CBD Oil For Degenerative Myelopathy In Dogs

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Is CBD oil safe for dogs? We define the terms and review the potential benefits, side effects, and proper dosages for canines. Upregulation of CB 2 receptors in reactive astrocytes in canine degenerative myelopathy, a disease model of amyotrophic lateral sclerosis This is an Open Access article distributed under the

What’s the Right Dosage of CBD Oil for Dogs?

Following the legalization of marijuana in many states, pet owners have shown increased interest in the benefits of marijuana and CBD oil for their canine companions. In this article, we will define CBD oil, explain how it differs from marijuana, and discuss health benefits, side effects, and dosage recommendations for dogs.

What Is CBD Oil?

The Cannabis plant has been around for thousands of years and is known to contain more than 450 chemicals and over 80 cannabinoids. A cannabinoid is a class of chemicals found in Cannabis that can cause various effects on the body. The effect of each cannabinoid can be different. The amount and concentration of each cannabinoid varies with the specific plant and strain of plant. CBD is derived from the hemp plant, which is a relative to the marijuana plant. Hemp is a type of Cannabis plant known to have more CBD than THC with a max of 0.3% THC.

The two most studied and available cannabinoids are Tetrahydrocannabinol (THC) and Cannabidiol (CBD). Cannabidiol (CBD) has limited or no psychotropic properties. CBD is thought to decrease anxiety, lessen nausea and vomiting, reduce seizure activity, control pain, and decrease inflammation. It is increasingly being used in both humans and dogs for a variety of medical and behavioral conditions.

CBD is one of the many components of marijuana but, when used by itself, it does not cause the “high” that is often associated with the plant. To date, there is no evidence that CBD can be addictive.

How Does CBD Oil Differ from Marijuana?

Marijuana, also known as Cannabis, is a drug derived from the Cannabis plant. It is classified as a psychoactive drug and used for recreational, spiritual, and medicinal purposes. As mentioned above, the Cannabis plant has many different cannabinoids, with tetrahydrocannabinol (THC) being the most potent and psychogenic cannabinoid. Marijuana can produce different physical and mental effects, including an altered sense of time and consciousness, changes in thought, relaxation, impaired body actions, increase in appetite, feelings of euphoria (in humans, referred to as a feeling of being “high” or “stoned”), and an increased awareness of sensation. Other effects may include delusions, anxiety, and/or paranoia.

Cannabis is used medically to treat nausea and vomiting, muscle spasms, seizures, chronic pain, anxiety, inappetence, as well as other medical problems. Learn more about the ingestion and toxicity of Tetrahydrocannabinol (THC) here.

Cannabis can be utilized by smoking or vaping, as extracts, or in food “edibles” such as brownies, cookies, and other baked goods. The effects of cannabis generally begin between 15 and 50 minutes of intake and last for two to seven hours, depending on the amount and concentration of the product.

Long-term effects are poorly defined. Some research suggests that long-term use can be associated with memory and cognitive problems, risk of schizophrenia, and risk of addiction.

As the medical benefits of marijuana are explored, increased interest has been placed on the plant and CBD products.

Because of the availability of marijuana, there has been an increase in exposure to pets. Learn about Marijuana Toxicity in Cats here.

How Is CBD Oil Made?

A process called CO2 extraction, which is pressurized carbon dioxide used to pull phytochemicals from a plant, allows for the removal of the desired cannabinoids. Some CBD products contain isolates or only certain cannabinoids, while others are described as being “full spectrum,” which means that the product contains all the cannabinoids natural to the plant. The full spectrum CBD products are high in CBD and extremely low in THC. Once CBD is extracted from the plant, it is often formulated and sold as an “oil.”

What Is the Difference Between Hemp Oil and CBD Oil?

It is important to note that CBD oil is different from hemp oil. Although both are derived from the hemp plant, hemp oil is made from hemp seeds and the end product contains no CBD. CBD is made from the flowers, stalks, and leaves of the hemp plant, where the cannabidiol is located. Each type of oil has its own medical benefits. Hemp oil is rich in minerals and vitamins and some pet owners use it as a vitamin supplement.

