Ultrasound pressure dependent cytokine and immune cell response lost in aged muscle

Abstract

Objective:

Therapeutic ultrasound remains a highly discussed topic in physical therapy due to uncertainty between treatment regimens and biological benefits. Its impact on aged populations, who are vulnerable to insufficient healing after muscle injury due to sarcopenia, is understudied. Despite the coupling between muscle inflammation and regeneration, research on the immune response after therapeutic ultrasound is limited. The objective of this study was to evaluate structure, inflammatory cytokine signaling and immune cell infiltration after therapeutic ultrasound in young and aging murine muscle.

Methods:

Young (6-weeks-old) and Adult (52-weeks-old) male and female mice non-injured gastrocnemii were treated with either low intensity pulsed ultrasound at 2 W/cm² (~0.243 MPa) or high intensity pulsed focused ultrasound at 554 W/cm² (~5.96 MPa). Cytokine expression was evaluated at 1-, 8-, and 24-hours, cell infiltration was measured via flow cytometry at 1- and 24-hours, and immunofluorescence assessed muscle fiber area, fibrosis and satellite cells at 24-hours after sonication.

Results:

Low intensity pulsed ultrasound induced an early, transient inflammatory response where IL-15 and macrophages (M2 > M1) were increased 1-hour post-sonication. High intensity pulsed focused ultrasound caused a late, extended immune response where MCP-1, neutrophils, monocytes, and macrophages (M1 > M2) were increased 24-hours post-sonication. Notably, these changes manifested solely in Young gastrocnemius. The Adult gastrocnemius exhibited decreased cytokine expression (IL-1α, IL-6, IL-15, M-CSF) and no alteration in immune cell recruitment post-sonication. There was no damage to muscle structure.

Conclusions:

Therapeutic ultrasound induced a pressure dependent inflammatory response that can augment or mitigate intrinsic muscle cytokine signaling and cell recruitment in adolescent or aged muscle, respectively.

Introduction

Muscle injuries are common in military combat, vehicle accidents, and sports. Elderly populations are vulnerable to muscle injuries because they have a decreased capacity for repair due to sarcopenia¹. Starting at age 40, humans experience a decline in skeletal muscle mass and function at a rate of ~1.5-2.0% and ~2.5-3.0% per year, respectively^{2, 3}. However, current treatment strategies primarily assessed young rodents (10-11-weeks-old mice, 8-35-weeks-old rats)^{4, 5, 6, 7, 8, 9, 10, 11} which correspond to ~5-21-year-old humans^12,13 and hence may not represent the aging patient population.

Current standards of care for muscle injury include physical therapy, electrical stimulation, and therapeutic ultrasound⁴. Even though ultrasound is a commonly used treatment modality, its effectiveness remains controversial¹⁴. Variation in injury models and ultrasound parameters (e.g., intensity, treatment initiation/frequency) contribute to this divergence in efficacy. Furthermore, there has been limited investigation into the effect of ultrasound on muscle immunology. A deeper understanding of the changes induced by ultrasound within the muscle microenvironment is required to use this as a method to enhance regeneration.

Inflammation in muscle is tightly coupled with regeneration¹⁵. In acute muscle damage, infiltrating neutrophils peak 1-day post-injury and initiate the clearing of impaired tissue^{15, 16}. Subsequently, macrophages are recruited to degrade cellular debris and secrete paracrine signals¹⁶. While the classification is overly simplified, infiltration of pro-inflammatory (M1) and anti-inflammatory (M2) macrophages peak 2- and 4-days post-injury, respectively¹⁵. M1 macrophage signaling (e.g., interleukin-one beta (IL-1β), IL-6, monocyte chemoattractant protein-one (MCP-1)) stimulates satellite cell proliferation^{15, 16, 17}. The switch to M2 macrophages is initiated by an increase in anti-inflammatory signaling (e.g., IL-10, monokine induced gamma interferon (MIG), interferon gamma induced protein-ten (IP-10)) which causes proliferating satellite cells to differentiate into myoblasts which subsequently fuse to form myotubes^{15, 18}. Muscle cytokine, chemokine, and trophic factor signaling (heretofore referred as cytokines) including IL-15 and MCP-1 enhance monocyte and macrophage recruitment^{15, 17, 18, 19}. Dysregulation of the complex and coordinated immune and myogenic response to injury can inhibit regeneration and promote fibrosis and satellite cell senescence^{17, 20}.

