RESUMEN
BACKGROUND: In this study, we evaluate the role of RGMa (Repulsive Guidance Molecule a) during peripheral nerve regeneration using the mouse median nerve model. METHODS: By real-time PCR and Western Blot analysis, we examined expression changes of RGMa mRNA and RGMa protein in neural tissue after transection and microsurgical repair of the mouse median nerve distal to the transection site. We evaluated histomorphometrical changes in neural tissue distal to the injury site and functional recovery of the grasping force after median nerve transection and repair in wild-type mice and RGMa+/- heterozygous mice. RESULTS: RT-PCR revealed a 1,8 fold increase of RGMa mRNA two weeks and a 4,4 fold increase of RGMa mRNA 3 weeks after nerve transection and repair in the nerve segment distal to the injury site. In Western blot analysis, we could show a high increase of RGMa in the nerve segment distal to the injury site at day 14. Histomorphometrical analysis showed significant differences between wild-type animals and heterozygous animals. The absolute number of myelinated fibres was significantly higher in operated heterozygous RGMa+/- animals compared to operated wildtye animals. Using the functional grasping test, we could demonstrate that peripheral nerve regeneration is significantly diminished in heterozygous RGMa+/- mice. CONCLUSIONS: Employing the mouse median nerve model in transgenic animals, we demonstrate that RGMa plays an important role during peripheral nerve regeneration.
Asunto(s)
Proteínas Ligadas a GPI/fisiología , Nervio Mediano/lesiones , Nervio Mediano/fisiopatología , Actividad Motora/fisiología , Regeneración Nerviosa/fisiología , Proteínas del Tejido Nervioso/fisiología , Traumatismos de los Nervios Periféricos/metabolismo , Animales , Conducta Animal/fisiología , Modelos Animales de Enfermedad , Proteínas Ligadas a GPI/metabolismo , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , ARN Mensajero/metabolismoRESUMEN
INTRODUCTION: In this study, we evaluated the role of the Netrin-1 receptor UNC5b (Uncoordinated), a neuronal guidance molecule, during peripheral nerve regeneration using the mouse median nerve model. MATERIALS AND METHODS: Using Western blot analysis, we examined the expression changes of UNC5b after transection and microsurgical repair of the mouse median nerve distal to the transection site. We evaluated the histomorphometrical changes and functional recovery of the grasping force after median nerve transection and repair in wild-type (WT) mice and UNC5b(+/-) heterozygous mice. RESULTS: In Western blot analysis, we could show a high increase of UNC5b in the nerve segment distal to the injury site at day 14. Histomorphometrical analysis did not show any significant differences between WT animals and heterozygous animals. Using the functional grasping test, we could demonstrate that peripheral nerve regeneration is significantly diminished in heterozygous UNC5b(+/-) mice. CONCLUSION: By using the mouse median nerve model in transgenic animals, we demonstrate that the Netrin-1 receptor UNC5b plays an important role during peripheral nerve regeneration.
Asunto(s)
Regeneración Nerviosa/fisiología , Receptores de Superficie Celular/biosíntesis , Animales , Nervio Mediano/fisiología , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Receptores de Netrina , Receptores de Superficie Celular/fisiología , Factores de TiempoRESUMEN
The neuronal guidance molecule netrin-1 is linked to the coordination of inflammatory responses. Given that mucosal surfaces are particularly prone to hypoxia-elicited inflammation, we sought to determine the function of netrin-1 in hypoxia-induced inflammation. We detected hypoxia-inducible factor 1alpha (HIF-1alpha)-dependent induction of expression of the gene encoding netrin-1 (Ntn1) in hypoxic epithelia. Neutrophil transepithelial migration studies showed that by engaging A2B adenosine receptor (A2BAR) on neutrophils, netrin-1 attenuated neutrophil transmigration. Exogenous netrin-1 suppressed hypoxia-elicited inflammation in wild-type but not in A2BAR-deficient mice, and inflammatory hypoxia was enhanced in Ntn1(+/-) mice relative to that in Ntn1(+/+) mice. Our studies demonstrate that HIF-1alpha-dependent induction of netrin-1 attenuates hypoxia-elicited inflammation at mucosal surfaces.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/metabolismo , Inflamación/inmunología , Factores de Crecimiento Nervioso/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Células CACO-2 , Quimiotaxis de Leucocito/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Hipoxia/complicaciones , Hipoxia/inmunología , Subunidad alfa del Factor 1 Inducible por Hipoxia/inmunología , Immunoblotting , Inmunohistoquímica , Inmunoprecipitación , Inflamación/genética , Inflamación/metabolismo , Ratones , Membrana Mucosa/inmunología , Membrana Mucosa/metabolismo , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/inmunología , Netrina-1 , Infiltración Neutrófila/inmunología , Receptores Purinérgicos P1/genética , Receptores Purinérgicos P1/inmunología , Receptores Purinérgicos P1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/inmunologíaRESUMEN
