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Viability of randomized skin flaps—an experimental study in rats
j o u r n a l o f s u r g i c a l r e s e a r c h j a n u a r y 2 0 1 7 ( 2 0 7 ) 9 2 e1 0 1
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Viability of randomized skin flapsdan experimental study in rats Nsingi N. Anto´nio, MD,a,* Andre´a Monte Alto Costa, PhD,b and Ruy G. Marques, MD, PhDc a
Post-Graduation Program in Physiopathology and Surgical Sciences, Department of General Surgery, Rio de Janeiro State University, Rio de Janeiro, Brazil b Laboratory of Tissue Repair, Institute of Biology Roberto Alcantara Gomes, Rio de Janeiro State University, Rio de Janeiro, Brazil c Laboratory of Experimental Surgery, Department of General Surgery, Rio de Janeiro State University, Rio de Janeiro, Brazil
article info
abstract
Article history:
Background: Randomized skin flaps are extensively used in plastic surgery, but the possi-
Received 28 January 2016
bility of necrosis has challenged their use. Several studies have been conducted aiming to
Received in revised form
find ways to reduce the occurrence of necrosis. We evaluated the effects of pentoxifylline
13 July 2016
(PTX) and hyaluronidase (HLD), each alone or combined, on randomized rat skin flaps.
Accepted 18 August 2016
Materials and methods: Fifty male Wistar rats were divided into five groups of 10 animals
Available online 31 August 2016
each: control I, control II, PTX, HLD, PTX-HLD. Substances were administered from the first to the 14th postoperative day. The necrotic area was measured on the seventh and 14th
Keywords:
postoperative day; the animals were killed on the 14th day, when samples were collected
Randomized skin flap
for histologic and immunohistochemical examination.
Viability
Results: On the seventh day, percentage of the necrotic area was significantly reduced in
Necrosis
PTX, HLD, and PTX-HLD animals compared with control groups. On 14th day, percentage of
Pentoxifylline
the necrotic area in PTX, HDL, and PTX-HLD groups was also significantly reduced
Hyaluronidase
compared with control groups. PTX and PTX-HLD showed a significant reduction in dermis cellularity, VV of macrophages, and myofibroblasts compared with control groups; PTX showed a significant enhancement of LV of blood vessels compared with all other groups. Conclusions: The use of each substance alone or combined increased flap viability compared with control groups. On the seventh day, PTX exhibited lower viability than HLD, whereas on the 14th day there was no difference between treated groups. PTX alone enhanced the LV of blood vessels, whereas PTX-HLD did not. However, PTX-HLD was more effective in decreasing the dermis cellularity and macrophage VV than HLD alone. ª 2016 Elsevier Inc. All rights reserved.
Introduction Randomized skin flaps are extensively used in plastic surgery, mainly for tissue reconstruction.1-4 However, the possible occurrence of necrosis appears to be the greatest
challenge regarding their use.1,3 Several factors are involved in the partial or total necrosis of randomized skin flaps, possibly related to the patient, such as metabolic alterations, tonus changes, vascular obstruction,4 or the flap confection itself.
* Corresponding author. Rua Cadete Ulisses Veiga 56/201, Sa˜o Cristo´va˜o, 20940-240 Rio de Janeiro, Brazil. Tel.: þ55 21 30210053. E-mail address: [email protected] (N.N. Anto´nio). 0022-4804/$ e see front matter ª 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2016.08.065
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The search for strategies to ensure greater viability of randomized skin flaps has driven investigations aiming not only to reduce harmful effects but also to expand mediators that promote wound healing.5-7 To this end, the action of various substances such as vasodilators, antioxidants, corticosteroids, and some enzymes has been studied.6,8,9 Pentoxifylline (PTX) is a nonselective phosphodiesterase inhibitor, a methylxanthine derivative10-13 whose molecular formula is C13H18N4O3, with a molecular weight of 278.31 Da. PTX is a drug with multiple pharmacologic properties,14 promotes relaxation of vascular smooth muscle, causing vasodilatation and preventing vascular spasm,15,16 enhances blood cell flexibility, including erythrocytes, favoring blood flow in peripheral vessels,9,14,17,18 promotes platelet disaggregation, and increases fibrinolysis, contributing to reduced blood viscosity.10,14,18-20 PTX modulates the immunologic activity by acting on cytokines, suppresses leukocyte hyperactivity by reducing superoxide release and neutrophil adhesion,17 inhibits the action of integrin and interleukin 2 (IL-2), preventing lymphocyte adhesion to endothelial cells,21 and reduces the synthesis of IL-1 and tumor necrosis factor a.14,18,20,22 It is used clinically to treat diseases of peripheral vessels.15,21,23,24 Hyaluronidase (HLD) is an enzyme of approximately 60,000 Da that acts on hyaluronic acid (HA) by temporarily depolarizing it and consequently reducing the viscosity of the intercellular medium, which in turn increases tissue permeability to other substances, whereas promoting the absorption of excess fluids, mobilizing edemas, and infiltrations.11,25 It is believed that this action increases the flow of interstitial fluid to the flap base, thus enhancing the drainage of metabolites from the flap extremity to more distant areas with normal vascularity.12,13 Considering the beneficial effect of both of these drugs on the rheological properties of organic fluids and on the wound healing process, based on different mechanisms of action, it has been hypothesized that the combined use of these drugs could increase the viability of randomized skin flaps. Thus, the aim of the present study was to evaluate the randomized skin flaps of rats treated with the combined use of these drugs compared with the separate use of each one.
Materials and methods The study was approved by the Ethics Committee on Animal Research of the Biology Institute Roberto Alcantara Gomes (University of Rio de Janeiro), under the CEUA/014/2015 protocol. All procedures strictly followed the care recommended by Brazilian law for the use of animals in research.26
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The animals were randomly divided into five groups of 10 animals each: (1) Control I (CTL-I): no treatment before or after the surgical procedure. (2) Control II (CTL-II): 0.25 mL of physiological saline solution (PSS) administered intraperitoneally (IP) twice a day, and 0.5 mL of the same solution administered intralesionally (Int.L) once a day. (3) PTX: 20 mg/kg PTX IP twice a day, and 0.5 mL PSS Int.L once a day. (4) HLD: 75 IU/d of HLD Int.L once a day, and 0.25 mL of PSS IP twice a day (5) PTX-HLD: same doses and routes of administration as described previously.
Anesthesia and trichotomy The animals were anesthetized with a combination of ketamine hydrochloride (150 mg/kg) and xylazine hydrochloride (15 mg/kg) at a 2:1 ratio, injected intramuscularly in the amount of 0.1 mL/100 g body weight. After anesthesia, the animals were submitted to trichotomy of the dorsal region.
