We have investigated whether a 1:1 combination of botanical extracts enriched in either Δ⁹-tetrahydrocannabinol (Δ⁹-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., CB₁ and CB₂ 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 CB₁ and CB₂ 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 CB₁ and CB₂ 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 CB₁ and/or CB₂ receptors with cannabinoid receptor-independent actions.

Effects of Phytocannabinoids on Malonate-Induced Striatal Damage

The first objective was to establish that we could produce the characteristic striatal damage associated with the local administration of malonate.

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Destroy user interface control10 As expected, the administration of malonate damaged the striatal parenchyma as indicated by the increased edema volume recorded in in vivo NMR imaging (F(2,19) = 8.86, p < 0.005; see Figure ​Figure1),1), the reduction in the number of surviving cells determined by Nissl staining (F(2,10) = 6.36, p < 0.05; see Figure ​Figure2),2), the increase in the level of degenerating cells determined by FluoroJade B staining (F(2,10) = 32.87, p < 0.0005; see Figure ​Figure2),2), and the extent of reactive microgliosis labeled with Iba-1 immunohistochemistry (F(2,12) = 4.652, p < 0.05; see Figure ​Figure3)3) and astrogliosis labeled with GFAP immunohistochemistry (F(2,11) = 9.25, p < 0.01; see Figure ​Figure3).3). Malonate toxicity also affects the expression of different markers related to inflammation (i.e., iNOS: F(2,11) = 5.78, p < 0.05) and neurotrophins (i.e., IGF-1: F(2,15) = 4.41, p < 0.05) (see Figure ​Figure4).4). These changes, although characteristic of a specific experimental model of HD, have also been found in HD patients using CSF, blood cell, and post-mortem brain samples,The following popper user interface control may not be accessible. Tab to the next button to revert the control to an accessible version.
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^{Destroy user interface control23} thus supporting the idea that glial activation associated with inflammatory events is part in the pathogenic process that occurs in HD patients. Lastly, malonate toxicity is also associated with a reduction in the expression of CB₁ receptors (F(2,25) = 4.863, p < 0.05; see Figure ​Figure4)4) an effect largely related to HD pathogenesis in different animal models.The following popper user interface control may not be accessible. Tab to the next button to revert the control to an accessible version.
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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 …

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 …

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 …

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 …

The administration of a 1:1 combination of Δ⁹-THC- and CBD-enriched botanical extracts that are present in Sativex attenuated, to a different extent, all these malonate-induced changes. For example, it slightly reduced the volume of edema measured with in vivo NMR imaging procedures 24 h after malonate application and 24 h before animal death (Figure ​(Figure1).1). These in vivo results correlated well with the data obtained after the histopathological analysis of animal brains. For example, the treatment with a Sativex-like combination of phytocannabinoids increased the number of Nissl-stained cells that had been reduced by malonate (see Figure ​Figure2),2), while reducing the high number of degenerating cells stained with FluoroJade-B (see Figure ​Figure2).2). In a similar way, the Sativex-like combination of phytocannabinoids attenuated the activation of glial elements caused by malonate (i.e., reactive microglia labeled with Iba-1, and astrogliosis labeled with GFAP; see Figure ​Figure3),3), which was particularly abundant and generalized along the whole striatal parenchyma in this experimental model. Lastly, the Sativex-like combination of phytocannabinoids attenuated the malonate-induced up-regulatory responses in iNOS (already described in ref (

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Destroy user interface control16)) and IGF-1 gene expression in the striatal parenchyma (see Figure ​Figure4),4), whereas it partially recovered the malonate-induced reduction in CB₁ receptors (see Figure ​Figure4). These4). These findings support the notion that the combination of both phytocannabinoid-botanical extracts may be effective as a neuroprotective therapy in malonate-lesioned rats as it was previously found when individual cannabinoid agonists (i.e., HU-308) were used in the same HD model.The following popper user interface control may not be accessible. Tab to the next button to revert the control to an accessible version.
^{Destroy user interface control10} In this study,The following popper user interface control may not be accessible. Tab to the next button to revert the control to an accessible version.
^{Destroy user interface control10} however, we found that CBD administered alone was not active against malonate-induced striatal damage, but, according to the present data, its combination with Δ⁹-THC resulted in highly positive effects presumably by enhancing the activity of this last phytocannabinoid at the CB₁ and CB₂ receptors (see below). The rationale for such combination has been largely discussed in previous reviews.The following popper user interface control may not be accessible. Tab to the next button to revert the control to an accessible version.
^{Destroy user interface control24} 

Involvement of CB₁ and/or CB₂ Receptors in the Beneficial Effects of Phytocannabinoids on Malonate-Induced Striatal Damage

In our previous study that used an experimental model of HD that primes oxidative injury and calpain activation as major causes of striatal damage, we detected protective effects of the Sativex-like combination of phytocannabinoids, that seemed to result from the antioxidant and cannabinoid receptor-independent properties of both phytocannabinoids rather than from any interaction with CB₁and/or CB₂ receptors. We wanted to see if this was also the case in the experimental model of HD used in the present study. To this end, we conducted additional experiments with selective antagonists for CB₁ and CB₂ receptor types, that is, SR141716 and AM630, respectively, combined with the Sativex-like mixture of phytocannabinoids. The same or similar antagonists had been already tested after being administered alone in the same animal model of HD, showing no effect in the case of CB₂ receptor blockade

