Local and Systemic Effects of Blood Flow Restriction Therapy in an Animal Model

Abstract

Background:

Blood flow restriction therapy (BFRT) has been increasingly applied to improve athletic performance and injury recovery. Validation of BFRT has lagged behind commercialization, and currently the mechanism by which this therapy acts is unknown. BFRT is one type of ischemic therapy, which involves exercising with blood flow restriction. Repetitive restriction of muscle blood flow (RRMBF) is another ischemic therapy type, which does not include exercise.

Hypothesis/Purpose:

The purpose was to develop a rat model of ischemic therapy, characterize changes to muscle contractility, and evaluate local and systemic biochemical and histologic responses of 2 ischemic therapy types. We hypothesized that ischemic therapy would improve muscle mass and strength as compared with the control group.

Study Design:

Controlled laboratory study.

Methods:

Four groups of 10 Sprague-Dawley rats were established: control, stimulation, RRMBF, and BFRT. One hindlimb of each subject underwent 8 treatment sessions over 4 weeks. To simulate exercise, the stimulation group underwent peroneal nerve stimulation for 2 minutes. The RRMBF group used a pneumatic cuff inflated to 100 mm Hg with a 48-minute protocol. The BFRT group involved 100–mm Hg pneumatic cuff inflation and peroneal nerve stimulation for a 5-minute protocol. Four methods of evaluation were performed: in vivo contractility testing, histology, immunohistochemistry, and ELISA. Analysis of variance with post hoc Tukey test and linear mixed effects modeling were used to compare the treatment groups.

Results:

There was no difference in muscle mass among groups (P = .40) or between hindlimbs (P = .73). In vivo contractility testing showed no difference in maximum contractile force among groups (P = .64) or between hindlimbs (P = .30). On histology, myocyte cross-sectional area was not different among groups (P = .55) or between hindlimbs (P = .44). Pax7 immunohistochemistry demonstrated no difference in muscle satellite cell density among groups (P = .06) or between hindlimbs (P = .046). ELISA demonstrated the RRMBF group as eliciting elevated GH levels as compared with the other groups (P < .001).

Conclusion:

Ischemic therapy did not induce gains in muscle mass, contractility strength, fiber cross-sectional area, or satellite cell density locally or systemically in this model, although the RRMBF group did have elevated GH levels on ELISA.

Clinical Relevance:

This animal model does not support ischemic therapy as a method to improve muscle mass, function, or satellite cell density.

Keywords

blood flow restriction ischemic therapy repetitive restriction of muscle blood flow ischemic preconditioning muscle hypertrophy satellite cell

Ischemic therapy is a rehabilitation treatment modality that was developed to improve patient muscle mass and performance.³⁰ It has been postulated to be useful in multiple applications, including recovery from injury, expediting postsurgical rehabilitation, and improving athletic ability. Commercialization of this technology has preceded scientific vetting of its biologic mechanisms, efficacy, and safety. Ischemic therapy has been widely incorporated into athletic training rooms and physical therapy clinics worldwide.

Ischemic therapy involves application of an occlusive device (pneumatic cuff or elastic band) at the proximal portion of an extremity for a prescribed period.^2,19,22,29 It was developed in Japan by Dr Yoshiaki Sato in the 1970s.⁷ There are 2 major types of musculoskeletal ischemic therapy: repetitive restriction of muscle blood flow (RRMBF) and blood flow restriction therapy (BFRT). RRMBF, also known as ischemic preconditioning, involves a cycle of restriction of blood flow, followed by a cycle of unrestricted blood flow for a set number of repetitions.¹⁷ BFRT involves a patient’s performing exercise with restricted blood flow, followed by a period of unrestricted blood flow for a set number of repetitions. BFRT has also been termed occlusive or KAATSU training.¹⁹ Some have reported as little as 20% of the muscle’s maximum contraction with BFRT can increase strength and induce muscle hypertrophy.^19,29,35

Although case series have demonstrated benefit, large randomized controlled trials that validate this therapy have not yet been performed.^‖ Inconsistent experimental conditions and unreported experimental design factors make comparison of these studies difficult. Basic science evaluation and validation of this technology has lagged behind commercialization, and to date there is no identified mechanism to explain the reported effects. Metabolic stress and mechanical tension have been hypothesized to be primary mechanisms leading to the hypertrophic effects of ischemic therapy.²⁸ It is further hypothesized that the following could be mechanistically involved: autocrine/paracrine interactions, reactive oxidative species (heat shock proteins and nitric oxide), cellular swelling, increased fiber recruitment, mechanotransduction, and muscle damage.²⁸ In this study, we developed a rat model of ischemic therapy, characterized changes to muscle contractility, and evaluated the local and systemic responses of 2 ischemic therapy types. We hypothesized that ischemic therapy would improve muscle mass and strength as compared with the control group.

