Ipsilesional Hippocampal GABA Is Elevated and Correlates With Cognitive Impairment and Maladaptive Neurogenesis After Cortical Stroke in Mice.

BACKGROUND: Cognitive dysfunction is a frequent stroke sequela, but its pathogenesis and treatment remain unresolved. Involvement of aberrant hippocampal neurogenesis and maladaptive circuitry remodeling has been proposed, but their mechanisms are unknown. Our aim was to evaluate potential underlying molecular/cellular events implicated. METHODS: Stroke was induced by permanent occlusion of the middle cerebral artery occlusion in 2-month-old C57BL/6 male mice. Hippocampal metabolites/neurotransmitters were analyzed longitudinally by in vivo magnetic resonance spectroscopy. Cognitive function was evaluated with the contextual fear conditioning test. Microglia, astrocytes, neuroblasts, interneurons, γ-aminobutyric acid (GABA), and c-fos were analyzed by immunofluorescence. RESULTS: Approximately 50% of mice exhibited progressive post-middle cerebral artery occlusion cognitive impairment. Notably, immature hippocampal neurons in the impaired group displayed more severe aberrant phenotypes than those from the nonimpaired group. Using magnetic resonance spectroscopy, significant bilateral changes in hippocampal metabolites, such as myo-inositol or N-acetylaspartic acid, were found that correlated, respectively, with numbers of glia and immature neuroblasts in the ischemic group. Importantly, some metabolites were specifically altered in the ipsilateral hippocampus suggesting its involvement in aberrant hippocampal neurogenesis and remodeling processes. Specifically, middle cerebral artery occlusion animals with higher hippocampal GABA levels displayed worse cognitive outcome. Implication of GABA in this setting was supported by the amelioration of ischemia-induced memory deficits and aberrant hippocampal neurogenesis after blocking pharmacologically GABAergic neurotransmission, an intervention which was ineffective when neurogenesis was inhibited. These data suggest that GABA exerts its detrimental effect, at least partly, by affecting morphology and integration of newborn neurons into the hippocampal circuits. CONCLUSIONS: Hippocampal GABAergic neurotransmission could be considered a novel diagnostic and therapeutic target for poststroke cognitive impairment.

Perinatal exposure to pesticides alters synaptic plasticity signaling and induces behavioral deficits associated with neurodevelopmental disorders.

Increasing evidence from animal and epidemiological studies indicates that perinatal exposure to pesticides cause developmental neurotoxicity and may increase the risk for psychiatric disorders such as autism and intellectual disability. However, the underlying pathogenic mechanisms remain largely elusive. This work was aimed at testing the hypothesis that developmental exposure to different classes of pesticides hijacks intracellular neuronal signaling contributing to synaptic and behavioral alterations associated with neurodevelopmental disorders (NDD). Low concentrations of organochlorine (dieldrin, endosulfan, and chlordane) and organophosphate (chlorpyrifos and its oxon metabolite) pesticides were chronically dosed ex vivo (organotypic rat hippocampal slices) or in vivo (perinatal exposure in rats), and then biochemical, electrophysiological, behavioral, and proteomic studies were performed. All the pesticides tested caused prolonged activation of MAPK/ERK pathway in a concentration-dependent manner. Additionally, some of them impaired metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD). In the case of the pesticide chlordane, the effect was attributed to chronic modulation of MAPK/ERK signaling. These synaptic alterations were reproduced following developmental in vivo exposure to chlordane and chlorpyrifos-oxon, and were also associated with prototypical behavioral phenotypes of NDD, including impaired motor development, increased anxiety, and social and memory deficits. Lastly, proteomic analysis revealed that these pesticides differentially regulate the expression of proteins in the hippocampus with pivotal roles in brain development and synaptic signaling, some of which are associated with NDD. Based on these results, we propose a novel mechanism of synaptic dysfunction, involving chronic overactivation of MAPK and impaired mGluR-LTD, shared by different pesticides which may have important implications for NDD.

