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Ann Thorac Surg 1999;68:1165-1170
© 1999 The Society of Thoracic Surgeons

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Original Articles

Do mature pulmonary lobes grow after transplantation into an immature recipient?

^a Department of Thoracic and Cardiovascular Surgery, Hannover Medical School, Hannover, Germany

Address reprint requests to Dr Schäfers, Department of Thoracic and Cardiovascular Surgery, University Hospitals Homburg, Kirrberger Str, 66424 Homburg/Saar, Germany
e-mail: chhjsc{at}med-rz.uni-sb.de

	Abstract

Top Abstract Introduction Material and methods Results Comment References

 
Background. The use of reduced-size adult lung transplants could help solve the profound pediatric donor lung shortage. However, adequate long-term function of the mature grafts requires growth in proportion to the recipient’s development.

Methods. Mature left lower lobes from adult mini-pigs (age: 7 months; mean body weight: 30 kg) were transplanted into 14-week-old piglets (mean body weight: 15 kg). By the end of the 14-week holding period, lungs of the recipients (n = 4) were harvested. After volumetric measurements, the lung morphology was studied using light microscopy, scanning, and transmission electron microscopy. Changes of alveolar airspace volume were determined using a computer aided image analysis system. Comparisons were made to age- and weight-matched controls.

Results. Volumetric studies showed no significant differences (p = 0.49) between the specific volume (mL/kg body weight) of lobar grafts and left lower lobes of adult controls. Morphologic studies showed marked structural differences between the grafts and the right native lungs of the recipients, with increased average alveolar diameter of the grafts. On light microscopy and scanning electron microscopy, alveoli appeared dilated and rounded compared to the normal polygonal shape in the controls. The computer generated semi-quantitative data of relative alveolar airspace volume tended to be higher in transplanted lobes.

Conclusions. The mature pulmonary lobar grafts have filled the growing left hemithorax of the developing recipient. Emphysema-like alterations of the grafts were observed without evidence of alveolar growth in the mature lobar transplants. Thus, it can be questioned whether mature pulmonary grafts can guarantee sufficient long-term gas exchange in growing recipients.

	Introduction

Top Abstract Introduction Material and methods Results Comment References

 
A more widespread application of lung transplantation has been limited by organ availability. The use of reduced-size adult lung transplants could potentially help to solve the profound pediatric donor lung shortage. In reduced-size transplantation, the defective lung of the pediatric recipient is replaced by a lobe of an adult lung, either cadaveric or living-related [1]. Of utmost importance in pediatric lobar transplantation is whether the grafts will grow with the somatic growth of the recipient. It is well known that physiologic lung volume does increase. There are, however, no exact data elucidating the mechanism of increased lung volume; that is, is it an increase in number of alveoli or simply an increase in alveolar size. The latter would be associated with a relative decrease in gas exchange surface area comparable to changes in emphysema.

The purpose of this experimental study was to examine whether or not an already mature lobe can grow by increasing the number of alveoli following transplantation into a developing recipient.

	Material and methods

Top Abstract Introduction Material and methods Results Comment References

 
All experiments were conducted with inbred female miniature pigs (Laboratories Dassel-Relliehausen, Relliehausen, Germany). Following left-sided pneumonectomy 6 miniature piglets (weight: 15 ± 2 kg; age: 14 weeks) received a left lower lobe transplant from adult miniature pig donors (weight: 30 ± 2 kg; age: 30 weeks)[2]. There were two weight- and age-matched control groups (piglet, adult, n = 4). All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Academy of Sciences and published by the National Institutes of Health (NIH publication No. 86-23, revised 1985).

Anesthesia and ventilation
After intramuscular premedication of azaperone (10 mg/kg) and atropine (0.025 mg/kg) the animals were intubated. The external jugular vein and the carotid artery were cannulated on the left side. The cannulas were used for the central venous application of drugs and intraoperative blood pressure monitoring. Metomidathydrochloride (25 mg/kg), pancuronium (0.1 mg/kg), and fentanyl (0.01 mg/kg) were used for induction of narcosis. For maintenance of intravenous anesthesia metomidathydrochloride (5 mg/kg), pancuronium (0.025 mg/kg), and fentanyl (0.0025 mg/kg) were given every 30 minutes.

