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j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 4 ( 2 0 1 3 ) 1 1 9 6e1 2 0 4

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Local shockwave-induced capillary recruitment improvessurvival of musculocutaneous flaps

Mickael Tobalem, MD,a,b,1 Reto Wettstein, MD,b,c,1 Brigitte Pittet-Cuenod, MD,a,b

Enrico Vigato, MD,a Hans-Gunther Machens, MD,d Jorn-Andreas Lohmeyer, MD,d

Farid Rezaeian, MD,a,d and Yves Harder, MDa,b,d,*a Faculty of Medicine, University of Geneva, Geneva, SwitzerlandbDivision of Plastic, Reconstructive, and Aesthetic Surgery, Geneva University Hospitals, Geneva, SwitzerlandcDepartment of Plastic, Reconstructive, Aesthetic, and Hand Surgery, University Hospital Basel, Basel, SwitzerlanddDepartment of Plastic Surgery and Hand Surgery, Klinikum rechts der Isar, Technische Universtat Munchen, Munich, Germany

a r t i c l e i n f o

Article history:

Received 21 December 2012

Received in revised form

26 February 2013

Accepted 13 March 2013

Available online 2 April 2013

Keywords:

Microcirculation and capillary

recruitment

Shockwave

Musculocutaneous tissue

Intravital epifluorescence

microscopy

Acute persistent ischemia

Preconditioning

Partly presented at the 45th Annual Congr* Corresponding author. Department of Plast

Ismaninger Strasse 22, D-81675 Munich, GerE-mail address: yves.harder@yahoo.com

1 The authors equally contributed to this w0022-4804/$ e see front matter ª 2013 Elsevhttp://dx.doi.org/10.1016/j.jss.2013.03.040

a b s t r a c t

Background: Shockwave (SW) application has been shown to limit flap necrosis. However,

the underlying microhemodynamic mechanisms remain unclear. Therefore, the objective

of this study was to analyze the effect of SW application on a microcirculatory level.

Methods: We treated 12 C57BL/6 mice with local SW application (500 shockwave impulses at

0.15 mJ/mm2) either 24 h before (preconditioning [PRE]) or 30 min after (postconditioning

[POST]) flap elevation. Animals with an untreated flap (CON) or without a flap served as

controls. We applied dorsal skinfold chambers to the animals and performed epifluor-

escence microscopy over a 10-d period to assess microcirculatory parameters (arteriolar

diameter, red blood cell velocity, blood flow, functional capillary density, and intercapillary

distance) as well as inflammation, apoptotic cell death, and necrosis.

Results: SW application significantly decreased tissue necrosis independently of the

application time point (PRE: 29% � 7%; POST: 25% � 7% versus CON: 47% � 2%; day 10, P <

0.05). Arteriolar diameter, red blood cell velocity, and blood flow were not statistically

significantly different among the 3 flap groups. However, SW (PRE and POST) resulted in an

early and persistent increase in functional capillary density and consequently decreased

intercapillary distance compared with CON and the group without a flap (P < 0.05). Also,

SW resulted in a significantly decreased inflammatory response (P < 0.05) and induced an

angiogenic response, as indicated by new functional microvessel formation observed 5

d after therapy.

Conclusions: Local SW application improved tissue survival by recruitment of sleeping

capillaries within the non ischemic tissue and maintenance of capillary perfusion within

the critically perfused tissue after induction of ischemia, which was independent of the

application time point. Neoangiogenesis occurred beyond the ischemic tolerance of the

tissue, and therefore does not seem to contribute to improved tissue survival.

ª 2013 Elsevier Inc. All rights reserved.

ess of the European Society for Surgical Research, Geneva, June 2010.ic Surgery and Hand Surgery, Klinikum rechts der Isar, Technische Universitat Munchen,many. Tel.: þ49 (0) 89 4140 2171; fax: þ49 (0) 4140 4869.(Y. Harder).ork.

ier Inc. All rights reserved.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 4 ( 2 0 1 3 ) 1 1 9 6e1 2 0 4 1197

1. Introduction a relative humidity of 60%e65% with a 12-h dayenight cycle

Tissue necrosis, particularly in the critically and often

randomly perfused distal flap areas, remains a significant

issue regarding morbidity of the patient, and eventually

increases the burden to the health care system [1].

The development of strategies that prevent ischemia and

its complications is an ongoing challenge in research. The

classic and still reliable way to prevent ischemic flap compli-

cations is surgical delay (i.e., the stepwise surgical interrup-

tion of the flap’s blood supply before transfer, to induce

a hypoxic environment that result in arteriogenesis and

angiogenesis) [2]. Yet, this approach is invasive and time

consuming, and therefore has not become routine in clinical

practice.

More recent interest has turned toward less invasive

protective measures of tissue conditioning that mimic the

effect of surgical delay, including ischemic preconditioning

[3e5], the administration of pharmacologic agents [6e9] and

growth factors [10e12], as well as physical stressors such as

local heat and cold [13e16]. The application of a nonspecific

sublethal cellular stress before or after surgery may therefore

represent an alternative to surgical delay [17].

