UPPER EXTREMITY ISCHEMIA IS SUPERIOR TO LOWEREXTREMITY ISCHEMIA FOR REMOTE ISCHEMIC CONDITIONINGOF ANTERO–LATERAL THIGH CUTANEOUS BLOOD FLOW

J. KOLBENSCHLAG,1* A. SOGORSKI,1 K. HARATI,1 A. DAIGELER,1 A. WIEBALCK,2 M. LEHNHARDT,1

N. KAPALSCHINSKI,1 and O. GOERTZ1

Remote ischemic conditioning (RIC) is known to improve microcirculation in various settings, but little is known about the impact of theamount of ischemic tissue mass or the limb itself. Since ischemia and subsequent necrosis of flaps is one of the most dreaded complica-tions in reconstructive surgery, adjuvant methods to improve microcirculation are desirable. We therefore performed a randomized trial tocompare the effect of arm versus leg ischemia for RIC of the cutaneous microcirculation of the antero–lateral thigh. Forty healthy volun-teers were randomized to undergo 5 min of ischemia of either the upper or lower extremity, followed by 10 min of reperfusion.Ischemiawas induced by a surgical tourniquet applied to the proximal limb, which was inflated to 250 mmHg for the upper and 300 mgHg for thelower extremity. This cycle was repeated a total of three times. Cutaneous microcirculation was assessed by combined laser dopplerspectrophotometry on the antero–lateral aspect of the thigh to measure cutaneous blood flow (BF), relative hemoglobin content (rHb), andoxygen saturation (StO2). Baseline measurements were performed for 10 min, after which the ischemia/reperfusion cycles were begun.Measurements were performed continuously and were afterwards pooled to obtain a mean value per minute. Both groups showed signifi-cant increases in all three measured parameters of cutaneous microcirculation after three cycles of ischemia/reperfusion when comparedto baseline (BF: 95.1% (P<0.001) and 27.9% (P 5 0.002); rHb: 9.4% (P<0.001) and 5.9% (P<0.001), StO2: 8.4% (P 5 0.045) and9.4% (P<0.001). When comparing both groups, BF was significantly higher in the arm group (P 5 0.019 after 11 min., P 5 0.009 after45 min). In conclusions, both ischemic conditioning of the upper and lower extremity is able to improve cutaneous BF on the ALT donorsite. However, RIC of the upper extremity seems to be a superior trigger for improvement of cutaneous BF. VC 2014 Wiley Periodicals, Inc.Microsurgery 00:000–000, 2014.

Tissue ischemia and the consecutive reperfusion injury

remain a major issue in medicine. Impaired tissue perfu-

sion can lead to necrosis and subsequent flap loss or

organ dysfunction, which can be further accentuated by

the so-called reperfusion injury.

