Peritoneal Free Autologous Fat Graft for the Control of Pulmonary Air Leaks in Emphysematous Rat Lungs Cristiano F. Andrade, MD, PhD, Eduardo Fontena, MD, Paulo F. G. Cardoso, MD, PhD, Raoni B. Pereira, MD, Gustavo Grun, MD, Luiz F. Forgiarini, BS, Jos e S. Moreira, MD, PhD, and Jos e C. Felicetti, MD, PhD ˇ

Hospital de Clínicas de Porto Alegre, Thoracic Surgery Department, and Santa Casa de Miseric ordia de Porto Alegre, Porto Alegre; Division of Thoracic Surgery, Heart Institute (InCor), Hospital das Clínicas, Faculdade de Medicina da Universidade de Sao Paulo, Sao Paulo; and Hospital Ernesto Dorneles, Porto Alegre, Brazil

Background. Persistent pulmonary air leak is the most frequent complication after lung resection, resulting in an increase in postoperative morbidity and mortality. We evaluated the viability, integration, and efficacy of a free peritoneal fat graft as a method for controlling air leak in normal and emphysematous rat lungs. Methods. Sixty Wistar rats were divided into two groups: elastase-produced lung emphysema (n [ 30) and control (normal) lungs (n [ 30). Pulmonary air leak was produced by puncture of the right lower lobe, and aerostasis was attempted by means of intrapulmonary injection of autologous free peritoneal fat graft. Rats in each group (n [ 6) were randomly allocated to subgroups and were sacrificed at 7, 14, 21, 30, and 60 days. Then, lungs were removed for histology, morphometry, vessel identification and counting, and immunohistochemistry for caspase 3, vascular endothelial growth factor, and factor VIII.

Results. Tissue integration of the free fat grafts was found in all animals in both groups. Vessels stained with India ink inside the fat grafts were present at all assessment periods in both groups. Vascular endothelial growth factor expression was significantly higher in all periods in the emphysema group compared with normal lungs (p < 0.001). There was a significant increase in caspase 3 expression in the emphysema group at 7, 21, 30, and 60 days (p < 0.001). Factor VIII showed a significant increase (p < 0.001) at 30 and 60 days in emphysematous lungs. Conclusions. The use of free peritoneal fat graft was able to control the air leaks in normal and emphysematous rat lungs, with persisting graft viability for as long as 60 days after implantation.


ersistent pulmonary air leak (PAL) can be defined as an air leak that lasts more than 7 days [1, 2]. It is the most frequent complication after lung resection and has an impact on postoperative morbidity and mortality [2–4]. Pulmonary air leak may also lead to empyema, pulmonary infection, and eventually, adult respiratory distress syndrome. The complications deriving from PAL prolong hospitalization and increase medical costs [5, 6]. A variety of preoperative conditions are associated with postoperative PAL. The most prevalent is chronic obstructive pulmonary disease, followed by other conditions such as diabetes mellitus, upper lobectomies, and incomplete fissures. The advances in surgical technique and postoperative care allow patients with moderate to severe emphysema to be considered as surgical candidates for lung resection [3]. To minimize the risk of postoperative PAL after pulmonary resection, the aerostasis must be meticulous,

particularly for chronic obstructive pulmonary disease patients [7]. In this regard, mechanical sutures, biological glue, bovine pericardium, injection of autologous blood, and aerostatic pulmonary suture have been used [8–10]. The association of pericardial fat pad with biological adhesives has also been described as an effective method for reducing the duration of PAL postoperatively [11]. In instances where intraoperative air leak was difficult to control and the building of a pedicled graft was not deemed feasible owing to the short extent of the pericardial fat pad, we used a free fat graft that worked surprisingly well for the control of PAL. Based on such serendipitous clinical observations, we designed an animal model of standardized pulmonary injury with air leak to put to test the efficacy, tissue integration, and viability of the free peritoneal fat graft for aerostasis in both normal and emphysematous rat lungs.

Accepted for publication March 13, 2014.

Material and Methods

Address correspondence to Dr Cardoso, Division of Thoracic Surgery, Heart Institute (InCor), Hospital das Clinicas, Faculty of Medicine, University of Sao Paulo, Rua Dr Eneas de Carvalho Aguiar 44, Bloco II, 7 Andar, Sao Paulo, SP 05403-000, Brazil; e-mail: [email protected].