Shockwave (SW) application has proved to be the treat-

ment of choice for kidney stones for many years. More

recently, SW application has been further applied in a variety

of clinical settings, including bone healing [18], tendinopathy

[19], and tissue conditioning [20e22]. Shockwaves are biphasic

high-energy acoustic waves that can be generated by elec-

trohydraulics. A high-voltage spark is discharged underwater,

causing vaporization and the release of acoustic waves,

characterized by a fast positive followed by a slower negative

wave. When applied to tissues, the acoustic waves are sug-

gested to act as transient micromechanical forces that induce

perturbations at the structural level of the cell, resulting in

various biologic effects. Recent studies show that SW treat-

ment induces the release of various macromolecules,

including vascular endothelial growth factor and endothelial

nitric oxide [23]. However, the precise microhemodynamic

mechanisms that lead to enhanced blood perfusion and

consequently to improved tissue survival have not yet been

completely elucidated [20e27]. Therefore, the present study

investigated the microvascular response to local SW applica-

tion in a musculocutaneous model of acute persistent

ischemia in mice.

2. Materials and methods

2.1. Animals

The experiments were conducted in accordance with the

Swiss legislation on the protection of animals. The Animal

Ethics Committee of the Canton of Geneva, Switzerland,

approved the experimental protocol. We used C57BL/6 mice

(age, 12e24 wk; body weight [bw], 24e28; Zootechnie, Centre

Medical Universitaire, Geneva, Switzerland) in the study. The

animals were housed one per cage at 22�C to 24�C and at

and had free access to standard pellet chow (Altromin, Lage,

Germany) as well as tap water ad libitum.

2.2. Anesthesia

We performed both surgery and repetitive intravital fluores-

cencemicroscopy under general anesthesia by intraperitoneal

injection of 0.1 mL saline solution per 10 g bw of a mixture of

90 mg ketamine hydrochloride/kg bw (Ketavet; Parke Davis,

Freiburg, Germany) and 25 mg dihydroxylidinothiazine

hydrochloride/kg bw (Rompun; Bayer; Zurich, Switzerland).

2.3. Induction of acute persistent ischemia inmusculocutaneous tissue

We used the dorsal skinfold chamber preparation for the

experiments [28]. It includes musculocutaneous tissue, con-

sisting of one layer of skin, subcutaneous tissue, and striated

muscle (i.e., panniculus carnosus). This tissue is either sub-

jected to normal perfusion (physiologic perfusion of mus-

culocutaneous tissue) [28] or acute persistent ischemia

(critical perfusion of musculocutaneous tissue) [29]. The

latter induces about 50% necrosis of the total flap surface if

kept untreated [29,30]. Briefly, after depilation, we raised

a flap 15 mm in length and 11 mm in width with a lateral

base (Fig. 1). We sutured the resulting flap back to the adja-

cent skin and then fixed it to the backside of the chamber’s

frame. Thereafter, we mounted the counterpart of the frame

and sealed the observation window with a coverglass for

subsequent microscopy. This window was placed onto the

flap’s center over the crucial zone of demarcation, which

allowed us to view approximately 45% of the total flap

surface (Fig. 1). Normal feeding and sleeping habits of the

animals indicated good tolerance of both surgery and the

chamber.

2.4. Shockwave application

We placed the anesthetized animals in a left lateral position.

We applied SW at the area of flap elevation (i.e., right flank)

either before (preconditioning) or after (postconditioning) flap

elevation. In the latter case, SW application was performed

after flap elevation, yet before final fixing of the chamber’s

frame. We applied SWs using an electrohydraulic shockwave

source with a cone-shaped probe (DermaPace; Sanuwave AG,

Lengwil, Switzerland). An ultrasound transmission gel

(Sanogel; Bad Camberg, Germany) was applied as contact

medium to the applicator between the SW probe and the

animal’s skin.

2.5. Intravital epi-illumination fluorescence microscopy

For in vivo microscopic analyses, which started after

a recovery period of 24 h, we placed anesthetized animals on

a custom-made Plexiglas frame; they received intravenous

retrobulbar injection of 0.05 mL fluorescein-isothiocyanate

(FITC)-labeled dextran (molecular weight 150,000; 50 mg/mL

Fig. 1 e Surgical procedure to elevate the random pattern flap and prepare the chamber displayed sequentially: (A) Flap

outlined on the skin (width to length[ 15 to 11mm) with its distal 2 mm extending to the contralateral side (midline: dotted

line). To study the back side of the flap’s tissue through the observation window, additional skin (hatched area) has to be

removed. (B) Elevation of the flap (transection of anterior and posterior pedicles) with a randomly arranged vascular

architecture through the flap’s base laterally (arrow). (C) Flap sutured back into the surrounding dorsal skin to guarantee

tightness. The black circle shows the inner diameter of the observation window centered on the flap. (D) Fully mounted

