Perfusion of the skin’s microcirculation after cold‐water immersion (10°C) and partial‐body cryotherapy (−135°C)

Abstract Background Investigations of the perfusion of the skin's microcirculation with laser speckle contrast imaging (LSCI) after cold treatments are rare. Therefore, the aim of this study was to compare the effects between cold‐water immersion (CWI) conduction and partial‐body cryotherapy (PBC) convection on perfusion of the microcirculation and skin temperature on the thigh. Materials and Methods Twenty healthy males were randomly allocated to CWI (10°C for 10 minutes) or PBC (−60°C for 30 seconds, −135°C for 2 minutes). Perfusion and skin temperature measurements were conducted on the anterior thigh region up to 60 minutes post‐treatment. Results Cold‐water immersion decreased perfusion of the microcirculation significantly compared to baseline values between 10 minutes (P = 0.003) and 30 minutes (P = 0.01) post‐treatment. PBC increased perfusion of the microcirculation and decreased skin temperature only at the first measurement interval (0 minute, both P = 0.01) post‐treatment. Additionally, local skin temperature was significantly decreased compared to baseline values only after CWI up to 30 minutes (P = 0.04) post‐treatment. Conclusion Cold‐water immersion reduced local skin microcirculation and skin temperature while PBC only slightly increased the perfusion of the microcirculation immediately after the treatment. For cooling purposes, the conduction method seems superior compared to the convection method, assessed with a LSCI device.

Cold-water immersion (CWI) is a popular technique, and its effectiveness to relieve pain has been studied extensively in earlier published studies with controversial results. 8,9 Another way of extracting heat from the body is the use of partial-body cryotherapy (PBC). During this treatment, participants stand in upright position in a cryocabin and cold air is produced through vaporization of liquid nitrogen. The treatment temperature of the vaporized air can reach up to −195°C with exposure times of up to 3 minutes. 10 Assessing skin microcirculation has become an easily accessible and representative indicator to understand microvascular function. 11 The observation of microvascular function has been highlighted to be of clinical importance as it plays a crucial role in physiological processes of tissue oxygenation and nutritional exchange. 12 It has been demonstrated that adequate tissue oxygen delivery is dependent on microcirculatory functioning, and thus, maintained tissue perfusion is a key indicator of injury and disease. 13 The pathophysiology of the skins' circulatory behavior has been studied in many popular fields such as peripheral artery diseases, 14 hypertension, 15 type 2 diabetes, 16 and primary aging. 17 Laser Doppler flowmetry (LDF) and laser Doppler perfusion imaging (LDPI) are commonly used, non-invasive methods to measure skin microcirculation. 11,18 Another established technology for assessing skin microcirculation is Tissue Viability (TiVi). 19,20 Laser speckle contrast imaging (LSCI) is a relatively new promising noncontact technology for assessing microcirculation of the skin. 21 A speckle pattern is an interference pattern produced by light reflected or scattered from different parts of the illuminated surface. 22 It is well known that cold applications lead initially to a strong vasoconstrictive reaction of the skin which contributes to the cold-induced analgesic effect. 4,23 Although this response is primarily triggered from local control systems, the sympathetic vasoconstrictor system is also involved in these processes. 24 The release of norepinephrine and other co-transmitters seem to contribute to the cold-induced vasoconstriction. 25,26 The narrowing of blood vessels is an effective way of the body to minimize heat loss although the extraction of heat is the primary mechanism of cold treatments to facilitate a therapeutically or medical effect. [27][28][29] Cutaneous vasoconstriction has been demonstrated to reduce skin temperature, 30 skin blood flow, 31 and nerve conduction velocity 3 and thus leading to pain relief. 4,32 Although cold water and cold air treatments are popular and competing pain-relieving strategies, to our knowledge, no direct comparison between CWI and PBC on the perfusion of the microcirculation of the skin has been published, using a LSCI device. Therefore, the aim of this study was to describe and compare the behavior of the perfusion of the microcirculation of the skin and the local skin temperature after CWI and PBC during a 60-minute follow-up period.