Preparation of the skin flap The dorsal area was submitted to antisepsis with an alcoholic chlorhexidine solution, and a 3-cm wide and 10-cm long skin flap with a caudal base was drawn with a marking pen according to a modification of the McFarlane model,10,27,28 at the level of a horizontal line connecting the two iliac crests. A flap consisting of skin, subcutaneous tissue, fleshy panicle, and superficial fascia was lifted and then repositioned in its original bed with the use of separate 4-0 nylon monofilament sutures.
Administration of the substances After the surgical procedure, the animals received PSS, PTX (PTX injectable solution; Vascer), and HLD (3000 UTR HLD; Biometil) at the same dose and by the same route of administration as described previously. Int.L administration was performed at the two cephalic thirds of the flap in the dermal and subdermal planes, and IP administration was carried out with introduction of the needle into the abdominal cavity. The substances were administered until the 14th day of the experiment, with each session being preceded by sedation of the animals with inhaled anesthetic sevoflurane (Sevocris; Crista´lia Produtos Quimicos Farmaceuticos Ltda, Itapira, Sao Paulo).
Animals and groups Sample collection The study was conducted on 50 male Wistar rats weighing 250-300 g from the vivarium of the Experimental Surgery Laboratory, Medical Science School, State University of Rio de Janeiro. The rats were housed in individual cages in a temperature-controlled room (22 C) on a 12-h light and12-h dark cycle, with free access to food and water.
The animals were killed with anesthetic overdoses on the 14th day of the experiment, and a tissue fragment was collected from the boundary area between necrotic and viable tissue for histology and immunohistochemistry examinations (Fig. 1).
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Fig. 1 e Tissue fragment collected from the boundary area between the viable and necrotic tissue. (Color version of figure is available online.)
Macroscopic analysis The necrotic area was delimited on the seventh and 14th days after surgery using a cellophane sheet. The data were processed with the Image J software (National Institute of Mental Health, Bethesda, MD) and are expressed as the percent necrotic area.
Fig. 2 e Top paneldpercent necrotic area of the different groups on the seventh day of the experiment determined by one-way analysis of variance and the Tukey multiple comparison test. Bottom paneldpercent necrotic area of the different groups on the 14th day of the experiment determined by one-way analysis of variance and the Tukey multiple comparison test. (Color version of figure is available online.)
Histologic analysis The sections were stained with hematoxylin-eosin and the images were digitized with a digital MIDI Pannoramic Viewer slide scanner (3D Histech Kft, Budapest, Hungary) at 40 magnification. The stereological point counting method with an M42 grid was used to calculate the cell volume density of the superficial and deep dermis by applying the following formula: VV ¼ PP/ PT.29 Ten random fields per animal were analyzed and the results are expressed as the percentage.
method was used to calculate the volumetric density of macrophages and myofibroblasts and the results are expressed as the percentage. For the quantification of blood vessels, 10 random fields per animal were captured using the image analysis Pannoramic Viewer software. Vessel count per test area (¼26,506.4 mm2) was obtained using an M 42 grid. The arithmetic mean was calculated and blood vessel length density (LV) was determined using the following formula: LV ¼ 2 QA, where QA is estimate of the objects number into a frame. The results are expressed as millimeters per cubic millimeter.29
Immunohistochemical analysis For immunohistochemical analysis, CD68 antibody was used for macrophage immunostaining, whereas an antibody against a-smooth muscle actin was used for myofibroblasts and blood vessels. Ten random fields per animal were captured using the Pannoramic image analysis software to quantify myofibroblasts and macrophages. The stereological point counting
Statistical analysis Data are reported as the mean standard deviation, and the presence or absence of normal distribution was determined by the D’Agostino and Pearson Omnibus N test. One-way ANOVA followed by the Tukey post test was used for parametric data, whereas the KruskaleWallis test with
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Fig. 3 e Skin flaps on the seventh postoperative day showing the difference in necrotic area between the various groups: first paneldcontrol group I; second paneldcontrol group II; third paneldpentoxifylline group; fourth paneldhyaluronidase group; and fifth paneldpentoxifylline D hyaluronidase group. (Color version of figure is available online).
Dunn’s post test was used for nonparametric data. The level of significance was set at P 0.05 in all analyses. All data were analyzed with the aid of the GraphPad Prism software (version 6.0 GraphPad Prism, San Diego, CA).
Results During the anesthetic procedure, one animal of the HLD group died, with a total of 49 rats being left, that is, only nine animals in the HLD group and 10 in each of the remaining groups.
Macroscopic results Statistical analysis of the percent necrotic area on day 7 of the experiment revealed that the CTL-I (M ¼ 48.17%; SD ¼ 9.18) and CTL-II (M ¼ 46.24%; SD ¼ 8.02) groups showed a higher percentage of the necrotic area compared with the PTX (M ¼ 33.16%; SD ¼ 7.75), HLD (M ¼ 21.4%; SD ¼ 7.13%), and PTX-HLD (M ¼ 28.04%; SD ¼ 10.50) (Fig. 2, top panel and Fig. 3). The second demarcation of necrotic skin area performed on day 14 of the experiment revealed that the PTX (M ¼ 41.91%; SD ¼ 6.15), HLD (M ¼ 43%; SD ¼ 4.71), and PTXHLD (M ¼ 40.56%; SD ¼ 8.67) groups showed a significant reduction of the percent necrotic area compared with the CTL-
I (M ¼ 53.61%; SD ¼ 5:58) and CTL-II (M ¼ 52.07%; SD ¼ 6.79) groups (Fig. 2, bottom panel and Fig. 4).
Histologic results The cell volumetric density of the superficial dermis of the PTX (M ¼ 17%; SD ¼ 5.16) and PTX-HLD (M ¼ 15.98%; SD ¼ 11.12) groups was lower than that of the CTL-I (M ¼ 34.54%; SD ¼ 10.54), CTL-II (M ¼ 26.76%; SD ¼ 10.74), and HLD (M ¼ 09.22, SD ¼ 8.86) groups. The VV of the PTX and PTX-HLD groups differed significantly from that of the CTL-I group (Fig. 5, top panel and Fig. 6). The PTX (M ¼ 11.71%; SD ¼ 4.65) and PTX-HLD (M ¼ 14.81%; SD ¼ 6.24) groups had lower cellularity in the deep dermis compared with the CTL-I (M ¼ 20.95%; SD ¼ 7.15), CTL-II (M ¼ 17.78%; SD ¼ 9.42), and HLD (M ¼ 15.31%; SD ¼ 5.12) groups. There was a significant difference between the PTX and CTL-I groups (Fig. 3, bottom panel and Fig. 7).
