ABSTRACT
Stroke is one of the most frequent causes of death and disability worldwide leading to a significant clinical and socioeconomic burden. Although different mechanisms are involved in the pathogenesis of stroke, inflammatory response occurs after ischemia and contributes to the expansion of brain injury. Platelet-activating factor receptor (PAF) plays crucial roles in both physiological and pathological conditions in the brain. PAF receptor (PAFR) may be expressed on cellular and nuclear membranes of various cell types, especially leukocytes, platelets, endothelial cells, neuronal cells and microglia. Herein, using mice lacking the PAFR receptor (PAFR(-/-)), we investigate a potential role for this receptor during experimental transient global cerebral ischemia and reperfusion (BCCAo). In PAFR deficiency, we observed a significant improvement in the neurological deficits, which were associated with a reduction of brain infarcted area as evaluated by triphenyltetrazolium chloride (TTC). Moreover, a decrease in the percentage of necrotic cavities areas and in the frequency of ischemic neurons was also found by employing histometric analysis. In addition, in PAFR(-/-) mice there was prevention of caspase-3 activation and decreased vascular permeability and brain edema. Decreased brain levels of the cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and the chemokine (C-X-C motif) ligand 1 (CXCL1) by ELISA were also detected in PAFR(-/-) BCCAo animals. Taken together, our results suggest that PAFR activation might be crucial for the global brain ischemia and reperfusion injury.
Subject(s)
Ischemic Attack, Transient/metabolism , Platelet Membrane Glycoproteins/metabolism , Receptors, G-Protein-Coupled/metabolism , Reperfusion Injury/metabolism , Animals , Blood-Brain Barrier/physiopathology , Brain Infarction/etiology , Caspase 3/metabolism , Cytokines/metabolism , Disease Models, Animal , Gene Expression Regulation/genetics , Ischemic Attack, Transient/complications , Ischemic Attack, Transient/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Nervous System Diseases/etiology , Platelet Membrane Glycoproteins/genetics , Receptors, G-Protein-Coupled/genetics , Reperfusion Injury/pathology , Statistics, NonparametricABSTRACT
Stroke is one of the most frequent causes of death and disability worldwide causing a major clinical and socioeconomic impact. Although the pathophysiology of brain ischemia and reperfusion is complex, the inflammatory process plays an important role in pathogenesis, contributing to the expansion of brain injury. The 5-lipoxygenase (5-LOX) is a key enzyme in the biosynthesis of the leukotrienes and has been implicated and in the central nervous system (CNS) disorders such as Alzheimer's disease and acute ischemic stroke. Zileuton, a selective 5-LOX inhibitor, has antiinflammatory properties and exerts an inhibitory effect on inflammatory diseases. The objective of this study was to evaluate the effects of blocking 5-LOX activity in a murine model of transient and global brain ischemia. Zileuton improved neurological deficits and significantly decrease volume and density of lesion, compared to vehicle-ischemic animals measured by magnetic resonance imaging (MRI). In addition, the blockage of 5-LOX reduced infarct area and histopathological changes. Furthermore, by enzyme immunoassay (ELISA) increased brain levels of tumor necrosis factor-alpha (TNFalpha), interferon-gamma (IFN-gamma), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), chemokine (C-X-C motif) ligand 1 (CXCL1), chemokine (C-C motif) ligand 3 (CCL3) and chemokine (C-C motif) ligand 5 (CCL5) were detected in the vehicle-ischemic group, whereas in Zileuton-ischemic group presented reduction of these mediators. The concentration of the antiinflammatory cytokine interleukin-10 (IL-10) was increased after 5-LOX inhibition. Our results suggest that Zileuton decreases brain damage and reduces inflammatory cytokines expression in the CNS which contributes, at least in part, to improve the neurological outcome of brain ischemia.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Brain Infarction/drug therapy , Encephalitis/drug therapy , Hydroxyurea/analogs & derivatives , Nervous System Diseases/complications , Analysis of Variance , Animals , Brain Infarction/etiology , Carotid Stenosis/complications , Cytokines/genetics , Cytokines/metabolism , Disease Models, Animal , Down-Regulation/drug effects , Encephalitis/etiology , Enzyme-Linked Immunosorbent Assay , Hydroxyurea/therapeutic use , Magnetic Resonance Imaging , Male , Mice , Mice, Inbred C57BL , Nervous System Diseases/etiologyABSTRACT
