Sativex-like combination of phytocannabinoids is neuroprotective in malonate-lesioned rats, an inflammatory model of Huntington's disease: role of CB1 and CB2 receptors

Abstract

We have investigated whether a 1:1 combination of botanical extracts enriched in either Δ(9)-tetrahydrocannabinol (Δ(9)-THC) or cannabidiol (CBD), which are the main constituents of the cannabis-based medicine Sativex, is neuroprotective in Huntington's disease (HD), using an experimental model of this disease generated by unilateral lesions of the striatum with the mitochondrial complex II inhibitor malonate. This toxin damages striatal neurons by mechanisms that primarily involve apoptosis and microglial activation. We monitored the extent of this damage and the possible preservation of the striatal parenchyma by treatment with a Sativex-like combination of phytocannabinoids using different histological and biochemical markers. Results were as follows: (i) malonate increased the volume of edema measured by in vivo NMR imaging and the Sativex-like combination of phytocannabinoids partially reduced this increase; (ii) malonate reduced the number of Nissl-stained cells, while enhancing the number of degenerating cells stained with FluoroJade-B, and the Sativex-like combination of phytocannabinoids reversed both effects; (iii) malonate caused a strong glial activation (i.e., reactive microglia labeled with Iba-1, and astrogliosis labeled with GFAP) and the Sativex-like combination of phytocannabinoids attenuated both responses; and (iv) malonate increased the expression of inducible nitric oxide synthase and the neurotrophin IGF-1, and both responses were attenuated after the treatment with the Sativex-like combination of phytocannabinoids. We also wanted to establish whether targets within the endocannabinoid system (i.e., CB(1) and CB(2) receptors) are involved in the beneficial effects induced in this model by the Sativex-like combination of phytocannabinoids. This we did using selective antagonists for both receptor types (i.e., SR141716 and AM630) combined with the Sativex-like phytocannabinoid combination. Our results indicated that the effects of this combination are blocked by these antagonists and hence that they do result from an activation of both CB(1) and CB(2) receptors. In summary, this study provides preclinical evidence in support of a beneficial effect of the cannabis-based medicine Sativex as a neuroprotective agent capable of delaying signs of disease progression in a proinflammatory model of HD, which adds to previous data obtained in models priming oxidative mechanisms of striatal injury. However, the interest here is that, in contrast with these previous data, we have now obtained evidence that both CB(1) and CB(2) receptors appear to be involved in the effects produced by a Sativex-like phytocannabinoid combination, thus stressing the broad-spectrum properties of Sativex that may combine activity at the CB(1) and/or CB(2) receptors with cannabinoid receptor-independent actions.

Keywords: CB1 and CB2 receptors; Huntington’s disease; Phytocannabinoids; basal ganglia; cannabidiol; malonate; neurodegeneration; neuroprotection; Δ9-tetrahydrocannabinol.

Figure 1

Volume of edema measured by in vivo NMR imaging procedures in the caudate-putamen of rats subjected to unilateral lesions with malonate and receiving two injections of Δ⁹-THC- and CBD-enriched botanical extracts combined in a Sativex-like ratio 1:1 (total cannabinoid dose equivalent to 3 mg/kg weight), or vehicle (Tween 80-saline). Details in the text. The 1:1 mixture Δ⁹-THC- and CBD-enriched botanical extracts is referred to as Sativex in this figure. Values are expressed as means ± SEM for 5–6 animals per group. Data were subjected to one-way analysis of variance followed by the Student–Newman–Keuls test (*p < 0.05, **p < 0.005 compared with controls). Representative in vivo NMR images for each experimental group are included in the left panel.

Figure 2

Nissl and FluoroJade B staining measured in the caudate-putamen of rats subjected to unilateral lesions with malonate and receiving two injections of Δ⁹-THC- and CBD-enriched botanical extracts combined in a Sativex-like ratio 1:1 (total cannabinoid dose equivalent to 3 mg/kg weight), or vehicle (Tween 80-saline). Details in the text. The 1:1 mixture Δ⁹-THC- and CBD-enriched botanical extracts is referred to as Sativex in this figure. Values correspond to percent over the contralateral nonlesioned side and are expressed as means ± SEM for 5–6 animals per group. Data were subjected to one-way analysis of variance followed by the Student–Newman–Keuls test (*p < 0.05, **p < 0.01, ***p < 0.005 compared with controls; ^#p < 0.05 compared with malonate + vehicle). Representative Nissl- and FluoroJade B-stained microphotographs for each experimental group are included in the left panel. Magnification = 20×.

Figure 3

Iba-1 and GFAP immunostainings measured in the caudate-putamen of rats subjected to unilateral lesions with malonate and receiving two injections of Δ⁹-THC- and CBD-enriched botanical extracts combined in a Sativex-like ratio 1:1 (total cannabinoid dose equivalent to 3 mg/kg weight), or vehicle (Tween 80-saline). Details in the text. The 1:1 mixture Δ⁹-THC- and CBD-enriched botanical extracts is referred to as Sativex in this figure. Values correspond to percent over the contralateral nonlesioned side and are expressed as means ± SEM for 3–4 animals per group. Data were subjected to one-way analysis of variance followed by the Student–Newman–Keuls test (*p < 0.05, **p < 0.01 compared with controls; ^#p < 0.05 compared with malonate + vehicle). Representative Iba-1 and GFAP immunostained microphotographs for each experimental group are included in the left panel. Magnification = 20×.

Figure 4

Gene expression for iNOS, IGF-1, and CB₁ receptors measured in the caudate-putamen of rats subjected to unilateral lesions with malonate and receiving two injections of Δ⁹-THC- and CBD-enriched botanical extracts combined in a Sativex-like ratio 1:1 (total cannabinoid dose equivalent to 3 mg/kg weight), or vehicle (Tween 80-saline). Details in the text. The 1:1 mixture Δ⁹-THC- and CBD-enriched botanical extracts is referred to as Sativex in this figure. Values correspond to percent over the contralateral nonlesioned side and are expressed as means ± SEM for 5–6 animals per group. Data were subjected to one-way analysis of variance followed by the Student–Newman–Keuls test (*p < 0.05 compared with controls; ^#p < 0.05 compared with malonate + vehicle).

Figure 5

Nissl staining measured in the caudate-putamen of rats subjected to unilateral lesions with malonate and receiving two injections of Δ⁹-THC- and CBD-enriched botanical extracts combined in a Sativex-like ratio 1:1 (total cannabinoid dose equivalent to 3 mg/kg weight), selective antagonists for the CB₁ (SR141716) and/or the CB₂ (AM630) receptor, or vehicle (Tween 80-saline). Details in the text. The 1:1 mixture Δ⁹-THC- and CBD-enriched botanical extracts is referred to as Sativex in this Figure. Values correspond to percent over the control group and are expressed as means ± SEM for 5–6 animals per group. Data were subjected to one-way analysis of variance followed by the Student–Newman–Keuls test (*p < 0.05, **p < 0.01 compared with controls; ^#p < 0.05 compared with malonate + Sativex).