chamber with coverglass and brown sealing band exceeding the device both laterally and on top of the chamber

immediately after the operation. The arrow heads show the demarcation line that develops within the window’s center.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 4 ( 2 0 1 3 ) 1 1 9 6e1 2 0 41198

saline; Sigma-Aldrich Chemie, Buchs, Switzerland) for intra-

vascular contrast enhancement of the plasma and 0.05 mL

rhodamine 6G (0.1 mg/mL saline; Sigma-Aldrich) for in vivo

labeling of circulating leukocytes. Subsequently, we posi-

tioned the animals under a Zeiss Axiotech microscope (Zeiss,

Feldbach, Switzerland) equipped with a 100-W mercury lamp

and filter sets for ultraviolet (330e390 nm excitation/>430-nm

emission wavelength) and blue (450e490 nm excitation,

> 520-nm emission wavelength) and green (530e560 nm

excitation/>580-nm emission wavelength) light.

We captured microscopic images by a charge-coupled

device video camera (Kappa CF 112; Zeiss) and transferred

them to a DVD recorder (LQ-MD 800; Panasonic, Lucerne,

Switzerland). All parameters were analyzed off-line using

a computer-assisted image analysis system (Cap-Image;

Zeintl Software; Heidelberg, Germany) [31]. Microscopy for

analysis ofmicrocirculation and tissuemorphologywithin the

skin and the striated muscle were performed at constant

room temperature of 23�C. Different objectives (�5, NA

(numerical aperture) ¼ 0.16; �20, NA ¼ 0.50; and �100,

NA ¼ 1.00) were used for recordings.

2.6. Planimetric, microcirculatory, and cellular analysis

We subdivided the window of the chamber into three hori-

zontal zones from the bottom to the top, which resulted in

a proximal, central, and distal zone of the flap. The proximal

area corresponds to the normally perfused base of the flap.

Untreated flaps consistently develop necrosis in the non-

perfused distal zone most remote from the vascular inflow.

The intercalated critically perfused central zone is the one of

interest because it can either undergo necrosis or be saved

[29,30].

At each observation point, we first scanned the tissue

within the chamber’s window at �5 magnification to deter-

mine the area of non-perfused but still vital (approximately

d 1 - 5) respectively necrotic tissue (approximately d 5 - 10), as

indicated by a complete lack of blood perfusion (i.e., the

absence of intravascular fluorescent dye). This area was

measured planimetrically and is given as a percentage of the

total window’s surface.

We selected easily identifiable branching patterns of

second- or third-order arterioles, accompanying collecting

venules, and capillary fields.Wemade video printouts of these

branching patterns using the �5 and �20 objective to relocate

the same vessels for repetitive measurements during the 10-d

observation period.

1. We analyzed red blood cell (RBC) velocity (millimeters per

second) using the line shift method based on the

measurement of the shift (millimeters) of an individual

intravascular gray-level pattern over time (seconds).

2. We measured arteriolar diameter (micrometers) perpen-

dicular to the vessel path.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 4 ( 2 0 1 3 ) 1 1 9 6e1 2 0 4 1199

3. Volumetric blood flow (picoliters per second) was

calculated in arterioles from RBC velocity and vessel

cross-sectional area (p * r2) according to the equation of

Gross and Aroesty [32] (i.e., Q ¼ V * p * r2, assuming

a cylindrical vessel geometry).

Fig. 2 e Skinmorphologyatday10aftersurgery inuntreated

animals without flap (A) and with flap preparation (BeD).

Note the clear demarcation zone within the critically

perfused area of untreated controls (B). Microvascular

dilation and remodeling cause the formation of a red fringe

(arrowheads), which separates the proximal vital zone from

the distal necrotic area (asterisk) (B). Preconditioning (C) and

postconditioning (D) with SW application considerably

ameliorate tissue survival. Magnification316. Time course

of necrosis in animals without a flap (baseline [black

squares]) and with an untreated flap (control [white

squares]), as well as in preconditionedmice (white circles)

and postconditionedmice (black circles). SW application of

either regimen showed significantly improved perfusion at

day 1, resulting in significantly reduced necrosis at day 10

(E). Mean ± SEM; *P< 0.05 versus untreated flap; #P< 0.05

versus animals without flap; n[ 6/group.

4. Functional capillary density (FCD) (centimeters per square

centimeters) was defined as the length of all RBC-perfused

capillaries per observation field.

5. We measured intercapillary distance (micrometers)

between in parallel arranged functional capillaries, that is

the distance of interstitial tissue between red blood cell

perfused capillaries (Fig. 4AeD).

