A mouse model of volumetric muscle loss and therapeutic scaffold implantation

Abstract

Skeletal myofibers naturally regenerate after damage; however, impaired muscle function can result in cases when a prominent portion of skeletal muscle mass is lost, for example, following traumatic muscle injury. Volumetric muscle loss can be modeled in mice using a surgical model of muscle ablation to study the pathology of volumetric muscle loss and to test experimental treatments, such as the implantation of acellular scaffolds, which promote de novo myogenesis and angiogenesis. Here we provide step-by-step instructions to perform full-thickness surgical ablation, using biopsy punches, and to remove a large volume of the tibialis anterior muscle of the lower limb in mice. This procedure results in a reduction in muscle mass and limited regeneration capacity; the approach is easy to reproduce and can also be applied to larger animal models. For therapeutic applications, we further explain how to implant bioscaffolds into the ablated muscle site. With adequate training and practice, the surgical procedure can be performed within 30 min.

Figure 1.. Schematic overview of creating VML injury to the TA muscle using a biopsy punch.

(a) Diagram of the tibialis anterior (TA) muscle. (b) An initial punch is made to create a full-thickness muscle defect. A spatula is placed behind the TA. (c) The resulting ablation is 2 mm in diameter and 4 mm deep. (d) A second biopsy punch is created adjacent to the first to expand the size of the ablation. (e) The ablated muscle volume derived from two punches is estimated by 2πr²h, where muscle thickness is represented by h=4 mm and r=punch radius, as indicated by the inset).

Figure 2.. Intraoperative images of the VML surgical procedure followed by scaffold implantation.

(a) A skin incision exposes the TA muscle. The ruler in photo depicts increments in mm. (b) The fascia is cut to expose the TA. The arrow indicates the fascia. (c) A metal spatula is placed directly beneath the TA muscle. (d) The morphology of the TA muscle is shown after the initial punch ablation, measuring 2 mm in diameter and 4 mm in depth. (e) The morphology of the TA is shown after a second biopsy punch ablation adjacent to the first. (f) A collagen scaffold is implanted into the region of ablation. (g) The muscle is closed with sutures. (h) Shown is the skin after closure with suture.

Figure 3.. Histological staining and force measurements of TA muscle at 21 days after muscle ablation (2-mm and 3-mm models).

(a) Torque measurements from tetanic contraction of the anterior crural muscles that include the TA muscle, based on ANOVA with Tukey’s post-test. Data is shown relative to that of the uninjured muscle (n=5, ****P<0.0001 for the indicated pair-wise comparison; ^####P<0.0001 when compared to the uninjured group). (b,c) Hematoxylin and eosin (H&E) staining of TA muscles after ablation using the 2-mm or 3-mm biopsy punch. (d) Cross-sectional region of the TA immediately post-surgery shows the muscle gap after biopsy punch, as denoted by the dotted lines. (e,f) Images show Masson’s trichrome staining of TA muscle using a 2-mm or 3 mm-sized biopsy punch. Arrows indicate the areas of fibrotic connective tissue and disorganized myofibers. Scale bar = 500 μm (d); 100 μm (b,c,e,f). See Source Data Figure 3 for raw data.

Figure 4.. Histological staining of TA muscle at 3 weeks after muscle ablation with collagen scaffold implantation.

(a,b) H&E stain of TA ablation using a 2-mm biopsy punch, followed by implantation of a nanofibrillar collagen scaffold into the defect. (c,d) Masson’s trichrome stain visualizes the nanofibrillar collagen scaffold in blue color after 3 weeks. The dotted line denotes the region of scaffold implantation. Scale bar = 500 μm.