The Effect of Ethanol Consumption on Composition and Morphology of Femur Cortical Bone in Wild-Type and ALDH2*2-Homozygous Mice

Abstract

ALDH2 inactivating mutation (ALDH2*2) is the most abundant mutation leading to bone morphological aberration. Osteoporosis has long been associated with changes in bone biomaterial in elderly populations. Such changes can be exacerbated with elevated ethanol consumption and in subjects with impaired ethanol metabolism, such as carriers of aldehyde dehydrogenase 2 (ALDH2)-deficient gene, ALDH2*2. So far, little is known about bone compositional changes besides a decrease in mineralization. Raman spectroscopic imaging has been utilized to study the changes in overall composition of C57BL/6 female femur bone sections, as well as in compound spatial distribution. Raman maps of bone sections were analyzed using multilinear regression with these four isolated components, resulting in maps of their relative distribution. A 15-week treatment of both wild-type (WT) and ALDH2*2/*2 mice with 20% ethanol in the drinking water resulted in a significantly lower mineral content (p < 0.05) in the bones. There was no significant change in mineral and collagen content due to the mutation alone (p > 0.4). Highly localized islets of elongated adipose tissue were observed on most maps. Elevated fat content was found in ALDH2*2 knock-in mice consuming ethanol (p < 0.0001) and this effect appeared cumulative. This work conclusively demonstrates that that osteocytes in femurs of older female mice accumulate fat, as has been previously theorized, and that fat accumulation is likely modulated by levels of acetaldehyde, the ethanol metabolite.

Keywords: Ethanol; Osteocytes; Osteoporosis; Raman spectroscopy; microCT.

Fig. 1

Example of Raman spectra from two spots within a bone section (left panel): raw data (black curve) and MLR fit (red curve) as a sum of purified spectra for the major sub-components of Raman spectra (right panel). Spot #1 is from an osteocyte location, spot #2 is typical of cell-free mineral/collagen matrix

Fig. 2

MLR fit maps for sections made from bones embedded in paraffin

Fig. 3

Selected MLR fit maps (two for each condition) of bone, mineral, bound proteoglycan and fat components for four different types of mouse femur samples: WT, WT and ethanol treatment, ALDH2*2/*2, ALDH2*2/*2 with ethanol treatment, as indicated. All vertical contrast scales are matched in each row. Green arrow marks typical osteocytes in WT and ALDH2*2/*2 samples, the latter also corresponds to the spot for the Raman spectrum of fat in Fig. S1a

Fig. 4.

Raman and fluorescence imaging was performed for regions in the middle of the cortical bone. Relative collagen, mineral, fat and bound proteoglycan content for four types of mouse femur samples: (a) collagen, mineral relative content for the individual groups, (b) fat relative content for the individual groups, (c) bound proteoglycan content for the individual groups. (d) Average normalized magnitude of fat signal inside osteocytes. Every data-point represents one osteocyte. Error bars are mean values ± SD. Number of individual animals is 9 for WT and 3 per other groups. Statistical significance was measured using non-parametric permutation tests (a-c) and t-tests with Welch’s adjustment for differing variances (d). Significance levels are marked as follows: * p < 0.05 (mineral WT to WT+EtOH), **p < 0.05 (proteoglycan WT to WT+EtOH)

Fig. 5.

Maps of Nile Red fluorescent stain for WT, WT+EtOH, ALDH2*2 and ALDH2*2+EtOH sections, showing elevated fat content in osteocytes for mice consuming alcohol and those with ALDH2*2 mutation. Scale bar is 50 μm. The quantified data is shown in Fig. 4d.

Fig. 6

(a) Bone mineral densities of 1 mm-long subsections along three WT bones, overlaid on top of front maximum intensity projection, top-view single X-Ray section is shown to the right of the graph (b) Bone mineral density for four sample types with symbols for every 1 mm section, showing the same trend as Raman data, but lack of statistical significance between groups, as measured by Wilcoxon independent test. Dashed line added as guide for the eye