Jacob Bell
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Delayed treatment with cannabidiol has a cerebroprotective action via a cannabinoid receptor-independent myeloperoxidase-inhibiting mechanism
Kazuhide Hayakawa1,
Kenichi Mishima1,
Masanori Nozako1,
Mai Hazekawa1,
Keiichi Irie1,
Masayuki Fujioka1,
Kensuke Orito4,
Kohji Abe3,
Nobuyoshi Hasebe3,
Nobuaki Egashira1,
Katsunori Iwasaki1,2,
Michihiro Fujiwara1
Article first published online: 9 MAR 2007
DOI: 10.1111/j.1471-4159.2007.04565.x
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Journal of Neurochemistry
Journal of Neurochemistry
Volume 102, Issue 5, pages 1488—1496, September 2007
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Keywords:
cannabidiol;
cannabinoid;
cerebral blood flow;
cerebral ischemia;
glutamate;
myeloperoxidase
Abstract
We examined the neuroprotective mechanism of cannabidiol, non-psychoactive component of marijuana, on the infarction in a 4 h mouse middle cerebral artery (MCA) occlusion model in comparison with Δ9-tetrahydrocannabinol (Δ9-THC). Release of glutamate in the cortex was measured at 2 h after MCA occlusion. Myeloperoxidase (MPO) and cerebral blood flow were measured at 1 h after reperfusion. In addition, infarct size and MPO were determined at 24 and 72 h after MCA occlusion. The neuroprotective effect of cannabidiol was not inhibited by either SR141716 or AM630. Both pre- and post-ischemic treatment with cannabidiol resulted in potent and long-lasting neuroprotection, whereas only pre-ischemic treatment with Δ9-THC reduced the infarction. Unlike Δ9-THC, cannabidiol did not affect the excess release of glutamate in the cortex after occlusion. Cannabidiol suppressed the decrease in cerebral blood flow by the failure of cerebral microcirculation after reperfusion and inhibited MPO activity in neutrophils. Furthermore, the number of MPO-immunopositive cells was reduced in the ipsilateral hemisphere in cannabidiol-treated group. Cannbidiol provides potent and long-lasting neuroprotection through an anti-inflammatory CB1 receptor-independent mechanism, suggesting that cannabidiol will have a palliative action and open new therapeutic possibilities for treating cerebrovascular disorders.
Abbreviations used
AM630
6-lodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl)(4-methoxyphenyl)methanone
cannabidiol
2-[1R,6R]-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-benzenediol
CBF
cerebral blood flow
ICAM-1
intracellular adhesion molecule-1
MCA
middle cerebral artery
MPO
myeloperoxidase
PBS
phosphate-buffered saline
SR141716
N-(piperidine-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride
Δ9-THC
Δ9-tetrahydrocannabinol
Cannabis contains about 60 different cannabinoids, including the psychoactive component Δ9-tetrahydrocannabinol (Δ9-THC) and other major non-psychoactive components such as cannabidiol, cannabinol, and cannabigerol. Δ9-THC has been demonstrated to produce hypothermia, neuroprotection, and tolerance (Hampson et al. 2000; Rubino et al. 2000; Wiley and Martin 2002; Braida et al. 2003; Leker et al. 2003; Hayakawa et al. 2004; Mishima et al. 2005). These effects are, at least in part, related to binding to the CB1 receptor. On the other hand, cannabidiol has a very low affinity (in the micromolar range) for CB1 and CB2 receptors and has been found to act as an anticonvulsant in animal models of epilepsy and in humans with epilepsy. Moreover, cannabidiol has been shown to have antispasmodic, anxiolytic, anti-nausea, and anti-rheumatoid properties (Mechoulam et al. 2002) and to be protective against N-methyl-d-aspartate and beta-amyloid peptide toxicity (Iuvone et al. 2004) and global and focal ischemic injury (Braida et al. 2003). Recently, it has also been reported that cannabidiol has the ability to enhance adenosine signaling through inhibition of uptake (Carrier et al. 2006). These actions are thought to be dependent on a new cannabinoid receptor, such as the G protein-coupled receptor (Begg et al. 2005; David et al. 2006; Munro et al. 1993), GPR55, abnormal-cannabidiol receptor rather than on the CB1 and CB2 receptors. Cannabidiol exerts a wide spectrum of effects, but the neuroprotective mechanism has not been fully explored.
Neutrophils, a type of leukocytes, are known to play a major role in inflammatory injury after transient ischemia (Kochanek and Hallenbeck 1992; Heinel et al. 1994). The progression and extent of brain injury resulting from cerebral ischemia is related to several reperfusion mechanisms, many of which involve post-injury inflammatory response elements. These inflammatory elements include the early neutrophil response (Barone et al. 1992; Chen et al. 1993; Matsuo et al. 1994; Zhang et al. 1994). Several lines of evidence have shown that neutrophils play an important role in the development of ischemic brain damage and indicate that the depletion of circulating neutrophils or the inhibition of neutrophil infiltration is thought to ameliorate cerebral ischemic injury (Jiang et al. 1995; Satoh et al. 1999; Phillips et al. 2000). Δ9-THC and other cannabinoids have been known to modulate inflammation. For example, Δ9-THC and cannabidiol suppress interleukin-1 and tumor necrosis factor (Watzl et al. 1991), and cannabinoids alter cytokine production in human immune cells (Srivastava et al. 1998). In addition, cannabinoids ablate the release of tumor necrosis factor-α in rat microglial cells stimulated with lypopolysaccharide (Facchinetti et al. 2003). However, no reports have yet been issued on the anti-neutrophil actions of Δ9-THC and cannabidiol.
In the present study, we investigated the effects of cannabidiol and Δ9-THC on ischemic brain damage induced by focal ischemia-reperfusion in mice. We measured and evaluated the effect of cannabidiol and Δ9-THC on the extracellular level of glutamate in the cortex using in vivo microdialysis. Moreover, the anti-neutrophil action was quantified by assaying myeloperoxidase (MPO) activity, which is mainly located in neutrophil primary granules.
Materials and methods
Animals
Male ddY mice (25—35 g; Kiwa Experimental Animal Laboratory, Wakayama, Japan) were kept under a 12 h light/dark cycle (lights on from 07:00 to 19:00 h) in an air-conditioned room (23 ± 2°C) with food (CE-2; Clea Japan, Tokyo, Japan) and water available ad libitum. All procedures regarding animal care and use were performed in compliance with the regulations established by the Experimental Animal Care and Use Committee of Fukuoka University, Fukuoka, Japan.
Focal cerebral ischemia
Focal cerebral ischemia was induced according to the method described in our previous study (Egashira et al. 2004; Mishima et al. 2005). Mice were anesthetized with 2% halothane and maintained thereafter with 1% halothane (Flosen; Takeda Chemical Industries, Osaka, Japan). After a midline neck incision, the left common and external carotid arteries were isolated and ligated. A nylon monofilament (8-0, Ethilon; Johnson & Johnson, Tokyo, Japan) coated with silicon resin (Xantopren; Heleus Dental Material, Osaka, Japan) was introduced through a small incision into the common carotid artery and advanced to a position 9 mm distal from the carotid bifurcation for occlusion of the middle cerebral artery (MCA). Following occlusion, we stopped the 1% halothane anesthesia. We confirmed occlusion of the MCA by examining forelimb flexion after awaking from halothane anesthesia. Four hours after occlusion, the mice were re-anesthetized with halothane and reperfusion was established by withdrawal of the filament.
2,3,5-Triphenyltetrazolium chloride staining
Twenty-four hours or 3 days after MCA occlusion, the animals were killed by decapitation. The brains were removed and sectioned coronally into four 2-mm slices using a mouse brain matrix. Slices were immediately stained with 2% 2,3,5-triphenyltetrazolium chloride (Sigma, St Louis, MO, USA). The border between the infarcted and non-infarcted tissue was outlined using an image analysis system (NIH Image, version 1.63, National Institutes of Health, Bethesda, MD, USA); the area of infarction was measured and the infarction volume was calculated.
Cerebral blood flow
Cerebral blood flow (CBF) was monitored by laser-Doppler flowmetry using the probe (diameter 0.5 mm) of a laser-Doppler flowmeter (ALF2100; Advance Co., Tokyo, Japan) inserted into the left cortex (anterior −0.22 mm; lateral 2.5 mm from bregma; depth 2 mm from the skull surface) through a guide cannula. CBF was measured during MCA occlusion and 1 h after reperfusion.