Immunohistochemical results The volumetric macrophage density of the PTX (M ¼ 10.59%; SD ¼ 5.13) and PTX-HLD (M ¼ 12.19%; SD ¼ 6.72) groups was lower than that of the CTL-I (M ¼ 26.55%; SD ¼ 4.90), CTL-II (M ¼ 26.79%; SD ¼ 2.89), and HLD (M ¼ 17:32%; SD ¼ 6.8) groups. There was a significant difference between the PTX
Fig. 4 e Skin flaps on the 14th postoperative day showing the difference in necrotic area between the various groups: first paneldcontrol group I; second paneldcontrol group II; third paneldpentoxifylline group; fourth paneldhyaluronidase group; and fifth paneldpentoxifylline D hyaluronidase group. (Color version of figure is available online.)
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Fig. 5 e Top paneldvolumetric cell density (VV) of the superficial dermis of the different groups analyzed by the KruskaleWallis and Dunn multiple comparison tests. Bottom paneldvolumetric cell density (VV) of the deep dermis of the different groups analyzed by the KruskaleWallis and Dunn multiple comparison tests. (Color version of figure is available online.)
and PTX-HLD groups and the CTL-I and CTL-II groups (Fig. 4, first panel and Figs. 8 and 9). The volumetric density of myofibroblasts was significantly lower in the PTX (M ¼ 2.86%; SD ¼ 1.11) and PTX-HLD (M ¼ 2.44%; SD ¼ 0.61) groups compared with the CTL-I (M ¼ 9.85%; SD ¼ 4.13) and CTL-II (M ¼ 7.32 %; SD ¼ 2.10) groups. The HLD group (M ¼ 4.29%; SD ¼ 2.04) showed a significantly lower myofibroblast VV than the CTL-I group (Fig. 4, second panel and Fig. 10). The length density of blood vessels (LV) of the CTL-I (M ¼ 182.5 mm/mm3; SD ¼ 43.34), CTL-II (M ¼ 238.8 mm/ mm3; SD ¼ 88.06), HLD (M ¼ 201.3 mm/mm3; SD ¼ 37.96), and PTX-HLD (M ¼ 240.0 mm/mm3; SD ¼ 32.95) groups was significantly lower when compared with the PTX group (M ¼ 413.8 mm/mm3; SD ¼ 65.89) (Fig. 4, third panel and Fig. 11).
Discussion The total or partial necrosis of randomized skin flaps continues to represent one of the greatest challenges in plastic surgery and several studies have been performed aiming to reduce its occurrence. In the present study, PTX and HLD alone or combined were used to increase the viability of randomized skin flaps. HLD is often used in plastic surgery, especially to treat complications secondary to cosmetic procedures such as those resulting from the use of a dermal filler with HA.11
Fig. 6 e Photomicrographs of the superficial dermis stained with hematoxylin-eosin (bar [ 50 mm), showing the cellularity: first paneldcontrol group I; second paneldcontrol group II; third paneldpentoxifylline group; fourth paneldhyaluronidase group; and fifth paneldpentoxifylline D hyaluronidase group. (Color version of figure is available online.)
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Fig. 8 e First paneldvolumetric macrophage density (VV) of the different groups analyzed by one-way analysis of variance (ANOVA) and the Tukey multiple comparison test. Second paneldvolumetric myofibroblast density (VV) of the different groups analyzed by one-way ANOVA and the Tukey multiple comparison test. Third paneldlength density (LV) of blood vessels of the different groups analyzed by one-way ANOVA and the Tukey multiple comparison test. (Color version of figure is available online.)
Fig. 7 e Photomicrographs of the deep dermis stained with hematoxylin-eosin (bar [ 50 mm), showing the cellularity: first paneldcontrol group I; second paneldcontrol group II; third paneldpentoxifylline group; fourth paneldhyaluronidase group; and fifth paneldpentoxifylline D hyaluronidase group. (Color version of figure is available online.)
Literature studies have evaluated the effect of this drug on randomized skin flaps, but none of them examined its effect for more than 7 d. However, all these studies have shown the ability of this drug to enhance the viability of randomized skin flaps in experimental models.5,30 Pimentel et al.,30 in a model of randomized rabbit skin flaps using an HLD dose of 1 mL (200 IU/mL) Int.L, once a day, starting in the immediate postoperative period and continuing until the seventh day of the experiment, obtained a decrease in the necrotic area. Similarly, Bogado et al.5 evaluated the effect of HLD on the viability of randomized skin flaps in rats using doses of 100 and 50 IU/d Int.L starting during
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Fig. 9 e Photomicrographs of sections labeled with CD68 antibody for immunohistochemical identification of macrophages (bar [ 50 mm): first paneldcontrol group I; second paneldcontrol group II; third paneldpentoxifylline group; fourth paneldhyaluronidase group; and fifth paneldpentoxifylline D hyaluronidase group. (Color version of figure is available online.)
Fig. 10 e Photomicrographs of sections labeled with asmooth muscle actin antibody for immunohistochemical identification of myofibroblasts (bar [ 50 mm): first paneldcontrol group I; second paneldcontrol group II; third paneldpentoxifylline group; fourth paneldhyaluronidase group; and fifth paneldpentoxifylline D hyaluronidase group. (Color version of figure is available online.)
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Fig. 11 e Photomicrographs of sections labeled with asmooth muscle actin antibody for immunohistochemical identification of blood vessels (bar [ 50 mm): first paneldcontrol group I; second paneldcontrol group II; third paneldpentoxifylline group; fourth paneldhyaluronidase group; and fifth paneldpentoxifylline D hyaluronidase group. (Color version of figure is available online.)
