Tumor angiogenesis  leads to pathological, dilated and tortuous
vascular network [2,3], that causes abnormal perfusion
of the hypoxic tumor area [4,5], leading to, at least in part,
resistance to conventional anti-cancer therapies [6-8].
Post-occlusive reactive hyperemia (PORH) is an ubiquitous
physiological vascular reaction [9,10], first described in the
skin , that consists of a hyperemia peak, occurring just
after the release of a temporary vascular occlusion, followed
by a progressive return to the pre-occlusion perfusion level.
Since tumor blood vessels are mostly functional and sensitive
to vasoactive agents [12-14], one can speculate that PORH
could increase tumor perfusion. In this line, PORH has been
recently described as a mean to enhance temporary and locally
the tumor perfusion and oxygenation levels thus increasing
the outcome of anticancer therapies in human . Tumor
PORH has also been recently demonstrated in human basal
cell carcinoma . In the present study, we set up a simple and reproducible
mouse model of leg PORH to test the effect of vascular occlusion
on a mouse syngenic B16 melanoma perfusion . In
addition, we used the laser speckle contrast imaging (LSCI),
a non-invasive and accurate technology to assess skin blood
flow . LSCI may provide a valuable tool to develop our
knowledge of tumor PORH phenomenon and therapeutic
Material and Methods
All experiments were conducted according to the local and
French veterinary guidelines and those formulated by the European
Community for experimental animal use (No. 07430).
8 weeks-old C57BL/6 mice were obtained from Janvier labs
(France). Syngeneic tumor cell line B16-F10-luc was cultured
in DMEM supplemented with 10% fetal calf serum, 1% antibiotics
(penicillin and streptomycin), and 2mM glutamine in
humidified incubator with 5% CO2 and 95% air.
Mice were anesthetized using 1.5 % isoflurane, since this anesthetic induces a modest effect on tumor perfusion and
oxygenation . It also ensures a perfect anesthesia without
perturbing motion during laser speckle measurement. Air was
used with a constant flow of 2L/min.
Footpad tumor implantation (Figure 1
Melanoma cells were harvested by trypsination and 106 B16-
F10-luc cells were suspended in 10 microliters of PBS. 10 microliters
per mice were then injected into the footpad of the
left leg of 10 anesthetized mice, using insulin needle (29G) oriented
towards the toes and inducing intradermal bubble in the
hairless area of the footpad. Tumor growth was observed every
three days. Tumor sizes were measured with an electronic caliper.
Laser speckle experiments were performed at 14 days after
the injection to allow easy and reproducible measurement of
tumor PO2 using fluorescence quenching oximetry probe (Oxford
Laser Speckle Contrast Imaging (LSCI) and conditions
for data acquisition (Figure 2
Cutaneous blood perfusion was recorded using a Laser Speckle
Contrast imaging system (PERICAM PSI system, high resolution
model, Perimed). The laser wavelength was 785 nm. The
laser head was positioned 10 cm above the skin and remained
perfectly motionless thanks to its fixing system onto the experiment
bench. The acquisition rate was 2 frames per second.
The temperature of the experimental room was monitored
at 22 ° C. Anesthetized mice were maintained in a decubitus
position on a laser speckle recording black carpet (Perimed).
Limbs were fixed with transparent tape, the left leg being fixed
in extension to allow its introduction into a lake tissue vascular
surgical tourniquet (Argyle vascular tourniquet, with a
surgical needle holder to tighten the tourniquet). The tail was
attached to the right side of the mouse, in order to avoid the
field of laser recording. Five minutes were required between
the beginning of anesthesia and that of data acquisition. Record
of laser speckle signal of the left leg required a perfect immobility
of the animal that was well-achieved using isoflurane
Measurements of Melanoma oxygen partial pressure
Fluorescence quenching oximetry probe measured
the tumor oxygen partial pressure (reference BF/
OT/E, Oxylite system, Oxford Optronics)  and was
placed into the tumor center through a 21G catheter.
1. Observation of perfusion in footpad healthy skin without
vascular occlusion: Ten mice were used. Skin perfusion was
evaluated without warming pad to avoid any detrimental effect
on skin blood flow during a long period of observation. We
first studied the skin perfusion of the left footpad for 20 minutes.
We also measured rectal temperature at the beginning of
the experiment, and after 20 minutes of observation.
