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The Vasodilator Effect of a Cream Containing 10% Menthol and 15% Methyl Salicylate on Random-Pattern Skin Flaps in Rats

November 2015
Archives of Plastic Surgery 42(6):695

November 2015
42(6):695

DOI:10.5999/aps.2015.42.6.695

License
CC BY-NC 3.0

Authors:

Utku Can Dölen

Northwestern Medicine

Gokhan Koca

S.B.Ü. Ankara Eğitim ve Araştırma Hastanesi

Onur Ertunç

T.C. Süleyman Demirel Üniversitesi

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Background It is still difficult to prevent partial or full-thickness flap necrosis. In this study, the effects of a cream containing menthol and methyl salicylate on the viability of randompattern skin flaps were studied. Methods Forty female Sprague-Dawley rats were divided into two equal groups. Caudally based dorsal random-pattern skin flaps were elevated, including the panniculus carnosus. In the study group, 1.5 mL of a cream containing menthol and methyl salicylate was applied to the skin of the flap, and saline solution (0.9%) was used in the control group. Upon completion of the experiment, flap necrosis was analyzed with imaging software and radionuclide scintigraphy. Histopathological measurements were made of the percentage of viable flaps, the number of vessels, and the width of the panniculus carnosus muscle. Results According to the photographic analysis, the mean viable flap surface area in the study group was larger than that in the control group (P=0.004). According to the scintigrams, no change in radioactivity uptake was seen in the study group (P>0.05). However, a significant decrease was observed in the control group (P=0.006). No statistically significant differences were observed between the groups in terms of the percentage of viable flaps, the number of vessels, or the width of the panniculus carnosus muscle (P>0.05). Conclusions Based on these results, it is certain that the cream did not reduce the viability of the flaps. Due to its vasodilatory effect, it can be used as a component of the dressing in reconstructive operations where skin perfusion is compromised.

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695

Original Article

INTRODUCTION

The introduction of flaps to plastic surgery had a major impact

on wound closure and the reconstruction of normal and func-

tional anatomical features. Local skin flaps provide an excellent

match in terms of color, texture, and thickness. However, it is

The Vasodilator Effect of a Cream Containing 10%

Menthol and 15% Methyl Salicylate on Random-

Pattern Skin Flaps in Rats

Utku Can Dölen1, Nezih Sungur2, Gökhan Koca3, Onur Ertunç4, Ayşe Tülay Bağcı Bosi5,

Uğur Koçer2, Meliha Korkmaz3

1Department of Plastic Surgery, Dr. Abdurrahman Yurtaslan Ankara Oncology Training and Research Hospital, Ankara; 2Plastic Surgery

Clinic, Ankara Training and Research Hospital, Ankara; 3Department of Nuclear Medicine, Ankara Training and Research Hospital,

Ankara; 4Department of Pathology, Medical School of Gazi University, Ankara; 5Department of Public Health, Medical School of Hacettepe

University, Ankara, Turkey

Background It is still difficult to prevent partial or full-thickness flap necrosis. In this study,

the effects of a cream containing menthol and methyl salicylate on the viability of random-

pattern skin flaps were studied.

Methods Forty female Sprague-Dawley rats were divided into two equal groups. Caudally

based dorsal random-pattern skin flaps were elevated, including the panniculus carnosus. In

the study group, 1.5 mL of a cream containing menthol and methyl salicylate was applied to

the skin of the flap, and saline solution (0.9%) was used in the control group. Upon

completion of the experiment, flap necrosis was analyzed with imaging software and

radionuclide scintigraphy. Histopathological measurements were made of the percentage of

viable flaps, the number of vessels, and the width of the panniculus carnosus muscle.

Results According to the photographic analysis, the mean viable flap surface area in the

study group was larger than that in the control group (P = 0.004). According to the

scintigrams, no change in radioactivity uptake was seen in the study group (P> 0.05).

However, a significant decrease was observed in the control group (P = 0.006). No statistically

significant differences were observed between the groups in terms of the percentage of

viable flaps, the number of vessels, or the width of the panniculus carnosus muscle (P > 0.05).

Conclusions Based on these results, it is certain that the cream did not reduce the viability of

the flaps. Due to its vasodilatory effect, it can be used as a component of the dressing in

reconstructive operations where skin perfusion is compromised.

Keywords Surgical flaps / Menthol / Methyl salicylate / Vasodilator agents / Perfusion

Correspondence: Utku Can Dölen

Department of Plastic Surgery, Dr.

Abdurrahman Yurtaslan Ankara

Oncology Training and Research

Hospital, Demetevler, Ankara 06200,

Turkey

Tel: +90-312-336-0909

Fax: +90-312-595-3656

E-mail: utkuchan@gmail.com

No potential conflict of interest relevant

to this article was reported.

Received: 10 Feb 2015 • Revised: 6 Jun 2015 • Accepted: 17 Aug 2015

pISSN: 2234-6163 • eISSN: 2234-6171 • http://dx.doi.org/10.5999/aps.2015.42.6.695 • Arch Plast Surg 2015;42:695-703

Dölen UC et al. Menthol methyl salicylate rat skin flap

696

not always possible to predict the viability of flaps, especially in

random-pattern skin flaps.

Partial or total flap loss may result in a new tissue defect at the

flap donor area, increasing the total defect area requiring recon-

struction. Such complications may increase the length of the

hospital stay, the cost of the treatment, and most importantly,

may damage the patient’s confidence and trust in his/her doc-

tor. In the literature, numerous surgical and pharmacological

methods of increasing flap viability have been described. Phar-

macological agents such as sympatholytics, vasodilators, calci-

um channel blockers, hemorheologics, prostaglandin inhibitors,

anticoagulants, glucocorticoids, and free radical scavengers have

been applied topically [1-3] and administered enterally or par-

enterally [4,5]. The use of some drugs with proven experimen-

tal efficacy has been restricted by side effects, high cost, and/or

poor availability.

Menthol is a natural and readily available agent used for a vast

range of purposes, including hygiene products, pharmaceuticals,

and as a flavor [6,7]. Menthol has antipruritic, antiseptic, anal-

gesic, anesthetic, and cooling effects when used topically. In ad-

dition, it has antibacterial, antiviral, and antifungal effects [7].

Calcium (Ca2+) channels play a major role in the regulation of

vascular tone. The transient receptor potential (TRP) channel

superfamily has non-voltage-gated cation-permeable channels.

The vanilloid-related (TRPV) and melastatin-related (TRPM)

TRP receptors are two major families that are responsible for

sensing acidity, osmolarity, mechanical forces, and temperature

in non-vascular tissues [8]. TRPM8, known as the menthol or

cold receptor, has been found in arterial smooth muscle. Limit-

ed and contradictory data exist regarding the effect of menthol

on vessels, with disagreement about whether it results in vasodi-

latation [8,9] or vasoconstriction [10-13].

Non-steroid anti-inflammatory drugs (NSAIDs) are the active

ingredients of topical analgesics that are widely used to relieve

muscle and joint pains. NSAIDs inhibit the cyclooxygenase en-

zyme that transforms arachidonic acid into various prostaglan-

dins. A decrease in the amount of prostaglandins, which activate

nociceptors, alleviates the pain. Methyl salicylate (MeSa) is hy-

drolyzed to salicylate, which has anti-inflammatory and analge-

sic effects [14]. In addition, it irreversibly inhibits platelet aggre-

gation [15] and increases local blood flow and the temperature

of the tissue.

