Effects of Riot Control Training on Systemic Microvascular Reactivity and Capillary Density

Effects of Riot Control Training on Systemic Microvascular Reactivity and Capillary Density Abstract Introduction The main aim of the present study is to evaluate the effects of strenuous exercise, related to special military training for riot control, on systemic microvascular endothelial function and skin capillary density. Materials and Methods Endothelium-dependent microvascular reactivity was evaluated in the forearm skin of healthy military trainees (age 23.4 ± 2.3 yr; n = 15) using laser speckle contrast imaging coupled with cutaneous acetylcholine (ACh) iontophoresis and post-occlusive reactive hyperemia (PORH). Functional capillary density was assessed using high-resolution, intra-vital color microscopy in the dorsum of the middle phalanx. Capillary recruitment (capillary reserve) was evaluated using PORH. Microcirculatory tests were performed before and after a 5-wk special military training for riot control. Results Microvascular endothelium-dependent vasodilatory responses were markedly and significantly reduced after training, compared with values obtained before training. The peak values of microvascular conductance obtained during iontophoresis of ACh or PORH before training (0.84 ± 0.22 and 0.94 ± 0.72 APU/mmHg, respectively) were markedly reduced after training (0.47 ± 0.11 and 0.71 ± 0.14 APU/mmHg; p < 0.0001 and p = 0.0037, respectively). Endothelium-dependent capillary recruitment was significantly reduced after training (before 101 ± 9 and after 95 ± 8 capillaries/mm2; p = 0.0007). Conclusions The present study showed that a 5-wk strenuous military training, performed in unfavorable climatic conditions, induces marked systemic microvascular dysfunction, mainly characterized by reduced endothelium-dependent microvascular vasodilation and blunted capillary recruitment. Introduction Currently, there is plenty of evidence that exercise is associated with several beneficial effects, including a reduction of mortality both in healthy subjects and in patients with cardiovascular disease.1–4 Regular exercise of moderate intensity induces an increase in vascular nitric oxide (NO) bioavailability, associated with a reduction of the production of reactive oxygen species (ROS), resulting in the improvement of vascular endothelial function.1 Nevertheless, high-intensity exercise may cause unfavorable disturbances in the NO/ROS balance and have the opposite effect.1 Endothelial function indeed appears to be impaired in individuals submitted to high-intensity, sustained aerobic training.5,6 For instance, elite Olympic athletes trained for aerobic sports show impaired flow-mediated dilation of the brachial artery, compared with non-trained, age- and sex-matched control subjects.7 Usually, military personnel are required to perform intensive physical training, including endurance and resistance exercise, to maintain high-level physical fitness and military skills for physically demanding tasks.8 Nevertheless, high-intensity physical effort during special military training – as for riot control – associated with unfavorable climatic conditions, such as high temperature and humidity, can also lead to adverse effects.9–11 For instance, the risk of exertional rhabdomyolysis and death in military personnel that engage consistently in regular and strenuous exercise of special military training is higher than in the civilian population.12 Riot control agents, which are frequently used in military settings, include o-chlorobenzylidene-malononitrile (CS), which is grouped with several other irritant agents referred to as “tear gas”13,14 and pepper (capsaicin) spray.15,16 These agents are generally supposed to be sub-lethal irritant incapacitants.17 The main toxic effects of these agents are related to respiratory, ocular, gastrointestinal, and cutaneous alterations.15 Nevertheless, cardiovascular effects, including tachycardia and transient hypertension, have been already described. These effects appear to be related to sensory autonomic reflexes, anxiety, pain, or psychological distress.18 Interestingly, it has already been reported that toxic symptoms after heavy exposure to CS in a field training setting can be disclosed by strenuous physical exercise.14 The physiological imbalances induced by all these factors involved in special military training may ultimately have an impact on the microcirculation, which is responsible for tissue perfusion. The non-invasive assessment of the cutaneous microcirculation using laser speckle contrast imaging (LSCI) is an innovative approach with proven usefulness for the investigation of the pathophysiology of cardiovascular and metabolic diseases.19 The cutaneous microcirculation is an accessible and representative vascular bed for the evaluation of systemic microcirculatory reactivity and capillary density, which are known to be closely correlated with cardiovascular and metabolic diseases.20,21 The present study aims to evaluate the effects of high, physically demanding military activities, related to special military training for riot control, associated with the exposure to irritant transient incapacitants, on systemic microvascular endothelial function and capillary density in young adults, using LSCI and skin video microscopy, respectively. Methods Study Design This observational study included military personnel from the Brazilian Air Force who completed a 5-wk special training period for riot control in Rio de Janeiro, Brazil. The study included 15 volunteers who consented to undergo evaluation of skin microvascular reactivity and capillary density and analyses of biochemical data. All were healthy according to routine medical examinations, physically active, and not in use of any medications or specific dietary regimens. The evaluation of microvascular reactivity and capillary density was performed before the 5-wk training and at the end of the training. Venous blood was collected before training and once a week during the 5-wk training period. All evaluations were performed in the morning, between 8 a.m. and 12 p.m., after a 12-hr fast. First, blood specimens were collected and then subjects rested for 20 min in a quiet environment with a constant temperature of 23 ± 1°C before the microvascular reactivity tests. The study was undertaken in accordance with the Helsinki Declaration of 1975, revised in 2000, and was approved by the Institutional Review Board (IRB) of the National Institute of Cardiology, Rio de Janeiro, Brazil, under protocol no. CAAE 49792515.6.0000.5272. All subjects read and signed an informed consent document approved by the IRB. Military Training Protocol The training protocol was designed by the Air Force authority. The present observational study did not include any interventional procedure. Military physical training, using military uniform, was conducted from Monday to Friday and consisted of two 20-min morning running sessions, with 20-min rest and free hydration in between, and performed with complete combat uniform and equipment, including the use of helmet and shield. At the end of running session each morning, the military training group was continuously exposed to toxic tear gas and pepper spray for 2 min. During 4 hr in the afternoon, the group was submitted to handling of warlike material and strategic movements to riot control, including successive running incursions, between 60 and 120 s, performed in the open field or climbing stairs, and exposure to the gases between 5 and 20 min without a mask. The running exercise was performed in warm (about 32°C) and humid (86% air relative humidity) conditions, typical of the summer season in Rio de Janeiro, Brazil. Evaluation of Skin Microvascular Flow and Reactivity Microvascular reactivity was evaluated using an LSCI system with a laser wavelength of 785 nm (PeriCam PSI System; Perimed, Järfälla, Sweden), which allowed non-invasive and continuous measurements of cutaneous microvascular perfusion changes, measured in arbitrary perfusion units (APU). Images were analyzed using PIMSoft software (Perimed, Järfälla, Sweden). Brachial systolic (SAP) and diastolic (DAP) blood pressures were measured twice immediately before the beginning of the recordings, using a mercury sphygmomanometer, and the mean values were recorded as the patients’ blood pressure. The mean arterial pressure (MAP) was calculated as DAP + 1/3 (SAP-DAP). One skin site on the ventral surface of the forearm was randomly chosen for the recordings. Hair, broken skin, areas of skin pigmentation, and visible veins were avoided, and two drug delivery electrodes were installed using adhesive discs (LI 611; Perimed, Järfälla, Sweden). The following two measurement areas were identified: a measurement area within the electrode (ACh) and another measurement area (baseline control) adjacent to the electrode. A vacuum cushion (AB Germa, Kristianstad, Sweden) was used to minimize recording artifacts generated by arm movements. ACh 2% w/v (Sigma Chemical CO, St. Louis, MO, USA) iontophoresis was performed using a micropharmacology system (PF 751 PeriIont USB Power Supply, Perimed, Sweden) using increasing anodal currents of 30, 60, 90, 120, 150, and 180 μA, which were administered for 10-s intervals spaced 1 min apart. The total charges for the above currents were 0.3, 0.6, 0.9, 1.2, 1.5, and 1.8 mC, respectively. The dispersive electrode was attached approximately 15 cm from the electrophoresis chamber. The pharmacological test results were expressed as peak values representing the maximal vasodilation observed after the highest ACh dose. Skin blood flow measurements in arbitrary perfusion units (APU) were divided by MAP to yield cutaneous vascular conductance (CVC) in APU/mmHg. Capillaroscopy by Intra-vital Microscopy Microcirculatory tests were performed in a room with a defined stable temperature (23 ± 1°C) after a 