Methods

Animal Subjects

Female Sprague-Dawley rats at 10 weeks of age were purchased from Charles River Laboratories. They were acclimated to the vivarium for 1 week before initiation of the study. The right hindlimb of each rat was assigned as the treatment limb, while the left hindlimb served as an internal control. Four treatment groups were established: control, stimulation, RRMBF, and BFRT. Group sizes were calculated to be 8 subjects per group based on an assumed 10% difference in muscle contractility between the treatment and control limbs (α = .05; power = 0.8; effect size, d = 1.6). An additional 2 rats were included per group to avoid any potential survival issues.

For all subjects, both hindlimbs were denuded. Isoflurane anesthesia was administered, and the health of the rat was continuously monitored during treatment. After completion of the treatment protocol, subcutaneous carprofen (5 mg/kg) was administered to each rat for pain control. On the basis of parameters reported in previous studies, all rats were treated twice per week for a total of 8 treatment sessions.^32,43 All analyses performed on samples were done in a blinded manner. All procedures were approved by our Institutional Animal Care and Use Committee (PRO00008129).

Treatment Groups

Control Group

Ten Sprague-Dawley rats were assigned to a control group for the study. These subjects underwent the hindlimb preparation, anesthesia, and postprocedure carprofen injection as described; however, no additional intervention was made.

Stimulation Group

Ten Sprague-Dawley rats were assigned to the stimulation group. A bipolar platinum wire electrode was placed around the common peroneal nerve at the level of the right knee joint. The S88X Dual Output Square Pulse Stimulator (Grass Instruments Astro-Med) was used in combination with a Stimulus Isolator (World Precision Instruments) to deliver a pulse waveform (ʎ = 40 Hz, 0.7-second stimulation, 2.3-second rest, I = 1 mA).^26,32 Surface stimulation of the peroneal nerve was verified visually by contraction of the anterior compartment musculature. Pilot testing had demonstrated that a stimulation frequency of 40 Hz produced a muscle contraction that was 50% of the maximum contraction (Figure 1). An ankle dorsiflexion blocking splint was applied to the lower leg to hold the foot in 20° of plantarflexion for isometric contraction of the anterior muscle compartment. The peroneal nerve was stimulated for 2 minutes using the aforementioned parameters (Figure 2).^32,33

RRMBF Group

Ten Sprague-Dawley rats were assigned to the RRMBF group. A 1.6-cm reusable digit/penile cuff (D.E. Hokanson Inc) was applied to the proximal hindlimb and inflated to 100 mm Hg using a handheld sphygmomanometer (D.E. Hokanson Inc).²⁵ The tourniquet was inflated for 5 minutes and then deflated for 3 minutes. This was repeated for 6 cycles, totaling 48 minutes (Figure 3).²⁶ Given the duration of anesthesia, these subjects received 5 mL of normal saline fluid support intraperitoneally.

BFRT Group

Ten Sprague-Dawley rats were assigned to the BFRT group. This group used the same parameters and equipment as the stimulation group; however, a 1.6-cm reusable cuff was applied to the proximal hindlimb. A total treatment of 5 minutes for the right hindlimb included the following: inflation of the thigh tourniquet at 100 mm Hg for 2 minutes, followed by surface stimulation of the peroneal nerve for 2 minutes and, finally, cuff deflation 1 minute after completion of nerve stimulation.^25,26,32 As in the stimulation group, the ankle dorsiflexion blocking splint was applied to the lower leg for isometric contraction of the anterior muscle compartment.

Evaluation Methods

In Vivo Muscle Contractility Testing

Tibialis anterior (TA) contractile properties were measured in vivo as previously described.¹⁵ In the anesthetized rats, the whole TA muscle was isolated from the surrounding tissues. The distal tendon insertion was released and secured to a servomotor lever arm (model 305B; Aurora Scientific Inc). The muscle was activated by stimulating the common peroneal nerve with a bipolar platinum wire electrode. The maximum isometric twitch was determined for each muscle by adjusting the voltage of a 0.2-millisecond stimulation pulse. The optimal muscle length (L₀) was determined by measuring the muscle length at the maximal twitch. To determine the maximum isometric tetanic force (P₀), the muscle was tensioned to L₀, and a 300-millisecond train of pulses was applied at increasing stimulation frequencies until a maximum force was achieved. TA fiber length (L_i) was calculated by multiplying L₀ by 0.45.³ To determine total fiber cross-sectional area, the mass of the TA was divided by the product of L_i and the density of mammalian skeletal muscle, 1.06 g/cm².¹⁵ To determine the specific force (specific P₀), P₀ was divided by the total fiber cross-sectional area.