Fig. 3 e Vasculardiameters of arterio- (a) venular (v) bundles

10 d after surgery displayed by intravital microscopy in

animalswithout a flap (A) andwith anuntreatedflaps (B), as

well as in SW-preconditioned (C) and postconditionedmice

(D). Note the comparable morphology of the vascular

diameter within the groups. Contrast enhancement with

(FITC-dextran 150,000. Magnification 380. Analysis of

arteriolar diameter over the 10-d observation period in

animals without a flap (baseline [black squares]) and with

an untreated flap (control [white squares]), as well as in

preconditioned (white circles) and postconditioned mice

(black circles) showing no diameter changes, either over

time or among groups (E). Mean ± SEM; n [ 6/group.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 4 ( 2 0 1 3 ) 1 1 9 6e1 2 0 41200

6. We characterized inflammatory response by leukocytee

endothelium interaction and analyzed it by counting the

total number of white blood cells that were intermittently

adhering along the endothelial lining of postcapillary

venules, passing a reference line within a 30-s period.

Leukocyte rolling is expressed as cells per minute per

millimeter circumference.

7. We identified apoptotic cells by the characteristics of nuclear

condensation, fragmentation, and margination; they are

given as number per square millimeter, using the DNA-

binding fluorochrome bisbenzimide H33342 (Sigma-Aldrich)

that was locally applied before coverglass application.

8. Angiogenic response included analysis of the density of all

newly formed, blood-perfused, capillary-like microvessels

developing perpendicularly to the preexisting capillaries.

Microvascular density (centimeters per square centimeters)

was calculated as the total length of all newly formed

microvessels within the observation area.

2.7. Experimental groups and protocol

We used six animals per group. For baseline (BL) values in

intact, physiologic, musculocutaneous tissue, we used animals

without a flap. Untreated flaps served as control (CON).

Shockwaves (DermaPace) were locally applied once either 24 h

before (preconditioning [PRE]) or 30min after (postconditioning

[POST]) induction of ischemia by flap elevation.

Both treatment groups received 500 impulses at a

frequency of 240/min and an energy flux density of

0.15 mJ/mm2 at 14 kV. Microhemodynamic parameters,

inflammation (leukocyte rolling), apoptotic cell death, and

angiogenesis were analyzed at days 1, 3, 5, 7, and 10 after

surgery. We killed all animals at the end of the experiment by

administering 1 mL of the anesthetic agent.

2.8. Statistical analysis

All values are expressed asmean� standard error of themean

(SEM). For comparison between individual time points, we

carried out analysis of variance for repeated measures,

Table 1 e Arteriolar RBC velocity and blood flowwithin the critflap elevation (induction of ischemia).

Group

1 3

RBC velocity (mm/s)

Baseline (non-ischemic) 1.7 � 0.2* 1.5 � 0.1*

Control (ischemic) 0.3 � 0.1y 0.3 � 0.1y

Preconditioning 0.5 � 0.1y 0.7 � 0.2y

Postconditioning 0.3 � 0.1y 0.5 � 0.1y

Blood flow (pL/s)

Baseline (non-ischemic) 2785 � 522* 2422 � 679*

Control (ischemic) 562 � 138y 697 � 152y

Preconditioning 1096 � 422y 1225 � 972

Postconditioning 1059 � 200y 1538 � 546

Baseline ¼ non-ischemic tissue; control ¼ untreated flap.

Values are mean � SEM.

* P < 0.05 versus control.yP < 0.05 versus baseline.

followed by the appropriate post hoc test, which included

measures to correct the a error according to Bonferroni

probabilities. Comparison between the groups included anal-

ysis of variance and StudenteNewmaneKeuls post hoc test.

Differences were considered significant at P< 0.05 (SigmaStat;

Jandel, San Rafael, CA).

3. Results

3.1. Tissue necrosis

In all flap animals, macroscopic tissue morphology at day 10

after induction of ischemia (Fig. 2) displayed a distinct

demarcation between vital tissue proximally and necrosis

distally, indicated by a red fringe, a zone of microvascular

dilation, and remodeling (Fig. 2BeD). At day 1, CON displayed

initial microcirculatory perfusion failure of 37% � 4% of the

window area. Persisting microcirculatory dysfunction resul-

ted in necrosis of 46% � 2% at day 10 (Fig. 2B and E). Either

regimen of SW application at day 1 after induction of ischemia

showed a significantly smaller area of non-perfused tissue of

2% � 2% (PRE) and 8% � 5% (POST), which resulted in signifi-

cantly reduced tissue necrosis at day 10 (PRE, 29%, � 7%; POST

25% � 7%: P < 0.05 versus CON) (Fig. 2CeE).

3.2. Arteriolar perfusion

We observed no changes in arteriolar diameter during the

10-d observation period in the critically perfused flap area

(Fig. 3), whereas RBC velocity and blood flowwere significantly

decreased in all experimental groups (CON, PRE, and POST)

compared with baseline (Table 1). Thus, animals receiving SW

application showed no difference in arteriolar blood flow

compared with CON.