We hypothesized that a combination of menthol and MeSa

could affect the viability of skin flaps due to the anti-inflamma-

tory properties of these compounds and their effects on blood

vessels. No similar previous studies were found in a thorough

review of the English-language, French-language, and Turkish-

language literature. In this experimental study, the effects of a

topical cream containing 10% menthol and 15% MeSa (Kamfo-

lin, Münir Şahin, Istanbul, Turkey) on the viability of dorsal ran-

dom-pattern skin flaps in rats were investigated.

METHODS

This research was conducted with the permission of the local

ethical committee and in accordance with international health

and medical research guidelines for animal welfare. The rats

were housed in separate cages, placed in an environmentally

controlled room, and fed with standard rat chow and tap water

ad libitum. Three days before surgery, 200 mg/kg of paracetamol

(Parol, Atabay, Istanbul, Turkey) was added to their water for

analgesia. All surgical procedures and imaging studies were per-

formed under general anesthesia, which was carried out with an

intraperitoneal injection of 75−100 mg/kg of ketamine hydro-

chloride (Ketalar, Eczacıbaşı, Istanbul, Turkey) and 10 mg/kg

of 2% xylazine (Rompun, Bayer, Leverkusen, Germany). The

dorsal hair was removed with an electric shaver and all surgical

procedures were carried out under sterile conditions by a single

surgeon (UCD).

Experimental protocol

Forty adult (three months old) female Sprague-Dawley rats

weighing 179–267 g were divided into two groups of 20 each.

Modified version of the McFarlane was performed. A caudally

based skin flap (10 cm × 3 cm) was marked on the shaved dor-

sum of the rat, starting from the line that connects the iliac spines.

The flap was elevated beneath the panniculus carnosus muscle

(PCM) in both groups, and the deep circumflex iliac artery and

perforator vessels were cauterized in order to ensure a random

pattern of blood circulation. The flaps were sutured back to their

donor site after the scintigraphic measurements were performed

(Fig. 1). A sterile silicone sheet (0.5 mm) was placed on the do-

nor site in order to isolate the flap from the underlying bed.

Study group (n=20)

1.5 mL (0.05 mL/cm2) of a cream containing 10% menthol and

15% MeSa was applied via massage on the skin of the flap im-

mediately after the skin closure and once a day for six days. Be-

fore each application, debris from the previous application was

removed with sterile isotonic saline solution (0.9%).

Control group (n=20)

Sterile isotonic saline solution (0.9%) was applied via massage

on the skin of the flap immediately after the skin closure and

once a day for six days, in order to control for the vasodilatory

effect of the massage [16].

Vol. 42 / No. 6 / November 2015

697

Weight analysis of the rats

All the rats were weighed before and after the experimental pro-

tocol with a digital scale in order to evaluate their physiological

response to the surgery and to the cream.

Clinical and photographic analysis

All flaps were evaluated daily. On the seventh postoperative day,

the rats were anesthetized and standardized photographs of the

flaps were taken with a high-resolution digital camera (Fujifilm

FinePix F80EXR, Fujifilm Holdings Corp., Beijing, China) be-

fore suture removal. These images were then uploaded to a

computer, and the surface areas of the entire flap and the ne-

crotic part of the flap were calculated in mm2 using Adobe Pho-

toshop CS5 software (Adobe Systems Inc., San Jose, CA, USA)

(Fig. 2). The surface area of the viable part of the flap was calcu-

lated by subtracting the surface area of the necrotic part of the

flap from the surface area of the entire flap. Comparisons were

made within and between the two groups.

Radionuclide scintigraphic analysis

On the first day of the experiment, scintigrams of the flaps were

taken before skin closure; on the seventh day, scintigrams were

obtained and the rats were then sacrificed. The rats were inject-

ed with 1 mCi (37 MBq) of technetium-99m pertechnetate

(Tc99m-PO4) in 0.1 mL of isotonic saline solution through the

tail vein. The rats were placed prone under a single-head gamma

camera equipped with a pinhole collimator (Siemens E.Cam,

Siemens Medical Solutions, Hoffman Estates, IL, USA). Images

of both groups were taken five minutes after injection and were

visualized for five minutes in a 256 ×256-pixel matrix. One lead

plate was placed under the flap and two other plates covered the

rest of the rat in order to obtain only the image of the flap. The

lead plates were covered with waterproof paper; separate sets of

waterproof paper were used in each rat in order to protect the

plates from radioactive contamination.

Tc99m-PO4-soaked cotton in a needle cap was placed at the

distal end of the flap in order to visualize the distal border of the

flap on the scintigram. After five seconds, it was removed and

the actual scintigram was obtained.

An experienced nuclear medicine specialist who was blinded

to the groups interpreted the scintigrams. On the computer, a

rectangle was drawn that enclosed the borders of the flap, en-

compassing the total region of interest (ROI). The total ROI

was divided into three equal ROIs: the proximal ROI (from the

pedicle) was referred to as ROI A, the middle third was referred

to as ROI B, and the distal third was referred to as ROI C (Fig.

3). The borders of the hyperemic parts of the flap were drawn

manually, using the free hand method, and the flap area was cal-

culated (Fig. 4). Those values were referred to as the manually

marked count of the viable flap area (MCFA). All results were

compared within each group and between the two groups.

The photographs and the scintigrams were merged digitally

with Adobe Photoshop CS5 software to better visualize the via-

ble part of the flap (Figs. 5, 6).

The dashed line represents the measured surface area.

Fig. 2. Measurement of the necrotic area of the flap

surface

(A) Elevation of a caudally based dorsal skin flap, (B) skin closure and application of 1.5 mL of cream on a rat in the study group.

Fig. 1. A modified McFarlane flap

A B

Dölen UC et al. Menthol methyl salicylate rat skin flap

698

Histopathologic assessment

On the seventh day, the rats were sacrificed with high-dose ket-

amine after scintigraphic imaging. The flaps were divided from

their base between the two iliac spines. The entire flap was fixed

in 10% formaldehyde solution. After routine hospital-specified

histopathological procedures, the paraffin-embedded speci-

mens were sliced longitudinally into 5-μm thick segments along

the long axis of the flap. All specimens were stained with hema-

toxylin and eosin and examined under light microscopy (Shang-

hai Optical Instrument Factory, Shanghai, China) by the same

pathologist who was kept blind to the groups.

The percentages of the viable and necrotic parts of each flap

were measured. The erythrocyte-containing capillary vessels

were then counted in the pedicle region, before the necrotic re-

The measurement of the manually marked count of the viable flap

area is shown by an asymmetric line that encloses the hyperemic

area of the flap. The rectangle represents the entire flap area. A

scintigram was taken (A) before and (B) after the experiment.

Fig. 4. Measurement of the manually marked count of the

viable flap area

A B

An image obtained after digitally combining the photographs and

the scintigrams. In the study group, (A) scintigrams taken before

the experiment showed little perfusion in the flap, while (B) scinti-

grams taken after the experiment showed almost no perfusion in

the flap.

Fig. 5. Combination of a scintigram and a photograph

A B

In the control group, (A) half of the flap was perfused before the

experiment, and (B) perfusion was only found in the pedicle region

after the experiment.

Fig. 6. Combination of a scintigram and a photograph

A B

Measurements of the ROIs on flaps in the (A, B) study and (C, D)

control groups. The flaps were divided into three ROIs, referred to as

ROI A, ROI B, and ROI C. Scintigrams (A, C) before and (B, D) after

the experiment. The white arrow shows the radionuclide-soaked

material that was used to identify the end of the flap.

