- Email: [email protected]
Buflomedil and pentoxifylline in the viability of dorsal cutaneous flaps of rats treated with nicotine
Journal of Plastic, Reconstructive & Aesthetic Surgery (2006) 59, 387–392
Buflomedil and pentoxifylline in the viability of dorsal cutaneous flaps of rats treated with nicotine Raul J. Mauad Jra,*, Maria H.M. Shimizua, Thais Mauadb, Erasmo M.C. de Tolosaa a
Department of Experimental Surgery, School of Medicine, Sao Paulo University, Av. Dr Arnaldo 455, 01246-903 Sao Paulo, SP, Brazil b Department of Pathology, School of Medicine, Sao Paulo University, Av. Dr Arnaldo 455, 01246-903 Sao Paulo, SP, Brazil Received 28 November 2004; accepted 11 July 2005
KEYWORDS Nicotine; Skin flap; Rat; Buflomedil; Pentoxifylline; Viability
Summary Background: Nicotine reduces skin-flap survival. Pharmacologic therapy may represent an alternative treatment strategy to counteract nicotine effects in the flap surgery setting. In this study, we have compared the isolated and associated actions of the vasoactive drugs buflomedil and pentoxifylline in the viability of dorsal cutaneous flaps of rats treated with subcutaneous doses of nicotine. Methods: The survival of modified McFarlane skin flaps was assessed on postoperative day 7. Nicotine group received 4 mg/kg nicotine during 40 days preoperatively and 7 days post-operatively. NicotineCbuflomedil group received nicotine and 6 mg/kg buflomedil 24 h pre-operatively and 7 days post-operatively. NicotineCpentoxifylline group received nicotine and 20 mg/kg pentoxifylline in 15 pre-operatively and 7 post-operatively days. NicotineCbuflomedilCpentoxifylline group received nicotine and both drugs administered as above. Control group received daily 1 ml normal saline during 40 days pre-operatively and 7 days postoperatively. Using image analysis, five different flap areas were quantified: Total, preserved, necrotic, ischaemic and viability. Viability areas comprised the sum of ischaemic and preserved areas. Results: Nicotine treated animals had lower percentage of viability areas (60.7%G 6.8) than the control group (73.7%G9.5), pZ0.016. The percentage of viability areas in the buflomedil (76.4%G11.4), pentoxifylline (74.2%G15.6) and buflomedilC pentoxifylline (74.0%G9.7) groups were larger than the nicotine group (pZ0.002, pZ0.011 and pZ0.012, respectively). There were no significant differences in the viability areas when drugs were used isolated or in association. We further demonstrated that the increase in the viability area of the buflomedil and pentoxifylline groups (isolated or in association) was due to increase in ischaemic areas.
* Corresponding author. Address: Rua Manoel Cebrian Ferrer, 60 Vila Clementino, CEP 04023-070 Sa ˜o Paulo-SP, Brazil. Tel.: C55 11 55496744; fax: C55 11 55493515. E-mail address: [email protected] (R.J. Mauad).
S0007-1226/$ - see front matter q 2005 The British Association of Plastic Surgeons. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.bjps.2005.07.005
388
R.J. Mauad et al. Conclusions: Both drugs equally increased flap survival in nicotine treated animals. Viability areas increased due to larger ischaemic areas, probably as a reflex of the action of these drugs in sites of partial circulatory deficit. q 2005 The British Association of Plastic Surgeons. Published by Elsevier Ltd. All rights reserved.
There is a body of clinical and experimental evidence that cigarette smoking, and especially nicotine, reduces skin-flap survival.1–4 The mechanism of action of nicotine in the pathogenesis of ischaemia and necrosis in cutaneous flaps seems to be related to factors such as a direct lesion of the endothelial cell, increase in the synthesis of catecholamines and alteration in the levels of prostaglandin and thromboxane A 2 . 5–7 In the human vasculature, Black et al. observed that acute exposure to nicotine amplified the mechanisms of vasoconstriction and impaired endothelium dependent skin vasorelaxation.8 The alterations linked to nicotine abuse can cause a 30–40% decrease in the blood flow of the peripheral vessels and, therefore, hinder the healing process.9 Buflomedil and pentoxifylline have been frequently used in clinical situations of peripheral circulatory deficit. Their isolated actions may represent an alternative to treat situations of circulatory deficit in different clinical situations, such as brain ischaemia, intermittent claudication and in the viability of flaps.10–13 Pentoxifylline, a methylxanthine, is a pharmacological alternative proposed for the treatment of intermittent claudication. It is one of the only two FDA-approved drugs available for this purpose.14 The results of the studies addressing its effects on exercise performance have been however still inconsistent.15 This drug seems to improve blood viscosity by decreasing serum levels of fibrinogen, inhibiting platelet aggregation, and increasing the deformability of erythrocytes and granulocytes.16,17 In most of the experimental studies, when used in 2– 3 weeks of pre-op schemes, it caused a significant reduction of necrosis of the skin flaps of rats.18,19 Buflomedil also represents a pharmacological alternative to treat situations of peripheral circulatory deficit, and it is currently available for use in the European countries. Meta-analyses studies related to intermittent claudication have found moderate effects of buflomedil on exercise performance tests.14 This drug acts by decreasing the peripheral vascular resistance with subsequent increase in the perfusion of impaired vascular beds of the microcirculation.20,21 Its administration, in the beginning of the pre-operative period, produces an increase in the viability of
flaps, as demonstrated in some experimental studies.22,23 Other studies, however, could not confirm these findings.24 Due to their mechanisms of action, both drugs could counteract the noxious effects of nicotine in the microcirculation. There is a single experimental study that has shown that pentoxifylline improved blood parameters and skin flap survival in rats previously submitted to administration of nicotine. 18 The effect of the administration of buflomedil in the survival of skin flaps of animals exposed to nicotine has been not investigated so far. Also, since these two drugs have different mechanisms of action in the microvasculature, it is possible to speculate that a combination of both could have an additive effect on the microvasculature. In fact, there is a report showing that the combination of pentoxifylline, buflomedil and defibrotide improved the walking autonomy in patients with obliterative arteriopathy of the legs.25 In this study, we have compared the action of the drugs buflomedil and pentoxifylline, isolated or in association, on the viability of dorsal cutaneous flaps of rats submitted to previous administration of nicotine.