Inflammaging is the chronic low-grade inflammation associated with aging that can accelerate tissue dysfunction^{16, 21, 22, 23}. Changes in elderly human muscle were reported to include increased pro-inflammatory gene expression and neutrophil infiltration, but decreased total macrophages²². Similarly in rodent models, pro-inflammatory gene expression was increased and cell composition was altered including increased neutrophils and decreased M1 macrophages and satellite cells in old (>87-weeks-old) compared to young (13-22-weeks-old) muscle^{21, 24, 25}. Structurally, old muscle displayed increased fibrosis and decreased weight compared to young^{21, 24}. Since inflammatory signaling and cell recruitment vary with age, investigation of therapeutic strategies in aged tissues is essential to optimize treatment parameters and regimens.

Therapeutic ultrasound including both low intensity pulsed ultrasound (LIPUS) or high intensity pulsed focused ultrasound (pFUS) has been used to enhance healing after musculoskeletal injury. The mechanical effects (i.e., acoustic radiation force or pressure) of ultrasound were previously shown to drive cellular changes^{26, 27, 28}. However, pre-clinical studies that utilized ultrasound to improve muscle regeneration after injury exhibited varying success⁴. In young contusion models, LIPUS with intensities ≥ 1 W/cm² exhibited increased cellularity and total protein within three weeks post-injury^{5, 6}, while intensities < 1 W/cm² did not change muscle mass, cellularity, nor myofiber area^{7, 8, 10}. However, one study using 0.6 W/cm² showed increased myofiber area and decreased fibrosis within three weeks after injury¹¹. In addition, CD86 and CD206 expression were decreased and increased, respectively¹¹. Previously, in non-injured hamstring, pFUS with 40 W acoustic power exhibited increased IL-9, IL-10, MCP-1, macrophage inflammatory protein-one alpha (MIP-1α), and vascular endothelial growth factor (VEGF) protein expression with no change in satellite cells 10-hours post-sonication²⁸. Overall, these variable outcomes make it difficult to discern the causality between the ultrasound regimens and potential biological benefits.

This study aims to evaluate inflammatory cytokines, immune cell infiltration and structural changes in Young (6-weeks-old; ~4-year-old human¹²) and Adult (52-weeks-old; ~48-year-old human¹²) non-injured murine gastrocnemii after either LIPUS (2 W/cm², ~0.243 MPa) or pFUS (554 W/cm², ~5.96 MPa). We hypothesized that the immune response will increase with increasing ultrasound intensity/pressure, but decrease with age, and muscle structure will only express a difference with age. While the ages chosen may not cover the geriatric population, they will discern what may initiate and drive the age-related factors affecting the response of muscle to ultrasound.

Materials and Methods

Animals and Study Design

A total of 294 Young (6-weeks-old) and Adult (52-weeks-old) male and female C57BL/6J mice from Charles River were used in this National Institute of Health Institutional Appropriate Care and Animal Use Committee approved study (Figure 1A). The right gastrocnemius of treated animals received a single sonication of either LIPUS (Figure 1B) or pFUS (Figure 1C). Control animals were not treated. Cytokine expression was measured at 1-, 8-, and 24-hours post-sonication (n = 5/age/sex/time/ultrasound + 5 female/age control + 4 male/age control; 138 total mice). Flow cytometry assessed cell recruitment at 1- and 24-hours post-sonication (n = 5/age/sex/time/ultrasound + 5/sex/age/time control; 120 total mice). Immunofluorescence analyzed muscle structure, satellite cells and fibrosis at 24-hours post-sonication (n = 3/age/sex/time/ultrasound + 3/age/sex/time control; 36 total mice). Both age and sex exhibited an effect on body and gastrocnemius weight (Supplemental Figure 1).

