Acceptable Warm Ischemia Time of Tracheal Grafts From Non–heart-beating Donors in Rats

Acceptable Warm Ischemia Time of Tracheal Grafts From Non– heart-beating Donors in Rats J.-Q. Han, K. Zhang, J. Cui, C. Liu, G.-B. Zhao and Y.-Z. Xin ABSTRACT Objectives. Warm ischemia (WI)-induced airway complications are common in clinical lung transplantation. However, the acceptable WI time of tracheal grafts from non– heartbeating donors (NHBDs) is unknown. The purpose of this study was to determine the acceptable WI time by observing tracheal epithelial regeneration among NHBD. Method. Forty-eight rats were randomly divided into four groups (each with 12 rats): WI-0 minutes (group A), WI-30 minutes (group B), WI-45 minutes (group C), and WI-60 minutes (group D). In each group, the tracheas from 6 rats were imbedded in the greater omentum of 6 other rats. Fourteen days later, the transplanted trachea was obtained from the recipient to evaluate epithelial thickness and regeneration. Six tracheas were obtained from living donors as a control group. Results. There were no significant differences in tracheal transplantation time (mean, 17.66 ⫾ 1.21 minutes). There were no significant differences in epithelial thickness and regeneration between the controls and groups A, B, and C (P ⬍ .05). Group D showed no normal epithelial structure of the trachea only with monolayer cells. Conclusions. The time limits of tolerance to WI of tracheal grafts from NHBDs may be 45 minutes. UNG transplantations from non– heart-beating donors (NHBDs) has been most widely studied in the last decade. Experimental studies have shown the acceptable warm ischemia (WI) of the lung to be at least 1 hour. With increasing experience in animal models, NHBDs have been used in clinical practice.1,2 To date, two groups in Spain have reported the use of uncontrolled donors, concluding that uncontrolled NHBDs represent a promising complementary source of lung donors for clinical transplantation.3 Recently, category III NHBD lungs account for 20% to 35% of patients in some lung transplant centers.4 Successful transplantation of lungs from NHBDs is based on the fact that lungs may remain viable after donor death. Respiration of lung parenchymal cells is achieved by diffusion from the air spaces.5 Airway ischemia is considered to be the most important factor in the development of bronchial complications. With improvements in organ preservation, surgical techniques, postoperative management, and immunosuppression, the incidence of anastomotic complications after lung transplantation has decreased to between 2.6% and 23.8%.6 Bronchial healing is one of the concerns in NHBD lung transplantation. If bronchi can normally heal, tracheal

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epithelia must have a tolerance to WI. Therefore, we sought to evaluate the acceptable WI time of tracheal grafts from NHBDs by observing epithelial regeneration of heterotopically placed trachea. MATERIALS AND METHODS All study methods were approved by our ethics committee. Fiftyfour syngeneic male Sprague-Dawley rats weighing 250 to 300 grams were used in this study. The 48 rats were divided into four groups, each with 12 rats according to the WI time. A control group, of 6 fresh tracheas obtained from living donors was compared with the other groups. The other groups of 6 tracheas each From the Department of Thoracic Surgery, the Fourth Affiliated Hospital of Harbin Medical University, Harbin, China. Supported by grants from academic leaders in Harbin (No.2011RFXYS077). The project: Study on type II alveolar epithelial cell apoptosis in non-heart-beating donor lung transplantation. Address reprint requests to Kai Zhang, Department of Thoracic Surgery, the Fourth Affiliated Hospital of Harbin Medical University, Harbin 150001, China. E-mail: yixuelunwenfuwu@ 126.com

0041-1345/11/$–see front matter doi:10.1016/j.transproceed.2011.09.070

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Transplantation Proceedings, 43, 3638 –3642 (2011)

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Recipient Preparation and Trachea Implantation Recipient rats were anesthetized in an induction chamber containing halothane followed by continuous anesthesia with 2% to 3% halothane in oxygen. After disinfection, the omentum was brought out through a 2-cm upper midline abdominal incision, and the stretched trachea imbedded in the greater omentum with fixation using two 6 – 0 Prolene stitches. The peritoneum and abdominal musculature were closed with 5– 0 Prolene interrupted sutures, the skin was closed with surgery stitch 2. After recovery from anesthesia, the caged rats were fed ad libitum with a standard diet and water.