How CBD Oil Works for Dogs

The endocannabinoid system (ECS) regulates and balances many body processes. In humans, it impacts immune responses, appetite, memory, mood, sleep, and even fertility. The ECS was discovered over 30 years ago as researchers worked to better understand THC. The ECS system is active in the body regardless of the use of cannabinoids and created to interact with naturally-occurring cannabinoids produced by the brain. These receptors can also interact with plant-based CBD products.

Cannabinoids bind with very specific membrane-bound receptors called the cannabinoid receptor type 1 (CB1) and cannabinoid receptor type 2 (CB2). Cannabinoid receptor type 1 (CB1) is found in the brain and (CB2) receptors are found in the immune system, such as within the spleen. Cannabidiol (CBD) has affinity for CB1 and CB2 receptors and may also work by altering the update of adenosine in the body to promote sleep and relaxation.

Health Benefits of CBD Oil for Dogs

In humans, CBD oil has been used to treat a variety of conditions. This includes anorexia and weight loss associated with human immunodeficiency virus (HIV), cachexia, cancer, chemotherapy-induced nausea and vomiting, degenerative neurological conditions, depression, dementia, dystonia, epilepsy, glaucoma, irritable bowel syndrome, pain, Parkinson’s disease, posttraumatic stress disorder (PTSD), schizophrenia, sleep disorders, spasticity associated with multiple sclerosis, traumatic brain injury, and Tourette’s syndrome.

CBD oil is used and prescribed for a variety of uses in dogs, many of which are extrapolated from human research and use.

The most common uses in dogs include:

  • Anxiety and Fear-Based Behaviors. Anxiety, such as separation anxiety, can affect some dogs, causing a wide range of symptoms that include pacing, circling, panting, escape behavior, persistent barking, aggression, tremors, chewing, and repetitive physical activity, such as digging, tail-chasing, and/or inappropriate urination and defecation. In humans, some believe that CBD decreases feelings of anxiety, and it is being used in dogs for the same purpose. Some research suggests that in humans, CBD may increase serotonin in the brain, which reduces feelings of anxiety and fear, and improves mood. Fear-based behaviors such as Noise phobias (thunderstorms) may also benefit from CBD oil.
  • Arthritis and Joint Pain. Arthritis is the inflammation of one or more joints that causes pain, stiffness, and decreased mobility. The most common use of CBD oil for dogs is for its anti-inflammatory effects. Inflammation is associated with many medical conditions. CBD is believed to interact with immune cell CB2 receptors, which decrease inflammation and pain.
  • Cancer. Some research suggests that CBD may have anti-tumor properties. Cancer is a disease caused by abnormal cells that divide uncontrollably and destroy body tissue. It is a very common illness in dogs and is the most common cause of death in senior dogs. Some cancers can be treated with surgery, while others are treated with drug therapies. The goal of cancer treatment is to remove the cancer (if possible) or decrease its growth. Many pet owners like CBD as part of their dog’s treatment, due to its low cost and lack of side effects when compared to chemotherapy. In humans, research is being conducted to document the use of CBD and explain how cannabinoids can slow the spread and growth of tumors. There are reports that suggest CBD can be useful in the treatment of breast cancer and colon cancer, and it may even enhance the effects of chemotherapy.
  • Homeostasis. It’s believed that CBD may create an environment of “homeostasis.” Homeostasis is a state of harmony (equilibrium) of internal and physical processes or a “state of perfect balance.” Research suggests that the endocannabinoid system may play some role in homeostasis. Diseases and inflammation develop when things are out of balance.
  • Nausea, Vomiting, and Appetite Stimulation. Nausea is a very common symptom of many different diseases and it can be a side effect of medications and chemotherapy. It decreases the appetite and is associated with weight loss, lethargy, lower activity levels, and dehydration. In human medicine, it is believed that CBD can control nausea and improve appetite.
  • Pain Management. It is believed that the endocannabinoid system is involved in pain management and, today, CBD is best known for its pain control capabilities. CBD has been shown during scientific trials to stop the absorption of anandamide, which is a brain chemical that depresses pain signals.
  • Seizure Disorders (epilepsy, etc.). One of the most popular discussions surrounding the use of CBD for dogs is related to seizures. Many believe that CBD has natural anticonvulsant properties, although it is unclear how this works and has only been documented in humans.

