Soft tissue manipulation enhances recovery of muscle mass in a disuse model of sarcopenia

Animals

Ten-week-old male Brown Norway rats were purchased from Charles River Labs (Wilmington, MA) and randomly assigned to the following groups: weight-bearing control (Controls) maintained in standard cages, or one of three groups subjected to 2-week hindlimb suspension (HLS) in custom-modified cages. Tails were secured via foam tape to a pulley that allowed movement around the cage and access to food and water ad lib; food was provided via floor feeding while water was provided via a bottle affixed to the cage wall. Cages were changed after 1 week. Following suspension, some animals were immediately euthanized (HLS group), whereas others were transferred to standard cages and allowed to re-ambulate for 8 days +/− instrument-assisted soft tissue manipulation (IASTM, see below). Euthanasia was completed by asphyxiation and bilateral pneumothorax. Blood and tissue samples were obtained immediately postmortem as follows: blood was collected with a 16-gague needle through cardiac puncture; distal portions of the left and right quadriceps were collected and stored in −80 °C; left and right gastrocnemii (all collected by JWL only to ensure consistency in technique) were weighed and dried as described below. All animal procedures were performed in alignment with a protocol approved by the Indiana University Institutional Animal Care & Use Committee and national standards.

Instrument-assisted soft tissue manipulation

A quantifiable soft tissue manipulation (QSTM) (Precision Care Technologies, Inc., Indianapolis, IN) handheld device (Q1-L) was utilized for manual delivery and standardization of localized force magnitude and stroke frequency application [24], 25]. The Q1-L treatment edge is similar to the GT3 device (Graston Technique, Indianapolis, IN) utilized for performing instrument-assisted soft tissue manipulation (IASTM). The treatment edge on the device, which is tooled to accommodate manipulation of small areas, was applied over four sessions beginning 1 day after release from HLS and then every other day thereafter. IASTM sessions were approximately 10 min in duration, with 5 min each to the upper and lower hindlimb, while the rats were under isoflurane-induced anesthesia (15.44063 ± 0.38 min average anesthesia per session). IASTM included applied curvilinear, longitudinal, and cross-fiber strokes at an average force of 2.72 N ± 0.11 N (i.e., 0.61 ± 0.02 lbs) at a frequency of 0.92 ± 0.12 Hz.

Muscle weights

Ipsilateral (right) and contralateral (left) gastrocnemii were weighed utilizing an analytical balance at two time points: (1) immediately following collection; and (2) after 12 days of drying at 55 °C.

Tissue homogenates

Quadriceps biopsies were homogenized in 1 X RIPA buffer (Cell Signaling, Danvers, MA) with 1 X Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA). After 10-min incubation on ice, samples were subjected to two 5-min cycles at full speed in a Bullet Blender (Next Advance, Troy, NY) then incubated for 30 min on ice. Samples were centrifuged at full speed for 15 min at 4 °C, and then the liquid phase was removed to a new tube. Protein concentration was determined utilizing a bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Waltham, MA) on a BioTek Synergy HTX plate reader (Agilent Technologies, Santa Clara, CA).

Serum collection

Whole blood was collected immediately postmortem and placed on ice. Following centrifugation at 1 k × gravity for 7 min at 4 °C, the liquid phase was removed to a new tube and stored at −80 °C. Protein concentration was determined utilizing a BCA Assay (Thermo Fisher Scientific, Waltham, MA) on a BioTek Synergy HTX plate reader (Agilent Technologies, Santa Clara, CA).

Cytokine arrays

A total of 500 µg total protein from ipsilateral quadriceps or serum was prepared utilizing equal amounts of protein for animals within a given group; specific numbers of animals are detailed in the respective figure or table legend. Pooled tissue homogenates and serum were analyzed utilizing the Proteome Profiler Rat XL Cytokine Array (R&D Systems, ARY030) as directed by the manufacturer with the following modification: the arrays were developed utilizing WesternBright Sirius reagent (Advansta, San Jose, CA) on a C-Digit scanner (LI-COR, Lincoln, NE). Signal densities were determined utilizing Image Studio software package (LI-COR). For each membrane, signal density for a given analyte’s duplicate spots were expressed as relative to the average density of the reference spots on the same membrane. Relative densities were then normalized to the respective control condition as detailed in the text and/or figure legend.

Statistical considerations

Statistical analyses were performed utilizing GraphPad Prism 10 as described in each respective figure legend or in the text. A p value of < 0.05 was considered significant.

Funding

This study was supported by a grant from the American Osteopathic Association (AOA, Award Number 21085846, issued to JWL) and intramural funds from Marian University (issued to JWL).

