The effects of different remote ischemic conditioning on ischemia-induced failure of microvascular circulation in humans

Abstract

BACKGROUND:

Intermittent ischemia in remote tissues can be applied before ischemic injury, during ischemic injury or at the beginning of reperfusion of an index organ ischemia. The aim of this study was to investigate the effect of Remote Ischemic Conditioning (RIC) of the leg on changes in ischemia-induced the microvascular functions of the arm.

MATERIAL AND METHODS:

Ischemic microvascular injury was induced by arm ischemia (20 min) and reperfusion in healthy, nonsmoker, male volunteers (ischemia group-ISC, n: 9). In another group of volunteers, to investigate the effects of remote organ ischemic conditioning 5 cycles of reperfusion followed by leg ischemia (each lasting 60 seconds) were applied either before (preRIC, n:11), or during (perRIC, n:12) or immediately after (postRIC, n:9) 20 minutes of arm ischemia. The microvascular flow of arm was assessed before and after ischemia using iontophoresis of the endothelium-derived nitric oxide (NO) releaser acetylcholine (ACh) and the endothelium-independent NO donor sodium nitroprusside (SNP). Changes in microvascular blood flow were measured using Laser Doppler imaging. The plasma level of biomarkers related to endothelial function such as nitric oxide (NO), asymmetric dimethylarginine (ADMA), total antioxidant capacity (TAC) and hydrogen sulphide (H₂S) were measured.

RESULTS:

No difference was determined between the groups in terms of age, BMI or blood biochemicals reflecting cardiovascular status. ACh caused a rise in microvascular blood flow in a charge dependent manner. The ACh-induced flow increase was not significantly depressed by ischemia and not affected by any of the types of RIC in the study subjects. The increase in SNP-induced microvascular flow was significantly decreased in the ISC, perRIC and postRIC groups, but not in the preRIC group. Plasma levels of NO, ADMA, TAC and H₂S were not changed by ischemia and RIC.

CONCLUSION:

These results suggested that microvascular perfusion of human forearm skin was elevated by either endothelium or drug-derived NO. The effect of ischemia and RIC on NO-induced flow increase was affected differently by different applications in the healthy young individuals. These complicated results are taken into consideration in experimental and therapeutic interventions.

Keywords

Ischemia-reperfusion remote ischemic conditioning microcirculation endothelial dysfunction

1 Introduction

Ischemic cardiovascular events remain the leading cause of morbidity and mortality in British and American people [1 –3]. Current treatment for acute ischemic events is timely reperfusion of the obstructed artery. However, reperfusion of the ischemic tissues itself will also induce injury, commonly referred to as ischemia- reperfusion (I/R) injury [4].

The prevention of I/R injury in cardiovascular diseases resulting in tissue damage has been important subject of research in the last few decades. In 1986, Murry et al. found that, unexpectedly, an I/R cycle consisting of a few brief episodes exhibited a protective effect against I/R injury in the myocardium [5]. The clinical adoption of this phenomenon, known as ischemic preconditioning, was delayed due to an inability to predict the timing of ischemia in daily practice. Ischemic preconditioning has been recognized as a major cardioprotective phenomenon. A protective technique known as Remote Ischemic Conditioning (RIC) characterized by temporary I/R episodes in the distant organ or leg has been developed in recent years [6, 7]. RIC eliminated the need for direct intervention in the heart for successful cardioprotection. In a recent study by Kolbenschlag et al. RIC has been shown to have a positive effect on cutaneous microcirculation in groups with a 10-minute long ischaemia interval [8].

The intermittent ischemia established in RIC can be applied before ischemic injury (preRIC), during ischemic injury (perRIC) or at the beginning of reperfusion (postRIC). The mechanism involved in RIC is still unclear. However, it has been suggested that these mechanisms begin with the release of humoral mediators and/or neural activation [9 –11]. It is also known that controlled reperfusion reduces tissue damage by reducing hematocrit, plasma and blood viscosity compared to full reperfusion [12]. Since the first discovery of RIC in the heart, its effectiveness has been investigated in several studies, with inconsistent results [13]. Recent studies have revealed the effectiveness of RIC against I/R injuries [14 –16]. However, the mechanism by which this is achieved and which method will give the best results are still unclear.

