Effect of antileukotriene treatment on capsular contracture: an experimental study

1 University of São Paulo, São Paulo, SP, Brazil. Introduction: 40% of patients undergoing radiotherapy after breast reconstruction by silicone prosthesis implant may develop prosthesis encapsulation. Several strategies have already been tested to prevent capsule contracture with unsatisfactory results. This study analyzed the effect of topical antileukotriene (AL) on capsular contracture formation in rats with silicone implants associated with irradiation. Methods: Silicone blocks were implanted in the dorsal region in 20 female rats Wistar with weights ranging from 200-250g. The animals were divided into two groups: control (injection of 0.9% saline solution into the tissue around the implant) and intervention group (injection of 10mg of AL into the tissue around the implant). Immediately after surgery, the animals were irradiated with a single dose of 10Gy. After two months, we collected capsule samples for histological analysis and gene expression analysis of the following biomarkers: iNOS, VEGF-a and MMP-9. Results: Vascular density was lower in the AL group when compared to the control group (55.4±30.0 vs. 81.8±26.7, p=0.05, respectively). Similarly, VEGF-a had the same behavior (control group 0.34±0.1 vs. group Al 0.02±0.001, p=0.04). Conclusion: This study suggested that treatment with AL decreases angiogenesis in animals submitted to silicone implants and underwent radiotherapy. ■ ABSTRACT

contracture, it should be replaced by another prosthesis implanted below the muscle. This procedure aims to avoid recurrence of contracture 4,5 .
For this reason, several techniques have been studied to prevent the occurrence of capsular contractures, such as the use of textured implants, implantation of the prosthesis in a submuscular pocket 4, use of medications such as corticosteroids, cyclosporine 5 and antileukotries 6,7 . All these therapeutic alternatives showed variable results without composing clinical evidence. Bastos et al. (2012) 8 showed positive results of using Montelukast, an antagonist of leukotrienes receptors, to prevent capsular contracture.

OBJECTIVE
This study will analyze the effect of Montelukast on animal models of radio-induced capsular contracture. Therefore, this study aimed to analyze the effect of the antileukotriene drug (Montelukast ® ) on capsular contracture formation in rats with silicone implants who underwent radiotherapy within two months.

INTRODUCTION
Breast implants are routinely used in cosmetic and restorative breast plastic surgery; according to the American Society of Plastic Surgery, in 2016, 290,467 implants for aesthetic purposes and 109,256 implants for repair procedures 1 . Although widely used, there is a risk of capsular contracture in 10.6% of operated cases 2 . However, several patients should undergo radiotherapy as local breast cancer treatment when performing reconstructive surgery, such as breast reconstruction. In these cases, the prevalence of this complication is greater than 40%, and the need for removal of the prosthesis is 15% 2 .

Surgical procedure
The animals were anesthetized by a solution composed of 100mg/kg of weight of ketamine hydrochloride (Ketamin ® , Cristália, Brazil) and 10mg/ kg of xylazine hydrochloride (Rompun ® , Bayer, Brazil) by intraperitoneal route.
After intraperitoneal anesthesia, the animals were positioned in ventral decubitus. Trichotomy was then performed in an area of 6cm 2 in the region of the midline of the spine just below the scapulae. Topical chlorhexidine was used for antisepsis in this region.
A 2cm horizontal incision was performed using a 15-inch blade scalpel. Pocket dissection of 6 cm 2 below the plane of the panniculus carnosus of the animal and hemostasis with an electric scalpel. Before performing the closing of the pocket was implanted a 2x2 cm silicone block. And then the animals were randomized into two groups: control (n=10), cream injection around the silicone block using 1ml of 0.09% saline solution; antileukotriene group (n=10), tissue injection around the silicone block using 1ml of antileukotriene (Singulair ® , Merck Sharp &Dohme, Campinas, Brazil) diluted in 0.9% saline solution.

Radiotherapy
All animals were submitted in the immediate postoperative period to a radiotherapy session. The radiation was made a specific irradiator for small animals (Rad Source RS2000, Quastar™), with an X-ray of 160KV of 25mAs.
The animals were positioned in lateral decubitus contained by an immobilizer that allowed the emission of rays in the tangential plane as used in patients. A 5mm lead plate was also inserted that had a window for the irradiation of the animal, ensuring treatment only in the desired region. A single dose of 10Gy was used in two opposite fields (5Gy per field) with a 6Gy/min card.