A study published in June of 2019 by the College of Veterinary Medicine and Biomedical Sciences at Colorado State University investigated “the effect of oral cannabidiol (CBD) administration in addition to conventional antiepileptic treatment on seizure frequency in dogs with idiopathic epilepsy.” Essentially, the study used 26 client-owned dogs with intractable idiopathic epilepsy. These 26 dogs were placed into two groups, one receiving CBD-infused oil twice a day for 3 months in addition to their normal seizure medications, and the other group receiving non-infused oil under the same conditions. The seizure activity, side effects, and blood CBD levels were compared for both groups.

Two dogs in the CBD group developed ataxia and were withdrawn from the study. This left 9 dogs in the CBD group and 7 in the placebo group. According to the study, “Dogs in the CBD group had a significant (median change, 33%) reduction in seizure frequency, compared with the placebo group. However, the proportion of dogs considered responders to treatment (≥ 50% decrease in seizure activity) was similar between both groups.

This data makes it hard to gauge the impact of CBD oil use for dogs. Ultimately, this study demanded additional research, specifically experimenting with higher doses of CBD to determine if that further influenced seizure activity.

Additional Uses and Potential Benefits of CBD Oil for Dogs

    are very common in dogs and cats because of food ingredients, pollens, molds, grass, and more. The anti-inflammatory properties of CBD may be beneficial to control the symptoms of itching and skin inflammation in some dogs.
  • Some veterinarians refer to Cognitive Dysfunction as “doggy Alzheimer’s,” since abnormal changes in the brain concerning cognitive function resemble those of humans with Alzheimer’s Disease. Signs of senile dementia may include reduced activity, sleeping more, lack of awareness of the environment, confusion, and difficulty walking. Some dogs will go into a room and stare at the wall or seem confused as to their surroundings. Research in human Alzheimer’s has suggested there may be benefits to giving CBD oil, although no research has been conclusive in veterinary medicine. The neuron processes that have been documented suggest that CBD may decrease inflammation of brain cells, promote new cell growth, and potentially decrease cells that contribute to amyloid production. is a slow, progressive spinal cord disorder most commonly seen in senior, large-breed dogs. It causes weakness and difficulty walking on the rear legs. Some believe the anti-inflammatory, pain control, and anti-anxiety properties of CBD benefit dogs with this disease. is a condition that results in high eye pressure, ultimately leading to vision loss. Treatment includes eye drops, oral medications, and surgery. Glaucoma can be genetic or caused by tumors, infections, secondary to cataracts, as well as other diseases. Human studies dating back to the 1970’s have looked at the benefits of cannabis on eye pressure and found it can reduce pressure up to 30%. How this impacts dogs is yet to be determined, but some advocates are hopeful. (IBD) is a group of gastrointestinal disorders that involve infiltration of the gastrointestinal tract by inflammatory cells. Common signs are persistent vomiting and diarrhea. The anti-inflammatory properties of CBD may make it beneficial to dogs with these disorders. associated with car or boat rides can occur in some dogs. The anti-nausea properties of CBD may help dogs with this condition. results from inflammation of the pancreas, characterized by activation of pancreatic enzymes that can cause the pancreas to begin digesting itself. It can be mild or life-threatening. Common signs include vomiting, diarrhea, lethargy, decreased appetite, abdominal pain, and weight loss. The anti-inflammatory and pain management attributes of CBD may help in treating this condition.
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CBD is also often recommended in addition to traditional therapies.

Side Effects of CBD Oil for Dogs

It is important that dog owners understand that adverse effects can occur with any drug or supplement. CBD is not psychotropic, which means it does not cause the symptoms noted with THC, like lethargy, listlessness, stumbling, glazed over eyes, and urinary incontinence, or the typical signs associated with the feeling of being “high.” There is evidence that CBD can increase a liver enzyme, the alkaline phosphatase (ALP), however the significance of this finding has yet to be determined. The overall toxicity of CBD oil in dogs appears to be limited.