Strategies to mitigate these effects have been

researched in various fields, most prominently in cardiol-

ogy. In 1986, Murry et al. showed that brief episodes of

coronary ischemia preceding an actual ischemic insult

were able to reduce the infarct size in a canine model.1

Seven years later, Przyklenk et al. found that ischemic

conditioning could also be applied at a remote vascular

bed to protect the myocardium.2 Thus, the term remote

ischemic conditioning (RIC) was coined and the way to

the clinical application of this technique was paved. In

addition to direct or remote ischemia, its timing in rela-

tion to the ischemia-reperfusion-injury lead to the con-

cepts of pre-, per-, and post-conditioning. The

mechanisms of these modalities are not yet fully under-

stood, but humoral factors like NO, TNF-a and reactive

oxygen species as well as neuronal factors seem to have

a great impact.3–5 The technique of RIC have found clin-

ical applications, most prominently in cardiac surgery,

percutaneous coronary interventions, and vascular

surgery.6–8

In reconstructive surgery, ischemia and reperfusion

injuries are the paramount problems associated with sur-

gical flaps in general and especially with free microvas-

cular transplants. While total flap loss rates are low and

are most often caused by technical issues or thrombosis

of the pedicle in coagulopathies, partial flap losses like

tip necrosis due to insufficient perfusion still pose a

major threat to the reconstructive outcome.9–11

There is a growing body of evidence that RIC can

improve the perfusion and therefore survival of surgical

flaps.3,12–17 However, studies considering the microcircula-

tion of flaps or potential flap donor sites in humans are

rare. To our knowledge, Kraemer et al. were the only

researchers to date to look into the effects of RIC on the

microcirculation of human skin at a potential flap donor

site.18 Also, while both upper and lower extremity were

used for RIC in various studies, their specific influence and

the impact of the amount of ischemic tissue on the effect

of RIC remains unclear.19,20 While a recent study found no

significant difference between the upper and lower extrem-

ity for remote ischemic postconditioning in patients under-

going percutaneous coronary intervention, the influence of

the extremity and therefore also the amount of ischemic tis-

sue on the cutaneous microcirculation remains unclear.21

1Department of Plastic Surgery, BG University Hospital Bergmannsheil,Hand Surgery, Burn Center, Ruhr University, Bochum, Germany2Department of Anesthesiology and Pain Therapy, Ruhrland Klinik, Univer-sity Hospital Essen, Essen, Germany.

*Correspondence to: Jonas Kolbenschlag, MD, BG University Hospital Berg-mannsheil, Department of Plastic Surgery, Burn Center, B€urkle-de-la-Camp-Platz 1, 44789 Bochum, Germany. E-mail: jonaskolbenschlag@gmail.comJ.K. and A.S. contributed equally to this work.

Received 23 June 2014; Revision accepted 17 September 2014; Accepted22 September 2014

Published online 00 Month 2014 in Wiley Online Library(wileyonlinelibrary.com). DOI: 10.1002/micr.22336

� 2014 Wiley Periodicals, Inc.

Therefore, we aimed to assess the influence of the

ischemic extremity and the ischemic tissue mass on the

acute RIC effects on the cutaneous microcirculation of a

common free flap donor site.

MATERIAL AND METHODS

After approval from the institutional ethics commit-

tee, healthy young volunteers were recruited. All subjects

were non-smokers without any history of known illnesses

or current medication. After screening for exclusion fac-

tors and obtaining the participants written informed con-

sent, a total of 40 volunteers were included in this study.

The demographic data for both groups is shown in Table

1, the included participants were between 19 and 40

years of age, with an BMI ranging from 18.8 to 30.9.

The participants were randomized into two groups:

one group underwent RIC of the leg, the other group of

the upper extremity.

Before measurements and conditioning, all partici-

pants were placed in a supine position in a temperature-

controlled room (21�C) without direct light exposure.

After resting for 15 min, blood pressure was measured

on the right upper arm. After this, baseline continuous

measurements were taken using the O2C device as

described below. These baseline measurements were per-

formed with the deflated tourniquet already attached to

the respective extremity to serve as an additional intrain-

dividual control for potential influences from the environ-

ment or the tourniquet itself. In both groups, RIC began

after those baseline measurements by occluding the blood

flow (BF) to either the upper or lower extremity for 5

min using surgical tourniquets. To achieve this occlusion,

250 mmHg, respectively 300 mmHg of pressure were

applied in the upper and lower extremity group. In all

participants, ischemia was verified by palpation and

doppler probe measurements of the dorsalis pedis artery

or the radial artery, respectively.

This phase of limb ischemia was then followed by a

reperfusion phase of 10 min before the next cycle was

applied, up to a total of three ischemia/reperfusion cycles

in both groups. Recordings were performed until the end

of the third reperfusion phase. All measurements were

performed by the same investigator.

In all cases, ischemia was applied to the extremity

contralateral to the leg on which the anterior lateral thigh

measurements were performed.

Ischemia was tolerated well by all but one partici-

pant. Because of increased discomfort during the second

leg ischemia, measurements had to be discontinued for

this volunteer. The data of this participant was removed

and replaced by measurements from an additional

participant.