Extracellular level of glutamate: in vivo microdialysis-HPLC measurements
The microdialysis experiments were performed in freely moving and unanesthetized mice for 2 h after occlusion. Microdialysis probes (Eicom, Kyoto, Japan) were implanted 1 day before cerebral ischemia. They consisted of U-shaped dialysis membranes composed of cellulose hollow fiber (each arm was 1-mm long, with an ID of 0.2 mm, OD of 0.22 mm, and a surface area of 0.69 mm2; molecular weight cut-off of 50 kDa) that were inserted into the distal ends of stainless steel tubing before implanting (coordinates; anterior −0.22 mm; lateral 2.5 mm from bregma; depth 2 mm from the skull surface). Each mouse with an implanted probe was placed in a Plexiglas chamber (50 × 35 × 35 cm; Clea, Japan). After the MCA had been occluded, the inlet of the probe was connected to a microsyringe driven by a microinfusion pump (ESP-64, Eicom), whereas the outlet end was connected to an autoinjector (EAS-20 autoinjector; Eicom) coupled to an HPLC-Electron Capture Detector system (LC EP-300; Eicom) by a Teflon tube (ID 0.1 mm and OD 0.4 mm). The probes were perfused at rate of 0.5 μL/min with buffered Ringer's solution (147 mmol/L NaCl, 4 mmol/L KCl, and 2.3 mmol/L CaCl2, pH 7.4). The ECD was set at +0.5 V (oxidation potential) against an Ag/AgCl reference analytical electrode. At the end of the experiments, coronal sections of the brain were examined to verify the probe sites.
Measurement of MPO activity
MPO activity in brain tissue was determined as an index of neutrophil accumulation, as previously described (Bradley et al. 1982). In brief, brains were rapidly removed and separated into right and left hemispheres. Wet weights were immediately recorded. Each sample was homogenized in 0.5 mL/100 mg tissue of 0.5% Hexadecyltri-Methylammonium Bromide in 50 mmol/L potassium phosphate buffer (pH 6.0) and centrifuged at 15 000 g for 2 min. The supernatant (50 μL) was mixed with 200 μL of potassium phosphate buffer (pH 6.0) containing 0.167 mg/mL o-dianisidine dihydrochloride and 0.05% hydrogen peroxide. Changes in absorbance at 450 nm were measured using a spectrophotometer. One mole of H2O2 gives a change in absorbance (ΔA) of 1.13 × 104/min. Therefore, 1 μmol H2O2 gives change in absorbance of 1.13 × 10−2/min. Because 1 U of MPO = 1 μmol H2O2 split, the change in absorbance due to 1 U of MPO is 1.13 × 10−2/min. So, the number of units of MPO in each brain sample was determined as ΔA/Δtime × 1.13 × 10−2. MPO activity in brain tissue was determined at 1 and 20 h after cerebral ischemia-reperfusion.
MPO immunohistochemistry
Four groups of mice (n = 3 in each group) were killed by decapitation after perfusion using saline and 4%p-formaldehyde at 3 days after MCA occlusion. After, brains were removed of fat and water by auto-degreasing unit (RH-12; SAKURA SEIKI CO. Tokyo, Japan) and were then embedded in paraffin. Subsequently, 5-μm thick sections were mounted on slides and processed for immunohistochemistry using DAKO LSAB kit (K0697; DAKO Inc., Carpinteria, CA, USA). After deparaffinization and rehydration, these sections were rinsed two times for 1 min with phosphate-buffered saline (PBS, pH7.4). Non-specific binding was blocked by PBS containing carrier protein and 15 mmol/L sodium azide for 5 min at 23 ± 2°C. The sections were incubated in the 1 : 400 dilution of rabbit polyclonal anti-MPO primary antibody (A0398; DAKO Inc.) overnight at 4°C. After treatment with 3% H2O2, the sections were then incubated with biotinylated anti-rabbit secondary antibody for 10 min at 23 ± 2°C. After rinsing two times for 1 min with PBS, the sections were incubated strept avidin solution for 10 min at 23 ± 2°C. Finally, they were treated with stable 3,3-diaminobenzidine tetrahydrochloride as a peroxidase substrate.
Rota-rod test in MCA occluded mice
Motor coordination was measured using the rota-rod test as described previously (Egashira et al. 2005). Mice were placed on a rotating rod (diameter: 3 cm; Neuroscience Inc., Tokyo, Japan) with a non-skid surface and the latency to fall was measured for up to 2 min. The rotating speed was 10 rpm.
Blood analysis
Physiological variables pH, Pco2, Po2, hematocrit, potassium and sodium, glucose, mean arterial pressure, heart rate, and body temperature were measured at 4 h after MCA occlusion.
Drug preparation and administration
Δ9-THC (isolated by Professor Y. Shoyama, Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, Japan), cannabidiol (Sigma-Aldrich, Tokyo, Japan), N-(piperidine-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716) (a generous gift from Sanofi Recherche, Montpellier, France), and 6-lodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl)(4-methoxyphenyl) methanone (AM630; Tocris, Bristol, UK) were dissolved in 1% Tween. Δ9-THC and cannabidiol were administered intraperitoneally (i.p.) immediately before and 3 h after MCA occlusion, immediately before, 3 h after and 4 h after MCA occlusion. Cannabidiol was also administered 1 and 2 h after reperfusion.
Statistical analysis
Results are expressed as the mean ± SEM. Multiple comparisons were evaluated by Tukey's test after a one-way anova. p < 0.05 was considered to be significant.
Results
The neuroprotective effects of Δ9-THC but not cannabidiol were inhibited by a CB1 antagonist
Both Δ9-THC 3, 10 mg/kg and cannabidiol 1, 3 mg/kg significantly reduced the infarct volume induced by MCA occlusion in mice [F(3, 28) = 10.004, p < 0.001, Δ9-THC 3,10 mg/kg, p < 0.01; F(3, 22) = 9.839, p < 0.001, cannabidiol 1, 3 mg/kg, p < 0.01, Table 1). Cannabidiol also prevented cerebral infarction at 3 days after MCA occlusion [F(2,16) = 4.303, p < 0.05, cannabidiol 3 mg/kg, p < 0.05 compared with vehicle, Fig. 1b]. Neither SR141716 (1 mg/kg) nor AM630 (1 mg/kg) alone changed the infarct volume [F(8,54) = 9.180, p < 0.0001, Δ9-THC (10 mg/kg), p < 0.01 compared with vehicle; cannabidiol (3 mg/kg), p < 0.01 compared with vehicle, Table 1]. The neuroprotective effect of 10 mg/kg Δ9-THC, but not that of 3 mg/kg cannabidiol, was inhibited by SR141716 at 1 mg/kg. By contrast, AM630, at either 1 mg/kg or 10 mg/kg (data not shown), had no effect on the neuroprotective effects of Δ9-THC (10 mg/kg) and cannabidiol (3 mg/kg).
Table 1. Effect of SR141716 and AM630 on the prevention of cerebralinfarction by treatment with Δ9-THC and cannabidiol Drag Infarct volume (mm3)
The neuroprotective effect of Δ9-THC was inhibited by SR141716 but not by AM630. Drugs were administered intraperitoneally (i.p.) immediately before and 3 h after MCA occlusion. Values are expressed as the mean ± SEM (n = 4—8). *p < 0.05, **p < 0.01 compared with vehicle; " " p < 0.01 compared with Δ9-THC 10 mg/kg (one-way anova test followed by Tukey's test). The neuroprotective effect of cannabidiol was not affected by either antagonist. Δ9-THC, Δ9-tetrahydrocannabinol; MCA, middle cerebral artery; AM630, 6-lodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl)(4-methoxyphenyl)methanone; SR141716, N-(piperidine-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride.
