RESULTS

Loss of cellular ion homeostasis was initiated by unilateral intrastriatal injection of 0.5 μl of the Na⁺/K⁺-ATPase inhibitor ouabain (1 mM) into 7- to 8-d-old Wistar rats (Lees et al., 1990; Lees, 1991; Lees and Leong, 1995; Stelmashook et al., 1999). Twelve animals received an additional injection with Δ⁹-THC (1 mg/kg, i.p.), and five animals received both Δ⁹-THC and the CB₁ antagonist SR141716 (1 mg/kg, i.p.) 30 min before ouabain injection.

ADC maps of brain tissue water, calculated from diffusion-weighted MR images acquired 15 min after ouabain injection, showed hypointense regions with reduced ADC values (∼0.67 × 10⁻³mm²/sec) in the ipsilateral hemisphere in all animals (Fig. 1). Normal ADC values (∼1.11 × 10⁻³mm²/sec) were measured in the contralateral hemisphere of the ouabain-injected rats (Fig. 1) and in the brain of the control animals, which received only vehicle (0.5 μl Tris-HCl; 40 mM; pH 7.4). The reduction in ADC values in the ipsilateral hemispheres after ouabain injection is considered to reflect neuronal swelling, i.e., cytotoxic edema, because of a relocation of part of the extracellular water into depolarized cells (Van Lookeren Campagne et al., 1994; Dijkhuizen et al., 1996). In this acute phase, the volume of brain tissue with cytotoxic edema was 22% smaller in the Δ⁹-THC group (p < 0.05) (Figs. 1,2). Coinjection of the CB₁receptor antagonist SR141716 completely abolished the Δ⁹-THC-induced effect (Figs. 1, 2). The same brain regions, including the caudate putamen, cortex, and hippocampus, were affected in all animals (Fig. 1).

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Fig. 1.

Three adjacent coronal ADC maps of neonatal rat brain 15 min after ouabain injection. a, No treatment;b, THC treatment; c, THC + SR141716 treatment. Hypointensities correlate to cytotoxic edema.

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Fig. 2.

Mean lesion volumes (±SE) of ouabain-injected rats on days 0 and 7, based on ADC and T₂ map analysis.

After 7 d, sharply delineated hyperintense regions were observed in the ADC maps (data not shown), indicative of the formation of vasogenic edema as well as tissue loss and ventricle dilatation. The volume of infarcted tissue as calculated from ADC maps was 36% smaller in Δ⁹-THC-treated rats (p < 0.01) (Fig. 2). SR141716 abolished the protective effect (p < 0.005) (Fig. 2). An 18% increase in infarct volume was observed in the CB₁-antagonist-treated rats compared with nontreated rats (Fig. 2). This trend did not reach statistical significance. Neuroprotection was observed in brain regions known to express CB₁ receptors, such as the hippocampus, caudate putamen, and cortex (Fernandez-Ruiz et al., 2000). Western blot analysis confirmed the presence of CB₁-like receptors, but as expected, not of CB₂-like receptors, in 7- and 14-day-old rat brains (data not shown).

The effects of Δ⁹-THC-treatment on neuronal damage after 7 d was also assessed using T₂-weighted imaging and verified with a histological procedure. T₂ maps demonstrated both hyperintensities and hypointensities (Fig.3). Both types of T₂ abnormalities indicate pathological changes. Hyperintense areas correspond to vasogenic edema, tissue loss, and ventricle dilatation, whereas hypointensities correlate to astrogliosis, i.e., phenotypic changes (hypertrophy) and proliferation of astroglial cells in response to neuronal injury (Fig.4) (Feuerstein et al., 1994; Van Lookeren Campagne et al., 1994). Lesion volumes, based on the combination of hyperintense and hypointense abnormalities on T₂maps, were reduced by 36% (p < 0.005) in Δ⁹-THC-treated rats compared with the control group (Figs. 2, 3). Infarct size based on T₂-hyperintense abnormalities was reduced by 35% in the Δ⁹-THC-treated group (p < 0.05) compared with the control animals (Figs. 2, 3). This effect could be blocked by the CB₁ antagonist (p < 0.05) (Figs. 2, 3). Conventional histology (Nissl and hematoxylin–eosin staining) showed the same lesion pattern on brain sections and confirmed the assessment made by ADC and T₂ map analysis (data not shown).

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Fig. 3.

Three adjacent coronal T₂ maps of neonatal rat brain 7 d after ouabain injection. a,No treatment; b, THC treatment; c, THC + SR141716 treatment. Hyperintensities correlate to ventricle dilatation, vasogenic edema, and tissue loss, whereas hypointensities correlate to astrogliosis.