Figure 1.

(A) Schematic of the in vivo experimental method timeline. Mouse gastrocnemii were sonicated with a single dose of either low intensity pulsed ultrasound (LIPUS) or high intensity pulsed focused ultrasound (pFUS). Gastrocnemii were harvested post-sonication at 1-, 8-, and 24-hours to evaluate cytokine expression, at 1- and 24-hours to assess cell infiltration via flow cytometry, and at 24-hours to analyze muscle structure via immunofluorescence. Schematics depict how the sonications were applied to the gastrocnemius using (B) LIPUS and (C) pFUS.

Therapeutic Ultrasound

Prior to ultrasound treatment, hair was removed from the right hindlimb. LIPUS was administered using a linear dual frequency transducer with a 5 cm² area (Sonicator 740, Mettler Electronics, Anaheim, CA, USA) operating at 1 MHz using the following parameters: 2 W/cm² intensity (~0.243 MPa), 10.0% duty cycle, and a 7-minute duration. The transducer was coupled to the right hindlimb using ultrasound gel and was centered on the gastrocnemius mid-belly. pFUS was administered using a 1.15 MHz transducer (VIFU 2000, Alpinion Medical, Bothell, WA, USA) with a 0.03 cm² focal area and 4.4 cm focal depth under ultrasound image guidance in degassed water at 37°C using the following parameters: 25 W input power (~16.6 W acoustic power, ~554 W/cm², ~5.96 MPa), 10.0% duty cycle, 5 Hz pulse repetition frequency and a 20-second duration per raster point. The entire right gastrocnemius was treated following a raster pattern with 2 mm elemental spacing. Intensities and pressures reported represent spatial-peak temporal-peak intensities and peak negative pressures, respectively.

Cytokine Expression

Gastrocnemii were harvested at 1-, 8-, and 24-hours post-sonication and flash frozen in liquid nitrogen. Muscle was homogenized in cold lysis buffer (Tris-buffered saline with 0.5% Tween-20 and EDTA-free Protease Inhibitor Cocktail (Roche, Basel, Switzerland)) using a Bead Ruptor Elite (Omni, Kennessaw, GA, USA). Insoluble material was removed by centrifugation. The total protein content of each sample was determined using a bicinchoninic acid assay (ThermoFisher, Rockville, MD, USA). A Milliplex Mouse 32 Cytokine Immunology Multiplex Assay (Millipore, Burlington, MA, USA) measured cytokine expression using a Luminex Bio-Plex 200 System (Bio-Rad, Hercules, CA, USA). To account for inter-plate variability, the study was designed for all samples at each age and ultrasound type to fit within one plate and controls were run on every plate. Data at each time point were normalized by subtracting the age- and sex-matched average control expression.

Flow Cytometry

Flow cytometry evaluated immune and endothelial cell populations in muscle after ultrasound (Table 1). Gastrocnemii were harvested at 1- and 24-hours post-sonication, minced and incubated for 1.5 hours in digestive solution (Ham’s F-10 with 10.0% horse serum and 700 U/mL collagenase type II). The digested muscle was centrifuged, resuspended in digestive solution (Ham’s F-10 with 10.0% horse serum, 100 U/mL collagenase type II, and 2 U/mL dispase II) and incubated for 30 minutes. The digested muscle was passed through a 20 G needle and strained through a 70 μm filter. The cells were centrifuged then resuspended in phosphate buffered saline. 5 x 10⁵ cells per sample were stained with Zombie Fixable Viability Dye for 15 minutes followed by primary antibodies for 25 minutes (Table 1) and then were fixed with 1.0% paraformaldehyde for 20 minutes. Beads (ArC Amine Reactive Compensation Beads (ThermoFisher), MACS Comp anti-Rat Beads (Miltenyi, Gaithersburg, MD, USA), and MACS Comp anti-REA Beads (Miltenyi)) were used for voltage gating and as compensation controls. Samples were run on a MACSQuant Analyzer 16 (Miltenyi). The flow cytometer was calibrated before each use. Data were analyzed using FlowJo software (FlowJo LLC, Asland, OR, USA) following the gating strategy outlined in Supplemental Figure 2A. All gating was performed based on unstained and fluorescence minus one controls (Supplemental Figure 3). The average cell viability for all groups was >70.0% (Supplemental Figure 2B). Data were presented as total cell count per mg to account for differences in gastrocnemius weight (Supplemental Figure 1): (gated cell count x (hemacytometer whole tissue cell count / flow cytometer total cell count)) / gastrocnemius weight.

