Eur J Cardiothorac Surg 1998;14:615-620
© 1998 Elsevier Science NL
High doses of hydrocortisone improved tracheal autograft revascularization1
Eugeniusz Jadczuk
Department of General Thoracic Surgery, Medical University of Gdansk, Gdansk, Poland
Received 29 November 1997;
received in revised form 13 May 1998;
accepted 7 July 1998.
Corresponding author. Department of General Thoracic Surgery, Medical University of Gdansk, 80-211 Gdansk ul. Debinki 7, Poland. Tel. +48-58-461194; Fax: +48-58-461194.
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Abstract
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Objective: Anti-inflammatory effects of steroid to revascularization of the tracheal autografts in pigs, with the use of pedicled muscle flaps of the abdominal rectus was evaluated. Methods: The research was done on 19 pigs. First group: eight pigs, no steroids were given. Second group: 11 pigs, a daily dose of 30 mg/kg hydrocortisone was given, starting on the day of operation. A segment of trachea ten rings long was skeletonized and excised, and reimplanted in the previous position. A muscle flap was sutured on the anterior aspect of the tracheal autograft. Results: All eight animals in the first group died from graft failure. In the second group receiving steroids, only two animals succumbed (18.1%). Nine animals recovered, and were put to sleep 30–42 days following surgery. The average diameter of the grafts was 85%. Microscopically, all structures of the trachea were preserved. Grafts perfusion was on average 80.6%. Conclusions: Two conditions have to be fulfilled for tracheal autografts to survive: One, well vascularising muscular flaps have to be employed, and second, a high dose of steroids must be given starting on the day of operation.
Key Words: Tracheal grafts revascularization • Steroids • Muscle flap
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Introduction
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Despite a long history and the spectacular achievements in thoracic surgery, the problem of long circumferential tracheal defect repair still remains unresolved.
The first attempts in this field involved procedures which used tracheal substitutes in the form of prosthetic grafts, autogenus material or a combination of the two. These attempts, however, proved unsuccessful, as prosthetic grafts induced granulation, fibrosis and obstruction of the lumen. Biological material underwent extensive fibrosis and destruction
[2]
[6]
[10]
[14].
Segmental circumferential tracheal resection with the creation of an end-to-end tracheo-tracheal anastomosis has been developed. It is now possible to resect as much as 60% of the trachea
[7].
This procedure, however, often proves insufficient, especially in the cases of tracheal cancer, congenital malformations and extensive tracheal scarring.
Palliative endotracheal intubation or various stenting procedures become necessary
[4]
[7]
[9]
[11]
[13]. In such cases trachea transplantation seems to be the procedure of choice.
In order to be viable, the transplanted trachea must be revascularised. The results however, are still too poor to recommend the procedure for routine clinical use
[6]
[12]. Administration of various angiogenic factors had no significant influence upon the revascularization of the tracheal transplant
[1]
[5].
The aim of this research is to evaluate the feasibility of tracheal autograft revascularisation procedure using a rectus muscle flap pedunculated on the internal thoracic and superior epigastric arteries, and the influence of high-dose hydrocortisone on survival of the tracheal autograft.
The construction of the experimental model closely resembles clinical conditions and is based on autotransplantation, which eliminates the influence of immunological factors on the course of revascularisation. The trachea transplantation can be accomplished through median sternotomy.
An analogy may be drawn between the process of wound healing and the pathophysiologic processes occurring on the interface of the ischemic tracheal graft and the vascularized muscle flap. Exaggerated inflammatory response may lead to destruction of the graft, and then overproduction of collagen may result in graft scarring. In order to prevent the exaggeration of the inflammatory response and the third phase of wound healing, the animals were started on hydrocortisone 30 mg/kg per day
[3].
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Methods
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The experiment was conducted in the Polish white pig. The animals came from three litters from the same mother, were bred in the same shed and weighed between 20 and 40 kg with an equal male-to-female ratio. General anaesthesia was performed with alternately administered thiopental in fractions of 100 mg and analgesic doses of fentanyl. Ventilation was maintained with a self-expanding sack.
All animals received humane care in compliance with the European Convention on Animal Care. The study was approved by the institutional ethics committee.
The median sternotomy approach was employed, which enabled separation of the internal thoracic and superior epigastric arteries with the rectus muscle and offered easy access to the cervical and mediastinal portions of the trachea. A segment of the trachea measuring ten tracheal cartilages was separated, resected and reimplanted in the original position. A rectus muscle flap with a good pulse on the internal thoracic artery was sutured to the anterior cartilagenous portion of the graft.
This uniform model was used in 19 pigs. The pigs were then divided into two groups. Group 1 consisted of eight pigs which did not receive hydrocortisone. Group 2 consisted of 11 pigs which received hydrocortisone 30 mg/kg per day, starting on the day of the procedure.
Clinically, the outcomes were classified as follows: (1).death due to graft failure (severe stridor necessitating putting the animal to sleep); (2) survival, graft-associated signs present (exertional stridor); (3) survival, graft-associated signs absent.