3.3. Capillary perfusion

At day 1, we observed significantly higher FCD in the proxi-

mal area of treated flaps (PRE, 271 � 23 cm/cm2 and POST,

ically perfused area of the flap at days 1, 3, 5, 7, and 10 after

Day

5 7 10

1.7 � 0.1* 1.9 � 0.1* 1.5 � 0.1*

0.5 � 0.2y 0.6 � 0.2y 0.4 � 0.2y

1.1 � 0.2y 0.8 � 0.2y 1 � 0.2*,y

0.6 � 0.2y 0.7 � 0.2y 0.6 � 0.2y

3726 � 1573* 3764 � 799* 2862 � 687

1051 � 271y 1324 � 324y 1442 � 437

2641 � 863 1728 � 915 2817 � 1494

2663 � 805 2854 � 1004 2793 � 1061

Fig. 4 e Functional capillary density (FCD) at day 1 after

surgery in animals without a flap (A) and with untreated

flaps (B), as well as in SW-preconditioned (C) and

postconditioned mice (D). Conditioned animals show

a similar pattern of horizontally arranged capillaries that

are almost all perfused and comparable to animals without

a flap (A, C, and D). Only untreated flaps display non-

perfused capillaries resulting in an increased intercapillary

distance (arrowheads) (B). Contrast enhancement with

FITC-dextran 150,000. Magnification 380. Time course of

FCDwithin the normally perfused tissue proximally (E) and

the critically perfused tissue distally (F) in animals without

a flap (baseline [black squares]), with an untreated flap

(control [white squares]), and with preconditioning (white

circles) and postconditioning (black circles). Note the

significantly higher FCD from day 1 onward within all mice

undergoing SW conditioning in the normally perfused

tissue (E). Mean ± SEM; *P < 0.05 versus untreated flap;#P < 0.05 versus animals without flap; n [ 6/group.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 4 ( 2 0 1 3 ) 1 1 9 6e1 2 0 4 1201

290 � 22 cm/cm2 versus BL,198 � 8 cm/cm2; and CON, 199 �9 cm/cm2, P< 0.05) (Fig. 4AeE). In the critically perfused areas,

untreated and postconditioned flaps showed significantly

decreased FCD compared with BL, and SW postconditioning

displayed significantly higher FCD compared with untrea-

ted controls. We saw no significant change in FCD between

SW preconditioning and BL. These differences in capillary

perfusion were maintained during the observation period

(Fig. 4AeF). The higher rate of patent capillaries in treated

animals was associated with a significantly decreased inter-

capillary distance compared with untreated ischemic controls

(Table 2).

3.4. Inflammatory response and apoptotic cell death

Compared with baseline, flap preparation induced an inflam-

matory response with leukocyte adherence and apoptosis.

However, animals receiving SW displayed a significantly

reduced inflammatory response (PRE, 2275 leukocytes/min/

mm circumference � 183 leukocytes/min/mm circumference

and POST, 1790 leukocytes/min/mm circumference � 408

leukocytes/min/mm circumference versus CON, 8233 leuko-

cytes/min/mm circumference � 1325 leukocytes/min/mm

circumference and BL, 1405 leukocytes/min/mm circumfer-

ence � 408 leukocytes/min/mm circumference; day 1; P < 0.05)

and a decrease in apoptotic cell death (PRE,1.1 cells/mm2 � 0.2

cells/mm2 and POST, 1.7 cells/mm2 � 0.6 cells/mm2 versus

CON, 3.1 cells/mm2 � 0.3 cells/mm2 and BL, 1.8 cells/mm2 � 0.2

cells/mm2; day 1; P < 0.05) in the critically perfused area of

the flap.

3.5. Angiogenic response

In control animals, capillaries within the critically perfused

zone of demarcation changed morphology toward corkscrew-

like shaped microvessels (i.e., microvessels with increased

tortuosity and irregular diameters, indicating microvascular

remodeling) (Fig. 5A). Shockwave application before and after

induction of ischemia was also associated with the induction

of perfused neo-capillaries, which originated from microvas-

cular buds and sprouts. They were oriented perpendicular to

the parallel arrangement of preexisting capillaries. We first

observed this angiogenic response on day 5 after flap

Table 2 e Intercapillary distance within the critically perfused area of the flap at days 1, 3, 5, 7 and 10 after induction ofischemia.

Group Day

1 3 5 7 10

Intercapillary distance (mm)

Baseline (non-ischemic) 30 � 4* 33 � 3* 39 � 5* 28 � 6* 32 � 2*

Control (ischemic) 48 � 3y 100 � 10y,z 88 � 9y,z 72 � 9y,z 65 � 6y,z

Preconditioning 28 � 1* 79 � 53 31 � 5* 51 � 23 29 � 1*

Postconditioning 31 � 5* 32 � 4* 39 � 7* 30 � 4* 30 � 6*

Baseline ¼ non-ischemic tissue; control ¼ untreated flap.

Data represent the intercapillary distance (mm). Values are mean � SEM.

* P < 0.05 versus control.yP < 0.05 versus baseline.zP < 0.05 versus day 1.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 4 ( 2 0 1 3 ) 1 1 9 6e1 2 0 41202

elevation. The density of newly formed capillaries was higher

after preconditioning than after postconditioning (Fig. 5BeE).