There are no clear research findings that indicate drug interactions when CBD is given with other medications such as anti-inflammatory drugs, steroids, and more.

In humans, the most common side effects of CBD oil include a dry mouth, drowsiness, and a slight drop in blood pressure. In dogs, it is more difficult to determine these effects and few adverse outcomes have been documented. Some dogs with dry mouths may lick their lips more often or drink more, dogs with lethargy may sleep more, and dogs with low blood pressure may appear weak, lethargic, or generally less active.

Impure CBD products may contain THC, which can cause psychotropic effects such as depression, lethargy, listlessness, loss of motor coordination or balance (stumbling), incontinence, low heart rate, low blood pressure, low body temperature, respiratory depression, dilated pupils and glazed over eyes, vocalization like crying or whining, agitation, drooling, vomiting, seizures, and coma. Some dogs may experience hallucinations and have increased sensory stimulation to noises or fast movements.

When giving CBD oils to your dog, make sure you monitor them closely for any abnormalities and notify your veterinarian of any problems.

The Controversy Over Using CBD Oil for Dogs

There is much controversy about the use of CBD oil products in veterinary medicine.

Issues include the following:

  • The U.S. Food and Drug Administration (USDA) has not approved CBD use for dogs.
  • It is unknown if CBD works for most medical conditions in dogs. Research is underway to determine its effect on pain and seizures. The most positive research to date, although limited, has been on the use of CBD in dogs with osteoarthritis.
  • There are substantial differences in the quality and purity of CBD products.
  • There is poor quality control of the production of CBD products. CBD is also unregulated by the Federal Government. This means that no agency is looking at the purity, concentration, quality, or label declarations. There are substantial differences in quality in various CBD products, specifically the presence of preservatives, insecticides, and additives. In fact, some products have been shown to contain little to no CBD oil.
  • Safe and therapeutic doses of CBD oil in dogs have not been established.
  • It is unknown how CBD interacts with other treatments or medications. There may be undocumented drug interactions that have yet to be determined.
  • Impure products may contain various amounts of THC, which can cause unwanted side effects.
  • Human CBD products may be in the form of baked goods or gummy candy and many contain an artificial sweetener known as Xylitol, which can be toxic to dogs. Learn more about Xylitol Toxicity in Dogs here.
  • It can be difficult to extrapolate the research to determine if CBD will work for a particular medical problem. Some CBD research studies conducted in dogs use very particular formulations and doses for specific conditions. This means you cannot assume a different formulation or dose will work.

When using CBD oil, it is recommended to obtain a high-quality product that is organic, free of preservatives, and additives. Ideally, it should be well documented that the product has been tested and free of THC.

Is CBD Legal and Can Vets Prescribe it?

Until recently, CBD was categorized by the Drug Enforcement Administration (DEA) as a Schedule 1 controlled substance, similar to heroin and morphine. This prohibited vets from prescribing CBD products.

In late 2018, the DEA announced that drugs including CBD with THC content below 0.1% were classified as Schedule 5 drugs, as long as they have been approved by the U.S. Food and Drug Administration (USDA). In many states, by law, CBD products can have no more than 0.3 percent (%) THC.

The laws surrounding the legality of veterinary prescription vary by state and have been the source of controversy and confusion. Even after the reclassification of CBD, many available products are not approved by the USDA and are still prohibited by some states.

What Is the Proper Dose of CBD Oil for Dogs?

The dose of CBD for dogs may vary. Please see your veterinarian for recommendations for the best quality and safest product, as well as the appropriate dose for your dog based on the condition you are trying to treat.

You can buy CBD in various formulations. CBD can be applied topically, but is more commonly given orally. The preferred formulation for most pet owners is an “oil,” which allows you to better regulate the amount your dog is receiving. Many products indicate dosage recommendations on their labels. The amount you give will be dependent on the concentration of CBD in the product.
CBD oil for dogs is most often dosed by body weight and given every 8 to 12 hours as needed. It is recommended to start with a low dosage and gradually increase until you receive the desired effects.

