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Alterations of endocannabinoids in cerebrospinal fluid of dogs with epileptic seizure disorder


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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. 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. 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.
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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
, Alexander A Zoerner
, Christina Brauer
, Stefan Engeli
, Dimitros Tsikas
and Andrea Tipold
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
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 Alzheimers, Parkinsons, and
Huntingtons disease, obesity, ischemic brain damage and
* Correspondence:
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 (, 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
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.
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.
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 (3.189.17) 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
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
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.
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.
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
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
and d
-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
-AEA), m/z 379 m/z
287 (2AG), and m/z 384 m/z287 (d
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-Wilks 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.
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.
This study was supported by the GKF (Gesellschaft für kynologischeForschung).
Author details
Department of Small Animal Medicine and Surgery, University of Veterinary
Medicine, Hannover, Germany.
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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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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... It may also serve in Veterinary Medicine for the treatment of domestic animals, in particular for dogs affected by different pathological conditions. This may include pathologies as osteoarthritis [19], atopic dermatitis [20], epilepsy [21], degenerative myelopathy [22], some neuroinflammatory diseases (meningitis-asteritis and intraspinal spirocercosis) [23], and others [24]. It is important to remark that the information collected in studies with dogs with these pathologies is not only important for the development of Sativex in Veterinary Medicine but also for their equivalents in the human pathologies, thus representing suitable translational models for studying specific human pathologies. ...
... Given the interest to evaluate whether this phytocannabinoid-based medicine has beneficial effects on different central and peripheral canine disorders, the pharmacokinetic profile of Sativex, when administered via sublingual delivery to naïve dogs, has been determined. This may help to determine the best dosage and timing for the future evaluation of Sativex in dogs affected by different pathologies (e.g., osteoarthritis [19], atopic dermatitis [20], epilepsy [21], degenerative myelopathy [22], some neuroinflammatory diseases (meningitis-asteritis and intraspinal spirocercosis) [23], and others [24]), although the effect that different pathologies may have on Sativex pharmacokinetics remains unknown. ...
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The phytocannabinoid-based medicine Sativex® is currently marketed for the treatment of spasticity and pain in multiple sclerosis patients and is being investigated for other central and peripheral pathological conditions. It may also serve in Veterinary Medicine for the treatment of domestic animals, in particular for dogs affected by different pathologies, including human-like pathological conditions. With the purpose of assessing different dosing paradigms for using Sativex in Veterinary Medicine, we investigated its pharmacokinetics when administered to naïve dogs via sublingual delivery. In the single dose arm of the study, adult Beagle dogs were treated with 3 consecutive sprays of Sativex, and blood samples were collected at 12 intervals up to 24 h later. In the multiple dose arm of the study, Beagle dogs received 3 sprays daily for 14 days, and blood samples were collected for 24 h post final dose. Blood was used to obtain plasma samples and to determine the levels of cannabidiol (CBD), Δ9-tetrahydrocannabinol (Δ9-THC) and its metabolite 11-hydroxy-Δ9-THC. Maximal plasma concentrations of both Δ9-THC (Cmax = 18.5 ng/mL) and CBD (Cmax = 10.5 ng/mL) were achieved 2 h after administration in the single dose condition and at 1 h in the multiple dose treatment (Δ9-THC: Cmax = 24.5 ng/mL; CBD: Cmax = 15.2 ng/mL). 11hydroxy-Δ9-THC, which is mainly formed in the liver from Δ9-THC, was almost undetected, which is consistent with the use of sublingual delivery. A potential progressive accumulation of both CBD and Δ9-THC was detected following repeated exposure, with maximum plasma concentrations for both cannabinoids being achieved following multiple dose. Neurological status, body temperature, respiratory rate and some hemodynamic parameters were also recorded in both conditions, but in general, no changes were observed. In conclusion, this study demonstrates that single or multiple dose sublingual administration of Sativex to naïve dogs results in the expected pharmacokinetic profile, with maximal levels of phytocannabinoids detected at 1–2 h and suggested progressive accumulation after the multiple dose treatment.