On gross examination, the grafts were classed as: (1) poor (diameter of less than 30% of control values, mucosa and tracheal cartilages grossly changed); (2) satisfactory (diameter of 50–80% of control values, mucosa and tracheal cartilages intact); (3) good (diameter 80–100% of control values, mucosa and tracheal cartilages intact).
Microscopically, three patterns were identified, each of them closely related to a particular clinical outcome: (1) early death (i.e. within the first 7 days of the procedure); (2) late death (i.e. between day 7 and 17); (3) survival (animals killed between day 30 and 42).
All specimens were examined by light microscopy. Specimens collected in the parts of the tracheal wall located beyond the graft served as control. The examined fragments of the graft included the middle portion of the graft. All the specimens had been fixed in buffered 4% formaldehyde, dehydrated in ethanol, cleared in xylene and infiltrated in melted paraffin. Apart from routine staining with hematoxylin and eosin, the preparations had been stained with Verhoff stain for elastic fibres and trichromatic Masson method for collagen and myofibrils.
In addition, square (10x10 mm) patches of graft mucosa from the `survival' animals were collected, spread on cork, fixed in 2% glutaraldehyde and 0.15 M sodium-cocodylo buffer, and studied by scanning electron microscopy.
The graft perfusion rate in the survival group was assessed using microaggregates of 99mTc-labelled human albumin administered into the left atrium. For this reason a resternotomy was performed under general anaesthesia and 99mTc-labelled albumin was injected into the left atrium. Five minutes later the animals were exsanguinated. The graft radioactivity per 1 g of tissue was estimated in a chamber and with a gamma camera, and recorded by means of static scintigraphy. Portions of the trachea beyond the graft served as a control. Graft perfusion was expressed as a graft-to-control radioactivity ratio measured in a 1 g sample.
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Results
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Trachea autotransplantation followed by graft revascularisation was performed in 19 pigs.
Animals in group 1 did not receive hydrocortisone. All eight died due to graft stricture: five pigs died within the first 7 days of the procedure (2 on days 4 and 5, one on day 6, two on day 7). The remaining three pigs died on days 14, 15 and 17 of the experiment.
Animals in the second group received hydrocortisone. Only two of the 11 pigs died, one on day 5 and the other on day 17. In all these cases the clinical course was similar. Stridor was the first sign to appear. Initially its intensity was low but in most cases suddenly increased after feeding. On the next day the animals were overtly dyspnoeic and assumed an upright position. The breathing effort steadily increased and reached the point at which they had to be put to death.
The remaining nine pigs in group 2 survived and between days 30 and 42 of the experiment were put to death. On day 17 one of these animals presented with a quiet post-exertional stridor. The stridor remained unchanged in intensity until day 35, when the animal was put to death (Table 1).
Eight out of 8 grafts in first group, and 2 out 11 grafts in second group were classed as poor (Table 2). The intraluminal diameter of the middle portion of all ten grafts was reduced to approximately 30% of control values. Seven of these animals died early and, on gross examination, in each graft, a slit-like stricture was revealed. The wall of each graft was thickened due to inflammatory infiltration but at the same time flaccid, anterior parts of the tracheal cartilages were bent towards the lumen as a result of infiltration of the pars membranacea. Also, the mucosa was detached, which contributed to lumen stricture. The rectus muscle flap was tightly attached to the cartilagenous part of the graft, and was viable.
The strictures found in grafts of the three animals which died on days 14, 15 and 17 were each caused by a formation of a circumferential scar, which reduced the intraluminal diameter down to, respectively, 28.6% (14 mm down to 4 mm), 30% (10 mm down to 3 mm) and 25% (12 mm down to 3 mm) of control values. The grafts were irregular in shape and appeared retracted, irregular deformities of the tracheal cartilages were present and the silvery-white mucosa displayed focal defects and areas covered with granulation tissue. The rectus muscle flap remained tightly joined to the graft. The internal thoracic artery was patent.
The remaining nine animals survived and between day 30 and 42 were all put to death. One graft was classed as satisfactory and the rest as good. The intraluminal diameter of the `satisfactory' graft was 8 mm, equivalent to 66.7% of preserved lumen (control diameter of 14 mm).
The intraluminal diameter of the `good' graft to the control was: 16/17 (94,1%), 14/17 (82,3%), 15/17 (88,2%), 14/14 (100%), 15/15 (100%), 8/12 (66,7%), 8/10 (80%), 14/17 (82,3%), 13/16 (81,2%). The mean value was 85%.
Examination of the graft specimens collected from the ten animals which died due to graft failure demonstrated two distinct histology patterns further referred to as the `early death' pattern and the `late death' pattern (Table 3).
In five cases of early death (i.e. death within the first 7 days of the procedure), microscopic examination revealed a pattern characteristic of necrotising inflammation, typified by the presence of a diffuse inflammatory infiltrate composed of neutrophils, eosinophils, lymphocytes and macrophages, which extended to almost all the layers of the graft (cartilages were spared). Foci of destruction of elastic fibres were present.
The muscle flaps in all cases out of pig number 6 receiving hydrocortisone were viable (
Fig. 1
).