Muscle Histology

After in vivo contractility testing, the deeply anesthetized rats were euthanized by pneumothorax, and the TA was harvested and grossly weighed. Immediately afterward, the samples were covered in Tissue-Tek OCT Compound (Sakura Finetek) and rapidly frozen in isopentane cooled in liquid nitrogen. The samples were axially sectioned at −20°C at a thickness of 12 μm. Tissue sections were air dried at room temperature, and fluorescent immunohistochemical staining was initiated the same day.

Two staining evaluations were performed to evaluate muscle satellite cells and muscle inflammation. Slides for satellite cell staining were fixed in 4% paraformaldehyde for 10 minutes at room temperature; endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 1 hour; and antigen retrieval was performed by incubation in sodium citrate buffer (10 mM, pH 6) for 10 minutes at 92°C. To stain for satellite cells, an antibody against Pax-7 was used (PAX7c, 1:100; DHSB) with DAPI (4′,6-diamidino-2-phenylindole; 2 µg/mL, D21490; Thermo Fisher Scientific) and wheat germ agglutinin (Alexa Fluor 488 conjugate, 5 µg/mL, W11261; Thermo Fisher Scientific) to counterstain cell nuclei and the myocyte basal lamina, respectively. For detection of the Pax7 primary antibody, we used a Goat Anti-mouse IgG Alexa Fluor 555 Tyramide SuperBoost Kit (B40913; Invitrogen) per the manufacturer’s recommendations.

Samples for the muscle inflammation evaluation were fixed in ice-cold acetone for 10 minutes at −20°C and then air dried. Slides were then blocked for 1 hour at room temperature in 10% goat serum prepared in phosphate-buffered saline before overnight incubation at 4°C with primary antibodies. For evaluation of muscle inflammation, wheat germ agglutinin (CF405S; Biotium), a CD68 antibody (ab31630, 1:50; Abcam), a CD163 antibody (sc-33560, 1:50; Santa Cruz Biotechnology), and an anti-granulocyte (HIS48) antibody (ab33760, 1:20; Abcam) were used to identify the myocyte basal lamina, CD68+ macrophages, CD163+ macrophages, and granulocytes, respectively. Immune cell antigen primary antibodies were detected using conjugated secondary antibodies, including Goat Anti-mouse IgG1 Alexa Fluor 488 (A21121, 1:500), Goat Anti-mouse IgM Alexa Fluor 555 (A21426, 1:500), and Goat Anti-rabbit IgG (H + L) Alexa Fluor 647 (A27040, 1:500; all from Thermo Fisher Scientific).

Imaging was performed using a Nikon A1 Confocal Inverted microscope (Nikon Corporation). MuscleJ Version 1.0.2 software (Fiji) was used for automated morphologic evaluation of myocyte cross-sectional area on stitched panoramic composites of the entire TA cross section.²³ Given the natural scarcity of satellite cells in resting muscle, the entire axial TA cross section was manually counted to determine the total number of muscle satellite cells present. Muscle satellite cells were defined here as Pax7 and DAPI double-positive nuclei that were located between the myofiber basal laminae. Manual satellite cell counts were normalized to the overall TA cross-sectional area as determined by MuscleJ and expressed as Pax7+ cells/mm². The inflammatory histologic evaluation was assessed qualitatively in this study.

ELISA Testing

Venous blood from each subject’s tail was collected at 4 time points during the study: before initial treatment session (t0), 15 minutes after initial treatment session (t1), before final treatment session (t2), and 15 minutes after final treatment session (t3). After collection, the blood coagulated for 30 minutes at room temperature and was then centrifuged to isolate the serum. After centrifugation, the serum was transferred to individual micropipette tubes and stored at −80°C until the time of testing. ELISA analysis was performed for all treatment groups and time points for 4 analytes: IGF-1 (MG100; R&D Systems), GH (KRC5311; Thermo Fisher Scientific), myostatin (DGDF80; R&D Systems), and VEGF (RRV00; R&D Systems).

Statistical Analysis

Prism Version 8.2.0 (GraphPad) was used for statistical analysis and visualization. Differences between hindlimbs and treatment groups were evaluated by analysis of variance with post hoc Tukey testing for contractility and histology evaluations. For the ELISA evaluation, a linear mixed effects model was used to analyze repeated measures time- and group-dependent concentrations. Four covariance structures were assessed for optimal data fit, and the unstructured covariance matrix was selected. All P values were adjusted using a modified Bonferroni correction. Significance was set at P < .05.