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Fig. 4.

GFAP staining of a brain section of a ouabain-injected rat. Markedly increased staining was observed in the thalamus, external capsule, and cortex of the injected hemisphere, whereas normal staining was seen in the contralateral hemisphere.

The hypointense regions on the T₂ maps corresponded to regions exhibiting increased staining for GFAP staining, which is typical of astrogliosis, on brain sections of ouabain-treated rats (Fig. 4). No indications were found for hemorrhage. Astrogliotic tissue constituted 40% of the lesion on T₂ maps and usually surrounded the edematous tissue and the dilatated ventricles (Fig. 3). The volume of astrogliotic tissue in Δ⁹-THC-treated rats was reduced by 37% compared with nontreated rats (p < 0.05). Importantly, this effect was not blocked by the CB₁ receptor antagonist (Fig.2).

DISCUSSION

In the brain at least 40% of the energy produced by mitochondrial respiration is required by the Na⁺/K⁺-ATPase to maintain ion gradients across the cell membranes. Energy levels in the brain can be compromised by a lack of glucose and oxygen or by defects in the respiratory chain such as occurring in stroke and Parkinson’s disease, respectively. Na⁺/K⁺-ATPase function is inhibited during energy failure. This may lead to a prolonged depolarization of the neuron, excessive release, and reversal of the uptake of excitatory amino acids, i.e., the induction of excitotoxicity (Dirnagl et al., 1999; Doble, 1999; Nicotera et al., 1999). Ouabain inhibits Na⁺/K⁺-ATPases and is a very potent neurotoxin that leads to pancellular necrosis and infarction (Lees, 1991). It is used to study the involvement of Na⁺/K⁺-ATPase in CNS pathology (Lees et al., 1990; Lees, 1991; Lees and Leong, 1994,1995; Stelmashook et al., 1999). Ouabain rapidly perturbs ion homeostasis and induces cell swelling and glutamate-dependent damage of cells, which can be prevented, at least in part, by blockade of the NMDA receptor (Lees et al., 1990; Lees, 1991; Lees and Leong, 1994,1995; Cousin et al., 1995; Greene and Greenamyre, 1996; Basarsky et al., 1999; Buckley et al., 1999; Doble, 1999; Stelmashook et al., 1999).

The diffusion-weighted MRI data acquired 15 min after the injection of ouabain showed that activation of the CB₁receptor by Δ⁹-THC attenuates in vivo cell swelling in an early phase after the induction of excitotoxicity. Activation of the CB₁ receptor on presynaptic neuron terminals can lead to inhibition of the Ca²⁺ influx via N-, and P/Q-type voltage-sensitive calcium channels, thereby preventing the release of glutamate and subsequent depolarization of other neurons (Shen et al., 1996; Shen and Thayer, 1998). Furthermore, cannabinoids can induce hyperpolarization via the CB₁-mediated activation of inward rectifying and A-type K⁺-channels (Deadwyler et al., 1993). Hyperpolarization raises the threshold to depolarization, which therefore may contribute to the observed reduction in the development of cytotoxic edema.

ADC and T₂ data acquired after 7 d demonstrated that Δ⁹-THC or its CB₁-active metabolite 11-HO-Δ⁹-THC reduced neuronal damage by 36%. Various mechanisms could underlie the following observed effects.

(1) Δ⁹-THC-induced hypothermia (Pertwee, 1997). In our experimental setup, the body temperature of the rats was externally controlled by an infrared lamp and a water-heated pad, making the contribution of cannabinoid-induced hypothermia to the protective effects unlikely.

(2) Anti-oxidative properties of Δ⁹-THC (Hampson et al., 1998). Anti-oxidant activity most likely does not play a major role in our model, because (1) the neuroprotective effects were blocked by the CB₁ antagonist, and (2) the dose of Δ⁹-THC (1 mg/kg) is low compared with the high dose of anti-oxidant (50–100 mg/kg) required for effective protection in other studies (Hara et al., 1990),

(3) Downregulation of brain-resident mast cells by activation of a CB₂-like receptor (Skaper et al., 1996a,b). This process is also unlikely to be effective in our model, because (1) the neuroprotective effects were blocked by SR141716, (2) Δ⁹-THC has been shown to have a low efficacy on the stimulation of the CB₂-receptor (Pertwee, 1997), and (3) a CB₂-like receptor could not be detected.

