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. 2015 Feb;71:106-14.
doi: 10.1016/j.bone.2014.10.008. Epub 2014 Oct 24.

Biomechanical Properties of Bone in a Mouse Model of Rett Syndrome

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Free PMC article

Biomechanical Properties of Bone in a Mouse Model of Rett Syndrome

Bushra Kamal et al. Bone. .
Free PMC article

Abstract

Rett syndrome (RTT) is an X-linked genetic disorder and a major cause of intellectual disability in girls. Mutations in the methyl-CpG binding protein 2 (MECP2) gene are the primary cause of the disorder. Despite the dominant neurological phenotypes, MECP2 is expressed ubiquitously throughout the body and a number of peripheral phenotypes such as scoliosis, reduced bone mineral density and skeletal fractures are also common and important clinical features of the disorder. In order to explore whether MeCP2 protein deficiency results in altered structural and functional properties of bone and to test the potential reversibility of any defects, we have conducted a series of histological, imaging and biomechanical tests of bone in a functional knockout mouse model of RTT. Both hemizygous Mecp2(stop/y) male mice in which Mecp2 is silenced in all cells and female Mecp2(stop/+) mice in which Mecp2 is silenced in ~50% of cells as a consequence of random X-chromosome inactivation, revealed significant reductions in cortical bone stiffness, microhardness and tensile modulus. Microstructural analysis also revealed alterations in both cortical and cancellous femoral bone between wild-type and MeCP2-deficient mice. Furthermore, unsilencing of Mecp2 in adult mice cre-mediated stop cassette deletion resulted in a restoration of biomechanical properties (stiffness, microhardness) towards wild-type levels. These results show that MeCP2-deficiency results in overt, but potentially reversible, alterations in the biomechanical integrity of bone and highlights the importance of targeting skeletal phenotypes in considering the development of pharmacological and gene-based therapies.

Keywords: Biomechanical properties; Cancellous bone; Cortical bone; MEPC2; Rett syndrome.

