Vanilloid VR₁ receptor is involved in rimonabant-induced neuroprotection

Abstract

1. Recently, a potential neuroprotective effect of rimonabant, independent of the CB₁ receptor interaction, has been proposed. In the present study, the role of transient receptor potential channel vanilloid subfamily member 1, named VR₁, on neuroprotective effect of rimonabant, on global cerebral ischemia in gerbils, was investigated.

2. Rimonabant (0.05–3 mg kg⁻¹), given i.p. 5 min after recirculation, dose dependently antagonized the ischemia-induced decrease in electroencephalographic (EEG) total spectral power and restored relative frequency band distribution 7 days after ischemia.

3. Rimonabant (0.125–0.5 mg kg⁻¹) fully prevented ischemia-induced hyperlocomotion 1 day after ischemia and memory impairment evaluated in a passive avoidance task, 3 days after ischemia.

4. At 7 days after ischemia, the survival of pyramidal cells, in the CA₁ subfield, was respectively 91 and 96%, in the animals given rimonabant 0.25 and 0.5 mg kg⁻¹, compared to the vehicle group. Higher doses were not protective.

5. The protection induced by rimonabant followed a bell-shaped curve, the maximal active doses being 0.25 and 0.5 mg kg⁻¹.

6. Capsazepine (0.01 mg kg⁻¹), a selective VR₁ vanilloid receptor antagonist, completely reversed rimonabant-induced neuroprotective effects against EEG flattening, memory impairment and CA₁ hippocampal neuronal loss.

7. These findings suggest that VR₁ vanilloid receptors are involved in rimonabant's neuroprotection even if other mechanisms can contribute to this effect.

Introduction

In addition to preventing the effects of cannabinoid agonists at CB₁ cannabinoid receptor (Pertwee, 1997; 1999; Nakamura-Palacios et al., 1999), rimonabant has been previously shown to possess a number of biochemical and behavioral effects when administered alone. For example, at normal pharmacological doses (1–5 mg kg⁻¹), rimonabant increases gastrointestinal motility and intestinal transit (Calignano et al., 1997; Colombo et al., 1998; Izzo et al., 1999), reduces food intake and enhances memory as well as produces conditioned place preference (reviewed in Chaperon & Thiebot, 1999). It is also effective in alleviating neuropathic pain in rats (Costa et al., 2005). At moderate to large doses (5–40 mg kg⁻¹), it produces head twitching and scratching in rodents (Aceto et al., 1995; Cook et al., 1998; Rubino et al., 1998; Darmani & Pandya, 2000; Darmani et al., 2003) and vomiting in the least shrew (Cryptotis parva) (Darmani, 2001; Darmani et al., 2003). The mechanisms by which rimonabant produces these effects are not yet fully elucidated. Furthermore, Hansen et al. (2002) also has provided strong evidence for an intrinsic neuroprotective effect of rimonabant on NMDA-damaged neurons, not mediated by CB₁ cannabinoid receptor activation. Rimonabant by itself attenuated the evoked release of [³H]glutamate from rodent hippocampal synaptosomes, suggesting a potential neuroprotective effect independent of the CB₁ receptor (Kofalvi et al., 2003). In a rat stroke model, rimonabant, given 30 min after initiation of permanent middle cerebral artery occlusion, reduced infarct volume approximately of 40% (Berger et al., 2004).

Pathological changes in brain temperature or pH, for example after a severe stroke, may influence the transient receptor potential channel vanilloid subfamily member 1 (TRPV1), known as VR₁ vanilloid receptor (Tominaga et al., 1998), suggesting an important role in events occurring after ischemia (like glutamate release and consequent activation of excitatory aminoacid receptors). VR₁ vanilloid receptor can be activated by endogenously generated compounds such as anandamide and lipoxygenase products which accumulate during brain injury (Marinelli et al., 2002). In addition, VR₁ is present in brain regions (hippocampus) that are highly susceptible to neurodegenerative insults, so this ion channel might contribute to the cellular processes involved in neuronal death (Mezey et al., 2000). The involvement of VR vanilloid receptor has been postulated in the protective effect of capsaicin (Veldhuis et al., 2003; Pegorini et al., 2005) in different ischemic models in rats and gerbils.

