David Fernández-López

Blood-brain barrier permeability is increased after acute adult stroke but not neonatal stroke in the rat

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 whether 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), gadolinium (III)-diethyltriaminepentaacetic acid tracers remained largely undisturbed 24 h 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 24 h (endothelial transcriptome, 31,042 total probe sets). Within significantly regulated 1266 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 matrix metalloproteinase-9 were among the key differences. The protein expression of collagen-IV, laminin, claudin-5, occludin, and zonula occludens protein 1 was also better preserved in neonatal rats. Neutrophil infiltration remained low in acutely injured neonates but neutralization of cytokine-induced neutrophil chemoattractant-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.

Cannabinoid Type 2 Receptor Activation Downregulates Stroke-Induced Classic and Alternative Brain Macrophage/Microglial Activation Concomitant to Neuroprotection

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)-1&bgr;, IL-6, tumor necrosis factor &agr; (TNF-&agr;), monocyte chemoattractant protein–1 (MCP-1), macrophage inflammatory peptide (MIP) –1&agr;, RANTES, inducible nitric oxide synthase (iNOS), cyclooxygenase-2, IL-4, IL-10, transforming growth factor &bgr; (TGF-&bgr;), 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-&agr;, MCP-1, MIP-1&agr;, RANTES, and iNOS) and alternative mediators/markers (IL-10, TGF-&bgr;, 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.

Synthesis of Lipoxin A4 by 5-Lipoxygenase Mediates PPARγ-Dependent, Neuroprotective Effects of Rosiglitazone in Experimental Stroke

Peroxisome proliferator-activated receptors gamma (PPARγ) are nuclear receptors with essential roles as transcriptional regulators of glucose and lipid homeostasis. PPARγ are also potent anti-inflammatory receptors, a property that contributes to the neuroprotective effects of PPARγ agonists in experimental stroke. The mechanism of these beneficial actions, however, is not fully elucidated. Therefore, we have explored further the actions of the PPARγ 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 A4 (LXA4) levels and inhibited MCAO-induced production of leukotriene B4 (LTB4). Furthermore, pharmacological inhibition and/or genetic deletion of 5-LO inhibited rosiglitazone-induced neuroprotection and downregulation of inflammatory gene expression, LXA4 synthesis and PPARγ transcriptional activity in rodents. Finally, LXA4 caused neuroprotection, which was partly inhibited by the PPARγ antagonist T0070907, and increased PPARγ transcriptional activity in isolated nuclei, showing for the first time that LXA4 has PPARγ 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 LTB4 to the synthesis of the proresolving LXA4. Our study suggests novel lines of study such as the interest of lipoxin-like anti-inflammatory drugs or the use of these molecules as prognostic and/or diagnostic markers for pathologies in which inflammation is involved, such as stroke.

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

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 μM), the CB1 agonist arachidonyl-2-chloroethylamide (ACEA) (50 μM), or the CB2 agonist JW133 (50 μM), alone or combined with the CB1 and CB2 receptor antagonist SR 141716 (50 μM) or SR 144528 (50 μM), 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 high-performance liquid chromatography (HPLC) and those of tumor necrosis factor α (TNF-α) 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-α release, and inducible nitric oxide synthase (iNOS) expression; WIN55212 inhibited all these actions. SR141716 and SR144528 inhibited the effect of R(+)-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-α release, and iNOS expression; this neuroprotective effect seemed to be mediated by CB1 and CB2 receptors.

The Cannabinoid Agonist Win55212 Reduces Brain Damage in an In Vivo Model of Hypoxic-Ischemic Encephalopathy in Newborn Rats