Hindlimb suspension results in skeletal muscle atrophy and changes in cytokine levels

Rats were randomly assigned to the weight-bearing control group (Controls) or the HLS group, wherein animals were suspended by their tail to remove loading of body weight from their hindlimbs (Figure 1A). After a 2-week suspension, animals were euthanized and tissue samples were collected for determining the effects of unloading on skeletal muscle mass and expression levels of select cytokines in both skeletal muscle and serum. This revealed that 2 weeks of unloading is associated with an approximately 48 % decrease in wet weight of the gastrocnemius (Figure 1B); raw weights of gastrocnemii may be found in Supplemental Figure 1.

Figure 1:

HLS results in skeletal muscle atrophy and changes in cytokine levels. (A) Images of HLS setup, wherein cages were modified with a bar and rolling pully (left) to which animal tails were affixed utilizing foam tape (right). (B) Mass of gastrocnemius immediately after sacrifice from weight-bearing controls (control) and animals subjected to 2-week HLS. Circles represent gastrocnemius mass from individual animals expressed as percent relative to the mean weight for the control group. Bar is mean ± standard error of the mean (SEM). n=5 for control and n=7 for HLS. Data were determined to be normally distributed by the Shapiro-Wilk test. * indicates p<0.05 by unpaired t-test. Raw weights obtained immediately after sacrifice and after 12 days of drying may be found in Supplemental Figures 1A and B. (C, D) membrane arrays utilizing quadricep homogenates obtained from control or HLS animals. Homogenates were pooled at equal ratios within groups for n=5 for control and n=6 for HLS. Red boxes in C indicate factors detailed in D, which are those altered by≥25 % in HLS compared to control. In D, data are expressed as fold change relative to control. Complete quantification may be found in Supplemental Table 1.

To examine the tissue-specific changes associated with HLS, homogenates from quadriceps biopsies were pooled within groups and subjected to membrane-based cytokine arrays that examine expression levels of nearly 80 targets simultaneously (Figure 1C and D). These assays detected modest changes in numerous cytokines with HLS (Supplemental Table 1), including 20 that were at least 25 % higher or lower compared to Controls (Figure 1C and D). Among these, the most strongly regulated cytokines were CTNF (1.69-fold increased), CCL22 (1.69-fold increased), and HGF (0.58-fold decreased). We next performed membrane arrays on serum pooled within groups to determine differences in circulating factors (Figure 2). This revealed HLS-associated changes in a greater number of serum factors than quadriceps, with levels of 34 targets at least 25 % higher or lower compared to Controls (Figure 2). Among these, the most strongly regulated cytokines were CCN1 (2.56-fold increased), CCL11 (2.15-fold increased), and FGF acidic (1.96-fold increased).

Figure 2:

Membrane arrays utilizing sera obtained from control or HLS animals. Sera were pooled at equal ratios within groups for n=5 for control and n=7 for HLS. Red boxes in (A) indicate factors detailed in (B), which are those altered by≥25 % in HLS compared to control. (B) Data are expressed as fold change relative to control. Complete quantification may be found in Supplemental Table 2.

STM enhances muscle mass recovery

We next sought to examine the effects of STM on muscle mass recovery following disuse atrophy. After 2 weeks of tail suspension, rats were randomly assigned to re-ambulate or re-ambulate plus four sessions of IASTM (Re-A+IASTM) delivered every other day to one hindlimb (Figure 3A and B). Details of the IASTM may be found in the Methods Section; briefly, under isoflurane anesthesia (average 15.44 ± 0.38 min total), an IASTM device was applied in a stroking pattern to the upper and lower hindlimb for 5 min each at an average force of 2.72 N ± 0.11 N (i.e., 0.61 ± 0.02 lbs) at a frequency of 0.92 ± 0.12 Hz. After 8 days, animals in both groups were euthanized. This revealed that Re-A alone is associated with approximately 50 % recovery of gastrocnemius wet weight, whereas Re-A+IASTM is associated with approximately 74 % recovery (Figure 3C); raw weights of gastrocnemii may be found in Supplemental Figure 1.

Figure 3:

STM enhances muscle mass recovery following disuse atrophy. (A) Schematic of timeline for animals subjected to HLS then 8 days of Re-A or Re-A+IASTM. IASTM was carried out every other day beginning the day following release from HLS for a total of four sessions (Tx). All animals were euthanized 8 days following HLS. (B) Images of the IASTM technique. (C) Mass of ipsilateral gastrocnemius immediately after sacrifice from Re-A and Re-A+IASTM animals. Circles represent gastrocnemius mass from individual animals expressed as percent relative to the mean lost in the HLS group. Bar is mean ± standard error of the mean (SEM). n=7 for Re-A and n=8 for Re-A+IASTM. Data were determined to be normally distributed by the Shapiro-Wilk test. * indicates p<0.05 by unpaired t-test. Raw weights obtained immediately after sacrifice and after 12 days of drying may be found in Supplemental Figures 1A and B.