Results

Four groups of 10 treatment randomized rats completed the study; each group underwent treatment over the course of 1 month. In vivo contractility testing and gross histology of the TA demonstrated no difference among the groups (P = .30-.83) with regard to the right and left hindlimbs (Figure 4, Table 1). When the percentage difference between the left and right hindlimbs was compared among groups (Figure 5), there was no difference with respect to muscle mass and maximum force (P = .3 and .73), but there was a significant difference in specific force (P = .01) between the BFRT and RRMBF groups on post hoc Tukey test (P < .01), with the BFRT group having a larger specific force. However, there was no difference between the RRMBF and BFRT groups and the control and stimulation groups (P = .14-.99).

Table 1

ANOVA Evaluation of the 4 Treatment Groups and In Vivo Contractility and Histologic Variables^a

	P Value
Variable: Hindlimb	ANOVA	Post Hoc Tukey
In vivo contractility variables
Maximum force
Right	.64
Left	.83
Right vs left^b	.30
Specific force
Right	.37
Left	.60
Right vs left^b	.01	<.01^c
Histology variables
Muscle mass
Right	.40
Left	.73
Right vs left^b	.73
Mean myocyte cross-sectional area
Right	.55
Left	.99
Right vs left^b	.44
Satellite cell count
Right	.06
Left	.049	.04^d
Right vs left^b	.046	—^e

ANOVA, analysis of variance; BFRT, blood flow restriction therapy; RRMBF, repetitive restriction of muscle blood flow.

Percentage difference.

RRMBF vs BFRT.

Control vs RRMBF.

No difference identified.

The mean myocyte cross-sectional area of the TA (Figure 6) demonstrated no difference among the treatment groups (P = .55-.99) with regard to the right hindlimb, left hindlimb, and percentage difference between the right and left hindlimbs (Table 1). Figure 7 demonstrates a representative image series of the satellite cell immunohistology used in this study. The muscle satellite cell counts demonstrated no difference with regard to the right hindlimb among the treatment groups. There was a significant difference among treatment groups for satellite cell density in the TA of the left internal control hindlimbs (P = .049) and in the percentage difference between the right and left hindlimbs (P = .046). With regard to the left hindlimb, the difference was between the control and RRMBF groups on post hoc Tukey test (P = .04), with the RRMBF group having more satellite cells than the contralateral control muscle. With regard to the percentage difference between hindlimbs, there was no identified difference on post hoc Tukey test, suggesting that this likely represented a type I error.

The inflammatory macrophage histologic evaluation was performed qualitatively, and only 1 finding was observed, with representative images available in Figure 8. Both hindlimbs in the control, stimulation, and RRMBF groups showed many resident CD68–, CD163+ macrophages surrounding the myocytes and very few scattered CD68+, CD163– macrophages (Figure 8, A-D). The right hindlimb of the BFRT group appeared notably different from the others (Figure 8, E-H). These subjects demonstrated many CD68+ macrophages, some of which coexpressed CD163. This finding was isolated to the treated hindlimb of the BFRT rats.

ELISA evaluation of the serum samples collected at 4 time points for the treatment groups is available in Figure 9. With regard to the IGF-1 ELISA evaluation, the RRMBF group at time points t3 and t2 had significantly lower IGF-1 concentrations than at time points t1 (P < .001) and t0 (P < .001), respectively. The RRMBF group also showed significantly lower IGF-1 concentrations at time points t0 and t2 than at time points t1 (P = .025) and t3 (P = .016). However, the control group also showed this trend with significantly lower IGF-1 concentrations at time points t0 and t2 than at time points t1 (P = .013) and t3 (P = .006). At t1, the RRMBF group had significantly larger IGF-1 concentrations than the control group (P = .041). The BFRT group at time points t3 and t2 had significantly lower IGF-1 concentrations than at time points t1 (P < .001) and t0 (P < .001). Additionally, the BFRT group had significantly lower IGF-1 concentrations at time points t2 (P = .004) and t3 (P = .032) than the stimulation group.

With regard to the GH ELISA evaluation, the RRMBF group at time points t3 and t2 had significantly larger GH concentrations than at time points t1 (P < .001) and t0 (P < .001), respectively. At t2, the RRMBF group had significantly larger GH concentrations than the stimulation group (P = .027). At t3, the RRMBF group had significantly larger GH concentrations than the BFRT (P = .001), stimulation (P < .001), and control (P < .001) groups. The stimulation group showed a decrease in GH concentration between time points t0 and t1 (P = .007). The BFRT group showed a decrease in GH concentration between time points t2 and t3 (P = .025).