Combined Laser Doppler Spectrophotometry

Continuous measurements of cutaneous blood flow

(BF in arbitrary units, AU), relative hemoglobin concen-

tration (rHb in AU) and oxygen saturation (StO2 in %)

were performed using the O2C device (LEA Medizin-

technik, Gießen, Germany).

This device applies combined laser doppler spectro-

photometry to simultaneously and continuously assess the

BF, the relative hemoglobin content (rHb) and the oxy-

gen saturation (StO2) in a depth of 2 mm, therefore rep-

resenting cutaneous microcirculation. The probe was

placed on the antero–lateral aspect of the thigh, at the

potential donor site of the antero–lateral thigh flap

(ALT). Its exact location was determined by drawing a

connecting line between the lateral patella and the ante-

rior superior iliac spine, with the probe being placed

halfway between the two anatomical landmarks. It was

taped to the leg in a standardized manner as proposed by

the manufacturer.

Measurements were performed continuously and were

afterwards pooled to obtain a mean value per minute to

correct for movement artifacts and other potential

influences.

Statistical Analysis

The extremity weight was calculated using the correc-

tion of the BMI formula for amputees as proposed by

Tzamaloukas et al.22 Statistical analysis was performed

using SPSS statistical package version 22.0 (IBM Corpo-

ration). The values for STO2 and rHb showed a normal

curve of distribution, therefore paired t-tests were used

for comparison to baseline and between groups. For BF,

data did not show a normal curve of distribution, there-

fore a nonparametric test was chosen to compare means

(Wilcoxon-rank-test) to baseline and between groups.

Results are displayed as mean 6 standard deviation (SD)

where applicable.

In addition to the absolute values, the relative

changes over the course of time are given as percentage

increase as compared to baseline (D%). An P-value of

<0.05 was considered to be statistically significant.

Table 1. Comparison of Demographic Data Between the Two

Groups

ARM LEG P

Age 26.25 6 4.76 23.95 6 2.65 0.069

Height 177.75 6 9.90 179.00 6 8.37 0.669

weight 72.70 6 14.02 77.70 6 12.85 0.247

BMI 22.80 6 2.67 24.00 6 2.79 0.162

Syst. BP 118.25 6 11.15 119.25 6 8.93 0.756

Dia. BP 76.75 6 4.94 75.00 6 7.43 0.386

Ext. Weight 4.82 6 0.92 14.55 6 2.40 <0.001

Male/female 14/6 6 0.47 15/5 6 0.44 0.731

BMI, body mass index, systl; BP, systolic blood pressure, dia; BP, diastolicblood pressure; ext. Weight, extremity weight.

2 Kolbenschlag et al.

Microsurgery DOI 10.1002/micr

RESULTS

There were no significant differences between the

two groups regarding age, BMI, blood pressure, or sex.

As expected, the calculated extremity weight was about

three times as high for the leg group, resulting in a sig-

nificant difference (P< 0.001, Table 1).

Blood Flow

Both groups started with a comparable BF without

significant differences. In the arm group, the BF was

already significantly improved after ten minutes when

compared to baseline (20.4% increase, P 5 0.048). It kept

rising throughout the ischemia/reperfusion cycles to end

up at a 95.1% increase after 45 min when compared to

baseline (P< 0.001). In the leg group, a significant

improvement in BF was first achieved after 25 min

(17.8% increase, P 5 0.006). It stayed significantly ele-

vated when compared to baseline, leading to a 27.9%

increase after 45 min (P 5 0.002). The first significant

difference between the two groups occurred at eleven

minutes, 6 min into the first reperfusion phase. Here, the

relative increase in flow for the arm group was 26.8%

and 10.7% for the leg group (P 5 0.019). After all three

ischemia/reperfusion cycles the difference between the

two groups became even more evident (95.1% increase

vs. 27.9%, P 5 0.009; Fig. 1, Table 2).

Relative Hemoglobin Content

There was no significant difference in the starting

values for both groups. Significant elevation of rHb was

found during the first reperfusion phase, at 7th min seven

in both groups (arm: 2.2% increase vs. baseline,

P 5 0.002; leg: 1.5% increase, P 5 0.009).