Lack of the aryl hydrocarbon receptor accelerates aging in mice.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor, largely known for its role in xenobiotic metabolism and detoxification as well as its crucial role as a regulator of inflammation. Here, we have compared a cohort wild-type and AhR-null mice along aging to study the relationship between this receptor and age-associated inflammation, termed as "inflammaging," both at a systemic and the CNS level. Our results show that AhR deficiency is associated with a premature aged phenotype, characterized by early inflammaging, as shown by an increase in plasma cytokines levels. The absence of AhR also promotes the appearance of brain aging anatomic features, such as the loss of the white matter integrity. In addition, AhR-/- mice present an earlier spatial memory impairment and an enhanced astrogliosis in the hippocampus when compared with their age-matched AhR+/+ controls. Importantly, we have found that AhR protein levels decrease with age in this brain structure, strongly suggesting a link between AhR and aging.-Bravo-Ferrer, I., Cuartero, M. I., Medina, V., Ahedo-Quero, D., Peña-Martínez, C., Pérez-Ruíz, A., Fernández-Valle, M. E., Hernández-Sánchez, C., Fernández-Salguero, P. M., Lizasoain, I., Moro, M. A. Lack of the aryl hydrocarbon receptor accelerates aging in mice.

Role of TLR4 (Toll-Like Receptor 4) in N1/N2 Neutrophil Programming After Stroke.

Background and Purpose- After stroke, the population of infiltrated neutrophils in the brain is heterogeneous, including a population of alternative neutrophils (N2) that express M2 phenotype markers. We explored the role of TLR4 (toll-like receptor 4) on neutrophil infiltration and polarization in this setting. Methods- Focal cerebral ischemia was induced by occlusion of the middle cerebral artery occlusion in TLR4-KO and WT (wild type) mice. Infarct size was measured by Nissl staining and magnetic resonance imaging. Leukocyte infiltration was quantified 48 hours after middle cerebral artery occlusion by immunofluorescence and flow cytometry. To elucidate mechanisms underlying TLR4-mediated N2 phenotype, a cDNA microarray analysis was performed in neutrophils isolated from blood 48 hours after stroke in WT and TLR4-KO mice. Results- As demonstrated previously, TLR4-deficient mice presented lesser infarct volumes than WT mice. TLR4-deficient mice showed higher density of infiltrated neutrophils 48 hours after stroke compared with WT mice, concomitantly to neuroprotection. Furthermore, cytometric and stereological analyses revealed an increased number of N2 neutrophils (YM1+ cells) into the ischemic core in TLR4-deficient mice, suggesting a protective effect of this neutrophil subset that was corroborated by depleting peripheral neutrophils or using mice with TLR4 genetically ablated in the myeloid lineage. Finally, cDNA microarray analysis in neutrophils, confirmed by quantitative polymerase chain reaction, showed that TLR4 modulates several pathways associated with ischemia-induced inflammation, migration of neutrophils into the parenchyma, and their functional priming, which might explain the opposite effect on outcome of the different neutrophil subsets. Conclusions- TLR4 deficiency increased the levels of alternative neutrophils (N2)-an effect associated with neuroprotection after stroke-supporting that modulation of neutrophil polarization is a major target of TLR4 and highlighting the crucial role of TLR4 at the peripheral level after stroke. Visual Overview- An online visual overview is available for this article.

TLR4-Binding DNA Aptamers Show a Protective Effect against Acute Stroke in Animal Models.