Materials and methods Animal management The study was conducted on five groups of 15 male Wistar rats, weighing 200–350 g each. The animals were kept in metabolic cages, fed with commercial rat diet (Nuvilab CR10) and offered water ad libitum. All animals received humane care in compliance with the guide for care and use of laboratory animals (NIH publication 85-23, revised 1985). This study was approved by the local Ethical Committee.
Study design Four groups of 15 animals (nZ60) received daily subcutaneous injections of 4 mg/kg of nicotine
Buflomedil and pentoxifylline in viability of dorsal cutaneous flaps of rats treated with nicotine (Nicotine-Sigma Chemical, St Louis, MO, USA) in 1 ml normal saline during 40 days pre-operatively and 7 days post-operatively. Due to the high toxicity of nicotine, subcutaneous injections were preferred because of slower absorption rates. The group nicotine (NIC) received only nicotine injections as above indicated. The group buflomedil (Gale ˆnica, Sao Paulo, Brazil) (NB) received one intraperitoneal (IP) injection of 6 mg/kg of buflomedil 24 h pre-operatively and seven daily injections post-operatively. The group pentoxifylline (Trental, Aventis Pharma, Sao Paulo, Brazil) (NP) received daily IP injections of 20 mg/kg pentoxifylline 15 pre-operatively days and seven 7 days postoperatively. The group NicotineCbuflomedilC pentoxifylline (NBP) received both drugs administered in an identical way as NB and NP. A fifth group (CTRL) of 15 animals received daily subcutaneous injections of 1 ml normal saline during 40 days preoperatively and 7 days post-operatively.
Figure 1 Dorsal cutaneous flap areas at the 7th postoperative day. PA, preserved area; NA, necrotic area; IA, ischaemic area; VA, viability area; TA, total area. Observe that the viability area was divided in ischaemic area and preserved area for quantification purposes.
389
Surgical procedure Dorsal flaps, based caudally, were raised at the end of 40 days of treatment.18,26 The randomised flap measured 3 cm of width and 10 cm of length.18 The flap was dissected and detached with its Panniculus carnosus and subsequently replaced on the dorsal area of the rat, sutured with simple stitches, keeping a distance of 0.5 cm between them. At the end of the 7 post-operatively day, animals were anesthetised with ether in an inhalation chamber and sacrificed.
Quantification At the end of the 7th post-operative day, the dorsal skin flaps were photographed with a digital camera (Sony DCR-PC 9) in a static table (Leica/Leitz-Reprovit-R), using constant parameters: Lighting in 458 in relation to the object, column height of 40 cm and distance of the object of 49.5 cm. Images were then transferred to a computer (Pentium 4 CPU 1.7 GHz). With the aid of an image analysis program (IMAGE TOOL UTHSCSA, The University of Texas Health Science Center in San Antonio, 1995–2002) the different areas of the flap were quantified after manual delineation. A 10 cm standardised reference measure was placed next to the image. For a detailed analysis of the cutaneous skin flaps, quantification of the viability area was subdivided according to two distinct morphological patterns, preserved and ischaemical. Therefore, five total areas were quantified: Total area (TA); preserved area (PA), defined as the image of the dorsal skin flap of the rat with normal skin characteristics; ischaemic area (IA), defined as the image of the dorsal skin flap of the rat with a mottled aspect; necrotic area (NA), defined as the image of the dorsal skin flap with total loss of its normal skin characteristics and dark color; and viability area (VA), defined as the sum of the areas of the preserved and ischaemic area. Values were expressed in percentage (%) of the different areas in relation to the total area. The same researcher, who was unaware of the animal treatment, carried out measurements. The final result was the mean of three consecutive measurements (Fig. 1).
Statistical analysis Results were presented as meanGSD. For the comparison among the different groups, the analysis of variance test (ANOVA) was used, followed by
390
R.J. Mauad et al.
the Tukey HSD post-test. The statistical package SPSS 10.0 (Chicago, IL, USA) was used for the statistical analysis. Differences at probability values of p!0.05 were considered to be statistically significant.
Results All animals presented the former defined areas in their cutaneous flaps. After 7 days post-operatively infections of the surgical wound and suture dehiscence have occurred in very few cases, and did not compromise the analysis. Table 1 shows the percentage values and the significance level among the different areas in relation to the NIC group. Animals treated with NIC had significantly lower percentage of viability areas (60.7%G6.8) than the CTRL group (73.7%G 9.5) (pZ0.02) with a significant increase in the NA (NICZ39.1G6.9, CTRLZ26.7G12.2, pZ0.019). The percentage of the viability areas in the NB (76.4%G11.4), NP (74.2%G15.6) and NBP groups (74.0G9.7) were significantly larger (pZ0.002, pZ 0.011, pZ0.012, respectively) than the NIC group. There was no difference in the percentage of viability areas among the NB, NP and NBP groups. The percentage of ischaemic areas in the NP (13.1%G8.2) and in the NBP group (16.5%G5.8) were significantly larger than the NIC group (5.8%G 5.7) (p!0.05), with a trend in the NB group (12.6%G5.6), pZ0.07. Necrotic areas were significantly smaller in the NP, NB and NBP when compared to the NIC group (p!0.004), with no differences among the groups that received the vasoactive drugs. There were no significant differences concerning the preserved areas among the groups. Table 1
Discussion In this study, we have shown that the isolated administration of the vasoactive drugs buflomedil and pentoxifylline increased skin flap survival in animals previously treated with nicotine, with no significant difference in the percentage of flap survival between the two drugs. We have also shown that the conjunct administration of buflomedil and pentoxifylline had no additive effect in increasing skin flap survival. To our knowledge, we are the first to investigate the action of buflomedil in animals previously treated with nicotine. The effect of pentoxifylline on the viability of cutaneous flap has been vastly investigated in experimental studies. Different doses, administration schemes and associations with other kind of drugs have been studied and several works report a beneficial effect on the protection against ischaemia and necrosis. This is especially observed when pentoxifylline is administered for a period of at least 15 days prior to the surgery for obtaining the flap.18,19 However, other studies could not find significant differences in skin flap survival models.27 Our results confirm the findings of Aker et al. that described an increase in flap survival in rats that received nicotine and were treated with pentoxifylline.18 In this study, we demonstrate that buflomedil increases skin flap survival in animals previously treated with nicotine. Buflomedil hydrochloride increases the perfusion of the brain blood vessels of the peripheral blood microcirculation by the combination of several pharmacological effects: Nonspecific inhibition of a-adrenergic receptors of blood vessel smooth muscles with a decrease of the peripheral vascular resistance, inhibition of platelet aggregation, increase in the deformation of the erythrocyte, decrease in blood viscosity and
Skin flap areas in relation to the total area at the 7th post-operatively day
nZ15
PA (%)
NA (%)
IA (%)
VA (%)
NIC NCB
55.4G6.5 63.8G15.2 n.s. 61.1G18.9 n.s. 57.5G10.7 n.s. 63.3G14.8 n.s.
