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R E S E A R C H A R T I C L E Open Access
Alterations of endocannabinoids in cerebrospinal
fluid of dogs with epileptic seizure disorder
Felix K Gesell
1*
, Alexander A Zoerner
2
, Christina Brauer
1
, Stefan Engeli
2
, Dimitros Tsikas
2
and Andrea Tipold
1
Abstract
Background: Epilepsy is one of the most common chronic neurological disorders in dogs characterized by
recurrent seizures. The endocannabinoid (EC) system plays a central role in suppressing pathologic neuronal
excitability and in controlling the spread of activity in an epileptic network. Endocannabinoids are released on
demand and their dysregulation has been described in several pathological conditions. Recurrent seizures may lead
to an adverse reorganization of the EC system and impairment of its protective effect. In the current study, we
tested the hypothesis that cerebrospinal fluid (CSF) concentrations of the endocannabinoids anandamide (AEA) and
2-arachidonoyl glycerol (2AG) are altered in epileptic dogs. Concentrations of AEA and total AG (sum of 2AG and
1AG) were measured in 40 dogs with idiopathic epilepsy and in 16 unaffected, healthy control dogs using liquid
chromatography combined with tandem mass spectrometry.
Results: AEA and total AG were measured at 4.94 (3.18 –9.17) pM and 1.43 (0.90 –1.92) nM in epileptic dogs and
at 3.19 (2.04 –4.28) pM and 1.76 (1.08 –2.69) nM in the control group, respectively (median, 25 –75% percentiles
in brackets). The AEA difference between epileptic and healthy dogs was statistically significant (p < 0.05). Values
correlated with seizure severity and duration of seizure activity. Dogs with cluster seizures and/or status epilepticus
and with seizure activity for more than six months displayed the highest EC concentrations.
Conclusion: In conclusion, we present the first endocannabinoid measurements in canine CSF and confirm the
hypothesis that the EC system is altered in canine idiopathic epilepsy.
Keywords: Endocannabinoids, Anandamide, 2-arachidonyl glycerol, Epilepsy, Cerebrospinal fluid, Canine
Background
Epilepsy is one of the most common neurological disor-
ders in dogs, characterized by recurrent seizures [1,2].
Based on underlying etiology epilepsy in dogs can be di-
agnosed as idiopathic or symptomatic [2,3]. In idiopathic
epilepsy, hereditary factors are responsible for recurrent
seizures [4]. Seizures reflect an abnormal hypersynchro-
nous electrical activity of neurons, caused by an imbal-
ance between excitation and inhibition in the brain [1].
Most dogs with idiopathic epilepsy suffer their first seiz-
ure between one and five years of age and although any
breed - including mix-breeds - can be affected, a genetic
basis for idiopathic epilepsy is suggested for a number of
breeds [5]. The prevalence of epilepsy in dogs has been
estimated in different studies to vary from 0.5 to 5% [1].
Using current treatment protocols a significant part of
epileptic dogs may still continue to suffer from seizures
[4-7]. Better understanding of the molecular pathogenesis
of seizure development would allow introducing new
treatment modalities.
The endocannabinoid (EC) system displays numerous
physiological functions [8]. Anandamide (AEA) and 2-
arachidonylglycerol (2AG), the two most studied endo-
cannabinoids, are endogenous lipid mediators that bind
to the G protein coupled cannabinoid receptors type 1
and type 2 (CB1 and CB2). CB1 is one of the most abun-
dant receptors in the mammalian brain and also present
in peripheral tissues [9]. Similary, CB2 is expressed in vari-
ous tissues, especially on cells of the immune system [10].
Endocannabinoids are involved in food intake, pain sensa-
tion and memory formation, amongst others [8,11]. EC
system dysregulation is connected to several pathological
conditions [10], such as Alzheimer’s, Parkinson’s, and
Huntington’s disease, obesity, ischemic brain damage and
* Correspondence: Felix.Gesell@tiho-hannover.de
1
Department of Small Animal Medicine and Surgery, University of Veterinary
Medicine, Hannover, Germany
Full list of author information is available at the end of the article
© 2013 Gesell et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Gesell et al. BMC Veterinary Research 2013, 9:262
http://www.biomedcentral.com/1746-6148/9/262
epileptic seizures [12-15]. Endocannabinoids are released
from the postsynaptic neurons and serve as retrograde
signaling molecules [16]. Generally, retrograde endocan-
nabinoid signaling leads to decreased synaptic transmis-
sion and might therefore be involved in maintaining the
seizure threshold. Consequently, endocannabinoid signal-
ing at glutamatergic synapses could have a beneficial effect
in epilepsy treatment [14]. The exact role of endocannabi-
noids in epilepsy pathophysiology is still unclear. A recent
study found significant decreased AEA concentrations in
cerebrospinal fluid (CSF) of human epileptic patients with
newly diagnosed seizures as compared to healthy controls.