Since Toll-like receptor 4 (TLR4) mediates brain damage after stroke, development of TLR4 antagonists is a promising therapeutic strategy for this disease. Our aim was to generate TLR4-blocking DNA aptamers to be used for stroke treatment. From a random oligonucleotide pool, we identified two aptamers (ApTLR#1R, ApTLR#4F) with high affinity for human TLR4 by systematic evolution of ligands by exponential enrichment (SELEX). Optimized truncated forms (ApTLR#1RT, ApTLR#4FT) were obtained. Our data demonstrate specific binding of both aptamers to human TLR4 as well as a TLR4 antagonistic effect. ApTLR#4F and ApTLR#4FT showed a long-lasting protective effect against brain injury induced by middle cerebral artery occlusion (MCAO), an effect that was absent in TLR4-deficient mice. Similar effects were obtained in other MCAO models, including in rat. Additionally, efficacy of ApTLR#4FT in a model of brain ischemia-reperfusion in rat supports the use of this aptamer in patients undergoing artery recanalization induced by pharmacological or mechanical interventions. The absence of major toxicology aspects and the good safety profile of the aptamers further encourage their future clinical positioning for stroke therapy and possibly other diseases in which TLR4 plays a deleterious role.

Toll-Like Receptor 4 Mediates Hemorrhagic Transformation After Delayed Tissue Plasminogen Activator Administration in In Situ Thromboembolic Stroke.

BACKGROUND AND PURPOSE: Hemorrhagic transformation is the main complication of revascularization therapies after stroke. Toll-like receptor 4 (TLR4) is implicated in cerebral damage and inflammation in stroke. This study was designed to determine the role of TLR4 in hemorrhagic transformation development after tissue plasminogen activator (tPA) administration. METHODS: Mice expressing (TLR4+/+) or lacking functional TLR4 (TLR4-/-) were subjected to middle cerebral artery occlusion using an in situ thromboembolic model by thrombin injection into the middle cerebral artery, and tPA (10 mg/kg) was administered 20 minutes or 3 hours after ischemia. Infarct size, hemorrhages, IgG extravasation, matrix metalloproteinase 9 expression, and neutrophil infiltration were assessed 24 hours after ischemia. RESULTS: In TLR4+/+, early reperfusion (tPA at 20 minutes) resulted infarct volume, whereas late recanalization (tPA at 3 hours) did not modify lesion size and increased the rate of the most severe hemorrhages. In TLR4-/- mice, both early and late reperfusion did not modify lesion size. Importantly, late tPA administration did not result in worse hemorrhages and in an increased bleeding area as occurred in TLR4+/+ group. In TLR4-/- animals, late reperfusion produced a lesser increase in matrix metalloproteinase 9 expression when compared with TLR4+/+ animals. CONCLUSIONS: Our results demonstrate TLR4 involvement in hemorrhagic transformation induced by delayed tPA administration, very likely by increasing matrix metalloproteinase 9 expression.

Cannabinoid Type-2 Receptor Drives Neurogenesis and Improves Functional Outcome After Stroke.

BACKGROUND AND PURPOSE: Stroke is a leading cause of adult disability characterized by physical, cognitive, and emotional disturbances. Unfortunately, pharmacological options are scarce. The cannabinoid type-2 receptor (CB2R) is neuroprotective in acute experimental stroke by anti-inflammatory mechanisms. However, its role in chronic stroke is still unknown. METHODS: Stroke was induced by permanent middle cerebral artery occlusion in mice; CB2R modulation was assessed by administering the CB2R agonist JWH133 ((6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran) or the CB2R antagonist SR144528 (N-[(1S)-endo-1,3,3-trimethylbicyclo-[2.2.1]-heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide) once daily from day 3 to the end of the experiment or by CB2R genetic deletion. Analysis of immunofluorescence-labeled brain sections, 5-bromo-2´-deoxyuridine (BrdU) staining, fluorescence-activated cell sorter analysis of brain cell suspensions, and behavioral tests were performed. RESULTS: SR144528 decreased neuroblast migration toward the boundary of the infarct area when compared with vehicle-treated mice 14 days after middle cerebral artery occlusion. Consistently, mice on this pharmacological treatment, like mice with CB2R genetic deletion, displayed a lower number of new neurons (NeuN+/BrdU+ cells) in peri-infarct cortex 28 days after stroke when compared with vehicle-treated group, an effect accompanied by a worse sensorimotor performance in behavioral tests. The CB2R agonist did not affect neurogenesis or outcome in vivo, but increased the migration of neural progenitor cells in vitro; the CB2R antagonist alone did not affect in vitro migration. CONCLUSIONS: Our data support that CB2R is fundamental for driving neuroblast migration and suggest that an endocannabinoid tone is required for poststroke neurogenesis by promoting neuroblast migration toward the injured brain tissue, increasing the number of new cortical neurons and, conceivably, enhancing motor functional recovery after stroke.