Dosage recommendations for CBD oils in dogs vary (as you will read below). A common starting dose recommendation is 0.1 to 0.2 mg/kilogram of body weight (0.05 to 0.1 mg/pound) once to twice daily.

Examples of Starting Doses by Weight:

  • 5 pounds to 10 pounds – approximately 0.25 mg to 1 mg per dog per day.
  • 11 to 20 pounds – approximately 0.5 mg to 2 mg per dog per day.
  • 21 to 40 pounds – approximately 1 mg to 4 mg per dog per day.
  • 41 to 60 pounds – approximately 2 mg to 6 mg per dog per day.
  • 61 to 80 pounds – approximately 3 mg to 8 mg per dog per day.
  • 81 to 100 pounds – approximately 4 mg to 10 mg per dog per day.
  • 101 to 120 pounds – approximately 5 mg to 12 mg per dog per day.
  • Over 121 pounds – use a combination of the above.

It is important to determine the mg of CBD per unit of the product to calculate this dose to give your dog.

ALWAYS SEE YOUR VETERINARIAN to determine the best quality and safest product, as well as the dose most appropriate for your dog and their condition.

A study conducted in 2018 by the Cornell University College of Veterinary Medicine evaluated the safety and pain control properties of CBD oil in dogs with osteoarthritis. Results suggested that CBD, when given twice daily at a dose of 2 or 8 milligrams (mg) per kilogram (kg) of body weight, increased comfort and activity (approximately 1 or 3.5 mg/pound).

Pet owners interested in CBD for their dogs may become frustrated by the lack of firm recommendations from their veterinarian. This is due to lack of research proving effectiveness, a vast range of quality and purity, laws that prohibit the use of CBD oil in some states, and the overall limited experience using it and seeing results. If CBD oil is recommended, it is also most commonly used in conjunction with traditional treatments. It is critical to obtain a high-quality product, start slowly, and get approval from your veterinarian.

Upregulation of CB2 receptors in reactive astrocytes in canine degenerative myelopathy, a disease model of amyotrophic lateral sclerosis

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

ABSTRACT

Targeting of the CB2 receptor results in neuroprotection in the SOD1 G93A mutant mouse model of amyotrophic lateral sclerosis (ALS). The neuroprotective effects of CB2 receptors are facilitated by their upregulation in the spinal cord of the mutant mice. Here, we investigated whether similar CB2 receptor upregulation, as well as parallel changes in other endocannabinoid elements, is evident in the spinal cord of dogs with degenerative myelopathy (DM), caused by 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 expression of the CB1 receptor (confirmed with CB1 receptor immunostaining) or 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, which was confirmed in the grey matter using CB2 receptor immunostaining. Using double-labelling immunofluorescence, CB2 receptor immunolabelling colocalized with GFAP but not Iba-1, indicating upregulation of CB2 receptors on astrocytes in DM-affected dogs. Our results demonstrate a marked upregulation of CB2 receptors in the spinal cord in canine DM, which is concentrated in activated astrocytes. Such receptors could be used as a potential target to enhance the neuroprotective effects exerted by these glial cells.

KEY WORDS: Cannabinoids, Endocannabinoid signaling, CB2 receptors, Canine degenerative myelopathy, Amyotrophic lateral sclerosis, SOD1, Activated astrocytes

INTRODUCTION

Amyotrophic lateral sclerosis (ALS) is a 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 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 drug approved by the US Food and Drug Administration (FDA), but it is 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 Δ 9 -tetrahydrocannabinol (Δ 9 -THC) delayed motor impairment and improved survival in the SOD-1 G93A 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 SOD1 G93A 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 is upregulated in reactive glia in post-mortem spinal cord tissue from ALS patients (Yiangou et al., 2006). Such elevation of CB2 receptors has been also described in SOD1 G93A 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 (F.E.P., 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 investigated in other disorders (Fernández-Ruiz et al., 2007, 2015; Iannotti et al., 2016 for review).