Endothelial cells have an important role in the regulation of vascular tone by secreting various vasoactive substances. Endothelium is particularly sensitive to I/R and dysfunction of these vital cells critically influences clinical outcomes after I/R [17]. Therefore, novel therapies to limit I/R injury as well as endothelial dysfunction are urgently needed. It is widely accepted that endothelium-derived NO represents the functional status of endothelial cells. Decreased bioavailability of NO in endothelial dysfunction is the result of inhibiting NO synthesis by I/R [18]. It has been reported that I/R-induced endothelial dysfunction is protected by RIC and NO has a central role in RIC [19 –21]. It has also been shown by Grau et al. that the cardioprotective effect of preRIC is associated with NO synthase activity and NO increase in red blood cells [22].

Although, several studies have examined the important role of NO in RIC, the effect of different RIC applications on ischemia-induced failure of NO-stimulated flow increase in the human microvascular bed has not yet been investigated. The aim of this study was to investigate the effect of leg RIC on endothelium and SNP-derived NO-induced elevation of the microvascular flow of the human forearm.

2 Methods

After approval from the institutional ethics committee, healthy young volunteers were recruited (The approval number of the ethical application: 154–4956). The study included 41 healthy, non-smoking, male subjects aged 21–28 years with no history of drug use or surgery and with no acute or chronic disease. Name and age details were recorded and informed consent was obtained from all participants. Height and weight measurements were then taken and recorded. Patients were placed in a supine position on the table where ischemia would be induced. The left arm was used for in vivo ischemia and for the assessment of microvascular functions, and the right leg for distant ischemic conditioning, as described previously [23, 24]. The right arm was used for blood specimen collection throughout the study. To induce I-R injury, a Hokanson cuff was then inflated to 50 mm Hg above SBP for 7.5 min.

The subjects were randomly seperated into 4 groups (Fig. 1).

Fig.1

Study protocol and groups. a:ischemia (ISC), b:pre-conditioning (preRIC), c:per-conditioning (perRIC), d:post-conditioning (postRIC).

Ischemia Group (ISC, n:9): A cuff placed on the left arm was inflated and left in position for 20 min. Reperfusion was then established by deflating the cuff. Another cuff was placed on the right leg in this group, but was not inflated.

Pre-Conditioning Group (preRIC; n:11): A cuff placed on the right leg was first inflated and deflated over 60 sec, five times, at 60-sec intervals. Immediately following this procedure on the leg, a cuff on the left arm was inflated and left in situ for 20 min. At the end of this period, reperfusion was initiated by deflating the cuff.

Perconditioning Group (perRIC; n:12): Ischemia was induced by inflating a cuff on the left arm, then after 10 mins, a cuff placed on the right leg was inflated and deflated over 60 sec, five times, at 60-sec intervals. At the end of this period, in other words at the end of 20-min arm ischemia, reperfusion was induced by deflating the cuff on the arm.

Post-Conditioning Group (postRIC, n:9): A cuff on the left arm was inflated and left in position for 20 min. Reperfusion was then established by deflating the cuff. Immediately after the start of reperfusion, post-conditioning was applied by a cuff placed on the right leg being inflated and deflated over 60 sec, five times, at 60-sec intervals.

2.1 Assessment of skin microvascular perfusion

To assess microvascular vasoreactivity, the endothelium-derived NO releaser acetylcholine (ACh) and the endothelium-independent sponteneous NO donor sodium nitroprusside (SNP) were both iontophoretically applied to collect measurements with a laser scanner. Iontophoresis is increasingly being used for the transdermal delivery of ACh and SNP for the assessment of microvascular reactivity. This technique is based on the fact that charged molecules migrate across the skin under the influence of an applied electrical field; thus the delivery of ionized drugs is dependent on the magnitude and duration of the applied current (current×time charge, in Coulombs). Laser Doppler imaging measures the dose-dependent changes in perfusion due to the iontophoresis.

