Morphological Alterations in Gastrocnemius and Soleus Muscles in Male and Female Mice in a Fibromyalgia Model

Abstract

Background: Fibromyalgia (FM) is a chronic musculoskeletal pain disorder, characterized by chronic widespread pain and bodily tenderness and is often accompanied by affective disturbances, however often with unknown etiology. According to recent reports, physical and psychological stress trigger FM. To develop new treatments for FM, experimental animal models for FM are needed to be development and characterized. Using a mouse model for FM including intermittent cold stress (ICS), we hypothesized that ICS leads to morphological alterations in skeletal muscles in mice.

Methods: Male and female ICS mice were kept under alternating temperature (4 °C/room temperature [22 °C]); mice constantly kept at room temperature served as control. After scarification, gastrocnemius and soleus muscles were removed and snap-frozen in liquid nitrogen-cooled isopentane or fixed for electron microscopy.

Results: In gastrocnemius/soleus muscles of male ICS mice, we found a 21.6% and 33.2% decrease of fiber cross sectional area (FCSA), which in soleus muscle concerns the loss of type IIa and IIx FCSA. This phenomenon was not seen in muscles of female ICS mice. However, this loss in male ICS mice was associated with an increase in gastrocnemius of the density of MIF+ (8.6%)-, MuRF+ (14.7%)-, Fbxo32+ (17.8%)-cells, a 12.1% loss of capillary contacts/muscle fiber as well as a 30.7% increase of damaged mitochondria in comparison with male control mice. Moreover, significant positive correlations exist among densities (n/mm(2)) of MIF+, MuRF+, Fbxo32+-cells in gastrocnemius/ soleus muscles of male ICS mice; these cell densities inversely correlate with FCSA especially in gastrocnemius muscle of male ICS mice.

Conclusion: The ICS-induced decrease of FCSA mainly concerns gastrocnemius muscle of male mice due to an increase of inflammatory and atrogenic cells. In soleus muscle of male ICS and soleus/gastrocnemius muscles of female ICS mice morphological alterations seem to occur not at all or delayed. The sex-specificity of findings, which is not easily reconciled with the epidemiology of FM (female predominance), implicate that gastrocnemius muscle of male ICS mice should preferentially be used for future investigations with FM. Moreover, we suggest to investigate morphological and/or molecular alterations at different time-points (up to two weeks) after ICS.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Fiber density (n/mm2) and fiber cross sectional area (μm2) of gastrocnemius (Gastro.) muscle.

Representative images of gastrocnemius muscle cross sections stained for myofibrillar ATPase after pre-incubation at pH 4.6 of male (♂) control (A) and ICS (B) are shown. Quantification of fiber type density (C) and fiber cross sectional area (D) are shown. Type IIa (white cross); Values are given as mean + SEM; *P ≤ 0.05, significance vs control and +P ≤ 0.05 vs male mice. N = 8–9 animals per group. Bar = 50 μm.

Fig 2. Fiber density (n/mm2) and fiber cross sectional area (μm2) of soleus muscle.

Representative images of soleus muscle cross sections stained for myofibrillar ATPase after preincubation at pH 4.6 of male (♂) control (A) and ICS (B) mice are shown. Quantification of fiber density (C), fiber cross sectional area (FCSA) (D), fiber type I FCSA (E), fiber type IIa FCSA (F) and fiber type IIx FCSA (G) are shown. Type I fibers are stained dark (white star); type IIx fibers are stained intermediate (white arrow head) and type IIa fibers are stained light-coloured (white cross). Values are given as mean + SEM. N = 8–9 animal per group. Bar = 50 μm.

Fig 3. Density (n/mm2) of MIF+ cells of gastrocnemius and soleus muscles.

Representative images of cross sections of male (♂) gastrocnemius muscle immunostained for MIF (A, C) are shown. Furthermore quantification of MIF+ cells of gastrocnemius (E) and soleus (F) muscles are shown. Male control (A, B), male ICS (C, D); Hoechst 33342 was used as a nuclear counterstain (B, D). MIF+ immunoreaction is indicated by black arrows (A, C) Values are given as mean + SEM; ++P <0.01 significance vs male ICS mice. N = 7–9 animals per group. Bar = 50 μm.

Fig 4. Density (n/mm2) of MuRF1+ and Fbxo32+ cells in mouse gastrocnemius and soleus muscles.

Representative images of cross sections of male (♂) gastrocnemius muscle immunostained for MuRF1 (A-D) and Fbxo32 (E-H) are shown. Furthermore, quantification of MuRF1+ and Fbxo32+ cells in gastrocnemius (I, K) and soleus (J, L), muscles are shown. MuRF1+ male control (A, B) and male ICS (C, D); Fbxo32+ male control (E, F) and male ICS (G, H). Hoechst 33342 was used as a nuclear counterstain (B, D, F, H). MuRF1+ and Fbxo32+ cells are indicated by black arrows. Values are given as mean + SEM; **P < 0.01, significance vs control and +P ≤ 0.05, ++P < 0.01 and +++P <0.001 vs male mice. N = 7–9 animals per group. Bar = 50 μm.

Fig 5. Capillary contacts/muscle fiber and density of capillaries (n/mm2) in cross sections of gastrocnemius and soleus muscles are shown.

Representative images of cross section of male gastrocnemius (A, B) and soleus (C, D) muscles stained with the endothelial marker BSI-B4-Lectin-HRP conjugated are shown. Furthermore, quantification of capillary contacts/muscle fiber in gastrocnemius (E) and soleus (F) muscles, as well as capillary density in gastrocnemius (G) and soleus (H) muscles are shown. Male control (A, C) and male ICS (B, D), capillaries are indicated by black arrows. Values are given as mean + SEM; *P ≤ 0.05 significance vs control and +P ≤ 0.05, ++P < 0.01, +++P < 0.001 vs male mice. N = 7–8 animals per group. Bar = 50 μm.

Fig 6. Mitochondrial density (n/mm2) and quality (in %) of mouse gastrocnemius and soleus muscles are shown.

Representative transmission electron microscopic images of male control gastrocnemius (A) and soleus (C), male ICS gastrocnemius (B) and soleus ICS (D) muscles. Quantification of mitochondrial density and quality in gastrocnemius (E, G) and soleus (F, H) muscles are shown. Mitochondria are indicated by white arrows. Representative example of intact (I, J) and damaged mitochondria (K, L). Mitochondrium with characteristic advanced feature of autophagy indicated with a black arrow (L). Values are given as mean + SEM; **P < 0.01, ***P < 0.001 significance vs control and +P ≤ 0.05, vs male mice. N = 5 animals per group. bar = 500 nm (A-D), (I-L) bar = 100 nm.