4. Discussion

A single local application of SW applied 24 h before or 30 min

after induction of ischemia significantly decreased necrosis.

This increased tissue survival primarily resulted from an SW-

induced recruitment of non-perfused capillaries and from the

maintenance of capillary perfusion. Shockwaves also atten-

uated the leukocytic inflammatory response and reduced

apoptotic cell death. Shockwave-induced neovascularization

does not seem to be protective.

The positive effect of SW application on tissue survival has

previously been demonstrated, and the optimal time point of

local SW application was within a certain time before or after

induction of ischemia [20,22,24,25]. Repetitive application or

accumulation of SWs before and after flap elevation has

shown to be less effective comparedwith a single cycle of SWs

[24]. It was hypothesized that repetitive SW application would

increase local energy density too much and eventually result

in localized tissue damage with restricted perfusion and

impaired neovascularization [20]. In the present study, we

applied a single preoperative or postoperative SW regimen

that was previously shown to be effective, to investigate the

effect of SW on microcirculation.

With the use of intravital fluorescence microscopyda

technique that allows both morphologic and dynamic

assessment of the microvasculature over timedwe bring

a new insight to SW-induced mechanisms promoting tissue

survival of ischemically endangered tissue. In contrast to

previous work [20,22,26] we observed no increase in arteriolar

blood flow of critically perfused skin (i.e., an increase in

arteriolar diameter and/or increase in arteriolar RBC velocity

after SW application). This discrepancy may have resulted

from the different methods used to measure perfusion; in

contrast to the intravital epifluorescence microscopy used in

this study, which allows for direct, selective visualization of

all microvessels, laser Doppler was applied in previous studies

[20,22,26]. Laser Dopplermeasures total blood flow and cannot

reflect blood flow distribution and distinguish between arte-

riolar and capillary perfusion [33].

Although flow conditions in arterioles were unchanged

after SW application compared with untreated ischemic

tissue, we observed maintained nutritive capillary perfusion

within the distal zone at risk of necrosis in treated animals.

We further observed that functional capillary density was

drastically increased after SW applicationwithin the proximal

area of the flap (i.e., the non-ischemic tissue area). This effect

seems to result from capillary recruitment. Several studies

support the theory that tissue is composed of a defined

number of non-perfused “sleeping” capillaries (capillary

reserve) that can be recruited without increasing arteriolar in-

flow to the challenged tissue, but by redistributing flow

predominantly to capillary structures [34,35]. Recently, Cal-

cagni et al. [36] demonstrated that SWs are able to recruit

capillaries within non-ischemic tissue. With the present

study, this phenomenon was observed for the first time in

ischemic musculocutaneous tissues. The tissue protective

effect was similar whether SWs were applied 24 h before or

30 min after the induction of ischemia.

Shockwave application in non-ischemic tissue margin-

ally induced both apoptotic cell death and inflammatory

response, which indicates that SW per se is associated

with mechanical stress [36]. However, this inflammatory

response was significantly decreased within the critically

perfused areas of the treated flaps, probably as a conse-

quence of better perfusion conditions compared with

untreated flaps.

Shockwave also induced an angiogenic response from day

5 onward. However, this response occurred beyond the

ischemic tolerance of the tissue (i.e., a point at which necrosis

is already fully demarcated) [37]. Neovascularization after SW

application has been shown to be mediated by early up-

regulation of angiogenesis-related growth factors such as

vascular endothelial growth factor [21e24,27]. It has further

been shown that preconditioning of muscular tissues with

SWs improved recruitment of circulating endothelial

progenitor cells into ischemic tissues via enhanced expression

of chemoattractant factors [38].

Intravital epifluorescence microscopy allows for viewing

dynamic and morphologic changes within the chamber’s

window. However, the window does not reflect the entire flap

surface, which is approximately double the size of the

window. Yet, if kept untreated, the zone of demarcation reli-

ably develops within the chamber’s window, which is

Fig. 5 e Perfused capillaries without morphologic changes

in animals without a flap (A). Microvascular remodeling

(i.e., dilated and tortuous microvessels [arrowheads])

within parallel arranged and perfused muscle capillaries

in the demarcation zone at day 7 after surgery in animals

with untreated flaps (B). SW-preconditioning (C) and

postconditioning (D) induced an angiogenic response

with development of new functional microvessels

(arrows) originating perpendicularly from the preexisting

capillaries. Contrast enhancement with FITC-dextran

150,000. Magnification 380. Time course of newly formed

microvessels density (C and D) in animals without a flap

(baseline [black squares]) and with untreated flap (control

[white squares]), as well as in preconditioned (white

circles) and postconditioned mice (black circles) (E). Note

the significantly increased number of newly formed

capillaries from day 7 onward in all mice receiving

SW application, irrespective of the administration time

point (C and D). Mean ± SEM; *P < 0.05 versus

untreated flap; #P < 0.05 versus animals without flap; n [

6/group.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 4 ( 2 0 1 3 ) 1 1 9 6e1 2 0 4 1203

localized between the proximal area of the flap that displays

physiologic perfusion conditions and the distal area of the flap

that develops complete necrosis. The percentage of tissue

survival presented in this study refers to the window surface

and not to the entire flap area; yet, this model has been

demonstrated to reliably represent the development of tissue

necrosis [6e8,15,29,30,37].

