ARTICLE |
Correspondence to: Daniel J. Garry, NB 11.118A, 5323 Harry Hines Blvd., U. of Texas Southwestern Medical Center, Dallas, TX 75390-8573. E-mail: daniel.garry@utsouthwestern.edu
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Summary |
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Hemoproteins are widely distributed among unicellular eukaryotes, plants, and animals. In addition to myoglobin and hemoglobin, a third hemoprotein, neuroglobin, has recently been isolated from vertebrate brain. Although the functional role of this novel member of the globin family remains unclear, neuroglobin contains a heme-binding domain and may participate in diverse processes such as oxygen transport, oxygen storage, nitric oxide detoxification, or modulation of terminal oxidase activity. In this study we utilized in situ hybridization (ISH) and RT-PCR analyses to examine the expression of neuroglobin in the normoxic and hypoxic murine brain. In the normoxic adult mouse, neuroglobin expression was observed in focal regions of the brain, including the lateral tegmental nuclei, the preoptic nucleus, amygdala, locus coeruleus, and nucleus of the solitary tract. Using ISH and RT-PCR techniques, no significant changes in neuroglobin expression in the adult murine brain was observed in response to chronic 10% oxygen. These results support the hypothesis that neuroglobin is a hemoprotein that is expressed in the brain and may have diverse functional roles. (J Histochem Cytochem 50:15911598, 2002)
Key Words: neuroglobin, mice, brain, in situ hybridization, hypoxia
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Introduction |
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Diverse organisms express cytoplasmic hemoproteins or tissue hemoglobins that reversibly bind oxygen and have been proposed to function in facilitated oxygen transport to sites of use (
Neuroglobin is a recently identified hemoprotein that is expressed in human, mouse, rat, zebrafish, and the pufferfish brain (
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Materials and Methods |
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Tissue Processing
In accordance with NIH and institutional guidelines for animal use, tissues were harvested from normoxic and hypoxic C57BL/6 adult male mice. Each mouse was anesthetized with Avertin and a thoracotomy was performed to expose the heart. After transcardiac perfusion with approximately 10 ml of ice-cold DEPCPBS, the mice were subsequently fixed with 10 ml of 4% paraformaldehyde/DEPCPBS (
Hypoxia Chamber
A plexiglass hypoxic chamber 2 x 2 x 2 feet was engineered and monitored continuously for oxygen and CO2 concentration, temperature, and humidity. Ascarite II (820 mesh; Thomas Scientific, Swedesboro, NJ) was utilized to scrub excess CO2 production while anhydrous calcium sulfate (Drierite; WA Hammond Drierite, Xenia, OH) was used to maintain the humidity in the chamber between 60 and 80%. In the hypoxic chamber, mice were individually housed in 15 x 32-cm cages and were maintained in a cycle of 12-hr light, and 12-hr dark. To maintain a constant hypoxic environment of 10% O2, a gas mixture of 10% O2 and 90% N2 was infused into the chamber at a rate of 1 liter/min. An outlet valve was kept open to prevent the accumulation of positive pressure in the chamber, and a 12-volt auxiliary case fan was inserted into the chamber to provide constant and steady circulation of air. The chamber was vented at a specified time period each week and the cages were cleaned. The hypoxic environment was re-established within a 1-hr period. Hematocrits were obtained before and after hypoxic exposure. Statistical analysis of data utilized Student's paired t-test.