No differences in 2AG concentrations were observed [17].
In another study, 2AG hippocampal tissue concentrations
were increased in a rat model of pilocarpine-induced sta-
tus epilepticus compared to control animals. The authors
concluded that seizure activity in otherwise healthy rats
increases endocannabinoid synthesis, possibly as a pro-
tective mechanism [18].
Although dogs appear to be an ideal model for the
study of human epileptic seizures [19], endocannabi-
noids have never been measured in canine CSF. We
therefore hypothesized that altered CSF concentrations
of AEA and 2AG are involved in the process of canine
epilepsy. In order to prove it, AEA and total arachidonyl
glycerol (total AG, i.e. the sum of 2AG and 1AG) in CSF
of 40 dogs with idiopathic epilepsy and in a control
group of 16 unaffected healthy dogs were measured.
Correlations between AEA and seizure severity and dis-
ease duration were calculated.
Results
AEA and total AG were detected and quantified in
nearly all of the CSF samples. Well in line with previous
endocannabinoid measurements in CSF and brain tissue
[17,20,21], concentrations were in the picomolar range
for AEA and in the nanomolar range for total AG. Ani-
mals suffering from idiopathic epilepsy had higher AEA
concentrations than control animals (median, 25 –75%
percentiles: 4.94, 3.18 –9.17 pM vs. 3.19, 2.04 –4.28 pM,
p = 0.033) (Table 1). No statistically significant difference
was observed for total AG concentration (Figure 1).
In a subgroup of dogs with severe seizures (n = 23, his-
tory of cluster seizures and/or status epilepticus), signifi-
cantly higher AEA concentration in CSF were measured
(median, 25 –75% percentiles: 8.05, 4.18 –12.80 pM)
than in dogs with single seizure episodes (n = 16, no clus-
ter seizures and no status epilepticus, median, 25 –75%
percentiles: 3.81, 1.77 –6.41 pM, p = 0.029). No signifi-
cant difference in total AG was observed (Figure 2).
Besides seizure severeness, disease duration was also
associated with higher AEA concentrations in CSF of
dogs that suffered from idiopathic epilepsy for longer
than six months (n = 16) presented with 8.30, 4.60 –
15.00 pM, whereas dogs with a history of idiopathic epi-
lepsy for shorter than six months (n = 20) presented
with 4.10, 2.99 –7.85 pM (p = 0.036). Total AG concen-
trations in these subgroups were again not significantly
different (Figure 3). Other calculated correlations included
age of the dogs, pretreated and untreated patients and
timepoint of CSF collection and did not reveal statistical
significant findings. However, the number of patients in
these subgroups was limited.
Discussion
Although epilepsy is one of the most common neuro-
logical diseases in dogs, a seizure free life cannot be
achieved in many patients by current treatment options.
Using well known antiepileptic drugs, approximately 25%
of dogs cannot be well controlled and continue to have
seizures [5,7]. Thus, in the authors opinion new treatment
options are needed and the controlled therapeutic modu-
lation of the EC system could represent one possibility in
order to treat those refractory epileptic patients. There-
fore, pathophysiological mechanisms of the EC system
have to be better understood.
EC system dysregulation appears to play a role in the
process of epilepsy [13]. In the central nervous system
(CNS), endocannabinoids are released on demand from
membrane phospholipid precursors at postsynaptic neu-
rons and bind to G-protein coupled cannabinoid receptors
Table 1 AEA levels and comparison of different subgroups
Groups Number of dogs AEA levels (pM) p -values
Dogs with idiopathic epilepsy (IE) 40 4.94 (3.18–9.17) p = 0.033
Control group 16 3.19 (2.04 –4.28)
Dogs with history of cluster seizures and/or status epilepticus 23 8.05 (4.18 –12.80) p = 0.029
Dogs with single generalized seizures 16 3.81 (1.77 –6.41)
Dogs with IE for > 6 months 16 8.30 (4.60 –15.00) p = 0.036
Dogs with IE for < 6 months 20 4.10 (2.99 –7.85)
IE, Idiopathic epilepsy.
Group differences of endocannabinoid concentrations were tested using the Wilcoxon-Test.
Differences were considered sta tistically significant when the p value was < 0.05.
AEA levels are presented as median and 25 –75% percentiles.
Gesell et al. BMC Veterinary Research 2013, 9:262 Page 2 of 6
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at the presynaptic neuron after retrograde diffusion [10].
The following signal cascade involves inhibition of cyclase
activity or different types of calcium channels and activa-
tion of certain potassium channels [9]. Overall, endocan-
nabinoid release leads to decreased synaptic transmission
and might therefore be involved in maintaining the seizure
threshold [14].