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the immediate postoperative period and continuing up to the seventh day after surgery and found a decreased necrotic area using both doses. The findings of these studies agree with those observed in the present study. We observed that the use of HLD alone was more efficient in increasing flap viability than PTX alone or control. However, there was no difference when compared with the use of PTX-HLD on the seventh day of the experiment. On the 14th day, there was no significant difference compared with the other treated groups. On the 14th day of the experiment, there was a greater increase in the necrotic area in the HLD group than in the other treated groups, which may indicate that the prolonged use of this drug is less efficient than its short-term use. Histologic and immunohistochemistry analysis showed that the use of HLD alone did not reduce the cellularity or the VV of macrophages. It also failed to increase the LV of blood vessels. These results corroborate the study of Fronza et al. using an experimental model of wound healing in rats and showing that HLD did not decrease cellularity. On the other hand, our results did not confirm the ability of HLD to stimulate angiogenesis, as demonstrated in the cited study.12 PTX alone showed relatively less efficiency than the use of HLD alone on the seventh day of the experiment, but on the 14th day there was no difference. This is probably because of the different mechanisms of action of these drugs. PTX enhances blood cell flexibility, including erythrocytes, favoring blood flow in peripheral vessels,9,14,17,18 promotes platelet disaggregation, and increases fibrinolysis, contributing to reduced blood viscosity.10,14,18-20 Perhaps these effects require more time to begin to manifest. HLD acts on HA by temporarily depolarizing it and consequently reducing the viscosity of the intercellular medium. In turn, this increases tissue permeability to other substances, whereas promoting the absorption of excess fluids, mobilizing edemas and infiltrations,11,25 thus enhancing the drainage of metabolites from the flap extremity to more distant areas with normal vascularity.12,13 Such mechanisms may explain the earlier onset of its action. PTX decreased the VV of macrophages and myofibroblasts, an effect that can be explained by its ability to inhibit the production of some proinflammatory substances such as integrin, IL-2,21 IL-1, and tumor necrosis factor a.14,18,20,22 It is important to emphasize that PTX has the ability to shift the macrophage phenotype from inflammatory to anti-inflammatory,31-33 probably improving flap healing. However, it is not clear how PTX increases the LV of blood vessels. Probably this effect is because of its ability to promote relaxation of vascular smooth muscle, vasodilatation, and prevention of vascular spasm.15,16 Several reports have shown the ability of PTX to increase the viability of randomized skin flaps, reducing the inflammatory infiltrates and increasing the LV of blood vessels. For instance, Roth et al. studied the effect of PTX on randomized rat skin flaps and showed a reduction in the necrotic area.15 Topalan et al. demonstrated a reduction in the necrotic area in rats using an IP dose of 25 mg/kg body weight once a day for 10 d, starting in the immediate postoperative period.23
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Nakagawa et al.34 showed that PTX reduced a pulmonary inflammatory infiltrate by attenuating the leukocytee endothelial interaction in an experimental model of hemorrhagic and septic shock in rats. Garcia et al.,35 in a study of inflammation in diabetic rats, showed that PTX decreased the inflammatory process by reducing the production of proinflammatory cytokines. Lucca et al.18 showed an increase in blood vessel numbers in a transverse rectus abdominis myocutaneous flap of rats treated with PTX. In a study by Shirazi et al.,36 PTX preserved the LV of renal and bladder blood vessels in rats. Concerning blood vessels, it seems that a combination of the drugs reversed the benefit of PTX alone. The reason behind this occurrence is not so clear and perhaps was because of the action of HLD on HA, decomposing it into short fragments (tetra- and hexasaccharides). This may be beneficial on a short-term basis for flap survival, but in the long-term the accumulation of these short fragments in the tissues can impair flap viability because of their inflammatory properties.37 This set of events can explain why the combined or separate use of HLD showed little benefit regarding flap viability when used during a long-term period, appearing to be most effective when used during a short-term period. The combined use of the two drugs did not enhance the effect of each drug used alone at each time point (seventh and 14th days of the experiment). Rather, it seems that the combination of the drugs reversed the benefit of PTX alone on LV of blood vessels. Thus, the expected synergistic effect aiming at a higher gain of viability of randomized skin flaps, taking into account the different mechanisms of action presented by these drugs, was not satisfactorily demonstrated in this study, probably because the maximum effect of each therapy was already achieved and at any point the mechanisms had a common point that did not allow a cumulative effect.
Conclusions In this experimental model, the use of each substance alone or of their combination increased flap viability compared with control. On the seventh day, the use of PTX alone showed lower viability than HLD but on the 14th day there was no difference between treated groups. PTX alone was more efficient in enhancing the LV of blood vessels and in reducing the VV of the deep dermis than when used in combination with HLD. On the other hand, the combined use of these drugs was more effective in decreasing the cellularity of the superficial dermis and of macrophage VV than the use of HLD alone.
Acknowledgment This study was partially financed with funds from the Foundation for Research Support of the State of Rio de Janeiro (FAPERJ). Author contributions: N.N.A. contributed toward conception and design of the study, acquisition of data, analysis and interpretation of data, drafting of the article, and final
approval of the version to be submitted. A.M.A.C. and R.G.M. contributed toward design of the study, analysis and interpretation of data, critical revision of the article, and final approval of the version to be submitted.
Disclosure The authors reported no financial interest in any of the products, devices, or drugs mentioned in this manuscript.
references
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16. Yessenow RS, Maves MD. The effect of pentoxifylline on random skin flap survival. Arch Otolaryngol Head Neck Surg. 1989;115:179e181. 17. Aziret M, Irkorucu O, Reyhan E, et al. The effects of vardenafil and pentoxifylline administration in an animal model of ischemic colitis. Clinics (Sao Paulo). 2014;69:763e769. 18. Lucca AF, Brasolin AG, Feitosa RG, Enokihara MM, Gomes HF, Ferreira LM. Histological modification in TRAM flap in rats treated with pentoxifylline. Act Cir Bras. 2014;29:34e37. 19. 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. 1997;100:66e71. 20. Hassan I, Dorjay K, Anwar P. Pentoxifylline and its applications in dermatology. Indian Dermatol Online J. 2014;5:510e516. 21. Goel PN, Gude RP. Pentoxifylline regulates the cellular adhesion and its allied receptors to extracellular matrix components in breast cancer cells. Biomed Pharmacother. 2014;68:93e99. 22. Garcia FA, Pinto SF, Cavalcante AF, et al. Pentoxifylline decreases glycemia levels and TNF-alpha, iNOS and COX-2 expressions in diabetic rat pancreas. Springerplus. 2014;3:283. 23. Topalan M, Arinci A, Erer M, Guvenc H. The effect of parenteral pentoxifylline therapy on skin flap survival. Eur J Plast Surg. 1993;16:33e35. 24. Rewale V, Prabhakar KR, Chitale AM. Pentoxifylline: a new armamentarium in diabetic foot ulcers. J Clin Diagn Res. 2014;8:84e86. 25. Grossman JA, McGonagle BA, Dowden RV, Dinner MI. The effect of hyaluronidase and dimethyl sulfoxide on experimental skin flap survival. Ann Plast Surg. 1983;11:223e226. 26. Marques RG, Morales MM, Petroianu A. Brazilian law for scientific use of animals. Act Cir Bras. 2009;24:69e74. 27. Syed SA, Tasaki Y, Fuji T, Hiano A, Kosbayashi K. A new experimental model: the vascular pedicle flap over the dorsal aspect (flank and hip) of the rat. Br J Plast Surg. 1992;45:23e25.
