The effect of Atorvastatin on Survival of Rat Ischemic Flap
Category of paper: Original
Short running: Atorvastatin for Viability of Ischemic Flap Jian-Xun Chena; Chia-Wei Chiub; Pin-Keng Shihc
aDepartment of Surgery, China Medical University Hospital, Taichung, Taiwan
bDepartment of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan
cDepartment of Plastic and Reconstructive Surgery, China Medical University Hospital, Taichung, Taiwan
Correspondence: Pin-Keng Shih , M.D.
Department of Plastic and Reconstructive Surgery, China Medical University Hospital, 2 Yuh-Der Road,Taichung City Taiwan 40447
Tel. +886 921 658 698, E-Mail: [email protected]
Management of skin avulsion with tissue exposure is a challenge for plastic surgeons. Clinical observations have suggested that longer survival of skin flap prevents further
contamination and infection. Less well known is the role of Atorvastatin in avulsion skin flap. Therefore, we attempted to determine whether Atorvastatin could alleviate avulsion skin flap in a rat model. Twenty male Sprague-Dawley rats were randomized into two groups: the Atorvastatin group and the control. Before operation, each rat received an initial blood perfusion scan as base-line data. Then, each rat received an operation of skin flap incision, elevation, and re-suturing to the original position under general anesthesia. Another blood perfusion scan was performed on each rat 30 min, 4 days, and 7 days postoperatively. On the 7th postoperative day, the necrotic area of skin flap was measured as the skin flap viability. The skin flap tissues at 2.5 and 5 cm distal to the skin flap base were collected for histopathological analysis, as well as measurement of vascular endothelial growth factor (VEGF) mRNA expression, and vascular density. Compared with 30 min post-operation, there was a significant increase in ratio of skin flap blood perfusion on the 4th and 7th days post-operation in both control and Atorvastatin groups (P < 0.05). Compared with the control group, there was a significant decrease in necrotic area, significant increase in ratio of skin flap blood perfusion on post-operation days 4 and 7, and significant increase in vascular density under high field at 2.5 cm distal to the base of skin flap in the Atorvastatin group (P < 0.05). The VEGF121 and VEGF165 mRNA expression at 2.5 cm distal to the base of skin flap differed significantly between the two groups (P < 0.05). Compared with the
control group, treatment of Atorvastatin improved skin flap blood perfusion, vascular density and necrotic area dependent on VEGF mRNA expression.
Key words: Atorvastatin; vascular endothelial growth factor; skin flap
處理皮瓣撕脫傷是整形外科醫生的挑戰，臨床觀察顯示較多的皮瓣存活可預
防傷口進一步污染和感染，Atorvastatin 在皮瓣撕脫傷的作用少為人知，因此，我 們試圖用大鼠模式了解是否 Atorvastatin 可以減輕撕脫皮瓣缺血的傷害。20 隻雄 性 Sprague-Dawley 大鼠隨機分為控制和 Atorvastatin 組。在實驗之前，每隻 大鼠接受第一次血液灌流掃描作為基準數據，然後每隻大鼠在全身麻醉下接受皮
瓣切開、剝離、再重新縫合的手術，於術後 30 分鐘、4 天、7 天分別進行另一次血液 灌流掃描。於術後 7 天，測量皮瓣壞死面積以及將距離皮瓣起點 2.5 厘米及 5 厘 米皮瓣組織收集進行組織病理學、血管內皮生長因子表現和血管密度的測量。跟 術後 30 分鐘相比，控制組和 Atorvastatin 組在術後第 4 和第 7 天皮瓣血液灌流比 例顯著增加（P <0.05）。與對照組相比，Atorvastatin 組在術後第 4 和第 7 天有 較少壞死區，皮瓣血液灌流比例顯著增加以及高倍視野下血管密度（距離皮瓣起 點 2.5厘米）顯著增加（P <0.05）。 血管內皮生長因子表 現（VEGF121和 VEGF165 mRNA）在距離皮瓣起點 2.5 厘米處表現量在兩組（P <0.05）間有顯著 不同。與對照組相比，Atorvastatin 藉由血管內皮生長因子 mRNA 表現有助於改 善皮瓣血液灌流、血管密度和壞死區比例。 關鍵字：皮瓣、血管內皮生長因子 Introduction
Skin abrasion wounds over a large area are common. Recovery of damaged skin over exposed tissue is beneficial not only for further tissue growth but it also reduces the risk of infection. Various studies have addressed ischemic skin flap survival. Vascular endothelial growth factor (by either pharmacological or gene facilitation) has shown great viability and neovascularization over ischemic skin flaps [1-3]. Other agents such
as captopril (angiotensin-converting enzyme inhibitor) [4], sildenafil (phosphodiesterase inhibitors) [5], and aspirin have been demonstrated to be beneficial for skin flap viability, although the detailed mechanisms are still unclear.