Table 1.

Methods for (i) flow cytometry cell population markers and (ii) flow cytometry and immunofluorescence antibodies.

Cell Type	Markers
Neutrophil	Viability CD45+ CD11b+ Ly6G+
Monocyte	Viability CD45+ CD11b+ CD43+
M1 Macrophage	Viability CD45+ CD11b+ Ly6G− F4/80+ CD86+
M2 Macrophage	Viability CD45+ CD11b+ Ly6G− F4/80+ CD206+
Endothelial Cell	Viability CD45− CD31+
Marker	Fluorphore	Catalog #	Dilution (μg/mL)	Use
Ly6G	BV570	127629a	1:33	Flow Cytometry
CD11b	BV605	101257a	1:33	Flow Cytometry
CD206	BV650	141723a	1:33	Flow Cytometry
CD86	VioBright 515	130-122-136b	1:50	Flow Cytometry
CD43	PE	130-112-887b	1:50	Flow Cytometry
CD45	PE-Vio615	130-110-666b	1:50	Flow Cytometry
CD31	PE-Vio770	130-111-542b	1:50	Flow Cytometry
F4/80	APC	130-116-547b	1:50	Flow Cytometry
Viability	NIR	423105a	1:5000	Flow Cytometry
Collagen Type I	-	Ab34710c	1:200 (5)	Immunofluorescence
Laminin	-	Ab11575c	1:200 (3.5)	Immunofluorescence
Pax7	-	Pax7d	1:1 (32)	Immunofluorescence
DAPI	358	D1306e	1:1000 (1)	Immunofluorescence
Donkey anti-Rabbit	488	Ab150073c	1:400 (5)	Immunofluorescence
Goat anti-Mouse	568	Ab175473c	1:400 (5)	Immunofluorescence

^(a)BioLegend, San Diego, CA, USA

^(b)Miltenyi, Gaithersburg, MD, USA

^(c)Abcam, Cambridge, United Kingdom

^(d)Developmental Studies Hybridoma Bank, Iowa City, IA, USA

^(e)Invitrogen, Waltham, MA, USA

Immunofluorescence and Histology

Skeletal muscle structure was quantified via myofiber cross-sectional area and collagen area fraction (indicative of fibrosis)²⁹. Gastrocnemii harvested at 24-hours post-sonication were mounted in tragacanth and flash frozen in liquid nitrogen cooled isopentane. A −22°C cryostat was used to cut 10 μm transverse sections. For each stain, three sections were cut per muscle. To quantitate myofiber cross-sectional area and satellite cells, sections were immunolabeled with primary antibodies for laminin and Pax7 (Table 1). Sections were incubated with 4.0% paraformaldehyde for 10 minutes, permeabilization solution (phosphate buffered saline with 0.1% TritonX-100) for 10 minutes, blocking solution (SuperBlock Blocking Buffer (ThermoFisher) with 40 μg/mL AffiniPure Fab Fragment Goat Anti-Mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA)) for 1-hour, and primary antibodies overnight at 4°C followed by secondary antibodies for 1-hour. Ten images per section were taken using a 40X oil objective for analysis. Myofiber cross-sectional area measurements and Pax7+ satellite cell counts were performed with a custom macro and the multi-point tool in ImageJ (National Institutes of Health, Bethesda, MD, USA), respectively. To measure collagen area fraction or fibrosis, sections were immunolabeled with primary antibody for collagen type I (Table 1). Sections were incubated with blocking solution (SuperBlock Blocking Buffer) for 1-hour and primary antibodies overnight at 4°C followed by secondary antibodies for 1-hour. Five images per section were taken using a 20X objective for analysis. Collagen area fraction was measured using an ImageJ pixel counter plug-in, where the number of positively stained pixels were counted and normalized to the total number of image pixels to calculate the percentage of positively (i.e., collagen) stained pixels²⁹. The percentage of collagen stained pixels is representative of the amount of collagen within the image. Antibody specificity and imaging settings were validated via no primary negative controls (Supplemental Figure 4). Sections were also stained with hematoxylin and eosin to view overall fiber morphology (Supplemental Figures 5–6).

