Endocannabinoids potently protect the newborn brain against AMPA-kainate receptor-mediated excitotoxic damage

Abstract

Brain lesions induced in newborn mice or rats by the glutamatergic agonists ibotenate (acting on NMDA and metabotropic receptors) or S-bromowillardiine (acting on AMPA-kainate receptors) mimic some aspects of white matter cysts and transcortical necrosis observed in human perinatal brain damage associated with cerebral palsy. Exogenous and endogenous cannabinoids have received increasing attention as potential neuroprotective agents in a number of neurodegenerative disorders of the adult. One recent study showed neuroprotection by the cannabinoid agonist WIN-55212 in a newborn rat model of acute severe asphyxia. The present study was designed to assess the neuroprotective effects of the endogenous cannabinoid anandamide using a well-defined rodent model of neonatal excitotoxic brain lesions. In this model, anandamide provided dose-dependent and long-lasting protection of developing white matter and cortical plate reducing the size of lesions induced by S-bromowillardiine. Anandamide had only marginal neuroprotective effect against ibotenate-induced cortical grey matter lesions. Anandamide-induced neuroprotection against AMPA-kainate receptor-mediated brain lesions were blocked by a CB1 antagonist but not by a CB2 antagonist. Furthermore, anandamide effects were mimicked by a CB1 agonist but not by a CB2 agonist. Real-time PCR confirmed the expression of CB1 receptors, but not CB2 receptors, in the untreated newborn neocortex. Finally, neuroprotective effects of anandamide in white matter involved increased survival of preoligodendrocytes and better preservation of myelination. The present study provides experimental support for the role of endocannabinoids as a candidate therapy for excitotoxic perinatal brain lesions.

Figure 1

TPM protects against S-bromowillardiine-induced brain lesions. Cresyl violet-stained sections showing brain lesions induced by S-bromowillardiine injected at P5 and studied at the age of P10. (a) Brain from a pup cotreated with intracerebral S-bromowillardiine and i.p. vehicle, showing the typical neuronal loss in layers II–VI (arrow) and the white matter cystic lesion (^*). (b) Brain from pup cotreated with intracerebral S-bromowillardiine and i.p. anandamide (10 mg kg⁻¹). LV, lateral ventricle. Bar=40 μm.

Figure 2

Anandamide has marginal effects on the size of ibotenate-induced lesions. Mouse pups were injected with intracerebral ibotenate on P5 and sacrificed on P10. Pups were injected with a single i.p. injection of vehicle, anandamide or anandamide+URB597 (0.3 mg kg⁻¹) immediately after ibotenate injection or with intracerebral MK-801 concomitantly with ibotenate. Bar represents mean length of the brain lesions at P10+s.e.m. Asterisks indicate statistically significant difference from black bars; ^**P<0.01; ^***P<0.001 in ANOVA with Bonferroni's multiple comparison tests.

Figure 3

Anandamide significantly mitigated S-bromowillardiine-induced lesions. Mouse or rat pups were injected with intracerebral S-bromowillardiine on P5. (a) Mouse pups were injected with a single i.p. injection of vehicle or anandamide immediately after S-bromowillardiine injection, or with intracerebral NBQX concomitantly with S-bromowillardiine. Pups were sacrificed on P10. (b) Mouse pups were injected with a single i.p. injection of vehicle or anandamide (Anand) immediately after S-bromowillardiine injection. Pups were killed on P30. (c) Rat pups were injected with a single i.p. injection of vehicle or anandamide (Anand) immediately after S-bromowillardiine injection. Pups were killed on P10. (d) Mouse pups were injected with a single i.p. injection of vehicle, ACPA, nabilone (Nab), or JWH133 (JWH) immediately after S-bromowillardiine injection. Pups were sacrificed on P10. (e) Mouse pups were injected with a single i.p. injection of drug of combination of drugs indicated on the X-axis immediately after S-bromowillardiine injection. Pups were killed on P10. (f) Mouse pups were injected with a single i.p. injection of anandamide administered at immediately, 4, 8, 12, or 24 h after S-bromowillardiine injection. Pups were sacrificed on P10. Bar represents mean length of the brain lesions at P10 or P30+s.e.m. Asterisks indicate statistically significant difference from black bars; ^*P<0.05; ^**P<0.01; ^***P<0.001 in ANOVA with Bonferroni's multiple comparison tests.

Figure 4

Effects of a single i.p. injection of anandamide (10 mg kg⁻¹) on S-bromowillardiine-induced changes in white matter myelin and pre-oligodendrocytes. (a, b) MBP immunostaining 9 days after S-bromowillardiine-vehicle (a) or S-bromowillardiine-anandamide (b) injection on P5. Bar=40 μm. (c, d) O4 immunostaining 24 h after S-bromowillardiine-vehicle (c) or S-bromowillardiine-anandamide (d) injection on P5. Arrows point to examples of O4-positive cells. Bar=20 μm. (e) Quantitative analysis of O4-stained cells in the periventricular white matter at the lesion site. Bars represent means±s.e.m. Asterisk indicates statistically significant difference from vehicle group in a Student's t-test; ^*P<0.05.

Figure 5

CB1 receptors are highly expressed in the P5 neocortex. (a) Real time PCR quantification of CB1 and CB2 receptor mRNAs in neocortices of untreated P5 mice. (b, c) Real time PCR quantification of CB1 (b) and CB2 (c) receptor mRNAs in neocortices of P5 mice intracerebrally injected 3 h earlier with PBS, ibotenate, or S-bromowillardiine. Data are presented as mean CB receptor/HPRT ratios+s.e.m. Asterisks indicate statistically significant difference from black bars; ^**P<0.01 in ANOVA with Bonferroni's multiple comparison tests.