Transplanted Tracheas Obtained from Recipient Rats After 14 days, recipients were anesthetized in an induction chamber containing halothane (3%), followed by continuous anesthesia with halothane (2% to 3%) in oxygen via a lateral abdominal incision we obtained the transplanted trachea for fixation in 4% paraform. The recipients were then exsanguinated.

Assessment Fig 1. Epithelial thickness in heterotopic tracheal isografts with different warm ischemia durations or without warm ischemia. Data are presented as mean ⫾ standard deviation. (Control: non-transplanted fresh tracheas; column A: 0-minute warm ischemia group; column B: 30-minute warm ischemia group; column C: 45-minute warm ischemia group; column D: 60minute warm ischemia group; * P ⬍.05 when compared with control group.) were obtained from 6 donors at room temperature (approximately 22 °C) and transplanted into the great omentum of 6 other recipients at 0 (group A), 30 (group B), 45 (group C) or 60 minutes (group D).

We sought to quantitate the viability of the heterotopic isografted trachea by measuring thickness and evaluating regeneration of the epithelium. The 3-␮m thick longitudinal sections cut beginning from the middle part of the transplanted tracheas were stained with hematoxylin and cosin. Eight sections including 4 from the cartilage rings and 4 from the tissue between them were randomly selected from each transplanted trachea. Epithelial regeneration was evaluated by a pathologist who was blinded to the experimental group. Along the longitudinal axis of each transplanted trachea. We randomly selected (8 samples with a length of 300 ␮m including 4 from cartilage rings and 4 from the tissue between them.

Epithelial Thickness In the 8 samples, we measured the length and area of epithelium as well as the thickness (thickness ⫽ area/length). The morphometry

Tracheas Obtained from Heart-Beating Donors in Group A Six rats were anesthetized by intraperitoneal injection of pentobarbital sodium (40 mg/kg) and heparin sodium (500 IU/kg). The upper trachea was exposed through a midline nuechal incision. Median sternotomy was performed to remove the thymus gland and to separate the right anterior vena cava, innominate artery, and aortic arch. The distal area of the trachea was dissected to its bifurcation. After both main bronchi were divided, the lower trachea was bluntly extracted after careful separation between the trachea and the esophagus. A 1.5-cm length of approximately 10 cartilaginous rings was excised to be flushed with 10 mL of isotonic saline solution. The trachea was then stretched to its original length on a metal approximately (U shaped, no. 4 surgical stainless steel wire, 22 gauges; Shanghai Puwei Medical Instrument, Shanghai, China). The tracheas were stored in wet gauze for 2 to 3 minutes during preparation of the recipient animals.

Tracheas Obtained from NHBDs in Group’s B, C, AND D Eighteen rats were sacrificed by intravenous injection of sodium pentobarbital (60 mg/kg), and administered intravenous heparin sodium (500 IU/kg) before cardiac arrest. Donors were placed in a supine position, fixed to a table. After 0 (group A), 30 (group B), 45 (group C) or 60 minutes (group D), the tracheas were obtained as previously described.

Fig 2. Epithelial regeneration score in heterotopic tracheal isografts with different warm ischemia-durations or without warm ischemia. Data are presented as mean ⫾ standard deviation. (*P ⬍.05 compared with control group).