Vehicle (8) 91.3 ± 3.5
Cannabidiol 0.1 mg/kg (6) 92.3 ± 9.6
Cannabidiol 1 mg/kg (6) 49.2 ± 10.3**
Cannabidiol 3 mg/kg (7) 51.4 ± 7.3**
Cannabidiol 3 mg/kg + SR141716 Img/kg (6) 55.0 ± 6.7**
Cannabidiol 3 mg/kg + AM630 Img/kg (6) 65.7 ± 7.8 *
Δ9-THC 1 mg/kg (8) 93.9 ± 9.3
Δ9-THC 3 mg/kg (8) 53.5 ± 10.4**
Δ9-THC 10 mg/kg (8) 46.7 ± 5.9**
Δ9-THC 10 mg/kg + SR141716 Img/kg (8) 84.7 ± 7.9" "
Δ9-THC 10 mg/kg + AM630 Img/kg (6) 58.3 ± 5.4*
SR141716 Img/kg (8) 91.1 ± 2.9
AM630 Img/kg (4) 84.0 ± 5.3
Figure 1. These are data of 3 days after 4 h middle cerebral artery (MCA) occlusion. (a) Myeloperoxidase immunohistochemistry (n = 3), (b) Infarct volume (n = 5—7), (c) Rota-rod test (n = 7—8). Cannabidiol but not Δ9-tetrahydrocannabinol (Δ9-THC) significantly reduced myeloperoxidase-immunopositive cells at striatum (a) scale bar = 100 μm. In addition, cannabidiol prevented the cerebral infarction, and Δ9-THC also prevented that but no significantly (b). Furthermore, cannabidiol significantly improved the motor coordination at 3 days after MCA occlusion, while Δ9-THC had no significantly improvement on the motor coordination at 10 rpm. Drugs were administered intraperitoneally (i.p.) immediately before and 3 h after MCA occlusion. Values are expressed as the mean ± SEM. *p < 0.05 compared with vehicle (one-way anova test followed by Tukey's test).
image
Δ9-THC and cannabidiol did not induce changes in physiological variable data
There were no significant differences in the physiological variables usually affected by cerebral ischemia (pH, Pco2, Po2, hematocrit, K and Na, glucose, mean arterial pressure, and heart rate) in the Δ9-THC- and cannabidiol-treated groups compared with vehicle-treated controls. However, Δ9-THC significantly decreased rectal temperature. [F(3,26) = 9.741, p < 0.001,Δ9-THC (10 mg/kg), p < 0.01 compared with vehicle] (Table 2).
Table 2. Physiological variable data 4 h after MCAo
Sham Vehicle Cannabidiol 3 mg/kg Δ9-THC 10 mg/kg
Data on physiological variables were obtained immediately before reperfusion, and drugs were administered immediately before and 3 h after cerebral ischemia. Values are expressed as the mean ± SEM (n = 4—8). **p < 0.01 compared with vehicle (one-way anova test followed by Tukey's test). MAP, mean arterial pressure; Δ9-THC, Δ9-tetrahydrocannabinol; Hct, hematocrit; MCAo, middle cerebral artery occlusion.
Physiological variables
pH (4) 7.3 ± 0.03 7.2 ± 0.02 7.2 ± 0.03 7.3 ± 0.04
pC02 (4) 49.7 ± 4.0 50.5 ± 2.7 44.4 ± 1.5 51.7 ± 3.2
p02 (4) 49.0 ± 8.9 57.3 ± 6.2 58.8 ± 6.9 60.6 ± 4.0
Hct (%) (4) 38.0 ± 1.1 38.1 ± 1.6 35.9 ± 1.7 35.1 ± 1.1
Na (mmol/L) (4) 150.6 ± 1.4 150.8 ± 2.1 147.9 ± 2.0 153.2 ± 6.0
K (mmol/L) (4) 4.5 ± 0.2 6.3 ± 1.5 5.0 ± 1.1 4.0 ± 0.7
Glucose (mg/dL) (8) 101.5 ± 2.9 91.4 ± 5.6 106.6 ± 6.2 111 ± 5.0
MAP (mmHg) (5) 78.2 ± 4.4 88.8 ± 5.4 84.5 ± 5.2 68.4 ± 5.2
Heart rate (beats/min) (5) 493.7 ± 34.6 529.7 ± 15.7 552.2 ± 10.2 492.3 ± 16.1
Body temperature (°C) (8) 36.6 ± 0.1 36.3 ± 0.3 36.1 ± 0.3 34.8 ± 0.2**
Pre- and post-ischemic treatment with cannabidiol but not Δ9-THC had a potent and a long-lasting neuroprotective effect
Only pre-ischemic treatment with Δ9-THC 10 mg/kg reduced the size of infarction, whereas both pre- and post-ischemic treatment with cannabidiol 3 mg/kg showed a potent and long-lasting neuroprotective effect [F(10,63) = 3.643, p < 0.001, Δ9-THC (10 mg/kg) immediately before and 3 h after MCA occlusion, p < 0.01;Δ9-THC (10 mg/kg) immediately before MCA occlusion, p < 0.05; cannabidiol (3 mg/kg) immediately before and 3 h after MCA occlusion, p < 0.01; cannabidiol (3 mg/kg) 4 h after occlusion, p < 0.05; cannabidiol (3 mg/kg) 1 h after reperfusion, p < 0.05; cannabidiol (3 mg/kg) 2 h after reperfusion, p < 0.01, Fig. 2].
Figure 2. Cannabidiol but not Δ9-tetrahydrocannabinol (Δ9-THC) had a potent and long-lasting neuroprotective effect when administered both pre- and post-ischemia. Only pre-ischemic treatment with Δ9-THC could reduce the size of infarction, whereas cannabidiol showed a potent and a long-lasting effect for both pre- and post-ischemic treatment. Group (A) before immediately and 3 h after middle cerebral artery occlusion (MCAo), Group (B) before immediately MCAo (Pre), Group (C) 3 h after MCAo, Group (D) 4 h after MCAo, Group (E) 1 h after reperfusion, and Group (F) 2 h after reperfusion. Values are expressed as the mean ± SEM (n = 7—8). *p < 0.05, **p < 0.01 compared with vehicle (one-way anova test followed by Tukey's test).
image
Δ9-THC but not cannabidiol inhibited excess release of glutamate after cerebral ischemia
Δ9-THC but not cannabidiol inhibited the release of glutamate in the cortex after MCA occlusion; this effect was inhibited by the CB1 receptor antagonist SR141716 [F(5,13) = 10.717, p < 0.001, Δ9-THC (10 mg/kg), p < 0.05; Δ9-THC (10 mg/kg) + (SR141716 1 mg/kg), p < 0.05, Fig. 3].
Figure 3. Δ9-Tetrahydrocannabinol (Δ9-THC) but not cannabidiol inhibited the release of glutamate in the cortex after middle cerebral artery (MCA) occlusion, an effect that was inhibited by the CB1 receptor antagonist SR141716. The data shows the average glutamate concentrations during the 2 h after MCA occlusion. Drugs were administered intraperitoneally (i.p.) immediately before MCA occlusion. Values are expressed as the mean ± SEM (n = 3). *p < 0.05 compared with vehicle; " p < 0.05 compared with Δ9-THC 10 mg/kg (one-way anova test followed by Tukey's test).
image
Cannabidiol suppressed the decrease in CBF due to the failure of cerebral microcirculation after reperfusion
Both cannabidiol and Δ9-THC increased the CBF during MCA occlusion, while only cannabidiol suppressed a decrease in CBF by the failure of cerebral microcirculation for 1 h after reperfusion [F(2,6) = 84.078, p < 0.0001, cannabidiol (3 mg/kg), p < 0.01, Fig. 4].
Figure 4. Both Cannabidiol and Δ9-tetrahydrocannabinol (Δ9-THC) increased the cerebral blood flow (CBF) during middle cerebral artery (MCA) occlusion, while only cannabidiol significantly suppressed the decrease in CBF due to the failure of cerebral microcirculation for 1 h after reperfusion. Drugs were administered intraperitoneally (i.p.) immediately before and 3 h after MCA occlusion. Values are expressed as the mean ± SEM (n = 3—4). **p < 0.01 compared with vehicle (one-way anova test followed by Tukey's test).
image
Cannabidiol inhibited MPO activity in neutrophils at 1 h and 20 h after the reperfusion
Cannabidiol, but not Δ9-THC, significantly inhibited MPO activity at 1 h after reperfusion. The effects of cannabidiol were not inhibited by either SR141716 (1 mg/kg) or AM630 (1 mg/kg) [F(7,33) = 4.596, p < 0.01, cannabidiol (3 mg/kg), p < 0.05; cannabidiol (3 mg/kg) + SR141716 (1 mg/kg), p < 0.05; cannabidiol (3 mg/kg) + AM630 (1 mg/kg), p < 0.05, Fig. 5a]. In addition, cannabidiol (3 mg/kg) inhibited MPO activity at 20 h after reperfusion, not only following treatment immediately before and 3 h after MCA occlusion, but also following a single treatment at 2 h after reperfusion [F(3,15) = 11.81, p < 0.001, cannabidiol (3 mg/kg) treatment before and after occlusion, p < 0.01; cannabidiol treatment after reperfusion, p < 0.01, Fig. 5b].