In the present study, all subjects fasted overnight and were asked to refrain from drinking any fluids except water before the measurements, which were collected in a temperature controlled room (23 ± 1°C). With the participant lying in the supine position, the left forearm anterior surface to be studied was cleaned with an alcohol wipe and allowed to dry before applying 2 perspex iontophoresis chambers (L 611, Perimed, Jarfalla, Sweden) to the surface of the arm 15 cm proximal to the elbow using double-sided adhesive rings. Hair, broken skin and superficial veins were avoided. The anodal chamber was filled with 0.25 ml 1% (w/v) acetylcholine chloride (ACh; Sigma–Aldrich Chemicals, UK), and the cathodal chamber was filled with 0.25 ml 1% (w/v) sodium nitroprusside (SNP; 60 mg, Nipruss, Adeka, Ankara, Turkey). Simultaneous drug delivery from each chamber was controlled by a battery powered constant current iontophoresis controller (Perilont 382 power supply, Perimed, Jarfalla, Sweden).

Drug iontophoresis and gaiter area cutaneous microvascular erythrocyte flux were assessed using a cumulative dose response protocol. This protocol was followed by incremental durations of drug delivery at a fixed current. Therefore, 0.1 mA was applied for 5, 10, 20, 40 and 80 seconds, which resulted in 0.5, 1, 2, 4 and 8 milliCoulombs (mC). This protocol was chosen to avoid non-specific vasodilation and to provide effective ACh and SNP delivery, as previously described by Henricson et al [25]. Non-invasive measurement of skin perfusion was performed via a laser Doppler imager (PeriScan PIM II, Perimed, Jarfalla, Sweden) with the following parameters: wavelength, 670 nm; power, 1 mW; and beam diameter, 1 mm. The technique is based on the Doppler shift imparted to the backscattered light by the motion of blood cells in the underlying tissue. The laser scans in a raster fashion over both chambers and through the coverslips. The backscattered light is collected by photodetectors and converted into a signal that is proportional to perfusion in arbitrary perfusion (flux) units (PU) and displayed as a color-coded image on a monitor. Perfusion measurements were obtained using the imager manufacturer’s image analysis software (LDPIwin software, Perimed, Jarfalla, Sweden) by outlining a region of interest around the internal circumference of the chamber. Before administration of the drug, we recorded four baseline images over a total duration of 120 seconds without current in the absence of the iontophoresis of any drug. Each iontophoretic drug application was followed by eight laser scans of 30 seconds each. Thus, each drug application was followed by 240 seconds of scanning. A total of 44 repetitive scans were taken; the first four were used as a control (i.e., prior to current administration) and were followed by the incremental time protocol described above.

The images were reviewed by an experienced observer blinded to the clinical data and groups. Following all baseline and 0.5, 1, 2, 4, 8 mC iontophoretic applications the values of the flow increase resulting from ACh and SNP stimulation were calculated using the percentage improvement formula. (Flow increase = [(mean value of 8 calculation in each charge–basal value)/basal value]×100).

2.2 Biochemical examinations

Blood samples were collected four times, before ischemia, at the end of 20-min ischemia and at the 20th and 45th mins of reperfusion, using a temporary catheter attached to one of the right forearm veins. The blood samples were collected, centrifuged and then seperated plasmas were frozen. Analysis was performed by another researcher blinded to the group distributions. Plasma NO, ADMA, TAC and H2S levels were measured in these specimens. Plasma nitrite levels were measured as a marker of NO production. NO levels were measured using the spectrophotometric method based on the Griess reaction [26]. Plasma TAC values were measured using a previously described method based on the reduction of Cu+2 to Cu+1 by antioxidants in plasma [27]. Neocuproine was used as a chromogenic agent and was determined spectrophotometrically at a wavelength of 455 nm. Plasma H₂S levels were measured using a previously described spectrophotometric method based on methylene blue absorption rates [28]. ADMA levels were measured using ELISA kits (Immunodiagnostic A.G., Germany).