With regard to the myostatin ELISA evaluation, the RRMBF (P = .024) and BFRT (P = .048) groups had a significantly lower myostatin concentration as compared with the control group at t1. Otherwise, there were no other differences among the 4 treatment groups at each time point. There were also no differences in myostatin concentration within each group between t0 and t2 or t1 and t3. However, all groups demonstrated a significant decrease in myostatin concentration between t0 and t1 (P < .001) and between t2 and t3 (P < .001).

With regard to the VEGF ELISA evaluation, there was significant variation in starting VEGF level among the treatment groups (P < .001). Many differences were identified between time points and treatment groups without any prominent pattern.

Discussion

The results of the current animal study do not support our hypothesis that ischemic therapy increases muscle mass and function, as we did not find gains in muscle mass, contractility, or fiber cross-sectional area locally or systemically, nor did we find increased levels of muscle satellite cell density in this model. Using a novel rat model of ischemic therapy, we performed the first comparison between 2 types of ischemic therapy: RRMBF and BFRT. This model was based on several studies that attempted to optimize protocols for ischemic therapy to induce muscle hypertrophy and gains in muscle strength.^{25,26,32,33,43} Reported successes of ischemic therapy in humans are limited to case series, which often evaluated multiple variables (muscle cross-sectional area, strength, power, total work),^{2,5,6,12,16,19} while others did not find BFRT to improve muscle mass and function.^4,9,14 While 2 previous rat model evaluations found BFRT and RRMBF to increase muscle mass, induce muscle hypertrophy, and increase fiber cross-sectional area as compared with control,^25,32 the results of this study do not support ischemic therapy as a means for improving muscle mass and function.

We specifically chose our stimulation group to have 50% maximum muscle contraction, as current American College of Sports Medicine guidelines suggest that 60% to 70% of maximum contraction is necessary to increase muscle strength, while studies in humans have demonstrated that as little as 20% of maximum contraction is needed with BFRT.²⁹ As expected, there was no difference between the stimulation (50% maximum contraction) and control groups’ muscle contractility and mass. Additionally, this level of muscle contraction allowed for evaluation of BFRT and isolation of its results from the effects of muscle stimulation alone. Overall, there was no difference among the BFRT, RRMBF, stimulation group, and control group with regard to muscle contractility and mass.

The reported number of treatments and duration of treatment with ischemic therapy vary widely within human and rat studies. The chosen frequency and duration of treatment in the present study were based on previous studies that demonstrated muscle hypertrophy with ischemic therapy.^32,33,43 We did not observe significant difference in muscle mass, strength, or cross-sectional area in the ischemic therapy groups as compared with the control group and stimulation group. Additionally, we did not observe significant satellite cell density differences in the ischemic therapy groups as compared with the control group and stimulation group, which would have been expected if the subjects were beginning to see the muscular effects of the treatment.

The mechanism by which ischemic therapy improves muscle quality is not understood.^{5,19,28,29,40} The following factors have been postulated to be involved: metabolic analyte accumulation (lactic acid), anabolic hormone release (GH, IGF-1, testosterone), reactive hyperemia, reactive oxidative species, heat shock proteins, muscular satellite cell activation, mRNA regulation (mTOR), protein synthesis regulators (myostatin), mechanoreceptors, muscle damage, nitric oxide, transcription factors regulation (FOXO), and various others.^{5,7,28,29,40,44} The immunohistochemistry evaluation in this study identified that CD68+ and CD163+ macrophages were present in the BFRT treatment hindlimb, which was unique to this treatment group. CD163 is a macrophage-specific protein that is upregulated in the context of acute and chronic inflammation.⁸ CD68 is a monocyte lineage glycoprotein that is involved in cellular immune functions, including traumatic injury or modified muscle use, and its role continues to be fully characterized.²⁷ This finding has not been reported and may represent a direction for establishing the mechanism of ischemic therapy.

ELISA evaluation in this study was performed to identify biochemical changes associated with ischemic therapy. Although the IGF-1 and GH pathways have traditionally been seen as a possible mechanism by which ischemic therapy enacts its effects, this is controversial, as other studies have shown that exercise-induced fluctuations are likely not sufficient to stimulate muscle hypertrophy.^5,20,29,42 Human studies using BFRT protocols have noted no change in IGF-1 as compared with controls.^2,5,14 It would be expected that after high-intensity exercise, there would be increased concentration of IGF-1, leading to muscle anabolism. In our study, the RRMBF group demonstrated a chronic decrease in IGF-1 levels after 1 month of treatment and acute decreases in IGF-1 levels after the initial and final treatment sessions. However, the control group also showed the trend of acute decreases in IGF-1 levels after the initial and final treatment sessions, which may indicate a fasting-related anesthesia effect. The BFRT group also demonstrated a chronic decrease in IGF-1 after 1 month of treatment. There was no difference in IGF-1 concentrations between groups at the final time points, which aligns with that found in human studies.