L-kynurenine/aryl hydrocarbon receptor pathway mediates brain damage after experimental stroke.

BACKGROUND: Aryl hydrocarbon receptor (AhR) is a transcription factor that belongs to the basic helix-loop-helix PAS (Per-Arnt-Sim homology domain) family known to mediate the toxic and carcinogenic effects of xenobiotics. Interestingly, AhR is widely expressed in the central nervous system, but its physiological and pathological roles are still unclear. METHODS AND RESULTS: To define the role of AhR in stroke, we used middle cerebral artery occlusion in mice and oxygen-glucose deprivation in rat cortical neurons. The results presented here show that the ischemic insult increases total and nuclear AhR levels and AhR transcriptional activity in neurons in vivo and in vitro. We also show that AhR has a causal role in acute ischemic damage because pharmacological or genetic loss-of-function approaches result in neuroprotection. Inhibition of cAMP response element-binding protein-dependent signaling may participate in the deleterious actions of AhR. Finally, we have also found that L-kynurenine, a tryptophan metabolite with AhR agonistic properties, is an endogenous ligand that mediates AhR activation in the brain after middle cerebral artery occlusion. CONCLUSIONS: Our data demonstrate that an L-kynurenine/AhR pathway mediates acute brain damage after stroke and open new possibilities for the diagnosis and treatment of this pathology.

Toll-like receptor 4 modulates cell migration and cortical neurogenesis after focal cerebral ischemia.

Toll-like receptor 4 (TLR4) mediates brain damage after stroke. Now our objective is to determine TLR4 involvement in stroke-induced neurogenesis. Stroke was induced by permanent middle cerebral artery occlusion in wild-type and TLR4-deficient mice. Stereological and densitometric analysis of immunofluorescence-labeled brain sections and FACS analysis of cell suspensions were performed. Our results show that subventricular zone (SVZ) cell proliferation after stroke depends on infarct size. Second, when comparing brains with similar lesions, TLR4 attenuated SVZ proliferation, as shown by a decrease in prominin-1(+)/EGFR(+)/nestin(-) cells (type-C cells) at 1-2 d, and in BrdU(+) cells at 7 d, in TLR4(+/+) vs. TLR4(-/-) mice. Interestingly, 7 d after the infarct, neuroblasts in TLR4(+/+) mice migrated farther distances, reaching areas closer to the lesion than those in TLR4-deficient mice. However, at 14 d, TLR4-deficient mice presented a higher number of neuroblasts in all migratory zones than the TLR4(+/+) counterparts, which suggests that TLR4 deficiency delays neuroblast migration. Consistently, TLR4(+/+) mice showed an increased number of interneurons (NeuN(+)/BrdU(+)/GAD67(+) cells) in peri-infarct cortex 14-28 d after stroke. Our data indicate that, despite a negative effect on SVZ cell proliferation, TLR4 plays an important role in stroke-induced neurogenesis by promoting neuroblasts migration and increasing the number of new cortical neurons after stroke.

Silent information regulator 1 protects the brain against cerebral ischemic damage.

