co-localized with GFAP
but not Iba-1, indicating up-regulation of CB2 receptors on astrocytes in DM-affected
dogs. In summary, our results demonstrated a marked up-regulation of CB2 receptors
occurring in the spinal cord in canine DM, which was concentrated in activated
astrocytes. Such receptors may be used as a potential target to enhance the
neuroprotective effects exerted by these glial cells.
Disease Models & Mechanisms • DMM • Advance article

Introduction
Amyotrophic lateral sclerosis (ALS) is progressive degeneration and loss of upper
and lower motor neurons in the brain and spinal cord, causing muscle weakness and
paralysis (Hardiman et al., 2011). In 1993, genetic studies identified the first
mutations in the copper-zinc superoxide dismutase gene (SOD1), which encodes for
a key antioxidant enzyme, SOD1 (Rosen et al., 1993). Mutations in SOD1 account
for 20% of genetic ALS and 2% of all ALS. More recently, similar studies have
identified mutations in other genes, such as TARDBP (TAR-DNA binding protein) and
FUS (fused in sarcoma), which encode proteins involved in pre-mRNA splicing,
transport and/or stability (Buratti and Baralle, 2010; Lagier-Tourenne et al., 2010),
and, in particular, the CCGGGG hexanucleotide expansion in the C9orf72 gene
which appears to account for up to 40% of genetic cases (Cruts et al., 2013). Their
pathogenic mechanisms, which differ, in part, from the toxicity associated with
mutations in SOD1, led to a novel molecular classification of ALS subtypes (Al-
Chalabi and Hardiman, 2013; Renton et al., 2014).
The ultimate goal in ALS is to develop novel therapeutics that will slow disease
progression. Rilutek has been the only FDA approved drug but limited in efficacy
(Habib and Mitsumoto, 2011). Recently cannabinoids have been shown to have
neuroprotective effects in transgenic rodent ALS models (Bilsland and Greensmith,
2008; de Lago et al., 2015, for review). Chronic treatment with the phytocannabinoid
9-tetrahydrocannabinol (9-THC) delayed motor impairment and improved survival
in the SOD-1G93A transgenic mouse (Raman et al., 2004). Other cannabinoid
compounds, including the less psychotropic plant-derived cannabinoid cannabinol
(Weydt et al., 2005), the non-selective synthetic agonist WIN55,212-2 (Bilsland et al.,
2006), and the selective cannabinoid receptor type-2 (CB2) agonist AM1241 (Kim et
al., 2006; Shoemaker et al., 2007), produced similar effects. Genetic or
pharmacological inhibition of fatty acid amide hydrolase (FAAH), one of the key
enzymes in endocannabinoid degradation, was also beneficial in SOD-1G93A
transgenic mice (Bilsland et al., 2006). The efficacy shown by compounds that target
the CB2 receptor (Kim et al., 2006; Shoemaker et al., 2007) appears to be facilitated
by the fact that this receptor was found to be up-regulated in reactive glia in post-
mortem spinal cord tissue from ALS patients (Yiangou et al., 2006). Such elevation of
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CB2 receptors has been also described in SOD1G93A transgenic mice (Shoemaker et
al., 2007; Moreno-Martet et al., 2014), and we recently found that the response
occurred predominantly in activated astrocytes recruited at lesion sites in the spinal
cord (Espejo-Porras et al., unpublished results). We have also described a similar
increase in CB2 receptors on reactive microglia in TDP-43 transgenic mice (Espejo-
Porras et al., 2015). Based on these studies, the CB2 receptor may be a novel target
in altering disease progression in ALS, given its effective control of glial influences
exerted on neurons, as has been investigated in other disorders (Fernández-Ruiz et
al., 2007, 2015; Iannotti et al., 2016 for review).