Figure 5. Cannabidiol, but not Δ9-tetrahydrocannabinol (Δ9-THC), significantly inhibited myeloperoxidase (MPO) activity at 1 h after reperfusion. The effects of cannabidiol were not inhibited by either SR141716 (1 mg/kg) or AM630 (1 mg/kg). Drugs were administered intraperitoneally (i.p.) immediately before and 3 h after middle cerebral artery (MCA) occlusion (a). In addition, 3 mg/kg cannabidiol also inhibited MPO activity at 20 h after reperfusion, not only when administered immediately before and 3 h after MCA occlusion, but also when solely administered at 2 h after the reperfusion (b). Values are expressed as the mean ± SEM (n = 5). *p < 0.05, **p < 0.01 compared with vehicle (one-way anova test followed by Tukey's test).
image
MPO immunohistochemistry
In the 3 days after 4 h MCA occlusion, immunohistochemistry showed that MPO-immunoreactive cells distributed in large quantities in and around the ischemic lesion. Positive cells were present in both ipsilateral striatum. MPO staining shows no positive cells in animals undergoing sham MCA occlusion. This indicates that the increased MPO staining is really caused by the accumulation of polymorphonuclear leukocytes (Fig. 1a). The quantity of MPO-immunopositive cells in the ipsilateral hemisphere in the cannabidiol-treated group appeared similar to that in the sham group. However, the number of immunopositive cells was not reduced in the ipsilateral hemisphere in Δ9-THC-treated group. The distribution of MPO immunostaining paralleled the results on MPO activity in each group.
Effects of cannabidiol on motor coordination in MCA occluded mice
In the rota-rod test for motor coordination, cannabidiol 3 mg/kg significantly improved the motor coordination at 3 days after MCA occlusion, while Δ9-THC 10 mg/kg had no significantly improvement on the motor coordination at 10 rpm [3 days after MCA occlusion; F(4,24) = 7.149, p < 0.001, cannabidiol 3 mg/kg, p < 0.05 compared with vehicle, Fig. 1c].
Discussion
Both Δ9-THC and cannabidiol significantly reduced the infarct volume in a mouse MCA occlusion model, and the neuroprotective effect of Δ9-THC was inhibited by the CB1 receptor antagonist SR141716, but not by the CB2 receptor antagonist AM630. Cannabidiol was not inhibited by either antagonist. In addition, only pre-ischemic treatment with Δ9-THC was able to reduce the size of infarction, whereas both pre- and post-ischemic treatment with cannabidiol showed more potent and more long-lasting neuroprotection than Δ9-THC. Δ9-THC but not cannabidiol inhibited the excess release of glutamate in the cortex after the occlusion, as measured by in vivo microdialysis; this effect of Δ9-THC was also inhibited by SR141716. Cannabidiol suppressed the decrease in CBF due to the failure of cerebral microcirculation after reperfusion. Cannabidiol also inhibited MPO activity in neutrophils after reperfusion. Moreover, cannabidiol inhibited MPO activity at 20 h after reperfusion. In addition, cannabidiol reduced MPO-immunopositive cells at 3 days after MCA occlusion. Thus, cannbidiol is a potent and long-lasting neuroprotectant and anti-inflammatory acting through a cannabinoid receptor-independent mechanism.
Δ9-THC is known to produce neuroprotection via the cannabinoid CB1 receptor. We have also reported that Δ9-THC prevented cerebral infarction through hypothermia acting the CB1 receptor (Hayakawa et al. 2004). On the other hand, cannabidiol, a non-psychoactive constituent of cannabis, has been shown to be protective against global and focal ischemic injury, in agreement with the present study (Molina-Holgado et al. 2002; Braida et al. 2003). However, the neuroprotective mechanism of cannabidiol remains unclear, but novel non-CB1 and non-CB2 receptors have been proposed, because cannabidiol, which has many pharmacological actions, has a very low affinity (in the micromolar range) for CB1 and CB2 receptors (Wiley and Martin 2002). In this study, Δ9-THC was shown to have a neuroprotective effect on cerebral injury induced by MCA occlusion acting via the CB1 receptor but not the CB2 receptor. On the contrary, cannabidiol was not inhibited by antagonists to either the CB1 or CB2 receptor. These results suggest that Δ9-THC exerts its neuroprotective action through the CB1 receptor, while cannabidiol prevents cerebral infarction via a CB1 and CB2 receptor-independent mechanism.
The neuroprotective effect of Δ9-THC was only evident with pre-ischemic, but not post-ischemic, treatment of MCA occlusion in mice. The pattern of excitatory amino acid efflux in different models of cerebral ischemia derives from the finding that a massive release of glutamate is considered to play a major role in inducing ischemic and post-ischemic cell death (Bullock et al. 1995). In fact, antagonists of glutamate receptors reduce the ischemic penumbra (Obrenovitch 1966; Obrenovitch and Richards 1995), and inhibitors of glutamate release exhibit cerebroprotective activity against ischemia/reperfusion-evoked injury (Molina-Holgado et al. 2002). The neuroprotective effect of Δ9-THC and other cannabinoids is related to the CB1 receptor-mediated inhibition of voltage-sensitive Ca2+ channels, which reduces Ca2+ influx, glutamate release, and excitotoxicity (Iuvone et al. 2004). In fact, the present study shows that Δ9-THC inhibits the release of glutamate and induced hypothermia. Moreover, the effects were inhibited by the CB1 receptor antagonist SR141716. Taken together, these findings suggest that the neuroprotective effect of Δ9-THC is induced only by pre-ischemic but not with post-ischemic treatment.
Both pre- and post-ischemic treatment with cannabidiol resulted in a potent and a long-lasting neuroprotective effect. It has been reported that warming increases pro-inflammatory factors such as leukocyte integrin expression and function on neutrophils and platelets (Forsyth and Levinsky 1990;Kurabayashi et al. 1997; Kochanek and Hallenbeck 1992), and significantly exacerbates functional and structural neurologic injury (Shum-Tim et al. 1998). The leukocytes then interact with intracellular adhesion molecule-1 (ICAM-1), adhere to endothelial cells, and migrate out of the vessels. Cannabidiol has been reported to reduce ICAM-1 expression in experimental diabetes (EI-Remessy et al. 2006). A previous study showed that the neuroprotective effect of cannabidiol is not inhibited by warming indicating that cannabidiol has a CB1 receptor-independent mechanism, unlike Δ9-THC (Hayakawa et al. 2004). Thus, cannabidiol might have a potent anti-inflammatory effect, inhibiting the migration of leukocytes, platelets, and neutrophils by reducing ICAM-1 expression. In this study, cannabidiol significantly inhibited MPO activity in neutrophils at 1 and 20 h after reperfusion via a cannabinoid receptor-independent mechanism. In addition, cannabidiol reduced MPO-immunopositive cells at 3 days after MCA occlusion. Moreover, because the focal cerebral ischemia-induced inflammation response occurs at a later stage than glutamate release (Dirnagl et al. 2003), cannabidiol might have a potent and long-lasting neuroprotective effect.
Inflammation is a critical process after stroke (Danton and Dietrich 2003). Studies have shown the over-expression of inflammatory factors such as ICAM-1, P-selectin, and E-selectin and the accumulation of inflammatory cells such as neutrophils, macrophages, and T-cells (Barone and Feuerstein 1999;Danton and Dietrich 2003; Zhang and Wang 2005). Moreover, these factors have also been known to cause the decrease in CBF due to the failure of cerebral microcirculation at 2—4 h after cerebral ischemia reperfusion (Jones et al. 1981; Garcia et al. 1994). In previously, cannabidiol has prevented cerebral infarction through an increase in CBF (Mishima et al. 2005). In the present study, we found that both cannabidiol and Δ9-THC increased the CBF during MCA occlusion, while only cannabidiol suppresses a decrease in CBF after reperfusion and that cannabidiol inhibits MPO activity in neutrophils via a cannabinoid receptor-independent mechanism. Cannabidiol has a very low affinity (in the micromolar range) for CB1 and CB2 receptors, so these actions might be dependent on a new receptor within the brain such as the G protein-coupled receptor, GPR55, abnormal-cannabidiol receptor.
In conclusion, the present study shows that cannabidiol has a profile of cerebroprotectant activity different from that of Δ9-THC. Cannabidiol, but not Δ9-THC, has a potent and long-lasting neuroprotective effect, when administered both pre- and post-ischemia, through a CB1 and CB2 receptor-independent mechanism. It is to be hoped that cannabidiol will have a palliative action and open new therapeutic vista for treating cerebrovascular disorders.
Acknowledgements
Part of this study was supported by a Grant-in-Aid for Scientific Research (No. 17590479) from the Ministry of Education, Science and Culture of Japan and the Advanced Materials Institute of Fukuoka University and The Naito Foundation.