20-min rest in the supine position. The dorsum of the non-dominant middle phalanx was used for image acquisition, while keeping the patient sitting comfortably. Room temperature was monitored and adjusted if necessary using air conditioning, considering that outside temperature was usually >25°C. The arm was positioned at the level of the heart and immobilized using a vacuum cushion (AB Germa, Kristianstad, Sweden). Capillary density, that is, the number of perfused capillaries per square millimeter of skin area, was assessed by high-resolution, intra-vital color microscopy (Moritex, Cambridge, UK), as previously described and validated by our research team.20,22,23 A video microscopy system was used, with an epi-illuminated fiber optic microscope containing a 100-W mercury vapor lamp light source and an M200 objective with a final magnification of 200×. Images were acquired and saved for posterior offline analysis using a semi-automatic integrated system (Microvision Instruments, Evry, France). For post-occlusive reactive hyperemia (PORH), a blood pressure cuff was then applied around the patient’s arm and inflated to suprasystolic pressure (50 mmHg greater than systolic arterial pressure) to completely interrupt the blood flow for 3 min. This time of occlusion has already been shown to effectively recruit capillaries in an endothelium-dependent manner. After cuff release, images were again acquired and recorded over the subsequent 60–90 s, during which time the maximal hyperemic response was expected to occur. The mean number of spontaneously perfused skin capillaries at rest was considered to represent the functional capillary density, as previously described.22,24 On the other hand, the number of perfused capillaries during post-occlusive reactive hyperemia was considered to represent functional capillary recruitment, resulting from the release of endothelial mediators and consequent arteriolar vasodilation.24 The mean capillary density for each patient was calculated as the arithmetic mean of visible (i.e., spontaneously perfused) capillaries in three contiguous microscopic fields of 1 mm2 each. Capillary counting was performed by two investigators blinded to the patients’ characteristics, and the final values of capillary density represent the mean of the individual counts. Reproducibility was assessed by examining an identical area of skin; intra-observer repeatability of data analysis was assessed by reading the same images blindly on two separate occasions (n = 15, coefficient of variability 4.3%). Statistical Analysis The results are presented as mean ± SD. Variables without a Gaussian distribution by Shapiro–Wilk normality test are presented as medians (25th–75th percentile). The comparisons between parameters obtained before and after training were performed using two-tailed paired Student’s t-tests or the Mann–Whitney tests, when appropriate. The dose-dependency of the effects of acetylcholine on microvascular vasodilation was tested using repeated measures ANOVA followed by the Dunnett’s multiples comparisons test. p-Values <0.05 were considered statistically significant. The identification of potential outliers was performed using the ROUT method (robust regression and outlier removal), which is based on the false discovery rate (FDR), with a specified value of Q = 1%. The statistical package used for the statistical analyses was Prism version 7.0 (GraphPad Software Inc., La Jolla, CA, USA). Results The anthropometric and hemodynamic parameters of the subjects before and after exercise are depicted in Table I. One subject developed exertional rhabdomyolysis on the second day of training and was excluded from the study. Laboratory data of the volunteers that completed training are shown in Table II. During training, creatine kinase, urea, creatinine, and C-reactive protein levels increased, declining after the end of training. Table I. Anthropometric and Hemodynamic Data Before or After Training Parameter  Before  After  p-Values  Age (yr)  23.4 ± 2.3  –  –  Weight (kg)  82.0 ± 10.2  82.6 ± 11.3  0.7529  Height (cm)  178.7 ± 7.7  –  –  Body mass index (kg/m2)  25.6 ± 2.2  25.9 ± 3.6  0.6306  Systolic arterial pressure (mmHg)  127 ± 7  127 ± 9  0.9117  Diastolic arterial pressure (mmHg)  68 ± 5  66 ± 5  0.1253  Mean arterial pressure (mmHg)  88 ± 5  87 ± 5  0.2790  Heart rate (bpm)  56 ± 9  58 ± 7  0.5229  Parameter  Before  After  p-Values  Age (yr)  23.4 ± 2.3  –  –  Weight (kg)  82.0 ± 10.2  82.6 ± 11.3  0.7529  Height (cm)  178.7 ± 7.7  –  –  Body mass index (kg/m2)  25.6 ± 2.2  25.9 ± 3.6  0.6306  Systolic arterial pressure (mmHg)  127 ± 7  127 ± 9  0.9117  Diastolic arterial pressure (mmHg)  68 ± 5  66 ± 5  0.1253  Mean arterial pressure (mmHg)  88 ± 5  87 ± 5  0.2790  Heart rate (bpm)  56 ± 9  58 ± 7  0.5229  The results are presented as the mean ± SD. p-Values were estimated using two-tailed paired t-tests. Table I. Anthropometric and Hemodynamic Data Before or After Training Parameter  Before  After  p-Values  Age (yr)  23.4 ± 2.3  –  –  Weight (kg)  82.0 ± 10.2  82.6 ± 11.3  0.7529  Height (cm)  178.7 ± 7.7  –  –  Body mass index (kg/m2)  25.6 ± 2.2  25.9 ± 3.6  0.6306  Systolic arterial pressure (mmHg)  127 ± 7  127 ± 9  0.9117  Diastolic arterial pressure (mmHg)  68 ± 5  66 ± 5  0.1253  Mean arterial pressure (mmHg)  88 ± 5  87 ± 5  0.2790  Heart rate (bpm)  56 ± 9  58 ± 7  0.5229  Parameter  Before  After  p-Values  Age (yr)  23.4 ± 2.3  –  –  Weight (kg)  82.0 ± 10.2  82.6 ± 11.3  0.7529  Height (cm)  178.7 ± 7.7  –  –  Body mass index (kg/m2)  25.6 ± 2.2  25.9 ± 3.6  0.6306  Systolic arterial pressure (mmHg)  127 ± 7  127 ± 9  0.9117  Diastolic arterial pressure (mmHg)  68 ± 5  66 ± 5  0.1253  Mean arterial pressure (mmHg)  88 ± 5  87 ± 5  0.2790  Heart rate (bpm)  56 ± 9  58 ± 7  0.5229  The results are presented as the mean ± SD. p-Values were estimated using two-tailed paired t-tests. Table II. Laboratory Testing of the Subjects Before, During, or After Training Parameters  Before  W1  W2  W3  W4  After  Reference Ranges  p-Values  Red blood cell count (106/μL)  4.9 ± 0.3  ND  ND  ND  ND  4.9 ± 0.2  4.5–6.2  0.8772   Hemoglobin (g/dL)  14.9 ± 0.8  ND  ND  ND  ND  14.8 ± 0.7  13.5–18.0  0.4363   Hematocrit (%)  45 ± 2  ND  ND  ND  ND  44 ± 3  40–54  0.6978  White blood cell count (μL) × 103  7.3 ± 1.2  ND  ND  ND  ND  6.9 ± 1.4  5–10  0.3353   Platelet count (×103/μL)  228 ± 55  ND  ND  ND  ND  216 ± 47  150–450  0.6816   Urea (mg/dL)  29 ± 7  43 ± 9***  34 ± 5  31 ± 11  32 ± 10  28 ± 7  15–40  0.0002   Creatinine (mg/dL)  0.9 ± 0.2  1. ± 0.1***  1.0 ± 0.1*  0.9 ± 0.1  0.9 ± 0.1  0.9 ± 0.1  0.6–1.2  0.0022   Calcium (mmol/L)  2.41 (2.37–2.45)  9.40**** (8.85–10.20)  9.60**** (9.35–9.85)  9.15**** (8.97–9.95)  9.45**** (9.22–9.67)  2.53*** (2.43–9.20)  2.23– 2.55  <0.0001   Magnesium (mg/dL)  2.0 ± 0.10  1.94 ± 0.28  1.99 ± 0.14  2.03 ± 0.21  2.1 ± 0.11  2.0 ± 0.16  1.6–2.6  0.4119   Sodium (mmol/L)  138 ± 1  137 ± 9  139 ± 6  136 ± 9  137 ± 4  137 ± 2  137–145  0.8130   Potassium (mmol/L)  4.2 ± 0.3  4.3 ± 1.1  3.9 ± 0.3  4.1 ± 0.3  4.2 ± 0.3  4.5 ± 0.2  3.6–5.0  0.1077  Creatine kinase-CK (U/L)  210 (112–298)  856** (434–1,095)  233 (138–296)  135 (94–245)  107 (95–144)  102 (80–210)  30–170  <0.0001   hs-CRP (mg/dL)  0.08 (0.03–0.27)  7.5**** (3.0–14.2)  0.65* (0.18–2.0)  0.08 (0.04–0.25)  0.10 (0.03–0.17)  0.25 (0.08–0.90)  <0.50  <0.0001  Parameters  Before  W1  W2  W3  W4  After  Reference Ranges  p-Values  Red blood cell count (106/μL)  4.9 ± 0.3  ND  ND  ND  ND  4.9 ± 0.2  4.5–6.2  0.8772   Hemoglobin (g/dL)  14.9 ± 0.8  ND  ND  ND  ND  14.8 ± 0.7  13.5–18.0  0.4363   Hematocrit (%)  45 ± 2  ND  ND  ND  ND  44 ± 3  40–54  0.6978  White blood cell count (μL) × 103  7.3 ± 1.2  ND  ND  ND  ND  6.9 ± 1.4  5–10  0.3353   Platelet count (×103/μL)  228 ± 55  ND  ND  ND  ND  216 ± 47  150–450  0.6816   Urea (mg/dL)  29 ± 7  43 ± 9***  34 ± 5  31 ± 11  32 ± 10  28 ± 7  15–40  0.0002   Creatinine (mg/dL)  0.9 ± 0.2  1. ± 0.1***  1.0 ± 0.1*  0.9 ± 0.1  0.9 ± 0.1  0.9 ± 0.1  0.6–1.2  0.0022   Calcium (mmol/L)  2.41 (2.37–2.45)  9.40**** (8.85–10.20)  9.60**** (9.35–9.85)  9.15**** (8.97–9.95)  9.45**** (9.22–9.67)  2.53*** (2.43–9.20)  2.23– 2.55  <0.0001   Magnesium (mg/dL)  2.0 ± 0.10  1.94 ± 0.28  1.99 ± 0.14  2.03 ± 0.21  2.1 ± 0.11  2.0 ± 0.16  1.6–2.6  0.4119   Sodium (mmol/L)  138 ± 1  137 ± 9  139 ± 6  136 ± 9  137 ± 4  137 ± 2  137–145  0.8130   Potassium (mmol/L)  4.2 ± 0.3  4.3 ± 1.1  3.9 ± 0.3  4.1 ± 0.3  4.2 ± 0.3  4.5 ± 0.2  3.6–5.0  0.1077  Creatine kinase-CK (U/L)  210 (112–298)  856** (434–1,095)  233 (138–296)  135 (94–245)  107 (95–144)  102 (80–210)  30–170  <0.0001   hs-CRP (mg/dL)  0.08 (0.03–0.27)  7.5**** (3.0–14.2)  0.65* (0.18–2.0)  0.08 (0.04–0.25)  0.10 (0.03–0.17)  0.25 (0.08–0.90)  <0.50  <0.0001  The results are presented as the mean ± SD. Values that did not follow a Gaussian distribution are presented as medians (25th–75th percentile) (Shapiro–Wilk normality test). p-Values were estimated using one-way ANOVA followed by Dunnett’s multiple comparisons test or Kruskal–Wallis test, as appropriate. p-Values in bold characters denote statistically significant differences. W, week; hs-CRP, high-sensitivity C-reactive protein; ND, not determined. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 compared with values before training. Table II. Laboratory Testing of the Subjects Before, During, or After Training Parameters  Before  W1  W2  W3  W4  After  Reference Ranges  p-Values  Red blood cell count (106/μL)  4.9 ± 0.3  ND  ND  ND  ND  4.9 ± 0.2  4.5–6.2  0.8772   Hemoglobin (g/dL)  14.9 ± 0.8  ND  ND  ND  ND  14.8 ± 0.7  13.5–18.0  0.4363   Hematocrit (%)  45 ± 2  ND  ND  ND  ND  44 ± 3  40–54  0.6978  White blood cell count (μL) × 103  7.3 ± 1.2  ND  ND  ND  ND  6.9 ± 1.4  5–10  0.3353   Platelet count (×103/μL)  228 ± 55  ND  ND  ND  ND  216 ± 47  150–450  0.6816   Urea (mg/dL)  29 ± 7  43 ± 9***  34 ± 5  31 ± 11  32 ± 10  28 ± 7  15–40  0.0002   Creatinine (mg/dL)  0.9 ± 0.2  1. ± 0.1***  1.0 ± 0.1*  0.9 ± 0.1  0.9 ± 0.1  0.9 ± 0.1  0.6–1.2  0.0022   Calcium (mmol/L)  2.41 (2.37–2.45)  9.40**** (8.85–10.20)  9.60**** (9.35–9.85)  9.15**** (8.97–9.95)  9.45**** (9.22–9.67)  2.53*** (2.43–9.20)  2.23– 2.55  <0.0001   Magnesium (mg/dL)  2.0 ± 0.10  1.94 ± 0.28  1.99 ± 0.14  2.03 ± 0.21  2.1 ± 0.11  2.0 ± 0.16  1.6–2.6  0.4119   Sodium (mmol/L)  138 ± 1  137 ± 9  139 ± 6  136 ± 9  137 ± 4  137 ± 2  137–145  0.8130   Potassium (mmol/L)  4.2 ± 0.3  4.3 ± 1.1  3.9 ± 0.3  4.1 ± 0.3  4.2 ± 0.3  4.5 ± 0.2  3.6–5.0  0.1077  Creatine kinase-CK (U/L)  210 (112–298)  856** (434–1,095)  233 (138–296)  135 (94–245)  107 (95–144)  102 (80–210)  30–170  <0.0001   hs-CRP (mg/dL)  0.08 (0.03–0.27)  7.5**** (3.0–14.2)  0.65* (0.18–2.0)  0.08 (0.04–0.25)  0.10 (0.03–0.17)  0.25 (0.08–0.90)  <0.50  <0.0001  Parameters  Before  W1  W2  W3  W4  After  Reference Ranges  p-Values  Red blood cell count (106/μL)  4.9 ± 0.3  ND  ND  ND  ND  4.9 ± 0.2  4.5–6.2  0.8772   Hemoglobin (g/dL)  14.9 ± 0.8  ND  ND  ND  ND  14.8 ± 0.7  13.5–18.0  0.4363   Hematocrit (%)  45 ± 2  ND  ND  ND  ND  44 ± 3  40–54  0.6978  White blood cell count (μL) × 103  7.3 ± 1.2  ND  ND  ND  ND  6.9 ± 1.4  5–10  0.3353   Platelet count (×103/μL)  228 ± 55  ND  ND  ND  ND  216 ± 47  150–450  0.6816   Urea (mg/dL)  29 ± 7  43 ± 9***  34 ± 5  31 ± 11  32 ± 10  28 ± 7  15–40  0.0002   Creatinine (mg/dL)  0.9 ± 0.2  1. ± 0.1***  1.0 ± 0.1*  0.9 ± 0.1  0.9 ± 0.1  0.9 ± 0.1  0.6–1.2  0.0022   Calcium (mmol/L)  2.41 (2.37–2.45)  9.40**** (8.85–10.20)  9.60**** (9.35–9.85)  9.15**** (8.97–9.95)  9.45**** (9.22–9.67)  2.53*** (2.43–9.20)  2.23– 2.55  <0.0001   Magnesium (mg/dL)  2.0 ± 0.10  1.94 ± 0.28  1.99 ± 0.14  2.03 ± 0.21  2.1 ± 0.11  2.0 ± 0.16  1.6–2.6  0.4119   Sodium (mmol/L)  138 ± 1  137 ± 9  139 ± 6  136 ± 9  137 ± 4  137 ± 2  137–145  0.8130   Potassium (mmol/L)  4.2 ± 0.3  4.3 ± 1.1  3.9 ± 0.3  4.1 ± 0.3  4.2 ± 0.3  4.5 ± 0.2  3.6–5.0  0.1077  Creatine kinase-CK (U/L)  210 (112–298)  856** (434–1,095)  233 (138–296)  135 (94–245)  107 (95–144)  102 (80–210)  30–170  <0.0001   hs-CRP (mg/dL)  0.08 (0.03–0.27)  7.5**** (3.0–14.2)  0.65* (0.18–2.0)  0.08 (0.04–0.25)  0.10 (0.03–0.17)  0.25 (0.08–0.90)  <0.50  <0.0001  The results are presented as the mean ± SD. Values that did not follow a Gaussian distribution are presented as medians (25th–75th percentile) (Shapiro–Wilk normality test). p-Values were estimated using one-way ANOVA followed by Dunnett’s multiple comparisons test or Kruskal–Wallis test, as appropriate. p-Values in bold characters denote statistically significant differences. W, week; hs-CRP, high-sensitivity C-reactive protein; ND, not determined. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 compared with values before training. Evaluation of Skin Microvascular Flow and Reactivity The skin iontophoresis of ACh induced significant increases in microvascular CVC both before (p < 0.0001) and after (p < 0.0001) training; however, microvascular vasodilatory responses were markedly and significantly reduced after training, compared with values obtained before training (Fig. 1). Figure 1. View largeDownload slide The effects of skin acetylcholine (ACh) iontophoresis on the cutaneous microvascular conductance (CVC, expressed in arbitrary perfusion units [APUs] divided by mean arterial pressure in mmHg) of the volunteers (n = 14) before and after strenuous exercise training. The values are expressed as means ± SD and were analyzed using either two-tailed paired Student’s t-tests or repeated measures analysis of variance, when appropriate. **p < 0.01; ***p < 0.001; ****p < 0.0001 compared with values measured before exercise training. #p < 0.05; ##p < 0.01; ###p < 0.001; ####p < 0.0001 compared with baseline values. Figure 1. View largeDownload slide The effects of skin acetylcholine (ACh) iontophoresis on the cutaneous microvascular conductance (CVC, expressed in arbitrary perfusion units [APUs] divided by mean arterial pressure in mmHg) of the volunteers (n = 14) before and after strenuous exercise training. The values are expressed as means ± SD and were analyzed using either two-tailed paired Student’s t-tests or repeated measures analysis of variance, when appropriate. **p < 0.01; ***p < 0.001; ****p < 0.0001 compared with values measured before exercise training. #p < 0.05; ##p < 0.01; ###p < 0.001; ####p < 0.0001 compared with baseline values. The basal skin microvascular flow values were not different before (16.2 ± 4.0 APU) or after training (15.5 ± 3.4 APU; p = 0.4191). Conversely, baseline microvascular conductance, which represents vasodilatory capability, was reduced after training from 0.22 ± 0.06 to 0.17 ± 0.04 APU/mmHg (p = 0.0057; Fig. 2A). Figure 2. View largeDownload slide (A) Baseline values, (B) peak effects, and (C) the maximal increases of the cutaneous microvascular conductance (CVC, expressed in arbitrary perfusion units [APUs] divided by mean arterial pressure in mmHg) elicited by skin ACh iontophoresis before and after exercise training. (D) The area under the curve of the microvascular vasodilation induced by skin ACh iontophoresis before and after exercise training. The amplitudes of the ACh responses are expressed as the peak CVC values minus the baseline CVC values. The values are expressed as means ± SD and were analyzed using two-tailed paired Student’s t-tests. Figure 2. View largeDownload slide (A) Baseline values, (B) peak effects, and (C) the maximal increases of the cutaneous microvascular conductance (CVC, expressed in arbitrary perfusion units [APUs] divided by mean arterial pressure in mmHg) elicited by skin ACh iontophoresis before and after exercise training. (D) The area under the curve of the microvascular vasodilation induced by skin ACh iontophoresis before and after exercise training. The amplitudes of the ACh responses are expressed as the peak CVC values minus the baseline CVC values. The values are expressed as means ± SD and were analyzed using two-tailed paired Student’s t-tests. The peak CVC values obtained during iontophoresis of ACh before training (0.84 ± 0.22 APU/mmHg) were also markedly reduced after training (0.47 ± 0.11 APU/mmHg; p < 0.0001; Fig. 2B). The increase (peak minus baseline) in CVC induced by ACh was also reduced after training (from 0.61 ± 0.20 to 0.29 ± 0.03 APU/mmHg; p < 0.0001; Fig. 2C). Moreover, area under the curve of endothelium-dependent microvascular vasodilation induced by ACh measured before training 18,510 ± 4,538 APU/s was reduced to 13,958 ± 3,733 APU/s (p = 0.0047; Fig. 2D) after training. The peak CVC values measured during post-occlusive reactive hyperemia (PORH) before training (0.94 ± 0.72 APU/mmHg) were also markedly reduced after training (0.71 ± 0.14 APU/mmHg; p = 0.0037; Fig. 3). The increase (peak minus baseline) in CVC induced by PORH was also reduced after training (from 0.61 ± 0.22 to 0.45 ± 0.13 APU/mmHg; p = 0.0149; Fig. 3). We did not identify any outlier in the data regarding microvascular reactivity. Figure 3. View largeDownload slide (A) Peak effects and (B) the maximal increases of the cutaneous microvascular conductance (CVC, expressed in arbitrary perfusion units [APUs] divided by mean arterial pressure in mmHg) elicited by post-occlusive reactive hyperemia (PORH) before and after exercise training. The amplitudes of the PORH responses are expressed as the peak CVC values minus the baseline CVC values. The values are expressed as means ± SD and were analyzed using two-tailed paired Student’s t-tests. Figure 3. View largeDownload slide (A) Peak effects and (B) the maximal increases of the cutaneous microvascular conductance (CVC, expressed in arbitrary perfusion units [APUs] divided by mean arterial pressure in mmHg) elicited by post-occlusive reactive hyperemia (PORH) before and after exercise training. The amplitudes of the PORH responses are expressed as the peak CVC values minus the baseline CVC values. The values are expressed as means ± SD and were analyzed using two-tailed paired Student’s t-tests. Capillary Density The basal mean skin capillary density of the volunteers was not different before and after training (before 95 ± 8 and after 94 ± 8 capillaries/mm2; p = 0.6489; Fig. 4). During PORH, which is considered as endothelium-dependent capillary recruitment, the capillary density was significantly reduced after training (before 101 ± 9 and after 95 ± 8 capillaries/mm2; p = 0.0007). Consequently, the increase in capillary number observed during PORH before training (5.5 [0.8–11.5] capillaries/mm2) was not detected after training (0.7 [−4.8 to 5.2] capillaries/mm2; Fig. 4). We did not identify any outlier in the data regarding skin capillary density. Figure 4. View largeDownload slide Functional capillary density at baseline (BASAL) and during post-occlusive reactive hyperemia (PORH) before and after exercise training. Inset: Increase in capillary number after PORH before and after exercise training. The values are expressed as means ± SD or medians (25th–75th percentile) and were analyzed using either two-tailed paired Student’s t-test or Wilcoxon-matched pairs signed rank test, as appropriate. **p < 0.01 compared with basal values before training. ###p < 0.001 compared with PORH values before training. Figure 4. View largeDownload slide Functional capillary density at baseline (BASAL) and during post-occlusive reactive hyperemia (PORH) before and after exercise training. Inset: Increase in capillary number after PORH before and after exercise training. The values are expressed as means ± SD or medians (25th–75th percentile) and were analyzed using either two-tailed paired Student’s t-test or Wilcoxon-matched pairs signed rank test, as appropriate. **p < 0.01 compared with basal values before training. ###p < 0.001 compared with PORH values before training. Discussion The main findings of the present study were that