Neonatal hypoxic-ischemic encephalopathy (NHIE) is a devastating condition for which effective therapeutic treatments are still unavailable. Cannabinoids emerge as neuroprotective substances in adult animal studies; therefore, we aimed herein to test whether cannabinoids might reduce brain damage induced by hypoxia-ischemia (HI) in newborn rats. Thus, 7-d-old Wistar rats (P7) were exposed to 8% O2 for 120 min after left carotid artery ligature, then received s.c. vehicle (VEH) (HI+VEH), the cannabinoid agonist WIN55212 (WIN) (0.1 mg/kg), or WIN with the CB1 or CB2 receptor antagonist SR141617 (SR1) (3 mg/kg) or SR141588 (SR2) (2 mg/kg). Brain damage was assessed by magnetic resonance imaging (MRI) at 1, 3, and 7 d after the insult. At the end of the experiment, MRI findings were corroborated by histology (Nissl staining). HI+VEH showed an area of cytotoxic and vasogenic edema at 24 h after the insult, then evolving to necrosis. HI+WIN showed a similar damaged area at 24 h after the insult, but the final necrotic area was reduced by 66%. Coadministration of either SR1 or SR2 reversed the effects of WIN. In conclusion, likely by activating CB1 and CB2 receptors, WIN afforded robust neuroprotection in newborn rats after HI.

Mechanisms of Perinatal Arterial Ischemic Stroke

The incidence of perinatal stroke is high, similar to that in the elderly, and produces a significant morbidity and severe long-term neurologic and cognitive deficits, including cerebral palsy, epilepsy, neuropsychological impairments, and behavioral disorders. Emerging clinical data and data from experimental models of cerebral ischemia in neonatal rodents have shown that the pathophysiology of perinatal brain damage is multifactorial. These studies have revealed that, far from just being a smaller version of the adult brain, the neonatal brain is unique with a very particular and age-dependent responsiveness to hypoxia–ischemia and focal arterial stroke. In this review, we discuss fundamental clinical aspects of perinatal stroke as well as some of the most recent and relevant findings regarding the susceptibility of specific brain cell populations to injury, the dynamics and the mechanisms of neuronal cell death in injured neonates, the responses of neonatal blood-brain barrier to stroke in relation to systemic and local inflammation, and the long-term effects of stroke on angiogenesis and neurogenesis. Finally, we address translational strategies currently being considered for neonatal stroke as well as treatments that might effectively enhance repair later after injury.

The seek of neuroprotection: introducing cannabinoids.

The cannabinoid system is constituted by some endogenous ligands (endocannabinoids), usually arachydonic acid derivatives, and their specific receptors. The endogenous cannabinoid system (ECS) is involved in the control of synaptic transmission, modulating memory, motivation, movement, nociception, appetite and thermoregulation. ECS also exert extraneural effects, mainly immunomodulation and vasodilation. Two cannabinoid receptors have been cloned so far: CB(1) receptors are expressed in the central nervous system (CNS) but can also be found in glial cells and in peripheral tissues; CB(1) receptors are Gi/o protein coupled receptors that modulate the activity of several plasma membrane proteins and intracellular signaling pathways. CB(2) receptors are also Gi/o protein-coupled receptors; although it is accepted that CB(2) receptors are not expressed in forebrain neurons, they have been described in activated glia. Some of the cannabinoids activate other receptors, for instance vanilloid receptors (TRPV1). Lately, the ECS is emerging as a natural system of neuroprotection. This consideration is based on some properties of cannabinoids as their vasodilatory effect, the inhibition of the release of excitotoxic amino acids and cytokines, and the modulation of oxidative stress and toxic production of nitric oxide. Such effects have been demonstrated in adult and newborn animal models of acute and chronic neurodegenerative conditions, and postulate cannabinoids as valuable neuroprotective agents. Patents related to cannabinoid receptors are also discussed.

Reduced infarct size and accumulation of microglia in rats treated with WIN 55,212-2 after neonatal stroke.

Cannabinoids have emerged as brain protective agents under neurodegenerative conditions. Many neuroprotective actions of cannabinoids depend on the activation of specific receptors, cannabinoid receptor type 1 (CB1R) and type 2 (CB2R). The aim of the present study was to determine whether the CB2R and CB1R agonist WIN 55,212-2 (WIN) protects neonatal brain against focal cerebral ischemia-reperfusion and whether anti-inflammatory mechanisms play a role in protection. Seven-day-old rats were subjected to 90-min middle cerebral artery occlusion (MCAO), and injured rats were identified by diffusion-weighted MRI during the occlusion. After reperfusion, rats were subcutaneously administered 1 mg/kg of WIN or vehicle twice daily until sacrifice. MCAO led to increased mRNA expression of CB2R (but not CB1R), chemokine receptors (CCR2 and CX3CR1), and cytokines (IL-1β and TNFα), as well as increased protein expression of chemokines MCP-1 and MIP-1α and microglial activation 24 h after MCAO. WIN administration significantly reduced microglial activation at this point and attenuated infarct volume and microglial accumulation and proliferation in the injured cortex 72 h after MCAO. Cumulatively, our results show that the cannabinoid agonist WIN protects against neonatal focal stroke in part due to inhibitory effects on microglia.

