This study was carried out in the Scientific Research Center of Fırat University Hospital, Elazığ, Turkey, from February to March 2009. All of the study protocols were approved by the Fırat University Ethics Committee on Animal Research. The animals were cared for in accordance with the Principles of Laboratory Animal Care of the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals formulated by the National Academy of Sciences (NIH publication 85-23, revised 1996).
Ten adult male New Zealand rabbits (aged 105 to 135 days, and weighing from 2800 to 3500g) were enrolled into this study. The animals were randomly divided into two groups of five each. The animals were kept in a wire top cage with free access to tap water. They were administered with standart food. Preoperatively each rabbit was kept off feed for a period of three hours before the induction of the anesthesia.
General anesthesia was obtained by intramuscular injection of 90mg/kg ketamine (Ketalar®, Pfizer, Turkey) and 10mg/kg Xylazine hydrochloride (Rompun®, Bayer, Turkey). Preoperatively the animals were given 50000 U/kg of intramuscular penicillin (Iecilline®, IE Ulagay, Turkey) as a primary prophylaxis to prevent any surgical infection. A line was maintained in one of the dorsal veins of the ear lobe through which mixture of serum solutions were passed if necessary. All the animals were operated under aseptic conditions, using spontaneous ventilation without an endotracheal tube. All the operations were performed by the same surgeon. Once asleep, the rabbit was fixed on a small table in supine position. The cervical area was shaved with a standart animal clipper. Then the surgical field was disinfected with povidone-iodine solution and draped in a standart fashion. A cervical longitudinal incision was performed, and after a thorough dissection of the subcutaneous and muscular planes, the trachea was exposed. Then the trachea was dissected and a segmental tracheal resection (5, 6, and 7th tracheal rings) was performed (Figure 1, and Figure 2).
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Figure 2: The view of the trachea after performing a three-segmental resection. |
End-to-end anastomoses were performed by using separated 5/0 silk sutures (Sterisilk®, SSM, Turkey) with full-thickness interrupted stitches all around the circumferential tracheal ring to the animals in the group I (Figure 3). The resected tracheal segments were used as fresh devascularized allografts. The excised allografts were immersed in a normal saline solution for not more than one hour, then they were transplanted to the animals in the group II. Anastomoses were performed by using the same suture material (Figure 4). Following an air-leak control using water, the incisions were closed with usual surgical methods.
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Figure 3: The end-to-end anastomosis procedure performed for the group I rabbits. |
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Figure 4: The transplantation procedure of the tracheal allograft for the group II rabbits. |
Tramodol 1 mg/kg (Contramal®, Abdi Ibrahim, Turkey) and penicillin 50000 U/kg (Iecilline®, IE Ulagay, Turkey) were administered intramuscularly twice per day during seven postoperative days. The rabbits were housed in separated cages and were given a normal diet. Food and water intake and weights of the animals were monitored and recorded daily. The animals were sacrificed on the planned day (20th postoperative day) with an intraperitoneal injection of 100 mg/kg sodium penthotal (Thiopental sodium®, IE Ulagay, Turkey).
Tracheal specimens including the entire anastomosis site were removed. Each trachea was resected one centimeter far from the anastomotic line. The average measurement of the calibre of the trachea in each rabbit was calculated, measuring the lateral (a) and anteroposterior (b) diameters at each anastomotic level, and (c) the average measurement of 5mm proximal and distally away from the anastomoses (normal level). The cross-sectional area (CSA) was calculated as described (8): CSA= (a/2) x (b/2) x Π. The CSA measurement in the anastomotic level was called “anastomotic CSA”, and the average of the CSA measured in the proximal and distal levels was called “normal CSA”. Every “anastomotic CSA”, and “normal CSA” values were compared in each group and the severity of the stenosis was noted according to the measurements of Hsieh et al (9): Very good: CSA 75-100%, Mild stenosis: CSA 50-74%, Important stenosis: CSA 30-49%, Critical stenosis: CSA 0-29%.
After the measurement, resected tracheal segments were taken to the Pathology Department of the Medical Faculty for further histopathological investigation. They were fixed with phormol at 10% and preserved in paraffin. In the macroscopic evaluation, any separation in the suture line, secretions in the luminal area, visible stenosis, and inflammatory changes were noted. Serial cuts were performed, stained with hematoxylin-eosin and viewed under a light microscope (NikonU-III multi-point sensor system, Japan), examining the wall integrity, the degree of inflammation in the mucosa, fibrosis, chondrogenesis, and foreign body reaction.
T-test was used for the comparison of numerical variables and the Fisher's exact test for the comparison of categorical variables. A p value of less than 0.05 was considered statistically significant.
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