strenuous exercise and gas exposure, related to special military training results for riot control in (1) marked decrease in endothelium-dependent systemic microvascular reactivity, (2) a reduction of endothelium-dependent systemic capillary recruitment, and (3) transient but significant increases in plasma urea, creatinine, calcium, and in the enzyme creatine kinase. It is well known that physical training is capable of inducing both beneficial and harmful alterations on vascular endothelial function.25,26 The evaluation of vascular reactivity is usually tested by means of the assessment of the capacity of endothelium-dependent vasoactive agents to induce vasodilation in resistance vessels, as physiologic endothelium-dependent vasodilation is essentially dependent on adequate NO bioavailability. On the other hand, endothelial dysfunction may result from the scavenging of NO by oxidized lipoproteins or superoxide radicals, which thus shortens the half-life of NO and thereby impairs NO-dependent vasodilation.26,27 High-intensity exercise – independently of the absolute intensity of exercise – may induce significant blunting of vascular endothelial function, by exposing the blood vessels to repeated bouts of oxidative stress, which is accompanied by a decreased level of plasma antioxidants and an increased production of free radicals.6,28,29 For instance, although increased in the plasma, the levels of reduced glutathione are decreased in the skeletal muscle of rats after exhaustive exercise.30 In fact, the protective effect of moderate-intensity exercise training, mainly represented by increased bioavailability of NO and consequent quenching of reactivity oxygen species (ROS), is lost when the increasing intensity of exercise results in massive increases of oxidative stress.5 We and others have already reported on the beneficial effects of moderate-intensity aerobic exercise on microvascular endothelial function both in animal31–33 and in human studies.34–37 In contrast, the present study showed that a 5-wk strenuous exercise training in a military setting, in young healthy individuals, induces a marked reduction of endothelium-dependent systemic microvascular reactivity. In fact, microvascular relaxation curves resulting from skin transdermal iontophoresis of acetylcholine showed a reduction of approximately 30% after training. Moreover, capillary recruitment induced by post-occlusive reactive hyperemia, which is dependent on physiological endothelium-dependent microvascular endothelial function, was blunted after military training. These results confirm the previously stated hypothesis that vascular function may be paradoxically impaired in healthy individuals by high levels of exercise.1 Exhaustive exercise may cause muscle damage, which can be evidenced as an increase in the plasma activity of cytosolic enzymes such as creatine kinase or lactate dehydrogenase, at least in part due to the increased production of free radicals.28 In the present study, plasma levels of creatine kinase were markedly increased (four-fold) in the first week of training and decreased thereafter. Chronically repeated small muscle injuries may trigger a systemic inflammatory response,38–41 while skeletal muscle lesions of greater magnitude caused by physical exercise are known as exertional rhabdomyolysis.42 In the present military training, one of the volunteers developed exertional rhabdomyolysis on the second day of training accompanied by highly increased plasma levels of creatine kinase (data not shown). Intense physical exercise is capable of acutely promoting muscle injuries associated with rupture of the cell membrane integrity, inflammation, and delayed-onset muscle pain.43 Moreover, this phenomenon is believed to occur predominantly during unusual strenuous exercise carried out uninterruptedly for several minutes, being associated with high production of reactive oxygen species.44 When physical exercise is prolonged and accomplished under adverse climatic conditions of high temperature and humidity, as in the military training described here, extreme exertion and dehydration may be associated with transitory skeletal muscle ATP deficiency.45 The irritant agents used in the military training described here, specifically tear gas and pepper spray, might also be putative contributors to detrimental vascular effects, as they activate the transient receptor potential (TRP) superfamily of cation channels, including the receptor subtypes vanilloid-1 and Ankyrin-1 (TRPV1 and TRPA1, respectively).15 TRP channels are involved in normal and pathophysiological responses in the blood vessels, mainly vascular smooth muscle contractility and endothelium-dependent vasodilation.46–48 Moreover, TRP can also be activated by substances produced at high levels during oxidative stress, such as ROS.47 Nevertheless, even if the activation of TRP channels is known to have cardiovascular effects, their specific roles in cardiovascular function remain to be elucidated.47 In conclusion, it is not prudent to speculate on the putative effects of the irritant agents on the microvascular effects described in the present report. The plasma levels of high-sensitivity C-reactive protein, an important clinical marker of systemic inflammation, were also markedly increased by military training, thus confirming the presence of a low-grade systemic inflammatory process described above. This alteration was accompanied by a five-fold increase in the plasma levels of calcium during the 4 wk of military training. One reasonable explanation for the increase in plasma calcium levels could be the hemoconcentration that probably occurred during the high-intensity training performed under unfavorable climatic conditions. In this context, it has already been reported that the increases in calcium plasma levels are accompanied by hemoconcentration after strenuous exercise in healthy volunteers.49,50 In conclusion, the present study showed that a 5-wk, high-intensity military training, performed in unfavorable climatic conditions, induces marked systemic microvascular dysfunction, mainly characterized by reduced endothelium-dependent microvascular vasodilation and blunted capillary recruitment. Acknowledgments The authors wish to thank nurse Marcio Marinho Gonzalez for his excellent technical assistance. We also wish to thank Major Manoel Gomes da Silva Neto de Queiroz, Captain Alexandre Fontoura da Silva, and Felipe Martins Peçanha of the Special Aeronautical Infantry Battalion of Rio de Janeiro, Brazil. Funding Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; ET, grant no. 303328/2013-4), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ; Eduardo Tibirica, grant no. E-26/102.981/2011). References 1 Durand MJ, Gutterman DD: Exercise and vascular function: how much is too much? Can J Physiol Pharmacol  2014; 92: 551– 7. Google Scholar CrossRef Search ADS PubMed  2 Naci H, Ioannidis JP: Comparative effectiveness of exercise and drug interventions on mortality outcomes: metaepidemiological study. BMJ  2013; 347: f5577. Google Scholar CrossRef Search ADS PubMed  3 Rush JW, Denniss SG, Graham DA: Vascular nitric oxide and oxidative stress: determinants of endothelial adaptations to cardiovascular disease and to physical activity. Can J Appl Physiol  2005; 30: 442– 74. Google Scholar CrossRef Search ADS PubMed  4 Ford ES, Caspersen CJ: Sedentary behaviour and cardiovascular disease: a review of prospective studies. Int J Epidemiol  2012; 41: 1338– 53. 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Google Scholar CrossRef Search ADS PubMed  19 Holowatz LA, Thompson-Torgerson CS, Kenney WL: The human cutaneous circulation as a model of generalized microvascular function. J Appl Physiol  1985; 2008( 105): 370– 2. 20 Kaiser SE, Sanjuliani AF, Estato V, Gomes MB, Tibirica E: Antihypertensive treatment improves microvascular rarefaction and reactivity in low-risk hypertensive individuals. Microcirculation  2013; 20: 703– 16. Google Scholar PubMed  21 Serne EH, de Jongh RT, Eringa EC, IJzerman RG, Stehouwer CD: Microvascular dysfunction: a potential pathophysiological role in the metabolic syndrome. Hypertension  2007; 50: 204– 11. Google Scholar CrossRef Search ADS PubMed  22 Debbabi H, Uzan L, Mourad JJ, Safar M, Levy BI, Tibirica E: Increased skin capillary density in treated essential hypertensive patients. Am J Hypertens  2006; 19: 477– 83. Google Scholar CrossRef Search ADS PubMed  23 Francischetti EA, Tibirica E, da Silva EG, Rodrigues E, Celoria BM, de Abreu VG: Skin capillary density and microvascular reactivity in obese subjects with and without metabolic syndrome. Microvasc Res  2011; 81: 325– 30. Google Scholar CrossRef Search ADS PubMed  24 Antonios TF, Rattray FE, Singer DR, Markandu ND, Mortimer PS, MacGregor GA: Maximization of skin capillaries during intravital video-microscopy in essential hypertension: comparison between venous congestion, reactive hyperaemia and core heat load tests. Clin Sci (Lond)  1999; 97: 523– 8. Google Scholar CrossRef Search ADS PubMed  25 Green DJ, Maiorana A, O’Driscoll G, Taylor R: Effect of exercise training on endothelium-derived nitric oxide function in humans. J Physiol  2004; 561: 1– 25. Google Scholar CrossRef Search ADS PubMed  26 Moncada S, Palmer RM, Higgs EA: The discovery of nitric oxide as the endogenous nitrovasodilator. Hypertension  1988; 12: 365– 72. 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Metab Syndr Relat Disord  2016; 14: 298– 304. Google Scholar CrossRef Search ADS PubMed  32 Machado MV, Vieira AB, da Conceicao FG, Nascimento AR, da Nobrega ACL, Tibirica E: Exercise training dose differentially alters muscle and heart capillary density and metabolic functions in an obese rat with metabolic syndrome. Exp Physiol  2017; 102( 12): 1716– 28. Google Scholar CrossRef Search ADS PubMed  33 Pereira F, de Moraes R, Tibirica E, Nobrega AC: Interval and continuous exercise training produce similar increases in skeletal muscle and left ventricle microvascular density in rats. Biomed Res Int  2013; 2013: 752817. Google Scholar CrossRef Search ADS PubMed  34 Borges JP, Mediano MF, Farinatti P, et al.  : The effects of unsupervised home-based exercise upon functional capacity after 6 months of discharge from cardiac rehabilitation: a retrospective observational study. J Phys Act Health  2016; 13: 1230– 35. 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Front Biosci  2007; 12: 4826– 38. Google Scholar CrossRef Search ADS PubMed  39 Pedersen BK, Ostrowski K, Rohde T, Bruunsgaard H: The cytokine response to strenuous exercise. Can J Physiol Pharmacol  1998; 76: 505– 11. Google Scholar CrossRef Search ADS PubMed  40 Purvis D, Gonsalves S, Deuster PA: Physiological and psychological fatigue in extreme conditions: overtraining and elite athletes. PM R  2010; 2: 442– 50. Google Scholar CrossRef Search ADS PubMed  41 Smith LL: Tissue trauma: the underlying cause of overtraining syndrome? J Strength Cond Res  2004; 18: 185– 93. Google Scholar PubMed  42 Scalco RS, Snoeck M, Quinlivan R, et al.  : Exertional rhabdomyolysis: physiological response or manifestation of an underlying myopathy? BMJ Open Sport Exerc Med  2016; 2: e000151. Google Scholar CrossRef Search ADS PubMed  43 Mizumura K, Taguchi T: Delayed onset muscle soreness: involvement of neurotrophic factors. J Physiol Sci  2016; 66: 43– 52. Google Scholar CrossRef Search ADS PubMed  44 Powers SK, Nelson WB, Hudson MB: Exercise-induced oxidative stress in humans: cause and consequences. Free Radic Biol Med  2011; 51: 942– 50. Google Scholar CrossRef Search ADS PubMed  45 Clausen T: Quantification of Na+,K+ pumps and their transport rate in skeletal muscle: functional significance. J Gen Physiol  2013; 142: 327– 45. Google Scholar CrossRef Search ADS PubMed  46 Fernandes ES, Fernandes MA, Keeble JE: The functions of TRPA1 and TRPV1: moving away from sensory nerves. Br J Pharmacol  2012; 166: 510– 21. Google Scholar CrossRef Search ADS PubMed  47 Earley S: TRPA1 channels in the vasculature. Br J Pharmacol  2012; 167: 13– 22. Google Scholar CrossRef Search ADS PubMed  48 Earley S, Brayden JE: Transient receptor potential channels in the vasculature. Physiol Rev  2015; 95: 645– 90. Google Scholar CrossRef Search ADS PubMed  49 Aloia JF, Rasulo P, Deftos LJ, Vaswani A, Yeh JK: Exercise-induced hypercalcemia and the calciotropic hormones. J Lab Clin Med  1985; 106: 229– 32. Google Scholar PubMed  50 Cunningham J, Segre GV, Slatopolsky E, Avioli LV: Effect of heavy exercise on mineral metabolism and calcium regulating hormones in humans. Calcif Tissue Int  1985; 37: 598– 601. Google Scholar CrossRef Search ADS PubMed  Author notes The views expressed in this article are solely those of the authors and do not reflect the official policy or position of the Brazilian Air Force or the Brazilian Government. © Association of Military Surgeons of the United States 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. 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Effects of Riot Control Training on Systemic Microvascular Reactivity and Capillary Density

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Abstract

Abstract Introduction The main aim of the present study is to evaluate the effects of strenuous exercise, related to special military training for riot control, on systemic microvascular endothelial function and skin capillary density. Materials and Methods Endothelium-dependent microvascular reactivity was evaluated in the forearm skin of healthy military trainees (age 23.4 ± 2.3 yr; n = 15) using laser speckle contrast imaging coupled with cutaneous acetylcholine (ACh) iontophoresis and post-occlusive reactive hyperemia (PORH). Functional capillary density was assessed using high-resolution, intra-vital color microscopy in the dorsum of the middle phalanx. Capillary recruitment (capillary reserve) was evaluated using PORH. Microcirculatory tests were performed before and after a 5-wk special military training for riot control. Results Microvascular endothelium-dependent vasodilatory responses were markedly and significantly reduced after training, compared with values obtained before training. The peak values of microvascular conductance obtained during iontophoresis of ACh or PORH before training (0.84 ± 0.22 and 0.94 ± 0.72 APU/mmHg, respectively) were markedly reduced after training (0.47 ± 0.11 and 0.71 ± 0.14 APU/mmHg; p < 0.0001 and p = 0.0037, respectively). Endothelium-dependent capillary recruitment was significantly reduced after training (before 101 ± 9 and after 95 ± 8 capillaries/mm2; p = 0.0007). Conclusions The present study showed that a 5-wk strenuous military training, performed in unfavorable climatic conditions, induces marked systemic microvascular dysfunction, mainly characterized by reduced endothelium-dependent microvascular vasodilation and blunted capillary recruitment. Introduction Currently, there is plenty of evidence that exercise is associated with several beneficial effects, including a reduction of mortality both in healthy subjects and in patients with cardiovascular disease.1–4 Regular exercise of moderate intensity induces an increase in vascular nitric oxide (NO) bioavailability, associated with a reduction of the production of reactive oxygen species (ROS), resulting in the improvement of vascular endothelial function.1 Nevertheless, high-intensity exercise may cause unfavorable disturbances in the NO/ROS balance and have the opposite effect.1 Endothelial function indeed appears to be impaired in individuals submitted to high-intensity, sustained aerobic training.5,6 For instance, elite Olympic athletes trained for aerobic sports show impaired flow-mediated dilation of the brachial artery, compared with non-trained, age- and sex-matched control subjects.7 Usually, military personnel are required to perform intensive physical training, including endurance and resistance exercise, to maintain high-level physical fitness and military skills for physically demanding tasks.8 Nevertheless, high-intensity physical effort during special military training – as for riot control – associated with unfavorable climatic conditions, such as high temperature and humidity, can also lead to adverse effects.9–11 For instance, the risk of exertional rhabdomyolysis and death in military personnel that engage consistently in regular and strenuous exercise of special military training is higher than in the civilian population.12 Riot control agents, which are frequently used in military settings, include o-chlorobenzylidene-malononitrile (CS), which is grouped with several other irritant agents referred to as “tear gas”13,14 and pepper (capsaicin) spray.15,16 These agents are generally supposed to be sub-lethal irritant incapacitants.17 The main toxic effects of these agents are related to respiratory, ocular, gastrointestinal, and cutaneous alterations.15 Nevertheless, cardiovascular effects, including tachycardia and transient hypertension, have been already described. These effects appear to be related to sensory autonomic reflexes, anxiety, pain, or psychological distress.18 Interestingly, it has already been reported that toxic symptoms after heavy exposure to CS in a field training setting can be disclosed by strenuous physical exercise.14 The physiological imbalances induced by all these factors involved in special military training may ultimately have an impact on the microcirculation, which is responsible for tissue perfusion. The non-invasive assessment of the cutaneous microcirculation using laser speckle contrast imaging (LSCI) is an innovative approach with proven usefulness for the investigation of the pathophysiology of cardiovascular and metabolic diseases.19 The cutaneous microcirculation is an accessible and representative vascular bed for the evaluation of systemic microcirculatory reactivity and capillary density, which are known to be closely correlated with cardiovascular and metabolic diseases.20,21 The present study aims to evaluate the effects of high, physically demanding military activities, related to special military training for riot control, associated with the exposure to irritant transient incapacitants, on systemic microvascular endothelial function and capillary density in young adults, using LSCI and skin video microscopy, respectively. Methods Study Design This observational study included military personnel from the Brazilian Air Force who completed a 5-wk special training period for riot control in Rio de Janeiro, Brazil. The study included 15 volunteers who consented to undergo evaluation of skin microvascular reactivity and capillary density and analyses of biochemical data. All were healthy according to routine medical examinations, physically active, and not in use of any medications or specific dietary regimens. The evaluation of microvascular reactivity and capillary density was performed before the 5-wk training and at the end of the training. Venous blood was collected before training and once a week during the 5-wk training period. All evaluations were performed in the morning, between 8 a.m. and 12 p.m., after a 12-hr fast. First, blood specimens were collected and then subjects rested for 20 min in a quiet environment with a constant temperature of 23 ± 1°C before the microvascular reactivity tests. The study was undertaken in accordance with the Helsinki Declaration of 1975, revised in 2000, and was approved by the Institutional Review Board (IRB) of the National Institute of Cardiology, Rio de Janeiro, Brazil, under protocol no. CAAE 49792515.6.0000.5272. All subjects read and signed an informed consent document approved by the IRB. Military Training Protocol The training protocol was designed by the Air Force authority. The present observational study did not include any interventional procedure. Military physical training, using