Cannabis Health Science Studies Index

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ANTI-INFLAMMATORY PROPERTIES  & Cannabis Studies

Overview

Anti-inflammatory or antiinflammatory refers to the property of a substance or treatment that reduces inflammation or swelling.

Science & Research

1981 - Study ~ Biological activity of cannabichromene, its homologs and isomers.

1988 - Study - Analgestic and anti-inflammatory activity of constittuents of cannabis sativa L.

1989 - Study ~ Antiinflammatory and antimicrobial compounds and compositions
United States Patent 4837228
.

1999 - Study ~ 1′,1′-Dimethylheptyl-Δ-8-tetrahydrocannabinol-11-oic Acid: A Novel, Orally Effective Cannabinoid with Analgesic and Anti-inflammatory Properties.

2000 - Study ~ Endocannabinoids and fatty acid amides in cancer, inflammation and related disorders.

2002 - Study ~ Antiinflammatory action of endocannabinoid palmitoylethanolamide and the synthetic cannabinoid nabilone in a model of acute inflammation in the rat

2003 - Study ~ Inhibition of Inflammatory Hyperalgesia by Activation of Peripheral CB2 Cannabinoid Receptors

2003 - StudyCannabidiol-transdermal delivery and anti-inflammatory effect in a murine model.

2004 - Study ~ New perspectives in the studies on endocannabinoid and cannabis: 2- arachidonoylglycerol as a possible novel mediator of inflammation

2004 - Study ~ Cannabinoids and neuroinflammation

2004 - Study ~ Inflammation and cancer IV. Colorectal cancer in inflammatory bowel disease: the role of inflammation.

2004 - Study ~ A novel synthetic, nonpsychoactive cannabinoid acid (HU-320) with antiinflammatory properties in murine collagen-induced arthritis.

2005 - Study ~ The cannabinoid receptor agonist WIN 55212-2 inhibits neurogenic inflammations in airway tissues.

2005 - Study ~ Ajulemic acid (IP-751): Synthesis, proof of principle, toxicity studies, and clinical trials

2005 - Study ~ Stimulation of cannabinoid receptor 2 (CB2) suppresses microglial activation

2005 - Study ~ Cannabinoids provide neuroprotection against 6-hydroxydopamine toxicity in vivo and in vitro: relevance to Parkinson's disease.

2005 - Study ~ Endogenous cannabinoid receptor agonists inhibit neurogenic inflammations in guinea pig airways.

2006 - Study ~ Role of the Cannabinoid System in Pain Control and Therapeutic Implications for the Management of Acute and Chronic Pain Episodes

2006 - Study ~ Involvement of the Cannabinoid CB2 Receptor and Its Endogenous Ligand 2- Arachidonoylglycerol in Oxazolone-Induced Contact Dermatitis in Mice

2006 - Study ~ The endocannabinoid anandamide protects neurons during CNS inflammation by induction of MKP-1 in microglial cells.

2006 - Study ~ Cannabinoid-Induced Immune Suppression and Modulation of Antigen-Presenting Cells

2006 - News ~ Anandamide, an endocannabinoid, protects neurons from inflammation after brain damage

2007 - Study ~ Endocannabinoid metabolism and uptake: novel targets for neuropathic and inflammatory pain

2007 - Study ~ Cannabinoid-2 receptor agonist HU-308 protects against hepatic ischemia/reperfusion injury by attenuating oxidative stress, inflammatory response, and apoptosis

2007 - Study ~ Cannabidiol in vivo blunts β-amyloid induced neuroinflammation by suppressing IL-1β and iNOS expression (Alzheimer's)

2007 - Study ~ Opposing control of cannabinoid receptor stimulation on amyloid-beta-induced reactive gliosis: in vitro and in vivo evidence.

2007 - Study ~ Cannabidiol displays unexpectedly high potency as an antagonist of CB1 and CB2 receptor agonists in vitro

2007 - Study ~ Honokiol, a natural plant product, inhibits inflammatory signals and alleviates inflammatory arthritis.

2007 - Study ~ Anti-inflammatory property of the cannabinoid agonist WIN-55212-2 in a rodent model of chronic brain inflammation

2007 - Study ~ The endocannabinoid system in targeting inflammatory neurodegenerative diseases

2007 - Study ~ Cannabinoid CB2 receptors: a therapeutic target for the treatment of inflammatory and neuropathic pain

2007 - Study ~ Cannabinoids and neuroprotection in motor-related disorders.

2007 - Study ~ A cannabinoid agonist differentially attenuates deep tissue hyperalgesia in animal models of cancer and inflammatory muscle pain.

2007 - Interview ~ Endocannabinoids, cannabinoid receptors and inflammatory stress: an interview with Dr. Pál Pacher

2007 - News ~ Constituents Of Hashish And Marijuana May Help To Fight Inflammation And Allergies

2007 - News ~ Pot Chemical May Curb Inflammation

2007 - News ~ Endocannabinoids appear to play important role in regulating inflammation

2007 - News ~ Hippies vindicated: Human-produced cannabinoids have anti-inflammatory powers

2008 - Study - Inflammation and aging: can endocannabinoids help?

2008 - Study - Cannabidiol in medicine: a review of its therapeutic potential in CNS disorders.

2008 - Study ~ Anti-inflammatory cannabinoids in diet.

2008 - Study ~ Cannabinoid receptors in acute and chronic complications of atherosclerosis.

2008 - Study ~ Cannabinoid receptor stimulation is anti-inflammatory and improves memory in old rats.

2008 - Study ~ Cannabinoid CB2 receptors in human brain inflammation.

2008- Study ~ Cannabinoid Modulation of Cutaneous A{delta} Nociceptors During Inflammation.

2008 - Study ~ Scientists are High on Idea that Cannabis Reduces Memory Impairment.

2008 - News - Marijuana reduces memory impairment.

2008 - News -  Why Cannabis Stems Inflammation.

2009 - Study - Cannabinoids, endocannabinoids, and related analogs in inflammation.

2009 - Study - Cannabidiol decreases bone resorption by inhibiting RANK/RANKL expression and pro-inflammatory cytokines during experimental periodontitis in rats.

2009 - Study ~ Emerging Role of the CB2 Cannabinoid Receptor in Immune Regulation and Therapeutic Prospects.

2009 - Study ~ Cannabinoid CB2 Receptor Potentiates Obesity-Associated Inflammation, Insulin Resistance and Hepatic Steatosis.

2009 - Study ~ Cannabinoids Δ9-Tetrahydrocannabinol and Cannabidiol Differentially Inhibit the Lipopolysaccharide-activated NF-κB and Interferon-β/STAT Proinflammatory Pathways in BV-2 Microglial Cells.

2009 - Study ~ Ajulemic acid, a synthetic cannabinoid, increases formation of the endogenous proresolving and anti-inflammatory eicosanoid, lipoxin A4.

2009 - Study ~ Cannabinoids as Therapeutic Agents for Ablating Neuroinflammatory Disease.

2009 - Study ~ Cannabidiol Attenuates Cisplatin-Induced Nephrotoxicity by Decreasing Oxidative/Nitrosative Stress, Inflammation, and Cell Death.

2009 - Study ~ Cannabinoids as novel anti-inflammatory drugs.

2009 - Study ~ The nonpsychotropic cannabinoid cannabidiol modulates and directly activates alpha-1 and alpha-1-Beta glycine receptor function.

2009 - Study ~ Endocannabinoid signalling as an anti-inflammatory therapeutic target in atherosclerosis: does it work?

2009 - Study ~ Cannabinoids attenuate the effects of aging upon neuroinflammation and neurogenesis.

2009 - News ~ How Hemp Seed Oil Can Help Your Arthritis.

2010 - Study ~ Cannabinoid-induced apoptosis in immune cells as a pathway to immunosuppression.

2010 - Study ~ Cannabinoids and Viral Infections.

2010 - Study ~ US Patent Application 20100222437 - COMPOSITION CONTAINING NON-PSYCHOTROPIC CANNABINOIDS FOR THE TREATMENT OF INFLAMMATORY DISEASES.

2010 - Study ~ The endocannabinoid system as a target for the treatment of neurodegenerative disease.

2010 - Study ~ The effects of Delta-tetrahydrocannabinol and cannabidiol alone and in combination on damage, inflammation and in vitro motility disturbances in rat colitis.

2010 - Study ~ Regulatory Role of Cannabinoid Receptor 1 in Stress-Induced Excitotoxicity and Neuroinflammation.

2010- Study ~ Acute administration of cannabidiol in vivo suppresses ischaemia-induced cardiac arrhythmias and reduces infarct size when given at reperfusion.

2010 - Study ~ WIN55212-2 ameliorates atherosclerosis associated with suppression of pro-inflammatory responses in ApoE-knockout mice.

2010 - Study ~ Beneficial effects of cannabinoids (CB) in a murine model of allergen-induced airway inflammation: Role of CB(1)/CB(2) receptors.

2010 - Study ~ Levels of endocannabinoids and palmitoylethanolamide and their pharmacological manipulation in chronic granulomatous inflammation in rats.

2010 - News ~ Hemp Oil Benefits for Skin.

2011 - Study ~ Cannabidiol reduces lipopolysaccharide-induced vascular changes and inflammation in the mouse brain: an intravital microscopy study.

2011 - Study ~ Cannabidiol Reduces Aβ-Induced Neuroinflammation and Promotes Hippocampal Neurogenesis through PPARγ Involvement.

2011 - Study ~ Evaluation of the Cyclooxygenase Inhibiting Effects of Six Major Cannabinoids Isolated from Cannabis sativa.

2011 - Study ~ Gut feelings about the endocannabinoid system.

2011 - StudyCannabidiol reduces intestinal inflammation through the control of neuroimmune axis.

2011 - Study ~ Local activation of cannabinoid CB1 receptors in the urinary bladder reduces the inflammation-induced sensitization of bladder afferents.

2011 - Study ~ Cannabinoid CB2 Receptors Contribute to Upregulation of β-endorphin in Inflamed Skin Tissues by Electroacupuncture.

2011 - Study ~ The Antinociceptive Effects of JWH-015 in Chronic Inflammatory Pain Are Produced by Nitric Oxide-cGMP-PKG-KATP Pathway Activation Mediated by Opioids.

2011 - Study ~ Cannabinoids and Innate Immunity: Taking a Toll on Neuroinflammation.

2011 - Study ~ Increasing endogenous 2-arachidonoylglycerol levels counteracts colitis and related systemic inflammation.

2011 - Study ~ Cannabidiol as an emergent therapeutic strategy for lessening the impact of inflammation on oxidative stress.

2011 - Study ~ GPR55 regulates cannabinoid 2 receptor-mediated responses in human neutrophils.

2011 - Study ~ Cannabinoid Receptor Type 1 Protects Nigrostriatal Dopaminergic Neurons against MPTP Neurotoxicity by Inhibiting Microglial Activation.

2011 - Study ~ Differential transcriptional profiles mediated by exposure to the cannabinoids cannabidiol and Δ(9) -tetrahydrocannabinol in BV-2 microglial cells.

2011 - Study ~ Cannabidiol protects against hepatic ischemia/reperfusion injury by attenuating inflammatory signaling and response, oxidative/nitrative stress, and cell death.

2011 - Study ~ Deletion of cannabinoid receptors 1 and 2 exacerbates APC function to increase inflammation and cellular immunity during influenza infection.

2011 - Study ~ A synthetic cannabinoid, CP55940, inhibits lipopolysaccharide-induced cytokine mRNA expression in a cannabinoid receptor-independent mechanism in rat cerebellar granule cells.

2011 - Study ~ Effects of cannabinoids and cannabinoid-enriched Cannabis extracts on TRP channels and endocannabinoid metabolic enzymes.

2011 - Study ~ Allergen Challenge Increases Anandamide in Bronchoalveolar Fluid of Patients With Allergic Asthma.

2011 - News ~ New metabolic pathway for controlling brain inflammation.

2012 - Study ~ Prolonged oral Cannabinoid Administration prevents Neuroinflammation, lowers beta-amyloid Levels and improves Cognitive Performance in Tg APP 2576 Mice.

2012 - Study ~ Mechanistic and Pharmacological Characterization of PF-04457845: A Highly Potent and Selective Fatty Acid Amide Hydrolase Inhibitor That Reduces Inflammatory and Noninflammatory Pain.