Rimonabant inhibits VR₁-mediated effect of anandamide on cytosolic Ca²⁺ concentration, although at concentrations higher than those required for cannabinoid receptor CB₁ antagonism (De Petrocellis et al., 2001).

The current study investigated the possible neuroprotective effect of rimonabant using a model of transient global cerebral ischemia in the gerbil. Capsazepine (CPZ), administered peripherally was used as vanilloid antagonist, to verify the role of VR₁. To quantify the ischemic damage, from 1 h to 7 days after reperfusion we measured different parameters known to be influenced by global cerebral ischemia: electroencephalographic (EEG) spectral power, spontaneous motor activity, memory function and hippocampal CA₁ neuronal count. We measured motor activity on day 1, since ischemia-induced hyperlocomotion is maximal 24 h after occlusion (Araki et al., 1986), memory function on day 3 according to Sala et al. (1997). Since the decrease in EEG has been related to pronounced damage of neurons on day 7 (Suzuki et al., 1983; Hunter et al., 1995; Peruche et al., 1995), EEG spectral power and neuronal counts were measured at this time.

Methods

Animals

Male Mongolian gerbils (Meriones unguiculatus) (Charles River, Calco, Como, Italy) weighing 60–80 g were housed singly in standard laboratory conditions: air-conditioned room (22±2°C), 12-h light/12-h dark light cycle, free access to food and water. The gerbils were allowed to acclimatize themselves to the environment for 1 week before surgical implantation of cortical electrodes. After implanting the EEG electrodes the gerbils were divided into different groups on the basis of the treatment assigned. Each animal received all tests.

All procedures were approved under Italian Governmental decree No. 32/2004.

Surgical procedure

Gerbils were anesthetised with an i.p. injection of chloral hydrate (450 mg kg⁻¹, Sigma, St Louis, MO, U.S.A.) dissolved in saline and given in a volume of 9 ml kg⁻¹. Four electrodes (Bilaney, Dusseldorf, Germany) were implanted for EEG recordings, as described elsewhere (Sala et al., 1997), on the right and left of the parieto-occipital cortex according to brain atlas coordinates (anterior +2, posterior –3, lateral 2, ventral 1.6 from bregma) (Loskota et al., 1974). Another electrode was inserted into the nasal bone as reference ground. The five electrodes were connected to a pedestal (Bilaney, Dusseldorf, Germany) and fixed with acrylic cement (Palavit, New Galetti and Rossi, Milan, Italy). The animals were allowed a week for recovery from surgery before starting the experiment.

EEG recording

Freely moving, awake gerbils were acclimatized in a sound-attenuated Faraday chamber, then their electroencephalogram (EEG) was recorded for 1 h a day, for 3 days, to determine the basal total and relative spectral power. Spectral powers between 0 and 25 Hz (0.2–4.0 Hz δ, 4.2–8.0 Hz ϑ, 8.2–13 Hz α, 13.2–25 Hz β) were evaluated using a resolution of 0.2 Hz. The signals were recorded and processed for fast Fourier transform spectral analysis by means of PC software (PowerLab, AD Instruments Pty Ltd, Australia). EEG recordings were also made during and 7 days after ischemia. Each 1-h spectral power was calculated as the mean of six 1-min recordings taken at 10-min intervals.

Cerebral ischemia

After basal EEG recordings, each gerbil was again lightly anesthetised with 2,2,2-tribromoethanol (200 mg kg⁻¹, 10 ml kg⁻¹ Sigma Aldrich, St Louis, MO, U.S.A.). Throughout surgery, body temperature was kept at 37°C with a heating lamp and 10-min ischemia was induced by bilateral common carotid arteries occlusion as previously described (Braida et al., 2000). The ischemia was verified qualitatively on paper by the complete flattening of the EEG. A group of animals (sham operated) underwent the same surgical procedure except that the carotid arteries were not clamped.

Locomotor activity

Spontaneous motor activity was evaluated as previously described (Braida et al., 2000) in an activity cage (43 cm long × 43 cm wide × 32 cm high: Ugo Basile, Varese, Italy), placed in a sound-attenuating room. The cage was fitted with two parallel horizontal infrared beams 2 cm off the floor. Cumulative horizontal movements were counted every 5 min for 30 min, 1 day after ischemia.