military uniform, was conducted from Monday to Friday and consisted of two 20-min morning running sessions, with 20-min rest and free hydration in between, and performed with complete combat uniform and equipment, including the use of helmet and shield. At the end of running session each morning, the military training group was continuously exposed to toxic tear gas and pepper spray for 2 min. During 4 hr in the afternoon, the group was submitted to handling of warlike material and strategic movements to riot control, including successive running incursions, between 60 and 120 s, performed in the open field or climbing stairs, and exposure to the gases between 5 and 20 min without a mask. The running exercise was performed in warm (about 32°C) and humid (86% air relative humidity) conditions, typical of the summer season in Rio de Janeiro, Brazil. Evaluation of Skin Microvascular Flow and Reactivity Microvascular reactivity was evaluated using an LSCI system with a laser wavelength of 785 nm (PeriCam PSI System; Perimed, Järfälla, Sweden), which allowed non-invasive and continuous measurements of cutaneous microvascular perfusion changes, measured in arbitrary perfusion units (APU). Images were analyzed using PIMSoft software (Perimed, Järfälla, Sweden). Brachial systolic (SAP) and diastolic (DAP) blood pressures were measured twice immediately before the beginning of the recordings, using a mercury sphygmomanometer, and the mean values were recorded as the patients’ blood pressure. The mean arterial pressure (MAP) was calculated as DAP + 1/3 (SAP-DAP). One skin site on the ventral surface of the forearm was randomly chosen for the recordings. Hair, broken skin, areas of skin pigmentation, and visible veins were avoided, and two drug delivery electrodes were installed using adhesive discs (LI 611; Perimed, Järfälla, Sweden). The following two measurement areas were identified: a measurement area within the electrode (ACh) and another measurement area (baseline control) adjacent to the electrode. A vacuum cushion (AB Germa, Kristianstad, Sweden) was used to minimize recording artifacts generated by arm movements. ACh 2% w/v (Sigma Chemical CO, St. Louis, MO, USA) iontophoresis was performed using a micropharmacology system (PF 751 PeriIont USB Power Supply, Perimed, Sweden) using increasing anodal currents of 30, 60, 90, 120, 150, and 180 μA, which were administered for 10-s intervals spaced 1 min apart. The total charges for the above currents were 0.3, 0.6, 0.9, 1.2, 1.5, and 1.8 mC, respectively. The dispersive electrode was attached approximately 15 cm from the electrophoresis chamber. The pharmacological test results were expressed as peak values representing the maximal vasodilation observed after the highest ACh dose. Skin blood flow measurements in arbitrary perfusion units (APU) were divided by MAP to yield cutaneous vascular conductance (CVC) in APU/mmHg. Capillaroscopy by Intra-vital Microscopy Microcirculatory tests were performed in a room with a defined stable temperature (23 ± 1°C) after a 20-min rest in the supine position. The dorsum of the non-dominant middle phalanx was used for image acquisition, while keeping the patient sitting comfortably. Room temperature was monitored and adjusted if necessary using air conditioning, considering that outside temperature was usually >25°C. The arm was positioned at the level of the heart and immobilized using a vacuum cushion (AB Germa, Kristianstad, Sweden). Capillary density, that is, the number of perfused capillaries per square millimeter of skin area, was assessed by high-resolution, intra-vital color microscopy (Moritex, Cambridge, UK), as previously described and validated by our research team.20,22,23 A video microscopy system was used, with an epi-illuminated fiber optic microscope containing a 100-W mercury vapor lamp light source and an M200 objective with a final magnification of 200×. Images were acquired and saved for posterior offline analysis using a semi-automatic integrated system (Microvision Instruments, Evry, France). For post-occlusive reactive hyperemia (PORH), a blood pressure cuff was then applied around the patient’s arm and inflated to suprasystolic pressure (50 mmHg greater than systolic arterial pressure) to completely interrupt the blood flow for 3 min. This time of occlusion has already been shown to effectively recruit capillaries in an endothelium-dependent manner. After cuff release, images were again acquired and recorded over the subsequent 60–90 s, during which time the maximal hyperemic response was expected to occur. The mean number of spontaneously perfused skin capillaries at rest was considered to represent the functional capillary density, as previously described.22,24 On the other hand, the number of perfused capillaries during post-occlusive reactive hyperemia was considered to represent functional capillary recruitment, resulting from the release of endothelial mediators and consequent arteriolar vasodilation.24 The mean capillary density for each patient was calculated as the arithmetic mean of visible (i.e., spontaneously perfused) capillaries in three contiguous microscopic fields of 1 mm2 each. Capillary counting was performed by two investigators blinded to the patients’ characteristics, and the final values of capillary density represent the mean of the individual counts. Reproducibility was assessed by examining an identical area of skin; intra-observer repeatability of data analysis was assessed by reading the same images blindly on two separate occasions (n = 15, coefficient of variability 4.3%). Statistical Analysis The results are presented as mean ± SD. Variables without a Gaussian distribution by Shapiro–Wilk normality test are presented as medians (25th–75th percentile). The comparisons between parameters obtained before and after training were performed using two-tailed paired Student’s t-tests or the Mann–Whitney tests, when appropriate. The dose-dependency of the effects of acetylcholine on microvascular vasodilation was tested using repeated measures ANOVA followed by the Dunnett’s multiples comparisons test. p-Values <0.05 were considered statistically significant. The identification of potential outliers was performed using the ROUT method (robust regression and outlier removal), which is based on the false discovery rate (FDR), with a specified value of Q = 1%. The statistical package used for the statistical analyses was Prism version 7.0 (GraphPad Software Inc., La Jolla, CA, USA). Results The anthropometric and hemodynamic parameters of the subjects before and after exercise are depicted in Table I. One subject developed exertional rhabdomyolysis on the second day of training and was excluded from the study. Laboratory data of the volunteers that completed training are shown in Table II. During training, creatine kinase, urea, creatinine, and C-reactive protein levels increased, declining after the end of training. Table I. Anthropometric and Hemodynamic Data Before or After Training Parameter  Before  After  p-Values  Age (yr)  23.4 ± 2.3  –  –  Weight (kg)  82.0 ± 10.2  82.6 ± 11.3  0.7529  Height (cm)  178.7 ± 7.7  –  –  Body mass index (kg/m2)  25.6 ± 2.2  25.9 ± 3.6  0.6306  Systolic arterial pressure (mmHg)  127 ± 7  127 ± 9  0.9117  Diastolic arterial pressure (mmHg)  68 ± 5  66 ± 5  0.1253  Mean arterial pressure (mmHg)  88 ± 5  87 ± 5  0.2790  Heart rate (bpm)  56 ± 9  58 ± 7  0.5229  Parameter  Before  After  p-Values  Age (yr)  23.4 ± 2.3  –  –  Weight (kg)  82.0 ± 10.2  82.6 ± 11.3  0.7529  Height (cm)  178.7 ± 7.7  –  –  Body mass index (kg/m2)  25.6 ± 2.2  25.9 ± 3.6  0.6306  Systolic arterial pressure (mmHg)  127 ± 7  127 ± 9  0.9117  Diastolic arterial pressure (mmHg)  68 ± 5  66 ± 5  0.1253  Mean arterial pressure (mmHg)  88 ± 5  87 ± 5  0.2790  Heart rate (bpm)  56 ± 9  58 ± 7  0.5229  The results are presented as the mean ± SD. p-Values were estimated using two-tailed paired t-tests. Table I. Anthropometric and Hemodynamic Data Before or After Training Parameter  Before  After  p-Values  Age (yr)  23.4 ± 2.3  –  –  Weight (kg)  82.0 ± 10.2  82.6 ± 11.3  0.7529  Height (cm)  178.7 ± 7.7  –  –  Body mass index (kg/m2)  25.6 ± 2.2  25.9 ± 3.6  0.6306  Systolic arterial pressure (mmHg)  127 ± 7  127 ± 9  0.9117  Diastolic arterial pressure (mmHg)  68 ± 5  66 ± 5  0.1253  Mean arterial pressure (mmHg)  88 ± 5  87 ± 5  0.2790  Heart rate (bpm)  56 ± 9  58 ± 7  0.5229  Parameter  Before  After  p-Values  Age (yr)  23.4 ± 2.3  –  –  Weight (kg)  82.0 ± 10.2  82.6 ± 11.3  0.7529  Height (cm)  178.7 ± 7.7  –  –  Body mass index (kg/m2)  25.6 ± 2.2  25.9 ± 3.6  0.6306  Systolic arterial pressure (mmHg)  127 ± 7  127 ± 9  0.9117  Diastolic arterial pressure (mmHg)  68 ± 5  66 ± 5  0.1253  Mean arterial pressure (mmHg)  88 ± 5  87 ± 5  0.2790  Heart rate (bpm)  56 ± 9  58 ± 7  0.5229  The results are presented as the mean ± SD. p-Values were estimated using two-tailed paired t-tests. Table II. Laboratory Testing of the Subjects Before, During, or After Training Parameters  Before  W1  W2  W3  W4  After  Reference Ranges  p-Values  Red blood cell count (106/μL)  4.9 ± 0.3  ND  ND  ND  ND  4.9 ± 0.2  4.5–6.2  0.8772   Hemoglobin (g/dL)  14.9 ± 0.8  ND  ND  ND  ND  14.8 ± 0.7  13.5–18.0  0.4363   Hematocrit (%)  45 ± 2  ND  ND  ND  ND  44 ± 3  40–54  0.6978  White blood cell count (μL) × 103  7.3 ± 1.2  ND  ND  ND  ND  6.9 ± 1.4  5–10  0.3353   Platelet count (×103/μL)  228 ± 55  ND  ND  ND  ND  216 ± 47  150–450  0.6816   Urea (mg/dL)  29 ± 7  43 ± 9***  34 ± 5  31 ± 11  32 ± 10  28 ± 7  15–40  0.0002   Creatinine (mg/dL)  0.9 ± 0.2  1. ± 0.1***  1.0 ± 0.1*  0.9 ± 0.1  0.9 ± 0.1  0.9 ± 0.1  0.6–1.2  0.0022   Calcium (mmol/L)  2.41 (2.37–2.45)  9.40**** (8.85–10.20)  9.60**** (9.35–9.85)  9.15**** (8.97–9.95)  9.45**** (9.22–9.67)  2.53*** (2.43–9.20)  2.23– 2.55  <0.0001   Magnesium (mg/dL)  2.0 ± 0.10  1.94 ± 0.28  1.99 ± 0.14  2.03 ± 0.21  2.1 ± 