Central versus peripheral mechanisms of cold-induced vasodilation: a study in the fingers and toes of people with paraplegia

Ethical approval and participants

The experimental protocol (ClinicalTrials.gov ID: NCT04215939) conformed to the standards set by the Declaration of Helsinki and was approved by the Bioethics Review Board of the University of Thessaly Department of Physical Education and Sport Science (protocol no.: 1320). Participants were requested to provide a medical history to exclude patients with Raynaud’s syndrome/phenomenon as well as those under prescription medication for hypertension or other drugs that could affect vasomotion. Three out of 14 participants (one able-bodied and two paraplegic participants) were smokers and were asked to refrain from smoking at least 10 h prior to each experimental session. Physiological data as well as the number of CIVDs from the six males and one female able-bodied participants have been previously published to investigate the cardiovascular stress and the characteristics of CIVD in women and men during cold water immersion (Tsoutsoubi et al. 2022). All volunteers were given a full explanation of all the procedures and signed a written informed consent prior to participating in the study.

The minimum required sample size for investigating “repeated-measures, within–between factors” was calculated using the results of a previous repeated-measures study (Daanen and Ducharme 1999) which assessed CIVDs in able-bodied individuals who were in thermoneutral, mildly hypothermic, and mildly hyperthermic conditions. Specifically, we calculated the effect size (f) (Cohen 1988) for the comparisons reported in the previously published study regarding minimum and maximum Tf among fingers as well as among participants (Daanen and Ducharme 1999). To ensure high statistical power, our calculation of the minimum required sample size was based on the lowest effect size identified. This was an effect size (f) equal to 0.5 [equating to an effect size (d) of 1.0] for the comparison of maximum Tf in the mild hypothermic (8.3 ± 1.7 °C) versus the thermoneutral (11 ± 3.2 °C) condition (Daanen and Ducharme 1999). Sample size calculations were conducted using G*Power 3.1.9.4 (Faul et al. 2007), while setting statistical power and α error probabilities at 0.90 and 0.05, respectively. Based on this process, six participants per group (12 participant in total) would provide sufficient power to detect differences between able-bodied and paraplegic individuals in our study. Therefore, six males and one female with paraplegia as well as an equivalent number of able-bodied males and female matched for age, body mass index, and body surface area (i.e., p > 0.05 between groups) participated in the study. The anthropometric characteristics of all 14 participants are shown in Table 1. The average time since injury of those with paraplegia was 13 ± 15 years (range 2–40 years), and all had complete motor paralysis of the legs with a diagnosis of ASIA A, following a clinical neurological exam which was registered in the public healthcare system. Of the paraplegic participants, one had suffered an injury in the T4, one in the T9, two in the T11, two in the T12, and one in the L1 (Fig. 1). Therefore, no neurological changes would be expected in the fingers of the paraplegic participants as the injury sites were below innervation to the hands/fingers.

Table 1 Anthropometric characteristics of the participants in the two groupsFig. 1figure 1

Level of injury for participants with paraplegia

Experimental protocol

The study included a familiarization session and three experimental sessions. During the familiarization session, participants were informed about all data collection procedures/equipment and underwent anthropometric and body composition (Dual-energy X-ray absorptiometry; (Lunar model DPX Madison, WI).) assessments. Thereafter, participants were requested to undergo three trials, in each being exposed to different environmental conditions inside a 32.5 m3 environmental chamber (HGX22G/190-4, ABB, Germany): cool environment (wet-bulb globe temperature: 10.8 °C; air temperature: 16 ± 1 °C; relative humidity: 45 ± 5%; air velocity: 0.2 m/s; solar radiation: 0 W/m2), neutral (wet-bulb globe temperature: 17.2 °C; air temperature: 23 ± 1 °C; relative humidity: 45 ± 5%; air velocity: 0.2 m/s; solar radiation: 0 W/m2), and hot (wet-bulb globe temperature: 27.2 °C; air temperature: 34 ± 1 °C; 45 ± 5% relative humidity; air velocity: 0.2 m/s; solar radiation: 0 W/m2) environments. The overall thermal stress experienced by the participants in our study was expressed by means of wet-bulb globe temperature which was found to be the most efficacious thermal stress indicator for assessing the physiological strain (Ioannou et al. 2022a, b, c). The experimental sessions were administered in a random order, based on a random allocation algorithm implemented in Excel Spreadsheets (Microsoft Office, Microsoft, Washington, USA).

