The immaturity of the CNS at birth greatly affects injury after stroke but the contribution of the blood-brain barrier (BBB) to the differential response to stroke in adults and neonates is poorly understood. We asked if the structure and function of the BBB is disrupted differently in neonatal and adult rats by transient middle cerebral artery occlusion. In adult rats, albumin leakage into injured regions was markedly increased during 2–24 h reperfusion but leakage remained low in the neonates. Functional assays employing intravascular tracers in the neonates showed that BBB permeability to both large (70-kDa dextran) and small (3-kDa dextran, Gd-DTPA) tracers remained largely undisturbed 24h after reperfusion. The profoundly different functional integrity of the BBB was associated with the largely nonoverlapping patterns of regulated genes in endothelial cells purified from injured and uninjured adult and neonatal brain at 24h (endothelial transcriptome, 31,042 total probe sets). Within significantly regulated 1,266 probe sets in injured adults and 361 probe sets in neonates, changes in the gene expression of the basal lamina components, adhesion molecules, the tight junction protein occludin, and MMP-9 were among the key differences. The protein expression of collagen-IV, laminin, claudin-5, occludin and ZO-1 was also better preserved in neonatal rats. Neutrophil infiltration remained low in acutely injured neonates but neutralization of CINC-1 in the systemic circulation enhanced neutrophil infiltration, BBB permeability and injury. The markedly more integrant BBB in neonatal brain than in adult brain after acute stroke may have major implications for the treatment of neonatal stroke.

Background and Purpose— Ischemic stroke continues to be one of the main causes of death worldwide. Inflammation accounts for a large part of damage in this pathology. The cannabinoid type 2 receptor (CB2R) has been proposed to have neuroprotective properties in neurological diseases. Therefore, our aim was to determine the effects of the activation of CB2R on infarct outcome and on ischemia-induced brain expression of classic and alternative markers of macrophage/microglial activation. Methods— Swiss wild-type and CB2R knockout male mice were subjected to a permanent middle cerebral artery occlusion. Mice were treated with either a CB2R agonist (JWH-133), with or without a CB2R antagonist (SR144528) or vehicle. Infarct outcome was determined by measuring infarct volume and neurological outcome. An additional group of animals was used to assess mRNA and protein expression of CB2R, interleukin (IL)-1β, IL-6, tumor necrosis factor α (TNF-α), monocyte chemoattractant protein–1 (MCP-1), macrophage inflammatory peptide (MIP) –1α, RANTES, inducible nitric oxide synthase (iNOS), cyclooxygenase-2, IL-4, IL-10, transforming growth factor β (TGF-β), arginase I, and Ym1. Results— Administration of JWH-133 significantly improved infarct outcome, as shown by a reduction in brain infarction and neurological impairment. This effect was reversed by the CB2R antagonist and was absent in CB2R knockout mice. Concomitantly, administration of JWH-133 led to a lower intensity of Iba1+ microglia/macrophages and a decrease in middle cerebral artery occlusion–induced gene expression of both classic (IL-6, TNF-α, MCP-1, MIP-1α, RANTES, and iNOS) and alternative mediators/markers (IL-10, TGF-β, and Ym1) of microglial/macrophage activation after permanent middle cerebral artery occlusion. Conclusions— The inhibitory effect of CB2R on the activation of different subpopulations of microglia/macrophages may account for the protective effect of the selective CB2R agonist JWH-133 after stroke.

1These authors contributed equally to this study.Abbreviations used: BCCAO, bilateral common carotid arteries occlusion; COX-2, cyclooxygenase-2; EAAT, excitatory amino acid transporter; IjB alpha, inhibitory kappa B alpha; iNOS, inducible nitric oxide synthase; IPC, ischemic preconditioning; IT, ischemic tolerance; LPS, lipopolysaccharide; MCA, middle cerebral artery; NF-jB, nuclear factor kappa B; pMCAO, permanent middle cerebral artery occlusion; TACE, TNF-alpha converting enzyme; TLR, toll-like receptor; TNF-a, tumor necrosis factor alpha. AbstractIt has been demonstrated that a short ischemic event (ischemic preconditioning, IPC) results in a subsequent resistance to severe ischemia (ischemic tolerance, IT). We have recently demonstrated the role of innate immunity and in particular of toll-like receptor (TLR) 4 in brain ischemia. Several evidences suggest that TLR4 might also be involved in IT. Therefore, we have now used an in vivo model of IPC to investigate whether TLR4 is involved in IT. A 6-min temporary bilateral common carotid arteries occlusion was used for focal IPC and it was performed on TLR4-deficient mice (C57BL/ 10ScNJ) and animals that express TLR4 normally (C57BL/ 10ScSn). To assess the ability of IPC to induce IT, permanent middle cerebral artery occlusion was performed 48 h after IPC. Stroke outcome was evaluated by determination of infarct volume and assessment of neurological scores. IPC caused neuroprotection as shown by a reduction in infarct volume and better outcome in mice expressing TLR4 normally. TLR4-deficient mice showed less IPC-induced neuroprotection than wild-type animals. Western blot analysis of tumor necrosis factor alpha (TNF-a), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) showed an up-regulation in the expression of these proteins in both substrains of mice measured 18, 24 and 48 h after IPC, being higher in mice with TLR4. Similarly, nuclear factor-kappa B (NF-jB) activation was observed 18, 24 and 48 h after IPC, being more intense in TLR4-expressing mice. These data demonstrate that TLR4 signalling is involved in brain tolerance as shown by the difference in the percentage of neuroprotection produced by IPC between ScSn and ScNJ (60% vs. 18%). The higher expression of TNF-a, iNOS and cyclooxygenase-2 and NF-jB activation in mice expressing TLR4 is likely to participate in this endogenous neuroprotective effect.

Peroxisome proliferator-activated receptors gamma (PPAR␥) are nuclear receptors with essential roles as transcriptional regulators of glucose and lipid homeostasis. PPAR␥ are also potent anti-inflammatory receptors, a property that contributes to the neuroprotective effects of PPAR␥ agonists in experimental stroke. The mechanism of these beneficial actions, however, is not fully elucidated. Therefore, we have explored further the actions of the PPAR␥ agonist rosiglitazone in experimental stroke induced by permanent middle cerebral artery occlusion (MCAO) in rodents. Rosiglitazone induced brain 5-lipoxygenase (5-LO) expression in ischemic rat brain, concomitantly with neuroprotection. Rosiglitazone also increased cerebral lipoxin A 4 (LXA 4 ) levels and inhibited MCAO-induced production of leukotriene B4 (LTB 4 ). Furthermore, pharmacological inhibition and/or genetic deletion of 5-LO inhibited rosiglitazone-induced neuroprotection and downregulation of inflammatory gene expression, LXA 4 synthesis and PPAR␥ transcriptional activity in rodents. Finally, LXA 4 caused neuroprotection, which was partly inhibited by the PPAR␥ antagonist T0070907, and increased PPAR␥ transcriptional activity in isolated nuclei, showing for the first time that LXA 4 has PPAR␥ agonistic actions. Altogether, our data illustrate that some effects of rosiglitazone are attributable to de novo synthesis of 5-LO, able to induce a switch from the synthesis of proinflammatory LTB 4 to the synthesis of the proresolving LXA 4 . Our study suggests novel lines of study suchastheinterestoflipoxin-likeanti-inflammatorydrugsortheuseofthesemoleculesasprognosticand/ordiagnosticmarkersforpathologies in which inflammation is involved, such as stroke.