The study brings new insight to the underlying micro-

hemodynamic mechanisms of SW therapy for tissue survival.

Tissue protection seems to be primarily mediated by early

redistribution of total blood flow that itself remains un-

changed. This flow redistribution was obtained by continu-

ously (PRE) or reperfused (POST) capillaries compared with

untreated flaps, and also by recruitment of “sleeping capil-

laries” comparedwith animals with physiologic perfusion (BL).

Furthermore, the ischemia-induced inflammatory response

is attenuated and apoptotic cell death is decreased after local

SW application. The onset of neovascularization occurs only

after demarcation of necrosis, and does not seem to protect

flap tissue.

Acknowledgment

This study was supported by the Swiss National Science

Foundation (grant 32003B-108408). The authors thank Sanu-

Wave AG, Baar, Switzerland, for supplying the Shockwave

device.

r e f e r e n c e s

[1] Wettstein R, Schurch R, Banic A, et al. Review of 197consecutive free flap reconstruction in the lower extremity. JPlast Reconstr Aesthet Surg 2008;61:772.

[2] Reinisch JF. The pathophysiology of skin flap circulation: thedelay phenomenon. Plast Reconstr Surg 1974;54:585.

[3] Mounsey RA, Pang CY, Boyd JB, Forrest C. Augmentation ofskeletal muscle survival in the latissimus dorsi porcinemodel using acute ischemic preconditioning. J Otolaryngol1992;21:315.

[4] Zahir KS, Syed SA, Zink JR, Restifo RJ, Thomson JG.Comparison of the effects of ischemic preconditioning andsurgical delay on pedicled musculocutaneous flap survival ina rat model. Ann Plast Surg 1998;40:422.

[5] Schlaudraff KU, Pepper MS, Tkatchouk EN, et al. Hypoxicpreconditioning increases skin oxygenation and viability butdoes not alter VEGF expression or vascular density. High AltMed Biol 2008;9:76.

[6] Harder Y, Contaldo C, Klenk J, Banic A, Jakob SM, Erni D.Preconditioning with monophosphoryl lipid A improvessurvival of critically ischemic tissue. Anesth Analg 2005;100:1786.

[7] Rezaeian F, Wettstein R, Amon M, et al. Erythropoietinprotects critically perfused flap tissue. Ann Surg 2008;248:919.

[8] Rezaeian F, Wettstein R, Scheuer C, et al. Ghrelin protectsmusculocutaneous tissue from ischemic necrosis byimproving microvascular perfusion. Am J Physiol Heart CircPhysiol 2012;302:603.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 4 ( 2 0 1 3 ) 1 1 9 6e1 2 0 41204

[9] Alizadeh N, Pittet B, Tenorio X, et al. Active-site inactivatedFVIIa decreases thrombosis and necrosis in a random skinflap model of acute ischemia. J Surg Res 2004;122:263.

[10] Zhang F, Waller W, Lineaweaver WC. Growth factors and flapsurvival. Microsurgery 2004;24:162.

[11] Zhang F, Yang F, Hu EC, Sones W, Lei M, Lineaweaver WC.Vascular endothelial growth factor therapy in improvementof skin paddle survival in a rat TRAM flap model. J ReconstrMicrosurg 2005;21:391.

[12] Rinsch C, Quinodoz P, Pittet B, et al. Delivery of FGF-2 but notVEGF by encapsulated genetically engineered myoblastsimproves survival and vascularization in a model of acuteskin flap ischemia. Gene Ther 2001;8:523.

[13] Salmi AM, Hong C, Futrell JW. Preoperative cooling andwarming of the donor site increase survival of skin flaps bythe mechanism of ischaemic preconditioning: anexperimental study in rats. Scand J Plast Reconstr Surg HandSurg 1999;33:163.

[14] Kubulus D, Amon M, Roesken F, Rucker M, Bauer I,Menger MD. Experimental cooling-induced preconditioningattenuates skin flap failure. Br J Surg 2005;92:1432.

[15] Harder Y, Amon M, Schramm R, et al. Heat shockpreconditioning reduces ischemic tissue necrosis by heatshock protein (HSP)-32-mediated improvement of themicrocirculation rather than induction of ischemictolerance. Ann Surg 2005;242:869.

[16] Harder Y, Contaldo C, Klenk J, Banic A, Jakob SM, Erni D.Improved skin flap survival after local heat preconditioningin pigs. J Surg Res 2004;119:100.