... On the other hand, their use to treat seizures in dog and rodent models has increased. Cerebrospinal fluid samples were obtained from dogs suffering from idiopathic epilepsy, where an increased level of anandamide and endocannabinoid was observed compared to healthy dogs (74). ...
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The use of cannabinoids in both veterinary and human medicine is controversial for legal and ethical reasons. Nonetheless, the availability and therapeutic use of naturally occurring or synthetic phytocannabinoids, such as Δ ⁹ -tetrahydrocannabidiol and cannabidiol, have been the focus of attention in studies regarding their medical uses. This review aims to examine the role of cannabinoids in pain modulation by analyzing scientific findings regarding the signaling pathways of the endocannabinoid system and discussing the analgesic effects of synthetic cannabinoids compared to cannabinoid extracts and the extent and involvement of their receptors. In animals, studies have shown the analgesic properties of these substances and the role of the cannabinoid binding −1 (CB1) and cannabinoid binding −2 (CB2) receptors in the endocannabinoid system to modulate acute, chronic and neuropathic pain. This system consists of three main components: endogenous ligands (anandamide and 2-arachidonoylglycerol), G protein-coupled receptors and enzymes that degrade and recycle the ligands. Evidence suggests that their interaction with CB1 receptors inhibits signaling in pain pathways and causes psychoactive effects. On the other hand, CB2 receptors are associated with anti-inflammatory and analgesic reactions and effects on the immune system. Cannabis extracts and their synthetic derivatives are an effective therapeutic tool that contributes to compassionate pain care and participates in its multimodal management. However, the endocannabinoid system interacts with different endogenous ligands and neurotransmitters, thus offering other therapeutic possibilities in dogs and cats, such is the case of those patients who suffer from seizures or epilepsy, contact and atopic dermatitis, degenerative myelopathies, asthma, diabetes and glaucoma, among other inflammatory diseases. Moreover, these compounds have been shown to possess antineoplastic, appetite-stimulating, and antiemetic properties. Ultimately, the study of the endocannabinoid system, its ligands, receptors, mechanism of action, and signaling, has contributed to the development of research that shows that hemp-derived and their synthetic derivatives are an effective therapeutic alternative in the multimodal management of pain in dogs and cats due to their ability to prevent peripheral and central sensitization.
... Additionally, dogs with idiopathic epilepsy with possible genetic background (Berendt and Gram, 1999;Thomas, 2010) have been shown to present increased AEA concentration in the cerebrospinal fluid than non-epileptic dogs, while dogs with severe chronic idiopathic epilepsy presented higher AEA concentration than dogs that experienced a single seizure episode (Gesell et al., 2013). Although these results support neuroplastic alterations in the endocannabinoid system of epileptic dogs similar to those detected in humans with temporal lobe epilepsy (Rocha et al., 2020) and in rodents with genetic absence epilepsy (Roebuck et al., 2021), possible changes in CB1 distribution, expression, and functionality, still remain to be assessed in epileptic dogs. ...
Currently, there is an urgent need to better comprehend neuroplastic alterations in cannabinoid receptor type 1 (CB1) and to understand the biological meaning of these alterations in epileptic disorders. The present study reviewed neuroplastic changes in CB1 distribution, expression, and functionality in animal models of epileptic seizures. Neuroplastic alterations in CB1 were consistently observed in chemical, genetic, electrical, and febrile seizure models. Most studies assessed changes in hippocampal and cortical CB1, while thalamic, hypothalamic, and brainstem nuclei were rarely investigated. Additionally, the relationship between CB1 alteration and the control of brain excitability through modulation of specific neuronal networks, such as nigrotectal and thalamocortical pathways and inhibitory projections to hippocampal pyramidal neurons were presented and discussed. Neuroplastic alterations in CB1 detected in animal models of epilepsy may reflect two different scenarios: (1) endogenous adaptations aimed to control neuronal hyperexcitability in epilepsy or (2) pathological alterations which facilitate neuronal hyperexcitability. Additionally, a better comprehension of neuroplastic and functional alterations in CB1 can improve pharmacological therapies for epilepsies.