Fig. 3. Measurements of the regions of interest (ROIs)

Vol. 42 / No. 6 / November 2015

699

gion, and at the transition zone between those regions. These

counts were performed at five different areas under high magni-

fication (400 × ) in each region in order to calculate the density

of capillary vessels per 1 mm2. The vessels were counted both at

the level of the papillary dermis and inferior to the PCM, and

the results were compared between the two groups (Fig. 7).

Microscopic images (40× ) of the flaps were photographed.

The thickness of PCM muscle was measured at the pedicle re-

gion, the necrotic region, and at the transition zone between

these regions with the help of Adobe Photoshop CS5. Muscle

thickness was measured in three different places in each region,

and the arithmetic average of these measurements was used as

the muscle thickness value for each region (Fig. 7).

Statistical analysis

Since the scintigraphy results were not normally distributed, a

base-10 logarithmic transformation was applied. The trans-

formed values were used for statistical analysis.

The Mann-Whitney U test was used to compare the necrotic,

viable, and total surface areas of the flaps, as calculated by photo-

graphic analysis. The ROIs and the MCFA were measured using

radionuclide scintigraphy, and the percentage of necrotic areas,

capillary vessel density, and the thickness of the PCM were ana-

lyzed by histopathology and compared between the study group

and the control group. The Wilcoxon signed-rank test was used

to compare the weight of the rats, the ROIs, and the MCFA with-

in the same group. The Kruskal-Wallis test was used to compare

the thickness of the PCM in different regions of the same flap.

Statistical analysis was performed using SPSS ver. 15.0 (SPSS

Inc., Chicago, IL, USA). Data were expressed as the mean±

standard deviation, and the cut-off point for statistical signifi-

cance was 0.05.

RESULTS

Two rats in the control group and one rat in the study group

died of unknown causes before the study was completed. Nei-

ther wounds nor systemic infections were observed in the sur-

viving rats.

Weight analysis of the rats

Before the experimental protocol, the median weight of the

study group was 227 g (range, 204–249 g), and that of the con-

trol group was 211 g (range, 193–233.3 g). On the seventh post-

operative day, the median weight of the study group was 205 g

(range, 183–227 g), and that of the control group was 195.5 g

(range, 182–213.8 g). No statistically significant difference was

observed between the two groups before (P= 0.13) and after

(P= 0.28) the experimental protocol. These figures show that

the rats were distributed randomly. The weight of the rats de-

creased to a statistically significant extent by the end of the ex-

periment in both the study (P<0.001) and control groups

(P = 0.001).

Clinical and photographic analysis

No statistically significant difference between the groups was

found in the total surface areas of the flaps (P= 0.6). This find-

ing indicates that similarly sized flaps were elevated in both

groups. The viable flap area of the study group (median, 15.8

cm2) was larger than that of the control group (median, 8.5

(A) Measurement of capillary vessel density (H&E, ×400). (B) Measurement of the thickness of the panniculus carnosus muscle (PCM) at three

different points in the necrotic region. An asterisk shows the dermal layer, the plus sign indicates the PCM, black arrows show the vessels, and

double arrows indicate the thickness of the PCM (H&E, ×40).

Fig. 7. Histopathology

A B

Dölen UC et al. Menthol methyl salicylate rat skin flap

700

cm2), which was a statistically significant difference (P= 0.004)

(Table 1, Fig. 8).

Radionuclide scintigraphic analysis

No statistical difference was observed in the values of ROI A be-

fore and after the experimental protocol in either the study

group (P= 0.51) or the control group (P= 0.09). A statistically

significant decrease was observed in the values of ROI B and

ROI C, both in the study group (P= 0.04 and P= 0.01, respec-

tively) and in the control group (P = 0.01 and P=0.00, respec-

tively).

A statistically significant decrease was found in the total ROI

of the control group after the experiment (P= 0.006). No statis-

tically significant difference was observed in the values of the to-

tal ROI of the study group (P=0.11). Despite the reduction in

both ROI B and ROI C in the study group, the total ROI did

not change significantly (Table 2).

No statistical differences were observed in all ROI values be-

tween the study and the control group before and after the ex-

perimental protocol. This finding shows that the pattern of

blood perfusion was similar among the flaps.

In the study group, the MCFA did not change after the experi-

mental protocol (P= 0.32). However, it decreased in the control

group (P =0.01) (Table 2).

No statistically significant difference was found in the pre-ex-

perimental scintigraphic values of the study group and the con-

trol group (MCFA, P =0.17). No statistically significant differ-

ence was found in the post-experimental scintigraphic values of

the two groups (MCFA, P= 0.11).

Histopathologic assessment

It was observed under the microscope that 36.18%± 20.79% of

the flaps in the study group and 28.22%± 10.31% of the flaps in

the control group survived. The difference between the two

groups with respect to the percentage of the viable flaps was not

statistically significant (P =0.18).

Capillary vessel density (the number of vessels in 1 mm2) did

not significantly differ between the study group and the control

group in the pedicle region (17 [range, 11–20] vs. 18 [range,

12.3–22], P= 0.94), the transitional region (21 [range, 15–25]

vs. 19.5 [range, 11–27], P = 0.78), and in the pre-necrotic region

(17 [range, 5–21] vs. 18 [range, 12–24.5], P = 0.22). Likewise,

no significant difference was found between the dermal papil-

lary layer and the layer inferior to the PCM.

The thickness of the PCM (μm) did not show a significant dif-

ference between the study and the control groups in the pedicle

region (212 [range, 163.1–235.2] vs. 210.2 [range, 192.4–

248.9], P= 0.60), the middle region (205.9 [range, 181.9–

244.2] vs. 181.1 [range, 156.8–236.9], P = 0.13), or in the ne-

Table 1. Distribution of the mean flap surface areas in the

study and the control group

Surface area of the

flap (cm2)

Study group

(n = 19)

Control group

(n = 18) P-valuea)

Viable 15.8 (10.2–19.4) 8.5 (5.9–12) 0.004b)

Necrotic 14.2 (11.1–19.6) 20.4 (17.8–23.1) 0.01b)

Total 30.1 (29.5–31.9) 29.9 (29.2–30.9) 0.60

A comparison of the control and study groups according to the median viable,

necrotic, and total flap areas. Values are presented as median (interquartile

range).

a)Mann-Whitney U test; b)Statistically significant.

Table 2. In-group comparison between pre- and post-experimental scintigraphic values

Scintigraphic values Study group (n=19) P-value Control group (n=18) P-value

Before After Before After

Region of interest A 3.7 (3.5–3.9) 3.7 (3.3–3.9) 0.51 3.6 (3.4–3.9) 3.5 (3.3–3.8) 0.09

Region of interest B 3.3 (3.2–3.6) 3.2 (2.8–3.4) 0.04a) 3.0 (2.6–3.2) 2.95 (2.6–3.2) 0.01a)

Region of interest C 3.1 (2.9–3.3) 2.7 (2.4–3.1) 0.01a) 3 (2.7–3.3) 2.5 (2.4–2.8) <0.01a)

Region of interest total 3.9 (3.8–4.2) 3.9 (3.5–4.1) 0.11 3.9 (3.7–4.1) 3.7 (3.5–3.9) 0.006a)

Manually marked count of viable flap area 3.9 (3.8–4.2) 3.8 (3.4–4) 0.06 3.8 (3.7–4.1) 3.7 (3.4–3.8) 0.01a)

Values are presented as median (interquartile range). P-value is calculated with Wilcoxon signed-rank test.

a)Statistically significant.

