Original article 721
Microvascular anastomosis using Ankaferd blood stopper: demonstration of long-term histopathologic effects on vascular tissue ¨ zlem T. C Bu¨lent Sac¸aka, Zeynep D. Akdenizb, Hakan S¸irinog˘luc, O ¸ ilingird, ¨ zhan B. C O ¸ elebilera, Feriha Ercand and Ayhan Numanog˘lua Ankaferd blood stopper (ABS) (Ankaferd I˙lac¸ Kozmetik A.S¸., Turkey) is a medicinal plant extract, which is used in Turkish traditional medicine as a haemostatic agent. The aim of this study was to investigate the haemostatic effect of ABS in preventing microvascular leakage on an anastomosis site and to look into its long-term impact on vascular tissue. Twenty-one Wistar albino rats were randomly divided into three groups. The animals in the second and third groups were pretreated with acetylsalicylic acid. All of the right femoral arteries were divided and anastomosed in an endto-end fashion. Following microvascular anastomosis, saline-soaked gauze tampons were applied in the first and second groups. In the third group, ABS-soaked tampons were applied to the anastomosis sites. The mean bleeding time of group 3 was significantly shorter than group 2 and group 1. Three weeks after the operation, there were aneurysms on all of the anastomosis sites in group 3 and none of the anastomoses were patent. Histologic examination demonstrated increased inflammatory cell infiltration, tunica media degeneration and contraction of
Introduction In order to reduce the amount of oozing following microvascular repair, either pressure is applied on the anastomosis site or sutures are added blindly. Each time the needle of the suture is passed through the vessel wall, endothelium is injured [1,2]. It has been demonstrated that tying the sutures around the vessel wall impairs nourishment of the tunica media [3,4]. Additionally, microvascular repair, especially replantation surgery, is a race against time. Therefore, surgeons are looking for methods to shorten the duration of anastomosis and to increase the likelihood of survival of free tissue transfer. Reducing the number of sutures is the most efficient way to reduce the anastomosis time. Yet, decreased number of sutures results in leakage and bleeding after releasing vascular clamps. Use of fibrin glue to safely minimize the number of sutures on the anastomosis site has been presented in several studies [5,6]. Ankaferd blood stopper (ABS) (Ankaferd I˙lac¸ Kozmetik A.S¸., Turkey) is a medicinal plant extract, which is used in Turkish traditional medicine as a haemostatic agent [7]. The use of this product was approved by the Turkish Ministry of Health in 2007. ABS is comprised of a mixture of Thymus vulgaris, Glycyrrhiza glabra, Vitis vinifera, Alpinia 0957-5235 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
tunica intima in group 3. This is the first study reporting the long-term effects of ABS on microvascular anastomosis. Contrary to previously reported studies, this agent is not appropriate for use on injured or anastomosed vessels. Blood Coagul Fibrinolysis 25:721–725 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. Blood Coagulation and Fibrinolysis 2014, 25:721–725 Keywords: Ankaferd, end-to-end anastomosis, haemostasis a Department of Plastic Reconstructive and Aesthetic Surgery, Marmara University Faculty of Medicine, ˙Istanbul, bDepartment of Plastic Reconstructive and Aesthetic Surgery, Adana Numune Training and Research Hospital, Adana, c Department of Plastic Reconstructive and Aesthetic Surgery, Kartal Training and Research Hospital and dDepartment of Histology and Embryology, Marmara University Faculty of Medicine, ˙Istanbul, Turkey
Correspondence to Zeynep D. Akdeniz, MD, Seyhan Uygulama Merkezi, Plastik Cerrahi Klinig˘i, C ¸ ukurova, Adana, 01170 Turkey Tel: +0090 5335766329; e-mail: [email protected] Received 24 September 2013 Revised 24 March 2014 Accepted 24 March 2014
officinarum and Urtica dioica plants. In an experimental study, it has been demonstrated that ABS application on an aortotomy wound provided haemostasis while preserving vascular patency [8]. The aim of this study is to investigate the effectiveness of ABS in providing haemostasis at an anastomosis site with vascular leakage because of either reduced number of sutures or a state of systemic coagulopathy and to look into its long-term impact on vascular tissue.
Materials and methods The study was approved by the Marmara University Ethics Committee of Experimental Animal Research. Twenty-one Wistar albino rats weighing approximately 250 g were used for the experiment. The animals were placed in separate cages and maintained on a 12/12 h light/dark cycle and fed ad libitum. Surgical procedure
The animals were randomly divided into three groups. All animals were anaesthesized with intraperitoneal injection of ketamine 90 mg/kg and xylazine 10 mg/kg. The rats were placed in supine position and right femoral arteries were exposed. Arteries were divided after placing DOI:10.1097/MBC.0000000000000135
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
722 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 7
the approximating clamps. In the first group (control) (n ¼ 7), arteries were anastomosed in an end-to-end fashion using four 10/0 microsutures placed 90 degrees apart. Microvascular clamps were released and salinesoaked gauze tampons were applied to the anastomosis sites. The animals in the second (n ¼ 7) and third groups (n ¼ 7) received 10 mg/kg oral acetylsalicylic acid (ASA) dissolved in isotonic saline via feeding catheters for 4 days. On the fifth day, right femoral arteries of the animals in the second group (ASA) were divided. Following the same technique of anastomosis described above, saline-soaked gauze tampons were applied to the anastomosis sites. In the third group (ASA þ ABS), following the division of right femoral arteries and same technique of anastomoses, ABS-soaked tampons were applied to the anastomosis sites.