... Areas of interest include use for osteoarthritis pain [88,[96][97][98], other types of pain (oncologic, neuropathic) [99], immune-mediated and inflammatory allergic disorders [100], cardio-vascular and respiratory conditions [101], and epilepsy [102,103]. According to the scientific literature review, cannabinoids are mainly used in the treatment of pain, especially osteoarthritis pain. ...
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As cannabis-derived products have become more available, veterinarians are seeing more cases of toxicosis. In addition, animal owners are having an increasing interest in using these products for their pets. This review looks at the situation in Europe and North America, the different types of cannabis and cannabis-derived products with historical examples of use in animals, and the cannabis industry. The existing regulatory framework for use in humans and animals as medicines and/or supplements was examined. Finally, a review of the clinical indications for which medicinal cannabis is authorised, a discussion of toxicosis, and recommendations and warnings around medical cannabis use are presented.
... The endocannabinoid AEA is known from preclinical [12] and clinical studies [9,48] to be involved in suppressing pathologic neuronal excitability and in controlling the spread of activity in an epileptic network [18,56]. Although seizures induced within ECT differ from epilepsy, our results of the negative correlation between AEA CSF levels and seizure quality fit into this established knowledge. ...
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For patients with depression treated with electroconvulsive therapy (ECT), the novel seizure quality index (SQI) can predict the risk of non-response (and non-remission)—as early as after the second ECT session—based the extent of several ictal parameters of the seizure. We aim to test several CSF markers on their ability to predict the degree of seizure quality, measured by the SQI to identify possible factors, that could explain some variability of the seizure quality. Baseline CSF levels of metabolites from the kynurenine pathway, markers of neurodegeneration (tau proteins, β-amyloids and neurogranin), elements of the innate immune system, endocannabinoids, sphingolipids, neurotrophic factors (VEGF) and Klotho were measured before ECT in patients with depression (n = 12) to identify possible correlations with the SQI by Pearson's partial correlation. Negative, linear relationships with the SQI for response were observed for CSF levels of T-tau (rpartial = − 0.69, p = 0.019), phosphatidylcholines (rpartial = − 0.52, p = 0.038) and IL-8 (rpartial = − 0.67, p = 0.047). Regarding the SQI for remission, a negative, linear relationship was noted with CSF levels of the endocannabinoid AEA (rpartial = − 0.70, p = 0.024) and CD163 (rpartial = − 0.68, p = 0.029). In sum, CSF Markers for the innate immune system, for neurodegeneration and from lipids were found to be associated with the SQI for response and remission after adjusting for age. Consistently, higher CSF levels of the markers were always associated with lower seizure quality. Based on these results, further research regarding the mechanism of seizure quality in ECT is suggested.
... For example, reduction in the striatum is accompanied by cognitive impairments and Alzheimer's disease [6,8]. Alteration of EC concentrations in the cerebrospinal fluid plays a role in epileptic seizure disorders [9,10]. Also, ECs are involved in a cascade leading to long-term depression of hippocampal synaptic functions [11]. ...
Aim: Solid-phase microextraction is proposed to measure concentrations of anandamide and 2-arachidonoyl glycerol in live rat brains in response to stress. Materials & methods: Solid-phase microextraction fibers were prepared from steel with 1.5 mm extraction coating. 24 male rats were divided into groups based on brain region, stria terminalis or posterior hypothalamus and loud noise or control groups. The fibers were desorbed in acetonitrile-water (75:25) and analyzed by ultraperformance LC–MS/MS. The linear range of the method was 0.05–50 ng/ml and the in vivo concentrations were found to be between 0.3 and 40 ng/ml. Conclusion: The new approach was successfully used to determine the concentrations of anandamide and 2-arachidonoyl glycerol in vivo and could be used in the future to measure other endogenous compounds.
