Recovery from volumetric muscle loss injury: A comparison between young and aged rats

Abstract

Termed volumetric muscle loss (VML), the bulk loss of skeletal muscle tissue either through trauma or surgery overwhelms the capacity for repair, leading to the formation of non-contractile scar tissue. The myogenic potential, along with other factors that influence wound repair are known to decline with age. In order to develop effective treatment strategies for VML injuries that are effective across a broad range of patient populations, it is necessary to understand how the response to VML injury is affected by aging. Towards this end, this study was conducted to compare the response of young and aged animal groups to a lower extremity VML injury. Young (3months, n=12) and aged (18months, n=8) male Fischer 344 rats underwent surgical VML injury of the tibialis anterior muscle. Three months after VML injury it was found that young TA muscle was on average 16% heavier than aged muscle when no VML injury was performed and 25% heavier when comparing VML treated young and aged animals (p<0.0001, p<0.0001). Peak contractile force for both the young and aged groups was found to decrease significantly following VML injury, producing 65% and 59% of the contralateral limbs' peak force, respectively (p<0.0001). However, there were no differences found for peak contractile force based on age, suggesting that VML affects muscle's ability to repair, regardless of age. In this study, we used the ratio of collagen I to MyoD expression as a metric for fibrosis vs. myogenesis. Decreasing fiber cross-sectional area with advancing age (p<0.005) coupled with the ratio of collagen I to MyoD expression, which increased with age, supports the thought that regeneration is impaired in the aged population in favor of fibrosis (p=0.0241). This impairment is also exacerbated by the contribution of VML injury, where a 77-fold increase in the ratio of collagen I to MyoD was observed in the aged group (p<0.0002). The aged animal model described in this study provides a tool for investigators exploring not only the development of VML injury strategies but also the effect of aging on muscle regeneration.

Keywords: Aging; Animal model; Musculoskeletal; Orthopedics; Tibialis anterior; VML.

Figure 1

Surgical VMLs are gross defect creations in the left TA muscle of the rat. Surgical site was cleaned and disinfected prior to a vertical incision that was made to expose underlying muscle (A). An 8mm biopsy punch was used to create a defect to a depth of 2mm and the amount of muscle removed is approximately equivalent to 20% of the TA weight (B). Surgical defects were created in the rat left hindlimb and the contralateral limbs were untouched, serving as internal controls. The surgical site was then sutured with 5-0 absorbable sutures (C).

Figure 2

Correlation of animal weight and TA weight. (A) Aged animal (n=8) terminal weight vs TA weight. (B) Young animal (n=12) terminal weight vs TA weight. Linear regression analysis performed for all cases provided information to approximate TA weight given animal weight. From the TA weight, the size of the defect (20% of TA weight) can be determined.

Figure 3

Young and aged Fisher 344 rat weight (g) prior to surgery and at the end of the study period. The younger animals experienced significant weight gain post-surgery, whereas the aged animals lost weight. Values are mean+SEM. * represents p<0.05 Student’s t-test for the young group.

Figure 4

Peak tetanic force measurements of Young and Aged rats via electrophysiology. (A) Percutaneous needle electrodes were placed in the anterior compartment of the TA to stimulate the peroneal nerve. The knee was stabilized and the foot was attached to a muscle lever system for isometric measurements. (B) In order to account for normal growth over the 3-month period post-surgery, contractile force measurements were normalized to body weight (N/kg). The contralateral normal limbs for both age groups yielded similar values as well as showing a similar decrease in force production after VML injury. Values are mean+SEM. * represents p<0.05 two-way ANOVA for normal vs VML injury.

Figure 5

Comparison of TA and EDL muscle weight between normal and VML injury groups between Young (n=12) and Aged (n=8) rats. Gross morphological images of young normal (A), young VML (B), aged normal (C), and aged VML (D) TA muscles. At the end of the 3 month study period, terminal weights for TA (E) and EDL (F) of both young and aged normal and VML groups were measured. Values are mean+SEM. # represents p<0.05 two-way ANOVA for young vs aged. * represents p<0.05 two-way ANOVA for normal vs VML injury.

Figure 6

Comparison of histological cross-sections of young normal (A, B) and VML (C, D), aged normal (E, F) and VML (G, H) TA muscles. The inset depicting the TA shows from where the histological cross-sections were taken. Masson’s trichrome stained sections imaged at 100X magnification (A, C, E, G) depict collagen in blue/purple colored regions whereas muscle is shown in red/pink. Sections stained for collagen I (green) and myosin heavy chain (MHC, red) at 100X magnification (B, D, F, H). * indicates the collagen I rich scar region in the young muscles treated with VML (D). ** indicates the VML region for the aged group which is characterized by diffuse collagen I deposition and disorganized myofiber bundles compared to the young VML group (H). Masson’s trichrome was stained using a commercially available kit. Primary antibodies used were anti-collagen I mouse IgG (1:500) and anti-myosin mouse IgG_2B (1:10). Secondary antibodies used were goat anti-mouse IgG (H+L) Alexa Fluor 488 and goat anti-mouse IgG_2B Alexa Fluor 594.

Figure 7

Representative TA cross-sections immunostained for Collagen I (A, green) and Collagen III (B, red). Stained images were then analyzed in ImageJ software to calculate % area of the stains as a function of cross-sectional area. No difference was found for collagen I area (C) but a decrease in collagen III area was found for the young group after VML injury (D). On the contrary, the aged group had an increase in collagen III following VML. Primary antibody stains used were anti-collagen I mouse IgG (1:500) and anti-collagen III rabbit polyclonal (1:500). Secondary antibody stains used were goat anti-mouse IgG (H+L) Alexa Fluor 488 and goat anti-rabbit IgG (H+L) Alexa Fluor 594. * represents p<0.05 two-way ANOVA for normal vs VML injury.

Figure 8

Rat TA muscle cross-sections of young (A) and aged (B) rats stained with H&E and imaged at 200X magnification. Average fiber cross-sectional areas were calculated using ImageJ software for young normal, young VML, aged normal, and aged VML groups (n=3, C). It was observed that fiber cross-sectional area decreases significantly with age, regardless of VML injury. # represents p<0.05 two-way ANOVA for young vs aged.

Figure 9

Normal gene expression compared to expression in response to VML. Expression of MyoD (A), Collagen I (B), Collagen III (C), ratio of Collagen I to MyoD (D), and ratio of Collagen I to Collagen III (E) are presented as fold-changes obtained using RT-PCR. Except for MyoD, which experienced only a treatment effect, interaction effects of age and treatment were found for all other genes/ratios tested. Values are mean+SEM. * represents p<0.05 two-way ANOVA for normal vs VML injury and # represents p<0.05 two-way ANOVA for young vs aged. Post-hoc analysis was performed upon finding significant interaction effects.