BACKGROUND AND PURPOSE: Sirtuin 1 (SIRT1) is a member of NAD+-dependent protein deacetylases implicated in a wide range of cellular functions and has beneficial properties in pathologies including ischemia/reperfusion processes and neurodegeneration. However, no direct evidence has been reported on the direct implication of SIRT1 in ischemic stroke. The aim of this study was to establish the role of SIRT1 in stroke using an experimental model in mice. METHODS: Wild-type and Sirt1-/- mice were subjected to permanent focal ischemia by permanent ligature. In another set of experiments, wild-type mice were treated intraperitoneally with vehicle, activator 3 (SIRT1 activator, 10 mg/kg), or sirtinol (SIRT1 inhibitor, 10 mg/kg) for 10 minutes, 24 hours, and 40 hours after ischemia. Brains were removed 48 hours after ischemia for determining the infarct volume. Neurological outcome was evaluated using the modified neurological severity score. RESULTS: Exposure to middle cerebral artery occlusion increased SIRT1 expression in neurons of the ipsilesional mouse brain cortex. Treatment of mice with activator 3 reduced infarct volume, whereas sirtinol increased ischemic injury. Sirt1-/- mice displayed larger infarct volumes after ischemia than their wild-type counterparts. In addition, SIRT1 inhibition/deletion was concomitant with increased acetylation of p53 and nuclear factor κB (p65). CONCLUSIONS: These results support the idea that SIRT1 plays an important role in neuroprotection against brain ischemia by deacetylation and subsequent inhibition of p53-induced and nuclear factor κB-induced inflammatory and apoptotic pathways.

Citicoline (CDP-choline) increases Sirtuin1 expression concomitant to neuroprotection in experimental stroke.

CDP-choline has shown neuroprotective effects in cerebral ischemia. In humans, although a recent trial International Citicoline Trial on Acute Stroke (ICTUS) has shown that global recovery is similar in CDP-choline and placebo groups, CDP-choline was shown to be more beneficial in some patients, such as those with moderate stroke severity and not treated with t-PA. Several mechanisms have been proposed to explain the beneficial actions of CDP-choline. We have now studied the participation of Sirtuin1 (SIRT1) in the neuroprotective actions of CDP-choline. Fischer rats and Sirt1⁻/⁻ mice were subjected to permanent focal ischemia. CDP-choline (0.2 or 2 g/kg), sirtinol (a SIRT1 inhibitor; 10 mg/kg), and resveratrol (a SIRT1 activator; 2.5 mg/kg) were administered intraperitoneally. Brains were removed 24 and 48 h after ischemia for western blot analysis and infarct volume determination. Treatment with CDP-choline increased SIRT1 protein levels in brain concomitantly to neuroprotection. Treatment with sirtinol blocked the reduction in infarct volume caused by CDP-choline, whereas resveratrol elicited a strong synergistic neuroprotective effect with CDP-choline. CDP-choline failed to reduce infarct volume in Sirt1⁻/⁻ mice. Our present results demonstrate a robust effect of CDP-choline like SIRT1 activator by up-regulating its expression. Our findings suggest that therapeutic strategies to activate SIRT1 may be useful in the treatment of stroke. Sirtuin 1 (SIRT1) is implicated in a wide range of cellular functions. Regarding stroke, there is no direct evidence. We have demonstrated that citicoline increases SIRT1 protein levels in brain concomitantly to neuroprotection. Citicoline fails to reduce infarct volume in Sirt1⁻/⁻ mice. Our findings suggest that therapeutic strategies acting on SIRT1 may be useful in the treatment of stroke.

Functional specialization of different PI3K isoforms for the control of neuronal architecture, synaptic plasticity, and cognition.

Neuronal connectivity and activity-dependent synaptic plasticity are fundamental properties that support brain function and cognitive performance. Phosphatidylinositol 3-kinase (PI3K) intracellular signaling controls multiple mechanisms mediating neuronal growth, synaptic structure, and plasticity. However, it is still unclear how these pleiotropic functions are integrated at molecular and cellular levels. To address this issue, we used neuron-specific virally delivered Cre expression to delete either p110α or p110ß (the two major catalytic isoforms of type I PI3K) from the hippocampus of adult mice. We found that dendritic and postsynaptic structures are almost exclusively supported by p110α activity, whereas p110ß controls neurotransmitter release and metabotropic glutamate receptor-dependent long-term depression at the presynaptic terminal. In addition to these separate functions, p110α and p110ß jointly contribute to N-methyl-d-aspartate receptor-dependent postsynaptic long-term potentiation. This molecular and functional specialization is reflected in different proteomes controlled by each isoform and in distinct behavioral alterations for learning/memory and sociability in mice lacking p110α or p110ß.