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Source: Delayed treatment with cannabidiol has a cerebroprotective action via a cannabinoid receptor-independent myeloperoxidase-inhibiting mechanism
Kazuhide Hayakawa1,
Kenichi Mishima1,
Masanori Nozako1,
Mai Hazekawa1,
Keiichi Irie1,
Masayuki Fujioka1,
Kensuke Orito4,
Kohji Abe3,
Nobuyoshi Hasebe3,
Nobuaki Egashira1,
Katsunori Iwasaki1,2,
Michihiro Fujiwara1
Article first published online: 9 MAR 2007
DOI: 10.1111/j.1471-4159.2007.04565.x
Issue
Journal of Neurochemistry
Journal of Neurochemistry
Volume 102, Issue 5, pages 1488—1496, September 2007
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Keywords:
cannabidiol;
cannabinoid;
cerebral blood flow;
cerebral ischemia;
glutamate;
myeloperoxidase
Abstract
We examined the neuroprotective mechanism of cannabidiol, non-psychoactive component of marijuana, on the infarction in a 4 h mouse middle cerebral artery (MCA) occlusion model in comparison with Δ9-tetrahydrocannabinol (Δ9-THC). Release of glutamate in the cortex was measured at 2 h after MCA occlusion. Myeloperoxidase (MPO) and cerebral blood flow were measured at 1 h after reperfusion. In addition, infarct size and MPO were determined at 24 and 72 h after MCA occlusion. The neuroprotective effect of cannabidiol was not inhibited by either SR141716 or AM630. Both pre- and post-ischemic treatment with cannabidiol resulted in potent and long-lasting neuroprotection, whereas only pre-ischemic treatment with Δ9-THC reduced the infarction. Unlike Δ9-THC, cannabidiol did not affect the excess release of glutamate in the cortex after occlusion. Cannabidiol suppressed the decrease in cerebral blood flow by the failure of cerebral microcirculation after reperfusion and inhibited MPO activity in neutrophils. Furthermore, the number of MPO-immunopositive cells was reduced in the ipsilateral hemisphere in cannabidiol-treated group. Cannbidiol provides potent and long-lasting neuroprotection through an anti-inflammatory CB1 receptor-independent mechanism, suggesting that cannabidiol will have a palliative action and open new therapeutic possibilities for treating cerebrovascular disorders.
Abbreviations used
AM630
6-lodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl)(4-methoxyphenyl)methanone
cannabidiol
2-[1R,6R]-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-benzenediol
CBF
cerebral blood flow
ICAM-1
intracellular adhesion molecule-1
MCA
middle cerebral artery
MPO
myeloperoxidase
PBS
phosphate-buffered saline
SR141716
N-(piperidine-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride
Δ9-THC
Δ9-tetrahydrocannabinol
Cannabis contains about 60 different cannabinoids, including the psychoactive component Δ9-tetrahydrocannabinol (Δ9-THC) and other major non-psychoactive components such as cannabidiol, cannabinol, and cannabigerol. Δ9-THC has been demonstrated to produce hypothermia, neuroprotection, and tolerance (Hampson et al. 2000; Rubino et al. 2000; Wiley and Martin 2002; Braida et al. 2003; Leker et al. 2003; Hayakawa et al. 2004; Mishima et al. 2005). These effects are, at least in part, related to binding to the CB1 receptor. On the other hand, cannabidiol has a very low affinity (in the micromolar range) for CB1 and CB2 receptors and has been found to act as an anticonvulsant in animal models of epilepsy and in humans with epilepsy. Moreover, cannabidiol has been shown to have antispasmodic, anxiolytic, anti-nausea, and anti-rheumatoid properties (Mechoulam et al. 2002) and to be protective against N-methyl-d-aspartate and beta-amyloid peptide toxicity (Iuvone et al. 2004) and global and focal ischemic injury (Braida et al. 2003). Recently, it has also been reported that cannabidiol has the ability to enhance adenosine signaling through inhibition of uptake (Carrier et al. 2006). These actions are thought to be dependent on a new cannabinoid receptor, such as the G protein-coupled receptor (Begg et al. 2005; David et al. 2006; Munro et al. 1993), GPR55, abnormal-cannabidiol receptor rather than on the CB1 and CB2 receptors. Cannabidiol exerts a wide spectrum of effects, but the neuroprotective mechanism has not been fully explored.
Neutrophils, a type of leukocytes, are known to play a major role in inflammatory injury after transient ischemia (Kochanek and Hallenbeck 1992; Heinel et al. 1994). The progression and extent of brain injury resulting from cerebral ischemia is related to several reperfusion mechanisms, many of which involve post-injury inflammatory response elements. These inflammatory elements include the early neutrophil response (Barone et al. 1992; Chen et al. 1993; Matsuo et al. 1994; Zhang et al. 1994). Several lines of evidence have shown that neutrophils play an important role in the development of ischemic brain damage and indicate that the depletion of circulating neutrophils or the inhibition of neutrophil infiltration is thought to ameliorate cerebral ischemic injury (Jiang et al. 1995; Satoh et al. 1999; Phillips et al. 2000). Δ9-THC and other cannabinoids have been known to modulate inflammation. For example, Δ9-THC and cannabidiol suppress interleukin-1 and tumor necrosis factor (Watzl et al. 1991), and cannabinoids alter cytokine production in human immune cells (Srivastava et al. 1998). In addition, cannabinoids ablate the release of tumor necrosis factor-α in rat microglial cells stimulated with lypopolysaccharide (Facchinetti et al. 2003). However, no reports have yet been issued on the anti-neutrophil actions of Δ9-THC and cannabidiol.
In the present study, we investigated the effects of cannabidiol and Δ9-THC on ischemic brain damage induced by focal ischemia-reperfusion in mice. We measured and evaluated the effect of cannabidiol and Δ9-THC on the extracellular level of glutamate in the cortex using in vivo microdialysis. Moreover, the anti-neutrophil action was quantified by assaying myeloperoxidase (MPO) activity, which is mainly located in neutrophil primary granules.
Materials and methods
Animals
Male ddY mice (25—35 g; Kiwa Experimental Animal Laboratory, Wakayama, Japan) were kept under a 12 h light/dark cycle (lights on from 07:00 to 19:00 h) in an air-conditioned room (23 ± 2°C) with food (CE-2; Clea Japan, Tokyo, Japan) and water available ad libitum. All procedures regarding animal care and use were performed in compliance with the regulations established by the Experimental Animal Care and Use Committee of Fukuoka University, Fukuoka, Japan.
Focal cerebral ischemia
Focal cerebral ischemia was induced according to the method described in our previous study (Egashira et al. 2004; Mishima et al. 2005). Mice were anesthetized with 2% halothane and maintained thereafter with 1% halothane (Flosen; Takeda Chemical Industries, Osaka, Japan). After a midline neck incision, the left common and external carotid arteries were isolated and ligated. A nylon monofilament (8-0, Ethilon; Johnson & Johnson, Tokyo, Japan) coated with silicon resin (Xantopren; Heleus Dental Material, Osaka, Japan) was introduced through a small incision into the common carotid artery and advanced to a position 9 mm distal from the carotid bifurcation for occlusion of the middle cerebral artery (MCA). Following occlusion, we stopped the 1% halothane anesthesia. We confirmed occlusion of the MCA by examining forelimb flexion after awaking from halothane anesthesia. Four hours after occlusion, the mice were re-anesthetized with halothane and reperfusion was established by withdrawal of the filament.
2,3,5-Triphenyltetrazolium chloride staining
Twenty-four hours or 3 days after MCA occlusion, the animals were killed by decapitation. The brains were removed and sectioned coronally into four 2-mm slices using a mouse brain matrix. Slices were immediately stained with 2% 2,3,5-triphenyltetrazolium chloride (Sigma, St Louis, MO, USA). The border between the infarcted and non-infarcted tissue was outlined using an image analysis system (NIH Image, version 1.63, National Institutes of Health, Bethesda, MD, USA); the area of infarction was measured and the infarction volume was calculated.
Cerebral blood flow
Cerebral blood flow (CBF) was monitored by laser-Doppler flowmetry using the probe (diameter 0.5 mm) of a laser-Doppler flowmeter (ALF2100; Advance Co., Tokyo, Japan) inserted into the left cortex (anterior −0.22 mm; lateral 2.5 mm from bregma; depth 2 mm from the skull surface) through a guide cannula. CBF was measured during MCA occlusion and 1 h after reperfusion.