(4) Closing of N- and P/Q-type calcium channels via a CB₁-receptor-mediated mechanism (Shen and Thayer, 1996; Di Marzo et al., 1998). Reduced influx of calcium decreases directly the activation of destructive pathways, e.g., it prevents the activation of neuronal NO-synthase (Hillard et al., 1999) and it reduces glutamatergic transmission, i.e., induction of excitotoxicity (Shen et al., 1996; Shen and Thayer, 1998). This CB₁-mediated mechanism is likely to dominate the observed neuroprotective effects in the late phase in our model.

Neuroprotection by Δ⁹-THC was observed in the hippocampus, striatum, and cortex. Western blots verified the presence of CB₁-receptors in neonatal rat brain. Previously, radioligand binding studies have demonstrated that CB receptors were expressed in the cerebral cortex, striatum, hippocampus, cerebellum, and brainstem at postnatal day 5 (Romero et al., 1997; Fernández-Ruiz et al., 2000). The presence of mRNA transcripts for the CB₁ receptor was also observed in some forebrain areas, such as the subventricular zone of the striatum, nucleus accumbens, and neocortex. The abundance of the mRNA transcripts was high at gestational day 21 but tended to wane to postnatal day 5 and disappeared at day 30 (Romero et al., 1997). Thus, these data support a CB₁-mediated neuroprotection.

The gliotic response to neuronal injury after ouabain injection has been reported in adult rats (Lees and Leong, 1995). We also observed astrogliosis surrounding vasogenic edema in our model. Δ⁹-THC treatment reduced the volume of brain tissue with astrogliosis. Although astrocytes express CB₁-like receptors sensitive to SR141716 (Fernandez-Ruiz et al., 2000), administration of the SR141716 did not block the reduction in astrogliotic tissue. Therefore, this process does not seem to be mediated by a CB₁-like receptor. Noteworthy, dexanabinol, a nonpsychotropic cannabinoid, inhibits tumor necrosis factor-α (TNF-α) release from astrocytes (Shohami et al., 1997). It is thought that TNF-α sets the stage for inflammatory reactions including glial cell activation and proliferation (Feuerstein et al., 1994). Δ⁹-THC is known to inhibit the release of TNF-α from immune cells (Klein et al., 1998). Thus, Δ⁹-THC or one of its metabolites might also inhibit the release of TNF-α from astrocytes (or immune cells) and reduce astrogliosis. Furthermore, nonpsychotropic cannabinoid metabolites inhibit prostaglandin synthesis (Burstein, 1999). Cyclooxygenase-2 activation has been shown to induce astrogliosis (Brambilla et al., 1999). Thus, it is also possible that nonpsychotropic metabolites of Δ⁹-THC reduce astrogliosis via this mechanism. Further research is required to investigate the mechanism of Δ⁹-THC-induced reduction of astrogliosis.

Our data may suggest that endogenous cannabinoids could be released on neuronal injury and protect neurons in the periphery of the infarct: on the ADC maps we observed a trend toward a larger infarct (+18%) in antagonist-treated rats compared with nontreated rats (Figs. 2, 3). It should be noted that tissue was considered to be pathological only in case ADC or T₂ values differed more than twice the SD of the mean value in the contralateral hemisphere. The periphery of the infarct with smaller changes in ADC or T₂is not incorporated in this way but may nevertheless have benefited from endogenous release of cannabinoids. Interestingly, the cortex was not severely damaged in the nontreated animals, whereas in the SR141716-injected animals this area was infarcted (Fig.3 a,c). It has been shown that glutamate-induced neurotoxicity leads to the formation of anandamide and its precursorN-acylphosphatidylethanolamine (Hansen et al., 1998, 2000). However, SR141716 is an inverse agonist. It is possible that SR141716 blocks constitutively active CB₁ receptors (Pertwee, 1997). Yet, Mechoulam and coworkers have found that the endogenous cannabinoid 2-AG is upregulated in the first hours after closed head injury in mice and that administration of 2-AG reduces edema formation via the CB₁ receptor, which strongly corroborates our findings (R. Mechoulam, personal communication).

In summary, we have shown that in an in vivo model of neurodegeneration Δ⁹-THC reduces neuronal damage via a CB₁-receptor-mediated mechanism. This holds in both the acute and late phase after induction of excitotoxicity. Δ⁹-THC inhibits astrogliosis via a non-CB₁-receptor-controlled mechanism. The results strengthen the concept that the endogenous cannabinoid system may serve to establish a defense system for the brain. This system may be functional in several neurodegenerative diseases in which excitotoxicity is thought to play a role, such as amyotrophic lateral sclerosis, Huntington’s and Parkinson’s diseases, and also in acute neuronal damage as found in stroke and traumatic brain injury. It is conceivable that the endogenous cannabinoid system can be exploited for therapeutic interventions in these types of primarily incurable diseases.