Human studies using BFRT protocols have noted no change in GH as compared with controls.^2,5,14 The RRMBF group demonstrated an increase in GH levels after 1 month of treatment; however, it did not show increases after a single training session. The RRMBF group had significantly larger GH levels than the other treatment groups at the end of 1 month of treatment. The stimulation group demonstrated a significant decrease in GH level between the start and end of the initial training session but no difference over the 1 month of treatment sessions. Similarly, the BFRT group demonstrated a significant decrease in GH level between the start and end of the final training session but no difference over the 1 month of treatment sessions. Overall, the RRMBF group demonstrated the greatest increase in GH concentration. The 2 groups that simulated exercise (stimulation and BFRT) demonstrated an acute drop in GH concentration after each treatment session. We postulate that the treatment-related differences represent normal fluctuations in GH concentration after low-intensity exercise. However, the BFRT, stimulation, and control groups were not different with regard to GH concentration, which aligns with human studies.

Human studies have noted decreased expression of myostatin after BFRT.¹⁶ Apart from 1 time point comparison in this study (RRMBF and BFRT vs control group), there were no significant differences among groups with respect to myostatin. However, there was a significant acute decrease in myostatin concentration within all groups after each treatment event. As this occurred in the control group and stimulation group as well as in the treatment groups, the significance of this observed difference is likely not clinically significant. Additionally, the evaluation of VEGF in this study was highly variable and may represent high natural variation within rats. Previous studies demonstrated no difference in VEGF concentrations with regard to RRMBF in a rat model.²⁶

Animal models of muscle hypertrophy have been found relevant to human application.¹⁸ Only a few ischemic therapy animal models have been developed, and they have also aimed to identify the mechanism of improving muscle mass and function. They have reported biochemical changes, but no clear mechanism has been identified. In a BFRT model, Nakajima et al²⁵ found that phosphorylation of ribosomal protein S6 was enhanced and that levels of peroxisome proliferator-activated receptor gamma coactivator 1α and glucose transporter 4 protein increased. They also found that BFRT increased gene expression of monocarboxylate transporter 1 more than exercise alone. Sudo et al³³ evaluated a BFRT model and found that S6K1 phosphorylation was significantly increased after eccentric exercise with and without blood flow restriction. Nakajima et al²⁶ evaluated an RRMBF rat model and found that RRMBF leads to decreased microvascular oxygen pressure and enhanced mTOR signaling. They found no effects on AMPK and MAP kinase phosphorylation, REDD1, myostatin, MuRF-1, FOXO3a, VEGF, and HIF-1α. Xu et al⁴³ performed a gene microarray analysis on a BFRT rat model and found that BFRT may promote muscle hypertrophy by increasing protein synthesis via the MAPK pathway and prevent muscle atrophy by repressing protein degradation via the ubiquitin proteasome and myostatin-Smad2/3 pathways.

The results of this animal model study do not support ischemic therapy as a means for improving muscle mass and function; however, this study is limited by our experimental conditions. We used a single strain of rats from a single laboratory, and it is possible that another strain could have a different response to hypertrophic stimuli. We also used female rats, and it is possible that male rats could give a larger hypertrophic response in future studies. Other variables could be optimized in future animal models to delineate whether any clinically meaningful effects exist and whether clinical application is practical, such as occlusion duration, occlusion pressure, stimulation duration, number of treatment repetitions, contraction strength, treatment duration, and number of treatment sessions. Other methods to provide an animal exercise, such as different muscle contraction types and treadmill walking, could be attempted in the future.

The validation of BFRT and RRMBF has lagged behind clinical application and commercial availability of these products. We have identified biochemical and immunohistochemical trends that may be involved with ischemic therapy, and this may guide further work. Additional research is required to validate this technology for human usage, including randomized controlled trials with larger samples sizes, before it is recommended to patients as an effective therapy option. Overall, this study represents a significant effort toward validating this technology by using multiple analysis techniques and an animal model; however, we have not found ischemic therapy to increase muscle mass, contraction force, myocyte cross-sectional area, and satellite cell density.

Footnotes

Acknowledgements

The authors thank Bonnie Nolan for her assistance with the animals used in this study and Dennis Claflin, PhD, for his assistance as an electronics consultant.

Submitted March 9, 2020; accepted June 3, 2020.

One or more of the authors has declared the following potential conflict of interest or source of funding: This study was funded by an institutional intramural faculty development grant. A.B. has received consulting fees from Arthrex, Smith & Nephew, and Flexion Therapeutics; royalties from Arthrex; and speaker fees from Arthrex and Smith & Nephew. AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto.