Fig. 1
Bleeding assay
Oozing was checked every 30 s by removing the tampon to record the time the bleeding stopped [9]. Patency of the anastomosis was determined and skin incision was primarily repaired using 5/0 silk suture after haemostasis was achieved. The animals were put under general anaesthesia to examine the anastomosis sites 3 weeks after the operation. The vessels containing the anastomoses with 1 cm of safety margin were removed for histological evaluation after the examinations. The animals were sacrificed with high-dose pentothal injection. Histological procedure
All specimens were fixed in 10% neutral formaldehyde solution. The specimens were embedded in paraffin blocks and cut into 5-mm thick sections following the dehydration process using graded ethanols. The sections were stained with haematoxylin and eosin (H&E) for morphological evaluation. All sections were examined under 400 magnification using Olympus BX51 photomicroscope (Tokyo, Japan) and photographed with Olympus DP2 camera.
Aneurysm formation 3 weeks after surgery.
used to compare two groups. The results with P < 0.05 were assumed statistically significant.
Results Duration of bleeding
Two animals from the third group died because of infection and were removed from the study. The duration of bleeding for animals in each group and the mean values are shown in Table 1. Statistical analysis showed a significant difference among three groups. When the groups were compared in two, there was no statistically significant difference between group 1 and group 2 (P > 0.05). When two ASA-treated groups (group 2 and 3) were compared, the mean bleeding time in group 3 was significantly shorter than group 2 (P < 0.05). The mean bleeding time of group 3 was again significantly shorter than group 1 (P < 0.05). This showed that ABS-soaked gas tampon effectively and significantly reduced the duration of bleeding.
Statistical analysis
Statistical analysis was carried out using MatLab software. The groups were compared using Kruskal–Wallis test. In cases of significance, Wilcoxon Rank Sum test was
Patency of blood flow
Blood flow was observed distal to all of the anastomoses immediately after microclamps were released. Table 2
Table 1
Clinical findings 3 weeks after the operation
Duration of bleeding for each group Control
1 2 3 4 5 6 7
Controla
ASAa
ASA þ ABSa
150 90 120 150 180 120 60
120 300 150 180 210 180 150
30 30 30 120 30
ABS, Ankaferd blood stopper; ASA, acetylsalicylic acid.
a
seconds.
1 2 3 4 5 6 7
Aneurysm
ASA þ ABS
ASA Patency þ þ þ þ þ þ þ
Aneurysm þ
Patency þ þ þ þ þ þ þ
Aneurysm þ þ þ þ þ
Patency
ABS, Ankaferd blood stopper; ASA, acetylsalicylic acid.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Microvascular anastomosis using Ankaferd blood stopper Sac¸ak et al. 723
Aneurysms were observed in all animals of group 3 three weeks after the operation (Fig. 1). None of the anastomoses were patent in this group. One animal had an aneurysm in group 2 and all anastomoses were patent in groups 1 and 2. Clinical findings are summarized in Table 2.
Fig. 2
(a)
Histological evaluation
Endothelium, inner elastic lamina, tunica media and tunica adventitia of muscular arterial tissues in group 1 had regular appearance. In group 2, the endothelium layer showed irregularities and erythrocyte aggregation was observed. Smooth muscle cells in tunica media layer showed degeneration. There were bleeding spots in tunica adventitia. The tunica intima was contracted and irregularities were observed on endothelial layer in group 3. Smooth muscle cell degeneration in tunica media was more prominent in group 3 than in group 2 (Fig. 2). Inflammatory cell infiltration was observed in group 3 (Fig. 3).
50 mm
(b)
Discussion It is known that the increased number of sutures and prolonged anastomosis jeopardize the success of microvascular repair [3,6]. Reducing the number of anastomotic sutures causes vascular leakage, which may lead to thrombosis and microsurgical failure. Surgeons may also encounter oozing from anatomosis sites in patients under antithrombotic or anticoagulant therapy. In order to reduce the number of sutures while preventing microvascular leakage on a microvascular anastomosis, different techniques, suture methods, devices and adhesives have been studied [10–17]. ABS is a Turkish traditional medicinal extract used as a haemostatic agent. This substance is a mixture of various plants each with different characteristics. Thymus vulgaris has antioxidative actions such as prevention of lipid peroxidation [18]. Urtica dioica causes vasodilation by inducing nitric oxide production by the endothelium [19]. Glycyrrhiza glabra decreases vascular endothelial growth factor production and cytokine-induced neovascularization [20]. It also shows anti-inflammatory, antithrombin, antiplatelet, antioxidant, antiatherosclerotic and antitumor activities [20]. Alpinia officinarum inhibits nitric oxide production [21]. Vitis vinifera has antiatherosclerotic effects [22]. This present study was designed to investigate whether this new agent will be successful in securely sealing a microvascular anastomosis while sparing the vessel from suture trauma. In an in-vitro study, Goker et al. have shown that the application of ABS induces a reaction between the agent and blood proteins causing a very rapid formation of an encapsulated protein network, which stimulates erythrocyte aggregation [7]. They have also demonstrated that
50 mm
(c)
50 mm
(a) Regular structure of endothelium (arrow), inner elastic lamina and adventitia () in group 1. A few degenerated smooth muscle cells can be seen (arrow head). (b) Partial irregularities and red blood cell attachments on the endothelium (arrow) in group 2. Vacuoles in tunica media showing smooth muscle cell degeneration (arrow head). Bleeding spots on tunica adventitia (). (c) Tunica intima is contracted and endothelium is irregular in group 3 (arrow). Increased number of degenerated smooth muscle cells in tunica media (arrow head). Bleeding spots on tunica adventitia () H&E staining, original magnification: 200.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
724 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 7
Fig. 3
Prominent inflammatory cell infiltration in tunica intima and tunica media in Group 3 (). H&E staining, original magnification: 200 (left) and 400 (right).