Statins are drugs for treatment of hypercholesterolemia which inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA). In addition to being a cholesterol-lowering class of drug, many studies have suggested that statins are of benefit in the prevention and treatment of various infections [6,7], as well as in wound strengthening and healing [8,9], angiogenesis [10-12] , and apoptosis [13]. It was reported that statins stimulated expression of several angiogenic mediators (VEGF, interleukin-8, angiopoietin (Ang)-1, Ang-2, eNOS, and hemoxidase (HO)-1) in an ischemic hind limb model [14]. Reports on the role of statins in preventing the progression of ischemic skin flap are rare. Therefore, in this study, we aimed to determine whether statins alleviated the necrotic area and blood perfusion of the rat ischemic skin flap and to examine the underlying mechanisms.
Material and Methods
Animals
All procedures were performed using Sprague-Dawley (SD) rats obtained from the National Science Council, and they were approved by the Institutional Animal Care and Use Committee of Kaohsiung Medical University.
Experimental groups
In this study, 20 SD (300-350 g) rats were divided into two groups. All experimental rats were fed and housed at Kaohsiung Medical University, following the approved guidelines. All rats were designed as follows:
Control group (n = 10): feed normal saline after surgery by lavage for 7 days;
Atorvastatin group (n=10): feeding Atorvastatin after surgery (10 mg/kg/day) by lavage for 7 days.
Atorvastatin (Lipitor ®; Abbott. Co. Ltd., Chicago, U.S.A) was purchased from Kaohsiung Medical University Hospital.
Surgical procedure
All surgical procedures were performed under Zoletil 50 anesthesia (Tiletamine and Zolazepam; Virbac. Co. Ltd., Carros Cedex, France; 5 mg/kg, intraperitoneal injection). Dorsal hair was shaved completely before operation. All rats had caudally based McFarlane-type dorsal skin flaps (3 × 10 cm) [15, 16]. Along the designated area, the skin including the panniculus carnosus muscle was incised and elevated vertically with the base over the bilateral iliac crest. To leave the skin flap with blood supply only from the pedicle, an elastic sheet of the same area was placed below the flap, and the flap was sutured with a 4-0 nylon suture back to its original location.
Measurement of microcirculation in skin flap
To assess the changes of blood flow in the flap, the Laser Doppler imager (MoorLDI2-2λ®, Moor Instruments, Millwey, UK) was used. Serial measurements of skin vascularity were taken at 4 time points: before operation, and 30 min, 4 days, and 7 days after operation respectively. The blood perfusion change rate
was expressed as percentage of value obtained at 30 min, 4 days or 7 days after operation/ value obtained before operation.
Evaluation of skin flap survival rate
The flap survival was evaluated on postoperative day 7. A millimetric paper template was used to record the necrotic area of each flap, and the necrotic area was calculated as the percentage of necrotic area to the whole flap.
Tissue harvest
All the rats were sacrificed on postoperative day 7. Skin samples 2.5 and 5.0 cm distal to the base of the pedicle were harvested and stored in liquid nitrogen and formalin for further reverse transcription polymerase chain reaction (RT-PCR) and histological assessment, respectively.
Measurement of VEGF mRNA expression
Total RNA was extracted from frozen skin sample with TRIzol (Invitrogen) according to the manufacturer’s instructions. Reverse transcription was preformed with SuperScriptIII (Invitrogen) according to the manufacturer’s instructions. Primers used for amplification of VEGF were as follows: forward 5’- TGC ACC CAC GAC AGA AGG GGA -3’, reverse 5’- TCA CCG CCT TGG CTT GTC ACA T -3’, GAPDH: forward 5’-GTA TGA CTC CAC TCA CGG CAA AT-3’, reverse 5’-GTA GAC TCC ACG ACA TAC TCA GCA C-3’ [17]. The PCR reaction for amplified VEGF and GAPDH was as follows: 95℃ 5 min, and than 30 cycle of 95℃ 15 sec, 58℃ 1 min and
72℃ for 1 min. The PCR product was analyzed by electrophoresis using 1.5% agarose gel; and the predicted lengths of the PCR products were 360 bp for VEGF121, 492 bp for VEGF165, 564 bp for VEGF189 and 152 bp for GAPDH, respectively.
