Skeletal muscle macrophage ablation in mice

Abstract

Macrophages are important mediators of skeletal muscle function in both healthy and diseased states. In vivo specific depletion of macrophages provides an experimental method to understand physiological and pathophysiological effects of macrophages. Systemic depletion of macrophages can deplete skeletal muscle macrophages but also alters systemic inflammatory responses and metabolism, which confounds the muscle specific effects of macrophage depletion. The primary aim of this manuscript is to evaluate two methods of murine intramuscular macrophage depletion in an acute lung injury-associated indirect skeletal muscle wasting mouse model. Adult C57BL/6 (WT) and Macrophage Fas-Induced Apoptosis (MaFIA, C57BL/6-Tg) mice received clodronate liposomes or the dimerization drug AP20187 through intramuscular injection of the tibialis anterior muscle compartment, respectively. Vehicle control was injected in the contralateral muscle. We demonstrate intramuscular AP20187 in the MaFIA mouse depletes macrophages but causes an infiltration of CD45 intermediate neutrophils. In contrast, intramuscular clodronate liposomes successfully depletes macrophages without an associated increase in CD45 intermediate cells. In conclusion, intramuscular clodronate is effective for selective depletion of muscle macrophages without eliciting acute inflammation seen with AP20187 in MaFIA mice. This technique is an important tool to study the functional roles of macrophages in skeletal muscle.

Keywords: Clodronate liposomes; Conditional macrophage depletion; Inflammation; Skeletal muscle; Transgenic mouse model.

Fig. 1.

A) Representative sample (2 muscles of 1 mouse) showing flow cytometry analysis of CD45⁺ cells isolated from TA muscle from MaFIA mice injected with intramuscular AP20187 (green) versus intramuscular vehicle control (grey). B) Representative sample (1 mouse muscle per panel) demonstrating flow cytometry analysis demonstrating the appearance of a large population of CD45^intermediate cells in AP20187 treatment compared to vehicle control treated muscle. C) Cell counts of the CD45^intermediate population in AP20187 treated versus vehicle control treated muscle. This experiment is representative of two independent experiments and n = 4. *p = 0.03 by Mann-Whitney test.

Fig. 2.

Intramuscular clodronate selectively depletes monocyte and macrophage muscle cell populations. A) Representative sample (1 mouse muscle per panel) showing flow cytometry analysis of CD45⁺ cells isolated from intramuscular clodronate treated compared to vehicle control mice. B) Total intramuscular CD 45⁺ cell populations in clodronate and vehicle treated mice. C) CD45⁺CD11b⁺CD11c⁻Ly6G⁻SSC^loLy6C^lo macrophages D) CD45⁺CD11b⁺CD11c⁻Ly6G⁻SSC^loLy6C^hi monocytes E) CD45⁺CD11b⁺CD11c⁻F4/80⁻Ly6G⁺ neutrophils, and F) CD45⁺CD11b⁺CD11c^hi dendritic cells in intramuscular clodronate or vehicle control treated mice. This experiment is representative of three independent experiments and n = 6. *p = 0.04 by Mann-Whitney test.