2012 - Study ~ The synthetic cannabinoid R(+)WIN55,212-2 augments interferon-β expression via peroxisome proliferator-activated receptor-α.

2012 - Study ~ Update on the role of cannabinoid receptors after ischemic stroke.

2012 - Study ~ Palmitoylethanolamide exerts neuroprotective effects in mixed neuroglial cultures and organotypic hippocampal slices via peroxisome proliferator-activated receptor-α.

2012 - Study ~ Cannabidiol, a non-psychotropic plant-derived cannabinoid, decreases inflammation in a ` murine model of acute lung injury: Role for the adenosine A(2A) receptor.

2012 - Study ~ Endocannabinoids limit excessive mast cell maturation and activation in human skin.

2012 - Study ~ The endocannabinoid system: a revolving plate in neuro-immune interaction in health and disease.

2012 - Study ~ Cannabinoid signalling regulates inflammation and energy balance: The importance of the brain-gut axis.

2012 - Study ~ The Role of Cannabinoids In Inflammatory Modulation of Allergic Respiratory Disorders, Inflammatory Pain and Ischemic Stroke.

2012 - Study ~ WIN55212-2 attenuates amyloid-beta-induced neuroinflammation in rats through activation of cannabinoid receptors and PPAR-γ pathway.

2012 - Study ~ Cannabidiol for neurodegenerative disorders: important new clinical applications for this phytocannabinoid?

2012 - Study ~ Cannabinoids suppress inflammatory and neuropathic pain by targeting α3 glycine receptors.

2012 - Study ~ Effects of palmitoylethanolamide on intestinal injury and inflammation caused by ischemia-reperfusion in mice.

2012 - Study ~ Differential migratory properties of monocytes isolated from human subjects naïve and non-naïve to Cannabis.

2012 - Study ~ Cannabinoid receptor-2-selective agonists improve recovery in experimental autoimmune encephalomyelitis.

2012 - Study ~ Anti-Inflammatory Effect of the Endocannabinoid Anandamide in Experimental Periodontitis and Stress in the Rat.

2012 - Study ~ Activation of cannabinoid receptor 2 attenuates leukocyte-endothelial cell interactions and blood-brain barrier dysfunction under inflammatory conditions.

2012 - Study ~ Palmitoylethanolamide is a new possible pharmacological treatment for the inflammation associated with trauma.

2012 - Study ~ Endocannabinoids alleviate proinflammatory conditions by modulating innate immune response in muller glia during inflammation.

2012 - Study ~ Cannabinoids inhibit peptidoglycan-induced phosphorylation of NF-κB and cell growth in U87MG human malignant glioma cells.

2012 - Study ~ Differential Modulation by Delta(9)-Tetrahydrocannabinol (∆ (9)-THC) of CD40 Ligand (CD40L) Expression in Activated Mouse Splenic CD4(+) T cells.

2012 - Study ~ Cannabidiol treatment ameliorates ischemia/reperfusion renal injury in rats

2012 - Study ~ A cannabinoid type 2 receptor agonist attenuates blood-brain barrier damage and
neurodegeneration in a murine model of traumatic brain injury.

2012 - Study ~ Activation of Cannabinoid Receptor 2 reduces inflammation in acute experimental
pancreatitis via intra-acinar activation of p38 and MK2-dependent mechanisms.

2012 - Study ~ N-acyl amines of docosahexaenoic acid and other n-3 polyunsatured fatty acids – From fishy endocannabinoids to potential leads

2012 - Study ~ N-arachidonoyl glycine induces macrophage apoptosis via GPR18

2012 - Study ~ Anti-Inflammatory Effect of the Endocannabinoid Anandamide in Experimental Periodontitis and Stress in the Rat

2012 - Study ~ Palmitoylethanolamide is a new possible pharmacological treatment for the inflammation associated with trauma .

2012 - Study ~ CD200-CD200R1 interaction contributes to neuroprotective effects of anandamide on experimentally induced inflammation

2012 - Study ~ Update on the endocannabinoid-mediated regulation of gelatinase release in arterial wall physiology and atherosclerotic pathophysiology.

2012 - Study ~ Cannabidiol (CBD) enhances lipopolysaccharide (LPS)-induced pulmonary inflammation in C57BL/6 mice.

2012 - Study ~ Cannabidiol reduces host immune response and prevents cognitive impairments in Wistar rats submitted to pneumococcal meningitis

2012 - Study ~ Electroacupuncture reduces the expression of proinflammatory cytokines in inflamed skin tissues through activation of cannabinoid CB2 receptors.

2013 - Study ~ Molecular evidence for the involvement of PPAR-δ and PPAR-γ in anti-inflammatory and neuroprotective activities of palmitoylethanolamide after spinal cord trauma

2013 - Study ~ The cannabinoid receptor type 2 as mediator of mesenchymal stromal cell immunosuppressive properties.

2013 - Study ~ Monoacylglycerol Lipase (MAGL) Inhibition Attenuates Acute Lung Injury in Mice.

2013 - Study ~ The monoacylglycerol lipase inhibitor JZL184 suppresses inflammatory pain in the mouse carrageenan model.

2013 - Study ~ The cannabinoid CB2 receptor-selective phytocannabinoid beta-caryophyllene exerts analgesic effects in mouse models of inflammatory and neuropathic pain

2013 - Study ~ The Dual Effect of Cannabinoid Receptor-1 Deficiency on the Murine Postoperative Ileus

2013 - Study ~ Effects on Immune Cells of a New 1,8-Naphthyridin-2-One Derivative and Its Analogues as Selective CB2 Agonists: Implications in Multiple Sclerosis

2013 - Study ~ Glia and Mast Cells as Targets for Palmitoylethanolamide, an Anti-inflammatory and Neuroprotective Lipid Mediator.

2013 - Study ~ Cannabinoid receptor modulation of the endothelial cell inflammatory response

2013 - Study ~ Cannabinoid CB2 receptors as novel target for inhibiting house dust mite induced allergic airway inflammation

2013 - Study ~ Palmitoylethanolamide is a New Possible Pharmacological Treatment for the Inflammation Associated with Trauma

2013 - Study ~ Amyotrophic Lateral Sclerosis Treatment with Ultramicronized Palmitoylethanolamide

2013 - Study ~ Cannabidiol provides long-lasting protection against the deleterious effects of inflammation in a viral model of multiple sclerosis: A role for A2A receptors.

2013 - Study ~ Controlling 2-arachidonoylglycerol metabolism as an anti-inflammatory strategy.

2013 - Study ~ Anti-inflammatory activity of topical THC in DNFB-mediated mouse allergic contact dermatitis independent of CB1 and CB2 receptors

2013 - Study ~ Prospects for cannabinoid therapies in viral encephalitis.

2013 - Study ~ Selective Activation of Cannabinoid Receptor 2 in Leukocytes Suppresses Their Engagement of the Brain Endothelium and Protects the Blood-Brain Barrier.

2013 - Study ~ The Influence of Cannabinoids on Generic Traits of Neurodegeneration.

2013 - Study ~ Cannabidiol in inflammatory bowel diseases: a brief overview.

2013 - Study ~ Cannabidiol provides long-lasting protection against the deleterious effects of inflammation in a viral model of multiple sclerosis: a role for A2A receptors.

2013 - Study ~ Long-term supplementation of honokiol and magnolol ameliorates body fat accumulation, insulin resistance, and adipose inflammation in high-fat fed mice.

2013 - Study ~ Magnolol inhibits LPS-induced inflammatory response in uterine epithelial cells : magnolol inhibits LPS-induced inflammatory response.

2013 - Study ~ Inhibition of fatty acid amide hydrolase (FAAH) as a novel therapeutic strategy in the treatment of pain and inflammatory diseases in the gastrointestinal tract

2013 - Study ~ Anti-inflammatory effects of Cannabinoid 2 Receptor activation in endotoxin-induced uveitis.

2013 - Study ~ The cannabinoid TRPA1 agonist cannabichromene inhibits nitric oxide production in macrophages and ameliorates murine colitis.

2013 - Study ~ Endocannabinoids: a unique opportunity to develop multitarget analgesics.

2013 - Study ~ Actions of the dual FAAH/MAGL inhibitor JZL195 in a murine inflammatory pain model.

2013 - News ~ Sending multiple sclerosis up in smoke

2013 - News ~ 5 Health Benefits Of Cannabichromene (CBC)

2013 - News ~ New Study: THC May Treat Inflammatory Diseases and Cancer By Altering Genes

2013 - News ~ Marijuana's Memory Paradox

2014 - Study ~ The endocannabinoid system: an emerging key player in inflammation.

2014 - Study ~ Trans-Caryophyllene Suppresses Hypoxia-Induced Neuroinflammatory Responses by Inhibiting NF-κB Activation in Microglia.

2014 - Study ~ Endocannabinoids affect innate immunity of Muller glia during HIV-1 Tat cytotoxicity.

2014 - News ~ Drugs Related to Cannabis Have Pain-Relieving Potential for Osteoarthritis


ANALGESIC AND ANTIINFLAMMATORY ACTIVITY OF CONSTITUENTS OF CANNABIS SATIVA L. (1988)

NFLAMMATION, Vol 12, No. 4, 1988 

E.A. Formukong, A.T. Evans, and F.J. Evans

Department of Pharmacognosy, The School of Pharmacy University of London,

29-39 Brunswick Square London, WC11N 1AX, England 

Abstract---Two extracts of Cannabis sativa herb, one being cannabinoid--free (ethanol) and the other containing the cannabinoids (petroleum), were shown to inhibit PBQ- induced writhing in mouse when given orally and also to antagonize tetradecanoylphorbol acetate (TPA) -induced erythema of mouse skin when applied topically. With the exception of cannabinol (CBN) and delta-1-tetrahydrocannabinol (delta-1-THC), the cannabinoids and olivetol (their biosynthetic precursor) demonstrated activity in the PBQ test exhibiting their maximal effect at doses of about 100 mcg/kg. Delta-1-THC only became maximally effective in doses of 10 mg/kg.

 

This higher dose corresponded to that which induced catalepsy and is indicative of a central action. CBN produce a 40% inhibition of PBQ-induced writhing. Cannabidiol (CBD) was the most effective of the cannabinoids at doses of 100 mcg/kg. Doses of cannabinoids that were effective in the analgesic test orally were used topically to antagonize TPA-induced erythema of skin. The fact that delta-1-THC and CBN were the least effective in this test suggests a structural relationship between analgesic activity and antiinflammatory activity among the cannabinoids related to their peripheral actions and separate from the central effects of delta-1-THC.  

INTRODUCTION  

Various preparations of Cannabis sativa have been employed for their medicinal effects, including antipyretic, antirheumatic, antiallergic, and analgesic purposes. Extracts of Cannabis have been shown to possess analgesic activity, and delta-1-tetrahydrocannabinol (delta-1-THC), the psychoactive component of Cannabis has also been shown to possess this activity in various models. In addition, cannabinol (CBN) but not cannabidiol (CBD) was shown to exhibit analgesic activity in vivo.

It is possible that the antiinflammatory and antiasthmatic properties of this herb are mediated through effects on arachidonate metabolism. However, constituents of Cannabis are known to stimulate and inhibit prostaglandin (PG) release by influencing enzymes of this pathway.

 

A cannabinoid or an extract of Cannabis with little or no central effects could be of use therapeutically. In this paper, we have examined the antiinflammatory potential of two extracts of Cannabis, pure cannabinoids and olivetol (a cannabinoid biosynthetic precursor) in two models of inflammation, in an attempt to separate on a structural basis the peripheral from the central action of these phenolic drugs.  

MATERIALS AND METHODS  

The folowing were used: aspirin (Sigma Chemical Co., Poole, Dorset.), tripotassium citrate (analytical grade), all cannabinoids except CBG (Sigma), and CBG (Makor Chemicals, Jerusalem, Israel).

Preparation of Drugs: PBQ Test. Cannabinoids and cannabis extracts were suspended in a 1% ethanolic solution containing 2.5% w/v Tween. Aspirin was dissolved in a 40 mg/ml solution of tripotassium citrate.

Phenyl Benzoquinone Writhing (PBQ) and Preparation of PBQ Solution. A 0.04% solution of PBQ was prepared immediately before use by dissolving PBQ in warm ethanol and diluting with water at 40 degrees C bringing the ethanolic concentration to 5%. The bottle was stoppered, foil paper wrapped around it, and the solution maintained at 34 degrees C. Deterioration of the solution occurs if left exposed to light and air.