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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. Most studies have been based on overexpression of specific human gene mutations. In this context, we have recently turned 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 breed, DM is characterized by degeneration in the white matter of the spinal cord and the peripheral nerves, which progresses to affect both upper and lower motor neurons (Coates and Wininger, 2010 for review). The disease appears at 8-14 years of age with an equivalent effect in both sexes that necessitates euthanasia (Coates and Wininger, 2010 for review). This canine pathology represents a unique opportunity to investigate ALS in a context much closer to the human pathology, using an animal species that are phylogenetically 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 note that such elements may result 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 ultimately in humans. The study was carried out with post-mortem tissues (spinal cords) from dogs affected by DM kindly provided by Dr Joan R. Coates (University of Missouri, Columbia, MO, USA) and 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 from species 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; mean±s.d.), 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 dogs 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 ).

Table 1.

Clinical, genetic and histopathological characteristics of DM-affected and control dogs whose spinal tissues were used in this study

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 ( Fig. 1 A,B). 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 ( Fig. 1 C,D). We also found microgliosis in both white and grey matter of the spinal cord, detected with DAB immunostaining for the microglial marker Iba-1 ( Fig. 2 A); Iba-1 levels were increased 2.5-fold in the grey matter of DM-affected dogs compared with levels in control dogs ( Fig. 2 B,C).

Nissl staining and glial activity in spinal cord sections of dogs with degenerative myelopathy (DM). Representative photomicrographs and quantification of Nissl staining (A,B) and GFAP immunofluorescence (C,D) in 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±s.e.m. for 6-7 animals per group. Data were analysed using the unpaired Student’s t-test (*P

Iba-1 distribution in spinal cord sections of DM-affected dogs. Representative photomicrographs of Iba-1 immunostaining using DAB (A) and Iba-1 immunofluorescence (B) and its quantification (C) in 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±s.e.m. for 5-7 animals per group. Data were analysed using the unpaired Student’s t-test (**P

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 there was a trend towards an elevation, there were no significant changes in expression of the CB1 receptor (CNR1), or FAAH, monoacylglcerol lipase (MAGL), N-arachidonoyl-phosphatidylethanolamine phospholipase D (NAPE-PLD) and diacylglycerol lipase (DAGL) enzymes between the two groups ( Fig. 3 A). We attempted to determine whether the trends detected for these five parameters may correspond to a greater effect 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 ( Fig. 3 B,C). This occurred despite the reduction in the number of motor neurons detected with Nissl staining.

Status of CB1 receptors in spinal samples of DM-affected dogs. Gene expression for the CB1 receptor (CNR1) and NAPE-PLD, DAGL, FAAH and MAGL measured by qPCR (A), and representative microphotographs for CB1 receptor immunostaining using DAB (C) and its quantification in the grey matter in the ventral horn (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±s.e.m. for 7-8 animals per group. Data were analysed using the unpaired Student’s t-test. Scale bar: 150 µm.

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 (CNR2) expression, measured by qPCR, in DM-affected dogs ( Fig. 4 A). 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 (2-fold increase; Fig. 4 B), as well as using DAB immunostaining, which showed that the number of CB2 receptors increased predominantly in the grey matter ( Fig. 5 A,B).

Gene expression and protein levels of CB2 receptor in DM-affected dogs. Gene expression of the CB2 receptor (CNR2) measured by qPCR (A), as well as western blot analysis for this receptor (B) in spinal cord samples of DM-affected and age-matched control dogs. Values correspond to % over control animals and are expressed as means±s.e.m. for 7 animals per group. Data were analysed using the unpaired Student’s t-test (*P

CB2 receptor immunostaining and quantification in DM-affected dogs. Representative photomicrographs for CB2 receptor immunostaining using DAB (A) and its quantification (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±s.e.m. for 5-6 animals per group. Boxed region in DM dog image is shown enlarged in panel below. Data were analysed using the unpaired Student’s t-test (*P2 receptor-positive cells.

