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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
Available online at w
journal homepage: www.JournalofSurgicalResearch.com
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.
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