Administration of Drugs. Male CDI male (Charles River) weighing 18-20 g were starved overnight for the experiment. Animals were placed in a thermostatically controlled environment maintained at 34 degrees C. Mice were orally administered test drug 20 min before the intraperitoneal injection of PBQ (4 mg/kg). Five minutes after injection, a hand tally counter was used to record the number of stretching movements for each mouse in a 5-min period. Control animals were only administered the vehicle. Note less than five animals were used per dose.

Statistical Analysis. Results are expressed as mean percentage inhibition of control (+SEM) in the case of PBQ test. IC-50s were obtained from graphs relating probit percentage inhibition (ordinate) against log dose (abscissa). The IC-50 is that dose of drug which would inhibit PBQ-induced writhing by 50%.

Tetradecanoyl phorbol-acetate-induced (TPA) Erythema of Mouse Ear. In order to exclude the possibility of a central mechanism of action (see Discussion), compounds also were tested for their ability to inhibit TPA-induced erythema on mouse ears in 100% of the animals was chosen as the challenging dose for inhibition studies, measured 4 h after application.

Test drugs were dissolved in ethanol and 5 ul applied to the inner ear of the mouse 15 min before the application of 1 mcg TPA in 5 ul acetone. Only one dose of test dug was used for this experiment, 100 mcg/mcl ethanols, except trifluoperazine at 1 mg/5 ul. The other ear acted as a control.

The results were expressed as percentage inhibition, taken to mean the complete suppression of erythema in the test animals, as described in reference 19. 

RESULTS 

PBQ-Induced Writhing. CBD, CBG, olivetol, ethanolic extract, and petroleum spirit extract produced significant inhibition at doses up to 10 mg/kg (Figures 1-3). CBN was only marginally active.

Delta-1-THC was fully effective only at concentrations above 10 mg/kg Figure 2).

The ethanolic and petroleum extract, CBD, olivetol, CBG, and cannflavon were more potent than aspirin. The petroleum spirit extract was about four times more potent than the ethanolic extract, which was virtually equipotent with CBD. Cannflavon, isolated from the ethanolic extract was 14 times less potent than the ethanolic extract of the dried herb.

There was a decline in response following the administration of doses greater than 0.1 mg/kg of some substances. This is most evident in the bell shaped dose-response curve of the petroleum spirit extract. The activity of the ethanolic extract and CBD was also found to decrease slightly at higher dose levels.

TPA-Induced Erythema. In general, the ability of compounds to inhibit TPA-induced erythema correlated well with their potency in the PBQ-writhing test. Thus, CBN and delta-1-THC were the least active followed by CBG, CBD, and cannflavon. Again, the extracts were the most active (Table 3). Twenty-four hours after application, the ethanolic extract still produced 16% inhibition of TPA-induced erythema of the animals. All other substances were without activity after 24 h.

All substances were more active than trifluoperazine, 1 mg/5ul, a known phorbol ester antagonist both in vivo and in vitro (20).  

DISCUSSION

The PBQ-induced writhing response is believed to be produced by the liberation of endogenous substance(s), notably metabolites of the arachidonic cascade. However, the PBQ test is not specific for weak analgesics such as the nonsteroidal antiinflammatory drugs, as it also detects centrally active analgesics.

Therefore, in the elucidation of the action of the cannabinoids as inflammatory drugs, it was necessary to perform more than one test. In this case, peripheral rather than central action was confirmed in the mouse ear erythema assay.

TPA-induced erythema was inhibited by the extracts cannflavon, cannabinoids, and olivetol. The activity of TPA has been shown to be dependent upon PG release in mouse epidermis and mouse peritoneal macrophages possibly via the initial stimulation of protein kinase C (for a review see reference. It has also been shown that compounds that show moderate to very potent antiinflammatory potential in standard in vivo inflammation models will also inhibit TPA-induced edema of the mouse ear, and phorbol-ester-induced erythema.

It is possible that the cannabinoids and their extracts are inhibiting both PBQ-induced writhing and TPA-induced erythema by effects on arachidonate release and metabolism. Cannabinoids and olivetol have been shown to inhibit PG mobilization and synthesis.

The noncannabinoid constituents of Cannabis, for example, cannflavon, have been shown to be mainly cyclooxygenase inhibitors. Cannabinoids, however, stimulate and inhibit phospholipase A2 (PLA2) activity, as well as inducing an inhibition of cyclooxygenase and lipoxygenase. The activity of Cannabis herb or resin is complex, in that activities can be demonstrated on at least three major enzymes of the arachidonate cascade.

The mechanism by which delta-1-THC inhibits PBQ-induced writhing may differ from that of the other substances. At concentrations greater than 10 mg/kg, delta-1-THC may be inhibiting PBQ-induced writhing by acting on central rather than peripheral functions.

It is possible that prostaglandins modulate certain inhibitory pathways in the brain, bringing about an increase in the pain threshold. This dose of delta-1-THC is capable of bringing about the cataleptic effect , which is a standard test for central involvement. Central analgesics have higher efficacies than peripheral ones, and this may explain the effectiveness of delta-1-THC.

The central involvement of delta-1-THC is perhaps the primary reason why delta-1-THC was recognized as an analgesic before other cannabinoids.

Our results suggest that the response of the ethanolic extract cannot be solely due to cannflavon. Other structurally related phenolic substances, known to be present in this complex extract, may account for the higher activity seen either due to cumulative or synergistic effects upon cyclooxygenase.

The activity of the petroleum ether extract is likely to be largely due to the presence of CBD and CBN. GLC analysis of the extract has shown that this extract contained 14.13% CBD, 9.08% CBN, and 6.68% delta-1-THC. On the basis of our results, it is possible to separate the centrally active cannabinoid delta-1-THC from peripherally active compounds of the herbal extracts. An attempt has been made to differentiate them structurally .

It can be seen that the olivetolic nucleus together with a free C-5 hydroxyl group are structural requirements for peripheral effects, involving both cyclooxygenase and lipoxygenase inhibition.

Substances possessing this structure possess antiinflammatory and analgesic activities without central hallucinogenic effects. Delta-1-THC and CBN, which are cyclized derivatives exhibiting no C-5 hydroxyl moiety, have little if any peripheral action.

The traditional use of Cannabis as an analgesic, antiasthmatic, and antirheumatic drug is well established. Our results would suggest that cultivation of Cannabis plants rich in CBD and other phenolic substances would be useful not only as fiber-producing plants but also for medicinal purposes in the treatment of certain inflammatory disorders. 

Acknowledgments----We are grateful to the Medicinal Research Council and the Government of Cameroon for financial support.  

REFERENCES 

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2. O.L. Davies, J. Raventos, and A.L. Walpole, 1946. A method for evaluation of analgesic activity using rats. Br. J. Pharmacol. 1: 255-264. 

3. Gill, E.W., W.D.M. Paton, and R.G. Pertwee, 1970. Preliminary experiments on the chemistry and pharmacology of Cannabis. Nature 228: 134-136. 

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8. White, H.L., and R.L. Tansik. 1980. Effects of delta-9-THC and cannabidiol on phospholipase and other enzymes regulating arachidonate metabolism. Prostaglandins Med. 4: 409-411,  

9. Burstein, S., and S.A. Hunter. 1978. Prostaglandins and Cannabis VI. Release of arachidonic acid from HeLa cells by delta-1-THC and other cannabinoids. Biochem. Pharmacol. 27: 1275-1280.  

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 12. Barrett, M.L., D.Gordon, and F.J. Evans. 1985. Isolation from Cannabis sativa L of cannflavin: A novel inhibitor of prostaglandin production. Biochem. Pharmacol. 34: 2019-2024.  

13. Evans, A.T., E.A. Formukong, and F.J. Evans. 1987. Activation of phospholipase A2 by cannabinoids. Lack of correlation with CNS effects. FEBS Lett. 211: 119-122.

 14. Evans, A.T., E.A. Formukong, and F.J. Evans. 1987. Actions of Cannabis constituents on enzymes of prostaglandin synthesis: Antiinflammatory potential. Biochm. Pharmacol. 36: 2035-2037.  

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Why Cannabis Stems Inflammation

ScienceDaily (July 22, 2008) — Cannabis has long been accredited with anti-inflammatory properties. ETH Zurich researchers, however, have now discovered that it is not only the familiar psychoactive substances that are responsible for this; a compound we take in every day in vegetable nutriment also plays a significant role.

People not only rate cannabis sativa L. highly because of its intoxicating effects; it has also long been used as a medicinal plant. Although the plant has been scrutinized for years, surprising new aspects keep cropping up.

For example, researchers from ETH Zurich and Bonn University examined a component in the plant’s essential oil that until then had largely been ignored and found it to have remarkable phar- macological effects.

The findings open up interesting perspectives, especially for the prevention and treatment of inflammations.

Completely different molecule structure

The hemp plant contains over 450 different substances, only three of which are responsible for its intoxicating effect. They activate the two receptors in the body CB1 and CB2. Whilst the CB1 receptor in the central nervous system influences perception, the CB2 receptor in the tissue plays a crucial role in inhibiting inflammation. If the receptor is activated, the cell releases fewer pro-inflammatory signal substances, or cytokines. The scientists have now discovered that the substance beta-carophyllene, which composes between 12 and 35 percent of the cannabis plant’s essential oil, activates the CB2 receptor selectively.

Unlike the three psychoactive substances, however, beta-carophyllene does not latch onto the CB1 receptor and consequently does not trigger the intoxicating effect. “Due to the various effects of cannabis, we had suspected for quite some time that other substances could come into play besides the psychoactive ones”, explains Jürg Gertsch from the Institute of Pharmaceutical Sciences at ETH Zurich. “However, astonishingly we didn’t know what substances these were until now.”

Gertsch finds it remarkable that beta-carophyllene has a very different molecule structure to that of the classical cannabinoids. “This is presumably why no one realized that the substance can also activate the CB2 receptor.”

The scientists were not only able to prove that beta-carophyllene binds with the CB2 receptor in vitro but also in animal tests, where they treated mice that were suffering from an inflammatory swelling on their paws with orally administered doses of the substance. The swelling declined in up to 70 percent of the animals, even for deep doses. For mice lacking the gene for the CB2 receptor, however, the substance did not make an impact.

Common substance

The results are encouraging for the prevention or treatment of ailments in which the CB2 receptor plays a positive role. However, Gertsch explains that we are still very much in the early stages on that score.

That said, the scientist can conceive that some day the compound will not only help heal certain forms of inflammation, but also be instrumental in treating chronic illnesses, such as liver cirrhosis, Morbus Crohn, osteoarthritis and arteriosclerosis.

In all of these diseases, the CB2 receptor and the associated endocannabinoid system play a crucial role.

The beauty is that beta-carophyllene is not only found in cannabis but also often in plants as a whole and we consume the substance in our diet.

The non-toxic compound, which incidentally has been used as a food additive for many years, can be found in spice plants like oregano, basil, cinnamon and black pepper. “Whether we have found a new link between the vegetable diet and the prevention of so-called lifestyle diseases in our study remains to be seen in future studies”, adds Gertsch.

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 Cannabis has long been recognized as a medicinal plant. Researchers from ETH Zurich and Bonn University have now established anti-inflammatory properties in hemp oil. (Credit: iStockphoto/Tatyana Ogryzko)


 

 

 

Cannabidiol decreases bone resorption by inhibiting RANK/RANKL expression and pro-inflammatory cytokines during experimental periodontitis in rats

Napimoga MH, Benatti BB, Lima FO, Alves PM, Campos AC, Pena-Dos-Santos DR, Severino FP, Cunha FQ, Guimarães FS 


Cannabidiol decreases bone resorption by inhibiting RANK/RANKL expression and pro-inflammatory cytokines during experimental periodontitis in rats. [Journal Article, Research Support, Non-U.S. Gov't]
Int Immunopharmacol 2009 Feb; 9(2):216-22.

 

Cannabidiol (CBD) is a cannabinoid component from Cannabis sativa that does not induce psychotomimetic effects and possess anti-inflammatory properties. In the present study we tested the effects of CBD in a periodontitis experimental model in rats.