‖

References 1, 2, 6, 9 -13, 19, 21, 24, 31, 34, 36 -41, .

References

Abe

Kearns

Sato

Muscle size and strength are increased following walk training with restricted venous blood flow from the leg muscle, KAATSU-walk training. J Appl Physiol (1985). 2006;100(5):1460-1466.

Behringer M, Behlau D, Montag JCK, McCourt ML, Mester J. Low-intensity sprint training with blood flow restriction improves 100-m dash. J Strength Cond Res. 2017;31(9):2462-2472.

Burkholder

Fingado

Baron

Lieber

RL.

Relationship between muscle fiber types and sizes and muscle architectural properties in the mouse hindlimb. J Morphol. 1994;221(2):177-190.

Colomer-Poveda

Romero-Arenas

Vera-Ibanez

Vinuela-Garcia

Marquez

Effects of 4 weeks of low-load unilateral resistance training, with and without blood flow restriction, on strength, thickness, V wave, and H reflex of the soleus muscle in men. Eur J Appl Physiol. 2017;117(7):1339-1347.

Cook

Brown

Deruisseau

Kanaley

Ploutz-Snyder

LL.

Skeletal muscle adaptations following blood flow-restricted training during 30 days of muscular unloading. J Appl Physiol (1985). 2010;109(2):341-349.

Cook

LaRoche

Villa

Barile

Manini

TM.

Blood flow restricted resistance training in older adults at risk of mobility limitations. Exp Gerontol. 2017;99:138-145.

Douris

Cogen

Fields

, et al. The effects of blood flow restriction training on functional improvements in an active single subject with Parkinson disease. Int J Sports Phys Ther. 2018;13(2):247-254.

Etzerodt

Moestrup

SK.

CD163 and inflammation: biological, diagnostic, and therapeutic aspects. Antioxid Redox Signal. 2013;18(17):2352-2363.

Fahs

Loenneke

Thiebaud

, et al. Muscular adaptations to fatiguing exercise with and without blood flow restriction. Clin Physiol Funct Imaging. 2015;35(3):167-176.

10.

Fujita

Abe

Drummond

, et al. Blood flow restriction during low-intensity resistance exercise increases S6K1 phosphorylation and muscle protein synthesis. J Appl Physiol (1985). 2007;103(3):903-910.

11.

Hughes

Rosenblatt

Haddad

, et al. Comparing the effectiveness of blood flow restriction and traditional heavy load resistance training in the post-surgery rehabilitation of anterior cruciate ligament reconstruction patients: a UK National Health Service randomised controlled trial. Sports Med. 2019;49(11):1787-1805.

12.

Hylden

Burns

Stinner

Owens

Blood flow restriction rehabilitation for extremity weakness: a case series. J Spec Oper Med. 2015;15(1):50-56.

13.

Kaijser

Sundberg

Eiken

, et al. Muscle oxidative capacity and work performance after training under local leg ischemia. J Appl Physiol (1985). 1990;69(2):785-787.

14.

Karabulut

Sherk

Bemben

MG.

Inflammation marker, damage marker and anabolic hormone responses to resistance training with vascular restriction in older males. Clin Physiol Funct Imaging. 2013;33(5):393-399.

15.

Larkin

Davis

Sims-Robinson

, et al. Skeletal muscle weakness due to deficiency of CuZn-superoxide dismutase is associated with loss of functional innervation. Am J Physiol Regul Integr Comp Physiol. 2011;301(5):R1400-R1407.

16.

Laurentino

Ugrinowitsch

Roschel

, et al. Strength training with blood flow restriction diminishes myostatin gene expression. Med Sci Sports Exerc. 2012;44(3):406-412.

17.

Loenneke

Fahs

Thiebaud

, et al. The acute muscle swelling effects of blood flow restriction. Acta Physiol Hung. 2012;99(4):400-410.

18.

Lowe

Alway

SE.

Animal models for inducing muscle hypertrophy: are they relevant for clinical applications in humans?

J Orthop Sports Phys Ther. 2002;32(2):36-43.

19.

Luebbers

Fry

Kriley

Butler

MS.

The effects of a 7-week practical blood flow restriction program on well-trained collegiate athletes. J Strength Cond Res. 2014;28(8):2270-2280.

20.

Manini

Yarrow

Buford

, et al. Growth hormone responses to acute resistance exercise with vascular restriction in young and old men. Growth Horm IGF Res. 2012;22(5):167-172.

21.

Mattar

Gualano

Perandini

, et al. Safety and possible effects of low-intensity resistance training associated with partial blood flow restriction in polymyositis and dermatomyositis. Arthritis Res Ther. 2014;16(5):473.