39.1G6.9 24.7G8.4 pZ0.004 23.9G15.5 pZ0.002 24.4G8.3 pZ0.003 26.7G12.2 pZ0.019
5.8G5.7 12.6G5.9 pZ0.07 13.1G8.2 0.047 16.5G5.8 pZ0.001 10.0G8.7 pZ0.48
60.7G6.8 76.4G11.4 pZ0.002 74.2G15.6 pZ0.011 74.0G9.7 pZ0.012 73.7G9.5 pZ0.016
NCP NCBCP CTRL
Values are compared to the NIC group and expressed as meanGSD. Each group had 15 animals. PA, preserved area; NA, necrotic area; IA, ischaemic area; VA, viability area; NIC, nicotine group; NCB, nicotineCbuflomedil group; NCP, nicotineCpentoxifyilline group; NCBCP, nicotineCbuflomedilCpentoxyfilline group; CTRL, control group; n.s., not significant.
Buflomedil and pentoxifylline in viability of dorsal cutaneous flaps of rats treated with nicotine increase in tissue oxygenation.11,20 Besides its direct action in the microcirculation, Toda et al., studying isolated dog arteries, demonstrated that buflomedil may have anti-nicotinic actions as well.21 As with pentoxifylline, the effects of this drug against flap ischaemia and necrosis have been previously researched, and some works report a beneficial effect of the use of this drug, comparable only to the process of autonomisation of flaps.22,23 However, controversial results have also been reported with buflomedil regarding skin flap survival.24 The discrepancies related to flap survival reported for both drugs may be partially explained by different administration and doses schemas. Pentoxifylline and buflomedil had similar effects on increasing viability areas in our study. Previous experimental studies comparing pentoxifylline and buflomedil were performed in skeletal muscles and on the hepatic microcirculation of the rat. While a most potent effect of buflomedil was observed in the hepatic microcirculation,28 no differences could be observed in vascularisation of the skeletal muscle.29 In clinical settings, both drugs seem to have similar efficacy.14 A possible advantage of buflomedil in the flap surgery setting is that it could be administered 24 h pre-operatively, while pentoxifylline would require longer pre-operatively administration schemes. In cases of emergence surgeries (such as digital reimplantation, trauma or oncological reconstruction) in smoking patients, this may represent an advantage. We could not demonstrate any addictive effect of pentoxifylline and buflomedil in increasing skin flap survival. We have not measured the mean arterial pressure and skin flap blood flow of the animals in our study. There is the possibility that combined pentoxifylline and buflomedil treatment may have caused some hypotension, hampering a possible beneficial addictive effect. The replacement of the traditional image capture models, associated to data processing computerised programs, has provided a great help to skin flap studies.30–32 The use of digital analysis allowed us to obtain the calculation of the different areas in a simplified, efficient and reproducible manner. The decomposition of the viability area into ischaemic and preserved areas allowed a better understanding of the action of buflomedil and pentoxifylline in the microcirculation of the flap. The increase in the viability area of the three groups was mainly due to an increase in the ischaemic area, since no differences were observed in the preserved areas. This effect is probably a reflex of the action of the drugs in sites of partial circulatory deficit aggravated by the nicotine. The final result was a reduction of the necrotic area and
391
an increase in the flap survival areas in the treated groups. In summary, we have demonstrated that buflomedil and pentoxifylline have a similar beneficial effect on the skin flap survival of rats previously treated with nicotine, with no addictive effect on the combination of both drugs. The recognition that the increase of the viability area is mainly due to an increase in the ischaemic areas of the flap illustrates the isolated effects of both drugs in areas of injured microcirculation. Complications of cigarette smoking in plastic surgeries continue to be a challenge to the plastic surgeon.4,7,8,33 Strikingly, two recent surveys in the US and the UK showed that the majority of plastic surgeons do perform procedures in smokers.34,35 Although no specific recommendations are available yet, smoking cessation for at least 4 weeks before elective surgical procedures should be required; the use of educational and psychological support should be stimulated.4 In urgent situations and in patients suspected of noncompliance, pharmacological therapy may represent an alternative strategy to counteract the effects of nicotine.4 Therefore, better understanding of the different pharmacological options is clearly desirable.
Acknowledgements We thank Sa ˜ o Paulo State Research Agency (Fundac ¸˜ ao de Amparo ` a Pesquisa do Estado de Sa ˜o Paulo—FAPESP) for the financial support. The authors also wish to thank Dr Luis Bernardo Froes and Dr Marisa Dolhnikoff for their support during the study.
References 1. Forrest CR, Pang CY, Lindsay WK. Pathogenesis of ischemic necrosis in random—pattern skin flaps induced by long-term low-dose nicotine treatment in the rat. Plast Reconstr Surg 1991;87:518–28. 2. Forrest CR, Xu N, Pang CY. Evidence for nicotine-induce skin flap ischemic necrosis in the pig. Can J Physiol Pharmacol 1994;72:30–8. 3. Forrest CR, Pang CY, Lindsay WK. Dose and time effects of nicotine treatment on the capillary blood flow and viability of random pattern skin flaps in the rat. Br J Plast Surg 1987; 40:295–9. 4. Krueger JK, Rohrich RJ. Clearing the smoke: The scientific rationale for tobacco abstention with plastic surgery. Plast Reconstr Surg 2001;108:1063–73. 5. Dintenfass L. Elevation of blood viscosity, aggregation of red cells, hematocrit values and fibrinogen levels with cigarette smokers. Med J Aust 1975;1:617–20.