Statistical Analysis

Preliminary analyses separated the sexes. Weights and flow cytometry data were analyzed with a three-way analysis of variance (ANOVA) for sex, time point and age with post-hoc Bonferroni comparisons between sexes and ages, to control, and over time. Cytokine data were analyzed with a two-way ANOVA for sex and time point with post-hoc Bonferroni comparisons between sexes, to control, and over time. Statistical comparisons were not made between ages because the samples were run on different plates. Immunofluorescence data were analyzed with a three-way ANOVA for sex, age, and sonication with post-hoc Bonferroni comparisons between sexes and ages, and to control.

Minimal, if any, sex differences were observed so the sexes were combined. Cytokine data were analyzed with a one-way ANOVA with post-hoc Bonferroni comparisons to control and over time. Flow cytometry data were analyzed with a two-way ANOVA for age and time point with post-hoc Bonferroni comparisons between ages, to control, and over time. Immunofluorescence data were analyzed with a two-way ANOVA for age and sonication with post-hoc Bonferroni comparisons between ages and to control. All statistical analyses were performed in GraphPad Prism (GraphPad Software Inc, La Jolla, CA, USA).

Results

Cytokine Expression

The sexes were combined because cytokine expression revealed minimal differences due to sex where 1/60 and 0/56 after LIPUS and 2/48 and 3/44 after pFUS occurred for Young and Adult gastrocnemii, respectively (Supplemental Tables 1–4). Not all cytokines were detected in each condition, hence the different total number of comparisons.

Cytokine expression was upregulated 1-hour post-LIPUS and 1-, 8-, and 24-hours post-pFUS in Young compared to control (Figure 2). Specifically, LIPUS increased IL-15 at 1-hour compared to control (p < 0.01). IL-9 and VEGF were increased at 1-hour compared to later time points (p < 0.05). After pFUS, VEGF at 1-hour, MIG at 8-hours, and MCP-1 at 24-hours were increased compared to control (p < 0.05). VEGF was also increased at 1-hour compared to subsequent time points (p < 0.05), while IP-10 and MCP-1 were increased at later time points compared to 1-hour (p < 0.01). IL-2 at 1- and 24-hours and IL-9 at 1-hour were decreased compared to control (p < 0.05).

Figure 2.

Cytokine expression at 1-, 8-, and 24-hours post-single sonication of (A) low intensity pulsed ultrasound (LIPUS) and (B) high intensity pulsed focused ultrasound (pFUS) in Young (6-weeks-old) and Adult (52-weeks-old) gastrocnemii. (X = not detected; * = different compared to age-matched control, # = decreased compared to the age-matched time point listed). (C) One-way ANOVA p values reported for each cytokine evaluated after single LIPUS and pFUS sonication in Young and Adult gastrocnemii. (bold = significant).

In Adult, 1-, 8-, and 24-hours after LIPUS or pFUS cytokine expression was downregulated compared to control (Figure 2). After LIPUS, IL-9 at 1-hour and IL-15 at 1-, 8-, and 24-hours were decreased compared to control (p < 0.05). After pFUS IL-1α, IL-2, IL-6, and macrophage colony stimulating factor (M-CSF) were decreased at 8- and 24-hours (p < 0.05) and IL-9 and MIP-1α were decreased at 1-, 8-, and 24-hours compared to control (p < 0.01).