coagulation factors II, V, VII, VIII, IX, X, XI and XIII were spared during the process and plasma fibrinogen activity, total protein, albumin and globulin levels decreased significantly. Kos¸ar et al. [23] have shown in an in-vivo study that this substance shortens the duration of bleeding in rats with antithrombotic drug-induced haemostatic abnormalities. ABS had produced the similar effects in this presented study. In an in-vivo study by Kandemir et al. [8], abdominal aortas of rats were wounded with an iris blade and either ABS-soaked or plain gauze tampons were applied to the vascular wounds. The authors reported that the mean bleeding time in ABS groups was significantly shorter than plain gauze groups. According to their macroscopic evaluation, no distal aortic occlusion was observed on days 1 and 7 after the operation. Dogan et al. [24] had topically applied ABS on coronary bypass suture lines of patients and reported that they required no revisions. ABS has been successfully used in clinical trials to control mucosal bleedings in upper gastrointestinal system [25] or in oral cavity during surgery [26]. These promising results led the authors of this study to investigate this agent’s effects on a microvascular anastomosis model. Previous studies had not demonstrated the agent’s longterm impact on vascular tissues or whether it would be successful in preventing anastomotic leakage in patients under antithrombotic or anticoagulant drugs. In this present study, topical application of ABS significantly shortened the duration of bleeding without compromising blood flow. However, these immediate successful results were not permanent. Three weeks after the operation, aneurysms were observed and blood flow distal to the anastomoses was occluded in all of the rats in the ABS group. Aneurysm formation over the microvascular anastomosis and its relation with tensile strength of the endothelial edges have been well studied [27,28]. We believe the clot formed by the protein network is not stable in the mid- and long-term mostly because of the inflammation induced by the agent. This protein network cannot provide the initial tensile strength, which may be
the main cause of aneurysm formation in the third group. Yet, further studies are needed to provide more detailed information on the relation of aneurysm formation and Ankaferd application in the long term. I˙s¸ler et al. [29] had investigated the effect of ABS on early bone healing and reported that inflammation was less in groups treated with ABS. They had attributed this effect to a component in the agent that might have anti-inflammatory activity. In contrast, there was prominent inflammatory cell infiltration following ABS administration and endothelium was contracted and irregular in our study. Conclusion
To our knowledge, this is the first study reporting the long-term effects of ABS in a microvascular model with histopathological evidence. Although there are published data on the application of ABS in clinical procedures, these studies are limited to early results, failing to demonstrate long-term adverse effects of the substance [24–26]. Our results demonstrate that contrary to previously reported studies [8,23], this agent is not appropriate for topical administration on injured or anastomosed vessel, causing aneurysm formation and occlusion of distal blood flow. Further studies can be designed to investigate the use of this agent with different microvascular incision or suture techniques.
Acknowledgements Conflicts of interest
There are no conflicts of interest.
References 1
2
3
4
Lemarie D, Mongeau J, Dorion D. Microvascular anastomosis using histoacryl glue and an intravascular soluble stent. J Otolaryngol 2000; 29:199–205. Cope C, Lee K, Stern H, Pennington D. Use of vascular closure staple applier for microvascular anastomosis in free-flap surgery. Plast Reconstr Surg 2000; 106:107–110. Padubidri AN, Browne E, Kononov A. Fibrin glue assisted end-to-side anastomosis of rat femoral vessels: comparison with conventional suture method. Ann Plast Surg 1996; 37:41–47. Bschorer R, Frerich B, Wolburg H, Gehrke G, Schwenzer N. Fibrin sealing and histometrical changes in conventionally sutured microvascular anastomoses. J Craniomaxillofac Surg 1993; 2:192–198.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Microvascular anastomosis using Ankaferd blood stopper Sac¸ak et al. 725
5
6
7
8
9
10
11 12
13
14
15
16
17