392 6. Renaud S, Blache D, Dumont E, Thevenon C, Wissendanger T. Platelet function after cigarette smoking in relation to nicotine and carbon monoxide. Clin Pharmacol Ther 1984; 36:389–95. 7. Krupski WC. The peripherical consequences of smoking. Ann Vasc Surg 1991;5:291–304. 8. Black CE, Huang N, Neligan PC, et al. Effect of nicotine on vasoconstrictor and vasodilator responses in human skin vasculature. Am J Physiol Regul Integr Comp Physiol 2001; 281:R1097–R104. 9. Mosely LH, Finseth F, Goody M. Nicotine and its effect on wound healing. Plast Reconstr Surg 1978;61:570–5. 10. Ward A, Clissold SP. Pentoxifylline: A review of its pharmacodynamic and pharmacokinetic properties, and its therapeutic efficacy. Drugs 1987;34:50–97. 11. Clissold SP, Lynch S, Sorkin EM. Buflomedil: A review of its pharmocokinetic properties and therapeutic efficacy in peripheral and cerebral vascular diseases. Drugs 1987;33: 430–60. 12. Yessenow RS, Maves MD. The effects of pentoxifylline on random skin flap survival. Arch Otolaryngol Head Neck Surg 1989;115:179–81. 13. Williams PB, Hankins DB, Pratt MF. Long-term pretreatment with pentoxifylline increases random skin flap survival. Arch Otolaryngol Head Neck Surg 1994;120:65–71. 14. Jacoby D, Mohler 3rd ER. Drug treatment of intermittent claudication. Drugs 2004;64:1657–70. 15. Dawson DL, Zheng Q, Worthy SA, Charles B, Bradley Jr DV. Failure of pentoxifylline or cilostazol to improve blood and plasma viscosity, fibrinogen, and erythrocyte deformability in claudication. Angiology 2002;53:509–20. 16. Mollitt DL, Poulos ND. The role of pentoxifylline in endotoxin-induced alterations of red cell deformability and whole blood viscosity in the neonate. J Pediatr Surg 1991; 26:572–4. 17. Mu ¨ller R, Lehrach F. Haemorheological role of platelet aggregation and hypercoagulability in microcirculation: Therapeutical approach with pentoxifylline. Pharmatherapeutica 1980;2:372–9. 18. Aker JS, Mancoll J, Lewis B, Colen LB. The effect of pentoxifylline on random-pattern skin-flap necrosis induced by nicotine treatment in the rat. Plast Reconstr Surg 1977; 100:66–71. 19. Roth AG, Briggs PC, Jones FR. Augmentation of skin flap survival by parenteral pentoxifylline. Br J Plast Surg 1988; 41:515–20. 20. Vanhoutte PM, Aarhus LL, Coen E, Lorenz RR, Rimele TJ, Verbeuren TJ. Effects of buflomedil on the responsiveness of canine vascular smooth muscle. J Pharmacol Exp Ther 1983; 227:613–20.
R.J. Mauad et al. 21. Toda N, Okunishi H, Okamura T, Miyazaki M. Effects of buflomedil on isolated dog arteries. Angiology 1983;32: 699–704. 22. Dias LC, Foutanos A, Carreira ˜o S, Pitanguy I. Influe ˆncia do buflomedil na viabilidade do retalho de pele. Rev Bras Cir 1990;80:49–55. 23. Galla TJ, Saetzler RK, Hammersen F, Messmer K. Increase in skin-flap survival by the vasoactive drug buflomedil. Plast Reconstr Surg 1991;87:130–6. 24. Quirinia A, Gottrup F, Viidik A. Failure of buflomedil to improve wound healing in ischaemic skin flaps. Scand J Plast Reconstr Surg Hand Surg 1996;30:81–7. 25. Marci M, Albiani B, Ricci M, Russo F. Treatment of chronic obliterative arteriopathy of the legs in the second Fontaine’s stage. Personal experience with a buflomedil–pentoxifylline– defibrotide combination. Clin Ter 1995;146:211–4. 26. McFarlane RM, DeYoung G, Henry RA. The design of a pedicle flap in the rat to study necrosis and its prevention. Plast Reconstr Surg 1965;35:177–82. 27. Freedman AM, Hyde GL, Luce EA. Failure of pentoxifylline to enhance skin flap survival in the rat. Ann Plast Surg 1989;1: 31–4. 28. Marteau P, Ballet F, Chretien Y, Rey C, Jaillon P, Poupon R. Effect of vasodilators on hepatic microcirculation: A study of the inhibition of norepinephrine-induced vasoconstriction in the isolated perfused rat liver. Hepatology 1988;8: 228–31. 29. Hudlicka O, Price S. Effects of torbafylline, pentoxifylline and buflomedil on vascularisation and fibre type of rat skeletal muscles subjected to limited blood supply. Br J Pharmacol 1990;99:786–90. 30. Akyurek M, Kayikcioglu A, Mavili ME, Safak T. Evaluation of skin flap survival in rats. Plast Reconstr Surg 1999;103: 337–8. 31. Chowdary RP, Moss M, Hugo NE. Use of computer analysis in flap research. Ann Plast Surg 1987;18:261–4. 32. Nichter LS, Sobieski MW, Morgan RF, Rodeheaver G, Edlich RF. Quantitation of skin-flap survival: A computerbased method. Plast Reconstr Surg 1984;73:684–6. 33. Akoz T, Akan M, Yildirim S. If you continue to smoke, we may have a problem: Smoking’s effects on plastic surgery. Aesthetic Plast Surg 2002;26:477–82. 34. Coberly DM, Krueger JK, Brown SA. Planning elective operations on patients who smoke: Survey of North American plastic surgeons. Plast Reconstr Surg 2002;109:350–5. 35. Rayatt S, White N, Jennings S, Matthews RN. Smoking and elective surgery: A survey of United Kingdom plastic surgery consultants. Plast Reconstr Surg 2004;114:605–6.