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Fig. 1. Pedunculated lobe of the rectus muscle of the abdomen of a pig not treated with steroids – died in the 7th day after the operation. A change in the staining of muscle fibres, between them the lumens of many capillary vessels. Staining using Masson's trichrome.
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In three cases of late death (i.e. death which occurred between days 14, 15 and 17 of the experiment), microscopic examination of the grafts identified a characteristic pattern of changes which included: proliferation of mucosal and adventitial fibrous connective tissue, focal destruction of elastic fibres, and within the rectus muscle flap: atrophic muscle fibres and marked vascularisation of connective tissue.
In group 2, which received hydrocortisone, only two pigs died. One pig died on day 5 and the other one on day 17. Microscopic examination revealed pattern characteristic for `early' and `late' death.
The remaining nine animals survived, microscopically the mucosal epithelium was intact, only focally metaplastic, mucous glands intact, collagen-to-elastic fibre ratio within the normal range. In all preparations of the grafts from the group which received hydrocortisone, an increased number of chondrocytes in the lacunae and extensive neovascularisation were noted (
Fig. 2
Fig. 3 Fig. 4
Fig. 5
).
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Fig. 2. Tracheal graft of pig no. 3 treated with steroids – put to sleep on the 42nd day after the operation. Normal mucous epithelium and under it well vascularized flaccid connective tissue with insignificant lymphocytic infiltrate. Staining using Masson's trichrome.
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Fig. 3. Tracheal graft of pig no. 3 treated with steroids – put to sleep on the 42nd day after the operation. Cartilage with small foci of calcification, in cartilagenous an increased number of chondrocytes. Staining using Masson's trichrome.
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Fig. 4. Pedunculated muscle lobe of pig no. 7 treated with steroids – put to sleep on the 37th day after the operation. Within well-vascularized connective tissue and adipose tissue are fragment of muscle fibres of variable staining in which striations and signs of nuclear proliferation are visible. Staining using Masson's trichrome.
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Fig. 5. Tracheal graft of pig no 8 treated with steroids – put to sleep on the 31st day of the experiment. Squamous cell epithelial metaplasia of the mucous epithelium is observed, under it a very large number of capillary vessels `contrasted' by erythrocytes. Staining using hematoxylin and eosin.
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Perfusion assessment
In all surviving subjects receiving hydrocortisone, graft and rectus muscle flap perfusion rates were measured (Table 4).
The mean perfusion rates in the graft and in the muscle flap were 93.9% and 145.7%, respectively.
The mucosal epithelium of surviving grafts was examined by scanning microscopy. The pictures varied, ranging from areas with a low number of ciliated cells to more abundant areas and resembling intact tracheal epithelium. In all examined cases the epithelium was intact (
Fig. 6
).
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Fig. 6. Scanning photograph of mucous membrane epithelium of a tracheal graft. Only a sparse number of cilliae are visible, however the epithelium is intact.
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Statistical analysis using non-parametric testing techniques revealed for Table 1Table 2: P=0.001 and for Table 3: P=0.002.
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Discussion
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Unsatisfactory results in the field of trachea transplantation are thought to be due to difficulties in tracheal revascularisation and to chronic rejection process. This, however, seems doubtful whenever the pattern of gross and microscopic changes in an autograft is compared with that seen in an allograft. Provided the same revascularisation method has in both cases been employed, few differences will be found
[6].
Easy healing of the bronchial anastomosis following lung transplantation is usually attributed to good operating technique or to choice of a better revascularisation flap than the previous time, often adding that such good results have been achieved despite the use of steroids.
The lack of concepts explaining changes occurring in the transplanted trachea has been a major cause of the developmental arrest in this area for several decades.
It seems quite reasonable to view tracheal graft healing process as being analogous to the wound healing process in which exaggerated inflammatory response and excess collagen formation may lead to destruction of the graft.
Thanks to such an approach, our proceedings are no longer limited to finding the right tissue with a blood supply optimal for tracheal graft revascularisation but we can also start thinking about pharmacological intervention in the process of healing
[3]. Glucocorticosteroids are very potent but their use to promote wound healing has never gained general acceptance. There were opinions that steroids might even complicate wound healing. They are avoided even following lung transplantation
[3]
[8].
Although the animals receiving hydrocortisone in this study were put to death only between days 30 and 42 of the experiment, it is quite probable that the grafts would not have undergone destruction. This assumption has been supported by two facts. Firstly, no gross or microscopic pathologic changes were detected in the survival grafts receiving hydrocortisone. All the grafts retained all their layers intact, the mucosa and tracheal cartilages in particular. Secondly, revascularisation of the graft was completely successful.
The results presented in this study prove that tracheal graft revasculation is feasible only if the revasculation pedicle flap and the right blood supply is chosen, and during the postoperative period the patient is started on high-dose hydrocortisone the day of the operation.
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Unlinked reference
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AUTHOR PLEASE CITE THE FOLLOWING REFERENCE
[15] IN TEXT.
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Footnotes
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Presented at the 11th Annual Meeting of the European Association for Cardio-thoracic Surgery, Copenhagen, Denmark, September 28 – October 1, 1997. 
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References
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Top
Abstract
Introduction