Medical use of Cannabis (or hemp) began thousands of years ago. In the 20th century, mechanisms of action were demonstrated with the discovery of its active substances, the phytocannabinoids, and its pharmacological targets, the endocannabinoid system. This system is composed of receptors, endogenous substances, and enzymes, and it participates in the modulation of physiological mechanisms in several species, including dogs. Studies indicate that changes in this system may contribute to the genesis of some diseases. Therefore, the use of substances that act on its components may help in the treatment of these diseases. The main phytocannabinoids described are Δ9−tetrahydrocannabinol (THC) and cannabidiol (CBD). In humans, the benefits of using CBD in several diseases have been demonstrated. The popularization of this type of treatment has also reached veterinary medicine, which on one hand was related to an increase in adverse event records, but on the other also allowed reports of anecdotal evidences of its effectiveness and safety in animals. Clinical studies published so far indicate that the use of CBD in dogs can be safe at given doses and can contribute to osteoarthritis and idiopathic epilepsy treatments. Clinical and pre-clinical studies and case reports were reviewed in this report to identify the main characteristics of hemp-based therapies in dogs, including its pharmacokinetics, pharmacodynamics, safety, and efficacy in the treatment of diseases.
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Kenevir Bitkisi ve Kannabinoid Üretiminde Biyoteknolojik Yöntemlerin Uygulanması Cannabis türleri gerek bütün bitki, gerekse metabolitleri ve yasal olduğu ölçüde hammadde olarak değişik ülke ve eyaletlerde çok farklı endüstrilerde farklı amaçlarla kullanılmaktadır. Bitkinin temel aktif bileşenleri arasında, kannabinoidler olmak üzere terpen ve fenolikler mevcuttur. Delta-9-tetrahidrokannabinol (THC), kannabidiol (CBD) ve kannabichromen (CBC) baskın kannabinoidler olmak üzere bunların yanında kannabigerol (CBG) ve kannabinol (CBN) gibi diğer kannabinoidler de bitkide tespit edilmiştir. Kannabinoidlerin içeriği ve bileşimi bitkinin cinsiyeti, yaşı ve gelişim evresi, iklim ve hava koşulları, hasat zamanı, yetiştirme yöntemleri, saklama koşulları gbi özelliklere bağlı olarak değişkenlik göstermekte olup büyük ölçekli tarla tarımında standardizasyonun kontrolü oldukça zordur. Bu nedenle alternatif üretim yöntemleri araştırılmıştır. Biyoteknolojik yöntemler bazı narkotik genotipler için umut verici olmakla birlikte lif türlerinin mikroçoğaltımı daha fazla araştırma gerektirmektedir. Mikroçoğaltım tekniklerinin geliştirilmesi, hem elit çeşitlerin elde edilmesi hem de ilaç ve gıda olarak kullanım dışında farklı amaçlı çeşitlerin üretilmesi için genetik modifikasyon çalışmalarında gerekli önemli bir adımdır. İlaçta ve gıdada kullanım amaçlı kannabinoid üretmek için kallus, hücre süspansiyon kültürleri ve kenevir saçak kök kültürleri izin verilen bölgelerde farklı gruplar tarafından başarıyla üretilmiştir. Şimdiye kadar kenevir doku kültürü alanında yapılan çalışmalar, kallus ve süspansiyon kültürlerinde kannabinoid üretiminde yüksek başarılar sağlanmamışken, adventif ve saçak kök kültürlerinde üretimde daha başarılı olunduğunu ortaya koymuştur. Yayınlanan çalışmalar pilot ölçekte olup, endüstriyel amaçlı kannabinoid üretimi için çalışmaların artırılmasına ihtiyaç duyulmaktadır.