2.3 Statistical analysis

Values were expressed as mean ± SEM. Statistical analysis was performed on SigmaPlot (Systat Software Inc., USA) version 11 for Windows software. Repeated-measures of two-way ANOVA was used for testing variations between the pre and post ischemia in cutaneous perfusion; ISC and RIC groups in Δ flow and ISC and RIC groups in blood level of biomarkers. A value of p < 0.05 was regarded as statistically significant. When p values were significant, comparison was performed with the Holm-Sidak test.

3 Results

No complications developed in any subject. The research protocol was applied to all individuals until the end of the study. There was no difference between the groups in terms of age, BMI or laboratory values reflecting cardiovascular status. The demographic data and blood biochemicals of the groups are shown in (Table 1).

Table 1
Demographic data and laboratory findings according to the groups

ISC preRIC perRIC postRIC

Age (Years) 24 (22–28) 25 (23–28) 23 (22–24) 23 (21–25)

BMI 24 (23–29) 23 (22–25) 25 (19–29) 25 (24–29)

Glucose (mg/dL) 88 (77–93) 78 (70–84) 87 (77–96) 83 (70–89)

Creatinine (mg/dL) 0.91 (0.88–0.99) 0.9 (0.8–1) 0.8 (0.79–0.9) 0.9 (0.7–1)

Cholesterol (mg/dL) 170 (160–180) 170 (141–199) 157 (122–168) 159 (143–193)

HDL (mg/dL) 46 (40–48) 39 (33–44) 42 (32–53) 39 (34–45)

LDL (mg/dL) 103 (95–105) 85 (70–117) 72 (61–84) 97 (71–118)

VLDL(mg/dL) 21 (16–29) 30 (19–39) 20 (10–38) 34 (23–39)

Triglycerides (mg/dL) 103 (77–141) 149 (95–197) 100 (55–192) 170 (118–190)

Bilirubin (mg/dL) 0.7 (0.6–0.8) 0.6 (0.4–1) 0.7 (0.4–1) 0.6 (0.5–1)

Homocysteine (mmol/L) 12 (10.4–13.9) 11 (10.4–14) 11 (10.5–16.2) 13 (11.7–16.9)

Hs CRP (mg/dL) 0.6 (0.5–1.7) 0.9 (0.7–2.4) 0.8 (0.7–2.7) 0.79 (0.7–2.1)

WBC (K/mm3) 6.6 (5.5–7.5) 7.5 (6.4–8.8) 5.2 (4.9–6.9) 6.8 (4.3–8.2)

Hemoglobin (g/dL) 15.1 (14.4–16.2) 14.9 (14.7–15.3) 15.4 (14.6–16.1) 15.5 (13.7–16.2)

	ISC	preRIC	perRIC	postRIC
Age (Years)	24 (22–28)	25 (23–28)	23 (22–24)	23 (21–25)
BMI	24 (23–29)	23 (22–25)	25 (19–29)	25 (24–29)
Glucose (mg/dL)	88 (77–93)	78 (70–84)	87 (77–96)	83 (70–89)
Creatinine (mg/dL)	0.91 (0.88–0.99)	0.9 (0.8–1)	0.8 (0.79–0.9)	0.9 (0.7–1)
Cholesterol (mg/dL)	170 (160–180)	170 (141–199)	157 (122–168)	159 (143–193)
HDL (mg/dL)	46 (40–48)	39 (33–44)	42 (32–53)	39 (34–45)
LDL (mg/dL)	103 (95–105)	85 (70–117)	72 (61–84)	97 (71–118)
VLDL(mg/dL)	21 (16–29)	30 (19–39)	20 (10–38)	34 (23–39)
Triglycerides (mg/dL)	103 (77–141)	149 (95–197)	100 (55–192)	170 (118–190)
Bilirubin (mg/dL)	0.7 (0.6–0.8)	0.6 (0.4–1)	0.7 (0.4–1)	0.6 (0.5–1)
Homocysteine (mmol/L)	12 (10.4–13.9)	11 (10.4–14)	11 (10.5–16.2)	13 (11.7–16.9)
Hs CRP (mg/dL)	0.6 (0.5–1.7)	0.9 (0.7–2.4)	0.8 (0.7–2.7)	0.79 (0.7–2.1)
WBC (K/mm3)	6.6 (5.5–7.5)	7.5 (6.4–8.8)	5.2 (4.9–6.9)	6.8 (4.3–8.2)
Hemoglobin (g/dL)	15.1 (14.4–16.2)	14.9 (14.7–15.3)	15.4 (14.6–16.1)	15.5 (13.7–16.2)

