RESUMO
Although infiltrating macrophages influence many pathological processes after spinal cord injury (SCI), the intrinsic molecular mechanisms that regulate their function are poorly understood. A major hurdle has been dissecting macrophage-specific functions from those in other cell types as well as understanding how their functions change over time. Therefore, we used the RiboTag method to obtain macrophage-specific mRNA directly from the injured spinal cord in mice and performed RNA sequencing to investigate their transcriptional profile. Our data show that at 7 d after SCI, macrophages are best described as foam cells, with lipid catabolism representing the main biological process, and canonical nuclear receptor pathways as their potential mediators. Genetic deletion of a lipoprotein receptor, CD36, reduces macrophage lipid content and improves lesion size and locomotor recovery. Therefore, we report the first macrophage-specific transcriptional profile after SCI and highlight the lipid catabolic pathway as an important macrophage function that can be therapeutically targeted after SCI.SIGNIFICANCE STATEMENT The intrinsic molecular mechanisms that regulate macrophage function after spinal cord injury (SCI) are poorly understood. We obtained macrophage-specific mRNA directly from the injured spinal cord and performed RNA sequencing to investigate their transcriptional profile. Our data show that at 7 d after SCI, macrophages are best described as foam cells, with lipid catabolism representing the main biological process and canonical nuclear receptor pathways as their potential mediators. Genetic deletion of a lipoprotein receptor, CD36, reduces macrophage lipid content and improves lesion size and locomotor recovery. Therefore, we report the first macrophage-specific transcriptional profile after SCI and highlight the lipid catabolic pathway as an important macrophage function that can be therapeutically targeted after SCI.
Assuntos
Metabolismo dos Lipídeos/fisiologia , Macrófagos/metabolismo , Traumatismos da Medula Espinal/patologia , Animais , Transplante de Medula Óssea , Antígenos CD36/genética , Antígenos CD36/metabolismo , Movimento Celular/genética , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/genética , Hemaglutininas/metabolismo , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/metabolismo , Metabolismo dos Lipídeos/genética , Locomoção , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Ribossômico/administração & dosagem , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Transdução de Sinais/genética , Traumatismos da Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/cirurgiaRESUMO
Spinal cord injury (SCI) leads to formation of a fibrotic scar that is inhibitory to axon regeneration. Recent evidence indicates that the fibrotic scar is formed by perivascular fibroblasts, but the mechanism by which they are recruited to the injury site is unknown. Using bone marrow transplantation in mouse model of spinal cord injury, we show that fibroblasts in the fibrotic scar are associated with hematogenous macrophages rather than microglia, which are limited to the surrounding astroglial scar. Depletion of hematogenous macrophages results in reduced fibroblast density and basal lamina formation that is associated with increased axonal growth in the fibrotic scar. Cytokine gene expression analysis after macrophage depletion indicates that decreased Tnfsf8, Tnfsf13 (tumor necrosis factor superfamily members) and increased BMP1-7 (bone morphogenetic proteins) expression may serve as anti-fibrotic mechanisms. Our study demonstrates that hematogenous macrophages are necessary for fibrotic scar formation and macrophage depletion results in changes in multiple cytokines that make the injury site less fibrotic and more conducive to axonal growth.
Assuntos
Axônios/fisiologia , Cicatriz/prevenção & controle , Macrófagos/fisiologia , Regeneração Nervosa/fisiologia , Traumatismos da Medula Espinal/fisiopatologia , Animais , Axônios/patologia , Membrana Basal/patologia , Membrana Basal/fisiopatologia , Transplante de Medula Óssea/métodos , Proteínas Morfogenéticas Ósseas/metabolismo , Ligante CD30/metabolismo , Cicatriz/patologia , Cicatriz/fisiopatologia , Modelos Animais de Doenças , Feminino , Fibroblastos/patologia , Fibroblastos/fisiologia , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Traumatismos da Medula Espinal/patologia , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismoRESUMO
Adenovirus preparations are used as vectors in a number of gene therapy clinical development programs. The success of commercial production of adenovirus will strongly depend on the development of methods to define the recombinant virus product by analysis as opposed to being defined by the manufacturing process. While most analytical techniques examine portions of the virus, e.g. proteins or DNA, ion-exchange chromatography has been used to separate intact virus at low efficiency. A free zone capillary electrophoretic method was developed for high-efficiency separations of adenovirus 5. Experimental conditions such as buffer pH and concentration were explored which produced a high-efficiency separation in less than 20 min. The virus band was identified by collection of CE fractions and examination using a cell based assay. Initially, a single virus peak is found in fresh virus samples. After as little as one freeze-thaw in 1 x phosphate-buffered saline with 2% sucrose, the active virus migrates as a regular series of peaks. The nature of the virus modification leading to the differing electrophoretic mobilities is presently under investigation.