Takato Abe

Neurovascular Protection by Ischemic Tolerance: Role of Nitric Oxide and Reactive Oxygen Species

Cerebral ischemic preconditioning or tolerance is a powerful neuroprotective phenomenon by which a sublethal injurious stimulus renders the brain resistant to a subsequent damaging ischemic insult. We used lipopolysaccharide (LPS) as a preconditioning stimulus in a mouse model of middle cerebral artery occlusion (MCAO) to examine whether improvements in cerebrovascular function contribute to the protective effect. Administration of LPS 24 h before MCAO reduced the infarct by 68% and improved ischemic cerebral blood flow (CBF) by 114% in brain areas spared from infarction. In addition, LPS prevented the dysfunction in cerebrovascular regulation induced by MCAO, as demonstrated by normalization of the increase in CBF produced by neural activity, hypercapnia, or by the endothelium-dependent vasodilator acetylcholine. These beneficial effects of LPS were not observed in mice lacking inducible nitric oxide synthase (iNOS) or the nox2 subunit of the superoxide-producing enzyme NADPH oxidase. LPS increased reactive oxygen species and the peroxynitrite marker 3-nitrotyrosine in wild-type mice but not in nox2 nulls. The peroxynitrite decomposition catalyst 5,10,15, 20-tetrakis(4-sulfonatophenyl)porphyrinato iron (III) attenuated LPS-induced nitration and counteracted the beneficial effects of LPS on infarct volume, ischemic CBF, and vascular reactivity. Thus, LPS preserves neurovascular function and ameliorates CBF in regions of the ischemic territory at risk for infarction. This effect is mediated by peroxynitrite formed from iNOS-derived NO and nox2-derived superoxide. The data indicate that preservation of cerebrovascular function is an essential component of ischemic tolerance and suggest that combining neuroprotection and vasoprotection may be a valuable strategy for treating ischemic brain injury.

Brain dendritic cells in ischemic stroke: time course, activation state, and origin

The immune response to stroke is comprised of inflammatory and regulatory processes. One cell type involved in both innate and adaptive immunity is the dendritic cell (DC). A DC population residing in the healthy brain (bDC) was identified using a transgenic mouse expressing enhanced yellow fluorescent protein (EYFP) under the promoter for the DC marker, CD11c (CD11c/EYFP Tg). To determine if bDC are involved in the immune response to cerebral ischemia, transient (40 min) middle cerebral artery occlusion (MCAO) followed by 6, 24, or 72 h reperfusion was conducted in CD11c/EYFP Tg mice. Our results demonstrated that DC accumulated in the ischemic hemisphere at 24 h post-MCAO-reperfusion, particularly in the border region of the infarct where T lymphocytes accrued. To distinguish resident bDC from the infiltrating peripheral DC, radiation chimeras [1. wild type (WT) hosts restored with CD11c/EYFP Tg bone marrow (BM) or 2. CD11c/EYFP Tg hosts restored with WT BM] were generated and examined by immunocytochemistry. These data confirmed that DC populating the core of the infarct at 72 h were of peripheral origin, whereas those in the border region were comprised primarily of resident bDC. The brain resident (CD45 intermediate) cells of CD11c/EYFP Tg mice were analyzed by flow cytometry. Compared to microglia, bDC displayed increased major histocompatibility class II (MHC II) and co-stimulatory molecules following MCAO-reperfusion. High levels of MHC II and the co-stimulatory molecule CD80 on bDC at 72 h corresponded to peak lymphocyte infiltration, and suggested a functional interaction between these two immune cell populations.

Key Role of CD36 in Toll-Like Receptor 2 Signaling in Cerebral Ischemia

Background and Purpose— Toll-like receptors (TLRs) and the scavenger receptor CD36 are key molecular sensors for the innate immune response to invading pathogens. However, these receptors may also recognize endogenous “danger signals” generated during brain injury, such as cerebral ischemia, and trigger a maladaptive inflammatory reaction. Indeed, CD36 and TLR2 and 4 are involved in the inflammation and related tissue damage caused by brain ischemia. Because CD36 may act as a coreceptor for TLR2 heterodimers (TLR2/1 or TLR2/6), we tested whether such interaction plays a role in ischemic brain injury. Methods— The TLR activators FSL-1 (TLR2/6), Pam3 (TLR2/1), or lipopolysaccharide (TLR4) were injected intracerebroventricularly into wild-type or CD36-null mice, and inflammatory gene expression was assessed in the brain. The effect of TLR activators on the infarct produced by transient middle cerebral artery occlusion was also studied. Results— The inflammatory response induced by TLR2/1 activation, but not TLR2/6 or TLR4 activation, was suppressed in CD36-null mice. Similarly, TLR2/1 activation failed to increase infarct volume in CD36-null mice, whereas TLR2/6 or TLR4 activation exacerbated postischemic inflammation and increased infarct volume. In contrast, the systemic inflammatory response evoked by TLR2/6 activation, but not by TLR2/1 activation, was suppressed in CD36-null mice. Conclusions— In the brain, TLR2/1 signaling requires CD36. The cooperative signaling of TLR2/1 and CD36 is a critical factor in the inflammatory response and tissue damage evoked by cerebral ischemia. Thus, suppression of CD36-TLR2/1 signaling could be a valuable approach to minimize postischemic inflammation and the attendant brain injury.