 

We also investigated possible mechanisms underlying these effects. Periodontal disease was induced by a ligature placed around the mandible first molars of each animal. Male Wistar rats were divided into 3 groups: control animals; ligature-induced animals treated with vehicle and ligature-induced animals treated with CBD (5 mg/kg, daily). Thirty days after the induction of periodontal disease the animals were sacrificed and mandibles and gingival tissues removed for further analysis.

 

Morphometrical analysis of alveolar bone loss demonstrated that CBD-treated animals presented a decreased alveolar bone loss and a lower expression of the activator of nuclear factor-kappaB ligand RANKL/RANK. Moreover, gingival tissues from the CBD-treated group showed decreased neutrophil migration (MPO assay) associated with lower interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha production. These results indicate that CBD may be useful to control bone resorption during progression of experimental periodontitis in rats.

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Cannabidiol in vivo blunts β-amyloid induced neuroinflammation by suppressing IL-1β and iNOS expression (Alzheimer's)

 
Background and purpose:
Pharmacological inhibition of beta-amyloid (Aβ) induced reactive gliosis may represent a novel rationale to develop drugs able to blunt neuronal damage and slow the course of Alzheimer's disease (AD). Cannabidiol (CBD), the main non-psychotropic natural cannabinoid, exerts in vitro a combination of neuroprotective effects in different models of Aβ neurotoxicity. The present study, performed in a mouse model of AD-related neuroinflammation, was aimed at confirming in vivo the previously reported antiinflammatory properties of CBD.
 
 
Experimental approach:
Mice were inoculated with human Aβ (1–42) peptide into the right dorsal hippocampus, and treated daily with vehicle or CBD (2.5 or 10 mg kg−1, i.p.) for 7 days. mRNA for glial fibrillary acidic protein (GFAP) was assessed by in situ hybridization. Protein expression of GFAP, inducible nitric oxide synthase (iNOS) and IL-1β was determined by immunofluorescence analysis. In addition, ELISA assay of IL-1β level and the measurement of NO were performed in dissected and homogenized ipsilateral hippocampi, derived from vehicle and Aβ inoculated mice, in the absence or presence of CBD.
 
 
Key results:
In contrast to vehicle, CBD dose-dependently and significantly inhibited GFAP mRNA and protein expression in Aβ injected animals. Moreover, under the same experimental conditions, CBD impaired iNOS and IL-1β protein expression, and the related NO and IL-1β release.
Conclusion and implications:
 
The results of the present study confirm in vivo anti-inflammatory actions of CBD, emphasizing the importance of this compound as a novel promising pharmacological tool capable of attenuating Aβ evoked neuroinflammatory responses.
 

Introduction
 
Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder (Koo et al., 1999) whose specific hallmarks are neurofibrillary tangles (Terry, 1963) and senile plaques (Braak and Braak, 1997). While neurofibrillary tangles result from the deposition of hyperphosphorylated tau proteins (Lee et al., 1991), senile plaques represent more complex extracellular lesions composed of a core of β-amyloid (Aβ) aggregates, surrounded by activated astrocytes and dystrophic neuritis (Itagaki et al., 1989; Cotman et al., 1996). At present, although biochemical events leading to Aβ neurotoxicity still remain unclear, proposed mechanisms include production of oxygen free radicals (Behl et al., 1994), changes in cytosolic calcium homeostasis (Ueda et al., 1997; Mattson, 2002) and activation of Wnt pathway as well as of the transcription nuclear factor NF-κB (Green and Peers, 2002; Caricasole et al., 2003). In addition to cytotoxic mechanisms directly affecting neurons, Aβ-induced glial cell activation, triggering inflammatory responses with subsequent release of neurotoxic cytokines, is present in the AD brain, contributing to the pathogenesis of disease (Craft et al., 2006). The possibility of interfering with this detrimental cycle by pharmacologically inhibiting reactive gliosis has been proposed as a novel rationale to develop drugs able to blunt neuronal damage and consequently slow the course of disease.
 
Cannabidiol (CBD), the main non-psychotropic component of the glandular hairs of Cannabis sativa, exhibits a plethora of actions including anti-convulsive, sedative, hypnotic, anti-psychotic, anti-nausea, anti-inflammatory and anti-hyperalgesic properties (Mechoulam et al., 2002; Costa et al., 2007). CBD has been proved to exert in vitro a combination of neuroprotective effects in Aβ-induced neurotoxicity, including anti-oxidant and anti-apoptotic effects (Iuvone et al., 2004), tau protein hyperphosphorylation inhibition through the Wnt pathway (Esposito et al., 2006a), and marked decrease of inducible nitric oxide synthase (iNOS) protein expression and nitrite production in Aβ-challenged differentiated rat neuronal cells (Esposito et al., 2006b).
 
 
In spite of the large amount of data describing the significant neuroprotective and anti-inflammatory properties of CBD in vitro, to date no evidence has been provided showing similar effects in vivo. To achieve this, the present study investigated the potential anti-inflammatory effect of CBD in a mouse model of AD-related neuroinflammation induced by the intrahippocampal injection of the human Aβ (1–42) fragment.

 

 

Methods
Animal care
Experiments were conducted in 3–5-months old C57BL/6J mice (35–40 g) (Harlan, Udine, Italy). Animals were housed under controlled illumination (12 h light/12 h dark cycle; light on 0600h) and standard environmental conditions (ambient temperature 20–22°C, humidity 55–60%) for at least 1 week before starting experiments. Food and water were available ad libitum. All surgery and experimental procedures were performed during the light cycle and were made according to the National Institutes of Health guidelines for the care and use of laboratory animals and to those of the Italian Ministry of Health (DL 116/92), and were approved by the local Institutional Animal Care and Use Committees. All efforts were made to reduce both animal number and suffering during the experiments.
 
 
Surgical preparation
 
 
Mice were anaesthetized with halothane (1–3%), placed in a stereotaxic frame, and injected with 10 ng of Aβ (1–42) (Tocris Cookson, Bristol, UK) or vehicle artificial cerebrospinal fluid (aCSF) into the right dorsal hippocampus, using the following coordinates relative to the bregma: AP=+2.0 mm; ML=−1.8 mm; DV=−2.3 mm. The flow was maintained at a constant value of 0.5 μl min−1, using a microdialysis pump and the needle was left in place for additional 5 min to allow for diffusion. Animals were kept on a warming pad until they had fully recovered from the anaesthetic and were kept in individual cages to prevent damage to the scalp sutures until they were killed for tissue processing.
 
 
Starting on the third day after surgery, mice were intraperitoneally (i.p.) treated daily with vehicle (Tocrisolve 100, Tocris Cookson) or CBD (Tocris Cookson) (2.5 or 10 mg kg−1) for 7 days. The doses of the drug were selected according to previous literature (Mechoulam et al., 2002), whereas the i.p. administration route was derived from the author's experience in such animal model of AD. Animals for in situ hybridization analysis were killed by cervical dislocation and brain were removed, snap-frozen on dry-ice, and stored at −80°C. Brains were mounted on Tissue Tek (Polysciences, PA, USA), and 14-μm-thick coronal sections were cut on a cryostat Microtome HM560 (Microm, Walldorf, Germany).
 
Sections were mounted onto frozen SuperFrost/Plus slides (Fisher Scientific, Schwerte, Germany), dried on a 42°C warming plate, and stored at −20°C until used. Animals for immunofluorescence analysis were killed and perfused with HEPES buffer containing protease inhibitors; brains were rapidly frozen in liquid N2. Tissue was cut on a freezing sliding microtome (Leica SM 2000 R, Milan, Italy) to obtain 30 μm sections collected in a 15 mM NaN3 phosphate-buffered saline (PBS) solution and stored at 4°C. For enzyme-linked immunosorbent assay (ELISA) experiments brains were bisected down the sagittal sulcus and the hippocampus was dissected out of the right side and quickly frozen in liquid N2.
 
 
In situ hybridization
 
Sections were fixed in ice-cold 4% paraformaldehyde for 20 min, rinsed in PBS, quenched for 15 min in 1% H2O2 methanol solution, rinsed in PBS, quenched for 8 min in 0.2 M HCl, rinsed in PBS, treated with proteinase K 20 μg ml−1 (Roche Molecular Diagnostics, Milan, Italy) in 50 mM Tris-HCl, 5 mM ethylene diamine tetra acetic acid (EDTA) (pH 8.0) for 10 min, rinsed in PBS, fixed in ice-cold 4% paraformaldehyde, incubated for 10 min in 0.1 M triethanolamine (pH 8.0) to which 1.2 ml acetic anhydride was added dropwise, rinsed in PBS, washed with 0.9% NaCl for 5 min, dehydrated in graded series of ethanol and air-dried. Hybridization was carried out in 100 μl of hybridization buffer containing specific sense or antisense 35S-labelled riboprobe for glial fibrillary acidic protein (GFAP; 70 000–100 000 c.p.m. μl−1). Hybridization buffer consisted of 50% deionized formamide, 20 mM Tris-HCl (pH 8.0), 0.3 M NaCl, 5 mM EDTA (pH 8.0), 10% dextran sulphate (Sigma, Milan, Italy), 0.02% Ficoll 400 (Sigma), 0.02% polyvinylpyrrolidone (PVP 40; Sigma), 0.02% bovine serum albumin (BSA; Sigma), 0.5 mg ml−1 tRNA (Roche Molecular Diagnostics), 0.2 mg ml−1 fragmented herring sperm DNA and 200 mM dithiothreitol. Before applying to the tissue the hybridization cocktail was denatured for 2 min at 95°C. Slides were incubated overnight at 54°C in a humidified chamber. Four high-stringency washes were carried out at 62°C with 5 × saline sodium citrate (SSC)/0.05% Tween-20 (Sigma), then with 50% formamide/2 × SSC/0.05% Tween-20, with 50% formamide/1 × SSC/0.05% Tween-20 and finally with 0.1 × SSC/0.05% Tween-20. Slides were dehydrated in graded ethanol series, air-dried and exposed to Biomax MR film (Scientific Imaging Systems, NY, USA). GFAP mRNA expression was semi-quantified by densitometric scanning of the Biomax film with a GS 700 imaging densitometer (Bio-Rad Laboratories, CA, USA) and a computer programme (Molecular Analyst, IBM, Milan, Italy).
 
 
Immunofluorescence
 
Brain coronal sections (30 μm) were fixed for 30 min in 4% paraformaldehyde, washed with PBS, and blocked for 15 min with 10% BSA. Sections were then incubated for 2 h with one of the following primary antibodies: monoclonal anti-GFAP (1:200, Lab Vision, CA, USA), monoclonal anti-IL-1β (1:100, Sigma) and monoclonal anti-iNOS (1:100, Sigma). Following PBS washing, sections were incubated in the dark for half an hour with Texas Red-conjugated or fluorescein isothiocyanate (FITC)-conjugated secondary antibody (1:200; AbCam, Cambridge, UK). After final PBS washing, sections were analysed with a Zeiss LSM 410 microscope equipped with a krypton/argon laser, dichroic beam splitters and barrier emission filters needed for triple labelling. Texas Red was excited at a wavelength of 568 nm and collected through a long pass filter (590LP). FITC was excited with a wavelength of 488 nm and collected with a narrow band filter (515–540BP). Texas Red and FITC were assigned to the red and green channels respectively of the generated RGB image.
 
 
Nitrite assay
 
NO was measured as nitrite (NO2) accumulated in the inoculated ipsilateral hippocampi. A spectrophotometer assay based on the Griess reaction was used (Di Rosa et al., 1990). Briefly, Griess reagent (1% sulphanilamide, 0.1% naphthylethylenediamine in H3PO4) was added to an equal volume of homogenized tissue supernatant and the absorbance at 550 nm was measured after 10 min. The NO2 concentration was thus determined using a standard curve of NaNO2 and referred to μg of homogenized hippocampal protein content according to BioRad assay method.
 
 
IL-1β assay
 
ELISA was used to quantify the presence of IL-1β in the supernatant of homogenized hippocampi ipsilateral to the inoculation site. A mouse IL-1β ELISA kit (R&D System, MN, USA) was used according to the manufacturer's recommendations. Briefly, 50 μl of standard, control buffer, or sample was combined with 50 μl of assay buffer in IL-1β antibody-coated wells on the ELISA plate and incubated at room temperature for 2 h. Wells were washed five times before the addition of 100 μl of the appropriate horseradish peroxidase conjugate and incubated for 2 h more. After a second wash cycle, 100 μl of hydrogen peroxide/tetramethylbenzidine substrate solution was added per well, and the plate was incubated for 30 min at room temperature in the dark. The reaction was stopped by addition of the hydrochloric acid solution provided in the kit. The absorbance at 450 nm was measured with a microreader (Bio-Rad Laboratories, 3550-UV) with wavelength correction at 570 nm.
 