Passive avoidance task

On the 3rd day after ischemia each gerbil was examined in the passive avoidance task. The apparatus (Ugo Basile, Varese, Italy) consisted of a box divided by a guillotine door into two compartments of the same size (22 cm long × 22 cm wide × 21 cm high) in which the floor was a stainless rod grid. One compartment was lit with a 10 W electric light bulb, while the other was dark. The step-through-type passive avoidance task was used, as described by Katoh et al. (1992), with some modifications. Animals were allowed to adapt for 10 min before the training, and retention was checked 1 day after the training. For the adaptation a gerbil was placed in the light compartment, and allowed to explore both compartments by leaving the guillotine door open for 5 min. After 10 min, training started during which each gerbil was placed in the light compartment and allowed to enter the dark. When the gerbil entered the dark compartment, the door automatically closed and an unavoidable scrambled foot shock (1.5 mA) was delivered for 5 s. Each gerbil was then allowed to stay there for 10 s. The procedure was immediately repeated twice. For the retention test, gerbils were placed in the light compartment and the latency to re-enter the dark compartment was recorded for up to 180 s.

Histology

At 7 days after the ischemic injury, all the gerbils were anesthetized with an overdose of chloral hydrate 5% and transcardially perfused with 4% paraformaldehyde (Sigma Aldrich, St Louis, MO, U.S.A.) for histological determination, as previously described (Braida et al., 2003). Brains were removed and placed in the same fixative overnight, then embedded in paraffin wax. Five serial 5-μm coronal hippocampal sections were cut at 1.5, 1.7 and 1.9 mm caudal to the bregma, using a microtome (Leica, Mod. RM2125RT, Solms, Germany), and stained with cresyl violet (Sigma Aldrich, St Louis, MO, U.S.A.). Neurons with a normal appearance in the pyramidal cell layer of the CA₁ sector were counted blind (from coded slides) in each section for each group.

Treatment

Gerbils submitted to ischemia were divided into 10 groups of five animals each, receiving acutely: vehicle s.c.+vehicle i.p.; vehicle s.c.+rimonabant (0.05, 0.125, 0.25, 0.5, 1 and 3 mg kg⁻¹ i.p.), (a kind gift from Sanofi Aventis, Montpellier, France); CPZ (0.01 mg kg⁻¹ s.c.) (Tocris Cookson Ltd, U.K.)+vehicle i.p.; CPZ (0.01 mg kg⁻¹ s.c.)+rimonabant (0.05, 0.125, 0.25, 0.5, 1 and 3 mg kg⁻¹ i.p.). CPZ was given 15 min before bilateral carotid occlusion and rimonabant was injected 5 min after recirculation. Vehicle was given 5 min before or 15 min after ischemia, either s.c. or i.p. Both drugs were dissolved in an appropriate vehicle (Tween-80, ethanol, saline, 1 : 1 : 8 for CPZ and cremophor, ethanol, saline 1 : 1 : 18 for rimonabant) and injected in a volume of 5 ml kg⁻¹ s.c. and i.p. The sham-operated group received the same volume of vehicle.

Statistical analysis

Data were presented as mean (±s.e.m.). Vehicle groups (s.c. and i.p) were pooled for analysis. Total EEG spectral data were expressed as the mean percentage difference from the preischemic value, and relative power as a percentage of the total. EEG, motor activity, latency time, and neuronal counts were analyzed by one-way ANOVA for multiple comparisons, followed by Tukey's or Student's t-test. The accepted level of significance was P<0.05. All statistical analyses were done using Prism version 4 software (GraphPad, U.S.A.).

Results

Physiological parameters – food and water consumption and body weight – were stable throughout the study in all groups (data not shown). Only animals treated with 3 mg kg⁻¹ showed a decrease of body weight (−20%) and a lethality of 20%.

EEG

A quantitative EEG analysis of gerbils treated with increasing doses of rimonabant is given in Figure 1. On day 7 there was a significant overall group effect, in terms of the percentages of preischemic mean total spectral power (F_[12,52]=23.32 P<0.0001, ANOVA). Post hoc analysis showed that, in comparison with sham-operated values, the vehicle group had an 84% decrease in EEG power. Rimonabant significantly antagonized the ischemia-induced EEG flattening at doses of 0.25 and 0.5 mg kg⁻¹. Higher doses (1 and 3 mg kg⁻¹) were ineffective. CPZ per se (0.01 mg kg⁻¹) did not cause any change in comparison with the vehicle group (data not shown). This dose was therefore chosen to study the antagonism. Pretreatment with CPZ significantly antagonized the protective effect of rimonabant alone at the doses of 0.25 and 0.5 mg kg⁻¹.