Experimental Model

Ó 2014 by The Society of Thoracic Surgeons Published by Elsevier Inc

(Ann Thorac Surg 2014;-:-–-) Ó 2014 by The Society of Thoracic Surgeons

A pilot study included 7 Wistar rats weighing 250 g to 300 g. They were anesthetized with intraperitoneal ketamine 0003-4975/$36.00



(50 mg/kg) and xylene (5 mg/kg), and intubated orotracheally (14G Abbocath; Abbott Laboratories, Columbia, MD). The animals were ventilated with room air with a volume-cycled ventilator (tidal volume 10 mL/kg, respiratory rate 85 breaths per minute, positive end-expiratory pressure [PEEP] 2 cmH2O [Harvard Rodent Ventilator, model 683; Harvard Apparatus, South Natick, MA). A small laparotomy was performed on the animals to extract a fragment of the omentum. The incision was closed with monofilament nonabsorbable suture. The animals were placed in left lateral decubitus position, and a right posterolateral thoracotomy on the fifth intercostal space was performed. The pulmonary ligament was released bluntly, exposing the base of the right lower lobe in which a 5 mm deep perforating wound was produced with a 14G needle. The presence of the air leak was confirmed by increasing the PEEP to 5 cmH2O (Fig 1A). The free fat graft was suctioned into the 14G catheter attached to a 1 mL syringe and injected into the pulmonary perforation previously created until a complete obliteration of the pulmonary wound was obtained (Fig 1B). The volume used of the free autologous fat graft was approximately 0.1 mL in each animal. Once the aerostatic effect of the free fat patch in the perforated area was verified by expanding the lung with 5 cmH2O PEEP, the chest was closed. Postoperative analgesia was given using buprenorphine (0.05 mg/kg intramuscularly), and 0.5 mL 1% lidocaine was administered subcutaneously around the incision site. All animals were given cefazolin preoperatively (15 mg/kg intramuscularly). The animals were humanely killed at different time points as described below. All animals received care according to the “Guide for the Care and Use of Laboratory Animals” (National Institutes of Health Publication 85-23, revised 1996).

Study Groups Animals were separated into two groups (emphysema and normal; n ¼ 30 each). Animals in the emphysema group received an intratracheal dose of 10 U porcine pancreatic elastase (Sigma-Aldrich, St. Louis, MO) diluted in 1 mL saline, whereas the control animals received the same volume of saline solution. Animals from both groups were observed for 30 consecutive days. After the procedure, animals in both groups Fig 1. Diaphragmatic aspect of the emphysematous right lower lobe inflated (positive end-expiratory pressure [PEEP] 5 cmH2O): (A) penetrating lung injury shows the air leak (arrow); (B) the same lobe with the fat graft inserted into the lung parenchyma, showing no air leak(arrow).

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were randomly divided into five subgroups (n ¼ 6 each) according to the time of sacrifice (7, 14, 21, 30, and 60 days, respectively). At the end of the observation period, animals were anesthetized, intubated, and mechanically ventilated in the same fashion as described previously. A median sternotomy was performed, and the right lung with free fat grafts was examined macroscopically. The pulmonary artery was cannulated through the right ventricle, the aorta was clamped, and the left atrium opened. The lungs were perfused with 20 mL saline at a 25 cmH2O pressure obtained by elevation of the perfusion solution. To verify the permeability of blood vessels in the graft, 3 animals in each subgroup received an additional 4 mL India ink through the pulmonary artery at the same infusion pressure. The heart-lung block was extracted, fixed in 10% formalin for 24 hours, embedded in paraffin, and sliced (5 mm) in a microtome (Leitz 1512; Ernst Leitz, Vienna, Austria). Slides were prepared with hematoxylin and eosin staining for histology and morphometry analysis.

Morphometry To quantify pulmonary emphysema we randomly selected 2 animals of each subgroup (n ¼ 12) from both groups. The morphometric analysis was performed using 25 random pictures taken in 100 high-power field. These pictures were adjusted to an axis of X-Y coordinates to count the septum pierced by the axis. This method helps establish the average values in which interalveolar septa intersect a series of grid lines.

Vessel Counting For confirming the presence of vessels within the free fat grafts, sections were performed from the slides stained with hematoxylin and eosin. Two blinded observers analyzed 10 fields randomly in the 6 animals of each subgroup (400 high-power field). The total number of counted vessels was represented as an average of each subgroup. Clusters of capillaries were counted as a single vessel.

Immunohistochemistry Each lung section was deparaffinized with xylene and rehydrated with graded alcohols. The sections were retrieved in a microwave oven (300 W) in citrate buffer (pH 6.0) for 10 minutes at 100 C and then incubated in

Ann Thorac Surg 2014;-:-–-

absolute methanol containing 3% hydrogen peroxide for 10 minutes at room temperature. The sections were sequentially preincubated with 10% normal rabbit serum for 10 minutes at room temperature and were then incubated with antibody for vascular endothelial growth factor (VEGF), factor VIII (Santa Cruz Biotechnology, Santa Cruz, CA), and caspase 3 (Cell Signaling Technology, Beverly, MA). After 120 minutes at room temperature, they were treated with EnVision reagent (Dako, Glostrup, Denmark) for 30 minutes. After incubation, they were washed three times with phosphate-buffered saline, and 3,39-diaminobenzidine was employed as a chromogen. The nuclei were lightly counterstained with hematoxylin solution. The primary antibodies were replaced with phosphate-buffered saline containing 0.1% bovine serum albumin as a negative control.