Bowen CV, Leach DH, Crosby NL, Reynolds R. Microvascular anastomoses. A comparative study of fibrinogen adhesive and interrupted suture techniques. Plast Reconstr Surg 1996; 97:792–800. Han SK, Kim SW, Kim WK. Microvascular anastomosis with minimal suture and fibrin glue: experimental and clinical study. Microsurgery 1998; 18:306–311. Goker H, Haznedaroglu IC, Ercetin S, Kirazli S, Akman U, Ozturk Y, et al. Haemostatic actions of the folkloric medicinal plant extract, Ankaferd blood stopper. J Int Med Res 2008; 36:163–170. Kandemir O, Buyukates M, Kandemir NO, Aktunc E, Gul AE, Gul S, et al. Demonstration of the histopathological and immunohistochemical effects of a novel haemostatic agent, Ankaferd Blood Stopper, on vascular tissue in a rat aortic bleeding model. J Cardiothorac Surg 2010; 5:110. Turan T, Ozc¸elik D, Kuran I, Sadikog˘lu B, Bas¸ L, San T, et al. Eversion with four sutures: an easy, fast, and reliable technique for microvascular anastomosis. Plast Reconstr Surg 2001; 107:463–470. Nataf P, Hinchliffe P, Manzo S, Simpson J, Kirsch WM, Zhu YH, et al. Facilitated vascular anastomosis: the one-shot device. Ann Thorac Surg 1998; 66:1041–1044. Lauritzen CA. A new and easier way to anastomose microvessels. Scand J Plast Reconstr Surg 1978; 12:291–294. Chen YX, Chen LE, Seaber A, Urbaniak JR. Comparison of continuous and interrupted suture techniques in microvascular anastomosis. J Hand Surg Am 2001; 26:530–539. Gu¨neren E, Odacı E, Yıldız L, Akbas¸ H, Erog˘lu L, Kaplan S. Use of longitudinal invaginating matrix sutures in microarterial sleeve anastomoses. Scand J Plast Reconstr Surg Hand Surg 2004; 38:1–4. Kirsch WM, Zhu YH, Hardesty RA, Chapolini R. A new method for microvascular anastomosis: report of experimental and clinical research. Am Surg 1992; 58:722–727. Karamu¨rsel S, Kayıkc¸ıog˘lu A, S¸afak T, Kec¸ik A, Su¨ru¨cu¨ S. A new technique for microvascular anastomosis: external metallic circle. Plast Reconstr Surg 1999; 104:1059–1065. Zhong C, Tang NX, Zheng CF, Xu YW, Wang TD. Experimental study on microvascular anastomosis using a dissolvable stent support in the lumen. Microsurgery 1991; 12:67–71. Schenck RT, Weinrib HB, Labanauskas IG. The external ring technique for microvascular anastomosis. J Hand Surg Am 1983; 8:105–108.
18
19
20
21
22
23
24 25
26
27 28
29
Lee SJ, Umano K, Shibamoto T, Lee KG. Identification of volatile components in basil (Ocimum basilicum L.) and thyme leaves (Thymus vulgaris L.) and their antioxidant properties. Food Chem 2007; 91:131–137. Testai L, Chericoni S, Calderone V, Nencioni G, Nieri P, Morelli I, et al. Cardiovascular effects of Urtica dioica L. (Urticaceae) roots extracts: in vitro and in vivo pharmacological studies. J Ethnopharmacol 2002; 81:105–109. Sheela ML, Ramakrishna MK, Salimath BP. Angiogenic and proliferative effects of the cytokine VEGF in Ehrlich ascites tumor cells is inhibited by Glycyrrhiza glabra. Int Immunopharmacol 2006; 6:494–498. Matsuda H, Ando S, Kato T, Morikawa T, Yoshikawa M. Inhibitors from the rhizomes of Alpina officinarum on production of nitric oxide in lipopolysaccharide-activated macrophages and the structural requirements of diarrylheptanoids for the activity. Bioorg med Chem 2006; 14:138–142. Yamakoshi J, Kataoka S, Koga T, Ariga T. Proanthocyanidin-rich extract from grape seeds attenuates the development of aortic atherosclerosis in cholesterol-fed rabbits. Atherosclerosis 1999; 142:139–149. Kosar A, Cipil HS, Kaya A, Uz B, Haznedaroglu IC, Goker H, et al. The efficacy of Ankaferd Blood Stopper in antithrombotic drug induced primary and secondary hemostatic abnormalities of a rat bleeding model. Blood Coagul Fibrinolysis 2009; 20:185–190. Dogan OF, Ozyurda U, Uymaz OK, Ercetin S. New anticoagulant for CABG surgery. Blood Coagul Fibrinolysis: ; 2008. Kurt M, Onal I, Akdogan M, Kekilli M, Arhan M, Sayilir A, et al. Ankaferd Blood Stopper for controlling gastrointestinal bleeding due to distinct benign lesions refractory to conventional antihemorrhagic measures. Can J Gastroenterol 2010; 24:380–384. Leblebisatan G, Bay A, Karakus SC, Kekilli M, Haznedarog˘lu IC. Topical Ankaferd hemostat application for the management of oral cavity bleedings in children with hemorrhagic diathesis. Blood Coagulation and Fibrinolysis 2012; 23:494–497. Moskovitz MJ, Bass L, Zhang L, Siebert JW. Microvascular anastomoses utilizing new intravascular stents. Ann Plast Surg 1994; 32:612–618. Maxwell GP, Szabo Z, Buncke HJ Jr. Aneurysms after microvascular anastomoses. Incidence and pathogenesis in experimental animals. Plast Reconstr Surg 1979; 63:824–829. ˙Is¸ler SC, Demircan S, Cakarer S, Cebi Z, Keskin C, Soluk M, et al. Effects of folk medicinal plant extract Ankaferd Blood Stopper on early bone healing. J Appl Oral Sci 2010; 18:409–414.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Microvascular anastomosis using Ankaferd blood stopper: demonstration of long-term histopathologic effects on vascular tissue ¨ zlem T. C Bu¨lent Sac¸aka, Zeynep D. Akdenizb, Hakan S¸irinog˘luc, O ¸ ilingird, ¨ zhan B. C O ¸ elebilera, Feriha Ercand and Ayhan Numanog˘lua Ankaferd blood stopper (ABS) (Ankaferd I˙lac¸ Kozmetik A.S¸., Turkey) is a medicinal plant extract, which is used in Turkish traditional medicine as a haemostatic agent. The aim of this study was to investigate the haemostatic effect of ABS in preventing microvascular leakage on an anastomosis site and to look into its long-term impact on vascular tissue. Twenty-one Wistar albino rats were randomly divided into three groups. The animals in the second and third groups were pretreated with acetylsalicylic acid. All of the right femoral arteries were divided and anastomosed in an endto-end fashion. Following microvascular anastomosis, saline-soaked gauze tampons were applied in the first and second groups. In the third group, ABS-soaked tampons were applied to the anastomosis sites. The mean bleeding time of group 3 was significantly shorter than group 2 and group 1. Three weeks after the operation, there were aneurysms on all of the anastomosis sites in group 3 and none of the anastomoses were patent. Histologic examination demonstrated increased inflammatory cell infiltration, tunica media degeneration and contraction of