Figure 1.

Cortically derived EEG total spectral power on day 7 after recirculation, as the difference (Δ%) from the preischemic value in freely moving, awake gerbils given increasing doses (mg kg⁻¹) of rimonabant, 5 min after recirculation, either alone or with capsazepine (CPZ) (0.01 mg kg⁻¹) s.c. 15 min before bilateral carotid occlusion. Each column represents the mean (±s.e.m.) of five animals. ^aP<0.05, ^bP<0.01, ^cP<0.001 compared with sham-operated animals; ^dP<0.001 compared with vehicle; ^eP<0.02 compared with rimonabant alone (one-way ANOVA followed by Tukey's test).

There was a treatment effect in the relative distribution of power over frequency bands (F_[12,52]=10.48, P<0.0001, ANOVA for δ) (F_[12,52]=9.60, P<0.0001, ANOVA for ϑ) (F_[12,52]=5.80, P<0.0001, ANOVA for α) (F_[12,52]=1.90, <0.05, ANOVA for β) 7 days after recirculation. Post hoc analysis indicated that ischemia significantly increased the relative EEG power density in the δ band, and reduced the ϑ and α frequency bands in the vehicle-treated group (Table 1). All the rimonabant doses significantly restored the power density except 3 mg kg⁻¹. Rimonabant increased the β frequency band, though not significantly, starting from 0.125 until 0.5 mg kg⁻¹. No differences were found between vehicle- and CPZ-treated gerbils. The dose of 3 mg kg⁻¹ significantly decreased α frequency band in comparison with sham, vehicle and CPZ alone groups.

Table 1.

Effect of rimonabant and capsazepine on spectral power distribution evaluated 7 days after ischemia, in gerbils

Drug dose (mg kg−1)	δ (0.2–4.0 Hz)	ϑ (4.2–8.0 Hz)	α (8.2–13 Hz)	β (13.2–25Hz)
Sham	45.00±3.00	44.00±2.00	10.00±0.40	1.00±0.30
Vehiclea	62.00±2.00**	32.00±2.20**	5.00±1.70**	1.50±0.50
Rimonabant (0.05)b	40.79±4.64$$	50.50±2.93$$$	8.50±1.50	0.75±0.05
Rimonabant (0.125)b	39.15±2.14$$	46.86±1.57$	12.35±1.47$$	1.80±0.37
Rimonabant (0.25)b	37.60±1.15$$$	49.60±1.15$$	11.20±1.15$$$	2.20±1.17
Rimonabant (0.5)b	38.33±2.18$$	45.33±1.20$	13.33±0.88$$	2.66±1.20
Rimonabant (1)	41.67±3.18$$	49.00±1.73$$$	8.67±1.20	1.00±0.50
Rimonabant (3)	71.67±3.69**	26.67±3.66**	0.83±0.17***,$	0.50±0.01
Capsazepine (0.01)	63.00±1.00**	31.00±2.50**	5.00±1.50**	1.00±0.50
Capsazepine (0.01)c+Rimonabant (0.05)	36.00±1.0$$$	48.00±1.0$$	15.50±0.3**,$$	0.50±0.20
Capsazepine (0.01)c+Rimonabant (0.125)	37.50±2.50$$	52.50±1.5$$$	9.50±3.50	0.75±0.25#
Capsazepine (0.01)c+Rimonabant (0.25)	47.00±9.50	44.00±5.6	9.00±3.80	0.70±0.20###
Capsazepine (0.01)c+Rimonabant (0.5)	37.50±2.5$$	50.50±3.5$$$	11.50±0.50$$	0.75±0.25###

^aVehicle was given 15 min before carotid occlusion or 5 min after recirculation.

^bRimonabant was administered i.p. 5 min after recirculation.

^cCapsazepine was injected s.c. 15 min before carotid occlusion. Each value represents mean (±s.e.m.) of five animals.

^**P<0.01 compared with sham-operated group;

^$P<0.05,

^$$P<0.01,

^$$$P<0.001 compared with the vehicle group and CPZ alone;

^#P<0.05,

^##P<0.01,

^###P<0.001 compared with rimonabant, alone (ANOVA followed by Tukey's test).

