COMPENSATORY LUNG GROWTH: LUNG PROTEIN,DNA AND RNA CONTENTS IN TRILOBECTOMIZED RATS

COMPENSATORY LUNG GROWTH: LUNG PROTEIN,DNA AND RNA CONTENTS IN TRILOBECTOMIZED RATS

Raul Lopes Ruiz Junior ¹

Roberto Carlos Burini ²

Antonio José Maria Cataneo¹

RUIZ Jr, R.L.; BURIRNI, R.C.; CAETANO, AJ.M. – Compensatory lung growth: lung protein, DNA and RNA contents in trilobectomized rats. Acta Cir. Bras, 13(1):18-25, 1998.

SUMMARY: Aiming at assessing compensatory lung growth after trilobectomy in rats, 3 groups of animals (control, thoracotomy and trilobectomy) were studied over 3 time intervals (7, 30 and 180 days post-operation). Protein, DNA and RNA contents in each lung were evaluated. The study of the left lung protein content reveals that compensatory growth ceased by day 30, whereas it continued to occur in the cranial lobe as long as 180 days post-operation. The lung DNA content in trilobectomized animals remained smaller than in the animals of the other groups demonstrating that compensatory growth was not brought about by hyperplasia. The lung RNA content in trilobectomized animals increased similarly to the lung protein content, demonstrating that the cells of the lung tissue must have had an increase in volume as no significant increase in their number occurred, as shown by the analysis of the lung DNA content. Therefore, it may be concluded that, in our experiment with adult animals, compensatory lung growth after trilobectomy in rats occurred due to an increase in the lung protein content and RNA content, suggesting a cellular volume increase (hypertrophy) and a probable increase in the intralveolar septs rather than an important cell multiplication.

HEADINGS: Compensatory Growth. Lung. Protein Content. DNA Content. RNA Content. Trilobectomy.

INTRODUCTION

The first experimental evidences that growth of the remaining lung occurs after resection of lung tissue, lobectomy and pneumonectomy were observed in the last century¹⁶.

This increase in size should be interpreted as compensatory growth of the remaining lung to compensate for the loss of volume and/or function rather than regeneration, as formerly believed. Regeneration implies restitution to normal, a phenomenon that occurs in the liver⁷ following partial hepatectomy.

Lung growth and kidney growth are very similar, cellular hypertrophy and hyperplasia occur, sometimes at the same time. However, hyperplasia is the major response.

It used to be thought that a compensatory hypertrophy took place after lung cells were stimulated (partial resection) during maturity¹⁵. Today, both physiologic and compensatory lung growth are known to have similar control mechanisms. These mechanisms may be: a) autocrine, inbuilt cellular mechanisms; b) paracrine, local specific, cellular secreted growth substances that affect other cells; c) endocrine, growth factors secreted from other organs (hormones)¹⁰.

Following resection, the remaining lung stretches to completely fill the pleural cavity. This stretch may be the starting factor of compensatory lung growth, which is characterized by hypertrophy in its initial phase, hyperplasia in its final phase and early collagen and elastin synthesis following resection¹.

The compensatory lung growth is influenced by some factors that may either change or stimulate this response. One of these factors is the dysanaptic growth between the lung parenchyma and airways. As the former is an extensile and very compliant structure its volume easily increases whereas the latter is more rigid and therefore less extensile.

The age of the experimental animals and the time of resection are factors that affect compensatory growth. The response is greater in younger animals. The sex of the animal is important when completely mature animals are used because males become considerably larger than females¹⁰.

Acute increase in blood flow may lead to increased lung growth and later remove inhibitors of cellular multiplication²⁷.

Both growth hormone and glucocorticoids may affect the compensatory response. BRODY & BUHAIN^4,5 observed that prior hypophysectomy prevented the expected increase in lung volume, in female pneumonectomized rats, BENNET et al.² found that bilateral adrenalectomy performed before pneumonectomy resulted in increase in DNA, RNA and protein in rats.

There are evidences for specific lung growth factors that might be involved in regulation of the postpneumonectomy response. This is suggested by the fact that, postpneumonectomy, there is increased DNA synthesis in the lung but not in the liver or kidneys of these animals⁶. SMITH et al.²⁵ found a factor that appeared in rabbit serum 9 days after pneumonectomy and was capable of stimulating incorporation of tritiated thymidine into DNA of cultured human type II pneumocytes. This factor was not detected in sham-operated control animals, demonstrating that there is a specific growth factor in the serum of animals submitted to compensatory lung growth stimulation after resection.