Astrocytic p38α MAPK drives NMDA receptor-dependent long-term depression and modulates long-term memory.

NMDA receptor-dependent long-term depression (LTD) in the hippocampus is a well-known form of synaptic plasticity that has been linked to different cognitive functions. The core mechanism for this form of plasticity is thought to be entirely neuronal. However, we now demonstrate that astrocytic activity drives LTD at CA3-CA1 synapses. We have found that LTD induction enhances astrocyte-to-neuron communication mediated by glutamate, and that Ca2+ signaling and SNARE-dependent vesicular release from the astrocyte are required for LTD expression. In addition, using optogenetic techniques, we show that low-frequency astrocytic activation, in the absence of presynaptic activity, is sufficient to induce postsynaptic AMPA receptor removal and LTD expression. Using cell-type-specific gene deletion, we show that astrocytic p38α MAPK is required for the increased astrocytic glutamate release and astrocyte-to-neuron communication during low-frequency stimulation. Accordingly, removal of astrocytic (but not neuronal) p38α abolishes LTD expression. Finally, this mechanism modulates long-term memory in vivo.

AhR Deletion Promotes Aberrant Morphogenesis and Synaptic Activity of Adult-Generated Granule Neurons and Impairs Hippocampus-Dependent Memory.

Newborn granule cells are continuously produced in the subgranular zone of dentate gyrus throughout life. Once these cells mature, they integrate into pre-existing circuits modulating hippocampus-dependent memory. Subsequently, mechanisms controlling generation and maturation of newborn cells are essential for proper hippocampal function. Therefore, we have studied the role of aryl hydrocarbon receptor (AhR), a ligand-activated bHLH-PAS transcription factor, in hippocampus-dependent memory and granule neuronal morphology and function using genetic loss-of-function approaches based on constitutive and inducible-nestin AhR-/- mice. The results presented here show that the impaired hippocampus-dependent memory in AhR absence is not due to its effects on neurogenesis but to aberrant dendritic arborization and an increased spine density, albeit with a lower number of mature mushrooms spines in newborn granule cells, a finding that is associated with an immature electrophysiological phenotype. Together, our data strongly suggest that AhR plays a pivotal role in the regulation of hippocampal function, by controlling hippocampal granule neuron morphology and synaptic maturation.

Complexity of the cell-cell interactions in the innate immune response after cerebral ischemia.

In response to brain ischemia a cascade of signals leads to the activation of the brain innate immune system and to the recruitment of blood borne derived cells to the ischemic tissue. These processes have been increasingly shown to play a role on stroke pathogenesis. Here, we discuss the key features of resident microglia and different leukocyte subsets implicated in cerebral ischemia with special emphasis of neutrophils, monocytes and microglia. We focus on how leukocytes are recruited to injured brain through a complex interplay between endothelial cells, platelets and leukocytes and describe different strategies used to inhibit their recruitment. Finally, we discuss the possible existence of different leukocyte subsets in the ischemic tissue and the repercussion of different myeloid phenotypes on stroke outcome. The knowledge of the nature of these heterogeneous cell-cell interactions may open new lines of investigation on new therapies to promote protective immune responses and tissue repair after cerebral ischemia or to block harmful responses. This article is part of a Special Issue entitled SI: Cell Interactions In Stroke.

Rational modulation of the innate immune system for neuroprotection in ischemic stroke.