Treatment with the VR₁ vanilloid receptor antagonist did not change the power spectral distribution obtained with rimonabant alone at all tested doses. However, in comparison with rimonabant alone, there was a significant decrease in the relative spectral power of the β band starting from the dose of 0.125 mg kg⁻¹.

Spontaneous locomotor activity

There were significant between-group changes in spontaneous locomotor activity 1 day after recirculation (F_[12,52]=10.09, P<0.0001, ANOVA) (Figure 2). As expected, the vehicle group showed a motor increase compared to the sham-operated group (Tukey's test). Post hoc Tukey's test showed that rimonabant completely antagonized ischemia-induced hyperlocomotion at all doses except the lowest and the two higher. CPZ (0.01 mg kg⁻¹), which per se did not affect ischemia-induced hyperlocomotion (data not shown), did not reverse the protective effect of rimonabant.

Figure 2.

Effect of increasing doses (mg kg⁻¹) of rimonabant given s.c. 5 min after recirculation either alone or with capsazepine (CPZ) 0.01 mg kg⁻¹ s.c. 15 min before bilateral carotid occlusion, on spontaneous motor activity evaluated for 30 min, 1 day after ischemia in gerbils. Each column represents the total horizontal counts (mean±s.e.m.) of five animals. ^aP<0.01, ^bP<0.001 compared with sham-operated animals; ^cP<0.01, ^dP<0.001 compared with vehicle; ^eP<0.001 compared with rimonabant alone (one-way ANOVA followed by Tukey's test).

Passive avoidance

The mean latency measured 3 days after recirculation differed significantly between groups (F_[12,52]=9.327, P<0.0001, ANOVA) (Figure 3). Post hoc analysis indicated that vehicle-treated gerbils suffered significant impairment, as shown by the decrease in mean escape latency in comparison with the sham-operated group. Rimonabant improved memory deficit only at 0.25 and 0.5 mg kg⁻¹. When CPZ was given in combination with rimonabant (0.25 and 0.5 mg kg⁻¹) complete antagonism was obtained (Tukey's test). CPZ alone did not affect the ischemia-induced mean escape latency (data not shown).

Figure 3.

Mean (±s.e.m.) escape latency in the passive avoidance task 3 days after ischemia. Increasing doses (mg kg⁻¹) of rimonabant were given s.c. 5 min after recirculation either alone or with capsazepine (CPZ) (0.01 mg kg⁻¹) s.c. 15 min before bilateral carotid occlusion. Each column represents the mean (±s.e.m.) of five animals. ^aP<0.01, ^bP<0.001 compared with sham-operated animals; ^cP<0.001 compared with vehicle; ^dP<0.05, ^eP<0.01 compared with rimonabant alone (one-way ANOVA followed by Tukey's test).

Histology

At 7 days after 10-min ischemia a difference was found in the number of surviving neurons in the CA₁ region (F_[5,24]=256.30, P<0.0001, ANOVA) (Figures 4 and 5). Histological examination of the hippocampus showed a mean loss of 80% of neuronal cells in vehicle-treated gerbils in comparison with the sham-operated group. Survival in the animals given rimonabant 0.25 and 0.5 mg kg⁻¹ was, respectively, 91 and 96%, compared to the vehicle group. Pretreatment with CPZ significantly antagonized rimonabant's neuroprotective effect. CPZ per se caused a mean loss of neuronal cells similar to vehicle (data not shown).

Figure 4.

Photomicrographs of the hippocampal CA₁ region of gerbils with or without 10-min ischemia, 7 days after recirculation. (a) CA₁ hippocampal region. (b) Sham-operated animal. (c) Ischemic animal treated with vehicle, 5 min after recirculation. (d) Ischemic animal treated with CPZ. (e–h) Capsazepine was given 5 min before bilateral carotid occlusion and rimonabant 5 min after recirculation. Doses are mg kg⁻¹. Bar=25 μm.

Figure 5.