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Destroy user interface control10 or even aggravating striatal damage in the case of SR141716.The following popper user interface control may not be accessible. Tab to the next button to revert the control to an accessible version.
^{Destroy user interface control25} In our present study, the blockade of CB₁ receptors with SR141716 or the blockade of CB₂ receptors with AM630 reversed the phytocannabinoid-induced decrease in the ability of malonate to attenuate Nissl-staining of cells (F(5,23) = 6.11, p < 0.005; see Figure ​Figure5).5). Interestingly, this reversal was no greater when both antagonists were administered together (see Figure ​Figure5).5). However, it is important to note that, although SR141716 and AM630, both separately and together, reversed the effects of the Sativex-like combination of Δ⁹-THC- and CBD-enriched botanical extracts, the lowest number of Nissl-stained cells was observed after the treatment with AM630, administered alone or in combination with SR141716. This finding, together with our previously published data showing that CB₂ receptors underwent an up-regulation in glial elements located in the striatal parenchyma after malonate,The following popper user interface control may not be accessible. Tab to the next button to revert the control to an accessible version.
^{Destroy user interface control10} whereas CB₁ receptors underwent a down-regulation in the same animal model in the present study (see Figure ​Figure4)4) and also in other models of HD,The following popper user interface control may not be accessible. Tab to the next button to revert the control to an accessible version.
^{Destroy user interface control8,}The following popper user interface control may not be accessible. Tab to the next button to revert the control to an accessible version.
^{Destroy user interface control16} suggests that even though Δ⁹-THC can activate both CB₁ and CB₂ receptors, the CB₂receptor plays a greater role than the CB₁ receptor in the protection from malonate-induced striatal damage that the Δ⁹-THC- and CBD-enriched botanical extracts produce. 

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 …

Animals and Surgery

Adult (12 week old; 350–400 g) male Sprague–Dawley rats (Harlan Ibérica, Barcelona, Spain) were used for the experiments. Animals were housed in a room with controlled photoperiod (08:00–20:00 light) and temperature (22 ± 1 °C) with free access to standard food and water. All experiments were conducted according to local and European rules (directive 86/609/EEC) and approved by the “Comité de Experimentación Animal” of our university. Rats were subjected to unilateral lesions of the striatum with the complex II inhibitor malonate, following a procedure described previously.

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Destroy user interface control10 Rats were injected stereotaxically (coordinates: +0.8 mm anterior, +2.9 mm lateral from the bregma, −4.5 mm ventral from the dura mater) into the left striatum with 2 M malonate (dissolved in PBS 0.1 M, pH = 7.4) in a volume of 1 μL. The contralateral striatum of each animal remained unaffected, when data generated in this experimental model are expressed as percent of the lesioned side over the corresponding nonlesioned side. 

Treatments and Sampling

We followed the same procedure used in previous studies with cannabinoids in malonate-lesioned rats.

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Destroy user interface control10,The following popper user interface control may not be accessible. Tab to the next button to revert the control to an accessible version.
Destroy user interface control25 Thus, 30 min before and 2 h after the intrastriatal injection of malonate, animals were treated with combinations of botanical extracts enriched with either Δ⁹-THC, kindly provided by GW Pharmaceuticals Ltd., Cambridgeshire, U.K. (Δ⁹-THC botanical extract contains 67.1% Δ⁹-THC, 0.3% CBD, 0.9% cannabigerol, 0.9% cannabichromene, and 1.9% other phytocannabinoids) or CBD, also provided by GW Pharmaceuticals Ltd., Cambridgeshire, U.K. (CBD botanical extract contains 64.8% CBD, 2.3% Δ⁹-THC, 1.1% cannabigerol, 3.0% cannabichromene, and 1.5% other phytocannabinoids). The total dose of cannabinoid administered was always 4.63 mg/kg (equivalent to 3 mg/kg of pure CBD + Δ⁹-THC), a dose within the range of effective doses of both compounds when they were administered in pure form in this and other experimental models of HD.The following popper user interface control may not be accessible. Tab to the next button to revert the control to an accessible version.
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^{Destroy user interface control11} This was also the effective dose in our previous study with Sativex-like combination of phytocannabinoids in another HD model.The following popper user interface control may not be accessible. Tab to the next button to revert the control to an accessible version.
^{Destroy user interface control16}Cannabinoids were prepared in Tween 80-saline solution (1:16) and they were administered i.p. Malonate-lesioned rats administered with vehicle, as well as sham-operated animals, were also included in this experiment. In a further experiment, rats injected with malonate, following the same procedure described above, and also injected with the 1:1 combination of Δ⁹-THC- and CBD-enriched botanical extracts used in the previous experiment, were coadministered (10 min before each injection of Sativex-like combination) with the CB₁ receptor antagonist SR141716 (1 mg/kg), kindly provided by Sanofi-Aventis (Montpellier, France) or the CB₂ receptor blocker AM630 (1 mg/kg), purchased from Tocris (Biogen Científica, Madrid, Spain), both prepared in Tween 80-saline (1:16). In both experiments, animals were killed 48 h after the administration of malonate and their brains were rapidly removed, the two striata dissected and frozen separately in 2-methylbutane cooled in dry ice, and stored at −80 °C for subsequent qRT-PCR analysis. Animals to be used for histological analysis were transcardially perfused with saline followed by fresh 4% paraformaldehyde [in 0.1 M phosphate buffered-saline (PBS)], and their brains were collected and postfixed overnight at 4 °C, and then immersed in antifreeze solution and stored at −20 °C for Nissl and FluoroJade B staining and immunohistochemical analysis. In all experiments, at least 5–6 animals were used per experimental group. 