Introduction In order to reduce the amount of oozing following microvascular repair, either pressure is applied on the anastomosis site or sutures are added blindly. Each time the needle of the suture is passed through the vessel wall, endothelium is injured [1,2]. It has been demonstrated that tying the sutures around the vessel wall impairs nourishment of the tunica media [3,4]. Additionally, microvascular repair, especially replantation surgery, is a race against time. Therefore, surgeons are looking for methods to shorten the duration of anastomosis and to increase the likelihood of survival of free tissue transfer. Reducing the number of sutures is the most efficient way to reduce the anastomosis time. Yet, decreased number of sutures results in leakage and bleeding after releasing vascular clamps. Use of fibrin glue to safely minimize the number of sutures on the anastomosis site has been presented in several studies [5,6]. Ankaferd blood stopper (ABS) (Ankaferd I˙lac¸ Kozmetik A.S¸., Turkey) is a medicinal plant extract, which is used in Turkish traditional medicine as a haemostatic agent [7]. The use of this product was approved by the Turkish Ministry of Health in 2007. ABS is comprised of a mixture of Thymus vulgaris, Glycyrrhiza glabra, Vitis vinifera, Alpinia 0957-5235 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
tunica intima in group 3. This is the first study reporting the long-term effects of ABS on microvascular anastomosis. Contrary to previously reported studies, this agent is not appropriate for use on injured or anastomosed vessels. Blood Coagul Fibrinolysis 25:721–725 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. Blood Coagulation and Fibrinolysis 2014, 25:721–725 Keywords: Ankaferd, end-to-end anastomosis, haemostasis a Department of Plastic Reconstructive and Aesthetic Surgery, Marmara University Faculty of Medicine, ˙Istanbul, bDepartment of Plastic Reconstructive and Aesthetic Surgery, Adana Numune Training and Research Hospital, Adana, c Department of Plastic Reconstructive and Aesthetic Surgery, Kartal Training and Research Hospital and dDepartment of Histology and Embryology, Marmara University Faculty of Medicine, ˙Istanbul, Turkey
Correspondence to Zeynep D. Akdeniz, MD, Seyhan Uygulama Merkezi, Plastik Cerrahi Klinig˘i, C ¸ ukurova, Adana, 01170 Turkey Tel: +0090 5335766329; e-mail: [email protected] Received 24 September 2013 Revised 24 March 2014 Accepted 24 March 2014
officinarum and Urtica dioica plants. In an experimental study, it has been demonstrated that ABS application on an aortotomy wound provided haemostasis while preserving vascular patency [8]. The aim of this study is to investigate the effectiveness of ABS in providing haemostasis at an anastomosis site with vascular leakage because of either reduced number of sutures or a state of systemic coagulopathy and to look into its long-term impact on vascular tissue.
Materials and methods The study was approved by the Marmara University Ethics Committee of Experimental Animal Research. Twenty-one Wistar albino rats weighing approximately 250 g were used for the experiment. The animals were placed in separate cages and maintained on a 12/12 h light/dark cycle and fed ad libitum. Surgical procedure
The animals were randomly divided into three groups. All animals were anaesthesized with intraperitoneal injection of ketamine 90 mg/kg and xylazine 10 mg/kg. The rats were placed in supine position and right femoral arteries were exposed. Arteries were divided after placing DOI:10.1097/MBC.0000000000000135
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
722 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 7
the approximating clamps. In the first group (control) (n ¼ 7), arteries were anastomosed in an end-to-end fashion using four 10/0 microsutures placed 90 degrees apart. Microvascular clamps were released and salinesoaked gauze tampons were applied to the anastomosis sites. The animals in the second (n ¼ 7) and third groups (n ¼ 7) received 10 mg/kg oral acetylsalicylic acid (ASA) dissolved in isotonic saline via feeding catheters for 4 days. On the fifth day, right femoral arteries of the animals in the second group (ASA) were divided. Following the same technique of anastomosis described above, saline-soaked gauze tampons were applied to the anastomosis sites. In the third group (ASA þ ABS), following the division of right femoral arteries and same technique of anastomoses, ABS-soaked tampons were applied to the anastomosis sites.