The legal market for recreational and medicinal cannabis for human consumption is growing worldwide. At the same time, marketing of cannabis products for use in pets is expanding. Yet, there is little research exploring the effects of cannabis use in veterinary medicine. This study used an anonymous, online survey to assess Canadian pet owners' reasons for purchasing cannabis products for their dogs, and their perceptions regarding efficacy of these treatments. Owners purchased cannabis products for treatment of pain, inflammation, and anxiety in dogs, and perceived these preparations to be equally or more effective than conventional medications. Most owners reported only minimal side effects in their dogs. Despite indicating comfort in discussing canine cannabis administration with their veterinarian, most owners relied on commercial websites for product information. The main reasons for choosing cannabis products were the ability to use as an adjuvant to other therapies, and the perception of it being a natural substance. Given this information, it is incumbent upon veterinarians to appropriately counsel their clients, and also to advocate for evidence-based studies to evaluate the efficacy of cannabis use in non-human species.
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Upon the identification of anandamide (AEA) in the porcine brain, numerous studies contributed to the current state of knowledge regarding all elements that form the "endocannabinoid system (ECS)."How this complex system of receptors, ligands, and enzymes is integrated in helping to regulate fundamental processes at level of central nervous and peripheral systems and how its regulation and dysregulation might counteract disturbances of such functions, is nowadays still under investigation. However, the most recent advances on the physiological distribution and functional role of ECS allowed the progress of various research tools aimed at the therapeutic exploitation of endocannabinoid (eCB) signaling, as well as the development of novel drugs with pharmacological advantages. Here, we shall briefly overview the metabolic and signal transduction pathways of the main eCBs representatives, AEA, and 2-arachidonoylglycerol (2-AG), and we will discuss the therapeutic potential of new ECS-oriented drugs.
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Advances in the understanding of the endogenous cannabinoid system have led to several therapeutic indications for new classes of compounds that enhance cannabinergic responses. Endocannabinoid levels are elevated during pathogenic conditions, and inhibitors of endocannabinoid inactivation promote such on-demand responses. The endocannabinoids anandamide and 2-arachidonoyl glycerol have been implicated in protective signaling against excitotoxic episodes, including seizures. To better understand modulatory pathways that can exploit such responses, we used the new generation compound AM6701 that blocks both the anandamide-deactivating enzyme fatty acid amide hydrolase (FAAH) and the 2-arachidonoyl glycerol-deactivating enzyme monoacylglycerol lipase (MAGL) with equal potency. Also studied was the structural isomer AM6702 which is 44-fold more potent for inhibiting FAAH versus MAGL. When applied before and during kainic acid (KA) exposure to cultured hippocampal slices, AM6701 protected against the resulting excitotoxic events of calpain-mediated cytoskeletal damage, loss of presynaptic and postsynaptic proteins, and pyknotic changes in neurons. The equipotent inhibitor was more effective than its close relative AM6702 at protecting against the neurodegenerative cascade assessed in the slice model. In vivo, AM6701 was also the more effective compound for reducing the severity of KA-induced seizures and protecting against behavioral deficits linked to seizure damage. Corresponding with the behavioral improvements, cytoskeletal and synaptic protection was elicited by AM6701, as found in the KA-treated hippocampal slice model. It is proposed that the influence of AM6701 on FAAH and MAGL exerts a synergistic action on the endocannabinoid system, thereby promoting the protective nature of cannabinergic signaling to offset excitotoxic brain injury.
