Effects of short-term hypoxia on neuroglobin levels and localization in mouse brain tissues.

Nerve cells are highly susceptible to ischemic and hypoxic injuries. The neuroglobin (Ngb), found in vertebrate nerve cells, has been suggested to protect nerve cells from ischemic episodes by a yet unknown mechanism. However, contradicting reports exist regarding localization and up-regulation of Ngb in response to hypoxia. The aim of the present study was to probe the distribution of Ngb proteins in mouse brain and retina by immunohistochemistry, and to quantify the levels of Ngb mRNA by reverse-transcription-polymerase chain reaction (RT-PCR) after short-term (2 h) exposure to 7.6% oxygen. We found Ngb to be present throughout the neocortex, most abundantly in the perirhinal, entorhinal and temporal cortical areas, the thalamus and hypothalamus, the choroid plexus, the olfactory bulb and the cranial nerve nuclei in the brainstem. Intense staining was observed in the mesencephalic central grey area and the Purkinje cells. Two-hour hypoxic exposure caused no detectable changes in staining intensity or spatial distribution of Ngb neither in the Purkinje cells nor in any other brain areas observed. The RT-PCR data supported the lack of differences in brain Ngb levels between normal and oxygen-deprived animals. In the retina, Ngb localization by immunohistochemistry was confined to the inner segments of the photoreceptors, the plexiform layers and the ganglion cells. Short-termed hypoxia did not change retinal Ngb levels as assessed by both techniques. The lack of Ngb up-regulation in the brain is consistent with results from previous long-term hypoxic experiments, suggesting that Ngb is not regulated by pure hypoxia in vivo.

Cytoglobin expression is upregulated in all tissues upon hypoxia: an in vitro and in vivo study by quantitative real-time PCR.

The vertebrate globin family has been extended with two members: neuroglobin and cytoglobin. We here investigate the changes of expression levels upon hypoxia of cytoglobin in parallel with neuroglobin, in vivo and in vitro, by using real-time quantitative PCR. Our data prove that cytoglobin is upregulated upon hypoxia in all tissues. The mechanism of induction of cytoglobin is regulated by the hypoxia-inducible factor 1, a posttranscriptionally regulated transcription factor controlling several hypoxia-inducible genes. The latter is argumented by: (1) cytoglobin is significantly upregulated upon hypoxia and this is dependent on the tissue and severity of hypoxia; (2) the regulation of cytoglobin expression in HIF-1 (+/-) knockout mice is affected; (3) the variations of the expression regulation are in the same manner as seen in the expression of our control gene VEGF, that is proven to be regulated by the HIF-1-pathway; and (4) cytoglobin promoter region contains HRE sites.

Hypoxia/ischemia and the regulation of neuroglobin and cytoglobin expression.

In analogy to hemoglobin (Hb) and myoglobin (Mb), neuroglobin (Ngb) and cytoglobin (Cygb) are supposed to be involved in oxygen (O2) storage and delivery. The Cygb gene harbours both conserved HREs and mRNA stabilization sites, strongly suggestive of an oxygen-dependent regulation. We examined the relative transcriptional changes of Ngb and Cygb in a situation of chronic hypoxia using real-time quantitative PCR. We could conclude that Cygb is a hypoxia-induced gene, which is transcriptionally upregulated during chronic hypoxia in a hippocampal neuronal cell line and in multiple murine metabolically active tissues. The mechanism of induction of Cygb is HIF-1alpha dependent. HIF-1 is unique among mammalian transcription factors with respect to the specificity and sensitivity of its induction by hypoxia. Ngb expression seems to be regulated using other response elements and is less influenced by hypoxia.