Buflomedil and pentoxifylline in the viability of dorsal cutaneous flaps of rats treated with nicotine Raul J. Mauad Jra,*, Maria H.M. Shimizua, Thais Mauadb, Erasmo M.C. de Tolosaa a
Department of Experimental Surgery, School of Medicine, Sao Paulo University, Av. Dr Arnaldo 455, 01246-903 Sao Paulo, SP, Brazil b Department of Pathology, School of Medicine, Sao Paulo University, Av. Dr Arnaldo 455, 01246-903 Sao Paulo, SP, Brazil Received 28 November 2004; accepted 11 July 2005
KEYWORDS Nicotine; Skin flap; Rat; Buflomedil; Pentoxifylline; Viability
Summary Background: Nicotine reduces skin-flap survival. Pharmacologic therapy may represent an alternative treatment strategy to counteract nicotine effects in the flap surgery setting. In this study, we have compared the isolated and associated actions of the vasoactive drugs buflomedil and pentoxifylline in the viability of dorsal cutaneous flaps of rats treated with subcutaneous doses of nicotine. Methods: The survival of modified McFarlane skin flaps was assessed on postoperative day 7. Nicotine group received 4 mg/kg nicotine during 40 days preoperatively and 7 days post-operatively. NicotineCbuflomedil group received nicotine and 6 mg/kg buflomedil 24 h pre-operatively and 7 days post-operatively. NicotineCpentoxifylline group received nicotine and 20 mg/kg pentoxifylline in 15 pre-operatively and 7 post-operatively days. NicotineCbuflomedilCpentoxifylline group received nicotine and both drugs administered as above. Control group received daily 1 ml normal saline during 40 days pre-operatively and 7 days postoperatively. Using image analysis, five different flap areas were quantified: Total, preserved, necrotic, ischaemic and viability. Viability areas comprised the sum of ischaemic and preserved areas. Results: Nicotine treated animals had lower percentage of viability areas (60.7%G 6.8) than the control group (73.7%G9.5), pZ0.016. The percentage of viability areas in the buflomedil (76.4%G11.4), pentoxifylline (74.2%G15.6) and buflomedilC pentoxifylline (74.0%G9.7) groups were larger than the nicotine group (pZ0.002, pZ0.011 and pZ0.012, respectively). There were no significant differences in the viability areas when drugs were used isolated or in association. We further demonstrated that the increase in the viability area of the buflomedil and pentoxifylline groups (isolated or in association) was due to increase in ischaemic areas.
* Corresponding author. Address: Rua Manoel Cebrian Ferrer, 60 Vila Clementino, CEP 04023-070 Sa ˜o Paulo-SP, Brazil. Tel.: C55 11 55496744; fax: C55 11 55493515. E-mail address: [email protected] (R.J. Mauad).
S0007-1226/$ - see front matter q 2005 The British Association of Plastic Surgeons. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.bjps.2005.07.005
388
R.J. Mauad et al. Conclusions: Both drugs equally increased flap survival in nicotine treated animals. Viability areas increased due to larger ischaemic areas, probably as a reflex of the action of these drugs in sites of partial circulatory deficit. q 2005 The British Association of Plastic Surgeons. Published by Elsevier Ltd. All rights reserved.
There is a body of clinical and experimental evidence that cigarette smoking, and especially nicotine, reduces skin-flap survival.1–4 The mechanism of action of nicotine in the pathogenesis of ischaemia and necrosis in cutaneous flaps seems to be related to factors such as a direct lesion of the endothelial cell, increase in the synthesis of catecholamines and alteration in the levels of prostaglandin and thromboxane A 2 . 5–7 In the human vasculature, Black et al. observed that acute exposure to nicotine amplified the mechanisms of vasoconstriction and impaired endothelium dependent skin vasorelaxation.8 The alterations linked to nicotine abuse can cause a 30–40% decrease in the blood flow of the peripheral vessels and, therefore, hinder the healing process.9 Buflomedil and pentoxifylline have been frequently used in clinical situations of peripheral circulatory deficit. Their isolated actions may represent an alternative to treat situations of circulatory deficit in different clinical situations, such as brain ischaemia, intermittent claudication and in the viability of flaps.10–13 Pentoxifylline, a methylxanthine, is a pharmacological alternative proposed for the treatment of intermittent claudication. It is one of the only two FDA-approved drugs available for this purpose.14 The results of the studies addressing its effects on exercise performance have been however still inconsistent.15 This drug seems to improve blood viscosity by decreasing serum levels of fibrinogen, inhibiting platelet aggregation, and increasing the deformability of erythrocytes and granulocytes.16,17 In most of the experimental studies, when used in 2– 3 weeks of pre-op schemes, it caused a significant reduction of necrosis of the skin flaps of rats.18,19 Buflomedil also represents a pharmacological alternative to treat situations of peripheral circulatory deficit, and it is currently available for use in the European countries. Meta-analyses studies related to intermittent claudication have found moderate effects of buflomedil on exercise performance tests.14 This drug acts by decreasing the peripheral vascular resistance with subsequent increase in the perfusion of impaired vascular beds of the microcirculation.20,21 Its administration, in the beginning of the pre-operative period, produces an increase in the viability of
flaps, as demonstrated in some experimental studies.22,23 Other studies, however, could not confirm these findings.24 Due to their mechanisms of action, both drugs could counteract the noxious effects of nicotine in the microcirculation. There is a single experimental study that has shown that pentoxifylline improved blood parameters and skin flap survival in rats previously submitted to administration of nicotine. 18 The effect of the administration of buflomedil in the survival of skin flaps of animals exposed to nicotine has been not investigated so far. Also, since these two drugs have different mechanisms of action in the microvasculature, it is possible to speculate that a combination of both could have an additive effect on the microvasculature. In fact, there is a report showing that the combination of pentoxifylline, buflomedil and defibrotide improved the walking autonomy in patients with obliterative arteriopathy of the legs.25 In this study, we have compared the action of the drugs buflomedil and pentoxifylline, isolated or in association, on the viability of dorsal cutaneous flaps of rats submitted to previous administration of nicotine.