Nuclear Factor-κB Activation and Postischemic Inflammation Are Suppressed in CD36-Null Mice after Middle Cerebral Artery Occlusion

CD36, a class-B scavenger receptor involved in multiple functions, including inflammatory signaling, may also contribute to ischemic brain injury through yet unidentified mechanisms. We investigated whether CD36 participates in the molecular events underlying the inflammatory reaction that accompanies cerebral ischemia and may contribute to the tissue damage. We found that activation of nuclear factor-κB, a transcription factor that coordinates postischemic gene expression, is attenuated in CD36-null mice subjected to middle cerebral artery occlusion. The infiltration of neutrophils and the glial reaction induced by cerebral ischemia were suppressed. Treatment with an inhibitor of inducible nitric oxide synthase, an enzyme that contributes to the tissue damage, reduced ischemic brain injury in wild-type mice, but not in CD36 nulls. In contrast to cerebral ischemia, the molecular and cellular inflammatory changes induced by intracerebroventricular injection of interleukin-1β were not attenuated in CD36-null mice. The findings unveil a novel role of CD36 in early molecular events leading to nuclear factor-κB activation and postischemic inflammation. Inhibition of CD36 signaling may be a valuable therapeutic approach to counteract the deleterious effects of postischemic inflammation.

The neuroprotective effect of prostaglandin E2 EP1 receptor inhibition has a wide therapeutic window, is sustained in time and is not sexually dimorphic

We investigated the preclinical characteristics of the neuroprotective effect of the prostaglandin E2 type 1 receptor (EP1) antagonist SC51089 in models of focal cerebral ischemia produced by occlusion of the mouse middle cerebral artery (MCA). We found that systemic administration of SC51089 (5 to 20 μg/kg; i.p.) reduces the brain injury produced by transient (−50% ± 8%; n = 12; P < 0.05) or permanent (−39% ± 7%; n = 12; P < 0.05) MCA occlusion. SC51089 was effective even when administered up to 12 h after ischemia. The protective effect was observed both in male and female mice and was sustained for at least 2 weeks after induction of ischemia. The reduction in injury volume was associated with an improvement in neurological function assessed by the Bederson deficit score, the hanging wire test and the corner test. The data provide proof of principle that EP1 receptor inhibition is a potentially valuable strategy for neuroprotection that deserves further preclinical investigation for therapeutic application in human stroke.

iNOS-Derived NO and Nox2-Derived Superoxide Confer Tolerance to Excitotoxic Brain Injury through Peroxynitrite

Sublethal injurious stimuli induce tolerance to subsequent lethal insults, a phenomenon termed preconditioning. Inducible nitric oxide synthase (iNOS) is essential for the preconditioning induced by transient bilateral common carotid artery occlusion (BCCAO) or by systemic administration of the endotoxin lipopolysaccharide (LPS). We used a model of brain injury produced by neocortical injection of N-methyl-d-aspartate (NMDA) to investigate the mechanisms by which iNOS-derived nitric oxide (NO) contributes to tolerance induced by LPS or BCCAO. We found that the tolerance is blocked by the iNOS inhibitor aminoguanidine, is not observed in iNOS-null mice, and is rescued by the NO donor DTPA NONOate. Lipopolysaccharide failed to induce preconditioning in mice lacking the nox2 subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, suggesting that superoxide derived from NADPH oxidase is needed for the induction of the tolerance. Because superoxide reacts with NO to form peroxynitrite, we investigated the role of peroxynitrite. We found that LPS induces the peroxynitrite marker 3-nitrotyrosine in cortical neurons and that the peroxynitrite decomposition catalyst FeTPPS abolishes LPS-induced preconditioning. These results suggest that the protective effect of iNOS-derived NO is mediated by peroxynitrite formed by the reaction of NO with NADPH oxidase-derived superoxide. Thus, peroxynitrite, in addition to its well-established deleterious role in ischemic brain injury and neurodegeneration, can also be beneficial by inducing tolerance to excitotoxicity.

Progranulin deficiency promotes post-ischemic blood-brain barrier disruption.