Fig. 8. Mean flap surface areas

Flap surface area (cm2)

Study group Control group

45% 31%

Necrotic

Viable

Vol. 42 / No. 6 / November 2015

701

crotic region (264.8 [range, 194.4–329.5] vs. 306.1 [range,

213.6–378.9], P = 0.83).

DISCUSSION

Flaps are the most important weapon in the arsenal of plastic

surgeons. Even though their use is as old as the history of hu-

mankind, some aspects of flap viability remain unknown. Sir

Harold Gillies said that “plastic surgery is a constant battle be-

tween blood supply and beauty,” and in this battle, the mission

of plastic surgeons is to elevate a flap without partial or total flap

loss. Especially in random-pattern flaps, blood perfusion is the

major issue that limits the size of the flap. Surgical delay is an ef-

ficient method, but requires multiple operations that not all pa-

tients can tolerate or accept. In contrast, pharmacologic agents

do not require additional surgery, but are often of limited utility

due to their high costs and restricted availability. Among these

agents, topical drugs are the most advantageous because they

can reach a therapeutic dose without causing any systemic side

effects. In addition, patients can easily use these drugs immedi-

ately after surgery, even if they are experiencing nausea, and they

can apply them by themselves at home without medical super-

vision. Topical agents require a skin permeation enhancer, such

as terpenes, in order to be absorbed through the cutaneous lay-

ers. Due to its lipophilic structure, menthol can easily pass the

stratum corneum and its chiral center enhances the absorption

of other molecules [17]. It has already been shown that menthol

facilitates and accelerates the absorption of MeSa [18].

A sterile silicone sheet was used to prevent the skin flap from

surviving as a graft [19,20]. We observed loose capsule forma-

tion and a small seroma around the sheet at the end of the ex-

periment. In the literature, the necrotic part of random-pattern

dorsal skin flaps has been reported to vary widely, from 22% to

50% [21]. In our study, the percentage of necrotic areas in the

flaps in the control group was found to be 69% in the photo-

graphic analysis and 72% in the histopathological analysis. We

interpreted this high percentage of necrosis as the result of the

silicone sheet, which prevented the flaps from undergoing plas-

matic imbibition and revascularization from the underlying bed.

We concluded that the cream did not cause any pain or irrita-

tion, because no significant differences were observed between

the two groups in terms of post-experimental weight and in the

consumption of water and food.

Johnson et al. [8] showed that rat blood vessels contain TRPM8

channels, which cause vasodilatation or vasoconstriction depen-

ding on the existing vascular tone. TRPM8 channels are also pre-

sent in human blood vessels and regulate vasomotor tone. It has

already been reported that Ca2+ channel blockers increase flap

survival [15,22] and if menthol uses the same pathway, it can be

hypothesized to increase flap viability [23].

Namer et al. [9] tested the effect of 40% menthol solution on

healthy humans. They used laser Doppler to show that menthol

increased the superficial blood flow in the area of the forearm

where menthol was applied. Olive et al. [10] compared the ef-

fects of a cream containing 3.5% menthol and ice on the brachial

artery of 12 healthy humans using ultrasound. Contrary to Na-

mer et al., they found that menthol decreased blood flow. Topp

et al. [24] also performed a similar study and found that both

3.5% menthol gel and 0.5 kg ice decreased the blood flow of the

radial artery without changing the diameter of the artery or the

pulse of the subjects. In another study, they investigated the ef-

fect of 3.5% menthol gel and a 10% menthol wipe on the thigh

after three isokinetic maximum voluntary muscular contrac-

tions of the quadriceps and hamstrings. They found that the ap-

plication of the gel decreased the blood flow in the popliteal ar-

tery, which had been increased after isokinetic exercises [11]. It

was hypothesized that topical menthol causes vasoconstriction

by inhibiting the nitric oxide system [13], and that its local cool-

ing property acts through a mechanism similar to that of cold

temperature sensations, which are perceived through α2c adren-

ergic receptors [12].

In addition to adrenergic vasoconstriction, platelet aggregation

in the microvascular system plays a major role in ischemic ne-

crosis of skin flaps. After trauma or surgery, thromboxane A2

(TXA2) released from the platelets causes vasoconstriction and

platelet aggregation. In normal conditions, prostacyclin released

from the vascular endothelium blocks the effect of TXA2. How-

ever, after vascular damage, the amount of TXA2 rises and that

of prostacyclin decreases, and the perfusion of the tissue is

therefore decreased. Salicylates, TXA2 inhibitors, and selective

TXA2 antagonists increase the viability of random-pattern skin

flaps [15,25]. MeSa, like all other salicylates, is a strong inhibitor

of prostaglandin synthase and platelet aggregation. We hypothe-

sized that MeSa would potentially increase flap survival through

this mechanism.

As described above, conflicting data exist regarding the effect

of menthol on vascular tone and blood flow. However, the stud-

ies showing a vasoconstrictive effect of menthol only investigat-

ed the blood flow of the major arteries and did not evaluate cu-

taneous blood flow. In our experiment, we indirectly observed

the blood circulation of the skin while investigating flap survival.

Photographic analysis showed that the cream increased flap via-

bility, although the number of the vessels did not change after

the experiment. The finding that the total ROI and MCFA of

the study group did not change, while the total ROI of the con-

trol group decreased after the experiment, may be attributed to

Dölen UC et al. Menthol methyl salicylate rat skin flap

702

the vasodilatory effect of the cream. This effect could possibly

be used in flap surgeries or replantations in order to increase the

perfusion of the flap. However, it was not found to increase the

overall viability of the flaps.

A limitation of our study was the absence of a control group

using a control cream instead of saline. Moreover, laser Doppler

or angiography could have provided more reliable results re-

garding the vasodilatory effect of the cream than scintigraphy,

which provides functional imaging of living cells. Immunologi-

cal stains could have shown neoangiogenesis more clearly.

Based on these results, it is certain that the cream containing

10% menthol and 15% methyl salicylate did not reduce the via-

bility of the flaps. Its vasodilatory effect means that it can be in-

corporated as part of the dressing in reconstructive operations

where skin perfusion is compromised. Nevertheless, further ex-

periments are necessary to determine the optimal concentra-

tions of menthol and MeSa (alone or in combination) for im-

proving flap viability.

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Short-term cutaneous vasodilatory and thermosensory effects of topical methyl salicylate

Article

Full-text available

Apr 2024

Introduction Methyl salicylate, the main compound of wintergreen oil, is widely used in topical applications. However, its vascular and thermosensory effects are not fully understood. The primary aim was to investigate the effects of topical methyl salicylate on skin temperature (Tskin), skin microcirculation (MCskin) and muscle oxygen saturation (SmO2) compared to a placebo gel. The secondary aim was to assess thermosensory responses (thermal sensation, thermal comfort) and to explore to which extent these sensations correspond to the physiological responses over time. Methods 21 healthy women (22.2 ± 2.9 years) participated in this single-blind, randomized controlled trial. Custom-made natural wintergreen oil (12.9%), containing methyl salicylate (>99%) and a placebo gel, 1 g each, were applied simultaneously to two paravertebral skin areas (5 cm × 10 cm, Th4-Th7). Tskin (infrared thermal imaging), MCskin (laser speckle contrast imaging) and SmO2 (deep tissue oxygenation monitoring) and thermosensation (Likert scales) were assessed at baseline (BL) and at 5-min intervals during a 45 min post-application period (T0-T45). Results Both gels caused an initial decrease in Tskin, with Tskin(min) at T5 for both methyl salicylate (BL-T5: Δ-3.36°C) and placebo (BL-T5: Δ-3.90°C), followed by a gradual increase (p < .001). Methyl salicylate gel resulted in significantly higher Tskin than placebo between T5 and T40 (p < .05). For methyl salicylate, MCskin increased, with MCskin(max) at T5 (BL-T5: Δ88.7%). For placebo, MCskin decreased (BL-T5: Δ-17.5%), with significantly lower values compared to methyl salicylate between T0 and T45 (p < .05). Both gels had minimal effects on SmO2, with no significant differences between methyl salicylate and placebo (p > .05). Thermal sensation responses to topical methyl salicylate ranged from “cool” to “hot”, with more intense sensations reported at T5. Discussion The findings indicate that topical methyl salicylate induces short-term cutaneous vasodilation, but it may not enhance skeletal muscle blood flow. This study highlights the complex sensory responses to its application, which may be based on the short-term modulation of thermosensitive transient receptor potential channels.