Vector Cloning
The heme-binding domain of mouse neuroglobin (Ngb) cDNA (157 bp) was amplified using RT-PCR. The neuroglobin primer set was designed from the sequence obtained from the National Center for Biotechnology Information Blast home page (http://www.ncbi.nlm.nih.gov/blast). The Ngb oligonucleotide primer set was purchased from Sigma-Genosys and sequences utilized for amplification were as follows: Ngb forward primer: 5'-GCTGCCTCTCTTCCAGTACAA- TGG-3'; Ngb reverse primer: 5'-GGTCAGGTACTCCTC- CAATGAAG-3'. Neuroglobin cDNA (157 bp) was gel-extracted using the Qiagen Gel Extraction Kit (Qiagen; Valencia, CA), ligated into the pCR II plasmid, and transformed utilizing the TA Cloning Kit-Dual Promoter (Invitrogen; Carlsbad, CA) (
RNA Isolation, Semiquantitative RT-PCR, and Real-time RT-PCR
Total RNA was isolated from adult normoxic and hypoxic (exposed to 10% oxygen for variable time periods) mouse brains utilizing the TriPure isolation kit (Roche Diagnostics; Indianapolis, IN), and reverse transcription (RT) was performed using Superscript II RNase H-Reverse Transcriptase (Invitrogen) to obtain cDNA. Briefly, 6 µg of total RNA was used in each reverse transcription reaction (60 µl). Two µl of diluted cDNA was used as a template for the PCR reaction in a 12-µl reaction volume. Semiquantitative RT-PCR utilizing RNA isolated from normoxic and hypoxic adult brains was performed as previously described (
To ensure equal loading between samples, RT-PCR of the various cDNA samples was performed using the forward and reverse primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH): GAPDH forward primer: 5'-GTGGCAAAGTGGAGATTGTTGCC-3'; GAPDH reverse primer: 5'-GATGATGACCCGTTTGGCTCC-3'. Real-time PCR was performed using the iCycler iQ Real-Time PCR Detection System (Bio-Rad Laboratories; Hercules, CA) with QuantiTect SYBR Green PCR Kit (Qiagen). The reaction was carried out in a final volume of 20 µl: 10 µl of 2 x SYBR Green PCR master mix, 0.3 µl of 20 µM forward primer, 0.3 µl of 20 µM reverse primer, 1 µl of cDNA template, and 8.4 µl water, for 40 cycles (20-sec denaturation at 95C, 20-sec annealing at 60C, and 30-sec elongation at 72C). The 102-bp amplicon was detected after each elongation step and was analyzed using the iCycler iQ software (Bio-Rad). A melting curve was obtained after completion of the cycles to verify the presence of a single amplicon. Data were expressed using the comparative threshold cycle (CT) method as an estimate of mRNA amount in the hypoxic tissue relative to reference control. Comparative expression level was 2-CT. The respective primer sequences used for real-time PCR included Ngb forward primer, 5'-TACAATGGCCGCCAGTTCT-3'; Ngb reverse primer, 5'-TGGTCACTGCAGCATCAATCA-3'.
Riboprobe Synthesis
Templates for antisense and sense riboprobes were linearized from cloned neuroglobinpCR II plasmid with the restriction enzymes, Xho I and BamH I, respectively. Radiolabeled neuroglobin RNA probes were synthesized from 500 ng of template by in vitro transcription (Maxiscript kit; Ambion, Austin, TX) in the presence of 60 µCi of [35S]-dUTP (Amersham; Piscataway, NJ) as previously described (
In Situ Hybridization
In situ hybridizations were performed according to procedures as previously described (
Microscopy and Photomicrography
Neuroglobin expression was visualized using a Leitz Laborlux-S microscope stand equipped with Plan-Apochromatic optics, a standard brightfield condenser, and a Mears low-magnification darkfield condenser. Photomicrographs were obtained with an Optronics VI-470 CCD camera and a Power Macintosh G3 equipped with a Scion CG-7 frame grabber and Scion Image 1.62 software.
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Results |
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This study was undertaken to define the spatial expression pattern for the novel hemoprotein, neuroglobin, in the adult murine brain. Fixed-frozen and paraffin-embedded brain sections were used for ISH in combination with radioactively labeled RNA probes to achieve enhanced sensitivity for this technique. Using these ISH techniques, neuroglobin mRNA expression was examined in adult male C57Bl/6 mice (n=3). Neuroglobin mRNA-positive neurons were localized to discrete nuclei in the adult brain (Fig 1). In the forebrain, neuroglobin was localized to the lateral septal nuclei (LSD, LSI, LSV), the median preoptic nucleus (MnPO), and the bed nucleus of the stria terminalis (BSTL). At the level of the optic chiasm, expression was observed in the subfornical organ (SFO) and was persistent in the medial preoptic area (MPA) and the bed nucleus of the stria terminalis (BSTL). A coronal section through the diencephelon revealed neuroglobin expression within the lateral habenula (LHb), central and basomedial amygdala (BMA), arcuate nucleus (Arc), lateral (LH), dorsomedial (DM), and ventromedial (VM) hypothalamus. Transcript expression in the midbrain was observed in the periaqueductal gray (PAG), sub-brachial nucleus (SubB), lateral tegmental nuclei (LDTg), deep gray of the colliculus (DpG), locus coeruleus (LC), and the nucleus of the solitary tract (Sol). The level of expression in these regions varied. Intense (++++) neuroglobin expression was observed in the lateral tegmental nuclei, median preoptic nucleus, and subbrachial nucleus. Moderate signals were detected in the bed nucleus of the stria terminalis, the subfornical organ, thalamus, arcuate nucleus, amygdala, and the locus coeruleus (Fig 1). No expression was observed in the cerbral cortex and Ammon's horn (Fig 1). Sections hybridized with the sense RNA probe were negative (Fig 2B vs Fig 2A). Examination of the signal at high magnification using both darkfield and brightfield microscopy revealed that grains were localized over neuronal cell bodies (Fig 2C and Fig 2D). No differences in expression patterns were noted when antisense neuroglobin riboprobes were hybridized to either frozen or paraffin sections of adult brain.