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Fig 3. Tracheal epithelium between cartilaginous rings. Structurally normal, mucocilliary epithelium are seen from A and B. C is characterized by multilayered epithelia with more secretory cells and less ciliated cells. The epithelium is made up of several layers of nucleated cells but cilia are not present in D. (Control: nontransplanted fresh tracheas; A: 0-minute warm ischemia group; B: 30-minute warm ischemia group; C: 45-minute warm ischemia group; D: 60-minute warm ischemia group; original magnification ⫻ 200.) was evaluated by pathological image analysis software (HPIAS100). The mean of 8 thickness values served as the thickness of each transplanted trachea.

we performed selected comparisons between WI and control groups. Statistical significance was established at P ⬍ .05.

Epithelial Regeneration

RESULTS

The epithelium was assessed according to the established grading system 7: 0, no epithelium only single non-confluent epithelial cells; 1, confluent single-layered nonciliated epithelium; 2 confluent multilayered nonciliated epithelium and 3 normal mucociliary epithelium.

Twenty-four rats underwent tracheal implantation with an operative survival of 100%. Two rats (one from group C and another from group D) experienced postoperative infections requiring exclusion from further histopathologic assessment. The epithelial thickness in each group is shown in Fig 1. The thickness of tracheal epithelium was significantly less among group D than the controls (P ⬍ .05). Epithelial thickness was not significantly different between the controls and groups A and B. Although there was a

Statistical Analysis Data are expressed as mean values ⫾ standard deviations. The statistical analysis was performed with SPSS 13.0. If an overall F test was significant upon one-way analysis of variance (ANOVA),

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Fig 4. Tracheal epithelium of cartilaginous part. Mucosal areas underlying cartilage rings seem to have significantly lower epithelial regeneration scores and epithelial thickness than inter-cartilage areas. Only a monolayer of epithelial cells is present in D. (Control: nontransplanted fresh tracheas; A: 0-minute warm ischemia group; B: 30-minute warm ischemia group; C: 45-minute warm ischemia group; D: 60-minute warm ischemia group; original magnification ⫻ 200.)

trend toward thinner epithelium in group C compared with the controls, the difference was not significant. The epithelial regeneration scores in each group are shown in Fig 2. The regeneration score was decreased with the extension of WI time. The regeneration scores were similar in groups A and B. There were no significant differences in regeneration scores between the controls and group C. However, the transplanted tracheas among group C were characterized by multilayered epithelia with more secretory cells and fewer ciliated cells (Fig 3C). Figure 4D shows almost complete destruction of the epithelium among those in group D. Mucosal areas underlying carti-

lage rings seemed to show lower epithelial regeneration scores and epithelial thickness than the intercartilage areas (Fig 4). DISCUSSION

The widespread application of lung transplantation is limited by the shortage of suitable donor organs. The use of NHBDs may resolve the problem. However, the use of lungs from uncontrolled NHBDs is limited by the short tolerable WI time and the deleterious effects of WI on bronchial healing. The lung is the only organ where the

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arterial blood supply is not restored routinely during engraftment. The donor airway depends on collateral connections between the pulmonary and bronchial circulations of the transplanted lung until there is adequate bronchial revascularization8. Egan et al1 have shown the possibility to use canine lungs from NHBDs after 1 hour of WI time for single-lung transplantation. In a swine model, Loche et al2 reported excellent gas exchange at 5 hours after transplantation when the lungs were retrieved from NHBDs at 90 minutes after death. However, these studies did not evaluate the long-term effects of WI on bronchial healing. Binns et al9 noted a higher incidence of anastomotic necrosis at 21 days after 1-hour WI of lung transplantations. In contrast, another study 10 did not observe differences in bronchial healing at 5 weeks after NHBD of single-lung transplantations in rats. A series of human NHBD lung transplantations reported no bronchial complications except fungal and Gram-negative infections.3 Therefore it is necessary to determine the acceptable WI time of tracheal grafts from NHBDs. In this study, all group C, grafts were viable showing no significant difference in viability compared with the controls. We concluded that the maximum allowable WI time of tracheas was 45 minutes. This finding was consistent with the observations of Binns et al,9 who reported good bronchial healing in 30-minute WI lungs but poor healing in 60-minute WI lungs. Impaired bronchial healing is consistent with ischemia injury.11 Mucosal areas underlying cartilage rings seemed to show lower tracheal viability in all of our group suggesting that intercartilaginous tissue has good revascularization. However, in our study, tracheas were studied at 14 days after transplantation. Perhaps, we identified the viability of the donor trachea early, but these findings could also represent an allowable tolerance of WI for adequate bronchial healing. Because tracheal cartilage tolerates longer WI,12 we did not evaluate its viability. Further lung transplantation experiments in large animals will be necessary to confirm the maximum allowable tolerance of warm ischemia for bronchial healing. The native anatomic variations in collateral blood flow from the pulmonary circulation play an important role in bronchial healing. However, the rat heterotopic tracheal isograft models allowed evaluation of tracheal viability without the influence of retrograde mucosal blood flow from the pulmonary circulation. The model was technically simple, reproducible, and inexpensive.