Extracellular level of glutamate: in vivo microdialysis-HPLC measurements
The microdialysis experiments were performed in freely moving and unanesthetized mice for 2 h after occlusion. Microdialysis probes (Eicom, Kyoto, Japan) were implanted 1 day before cerebral ischemia. They consisted of U-shaped dialysis membranes composed of cellulose hollow fiber (each arm was 1-mm long, with an ID of 0.2 mm, OD of 0.22 mm, and a surface area of 0.69 mm2; molecular weight cut-off of 50 kDa) that were inserted into the distal ends of stainless steel tubing before implanting (coordinates; anterior −0.22 mm; lateral 2.5 mm from bregma; depth 2 mm from the skull surface). Each mouse with an implanted probe was placed in a Plexiglas chamber (50 × 35 × 35 cm; Clea, Japan). After the MCA had been occluded, the inlet of the probe was connected to a microsyringe driven by a microinfusion pump (ESP-64, Eicom), whereas the outlet end was connected to an autoinjector (EAS-20 autoinjector; Eicom) coupled to an HPLC-Electron Capture Detector system (LC EP-300; Eicom) by a Teflon tube (ID 0.1 mm and OD 0.4 mm). The probes were perfused at rate of 0.5 μL/min with buffered Ringer's solution (147 mmol/L NaCl, 4 mmol/L KCl, and 2.3 mmol/L CaCl2, pH 7.4). The ECD was set at +0.5 V (oxidation potential) against an Ag/AgCl reference analytical electrode. At the end of the experiments, coronal sections of the brain were examined to verify the probe sites.
Measurement of MPO activity
MPO activity in brain tissue was determined as an index of neutrophil accumulation, as previously described (Bradley et al. 1982). In brief, brains were rapidly removed and separated into right and left hemispheres. Wet weights were immediately recorded. Each sample was homogenized in 0.5 mL/100 mg tissue of 0.5% Hexadecyltri-Methylammonium Bromide in 50 mmol/L potassium phosphate buffer (pH 6.0) and centrifuged at 15 000 g for 2 min. The supernatant (50 μL) was mixed with 200 μL of potassium phosphate buffer (pH 6.0) containing 0.167 mg/mL o-dianisidine dihydrochloride and 0.05% hydrogen peroxide. Changes in absorbance at 450 nm were measured using a spectrophotometer. One mole of H2O2 gives a change in absorbance (ΔA) of 1.13 × 104/min. Therefore, 1 μmol H2O2 gives change in absorbance of 1.13 × 10−2/min. Because 1 U of MPO = 1 μmol H2O2 split, the change in absorbance due to 1 U of MPO is 1.13 × 10−2/min. So, the number of units of MPO in each brain sample was determined as ΔA/Δtime × 1.13 × 10−2. MPO activity in brain tissue was determined at 1 and 20 h after cerebral ischemia-reperfusion.
MPO immunohistochemistry
Four groups of mice (n = 3 in each group) were killed by decapitation after perfusion using saline and 4%p-formaldehyde at 3 days after MCA occlusion. After, brains were removed of fat and water by auto-degreasing unit (RH-12; SAKURA SEIKI CO. Tokyo, Japan) and were then embedded in paraffin. Subsequently, 5-μm thick sections were mounted on slides and processed for immunohistochemistry using DAKO LSAB kit (K0697; DAKO Inc., Carpinteria, CA, USA). After deparaffinization and rehydration, these sections were rinsed two times for 1 min with phosphate-buffered saline (PBS, pH7.4). Non-specific binding was blocked by PBS containing carrier protein and 15 mmol/L sodium azide for 5 min at 23 ± 2°C. The sections were incubated in the 1 : 400 dilution of rabbit polyclonal anti-MPO primary antibody (A0398; DAKO Inc.) overnight at 4°C. After treatment with 3% H2O2, the sections were then incubated with biotinylated anti-rabbit secondary antibody for 10 min at 23 ± 2°C. After rinsing two times for 1 min with PBS, the sections were incubated strept avidin solution for 10 min at 23 ± 2°C. Finally, they were treated with stable 3,3-diaminobenzidine tetrahydrochloride as a peroxidase substrate.
Rota-rod test in MCA occluded mice
Motor coordination was measured using the rota-rod test as described previously (Egashira et al. 2005). Mice were placed on a rotating rod (diameter: 3 cm; Neuroscience Inc., Tokyo, Japan) with a non-skid surface and the latency to fall was measured for up to 2 min. The rotating speed was 10 rpm.
Blood analysis
Physiological variables pH, Pco2, Po2, hematocrit, potassium and sodium, glucose, mean arterial pressure, heart rate, and body temperature were measured at 4 h after MCA occlusion.
Drug preparation and administration
Δ9-THC (isolated by Professor Y. Shoyama, Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, Japan), cannabidiol (Sigma-Aldrich, Tokyo, Japan), N-(piperidine-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716) (a generous gift from Sanofi Recherche, Montpellier, France), and 6-lodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl)(4-methoxyphenyl) methanone (AM630; Tocris, Bristol, UK) were dissolved in 1% Tween. Δ9-THC and cannabidiol were administered intraperitoneally (i.p.) immediately before and 3 h after MCA occlusion, immediately before, 3 h after and 4 h after MCA occlusion. Cannabidiol was also administered 1 and 2 h after reperfusion.
Statistical analysis
Results are expressed as the mean ± SEM. Multiple comparisons were evaluated by Tukey's test after a one-way anova. p < 0.05 was considered to be significant.
Results
The neuroprotective effects of Δ9-THC but not cannabidiol were inhibited by a CB1 antagonist
Both Δ9-THC 3, 10 mg/kg and cannabidiol 1, 3 mg/kg significantly reduced the infarct volume induced by MCA occlusion in mice [F(3, 28) = 10.004, p < 0.001, Δ9-THC 3,10 mg/kg, p < 0.01; F(3, 22) = 9.839, p < 0.001, cannabidiol 1, 3 mg/kg, p < 0.01, Table 1). Cannabidiol also prevented cerebral infarction at 3 days after MCA occlusion [F(2,16) = 4.303, p < 0.05, cannabidiol 3 mg/kg, p < 0.05 compared with vehicle, Fig. 1b]. Neither SR141716 (1 mg/kg) nor AM630 (1 mg/kg) alone changed the infarct volume [F(8,54) = 9.180, p < 0.0001, Δ9-THC (10 mg/kg), p < 0.01 compared with vehicle; cannabidiol (3 mg/kg), p < 0.01 compared with vehicle, Table 1]. The neuroprotective effect of 10 mg/kg Δ9-THC, but not that of 3 mg/kg cannabidiol, was inhibited by SR141716 at 1 mg/kg. By contrast, AM630, at either 1 mg/kg or 10 mg/kg (data not shown), had no effect on the neuroprotective effects of Δ9-THC (10 mg/kg) and cannabidiol (3 mg/kg).
Table 1. Effect of SR141716 and AM630 on the prevention of cerebralinfarction by treatment with Δ9-THC and cannabidiol Drag Infarct volume (mm3)
The neuroprotective effect of Δ9-THC was inhibited by SR141716 but not by AM630. Drugs were administered intraperitoneally (i.p.) immediately before and 3 h after MCA occlusion. Values are expressed as the mean ± SEM (n = 4—8). *p < 0.05, **p < 0.01 compared with vehicle; " " p < 0.01 compared with Δ9-THC 10 mg/kg (one-way anova test followed by Tukey's test). The neuroprotective effect of cannabidiol was not affected by either antagonist. Δ9-THC, Δ9-tetrahydrocannabinol; MCA, middle cerebral artery; AM630, 6-lodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl)(4-methoxyphenyl)methanone; SR141716, N-(piperidine-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride.