Cell Recruitment and Infiltration

Flow cytometry revealed post-LIPUS 5/15 and 3/15 and post-pFUS 1/15 and 0/15 differences due sex in Young and Adult, respectively (Supplemental Tables 5–6). Only one pattern emerged following LIPUS at 1- and 24-hours, males exhibited increased endothelial cells compared to females in Young and Adult. However, at these time points there were no differences compared to controls. Therefore, the sexes were combined.

In Young, cell recruitment and infiltration peaked at 1-hour after LIPUS and at 24-hours after pFUS (Figure 3). After LIPUS M1 and M2 macrophages were increased at 1-hour compared to control and 24-hours (p < 0.01). After pFUS neutrophils, monocytes, and M1 and M2 macrophages were increased at 24-hours compared to control and 1-hour (p < 0.001). Endothelial cells were decreased at 24-hours after pFUS compared to control (p < 0.01).

Figure 3.

Flow cytometry at 1- and 24-hours post-single sonication of (A-E) low intensity pulsed ultrasound (LIPUS) and (F-J) high intensity pulsed focused ultrasound (pFUS) in Young (6-weeks-old) and Adult (52-weeks-old) gastrocnemii. The cell types evaluated include (A,F) neutrophils, (B,G) monocytes, (C,H) M1 macrophages, (D,I) M2 macrophages, and (E,J) endothelial cells. (C = Control; * = different compared to age-matched control, ‡ = difference between age-matched time points, # = difference between time point-matched ages). (K) Two-way ANOVA for age and time point, p values reported for each cell type evaluated after single LIPUS or pFUS sonication. (bold = significant).

In Adult, no significant changes in cell populations happened over time after sonication but there were differences due to age (Figure 3). In the control and at 1- and 24-hours after LIPUS, monocytes and M1 and M2 macrophages were decreased in Adult compared to Young (p < 0.001). Neutrophils, monocytes, and M1 and M2 macrophages were decreased 24-hours post-pFUS in Adult compared to Young (p < 0.001). M2 macrophages and endothelial cells were also decreased in Adult controls compared Young (p < 0.01).

Gastrocnemius Structure, Satellite Cells, and Fibrosis

There were no differences due to sex for myofiber cross-sectional area, Pax7+ satellite cell counts, collagen area fraction, or morphology so the sexes were combined (Supplemental Figures 5–8).

Gastrocnemius structure, satellite cells and fibrosis were not affected 24-hours after sonication but there were differences due to age. In the control and at 24-hours after LIPUS or pFUS, myofiber cross-sectional area (Figure 4; p < 0.001) and collagen area fraction (Figure 5; p < 0.01) were increased, while Pax7+ satellite cells were decreased in Adult compared to Young (Figure 4; p < 0.01).

Figure 4.

(A) Representative immunofluorescence at 24-hours post-single sonication of low intensity pulsed ultrasound (LIPUS) and high intensity pulsed focused ultrasound (pFUS) in Young (6-weeks-old) and Adult (52-weeks-old) female gastrocnemii. Laminin was green, Pax7 was red, and DAPI was blue. (white triangle = Pax7+ satellite cell, scalebar = 10 μm). From these images (B) myofiber cross-sectional area was measured and (C) Pax7+ cells were counted. (# = difference between sonication-matched ages). (D) Two-way ANOVA for age and sonication, p values reported for each measurement. (bold = significant).

Figure 5.

(A) Representative immunofluorescence at 24-hours post-single sonication of low intensity pulsed ultrasound (LIPUS) and high intensity pulsed focused ultrasound (pFUS) in Young (6-weeks-old) and Adult (52-weeks-old) female gastrocnemii. Collagen type I was green and DAPI was blue. (scalebar = 50 μm). From these images (B) collagen area fraction was measured as an indicator of muscle fibrosis. (# = difference between sonication-matched ages). (C) Two-way ANOVA for age and sonication, p values reported for each measurement. (bold = significant).