Extracellular ATP liberated during hypoxia and inflammation can either signal directly on purinergic receptors or can activate adenosine receptors following phosphohydrolysis to adenosine. Given the association of polymorphonuclear leukocytes (PMNs) with adenine-nucleotide/nucleoside signaling in the inflammatory milieu, we hypothesized that PMNs are a source of extracellular ATP. Initial studies using high-performance liquid chromatography and luminometric ATP detection assays revealed that PMNs release ATP through activation-dependent pathways. In vitro models of endothelial barrier function and neutrophil/endothelial adhesion indicated that PMN-derived ATP signals through endothelial adenosine receptors, thereby promoting endothelial barrier function and attenuating PMN/endothelial adhesion. Metabolism of extracellular ATP to adenosine required PMNs, and studies addressing these metabolic steps revealed that PMN express surface ecto-apyrase (CD39). In fact, studies with PMNs derived from cd39(-/-) mice showed significantly increased levels of extracellular ATP and lack of ATP dissipation from their supernatants. After excluding lytic ATP release, we used pharmacological strategies to reveal a potential mechanism involved in PMN-dependent ATP release (eg, verapamil, dipyridamole, brefeldin A, 18-alpha-glycyrrhetinic acid, connexin-mimetic peptides). These studies showed that PMN ATP release occurs through connexin 43 (Cx43) hemichannels in a protein/phosphatase-A-dependent manner. Findings in human PMNs were confirmed in PMNs derived from induced Cx43(-/-) mice, whereby activated PMNs release less than 15% of ATP relative to littermate controls, whereas Cx43 heterozygote PMNs were intermediate in their capacity for ATP release (P<0.01). Taken together, our results identify a previously unappreciated role for Cx43 in activated PMN ATP release, therein contributing to the innate metabolic control of the inflammatory milieu.
Asunto(s)
Adenosina Trifosfato/fisiología , Conexina 43/fisiología , Células Endoteliales/fisiología , Activación Neutrófila/fisiología , Neutrófilos/fisiología , Adenosina/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Antígenos CD/metabolismo , Apirasa/deficiencia , Apirasa/metabolismo , Conexina 43/deficiencia , Conexina 43/metabolismo , Células Endoteliales/metabolismo , Humanos , Ratones , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , FosforilaciónRESUMEN
Interactions between the vascular endothelium and polymorphonuclear leukocytes (PMNs) are central to PMN emigration into inflamed tissues, and to neutrophil-endothelial crosstalk pathways that modulate inflammatory responses and vascular barrier function. For example, during episodes of inflammation, the transendothelial migration (TEM) of PMNs potentially disturbs vascular barrier and gives rise to intravascular fluid extravasation and edema. However, because of the close special relationship between PMNs and the vascular endothelium, TEM creates an ideal situation for neutrophil-endothelial crosstalk. While investigating innate mechanisms to dampen intravascular fluid loss and edema occurring during TEM, we observed that PMNs release adenine nucleotides after activation (adenosine triphosphate [ATP] and adenosine monophosphate [AMP]). ATP and AMP are metabolized by endothelial cell-surface enzymes, the ecto-apyrase (CD39, metabolizes ATP to AMP) and the 5'-ecto-nucleotidase (CD73, metabolizes AMP to adenosine). Adenosine generated in this fashion can activate endothelial adenosine receptors, leading to increases in intracellular cyclic AMP and resealing of the endothelial junctions, thereby promoting vascular barrier function. This crosstalk pathway provides an endogenous mechanism to dampen vascular leak syndrome during neutrophil-endothelial interaction. In other words, during TEM, neutrophils close the door behind them.