The animals were ventilated mechanically (Bennett MA-1B; Nellcor Puritan Bennett/Mallinckrodt, St. Louis, MO). Tidal volume was 15 mL/kg. The respiratory rate was 8–12 per minute. An inspiratory oxygen fraction of 50% was chosen.

Donor operation
After medial sternotomy and snugging of the inferior vena cava, the pericardium was incised in total length. The superior vena cava and the ascending aorta were separated. Heparin (300 IE/kg) and epoprostenol (10 µg/kg) were applied intravenously and a perfusion catheter was placed in the pulmonary trunk. After clamping, both venae cavae, the azygos vein, and the ascending aorta, the left atrial appendage was incised. The pulmonary arteries were perfused with modified Euro-Collins solution (4 °C) under a constant pressure of 30 cm H₂O. The lungs and the heart were removed en-bloc and the left lower lobes were excised.

Recipient operation
The animals underwent a left-sided lateral thoracotomy (5th intercostal space). After preparation and clamping of left pulmonary artery, pulmonary veins and main bronchus, a pneumonectomy was performed. First pulmonary veins of the lobar transplant and atrium of the recipient were anastomosed by continuous suture (5-0 Prolene, Ethicon, Somerville, NJ). The same technique was used for the pulmonary artery anastomosis. Finally, the left lower lobe bronchus was anastomosed to the left main bronchus using single absorbable sutures (6-0 PDS, Ethicon). Average ischemic time was 120 ± 15 min.

After placement of a chest drain the thoracotomy was closed in typical fashion. The central venous line was tunneled through the skin of the neck. The catheter was used for drug application in the first 10 postoperative days. The animals were extubated 4 ± 2 hours after skin closure.

Immunosuppression and animal holding
Intraoperatively intravenous triple-drug immunosuppression was given intravenously (cyclosporine 5 mg/kg, azathioprine 2 mg/kg, and methylprednisolone 12.5 mg/kg). Oxytetracycline (10 mg/kg IV) was administered for prophylaxis of infectious complications.

Postoperatively, the oral dosage of cyclosporine (~ 20 mg/kg/per day) was adjusted to trough whole blood levels (goal: 250 ng/ml). Animals received 2 mg/kg/per day azathioprine. Prednisolone (0.4 mg/kg/per day) was given in the first 4 postoperative weeks. The dosage was then tapered to 0.2 mg/kg/per day. On postoperative days 8, 9, and 10 methylprednisolone (10 mg/kg) was administered intravenously for rejection prophylaxis.

During postoperative days 1–10 doxycycline (2 mg/kg/per day) was given. The animals were held in separate boxes. They received standard feeding and had free access to water.

All 6 transplanted animals survived the perioperative period. Two recipients had to be excluded from further analysis because of death from pneumonia at 8 and 11 weeks, respectively.

Preparation and morphologic analysis
A bilateral pneumothorax was produced through an abdominal incision and perforation of both diaphragms. Reinflation and fixation of the lungs was obtained by instillation of a K-phosphate buffered glutaraldehyde solution (pH 7.40; total osmolarity 350 mOsm) through an endotracheal tube under a constant pressure of 25 cm H₂O above the tracheal level. After median sternotomy the heart and lungs were removed en-bloc. The heart was excised and the lungs were split into their lobes.

The volumes of the lobes were determined by means of the water displacement method according to Scherle [3]. Specimens for light microscopy (LM) and electron microscopy (EM) were cut out of the lower lobes of the intravitally fixed lungs.

The specimens for LM-analysis were embedded in paraffin blocks, slices of 4 µm thickness were cut and stained with hematoxylin-eosin. The LM analyses were carried out on a Reichert-Jung Polyvar microscope (Reichert-Jung, Vienna, Austria) using two different magnifications (15x, 60x).

The tissue blocks for scanning EM analysis [4] were dehydrated in a graded ethanol series. The blocks were immersed in liquid nitrogen and fractured in half with a razor blade. After critical point drying with CO₂ the specimens were mounted on stubs with the fracture face above and sputtered with gold to a layer thickness of 10 nm. The scanning electron micrographs were recorded on a Philips SEM 500 at 25 kV (Philips AG, Eindhoven, The Netherlands). The magnification used was 80x and 160x.