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28. Dunn RM, Mancoll J. Flap models in the rat: a review and reappraisal. Plast Reconstr Surg. 1992;90:319e328. 29. Mandarim-de-Lacerda CA. Stereological tools in biomedical research. An Acad Bras Cienc. 2003;75:469e486. 30. Pimentel LA, Goldenberg RC. Injec¸a˜o local de hialuronidase para aumentar a sobreviveˆncia de retalhos cutaˆneosdEstudo experimental. Rev Bras Cir Plast. 1999;14:49e58. 31. Sunil VR, Vayas KN, Cervelli JA, et al. Pentoxifylline attenuates nitrogen mustard-induced acute lung injury, oxidative stress and inflammation. Exp Mol Pathol. 2014;97:89e98. 32. Banfi C, Sironi L, Simoni G, et al. Pentoxifylline prevents spontaneous brain ischemia in stroke-prone rats. J Pharmacol Exp Ther. 2004;310:890e895. 33. Chen YM, Chien CT, Hu-Tsai MI, et al. Pentoxifylline attenuates experimental mesangial proliferative glomerulonephritis. Kidney Int. 1999;56:932e943. 34. Nakagawa NK, Cruz RJ, Aikawa P, et al. Pentoxifylline attenuates leukocyte-endothelial interactions in a twohit model of shock and sepsis. J Surg Res. 2015;193:421e428. 35. Garcia FA, Rebouc¸as JF, Balbino TQ, et al. Pentoxifylline reduces the inflammatory process in diabetic rats relationship with decreases of pro-inflammatory cytokines and inducible nitric oxide synthase. J Inflamm (Lond). 2015;12:1e10. 36. Shirazi M, Soltani MR, Jahanabadi Z, Abdollahifar MA, Tanideh N, Noorafshan A. Stereological comparison of the effects of pentoxifylline, captopril, simvastatin, and tamoxifen on kidney and bladder structure after partial urethral obstruction in rats. Korean J Urol. 2014;55:756e763. 37. Stern R. Review: devising a pathway for hyaluronan catabolism: are we there yet? Glycobiology. 2003;13:105e115.
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Viability of randomized skin flapsdan experimental study in rats Nsingi N. Anto´nio, MD,a,* Andre´a Monte Alto Costa, PhD,b and Ruy G. Marques, MD, PhDc a
Post-Graduation Program in Physiopathology and Surgical Sciences, Department of General Surgery, Rio de Janeiro State University, Rio de Janeiro, Brazil b Laboratory of Tissue Repair, Institute of Biology Roberto Alcantara Gomes, Rio de Janeiro State University, Rio de Janeiro, Brazil c Laboratory of Experimental Surgery, Department of General Surgery, Rio de Janeiro State University, Rio de Janeiro, Brazil
article info
abstract
Article history:
Background: Randomized skin flaps are extensively used in plastic surgery, but the possi-
Received 28 January 2016
bility of necrosis has challenged their use. Several studies have been conducted aiming to
Received in revised form
find ways to reduce the occurrence of necrosis. We evaluated the effects of pentoxifylline
13 July 2016
(PTX) and hyaluronidase (HLD), each alone or combined, on randomized rat skin flaps.
Accepted 18 August 2016
Materials and methods: Fifty male Wistar rats were divided into five groups of 10 animals
Available online 31 August 2016
each: control I, control II, PTX, HLD, PTX-HLD. Substances were administered from the first to the 14th postoperative day. The necrotic area was measured on the seventh and 14th
Keywords:
postoperative day; the animals were killed on the 14th day, when samples were collected
Randomized skin flap
for histologic and immunohistochemical examination.
Viability
Results: On the seventh day, percentage of the necrotic area was significantly reduced in
Necrosis
PTX, HLD, and PTX-HLD animals compared with control groups. On 14th day, percentage of
Pentoxifylline
the necrotic area in PTX, HDL, and PTX-HLD groups was also significantly reduced
Hyaluronidase
compared with control groups. PTX and PTX-HLD showed a significant reduction in dermis cellularity, VV of macrophages, and myofibroblasts compared with control groups; PTX showed a significant enhancement of LV of blood vessels compared with all other groups. Conclusions: The use of each substance alone or combined increased flap viability compared with control groups. On the seventh day, PTX exhibited lower viability than HLD, whereas on the 14th day there was no difference between treated groups. PTX alone enhanced the LV of blood vessels, whereas PTX-HLD did not. However, PTX-HLD was more effective in decreasing the dermis cellularity and macrophage VV than HLD alone. ª 2016 Elsevier Inc. All rights reserved.
Introduction Randomized skin flaps are extensively used in plastic surgery, mainly for tissue reconstruction.1-4 However, the possible occurrence of necrosis appears to be the greatest
challenge regarding their use.1,3 Several factors are involved in the partial or total necrosis of randomized skin flaps, possibly related to the patient, such as metabolic alterations, tonus changes, vascular obstruction,4 or the flap confection itself.
* Corresponding author. Rua Cadete Ulisses Veiga 56/201, Sa˜o Cristo´va˜o, 20940-240 Rio de Janeiro, Brazil. Tel.: þ55 21 30210053. E-mail address: [email protected] (N.N. Anto´nio). 0022-4804/$ e see front matter ª 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2016.08.065
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The search for strategies to ensure greater viability of randomized skin flaps has driven investigations aiming not only to reduce harmful effects but also to expand mediators that promote wound healing.5-7 To this end, the action of various substances such as vasodilators, antioxidants, corticosteroids, and some enzymes has been studied.6,8,9 Pentoxifylline (PTX) is a nonselective phosphodiesterase inhibitor, a methylxanthine derivative10-13 whose molecular formula is C13H18N4O3, with a molecular weight of 278.31 Da. PTX is a drug with multiple pharmacologic properties,14 promotes relaxation of vascular smooth muscle, causing vasodilatation and preventing vascular spasm,15,16 enhances blood cell flexibility, including erythrocytes, favoring blood flow in peripheral vessels,9,14,17,18 promotes platelet disaggregation, and increases fibrinolysis, contributing to reduced blood viscosity.10,14,18-20 PTX modulates the immunologic activity by acting on cytokines, suppresses leukocyte hyperactivity by reducing superoxide release and neutrophil adhesion,17 inhibits the action of integrin and interleukin 2 (IL-2), preventing lymphocyte adhesion to endothelial cells,21 and reduces the synthesis of IL-1 and tumor necrosis factor a.14,18,20,22 It is used clinically to treat diseases of peripheral vessels.15,21,23,24 Hyaluronidase (HLD) is an enzyme of approximately 60,000 Da that acts on hyaluronic acid (HA) by temporarily depolarizing it and consequently reducing the viscosity of the intercellular medium, which in turn increases tissue permeability to other substances, whereas promoting the absorption of excess fluids, mobilizing edemas, and infiltrations.11,25 It is believed that this action increases the flow of interstitial fluid to the flap base, thus enhancing the drainage of metabolites from the flap extremity to more distant areas with normal vascularity.12,13 Considering the beneficial effect of both of these drugs on the rheological properties of organic fluids and on the wound healing process, based on different mechanisms of action, it has been hypothesized that the combined use of these drugs could increase the viability of randomized skin flaps. Thus, the aim of the present study was to evaluate the randomized skin flaps of rats treated with the combined use of these drugs compared with the separate use of each one.