In a human study examining newly-diagnosed epileptic
patients, CSF concentrations of AEA were reduced signifi-
cantly in affected patients compared to healthy controls
[17]. In contrast, AEA concentrations in affected dogs
were increased compared to the control group. We not
only studied newly-diagnosed epileptic dogs but also dogs
with a longer history of epilepsy. AEA concentrations in
dogs with a seizure-onset more than six months prior to
CSF-sampling were increased compared to those dogs
with a seizure-onset within the last six months. We there-
fore suggest that EC system changes occur slowly and
represent a counterregulatory mechanism to the patho-
logical changes associated with epilepsy. The fact that only
newly-diagnosed patients were included in the human
study [17] may explain the contradictory findings. A limi-
tation of the current study is that the number of patients
was limited in the different subgroups.
In an in vivo study of neonatal rats with widespread
neurodegeneration [20], the authors concluded that
pathological events provide a stimulus for anandamide
formation, finally leading to increased AEA concentrations.
This interpretation supports our findings of increased
AEA concentrations in the CSF of epileptic dogs.
Not only the concentration of endocannabinoids but
also the density of the endocannabinoid receptors seems
to be altered in the CNS of epileptic patients. CB1 re-
ceptor mRNA expression was down regulated in the hu-
man hippocampus of epileptic patients compared to
healthy controls [22]. This down regulation confirms
Figure 1 AEA and total AG concentrations of 40 dogs with idiopathic epilepsy and 16 control dogs, statistic was calculated using the
Wilcoxon-Test, central lines of the box represent the median, upper and lower limits of the box represent the 75 th and
25 th percentiles.
Figure 2 AEA and total AG concentrations of 16 dogs with single seizures and 23 dogs with cluster seizures and/or status epilepticus,
statistic was calculated using the Wilcoxon-Test, central lines of the box represent the median, upper and lower limits of the box
represent the 75 th and 25 th percentiles.
Gesell et al. BMC Veterinary Research 2013, 9:262 Page 3 of 6
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the hypothesis that protective endocannabinoid signalling
is diminished in epileptic patients and may result in the
incapability of increased AEA concentrations to inhibit
pathological hyperexcitability.
In another study with pilocarpine-induced epilepsy in
mice, CB1-receptor down regulation in the hippocampus
was only found in the acute phase, whereas in the chronic
phase, an upregulation was observed [8].
Dogs with severe seizures including cluster seizures
and/or status epilepticus, had significant higher AEA CSF
concentrations compared to dogs with single seizure
events. We suggest that severe seizures increase cellular
AEA release in order to control these seizures. If elevated
AEA concentrations and an activation of the EC system
were partially responsible for the disease, elevated AEA
concentrations would have been expected to occur in all
epileptic dogs including dogs with single seizure events.
Disease duration also influenced CSF endocannabinoid
concentrations. A higher concentration of AEA was mea-
sured in the CSF of dogs that suffered from idiopathic epi-
lepsy for longer than six months compared to the dogs
with idiopathic epilepsy for less than six months. We sug-
gest that more AEA is released over time in order to con-
trol the seizures and therefore dogs with longer disease
duration show higher CSF endocannabinoid concentra-
tions. We would expect elevated AEA concentrations to
occur in all epileptic dogs including newly diagnosed dogs,
if elevated AEA concentrations and an activation of the
EC system contributed to the development of the disease.
Despite the observed elevated AEA concentration in ani-
mals suffering for less than six months from epilepsy com-
pared to the control animals, the further increase of AEA
over time suggests a counterregulation of the EC system
or seizure activity itself is leading to AEA increase.
Another interesting aspect would be the exact time
elapsing between the last seizure and CSF sampling. Be-
cause of the retrospective character of our study it was
not possible to investigate this aspect. We would hypo-
thesize that immediately after the seizure event the endo-
cannabinoid concentration is at its highest point. Such a
suggestion would support our results and our hypothesis
that ECs are released as a counterregulatory mechanism
in order to control prolonged seizure events.
Conclusion
In conclusion, we demonstrated an elevation of CSF AEA
concentrations in dogs with idiopathic epilepsy. The high-
est AEA concentrations were found in dogs with severe
seizures and a long disease history. Possibly, the activation
of the EC system serves as a counter-mechanism in order
to regulate the seizure-threshold in epilepsy. However, the
EC system can either alter or be altered by seizure activity,
so that further, prospective studies are warranted to in-
vestigate pathological mechanisms. Despite endocanna-
binoids can be synthesized “on demand”,theECsystem
should be considered for development of new treatment
strategies against epilepsy.
Methods
This retrospective analysis included data and CSF sam-
ples of patients of the Department of Small Animal
Medicine and Surgery of the University of Veterinary
Medicine Hannover collected between January 2010 and
February 2012. The study was conducted in accordance
with the ethical rules of the university and approved by
the national authority (number 33.9-42502-05-12A214).