The tissue blocks for transmission EM analysis were fixed in sodium-cacodylate buffered 1% OsO4 solution (pH 7.4; osmolarity 350 mOsm). The blocks were block-stained with uranyl-acetate. After dehydration in graded alcohol series the blocks were embedded in Epon. On a Reichert Microtome (Reichert, Vienna, Austria), sections of 80 nm thickness were obtained. Transmission electron micrographs were taken with a Philips-EM 300 (Philips AG, Eindhoven, The Netherlands) operated at 60 kV.

Semiquantitative analysis
For semiquantitative analysis of the relative alveolar airspace volume, representative portions of the LM sections were photographed on slides (24 x 36 mm) using a magnification of 10x. Sixteen observation units (0.6 x 0.6 mm; 0.36 mm² of original area on the section) of each slide were investigated using a computer-assisted image processing system (CapImage, Zeintl, Heidelberg, Germany). Parameters measured were airspace volume versus parenchymal volume in the alveolar region. We present here quantitative tendencies that correspond closely to the structural characteristics of the observed lung tissue.

Statistical analyses
Data of body weight and lobar volumes were expressed as means ± standard deviations. For statistical analysis, data were compared using Student’s t-test for paired or unpaired data when applicable. A probability value (p) equal to or less than 0.05 was taken to indicate a statistically significant difference between the values.

	Results

Top Abstract Introduction Material and methods Results Comment References

 
All animals in the study group doubled their weight in the 14-week holding period following left lower lobe transplantation; ie, the transplanted animals grew normally compared to nontransplanted animals. There was a normal increase in chest circumference of the transplanted pigs compared to controls.

The chest roentgenogram showed an expansion of the mature lobar grafts in the growing thoracic cage of all recipients (Fig 1). No mediastinal shift was seen. All lobar transplants had normal radiologic appearance at the end of the holding period.

View larger version (83K): [in this window] [in a new window]   Fig 1. Chest roentgenogram of transplanted animal No. 2. (A) (left panel), Postoperative day 27. (B) (right panel), Postoperative day 98. The lobar graft filled the left hemithorax.

View larger version (96K): [in this window] [in a new window]   Fig 2. Hematoxylin and eosin stained tissue sections. (A) Low-power view of (native) right lung parenchyma. Original magnification x36. (B) Parenchyma of left lower lobe transplant. A marked increase in alveolar airspace can be observed. Original magnification x36, bar = 500 µm.

View larger version (118K): [in this window] [in a new window]   Fig 3. Scanning electron micrographs. (A) (Native) right lung parenchyma. Original magnification x104. (B) Parenchyma of left lower lobe transplant. Note the increase in alveolar diameter. Original magnification x104, bar = 100 µm.

View larger version (76K): [in this window] [in a new window]   Fig 4. Transmission electron micrographs. (A) (Native) right lung parenchyma. Original magnification x4338. (B) Parenchyma of left lower lobe transplant. No ultrastructural alterations of air-blood barrier can be observed. Original magnification x4338, bar = 3 µm.

View larger version (19K): [in this window] [in a new window]   Fig 5. Relative airspace (%) of alveolar region. (Control = adult control; Tx = transplanted animals.)

	Comment

Top Abstract Introduction Material and methods Results Comment References

 
The most important problem limiting a more widespread application of lung transplantation, particularly in pediatric patients, is the lack of suitable lung donors. The concept of lobar transplantation is a possible solution to the problem of donor lung shortage frequently caused by a recipient to donor size disparity.

There is growing clinical experience with lobar lung transplantation, either cadaveric or living-related [5, 6] with good results in older children or adults. However, the question remains whether there will be growth of the mature lobar grafts following transplantation into an immature recipient of small size. The purpose of this experimental study was to investigate the growth potential of a mature lobe placed as an entire lung into a growing recipient.

Except for the right middle lobe all lobes are usable for lobar transplantation. However, the lower lobes, especially the left one, appear to be better suited, for anatomical reasons.