Double-labelling analyses to identify the CB2 receptor-positive cellular substrates

Examination of the morphology of those cells positive for the CB2 receptor in DAB immunostaining ( Fig. 5 A) suggested they were glial cells. We wanted to confirm this by using double-labelling immunofluorescence analysis. We found that CB2 receptor immunolabelling colocalized with GFAP immunofluorescence ( Fig. 6 ), thus indicating that the upregulation of CB2 receptors 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 microglial cells in the spinal cord of DM-affected dogs ( Fig. 7 ).

Double immunofluorescence analysis for CB2 receptor and GFAP in spinal cord sections of DM-affected dogs. Representative photomicrographs showing 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). Scale bar: 50 µm. Arrows indicate cells labelled with the antibodies for the two markers.

Double immunofluorescence analysis for CB2 receptor and Iba-1 in DM-affected dogs. Representative photomicrographs showing 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). Scale bar: 50 µm.

DISCUSSION

Our study addressed changes in specific endocannabinoid elements in canine DM, a disease of older dogs with similarities to ALS (Coates and Wininger, 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 have been investigated in the context of an important neurodegenerative disorder occurring in dogs. The benefits of such an investigation could result in the development of cannabinoid-based therapies for human ALS, but these studies may also serve as a first step in a cannabinoid-based pharmacotherapy useful for 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 important to mention that our study demonstrates no loss of CB1 receptors, which are typically located in neurons, despite the loss of motor neurons occurring in canine DM. This suggests that, contrary to other neurodegenerative conditions in humans, which suffer a profound loss of neuronal CB1 receptors, e.g. Huntington’s disease (Fernández-Ruiz et al., 2015), the CB1 receptor may serve as a potential target in canine DM (shown here) and also in human ALS (de Lago et al., 2015).

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As we report for canine DM here, the CB2 receptor also becomes strongly upregulated in activated glia in response to neuronal damage 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., 2006). The response is not exclusive to ALS but is also observed in other acute or chronic neurodegenerative disorders (e.g. ischemia, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease; reviewed in Fernández-Ruiz et al., 2007, 2015; Iannotti et al., 2016). These findings support the idea that the rise in CB2 receptors in activated glial elements is an endogenous response of endocannabinoid signalling aimed at protecting neurons against cytotoxic insults, as well as restoring neuronal homeostasis and integrity (Pacher and Mechoulam, 2011; de Lago et al., 2015; Fernández-Ruiz et al., 2015).

Results of our study further demonstrated that the elevation of CB2 receptors occurs in activated astrocytes rather than in microglial cells. This finding has been previously described in the spinal cords of transgenic SOD1 mice (F.E.P., unpublished results). In other transgenic models of ALS, e.g. TDP-43 transgenic mice, the upregulatory response of these receptors occurs 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 disease, the overexpression of CB2 receptors occurs in both activated astrocytes and reactive microglia (Benito et al., 2007; Sagredo et al., 2009). The increase in CB2 receptors in activated glial elements may be related to the influence of these cells on neuronal homeostasis, for example, by enhancing metabolic support and glutamate reuptake activity exerted by astrocytes (Fernández-Ruiz et al., 2015 for review), by facilitating the transfer of microglial cells from M1 to 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., 2015 for review). These possibilities place the receptor in a promising position for the development of novel therapies. In light 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 selective CB2 receptor activation in these glial cells during the progression of this canine disease.

In conclusion, our results demonstrated a marked upregulation of CB2 receptors occurring in the spinal cord of dogs affected by DM. Such upregulation occurred in the 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.