Ambient oxygen has a profound effect on compensatory lung growth and normal lung growth. The transient hypoxia that may occur in the immediated postoperative would act as a trigger for compensatory growth. However, NATTIE et al.²⁰ failed to demonstrate this fact and concluded that hypoxic stress was unlikely to be the sole determinant of postpneumonectomy growth.

Ozone exposure may induce changes in mechanical properties of the alveolar wall (maximal extensibility visoelastic properties). Compensatory growth may confer some protection from these alveolar changes¹⁸.

Administration of endotoxin accelerates postpneumonectomy lung growth²⁶ by an unknown mechanism. However, it is believed that the increase in metabolism affects the compensatory response

Most investigators have demonstrated that compensatory growth is more complete and faster in young animals^8,12,17,20. However, Wandel et al.²⁹ demonstrated that, in the adult rat, there was compensation after two lung lobes were removed, similarly to young rats⁹.

It has been demonstrated there is a restitution of gas exchanges following the removal of the lung parenchyma. According to some investigators this is due to an increase in the alveolar number in the remaining lung^12,17,20. Some others assume there is a lengthening of the alveolar walls which enlarges the gas exchange surface but does not increase the alveolar number^3,8,24 or that the increase in the gas exchange surface occurs also through the openings of previously colapsed alveoli²¹.

Nevertheless, investigators agree that after experimental lung ablation, the remaining lung mass and volume increase ^{1,8,11,12,13,20,21,23,24,27,29}; as well as DNA, RNA and proteins, suggesting cell multiplication^1,8,13,22.

Cataneo et al.¹¹ observed that 7 days after approximately 55% of the lung tissue was removed from adult rats, the total pulmonary weight was completely restored and at day 30 post-surgery the same occurred to volume.

By comparing the large number of investigations related to liver and kidney resections with the few studies about compensatory growth after lung resection, it can be observed that the latter has been little investigated, thus the phenomena related to it are not well known²⁸.

The purpose of this work was to study the lung protein, DNA and RNA contents in the lung compensatory growth after trilobectomy, by measuring the proteins, DNA and RNA found in the remaining lung of healthy, well-nourished animals, after approximately 55% of the lung tissue was removed, at different intervals (7, 30 and 180 days post-surgery).

METHOD

For this investigation, 90 male, 75-day old, healthy Wistar rats, weighing approximately 170g were obtained from the UNESP Central Vivarium - Campus of Botucatu. All animals were kept at the Vivarium of the Experimental Surgery and Surgical Techniques Laboratory, pertaining to the Department of Surgery and Orthopedics of the Botucatu Medical School - UNESP, inside a closed room with artificial 12-h illumination. The animals, housed in individual metallic cages, were fed on commercial food and had free access to water, throughout the experiment. They fasted 24 hours prior to anesthesia/surgery.

After an initial 14-day adaptation period, the animals were randomly divided into 3 groups, which received 3 different types of treatment: control (G1) - non-operated animals, kept under the same conditions of the other groups; thoracotomy G2) - animals submitted to sham operation, which involved all procedures except lung removal; and, trilobectomized (G3) - animals that had 3 of their lung lobes excised.

Each group was randomly subdivided into 3 subgroups (A, B, C) that were sacrificed at day 7, 30 and 180 after treatment, respectively.

NOTE: Whenever an animal died, it was replaced by another in similar conditions, so that the number of rats remained the same throughout the experiment. All the animals and their lungs were weighed.

General anesthesia was induced by an intraperitoneal injection of sodium pentobarbital (30mg/kg weight). Airways were maintained by orotracheal intubation with an 8.2cm-long PE160 polyethylene tube.

Observing the rules of cleanness (tricothomy and iodized alcohol), trilobectomy was performed. The chest was opened by dissection through the seventh intercostal space on the right. The hilum of the caudal lobe was exposed and tied with a 10-cotton suture. The same procedure was used with the middle and accessory lobes. Air was expelled from the thoracic space by hyperinflating the remaining lung at the last stitch of the muscular wall. Ribs, subcutaneous tissue and skin were closed with continuous 10-cotton suture. Ventilation was manually provided by AMBU (Automatical Manual Breathing Unit).

In order to collect material, animals were weighed (Filizzola® Scales) and anesthetized as previously described. Exsanguination was performed through vena cava inferior and abdominal aorta.