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The endocannabinoid system plays a central role in retrograde synaptic communication and may control the spread of activity in an epileptic network. Using the pilocarpine model of temporal lobe epilepsy we examined the expression pattern of the Type 1 cannabinoid receptor (CB1-R) in the hippocampi of CD1 mice at survival times of 2 hours, 1 day, 3 days and 2 months (acute, latent and chronic phases). Based on the behavioral signs of the acute seizures, animals were classified as "weakly" or "strongly" epileptic using the modified Racine scale. Mice of the weak group had mild seizures, whereas seizures in the strong group were frequent with intense motor symptoms and the majority of these animals developed sclerosis in the chronic phase. In control samples the most intense staining of CB1-R-positive fibers was found in the molecular layer of the dentate gyrus and in str. pyramidale of the cornu Ammonis. In weak animals no significant changes were seen at any survival time compared to controls. In strong animals, however, in the acute phase, a massive reduction in CB1-R-stained terminals occurred in the hippocampus. In the latent phase CB1-R immunoreactivity gradually recovered. In the chronic phase, CB1-immunostaining in sclerotic samples was stronger throughout the hippocampus. Quantitative electron microscopic analysis showed an increase in the number of CB1-R-positive terminals in the dentate gyrus. Moreover, the number of immunogold particles significantly increased in GABAergic terminals. Our results suggest a proconvulsive downregulation of CB1 receptors in the acute phase most probably due to receptor internalization, followed by compensatory upregulation and sprouting in the chronic phase of epilepsy. In conclusion, the changes in CB1 receptor expression pattern revealed in this study are associated with the severity of hippocampal injury initiated by acute seizures that ultimately leads to sclerosis in the vulnerable regions in the chronic phase.
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There has been much interest in utilizing the dog as a genetic model for common human diseases. Both dogs and humans suffer from naturally occurring epilepsies that share many clinical characteristics. Investigations of inherited human epilepsies have led to the discovery of several mutated genes involved in this disease; however, the vast majority of human epilepsies remain unexplained. Mouse models of epilepsy exist, including single-gene spontaneous and knockout models, but, similar to humans, other, polygenic models have been more difficult to discern. This appears to also be the case in canine epilepsy genetics. There are two forms of canine epilepsies for which gene mutations have been described to date: the progressive myoclonic epilepsies (PMEs) and idiopathic epilepsy (IE). Gene discovery in the PMEs has been more successful, with eight known genes; six of these are orthologous to corresponding human disorders, while two are novel genes that can now be used as candidates for human studies. Only one IE gene has been described in dogs, an LGI2 mutation in Lagotto Romagnolos with a focal, juvenile remitting epilepsy. This gene is also a novel candidate for human remitting childhood epilepsy studies. The majority of studies of dog breeds with IE, however, have either failed to identify any genes or loci of interest, or, as in complex mouse and human IEs, have identified multiple QTLs. There is still tremendous promise in the ongoing canine epilepsy studies, but if canine IEs prove to be as genetically complex as human and murine IEs, then deciphering the bases of these canine epilepsies will continue to be challenging.
Seizures are the most common neurologic condition encountered in small animal practice and arise from an imbalance of excitatory and inhibitory mechanisms in the brain. Epilepsy refers to recurrent seizures of any cause. Successful management of epilepsy requires knowledge of the pharmacologic properties of available antiepileptic medications, regular patient evaluations to assess response to therapy and monitor for adverse effects, and thorough client education to ensure that goals and expectations of therapy are understood. Recommendations for emergency care of seizures at home should be provided for patients with seizures that are not controlled with maintenance antiepileptic therapy.
Endogenous cannabinoid receptor ligands (endocannabinoids) may rescue neurons from glutamate excitotoxicity. As these substances also accumulate in cultured immature neurons following neuronal damage, elevated endocannabinoid concentrations may be interpreted as a putative neuroprotective response. However, it is not known how glutamatergic insults affect in vivo endocannabinoid homeostasis, i.e. N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG), as well as other constituents of their lipid families, N-acylethanolamines (NAEs) and 2-monoacylglycerols (2-MAGs), respectively. Here we employed three in vivo neonatal rat models characterized by widespread neurodegeneration as a consequence of altered glutamatergic neurotransmission and assessed changes in endocannabinoid homeostasis. A 46-fold increase of cortical NAE concentrations (anandamide, 13-fold) was noted 24 h after intracerebral NMDA injection, while less severe insults triggered by mild concussive head trauma or NMDA receptor blockade produced a less pronounced NAE accumulation. By contrast, levels of 2-AG and other 2-MAGs were virtually unaffected by the insults employed, rendering it likely that key enzymes in biosynthetic pathways of the two different endocannabinoid structures are not equally associated to intracellular events that cause neuronal damage in vivo. Analysis of cannabinoid CB1 receptor mRNA expression and binding capacity revealed that cortical subfields exhibited an up-regulation of these parameters following mild concussive head trauma and exposure to NMDA receptor blockade. This may suggest that mild to moderate brain injury may trigger elevated endocannabinoid activity via concomitant increase of anandamide levels, but not 2-AG, and CB1 receptor density.