[17] Harder Y, Amon M, Laschke MW, et al. An old dreamrevitalised: preconditioning strategies to protect surgicalflaps from critical ischaemia and ischaemia-reperfusioninjury. J Plast Reconstr Aesthet Surg 2008;61:503.

[18] Alvarez RG, Cincere B, Channappa C, et al. Extracorporealshock wave treatment of non- or delayed union of proximalmetatarsal fractures. Foot Ankle Int 2011;32:746.

[19] Wang CJ, Weng LH, Chou WY, et al. Extracorporeal shockwave therapy enhances early tendon-bone healing andreduces bone tunnel enlargement in hamstring autograftanterior cruciate ligament reconstruction. Am J Sports Med2012;40:NP14.

[20] Kuo YR, Wu WS, Hsieh YL, et al. Extracorporeal shock waveenhanced extended skin flap tissue survival via increase oftopical blood perfusion and associated with suppression oftissue pro-inflammation. J Surg Res 2007;143:385.

[21] Meirer R, Brunner A, Deibl M, Oehlbauer M, Piza-Katzer H,Kamelger FS. Shock wave therapy reduces necrotic flapzones and induces VEGF expression in animal epigastric skinflap model. J Reconstr Microsurg 2007;23:231.

[22] Mittermayr R, Hartinger J, Antonic V, et al. Extracorporealshock wave therapy (ESWT) minimizes ischemic tissuenecrosis irrespective of application time and promotes tissuerevascularization by stimulating angiogenesis. Ann Surg2011;253:1024.

[23] Qureshi AA, Ross KM, Ogawa R, Orgill DP. Shock wavetherapy in wound healing. Plast Reconstr Surg 2011;128:721.

[24] Reichenberger MA, Keil H, Mueller W, et al. Optimal timing ofextracorporeal shock wave treatment to protect ischemictissue. Ann Plast Surg 2011;67:539.

[25] Kuo YR, Wang CT, Wang FS, Yang KD, Chiang YC, Wang CJ.Extracorporeal shock wave tretment modulates skinfibroblast recruitment and leukocyte infiltration forenhancing extended skin-flap survival. Wound Repair Regen2009;17:80.

[26] Yan X, Zeng B, Chai Y, Luo C, Li X. Improvement of bloodflow, expression of nitric oxide, and vascular endothelialgrowth factor by low-energy shockwave therapy in random-pattern skin flap model. Ann Plast Surg 2008;61:646.

[27] Keil H, Mueller W, Herold-Mende C, et al. Preoperative shockwave treatment enhances ischemic tissue survival, bloodflow and angiogenesis in a rat skin flap model. Int J Surg2011;9:292.

[28] Lehr HA, Leunig M, Menger MD, Nolte D, Messmer K. Dorsalskinfold chamber technique for intravital microscopy innude mice. Am J Pathol 1993;143:1055.

[29] Harder Y, Amon M, Erni D, Menger MD. Evolution of ischemictissue injury in a random pattern flap: a new mouse modelusing intravital microscopy. J Surg Res 2004;121:197.

[30] Harder Y, Amon M, Georgi M, Banic A, Erni D, Menger MD.Evolution of a “falx lunatica” in demarcation of criticallyischemic myocutaneous tissue. Am J Physiol Heart CircPhysiol 2005;288:H1224.

[31] Klyscz T, Hahn M, Rassner G, Junger M. [The technicalconstruction of a computer-supported measuring unit for in-vivo capillary pressure measurement in human nail foldcapillaries]. Biomed Tech (Berl) 1997;42:310.

[32] Gross JF, Aroesty J. Mathematical models of capillary flow:a critical review. Biorheology 1972;9:225.

[33] Coggins M, Lindner J, Rattigan S, et al. Physiologichyperinsulinemia enhances human skeletal muscleperfusion by capillary recruitment. Diabetes 2001;50:2682.

[34] Clark MG, Rattigan MG, Barrett EJ, Vincent MA. Point: there iscapillary recruitment in active skeletal muscle duringexercise. J Appl Physiol 2008;104:889.

[35] Wettstein R, Tsai AG, Erni D, Lukyanov AN, Torchilin VP,Intaglietta M. Improving microcirculation is more effectivethan substitution of red blood cells to correct metabolicdisorder in experimental hemorrhagic shock. Shock 2004;21:235.

[36] Calcagni M, Chen F, Hogger DC, et al. Microvascular responseto shock wave application in striated skin muscle. J Surg Res2010;171:347.

[37] Rezaeian F, Wettstein R, Egger JF, et al. Erythropoietin-induced upregulation of endothelial nitric oxide synthasebut not vascular endothelial growth factor preventsmusculocutaneous tissue from ischemic damage. Lab Invest2010;90:40.

[38] Aicher A, Heeschen C, Sasaki KI, Urbich C, Zeiher AM,Dimmeler S. Low-energy shock wave for enhancingrecruitment of endothelial progenitor cells: a new modalityto increase efficacy of cell therapy in chronic hind limbischemia. Circulation 2006;114:2823.