Ferritin as a reporter gene for MRI: chronic liver over expression of H-ferritin during dietary iron supplementation and aging

Abstract

The iron storage protein, ferritin, provides an important endogenous MRI contrast that can be used to determine the level of tissue iron. In recent years the impact of modulating ferritin expression on MRI contrast and relaxation rates was evaluated by several groups, using genetically modified cells, viral gene transfer and transgenic animals. This paper reports the follow-up of transgenic mice that chronically over-expressed the heavy chain of ferritin (h-ferritin) in liver hepatocytes (liver-hfer mice) over a period of 2 years, with the aim of investigating the long-term effects of elevated level of h-ferritin on MR signal and on the well-being of the mice. Analysis revealed that aging liver-hfer mice, exposed to chronic elevated expression of h-ferritin, have increased R(2) values compared to WT. As expected for ferritin, R(2) difference was strongly enhanced at high magnetic field. Histological analysis of these mice did not reveal liver changes with prolonged over expression of ferritin, and no differences could be detected in other organs. Furthermore, dietary iron supplementation significantly affected MRI contrast, without affecting animal wellbeing, for both wildtype and ferritin over expressing transgenic mice. These results suggest the safety of ferritin over-expression, and support the use of h-ferritin as a reporter gene for MRI.

Figure 1. MRI gray scale image of mice with regular diet and High iron diet

4.7T and 9.4T MR images of WT mice (A-D) and liver-hfer mice (E-H) with constitutive transgene expression (2 years old) that were treated with normal diet and high iron diet (TR=2 s, TE=11 ms).

Figure 2. R₂ values according to ROI measurements in the liver of the different groups at 4.7T magnet

Each group was scanned at a 4.7T magnet during 18 months of normal and high iron diet, (A) ROI analysis showed significant difference in R₂ between the groups that were treated with high iron diet to groups with regular diet at all time points except for the last time point (*p<0.05 unpaired 2 tail Ttest). Numbers at the top of each bar state the mice age in months (iron supplementation diet was initiated when mice were 2 months old). (B) Analysis of the distribution of R₂ values derived from R₂ maps showed the spatial distribution of 4.7T R₂ values in the liver of 9 months old mice. Overexpression of h-ferritin resulted in elevated R₂ values. Wild type mice (gray), liver-hfer mice (green).

Figure 3. Field dependence of R₂ in liver-hfer mice

Overlay of R₂ color maps on a gray scale anatomical images (TE=6.4 ms) of WT mice (A, C) and liver-hfer mice (B, D) (age 24 months). Mice at the age of 24 months were imaged at two magnetic fields, 4.7T and 9.4T. (E, F) ROI analysis reveals significant difference in R₂ between WT and liver-hfer mice that were treated with regular diet and scanned at 9.4T (*p<0.05 unpaired 2 tail Ttest).

Figure 4. Histological evaluation of the liver

Liver sections that were taken from representative mice at the age of 20 months were stained with eosin hematoxylin (A-B, E-F) and by Prussian blue, which stains ferric ions (Fe+3) in bright blue color (C-D, G-H). (A, C) WT mice with normal diet, (B, D) liver-hfer mice with normal diet, (E, G) WT mice with high iron diet, (F, H) liver-hfer mice with high iron diet (arrows, hemosiderin deposits).

Figure 5. Histological evaluation of liver-hfer and WT mice

Different organs sections were taken from representative mice from each group at the age of 20 months and stained with eosin hematoxylin . (A-D) Brain, (E-H) Heart, (I-L) Kidney, (M-P) Spleen (arrows, hemosiderin deposits).

Figure 6. Iron levels evaluation by Prussian blue stain

Different organs sections were taken from representative mice from each group at the age of 20 months and stained by Prussian blue. (A-D) Brain, (E-H) Heart, (I-L) Kidney, (M-P) Spleen.

Figure 7. Iron mapping of the liver using scanning electron microscope (SEM)

Liver sections that were taken from representative mice at the age of 24 months were mapped for iron levels. (A-D) Livers of mice that were raised with normal diet (A, D back scattered images, B-C iron maps). (E-H) Livers of mice that were raised with high iron diet (E,H back scattered images, F-G iron maps). (I-L) magnification of the white square area. Scale bar for A-H equals 100μm, Scale bar for I-L equals 50μm.

Figure 8. Transmission electron microscopy images of the liver

Liver sections that were taken from representative mice at the age of 24 months were examined by Transmission electron microscope. (A) WT mice with normal diet (B) liver-hfer mice with normal diet (C) WT mice with high iron diet (D) liver-hfer mice with high iron diet. Large hemosiderin clusters are observed in mice that were raised on high iron diet (C, D arrows marking). Scale bar equals 200nm.