Teve como objetivo estudar duas moléculas antiparasíticas (gomesina e fosfolipase A2 (PLA2m)) como candidatas à expressão em mosquitos. O peptídeo antimicrobiano gomesina da aranha Acanthoscurria gomesiana, foi capaz de inibir o desenvolvimento das formas sanguíneas das cepas W2 e 3D7-GFP de P. falciparum, com valores de IC50 de 75,8 e86,6 (...). Anteriormente, experimentos in vitro, mostraram que aenzima fosfolipase A2 (PLA2) isolada do veneno de abelha apresentou ação inibitória sobre o desenvolvimento de P. falciparum e P. gallinaceum, mas apresentou efeitos deletérios em mosquitos transgênicos, quando na sua forma ativa. Neste trabalho, expressamos em bactérias e purificamos uma forma mutante da proteína (PLA2m). Na concentração de 0,1(...) a proteína recombinante foi capaz de reduzir em até 79 por cento o número de oocistosde P. gallinaceum em mosquitos Aedes fluviatilis, quando adicionada ao sangue de aves domésticas (Gallus gallus domesticus) infectadas. Para a geração de mosquitostransgênicos utilizamos o promotor da proteína 1 da matriz peritrófica de An. gambiae (AgPer1) para dirigir a expressão do gene da PLA2m. A construção do gene híbrido(AgPer1/PLA2m) foi inserida no elemento de transposição piggyBac, que contém como marcador, o gene da proteína verde fluorescente melhorada (EGFP). Pela microinjeção de770 embriões, foram formadas 15 famílias e, após a seleção de cerca de 22.000 larvas foram obtidas quatro linhagens transgênicas, representando uma eficiência na transformação de 27 por cento. A expressão do EGFP foi observada no tubo neural e olhos de larva, pupa e adulto. Utilizando iniciadores específicos, confirmamos, pela PCR, a presença dos genes EGFP (700pb) e PLA2m (500bp). A produção do RNAm da PLA2m foi específicano intestino de fêmeas, não variando após a alimentação sanguínea. Por microscopia confocal detectamos a presença da proteína PLA2m no intestino dos mosquitostransgênicos. Além disso, ensaios de bloqueio ao parasita mostrar.
Subject(s)
Anopheles , Malaria, Avian , Aedes , Oocysts , Plasmodium , Polymerase Chain ReactionABSTRACT
The technique to generate transgenic mosquitoes requires adaptation for each target species because of aspects related to species biology, sensitivity to manipulation and rearing conditions. Here we tested different parameters on the microinjection procedure in order to obtain a transgenic Neotropical mosquito species. By using a transposon-based strategy we were able to successfully transform Aedes fluviatilis (Lutz), which can be used as an avian malaria model. These results demonstrate the usefulness of the piggyBac transposable element as a transformation vector for Neotropical mosquito species and opens up new research frontiers for South American mosquito vectors.
Subject(s)
Aedes/genetics , DNA Transposable Elements/genetics , Gene Transfer Techniques , Insect Vectors/genetics , Transformation, Genetic/genetics , Animals , Female , Genes, Insect , Germ Cells , Male , MicroinjectionsABSTRACT
The technique to generate transgenic mosquitoes requires adaptation for each target species because of aspects related to species biology, sensitivity to manipulation and rearing conditions. Here we tested different parameters on the microinjection procedure in order to obtain a transgenic Neotropical mosquito species. By using a transposon-based strategy we were able to successfully transform Aedes fluviatilis (Lutz), which can be used as an avian malaria model. These results demonstrate the usefulness of the piggyBac transposable element as a transformation vector for Neotropical mosquito species and opens up new research frontiers for South American mosquito vectors.