Fourty dogs with idiopathic epilepsy and 16 healthy dogs
were included in this study. The group of dogs with idio-
pathic epilepsy included 19 females (13 were neutered) and
21 males (7 were sterilized), ranged between 10 months
and 11 years of age (median of 3 years). The group included
26 different breeds dominating mix breed (6/40) and
Labrador retriever (4/40). The healthy group included 4
females (1 was neutered) and 12 males (1 was sterilized),
Figure 3 AEA and total AG concentrations of 20 dogs with idiopathic epilepsy for less than 6 months and 16 dogs with idiopathic
epilepsy for more than 6 months, statistic was calculated using the Wilcoxon-Test, central lines of the box represent the median, upper
and lower limits of the box represent the 75 th and 25 th percentiles.
Gesell et al. BMC Veterinary Research 2013, 9:262 Page 4 of 6
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ranged between 7 months and 8 years of age (median of
1 ½ years). The group included 7 different breeds dom-
inating the Beagles (9/16).
CSF samples without blood contamination had been
collected from all patients, frozen and stored at −20°C
until analysis. Patient history was analyzed from clinical
records and included age at epilepsy onset, seizure fre-
quency, previous therapy and information about occur-
rence of status epilepticus or cluster seizures. Idiopathic
epilepsy had been diagnosed on the basis of routine clin-
ical and neurological examination. No abnormalities on
hemogram, chemistry profile and magnetic resonance
imaging (MRI) were detected. All dogs with idiopathic
epilepsy had routine CSF parameters in physiological
ranges relating to the cell count (0 –3 cells/μl), glucose
(60 –80% of the blood glucose concentration) and pro-
tein (less than 25 mg/dl) after suboccipital puncture
[23]. Clinical and neurological examinations in all 16
healthy control dogs revealed no pathological findings
and no history of seizures in the past.
AEA and total AG were measured by stable-isotope
dilution liquid chromatography combined with tandem
mass spectrometry (LC-MS/MS) as previously described
[24]. To improve extraction efficacy, hydroxypropyl-β-
cyclodextrine (10% w/v) was added after thawing the
CSF samples on ice prior to liquid-liquid extraction. To
500 μl of each sample the internal standards d
4
-AEA
and d
5
-2AG were added to a final concentration of 1.0 nM
and 0.9 nM, respectively. Liquid-liquid extraction was per-
formed by adding 500 μl toluene and shaking for 2 × 20 s
at 5000 rpm in a PreCellyshomogenisator. After centrifu-
gation (5 min, 4500 × g, 4°C) the organic phase was sepa-
rated and evaporated to dryness by a gentle stream of
nitrogen. The residues were reconstituted in 50 μleluent
for LC analysis (25% water, 75% methanol). 25 μlofthe
solution was injected into the Waters ACQUITY/XEVO
TQ-MS LC-MS/MS system. Chromatographic separation
took place on a Waters ACQUITY BEH C18 reversed
phase column (100 mm × 2.1 mm ID, 1.7 μM particle size).
The following transitions were monitored: m/z348 →m/z
62 (AEA), m/z352 →m/z66 (d
4
-AEA), m/z 379 →m/z
287 (2AG), and m/z 384 →m/z287 (d
5
-2AG).
Because of the fast isomerization of 2AG to the bio-
logical inactive 1AG, quantification of 2AG is difficult to
accomplish [21]. The available CSF samples in this study
already contained a large portion of 1AG as compared
to 2AG. Therefore, 2AG was calculated and referred to
as the total AG concentration using the sum of the 2AG
and 1AG peak areas in the acquired chromatograms.
Statistical analysis of data was performed by SAS 9.2.
Due to non-gaussian data distribution as determined by
the Shapiro-Wilk’s W test, group differences of endocan-
nabinoid concentrations were tested using the Wilcoxon-
Test. Differences were considered statistically significant
when the p value was <0.05. All data are presented as me-
dian and 25 –75% percentiles.
Competing interests
The authors declare that they have no competing interest.
Authors’contributions
All authors helped to draft the manuscript. FKG, AT and AAZ conceived of
the study, and participated in its design and coordination. All authors read
and approved the final manuscript.
Acknowledgement
This study was supported by the GKF (Gesellschaft für kynologischeForschung).
Author details
1
Department of Small Animal Medicine and Surgery, University of Veterinary
Medicine, Hannover, Germany.
2
Institute for Clinical Pharmacology, Hannover
Medicine School, Hannover, Germany.
Received: 8 July 2013 Accepted: 19 December 2013
Published: 26 December 2013
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doi:10.1186/1746-6148-9-262
Cite this article as: Gesell et al.:Alterations of endocannabinoids in
cerebrospinal fluid of dogs with epileptic seizure disorder. BMC
Veterinary Research 2013 9:262.
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