STM alters cytokine levels in skeletal muscle and serum

To examine the tissue-specific changes associated with IASTM, homogenates from treated (i.e., ipsilateral) quadriceps biopsies were pooled within groups and subjected to membrane-based cytokine arrays (Figure 4). These assays detected changes in numerous cytokines with IASTM (Supplemental Table 2), including 49 that were at least 25 % higher or lower compared to Re-A alone. The 20 most strongly regulated cytokines are detailed in Figure 4B, with CTNF (4.72-fold increased), MMP-9 (2.30-fold increased), and clusterin (2.12-fold increased) representing the three largest changes compared to the Re-A group.

Figure 4:

Membrane arrays utilizing quadricep homogenates obtained from animals subjected to HLS then 8 days of Re-A or Re-A+IASTM. Homogenates were pooled at equal ratios within groups for n=7 for Re-A and n=8 for Re-A+IASTM. Red boxes in (A) indicate factors detailed in B, which are those altered by≥25 % in HLS compared to control. (B) Data are expressed as fold change relative to control. Complete quantification may be found in Supplemental Table 3.

We next utilized membrane arrays to determine the effects of STM on serum factors, which revealed changes in the levels of 53 targets at least 25 % higher or lower compared to Re-A alone (Figure 5). The 20 most strongly regulated cytokines are detailed in Figure 5B, with CXCL2 (2.78-fold increased), Galectin-1 (2.65-fold increased), and Galectin-3 (2.63-fold increased) representing the three largest changes compared to the Re-A group.

Figure 5:

Membrane arrays utilizing sera homogenates obtained from animals subjected to HLS then 8 days of Re-A or Re-A+IASTM. Homogenates were pooled at equal ratios within groups for n=7 for Re-A and n=8 for Re-A+IASTM. Red boxes in (A) indicate factors detailed in (B), which are those altered by≥25 % in HLS compared to control. (B) Data are expressed as fold change relative to control. Complete quantification may be found in Supplemental Table 4.

Figure 6:

Mass of contralateral gastrocnemius immediately after sacrifice from Re-A and Re-A+IASTM animals. Circles represent gastrocnemius mass from individual animals expressed as percent relative to the mean lost in the HLS group. Bar is mean ± standard error of the mean (SEM). n=7 for Re-A and n=8 for Re-A+IASTM. Data were determined to be normally distributed by the Shapiro-Wilk test. * indicates p<0.05 by unpaired t-test. Raw weights obtained immediately after sacrifice and after 12 days of drying may be found in Supplemental Figures 1C and D.

Crossover effect of STM on muscle mass recovery

Given the changes in systemic factors observed in rats subjected to IASTM, we examined the possibility for crossover effects on muscle mass recovery in contralateral hindlimbs. Analysis of gastrocnemius wet weight indicates that recovery of muscle mass is enhanced by IASTM in contralateral limbs, with Re-A alone associated with approximately 52 % recovery, whereas Re-A+IASTM was associated with approximately 79 % recovery (Figure 6); raw weights of contralateral gastrocnemii may be found in Supplemental Figure 1.

Identification of putative therapeutic targets in disuse atrophy regulated by IASTM

Our analyses above were normalized in such a manner to aid in clear identification of factors altered by HLS compared to controls (Figures 1 and 2) or by IASTM compared to Re-A alone (Figures 4 and 5). However, this statistical method is not ideal to identify those factors that may play a role in the therapeutic benefit of IASTM on the recovery of muscle mass following disuse atrophy.

To aid in this identification, we normalized the membrane arrays to the weight-bearing condition and filtered for the following: Group 1) factors that were increased/decreased by HLS, regulated in the opposite direction by Re-A, and further regulated by Re-A+IASTM; Group 2) factors that were increased/decreased by HLS, unchanged by Re-A alone, but regulated by Re-A+IASTM in an opposite direction than HLS; Group 3) factors that were regulated in opposite directions by Re-A vs. Re-A+IASTM; Group 4) factors that were unchanged by HLS or Re-A but increased/decreased by Re-A+IASTM. A threshold of 25 % increase or decrease was utilized for all analyses.

Application of these criteria to homogenates from ipsilateral quadriceps may be found in Supplemental Table 3, and the 22 factors that meet any set of conditions are highlighted in Table 1. Similarly, application of these criteria to the full panel of serum factors may be found in Supplemental Table 4, and the 35 factors that meet any set of conditions are highlighted in Table 2.