Materials and methods Animal management The study was conducted on five groups of 15 male Wistar rats, weighing 200–350 g each. The animals were kept in metabolic cages, fed with commercial rat diet (Nuvilab CR10) and offered water ad libitum. All animals received humane care in compliance with the guide for care and use of laboratory animals (NIH publication 85-23, revised 1985). This study was approved by the local Ethical Committee.
Study design Four groups of 15 animals (nZ60) received daily subcutaneous injections of 4 mg/kg of nicotine
Buflomedil and pentoxifylline in viability of dorsal cutaneous flaps of rats treated with nicotine (Nicotine-Sigma Chemical, St Louis, MO, USA) in 1 ml normal saline during 40 days pre-operatively and 7 days post-operatively. Due to the high toxicity of nicotine, subcutaneous injections were preferred because of slower absorption rates. The group nicotine (NIC) received only nicotine injections as above indicated. The group buflomedil (Gale ˆnica, Sao Paulo, Brazil) (NB) received one intraperitoneal (IP) injection of 6 mg/kg of buflomedil 24 h pre-operatively and seven daily injections post-operatively. The group pentoxifylline (Trental, Aventis Pharma, Sao Paulo, Brazil) (NP) received daily IP injections of 20 mg/kg pentoxifylline 15 pre-operatively days and seven 7 days postoperatively. The group NicotineCbuflomedilC pentoxifylline (NBP) received both drugs administered in an identical way as NB and NP. A fifth group (CTRL) of 15 animals received daily subcutaneous injections of 1 ml normal saline during 40 days preoperatively and 7 days post-operatively.
Figure 1 Dorsal cutaneous flap areas at the 7th postoperative day. PA, preserved area; NA, necrotic area; IA, ischaemic area; VA, viability area; TA, total area. Observe that the viability area was divided in ischaemic area and preserved area for quantification purposes.
389
Surgical procedure Dorsal flaps, based caudally, were raised at the end of 40 days of treatment.18,26 The randomised flap measured 3 cm of width and 10 cm of length.18 The flap was dissected and detached with its Panniculus carnosus and subsequently replaced on the dorsal area of the rat, sutured with simple stitches, keeping a distance of 0.5 cm between them. At the end of the 7 post-operatively day, animals were anesthetised with ether in an inhalation chamber and sacrificed.
Quantification At the end of the 7th post-operative day, the dorsal skin flaps were photographed with a digital camera (Sony DCR-PC 9) in a static table (Leica/Leitz-Reprovit-R), using constant parameters: Lighting in 458 in relation to the object, column height of 40 cm and distance of the object of 49.5 cm. Images were then transferred to a computer (Pentium 4 CPU 1.7 GHz). With the aid of an image analysis program (IMAGE TOOL UTHSCSA, The University of Texas Health Science Center in San Antonio, 1995–2002) the different areas of the flap were quantified after manual delineation. A 10 cm standardised reference measure was placed next to the image. For a detailed analysis of the cutaneous skin flaps, quantification of the viability area was subdivided according to two distinct morphological patterns, preserved and ischaemical. Therefore, five total areas were quantified: Total area (TA); preserved area (PA), defined as the image of the dorsal skin flap of the rat with normal skin characteristics; ischaemic area (IA), defined as the image of the dorsal skin flap of the rat with a mottled aspect; necrotic area (NA), defined as the image of the dorsal skin flap with total loss of its normal skin characteristics and dark color; and viability area (VA), defined as the sum of the areas of the preserved and ischaemic area. Values were expressed in percentage (%) of the different areas in relation to the total area. The same researcher, who was unaware of the animal treatment, carried out measurements. The final result was the mean of three consecutive measurements (Fig. 1).
Statistical analysis Results were presented as meanGSD. For the comparison among the different groups, the analysis of variance test (ANOVA) was used, followed by
390
R.J. Mauad et al.
the Tukey HSD post-test. The statistical package SPSS 10.0 (Chicago, IL, USA) was used for the statistical analysis. Differences at probability values of p!0.05 were considered to be statistically significant.
Results All animals presented the former defined areas in their cutaneous flaps. After 7 days post-operatively infections of the surgical wound and suture dehiscence have occurred in very few cases, and did not compromise the analysis. Table 1 shows the percentage values and the significance level among the different areas in relation to the NIC group. Animals treated with NIC had significantly lower percentage of viability areas (60.7%G6.8) than the CTRL group (73.7%G 9.5) (pZ0.02) with a significant increase in the NA (NICZ39.1G6.9, CTRLZ26.7G12.2, pZ0.019). The percentage of the viability areas in the NB (76.4%G11.4), NP (74.2%G15.6) and NBP groups (74.0G9.7) were significantly larger (pZ0.002, pZ 0.011, pZ0.012, respectively) than the NIC group. There was no difference in the percentage of viability areas among the NB, NP and NBP groups. The percentage of ischaemic areas in the NP (13.1%G8.2) and in the NBP group (16.5%G5.8) were significantly larger than the NIC group (5.8%G 5.7) (p!0.05), with a trend in the NB group (12.6%G5.6), pZ0.07. Necrotic areas were significantly smaller in the NP, NB and NBP when compared to the NIC group (p!0.004), with no differences among the groups that received the vasoactive drugs. There were no significant differences concerning the preserved areas among the groups. Table 1
Discussion In this study, we have shown that the isolated administration of the vasoactive drugs buflomedil and pentoxifylline increased skin flap survival in animals previously treated with nicotine, with no significant difference in the percentage of flap survival between the two drugs. We have also shown that the conjunct administration of buflomedil and pentoxifylline had no additive effect in increasing skin flap survival. To our knowledge, we are the first to investigate the action of buflomedil in animals previously treated with nicotine. The effect of pentoxifylline on the viability of cutaneous flap has been vastly investigated in experimental studies. Different doses, administration schemes and associations with other kind of drugs have been studied and several works report a beneficial effect on the protection against ischaemia and necrosis. This is especially observed when pentoxifylline is administered for a period of at least 15 days prior to the surgery for obtaining the flap.18,19 However, other studies could not find significant differences in skin flap survival models.27 Our results confirm the findings of Aker et al. that described an increase in flap survival in rats that received nicotine and were treated with pentoxifylline.18 In this study, we demonstrate that buflomedil increases skin flap survival in animals previously treated with nicotine. Buflomedil hydrochloride increases the perfusion of the brain blood vessels of the peripheral blood microcirculation by the combination of several pharmacological effects: Nonspecific inhibition of a-adrenergic receptors of blood vessel smooth muscles with a decrease of the peripheral vascular resistance, inhibition of platelet aggregation, increase in the deformation of the erythrocyte, decrease in blood viscosity and
Skin flap areas in relation to the total area at the 7th post-operatively day
nZ15
PA (%)
NA (%)
IA (%)
VA (%)
NIC NCB
55.4G6.5 63.8G15.2 n.s. 61.1G18.9 n.s. 57.5G10.7 n.s. 63.3G14.8 n.s.