0.11  2.0 ± 0.16  1.6–2.6  0.4119   Sodium (mmol/L)  138 ± 1  137 ± 9  139 ± 6  136 ± 9  137 ± 4  137 ± 2  137–145  0.8130   Potassium (mmol/L)  4.2 ± 0.3  4.3 ± 1.1  3.9 ± 0.3  4.1 ± 0.3  4.2 ± 0.3  4.5 ± 0.2  3.6–5.0  0.1077  Creatine kinase-CK (U/L)  210 (112–298)  856** (434–1,095)  233 (138–296)  135 (94–245)  107 (95–144)  102 (80–210)  30–170  <0.0001   hs-CRP (mg/dL)  0.08 (0.03–0.27)  7.5**** (3.0–14.2)  0.65* (0.18–2.0)  0.08 (0.04–0.25)  0.10 (0.03–0.17)  0.25 (0.08–0.90)  <0.50  <0.0001  Parameters  Before  W1  W2  W3  W4  After  Reference Ranges  p-Values  Red blood cell count (106/μL)  4.9 ± 0.3  ND  ND  ND  ND  4.9 ± 0.2  4.5–6.2  0.8772   Hemoglobin (g/dL)  14.9 ± 0.8  ND  ND  ND  ND  14.8 ± 0.7  13.5–18.0  0.4363   Hematocrit (%)  45 ± 2  ND  ND  ND  ND  44 ± 3  40–54  0.6978  White blood cell count (μL) × 103  7.3 ± 1.2  ND  ND  ND  ND  6.9 ± 1.4  5–10  0.3353   Platelet count (×103/μL)  228 ± 55  ND  ND  ND  ND  216 ± 47  150–450  0.6816   Urea (mg/dL)  29 ± 7  43 ± 9***  34 ± 5  31 ± 11  32 ± 10  28 ± 7  15–40  0.0002   Creatinine (mg/dL)  0.9 ± 0.2  1. ± 0.1***  1.0 ± 0.1*  0.9 ± 0.1  0.9 ± 0.1  0.9 ± 0.1  0.6–1.2  0.0022   Calcium (mmol/L)  2.41 (2.37–2.45)  9.40**** (8.85–10.20)  9.60**** (9.35–9.85)  9.15**** (8.97–9.95)  9.45**** (9.22–9.67)  2.53*** (2.43–9.20)  2.23– 2.55  <0.0001   Magnesium (mg/dL)  2.0 ± 0.10  1.94 ± 0.28  1.99 ± 0.14  2.03 ± 0.21  2.1 ± 0.11  2.0 ± 0.16  1.6–2.6  0.4119   Sodium (mmol/L)  138 ± 1  137 ± 9  139 ± 6  136 ± 9  137 ± 4  137 ± 2  137–145  0.8130   Potassium (mmol/L)  4.2 ± 0.3  4.3 ± 1.1  3.9 ± 0.3  4.1 ± 0.3  4.2 ± 0.3  4.5 ± 0.2  3.6–5.0  0.1077  Creatine kinase-CK (U/L)  210 (112–298)  856** (434–1,095)  233 (138–296)  135 (94–245)  107 (95–144)  102 (80–210)  30–170  <0.0001   hs-CRP (mg/dL)  0.08 (0.03–0.27)  7.5**** (3.0–14.2)  0.65* (0.18–2.0)  0.08 (0.04–0.25)  0.10 (0.03–0.17)  0.25 (0.08–0.90)  <0.50  <0.0001  The results are presented as the mean ± SD. Values that did not follow a Gaussian distribution are presented as medians (25th–75th percentile) (Shapiro–Wilk normality test). p-Values were estimated using one-way ANOVA followed by Dunnett’s multiple comparisons test or Kruskal–Wallis test, as appropriate. p-Values in bold characters denote statistically significant differences. W, week; hs-CRP, high-sensitivity C-reactive protein; ND, not determined. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 compared with values before training. Table II. Laboratory Testing of the Subjects Before, During, or After Training Parameters  Before  W1  W2  W3  W4  After  Reference Ranges  p-Values  Red blood cell count (106/μL)  4.9 ± 0.3  ND  ND  ND  ND  4.9 ± 0.2  4.5–6.2  0.8772   Hemoglobin (g/dL)  14.9 ± 0.8  ND  ND  ND  ND  14.8 ± 0.7  13.5–18.0  0.4363   Hematocrit (%)  45 ± 2  ND  ND  ND  ND  44 ± 3  40–54  0.6978  White blood cell count (μL) × 103  7.3 ± 1.2  ND  ND  ND  ND  6.9 ± 1.4  5–10  0.3353   Platelet count (×103/μL)  228 ± 55  ND  ND  ND  ND  216 ± 47  150–450  0.6816   Urea (mg/dL)  29 ± 7  43 ± 9***  34 ± 5  31 ± 11  32 ± 10  28 ± 7  15–40  0.0002   Creatinine (mg/dL)  0.9 ± 0.2  1. ± 0.1***  1.0 ± 0.1*  0.9 ± 0.1  0.9 ± 0.1  0.9 ± 0.1  0.6–1.2  0.0022   Calcium (mmol/L)  2.41 (2.37–2.45)  9.40**** (8.85–10.20)  9.60**** (9.35–9.85)  9.15**** (8.97–9.95)  9.45**** (9.22–9.67)  2.53*** (2.43–9.20)  2.23– 2.55  <0.0001   Magnesium (mg/dL)  2.0 ± 0.10  1.94 ± 0.28  1.99 ± 0.14  2.03 ± 0.21  2.1 ± 0.11  2.0 ± 0.16  1.6–2.6  0.4119   Sodium (mmol/L)  138 ± 1  137 ± 9  139 ± 6  136 ± 9  137 ± 4  137 ± 2  137–145  0.8130   Potassium (mmol/L)  4.2 ± 0.3  4.3 ± 1.1  3.9 ± 0.3  4.1 ± 0.3  4.2 ± 0.3  4.5 ± 0.2  3.6–5.0  0.1077  Creatine kinase-CK (U/L)  210 (112–298)  856** (434–1,095)  233 (138–296)  135 (94–245)  107 (95–144)  102 (80–210)  30–170  <0.0001   hs-CRP (mg/dL)  0.08 (0.03–0.27)  7.5**** (3.0–14.2)  0.65* (0.18–2.0)  0.08 (0.04–0.25)  0.10 (0.03–0.17)  0.25 (0.08–0.90)  <0.50  <0.0001  Parameters  Before  W1  W2  W3  W4  After  Reference Ranges  p-Values  Red blood cell count (106/μL)  4.9 ± 0.3  ND  ND  ND  ND  4.9 ± 0.2  4.5–6.2  0.8772   Hemoglobin (g/dL)  14.9 ± 0.8  ND  ND  ND  ND  14.8 ± 0.7  13.5–18.0  0.4363   Hematocrit (%)  45 ± 2  ND  ND  ND  ND  44 ± 3  40–54  0.6978  White blood cell count (μL) × 103  7.3 ± 1.2  ND  ND  ND  ND  6.9 ± 1.4  5–10  0.3353   Platelet count (×103/μL)  228 ± 55  ND  ND  ND  ND  216 ± 47  150–450  0.6816   Urea (mg/dL)  29 ± 7  43 ± 9***  34 ± 5  31 ± 11  32 ± 10  28 ± 7  15–40  0.0002   Creatinine (mg/dL)  0.9 ± 0.2  1. ± 0.1***  1.0 ± 0.1*  0.9 ± 0.1  0.9 ± 0.1  0.9 ± 0.1  0.6–1.2  0.0022   Calcium (mmol/L)  2.41 (2.37–2.45)  9.40**** (8.85–10.20)  9.60**** (9.35–9.85)  9.15**** (8.97–9.95)  9.45**** (9.22–9.67)  2.53*** (2.43–9.20)  2.23– 2.55  <0.0001   Magnesium (mg/dL)  2.0 ± 0.10  1.94 ± 0.28  1.99 ± 0.14  2.03 ± 0.21  2.1 ± 0.11  2.0 ± 0.16  1.6–2.6  0.4119   Sodium (mmol/L)  138 ± 1  137 ± 9  139 ± 6  136 ± 9  137 ± 4  137 ± 2  137–145  0.8130   Potassium (mmol/L)  4.2 ± 0.3  4.3 ± 1.1  3.9 ± 0.3  4.1 ± 0.3  4.2 ± 0.3  4.5 ± 0.2  3.6–5.0  0.1077  Creatine kinase-CK (U/L)  210 (112–298)  856** (434–1,095)  233 (138–296)  135 (94–245)  107 (95–144)  102 (80–210)  30–170  <0.0001   hs-CRP (mg/dL)  0.08 (0.03–0.27)  7.5**** (3.0–14.2)  0.65* (0.18–2.0)  0.08 (0.04–0.25)  0.10 (0.03–0.17)  0.25 (0.08–0.90)  <0.50  <0.0001  The results are presented as the mean ± SD. Values that did not follow a Gaussian distribution are presented as medians (25th–75th percentile) (Shapiro–Wilk normality test). p-Values were estimated using one-way ANOVA followed by Dunnett’s multiple comparisons test or Kruskal–Wallis test, as appropriate. p-Values in bold characters denote statistically significant differences. W, week; hs-CRP, high-sensitivity C-reactive protein; ND, not determined. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 compared with values before training. Evaluation of Skin Microvascular Flow and Reactivity The skin iontophoresis of ACh induced significant increases in microvascular CVC both before (p < 0.0001) and after (p < 0.0001) training; however, microvascular vasodilatory responses were markedly and significantly reduced after training, compared with values obtained before training (Fig. 1). Figure 1. View largeDownload slide The effects of skin acetylcholine (ACh) iontophoresis on the cutaneous microvascular conductance (CVC, expressed in arbitrary perfusion units [APUs] divided by mean arterial pressure in mmHg) of the volunteers (n = 14) before and after strenuous exercise training. The values are expressed as means ± SD and were analyzed using either two-tailed paired Student’s t-tests or repeated measures analysis of variance, when appropriate. **p < 0.01; ***p < 0.001; ****p < 0.0001 compared with values measured before exercise training. #p < 0.05; ##p < 0.01; ###p < 0.001; ####p < 0.0001 compared with baseline values. Figure 1. View largeDownload slide The effects of skin acetylcholine (ACh) iontophoresis on the cutaneous microvascular conductance (CVC, expressed in arbitrary perfusion units [APUs] divided by mean arterial pressure in mmHg) of the volunteers (n = 14) before and after strenuous exercise training. The values are expressed as means ± SD and were analyzed using either two-tailed paired Student’s t-tests or repeated measures analysis of variance, when appropriate. **p < 0.01; ***p < 0.001; ****p < 0.0001 compared with values measured before exercise training. #p < 0.05; ##p < 0.01; ###p < 0.001; ####p < 0.0001 compared with baseline values. The basal skin microvascular flow values were not different before (16.2 ± 4.0 APU) or after training (15.5 ± 3.4 APU; p = 0.4191). Conversely, baseline microvascular conductance, which represents vasodilatory capability, was reduced after training from 0.22 ± 0.06 to 0.17 ± 0.04 APU/mmHg (p = 0.0057; Fig. 2A). Figure 2. View largeDownload slide (A) Baseline values, (B) peak effects, and (C) the maximal increases of the cutaneous microvascular conductance (CVC, expressed in arbitrary perfusion units [APUs] divided by mean arterial pressure in mmHg) elicited by skin ACh iontophoresis before and after exercise training. (D) The area under the curve of the microvascular vasodilation induced by skin ACh iontophoresis before and after exercise training. The amplitudes of the ACh responses are expressed as the peak CVC values minus the baseline CVC values. The values are expressed as means ± SD and were analyzed using two-tailed paired Student’s t-tests. Figure 2. View largeDownload slide (A) Baseline values, (B) peak effects, and (C) the maximal increases of the cutaneous microvascular conductance (CVC, expressed in arbitrary perfusion units [APUs] divided by mean arterial pressure in mmHg) elicited by skin ACh iontophoresis before and after exercise training. (D) The area under the curve of the microvascular vasodilation induced by skin ACh iontophoresis before and after exercise training. The amplitudes of the ACh responses are expressed as the peak CVC values minus the baseline CVC values. The values are expressed as means ± SD and were analyzed using two-tailed paired Student’s t-tests. The peak CVC values obtained during iontophoresis of ACh before training (0.84 ± 0.22 APU/mmHg) were also markedly reduced after training (0.47 ± 0.11 APU/mmHg; p < 0.0001; Fig. 2B). The increase (peak minus baseline) in CVC induced by ACh was also reduced after training (from 0.61 ± 0.20 to 0.29 ± 0.03 APU/mmHg; p < 0.0001; Fig. 2C). Moreover, area under the curve of endothelium-dependent microvascular vasodilation induced by ACh measured before training 18,510 ± 4,538 APU/s was reduced to 13,958 ± 3,733 APU/s (p = 0.0047; Fig. 2D) after training. The peak CVC values measured during post-occlusive reactive hyperemia (PORH) before training (0.94 ± 0.72 APU/mmHg) were also markedly reduced after training (0.71 ± 0.14 APU/mmHg; p = 0.0037; Fig. 3). The increase (peak minus baseline) in CVC induced by PORH was