Materials and methods The study was approved by the Ethics Committee on Animal Research of the Biology Institute Roberto Alcantara Gomes (University of Rio de Janeiro), under the CEUA/014/2015 protocol. All procedures strictly followed the care recommended by Brazilian law for the use of animals in research.26
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The animals were randomly divided into five groups of 10 animals each: (1) Control I (CTL-I): no treatment before or after the surgical procedure. (2) Control II (CTL-II): 0.25 mL of physiological saline solution (PSS) administered intraperitoneally (IP) twice a day, and 0.5 mL of the same solution administered intralesionally (Int.L) once a day. (3) PTX: 20 mg/kg PTX IP twice a day, and 0.5 mL PSS Int.L once a day. (4) HLD: 75 IU/d of HLD Int.L once a day, and 0.25 mL of PSS IP twice a day (5) PTX-HLD: same doses and routes of administration as described previously.
Anesthesia and trichotomy The animals were anesthetized with a combination of ketamine hydrochloride (150 mg/kg) and xylazine hydrochloride (15 mg/kg) at a 2:1 ratio, injected intramuscularly in the amount of 0.1 mL/100 g body weight. After anesthesia, the animals were submitted to trichotomy of the dorsal region.
Preparation of the skin flap The dorsal area was submitted to antisepsis with an alcoholic chlorhexidine solution, and a 3-cm wide and 10-cm long skin flap with a caudal base was drawn with a marking pen according to a modification of the McFarlane model,10,27,28 at the level of a horizontal line connecting the two iliac crests. A flap consisting of skin, subcutaneous tissue, fleshy panicle, and superficial fascia was lifted and then repositioned in its original bed with the use of separate 4-0 nylon monofilament sutures.
Administration of the substances After the surgical procedure, the animals received PSS, PTX (PTX injectable solution; Vascer), and HLD (3000 UTR HLD; Biometil) at the same dose and by the same route of administration as described previously. Int.L administration was performed at the two cephalic thirds of the flap in the dermal and subdermal planes, and IP administration was carried out with introduction of the needle into the abdominal cavity. The substances were administered until the 14th day of the experiment, with each session being preceded by sedation of the animals with inhaled anesthetic sevoflurane (Sevocris; Crista´lia Produtos Quimicos Farmaceuticos Ltda, Itapira, Sao Paulo).
Animals and groups Sample collection The study was conducted on 50 male Wistar rats weighing 250-300 g from the vivarium of the Experimental Surgery Laboratory, Medical Science School, State University of Rio de Janeiro. The rats were housed in individual cages in a temperature-controlled room (22 C) on a 12-h light and12-h dark cycle, with free access to food and water.
The animals were killed with anesthetic overdoses on the 14th day of the experiment, and a tissue fragment was collected from the boundary area between necrotic and viable tissue for histology and immunohistochemistry examinations (Fig. 1).
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Fig. 1 e Tissue fragment collected from the boundary area between the viable and necrotic tissue. (Color version of figure is available online.)
Macroscopic analysis The necrotic area was delimited on the seventh and 14th days after surgery using a cellophane sheet. The data were processed with the Image J software (National Institute of Mental Health, Bethesda, MD) and are expressed as the percent necrotic area.
Fig. 2 e Top paneldpercent necrotic area of the different groups on the seventh day of the experiment determined by one-way analysis of variance and the Tukey multiple comparison test. Bottom paneldpercent necrotic area of the different groups on the 14th day of the experiment determined by one-way analysis of variance and the Tukey multiple comparison test. (Color version of figure is available online.)
Histologic analysis The sections were stained with hematoxylin-eosin and the images were digitized with a digital MIDI Pannoramic Viewer slide scanner (3D Histech Kft, Budapest, Hungary) at 40 magnification. The stereological point counting method with an M42 grid was used to calculate the cell volume density of the superficial and deep dermis by applying the following formula: VV ¼ PP/ PT.29 Ten random fields per animal were analyzed and the results are expressed as the percentage.
method was used to calculate the volumetric density of macrophages and myofibroblasts and the results are expressed as the percentage. For the quantification of blood vessels, 10 random fields per animal were captured using the image analysis Pannoramic Viewer software. Vessel count per test area (¼26,506.4 mm2) was obtained using an M 42 grid. The arithmetic mean was calculated and blood vessel length density (LV) was determined using the following formula: LV ¼ 2 QA, where QA is estimate of the objects number into a frame. The results are expressed as millimeters per cubic millimeter.29
Immunohistochemical analysis For immunohistochemical analysis, CD68 antibody was used for macrophage immunostaining, whereas an antibody against a-smooth muscle actin was used for myofibroblasts and blood vessels. Ten random fields per animal were captured using the Pannoramic image analysis software to quantify myofibroblasts and macrophages. The stereological point counting
Statistical analysis Data are reported as the mean standard deviation, and the presence or absence of normal distribution was determined by the D’Agostino and Pearson Omnibus N test. One-way ANOVA followed by the Tukey post test was used for parametric data, whereas the KruskaleWallis test with
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Fig. 3 e Skin flaps on the seventh postoperative day showing the difference in necrotic area between the various groups: first paneldcontrol group I; second paneldcontrol group II; third paneldpentoxifylline group; fourth paneldhyaluronidase group; and fifth paneldpentoxifylline D hyaluronidase group. (Color version of figure is available online).
Dunn’s post test was used for nonparametric data. The level of significance was set at P 0.05 in all analyses. All data were analyzed with the aid of the GraphPad Prism software (version 6.0 GraphPad Prism, San Diego, CA).
Results During the anesthetic procedure, one animal of the HLD group died, with a total of 49 rats being left, that is, only nine animals in the HLD group and 10 in each of the remaining groups.