No parameters were significantly different between the groups. BMI: Body mass index, HDL: High density lipoprotein, LDL: Low density lipoprotein, VLDL: Very low density lipoprotein, hs-CRP: high sensitive c reactive protein, WBC: White blood cell. Values are expressed as mean ± SEM (n = 9–12).

The administration of ACh caused an endothelium-derived NO dependent perfusion increase in a current-dependent manner (Fig. 2). ACh-stimulated flow elevation was reduced by ischemia, but pre-reperfusion values (PRE) were not statistically different from the values measured post-reperfusion (POST) in all groups. In addition, Δ values (PRE-POST) of RIC groups were not different from those of the ISC group (Fig. 2).

Fig.2

The effect of acethylcholine on skin microvascular circulation of the forearm. The ACh-stimulated endothelium-dependent increase in microvascular perfusion was not significantly decreased in the post-ischemic period in all groups. Values are expressed as mean ± SEM (n = 6–10).

SNP is a spontaneous NO donor and stimulates endothelium-independent vasodilation. The analysis of dilation induced with the administration of SNP showed that arm ischemia led to a significant impairment of flow arising with SNP in the ISC group (191.06 ± 46.85 PRE and 47.29 ± 13.33 POST for 80 mC) (p < 0.05) (Fig. 3). Similarly, PRE and POST values of the perRIC and postRIC groups were also significantly different (208.90 ± 24.19 PRE and 75.84 ± 11.01 POST in perRIC and 176.16 ± 34.89 PRE and 75.46 ± 13.03 POST in postRIC for 80 mC) (p < 0.05) (Fig. 3). There was no difference in terms of PRE and POST in the preRIC group. However, Δ values (PRE-POST) of the ISC group were not different from the Δ values of the RIC groups (Fig. 3).

Fig.3

The effect of SNP on skin microvascular circulation of the forearm. The SNP-stimulated endothelium-independent increase in microvascular perfusion was significantly attenuated in the ISC, perRIC and postRIC groups (P < 0.05). Differences from pre-ischemic values (*). Values are expressed as mean ± SEM (n = 6–10).

The blood levels of biomarkers related to endothelial functions are shown in Table 2. No significant variation was determined between the ISC and RIC groups in terms of the biomarkers NO, ADMA, TAC and H₂S plasma values at times 0 (pre-ischemia), 1 (start of reperfusion at the end of ischemia), 2 (pre-iontophoresis after reperfusion) and 3 (post-iontophoresis after reperfusion) (Table 2).