The endocannabinoids anandamide (arachidonoyl ethanolamide, AEA) and 2-arachidonoyl glycerol (2AG) are physiologically occurring, biologically active compounds on CB1 and CB2 receptors with multiple physiological functions. AEA and 2AG have been identified and quantified in many mammalian biological fluids and tissues, such as human plasma, adipocytes, tissues and tissue microdialysates, at concentrations in the picomolar-to-nanomolar range under basal conditions. In this article, recently published chromatographic and mass spectrometric analytical methods, i.e., HPLC with fluorescence or ultraviolet detection, LC–MS, LC–MS/MS, GC–MS and GC–MS/MS, are reviewed and discussed, notably from the quantitative point of view. We focus on and emphasize the particular importance of blood sampling, sample storage and work-up including solvent and solid-phase extraction and derivatization procedures, matrix-effects, and stability of analytes. As 2AG spontaneously isomerizes to its CB1/CB2 receptors biologically inactive 1-arachidonoyl glycerol (1AG) by acyl migration, this phenomenon and its particular importance for accurate quantification of 2AG are discussed in detail. Due to the electrical neutrality of AEA and 2AG their solvent extraction by toluene offers the least matrix-effect and minimum isomerization. LC–MS/MS is the most frequently used analytical technique for AEA and 2AG. At present, the utility of the GC–MS/MS methodology seems to be limited to AEA measurement in human plasma, bronchoalveolar liquid (BAL) and microdialysate samples. Despite great instrumental advances in the LC–MS/MS methodology, sampling and sample treatment remains one of the most crucial analytical steps in 2AG analysis. Extension of the LC–MS/MS methodology, for instance to microdialysate and BAL samples from clinical studies, is a big analytical challenge in endocannabinoid analysis in clinical settings. Currently available LC–MS/MS and GC–MS/MS methods should be useful to investigate the metabolism of AEA and 2AG beyond hydrolysis, i.e., by β- and ω-oxidation pathways. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.Highlights► We provide an extensive review covering quantitative endocannabinoid analysis. ► It provides a broad overview of methodologies published in the last 30 years. ► The molecules anandamide, 2-arachidonoyl glycerol, virodhamine, 2-arachidonyl glycerol ether and arachidonoyl dopamide are discussed. ► Mass spectrometric approaches of endocannabinoid analysis are described in detail.
Phytocannabinoids isolated from the cannabis plant have broad potential in medicine that has been well recognized for many centuries. It is presumed that these lipid soluble signaling molecules exert their effects in both the central and peripheral nervous system in large part through direct interaction with metabotropic cannabinoid receptors. These same receptors are also targeted by a variety of endogenous cannabinoids including 2-arachidonoyl glycerol and anandamide. Significant effort over the last decade has produced an enormous advance in our understanding of both the cellular and the synaptic physiology of endogenous lipid signaling systems. This increase in knowledge has left us better prepared to carefully evaluate the potential for both natural and synthetic cannabinoids in the treatment of a variety of neurological disorders. In the case of epilepsy, long standing interest in therapeutic approaches that target endogenous cannabinoid signaling systems are, for the most part, not well justified by available clinical data from human epileptics. Nevertheless, basic science experiments have clearly indicated a key role for endogenous cannabinoid signaling systems in moment to moment regulation of neuronal excitability. Further it has become clear that these systems can both alter and be altered by epileptiform activity in a wide range of in vitro and in vivo models of epilepsy. Collectively these observations suggest clear potential for effective therapeutic modulation of endogenous cannabinoid signaling systems in the treatment of human epilepsy, and in fact, further highlight key obstacles that would need to be addressed to reach that goal.