Fig. 1
Bleeding assay
Oozing was checked every 30 s by removing the tampon to record the time the bleeding stopped [9]. Patency of the anastomosis was determined and skin incision was primarily repaired using 5/0 silk suture after haemostasis was achieved. The animals were put under general anaesthesia to examine the anastomosis sites 3 weeks after the operation. The vessels containing the anastomoses with 1 cm of safety margin were removed for histological evaluation after the examinations. The animals were sacrificed with high-dose pentothal injection. Histological procedure
All specimens were fixed in 10% neutral formaldehyde solution. The specimens were embedded in paraffin blocks and cut into 5-mm thick sections following the dehydration process using graded ethanols. The sections were stained with haematoxylin and eosin (H&E) for morphological evaluation. All sections were examined under 400 magnification using Olympus BX51 photomicroscope (Tokyo, Japan) and photographed with Olympus DP2 camera.
Aneurysm formation 3 weeks after surgery.
used to compare two groups. The results with P < 0.05 were assumed statistically significant.
Results Duration of bleeding
Two animals from the third group died because of infection and were removed from the study. The duration of bleeding for animals in each group and the mean values are shown in Table 1. Statistical analysis showed a significant difference among three groups. When the groups were compared in two, there was no statistically significant difference between group 1 and group 2 (P > 0.05). When two ASA-treated groups (group 2 and 3) were compared, the mean bleeding time in group 3 was significantly shorter than group 2 (P < 0.05). The mean bleeding time of group 3 was again significantly shorter than group 1 (P < 0.05). This showed that ABS-soaked gas tampon effectively and significantly reduced the duration of bleeding.
Statistical analysis
Statistical analysis was carried out using MatLab software. The groups were compared using Kruskal–Wallis test. In cases of significance, Wilcoxon Rank Sum test was
Patency of blood flow
Blood flow was observed distal to all of the anastomoses immediately after microclamps were released. Table 2
Table 1
Clinical findings 3 weeks after the operation
Duration of bleeding for each group Control
1 2 3 4 5 6 7
Controla
ASAa
ASA þ ABSa
150 90 120 150 180 120 60
120 300 150 180 210 180 150
30 30 30 120 30
ABS, Ankaferd blood stopper; ASA, acetylsalicylic acid.
a
seconds.
1 2 3 4 5 6 7
Aneurysm
ASA þ ABS
ASA Patency þ þ þ þ þ þ þ
Aneurysm þ
Patency þ þ þ þ þ þ þ
Aneurysm þ þ þ þ þ
Patency
ABS, Ankaferd blood stopper; ASA, acetylsalicylic acid.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Microvascular anastomosis using Ankaferd blood stopper Sac¸ak et al. 723
Aneurysms were observed in all animals of group 3 three weeks after the operation (Fig. 1). None of the anastomoses were patent in this group. One animal had an aneurysm in group 2 and all anastomoses were patent in groups 1 and 2. Clinical findings are summarized in Table 2.
Fig. 2
(a)
Histological evaluation
Endothelium, inner elastic lamina, tunica media and tunica adventitia of muscular arterial tissues in group 1 had regular appearance. In group 2, the endothelium layer showed irregularities and erythrocyte aggregation was observed. Smooth muscle cells in tunica media layer showed degeneration. There were bleeding spots in tunica adventitia. The tunica intima was contracted and irregularities were observed on endothelial layer in group 3. Smooth muscle cell degeneration in tunica media was more prominent in group 3 than in group 2 (Fig. 2). Inflammatory cell infiltration was observed in group 3 (Fig. 3).
50 mm
(b)
Discussion It is known that the increased number of sutures and prolonged anastomosis jeopardize the success of microvascular repair [3,6]. Reducing the number of anastomotic sutures causes vascular leakage, which may lead to thrombosis and microsurgical failure. Surgeons may also encounter oozing from anatomosis sites in patients under antithrombotic or anticoagulant therapy. In order to reduce the number of sutures while preventing microvascular leakage on a microvascular anastomosis, different techniques, suture methods, devices and adhesives have been studied [10–17]. ABS is a Turkish traditional medicinal extract used as a haemostatic agent. This substance is a mixture of various plants each with different characteristics. Thymus vulgaris has antioxidative actions such as prevention of lipid peroxidation [18]. Urtica dioica causes vasodilation by inducing nitric oxide production by the endothelium [19]. Glycyrrhiza glabra decreases vascular endothelial growth factor production and cytokine-induced neovascularization [20]. It also shows anti-inflammatory, antithrombin, antiplatelet, antioxidant, antiatherosclerotic and antitumor activities [20]. Alpinia officinarum inhibits nitric oxide production [21]. Vitis vinifera has antiatherosclerotic effects [22]. This present study was designed to investigate whether this new agent will be successful in securely sealing a microvascular anastomosis while sparing the vessel from suture trauma. In an in-vitro study, Goker et al. have shown that the application of ABS induces a reaction between the agent and blood proteins causing a very rapid formation of an encapsulated protein network, which stimulates erythrocyte aggregation [7]. They have also demonstrated that
50 mm
(c)
50 mm
(a) Regular structure of endothelium (arrow), inner elastic lamina and adventitia () in group 1. A few degenerated smooth muscle cells can be seen (arrow head). (b) Partial irregularities and red blood cell attachments on the endothelium (arrow) in group 2. Vacuoles in tunica media showing smooth muscle cell degeneration (arrow head). Bleeding spots on tunica adventitia (). (c) Tunica intima is contracted and endothelium is irregular in group 3 (arrow). Increased number of degenerated smooth muscle cells in tunica media (arrow head). Bleeding spots on tunica adventitia () H&E staining, original magnification: 200.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
724 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 7
Fig. 3
Prominent inflammatory cell infiltration in tunica intima and tunica media in Group 3 (). H&E staining, original magnification: 200 (left) and 400 (right).