Figures

Fig. 1
Fig. 1
Experimental design of tamoxifen regime (rescuing) of Mecp2stop/y, CreER. Experimental design of the current study showing treatment (A) and sampling phases (B) in male mouse comparison cohorts. Wild-type (Wt) , Mecp2stop/y (non rescue) and Mecp2stop/y, CreER (rescue) were given one injection of tamoxifen (100 mg/kg) per week for 3 weeks (age 6–8 weeks) then followed by 4 daily injections in consecutive days in the 4th week (age 9 weeks). Mice were then culled at 14 weeks and bones sampled for imaging, histology and biomechanical testing.
Fig. 2
Fig. 2
MeCP2 is expressed widely in bone tissues. (A) Low power and (ii–iv) high power micrographs of transverse sections taken from mid shaft mouse femur showing GFP expression in all DAPI-labelled nuclei in a male Mecp2+/y mouse in which the native MeCP2 is tagged with a C-terminal GFP. Note that MeCP2 is restricted to the nucleus of osteocytes as indicated by the complete overlap with DAPI staining but present in all nuclei. (B) GFP expression is not observed in mice in which MeCP2 expression is functionally silenced by a neo-stop cassette. (C) Low power (i) and (ii–iv) high power micrographs showing mosaic expression of GFP-tagged MeCP2 protein in ~ 50% of DAPI positive nuclei in a female heterozygous Mecp2+/stop mouse in which one Mecp2 allele is functionally silenced. All scale bars: 100 μm.
Fig. 3
Fig. 3
Three-point bending test reveals mechanical deficits in the tibia of male MeCP2-deficient mice. (A) Mouse tibia were placed on the posterior surface across two supporting bars with a distance of 8 mm apart and a load was applied to the anterior surface of shaft until the bone fractured. In male mice the measures of (B) cortical bone stiffness (p < 0.01; one way ANOVA with Tukey's post hoc test; n = 5 tibia per genotype), (C) ultimate load (p < 0.01) and (D) Young's modulus (p < 0.05) were significantly reduced in Mecp2stop/y (Stop) mice as compared to wild-type (Wt), and genetically rescued Mecp2stop/y; CreER (Rescue) mice. Abbreviation: ns = not significant; *p < 0.05, **p < 0.01. Plots show mean ± S.D.
Fig. 4
Fig. 4
Three-point bending test reveals no mechanical deficits in the tibia of heterozygous Mecp2+/stop mice. Bar plot showing measures of (A) cortical bone stiffness, (B) ultimate load and (C) Young's modulus in female wild-type (Wt), Mecp2stop/+ (Stop) and Mecp2stop/+ (Rescue) mice (one way ANOVA with Tukey's post hoc test; n = 3–5 per genotype). Abbreviation: ns = not significant; plots show mean ± S.D.
Fig. 5
Fig. 5
Microindentation hardness test reveals a significant but reversible reduction in cortical bone hardness in MeCP2-deficient mice. (A). Mouse femur (i) was sectioned at mid-shaft, mounted, polished and assessed by microindentation (ii; indentation marks shown by arrows). B. Microindentation test in male mice revealed a significant reduced cortical bone hardness in Mecp2stop/y (Stop) mice when compared with the wild-type (Wt) controls (p < 0.05, one way ANOVA with Tukey's post hoc test, n = 5 femurs per genotype). In contrast, microhardness measures in rescued Mecp2stop/y, CreER (Rescue) mice were not different from controls (p > 0.05; n = 5 femurs). (C) Microindentation hardness test results in female mice showed a similar pattern with reduced cortical bone hardness in Mecp2+/stop (Stop) mice when compared with wild-type (Wt) controls and rescued mice (n = 3–5 femurs per genotype, one way ANOVA with Tukey's post hoc test). Abbreviations: ns = not significant (* = p < 0.05, ** = p < 0.01, *** = p < 0.001). Plots show mean ± S.D.
Fig. 6
Fig. 6
Femoral neck test revealed no alteration in bio-mechanical properties in MeCP2-deficient mice. (A) Image showing femoral neck test in which load was applied downward onto the femoral head until the femoral neck fractured. Results in male mice showed no significant difference in measurements of (B) bone stiffness or (C) ultimate load between wild-type (Wt), Mecp2stop/y (Stop) and Mecp2stop/y, CreER (Rescue) mice (p > 0.05; one way ANOVA; n = 5 per genotype). Plots show mean ± S.D. Abbreviations: ns = not significant.
Fig. 7
Fig. 7
Femoral neck test revealed no alteration in bio-mechanical properties in female MeCP2-deficient mice. Bar plots showing no significant difference in measures of (A) bone stiffness or (B) ultimate load between wild-type (Wt), Mecp2stop/+ (Stop) and Mecp2stop/+, CreER (Rescue) mice (all p > 0.05; ANOVA; n = 3–5 per genotype). Plots show mean ± S.D. Abbreviations: ns = not significant.
Fig. 8
Fig. 8
Sirius red staining revealed a reduced collagen content in MeCP2-deficient mice. (Ai) Longitudinal section of proximal femur stained with sirius red to reveal collagen. Regions of interest (ROI) from shaft of proximal femur were selected for quantification. Collagen content was measured in (ii) wild-type (Wt), (iii) Mecp2stop/y (Stop) and (iv) Mecp2stop/y, CreER (rescue) comparison groups. (B) Bar chart showed that % Collagen content was reduced in Mecp2stop/y (Stop) mice as compared to wild-type (Wt; p < 0.01). Abbreviations: ns = not significant (* = p < 0.05, ** = p < 0.01; one way ANOVA with Tukey's post hoc test). Plots show mean ± S.D.
Fig. 9
Fig. 9
Scanning electron microscopy reveals pitted cortical bone and altered trabecular structure in distal femur of male MeCP2-deficient mice. Scanning electron micrographs of distal femur in (Ai) wild-type (Wt) and (Bi) Mecp2stop/y (Stop). Higher powered images of cortical (ii) and metaphyseal (iii) regions (areas indicated in A) reveal a more porous structure in cortical bone (arrows in Bi indicate pores) and a sparse trabecular structure in Mecp2stop/y mice when compared with representative with Wt controls. (Ci–iii) Representative micrograph from a Mecp2stop/y, CreER (Rescue) mouse.
Fig. 10
Fig. 10
μCT scans of L5 vertebrae revealed thinner trabecular mass in MeCP2-deficient mice. (A) μCT image showing trabecular region of interest (ROI) selected within the 5th Lumbar vertebral body. (B–D) Micrographs showing representative trabecular samples from wild-type (Wt), Mecp2stop/y (Stop) and Mecp2stop/y, CreER (Rescue) mice. (E) Bar plot showing quantitative analysis of trabecular thickness (arrows in B–D). Note the reduced thickness in Mecp2stop/y samples (p < 0.05; n = 7 per genotype). Scale bar: A, 200 μm; B–D, 50 μm. Abbreviations: ns = not significant (* = p < 0.05, ** = p < 0.01; one way ANOVA with Tukey's post hoc test). Plots show mean ± S.D.

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