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In conclusion, WI can reduce the viability of the tracheal grafts. Epithelial regeneration of 45-minute WI tracheal grafts was not significantly worse than that of nonischemic tracheal grafts. We therefore concluded that to ensure adequate bronchial healing, lungs from NHBDs must be obtained within 45-minutes WI. It is necessary to investigate whether 45-minute WI lungs from large animal NHBDs show normal bronchial healing. ACKNOWLEDGMENT The authors thank Juan Wang for her help with the statistical analysis.

REFERENCES 1. Egan TM, Lambert Cj Jr, Reddich R: A strategy to increase the donor pool: the use of cadaver lungs for transplantation. Ann Thorac Surg 52:1113, 1991 2. Loehe F, Mueller C, Annecke T, et al: Pulmonary graft function after long-term preservation of non-heart-beatingdonors lungs. Ann Thorac Surg 69:1556, 2000 3. De Antonio DG, Marcos R, Laporta R, et al: Results of clinical lung transplant from uncontrolled non-heart-beating donors. J heart Lung Transplant 26:529, 2007 4. De Vleeschauwer S, Van Raemdonck D, Vanaudenaerde B, et al: Early outcome after lung transplantation fromnon-heartbeating donors is comparable to heart-beating donors. J Heart Lung Transplant 28:380, 2009 5. Van Raemdonck DEM, Rega FR, Neyrinck AP, et al: Nonheart-beating donors. Semin Thorac Cardiovasc Surg 16:309, 2004 6. Ruttmann E, Ulmer H, Marchese M, et al: Evaluation of factors damaging the bronchial wall in lung transplantation. J Heart Lung Transplant 24:275, 2005 7. Myer E, Cardoso PFG, Puskas JD, et al: The effect of basic fibroblast growth factor and omentopexy on revascularization and epithelial regeneration of heterotopic rat tracheal isografts. J Thorac Cardiovasc Surg 104:180, 1992 8. Ramirez J, Patterson GA: Airway complications after lung transplantation. Semin Thorac Cardiovasc Surg 4:147, 1992 9. Binns OA, DeLima NF, Buchanan SA, et al: Impaired bronchial healing after lung donation from non-heart-beating donors. J Heart Lung Transplant 15:1084, 1996 10. Wierup P, Anderson C, Janciauskas D, et al: Bronchial healing, lung parenchymal histology, and blood gases one month after transplantation of lung topically cooled for 2 hours in the non-heart-beating cadaver. J Heart Lung Transplant 19:270, 2000 11. Buchanan SA, DeLima NF, Binns OA, et al: Pulmonary function after non-heart-beating lung donation in a survival model. Ann Thorac Surg 60:38, 1995 12. Kushibe K, Tojo T, Sakaguchi H, et al: Assessment of cartilage viability in the cryopreserved tracheal allograft by measurement of Na(2)(35)SO(4) incorporation. Transplant Proc 32: 1655, 2000