Over the course of time, rHb increased steadily in

both groups, resulting in a 9.4% increase for the arm and

5.9% increase and leg group after 45 min. Although

these increases were significant when compared to base-

lines (P< 0.001 for both), they did not differ signifi-

cantly between the two groups (Fig. 2, Table 3).

Oxygen Saturation

As for the parameters discussed above, the starting

values for both groups did not differ significantly. For

the leg group, a significant increase in SO2 when com-

pared to baseline was observed after 8 min (2.9%

increase, P 5 0.009). In the arm group, the first signifi-

cant increase of SO2 was seen only after 45 min (8.4%

increase, P 5 0.045). Although the increase in SO2

occurred earlier and more pronounced in the leg group,

there was no significant difference between the two

groups over the course of time (P 5 0.884) (Fig. 3, Table

4).

DISCUSSION

The combined laser doppler spectrophotometry uti-

lized in this study has been regularly used.18,23–28

As shown in previous works, RIC is capable of

improving the cutaneous microcirculation.18 This effect

was most pronounced for the BF, which was increased

Figure 1. Course of cutaneous blood flow in AU over time for both

groups. The five minutes of ischemia are grayed out. Note the ear-

lier significant increase in flow for the arm group (P 5 0.048 after

10 min vs. P 5 0.006 after 25 min) and the significant higher blood

flow in the arm group when compared to the leg group after three

cycles of ischemia/reperfusion (*P 5 0.009 after 45 min).

(AU 5 arbitrary units).

Table 2. Comparison of the Cutaneous Blood Flow Over the Course of Time for Both Groups

Time

ARM LEG

P ARM vs. LEGAU D% P vs. BL AU D% P vs. BL

Baseline(BL) 21.30 6 12.58 – – 26.00 6 21.49 – – –

10 min 25.64 6 18.67 20.4 0.048* 28.50 6 26.96 9.6 0.099 0.071

11 min 27.01 6 19.11 26.8 0.006 28.77 6 26.61 10.7 0.062 0.019a

15 min 29.39 6 21.76 38.0 0.001 29.34 6 28.39 12.8 0.100 0.019

25 min 33.05 6 25.74 55.2 0.001 30.62 6 27.69 17.8 0.006a 0.014

30 min 35.76 6 25.96 67.9 < 0.001 31.47 6 28.36 21.0 0.006 0.005

45 min 41.56 6 34.17 95.1 < 0.001 33.26 6 27.89 27.9 0.002 0.009

AU, abitrary units; D%, is the percentage increase versus baseline measurements.aMarks the first point in time at which there was a significant increase versus baseline or a significant difference between groups.

RIC Extremity 3

Microsurgery DOI 10.1002/micr

nearly twofold in the arm group and about 1.3-fold in

the leg group. The increases seen in rHb and SO2 were

less prominent, but nonetheless statistically significant.

This matches the findings of Kraemer et al. except for

the rHb, which showed a significant reduction during the

second reperfusion phase, but no significant difference

after 50 min.18

The fact that BF was the most improved parameter

fits in with the previous findings that describe increased

flap survival, since an increased BF leads to a better

nutritional supply. Cutaneous BF is influenced by a num-

ber of elements such as the autonomous nervous system

and humoral factors and can therefore be controlled and

modulated in various ways.29 While cutaneous BF is sub-

jected to direct control, the changes in rHb and SO2

result from the interaction between the BF and the per-

fused tissue. After the increase of BF, the vascular bed

receives an additional inflow of hemoglobin and therefore

an increased oxygen supply. Since the measured parame-

ters primarily represent the venous system, these values

depict the nutritional state of the local tissue. As the met-

abolic demand of the local tissue was not significantly

altered in our study, the increased inflow of hemoglobin

resulted in a slight increase in SO2 and rHb. In tissue

with increased metabolic demand, the SO2 would be

reduced due to increased oxygen consumption. While the

SO2 was more than sufficient in the healthy volunteers

examined in this study, in a surgical pedicled or free flap

the distal part might already have a shortage of oxygen

supply due to the compromised vascular network, result-

ing in a lower SO2 and might therefore benefit from this

additional oxygen supply. Therefore, RIC might success-

fully be applied in flap surgery. However, one must take

into account that this study was conducted on a potential

donor site with an intact vascular bed.

