Determination of a Critical Size Threshold for Volumetric Muscle Loss in the Mouse Quadriceps

Abstract

The goal of this study was to determine the threshold for a critically sized, nonhealing muscle defect by characterizing key components in the balance between fibrosis and regeneration as a function of injury size in the mouse quadriceps. There is currently limited understanding of what leads to a critically sized muscle defect and which muscle regenerative components are functionally impaired. With the substantial increase in preclinical VML models as testbeds for tissue engineering therapeutics, defining the critical threshold for VML injuries will be instrumental in characterizing therapeutic efficacy and potential for subsequent translation.

Keywords: neuromuscular junction; satellite cells; skeletal muscle; volumetric muscle loss.

FIG. 1.

Various sized VML injuries in mouse quadriceps. (A) Schematic representation of 2, 3, and 4 mm biopsy punch injuries to the mouse left quadriceps with respect to component muscle groups. (B) Mouse quadriceps after removal of biopsied muscle tissue. From left to right, 2, 3, and 4 mm biopsy punches. Scale bars represent 5 mm. (C) The wet weights of biopsied quadriceps muscle normalized to the contralateral control as plotted for each biopsy punch size. Mean percentages of contralateral control are 4.44, 15.5, and 32.2 for 2, 3, and 4 mm injuries, respectively (n = 12 per injury size, error bars indicate mean ± standard error of the mean (SEM), ****p < 0.0001 after one-way ANOVA and Tukey's test). (D) Wet weight of the injured quadriceps 7, 14, and 28 days postinjury normalized to total body weight. Day 0 values are calculated as an average of the defect wet weight subtracted from its respective Day 7, 14, and 28 contralateral control quadriceps wet weight and normalized to the body weight of the animal, mean is plotted ± SEM. The dotted line shows the average value of contralateral control quadriceps normalized to body weight (n = 4 for each group, ^#p < 0.01, ^†p < 0.0001 as compared with the control after two-way ANOVA and Tukey's test post hoc). RF, rectus femoris; VL, vastus lateralis; VM, vastus medialis; VI, vastus intermedius; VML, volumetric muscle loss. Color images are available online.

FIG. 2.

H&E staining of each injury size at various time points. Images of quadriceps cross-sections, stained with H&E. From left to right are shown representative images of a contralateral control, 2 mm injury, 3 mm injury, and 4 mm injury, from 14-day (A) and 28-day (B) time points. Scale bars represent 200 μm. H&E, hematoxylin and eosin. Color images are available online.

FIG. 3.

Assessment of fibrotic response at each injury size. Quadriceps cross-sections 14 (A, C, E, G) or 28 (B, D, F, H)-day time points postinjury. For each set of images (A–H) the left-hand image is a cross-section stained with CD68 antibody (shown in red) and imaged on a 2-photon scanning confocal microscope (at 810 nm) for second harmonic generation imaging of collagen (shown in blue), and the right-hand image is a colorimetric image of a cross-section stained with Gormori's Trichrome. The sets of images are representative sections from contralateral control (A, B), 2 mm injury (C, D), 3 mm injury (E, F), or 4 mm injury (G, H) samples. Scale bars represent 200 μm. Color images are available online.

FIG. 4.

Visualization and quantification of fiber cross-sectional area in each injury size. Representative immunofluorescence images of each time point and injury size: (A) contralateral control, (B, C, D) 14-day time point of 2, 3, and 4 mm injuries, respectively, and (E, F, G) 28-day time point of 2, 3, and 4 mm injuries, respectively. Each of the sections were stained for dystrophin (green), DAPI (blue), and eMHC (red). Scale bars represent 100 μm. Cross-sectional area was quantified using stitched images of the entire cross-section from the dystrophin channel. The measured cross-sectional areas used to create cumulative histograms 14 (H) and 28 (I)-day time points. Data were categorized by injury size. Each category (control, 2, 3, and 4 mm) had n = 4 individual animals with three replicate slides per animal: one replicate from the beginning, one from the middle, and one from the end of the damaged area. In the case of the control, replicate slides were taken from the corresponding areas to the damaged samples. Fibers with centrally located nuclei were quantified at 14 and 28 days postinjury (J). Five representative areas from each of the same replicate slides used for cross-sectional area quantification were used to count centrally located nuclei per area. All data points shown, with bars representing mean ± SEM. Two-way ANOVA performed with Tukey's test post hoc, significance for p < 0.05. **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant. Color images are available online.

FIG. 5.

Whole-mounted sections of neuromuscular junctions in VML compared with the contralateral control. NMJs were quantified by three classifications (A, B) as either group 1, 2, or 3 in Thy1-YFP mice 14 or 28 days post 3 mm VML injury. Group 1 NMJs were normal, pretzel-like morphology, group 2 NMJs were abnormal, fragmented morphology, and group 3 were newly forming AChR clusters. (A) Shows mean raw count data ± SEM for each animal and time point. (B) Depicts the same data as in (A), but displayed as a percentage of total number of NMJs in each category. Representative maximum intensity projections of z-stacks taken from the control (left) and injured (right) experimental groups are shown for 14 (C) and 28 (D)-day time points. Each image shows only the BTX channel from the image to visualize the morphology of the postsynaptic AChRs. The BTX channel was used for quantification. For images including GFP channel, see Supplementary Figure S3. All scale bars are 50 μm. NMJ, neuromuscular junctions; BTX, α-bungarotoxin; AchR, acetylcholine receptor. Color images are available online.

FIG. 6.

Micro-CT and IHC analysis of vasculature in VML compared with control quadriceps. (A) Reconstructed 3D heat map of μCT images from Microfil perfused contralateral control and 3 mm injured quadriceps, left to right, respectively, from the same animal. (B) Reconstructed 3D heat map of the middle third of the same samples as in (A). Color scale is from 0.000 to 0.315 mm for vascular diameter, length scale bar is 1 mm. The middle third of each sample is what was quantitatively analyzed in (C, D). (C) Total perfused vascular volume for the middle third of each sample shown in a pairwise comparison to match injured and control from the same animal. (D) Histogram counting the number of vessels in each diameter bin. The bins represent the resolution of the measurement itself (e.g., vessels between 0 and 21 μm are placed in the 21 μm bin). Counts are shown as mean ± SEM from five samples. For vascular volume (C) statistical analysis, a paired, two-tailed t-test was performed. For comparison of injured and control values in each bin of the vascular diameter histogram (D) a two-way repeated-measures ANOVA was performed with Sidak post hoc, *p < 0.05, †p < 0.0001. (E) Images taken of quadriceps cross-sections 28 days after a 3 mm injury (right) and its contralateral control (left). Staining was done for phalloidin (gray), nuclei (blue), and vWF (red). Color images are available online.