A challenge of preclinical studies of novel neuroprotective agents in ALS is poor
translation of therapeutic success in small animal (e.g. rodents, zebrafish, flies,
nematodes) to human ALS patients. In most of cases, they were based on the over-
expression of specific human gene mutations. In this context, we have recently paid
attention to canine degenerative myelopathy (DM), a multisystem central and
peripheral axonopathy described in dogs in 1973 (Averill, 1973), with an overall
prevalence of 0.19% (Coates and Wininger, 2010 for review), which shares
pathogenic mechanisms with some forms of human ALS, including mutations in
SOD1 as one of the major causes of the disease (Awano et al., 2009). With some
differences depending on the type of breeds, DM is characterized by degeneration in
the white matter of the spinal cord and the peripheral nerves, then affecting both
upper and lower motor neurons (Coates and Wininger, 2010 for review). The disease
appears at 8-14 years of age with an equivalent affectation in both genders and
necessitating euthanasia (Coates and Wininger, 2010 for review). This canine
pathology represents a unique opportunity to investigate ALS in a context much more
close to the human pathology, using animal species which, in the phylogeny, are
closest to humans, and in which the disease occurs spontaneously. Our objective in
the present study has been to investigate the changes that the development of DM
produces in endocannabinoid elements in those CNS sites (spinal cord) most
affected in this disease. It is important to remark that such elements may derive in
potential targets for a pharmacological therapy with cannabinoid-based therapies
(e.g. Sativex) aimed at delaying/arresting the progression of the disease in these
dogs, and furtherly in humans. The study has been carried out with post-mortem
tissues (spinal cords) from dogs affected by DM kindly provided by Dr. Joan R.
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Coates (University of Missouri, Columbia, MO, USA), adequately classified in
different disease stages (Coates and Wininger, 2010). All DM tissues included the
necessary clinical, genetic and neuropathological information, and they were
accompanied by adequate matched control tissues. Both DM-affected and control
tissues were used for analysis of endocannabinoid receptors and enzymes using
biochemical (qPCR, Western blot) and, in some cases, histological
(immunohistochemistry) procedures, including the use of double
immunofluorescence staining to identify the cellular substrates in which the changes
in endocannabinoid elements (CB2 receptors) take place.
Results
Validation of the expected histopathological deterioration in DM-affected dogs
The data provided by the biobank confirmed that all tissues obtained from DM-
affected dogs had a clinical diagnosis of DM in all cases supported by the genetic
analysis which confirmed the presence of the SOD1 mutation. They corresponded to
two different breeds, which are within the most affected by this disease (Coates and
Wininger, 2010), and animals were all euthanized in an age interval of 9-13.6 years
(11.8 ± 0.6), with a grade of the disease of 1-3 (2.2 ± 0.3). DM-affected dogs included
6 spayed females and 2 castrated males (see details in Table 1). The control tissues
were selected from normal dogs or with no clinical diagnosis of DM. All control dogs
were homozygous wild-type and age-matched (8-13.6 years; 10.0 ± 0.8). They
included 6 females, 1 of them spayed, and 1 castrated male (see details in Table 1).
We found a significant reduction in the number of Nissl-stained cell bodies
corresponding to lower motor neurons located in the ventral horn of DM-affected
spinal cords (Figures 1A and 1B). The neuronal loss was accompanied by an intense
glial reactivity in the affected areas, in particular we detected a 3-fold increase in
GFAP immunolabelling in the spinal grey matter (Figures 1C and 1D). We also found
microgliosis in both white and grey matter of the spinal cord, detected with DAB
immunostaining for the microglial marker Iba-1 (Figure 2A), which we quantitated in a
2.5-fold elevation in the grey matter using Iba-1 immunofluorescence (Figures 2B
and 2C).