 
Statistical analysis
 
Results were expressed as mean±s.e.m. of experiments. Statistical analysis was performed using analysis of variance, and multiple comparisons were performed by Bonferroni's test, with P<0.05 considered significant.

 

Results
 
 
CBD effects on GFAP mRNA expression in Aβ inoculated mice
The induction of mRNA for GFAP protein 10 days following intrahippocampal injection of Aβ (1–42) (10 μg ml−1) was examined. As shown in Figure 1, GFAP mRNA, as measured by densitometry, was significantly increased by Aβ treatment in comparison with mice hippocampi injected with vehicle (+883±12%). CBD (2.5 or 10 mg kg−1) dose-dependently and significantly inhibited (−31.3±4.1 and −81±6.7% respectively) GFAP mRNA expression versus Aβ-injected animals i.p. treated with vehicle. Negligible or no increase in GFAP mRNA was observed following treatment with Aβ (42–1) reverse peptide or CBD alone (data not shown).
 
 
Figure 1
Figure 1
Effects of cannabidiol (CBD) (intraperitoneal (i.p.) treatment for 7 consecutive days) on glial fibrillary acidic protein (GFAP) mRNA in mouse hippocampus. Upper panel: Dark-field photomicrographs showing the distribution of GFAP mRNA as detected by (more ...)
 
 
CBD effects on GFAP, iNOS and IL-1β protein expression in Aβ inoculated mice
Immunofluorescence analysis was aimed at estimating the effect of CBD treatment on the expression of inflammatory proteins 10 days following Aβ (1–42) (10 μg ml−1) injection into mouse hippocampi. As shown in Figures 2, ,33 and and4,4, the number of GFAP, iNOS and IL-1β-positive cells was significantly increased by Aβ (+407±34, +1025±68, and +1288±16% respectively) versus vehicle-inoculated hippocampi. CBD (2.5 or 10 mg kg−1) treatment dose-dependently and significantly inhibited the number of cells positive for GFAP (−30±3.12 and −64.14±6.2% respectively), iNOS (−33.3±5.2 and −61.5±4.25% respectively) or IL-1β (−30.5±5.7 and −68±4.23% respectively), in comparison with animals given Aβ and injected with CBD vehicle. Also in this case, negligible or no increase in GFAP, iNOS and IL-1β was observed following treatment with Aβ (42–1) reverse peptide or CBD alone (data not shown).
 
 
Figure 2
Figure 2
Effects of cannabidiol (CBD) (intraperitoneal (i.p.) treatment for 7 consecutive days) on glial fibrillary acidic protein (GFAP) in mouse hippocampus. Upper panel:Representative photomicrographs showing GFAP immunoreactive cells in: (a) vehicle inoculated (more ...)
 
 
Figure 3
 
 
Effects of cannabidiol (CBD) (intraperitoneal (i.p.) treatment for 7 consecutive days) on inducible nitric oxide synthase (iNOS) in mouse hippocampus. Upper panel: Representative photomicrographs showing iNOS immunoreactive cells in: (a) vehicle inoculated (more ...)
 
 
Figure 4
 
 
Effects of cannabidiol (CBD) (intraperitoneal (i.p.) treatment for 7 consecutive days) on IL-1β in mouse hippocampus. Upper panel: Representative photomicrographs showing IL-1β immunoreactive cells in: (a) vehicle inoculated mice (control), (more ...)
CBD effects on NO release in hippocampal homogenates
The release of NO was evaluated by measurement of its stable metabolite (NO2) in homogenized ipsilateral hippocampi 10 days after Aβ (1–42) (10 μg ml−1) injection. As shown in Figure 5 NO2 levels were significantly increased by Aβ injection in comparison with vehicle-inoculated hippocampi (+525±30%). CBD (2.5 or 10 mg kg−1) treatment dose dependently and significantly inhibited NO2 release in tissue homogenates (−30±1 and −51±3.71% respectively) compared with those from mice injected with vehicle.
 
 
Figure 5
 
 
Effects of cannabidiol (CBD) (2.5 or 10 mg kg−1 intraperitoneal (i.p.) for 7 consecutive days) on nitrite (NO2) level in mouse hippocampal homogenates 10 days after Aβ (1–42) (10 μg ml (more ...)
CBD effects on IL-1β levels in hippocampal homogenates
ELISA assay was performed on homogenized ipsilateral hippocampi 10 days following Aβ (1–42) (10 μg ml−1) injection to evaluate the effect of CBD treatment on IL-1β release. As shown in Figure 6, IL-1β level was significantly increased by Aβ injection in comparison with vehicle-inoculated hippocampi (+900±60%). Treatment with CBD (2.5 or 10 mg kg−1) dose-dependently and significantly inhibited IL-1β release in tissue homogenates (−30±3 and −46.7±4% respectively) when compared with homogenates derived from vehicle-treated animals.
 
 
Figure 6
 
 
Effects of cannabidiol (CBD) (2.5 or 10 mg kg−1 intraperitoneal (i.p.) for 7 consecutive days) on IL-1β level in hippocampal homogenates 10 days after Aβ (1–42) (10 μg ml−1 (more ...)

Discussion and conclusions
The urgent need for novel strategies for AD is apparent with the realization that the currently approved therapies are only palliative without significant and substantial disease modifying effects (Turner, 2006). In contrast, the present study suggests that CBD, here investigated with a primary focus on glial pathways, exhibits a potential to delay effectively the onset and progression of Aβ neurotoxicity. Actually, the current results provide evidence that CBD causes a clear-cut reduction of the transcription and expression of glial pro-inflammatory molecules in the hippocampus of an in vivo model of Aβ-induced neuroinflammation. They suggest CBD may be regarded as a promising tool able to affect the course of Aβ-related neuropathology, by reducing Aβ-generated reactive gliosis and subsequent neuroinflammatory responses, in addition to the previously demonstrated protective effects directly affecting neurons (Iuvone et al., 2004; Esposito et al., 2006a, 2006b).
 
 
Indeed, the increasing body of immunohistological and molecular findings, showing that inflammatory processes are pre-eminent and constant aspects of the neuropathology generated by the Aβ toxicity, supports the notion that the previously under-appreciated glial activation plays a critical role in the pathogenesis of brain lesions subsequent to Aβ deposition (Craft et al., 2006). Although acute activation of glial cells may have important beneficial effects in the recovery of the CNS from a variety of insults, it is believed that a persistent activation amplifies inflammatory responses leading to a worsening of the consequences of injury (Ralay Ranaivo et al., 2006). In the scenario of reactive gliosis, the main features are astrocytic hypertrophy and proliferation, along with a marked overexpression of the intermediate filament proteins, such as GFAP, the best known hallmark of activated astrocytes (O'Callaghan and Sriram, 2005).
 
 
The present investigation focuses the ability of this phytocannabinoid, CBD, to negatively modulate GFAP transcription and expression as well as to significantly reduce IL-1β and iNOS upregulation, which importantly contribute to disease progression, through the propagation of inflammation and oxidative stress. Among the many active substances produced by Aβ stimulated microglia, IL-1β has proved to be substantially implicated in the cytokine cycle of cellular and molecular events responsible for the neurodegenerative consequences (Griffin et al., 1998). These include synthesis and processing of amyloid precursor protein (Buxbaum et al., 1992; Mrak and Griffin, 2000), as well as astrocyte activation with a subsequent iNOS overexpression and excessive production of NO (Das and Potter, 1995; Sheng et al., 1996). Increasing amounts of NO, a short-lived and diffusible free radical involved in all reported neuroinflammatory and neurodegenerative conditions (Murphy, 2000), accelerate neuronal protein nitration and cause a marked increase in tau protein hyperphosphorylation (Saez et al., 2004), encouraging the detrimental progression of Aβ-related pathology (Nathan et al., 2005).
 
 
Therefore, in this context where inflammatory pathways are believed to play relevant roles as driving forces of the Aβ-induced injury, they are identified as potential modulators of the neuronal damage and are reported as neuronal targets for effective therapeutic interventions. The present investigation provides the first evidence that substantial components of the neuroinflammatory response, set in motion by Aβ deposition and allowing for progression of neuropathology, are suppressed in vivo by CBD. The current data confirm and further reinforces the view that CBD can exhibit protective effects in models of neuroinflammation/neurodegeneration.
 
 
The seminal work describing CBD neuroprotective properties demonstrated its ability to protect cortical neurons in culture against glutamate-induced neurotoxicity. Such effects were found to be not antagonized by the established CB1 antagonist SR141716A, suggesting that they were independent of CB1 cannabinoid receptor involvement. Later CBD was shown to prevent Aβ-induced toxicity in PC12 pheocromocytoma cells, increasing survival while decreasing reactive oxygen species production, lipid peroxidation, caspas-3 levels, DNA fragmentation and intracellular calcium (Iuvone et al., 2004). In addition to this combination of anti-oxidant, anti-inflammatory and anti-apoptotic effects, subsequent studies, carried out under the same experimental conditions, demonstrated that CBD was able to operate as a Wnt/β-catenin pathway rescuer, inhibiting Aβ-induced tau protein hyperphosphorylation while attenuating iNOS protein expression and NO production (Esposito et al., 2006b). Such a wide range of effects on pathophysiological processes implicated in neuroinflammatory/neurodegenerative diseases appears truly intriguing and encourages the clinical applicability of CBD for therapeutic use.
 
 
Its antioxidant and neuroprotective actions are presumably related in part to a potential as a scavenger of free radicals due to its structural characteristics (Hampson et al., 2000), although there is room for alternative mechanisms. Ruling out the possibility that transient receptor potential vanilloid type 1 channels may be involved in the suppression of reactive gliosis exerted by CBD (personal data), a potential involvement of the CB2 receptor might be taken into account. The recently provided in vitro evidence that CBD can display CB2 receptor inverse agonist properties (Thomas et al., 2007) might offer an explanation of the anti-neuroinflammatory effects we have shown here. In Aβ neurotoxicity, several results have related CB2 receptors to events involved in the progression of brain damage by affecting reactive gliosis at neuroinflammatory lesion sites (Walter and Stella, 2004). Further, we have recently reported that, in a rodent model of Aβ-induced reactive gliosis, CB2 receptors were overexpressed (van der Stelt et al., 2006), paralleling the changes in cannabinoid receptor expression occurring in AD brain, where, in astrocyte-associated plaques, CB2 receptors were also found to be up-regulated (Ramirez et al., 2005). Interestingly, some of our recent unpublished results suggest that pharmacological interactions at glial CB1 and CB2 receptors result in a marked and opposite regulation of reactive astroglial response, with CB2 receptor blockade suppressing astroglial activation. These findings would imply a function for CB2 receptors in the regulation of CBD actions and would encourage further study of how pharmacological interactions at this receptor could influence the effects of CBD. Although more research will be needed to elucidate fully the molecular mechanisms implicated in the CBD actions described in this paper, the current data showed that the early administration of CBD markedly attenuated in vivo the reactive gliosis induced by Aβ injury.
 
The relevance of these results stems from the fact that a proper control of glial cell function, which is compromised by the persistence of inflammatory events, is critical to provide an environment capable of ensuring neuronal survival and function. For this reason, on the basis of the present results, CBD, a drug well tolerated in humans, may be regarded as an attractive medical alternative for the treatment of AD, because of its lack of psychoactive and cognitive effects.
Acknowledgments
This work was supported by FIRB2006.
Abbreviations
 
Aβbeta amyloid
 
ADAlzheimer's disease
 
CBDcannabidiol
 
i.p.intraperitoneally
 
GFAPglial fibrillary acidic protein
 
iNOSinducible nitric oxide synthase
 
IL-1βinterleukin 1 beta
 
ELISAenzyme linked immunosorbent assay

Notes
Conflict of interest
The authors state no conflict of interest.