By raising the flap and reducing its vascular supply

to the pedicle the tissue is depraved of its compensatory

mechanism. In addition, the flap itself is denervated in

the progress, since the nerve fibers from the surrounding

tissue are cut. In free flaps, even the innervation of the

vascular pedicle is severed. Therefore, in free flaps even

more than in pedicled flaps, direct neuronal modulation

of the perfusion might be hampered. However, based on

the existing data from animal models, humoral transmit-

ters, either released due to vascular stress, ischemia, or

as end effectors of neuronal regulation, seem to be

responsible for the regulation of BF and also the protec-

tive effects of RIC.12–16,30 Therefore, further research

concerning the possibly different reactions to an RIC

stimulus of pedicled and free-flaps as compared to local

skin seems warranted, especially in relation to the timing

of conditioning.

Considering the mechanisms of RIC, a variety of trig-

gers, mediators and end effectors are discussed.31 Among

those, NO and oxygen radicals seem to act as such

potential triggers.3,31,32 Because of the high oxygen con-

sumption of skeletal muscle, its ischemia tolerance is

limited, resulting in the accumulation of oxygen radicals

and endproducts of ischemic metabolism.33–35 During

reperfusion, these substances are washed out and together

with reactive leukocyte activation result in the so-called

reperfusion injury.12

In addition, the postocclusive reactive hyperemia

(PORH) leads to an increased vascular shear and stretch,

resulting in the release of NO.36

Since the magnitude of the release of the substances

aforementioned correlates with the amount of ischemic

Figure 2. Course of relative hemoglobin content in AU over time for

both groups. Note the early significant increase in rHb when com-

pared to baseline for both groups. The difference between groups

after 45 min showed a nonstatistically significant trend (P 5 0.074).

(AU 5 arbitrary units).

Figure 3. Course of oxygen saturation over time for both groups.

While both groups showed a significant increase when compared

to baseline, this effect was obtained earlier in the leg group. There

was no significant difference between the two groups.

(% 5 percentage of oxygen saturation).

4 Kolbenschlag et al.

Microsurgery DOI 10.1002/micr

tissue, one might hypothesize that a higher mass of

ischemic tissue could lead to an increase in the effect of

RIC. However, our results show that the opposite seems

to be the case, since the amount of ischemic tissue in the

leg group was about three times as high as in the arm

group. Therefore, one could hypothesize that there might

be a certain threshold that triggers an RIC stimulus and

after which further activation does not result in an

increased response.

Another possible explanation could be that the ische-

mia of the upper extremity acts as a more potent trigger

of the autonomous nervous system. Various works show

differences between the autonomic reactions of the upper

and lower extremity. Mental stress for example seems to

lead to a more pronounced vascular change in the fore-

arm when compared to the calf, although these results

seem to be modified by various factors, including gen-

der.37 This further contributes to the evidence that a

combination of neuronal and humoral factors seem to be

involved in RIC.38

The stress and relief from inflating and deflating the

tourniquet itself could also contribute to an autonomic

response, potentially influencing the perfusion by trigger-

ing a pain-induced “fight-or-flight-response.” Since the

ischemic cycling was tolerated well by all participants in

the arm group and by all but one volunteer in the leg

group, this stimulus seems to be distributed equally

between groups. If one would assume the leg ischemia to

be more painful based on this one drop-out, it should

lead to a more pronounced perfusion in the leg group,

which is not the case. However, additional research to

identify a potential correlation between a pain stimulus

and perfusion changes seem warranted.

