Trial design
This is a randomized, parallel, placebo-controlled, double-blind, exploratory, 1:1 randomized clinical trial in one of two study groups, namely: remote ischemic conditioning or placebo.
The study protocol follows the SPIRIT 2013 checklist (Standard Protocol Items: Recommendations for International Trials) (33) (Supplementary File 1) and the TIDieR (Template for Intervention Description and Replication) (34), so that the information and quality of reports of interventions are well described (34).
Eligibility Criteria
Women with knee osteoarthritis aged 45 years or older and younger than 60 years will be included in the study if they have unilateral or bilateral knee osteoarthritis (35–37) diagnosed according to American College of Rheumatology criteria (38) which are: criteria for clinical and radiographic diagnosis (having knee pain, osteophytes and having at least one of the three items - age greater than 50 years; stiffness lasting less than 30 minutes and/or crepitus) or criteria for clinical diagnosis (pain in the knee and having at least three of the six items - age greater than 50 years; stiffness lasting less than 30 minutes; crepitus; bone enlargement; bone tenderness; no palpable heat).
In addition, participants who present systemic rheumatic diseases (fibromyalgia, rheumatoid arthritis and systemic lupus erythematosus); congestive heart failure, peripheral vascular disease, systolic blood pressure greater than 160 or less than 100 mmHg or diastolic blood pressure greater than 100 mmHg, deep vein thrombosis, a history of acute myocardial infarction and stroke; diabetes mellitus; respiratory disease; a history of knee surgery; will not be included, as well as participants who have high levels of physical activity assessed by the Habitual Physical Activity Questionnaire (39, 40), are alcoholics, smokers, use drugs that influence cardiac autonomic modulation (beta-blockers) and have one or more predisposing risk factors for thromboembolism (41).
Participants who, in the 24 hours before the test, used anti-inflammatory drugs or analgesics, performed physical activity, consumed alcohol, performed therapeutic treatments for pain relief, had errors in capturing the heart rate beat by beat (RR intervals) and wished to withdraw from the study will be excluded. In addition, participants may choose to stop performing the intervention for any reason. The flowchart of the study design and the composition of the groups is illustrated in Fig. 1.
Recruitment
Participants will be recruited through advertising folders on the institution's premises, health centers and social networks. These procedures are recommended by Treweek et al. (42) as strategies to improve the recruitment of participants.
All participants will undergo an initial telephone screening to determine eligibility for inclusion in the study. The following data will be obtained as part of the screening: age, self-reported weight, brief health history including symptoms of knee osteoarthritis (knee joint pain and stiffness), patient comorbidities, and medications in use. Afterwards, the participants will answer the Habitual Physical Activity Questionnaire (40, 41) that assesses the level of physical activity. Those who claim to have knee pain and meet the study inclusion criteria will be invited to remain in the study.
Initial assessment data will also be used for sample characterization purposes. If eligible and willing to participate in the study, a time will be scheduled for the participant to attend the Sports Physiotherapy Laboratory. To increase the participants’ adherence to the intervention, telephone contact will be made daily to encourage them to remain in the study, and thus avoid losses.
All participants will go through an informed consent process that will include the delivery of written information about the need and the overall benefit of the study, followed by discussion with a researcher. This discussion will include verifying their understanding of the benefits and risks of participation and ensuring that participants accept that treatment will be randomly allocated, regardless of any personal preferences they may have. If the participant agrees to participate, a consent form is signed. It is worth noting that this study will not perform laboratory evaluation and storage of biological specimens for genetic or molecular analysis at this time or for future use.
Randomization And Blinding
Participants who provide written informed consent will be randomly allocated to either placebo remote ischemic conditioning or remote ischemic conditioning. The randomization sequence will be elaborated using software (Microsoft Office Excel 2007) and will be placed in sequential numbering in opaque and sealed envelopes. A researcher who will not have contact with the participants and evaluators will generate the allocation sequence (assign the participants to the interventions). Evaluators, participants and data analysts will be blinded to the allocation of groups. At the end of the survey, to disseminate the results to the participants, the allocation group may be revealed.
There is a Trial Steering Committee that meets weekly to provide daily support for the study. Any changes to the protocol will be notified to the ethics committee and an update will be made to the clinical trial registry.
Protective Measures Against Covid-19
For the safety of the participants and evaluators involved, protective measures will be taken against COVID-19: social distancing, evaluation of only one participant will be carried out at a time, and only the strictly necessary staff will remain on site. In addition, gel alcohol will be made available for participants and for the hygiene of equipment and the use of face masks will be mandatory for all present on site.
As a protective measure, participants will be asked over the phone about COVID-19 specific symptoms, such as tiredness, cough, sore throat, runny nose, fever, loss of smell or taste buds) on the day before they arrive at the collection site to ensure greater safety and if symptoms are present, they will be instructed to seek the nearest health center or hospital for immediate care (43).