Histopathological analysis
In each group, the harvested tissue was fixed in 10% buffered formalin and 5-mm section was prepared from paraffin-embedded tissues. Paraffin sections (5-μm thick) were cut and stained with haematoxylin-eosin (H&E) under standard histological methods. Histopathological analysis was carried out by a pathologist blinded to the research groups.
Vascular density
Quantitative assessment of neovascularization was executed under x400 magnification of the light microscope. The number of vessels within the dermis level under 5 random high power fields was calculated. An average number of vessels of all fields represented vascular density.
Statistical Analysis
The data were expressed as mean ± SD. Comparisons of ratio of skin flap blood perfusion at different time points in both control and Atorvastatin groups were determined by one-way analysis of variance (ANOVA), followed by Tukey’s test for post hoc comparisons. Comparisons of other parameters between control and Atorvastatin groups were performed by Mann-Whitney test. A p value of less than 0.05
was considered statistically significant. GraphPad Prism 5.0 was used for all statistics.
Results
Effect of Atorvastatin on viability of skin flap
The effect of Atorvastatin on viability of skin flap is illustrated in Fig. 1. The percentages of necrotic area of control and Atorvastatin groups were 66.08±4.36 % and 43.83±13.12 %, respectively. There was a significant difference between the groups (p < 0.05). Figures 1A and 1B show representative skin flap viability on postoperatiiv day 7 in control and Atorvastatin-treated rats, respectively.
Effect of Atorvastatin on blood perfusion of skin flap
The effect of Atorvastatin on blood perfusion of skin flap is illustrated in Fig. 2. In the control group, the blood perfusion ratios 30 min, 4 days and 7 days post-operation were 0.273±0.067, 0.548±0.148, and 0.493±0.193, respectively (Fig. 2B). There was a significant increase in blood perfusion ratio on postoperative day 4 and day 7 compared with 30 min post-operation in the control group (p < 0.05). Figure 2A shows
representative blood perfusion of skin flap in the control group before operation, and 30 min, 4 days and 7 days post-operation.
In the Atorvastatin group, the blood perfusion ratios 30 min, 4 days and 7 days after operation were 0.477±0.208, 0.881±0.279, and 0.905±0.261, respectively (Fig. 2D). There was a significant increase in blood perfusion ratio on postoperative day 4 and day 7 compared with 30 min after operation in the control group (p < 0.05). Figure 2A shows representative blood perfusion of skin flap in the Atorvastatin group before operation, and 30 min, 4 days and 7 days after operation.
Compared with the control group, there was a significant increase in blood perfusion ratio in the Atorvastatin group on postoperative day 4 and day 7 (p < 0.05; Fig. 3).
Effect of Atorvastatin on VEGF mRNA expression
The effect of Atorvastatin on VEGF mRNA expression is illustrated in Fig. 4. Figures 4A and 4B show VEGF mRNA expression at 2.5 cm and 5.0 cm distal to the base of the skin flap respectively. The ratio of VEGF121/GAPDH mRNA expression at 2.5 cm distal to the base of the skin flap was 0.223±0.042 and 0.431±0.079 in control and Atorvastatin groups, respectively. The ratio of VEGF165/GAPDH mRNA expression at 2.5 cm distal to the base of the skin flap was 0.151±0.042 and 0.271±0.036 in control and Atorvastatin groups, respectively. The ratio of VEGF121/GAPDH mRNA and VEGF165/GAPDH mRNA expression at 2.5 cm distal to the base of the skin flap showed
significant difference between control and Atorvastatin groups (p < 0.05).