MATERIALS AND METHODS

Management of the post-mortem 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 (Averill, 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 were excluded from the study. The spinal cord segments were examined for the presence of SOD1-immunoreactive aggregates within ventral horn motor neurons (Avano 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 faecal 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 faecal incontinence; and (iv) Stage 4 (lower motor neuron tetraplegia and brainstem signs): flaccid tetraplegia, difficulty with swallowing and tongue movements, reduced to absent cutaneous trunci reflex, generalized and severe loss of muscle mass, and urinary and faecal incontinence (see details in Coates and Wininger, 2010). All DM tissues were accompanied by details of symptoms, 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, including double immunofluorescence staining to identify the cellular substrates in endocannabinoid elements (CB2 receptors). 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). The total amount of RNA extracted was quantified 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 total RNA using a commercial kit (RNeasy Mini Quantitect Reverse Transcription, Qiagen). 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) to quantify mRNA levels for CB1 receptor (Cf02716352_u1), CB2 receptor (Cf02696139_s1), DAGL (Cf02705627_m1), FAAH (Cf02648944_m1) and MAGL (Cf02662432_m1). For NAPE-PLD, we used a custom-designed assay (Custom Plus TaqMan RNA Assay Design, Applied Biosystems). In all cases, we used GAPDH expression (Cf04419463_gH) as an endogenous control gene for normalization. The PCR assay was performed using the 7300 Fast Real-Time PCR System (Applied Biosystems) and the threshold cycle (Ct) was calculated by the instrument’s software (7300 Fast System, Applied Biosystems). 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 protein was 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, pH 6.8) and loaded onto a 12% acrylamide gel (Bio-Rad), and then transferred to a PVDF membrane (Immobilon-P, Millipore) using mini Trans-Blot Electrophoretic transfer cell (Bio-Rad). Membranes were blocked with 5% non-fat milk and incubated overnight at 4°C with a mouse anti-CB2 receptor antibody (1:200; Santa Cruz Biotechnology, SC-293188), followed by a second incubation during 2 h at room temperature with an ECL horseradish peroxidase-linked whole secondary antibody (GE Healthcare) at a 1:5000 dilution. Reactive bands were detected by chemiluminescence with the Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare). Images were analysed on a ChemiDoc station with Quantity One software (Bio-Rad). 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 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 20× objective under the same conditions of light, brightness and contrast. Four images coming from at least three sections per animal were analysed. The final value is the mean for all animals included in each experimental group.

Immunohistochemistry

Slices were pre-incubated for 20 min in 0.1 M PBS with 0.1% Triton X-100, pH 7.4, and subjected to endogenous peroxidase blockade by incubation for 1 h at room temperature in peroxidase blocking solution (Dako). Then, they were incubated in 0.1 M PBS with 0.01% Triton X-100, pH 7.4, with one of the following primary antibodies: (i) polyclonal anti-rabbit Iba-1 antibody (1:500; Wako Chemicals, #019-19741); (ii) polyclonal anti-rabbit CB1 receptor (1:400; Thermo Fisher Scientific, PA1-743) and (iii) polyclonal anti-goat CB2 receptor antibody (1:100; Santa Cruz Biotechnology, SC10076). Samples were incubated overnight at 4°C, then sections were washed in 0.1 M PBS and incubated for 2 h at room temperature with the appropriate biotin-conjugated anti-goat or anti-rabbit (1:200; Vector Laboratories) secondary antibodies. The Vectastain Elite ABC kit (Vector Laboratories) and a DAB substrate-chromogen system (Dako) 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 h with Tris-buffered saline with 1% Triton X-100 (pH 7.5). Then, sections were sequentially incubated overnight at 4°C with a polyclonal anti-Iba-1 (1:500; Wako Chemicals, #019-19741) or polyclonal anti-GFAP (1:200; Dako, Z0334), followed by washing in Tris-buffered saline and a further incubation (at 37°C for 2 h) with an Alexa Fluor 488 anti-rabbit antibody conjugate made in donkey (1:200; Invitrogen), rendering green fluorescence for anti-Iba-1 or anti-GFAP. Immunofluorescence was quantified using a SP5 Leica confocal microscope and ImageJ software (US National Institutes of Health, Bethesda, Maryland, USA). For double-labelling studies, sections were then washed again and incubated overnight at 4°C with a polyclonal anti-CB2 receptor (1:100; Santa Cruz Biotechnology, SC-10076). This was followed by washing in Tris-buffered saline and a further incubation (at room temperature for 2 h) with a biotin-conjugated anti-goat (1:200; Vector Laboratories) secondary antibody, followed by a new washing and an incubation (at 37°C for 2 h) 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) 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 sequentially 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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