also reduced after training (from 0.61 ± 0.22 to 0.45 ± 0.13 APU/mmHg; p = 0.0149; Fig. 3). We did not identify any outlier in the data regarding microvascular reactivity. Figure 3. View largeDownload slide (A) Peak effects and (B) the maximal increases of the cutaneous microvascular conductance (CVC, expressed in arbitrary perfusion units [APUs] divided by mean arterial pressure in mmHg) elicited by post-occlusive reactive hyperemia (PORH) before and after exercise training. The amplitudes of the PORH responses are expressed as the peak CVC values minus the baseline CVC values. The values are expressed as means ± SD and were analyzed using two-tailed paired Student’s t-tests. Figure 3. View largeDownload slide (A) Peak effects and (B) the maximal increases of the cutaneous microvascular conductance (CVC, expressed in arbitrary perfusion units [APUs] divided by mean arterial pressure in mmHg) elicited by post-occlusive reactive hyperemia (PORH) before and after exercise training. The amplitudes of the PORH responses are expressed as the peak CVC values minus the baseline CVC values. The values are expressed as means ± SD and were analyzed using two-tailed paired Student’s t-tests. Capillary Density The basal mean skin capillary density of the volunteers was not different before and after training (before 95 ± 8 and after 94 ± 8 capillaries/mm2; p = 0.6489; Fig. 4). During PORH, which is considered as endothelium-dependent capillary recruitment, the capillary density was significantly reduced after training (before 101 ± 9 and after 95 ± 8 capillaries/mm2; p = 0.0007). Consequently, the increase in capillary number observed during PORH before training (5.5 [0.8–11.5] capillaries/mm2) was not detected after training (0.7 [−4.8 to 5.2] capillaries/mm2; Fig. 4). We did not identify any outlier in the data regarding skin capillary density. Figure 4. View largeDownload slide Functional capillary density at baseline (BASAL) and during post-occlusive reactive hyperemia (PORH) before and after exercise training. Inset: Increase in capillary number after PORH before and after exercise training. The values are expressed as means ± SD or medians (25th–75th percentile) and were analyzed using either two-tailed paired Student’s t-test or Wilcoxon-matched pairs signed rank test, as appropriate. **p < 0.01 compared with basal values before training. ###p < 0.001 compared with PORH values before training. Figure 4. View largeDownload slide Functional capillary density at baseline (BASAL) and during post-occlusive reactive hyperemia (PORH) before and after exercise training. Inset: Increase in capillary number after PORH before and after exercise training. The values are expressed as means ± SD or medians (25th–75th percentile) and were analyzed using either two-tailed paired Student’s t-test or Wilcoxon-matched pairs signed rank test, as appropriate. **p < 0.01 compared with basal values before training. ###p < 0.001 compared with PORH values before training. Discussion The main findings of the present study were that strenuous exercise and gas exposure, related to special military training results for riot control in (1) marked decrease in endothelium-dependent systemic microvascular reactivity, (2) a reduction of endothelium-dependent systemic capillary recruitment, and (3) transient but significant increases in plasma urea, creatinine, calcium, and in the enzyme creatine kinase. It is well known that physical training is capable of inducing both beneficial and harmful alterations on vascular endothelial function.25,26 The evaluation of vascular reactivity is usually tested by means of the assessment of the capacity of endothelium-dependent vasoactive agents to induce vasodilation in resistance vessels, as physiologic endothelium-dependent vasodilation is essentially dependent on adequate NO bioavailability. On the other hand, endothelial dysfunction may result from the scavenging of NO by oxidized lipoproteins or superoxide radicals, which thus shortens the half-life of NO and thereby impairs NO-dependent vasodilation.26,27 High-intensity exercise – independently of the absolute intensity of exercise – may induce significant blunting of vascular endothelial function, by exposing the blood vessels to repeated bouts of oxidative stress, which is accompanied by a decreased level of plasma antioxidants and an increased production of free radicals.6,28,29 For instance, although increased in the plasma, the levels of reduced glutathione are decreased in the skeletal muscle of rats after exhaustive exercise.30 In fact, the protective effect of moderate-intensity exercise training, mainly represented by increased bioavailability of NO and consequent quenching of reactivity oxygen species (ROS), is lost when the increasing intensity of exercise results in massive increases of oxidative stress.5 We and others have already reported on the beneficial effects of moderate-intensity aerobic exercise on microvascular endothelial function both in animal31–33 and in human studies.34–37 In contrast, the present study showed that a 5-wk strenuous exercise training in a military setting, in young healthy individuals, induces a marked reduction of endothelium-dependent systemic microvascular reactivity. In fact, microvascular relaxation curves resulting from skin transdermal iontophoresis of acetylcholine showed a reduction of approximately 30% after training. Moreover, capillary recruitment induced by post-occlusive reactive hyperemia, which is dependent on physiological endothelium-dependent microvascular endothelial function, was blunted after military training. These results confirm the previously stated hypothesis that vascular function may be paradoxically impaired in healthy individuals by high levels of exercise.1 Exhaustive exercise may cause muscle damage, which can be evidenced as an increase in the plasma activity of cytosolic enzymes such as creatine kinase or lactate dehydrogenase, at least in part due to the increased production of free radicals.28 In the present study, plasma levels of creatine kinase were markedly increased (four-fold) in the first week of training and decreased thereafter. Chronically repeated small muscle injuries may trigger a systemic inflammatory response,38–41 while skeletal muscle lesions of greater magnitude caused by physical exercise are known as exertional rhabdomyolysis.42 In the present military training, one of the volunteers developed exertional rhabdomyolysis on the second day of training accompanied by highly increased plasma levels of creatine kinase (data not shown). Intense physical exercise is capable of acutely promoting muscle injuries associated with rupture of the cell membrane integrity, inflammation, and delayed-onset muscle pain.43 Moreover, this phenomenon is believed to occur predominantly during unusual strenuous exercise carried out uninterruptedly for several minutes, being associated with high production of reactive oxygen species.44 When physical exercise is prolonged and accomplished under adverse climatic conditions of high temperature and humidity, as in the military training described here, extreme exertion and dehydration may be associated with transitory skeletal muscle ATP deficiency.45 The irritant agents used in the military training described here, specifically tear gas and pepper spray, might also be putative contributors to detrimental vascular effects, as they activate the transient receptor potential (TRP) superfamily of cation channels, including the receptor subtypes vanilloid-1 and Ankyrin-1 (TRPV1 and TRPA1, respectively).15 TRP channels are involved in normal and pathophysiological responses in the blood vessels, mainly vascular smooth muscle contractility and endothelium-dependent vasodilation.46–48 Moreover, TRP can also be activated by substances produced at high levels during oxidative stress, such as ROS.47 Nevertheless, even if the activation of TRP channels is known to have cardiovascular effects, their specific roles in cardiovascular function remain to be elucidated.47 In conclusion, it is not prudent to speculate on the putative effects of the irritant agents on the microvascular effects described in the present report. The plasma levels of high-sensitivity C-reactive protein, an important clinical marker of systemic inflammation, were also markedly increased by military training, thus confirming the presence of a low-grade systemic inflammatory process described above. This alteration was accompanied by a five-fold increase in the plasma levels of calcium during the 4 wk of military training. One reasonable explanation for the increase in plasma calcium levels could be the hemoconcentration that probably occurred during the high-intensity training performed under unfavorable climatic conditions. In this context, it has already been reported that the increases in calcium plasma levels are accompanied by hemoconcentration after strenuous exercise in healthy volunteers.49,50 In conclusion, the present study showed that a 5-wk, high-intensity military training, performed in unfavorable climatic conditions, induces marked systemic microvascular dysfunction, mainly characterized by reduced endothelium-dependent microvascular vasodilation and blunted capillary recruitment. Acknowledgments The authors wish to thank nurse Marcio Marinho Gonzalez for his excellent technical assistance. We also wish to thank Major Manoel Gomes da Silva Neto de Queiroz, Captain Alexandre Fontoura da Silva, and Felipe Martins Peçanha of the Special Aeronautical Infantry Battalion of Rio de Janeiro, Brazil. Funding Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; ET, grant no. 303328/2013-4), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ; Eduardo Tibirica, grant no. 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Military MedicineOxford University Press

Published: Mar 14, 2018

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