Table 2

Plasma levels of NO, ADMA, TAC and H2S

	ISC	preRIC	perRIC	postRIC
(μM)
Nitrite 0	44.31 ± 10.47	50.09 ± 5.05	46.98 ± 4.76	48.24 ± 5.47
Nitrite 1	40.16 ± 10.64	44.05 ± 5.97	44.83 ± 2.87	45.55 ± 4.98
Nitrite 2	43.84 ± 11.31	45.83 ± 3.82	44.89 ± 3.38	44.87 ± 6.21
Nitrite 3	35.83 ± 8.39	40.85 ± 4.29	47.29 ± 3.52	45.72 ± 6.41
(μM)
ADMA 0	407.17 ± 66.87	521.75 ± 50.59	495.56 ± 52.53	447.13 ± 58.36
ADMA 1	637.83 ± 177.38	523.57 ± 29.80	607.67 ± 100.26	389.00 ± 59.48
ADMA 2	489.00 ± 85.18	453.88 ± 69.04	482.38 ± 80.17	453.71 ± 44.98
ADMA 3	437.00 ± 97.78	433.00 ± 64.49	463.22 ± 74.47	425.75 ± 86.81
(μM)
TAC 0	645.88 ± 90.78	728.42 ± 41.28	671.90 ± 41.20	685.59 ± 61.61
TAC 1	628.81 ± 55.87	733.90 ± 39.11	597.98 ± 48.59	598.47 ± 38.29
TAC 2	593.68 ± 51.10	724.70 ± 39.20	611.52 ± 38.72	608.90 ± 53.81
TAC 3	590.06 ± 48.49	726.59 ± 65.02	588.81 ± 12.24	774.86 ± 95.56
(μM)
H₂S 0	77.03 ± 27.82	80.93 ± 15.24	76.40 ± 29.40	116.03 ± 34.47
H₂S 1	68.12 ± 19.30	62.67 ± 18.94	115.11 ± 27.33	73.68 ± 16.80
H₂S 2	169.65 ± 47.65	76.27 ± 23.23	207.51 ± 81.99	131.94 ± 32.60
H₂S 3	102.32 ± 33.03	94.28 ± 19.62	148.87 ± 41.97	106.47 ± 28.12

The plasma Nitrite (NO), ADMA, TAC and H₂S levels were not significantly different within the groups (0:pre-ischemia, 1:starting of reperfusion at the end of ischemia, 2:pre-iontophoresis after reperfusion and 3:post-iontophoresis after reperfusion. Values are expressed as mean ± SEM (n = 6–10).

4 Discussion

For the last two decades, studies have focused on identifying mechanism of protective effect of the RIC methods and their targets. In addition, it has been demonstrated that the RIC effect occurred due to neural, humoral, and occasionally both mechanisms [7]. Vascular endothelium is an important effector organ for the investigation of the I/R injury and protective effect of the RIC methods because it is a functional organ and it can give insight regarding the functions of all the organs. Therefore, in our study, we aimed to investigate the effect of IR damage and preventive effects of different RIC methods, which have complex mechanisms, through microvascular endothelial functions.

The results of this study demonstrate that despite a remarkable trend in diminished microvascular vasodilation after arm reperfusion, there was no significant difference between pre-reperfusion and post-reperfusion values for endothelium-derived NO dependent vasodilation in ischemia group. Also, all three remote ischemic conditioning techniques did not cause and significant change in vasodilation response. On the other hand, arm ischemia led to diminished vasodilation response in endothelium-independent vasodilation and only preconditioning was associated with some amelioration of postischemic vasodilation response. When effects of ischemia and remote conditioning were evaluated using biomarkers related to endothelial functions, there was no difference in terms of either worsening or improvement in endothelial functions using this experimental model.

Several studies in different models demonstrated that I/R caused endothelial dysfunction, which can be ameliorated by different ischemic conditioning techniques. Loukogeorgakis et al., using brachial artery flow-mediated dilation (FMD), showed that I/R caused endothelial dysfunction, which was prevented by postRIC applied as 10-second cycles of reperfusion/ischemia and 30-second cycles of reperfusion/ischemia immediately at the onset of reperfusion [29]. Kharbanda et al. made assessments with radial artery FMD and reported that preRIC (three 5-minute episodes of ischemia) prevented endothelial dysfunction and neutrophil activation [24]. However, Dragoni et al., using radial artery endothelium-dependent FMD, showed that postRIC did not limit post-ischemic endothelial dysfunction in a human in vivo fore-arm ischemia model [30]. Such studies investigating the effect of I/R injury and RIC on I/R evaluated macrovascular endothelial functions and showed impairment after I/R in conduit arteries using FMD method as well as prevention of such impairment with some RIC methods.