For each session, participants arrived at the same time of the day. They were requested to refrain from caffeine for at least two hours, from food for at least three hours, and from alcohol and exercise for at least 12 h prior to experiment. Upon arrival, participants dressed down to a long-sleeve shirt and a pair of pants 100% cotton (Fig. 2). The room temperature in the lab where the participants changed clothes before entering the environmental chamber was thermoneutral (wet-bulb globe temperature: 16.3 °C; air temperature: 22 °C; relative humidity: 45%; air velocity: 0.2 m/s; solar radiation: 0 W/m2). The time spent in this environment (for dressing down and to ask any further questions) was about 10–15 min. The estimated clothing insulation was 0.67 clo for males (underwear 0.04; long-sleeve shirt 0.29; pants: 0.34) and 0.69 clo for females (underwear 0.04; bra: 0.02; long-sleeve shirt 0.29; pants: 0.34). (American Society of Heating 2004; Ioannou et al. 2019). Thereafter, participants entered the environmental chamber and instrumentation took place for 10–15 min. Once all sensors were applied, participants were requested to relax seated for 20 min with their hands supported at the level of the heart (Fig. 2). After the baseline period, participants were asked to simultaneously immerse their left hand (up to the ulnar styloid process) and left foot (up to the lateral malleolus) in warm water (35 ± 1 °C) for five minutes to ensure similar starting local tissue temperature (Daanen et al. 2012). Thereafter, the left hand and foot were immersed in cold water (8 ± 1 °C) for 40 min (Fig. 2). After the end of the cold water immersion period, the left hand and foot were removed from the water and participants remained seated for an additional five minutes to monitor the recovery phase. The total duration of the experiment was 70 min: 20-min baseline period, 5-min warm water immersion, 40-min cold water immersion, and 5-min recovery.

Fig. 2figure 2

An able-bodied participant during the data collection. The right sleeve of the shirt was cut at elbow height to avoid constricting blood flow. The blood pressure cuff was comfortably placed at the upper arm without constricting blood flow except when assessing arterial blood pressure

The water tanks used for the warm immersion were 0.58 m3 for the hand and 0.85 m3 for the foot. The water tanks used for the cold immersion were 0.91 m3. Water temperature was controlled within 1 °C using an air-to-water heat pump (model KF120-B, Dongguan, China) which was adjusted to continuously circulate the water to ensure uniform exposure. During the water immersion periods, the left foot was placed in a water tank on the floor, and the right foot rested on the floor (Fig. 2). Water-permeable mesh fabrics were used to line the bottom of the water tanks and the floor to ensure that the immersed hand and foot made no contact with the bottom of the water tanks, and that the non-immersed hand and foot made no contact with the floor of the environmental chamber (Fig. 2).

Measurements

Gastro-intestinal (Tgi) and mean skin (Tsk) temperature (both assessed in 1-min intervals), as well as finger temperature (Tf), toe temperature (Tt), skin blood flow (SkBF), sweat rate, and heart rate were continuously monitored. Raw data from these variables—assessed in 0.05 s (for SkBF), 1 s (for heart rate), 8 s (for Tf and Tt), and 10 s (for sweat rate) intervals—were used to provide 1-min averages which were used for all statistical analyses. In addition, 20-s averages were calculated for Tf, Tt, and SkBF and were used to identify CIVD reactions (Fig. 3). In terms of the remaining measurements, arterial blood pressure was assessed on the arm of the non-immersed hand every 10 min during baseline, at the end of the warm immersion, every 10 min during cold water immersion, and at the end of the recovery period (Fig. 3). Thermal comfort and thermal sensation were recorded every 10 min during baseline and every 5 min during cold water immersion. Pain sensation was assessed at the start of cold immersion and then every five minutes (Fig. 3). Tactile sensitivity of the immersed limbs was tested at the end of the baseline period, at the end of the warm water immersion, as well as at minutes 5 and 40 of the cold water immersion (for the latter two, the hand and foot were briefly removed from the water) (Fig. 3).

Fig. 3figure 3

Physiological and perceptual measurements during the experimental protocol

Gastro-intestinal temperature (Tgi) was monitored using telemetric capsules (BodyCap, Caen, France) as an indicator of gastrointestinal (core) temperature. Although the time following ingestion does not significantly influence the validity of telemetry capsule measurements of core temperature in the absence of fluid consumption (Notley et al. 2021), the capsules were always ingested at the same time before the start of the protocol for each participant. Skin temperature from four sites (arm, chest, leg and thigh) was recorded using wireless thermistors (iButtons type DS1921H, Maxim/Dallas Semiconductor Corp., USA) and was used to calculate Tsk according to Ramanathan [Tsk = 0.3(chest + arm) + 0.2(thigh + leg)] (Ramanathan 1964). Mean body temperature (Tb) was calculated by Burton in 1935: Tb = 0.64 × Tgi + 0.36 × Tsk (Burton 1935).