Post-treatment with metformin improves random skin flap survival through promoting angiogenesis in rats

Article

Full-text available

Jun 2022

Skin flap necrosis has been remained as an unsolved problem in plastic and reconstructive surgeries. Here, we explored the effects of metformin post-treatment on random skin flap survival in rats. An 8.00 × 2.00 cm dorsal skin flap was created in 24 rats and they were then divided into three groups (n = 8) including Control, metformin (Met) 50.00 mg kg-1 and Met 100 mg kg-1. All animals were administrated orally until seven days after flap surgery. Flap survival, the number of blood vessels and mast cells in the flap tissues were analyzed. Vascular endothelial growth factor (VEGF) expression levels in flap tissues was also determined using immunohistochemical methods. The percentage of survival area in Met 50.00 mg kg-1 and Met 100 mg kg-1 groups were significantly higher compared to control. The blood vessel density and the VEGF positive cells in the viable areas of flaps showed a significant increase in Met 50.00 mg kg-1 group compared to control group. The results of this study suggested that treatment with metformin, especially with low dose following skin flap surgery was effective in improving the flap survival and increasing the neovascularization in the flaps tissues of rats.

Standardized Pre-clinical Surgical Animal Model Protocol to Investigate the Cellular and Molecular Mechanisms of Ischemic Flap Healing

Article

Full-text available

Jan 2024
BIOL PROCED ONLINE

Background Some of the most complex surgical interventions to treat trauma and cancer include the use of locoregional pedicled and free autologous tissue transfer flaps. While the techniques used for these reconstructive surgery procedures have improved over time, flap complications and even failure remain a significant clinical challenge. Animal models are useful in studying the pathophysiology of ischemic flaps, but when repeatability is a primary focus of a study, conventional in-vivo designs, where one randomized subset of animals serves as a treatment group while a second subset serves as a control, are at a disadvantage instigated by greater subject-to-subject variability. Our goal was to provide a step-by-step methodological protocol for creating an alternative standardized, more economical, and transferable pre-clinical animal research model of excisional full-thickness wound healing following a simulated autologous tissue transfer which includes the primary ischemia, reperfusion, and secondary ischemia events with the latter mimicking flap salvage procedure. Results Unlike in the most frequently used classical unilateral McFarlane’s caudally based dorsal random pattern skin flap model, in the herein described bilateral epigastric fasciocutaneous advancement flap (BEFAF) model, one flap heals under normal and a contralateral flap—under perturbed conditions or both flaps heal under conditions that vary by one within-subjects factor. We discuss the advantages and limitations of the proposed experimental approach and, as a part of model validation, provide the examples of its use in laboratory rat (Rattus norvegicus) axial pattern flap healing studies. Conclusions This technically challenging but feasible reconstructive surgery model eliminates inter-subject variability, while concomitantly minimizing the number of animals needed to achieve adequate statistical power. BEFAFs may be used to investigate the spatiotemporal cellular and molecular responses to complex tissue injury, interventions simulating clinically relevant flap complications (e.g., vascular thrombosis) as well as prophylactic, therapeutic or surgical treatment (e.g., flap delay) strategies in the presence or absence of confounding risk factors (e.g., substance abuse, irradiation, diabetes) or favorable wound-healing promoting activities (e.g., exercise). Detailed visual instructions in BEFAF protocol may serve as an aid for teaching medical or academic researchers basic vascular microsurgery techniques that focus on precision, tremor management and magnification. Graphical Abstract

Topical Analgesic Containing Methyl Salicylate and L-Menthol Accelerates Heat Loss During Skin Cooling for Exercise-Induced Hyperthermia

Article

Full-text available

Jul 2022

Hyperthermia impairs physical performance and, when prolonged, results in heat stroke or other illnesses. While extensive research has investigated the effectiveness of various cooling strategies, including cold water immersion and ice-suit, there has been little work focused on overcoming the cutaneous vasoconstriction response to external cold stimulation, which can reduce the effectiveness of these treatments. Over-the-counter (OTC) topical analgesics have been utilized for the treatment of muscle pain for decades; however, to date no research has examined the possibility of taking advantage of their vasodilatory functions in the context of skin cooling. We tested whether an OTC analgesic cream containing 20% methyl salicylate and 6% L-menthol, known cutaneous vasodilators, applied to the skin during skin cooling accelerates heat loss in exercise-induced hyperthermia. Firstly, we found that cutaneous application of OTC topical analgesic cream can attenuate cold-induced vasoconstriction and enhance heat loss during local skin cooling. We also revealed that core body heat loss, as measured by an ingestible telemetry sensor, could be accelerated by cutaneous application of analgesic cream during ice-suit cooling in exercise-induced hyperthermia. A blunted blood pressure response was observed during cooling with the analgesic cream application. Given the safety profile and affordability of topical cutaneous analgesics containing vasodilatory agents, our results suggest that they can be an effective and practical tool for enhancing the cooling effects of skin cooling for hyperthermia.

The outcomes of dexmedetomidine and calcitriol on flap viability 1

Article

Full-text available

Sep 2020
ACTA CIR BRAS

Purpose: To evaluate protective effects of dexmedetomidine, calcitriol and their combination. Methods: Forty Wistar-albino rats were divided into 4 groups; group of Sham (Group Sham); group of dexmedetomidine (Group DEX); group of calcitriol (Group CAL) and group of dexmedetomidineandcalcitriol (Group DEX-CAL). Photographic analysis was used for macroscopic analysis and perfusion analyses were evaluated by scintigraphy. Additionally, tissue malondialdehyde (MDA) and total oxidant status (TOS) and total antioxidant activity (TAS) were recorded and oxidative stress index (OSI) was calculated. Each flap was assessed by histopathology. Results: Compared to Group Sham, the viable flap areas were higher in all treatment groups both by photographic image analyses and perfusion analyses (p<0.05). Group DEX-CAL had the highest viable flap percentage both in scintigraphic and photographic analyses; whereas Group Sham had the lowest viable flap percentage. Similarly, TAS and MDA levels were elevated and TOS levels were declined in all treatment groups compared to Group Sham (p<0.005). Histopathological analysis at flap demarcation zone confirmed neovascularization was significantly higher and edema, necrosis and inflammation were significantly lower in all treatment groups compared to Group Sham. Conclusion: The outcomes show that additional premedication with either dexmedetomidine or calcitriol or their combination reduces ischemia-reperfusion injury of flap area and show significant increase in the percentage of viable flap tissue.