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To determine whether chronic hypoxia results in altered expression of neuroglobin (i.e., global expression of neuroglobin), mice were exposed to 10% hypoxia for periods of up to 2 weeks. Although the mice responded by mounting a polycythemic response to chronic hypoxia [hematocrit of wild-type normoxic mice 51 ± 2% (n=3); hematocrit of 2-week hypoxic mice 71 ± 3% (n=3), p<0.005] there was no significant increase in neuroglobin transcript expression involving additional nuclei using ISH (Fig 3). RT-PCR analysis was undertaken for enhanced examination of neuroglobin expression in response to a significant hypoxic challenge. Utilizing RT-PCR analysis and primers spanning an intron, we observed that the neuroglobin transcript increased with postnatal development (data not shown) and remained relatively constant with exposure to hypoxic conditions (Fig 4).
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Discussion |
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The present study defines the spatial pattern of expression of the endogenous neuroglobin gene in the adult mouse. Neuroglobin gene expression is expressed in focal regions of the adult brain. Within these focal regions of the central nervous system (CNS), neuroglobin mRNA expression is observed in neuronal cells and is qualitatively absent in the vasculature.
Neuroglobin is the third known tissue hemoglobin and is expressed predominantly in the vertebrate brain (
Previous studies support a role for neuroglobin in the storage or transport of oxygen (24 hr, 95% N2/5% CO2) or pharmacological agents such as cobalt chloride or deferoxamine, which promote HIF-1
and the hypoxia-inducible gene program (
In this study we did not observe expanded neuroglobin expression in response to a chronic 10% hypoxic challenge using RT-PCR and ISH techniques. We cannot exclude the possibility that neuroglobin expression is transiently increased during the acute phase (<24 hr) after exposure to 10% oxygen. In addition, it is possible that RT-PCR analysis using total RNA isolated from the normoxic and hypoxic brains would not reveal localized changes in specific regions. We also recognize that, as for other hypoxia-inducible factors (i.e., HIF-1), the possibility exists that neuroglobin is regulated at the translational level in response to a hypoxic challenge. Alternatively, the absence of altered gene expression in response to chronic hypoxia may be due to alternative functions for neuroglobin in vivo, such as a role as a nitric oxide scavenger or as a modulator of terminal oxidases (
Neuroglobin is the first example of a hexacoordinate hemoglobin in vertebrates (
In this study we observed persistent focal expression of neuroglobin in the adult brain. These regions (e.g., parabrachial complex, locus coeruleus, PAG, NTS) are responsive to both hypercapnic and hypoxic stimuli (
Previous studies have reported regional localization of neuroglobin in neurons associated with Ammon's horn, as well as the cerebral cortex, in the rat (
Neuroglobin is a hexacoordinated hemoglobin that is persistently expressed in the vertebrate brain (
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Acknowledgments |
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Supported by grants from the American Heart Association, the National Institutes of Health, and the D.W. Reynolds Foundation. This research was conducted while Dr P.P.A. Mammen was a Pfizer Postdoctoral Fellow.
We thank Drs Robert P. Elde (University of Minnesota; Minneapolis, MN) and Martin Wessendorf (University of Minnesota) for helpful discussions throughout the course of this study. Technical assistance for these studies was provided by Chris Pomajzl and Jeff Stark.
Received for publication May 8, 2002; accepted July 3, 2002.
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