Study Design
Data collection will be carried out at Sports Physiotherapy Laboratory at Faculdade de Ciências e Tecnologia/UNESP in Presidente Prudente, São Paulo, Brazil. All procedures will be performed under standardized conditions (temperature: 21–23°C; relative humidity: 40–60%).
Each participant will attend the laboratory on two days separated by one week. On the first day they will be evaluated for anthropometric characteristics, using a scale (Tanita BC 554, Iron Man/Inner, Arlington Heights Illinois, USA) and a stadiometer (Sany - American Medical do Brasil, São Paulo, Brazil) and, later, the body mass index (BMI) will be calculated. Blood pressure measurements will also be performed (44) and the Knee injury and Osteoarthritis Outcome Score (KOOS) questionnaire (45) will be applied. After these initial procedures, a 10-minute rest will be performed and, in the sequence, the evaluation of total occlusion pressure (TOP) will be performed. After that, the initial CPM test will be performed, followed by the RIC and finally the CPM test again. RR intervals will be captured from the initial 10 minutes of rest to the end of the complete CPM protocol.
On the second day, only the RR intervals will be captured before the RIC (15' at rest), during and after (15' at the end) the RIC. The study design is outlined in Fig. 2.
Interventions
In both study groups, the TOP will be determined: first, participants will be asked to remain at rest in the supine position for ten minutes in an acclimatized room (21–23°C) and silent (46, 47). To determine the TOP, a vascular Doppler (DF-7000 V; Medpej, Ribeirão Preto, São Paulo, Brazil) will be used, which will be positioned over the posterior tibial artery (located in the middle distance between the medial malleolus of the tibia and the Achilles tendon) to capture the auscultatory pulse (48).
A cuff (velcro; 12.5 cm wide and 84 cm long and inflatable chamber 7 cm wide and 52 cm long, Cardiomed, Curitiba, Paraná, Brazil) will be positioned in the region of the inguinal fold with the inflatable region of the cuff in the medial portion of the thigh covering the femoral artery (49) of the lower limb with the greatest complaint of knee pain (48, 49) and then it will be progressively inflated waiting 30 seconds every 50 mmHg and completely deflating and pausing for 10 seconds, and 40 mmHg will be added to the initial 50 mmHg (total of 90 mmHg).
The sequence will be repeated, always adding the 40 mmHg until the auscultatory pulse of the tibial artery is completely interrupted. Then wait 30 seconds and slowly deflate until the auscultatory pulse returns. The POT will be defined when the lowest pressure capable of completely obstructing blood flow is reached (48, 50, 51, 52). A wider cuff was chosen, as it has been proven that the width of the cuff has a great influence on the pressure needed to achieve full restriction of blood flow (52). The intervention and placebo groups will be different as described below.
Remote Ischemic Conditioning
The remote ischemic conditioning protocol (RIC) will be applied to the proximal region of the thigh of the limb with knee osteoarthritis and if both knees are affected, it will be applied to the knee with the greatest pain complaint (35). Participants will be relaxed and comfortably positioned in the supine position. The same cuff used to determine TOP will be used and the protocol will consist of four cycles of total ischemia (according to the TOP value determined individually) of five minutes, followed immediately by four cycles of five minutes of vascular reperfusion (0 mmHg), totaling 40 minutes as shown in Fig. 3.
Remote Ischemic Conditioning Placebo
The remote ischemic conditioning placebo protocol will also be performed on the lower limb with knee osteoarthritis and if both knees are affected, it will be applied on the knee with the greatest pain complaint. Participants will perform a protocol similar to that of RIC, but during the four cycles of five minutes of occlusion, the cuffs will be inflated only with 10 mmHg so as not to cause arterial or venous occlusion alternating with four cycles of five minutes of reperfusion (0 mmHg) (See Fig. 3) (53, 54).
Outcomes
Conditioned Pain Modulation
CPM is a dynamic measure capable of evaluating endogenous pain inhibition using a “pain-by-pain inhibition” model in which pain in a local area (test stimulus) is inhibited by a second concomitant pain (conditioning stimulus) (3, 55). In this study, the test stimulus will be performed through the pressure pain threshold using the algometer and the conditioning stimulus will be performed through the immersion of the hand in cold water, using the cold pressor test (CPT).