Immunohistochemical Scoring Semiquantitative analysis was carried out using a Nikon Labophot optical microscope (Nikon, Tokyo, Japan). Percentage of expression in stained pixels was determined by histomorphometric analysis using a digital camera connected to an image capture board of a computer running Adobe Photoshop CS3 Extended version 10.0 software (Adobe Systems, San Jose, CA). From each slide, 10 images were captured from randomly selected high-power fields. Every image exhibited on the monitor was adjusted to the same threshold level, and the area of the structures was measured in pixels. The total number of pixels per image remained constant in all fields. The percentage of expression in stained pixels in each image was then calculated using the ratio of the stained pixels area to the total amount of pixels per image. The stained pixels were selected using the software and were calibrated with a positive control. A pathologist blinded to the study groups performed the histologic examination.

Statistical Analysis The statistical analysis was performed using SPSS version 19 (IBM Corporation, Armonk, NY). The Student t test was used for comparison between groups and two-way analysis of variance for within and between group analyses. Multiple comparison adjustment was performed using the Bonferroni test. The results are presented as mean and standard deviation of the mean, and significance is defined as p less than 0.05. Correlation was tested



using the Pearson correlation coefficient at a p less than 0.01 level of significance.

Results Overall perioperative mortality was 3 animals per group. The animals were replaced to keep 6 animals per group. All animals survived the observation period. Tissue integration of the free autologous fat graft was observed in all animals in both groups at all times. There was a significant reduction in the number of septa in the emphysema group (p < 0.001). All animals from the different groups at all different times showed vessels stained with India ink inside the fat graft (Fig 2). There were no significant differences in the number of vessels in the fat graft in both groups when comparing at 7 days (control 9.25  4.5 vessels; emphysema 6.17  2.52) and 60 days (control 8.42  2.84 vessels; emphysema 5.83  2.69) after implantation. The findings for VEGF, caspase 3, and factor VIII are summarized in Table 1. The VEGF was present in all the free fat grafts at all time periods. Expression of VEGF was significantly higher at all times in the emphysema lungs compared with normal lungs. At 30 and 60 days in the emphysema group, there was a significant decrease in VEGF expression compared with the earlier periods (p < 0.001). There was a steady increase in caspase 3 expression in the emphysema group over time, and it was significantly higher than in the control group at 7, 21, 30, and 60 days (p < 0.001). The immunohistochemical analysis of factor VIII for the assessment of neovascularization showed a significant increase in vascular density at 30 and 60 days after free fat graft implantation in emphysematous lungs compared with the other subgroups. A positive correlation was found between the increase in caspase 3 and factor VIII in the emphysema lungs (p < 0,001). The decrease in VEGF expression observed at 30 and 60 days correlated inversely with the rise in both caspase 3 (p < 0,001) and factor VIII (p < 0,001) in emphysema lungs (Fig 3).

Comment This study shows the integration and viability of free fat grafts into the rat lung parenchyma for as long as 60 days after its implantation. Fig 2. Emphysematous rat lungs showing the fat graft vessels stained with India ink (arrows) at (A) 7 days and at (B) 60 days. (Hematoxylin and eosin: 100 high-power field.)

Factor VIII

Caspase 3

VEGF ¼ vascular endothelial growth factor.

0.29 0.82 1.03 1.29 3.19 2.86 Control Emphysema Control Emphysema Control Emphysema VEGF

12.59 39.25 12.66 16.69 34.52 33.48

Group (n ¼ 6) Variable

Values are mean  SD of stained pixels. Vascular endothelial growth factor (VEGF) expression was higher in the emphysema group than the control at all times and decreased after 30 and 60 days in the emphysema group (p < 0.001) although it remained higher than controls. Caspase 3 expression increased significantly at 7, 21, 30, and 60 days in comparison with control lungs (p < 0.001). Factor VIII expression significantly increased at 30 and 60 days in the emphysematous lungs when compared with all time periods of normal lungs (n ¼ 6 animals per group).

Peritoneal free autologous fat graft for the control of pulmonary air leaks in emphysematous rat lungs.

Persistent pulmonary air leak is the most frequent complication after lung resection, resulting in an increase in postoperative morbidity and mortalit...
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