39.1G6.9 24.7G8.4 pZ0.004 23.9G15.5 pZ0.002 24.4G8.3 pZ0.003 26.7G12.2 pZ0.019
5.8G5.7 12.6G5.9 pZ0.07 13.1G8.2 0.047 16.5G5.8 pZ0.001 10.0G8.7 pZ0.48
60.7G6.8 76.4G11.4 pZ0.002 74.2G15.6 pZ0.011 74.0G9.7 pZ0.012 73.7G9.5 pZ0.016
NCP NCBCP CTRL
Values are compared to the NIC group and expressed as meanGSD. Each group had 15 animals. PA, preserved area; NA, necrotic area; IA, ischaemic area; VA, viability area; NIC, nicotine group; NCB, nicotineCbuflomedil group; NCP, nicotineCpentoxifyilline group; NCBCP, nicotineCbuflomedilCpentoxyfilline group; CTRL, control group; n.s., not significant.
Buflomedil and pentoxifylline in viability of dorsal cutaneous flaps of rats treated with nicotine increase in tissue oxygenation.11,20 Besides its direct action in the microcirculation, Toda et al., studying isolated dog arteries, demonstrated that buflomedil may have anti-nicotinic actions as well.21 As with pentoxifylline, the effects of this drug against flap ischaemia and necrosis have been previously researched, and some works report a beneficial effect of the use of this drug, comparable only to the process of autonomisation of flaps.22,23 However, controversial results have also been reported with buflomedil regarding skin flap survival.24 The discrepancies related to flap survival reported for both drugs may be partially explained by different administration and doses schemas. Pentoxifylline and buflomedil had similar effects on increasing viability areas in our study. Previous experimental studies comparing pentoxifylline and buflomedil were performed in skeletal muscles and on the hepatic microcirculation of the rat. While a most potent effect of buflomedil was observed in the hepatic microcirculation,28 no differences could be observed in vascularisation of the skeletal muscle.29 In clinical settings, both drugs seem to have similar efficacy.14 A possible advantage of buflomedil in the flap surgery setting is that it could be administered 24 h pre-operatively, while pentoxifylline would require longer pre-operatively administration schemes. In cases of emergence surgeries (such as digital reimplantation, trauma or oncological reconstruction) in smoking patients, this may represent an advantage. We could not demonstrate any addictive effect of pentoxifylline and buflomedil in increasing skin flap survival. We have not measured the mean arterial pressure and skin flap blood flow of the animals in our study. There is the possibility that combined pentoxifylline and buflomedil treatment may have caused some hypotension, hampering a possible beneficial addictive effect. The replacement of the traditional image capture models, associated to data processing computerised programs, has provided a great help to skin flap studies.30–32 The use of digital analysis allowed us to obtain the calculation of the different areas in a simplified, efficient and reproducible manner. The decomposition of the viability area into ischaemic and preserved areas allowed a better understanding of the action of buflomedil and pentoxifylline in the microcirculation of the flap. The increase in the viability area of the three groups was mainly due to an increase in the ischaemic area, since no differences were observed in the preserved areas. This effect is probably a reflex of the action of the drugs in sites of partial circulatory deficit aggravated by the nicotine. The final result was a reduction of the necrotic area and
391
an increase in the flap survival areas in the treated groups. In summary, we have demonstrated that buflomedil and pentoxifylline have a similar beneficial effect on the skin flap survival of rats previously treated with nicotine, with no addictive effect on the combination of both drugs. The recognition that the increase of the viability area is mainly due to an increase in the ischaemic areas of the flap illustrates the isolated effects of both drugs in areas of injured microcirculation. Complications of cigarette smoking in plastic surgeries continue to be a challenge to the plastic surgeon.4,7,8,33 Strikingly, two recent surveys in the US and the UK showed that the majority of plastic surgeons do perform procedures in smokers.34,35 Although no specific recommendations are available yet, smoking cessation for at least 4 weeks before elective surgical procedures should be required; the use of educational and psychological support should be stimulated.4 In urgent situations and in patients suspected of noncompliance, pharmacological therapy may represent an alternative strategy to counteract the effects of nicotine.4 Therefore, better understanding of the different pharmacological options is clearly desirable.
Acknowledgements We thank Sa ˜ o Paulo State Research Agency (Fundac ¸˜ ao de Amparo ` a Pesquisa do Estado de Sa ˜o Paulo—FAPESP) for the financial support. The authors also wish to thank Dr Luis Bernardo Froes and Dr Marisa Dolhnikoff for their support during the study.
References 1. Forrest CR, Pang CY, Lindsay WK. Pathogenesis of ischemic necrosis in random—pattern skin flaps induced by long-term low-dose nicotine treatment in the rat. Plast Reconstr Surg 1991;87:518–28. 2. Forrest CR, Xu N, Pang CY. Evidence for nicotine-induce skin flap ischemic necrosis in the pig. Can J Physiol Pharmacol 1994;72:30–8. 3. Forrest CR, Pang CY, Lindsay WK. Dose and time effects of nicotine treatment on the capillary blood flow and viability of random pattern skin flaps in the rat. Br J Plast Surg 1987; 40:295–9. 4. Krueger JK, Rohrich RJ. Clearing the smoke: The scientific rationale for tobacco abstention with plastic surgery. Plast Reconstr Surg 2001;108:1063–73. 5. Dintenfass L. Elevation of blood viscosity, aggregation of red cells, hematocrit values and fibrinogen levels with cigarette smokers. Med J Aust 1975;1:617–20.