The innate immune system plays a dualistic role in the evolution of ischemic brain damage and has also been implicated in ischemic tolerance produced by different conditioning stimuli. Early after ischemia, perivascular astrocytes release cytokines and activate metalloproteases (MMPs) that contribute to blood-brain barrier (BBB) disruption and vasogenic oedema; whereas at later stages, they provide extracellular glutamate uptake, BBB regeneration and neurotrophic factors release. Similarly, early activation of microglia contributes to ischemic brain injury via the production of inflammatory cytokines, including tumor necrosis factor (TNF) and interleukin (IL)-1, reactive oxygen and nitrogen species and proteases. Nevertheless, microglia also contributes to the resolution of inflammation, by releasing IL-10 and tumor growth factor (TGF)-ß, and to the late reparative processes by phagocytic activity and growth factors production. Indeed, after ischemia, microglia/macrophages differentiate toward several phenotypes: the M1 pro-inflammatory phenotype is classically activated via toll-like receptors or interferon-γ, whereas M2 phenotypes are alternatively activated by regulatory mediators, such as ILs 4, 10, 13, or TGF-ß. Thus, immune cells exert a dualistic role on the evolution of ischemic brain damage, since the classic phenotypes promote injury, whereas alternatively activated M2 macrophages or N2 neutrophils prompt tissue remodeling and repair. Moreover, a subdued activation of the immune system has been involved in ischemic tolerance, since different preconditioning stimuli act via modulation of inflammatory mediators, including toll-like receptors and cytokine signaling pathways. This further underscores that the immuno-modulatory approach for the treatment of ischemic stroke should be aimed at blocking the detrimental effects, while promoting the beneficial responses of the immune reaction.

Rosiglitazone-induced CD36 up-regulation resolves inflammation by PPARγ and 5-LO-dependent pathways.

PPARγ-achieved neuroprotection in experimental stroke has been explained by the inhibition of inflammatory genes, an action in which 5-LO, Alox5, is involved. In addition, PPARγ is known to promote the expression of CD36, a scavenger receptor that binds lipoproteins and mediates bacterial recognition and also phagocytosis. As phagocytic clearance of neutrophils is a requisite for resolution of the inflammatory response, PPARγ-induced CD36 expression might help to limit inflammatory tissue injury in stroke, an effect in which 5-LO might also be involved. Homogenates, sections, and cellular suspensions were prepared from brains of WT and Alox5(-/-) mice exposed to distal pMCAO. BMMs were obtained from Lys-M Cre(+) PPARγ(f/f) and Lys-M Cre(-) PPARγ(f/f) mice. Stereological counting of double-immunofluorescence-labeled brain sections and FACS analysis of cell suspensions was performed. In vivo and in vitro phagocytosis of neutrophils by microglia/macrophages was analyzed. PPARγ activation with RSG induced CD36 expression in resident microglia. This process was mediated by the 5-LO gene, which is induced in neurons by PPARγ activation and at least by one of its products--LXA4--which induced CD36 independently of PPARγ. Moreover, CD36 expression helped resolution of inflammation through phagocytosis, concomitantly to neuroprotection. Based on these findings, in addition to a direct modulation by PPARγ, we propose in brain a paracrine model by which products generated by neuronal 5-LO, such as LXA4, increase the microglial expression of CD36 and promote tissue repair in pathologies with an inflammatory component, such as stroke.

Neurological tests for functional outcome assessment in rodent models of ischaemic stroke.

A critical aspect in all models is the assessment of the final outcome of the modelling procedure. In the case of a focal ischaemic brain injury, apart from the determination of the size of the lesion, another valuable tool is the evaluation of the final functional deficit. Indeed, ischaemic damage leads to the appearance of different degrees of sensoriomotor and cognitive impairments, which may yield useful information on location and size of the lesion and on the efficacy of neuroprotective treatments after the acute injury. In addition, the magnitude of these impairments may also be useful to predict final outcome and to evaluate neuro-restorative therapies in a long-term scenario. To this aim, a wide range of tests has been developed which allow the quantification of all these neurological symptoms. This review intends to compile the most useful behavioural tests designed to assess neurological symptoms in studies of focal experimental cerebral ischemia in rodents induced by middle cerebral artery occlusion, the most commonly used model of ischaemic stroke.