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Cannabidiol displays unexpectedly high potency as an antagonist of CB1 and CB2 receptor agonists in vitro

 
Background and purpose:
 
A nonpsychoactive constituent of the cannabis plant, cannabidiol has been demonstrated to have low affinity for both cannabinoid CB1 and CB2 receptors. We have shown previously that cannabidiol can enhance electrically evoked contractions of the mouse vas deferens, suggestive of inverse agonism. We have also shown that cannabidiol can antagonize cannabinoid receptor agonists in this tissue with a greater potency than we would expect from its poor affinity for cannabinoid receptors. This study aimed to investigate whether these properties of cannabidiol extend to CB1 receptors expressed in mouse brain and to human CB2 receptors that have been transfected into CHO cells.
 
Experimental approach:
 
The [35S]GTPγS binding assay was used to determine both the efficacy of cannabidiol and the ability of cannabidiol to antagonize cannabinoid receptor agonists (CP55940 and R-(+)-WIN55212) at the mouse CB1 and the human CB2 receptor.
 
Key results:
 
This paper reports firstly that cannabidiol displays inverse agonism at the human CB2 receptor. Secondly, we demonstrate that cannabidiol is a high potency antagonist of cannabinoid receptor agonists in mouse brain and in membranes from CHO cells transfected with human CB2 receptors.
Conclusions and implications:
 
This study has provided the first evidence that cannabidiol can display CB2 receptor inverse agonism, an action that appears to be responsible for its antagonism of CP55940 at the human CB2 receptor. The ability of cannabidiol to behave as a CB2 receptor inverse agonist may contribute to its documented anti-inflammatory properties.
 
Keywords: cannabidiol, CP55940, O-2654, inverse agonism, neutral antagonism, GTPγS assay, CB1 receptor, CB2 receptor, mouse brain... more...

 

 

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Antiinflammatory action of endocannabinoid palmitoylethanolamide and the synthetic cannabinoid nabilone in a model of acute inflammation in the rat

  • The antiinflammatory activity of synthetic cannabinoid nabilone in the rat model of carrageenan-induced acute hindpaw inflammation was compared with that of the endocannabinoid palmitoylethanolamide and the nonsteroidal antiinflammatory drug indomethacin.
  • Preliminary experiments in rats used a tetrad of behavioural tests, specific for tetrahydrocannabinol-type activity in the CNS. These showed that the oral dose of nabilone 2.5 mg kg−1 had no cannabinoid psychoactivity.
  • Intraplantar injection of carrageenan (1% w v−1) elicited a time-dependent increase in paw volume and thermal hyperalgesia.
  • Nabilone (0.75, 1.5, 2.5 mg kg−1, p.o.), given 1 h before carrageenan, reduced the development of oedema and the associated hyperalgesia in a dose-related manner. Nabilone 2.5 mg kg−1, palmitoylethanolamide 10 mg kg−1 and indomethacin 5 mg kg−1, given p.o. 1 h before carrageenan, also reduced the inflammatory parameters in a time-dependent manner.
  • The selective CB2 cannabinoid receptor antagonist {N-[(1S)-endo-1,3,3-trimethyl bicyclo [2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)pyrazole-3 carboxamide} (SR 144528), 3 mg kg−1 p.o. 1 h before nabilone and palmitoylethanolamide, prevented the anti-oedema and antihyperalgesic effects of the two cannabinoid agonists 3 h after carrageenan.
  • Our findings show the antiinflammatory effect of nabilone and confirm that of palmitoylethanolamide indicating that these actions are mediated by an uncharacterized CB2-like cannabinoid receptor....read more...



Inflammation and aging: can endocannabinoids help?

Aging often leads to cognitive decline due to neurodegenerative process in the brain. As people live longer, a growing concern exist linked to long-term, slowly debilitating diseases that have not yet found a cure, such as Alzheimer’s disease. Recently, the role of neuroinflammation has attracted attention due to its slow onset, chronic nature and its possible role in the development of many different neurodegenerative diseases. In the future, treatment of chronic neuroinflammation may help counteract aspects of neurodegenerative disease. Our recent studies have focused upon the endocannabinoid system for its unique effects on the expression of neuroinflammation. The basis for the manipulation of the endocannabinoid system in the brain in combination with existing treatments for Alzheimer’s disease will be discussed in this review....more...

 

 

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Anti-inflammatory property of the cannabinoid agonist WIN-55212-2 in a rodent model of chronic

Cannabinoid receptors (CBr) stimulation induces numerous central and peripheral effects. A

growing interest in the beneficial properties of manipulating the endocannabinoid system has lead to the possible involvement of CBr in the control of brain inflammation. In the present study we examined the effect of the CBr agonist, (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo[1,2,3-de]-1,4benzoxazin-6-yl]-1-naphthalenyl-methanone mesylate (WIN-55212-2), on microglial activation and spatial memory performance, using a well characterized animal model of chronic brain inflammation produced by the infusion of lipopolysaccharide (LPS, 250 ng/hr for 3 weeks) into the 4th ventricle of young rats. WIN-55212-2 (0.5 or 1.0 mg/kg/day, i.p.) was administered for three weeks. During the third week of treatment, spatial memory ability was examined using the Morris water-maze task. We found that 0.5 and 1 mg/kg WIN-55212-2 reduced the number of LPS-activated microglia, while 1 mg/kg WIN-55212-2 potentiated the LPS-induced impairment of performance in the watermaze task. CB1 receptors were not expressed by microglia and astrocytes, suggesting an indirect effect of WIN on microglia activation and memory impairment. Our results emphasize the potential use of CBr agonists in the regulation of inflammatory processes within the brain; this knowledge may lead to the use of CBr agonists in the treatment of neurodegenerative diseases associated with chronic neuroinflammation, such as Alzheimer disease.

 

Keywords: cannabinoid receptors, inflammation, activated microglia, Alzheimer’s disease, LPS, spatial memory......Microglial cells play a pivotal role as immune effectors in the central nervous system and may participate in the initiation and progression of neurological disorders, such as Alzheimer’s disease (AD), Parkinson’s disease and multiple sclerosis by releasing harmful molecules such as pro-inflammatory cytokines, reactive oxygen species or complement proteins (Akiyama et al., 2000; Kim and de Vellis, 2005). Many of the pathological, immunological, biochemical and behavioral changes seen in these and other neurodegenerative diseases can be reproduced in young rats by chronic infusion of lipopolysaccharide (LPS) into the 4th ventricle (Hauss-Wegrzyniak et al., 1998, Hauss-Wegrzyniak et al., 1999). Chronic infusion of LPS results in the activation of microglia within hippocampus and entorhinal cortex, brain regions involved in learning and memory formation (Hauss-Wegrzyniak et al., 1998; Rosi et al., 2004). Chronic brain inflammation is associated with impaired spatial memory, impaired induction of long-term potentiation, a loss of N-methyl-d-aspartate (NMDA) receptors, astrocytosis, elevated cytokines and related pro-inflammatory proteins and transcription factors (Hauss-Wegrzyniak et al., 1998, Hauss-Wegrzyniak et al., 1999; Rosi et al., 2004).
 
 
The endocannabinoid system may regulate many aspects of the brain’s inflammatory response, including the release of pro-inflammatory cytokines and modulation of microglial activation (Neumann, 2001; Klein, 2005). The endocannabinoid system is comprised of two G-protein-coupled receptors designated as CB1 and CB2, although not all endocannabinoid effects can be explained only by these two receptors (Begg et al., 2005). CB1 receptors are expressed in the brain and are responsible for most of the behavioral effects of the cannabinoids. CB2 receptors are expressed by immune and hematopoietic cells peripherally (Begg et al., 2005), and seem to be expressed on neurons in the brainstem and the brain (Van Sickle et al., 2005; Gong et al., 2006; Onaivi et al. 2006) although their presence in the brain is controversial (Munro et al., 1993). Two endogenous ligands for these receptors, arachydonylethanolamine and 2-arachidonoylglycerol (Stella, 2004), influence immune responses by inhibiting cytokine release and other anti-inflammatory actions (Klein et al., 2003; Klein, 2005). Microglia also express CBr and release cytokines in response to exposure to LPS or beta-amyloid protein; this effect can be inhibited by prior cannabinoid treatment (Facchinetti et al., 2003; Ramirez et al., 2005; Sheng et al., 2005). Astrocytes may also synthesize and release endocannabinoids (Walter et al., 2002). CB1 receptors have been widely studied because of their role in the psychoactive effects of the cannabis sativa plant (Δ9-etrahydrocannabinol or Δ9-THC). Δ9-THC can impair performance in rats, mice or monkeys under multiple experimental conditions (Castellano et al., 2003). Therefore, in the current study, we investigated the effect of a CBr agonist on microglial activation and spatial memory in a rodent model of chronic brain inflammation induced by LPS infusion into the 4th ventricle. We used the number of immunoreactive microglia (activated) as a biomarker of brain inflammation (Hauss-Wegrzyniak et al., 1998, Hauss-Wegrzyniak et al., 1999; Rosi et al., 2004, Rosi et al., 2005) to evaluate the potential anti-inflammatory properties of the WIN 55-212-2 compound.

EXPERIMENTAL PROCEDURES
 
 
Subjects and surgical procedures
Fifty-four young (3 months old) male F-344 rats (Harlan Sprague-Dawley, Indianapolis, IN) were singly housed in Plexiglas cages with free access to food and water. The rats were maintained on a 12/12-h light-dark cycle in a temperature-controlled room (22°C) with lights off at 0800. All rats were given health checks, handled upon arrival and allowed at least one week to adapt to their new environment prior to surgery. Artificial cerebrospinal fluid (aCSF n=26) or LPS (Sigma, St-Louis, MO, E. coli, serotype 055:B5, TCA extraction, 1.0 mg/ml dissolved in aCSF, n=28) were chronically infused for 21 days through a cannula implanted into the 4th ventricle that was attached (via Tygon tubing, 0.06 O.D., and an osmotic pump connect, Model 3280P, Plastics One, Roanoke, VA) to an osmotic minipump (Alzet, Cupertino, CA, model #2004, to deliver 0.25 μl/h, Hauss-Wegrzyniak et al., 1998). The aCSF vehicle contained (in mM) 140 NaCl; 3.0 KCl; 2.5 CaCl2; 1.0 MgCl2; 1.2 Na2HPO4, adjusted to pH 7.4. Rats infused with either aCSF or LPS were also administered daily the synthetic cannabinoid CB1/CB2 receptor agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo[1,2,3-de]-1,4benzoxazin-6-yl]-1-naphthalenyl-methanone mesylate (WIN-55212-2, Sigma, St-Louis, MO, 0.5 or 1.0 mg/kg, i.p.) or vehicle (Dimethylsulfoxide (100%), DMSO, Sigma, St-Louis, MO). Rats were assigned to one of the following six groups: aCSF + vehicle, LPS+ vehicle, aCSF + WIN (0.5 mg/kg), LPS + WIN (0.5mg/kg), aCSF + WIN (1 mg/kg), LPS + WIN (1 mg/kg). Drug or vehicle administration began the first day after surgery and continued throughout the behavioral testing, which began on day 14 of the LPS or aCSF infusion.
 
 
Behavioral Testing
Spatial learning ability was assessed using a 185 cm diameter water maze with white walls. The water was maintained at 26-28°C and made opaque by adding white, non-toxic, paint. The pool was in the center of a 2.3 x 2.73 x 2.5m room with multiple visual stimuli on the wall as distal cues, and a chair and a metal board against the wall of the pool as proximal cues. The circular escape platform was 11.5 cm in diameter. For the spatial (hidden-platform) version of the water task, a circular escape platform was present in a constant location, submerged about 2.5 cm below the water surface. The rats were tracked by an overhead video camera connected to a VP114 tracking unit (HVS Image, England). Custom software was used to store and analyze each rat’s latency to find the submerged platform during each trial.
 
 
Each rat performed three training blocks per day (two training trials per block) for 4 days (24 trials total), with a 60-min inter-block interval. On each trial, the rat was released into the water from one of seven locations spaced evenly at the side of the pool, which varied randomly from trial to trial. After the rat found the escape platform or swam for a maximum of 60 sec, it was allowed to remain on the platform for 30 sec. To control for possible drug-induced deficits in visual acuity and swimming ability, the same rats were also tested on a second version of this task. In this version, a visible platform raised 2 cm above the surface of the water was moved randomly to one of four locations in the tank after each trial. A total of 4 visible-platform trials were performed. Drug administration was performed 20 minutes prior to the behavioral testing. The results, i.e. latency (sec) to find the hidden platform, were analyzed by ANOVA followed by post hoc comparisons according to the method of Bonferonni/Dunn.
 