Histological analysis
After two months, the animals were euthanized by intraperitoneal injection of ketamine hydrochloride (Ketamin ® , Cristália, Brazil) at a dose of 180mg/kg of weight, associated with xylazine (Rompun ® , Bayer, Brazil) at a dose of 15mg/kg and then placed in a 100% CO chamber.
Samples of the capsule were collected around the silicone block through an incision in the back of the animals. Part of the samples was fixed with formalin 4% for 24h and then colored with hematoxylin-eosin (HE) and picrosirius for microscopy analysis (Nikon eclipse E600 ® , Japan) in magnification of 200 and 400 times.
The hematoxylin-eosin-stained slides analyzed the presence of inflammatory cells and vascular density (arteriole count) in 10 fields along the histological sections. The slides stained by the picrosirius method graphically demonstrated collagen deposition along with the capsule around the silicone block.

Total RNA extraction
Panniculus carnosus samples were macerated using the Tissue Lyser LT device (Qiagen, Germantown, USA). 1.0ml Trizol® (Invitrogen-Life Technologies, Carlsbad, USA) and stainless-steel beads were added to microcentrifuge tubes with samples. Fragmentation was performed for 6 minutes at 50Hz.
After the removal of the spheres, 0.2 ml of chloroform (Merck) was added. The samples were centrifuged for 15 minutes at 12,000rpm at 4ºC. After centrifugation, the aqueous phase was transferred to a new microcentrifuge tube, and 0.5ml of icy isopropyl alcohol (Merck) was added for RNA precipitation. The samples were incubated at room temperature for 10 minutes and then centrifuged at 12,000rpm for 10 minutes at 4ºC. The supernatant was discarded, and the RNA precipitate was washed with 1.0ml of 75% ethanol. The RNA pellet was resuspended in 50 to 100μL of sterile ultrapure water free of DNase/RNase (Invitrogen-Life Technologies, Carlsbad, USA).
The concentration of the extracted RNAs was determined in a NanoDrop™ ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, USA). The degree of purity was evaluated using the 260/280nm ratio, using only RNAs whose ratio was ≥1.8. To analyze the integrity of the RNAs, agarose gel electrophoresis was performed to verify the 28S and 18S bands. The extracted RNAs were stored at -80°C until use.

cDNA Synthesis
For cDNA synthesis from the total RNA, the High Capacity RNA-to-cDNA (AppliedBiosystems) kit was used in GeneAmp 2400 thermocycler (Applied Biosystems). In the final volume of 20μL: 1.0μL of enzyme mix; 10.0μL of RT buffer; 20μL q.s. of sterile ultrapure water free of DNase/RNase and total RNA (500ng). For reaction and reaction stoppage, the tubes were incubated at 37ºC for 60 minutes and 95ºC for 5 minutes, respectively. cDNA samples were stored at -20ºC until use.

Real-time polymerase chain reaction (qRT-PCR)
The analysis of the expression of the mRNA levels of the genes of interest was performed by qRT-PCR in the StepOnePlus TM (AppliedBiosystems) thermocycler with the TaqMan ® Gene Expression Assays (Applied Biosystems) system.
QRT-PCR was performed in duplicate for each sample using: 10.0μL TaqMan® Universal Master Mix II 2X; 1μL TaqMan® Gene Expression Assay 20X and 4μL of diluted cDNA (dilution 1:5) in the final volume of 20μL, in plates of 96 wells covered with an optical sealant.
The reaction conditions were 50ºC for 2 minutes, 95 for 10 minutes, followed by 40 cycles at 95ºC for 15 seconds and 60ºC for 1 minute.
For the calculation of the expression level of each target gene, GenEx Standard 6.1 (MultiD Analyses AB)software was used, which uses the 2-delta delta Ct method for relative quantification, where Ct (threshold cycle) is the real-time PCR cycle, in which amplification reaches the logarithmic phase, where delta Ct is the difference of expression between the target gene and endogenous control of a given sample, and delta delta Ct corresponds to the difference between the delta Ct of the sample and the delta Ct of the control.

Statistical analysis
The variables were submitted to descriptive analysis. After checking the distribution and variances of the data, the two groups were compared using the Wilcoxon ranksum test (non-normal distribution), considering an alpha p of 0.05. Stata software version 14 (StataCorp., 2015) was used. Stata Statistical Software: Release 14. College Station, TX: StataCorp LP, USA).

RESULTS
Three animals in the control group and one in the AL group presented dehiscence and extrusion of the silicone block after four weeks of irradiation.