Using surgical tape (3 M Transpore Tape, 3 M Canada), six ceramic chip skin thermistors (MA-100, Thermometrics) were attached on the lower part of the pad on the 2nd finger (i.e., index finger) of both hands and on the lower part of the pad on the 1st, 3rd, and 5th toes of the immersed foot, as well as at on the 1st toe of the non-immersed foot. Data were recorded using a data logger (Smartreader 8 Plus, ACR, Vancouver, Canada).

Skin blood flow (SkBF) was monitored with a laser Doppler flowmeter (PF4000 LDPM, Perimed, Stockholm, Sweden, PF5010 LDPM, Perimed, Stockholm, Sweden) at the pad of the 2nd finger in each hand as well as at the distal edge of the 1st toe of each foot. The probe (PR 407 small straight probe, Perimed) on the non-immersed 2nd finger was held in place with a plastic mini holder (diameter: 5 mm; PH 07-5, Perimed), which was fixed to the skin using double-sided adhesive strips (PF 105-3, Perimed) without constricting the finger. All other probes (413 Integrating Probe, Perimed, Stockholm, Sweden) were held in place with a plastic holder (PH 13, Perimed, Stockholm, Sweden). The SkBF data were expressed as absolute values in perfusion units (PU).

Sweat rate was measured at the forehead and the belly of the gastrocnemius using the ventilated capsule method (SFM4100, Sensirion, Staefa, Switzerland). Heart rate was monitored using a wireless heart rate system (Polar Team2, Polar Electro Oy, Kempele, Finland). Arterial blood pressure was assessed on the arm of the non-immersed hand (Omron Healthcare, M6 comfort, Kyoto, Japan). Whole body thermal comfort (from 1 = comfortable to 5 = extremely uncomfortable) and thermal sensation (from −3 = cold to + 3 = hot) were recorded using standard scales (Gagge et al. 1967). Pain intensity (from 1 = no pain to 10 = worst pain imaginable), as well as pain distress [in Likert (from 1 = no pain to 10 = unbearable pain) and visual analog scale (no pain to unbearable pain)] were recorded using standardized scales (Daanen et al. 2012). Tactile sensitivity of the tip of the middle finger (for all participants) and of the tip of the second toe (for able-bodied individuals) of the immersed limbs was assessed using Semmes–Weinstein monofilaments and a digital esthesiometer, based on the previous methods (Daanen et al. 2012; Sapa et al. 2019). For both measurements, lower values indicate increased tactile sensitivity.

Statistical analysis

The 20-s average values for Tf and Tt were used to visually identify CIVDs based on previous criteria (Daanen 2003; Daanen et al. 2012) as follows:

1.

Number of waves (N): The number of each wave that fulfill the criterium of an increase of at least 1 °C.

2.

Minimum temperature (Tmin): the lowest temperature just before the start of CIVD in °C.

3.

Maximum temperature (Tmax): the highest temperature during the CIVD in °C.

4.

Onset time (Tonset): the time from immersion to Tmin in minutes.

5.

Peak time (Tpeak): the time to reach the maximum temperature (time at Tmax minus time at Tmin) in minutes.

6.

Average temperature (Tavg): the average temperature during the cold water immersion period without the first five minutes of the cold immersion in °C.

7.

Temperature amplitude (ΔT): the difference between Tmin and Tmax in °C.

A Shapiro–Wilks test was used to test the normality assumption in continuous variables, demonstrating that they were distributed normally. Chi-square tests were used to compare the frequency of CIVDs across the three different environments, the fingers/toes, as well as between the hand and the foot. Since the aim of the study was to identify differences between matched groups the mean data for each phase were used to perform paired sample t-tests as well as effect sizes (d) to detect differences between the participants with paraplegia and able-bodied controls across all the collected variables [Tf, Tt, SkBF, Tsk, Tgi, arm/chest/leg/thigh skin temperature, perceptual scales (thermal comfort, thermal sensation, pain), systolic blood pressure, diastolic blood pressure, heart rate]. The comparisons were performed separately for each phase of the protocol (baseline, warm immersion, cold immersion, recovery). The level of statistical significance in the t-tests was adjusted for multiple comparisons using the Bonferroni correction, resulting in a level of significance of 0.008. The magnitude of Cohen’s (d) effect sizes was determined as follows: d (0.01) = very small; d (0.2) = small; d (0.5) = medium; d (0.8) = large; d (1.2) = very large; and d (2.0) = huge (Sawilowsky 2009). Finally, in participants with paraplegia, we also computed correlation coefficients for the relationship between the vertebrae number (with C1 coded as “1” and L5 coded as “25”) and all the collected variables to investigate if the level of injury is linked with the severity of changes in the physiological and perceptual data. Effect sizes (d) were computed with Excel Spreadsheets (Microsoft Office, Microsoft, Washington, USA) and all other analyses were conducted with SPSS v27.0 (IBM, Armonk, NY, USA).

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