coagulation factors II, V, VII, VIII, IX, X, XI and XIII were spared during the process and plasma fibrinogen activity, total protein, albumin and globulin levels decreased significantly. Kos¸ar et al. [23] have shown in an in-vivo study that this substance shortens the duration of bleeding in rats with antithrombotic drug-induced haemostatic abnormalities. ABS had produced the similar effects in this presented study. In an in-vivo study by Kandemir et al. [8], abdominal aortas of rats were wounded with an iris blade and either ABS-soaked or plain gauze tampons were applied to the vascular wounds. The authors reported that the mean bleeding time in ABS groups was significantly shorter than plain gauze groups. According to their macroscopic evaluation, no distal aortic occlusion was observed on days 1 and 7 after the operation. Dogan et al. [24] had topically applied ABS on coronary bypass suture lines of patients and reported that they required no revisions. ABS has been successfully used in clinical trials to control mucosal bleedings in upper gastrointestinal system [25] or in oral cavity during surgery [26]. These promising results led the authors of this study to investigate this agent’s effects on a microvascular anastomosis model. Previous studies had not demonstrated the agent’s longterm impact on vascular tissues or whether it would be successful in preventing anastomotic leakage in patients under antithrombotic or anticoagulant drugs. In this present study, topical application of ABS significantly shortened the duration of bleeding without compromising blood flow. However, these immediate successful results were not permanent. Three weeks after the operation, aneurysms were observed and blood flow distal to the anastomoses was occluded in all of the rats in the ABS group. Aneurysm formation over the microvascular anastomosis and its relation with tensile strength of the endothelial edges have been well studied [27,28]. We believe the clot formed by the protein network is not stable in the mid- and long-term mostly because of the inflammation induced by the agent. This protein network cannot provide the initial tensile strength, which may be
the main cause of aneurysm formation in the third group. Yet, further studies are needed to provide more detailed information on the relation of aneurysm formation and Ankaferd application in the long term. I˙s¸ler et al. [29] had investigated the effect of ABS on early bone healing and reported that inflammation was less in groups treated with ABS. They had attributed this effect to a component in the agent that might have anti-inflammatory activity. In contrast, there was prominent inflammatory cell infiltration following ABS administration and endothelium was contracted and irregular in our study. Conclusion
To our knowledge, this is the first study reporting the long-term effects of ABS in a microvascular model with histopathological evidence. Although there are published data on the application of ABS in clinical procedures, these studies are limited to early results, failing to demonstrate long-term adverse effects of the substance [24–26]. Our results demonstrate that contrary to previously reported studies [8,23], this agent is not appropriate for topical administration on injured or anastomosed vessel, causing aneurysm formation and occlusion of distal blood flow. Further studies can be designed to investigate the use of this agent with different microvascular incision or suture techniques.
Acknowledgements Conflicts of interest
There are no conflicts of interest.
References 1
2
3
4
Lemarie D, Mongeau J, Dorion D. Microvascular anastomosis using histoacryl glue and an intravascular soluble stent. J Otolaryngol 2000; 29:199–205. Cope C, Lee K, Stern H, Pennington D. Use of vascular closure staple applier for microvascular anastomosis in free-flap surgery. Plast Reconstr Surg 2000; 106:107–110. Padubidri AN, Browne E, Kononov A. Fibrin glue assisted end-to-side anastomosis of rat femoral vessels: comparison with conventional suture method. Ann Plast Surg 1996; 37:41–47. Bschorer R, Frerich B, Wolburg H, Gehrke G, Schwenzer N. Fibrin sealing and histometrical changes in conventionally sutured microvascular anastomoses. J Craniomaxillofac Surg 1993; 2:192–198.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Microvascular anastomosis using Ankaferd blood stopper Sac¸ak et al. 725
5
6
7
8
9
10
11 12
13
14
15
16
17