First, the markings of the places where the test stimuli will be performed on the participant will be carried out. The participant will be in dorsal decubitus with approximately 20° of hip and knee flexion with the lower limb comfortably supported and markings will be performed in eight locations on both knees: site 1: 2 cm distal to the lower medial border of the patella; site 2: 2 cm distal to the lower lateral border of the patella; site 3: 3 cm lateral to the midpoint on the lateral edge of the patella; site 4: 2 cm proximal to the upper lateral border of the patella; site 5: 2 cm proximal to the upper edge of the patella; site 6: 2 cm proximal to the upper medial border of the patella; site 7: 3 cm medial to the midpoint on the medial border of the patella; site 8: in the center of the patella (56, 57) and in the joint space between the first and second metacarpophalangeal joints (thenar eminence region of the hand) on the contralateral side (35).
These marked sites will receive the test stimulus through the pressure pain threshold test using a pressure algometer which is a reliable and validated instrument (FDX 50/220; Wagner instruments, Greenwich, Connecticut, USA) which has a tip of 1 cm2 (58).
The evaluator will apply pressure perpendicularly to the participant's skin until she reports the change from pressure sensation to painful sensation (35). The procedure will be performed three times at each site and the mean values in kgf/cm2 will be calculated to determine the pressure pain threshold. An interval of 20 seconds will be maintained between applications to avoid sensitization of pain responses (35).
After each measurement of the pressure pain threshold, pain will be asked using a visual analogue scale (VAS) that ranges from 0 (no pain) to 100 (worst imaginable pain). VAS will also be used to measure knee pain at rest and during CPT (5, 35, 57).
After baseline pressure pain threshold measurements, the CPT (conditioning stimulus) (5, 35, 59) will be applied. Participants will be asked to dip their hand in a container of cold water and ice for up to 1 minute. The temperature will be monitored by thermometer (model 5130, Incoterm) and maintained between 1ºC and 4ºC (35). Soon after, the pressure pain threshold measurements (test stimulus) will be performed in the same places.
MCD will be quantified by subtracting the second mean pressure pain threshold value (after CPT) from the first mean pressure pain threshold value (before CPT) [CPM = baseline pressure pain threshold – pressure pain threshold Final]. Thus, negative CPM values mean CPM inhibitory activity (higher pressure pain threshold after CPT), while positive values indicate CPM facilitating activity (lower pressure pain threshold after CPT) (3, 35, 59). The percentage of change will also be calculated using the following formula: [(pain threshold at final pressure – pain threshold at baseline pressure) / pain threshold at final pressure) x 100]. This entire protocol will be repeated after the RIC.
Cardiac Autonomic Modulation
The collection of data will be carried out individually and the participants will be instructed to remain silent, awake, at rest and in the supine position. The movement of people on site will be restricted, in order to reduce interference during collection. Participants will be instructed not to consume ANS stimulant substances such as alcoholic beverages, coffee, tea, soft drinks and chocolate drinks during the 24 hours prior to capturing the RR intervals.
The assessment of cardiac autonomic modulation will be performed through HRV by capturing the RR intervals using the heart rate monitor (Polar Electro Oy, Kempele, Finland – model V800) which is a validated equipment (60).
Indices obtained through linear methods, in the time domain (Mean RR, SDNN and rMSSD), frequency (LF and HF) and the Poincaré plot (SD1 and SD2) will be analyzed (61, 62). In addition, RR intervals will be analyzed to obtain the 30-second rMSSD that represents vagal reentry. All HRV indices will be obtained using the software Kubios HRV – version 3.2 (The Biomedical Signal Analysis Group, Department of Applied Physics, University of Kuopio, Finland) (63).
For this analysis, the time series of RR intervals will be initially submitted to a digital filtering moderated by the software Kubios HRV - version 3.2 (63) complemented by a manual filtering, to eliminate premature ectopic beats and artifacts, and only series with more than 95% of sinus beats will be included in the study (61). Through the visual analysis of the time series, the absence of artifacts or ectopic beats that may interfere with the HRV analysis will be observed.
The series of RR intervals will be analyzed in two different moments: i. during the performance of the CPM tests and, ii. during the evaluation of the RIC. In order to obtain these analyses, on the first day of intervention, the following moments will be analyzed: M1 (10' basal at rest before TOP), M2 (test stimuli through the algometer), M3 (conditioning stimulus, through the CPT), M4 (test stimuli through the algometer), M5 (during RIC), M6 (test stimuli through the algometer), M7 (conditioning stimulus, through the CPT), M8 (test stimuli through the algometer). In these stretches, 256 consecutive RR intervals (62) will be obtained. It is worth noting that at moments M3 and M7, only vagal reentry with a rMSSD of 30 seconds will be analyzed due to the short time to capture the RR intervals.
On the second day of intervention, the Pre-RIC (15' baseline before RIC), RIC (during RIC) and Post-RIC (15' after RIC) moments will be analyzed, with 1000 consecutive RR intervals being obtained for this analysis (62).