The ratio of VEGF121/GAPDH mRNA expression at 5.0 cm distal to the base of the skin flap was 0.105±0.091 and 0.232±0.093 in control and Atorvastatin groups, respectively. The ratio of VEGF165/GAPDH mRNA expression at 5.0 cm distal to the base of the skin flap was 0.080±0.060 and 0.093±0.040 in control and Atorvastatin groups, respectively. The ratio of VEGF121/GAPDH mRNA and VEGF165/GAPDH mRNA expression at 5.0 cm distal to the base of the skin flap showed no significant difference between control and Atorvastatin groups.
Effect of Atorvastatin on vascular density of skin flap
The effect of Atorvastatin on vascular density of skin flap is illustrated in Fig. 5. Figures 5A and 5B show representative histopathological pictures at 2.5 cm distal to the base of the skin flap in control and Atorvastatin-treated rats, respectively. Figures 5C and 5D show representative histopathological pictures at 5.0 cm distal to the base of the skin flap in control and Atorvastatin-treated rats, respectively. The average vascular density under high field at 2.5 cm distal to the base of the skin flap was 1.5±1.12 and 3.86±0.82 in control and Atorvastatin groups, respectively (Figure 5E). There was a significant difference between groups at 2.5 cm distal to the base of the skin flap (p < 0.05). The average vascular density under high field at 5.0 cm distal to the base of the skin flap was 0.45±0.88 and 0.32±0.36 in control and Atorvastatin groups, respectively
(Figure 5F). There was no significant difference between groups at 5.0 cm distal to the base of the skin flap (p > 0.05).
Discussion
Ischaemic flap treated with VEGF gene therapy showed improved ischaemic flap survival and neoangiogenesis [1,2]. Agents such as Sildenafil, celecoxib, and angiotensin-converting enzyme inhibitor (ACEI) were proven to improve ischemic flaps by induction of VEGF [4,5,18]. Some studies have suggested statins in different doses seem to produce biphasic effects in angiogenesis. In a review study presented by Hindler et al., the authors suggested that low doses of statins facilitate tumor angiogenesis by stimulation of protein kinase B and/or activation of endothelial nitric oxide synthase, and high doses of statins inhibit angiogenesis by inhibition of capillary tube formation and/or decreased endothelial vascular growth factor release [19]. In another inflammation-related study, the angiogenesis was enhanced with low-dose statin therapy (0.5 mg/kg/day) but was significantly inhibited with high concentrations of Cerivastatin or Atorvastatin (2.5 mg/kg/day) [20]. The result of our study suggested that Atorvastatin (10 mg/kg/day) improved flap necrotic area in rat skin flap models when compared with the control group, but the dose in this study could be considered
high-dose statin therapy compared with previous studies. However, one study suggested that Atorvastatin (10 mg/kg/day) strongly induced angiogenesis in ischemic hind limbs [14]. Therefore, the definite borderline of high/low dose of statins seem to have no standard. More related studies are necessary.
The result of this study showed better blood perfusion of ischaemic skin flap on postoperative days 4 and 7 than 30 min after operation in both control and Atorvastatin groups. In a similar study presented by Lindenblat et al., capillary widening in the wound bed appeared at day 1 after grafting and increased until day 4. The maximum expression of hypoxic-inducible factor-1α (HIF-1α) appeared within the first 48 hours, but the peak of VEGF expression occurred at 72 hours [21]. Multiple HIF-1 target genes have been shown to modulate angiogenesis by promoting the mitogenic and migratory activities of endothelial cells [22]. Lindenblat’s finding may explain how the ischemic skin flap restored blood perfusion in the animal model.
In Matsumura et al.’s study, Atorvastatin strongly induced angiogenesis with increases in expressions of VEGF, interleukin-8, angiopoietin (Ang)-1, Ang-2, eNOS, and hemoxidase (HO)-1 proteins in ischaemic hind limbs [14]. Similar results were also proven in an in vitro study, which suggested that Atorvastatin stimulated the expression of Ang-2, eNOS in human umbilical endothelial cells [23]. The findings in our study showed that Atorvastatin significantly increased VEGF mRNA expression on
postoperative day 7 as compared with those without treatments, which matched the finding of improved blood perfusion and vascular density.
In Uygur et al.’s study, tissue was harvested from 1cm, 3cm, 5cm distal to the base of the flap [24]. We considered that there should be no differences between control and experimental group at 1 cm distal to the base of the flap, which was the reason for our modification to tissue harvest from 2.5cm and 5cm distal to the base of the flap.