392 6. Renaud S, Blache D, Dumont E, Thevenon C, Wissendanger T. Platelet function after cigarette smoking in relation to nicotine and carbon monoxide. Clin Pharmacol Ther 1984; 36:389–95. 7. Krupski WC. The peripherical consequences of smoking. Ann Vasc Surg 1991;5:291–304. 8. Black CE, Huang N, Neligan PC, et al. Effect of nicotine on vasoconstrictor and vasodilator responses in human skin vasculature. Am J Physiol Regul Integr Comp Physiol 2001; 281:R1097–R104. 9. Mosely LH, Finseth F, Goody M. Nicotine and its effect on wound healing. Plast Reconstr Surg 1978;61:570–5. 10. Ward A, Clissold SP. Pentoxifylline: A review of its pharmacodynamic and pharmacokinetic properties, and its therapeutic efficacy. Drugs 1987;34:50–97. 11. Clissold SP, Lynch S, Sorkin EM. Buflomedil: A review of its pharmocokinetic properties and therapeutic efficacy in peripheral and cerebral vascular diseases. Drugs 1987;33: 430–60. 12. Yessenow RS, Maves MD. The effects of pentoxifylline on random skin flap survival. Arch Otolaryngol Head Neck Surg 1989;115:179–81. 13. Williams PB, Hankins DB, Pratt MF. Long-term pretreatment with pentoxifylline increases random skin flap survival. Arch Otolaryngol Head Neck Surg 1994;120:65–71. 14. Jacoby D, Mohler 3rd ER. Drug treatment of intermittent claudication. Drugs 2004;64:1657–70. 15. Dawson DL, Zheng Q, Worthy SA, Charles B, Bradley Jr DV. Failure of pentoxifylline or cilostazol to improve blood and plasma viscosity, fibrinogen, and erythrocyte deformability in claudication. Angiology 2002;53:509–20. 16. Mollitt DL, Poulos ND. The role of pentoxifylline in endotoxin-induced alterations of red cell deformability and whole blood viscosity in the neonate. J Pediatr Surg 1991; 26:572–4. 17. Mu ¨ller R, Lehrach F. Haemorheological role of platelet aggregation and hypercoagulability in microcirculation: Therapeutical approach with pentoxifylline. Pharmatherapeutica 1980;2:372–9. 18. Aker JS, Mancoll J, Lewis B, Colen LB. The effect of pentoxifylline on random-pattern skin-flap necrosis induced by nicotine treatment in the rat. Plast Reconstr Surg 1977; 100:66–71. 19. Roth AG, Briggs PC, Jones FR. Augmentation of skin flap survival by parenteral pentoxifylline. Br J Plast Surg 1988; 41:515–20. 20. Vanhoutte PM, Aarhus LL, Coen E, Lorenz RR, Rimele TJ, Verbeuren TJ. Effects of buflomedil on the responsiveness of canine vascular smooth muscle. J Pharmacol Exp Ther 1983; 227:613–20.
R.J. Mauad et al. 21. Toda N, Okunishi H, Okamura T, Miyazaki M. Effects of buflomedil on isolated dog arteries. Angiology 1983;32: 699–704. 22. Dias LC, Foutanos A, Carreira ˜o S, Pitanguy I. Influe ˆncia do buflomedil na viabilidade do retalho de pele. Rev Bras Cir 1990;80:49–55. 23. Galla TJ, Saetzler RK, Hammersen F, Messmer K. Increase in skin-flap survival by the vasoactive drug buflomedil. Plast Reconstr Surg 1991;87:130–6. 24. Quirinia A, Gottrup F, Viidik A. Failure of buflomedil to improve wound healing in ischaemic skin flaps. Scand J Plast Reconstr Surg Hand Surg 1996;30:81–7. 25. Marci M, Albiani B, Ricci M, Russo F. Treatment of chronic obliterative arteriopathy of the legs in the second Fontaine’s stage. Personal experience with a buflomedil–pentoxifylline– defibrotide combination. Clin Ter 1995;146:211–4. 26. McFarlane RM, DeYoung G, Henry RA. The design of a pedicle flap in the rat to study necrosis and its prevention. Plast Reconstr Surg 1965;35:177–82. 27. Freedman AM, Hyde GL, Luce EA. Failure of pentoxifylline to enhance skin flap survival in the rat. Ann Plast Surg 1989;1: 31–4. 28. Marteau P, Ballet F, Chretien Y, Rey C, Jaillon P, Poupon R. Effect of vasodilators on hepatic microcirculation: A study of the inhibition of norepinephrine-induced vasoconstriction in the isolated perfused rat liver. Hepatology 1988;8: 228–31. 29. Hudlicka O, Price S. Effects of torbafylline, pentoxifylline and buflomedil on vascularisation and fibre type of rat skeletal muscles subjected to limited blood supply. Br J Pharmacol 1990;99:786–90. 30. Akyurek M, Kayikcioglu A, Mavili ME, Safak T. Evaluation of skin flap survival in rats. Plast Reconstr Surg 1999;103: 337–8. 31. Chowdary RP, Moss M, Hugo NE. Use of computer analysis in flap research. Ann Plast Surg 1987;18:261–4. 32. Nichter LS, Sobieski MW, Morgan RF, Rodeheaver G, Edlich RF. Quantitation of skin-flap survival: A computerbased method. Plast Reconstr Surg 1984;73:684–6. 33. Akoz T, Akan M, Yildirim S. If you continue to smoke, we may have a problem: Smoking’s effects on plastic surgery. Aesthetic Plast Surg 2002;26:477–82. 34. Coberly DM, Krueger JK, Brown SA. Planning elective operations on patients who smoke: Survey of North American plastic surgeons. Plast Reconstr Surg 2002;109:350–5. 35. Rayatt S, White N, Jennings S, Matthews RN. Smoking and elective surgery: A survey of United Kingdom plastic surgery consultants. Plast Reconstr Surg 2004;114:605–6.