The experimental model in this study was left lower mature lobe transplantation in miniature piglets following left-sided pneumonectomy.

Radiologic and volumetric data showed that the mature lobar transplant adequately filled the growing thoracic cage of the developing recipients. Whether this lung volume increase represents dilatation of existing airspaces or growth (ie, subseptation) of alveoli was answered by morphological techniques (LM and EM).

Morphological analysis of the lobar transplants showed a marked increase in alveolar diameter. The emphysema-like alterations of the grafts may be due to passive dilatation. A potential influence of early changes consistent with obliterative bronchiolitis cannot be ruled out, even though bronchiolar histology appeared generally normal. Growth of alveoli in the mature lobar transplants could not be demonstrated by this study.

Quantitative tendencies obtained from computer assisted image analysis supported the qualitative morphologic data. We observed a striking increase of alveolar airspace volume in the mature lobar transplants compared to the native right lungs.

Finally, a transmission electron microscopy analysis of the air-blood barrier was performed. No ultrastructural changes were seen in endothelial and epithelial lining of alveoli of the left lower lobes at the end of the 14-week holding period following transplantation. Thus the structure of the air-blood barrier appeared intact in the face of immunosuppressive drugs.

From our experimental data several conclusions can be drawn. Reduced-size lung transplants in mini-pigs increase in size following unilateral transplantation into a growing recipient. This is due to an increase in the size of the alveoli but not due to an increase in number. Thus the increase in alveolar surface area in the mature lobar grafts is small following transplantation into an immature recipient.

We reviewed the literature about growth of immature and mature lungs [7, 8] and lung transplants: There are reports of growth potential of immature lung transplants in animals [9]. The data indicated normal growth of pulmonary arteries and bronchi in the transplanted whole lungs. However, growth of lung parenchyma was not examined.

In animal models, functional and morphologic studies of reduced-size mature lobar transplantation have been reported. Kern and coworkers published the results of a study about the growth potential of porcine reduced-size mature pulmonary lobar transplants [10]. They reported an increase of volume of the mature lobar grafts after transplantation into growing pigs. No significant increase in the gas exchanging area in the transplanted lobes compared to control lobes was seen. These results are consistent with the results of our own experimental study.

However, no significant differences in alveolar size and number between the transplanted and control lobes were seen. Thus the mechanism of the volume increase of the transplanted mature lobes by the end of the holding period remained unclear. This is an important difference to the results of our study, because we found hyperinflation of existing airspaces (compensatory emphysema) in the transplanted lobes with significant increase in alveolar size.

To date no data exist concerning long-term function of mature lobar grafts after transplantation into growing human recipients. We have not sufficiently answered the question whether new alveoli can form in healthy mature human lungs. The studies on pulmonary reactions to lobectomy and pneumonectomy are mostly based on lung function tests. Lung function tests showed an increase of functional residual capacity of the remaining mature lung tissue following lung resections [11]. Only in a few cases were anatomic and morphometric analyses of lung structure performed. Dunnill morphometrically examined the lung of an adult 10 years after pneumonectomy because of bronchial carcinoma [12]. He found hyperinflation of the residual lung parenchyma, which he called compensatory emphysema. All data on adaptive lung reactions have to be interpreted with caution because lung resections are not performed on healthy lungs. However, it appears more likely that there is no potential for adaptive growth in mature human lungs.

In our experimental study, investigating the growth potential of mature lobar transplants, emphysema-like alterations of graft parenchyma could be demonstrated. This experimental study (like others before) has limitations because the normally functioning contralateral lung of the transplanted animals was in place. Because dilatation of alveoli is only accompanied by a small increase of alveolar surface area compared to growth (ie, formation of new septa), it can be questioned whether mature pulmonary lobar transplants can guarantee sufficient oxygen supply over the long term for growing immature recipients. Thus, application of reduced-size lung transplantations in neonates and small children may be of only relatively short-term benefit.

	Acknowledgments

 
We thank Michael D. Menger, MD, of the Department of Experimental Surgery, University Hospitals Homburg for his assistance. Part of this work was supported by Swiss National Science Foundation Grant No. 31.45831.95.

	References

Top Abstract Introduction Material and methods Results Comment References

 

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