Novel, standardized pre-clinical surgical animal model to investigate the cellular and molecular mechanisms of ischemic flap healing

Preprint

Full-text available

Jun 2023

Background Some of the most complex surgical interventions to treat trauma and cancer include the use of locoregional pedicled flaps and free autologous tissue transfer. While the techniques used for these reconstructive surgery procedures have improved over time, flap complications and even failure remain a significant clinical challenge. Animal models are useful in studying the pathophysiology of ischemic flaps, but when repeatability is a primary focus of a study, conventional in-vivo designs, where one randomized subset of animals serves as a treatment group while a second subset serves as a control, are at a disadvantage instigated by greater subject-to-subject variability. Results We present a novel, economical, and standardized pre-clinical animal model of excisional full-thickness wound closure and healing using axial pattern flaps that incorporates an anatomically named vascular pedicle. In this bilateral epigastric fasciocutaneous advancement flap (BEFAF) model, which simulates an actual clinical procedure of autologous tissue transfer, one flap heals under normal and a contralateral flap - under perturbed conditions or both flaps heal under conditions that vary by one within-subjects factor. Although we describe step-by-step methodological approach how to create such flaps as well as collect, process, and analyze flap survival data in larger size rodents, specifically, the rats (Rattus norvegicus), the BEFAF model is transferable and could be implemented in any mammalian species. We also stress the importance of taking and presenting high resolution histological stain images of the flaps that are indispensable for diagnostic result interpretation in the full-thickness wound healing studies. Conclusions BEFAFs may be used to investigate the spatiotemporal cellular and molecular responses to complex tissue injury and interventions simulating clinically relevant flap complications (e.g., secondary arterial, venous, or mixed ischemia) and therapeutic or surgical treatments (e.g., flap delay) in the presence or absence of confounding risk factors (e.g., substance abuse, irradiation, diabetes) or favorable wound-healing promoting activities (e.g., exercise). This technically challenging but feasible reconstructive surgery model eliminates inter-subject variability, while concomitantly minimizing the number of animals needed to achieve adequate statistical power. BEFAF also serves as an aid for teaching basic vascular microsurgery techniques that focus on precision, tremor management and magnification.

Effects of cutaneous administration of an over-the-counter menthol cream during temperate-water immersion for exercise-induced hyperthermia in men

Article

Full-text available

May 2023

Introduction: Hyperthermia impairs various physiological functions and physical performance. We examined the effects of cutaneous administration with an over-the-counter (OTC) analgesic cream containing 20% methyl salicylate and 6% L-menthol during temperate-water immersion (TWI) for exercise-induced hyperthermia. Methods: In a randomized crossover design, twelve healthy males participated in both of two experiments. Firstly, participants underwent a 15-min TWI at 20°C with (CREAM) or without (CON) cutaneous application of an analgesic cream. Cutaneous vascular conductance (CVC) was measured using laser doppler flowmetry during TWI. In a subsequent experiment, same participants performed a 30-min strenuous interval exercise in a heated (35°C) environment to induce hyperthermia (~39°C), which was followed by 15 min of TWI. Results: Core body temperature, as measured by an ingestible telemetry sensor, and mean arterial pressure (MAP) were measured. CVC and %CVC (% baseline) were higher during TWI in CREAM than in CON (Condition effect: p = 0.0053 and p = 0.0010). An additional experiment revealed that core body heat loss during TWI was greater in CREAM than in CON (Cooling rate: CON 0.070 ± 0.020 vs. CREAM 0.084°C ± 0.026°C/min, p = 0.0039). A more attenuated MAP response was observed during TWI in CREAM than in CON (Condition effect: p = 0.0007). Conclusion: An OTC analgesic cream containing L-menthol and MS augmented cooling effects when cutaneously applied during TWI in exercise-induced hyperthermia. This was, at least in part, due to the counteractive vasodilatory effect of the analgesic cream. The cutaneous application of OTC analgesic cream may therefore provide a safe, accessible, and affordable means of enhancing the cooling effects of TWI.

Remodelling of the superficial vascular network of skin flaps in rats, following a vasodilatory cream application, before elevation

Article

Feb 2022

Flap necrosis on random pattern skin flaps continues to be a challenge. In this study, we evaluated whether topical application of a vasodilator substance (the prostaglandin PGI2 analogue, Iloprost®, in cream form) as pre-treatment, would increase blood flow and improve flap viability. Wistar rats randomly allocated into four groups with 7 rats per group and two flaps 4cm × 2cm in the same rat i.e. 56 flaps were developed. Flaps on the wright side received pre-treatment with Cream in different drug concentrations, 2.5μgr/gr, 5 μgr/gr, and 10μgr/gr and 20 μgr/gr containing the active factor Iloprost®® ZK 36,374(M. W 360.5) prepared with white petrolatum as a base. Flaps on the left side received placebo cream (white petrolatum). After 10 days of flap pre-treatment, evaluation of blood flow by laser Doppler flowmetry (LDF) were recorded, then flap elevation and re suturing back were performed. After 7 days we estimated flap viability on digital imaging and the percentages of flap survival estimated. Means and standard deviations were used to describe blood flow measurements and survival percentages. The significance was set at 0.05 in all cases and the analysis were carried out with the use of the SPSS v23.0. Furthermore, we performed dynamic analyses of circulation using the radioisotope 99mTc which confirmed hyperaemia of the treated areas relative to that observed in control areas. These findings demonstrated that pretreatment of skin flaps with Iloprost® cream for 10 days prior to elevation increased blood flow in the flap and improved their overall survival rate.

Standardization of the Rat Dorsal Random Pattern (McFarlane) Flap Model and Evaluation of the Pharmacological Agents Aiming to Salvage Partial Flap Necrosis: A Systematic Review and a Meta-analysis

Article

May 2021

Introduction: Partial flap necrosis is a common complication after surgery. McFarlane flap model has been used for assessment of various agents' effects on random flap survival. The aim of this study was to review the methodology of studies using this flap model and reveal the most successful agents. Materials and methods: PubMed, Scopus, and Web of Science databases were screened for words "McFarlane flap," "flap survival," and ("flap" and "rat") by using time limits between 1965 and 2019. A total of 71 original articles were reviewed. Dimensions and base (cranial/caudal) of the flap, treatment protocol, follow-up period, and survival rates were extracted. Modified survival rates were calculated. Coefficients of variation of cranial/caudally based control group flaps and most commonly used flap models were calculated to assess interstudy variability. Results: A total of 165 different treatment regimens were studied. One-hundred twelve regimens (67.9%) were found to increase flap survival. Most common flap dimensions were 9 cm × 3 cm, followed by 10 cm × 3 cm, 8 cm × 2 cm and 6 cm × 2 cm. Studies using caudally based flaps showed less interstudy variability, but survival rates were similar. Pentoxifylline, sildenafil, chlorpromazine, phenoxybenzamine, and phentolamine were reported to be successful in multiple studies. Conclusions: There are numerous agents found to be effective for treatment of partial flap necrosis, but further clinical research is needed. To overcome standardization problems, use of commonly used flap dimensions with a caudal base and interpretation of results after 7 days of follow-up seems appropriate.