Macroscopic results Statistical analysis of the percent necrotic area on day 7 of the experiment revealed that the CTL-I (M ¼ 48.17%; SD ¼ 9.18) and CTL-II (M ¼ 46.24%; SD ¼ 8.02) groups showed a higher percentage of the necrotic area compared with the PTX (M ¼ 33.16%; SD ¼ 7.75), HLD (M ¼ 21.4%; SD ¼ 7.13%), and PTX-HLD (M ¼ 28.04%; SD ¼ 10.50) (Fig. 2, top panel and Fig. 3). The second demarcation of necrotic skin area performed on day 14 of the experiment revealed that the PTX (M ¼ 41.91%; SD ¼ 6.15), HLD (M ¼ 43%; SD ¼ 4.71), and PTXHLD (M ¼ 40.56%; SD ¼ 8.67) groups showed a significant reduction of the percent necrotic area compared with the CTL-
I (M ¼ 53.61%; SD ¼ 5:58) and CTL-II (M ¼ 52.07%; SD ¼ 6.79) groups (Fig. 2, bottom panel and Fig. 4).
Histologic results The cell volumetric density of the superficial dermis of the PTX (M ¼ 17%; SD ¼ 5.16) and PTX-HLD (M ¼ 15.98%; SD ¼ 11.12) groups was lower than that of the CTL-I (M ¼ 34.54%; SD ¼ 10.54), CTL-II (M ¼ 26.76%; SD ¼ 10.74), and HLD (M ¼ 09.22, SD ¼ 8.86) groups. The VV of the PTX and PTX-HLD groups differed significantly from that of the CTL-I group (Fig. 5, top panel and Fig. 6). The PTX (M ¼ 11.71%; SD ¼ 4.65) and PTX-HLD (M ¼ 14.81%; SD ¼ 6.24) groups had lower cellularity in the deep dermis compared with the CTL-I (M ¼ 20.95%; SD ¼ 7.15), CTL-II (M ¼ 17.78%; SD ¼ 9.42), and HLD (M ¼ 15.31%; SD ¼ 5.12) groups. There was a significant difference between the PTX and CTL-I groups (Fig. 3, bottom panel and Fig. 7).
Immunohistochemical results The volumetric macrophage density of the PTX (M ¼ 10.59%; SD ¼ 5.13) and PTX-HLD (M ¼ 12.19%; SD ¼ 6.72) groups was lower than that of the CTL-I (M ¼ 26.55%; SD ¼ 4.90), CTL-II (M ¼ 26.79%; SD ¼ 2.89), and HLD (M ¼ 17:32%; SD ¼ 6.8) groups. There was a significant difference between the PTX
Fig. 4 e Skin flaps on the 14th postoperative day showing the difference in necrotic area between the various groups: first paneldcontrol group I; second paneldcontrol group II; third paneldpentoxifylline group; fourth paneldhyaluronidase group; and fifth paneldpentoxifylline D hyaluronidase group. (Color version of figure is available online.)
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Fig. 5 e Top paneldvolumetric cell density (VV) of the superficial dermis of the different groups analyzed by the KruskaleWallis and Dunn multiple comparison tests. Bottom paneldvolumetric cell density (VV) of the deep dermis of the different groups analyzed by the KruskaleWallis and Dunn multiple comparison tests. (Color version of figure is available online.)
and PTX-HLD groups and the CTL-I and CTL-II groups (Fig. 4, first panel and Figs. 8 and 9). The volumetric density of myofibroblasts was significantly lower in the PTX (M ¼ 2.86%; SD ¼ 1.11) and PTX-HLD (M ¼ 2.44%; SD ¼ 0.61) groups compared with the CTL-I (M ¼ 9.85%; SD ¼ 4.13) and CTL-II (M ¼ 7.32 %; SD ¼ 2.10) groups. The HLD group (M ¼ 4.29%; SD ¼ 2.04) showed a significantly lower myofibroblast VV than the CTL-I group (Fig. 4, second panel and Fig. 10). The length density of blood vessels (LV) of the CTL-I (M ¼ 182.5 mm/mm3; SD ¼ 43.34), CTL-II (M ¼ 238.8 mm/ mm3; SD ¼ 88.06), HLD (M ¼ 201.3 mm/mm3; SD ¼ 37.96), and PTX-HLD (M ¼ 240.0 mm/mm3; SD ¼ 32.95) groups was significantly lower when compared with the PTX group (M ¼ 413.8 mm/mm3; SD ¼ 65.89) (Fig. 4, third panel and Fig. 11).
Discussion The total or partial necrosis of randomized skin flaps continues to represent one of the greatest challenges in plastic surgery and several studies have been performed aiming to reduce its occurrence. In the present study, PTX and HLD alone or combined were used to increase the viability of randomized skin flaps. HLD is often used in plastic surgery, especially to treat complications secondary to cosmetic procedures such as those resulting from the use of a dermal filler with HA.11
Fig. 6 e Photomicrographs of the superficial dermis stained with hematoxylin-eosin (bar [ 50 mm), showing the cellularity: first paneldcontrol group I; second paneldcontrol group II; third paneldpentoxifylline group; fourth paneldhyaluronidase group; and fifth paneldpentoxifylline D hyaluronidase group. (Color version of figure is available online.)
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Fig. 8 e First paneldvolumetric macrophage density (VV) of the different groups analyzed by one-way analysis of variance (ANOVA) and the Tukey multiple comparison test. Second paneldvolumetric myofibroblast density (VV) of the different groups analyzed by one-way ANOVA and the Tukey multiple comparison test. Third paneldlength density (LV) of blood vessels of the different groups analyzed by one-way ANOVA and the Tukey multiple comparison test. (Color version of figure is available online.)
Fig. 7 e Photomicrographs of the deep dermis stained with hematoxylin-eosin (bar [ 50 mm), showing the cellularity: first paneldcontrol group I; second paneldcontrol group II; third paneldpentoxifylline group; fourth paneldhyaluronidase group; and fifth paneldpentoxifylline D hyaluronidase group. (Color version of figure is available online.)
Literature studies have evaluated the effect of this drug on randomized skin flaps, but none of them examined its effect for more than 7 d. However, all these studies have shown the ability of this drug to enhance the viability of randomized skin flaps in experimental models.5,30 Pimentel et al.,30 in a model of randomized rabbit skin flaps using an HLD dose of 1 mL (200 IU/mL) Int.L, once a day, starting in the immediate postoperative period and continuing until the seventh day of the experiment, obtained a decrease in the necrotic area. Similarly, Bogado et al.5 evaluated the effect of HLD on the viability of randomized skin flaps in rats using doses of 100 and 50 IU/d Int.L starting during
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Fig. 9 e Photomicrographs of sections labeled with CD68 antibody for immunohistochemical identification of macrophages (bar [ 50 mm): first paneldcontrol group I; second paneldcontrol group II; third paneldpentoxifylline group; fourth paneldhyaluronidase group; and fifth paneldpentoxifylline D hyaluronidase group. (Color version of figure is available online.)