22.

Mattocks

Jessee

Mouser

, et al. The application of blood flow restriction: lessons from the laboratory. Curr Sports Med Rep. 2018;17(4):129-134.

23.

Mayeuf-Louchart

Hardy

Thorel

, et al. MuscleJ: a high-content analysis method to study skeletal muscle with a new Fiji tool. Skeletal Muscle. 2018;8(1):25.

24.

Moore

Burgomaster

Schofield

, et al. Neuromuscular adaptations in human muscle following low intensity resistance training with vascular occlusion. Eur J Appl Physiol. 2004;92(4-5):399-406.

25.

Nakajima

Koide

Yasuda

, et al. Muscle hypertrophy following blood flow-restricted, low-force isometric electrical stimulation in rat tibialis anterior: role for muscle hypoxia. J Appl Physiol (1985). 2018;125(1):134-145.

26.

Nakajima

Yasuda

Koide

, et al. Repetitive restriction of muscle blood flow enhances mTOR signaling pathways in a rat model. Heart Vessels. 2016;31(10):1685-1695.

27.

Okada

Arai

Itoh

, et al. CD68 on rat macrophages binds tightly to S100A8 and S100A9 and helps to regulate the cells’ immune functions. J Leukoc Biol. 2016;100(5):1093-1104.

28.

Pearson

Hussain

SR.

A review on the mechanisms of blood-flow restriction resistance training–induced muscle hypertrophy. Sports Med (Auckland, NZ). 2015;45(2):187-200.

29.

Pope

Willardson

Schoenfeld

BJ.

Exercise and blood flow restriction. J Strength Cond Res. 2013;27(10):2914-2926.

30.

Ramme

Rourke

Larson

Bedi

Ischemic therapy in musculoskeletal medicine. Am J Sports Med. 2020;48(12):3112-3120.

31.

Sousa

Neto

Santos

, et al. Effects of strength training with blood flow restriction on torque, muscle activation and local muscular endurance in healthy subjects. Biol Sport. 2017;34(1):83-90.

32.

Sudo

Ando

Kano

Repeated blood flow restriction induces muscle fiber hypertrophy. Muscle Nerve. 2017;55(2):274-276.

33.

Sudo

Ando

Poole

Kano

Blood flow restriction prevents muscle damage but not protein synthesis signaling following eccentric contractions. Physiol Rep. 2015;3(7):e12449.

34.

Sumide

Sakuraba

Sawaki

Ohmura

Tamura

Effect of resistance exercise training combined with relatively low vascular occlusion. J Sci Med Sport. 2009;12(1):107-112.

35.

Tabata

Suzuki

Azuma

Matsumoto

Rhabdomyolysis after performing blood flow restriction training: a case report. J Strength Cond Res. 2016;30(7):2064-2068.

36.

Takada

Okita

Suga

, et al. Low-intensity exercise can increase muscle mass and strength proportionally to enhanced metabolic stress under ischemic conditions. J Appl Physiol (1985). 2012;113(2):199-205.

37.

Takarada

Sato

Ishii

Effects of resistance exercise combined with vascular occlusion on muscle function in athletes. Eur J Appl Physiol. 2002;86(4):308-314.

38.

Takarada

Takazawa

Sato

, et al. Effects of resistance exercise combined with moderate vascular occlusion on muscular function in humans. J Appl Physiol (1985). 2000;88(6):2097-2106.

39.

Takarada

Tsuruta

Ishii

Cooperative effects of exercise and occlusive stimuli on muscular function in low-intensity resistance exercise with moderate vascular occlusion. Jpn J Physiol. 2004;54(6):585-592.

40.

Tennent

Burns

Johnson

Owens

Hylden

CM.

Blood flow restriction training for postoperative lower-extremity weakness: a report of three cases. Curr Sports Med Rep. 2018;17(4):119-122.

41.

Tennent

Hylden

Johnson

, et al. Blood flow restriction training after knee arthroscopy: a randomized controlled pilot study. Clin J Sport Med. 2017;27(3):245-252.

42.

West

Phillips

SM.

Anabolic processes in human skeletal muscle: restoring the identities of growth hormone and testosterone. Phys Sportsmed. 2010;38(3):97-104.

43.

Liu

Chen

, et al. Transcriptional profiling of rat skeletal muscle hypertrophy under restriction of blood flow. Gene. 2016;594(2):229-237.

44.

Yow

Tennent

Dowd

Loenneke

Owens

JG.

Blood flow restriction training after Achilles tendon rupture. J Foot Ankle Surg. 2018;57(3):635-638.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB