Postnatal hyperoxia exposure differentially affects hepatocytes and liver haemopoietic cells in newborn rats

Abstract

Premature newborns are frequently exposed to hyperoxic conditions and experimental data indicate modulation of liver metabolism by hyperoxia in the first postnatal period. Conversely, nothing is known about possible modulation of growth factors and signaling molecules involved in other hyperoxic responses and no data are available about the effects of hyperoxia in postnatal liver haematopoiesis. The aim of the study was to analyse the effects of hyperoxia in the liver tissue (hepatocytes and haemopoietic cells) and to investigate possible changes in the expression of Vascular Endothelial Growth Factor (VEGF), Matrix Metalloproteinase 9 (MMP-9), Hypoxia-Inducible Factor-1α (HIF-1α), endothelial Nitric Oxide Synthase (eNOS), and Nuclear Factor-kB (NF-kB). Experimental design of the study involved exposure of newborn rats to room air (controls), 60% O2 (moderate hyperoxia), or 95% O2 (severe hyperoxia) for the first two postnatal weeks. Immunohistochemical and Western blot analyses were performed. Severe hyperoxia increased hepatocyte apoptosis and MMP-9 expression and decreased VEGF expression. Reduced content in reticular fibers was found in moderate and severe hyperoxia. Some other changes were specifically produced in hepatocytes by moderate hyperoxia, i.e., upregulation of HIF-1α and downregulation of eNOS and NF-kB. Postnatal severe hyperoxia exposure increased liver haemopoiesis and upregulated the expression of VEGF (both moderate and severe hyperoxia) and eNOS (severe hyperoxia) in haemopoietic cells. In conclusion, our study showed different effects of hyperoxia on hepatocytes and haemopoietic cells and differential involvement of the above factors. The involvement of VEGF and eNOS in the liver haemopoietic response to hyperoxia may be hypothesized.

Figure 1. Severe hyperoxia increases liver haemopoiesis.

A) Haematoxylin-eosin staining of neonatal rat livers exposed to moderate and severe hyperoxia. a) ambient air; b) 60% hyperoxia; c) 95% hyperoxia. Arrows indicate haemopoietic foci. Scale bar: 200 µm, magnification 10x. B) Anti-ki67 immunohistochemistry showing diffuse positivity of haemopoietic cells. Scale bar: 50 µm, magnification 40x. C) Anti-CD34 immunohistochemistry showing diffuse positivity of haemopoietic cells. Scale bar: 50 µm, magnification 63x. D) Anti-CD45 immunohistochemistry showing diffuse positivity of haemopoietic cells. Scale bar: 50 µm, magnification 63x. E) Graphic representation of the mean values of the percentage area of liver haemopoietic foci (±SD) determined in ten fields for each of three slides per sample. ** = p<0.01.

Figure 2. Severe hyperoxia increases hepatocyte apoptosis.

A) TUNEL detection of apoptotic nuclei (arrow) in neonatal rat livers exposed to moderate and severe hyperoxia. a) ambient air; b) 60% hyperoxia; c) 95% hyperoxia; d) positive control; e) negative control. Scale bar: 50 µm, magnification 40x. B) Graphic representation of the percentage of TUNEL positive nuclei (±SD) determined by direct visual counting of ten fields for each of three slides per sample. ** = p<0.01.

Figure 3. Moderate and severe hyperoxia reduces the amount of reticular fibers in rat liver.

A) Silver staining of neonatal rat livers exposed to moderate and severe hyperoxia. Reticular fibers are labeled by black stain. In livers of rats exposed to moderate and severe hyperoxia a lower amount of reticular fibers is visible. a) ambient air; b) 60% hyperoxia; c) 95% hyperoxia. Scale bar: 50 µm, magnification 40x. B) Graphic representation of the percentage area of reticular fibers (±SD); densitometric analysis determined by quantifying thresholded area for black color in ten fields for each of three slides per sample. * = p<0.05.

Figure 4. Severe hyperoxia increases liver expression of MMP-9.

Western blotting analysis of MMP-9 expression in neonatal rat livers exposed to moderate and severe hyperoxia. a) ambient air; b) 60% hyperoxia; c) 95% hyperoxia. Each membrane has been probed with anti-tubulin antibody to verify loading evenness. Data are the densitometric measurements of protein bands expressed as mean values of Integrated Optical Intensity (IOI) (±SD).

Figure 5. Severe hyperoxia decreases the number of hepatocytes immunostained for VEGF.

A) Immunohistochemical detection of VEGF expression in neonatal rat livers exposed to moderate and severe hyperoxia. a) ambient air; b) 60% hyperoxia; c) 95% hyperoxia; d) negative control; scale bar: 50 µm, magnification 40x. B) Graphic representation of the percentage of VEGF positive nuclei (±SD); densitometric analysis determined by direct visual counting of ten fields for each of three slides per sample. * = p<0.05; ** = p<0.01.

Figure 6. Moderate and severe hyperoxia increases VEGF expression in liver haemopoietic foci.

A) Immunohistochemical detection of VEGF-positive haemopoietic cells in neonatal rat livers exposed to moderate and severe hyperoxia. a) ambient air; b) 60% hyperoxia; c) 95% hyperoxia; d) negative control; scale bar: 50 µm, magnification 40x. B) Graphic representation of VEGF-positive haemopoietic cells percentages (±SD); densitometric analysis determined by direct visual counting of ten fields for each of three slides per sample. ** = p<0.01.

Figure 7. Moderate hyperoxia increases the number of HIF-1α-positive hepatocytes with respect to normoxia and severe hyperoxia.

A) Immunohistochemical detection of HIF-1α expression in neonatal rat livers exposed to moderate and severe hyperoxia. a) ambient air; b) 60% hyperoxia; c) 95% hyperoxia; d) negative control; scale bar: 50 µm, magnification 40x. B) Graphic representation of the percentages of HIF-1α positive nuclei (±SD); densitometric analysis determined by direct visual counting of ten fields for each of three slides per sample. ** = p<0.01.

Figure 8. Moderate hyperoxia reduces hepatocyte eNOS expression with respect to normoxia and severe hyperoxia.

A) Immunohistochemical detection of eNOS expression in neonatal rat livers exposed to moderate and severe hyperoxia. a) ambient air; b) 60% hyperoxia; c) 95% hyperoxia; d) negative control; scale bar: 50 µm, magnification 40x. B) Graphic representation of the percentage of eNOS positive area (±SD); densitometric analysis determined by direct visual counting of ten fields for each of three slides per sample. * = p<0.05. C) Western blotting analysis of eNOS expression in neonatal rats. Each membrane has been probed with anti β-tubulin antibody to verify loading evenness. The most representative out of three separate experiments is shown. Data are the densitometric measurements of protein bands expressed as mean values of Integrated Optical Intensity (IOI) (±SD).

Figure 9. Severe hyperoxia increases eNOS expression in haemopoietic foci.

A) Immunohistochemical detection of eNOS-positive haemopoietic cells (arrows) in neonatal rat livers exposed to moderate and severe hyperoxia. a) ambient air; b) 60% hyperoxia; c) 95% hyperoxia; d) negative control; scale bar: 50 µm, magnification 40x. B) Graphic representation of the percentages of eNOS-positive haemopoietic cells (±SD); densitometric analysis determined by direct visual counting of ten fields for each of three slides per sample. ** = p<0.01.

Figure 10. Moderate hyperoxia reduces the number of NF-kB positive hepatocytes with respect to normoxia and severe hyperoxia.

A) Immunohistochemical detection of Nfκb expression in neonatal rat livers exposed to moderate and severe hyperoxia. a) ambient air; b) 60% hyperoxia; c) 95% hyperoxia; d) negative control; scale bar: 50 µm, magnification 40x. B) Graphic representation of the percentages of NF-kB positive nuclei (±SD); densitometric analysis determined by direct visual counting of ten fields for each of three slides per sample. * = p<0.05; ** = p<0.01.