| Participants and design
In this randomized trial, a total of n = 20 healthy Caucasian males (age: 26.7 ± 3.9 years, height: 177.4 ± 8.7 cm, weight: 76.1 ± 6.2 kg) voluntarily participated. All participants were non-smokers and were regularly involved in physical endurance training (running and cycling).
All participants were screened for eligibility and excluded in case of skin abnormalities (eg, scars on the measurement site and psoriatic skin), or in case, they were allergic to cold (including Raynaud's disease), had cardiovascular diseases, or took any medication. On the day of the measurements, the participants were randomly allocated (by drawing lots) into either the CWI (n = 10) or the PBC group (n = 10). The participant's characteristics can be observed in Table 1. Participants were informed about the study content and protocol which was approved by the local ethical committee of Zurich (PB_2016-01125) in accordance with the Declaration of Helsinki (ICH-GCP).

| Experimental design
On the day of the experiment, participants were acclimatized for the duration of 20 minutes to the laboratory conditions of 21 ± 1°C and relative humidity of 40 ± 5%. During this period, participants were in supine position, wearing only swimming trunks. A region of interest (ROI) of 21 cm 2 , measured from the anterior patellar base in proximal direction, was clearly marked during the acclimatization period, to obtain valid microcirculation results of the left anterior thigh.
Additionally, a single thermochron was taped (3M, Tegaderm) on the mid-section of the right anterior thigh to observe the effects on the local skin temperature. After the acclimatization period, the baseline measurements were conducted. After the baseline measurements, the participants were either immersed in cold water or entered the cryocabin. The follow-up measurements were performed after the treatments (0 minute) and in 10-minute intervals up to 60 minutes post-treatment.  instructed to breathe normally and not to talk during the measurements. To obtain reliable data, the distance between the measured skin area and the LSCI device was controlled with the aiming laser function. These two lasers converge into a single point at a distance of 25 cm between the surface and the LSCI device. To receive highresolution images (752 × 580 pixels), the temporal filter was set to 25 frames (1 s/frame) with an interval of 5 seconds.

| Measurement of the local skin temperature
Skin temperature of the thigh was measured with the iButton system (Maxim Integrated) in supine position. The thermochron (DSL1922L) was placed on the mid-section of the right frontal thigh (midway between the proximal patella and the inguinal crease) to obtain continuous information about the local skin temperature. The iButton data logger system can be used to obtain a valid measurement of human skin temperature. 33

| Cold treatments
After the acclimatization and baseline measurements were conducted, the participants received one of the two possible cold treatments in a randomized order.
During the CWI, participants were immersed up to the sternal level in cold water (10 ± 1°C). The water temperature was con-

| Statistical analysis
All data were checked for normality using the Shapiro-Wilk test, al- Post hoc pairwise comparisons with Bonferroni correction were used to assess within-group differences. The observed power is expressed as 1−ß, and the significance level was set at α = 0.05. All statistical analyses were performed in SPSS (SPSS Inc) version 24.0.

| D ISCUSS I ON
The aim of this study was to describe and compare the behavior of the perfusion of the microcirculation of the skin and the local skin temperature after CWI and PBC during a 60-minute followup period. The primary findings of this study are that CWI led to cold-induced vasoconstriction which was not observed after PBC inter-day reproducibility due to good temporal and spatial resolutions. [38][39][40] Consequently, LSCI technology can investigate wide skin areas and fast changes of skin microcirculation compared to LDF devices. 38,40 Similarly to the results from Mawhinney et al are those obtained from another research group by Costello et al. 41 These authors demonstrated that the red blood cell concentration was also de- Beside the different approaches to assess the cutaneous microcirculatory properties, the differences in cooling methods might have affected the results, as described elsewhere. 34,43 In the present study, local skin temperature of the thigh was measured with a conductive thermo-button system, which demon-

| CON CLUS ION
The results of the current study show that CWI decreases local perfusion of the microcirculation of the skin to a greater extent than a PBC treatment, measured with a contactless LSCI. This study suggests that the use of skin contact-free lasers provides similar results to traditional methods with skin contact, in the research field of cryotherapy.

ACK N OWLED G EM ENT
Thanks to the "Thim van der Laan" foundation for the financial support.
F I G U R E 2 Skin temperature of the right anterior thigh in function of time. Values are means ± SD. *P < 0.05 within-group decrease compared to baseline. **P < 0.05 between CWI and PBC O RCI D Erich Hohenauer https://orcid.org/0000-0002-2101-0211