Histological analysis
The analysis by the histomorphometry method showed no difference in the number of inflammatory cells. However, there was a decrease in vascular density in the group treated with AL when compared to the control group (55.4±30.0 (AL) vs. 81.8±26.7 (control); p=0.05) ( Table 1 and Figure 1).
The slides colored by picrosirius showed a higher amount of collagen in the control group ( Figure 2). Table 1. Analysis by histomorphometry refers to inflammatory cells and vascular density in the control and AL groups.   Analysis of gene expression of biomarkers (iNOS, VEGF-a, MMP-9) The AL group showed a lower amount of VEGF-a when compared to the control group (0.02±0.001 vs. 0.34±0.1, respectively, p=0.04) ( Table 2).

DISCUSSION
Leukotrienes are lipids (eicosanoid) belonging to the family of inflammation-mediating substances. Act on smooth muscle contraction (bronchoconstriction) and increasing microvascular permeability perpetuating the inflammatory reaction 9 .
Camargo CP et al.
www.rbcp.org.br Table 2. Gene expression of iNos, VEGF-a, ICAM-1 and MMP-9 in the control and AL groups. Antileukotrienes, therefore, have the function of reducing and preventing smooth muscle contraction and reducing vascular permeability. Although the mechanism of action has not yet been elucidated, several studies have demonstrated the beneficial effect of antileukotrienes in inhibiting or minimizing capsular contracture after breast alloplastic implants [6][7][8]10 .
Our hypothesis was to analyze the effect of Al on the local inflammatory reaction (decrease in late neoangiogenesis and decrease in inflammatory cells) and inhibition of fibrosis formation. To enhance the results of this study and to pet the clinical situation of treatment adjunct to breast cancer reconstruction, all animals received a dose of irradiation immediately after surgery.
This study demonstrated a 50% decrease in inflammatory cell count in the Al group (4.4±4.0) compared to the control group (8.25±5.9). Although it did not reach significance, there is a tendency to observe less inflammatory reaction in the AL group. In the histological analysis regarding vascular density, this study demonstrated a lower count of neoformed vessels in the AL group when purchasing with the control group (55.4±30.0 vs. 81.8±26.7, respectively; p=0.05). These two findings suggest decreased chronic inflammation in the silicone implant capsule. Kang et al. (2015) 11 analyzed the use of Zafirlukast in the formation of silicone implant capsules in rabbits. These authors demonstrated a similar result to our study; animals treated with Al showed decreased capsular contracture after two months of follow-up. Still, Kang et al. (2015) 11 also analyzed the inflammatory reaction around the silicone implant. Again, this group demonstrated a decrease in macrophages, as presented in our study.
Still, Kang et al. (2015) 11 studied an intermediate outcome measuring the gene expression of myofibroblasts. The authors showed a decrease in the expression of the myofibroblast gene in the AL-treated group compared to the control group. Based on these literature findings, our group explored other pathways responsible for the mechanism of action of AL.
The expression of the iNOS gene was higher in the control group than in the AL group (0.013±0.01 and 0.005±0.001, respectively) (p=0.08), but in this study, this difference showed a tendency to significance. The action of nitric oxide inducible synthetase (iNOS)refers to an enzyme in the inflammatory response mediated by macrophages. And, consequently, the increase in nitric oxide (NO) produced by macrophages induces a pro-inflammatory state. Therefore, the decrease in gene expression in the AL group suggests an antiinflammatory effect on the silicone implant capsule. As demonstrated by McNeill et al. (2015) 12 . According to this study, the dissection of macrophages, according to the free radical pathway, demonstrated a decrease in the inflammatory process due to the inhibition of iNOS 11,12 . Another biomarker studied by McNeill et al. (2015) 12 was VEFG-a as a possible factor contributing to the mechanism of action to the anti-inflammatory effect of AL. In our study, the gene expression of VEGF-a decreased in the group treated with AL compared to the control group. Reflecting on this outcome, the decrease in the long-term neoangiogenesis marker (2 months) may demonstrate the transition of phenotype 1 macrophages to phenotype 2; there is the resolution of the inflammatory process 13 . Another intermediate outcome analyzed in this study was the gene expression of MMP-9, which corresponds to gelatinase metabolism (extracellular matrix component). In addition, several authors have analyzed other metalloproteinases, intending to study collagen metabolism in tissue regeneration 14,15 .
This study found no difference in gelatinase (MMP-9) between groups. However, this study presented some limitations: the anatomical structure of the mine model adopted in this study presented some differences (panniculus carnosus) concerning humans.

CONCLUSION
This study suggests that the treatment with AL decreases angiogenesis in animals submitted to silicone implants and underwent radiotherapy

COLLABORATIONS CPC
Analysis and/or data interpretation, Conception and design study, Conceptualization, Final manuscript approval, Formal Analysis, Methodology, Writing -Review & Editing.