Although our study seemed to have a similar methodology with a limited number of previous studies on humans, it differed from them in some aspects. As far as we know, this is the first study to evaluate the protective effect of I/R damage and different RIC methods on the microvascular system using iontophoretic laser Doppler flowmeter. In our study, we did not observe young adult male subjects suffer any impairment in endothelium-dependent microvascular function after I/R. Therefore, we could not show the protective effect of RIC applications. This may be due to the fact that free radicals in the macrovascular compartment after acute I/R can not be fully transmitted to the microvascular circulation by the precapillary sphincter. The precapillary sphincter is a small cuff of smooth muscle that is usually not innervated but is very responsive to local tissue conditions. Relaxation or contraction of the precapillary sphincter may modulate tissue blood flow [31]. The fact that our group’s previous study showed that microvascular functions were disrupted particularly in elderly and chronic peripheral arterial patients suggest that precapillary sphincter function plays a significant role in ischemic events [32].

In our study, the sensitive structure of ACh, which is highly labile and affected by external environment, may also have affected endothelium-dependent relaxation responses. However, the lack of any changes in plasma biomarkers after I/R has put us away from the idea that ACh liability is the reason for not disrupting ACh responses. Being no change in endothelium-dependent relaxation responses may also be related to the duration of ischemia and reperfusion. Prolonged ischemia durations may be a contributing factor in the transmission of the impairment in macrovascular functions to the microvascular endothelial system. Thus, the ACh-dependent responses we examined in the 45th minute of reperfusion may be deteriorating in the late period.

Interestingly, in our study, we found deterioration after I/R in SNP relaxation responses, which are exogeneous NO sources. In fact, impairment in SNP responses without deterioration in ACh-dependent responses after I/R suggests that a different mechanism comes into play. SNP causes strong vasodilatation in the environment due to spontaneous release of NO. Thus SNP-induced vasodilation is much more potent than endothelium derived endogeneous NO stimulated by ACh. However, endogeneous or exogeneous formed NO has a very short half-life. The intravascular half-life of NO is approximately 2 milli second, while the extravascular half-life will range from 0.09 to >2 seconds [33]. On the other hand, spontaneous conversion of SNP to NO in the body is dependent on a redox modulation. Thus, oxidative stress induced by I/R may impair SNP-induced endothelium-independent relaxation by inhibiting the conversion of SNP into NO [34, 35]. These results suggest that because of oxidative stress, the cutaneous microvascular circulation in ischemia is differently affected from endogeneous NO releaser and exogeneous NO donor.

Previous studies showed that RIC methods improve macrovascular endothelial functions in I/R injury [30, 32]. However, we could not clearly assess the effect of RIC methods on I/R injury in our study of microvascular responses. This may be explained by the masking of the protective effect of the RIC methods in the absence of any deterioration of the ACh responses. In fact, the occurrence of PreRIC’s protective effect on impaired SNP responses after I/R supports this hypothesis. However, the results of this study show that arm ischemia and RIC in young, healthy people have no effect on plasma NO, ADMA, TAC, and H2S levels. Therefore, it can be assumed that the extremities will not be affected by ischemia and RIC in a young, healthy person as regards to biomarkers associated with endothelial functions in the systemic circulation.

The limitations of our study may include the limited number of subjects in the groups, the timing of evaluation of the cutaneous microvascular response following reperfusion, and the magnitute of remote conditioning as well as the sensitivity and accuracy of the method that we used to investigate microvascular function. To reduce the high rate of deviation in our results required a larger study population, but disturbing nature of ischemic method limited enrolling number of volunteers.

In conclusion, all these findings suggest that the microvascular circulation dynamics are different in contrast to studies evaluating the effects of I/R injury and RIC on macrovascular endothelial functions. In healthy young adults, we showed that SNP-induced cutaneous microvascular responses are suppressed while endothelium-based NO-induced responses did not manifest any changes. In addition, we showed in this young adult population that blood levels associated with endothelial function of biological markers are not affected by ischemia and RIC. These findings may have important implications for the comparison of young-old and healthy patient skin microcirculation stimulated by endogeneous and exogeneous NO in ischemia and RIC situations. More studies are needed to clarify the effects of I/R injury and RIC methods which have quite complex mechanisms on microvascular functions.

Conflict of interest

The authors have no conflict of interest.

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