Discussion

Muscle injuries occur during daily and vocational activities at any age, but elderly populations are particularly susceptible due to sarcopenia¹. While current standards of care for muscle injury include therapeutic ultrasound, its ability to improve clinical outcomes remains unclear because previous results were often conflicting and arduous to interpret due to the varied experimental designs^{4, 14}. Previous pre-clinical work focused on the treatment response of young rodent models^{4, 5, 6, 7, 8, 9, 10, 11} but these results cannot be extrapolated to represent the aging patient population. Despite the well-established connection between inflammation and muscle regeneration¹⁵, few studies investigated how ultrasound affects muscle immunology. The goal of this study was to evaluate inflammation and structural changes in Young and Adult non-injured gastrocnemii after either LIPUS or pFUS.

In Young, an early temporary inflammatory response was induced by LIPUS. IL-15, a muscle pro-inflammatory signal that enhances cell recruitment¹⁹, was increased at 1-hour after LIPUS compared to control (Figure 2A) and likely triggered the increase in M1 and M2 macrophages (Figure 3C–D). While the ratio of M1 to M2 macrophages did not change with sonication, there were consistently more M2 macrophages. Previously LIPUS was shown to increase protein expression for CD206 (M2 macrophage marker)¹¹. In the current study, LIPUS did not affect muscle fiber cross-sectional area, collagen area fraction (fibrosis), or Pax7+ satellite cells in Young 24-hours post-sonication (Figures 4–5). Previous work reported that three consecutive days of LIPUS post-injury increased the number of satellite cells⁵. Since the acute inflammatory response induced by a single sonication was not enough to increase satellite cells, a repetitive treatment may be needed. In general, LIPUS represents a transient bimodal treatment strategy with pro-inflammatory signaling and anti-inflammatory cell recruitment.

pFUS caused a continuous response that was not resolved before the end of the evaluation period in Young. While VEGF was upregulated 1-hour post-sonication (Figure 2B), the subsequent upregulation of MIG at 8-hours likely prevented an increase in endothelial cells (Figure 3J) because MIG inhibits endothelial cell chemotaxis and angiogenesis¹⁸. Increased MCP-1 (Figure 2B), an inflammatory cell chemoattractant^{15, 17}, corresponded to increased neutrophils, monocytes, and M1 and M2 macrophages compared to control 24-hours post-sonication (Figure 3F–I). The ratio of macrophage phenotypes switched from M2- to M1-dominated 24-hours after pFUS. pFUS did not alter muscle structure or satellite cells in Young 24-hours post-sonication (Figures 4–5). Previous literature using higher intensity/pressure pFUS also exhibited similar results²⁸. Thus, pFUS represents an extended treatment strategy dominated by pro-inflammatory signaling and cell recruitment.

In Adult, therapeutic ultrasound did not elicit the same response. Cytokine expression was downregulated after LIPUS and pFUS in Adult compared to control (Figure 2). While not statistically evaluated due to limits of the assay, cytokine expression in Adult control compared to Young was at least two times greater for IL-1α, IL-6, IL-15, MCP-1 and M-CSF (Supplemental Tables 1–4). Age has been associated with increased cytokine expression when comparing geriatric to adolescent rodent muscle^{21, 24, 30}. Thus, in the current study, Adult control cytokine expression presented elevated baseline pro-inflammatory signaling compared to Young. This inflammaging response may contribute to the development of muscle atrophy over time³¹. Interestingly, therapeutic ultrasound suppressed this naturally elevated cytokine expression in Adult.