Change in total anti-oxidant capacity (TAC) of tissue, a new method for improving dermal wound healing

Article

Full-text available

Jul 2019

in determining the physiological or physio-pathological function of oxygen reactive spices. Release of reactive oxygen species (ROS) in dermal wounds, leads to necrosis as well as apoptosis and tissue loss. Management of the risk factors for tissue loss due to flap failure can reduce risk factors with resultant improvement in the survival and efficacy of the flaps. Materials and Methods: 40 Sprauge-Dawkey rats were divided into 4 groups (1) sham group with an 8×3 cm skin cut without skin elevation (2) Flap group with an 8×3 cm skin cut with skin elevation (3) cymophenol group treated with cymophenol (25 mg/kg/day) (4) cymophenol group treated with cymophenol (50 mg/kg/day) after surgery. After one week, percentage of the living tissue, superoxide dismutase (SOD) activity and amount of malondialdehyde (MDA) were measured in the groups and the proximity results were extracted. Using GraphPad Prism 6 software, data were analyzed and compared by ANOVA and Tukey test. Results: Cymophenol application decreased the amount of malondialdehyde (p

Topical Menthol, Ice, Peripheral Blood Flow, and Perceived Discomfort

Article

Full-text available

Mar 2013
J ATHL TRAINING

Context: Injury management commonly includes decreasing arterial blood flow to the affected site in an attempt to reduce microvascular blood flow and edema and limit the induction of inflammation. Applied separately, ice and menthol gel decrease arterial blood flow, but the combined effects of ice and menthol gel on arterial blood flow are unknown. Objectives: To compare radial artery blood flow, arterial diameter, and perceived discomfort before and after the application of 1 of 4 treatment conditions. Design: Experimental crossover design. Setting: Clinical laboratory. PARTICIPANTS OR OTHER PARTICIPANTS: Ten healthy men, 9 healthy women (mean age = 25.68 years, mean height = 1.73 m, mean weight = 76.73 kg). Intervention(s): Four treatment conditions were randomly applied for 20 minutes to the right forearm of participants on 4 different days separated by at least 24 hours: (1) 3.5 mL menthol gel, (2) 0.5 kg of crushed ice, (3) 3.5 mL of menthol gel and 0.5 kg of crushed ice, or (4) no treatment (control). Main outcome measure(s): Using high-resolution ultrasound, we measured right radial artery diameter (cm) and blood flow (mL/min) every 5 minutes for 20 minutes after the treatment was applied. Discomfort with the treatment was documented using a 1-to-10 intensity scale. Results: Radial artery blood flow decreased (P < .05) from baseline in the ice (-20% to -24%), menthol (-17% to -24%), and ice and menthol (-36% to -39%) treatments but not in the control (3% to 9%) at 5, 10, and 15 minutes. At 20 minutes after baseline, only the ice (-27%) and combined ice and menthol (-38%) treatments exhibited reductions in blood flow (P < .05). Discomfort was less with menthol than with the ice treatment at 5, 10, and 20 minutes after application (P < .05). Arterial diameter and heart rate did not change. Conclusions: The application of 3.5 mL of menthol was similar to the application of 0.5 kg of crushed ice in reducing peripheral blood flood. Combining crushed ice with menthol appeared to have an additive effect on reducing blood flow.

Effect of topical menthol on ipsilateral and contralateral superficial blood flow following a bout of maximum voluntary muscle contraction

Article

Full-text available

Jun 2011
Int J Sports Phys Ther

Various doses of topical menthol are commonly applied prior to, during, and after exercise to relieve pain although there is limited empirical evidence examining the physiological effects of this treatment. The purpose of this study was to examine the effects of two different doses of menthol (3.5% and 10%) on blood flow and arterial diameter before and after an acute bout of three isokinetic maximum voluntary muscular contraction (MVMC) of the quadriceps and hamstrings. Blood flow and arterial diameter of the right and left popliteal arteries were measured with an ultrasound Doppler prior to and after subjects completed 1 set of 3 MVMC isokinetic knee extension/flexion exercises. Immediately following this exercise one of three different treatment conditions was randomly applied to the right thigh only; 3.5% menthol gel, 10% menthol wipe, or a control condition. Five minutes following the treatment application blood flow through both right and left popliteal arteries was reassessed. This procedure was completed once per week until each of the 16 subjects was exposed to each treatment condition. Repeated measures ANOVA with post hoc analysis indicated that both menthol dosages resulted in significant decreases in popliteal blood flow on the right (-19.60 to -8.39%) and left sides (-14.72 to -5.4%) while the control condition demonstrated an increase in blood flow bilaterally (+26.40 to +15.19%) as a result of the MVMC exercise. The right popliteal arterial diameter was also significantly reduced as a result of both menthol dosages (-5.73 to -6.73%) and increased under the control condition (+6.67%). These results indicate that topical menthol has a rapid effect on reducing ipsilateral and contralateral arterial blood flow as well as ipsilateral arterial diameter. 2a.

Transient receptor potential melastatin 8 channel involvement in the regulation of vascular tone

Article

Full-text available

May 2009

The transient receptor potential melastatin 8 (TRPM8) channel has been characterized as a cold and menthol receptor expressed in a subpopulation of sensory neurons but was recently identified in other tissues, including the respiratory tract, urinary system, and vasculature. Thus TRPM8 may play multiple functional roles, likely to be in a tissue- and activation state-dependent manner. We examined the TRPM8 channel presence in large arteries from rats and the functional consequences of their activation. We also aimed to examine whether these channels contribute to control of conscious human skin blood flow. TRPM8 mRNA and protein were detected in rat tail, femoral and mesenteric arteries, and thoracic aorta. This was confirmed in single isolated vascular myocytes by immunocytochemistry. Isometric contraction studies on endothelium-denuded relaxed rat vessels found small contractions on application of the TRPM8-specific agonist menthol (300 microM). However, both menthol and another agonist icilin (50 microM) caused relaxation of vessels precontracted with KCl (60 mM) or the alpha-adrenoceptor agonist phenylephrine (2 microM) and a reduction in sympathetic nerve-mediated contraction. These effects were antagonized by bromoenol lactone treatment, suggesting the involvement of Ca(2+)-independent phospholipase A(2) activation in TRPM8-mediated vasodilatation. In thoracic aorta with intact endothelium, menthol-induced inhibition of KCl-induced contraction was enhanced. This was unaltered by preincubation with either N(omega)-nitro-l-arginine methyl ester (l-NAME; 100 nM), a nitric oxide synthase inhibitor, or the ACh receptor antagonist atropine (1 microM). Application of menthol (3% solution, topical application) to skin caused increased blood flow in conscious humans, as measured by laser Doppler fluximetry. Vasodilatation was markedly reduced or abolished by prior application of l-NAME (passive application, 10 mM) or atropine (iontophoretic application, 100 nM, 30 s at 70 microA). We conclude that TRPM8 channels are present in rat artery vascular smooth muscle and on activation cause vasoconstriction or vasodilatation, dependent on previous vasomotor tone. TRPM8 channels may also contribute to human cutaneous vasculature control, likely with the involvement of additional neuronal mechanisms.

Menthol: A simple monoterpene with remarkable biological properties

Article

Sep 2013

Menthol relaxes rat aortae, mesenteric and coronary arteries by inhibiting calcium influx

Article

Feb 2013

Menthol, a naturally occurring compound in mint, is known to give cold sensation. However, previous findings about its pharmacological activity in blood vessels are full of paradox. The present study investigated the action of menthol on vascular reactivity in different arteries isolated from male Sprague-Dawley rats, e.g. aortae, main mesenteric arteries and coronary arteries. The arterial segments were suspended in organ bath or in wire myograph for measurement of isometric force. Menthol concentration-dependently relaxed all three arteries with similar relaxing sensitivity in arteries contracted by different contractors, KCl, U46619 (9,11-dideoxy-9α,11α-methanoepoxy Prostaglandin F(2)α) and phenylephrine. Menthol-induced relaxations were unaffected by nitric oxide synthase inhibitor L-NAME, soluble guanylyl cyclase inhibitor ODQ, or mechanical removal of endothelium. Menthol also concentration-dependently suppressed 60mM KCl-induced constriction and CaCl(2)-induced contraction in Ca(2+)-free K(+)-containing solution. Calcium fluorescent imaging using fluo-4 showed that 10min-incubation with 1mM menthol inhibited 60mM KCl-induced Ca(2+) influx in rat aortic smooth muscle cell line A7r5 and vascular smooth muscle of coronary arteries. To conclude, menthol induces relaxation and inhibits contraction in rat aortae, mesenteric and coronary arteries primarily through inhibiting Ca(2+) influx via nifedipine-sensitive Ca(2+) channels in vascular smooth muscle.