Since we only measured the short-term response of

cutaneous microcirculation to RIC, the mid- and long-

term effects remains unclear. RIC is known to have an

early and a late window of effect, with the former being

primarily triggered by the immediate effects of humoral

and neuronal factors and the later being based on

changes in gene expressions that lead to a down-

regulation of proinflammatory genes and can also modify

neutrophil function. These effects were shown to last up

to ten days after RIC.38,39

Also, since we performed RIC of a potential flap

donor site, the effects seen in this study have to be

considered as “pre-conditioning.” Although various ani-

mal studies have shown positive effects of precondi-

tioning on flap survival, to our knowledge, none such

data exists for humans.13,14,40 In addition, in many

studies, “preconditioning” in relation to flap surgery

was defined as an RIC stimulus after flap elevation but

before flap ischemia, in contrast to the stimulus in our

study, which was applied to a healthy and intact vascu-

lar bed.14,40 In a rat muscle flap model, Carroll et al.

also found beneficial effects for acute ischemic condi-

tioning prior to flap elevation, therefore it seems appro-

priate to assume a positive effect of preconditioning in

a clinical setting.41

Table 4. Comparison of the Oxygen Saturation Between the Two Groups Over the Course of Time

Time

ARM LEG

P ARM vs. LEG% D% P vs. BL % D% P vs. BL

Baseline(BL) 51.90 6 8.78 – – 46.50 6 12.85 – – –

8 min 52.94 6 10.64 2.0 0.269 47.84 6 13.37 2.9 0.009a 0.554

15 min 53.29 6 9.54 2.7 0.337 49.19 6 13.20 5.8 <0.001 0.506

30 min 55.39 6 10.68 6.7 0.099 49.98 6 13.38 7.5 <0.001 0.839

45 min 56.27 6 11.16 8.4 0.045a 50.87 6 13.50 9.4 <0.001 0.884

%, percentage of oxygen saturation; D%, is the percentage increase versus baseline measurements.aMarks the first point in time at which there was a significant increase versus baseline.

Table 3. Comparison of the Relative Hemoglobin Content Over the Course of Time

Time

ARM LEG

P ARM vs. LEGAU D% P vs. BL AU D% P vs. BL

Baseline(BL) 65.50 6 6.77 – – 68.35 6 6.84 – – –

7 min 66.96 6 7.42 2.2 0.002a 69.37 6 6.94 1.5 0.009a 0.312

15 min 68.57 6 6.87 4.7 <0.001 70.67 6 7.01 3.4 <0.001 0.385

30 min 70.88 6 8.28 8.2 <0.001 71.75 6 7.19 5.0 <0.001 0.088

45 min 71.67 6 8.90 9.4 <0.001 72.36 6 7.23 5.9 <0.001 0.074

AU, arbitrary units; D%, is the percentage increase vs. baseline measurements.aMarks the first point in time at which there was a significant increase vs. baseline.

RIC Extremity 5

Microsurgery DOI 10.1002/micr

However, further studies need to look into the effect

of RIC and its timing (especially pre- and post-condition-

ing) on surgical flaps in a clinical setting. Especially, the

role of preoperative conditioning and late postcondition-

ing (after surgery) should be evaluated, since they can

easily applied in a clinical setting without prolonging the

duration of the surgery.

CONCLUSION

Ischemia of both the upper and lower extremity is

able to improve cutaneous microcirculation of the

antero–lateral thigh via RIC. The cutaneous BF improved

superior after ischemia of the arm when compared to

ischemia of the leg. Therefore, if possible, the upper

extremity should be used for RIC purposes. The amount

of ischemic tissue seems to have little impact on the

effect of RIC. This might suggest a certain role of neuro-

nal factors that warrants further research.

ACKNOWLEDGMENTS

The author thank the participants of this study for volun-

teering as well as Professor Hans Trampisch and Renate

Klaaßen-Mielke of the Department of Medical Infor-

matics, Biometry and Epidemiology, University of

Bochum for their help considering the statistical analysis.

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Microsurgery DOI 10.1002/micr