Loss-of-function mutations of progranulin (PGRN) have been linked to frontotemporal dementia, but little is known about the effects of PGRN deficiency on the brain in health and disease. PGRN has been implicated in neurovascular development, inflammation, and Wnt signaling, a pathway involved in the formation of the blood–brain barrier (BBB). Because BBB alterations and inflammation contribute to ischemic brain injury, we examined the role of PGRN in the brain damage produced by ischemia-reperfusion. PGRN+/− and PGRN−/− mice underwent middle cerebral artery occlusion (MCAO) with monitoring of cerebral blood flow. Infarct volume and motor deficits were assessed 72 h later. Post-ischemic inflammation was examined by expression of inflammatory genes and flow cytometry. BBB structure and permeability were examined by electron microscopy (EM) and Evans blue (EB) extravasation, respectively. MCAO resulted in ∼60% larger infarcts in PGRN+/− and PGRN−/− mice, an effect independent of hemodynamic factors or post-ischemic inflammation. Rather, massive hemorrhages and post-ischemic BBB disruption were observed, unrelated to degradation of tight junction (TJ) proteins or matrix metalloproteinases (MMPs). By EM, TJ were 30–52% shorter, fewer, and less interlocking, suggesting a weaker seal between endothelial cells. Intracerebral injection of platelet-derived growth factor-CC (PDGF-CC), which increases BBB permeability, resulted in a more severe BBB breakdown in PGRN+/− and PGRN−/− than wild-type mice. We describe a previously unrecognized involvement of PGRN in the expression of key ultrastructural features of the BBB. Such a novel vasoprotective role of PGRN may contribute to brain dysfunction and damage in conditions associated with reduced PGRN function.

Purinergic signaling induces cyclooxygenase-1-dependent prostanoid synthesis in microglia: roles in the outcome of excitotoxic brain injury.

Cyclooxygenases (COX) are prostanoid synthesizing enzymes constitutively expressed in the brain that contribute to excitotoxic neuronal cell death. While the neurotoxic role of COX-2 is well established and has been linked to prostaglandin E2 synthesis, the role of COX-1 is not clearly understood. In a model of N-Methyl-D-aspartic acid (NMDA) induced excitotoxicity in the mouse cerebral cortex we found a distinctive temporal profile of COX-1 and COX-2 activation where COX-1, located in microglia, is responsible for the early phase of prostaglandin E2 synthesis (10 minutes after NMDA), while both COX-1 and COX-2 contribute to the second phase (3–24 hours after NMDA). Microglial COX-1 is strongly activated by ATP but not excitatory neurotransmitters or the Toll-like receptor 4 ligand bacterial lipopolysaccharide. ATP induced microglial COX-1 dependent prostaglandin E2 synthesis is dependent on P2X7 receptors, extracellular Ca2+ and cytoplasmic phospholipase A2. NMDA receptor activation induces ATP release from cultured neurons leading to microglial P2X7 receptor activation and COX-1 dependent prostaglandin E2 synthesis in mixed microglial-neuronal cultures. Pharmacological inhibition of COX-1 has no effect on the cortical lesion produced by NMDA, but counteracts the neuroprotection exerted by inhibition of COX-2 or observed in mice lacking the prostaglandin E2 receptor type 1. Similarly, the neuroprotection exerted by the prostaglandin E2 receptor type 2 agonist butaprost is not observed after COX-1 inhibition. P2X7 receptors contribute to NMDA induced prostaglandin E2 production in vivo and blockage of P2X7 receptors reverses the neuroprotection offered by COX-2 inhibition. These findings suggest that purinergic signaling in microglia triggered by neuronal ATP modulates excitotoxic cortical lesion by regulating COX-1 dependent prostanoid production and unveil a previously unrecognized protective role of microglial COX-1 in excitotoxic brain injury.

Lipoprotein Receptor–Related Protein-6 Protects the Brain From Ischemic Injury

Background and Purpose— Loss-of-function mutations of the lipoprotein receptor–related protein-6 (LRP6), a coreceptor in the Wingless-related integration site-&bgr;−catenin prosurvival pathway, have been implicated in myocardial ischemia and neurodegeneration. However, it remains to be established whether LRP6 is also involved in ischemic brain injury. We used LRP6+/− mice to examine the role of this receptor in the mechanisms of focal cerebral ischemia. Methods— Focal cerebral ischemia was induced by transient occlusion of the middle cerebral artery. Motor deficits and infarct volume were assessed 3 days later. Glycogen-synthase-kinase-3&bgr; (GSK-3&bgr;) phosphorylation was examined by Western blotting with phosphospecific antibodies, and the mitochondrial membrane potential changes induced by Ca2+ were also assessed. Results— LRP6+/− mice have larger stroke and more severe motor deficits, effects that were independent of intraischemic cerebral blood flow, vascular factors, or cytosolic &bgr;-catenin levels. Rather, LRP6 haploinsufficiency increased the activating phosphorylation and decreased the inhibitory phosphorylation of GSK-3&bgr;, a kinase involved in proinflammatory signaling and mitochondrial dysfunction. Accordingly, postischemic inflammatory gene expression was enhanced in LRP6+/− mice. Furthermore, the association of mitochondria with activated GSK-3&bgr; was increased in LRP6+/− mice, resulting in a reduction in the Ca2+ handling ability of mitochondria. The mitochondrial dysfunction was reversed by pharmacological inhibition of GSK-3&bgr;. Conclusions— LRP6 activates an endogenous neuroprotective pathway that acts independently of &bgr;-catenin by controlling GSK-3&bgr; activity and preventing its deleterious mitochondrial and proinflammatory effects. The findings raise the possibility that emerging treatment strategies for diseases attributable to LRP6 loss-of-function mutations could also lead to new therapeutic avenues for ischemic stroke.