 
Histological procedures
After behavioral testing was completed, each rat was deeply anesthetized with isoflurane and prepared for a transcardiac perfusion of the brain with cold saline containing 1 U/ml heparin, followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Brains were then removed and the placement of the cannula in the 4th ventricle was confirmed. The brains were then post-fixed one hour in the same fixative and then stored (4°C) in phosphate buffer saline (PBS), pH 7.4. Free-obtained using a vibratome from perfused tissues for staining with standard avidin/biotin peroxydase or fluorescence labeling methods. The monoclonal antibody OX-6 (final dilution 1:400, Pharmigen, San Diego, CA) was used to visualize activated microglia cells. This antibody is directed against Class II major histocompatibility complex (MHC II) antigen. After quenching endogenous peroxydase/activity and blocking nonspecific binding, the sections were incubated (4°C) overnight with primary antibodies directed against the specific epitope (MCH II). Thereafter, the sections were incubated for 2h (22°C) with the secondary monoclonal antibody, rat adsorbed biotinylated horse anti-mouse immunoglobulin G (final dilution 1:200, Vector, Burlingame, CA). Sections were than incubated for 1h (22°C) with avidin-biotinylated horseradish peroxydase (Vectastain, Elite ABC kit, Vector, Burlingame, CA). After washing again in PBS, the sections were incubated with 0.05% 3, 3’-diaminobenzidine tetrahydrochloride (Vector, Burlingame, CA) as chromogen. The reaction was stopped by washing the section with buffer. No staining was detected in the absence of the primary or secondary antibodies. Sections were mounted on slides, air-dried and coverslipped with cytoseal (Allan Scientific, Kalamazoo, MI) mounting medium. The location of immunohistochemically-defined cells was examined by light microscopy. Quantification of cell density in the reconstructed hippocampal coronal sections was assessed with MetaMorph imaging software (Universal Image Corporation, West Chester, PA). Briefly, areas of interest were determined as previously reported in detail (Rosi et al., 2005), their surface measured, and the immunoreactive cells numerated, allowing use to determine a number of immunoreactive cells by millimeter square in the areas of interest.
 
 
A polyclonal antibody directed against the first ninety-nine amino-acid residue from the human CB1 (final dilution 1:500, Affinity Bioreagents, Golden, CO) was used to visualize CB1 receptors. After quenching endogenous peroxydase/activity and blocking nonspecific binding, the sections were rinsed in 0.1 M TRIS buffer (TB), pH 7.4, for 15min, in 0.1 M Tris buffered saline (TBS), pH 7.4, for 15 min, blocked using 2% avidin in TBS for 30 min, rinsed in TBS for 30 min, blocked using 2% biotin for 30min, and finally rinsed in TBS for 30 min. The tissue sections were then incubated in the anti-CB1 (diluted 1:500) overnight at 4°C. The antibody was diluted in a solution containing 0.1% Triton X-100 and 1% NGS in 0.1 M TBS. These sections were then rinsed in TBS for 60 min and incubated in biotinylated goat anti-rabbit IgG (diluted 1:200) for 90 min at room temperature. The sections were rinsed with TBS for 60 min and incubated for 1h (22°C) with avidin-biotinylated horseradish peroxydase (Vectastain, Elite ABC kit, Vector, Burlingame, CA). The sections were then rinsed with TBS for 30 min and then incubated with 0.05% 3,3’-diaminobenzidine tetrahydrochloride as chromogen. The reaction was stopped by washing the section with buffer. No staining was detected in the absence of the primary or secondary antibodies. Sections were mounted on slides, air-dried for 24 h.Counter staining (Cresyl Violet) was performed before slides were cover-slipped with cytoseal (Allan Scientific, Kalamazoo, MI) mounting medium.
 
 
Double immunofluorescence staining
Free floating sections were mounted on slides and air-dried under a hound. The tissues were than processed as described in details in Rosi et al., 2005. After washing in TBS solution the polyclonal rabbit anti-CB1 (Affinity Bioreagents, Golden, CO, dilution 1:500) was apply. After 48 h of incubation at 4°C, the sections were incubated for 2 h at room temperature with the secondary anti-rabbit biotinylated antibody (Vector, Burlingame, CA), followed by incubation with Avidin+Biotin amplification system (Vector, Burlingame, CA) for 45 minutes. The staining was visualized using the TSA fluorescence system CY3 (PerkinElmer Life Sciences, Emeryville, CA). After washing in TBS solution, the tissue was quenched and blocked again and incubated with the monoclonal antibody OX-6 (Pharmigen, San Diego, CA, final dilution 1:400) for 24 h or with the monoclonal antibody anti-GFAP (Chemicon, Temecula, CA, final dilution 1:500) for 24h. Before applying the biotinylated monoclonal secondary rat adsorbed antibody (Vector, Burlingame, CA) for 2 h, the tissue was incubated with Avidin Biotin Blocking Kit (Vector, Burlingame, CA) for 30 min to block cross reaction with the primary staining. Following treatment with an Avidin+Biotin amplification system (Vector, Burlingame, CA), the staining was visualized with TSA fluorescence system CY5 (PerkinElmer Life Sciences, Emeryville, CA) and the nuclei were counterstained with Sytox-Green (Molecular Probes, Eugene, OR). No staining was detected in the absence of the primary or secondary antibodies.

RESULTS
Chronic infusion of LPS and WIN-55212-2 injections were well tolerated by all rats: they gained weight normally for the duration of the study.
Behavior
No significant difference in locomotor activity (swim speed) was found across groups, p>0.1. An ANOVA performed on the latency results obtained in the water maze task revealed an overall main effect of testing day (F5, 218=16.057, p<0.0001) for all groups (See Figure 1) and an overall group effect (LPS versus aCSF) upon latency for each day of testing (F1, 328=11.367, p=0.0008 for day1; F1, 328=85.681, p<0.0001 for day2; F1,328=109.756, p<0.0001 for day3; F1, 328=96.25, p<0.0001 for day 4). There was no significant effect of treatment except for day 3 (F2, 328=5.788, p=0.0034). There was a significant interaction between group and drug on days 2 and 3 (F2, 328=3.982, p=0.0196 for day 2; F2, 328=12.641, p<0.0001 for day 3). Post-hoc analyses of each testing day revealed a significant treatment effect on day 1, where LPS+WIN 1 rats were significantly impaired compared to the aCSF+WIN 1 rats (p=0.0015). On days 2 and 3, all LPS-infused rats were significantly (p<0.0033) impaired, as compared to their respective control groups. On day 4, all LPS-infused rats were significantly (p<0.0033) impaired as compared to their respective aCSF controls. There was a significant interaction between treatment and LPS-infused animals (F2, 113=5.026, p=0.081). Post-hoc analyses of each testing day revealed a significant treatment effect on days 2 and 3; performance of LPS+WIN 1 rats was significantly (p<0.0033) worse as compared to the LPS+vehicle rats on those both days. Overall, the drug treatment (0.5 or 1 mg/kg) did not significantly impair performance of aCSF-infused rats and did not attenuate the impairment induced by the LPS infusion. The 1 mg/kg treatment, that did not cause any impairment in aCSF-infused rats, but did worsen the impairment observed in LPS-infused rats, demonstrating an interaction between inflammation and the highest dose of the drug used.
 
 
Figure 1
Figure 1
Water maze performance. On days 2 and 3, all LPS-infused animals (closed triangles, squares and circles) were significantly impaired (*p<0.0033, Bonferroni/Dunn post hoc test) compared to their control groups. On days 2 and 3, LPS+WIN 1 rats were (more ...)
Histology
Immunostaining for OX-6 (Figure 2A) revealed numerous highly activated microglia cells distributed throughout the hippocampus of LPS-infused rats (Figure 2A d). The activated microglia had a characteristic bushy morphology with increased cell body size and contracted and ramified processes (Figure 2A d). Rats infused with aCSF had very few mildly activated microglia evenly scattered throughout the brain, consistent with results from previous studies (Hauss-Wegrzyniak et al., 1998; Rosi et al., 2005). No difference in staining was evident between the aCSF group and the aCSF rats injected with either dose of WIN (Figure 2A a-c). In LPS-infused rats that were also treated with WIN (Figure 2A e-f) the OX-6 antibody stained fewer activated microglial cells.
 
 
Figure 2
Figure 2
(A.) Activated microglia in the dentate gyrus. Note the diminution of activated microglia cells in the dentate gyrus of animals treated with either doses of WIN 55212-2 (e,f) compare to the LPS+vehicle group (d) (B.) Density of activated microglial cells (more ...)
Activated microglia cell counts
The number of activated microglia per millimeter square was determined in 4 different brain regions: dentate gyrus (DG), CA3 and CA1 regions of the hippocampus and the entorhinal cortex (EC) (Figure 2B). These brain regions were examined for their known implication in spatial learning (Nadel and Land, 2000). An ANOVA of the data revealed an overall main region effect (F5,210=16.057, p<0.0001) for all groups and an overall main effect of the infusion of LPS in each region examined (F5,48=32.557, p<0.0001 for DG; F5,48=23.552, p<0.0001 for CA3; F5,48=3.828, p=0.0053 for CA1;F5,48=19.308, p<0.0001 for EC).
 
 
In the DG, CA3 and EC, all LPS infused rats had a significantly (p<0.0033) higher density of activated microglia compared to their respective control groups, consistent with our previous results (Rosi et al., 2005). In the DG and CA3 the number of activated microglia was significantly (p<0.0033) reduced in LPS-infused rats given either dose of WIN, as compared to rats in the LPS+vehicle group (DG, 41.4% reduction for 0.5mg/kg and 40.6% reduction for 1mg/kg; CA3, 43.7% reduction for WIN 0.5mg/kg and 49.4% reduction for WIN 1mg/kg). In the EC, despite a 24.2% reduction for WIN 0.5mg/kg and 32.4% reduction for WIN 1mg/kg, no significant difference was found between the LPS-infused groups. In the CA1 region of the hippocampus, there was a significant (p<0.0033) difference only between the aCSF and LPS+vehicle groups. Overall, the effects of WIN were not dose-dependent in the DG and CA3 and not significant within the CA1 or EC brain regions.
 
 
CB1 receptors
The distribution of the CB1 receptors observed following our staining is in accordance with previous studies (Tsou et al., 1998) (Figure 3a). Neuronal CB1 immunoreactivity was found in the hippocampus, striatum, amygdala as well as in the somatosensory, cingulate and entorhinal cortices. The apparent density of immunoreactive cells in the areas of interest (DG, CA3, CA1, or EC) did not vary across groups.
Figure 3
Figure 3
Immunoreactivity (IR) of CBr in the CA3 region of the hippocampus. (a) CB1-IR (DAB stain) is seen in the cytoplasm of all CA3 neuronal cell bodies (counterstaining with cresyl violet). (b) CB1-IR (red) and MCH-II-IR (green) do no co-localize, as indicated (more ...)
No co-localization between CB1 receptors and activated microglial cells
Double-immunofluorescence staining for CB1 and activated microglial cells performed on the brains of all LPS-infused groups did not show any co-localization (Figure 3b). These results indicate that CB1 receptors are not present on activated microglial cells in response to LPS infusion or treatment with a CBr agonist.
No co-localization between CB1 and astrocytes
Double-immunofluorescence staining for CB1 and astrocytes performed on the brains of all LPS-infused groups did not show any co-localization (Figure 3c). These results indicate that CB1 are not present on astrocytes in response to LPS infusion or treatment with a CBr agonist.

