RESUMO
The ventilatory response to hypoxia is mediated by peripheral inputs arising from the arterial chemoreceptors. In their absence, hypoxic adaptation can be achieved, possibly as a result of central cellular reorganization. To study this reorganization, we used chemodenervated rats to investigate the expression and localization of vascular endothelial growth factor (VEGF) in the brainstem. VEGF is a target gene of hypoxia-inducible factor (HIF) that is responsible for the morphofunctional remodeling induced by hypoxia. Intact and chemodenervated rats were subjected to normoxia or hypoxia for 6 hr (10% O(2) in N(2)). VEGF protein was quantified in micropunches of brainstem tissue. Only chemodenervated animals showed an increased VEGF expression in response to hypoxia, whereas, in normoxia, VEGF expression was not modified by chemodenervation. The same hypoxic condition was repeated for 8 days before immunocytochemical staining with anti-VEGF; antiglial fibrillary acidic protein (GFAP), a marker of astrocytes; and anti-rat endothelial cell antigen-1 (anti-RECA-1) that recognizes endothelial cells. Confocal analysis showed a cellular colocalization of GFAP and VEGF, indicating that VEGF was overexpressed predominantly in astrocytes. Increased RECA-1 immunolabeling indicated an enhanced angiogenesis in chemodenervated rats subjected to hypoxia. These results indicate that glial cells and the vascular network contribute to the brainstem remodeling. The peripheral chemodenervation reveals a central O(2) chemosensitivity involving a cascade of gene expression triggered by hypoxia, which in intact animals may act synergically with peripheral chemosensory inputs.
Assuntos
Tronco Encefálico/metabolismo , Regulação da Expressão Gênica/fisiologia , Hipóxia/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Análise de Variância , Animais , Antígenos de Superfície/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Proteína Glial Fibrilar Ácida/metabolismo , Nervo Glossofaríngeo/fisiopatologia , Traumatismos do Nervo Glossofaríngeo , Masculino , Glicoproteínas de Membrana/metabolismo , Ratos , Ratos Sprague-Dawley , Rizotomia/métodos , Estatísticas não ParamétricasRESUMO
Mutations of genes encoding Phox2a or Phox2b transcription factors induce modifications of different brainstem neuronal networks. Such modifications are associated with defects in breathing behavior at birth. In particular, an abnormal breathing frequency is observed in Phox2a-/- mutant mice, resulting from abnormal development of the locus coeruleus (LC) nucleus. However, the role of Phox2a proteins in the establishment of respiratory neuronal pathways is unknown, largely because mutants die shortly after birth. In the present study, we examined the effects of a haploinsufficiency of the Phox2a gene. Phox2a heterozygotes survive and exhibit a significantly larger inspiratory volume both during normoxic breathing and in response to hypoxia and a delayed maturation of inspiratory duration compared to wild-type animals. This phenotype accompanied by an unaltered frequency is evident at birth and persists until at least postnatal day 10. Morphological analyses of Phox2a+/- animals revealed no anomaly in the LC region, but highlighted an increase in the number of cells expressing tyrosine hydroxylase enzyme, a marker of chemoafferent neurons, in the petrosal sensory ganglion. These data indicate that Phox2a plays a critical role in the ontogeny of the reflex control of inspiration.
Assuntos
Proteínas de Homeodomínio/genética , Camundongos Knockout/anormalidades , Transtornos Respiratórios/genética , Transtornos Respiratórios/patologia , Fatores Etários , Análise de Variância , Animais , Animais Recém-Nascidos , Contagem de Células/métodos , Hipóxia/genética , Hipóxia/fisiopatologia , Imuno-Histoquímica , Técnicas In Vitro , Locus Cerúleo/metabolismo , Locus Cerúleo/patologia , Camundongos , Pletismografia/métodos , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Studies of the sites and mechanisms involved in mammalian respiratory rhythm generation point to two clusters of rhythmic neurons forming a coupled oscillator network within the brainstem. The location of these oscillators, the pre-Bötzinger complex (preBötC) at vagal level, and the para-facial respiratory group at facial level, probably result from regional patterning schemes specifying neural types in the hindbrain during embryogenesis. Here, we report evidence that the preBötC oscillator (i) is first active at embryonic stages, (ii) originates in the post-otic hindbrain neural tube and (iii) requires the glutamate vesicular transporter 2 for rhythm generation.
Assuntos
Embrião de Mamíferos/fisiologia , Bulbo/fisiologia , Periodicidade , Centro Respiratório/fisiologia , Animais , Camundongos/embriologia , Proteína Vesicular 2 de Transporte de Glutamato/metabolismoRESUMO
The chromosome locations of 368 human Kruppel-type zinc finger (ZNF) PAC clones were physically mapped by FISH to human chromosomes in support of recent efforts of assigning KOX cDNAs (KOX1-KOX32) to zinc finger gene clusters. Recent mapping results were validated and confirmed by sequence comparisons to zinc finger gene sequences automatically annotated in EnsEMBL. In toto, 799 Kruppel-type zinc finger genes have been annotated in EnsEMBL of which 290 genes are found to encode KRAB domains. Sequence homologies of the zinc finger domains were used to establish phylogenic trees of KOX zinc finger genes as well as of all KRAB containing human zinc finger and KOX genes documenting the evolution of KRAB zinc finger genes late in primate evolution. A list of 368 assigned ZNF PAC clones is available under http://www.pzr.uni-rostock.de/supplements.