Effect of increasing doses of rimonabant (mg kg⁻¹) on neuronal counts 7 days after reperfusion in the CA₁ region of the hippocampus of sham-operated or ischemic gerbils. Rimonabant was given i.p. 5 min after recirculation either alone or with capsazepine (CPZ) (0.01 mg kg⁻¹) s.c. 15 min before bilateral carotid arteries occlusion. Each column represents the mean (±s.e.m.) of five hippocampal sections from the same coronal plane for each animal. n=5 for each group. ^aP<0.001 compared with sham-operated animals; ^bP<0.001 compared with vehicle; ^cP<0.001 compared with rimonabant 0.25 and 0.5 mg kg^–1; ^dP<0.001 compared with corresponding rimonabant alone (one-way ANOVA followed by Tukey's test).

Discussion

The main finding of the present study was that postischemic treatment with the CB₁ cannabinoid receptor antagonist, rimonabant, facilitated neuroprotection, quantified in terms of complete recovery of total and relative spectral power, spontaneous motor activity, memory function and hippocampal CA₁ neuronal density.

EEG and behavioral findings followed a bell-shaped curve, the dose of 0.25 and 0.5 mg kg⁻¹ being protective and the higher being ineffective. A similar trend has been also observed after treatment with some exogenous cannabinoids (including anandamide) for ambulation, defecation and analgesia (Sulcova et al., 1998) neuroprotective (Braida et al., 2003) and rewarding effects (Braida et al., 2004) and also for the head twitch and scratching induced by rimonabant itself (Darmani et al., 2003). The involvement of more than one receptor cannot be excluded to explain the observed biphasic effect. However, the exact mechanism is still to be elucidated.

Some cognitive and sensorimotor deficits have been seen in several human pathologies (heart attack and coronary artery bypass surgery) (Hunter et al., 1998) similar to those observed in our gerbils submitted to global cerebral ischemia and in other reports (Suzuki et al., 1983; Araki et al., 1986; Hunter et al., 1995; Peruche et al., 1995; Sala et al., 1997). Furthermore, the histopathology in the gerbil is similar to that in the hippocampal CA₁ region of human brain after cardiac arrest (Hunter et al., 1995).

Rimonabant prevented ischemia-induced damage, evaluated from the EEG, memory and histology, at doses of 0.25 and 0.5 mg kg⁻¹, with hyperlocomotion starting from 0.125 mg kg⁻¹. Higher doses (1 and 3 mg kg⁻¹) were ineffective in all the parameters. In addition, the dose as 3 mg kg⁻¹ decreased body weight and increased lethality suggesting some degree of toxicity of the drug in this species.

The observed protection indicates that the compound may contrast the cascade of pathological events that lead to neuronal death. Hippocampal CA₁ is the most selectively vulnerable sector to reduced cerebral blood flow (Schmidt-Kastner & Freund, 1991). The survival of CA₁ neurons 7 days after ischemia with rimonabant (0.25 and 0.5 mg kg⁻¹) may explain the protection against EEG flattening, which has been related to severe neuronal damage (Peruche et al., 1995).

There was an increase in the relative power of the δ band in vehicle-treated gerbils, which can be interpreted as a sign of synchronization of neuronal activity (Frigeni et al., 2001). In contrast, ϑ and α frequency bands decreased. Similar EEG changes have been reported for different kinds of focal brain ischemia in different animal species (Gloor et al., 1977; Hossmann & Schuier, 1980; Kataoka et al., 1987). Rimonabant, at all the tested doses, except the highest, caused a significant recovery of the spectral power distribution toward basal values, suggesting recovery of neuronal activity. The cannabinoid antagonist increased the β frequency band, though not significantly. This might reflect the fact that rimonabant per se increased the time spent in wakefulness in freely moving rats (Santucci et al., 1996).

The protection against motor hyperactivity in rimonabant-treated gerbils suggests that the compound reversed the ischemia-induced inability to form spatial maps (O'Neill & Clemens, 2000) because of the loss of CA₁ neurons. We can exclude any stimulating effect of rimonabant, as shown by Compton et al. (1996), since the evaluation was carried out 24 h after treatment.

Rimonabant prevented ischemia-induced memory impairment, since it promoted a survival of CA₁ pyramidal cells, the most vulnerable to hypoxic insult and involved in learning and memory function (LaPoncin-Lafitte et al., 1981; Kiyota et al., 1985). This memory disturbance has been clinically demonstrated during cardiac arrest as revealed by an amnesic syndrome characterized by impaired learning and memory of events after the injury (Volpe & Hirst, 1983).