DISCUSSION
The results demonstrate that a CB1/CB2 agonist, WIN-55212-2, prevents microglial cell activation during LPS-induced chronic neuroinflammation in young rats. The effects of this agonist were not dependent on direct CB1 receptors stimulation of microglia or astrocytes, were region dependant and did not reverse the LPS-induced impairment in a spatial memory task. The neurodegeneration associated with AD may result from prolonged activation of microglia and a chronic elevation of cytokines and nitric oxide (Akiyama et al., 2000; Streit, 2004) leading to a cascade of self-propagating cellular events that alters the expression of cannabinoid receptors (Minghetti and Levi, 1998; Bernardino et al., 2005; Klein, 2005). Endocannabinoids are implicated in the modulation of the central inflammatory response by neurons and glia (Klein et al., 2003; Klein, 2005) and may have a neuroprotective role in several neuroinflammation related diseases (Klein, 2005). Cannabinoid agonists can prevent the activation of microglia by β-amyloid, reduce the subsequent release of TNF-α(Ramirez et al., 2005), and attenuate the induction and release of nitric oxide by cultured microglia (Waksman et al., 1999). An inhibition of glutamate release by cannabinoids and the subsequent reduction of the calcium influx via NMDA channels (Piomelli, 2003; Takahashi and Castillo, 2006) have also been demonstrated. Additionally, cannabinoids can attenuate oxidative stress and subsequent toxicity (Hampson and Grimaldi, 2001) and induce the expression of brain-derived neurotrophic factor following the infusion of kainic acid (Marsicano et al., 2003).
CBr are expressed in senile plaques (Benito et al., 2003; Ramirez et al., 2005) and the number of CB1 receptor-immunoreactive neurons is greatly reduced in areas of microglial activation in the AD brain (Ramirez et al., 2005).
 
 
In contrast, we did not find evidence for changes in CB1 receptor expression on hippocampal neurons in response to brain inflammation or cannabinoid receptor stimulation. This absence of changes in CB1 receptors on neurons in our model may be related to the fact that the chronic infusion of LPS into young rats does not induce senile plaque formation (Hauss-Wegrzyniak et al., 1998) or neuronal loss in the hippocampus (Rosi et al., 2005), important factors that likely influence the expression of CBr-positive neuron in post-mortem studies on AD brains from aged humans. The LPS infusion into the 4th ventricle produces widespread activation of glia and impaired performance in the water maze task (Hauss-Wegrzyniak et al., 1998). In contrast, an infusion of amyloid into a lateral ventricle (Ramirez et al., 2005) produced a more localized and limited glial activation (frontal cortex) and performance impairment that responded positively to treatment with WIN 55-212-2 (Ramirez et al., 2005). This difference may explain why WIN 55-212-2 in our model did not reverse the impairment induced by LPS in the 4th ventricle.
 
 
In the current study, CB1 receptors were not co-localized with MCH II-positive microglia or GFAP-positive astrocytes within the hippocampus; the presence of these receptors appeared to be solely neuronal (Figure 3). This suggests an indirect role of CBr upon microglia that is linked to the modulation of neuronal activity by stimulation of the endocannabinoid system. Our results are consistent with previous in vivo findings (Tsou et al., 1998) that describe CBr receptors located on hippocampal neurons, and their modulator role upon glutamatergic and GABAergic function (Takahashi and Castillo, 2006; Katona et al., 1999). CBr are present on astrocytes and microglial cells taken from humans, monkeys, rats and mice when studied in vitro (Stella 2004). The contradictory findings we report may be related to many factors, such as the different species that have been studied, the microenvironment of the cells (in vivo vs in vitro), antibody sensitivity and selectivity, and the age or pathological condition under examination. Further experiments must be performed to determine clearly the characteristic and changes of the endocannabinoid system in our model and thus explain our present findings.
 
 
We have previously speculated that LPS induces a cascade of inflammatory processes that leads to an elevation in extracellular glutamate and activation of NMDA receptors (Wenk et al., 2006). The selective antagonism of NMDA receptors reduced microglia activation (Rosi et al., 2006) similar to that reported in the current study using WIN-55212-2. Taken together these findings are consistent with the hypothesis that the ability of endocannabinoids to reduce the release of glutamate within the hippocampus underlies the reduction of microglia activation by WIN-55212-2.
 
 
Interestingly, our results demonstrated an interaction between the presence of brain inflammation and the actions of the cannabinoid agonist at a dose (1 mg/kg/day i.p. of WIN 55-212-2) that did not impair performance in the control rats. Surprisingly, those doses of agonist (0.5 and 1 mg/kg/day i.p. of WIN 55-212-2) also did not improve performance, as might be expected given the results of our previous investigations using more typical anti-inflammatory drugs (Hauss-Wegrzyniak et al., 1999). We speculate that this was because the WIN 55-212-2 treatment was not able to reverse totally the activation of microglia that had been induced by the chronic LPS infusion; in the current study, the reduction in the number of activated microglia was only about 40-50%, in contrast to the ability of an non-steroidal anti-inflammatory drug’s ability to reduce the number of activated microglia almost completely. This partial reduction in microglia activation might thus not be sufficient to reverse the spatial memory impairment produced by the LPS infusion. This result is of particular importance with regard to patients suffering with a disease associated with brain inflammation, e.g. AD, Parkinson’s disease or multiple sclerosis, who are also using marijuana.
 
 
The current report is the first to our knowledge to demonstrate the modulatory role of cannabinoids in an animal model of chronic neuroinflammation, pointing out the effectiveness of a CBr agonist on the consequences of LPS mediated neuroinflammation at a dose (0.5 mg/kg/day i.p. of WIN-55212-2) that does not impair performance in a patial memory task. These results further advocate for the manipulation of the endocannabinoid system to diminish the consequences of neuroinflammation in progression of AD and others inflammation-related diseases (Klein, 2005).
 
 
Acknowledgments
We thank A. Sy, P. Raniero, F. Cerbai for technical assistance and Dr V. Ramirez-Amaya for critical experimental procedure assistance.
List of abbreviations
 
Δ9-THCΔ9-tetrahydrocannabinol
 
aCSFartificial cerebral spinal fluid
 
ADAlzheimer’s disease
 
CB1cannabinoid receptor 1
 
CB2cannabinoid receptor 2
 
CBrcannabinoid receptors
 
DGDentate gyrus
 
ECEntorhinal cortex
 
LPSLipopolysaccharide
 
NMDAN-methyl-d-aspartate
 
PBSphosphate buffer saline
 
TBSTris buffer saline
 
WIN-55212-2(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo[1,2,3-de]-1,4benzoxazin-6-yl]-1-naphthalenyl-methanone mesylate

 
Financial support:This study has been supported by AG10546 (GLW).
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Section editor: (j) Systems Neuroscience

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Cannabidiol in medicine: a review of its therapeutic potential in CNS disorders

Scuderi C, Filippis DD, Iuvone T, Blasio A, Steardo A, Esposito G 
Cannabidiol in medicine: a review of its therapeutic potential in CNS disorders. [JOURNAL ARTICLE]
Phytother Res 2008 Oct 9.


Cannabidiol (CBD) is the main non-psychotropic component of the glandular hairs of Cannabis sativa. It displays a plethora of actions including anticonvulsive, sedative, hypnotic, antipsychotic, antiinflammatory and neuroprotective properties. However, it is well established that CBD produces its biological effects without exerting significant intrinsic activity upon cannabinoid receptors. For this reason, CBD lacks the unwanted psychotropic effects characteristic of marijuana derivatives, so representing one of the bioactive constituents of Cannabis sativa with the highest potential for therapeutic use.The present review reports the pharmacological profile of CBD and summarizes results from preclinical and clinical studies utilizing CBD, alone or in combination with other phytocannabinoids, for the treatment of a number of CNS disorders. Copyright (c) 2008 John Wiley & Sons, Ltd.

 

 

 

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Cannabinoids, endocannabinoids, and related analogs in inflammation

Burstein SH, Zurier RB.

Department of Biochemistry & Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation St., Worcester, Massachusetts 01605, USA. [email protected]

Abstract

This review covers reports published in the last 5 years on the anti-inflammatory activities of all classes of cannabinoids, including phytocannabinoids such as tetrahydrocannabinol and cannabidiol, synthetic analogs such as ajulemic acid and nabilone, the endogenous cannabinoids anandamide and related compounds, namely, the elmiric acids, and finally, noncannabinoid components of Cannabis that show anti-inflammatory action. It is intended to be an update on the topic of the involvement of cannabinoids in the process of inflammation.

 

A possible mechanism for these actions is suggested involving increased production of eicosanoids that promote the resolution of inflammation. This differentiates these cannabinoids from cyclooxygenase-2 inhibitors that suppress the synthesis of eicosanoids that promote the induction of the inflammatory process.

 

 

 

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Marijuana reduces memory impairment

The more research they do, the more evidence Ohio State University scientists find that specific elements of marijuana can be good for the aging brain by reducing inflammation there and possibly even stimulating the formation of new brain cells.

 

 The research suggests that the development of a legal drug that contains certain properties similar to those in marijuana might help prevent or delay the onset of Alzheimer's disease. Though the exact cause of Alzheimer's remains unknown, chronic inflammation in the brain is believed to contribute to memory impairment.

Any new drug's properties would resemble those of tetrahydrocannabinol, or THC, the main psychoactive substance in the cannabis plant, but would not share its high-producing effects. THC joins nicotine, alcohol and caffeine as agents that, in moderation, have shown some protection against inflammation in the brain that might translate to better memory late in life.

"It's not that everything immoral is good for the brain. It's just that there are some substances that millions of people for thousands of years have used in billions of doses, and we're noticing there's a little signal above all the noise," said Gary Wenk, professor of psychology at Ohio State and principal investigator on the research.

Wenk's work has already shown that a THC-like synthetic drug can improve memory in animals. Now his team is trying to find out exactly how it works in the brain.

The most recent research on rats indicates that at least three receptors in the brain are activated by the synthetic drug, which is similar to marijuana. These receptors are proteins within the brain's endocannabinoid system, which is involved in memory as well as physiological processes associated with appetite, mood and pain response.

This research is also showing that receptors in this system can influence brain inflammation and the production of new neurons, or brain cells.

"When we're young, we reproduce neurons and our memory works fine. When we age, the process slows down, so we have a decrease in new cell formation in normal aging. You need those cells to come back and help form new memories, and we found that this THC-like agent can influence creation of those cells," said Yannick Marchalant, a study coauthor and research assistant professor of psychology at Ohio State.

Marchalant described the research in a poster presentation Wednesday (11/19) at the Society for Neuroscience meeting in Washington, D.C.

Knowing exactly how any of these compounds work in the brain can make it easier for drug designers to target specific systems with agents that will offer the most effective anti-aging benefits, said Wenk, who is also a professor of neuroscience and molecular virology, immunology and medical genetics.

"Could people smoke marijuana to prevent Alzheimer's disease if the disease is in their family? We're not saying that, but it might actually work. What we are saying is it appears that a safe, legal substance that mimics those important properties of marijuana can work on receptors in the brain to prevent memory impairments in aging. So that's really hopeful," Wenk said.

One thing is clear from the studies: Once memory impairment is evident, the treatment is not effective. Reducing inflammation and preserving or generating neurons must occur before the memory loss is obvious, Wenk said.

Marchalant led a study on old rats using the synthetic drug, called WIN-55212-2 (WIN), which is not used in humans because of its high potency to induce psychoactive effects.

The researchers used a pump under the skin to give the rats a constant dose of WIN for three weeks – a dose low enough to induce no psychoactive effects on the animals. A control group of rats received no intervention. In follow-up memory tests, in which rats were placed in a small swimming pool to determine how well they use visual cues to find a platform hidden under the surface of the water, the treated rats did better than the control rats in learning and remembering how to find the hidden platform.

"Old rats are not very good at that task. They can learn, but it takes them more time to find the platform. When we gave them the drug, it made them a little better at that task," Marchalant said.

In some rats, Marchalant combined the WIN with compounds that are known to block specific receptors, which then offers hints at which receptors WIN is activating. The results indicated the WIN lowered the rats' brain inflammation in the hippocampus by acting on what is called the TRPV1 receptor. The hippocampus is responsible for short-term memory.

With the same intervention technique, the researchers also determined that WIN acts on receptors known as CB1 and CB2, leading to the generation of new brain cells – a process known as neurogenesis. Those results led the scientists to speculate that the combination of lowered inflammation and neurogenesis is the reason the rats' memory improved after treatment with WIN.

The researchers are continuing to study the endocannabinoid system's role in regulating inflammation and neuron development. They are trying to zero in on the receptors that must be activated to produce the most benefits from any newly developed drug.

What they already know is THC alone isn't the answer.

"The end goal is not to recommend the use of THC in humans to reduce Alzheimer's," Marchalant said. "We need to find exactly which receptors are most crucial, and ideally lead to the development of drugs that specifically activate those receptors. We hope a compound can be found that can target both inflammation and neurogenesis, which would be the most efficient way to produce the best effects."

The National Institutes of Health supported this work.

 November 20, 2008 sourced from Ohio State University - http://osu.edu/)

 

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