Since the pro-inflammatory paracrine signals were decreased, there was a corresponding decrease in inflammatory cell recruitment in Adult following therapeutic ultrasound. There were no changes over time in the immune cell response compared to control following LIPUS or pFUS (Figure 3). Age was the only influencing factor, where monocytes and M1/M2 macrophages after LIPUS (Figure 3B–D) and neutrophils, monocytes, and M1/M2 macrophages after pFUS (Figure 3F–I) were decreased in Adult compared to Young. Previous studies comparing geriatric to adolescent rodent muscle exhibited increased neutrophils, decreased M1 macrophages, and no change in M2 macrophages^{21, 24, 25}. While lacking the increased neutrophil response, the decreased monocytes and macrophages in Adult may represent the altered cell composition in muscle due to age. Over time this pro-inflammatory microenvironment and decreased monocyte/macrophage response may drive the increased neutrophil expression observed in geriatric muscle. Overall, the same sonication regimen used in Young increased the immune response as compared to Adult where it was decreased. Hence, the decreased inflammaging in Adult after sonication may ultimately mitigate aged-induced muscle atrophy. This proposes new applications of therapeutic ultrasound outside of muscle injury within the adult and geriatric patient populations in clinic.

Age dominated over the effect of therapeutic ultrasound within muscle structure. After a single sonication, no damage was found in Adult (Figures 4–5). Similar to previous reports in geriatric rodent muscle^{20, 24}, Adult exhibited decreased satellite cells and increased fibrosis compared to Young (Figures 4–5). However, unlike geriatric muscle, the Adult gastrocnemius was not sarcopenic as the muscle weight and fiber area were increased compared to Young (Figure 4, Supplemental Figure 1)². Hence, the increased pro-inflammatory cytokine signaling and altered immune/satellite cell composition with aging preceded changes to muscle weight and fiber area. Therefore, inflammaging most likely proceeds and contributes to muscle atrophy in adult and geriatric populations^{23, 24}.

Several remaining questions and/or study limitations provide motivation for future work. First, only the response of non-injured muscle to therapeutic ultrasound was evaluated in this study. The goal of these initial experiments was to understand the baseline inflammatory response at various ages and ensure that no structural damage ocurred. The short-term inflammatory response induced by LIPUS represents a potentially viable treatment to boost the natural healing/immune response after muscle injury. Future studies will utilize LIPUS to enhance regeneration in a muscle contusion injury model. Second, this study only assessed muscle response following a single sonication, yet ultrasound is typically a repetitive treatment in clinic. Future investigation will include a treatment plan to discern the cumulative effect of repeated sonications. Third, the effects of ultrasound were only measured throughout the first 24-hours after sonication. Additional time points will be the purview of future experiments. Lastly, the individual effects of acoustic radiation force, acoustic cavitation, and heat from therapeutic ultrasound were not investigated. However, previously in muscle, cavitation signatures like increased half harmonic or broadband emissions were only detectable at 1.125 MHz when peak negative pressure was > 6 MPa³². Therefore, it is possible that a combination of these effects (e.g., acoustic radiation force and endogenous cavitation) may contribute to the biological response.

Conclusion

The goal of this study was to evaluate inflammation and structural changes in Young and Adult non-injured gastrocnemii after either LIPUS or pFUS to better inform treatment design. LIPUS caused an early, transient response with pro- and anti-inflammatory effects. pFUS induced a late, extended response dominated by pro-inflammatory signaling and cell recruitment. However, the ultrasound driven immune response only occurred in Young and was lost in Adult. While in this study Adult gastrocnemius was not sarcopenic, it expressed increased baseline pro-inflammatory cytokines, a hallmark of aging muscle, which were decreased after LIPUS or pFUS sonication. Hence the same ultrasound parameters in different populations could be utilized to achieve distinct goals. For example, in younger muscle it could be used to enhance regeneration while in aged muscle it could be used to prevent atrophy. This highlights the importance that every study design needs to reflect the patient population of interest to accurately evaluate treatment outcomes. Neither LIPUS nor pFUS caused damage to muscle structure at either age. In conclusion, ultrasound caused a pressure dependent immune response that can be used to enhance or minimize intrinsic muscle signaling and cell recruitment in adolescent or aged muscle, respectively.

Data Availability Statement

The data presented within this manuscript are available from the corresponding author upon request.