Quantitative Analysis Of Rat Dorsal Root Ganglion Neurons Cultured On Microelectrode Arrays Based On Fluorescence Microscopy Image Processing.

Microelectrode Arrays (MEA) are devices for long term electrophysiological recording of extracellular spontaneous or evocated activities on in vitro neuron culture. This work proposes and develops a framework for quantitative and morphological analysis of neuron cultures on MEAs, by processing their corresponding images, acquired by fluorescence microscopy. The neurons are segmented from the fluorescence channel images using a combination of segmentation by thresholding, watershed transform, and object classification. The positioning of microelectrodes is obtained from the transmitted light channel images using the circular Hough transform. The proposed method was applied to images of dissociated culture of rat dorsal root ganglion (DRG) neuronal cells. The morphological and topological quantitative analysis carried out produced information regarding the state of culture, such as population count, neuron-to-neuron and neuron-to-microelectrode distances, soma morphologies, neuron sizes, neuron and microelectrode spatial distributions. Most of the analysis of microscopy images taken from neuronal cultures on MEA only consider simple qualitative analysis. Also, the proposed framework aims to standardize the image processing and to compute quantitative useful measures for integrated image-signal studies and further computational simulations. As results show, the implemented microelectrode identification method is robust and so are the implemented neuron segmentation and classification one (with a correct segmentation rate up to 84%). The quantitative information retrieved by the method is highly relevant to assist the integrated signal-image study of recorded electrophysiological signals as well as the physical aspects of the neuron culture on MEA. Although the experiments deal with DRG cell images, cortical and hippocampal cell images could also be processed with small adjustments in the image processing parameter estimation.25155003

Peripheral Inflammatory Hyperalgesia Depends on P2X7 Receptors in Satellite Glial Cells.

Peripheral inflammatory hyperalgesia depends on the sensitization of primary nociceptive neurons. Inflammation drives molecular alterations not only locally but also in the dorsal root ganglion (DRG) where interleukin-1 beta (IL-1β) and purinoceptors are upregulated. Activation of the P2X7 purinoceptors by ATP is essential for IL-1β maturation and release. At the DRG, P2X7R are expressed by satellite glial cells (SGCs) surrounding sensory neurons soma. Although SGCs have no projections outside the sensory ganglia these cells affect pain signaling through intercellular communication. Therefore, here we investigated whether activation of P2X7R by ATP and the subsequent release of IL-1β in DRG participate in peripheral inflammatory hyperalgesia. Immunofluorescent images confirmed the expression of P2X7R and IL-1β in SGCs of the DRG. The function of P2X7R was then verified using a selective antagonist, A-740003, or antisense for P2X7R administered in the L5-DRG. Inflammation was induced by CFA, carrageenan, IL-1β, or PGE2 administered in rat's hind paw. Blockage of P2X7R at the DRG reduced the mechanical hyperalgesia induced by CFA, and prevented the mechanical hyperalgesia induced by carrageenan or IL-1β, but not PGE2. It was also found an increase in P2X7 mRNA expression at the DRG after peripheral inflammation. IL-1β production was also increased by inflammatory stimuli in vivo and in vitro, using SGC-enriched cultures stimulated with LPS. In LPS-stimulated cultures, activation of P2X7R by BzATP induced the release of IL-1β, which was blocked by A-740003. In summary, our data suggest that peripheral inflammation leads to the activation of P2X7R expressed by SGCs at the DRG. Then, ATP-induced activation of P2X7R mediates the release of IL-1β from SGC. This evidence places the SGC as an active player in the establishment of peripheral inflammatory hyperalgesia and highlights the importance of the events in DRG for the treatment of inflammatory diseases

Down-regulation of CXCR2 on neutrophils in severe sepsis is mediated by inducible nitric oxide synthase-derived nitric oxide.

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2015-04-17T18:22:40Z No. of bitstreams: 1 Santos FR Down-regulation of....pdf: 1145084 bytes, checksum: 28d28253aec81401016829d03b4d7378 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2015-04-17T18:40:45Z (GMT) No. of bitstreams: 1 Santos FR Down-regulation of....pdf: 1145084 bytes, checksum: 28d28253aec81401016829d03b4d7378 (MD5)Made available in DSpace on 2015-04-17T18:40:45Z (GMT). No. of bitstreams: 1 Santos FR Down-regulation of....pdf: 1145084 bytes, checksum: 28d28253aec81401016829d03b4d7378 (MD5) Previous issue date: 2007Universidade de São Paulo. Faculdade de Medicina. Departamento de Farmacologia. Ribeirão Preto, SP, Brasil / University of Santa Cruz. Department of Health. Santa Cruz, BA, BrasilUniversidade de São Paulo. Faculdade de Medicina. Departamento de Farmacologia. Ribeirão Preto, SP, BrasilUniversity of Santa Cruz. Department of Health. Santa Cruz, BA, BrasilUniversidade de São Paulo. Faculdade de Medicina. Departamento de Farmacologia. Ribeirão Preto, SP, BrasilUniversidade de São Paulo. Faculdade de Medicina. Departamento de Farmacologia. Ribeirão Preto, SP, BrasilUniversidade de São Paulo. Faculdade de Medicina. Departamento de Farmacologia. Ribeirão Preto, SP, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Laboratorio de Engenharia Tecidual e Imunofarmacologia . Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Laboratorio de Engenharia Tecidual e Imunofarmacologia . Salvador, BA, BrasilUniversidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, BrasilUniversidade de São Paulo. Faculdade de Medicina. Departamento de Farmacologia. Ribeirão Preto, SP, BrasilRATIONALE: The failure of neutrophils to migrate to an infection focus during severe sepsis is an important determinant of the inability of a host to deal with an infectious insult. Our laboratory has shown that inducible nitric oxide synthase (iNOS) induction and NO production contribute to the failure of neutrophils to migrate in the context of sepsis. OBJECTIVES AND METHODS: We investigated whether CXCR2 expression contributed to the failure of neutrophils to migrate during severe sepsis and the role of NO in modulating CXCR2 expression on neutrophils in mice subjected to nonsevere (NS) or severe (S) cecal ligation and puncture (CLP). RESULTS: Neutrophil migration to the infection focus was deficient in S-CLP mice, a phenomenon prevented by pharmacologic (aminoguanidine, l-canavanine) or genetic (iNOS gene deletion) inhibition of iNOS. The expression of CXCR2 on neutrophils from S-CLP mice was significantly reduced when compared with neutrophils from NS-CLP or sham-operated mice. CXCR2 expression was reestablished by pharmacologic and genetic inhibition of iNOS. Immunofluorescence and confocal analysis revealed that iNOS blockade reduced neutrophil CXCR2 internalization. Adhesion and emigration of neutrophils in macrophage inflammatory protein-2-stimulated mesentery microcirculation were reduced in S-CLP mice, compared with NS-CLP mice, and reestablished by pretreatment with aminoguanidine or l-canavanine. The NO donor S-nitroso-N-acetyl-d,l-penicillamine inhibited CXCL8-induced human neutrophil chemotaxis and CXCR2 expression on human and murine neutrophils. CONCLUSION: These results highlight evidences that the failure of neutrophils to migrate to an infection focus during severe sepsis is associated with excessive NO production and NO-dependent regulation of the expression of CXCR2 on the neutrophil surfac