There were some limitations in this study. One is that the dose used was higher than that used in humans. No study has found that the dose of Atorvastatin (10 mg/kg/day) is strongly associated with impaired liver function, but studies have reported liver disease after Atorvastatin treatment [25]. In addition, although the VEGF mRNA expression induction by statins is demonstrated in our study, further time- and dose-dependent studies of statins are desirable.
The flap necrotic area and blood perfusion in this study were designed for gross observation of this flap. We found that the Atorvastatin does decrease the necrotic area (avascular zone) compared with the control group, as demonstrated by the blood perfusion scan. However, the perfusion scan showed non-homogenous distribution of vessel over all flap. Therefore, we harvested tissue 2.5cm and 5 cm distal to the base of the flap to observe the vascular density and the expression of VEGF mRNA. We found that there is no difference between Atorvastatin and control groups at 5 cm distal to the
base of the flap but that there is significant difference at 2.5 cm distal to the base of the flap. These results suggested that the increased vascular density and VEGF mRNA expression in proximal site of Atorvastatin group could supply more reliable flap area although no differences were noted in vascular density and VEGF mRNA expression in the control portion.
In conclusion, compared with the control group, Atorvastatin improved skin flap blood perfusion, vascular density and necrotic area via VEGF-dependent pathway.
Disclosure:
The authors declare that they have no competing financial interests, commercial associations, or financial disclosures that might pose or create a conflict of interest with information presented in this article.
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Figure legends:
Figure 1. Effect of Atorvastatin on survival of skin flap on postoperative day 7. (A) and (B) are representative pictures of skin flap in control and Atorvastatin groups respectively. Ratio of necrotic/original area in both groups is illustrated in (C). Each bar represents the mean ratio of necrotic area ± SD of the data pooled from each group (n=10). *p < 0.05-significance of the difference of ratio of necrotic area between control and Atorvastatin groups.
Figure 2. Effect of Atorvastatin on skin flap blood perfusion at different time points. (A) and (C) are representative skin flap blood perfusions at different time points (Pre OP, Post OP +30 min, +4 days, and +7 days) in control and Atorvastatin groups respectively. (B) and (D) are ratios (Post OP/Pre OP) of blood perfusion in control (n=10) and Atorvastatin groups (n=10) respectively. Each bar represents the mean ratio of blood perfusion ± SD of the data pooled from each group. *p < 0.05, **p < 0.05 – significance of the difference of ratio of blood perfusion between 30 min and 4 days postoperation or significance of the difference of ratio of blood perfusion between 30 min and 7 days postoperation respectively.
Figure 3. Time course of ratio of skin flap blood perfusion between 2 groups (n=10). *p < 0.05 - significance of the difference of ratio of skin flap blood perfusion between control and Atorvastatin groups.
Figure 4. Semiquantitative RT-PCR of The VEGF121 and VEGF165 at 2.5 and 5.0 cm distal to the baseline of the skin flap on postoperative day 7. The RT-PCR of VEGF121 at 2.5 and 5.0 cm distal to the base of the skin flap in both control and Atorvastatin groups is presented in (A). The RT-PCR of VEGF165 at 2.5 and 5.0 cm distal to the base of the skin flap in both control and Atorvastatin groups is presented in (B). Each bar represents the mean ratio of VEGF/GAPDH mRNA expression ± SD of the data pooled from each group (n=10). GAPDH was used as internal control.
Figure 5. Effect of Atorvastatin on vascular density of skin flap at different locations. Morphological changes of skin flap (HE staining, original magnification ×10) are presented in (A), (B), (C) and (D). (A): 2.5 cm distal to the base of skin flap in
Atorvastatin group: obviously patent vessels. (B): 2.5 cm distal to the base of skin flap in control group: patent vessels decreased compared with (A). (C): 5.0 cm distal to the base of skin flap in Atorvastatin group: patent vessels obviously decreased compared with (A). (D): 5.0 cm distal to the base of skin flap in control group: rare vessels available. Each arrow points to patent vessel. (E) and (F) are average vascular densities under high field (magnification ×40) in control (n=10) and Atorvastatin group (n=10), respectively. Each bar represents the mean average vascular density ± SD of the data pooled from each group. *p < 0.05-significance of the difference of average vascular density between control and Atorvastatin groups.