Better Survival of Random Pattern Skin Flaps Through the Use of Epigallocatechin Gallate

Article

Sep 2012
DERMATOL SURG

Background: Surgical skin flaps are useful, but flap necrosis is a common problem. Epigallocatechin gallate (EGCG), the major polyphenol of green tea, has been investigated as a neovascularization agent to counteract necrosis. Objectives: To evaluate the effect of EGCG on random pattern skin flap survival. Materials and methods: The rat dorsal skin flap model was employed. One hundred rats were divided into five groups of equal size. Experimental Group A received local injection of EGCG to the flap, experimental Group B received topical application of EGCG, control Group C received local injection of saline, control Group D received topical application of an acetone-olive oil mixture, and control Group E received no treatment. The surviving area of the flap, regional blood perfusion, capillary density, and vascular endothelial growth factor (VEGF) expression were evaluated. Results: We observed a larger area of skin flap survival in Groups A and B than in the control groups. Doppler measurement showed greater perfusion in Groups A and B, and the capillary density and VEGF expression were significantly higher in the experimental animals. Conclusion: The EGCG treatment enhanced neovascularization and regional perfusion and, as a result, improved skin flap survival.

The effect of capsaicin ointment on skin for the survival of a cutaneous flap

Article

Mar 2001

Recent studies have shown that capsaicin has three major effects: vasodilation, platelet disaggregation and pain reduction. In this study, vasodilation and platelet disaggregation effects were compared using an ointment (Surgilube) containing capsaicin. Forty rats were divided into four groups of 10. A distally-based dorsal skin flap was raised and sutured back into place. The circulatory territory of the flap was measured by a fluorescein technique. In group A, Surgilube was applied on and around the flap as a control for the surgical technique and ointment base. Surgilube containing capsaicin (0.2%) was applied on the surface of the flap in group B to observe platelet disaggregation effects, and around the perimeter (avoiding the incision) of the flap in group C to observe vasodilation effects. Group D received the capsaicin ointment both on and around the flap. Survival distance in millimeters was calculated as the difference from the distal end of the fluorescein stain to the distal margin of flap survival at 7 days after surgery. Analysis for difference among the groups was assessed by the method of Kruskall-Wallis followed by Mann Whitney tests for differences between the two group. The survival distances (mm) for the groups were, in order of better survival, group C (19.9), group D (14.6), group B (9.7) and group A (7.2). The improvement in survival related to the control group was statistically significant for group C (P=0.002), with group D also approaching significance (P=0.052). Furthermore, the survival distance of group C was significantly better than that of group B (P=0.004). Capsaicin was most effective in promoting skin flap survival when applied around the flap. This suggests that the vasodilation effect in flap survival is stronger than that of platelet disaggregation.

Microencapsulated Myoblasts Transduced by the Vascular Endothelial Growth Factor (VEGF) Gene for the Ischemic Skin Flap

Article

Oct 2010
AESTHET PLAST SURG

This experimental study aimed to explore the influence of locally administered microencapsulated vascular endothelial growth factor (VEGF)-secreting myoblasts on the survival of the ischemic skin flap in rats and to elucidate the underlying molecular mechanism. The pcDNA6/His A-VEGF165 plasmid was constructed, amplified, and transfected into myoblasts. Cells then were encapsulated in a sodium alginate-barium chloride microcapsule. The study investigated 64 Wistar rats (males and females). Two symmetric 2 × 10-cm, full-thickness dorsal ischemic skin flaps were elevated on each rat. One flap was used as the experiment area, and the other was used as a control. The microencapsulated VEGF-secreting myoblasts were injected into the right flap of the rat on preoperative days 0, 2, 4, and 7. The left flap in each animal was injected with the encapsulated untransfected cells. The 64 rats were randomly divided into four groups of 16 rats each. The effect of the experimental group was significantly better than that of the control group. The experimental group had a certain time-dependent effect. Microencapsulated VEGF-secreting-myoblasts may be a promising therapy for ischemic flaps in rats.

Vascular Conductance is Reduced After Menthol or Cold Application

Article

Sep 2010

To compare the effects of commercially sold menthol (3.5%) ointment and cold application on blood flow in the forearm. : Prospective counterbalanced design. University research laboratory. Twelve (6 men and 6 women) college-aged students. Each participant had blood flow measured in the brachial artery for 5 minutes before and 10 minutes after menthol ointment or cold application to the forearm. Blood velocity, arterial diameter size, and blood pressure were recorded during testing procedures. Vascular conductance was calculated based on these measures and used to describe limb blood flow. We observed a significant reduction (35%; P = 0.004) in vascular conductance within 60 seconds of menthol and cold application to the forearm. Vascular conductance remained significantly reduced for 10 minutes by approximately 19% after both menthol and cold application [F(2.313, 43.594) = 10.328, P < 0.0001]. There was no significant difference between conditions [F(1, 19) = 0.000, P = 0.945]. The application of a 3.5% menthol ointment significantly reduces conductance in the brachial artery within 60 seconds of application, and this effect is maintained for at least 10 minutes after application. The overall decline in conductance is similar between menthol ointment and cold application.

The Effect of Calcium Channel Blockers on Smoking-Induced Skin Flap Necrosis

Article

Mar 2010

Calcium channel blockers have been shown experimentally to reverse many of the effects of nicotine. The purpose of this study was to assess the effect of calcium channel blockers on smoking-induced skin flap necrosis. Forty male albino Wistar rats were divided into four groups. Groups A, B, and C were treated in a controlled smoking chamber for 20 minutes daily for 21 days. On day 14, caudally based dorsal skin flaps (3 x 10 cm) were created. On days 14 through 21, group B animals received verapamil (20 mg/kg/day) by gavage. Group C received nifedipine (10 mg/kg/day). On day 21, standardized photographs were taken and flap survival areas determined. Urine cotinine concentrations were measured on days 14 and 21. The mean cotinine level at surgery was 161 ng/ml in group A (smoking), 149 ng/ml in group B (verapamil), and 168 ng/ml in group C (nifedipine). These differences were not statistically significant. Cotinine concentration at surgery for group D (no smoking) was less than 10 ng/ml. The mean flap survival in group D was 79.1 percent, compared with 63.7 percent in group A (p = 0.003). The mean flap survival in group B (verapamil) was 72.8 percent, compared with 73.7 percent in group C (nifedipine). Both values were significantly greater than in group A (p = 0.04 and p = 0.008, respectively). In this study, enteral calcium channel blockers were associated with a statistically significant improvement in flap survival compared with untreated animals with an equivalent smoke exposure. Calcium channel blockers may reduce perioperative risk in active smokers who require skin flap surgery.

The Vasodilator Effect of a Cream Containing 10% Menthol and 15% Methyl Salicylate on Random-Pattern Skin Flaps in Rats

Abstract

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