2. Post- occlusive reactive hyperemia study in footpad
healthy skin: In a second step, we studied the healthy skin
reaction to leg vascular occlusion through tourniquet, and
the influence of the occlusion time (30 seconds or 3 minutes
of occlusion) on the characteristics of PORH assessed by laser
speckle contrast imaging. After placing the mouse on the
black carpet, laser speckle recording was started, and a basic
reference measure was obtained in the first 30 seconds. Then,
vascular occlusion of the left leg was performed at the groin
through the surgical tourniquet. The arrest of perfusion was
controlled directly by LSCI. Unclamping was accompanied by
immediate reperfusion. The perfusion was then measured until
return to stable levels. 30 seconds and 3 minutes occlusion
were realized on the same mice, with one day interval. The
same group of 10 mice then received injection of B16 melanoma
cells in the left footpad.
3. Observation of perfusion in footpad B16 melanoma without
vascular occlusion: Fourteen days after the injection, we observed the melanoma perfusion during 20 minutes, to identify
the ideal timing of vascular occlusion induction. The ideal
moment is the time corresponding to the hypo-perfused state
of the melanoma in order to have a less vasodilated state of
tumor vasculature. This allowed us to reveal the maximal potential
of vasodilatation induced by PORH. A drop in tumor
perfusion was observed during the first 5 minutes leading to
set the time of vascular occlusion at 5 minutes after the start of
the laser speckle recording.
4. Post- occlusive reactive hyperemia study in footpad B16
melanoma: Next, vascular occlusion was induced for 3 minutes,
since this duration has been shown to induce a marked
hyperemia reaction in healthy skin.
5. Melanoma oxygen partial pressure (PO2) and tumor
size caliper measurement: At the end of the LSCI recording,
steady tumor PO2 (mmHg) was measured 10 minutes after the
introduction of oxylite system oximetry probe in the tumor
center in one point. Then, tumor size was measured by caliper
electronic and tumor volume (mm3) calculated using the
formula of an ellipsoid tumor volume (V = L x l2 x 0.52). Mice
were then sacrificed and footpad tumors were opened with
LSCA Data analysis (Figure 3
Data were digitalized, stored on a computer and analyzed offline
with a specific analysis software (Pimsoft, Perimed). Perfusion
was expressed as perfusion unit (Perfusion Unit, PU).
i. Selection of Region Of Interest (ROI): Healthy skin ROI
is the region of the footpad located externally to the vessels
of the leg and was designed as a quadrilateral shape starting
near knee and ended at the bifurcation of the footpad vessels.
Healthy skin ROI was selected in mice without tumor only.
Tumor ROI appeared as hyper-perfused area, at least in the
periphery, and sometimes with central hypoperfused necrotic
area. Tumor ROI was drawn as a free form.
ii. Selection of Time Of Interest (TOI) periods, and description
of post- occlusive reactive hyperemia data: All TOI periods
lasted 10 seconds and corresponded to stable ROI. TOI
were placed every 5 minutes. Basal pre-occlusion TOI was determined
just before the beginning of the vascular clamping. Hyperemia peak TOI was centered at the time of the hyperemia
peak. Hyperemia percentage was calculated as: hyperemia
peak - pre-occlusion value/pre-occlusion value. The value
of tumor perfusion always returned to its pre-occlusion level.
The time to peak was the time in seconds between the occlusion
release and the hyperemia peak. The hyperemia duration
corresponds to the time between occlusion release and the
return to the pre-occlusion perfusion value. The repayment /
debt ratio was the ratio of the AUC (Area Under Curve) of the
post-occlusive hyperemia TOI and the AUC of perfusion debt
during vascular occlusion.
All analyses were performed with R software. We used Mann-
Whitney and Kruskall-Wallis tests to compare the medians between
two groups or more. Wilcoxon’s matched pairs, signed
ranks and Friedman’s tests were used for paired comparisons.
Correlations between the entire tumor's percentage and variables
of interest were assessed with Spearman correlation test.
Significance was accepted at p< 0.05. Results are expressed as
the median and interquartile in parentheses.