In Vivo NMR Imaging Procedures

In the first experiment consisting of injections of Sativex-like combinations of phytocannabinoids to malonate-lesioned rats, animals from the three experimental groups were used, at 24 h after malonate lesion and 24 h before animal death, for in vivo NMR imaging studies in which the volume of striatal edema was calculated. Experiments were performed at the Nuclear Magnetic Resonance Centre of Complutense University (Madrid, Spain) using a BIOSPEC BMT 47/40 (Bruker, Ettlingen, Germany) operating at 4.7 T, equipped with a 12 cm actively shielded gradient system. Rats were anesthetized with oxygen:isoflurane and subsequently placed in prone position inside a cradle. The animal head was immobilized and placed underneath a 4 cm surface coil. A respiration sensor was used to control the animals. First global shimmer was assessed, and then three gradient-echo scout images in axial, sagittal, and coronal directions were acquired (TR/TE = 100/3.2 ms, matrix = 128 × 128). A 3D fast spin–echo experiment with axial slice orientation was subsequently performed using the following acquisition parameters: TR = 3000 ms, effective TE = 86.5 ms, NA = 2, FOV = 2.56 × 2.56 × 1.28 cm³, matrix size = 256 × 128 × 32. The reconstructed matrix size was 256 × 256 × 32. The total time of the acquisition experiment was 27 min.

Histological Analyses

Coronal brain sections (25 μm thick) were obtained with a vibratome and collected as floating slices at the level of the caudate-putamen. They were used for Nissl (see details in ref (

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Destroy user interface control27)) and FluoroJade B (see details in ref (The following popper user interface control may not be accessible. Tab to the next button to revert the control to an accessible version.
Destroy user interface control10)) staining, which permitted determination of the effects of particular treatments on cell number, and for immunohistochemical analysis of Iba-1, a marker of microglial cells, and GFAP, a marker of astrocytes. For immunohistochemistry, sections were incubated overnight at 30 °C with (i) monoclonal antirat Iba-1 antibody (Wako, Neuss, Germany) used at 1:300, or (ii) monoclonal antirat GFAP (Sigma-Aldrich, Madrid, Spain) used at 1:400. After incubation with the corresponding primary antibody, sections were washed in 0.1 M PBS and incubated for 2 h at 37 °C with a mouse highly cross-adsorbed AlexaFluor 488 secondary antibody (Invitrogen, Carlsbad, CA) for GFAP immunostaining, and with a rabbit biotinylated secondary antibody (Sigma-Aldrich, Madrid, Spain) followed by incubation for 2 additional hours with streptavidin 488 (Molecular Probes, Paisley, U.K.) for Iba-1 immunostaining. Both secondary antibodies were used at 1:200. Negative control sections were obtained using the same protocol with omission of the primary antibody. All sections for each immunohistochemical procedure were processed at the same time and under the same conditions. A Nikon Eclipse 90i confocal microscope and a Nikon DXM 1200F camera were used for slide observation and photography, and all image processing, including cell counting, was done using ImageJ, the software developed and freely distributed by the U.S. National Institutes of Health (Bethesda, MD). For this purpose, multiple sections, selected from levels located approximately 200 μm from the middle of the lesion, were obtained from each brain and used to generate a mean value per subject. 

Real Time qRT-PCR Analysis

Total RNA was isolated from striata using RNATidy reagent (AppliChem. Inc., Cheshire, CT). The total amount of RNA extracted was quantitated by spectrometry at 260 nm and its purity from the ratio between the absorbance values at 260 and 280 nm, whereas its integrity was confirmed in agarose geles. After genomic DNA was removed (to eliminate DNA contamination), single-stranded cDNA was synthesized from 1 μg of total RNA using a commercial kit (Rneasy Mini Quantitect Reverse Transcription, Qiagen, Izasa, Madrid, Spain). The reaction mixture was kept frozen at −20 °C until enzymatic amplification. Quantitative RT-PCR assays were performed using TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA) to quantify mRNA levels for IGF-1 (ref Rn99999087_m1), iNOS (ref Rn00561646_m1), or CB₁ receptor (ref Rn00562880_m1), using β-actin expression (ref Rn00667869_m1) as an endogenous control gene for normalization. The PCR assay was performed using the 7300 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA), and the threshold cycle was calculated by the instrument’s software (7300 Fast System, Applied Biosystems, Foster City, CA).