Fig. 10 e Photomicrographs of sections labeled with asmooth muscle actin antibody for immunohistochemical identification of myofibroblasts (bar [ 50 mm): first paneldcontrol group I; second paneldcontrol group II; third paneldpentoxifylline group; fourth paneldhyaluronidase group; and fifth paneldpentoxifylline D hyaluronidase group. (Color version of figure is available online.)
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Fig. 11 e Photomicrographs of sections labeled with asmooth muscle actin antibody for immunohistochemical identification of blood vessels (bar [ 50 mm): first paneldcontrol group I; second paneldcontrol group II; third paneldpentoxifylline group; fourth paneldhyaluronidase group; and fifth paneldpentoxifylline D hyaluronidase group. (Color version of figure is available online.)
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the immediate postoperative period and continuing up to the seventh day after surgery and found a decreased necrotic area using both doses. The findings of these studies agree with those observed in the present study. We observed that the use of HLD alone was more efficient in increasing flap viability than PTX alone or control. However, there was no difference when compared with the use of PTX-HLD on the seventh day of the experiment. On the 14th day, there was no significant difference compared with the other treated groups. On the 14th day of the experiment, there was a greater increase in the necrotic area in the HLD group than in the other treated groups, which may indicate that the prolonged use of this drug is less efficient than its short-term use. Histologic and immunohistochemistry analysis showed that the use of HLD alone did not reduce the cellularity or the VV of macrophages. It also failed to increase the LV of blood vessels. These results corroborate the study of Fronza et al. using an experimental model of wound healing in rats and showing that HLD did not decrease cellularity. On the other hand, our results did not confirm the ability of HLD to stimulate angiogenesis, as demonstrated in the cited study.12 PTX alone showed relatively less efficiency than the use of HLD alone on the seventh day of the experiment, but on the 14th day there was no difference. This is probably because of the different mechanisms of action of these drugs. PTX enhances blood cell flexibility, including erythrocytes, favoring blood flow in peripheral vessels,9,14,17,18 promotes platelet disaggregation, and increases fibrinolysis, contributing to reduced blood viscosity.10,14,18-20 Perhaps these effects require more time to begin to manifest. HLD acts on HA by temporarily depolarizing it and consequently reducing the viscosity of the intercellular medium. In turn, this increases tissue permeability to other substances, whereas promoting the absorption of excess fluids, mobilizing edemas and infiltrations,11,25 thus enhancing the drainage of metabolites from the flap extremity to more distant areas with normal vascularity.12,13 Such mechanisms may explain the earlier onset of its action. PTX decreased the VV of macrophages and myofibroblasts, an effect that can be explained by its ability to inhibit the production of some proinflammatory substances such as integrin, IL-2,21 IL-1, and tumor necrosis factor a.14,18,20,22 It is important to emphasize that PTX has the ability to shift the macrophage phenotype from inflammatory to anti-inflammatory,31-33 probably improving flap healing. However, it is not clear how PTX increases the LV of blood vessels. Probably this effect is because of its ability to promote relaxation of vascular smooth muscle, vasodilatation, and prevention of vascular spasm.15,16 Several reports have shown the ability of PTX to increase the viability of randomized skin flaps, reducing the inflammatory infiltrates and increasing the LV of blood vessels. For instance, Roth et al. studied the effect of PTX on randomized rat skin flaps and showed a reduction in the necrotic area.15 Topalan et al. demonstrated a reduction in the necrotic area in rats using an IP dose of 25 mg/kg body weight once a day for 10 d, starting in the immediate postoperative period.23
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Nakagawa et al.34 showed that PTX reduced a pulmonary inflammatory infiltrate by attenuating the leukocytee endothelial interaction in an experimental model of hemorrhagic and septic shock in rats. Garcia et al.,35 in a study of inflammation in diabetic rats, showed that PTX decreased the inflammatory process by reducing the production of proinflammatory cytokines. Lucca et al.18 showed an increase in blood vessel numbers in a transverse rectus abdominis myocutaneous flap of rats treated with PTX. In a study by Shirazi et al.,36 PTX preserved the LV of renal and bladder blood vessels in rats. Concerning blood vessels, it seems that a combination of the drugs reversed the benefit of PTX alone. The reason behind this occurrence is not so clear and perhaps was because of the action of HLD on HA, decomposing it into short fragments (tetra- and hexasaccharides). This may be beneficial on a short-term basis for flap survival, but in the long-term the accumulation of these short fragments in the tissues can impair flap viability because of their inflammatory properties.37 This set of events can explain why the combined or separate use of HLD showed little benefit regarding flap viability when used during a long-term period, appearing to be most effective when used during a short-term period. The combined use of the two drugs did not enhance the effect of each drug used alone at each time point (seventh and 14th days of the experiment). Rather, it seems that the combination of the drugs reversed the benefit of PTX alone on LV of blood vessels. Thus, the expected synergistic effect aiming at a higher gain of viability of randomized skin flaps, taking into account the different mechanisms of action presented by these drugs, was not satisfactorily demonstrated in this study, probably because the maximum effect of each therapy was already achieved and at any point the mechanisms had a common point that did not allow a cumulative effect.
Conclusions In this experimental model, the use of each substance alone or of their combination increased flap viability compared with control. On the seventh day, the use of PTX alone showed lower viability than HLD but on the 14th day there was no difference between treated groups. PTX alone was more efficient in enhancing the LV of blood vessels and in reducing the VV of the deep dermis than when used in combination with HLD. On the other hand, the combined use of these drugs was more effective in decreasing the cellularity of the superficial dermis and of macrophage VV than the use of HLD alone.
Acknowledgment This study was partially financed with funds from the Foundation for Research Support of the State of Rio de Janeiro (FAPERJ). Author contributions: N.N.A. contributed toward conception and design of the study, acquisition of data, analysis and interpretation of data, drafting of the article, and final
approval of the version to be submitted. A.M.A.C. and R.G.M. contributed toward design of the study, analysis and interpretation of data, critical revision of the article, and final approval of the version to be submitted.
Disclosure The authors reported no financial interest in any of the products, devices, or drugs mentioned in this manuscript.
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