Footpad skin perfusion was stable during 20 min recording
even without warming pad(Figure 4
, Table 1
Skin perfusion was stable over 20 minutes, with no significant
difference between the basal perfusion and the end of measurement
(p = 0.77). However, there was a fall in perfusion recording
at the onset of recording (from 36.85 PU (5) at 0 minutes,
to 32.25 PU (4.4) at 5 minutes) (p = 0.006), and a rise of
the perfusion at the end of recording (from 29.65 PU (12.8) at
15 minutes, to 31.95 PU (15.9) at 20 minutes) (p = 0.002). The
initial temperature of 34.4 °C (0, 8) dropped to 27.8 ° C (1.5) in
20 minutes, with consequent loss of 7.1 ° C (2.5) (p =0.002), in
contrast to the robust preservation of the footpad skin perfusion
during 20 minutes. Therefore, absence of warming pad
during experiments did not markedly impact the basal skin
perfusion in our experimental conditions.
Post-occlusive reactive hyperemia values increased with vascular
occlusion duration, as in human skin PORH (Figure 5
Healthy footpad skin ROI size (p=0.38) and basal perfusion
(p=0.12) were equivalent before 30 seconds or 3 minutes of
occlusion. PORH values increased between 30 seconds and
3 minutes of occlusion. Hence, hyperemia percentage raised
strongly from 105% (24) to 160% (42.4) (p=0.002). Hyperemia
peak increased from 74.5 PU (6.1) to 78.8 PU (24.3) but did not
reach statistical significance (p=0.09). Time to peak also markedly
increased from 16.5 seconds (3.3) to 24.5 seconds (8.3)
(p=0.006) as well as hyperemia duration that was enhanced
from 46.5 seconds (14.8) to 146 seconds (41) (p=0.0002). As expected, the repayment/debt ratio decreased between 30
seconds and 3 minutes of occlusion, from 229.6% (76.9) to
125.7% (37.3) (p=0.0007), respectively. Indeed, perfusion debt
increased much more (1048.8 to 5875.2) than perfusion repayment
(2476 to 7092) according to the duration of occlusion
duration. Thus, like in human, PORH occurred in mouse skin
and the intensity and the duration of hyperemia was related to
the duration of occlusion [9,10].
Melanoma perfusion was higher than that of healthy skin
and decreased during the first 5 minutes of LSCA measurement
Tumor perfusion was higher than that of healthy skin with PU
of 108.4 (17.2) versus 40 (6.6) (p = 0.002), respectively, revealing
the intense melanoma tumor angiogenesis  (Table 1,
Figure 5). Tumor perfusion decreased rapidly and significantly
from 108.4 PU (17.2) to 89.2 PU (22.5) (p = 0.002) after 5 minutes
of recording and reached 88.7 PU (28.3) at 20 minutes (p
= 0.03 versus T0). This drop may reflect the well-know tumor
perfusion instability, particularly during anesthesia [22,23].
There was no significant difference in tumor perfusion between
5 minutes and 10 minutes (p = 0.084). Therefore, we
have decided to wait only 5 minutes after the start of tumor
perfusion recording to induce vascular occlusion in order to
have the less dilated tumor micro-vascular network before occlusion
and to avoid negative impact on tumor PORH amplitude.
Post-occlusive reactive hyperemia occurred in melanoma,
with different characteristics compared to healthy skin (Figure 6
, Table 2
Post -occlusive hyperemia was found in each tumor. The median
percentage of hyperemia was 48% (43.4) % versus 160%
(42.4) for healthy skin (p = 0.001). All data were different between
the tumor and the healthy skin, revealing a particular
vascular reactivity in the melanoma. Peak hyperemia was higher
(PU 131.7 (71.4) versus 78.8 PU (24.3), p = 0.02), the time to
peak was longer (46.5 seconds (26.8) versus 24.5 seconds (8.3),
p < 0.001) as well as the duration of hyperemia (248.5 seconds
(67.5) versus 146 seconds (41), p = 0.001) in tumor versus
healthy skin, respectively. Concomitantly to the increase of the
duration of hyperemia, the repayment/debt ratio was higher in
the tumor when compared to that of healthy skin (143% (47.5)
versus 125.7% (37.3), p = 0.04, respectively). Secondary to the
3 minutes vascular occlusion, tumor vasodilatation was thus
feebler (with a weaker PORH percentage), but longer than in
healthy skin vessels (thus with a lengthier hyperemia).
