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1. ENHANCEMENT OF THE SKIN FLAP SURVIVAL IN
MICE APPLYING TOPICALAND SYSTEMIC
ATORVASTATINE:A RANDOMIZED CLINICAL
TRIAL STUDY

2. INTRODUCTION
• The randomly designed skin flap is used extensively in reconstructive
surgeries because of its convenience and resilience.
• There is a growing body of evidence suggesting that the flap survival
related to three main factors including adequate blood supply,
inflammatory reactions, and oxidative stress.
• To improve skin flap survival rates, a variety of pharmacological agents,
including antioxidants, anti-inflammatory drugs, and vasodilators, have
been studied. Commonly their application is based on the surgeon's
preference.

3. • Statins were described as competitive inhibitors of 3-Hydroxy-
methylglutaryl coenzyme A (HMG-CoA) reductase and well known as a
valuable inhibitor of cholesterol synthesis.
• Due to Statin's anti-inflammatory, anti-oxidant, and angiogenesis effects,
we hypothesized that statin might affect the survival of random skin
flaps.
• Taking into understanding of atorvastatin various pharmacological
forms, a study was designed to further evaluate the effect of atorvastatin
use with 3 standard doses (systemic and topical form) in flap survival in
mice.

4. MATERIAL AND METHODS
• The present study is a randomised clinical trial in 100 healthy adult male mice
weighing 30-35 gms , at the age of 7–8 weeks. at Shahid Beheshti University of
Medical Sciences, Tehran, Iran .
• The subjects were randomly divided into four main groups, characterized as:
Group 1 or control group, group 2 or vehicle group, group 3,and group 4 as
intervention groups (systemic atorvastatin and topical atorvastatin). Each main
group included some subgroups , a total of ten subgroups of ten (subgroup
1to10). All subjects underwent one skin flap surgery by a single-blinded surgeon.

5. PROCEDURE
Mice were anesthetized by intramuscular administration of 90 mg/kg of
body weight ketamine (Ketamin 10% Alfasan LAB, Woerden, Holland) as
well as9 mg/kg of body weight Xylasein (Xylazin 2%, Alfasan LAB,
Woerden, Holland).
The prophylactic antibiotic was administered using 60 mg/kg of body
weight intramuscular cefazolin injection. After the depth of anesthesia
was confirmed through the pinch flexion withdrawal test, surgery was
started using an aseptic method.

6. The flap was incised with a scalpel blade (number 15) and
elevated in a plane superficial to the deep muscular fascia (Fig.
2). Afterward, an impermeable sterile plastic sheet was placed
between the flap and its donor site. Then the flap was sutured in
place using simple interrupted Nylon 5-0 sutures.
A random pattern skin flap designed based on the modified
McFarlane method. A rectangular template with standard
dimensions (3 x 5 cm) was utilized and the hip joint was chosen
as the marker for the centre of flap base (Fig. 1).

7. • The first main group (control group) included 2 subgroups, saline
was injected intraperitoneally once a day in subgroup 1 (n ¼ 10)
and applied topically once a day in the subgroup 2 (n ¼ 10).
• In the second main group (vehicle group) that included 2
subgroups, carboxymethyl cellulose (the vehicle for atorvastatin
injection) was injected intraperitoneally daily for two weeks in
subgroup 3 (n ¼ 10) and Vaseline (the vehicle in atorvastatin
ointment) applied topically once a day in subgroup 4 (n ¼ 10).

8. In the post-operation period, the daily visit was done by the surgeon and a veterinarian to
observe the general condition and skin flap appearance. At the end of this two-weeks, the
flap surfaces were evaluated. The demarcation was done between the viable tissue featured
as warm, soft, and pink skin and the necrotic area characterized by cool, stiff, and dark
skin.
Afterward, the digital photography was taken from flap along with the graded ruler (Nikon
L300, Macro lens 60 mm magnification 1:10, 80 cm distance).The necrotic surface was
measured using Image J v.1.45g (NIH USA) software. The measurements were computed
in cm2 and recorded by a single investigator who was unaware of the type of intervention.
The collected data were analysed by SPSS 16.5 software. The independent t-test applied to
compare the means surface of the necrotic zone between groups. The significance level
was fixed in 0.05 (<0.05).

9. RESULTS
After 2 weeks, one mouse from the topical saline group (control group), and
one mice from 5mg/kg systemic atorvastatin group (intervention group) were
expired. All mortality was spontaneous without any prior reported illness. No
flap related post-surgical complication was seen in other remaining
subgroups. In the present study, the mean necrotized surface extent from 11.6
+/- 1.4cm2 in the control group (topical saline) to 4.1 +/- 1.2 cm2 in the
systemic atorvastatin (10 mg/kg) group.

12. DISCUSSION
• As the pathophysiologic characteristics of random-pattern skin
flap necrosis become more clearly defined, pharmacologic
treatment of flap ischemia has become an area of increasing
interest.
• Various drugs have been studied to prevent or reverse the skin
flap ischemia and necrosis phenomenon. These drugs are
generally divided into 8 main groups, including vasodilator
drugs, blood diluents, or sympatholytic drugs, calcium channel
blockers, anticoagulants, prostaglandin inhibitors,
glucocorticoids, and free radicals’ scavengers.

13. • Today, statins are widely applied in cardiovascular diseases. Recent
studies have shown that, in addition to lowering cholesterol and
triglyceride, these drugs have anti-inflammatory, vasodilatory,
antioxidant and angiogenesis effects as well and stimulate the
production of angiogenetic factors such as interleukin 8,
angiopoietin, and hemoxidase.
• In a study conducted by Matsumura et al., atorvastatin increased
the expression of vascular endothelial growth factors (VEGF),
interleukin-8, angiopoietin 1 (Ang 1) and angiopoietin 2 (Ang 2)
in endothelial cells .

14. • Hindler et al. showed that atorvastatin has a biphasic effect on
angiogenesis. Low doses atorvastatin causes angiogenesis by
activating protein kinase B (PKB) or nitric oxide pathway, but in
high doses, atorvastatin impedes angiogenesis by inhibiting
vascular growth factors.
• In another study conducted by Chen et al., atorvastatin (10 mg/kg/day)
increased the expression of VEGF mRNA resulting in elevating blood flow
in the flap on 4th and 7th post-operative days.
• On the other hand, high doses of atorvastatin may lead to systemic
complications, including hepatotoxicity in humans .

15. • This study showed that atorvastatin in three different doses (1, 5, and
10 mg/kg/day) reduced necrotized areas compared to the control
group.
• This promising result can be a positive step toward conducting other
research studies in this regard. Therefore, it seems that further
research studies are needed to confirm or deny the effect of high
doses of atorvastatin on flap survival.
• Total mortality was around 2% in this study. According to a survey
of animal studies examining cause of mortality, the frequency of
unknown death was estimated to be between 7% and 17% . Thus,
mortality (2 out of 100) seems to be acceptable.

16. • Considering the study data analysis, systemic administration of
atorvastatin can significantly increase the survival of random skin
flap in mice , still trials need to be done in humans
• However, the systemic application of atorvastatin seems to be more
effective than topical administration, atorvastatin can also increase
skin flap survival when applied topically.
• Due to the low cost and the fewer side effects of atorvastatin, it can
be advised when the skin flap necrosis or ischemia is likely to occur.
CONCLUSION

17. LIMITATIONS
• No proven human study