The Cannabinoid WIN55212-2 Promotes Neural Repair After Neonatal Hypoxia–Ischemia

Background and Purpose— The endocannabinoid system has been involved in the modulation of neural stem cells proliferation, survival and differentiation as well as in the generation of new oligodendrocyte progenitors in the postnatal brain. The present work aims to test the effect of the synthetic Type 1 and Type 2 cannabinoid receptor agonist WIN55212-2 on these processes in the context of neonatal rat brain hypoxia–ischemia (HI). Methods— P7 Wistar rats were subjected to HI and treated either with WIN55212-2 (1 mg/kg) or vehicle twice daily for 7 days after HI and euthanized at 1, 2, 7, 14, or 28 days to explore white matter injury progression and the neurogenic response in the subventricular zone after HI. Results— Our findings reveal that WIN55212-2 promotes remyelination of the injured external capsule, increasing the number of NG2+ early oligodendrocyte progenitors 7 days after HI in this area and the number of APC+ mature oligodendrocytes in the injured striatum 14 and 28 days after HI. WIN55212-2 also increases cell proliferation and protein expression of the neuroblast marker doublecortin in the subventricular zone 7 days after neonatal HI as well as the number of newly generated neuroblasts (5-bromodeoxyuridine+/doublecortin+ cells) in the ipsilateral striatum 14 days after HI. Conclusions— Our results suggest that the activation of the endocannabinoid system promotes white and gray matter recovery after neonatal HI injury.

Microglial Cells Prevent Hemorrhage in Neonatal Focal Arterial Stroke

Perinatal stroke leads to significant morbidity and long-term neurological and cognitive deficits. The pathophysiological mechanisms of brain damage depend on brain maturation at the time of stroke. To understand whether microglial cells limit injury after neonatal stroke by preserving neurovascular integrity, we subjected postnatal day 7 (P7) rats depleted of microglial cells, rats with inhibited microglial TGFbr2/ALK5 signaling, and corresponding controls, to transient middle cerebral artery occlusion (tMCAO). Microglial depletion by intracerebral injection of liposome-encapsulated clodronate at P5 significantly reduced vessel coverage and triggered hemorrhages in injured regions 24 h after tMCAO. Lack of microglia did not alter expression or intracellular redistribution of several tight junction proteins, did not affect degradation of collagen IV induced by the tMCAO, but altered cell types producing TGFβ1 and the phosphorylation and intracellular distribution of SMAD2/3. Selective inhibition of TGFbr2/ALK5 signaling in microglia via intracerebral liposome-encapsulated SB-431542 delivery triggered hemorrhages after tMCAO, demonstrating that TGFβ1/TGFbr2/ALK5 signaling in microglia protects from hemorrhages. Consistent with observations in neonatal rats, depletion of microglia before tMCAO in P9 Cx3cr1GFP/+/Ccr2RFP/+ mice exacerbated injury and induced hemorrhages at 24 h. The effects were independent of infiltration of Ccr2RFP/+ monocytes into injured regions. Cumulatively, in two species, we show that microglial cells protect neonatal brain from hemorrhage after acute ischemic stroke. SIGNIFICANCE STATEMENT The pathophysiological mechanisms of brain damage depend on brain maturation at the time of stroke. We assessed whether microglial cells preserve neurovascular integrity after neonatal stroke. In neonatal rats, microglial depletion or pharmacological inhibition of TGFbr2/ALK5 signaling in microglia triggered hemorrhages in injured regions. The effect was not associated with additional changes in expression or intracellular redistribution of several tight junction proteins or collagen IV degradation induced by stroke. Consistent with observations in neonatal rats, microglial depletion in neonatal mice exacerbated stroke injury and induced hemorrhages. The effects were independent of infiltration of monocytes into injured regions. Thus, microglia protect neonatal brain from ischemia-induced hemorrhages, and this effect is consistent across two species.