Melanoma volume correlated negatively with post-occlusive
reactive hyperemia (Figure 7
Tumor volume (45.3 mm3 (46.1)) and surface (29.8 mm2
(16.6)) were negatively correlated with the hyperemia percentage
(r = -0.75, p = 0.01 and r = -0.83, p=0.003, respectively).
Central necrosis is generally proportional to tumor growth
and reflected the degree of aggressive tumor. It is likely that
necrosis also reduced the vasoactive potential of a tumor,
dampening the PORH phenomenon. Accordingly, the melanoma
were strongly anoxic and necrotic, with a median PO2
of 0.75mmHg (10), and a visual aspect of black soft necrosis
during tumor opening after the sacrifice of the mice.
In the present study, we have developed a simple and reliable
mouse model of footpad PORH using a surgical tourniquet
and a laser speckle contrast imaging system. Even without
warming pad, the healthy skin perfusion remained relatively
steady. Such mouse model of PORH displays the same characteristics
than that of human skin or others organs (9,10). Hence, our mouse model may constitute a guileless and useful
replica of skin PORH that may be of high interest for further
analysis of molecular and cellular mechanisms of PORH. Interestingly,
we also created a footpad B16 melanoma model.
Implantation was always successful (10/10), with rapid growth
over 14 days. Without the need to shave the footpad, melanoma
perfusion was directly assessed with laser speckle contrast
imaging, allowing a direct and easy measure of PORH in
melanoma through a non-invasive way. Footpad constitutes a
very useful location in order to test the effect of PORH on different
type of murine or xenograft-derived cell lines, especially
during radiotherapy and/or chemotherapy treatment.
This model will also permit to assess the diagnostic value of
tumor vascular reactivity to temporary vascular occlusion,
since tumor vascular immaturity is often related to tumor
aggressiveness. Accordingly, in our study, melanoma hyperemia
percentage was inversely correlated to melanoma
size (p=0.01), that is linked to aggressive state of the tumor.
This study is also the first demonstration of the existence of
PORH in an aggressive malignant tumor. Furthermore, it was
the first demonstration of a significant PORH in a melanoma
with marked central necrotic area suggesting that hyperemia
can occur in aggressive tumor and may cause increases in perfusion
and oxygenation in the peripheral zone of the tumor.
Interestingly, such area display high resistance to radiotherapy
and/or chemotherapy treatments [6,8]. However, the lacks of
tools required to study spatially, in a hypoxic zone, the rapid
variations of perfusion and oxygenation that occur during
PORH dampen research on this specific topic.
In our experimental conditions, PORH in B16 melanoma was
different from that of healthy skin suggesting that the vessels
irrigating the tumor have a passive or active role in tumor
PORH phenomenon. In correlation with results obtained after
laser speckle contrast imaging, extensive study of tumor vessels
architecture and network  (including micro-vessels
density, vessels diameter, vascular surface or pericyte coverage),
as well as intravital vascular imaging at different time of
tumor growth will be useful to understand the respective role
of intrinsic or extrinsic tumor vessels.
One can then speculate that post-occlusive reactive hyperemia
may become an useful clinical method [15,16], not only in
patients with arm or leg tumors as illustrated by the mouse
footpad melanoma model, but also in internal organs tumors.
Indeed, the application of intravascular temporal occlusion
of tumor vascular supply through balloon catheters technology
(or perivascular occlusion with a vascular occluder) may
increase solid malignant tumors perfusion and oxygenation
during chemotherapy or radiotherapy, and, subsequently
We have developed a simple and reproducible murine model
of skin PORH using laser speckle contrast imaging technology
that will foster further analysis of PORH in healthy footpad
skin. Significant PORH was also demonstrated in B16 melanoma
with a median hyperemia percentage of 48% at the surface
of necrotic tumors, suggesting a putative therapeutic potential, even in locally advanced tumors. Additional experiments
using laser speckle contrast imaging, histological and intravital
imaging are required to confirm and develop our results.
Conflict of Interest and Disclosure Statement
Julien Reyal has filed a patent in cooperation with the Agency
for Medical Innovations (GmbH, A.M.I., Austria) in 2010, and
is registered as a co-inventor of this patent, describing a portal
vein vascular occluder for another application that could
eventually be modified and used clinically for tumoral PORH
(Patent Application WO/2010/102661). Others authors did
not have any conflict of interest.
Julien Reyal was supported by a Master grant from the Fondation
pour la Recherche Médicale (FRM).