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. 2015 Oct 21;23:48.
doi: 10.1186/s40199-015-0131-8.

A New Formulation of Cannabidiol in Cream Shows Therapeutic Effects in a Mouse Model of Experimental Autoimmune Encephalomyelitis

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Free PMC article

A New Formulation of Cannabidiol in Cream Shows Therapeutic Effects in a Mouse Model of Experimental Autoimmune Encephalomyelitis

Sabrina Giacoppo et al. Daru. .
Free PMC article

Abstract

Background: The present study was designed to investigate the efficacy of a new formulation of alone, purified cannabidiol (CBD) (>98 %), the main non-psychotropic cannabinoid of Cannabis sativa, as a topical treatment in an experimental model of autoimmune encephalomyelitis (EAE), the most commonly used model for multiple sclerosis (MS). Particularly, we evaluated whether administration of a topical 1 % CBD-cream, given at the time of symptomatic disease onset, could affect the EAE progression and if this treatment could also recover paralysis of hind limbs, qualifying topical-CBD for the symptomatic treatment of MS.

Methods: In order to have a preparation of 1 % of CBD-cream, pure CBD have been solubilized in propylene glycoland basic dense cream O/A. EAE was induced by immunization with myelin oligodendroglial glycoprotein peptide (MOG35-55) in C57BL/6 mice. After EAE onset, mice were allocated into several experimental groups (Naïve, EAE, EAE-1 % CBD-cream, EAE-vehicle cream, CTRL-1 % CBD-cream, CTRL-vehicle cream). Mice were observed daily for signs of EAE and weight loss. At the sacrifice of the animals, which occurred at the 28(th) day from EAE-induction, spinal cord and spleen tissues were collected in order to perform histological evaluation, immunohistochemistry and western blotting analysis.

Results: Achieved results surprisingly show that daily treatment with topical 1 % CBD-cream may exert neuroprotective effects against EAE, diminishing clinical disease score (mean of 5.0 in EAE mice vs 1.5 in EAE + CBD-cream), by recovering of paralysis of hind limbs and by ameliorating histological score typical of disease (lymphocytic infiltration and demyelination) in spinal cord tissues. Also, 1 % CBD-cream is able to counteract the EAE-induced damage reducing release of CD4 and CD8α T cells (spleen tissue localization was quantified about 10,69 % and 35,96 % of positive staining respectively in EAE mice) and expression of the main pro-inflammatory cytokines as well as several other direct or indirect markers of inflammation (p-selectin, IL-10, GFAP, Foxp3, TGF-β, IFN-γ), oxidative injury (Nitrotyrosine, iNOS, PARP) and apoptosis (Cleaved caspase 3).

Conclusion: All these data suggest an interesting new profile of CBD that could lead to its introduction in the clinical management of MS and its associated symptoms at least in association with current conventional therapy.

Figures

Fig. 1
Fig. 1
Panel a shows timeline of experimental design. EAE was induced on the 0th day. The disease onset occurred on the 14th day simultaneously daily treatment with CBD-cream was started and protracted until the day of sacrifice, which occurred at the 28th day. Mice were immunized with MOG35–55 and monitored for clinical disease score of EAE (b) and body weight variations (x). Data have been expressed as mean ± SEM of all measurements of each experimental group. A p value < 0.05 was considered statistically significant. *p < 0.03 vs NAIVE, **p < 0.0011 vs NAÏVE (b). ****p < 0.0001 vs NAIVE, °°p < 0.0016, °°°°p < 0.0001 vs CBD-cream (c). Panel d displays score of sensibility measured by needle test ****p < 0.0001 vs NAÏVE, °°p < 0.0011, °°°p < 0.004, °°°°p < 0.0001 vs CBD-cream
Fig. 2
Fig. 2
LFB staining compared EAE group (a:10x, A1 magnification:40x) to EAE+ 1 % CBD-cream (b:10x, B1 magnification:40x). May-Grunwald Giemsa staining for EAE mice (c:10x, C1 magnification:40x) compared to mice treated with 1 % CBD-cream (d:10x, D1 magnification:40x). Immunohistochemical evaluation for Foxp3 in EAE (e:20x, E1 magnification:40x) and in EAE + 1 % CBD-cream mice (f:20x, F1 magnification:40x). Immunohistochemical evaluation for GFAP in EAE (g:10x, G1 magnification:40x) and in EAE + 1 % CBD-cream mice (h:10x, H1 magnification:40x)
Fig. 3
Fig. 3
Immunohistochemical analysis for CD4 in spleen tissues from EAE mice (a:10x, A1 magnification:40x) and mice treated with 1 % CBD-cream (b:10x, B1 magnification:40x). Immunohistochemical image for CD8α localization of EAE mice (c:10x, C1 magnification:40x) compared to CBD topical treated mice (d:10x, D1 magnification:40x) in spleen tissues
Fig. 4
Fig. 4
Comparision of p-selectin immunohistochemical localization between EAE mice (a:10x, A1 magnification:40x) and EAE treated mice with 1 % CBD-cream (b:10x, B1 magnification:40x). Immunohistochemical analysis for IL-1β in spinal cord tissues from EAE mice (c:10x, C1 magnification:40x) and mice treated with 1 % CBD-cream (d:10x, D1 magnification:40x). Western blot analysis for TNF-α was showed in e. β-actin was used as internal control. ****p < 0.0001 vs EAE, ***p < 0.0004, ***p < 0.0010 vs EAE + 1 % CBD-cream
Fig. 5
Fig. 5
Western blot analysis for IL-6 was showed in a. ****p < 0.0001 vs EAE, ****p < 0.0001 vs EAE + 1 % CBD-cream. Western blot analysis for TGF-β (b). ***p < 0.0004 vs EAE, ***p < 0.0004 vs EAE + 1 % CBD-cream. In c was displayed western blot analysis for IFN-γ. **p < 0.0011 vs EAE, ***p < 0.0008 vs EAE + 1 % CBD-cream. Western blot analysis for IL-10 (d). *p < 0.0326, *p < 0.0415 vs EAE + 1 % CBD-cream. In e was displayed western blot analysis for GFAP **p < 0.0016 vs EAE, ***p < 0.0009 vs EAE + 1 % CBD-cream. ND not detectable
Fig. 6
Fig. 6
Immunohistochemical image for nitrotyrosine localization of EAE mice (a:10x, A1 magnification:40x) compared to CBD topical treated mice (b:10x, B1 magnification:40x). Immunohistochemical evaluation for iNOS in EAE (c:10x, C1 magnification:40x) and in EAE + 1 % CBD-cream mice (d:10x, D1 magnification:40x). Immunohistochemical analysis for PARP in spinal cord tissues from EAE mice (e) and mice treated with 1 % CBD-cream (f). Panel g shows western blot analysis for Cleaved-caspase 3. GAPDH was used as internal control. ****p < 0.0001 vs EAE, *p < 0.0244, ****p < 0.0001 vs. EAE + CBD-cream. ND not detectable

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