Bowen CV, Leach DH, Crosby NL, Reynolds R. Microvascular anastomoses. A comparative study of fibrinogen adhesive and interrupted suture techniques. Plast Reconstr Surg 1996; 97:792–800. Han SK, Kim SW, Kim WK. Microvascular anastomosis with minimal suture and fibrin glue: experimental and clinical study. Microsurgery 1998; 18:306–311. Goker H, Haznedaroglu IC, Ercetin S, Kirazli S, Akman U, Ozturk Y, et al. Haemostatic actions of the folkloric medicinal plant extract, Ankaferd blood stopper. J Int Med Res 2008; 36:163–170. Kandemir O, Buyukates M, Kandemir NO, Aktunc E, Gul AE, Gul S, et al. Demonstration of the histopathological and immunohistochemical effects of a novel haemostatic agent, Ankaferd Blood Stopper, on vascular tissue in a rat aortic bleeding model. J Cardiothorac Surg 2010; 5:110. Turan T, Ozc¸elik D, Kuran I, Sadikog˘lu B, Bas¸ L, San T, et al. Eversion with four sutures: an easy, fast, and reliable technique for microvascular anastomosis. Plast Reconstr Surg 2001; 107:463–470. Nataf P, Hinchliffe P, Manzo S, Simpson J, Kirsch WM, Zhu YH, et al. Facilitated vascular anastomosis: the one-shot device. Ann Thorac Surg 1998; 66:1041–1044. Lauritzen CA. A new and easier way to anastomose microvessels. Scand J Plast Reconstr Surg 1978; 12:291–294. Chen YX, Chen LE, Seaber A, Urbaniak JR. Comparison of continuous and interrupted suture techniques in microvascular anastomosis. J Hand Surg Am 2001; 26:530–539. Gu¨neren E, Odacı E, Yıldız L, Akbas¸ H, Erog˘lu L, Kaplan S. Use of longitudinal invaginating matrix sutures in microarterial sleeve anastomoses. Scand J Plast Reconstr Surg Hand Surg 2004; 38:1–4. Kirsch WM, Zhu YH, Hardesty RA, Chapolini R. A new method for microvascular anastomosis: report of experimental and clinical research. Am Surg 1992; 58:722–727. Karamu¨rsel S, Kayıkc¸ıog˘lu A, S¸afak T, Kec¸ik A, Su¨ru¨cu¨ S. A new technique for microvascular anastomosis: external metallic circle. Plast Reconstr Surg 1999; 104:1059–1065. Zhong C, Tang NX, Zheng CF, Xu YW, Wang TD. Experimental study on microvascular anastomosis using a dissolvable stent support in the lumen. Microsurgery 1991; 12:67–71. Schenck RT, Weinrib HB, Labanauskas IG. The external ring technique for microvascular anastomosis. J Hand Surg Am 1983; 8:105–108.
18
19
20
21
22
23
24 25
26
27 28
29
Lee SJ, Umano K, Shibamoto T, Lee KG. Identification of volatile components in basil (Ocimum basilicum L.) and thyme leaves (Thymus vulgaris L.) and their antioxidant properties. Food Chem 2007; 91:131–137. Testai L, Chericoni S, Calderone V, Nencioni G, Nieri P, Morelli I, et al. Cardiovascular effects of Urtica dioica L. (Urticaceae) roots extracts: in vitro and in vivo pharmacological studies. J Ethnopharmacol 2002; 81:105–109. Sheela ML, Ramakrishna MK, Salimath BP. Angiogenic and proliferative effects of the cytokine VEGF in Ehrlich ascites tumor cells is inhibited by Glycyrrhiza glabra. Int Immunopharmacol 2006; 6:494–498. Matsuda H, Ando S, Kato T, Morikawa T, Yoshikawa M. Inhibitors from the rhizomes of Alpina officinarum on production of nitric oxide in lipopolysaccharide-activated macrophages and the structural requirements of diarrylheptanoids for the activity. Bioorg med Chem 2006; 14:138–142. Yamakoshi J, Kataoka S, Koga T, Ariga T. Proanthocyanidin-rich extract from grape seeds attenuates the development of aortic atherosclerosis in cholesterol-fed rabbits. Atherosclerosis 1999; 142:139–149. Kosar A, Cipil HS, Kaya A, Uz B, Haznedaroglu IC, Goker H, et al. The efficacy of Ankaferd Blood Stopper in antithrombotic drug induced primary and secondary hemostatic abnormalities of a rat bleeding model. Blood Coagul Fibrinolysis 2009; 20:185–190. Dogan OF, Ozyurda U, Uymaz OK, Ercetin S. New anticoagulant for CABG surgery. Blood Coagul Fibrinolysis: ; 2008. Kurt M, Onal I, Akdogan M, Kekilli M, Arhan M, Sayilir A, et al. Ankaferd Blood Stopper for controlling gastrointestinal bleeding due to distinct benign lesions refractory to conventional antihemorrhagic measures. Can J Gastroenterol 2010; 24:380–384. Leblebisatan G, Bay A, Karakus SC, Kekilli M, Haznedarog˘lu IC. Topical Ankaferd hemostat application for the management of oral cavity bleedings in children with hemorrhagic diathesis. Blood Coagulation and Fibrinolysis 2012; 23:494–497. Moskovitz MJ, Bass L, Zhang L, Siebert JW. Microvascular anastomoses utilizing new intravascular stents. Ann Plast Surg 1994; 32:612–618. Maxwell GP, Szabo Z, Buncke HJ Jr. Aneurysms after microvascular anastomoses. Incidence and pathogenesis in experimental animals. Plast Reconstr Surg 1979; 63:824–829. ˙Is¸ler SC, Demircan S, Cakarer S, Cebi Z, Keskin C, Soluk M, et al. Effects of folk medicinal plant extract Ankaferd Blood Stopper on early bone healing. J Appl Oral Sci 2010; 18:409–414.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