In agreement with our findings, showing a protective effect of rimonabant, there was a significant reduction of infarct size in rat pups, pretreated with rimonabant, in response to NMDA receptor-induced neurotoxicity (Hansen et al., 2002). Rimonabant also attenuated the evoked [³H]glutamate release from rodent hippocampal synaptosomes with a mechanism independent of the CB₁ receptor (Kofalvi et al., 2003). Berger et al. (2004) showed that treatment with rimonabant, 30 min after permanent middle cerebral artery occlusion, had no significant effect on glutamate release or anandamide levels but reduced infarct volume at 5 h by approximately 40%, suggesting a mechanism other than the activation of cannabinoid receptors. Rimonabant appears to have also its own signalling potential in that it stimulates ERK phosphorylation and, to a lesser degree, AP-1 activity (Berdyshev et al., 2001). ERK activation is a crucial common pathway in the initial stages of stroke pathophysiology regulating cell survival or death.

Pretreatment with CPZ reversed the protective effects of rimonabant, suggesting an involvement of the VR₁ receptor in neuroprotection against ischemic damage. The fact that CPZ did not completely reverse the protective effect of rimonabant on spontaneous motor activity and spectral power distribution, may be attributed to the employed low dose.

Endogenously generated compounds such as anandamide and lipoxygenase products which accumulate during brain injury (Marinelli et al., 2002) have been found to activate both VR₁ vanilloid and CB₁ cannabinoid receptors (Zygmunt et al., 1999; De Petrocellis et al., 2001; Hermann et al., 2003) (even if with different affinity). Both receptors are coexpressed in brain areas including hippocampus which is highly susceptible to neurodegenerative insults (Mezey et al., 2000).

We suggested that, in presence of rimonabant, which blocks CB₁ cannabinoid receptors as antagonist (Rinaldi-Carmona et al., 1994), the increased N-acylethanolamines, including anandamide, can activate and quickly desensitize VR₁ receptors, as already suggested by Veldhuis et al. (2003). This study shows that arvanil, a synthetic AEA analog, attenuated excitotoxic brain injury and cotreatment of arvanil with CPZ reduced the neuroprotective effect of arvanil, indicative of VR₁-mediated neuroprotection. However, it is worthy to note that Veldhuis et al. (2003) also showed that CPZ alone reduced brain injury caused by excitotoxicity. This neuroprotective action of CPZ may imply VR₁-mediated neurotoxicity, arguing against a VR₁-mediated neuroprotection, although the neuroprotection of CPZ is not always via an action on VR₁ (Ray et al., 2003). This apparent discrepancy (neurotoxicity versus neuroprotection) is probably a result of the use of different experimental conditions as proposed by Kim et al. (2005).

Alternatively rimonabant might act as a partial and weak agonist and, subsequently, as a competitive antagonist for VR₁ receptors (De Petrocellis et al., 2001). However, it seems unlikely since rimonabant activates VR₁ receptors at least in vitro at concentrations >5 μM against >1 μM to activate CB₁ receptors (De Petrocellis et al., 2001). Anandamide has been also shown to directly inhibit T-type calcium channels (Chemin et al., 2001), preventing neuronal damage.

Finally, we cannot exclude the possibility that rimonabant acts on glutamate release with a mechanism independent from CB₁ cannabinoid receptor, as reported in vitro in hippocampal synaptosomes of rats and mice by Kofalvi et al. (2003).

A further explanation for the neuroprotection operated by rimonabant may be the presence, in the CNS, of an as yet undefined CBn/VRn receptor, sensitive also to capsaicin, anandamide, arvanil as already suggested by different authors (Brooks et al., 2002; Hajos & Freund, 2002; Veldhuis et al., 2003; Pegorini et al., 2005).

In conclusion, the present data support a potential therapeutic effect of rimonabant as an anti-ischemic drug, as shown by its ability to antagonize ischemia-induced EEG flattening, hyperlocomotion, memory impairment, and CA₁ hippocampal neuronal loss, when given 5 min after recirculation. This effect was inhibited by CPZ, suggesting that the block operated by CB₁ cannabinoid receptor may promote activation of VR₁ receptors by endogenously generated compounds such as anandamide and lipoxygenase products with subsequent desensitization.

Abbreviations

ANOVA

analysis of variance

CNS

central nervous system

CPZ

capsazepine

EEG

electroencephalographic

TRPV1

transient receptor potential channel vanilloid subfamily member 1

VR₁

vanilloid receptor