Time Domain
For analysis of HRV in the time domain, the Mean RR, SDNN and rMSSD (62–65) indices will be used. The Mean RR index corresponds to the means of all RR intervals (61, 62). The SDNN index represents the standard deviation of all normal RR intervals recorded in a time interval, expressed in milliseconds (ms) and is considered a measure of global variability, that is, it evaluates both sympathetic and parasympathetic modulation (61, 62). The rMSSD index corresponds to the root mean square of the differences between adjacent normal RR intervals in a time interval expressed in ms and assesses the behavior of parasympathetic autonomic modulation (61, 62).
Frequency Domain
In the frequency domain, the following will be used: the low frequency spectral component (LF - frequency between 0.04 and 0.15Hz) index that evaluates the sympathetic and parasympathetic behavior, with sympathetic predominance and the high frequency component (HF - frequency between 0.15 to 0.4Hz), which is related as a marker of vagal modulation, in normalized units (nu) and in milliseconds squared (ms2) (61, 62). The spectral indices will be calculated from a tachogram using the Fast Fourier Transform (FFT) algorithm (61, 62).
Poincaré plot
The Poincaré plot indices, SD1 and SD2, will be used for a quantitative analysis (62). The Poincaré plot represents a time series within a Cartesian plane which allows each RR interval to be correlated with the previous interval. It is a geometric method for analysis in which each point is represented on the x-axis (horizontal/abscissa) by the preceding normal RR interval, and on the y-axis (vertical/ordinate) by the following RR interval (61, 62). SD1 represents the dispersion of points perpendicular to the line of identity and appears to be an index of instantaneous recording of beat-to-beat variability, representing vagal modulation (61, 62). SD2, on the other hand, represents the dispersion of points along the identity line and represents the HRV in long-term records, considered a marker of sympathetic and parasympathetic modulation (61, 62, 66).
Vagal reentry
Series of RR intervals of the last 30 seconds of all moments (M1-M11) will be selected in order to compare the moments and assess vagal reentry, as observed in the studies by Imai et al. (67) and Goldberger et al. (68).
Sample Size
The sample size calculation was based on the study by Schliessbach et al. (68), using the CPM test variable. The software G*Power 3.1.9.4 and reference value of 98 ± 103 kPa were used, using a two-tailed hypothesis test with test power of 80% and a significance level of 5%. Eventually, 19 participants per group were stipulated. A possible sample loss of 15% was considered so three participants will be added to each group, totaling 44 individuals divided into two groups (22 participants per group).
Statistical analysis
Statistical analysis will be conducted using SPSS software (version 18; SPSS Inc. Chicago, IL, USA). Data normality will be verified by the Kolmogorov Smirnov test, if normality is detected, the sample characterization variables will be presented as mean and standard deviation/ if not, in median and interquartile range. To compare the characterization of the sample, Student's t test will be used for independent samples.
Descriptive data of outcomes will be presented in mean, standard deviation and confidence interval values. Comparisons of outcomes between the groups studied and the moments will be performed using Generalized Linear Mixed Models adjusted to the data with Bonferroni post-test when the main analyzes are significant. In addition, the effects of the groups studied will be verified for all outcomes evaluated by calculating the effect size (ES) through Cohen's d, which will be considered as “null” (< 0.2), “small” (≥ 0.2), “moderate” (≥ 0.6), “large” (≥ 1.2) or “very large” (≥ 2.0) (70). Intention to treat will be used and, for data imputation, a mixed linear model will be used.
To correlate CPM and cardiac autonomic modulation, the Pearson or Spearman correlation test will be used according to the normality of the data. According to the parameters of Portney and Watkins (71), the correlation magnitude of 0.00 to 0.19 is considered to be very weak; 0.20 to 0.39 weak correlation; 0.40 to 0.69 moderate correlation; 0.70 to 0.89 strong correlation and 0.90 to 1.00 very strong correlation (71). All analyzes will assume a significance level of p < 0.05.
The external evaluator will enter the data into the database for screening, randomization and statistical analysis. Dual data entry in electronic format will be used. Data integrity will be monitored regularly by examining data files for omissions and errors. Participants will receive an anonymous study ID to protect confidentiality, and only study investigators will have access to the final study dataset. The spreadsheets containing the raw numerical data of the data generated in this study will be stored in their entirety initially on two external hard drives and two online clouds. Any data needed to support the protocol can be provided upon request.
Adverse Event Reporting And Harms
Monitoring all of the described variables minimizes any risk of serious injury or cardiovascular complications during remote ischemic conditioning, i.e., if the participant experiences sensations such as dizziness, pallor, intense sweating, excessive blood pressure increase, pain, or any signs or symptoms, the procedure will be stopped immediately. Pressure from the cuff in the thigh region can generate discomfort and a feeling of tightness, but these symptoms disappear when the pressure is removed and as described above, all necessary care will be taken. All these variables will be noted in the data collection.