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Changes in the endocannabinoid receptors and enzymes in DM-affected dogs
Next, we investigated possible differences between DM-affected dogs and control
animals in the expression of endocannabinoid genes measured by qPCR. Although
certain trends towards an elevation may be apparently appreciated, there were no
significant changes in the CB1 receptor, as well as in FAAH, monoacylglcerol lipase
(MAGL), N-arachidonoyl-phosphatidylethanolamine phospholipase D (NAPE-PLD),
diacylglycerol lipase (DAGL) enzymes between the two groups (Figure 3A). We
attempted to determine whether the trends detected for these five parameters may
correspond to a greater affectation in DM dogs with advanced disease, but we did
not find any statistically significant correlation (data not shown). They were not
related to gender-dependent differences (data not shown). The absence of changes
in CB1 receptor gene expression was observed at the protein level using DAB
immunostaining in the grey matter (Figures 3B and 3C). This happened despite the
reduction in the number of motor neurons described before with Nissl staining.
Next, we investigated the CB2 receptor, an endocannabinoid element that is
frequently altered in conditions of neurodegeneration (Fernández-Ruiz et al., 2007,
2015; Iannotti et al., 2016), including ALS (Yiangou et al., 2006; Shoemaker et al.,
2007; Moreno-Martet et al., 2014; Espejo-Porras et al., 2015). First, we detected an
increase of more than 2-fold in CB2 receptor expression, measured by qPCR, in DM-
affected dogs (Figure 4A). We also investigated whether this increase occurred
predominantly in the tissues obtained from DM-affected dogs at the intermediate and
advanced stages, but we did not find any significant correlation between both
variables (data not shown). This increase in gene expression was confirmed at the
protein level using Western blotting first (2-fold increase; Figure 4B), as well as using
DAB immunostaining which showed that the elevation of CB2 receptors occurred
predominantly in the grey matter (Figures 5A and 5B).
Double-labelling analyses to identify the CB2 receptor-positive cellular substrates
The examination of the morphology of those cells positive for the CB2 receptor in
DAB immunostaining (Figure 5A) suggested they should be glial cells. We wanted to
confirm this fact by using double-labelling immunofluorescence analysis. We found
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that CB2 receptor immunolabelling colocalized with GFAP immunofluorescence
(Figure 6), thus indicating that the up-regulation of CB2 receptor in the spinal cord of
DM-affected dogs occurred in reactive astrocytes. Similar double-labelling
immunofluorescence with Iba-1 did not detect any colocalization with the CB2
receptor immunostaining, indicating that the receptor is not located in the microglial
cells in the spinal cord of DM-affected dogs (Figure 7).
Discussion
Our study addressed changes in specific endocannabinoid elements in canine DM, a
disease of older dogs with similarities to ALS (Coates and Winninger, 2010 for
review). The endocannabinoid system has been previously investigated in different
regions of the canine brain (Pirone et al., 2016), but this is the first time that these
elements are investigated in the context of an important neurodegenerative disorder
occurring in dogs. The benefits that such investigation may have would impact in a
better development of cannabinoid-based therapies for human ALS, but also these
studies may serve as a first step in the process to have also a cannabinoid-based
pharmacotherapy useful in the veterinary medicine. Our study has investigated the
six endocannabinoid elements commonly recognized to develop pharmacological
therapies, and has identified the CB2 receptor as promising potential target. It is also
important to mention that our study demonstrates no losses of CB1 receptors, which
are typically located in neurons, despite the losses of motor neurons occurring in the
disease. This supports that, contrarily to other neurodegenerative conditions in
humans with profound losses of neuronal CB1 receptors, e.g. Huntington’s disease
(Fernández-Ruiz et al., 2015), this receptor may also serve as a potential target in
canine DM (shown here) and also in human ALS (de Lago et al., 2015).
Regarding the CB2 receptor, what we found here has been similarly observed in
transgenic ALS rodent models (Shoemaker et al., 2007; Moreno-Martet et al., 2014;
Espejo-Porras et al., 2015) and ALS patients (Yiangou et al., 2016), that the receptor
becomes strongly up-regulated in activated glia in response to neuronal damage.
The response is not exclusive of ALS but also observed in other acute or chronic
neurodegenerative disorders (e.g. ischemia, Alzheimer’s disease, Parkinson’s
disease, Huntington’s chorea; reviewed in Fernández-Ruiz et al., 2007, 2015; Iannotti
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et al., 2016). These findings support that the elevation of CB2 receptors in activated
glial elements is an endogenous response of the endocannabinoid signaling aimed at
protecting neurons against cytotoxic insults, as well as at restoring neuronal
homeostasis and integrity (Pacher and Mechoulam, 2011; de Lago et al., 2015;
Fernández-Ruiz et al., 2015).
Result of our study further demonstrated that the elevation of CB2 receptors occurred
in activated astrocytes rather than in microglial cells. This finding has been previously
described in the spinal cords of transgenic SOD1 mice (Espejo-Porras et al.,
unpublished results). In other transgenic models of ALS, e.g. TDP-43 transgenic
mice, the up-regulatory response of these receptors occurred predominantly in
reactive microglial cells (Espejo-Porras et al., 2015) and in tissues of human ALS
patients (Yiangou et al., 2006). In multiple sclerosis and Huntington’s chorea, the
overexpression of CB2 receptors occurred in both activated astrocytes and reactive
microglia (Benito et al., 2007; Sagredo et al., 2009). The CB2 receptor elevations in
activated glial elements may be related to the control of the influence of these cells
on neuronal homeostasis, for example, by enhancing the metabolic support and the
glutamate reuptake activity exerted by astrocytes (Fernández-Ruiz et al., 2015 for
review), by facilitating the transfer of microglial cells from the M1 to the M2
phenotypes (Mecha et al., 2016 for review), or by attenuating the generation of
proinflammatory cytokines, chemokines, nitric oxide and reactive oxygen species by
either astrocytes or microglial cells when they become activated (Fernández-Ruiz et
al., for review). These potentialities situate this receptor in a promising position to
serve for the development of novel therapies. In the case of our present study, and
given their preferential location in activated astrocytes, we will need to conduct
additional research aimed at investigating the consequences of the selective CB2
receptor activation in these glial cells during the progression of this canine disease.
In conclusion, our results demonstrated a marked up-regulation of CB2 receptors
occurring in the spinal cord of dogs affected by DM. Such up-regulation occurred in
absence of changes in other endocannabinoid elements and was concentrated in
activated astrocytes, then becoming a potential target to enhance the protective
effects exerted by these glial cells to improve neuronal homeostasis and integrity.
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Materials and Methods
Management of the postmortem tissues
All experiments were conducted on post-mortem spinal cord tissues collected from
DM affected and unaffected dogs. All tissues (formalin-fixed tissues for routine
histopathology and frozen tissues for qPCR and Western blotting) were provided by
Dr. Joan R. Coates (Department of Veterinary Medicine and Surgery, College of
Veterinary Medicine, University of Missouri, Columbia, MO, USA). Protocols for
tissue collection were approved by the University of Missouri Animal Care and Use
Committee.
Tissues provided included those of DM affected dogs and age-matched controls and
accompanied by adequate clinical and genetic testing information (see details in
Table 1). DM diagnoses were confirmed histopathologically by assessing the mid- to
lower thoracic spinal cord segment for evidence of myelinated axon loss and
pronounced astrogliosis in the dorsal portion of the lateral funiculus (Awerill, 1973;
March et al., 2009). Dogs that had exhibited clinical signs of DM but did not show the
typical histopathology were presumed to have another cause for the myelopathy and
excluded from the study. The spinal cord segments were examined for the presence
of SOD1 immunoreactive aggregates within ventral horn motor neurons (Awano et
al., 2009). Dogs that had not exhibited any clinical signs of DM prior to euthanasia
and whose thoracic spinal cords were histologically normal were used as controls.
Tissues from DM-affected dogs were sorted by different stages of disease
progression characterized at origin according to the following clinical and
histopathological characteristics: (i) Stage 1 (upper motor neuron paraparesis):
progressive general propioceptive ataxia and asymmetric spastic paraparesis, but
intact spinal reflexes; (ii) Stage 2 (non-ambulatory paraparesis to paraplegia): mild to
moderate loss of muscle mass, reduced to absent spinal reflexes in pelvic limbs, and
possible urinary and fecal incontinence; (iii) Stage 3 (lower motor neuron paraplegia
to thoracic limb paresis): signs of thoracic limb paresis, flaccid paraplegia, severe
loss of muscle mass in pelvic limbs, and urinary and fecal incontinence; and (iv)
Stage 4 (lower motor neuron tetraplegia and brainstem signs): flaccid tetraplegia,
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difficulty with swallowing and tongue movements, reduced to absent cutaneous trunci
reflex, generalized and severe loss of muscle mass, and urinary and fecal
incontinence (see details in Coates and Wininger, 2010). All DM tissues were
accompanied by the necessary signalment, genotype and clinical diagnosis (see
details in Table 1). Tissue studies confirm the loss of motor neurons using Nissl
staining and accompanied by analysis of glial reactivity using GFAP and Iba-1
immunostaining. Next, we investigated the status of endocannabinoid receptors and
enzymes using biochemical (qPCR, Western blot) and, in some cases,
immunostaining procedures, included double immunofluorescence staining to identify
the cellular substrates in endocannabinoid elements (CB2 receptors) take place. For
all measures, tissues used corresponded to 7-8 different animals per experimental
group.
Real time qRT-PCR analysis
Total RNA was extracted from spinal cord samples (from T7 to T10) using TRI
Reagent (Sigma Chem., Madrid, Spain). The total amount of RNA extracted was
quantitated by spectrometry at 260 nm and its purity was evaluated by the ratio
between the absorbance values at 260 and 280 nm, whereas its integrity was
confirmed in agarose gels. To prevent genomic DNA contamination, DNA was
removed and single-stranded complementary DNA was synthesized from 0.6 μg of
total RNA using a commercial kit (Rneasy Mini Quantitect Reverse Transcription,
Qiagen, Izasa, Madrid, Spain). The reaction mixture was kept frozen at -80C until
enzymatic amplification. Quantitative real-time PCR assays were performed using
TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, U.S.A.) to
quantify mRNA levels for CB1 receptor (ref. Cf02716352_u1), CB2 receptor (ref.
Cf02696139_s1), DAGL (Cf02705627_m1), FAAH (ref. Cf02648944_m1) and MAGL
(ref. Cf02662432_m1). For NAPE-PLD, we used one custom designed assay
(Custom Plus Taqman RNA Assay Design, Applied Biosystems, Foster City, CA,
USA). In alls cases, we used GAPDH expression (ref. Cf04419463_gH) as an
endogenous control gene for normalization. The PCR assay was performed using the
7300 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and
the threshold cycle (Ct) was calculated by the instrument’s software (7300 Fast
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System, Applied Biosystems, Foster City, CA, USA). Values were normalized as
percentages over the control group.
Western blot analysis
Purified protein fractions were isolated using ice-cold RIPA buffer. Then, 20 μg of
protein were boiled for 5 min in Laemmli SDS loading buffer (10% glycerol, 5% SDS,
5% β-mercaptoethanol, 0.01% bromophenol blue and 125 mM TRIS-HCl, at pH 6.8)
and loaded onto a 12% acrylamide gel (Bio-Rad Laboratories, Hercules, CA, USA),
and then transferred to a PVDF membrane (Immobilon-P, Millipore, Bedford, MA,
USA) using mini Trans-Blot Electrophoretic transfer cell (Bio-Rad Laboratories,
Hercules, CA, USA). Membranes were blocked with 5% non-fat milk and incubated
overnight at 4°C with the mouse anti-CB2 receptor antibody (Santa Cruz
Biotechnology, Santa Cruz, CA, USA), followed by a second incubation during 2
hours at room temperature with an ECL™ Horseradish Peroxidase-linked whole
secondary antibody (GE Healthcare UK Limited, Buckinghamshire, UK) at a 1:5000
dilution. Reactive bands were detected by chemiluminescence with the Amersham™
ECL™ Prime Western Blotting Detection Reagent (GE Healthcare UK Limited,
Buckinghamshire, UK). Images were analyzed on a ChemiDoc station with Quantity
one software (Bio-Rad Laboratories, Madrid, Spain). Data were calculated as the
ratio between the optical densities of the specific protein band and the housekeeping
protein GAPDH, and they were normalized as percentages over the control group.
Histological procedures
Tissue slicing. Fixed spinal cords were sliced with a cryostat at the thoracic level,
always between T7-T10, which correspond to the spinal level in which the axonal
degeneration was most severe (Coates and Wininger, 2010). Coronal sections (20
μm thick) were collected on gelatin-coated slides. Sections were used for procedures
of Nissl-staining, immunohistochemistry and immunofluorescence.
Nissl staining. Slices were used for Nissl staining using cresyl violet, as previously
described (Alvarez et al., 2008). A Leica DMRB microscope (Leica, Wetzlar,
Germany) and a DFC300FX camera (Leica) were used for the observation and
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photography of the slides, respectively. For counting the number of Nissl-stained
large motor neurons in the anterior horn, high resolution photomicrographs were
taken with the 20x objective under the same conditions of light, brightness and
contrast. Four images coming from at least 3 sections per animal were analyzed. The
final value is the mean for all animals included in each experimental group.
Immunohistochemistry. Slices were preincubated for 20 min in 0.1M PBS with 0.1%
Triton X-100, pH 7.4, and subjected to endogenous peroxidase blockade by 1 hour
incubation at room temperature in peroxidase blocking solution (Dako Cytomation,
Glostrup, Denmark). Then, they were incubated in 0.1M PBS with 0.01% Triton X-
100, pH 7.4, with one of the following primary antibodies: (i) polyclonal anti-rabbit Iba-
1 antibody (Wako Chemicals, Richmond, VI, USA) used at 1/500; (ii) polyclonal anti-
rabbit CB1 receptor (Thermo Scientific, MA, USA) used at 1/400; and (iii) polyclonal
anti-goat CB2 receptor antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA)
used at 1/100. Incubation was prolonged overnight at 4ºC, then sections were
washed in 0.1M PBS and incubated for 2 hours at room temperature with the
appropriate biotin-conjugated anti-goat or anti-rabbit (1:200; Vector Laboratories,
Burlingame, CA, USA) secondary antibodies. Vectastain® Elite ABC kit (Vector
Laboratories, Burlingame, CA, USA) and a DAB substrate–chromogen system (Dako
Cytomation, Glostrup, Denmark) were used to obtain a visible reaction product.
Negative control sections were obtained using the same protocol with omission of the
primary antibody. All sections for each immunohistochemical procedure were
processed at the same time and under the same conditions. A Leica DMRB
microscope (Leica, Wetzlar, Germany) and a DFC300FX camera (Leica) were used
for slide observation and photography.
Immunofluorescence. Quantification of GFAP and Iba-1 immunoreactivity was also
carried out using immunofluorescence, and this procedure was also used for double-
labelling studies. Slices were preincubated for 1 hour with Tris-buffered saline with
1% Triton X-100 (pH 7.5). Then, sections were sequentially incubated overnight at
4ºC with a polyclonal anti-Iba-1 (1/500; Wako Chemicals, Richmond, VI, USA) or
polyclonal anti-GFAP (1/200; Dako Cytomation, Glostrup, Denmark), followed by
washing in Tris-buffered saline and a new incubation (at 37ºC for 2 hours) with an
Alexa 488 anti-rabbit antibody conjugate made in donkey (1/200; Invitrogen,
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Carlsbad, CA, USA), rendering green fluorescence for anti-Iba-1 or anti-GFAP. The
immunofluorescence was quantified using a SP5 Leica confocal microscope and the
ImageJ software (U.S. National Institutes of Health, Bethesda, Maryland, USA). For
double-labelling studies, sections were then washed again and incubated overnight
at 4ºC with a polyclonal anti-CB2 receptor (1/100; Santa Cruz Biotechnology, Santa
Cruz, CA, USA). This was followed by washing in Tris-buffered saline and a further
incubation (at room temperature for 2 hours) with a biotin-conjugated anti-goat
(1:200; Vector Laboratories, Burlingame, CA, USA) secondary antibody, followed by
a new washing and an incubation (at 37ºC for 2 hours) with red streptavidin (Vector
Laboratories, Burlingame, CA, USA) rendering red fluorescence for anti-CB2 receptor.
Sections were counter-stained with nuclear stain TOPRO-3-iodide (Molecular
Probes, Eugene, OR, USA) to visualize cell nuclei. To quench endogenous
autofluorescence, tissue sections were also treated with 0.5% Sudan Black
(Merck, Darmstadt, Germany) in 70% ethanol for 1 min and differentiated with
70% ethanol (Schnell et al., 1999). A Leica TCS SP5 microscope was used for slide
observation and photography. Differential visualization of the fluorophores was
accomplished through the use of specific filter combinations. Samples were scanned
sequentialy to avoid any potential for bleed through of fluorophores.
Statistics
Data were assessed by unpaired Student’s t-test or one-way ANOVA followed by the
Student-Newman-Keuls test, as required.
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Acknowledgements
The authors are indebted to Yolanda García-Movellán for administrative assistance.
Authors’ contribution:
 Study design: Eva de Lago, Carmen Pérez-Díaz and Javier Fernández-Ruiz
 Sample collection and clinical/genetic characterization: Joan R. Coates
 Sample analysis: María Fernández-Trapero, Francisco Espejo-Porras and
Carmen Rodríguez-Cueto
 Data interpretation (including statistical assessment): Eva de Lago and Javier
Fernández-Ruiz
 Manuscript draft: Javier Fernández-Ruiz (revised, corrected and approved by
all authors)
Disclosure of potential conflicts of interest
Authors declare that they have no conflicts of interest in relation with this submission.
Funding
This work was supported by grants from CIBERNED (CB06/05/0089), MINECO
(SAF2012/39173 and SAF2015-68580-C2-1-R), and GW Pharmaceuticals Ltd.
These agencies had no further role in study design, collection, analysis and
interpretation of the data, in the writing of the report, or in the decision to submit the
paper for publication. Francisco Espejo-Porras is a predoctoral fellow supported by
the MINECO (FPI Programme).
Disease Models & Mechanisms • DMM • Advance article

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Table
Table 1. Clinical, genetic and histopathological characteristics of DM-affected and
control dogs whose spinal tissues have been used in this study.
UCM
animal
code
Genotype
Age at
death
(years)
Gender
Breed
Diagnosis
Disease
grade
DM-affected dogs
DM#1
A/A
12
Spayed female
Boxer
DM
2
DM#2
A/A
11
Spayed female
Boxer
DM
1.5
DM#3
A/A
9
Spayed female
Boxer
DM
1
DM#4
A/A
12.78
Spayed female
Pembroke Welsh Corgi
DM
3
DM#5
A/A
12.58
Castrated male
Pembroke Welsh Corgi
DM
2.5-3
DM#6
A/A
13.6
Spayed female
Pembroke Welsh Corgi
DM
3 (early)
DM#7
A/A
11.6
Castrated male
Pembroke Welsh Corgi
DM
2
DM#8
A/A
11.7
Spayed female
Pembroke Welsh Corgi
Severe DM and disc
herniation 2 years prior
3
Control dogs
CT#1
G/G
10.5
Castrated male
German Shepherd
spinal cord
histopathology normal
NA
CT#2
G/G
13.6
Female
Pit Bull Mix
spinal cord
histopathology normal
NA
CT#3
G/G
11.8
Spayed female
Rhodesian Rodgeback
spinal cord
histopathology normal
NA
CT#4
G/G
8.5
Female
Beagle
spinal cord
histopathology normal
NA
CT#5
G/G
8
Female
Beagle
spinal cord
histopathology normal
NA
CT#6
G/G
9.6
Female
Beagle
spinal cord
histopathology normal
NA
CT#7
G/G
8
Female
Beagle
spinal cord
histopathology normal
NA
Disease Models & Mechanisms • DMM • Advance article

Figures
Figure 1. Representative microphotographs and quantification for Nissl staining
(panels A and B, respectively) and GFAP immunofluorescence (panels C and D,
respectively) in the spinal cord sections (grey matter in the ventral horn at T7-T10) of
DM-affected and age-matched control dogs. Values are expressed as means  SEM
for 6-7 animals per group. Data were analyzed using the unpaired Student’s t-test
(*p<0.05, **p<0.01 compared to control animals). Bar scale = 300 µm (Nissl staining)
and 200 µm (GFAP immunofluorescence)
Disease Models & Mechanisms • DMM • Advance article

Figure 2. Representative microphotographs for Iba-1 immunostaining using DAB
(panel A), as well as for Iba-1 immunofluorescence (panel B) and its quantification
(panel C), in the spinal cord sections (grey matter in the ventral horn and white
matter in the dorsal area, both at T7-T10) of DM-affected and age-matched control
dogs. Values are expressed as means  SEM for 5-7 animals per group. Data were
analyzed using the unpaired Student’s t-test (**p<0.01 compared to control animals).
Bar scale = 300 µm for DAB immunostaining and 200 µm for immunofluorescence.
Disease Models & Mechanisms • DMM • Advance article

Figure 3. Gene expression for the CB1 receptor and the NAPE-PLD, DAGL, FAAH
and MAGL enzymes measured by qPCR (panel A), and representative
microphotographs for CB1 receptor immunostaining using DAB (panel C) and its
quantification in the grey matter in the ventral horn (panel B), in the spinal cord
samples (for qPCR) or T7-T10 sections (for immunostaining) of DM-affected and
age-matched control dogs. Values are expressed as means  SEM for 7-8 animals
per group. Data were analyzed using the unpaired Student’s t-test. Bar scale = 150
µm.
Disease Models & Mechanisms • DMM • Advance article

Figure 4. Gene expression for the CB2 receptor measured by qPCR (panel A), as
well as Western blot analysis for this receptor (panel B) in spinal cord samples of
DM-affected and age-matched control dogs. Values correspond to % over control
animals and are expressed as means  SEM for 7 animals per group. Data were
analyzed using the unpaired Student’s t-test (*p<0.05 compared to control animals).
Disease Models & Mechanisms • DMM • Advance article

Figure 5. Representative microphotographs for CB2 receptor immunostaining using
DAB (panel A) and its quantification (panel B) in the grey matter of the ventral horn in
T7-T10 spinal cord sections of DM-affected and age-matched control dogs. Values
are expressed as means  SEM for 5-6 animals per group. Data were analyzed using
the unpaired Student’s t-test (*p<0.05 compared to control animals). Bar scale = 150
µm and 50 µm for the detail. Arrows indicate the CB2 receptor-positive cells.
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Figure 6. Representative microphotographs corresponding to a double
immunofluorescence analysis for the CB2 receptor and GFAP, using TOPRO-3 for
labelling cell nuclei, in the grey matter of the ventral horn in T7-T10 spinal cord
sections of DM-affected and age-matched control dogs (n=3/group). Bar scale = 50
µm. Arrows indicate cells labelled with the antibodies for the two markers.
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Figure 7. Representative microphotographs corresponding to a double
immunofluorescence analysis for the CB2 receptor and Iba-1, using TOPRO-3 for
labelling cell nuclei, in the grey matter of the ventral horn in T7-T10 spinal cord
sections of DM-affected and age-matched control dogs (n=3/group). Bar scale = 50
µm.
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