Vehicle (8) 91.3 ± 3.5
Cannabidiol 0.1 mg/kg (6) 92.3 ± 9.6
Cannabidiol 1 mg/kg (6) 49.2 ± 10.3**
Cannabidiol 3 mg/kg (7) 51.4 ± 7.3**
Cannabidiol 3 mg/kg + SR141716 Img/kg (6) 55.0 ± 6.7**
Cannabidiol 3 mg/kg + AM630 Img/kg (6) 65.7 ± 7.8 *
Δ9-THC 1 mg/kg (8) 93.9 ± 9.3
Δ9-THC 3 mg/kg (8) 53.5 ± 10.4**
Δ9-THC 10 mg/kg (8) 46.7 ± 5.9**
Δ9-THC 10 mg/kg + SR141716 Img/kg (8) 84.7 ± 7.9" "
Δ9-THC 10 mg/kg + AM630 Img/kg (6) 58.3 ± 5.4*
SR141716 Img/kg (8) 91.1 ± 2.9
AM630 Img/kg (4) 84.0 ± 5.3
Figure 1. These are data of 3 days after 4 h middle cerebral artery (MCA) occlusion. (a) Myeloperoxidase immunohistochemistry (n = 3), (b) Infarct volume (n = 5—7), (c) Rota-rod test (n = 7—8). Cannabidiol but not Δ9-tetrahydrocannabinol (Δ9-THC) significantly reduced myeloperoxidase-immunopositive cells at striatum (a) scale bar = 100 μm. In addition, cannabidiol prevented the cerebral infarction, and Δ9-THC also prevented that but no significantly (b). Furthermore, cannabidiol significantly improved the motor coordination at 3 days after MCA occlusion, while Δ9-THC had no significantly improvement on the motor coordination at 10 rpm. Drugs were administered intraperitoneally (i.p.) immediately before and 3 h after MCA occlusion. Values are expressed as the mean ± SEM. *p < 0.05 compared with vehicle (one-way anova test followed by Tukey's test).
image
Δ9-THC and cannabidiol did not induce changes in physiological variable data
There were no significant differences in the physiological variables usually affected by cerebral ischemia (pH, Pco2, Po2, hematocrit, K and Na, glucose, mean arterial pressure, and heart rate) in the Δ9-THC- and cannabidiol-treated groups compared with vehicle-treated controls. However, Δ9-THC significantly decreased rectal temperature. [F(3,26) = 9.741, p < 0.001,Δ9-THC (10 mg/kg), p < 0.01 compared with vehicle] (Table 2).
Table 2. Physiological variable data 4 h after MCAo
Sham Vehicle Cannabidiol 3 mg/kg Δ9-THC 10 mg/kg
Data on physiological variables were obtained immediately before reperfusion, and drugs were administered immediately before and 3 h after cerebral ischemia. Values are expressed as the mean ± SEM (n = 4—8). **p < 0.01 compared with vehicle (one-way anova test followed by Tukey's test). MAP, mean arterial pressure; Δ9-THC, Δ9-tetrahydrocannabinol; Hct, hematocrit; MCAo, middle cerebral artery occlusion.
Physiological variables
pH (4) 7.3 ± 0.03 7.2 ± 0.02 7.2 ± 0.03 7.3 ± 0.04
pC02 (4) 49.7 ± 4.0 50.5 ± 2.7 44.4 ± 1.5 51.7 ± 3.2
p02 (4) 49.0 ± 8.9 57.3 ± 6.2 58.8 ± 6.9 60.6 ± 4.0
Hct (%) (4) 38.0 ± 1.1 38.1 ± 1.6 35.9 ± 1.7 35.1 ± 1.1
Na (mmol/L) (4) 150.6 ± 1.4 150.8 ± 2.1 147.9 ± 2.0 153.2 ± 6.0
K (mmol/L) (4) 4.5 ± 0.2 6.3 ± 1.5 5.0 ± 1.1 4.0 ± 0.7
Glucose (mg/dL) (8) 101.5 ± 2.9 91.4 ± 5.6 106.6 ± 6.2 111 ± 5.0
MAP (mmHg) (5) 78.2 ± 4.4 88.8 ± 5.4 84.5 ± 5.2 68.4 ± 5.2
Heart rate (beats/min) (5) 493.7 ± 34.6 529.7 ± 15.7 552.2 ± 10.2 492.3 ± 16.1
Body temperature (°C) (8) 36.6 ± 0.1 36.3 ± 0.3 36.1 ± 0.3 34.8 ± 0.2**
Pre- and post-ischemic treatment with cannabidiol but not Δ9-THC had a potent and a long-lasting neuroprotective effect
Only pre-ischemic treatment with Δ9-THC 10 mg/kg reduced the size of infarction, whereas both pre- and post-ischemic treatment with cannabidiol 3 mg/kg showed a potent and long-lasting neuroprotective effect [F(10,63) = 3.643, p < 0.001, Δ9-THC (10 mg/kg) immediately before and 3 h after MCA occlusion, p < 0.01;Δ9-THC (10 mg/kg) immediately before MCA occlusion, p < 0.05; cannabidiol (3 mg/kg) immediately before and 3 h after MCA occlusion, p < 0.01; cannabidiol (3 mg/kg) 4 h after occlusion, p < 0.05; cannabidiol (3 mg/kg) 1 h after reperfusion, p < 0.05; cannabidiol (3 mg/kg) 2 h after reperfusion, p < 0.01, Fig. 2].
Figure 2. Cannabidiol but not Δ9-tetrahydrocannabinol (Δ9-THC) had a potent and long-lasting neuroprotective effect when administered both pre- and post-ischemia. Only pre-ischemic treatment with Δ9-THC could reduce the size of infarction, whereas cannabidiol showed a potent and a long-lasting effect for both pre- and post-ischemic treatment. Group (A) before immediately and 3 h after middle cerebral artery occlusion (MCAo), Group (B) before immediately MCAo (Pre), Group (C) 3 h after MCAo, Group (D) 4 h after MCAo, Group (E) 1 h after reperfusion, and Group (F) 2 h after reperfusion. Values are expressed as the mean ± SEM (n = 7—8). *p < 0.05, **p < 0.01 compared with vehicle (one-way anova test followed by Tukey's test).
image
Δ9-THC but not cannabidiol inhibited excess release of glutamate after cerebral ischemia
Δ9-THC but not cannabidiol inhibited the release of glutamate in the cortex after MCA occlusion; this effect was inhibited by the CB1 receptor antagonist SR141716 [F(5,13) = 10.717, p < 0.001, Δ9-THC (10 mg/kg), p < 0.05; Δ9-THC (10 mg/kg) + (SR141716 1 mg/kg), p < 0.05, Fig. 3].
Figure 3. Δ9-Tetrahydrocannabinol (Δ9-THC) but not cannabidiol inhibited the release of glutamate in the cortex after middle cerebral artery (MCA) occlusion, an effect that was inhibited by the CB1 receptor antagonist SR141716. The data shows the average glutamate concentrations during the 2 h after MCA occlusion. Drugs were administered intraperitoneally (i.p.) immediately before MCA occlusion. Values are expressed as the mean ± SEM (n = 3). *p < 0.05 compared with vehicle; " p < 0.05 compared with Δ9-THC 10 mg/kg (one-way anova test followed by Tukey's test).
image
Cannabidiol suppressed the decrease in CBF due to the failure of cerebral microcirculation after reperfusion
Both cannabidiol and Δ9-THC increased the CBF during MCA occlusion, while only cannabidiol suppressed a decrease in CBF by the failure of cerebral microcirculation for 1 h after reperfusion [F(2,6) = 84.078, p < 0.0001, cannabidiol (3 mg/kg), p < 0.01, Fig. 4].
Figure 4. Both Cannabidiol and Δ9-tetrahydrocannabinol (Δ9-THC) increased the cerebral blood flow (CBF) during middle cerebral artery (MCA) occlusion, while only cannabidiol significantly suppressed the decrease in CBF due to the failure of cerebral microcirculation for 1 h after reperfusion. Drugs were administered intraperitoneally (i.p.) immediately before and 3 h after MCA occlusion. Values are expressed as the mean ± SEM (n = 3—4). **p < 0.01 compared with vehicle (one-way anova test followed by Tukey's test).
image
Cannabidiol inhibited MPO activity in neutrophils at 1 h and 20 h after the reperfusion
Cannabidiol, but not Δ9-THC, significantly inhibited MPO activity at 1 h after reperfusion. The effects of cannabidiol were not inhibited by either SR141716 (1 mg/kg) or AM630 (1 mg/kg) [F(7,33) = 4.596, p < 0.01, cannabidiol (3 mg/kg), p < 0.05; cannabidiol (3 mg/kg) + SR141716 (1 mg/kg), p < 0.05; cannabidiol (3 mg/kg) + AM630 (1 mg/kg), p < 0.05, Fig. 5a]. In addition, cannabidiol (3 mg/kg) inhibited MPO activity at 20 h after reperfusion, not only following treatment immediately before and 3 h after MCA occlusion, but also following a single treatment at 2 h after reperfusion [F(3,15) = 11.81, p < 0.001, cannabidiol (3 mg/kg) treatment before and after occlusion, p < 0.01; cannabidiol treatment after reperfusion, p < 0.01, Fig. 5b].
