Characterization of the Neuroprotective Effect of the Cannabinoid Agonist WIN-55212 in an In Vitro Model of Hypoxic-Ischemic Brain Damage in Newborn Rats

Abstract

Brain slices from 7-d-old Wistar rats were exposed to oxygen-glucose deprivation (OGD) for 30 min. OGD slices were incubated with vehicle or with the CB1/CB2 cannabinoid agonist WIN55212 (50 M), the CB1 agonist arachidonyl-2-chloroethylamide (ACEA) (50 M), or the CB2 agonist JW133 (50 M), alone or combined with the CB1 and CB2 receptor antagonist SR 141716 (50 M) or SR 144528 (50 M), respectively. Neuronal damage was assessed by histologic analysis and spectrophotometric determination of lactate dehydrogenase (LDH) efflux into the incubation medium. Additionally, medium glutamate levels were determined by highperformance liquid chromatography (HPLC) and those of tumor necrosis factor ␣ (TNF-␣) by enzyme-linked immunosorbent assay. Finally, inducible nitric oxide synthase (iNOS) and CB1/CB2 receptor expression were determined in slices homogenate by Western blot. Both CB1 and CB2 receptors were expressed in slices. OGD increased CB1 expression, cellular damage, LDH efflux, glutamate and TNF-␣ release, and inducible nitric oxide synthase (iNOS) expression; WIN55212 inhibited all these actions. SR141716 and SR144528 inhibited the effect of R(ϩ)-WIN-55212-2 (WIN), as well as the reduction of LDH efflux by ACEA and JW133, respectively. In conclusion, WIN55212 afforded robust neuroprotection in the forebrain slices exposed to OGD, by acting on glutamatergic excitotoxicity, TNF-␣ release, and iNOS expression; this neuroprotective effect seemed to be mediated by CB1 and CB2 receptors. P erinatal hypoxia-ischemia remains the single most important cause of brain injury in the newborn, leading to death or lifelong sequelae (1,2). The complexities of neonatal hypoxic-ischemic encephalopathy (NHIE) pathophysiology suggest that successful neuroprotection could be achieved only with a multitherapeutic approach (2). In recent years, interest in the neuroprotective possibilities of cannabinoids has grown (3-5). Endocannabinoids emerge as natural brain protective substances in different damaging situations (3,4); in newborn rats, enhanced levels of cannabinoids have been observed in the brain after acute excitotoxic insult (6). Exogenous cannabinoid agonists are neuroprotective in different paradigms of brain injury (7,8); they inhibit intracellular calcium influx, reduce glutamate and TNF-␣ release, decrease stimulated iNOS expression, induce hypothermia, and exert immunomodulatory and antioxidant actions (3-5,9,10). Some of these effects are dependent on the activation of the principal brain cannabinoid receptors, the CB1 receptors, and others are dependent on the molecular properties of the cannabinoid or the activation of non-CB1 receptors (3). There are few studies regarding the possible neuroprotective effect of cannabinoids in newborns. Early studies describe that the administration of anandamide (11) or the cannabinoid agonist ⌬ (9)-tetrahydrocannabinol (12) affords neuroprotection in a newborn rat model with excitotoxic neuronal injury. It has also been demonstrated that the cannabinoid agonist WIN, prevents ea...

Cannabinoids in Neurodegenerative Disorders and Stroke/Brain Trauma: From Preclinical Models to Clinical Applications

“…This evidence derives from studies that were initiated >10 years ago using newborn rat forebrain slices subjected to oxygen glucose deprivation and exposed to the CB 1 R/CB 2 R agonist WIN55212-2, which reduced cell death, decreasing glutamate and cytokine release, as well as inducible nitric oxide synthase expression, effects that were abolished by either CB 1 R or CB 2 R antagonists [65]. In newborn rats exposed to severe anoxia or to acute hypoxiaischemia [66,67], postinsult administration of WIN55212-2 afforded a strong neuroprotective effect, abolished by either CB 1 R or CB 2 R antagonists, too, as well as increasing neuronal and oligodendroglial cell proliferation in the subventricular zone 7 days after neonatal HI in rats [68].…”

The Cannabinoid CB2 Receptor as a Target for Inflammation-Dependent Neurodegeneration

“…Second, CB1 is involved in the regulation of vasodilation, both directly through vascular CB1 receptors and indirectly through the inhibition of the vasoconstrictor endothelin-1 [60,71]. Third, CB1 receptors are known to regulate the release of pro-inflammatory factors such as NO and TNF-in the acute phase of injury [18,55].…”

Cannabinoid as a neuroprotective strategy in perinatal hypoxic-ischemic injury

“…Moreover, the use of CB2 receptor agonists has revealed promising results in different paradigms of neonatal hypoxic-ischemic brain injury [133,150] , reducing cell death, accompanied by modulations of glutamate release, cytokine production, and cyclooxygenase-2 and iNOS expression, supporting the hypothesis that the protective effect of CB2 receptor relies mostly on its anti-inflammatory effects. This provides new hints on its possible use as a neuroprotective target after perinatal asphyxia.…”

“…Various studies have elucidated that cannabinoids can inhibit intracellular calcium influx, reduce the release of glutamate and tumor necrosis factor-alpha (TNF-α), decrease stimulated inducible nitric oxide synthase (iNOS) expression, induce hypothermia, and exert immunomodulatory and antioxidant actions [129][130][131][132][133][134] .…”

“…1, unpublished data), an early and sensitive biochemical marker of cerebral injury in term newborn infants with hypoxic-ischemic encephalopathy [136][137][138][139][140] . Another group reported that WIN also had a neuroprotective effect on 7-d- old rat forebrain slices exposed to oxygen-glucose deprivation, based on the reductions of glutamate release, TNF-α release and iNOS expression [133] . Lately, this group demonstrated that WIN successfully reduced brain injury [141] and enhanced subventricular zone cell proliferation, oligodendrogenesis, white matter remyelination, and neuroblast generation in newborn rats [142] .…”