Figure 5. Cannabidiol, but not Δ9-tetrahydrocannabinol (Δ9-THC), significantly inhibited myeloperoxidase (MPO) activity at 1 h after reperfusion. The effects of cannabidiol were not inhibited by either SR141716 (1 mg/kg) or AM630 (1 mg/kg). Drugs were administered intraperitoneally (i.p.) immediately before and 3 h after middle cerebral artery (MCA) occlusion (a). In addition, 3 mg/kg cannabidiol also inhibited MPO activity at 20 h after reperfusion, not only when administered immediately before and 3 h after MCA occlusion, but also when solely administered at 2 h after the reperfusion (b). Values are expressed as the mean ± SEM (n = 5). *p < 0.05, **p < 0.01 compared with vehicle (one-way anova test followed by Tukey's test).
image
MPO immunohistochemistry
In the 3 days after 4 h MCA occlusion, immunohistochemistry showed that MPO-immunoreactive cells distributed in large quantities in and around the ischemic lesion. Positive cells were present in both ipsilateral striatum. MPO staining shows no positive cells in animals undergoing sham MCA occlusion. This indicates that the increased MPO staining is really caused by the accumulation of polymorphonuclear leukocytes (Fig. 1a). The quantity of MPO-immunopositive cells in the ipsilateral hemisphere in the cannabidiol-treated group appeared similar to that in the sham group. However, the number of immunopositive cells was not reduced in the ipsilateral hemisphere in Δ9-THC-treated group. The distribution of MPO immunostaining paralleled the results on MPO activity in each group.
Effects of cannabidiol on motor coordination in MCA occluded mice
In the rota-rod test for motor coordination, cannabidiol 3 mg/kg significantly improved the motor coordination at 3 days after MCA occlusion, while Δ9-THC 10 mg/kg had no significantly improvement on the motor coordination at 10 rpm [3 days after MCA occlusion; F(4,24) = 7.149, p < 0.001, cannabidiol 3 mg/kg, p < 0.05 compared with vehicle, Fig. 1c].
Discussion
Both Δ9-THC and cannabidiol significantly reduced the infarct volume in a mouse MCA occlusion model, and the neuroprotective effect of Δ9-THC was inhibited by the CB1 receptor antagonist SR141716, but not by the CB2 receptor antagonist AM630. Cannabidiol was not inhibited by either antagonist. In addition, only pre-ischemic treatment with Δ9-THC was able to reduce the size of infarction, whereas both pre- and post-ischemic treatment with cannabidiol showed more potent and more long-lasting neuroprotection than Δ9-THC. Δ9-THC but not cannabidiol inhibited the excess release of glutamate in the cortex after the occlusion, as measured by in vivo microdialysis; this effect of Δ9-THC was also inhibited by SR141716. Cannabidiol suppressed the decrease in CBF due to the failure of cerebral microcirculation after reperfusion. Cannabidiol also inhibited MPO activity in neutrophils after reperfusion. Moreover, cannabidiol inhibited MPO activity at 20 h after reperfusion. In addition, cannabidiol reduced MPO-immunopositive cells at 3 days after MCA occlusion. Thus, cannbidiol is a potent and long-lasting neuroprotectant and anti-inflammatory acting through a cannabinoid receptor-independent mechanism.
Δ9-THC is known to produce neuroprotection via the cannabinoid CB1 receptor. We have also reported that Δ9-THC prevented cerebral infarction through hypothermia acting the CB1 receptor (Hayakawa et al. 2004). On the other hand, cannabidiol, a non-psychoactive constituent of cannabis, has been shown to be protective against global and focal ischemic injury, in agreement with the present study (Molina-Holgado et al. 2002; Braida et al. 2003). However, the neuroprotective mechanism of cannabidiol remains unclear, but novel non-CB1 and non-CB2 receptors have been proposed, because cannabidiol, which has many pharmacological actions, has a very low affinity (in the micromolar range) for CB1 and CB2 receptors (Wiley and Martin 2002). In this study, Δ9-THC was shown to have a neuroprotective effect on cerebral injury induced by MCA occlusion acting via the CB1 receptor but not the CB2 receptor. On the contrary, cannabidiol was not inhibited by antagonists to either the CB1 or CB2 receptor. These results suggest that Δ9-THC exerts its neuroprotective action through the CB1 receptor, while cannabidiol prevents cerebral infarction via a CB1 and CB2 receptor-independent mechanism.
The neuroprotective effect of Δ9-THC was only evident with pre-ischemic, but not post-ischemic, treatment of MCA occlusion in mice. The pattern of excitatory amino acid efflux in different models of cerebral ischemia derives from the finding that a massive release of glutamate is considered to play a major role in inducing ischemic and post-ischemic cell death (Bullock et al. 1995). In fact, antagonists of glutamate receptors reduce the ischemic penumbra (Obrenovitch 1966; Obrenovitch and Richards 1995), and inhibitors of glutamate release exhibit cerebroprotective activity against ischemia/reperfusion-evoked injury (Molina-Holgado et al. 2002). The neuroprotective effect of Δ9-THC and other cannabinoids is related to the CB1 receptor-mediated inhibition of voltage-sensitive Ca2+ channels, which reduces Ca2+ influx, glutamate release, and excitotoxicity (Iuvone et al. 2004). In fact, the present study shows that Δ9-THC inhibits the release of glutamate and induced hypothermia. Moreover, the effects were inhibited by the CB1 receptor antagonist SR141716. Taken together, these findings suggest that the neuroprotective effect of Δ9-THC is induced only by pre-ischemic but not with post-ischemic treatment.
Both pre- and post-ischemic treatment with cannabidiol resulted in a potent and a long-lasting neuroprotective effect. It has been reported that warming increases pro-inflammatory factors such as leukocyte integrin expression and function on neutrophils and platelets (Forsyth and Levinsky 1990;Kurabayashi et al. 1997; Kochanek and Hallenbeck 1992), and significantly exacerbates functional and structural neurologic injury (Shum-Tim et al. 1998). The leukocytes then interact with intracellular adhesion molecule-1 (ICAM-1), adhere to endothelial cells, and migrate out of the vessels. Cannabidiol has been reported to reduce ICAM-1 expression in experimental diabetes (EI-Remessy et al. 2006). A previous study showed that the neuroprotective effect of cannabidiol is not inhibited by warming indicating that cannabidiol has a CB1 receptor-independent mechanism, unlike Δ9-THC (Hayakawa et al. 2004). Thus, cannabidiol might have a potent anti-inflammatory effect, inhibiting the migration of leukocytes, platelets, and neutrophils by reducing ICAM-1 expression. In this study, cannabidiol significantly inhibited MPO activity in neutrophils at 1 and 20 h after reperfusion via a cannabinoid receptor-independent mechanism. In addition, cannabidiol reduced MPO-immunopositive cells at 3 days after MCA occlusion. Moreover, because the focal cerebral ischemia-induced inflammation response occurs at a later stage than glutamate release (Dirnagl et al. 2003), cannabidiol might have a potent and long-lasting neuroprotective effect.
Inflammation is a critical process after stroke (Danton and Dietrich 2003). Studies have shown the over-expression of inflammatory factors such as ICAM-1, P-selectin, and E-selectin and the accumulation of inflammatory cells such as neutrophils, macrophages, and T-cells (Barone and Feuerstein 1999;Danton and Dietrich 2003; Zhang and Wang 2005). Moreover, these factors have also been known to cause the decrease in CBF due to the failure of cerebral microcirculation at 2—4 h after cerebral ischemia reperfusion (Jones et al. 1981; Garcia et al. 1994). In previously, cannabidiol has prevented cerebral infarction through an increase in CBF (Mishima et al. 2005). In the present study, we found that both cannabidiol and Δ9-THC increased the CBF during MCA occlusion, while only cannabidiol suppresses a decrease in CBF after reperfusion and that cannabidiol inhibits MPO activity in neutrophils via a cannabinoid receptor-independent mechanism. Cannabidiol has a very low affinity (in the micromolar range) for CB1 and CB2 receptors, so these actions might be dependent on a new receptor within the brain such as the G protein-coupled receptor, GPR55, abnormal-cannabidiol receptor.
In conclusion, the present study shows that cannabidiol has a profile of cerebroprotectant activity different from that of Δ9-THC. Cannabidiol, but not Δ9-THC, has a potent and long-lasting neuroprotective effect, when administered both pre- and post-ischemia, through a CB1 and CB2 receptor-independent mechanism. It is to be hoped that cannabidiol will have a palliative action and open new therapeutic vista for treating cerebrovascular disorders.
Acknowledgements
Part of this study was supported by a Grant-in-Aid for Scientific Research (No. 17590479) from the Ministry of Education, Science and Culture of Japan and the Advanced Materials Institute of Fukuoka University and The Naito Foundation.
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Source: Delayed treatment with cannabidiol has a cerebroprotective action via a cannabinoid receptor-independent myeloperoxidase-inhibiting mechanism