By resecting the sternum, the heart and lungs were removed from the chest and lungs were carefully dissected. The organs were weighed, wrapped in aluminum and stored at -10^oC for subsequent measurement of the lung protein content by using Total Nitrogen, accordingly to the Micro-Kjldahl (1965); lung DNA content (m g) by the Diphenylamine Method of Burton (1956), modified by GILES & MYERS¹⁴; and lung RNA content (m g) by the method of Schmidt & Thannhauser (1945) modified by MUNRO & FLECK¹⁹.

Randomized Factorial Analysis of Variance³² was used in order to assess changes of control with time with the following tests: the interactions between groups and time, group effects, time effects, the means of each group at the same time and the changes with time in each group, followed by Tukey test when a significant difference was indicated. Differences were considered significant when p<0.05.

RESULTS

Results are illustrated by the graphs below. Time intervals (A,B,C) are plotted along the x axis and mean values of lung protein, DNA and RNA contents are plotted along the y axis.

1. Cranial lobe protein content (m g):

Comment: The protein content of the cranial lobe was more elevated in the trilobectomized animals than in the other groups over all the intervals and this difference increased with time.

2. Left lung protein content (m g):

Comments: The protein content present in the left lung was more elevated in trilobectomized animals than in the other groups over all time intervals.

3. Right lung protein content (m g):

Comments: The protein content present in the right lung was less in trilobectomized animals than in the other groups over all time intervals.

4. Total (left + right) lung protein content (m g):

Comments: The total lung protein content was similar in all the groups over all time intervals studied.

5. Cranial lobe DNA content (m g):

Comments: No significant difference was observed among the groups over all time intervals studied.

6. Left lung DNA content (m g):

Comments: The DNA content of the left lung was more elevated in the trilobectomized animals only by day 30.

7. Right lung DNA content (m g):

Comments: The DNA content of the right lung was reduced in the trilobectomized animals over all time intervals studied.

8. Total (left + right) lung DNA content (m g):

Comments: The lung DNA content was less in trilobectomized animals over all time intervals studied.

9. Cranial lobe RNA content (m g):

Comments: The RNA content in the cranial lobe was more elevated in trilobectomized animals over all time intervals studied.

10. Left lung RNA content (m g):

Comments: The RNA content in the left lung was more elevated in trilobectomized animals over all time intervals studied.

11.Right lung RNA content (m g):

Comments: The RNA content in the right lung was less in trilobectomized animals over all time intervals studied.

12. Total (left + right) lung RNA content (m g):

Comments: The total lung RNA content was more elevated in trilobectomized animals over all time intervals studied.

DISCUSSION

1. Lung Protein Content

In trilobectomized animals the protein content of the cranial lobe was more elevated than in the other groups over all intervals. However, this increase was not enough to compensate for the loss of the excised lobes because the protein content of the right lung remained the smallest in the trilobectomized animals.

Total protein was more elevated in the left lung of trilobectomized animals over all intervals investigated.

Upon analysis of both lungs, no significant difference was observed in the total lung protein content of the groups over all the periods studied, demonstrating that in trilobectomized animals the increased protein content of both cranial lobe and left lung compensated for the quantity found in the whole lung of the other groups. Therefore, compensatory growth occurred in both the ipsilateral and contralateral lung.

The total lung protein content progressively increased in trilobectomized animals as a result of the large mass increase of the cranial lobe, which was around 2.5-fold by day 180.

By day 180 the protein content in the cranial lobe was more elevated (2.5-fold) in trilobectomized animals than in the animals of the other groups.

As significant increases of protein content occurred more frequently in the cranial lobe of trilobectomized animals than in the other groups, it may be stated that its compensatory growth continued even after 30 days, and that it is persistent, as demonstrated by CATANEO et al.¹¹. The exact moment it ceased could not be determined, it might have happened between day 30 and day 180, or might have continued after day 180.

The protein content in the left lung increased more in trilobectomized animals as compared with the other groups as long as 30 days post-operation. From that day on all groups presented similar increases, in contrast to the cranial lobe; Therefore, compensatory growth did not persist as reported by CATANEO et al.¹¹.

The compensatory lung growth of the cranial lobe was different from that of the left lung, probably due to the mechanical factor which continued to stimulate the cranial lobe to grow until it completed the pleural cavity, in contrast to the left lung that already fills it completely.

Different results may be achieved in different studies. The age of the animal, among other factors, may account for these differences, as it is quite known that in young animals the response after lung resection is complete, with an effective increase in the total lung protein. This has happened to older animals though not so intensely¹⁷.

WATKINS & RANNELS³⁰ obtained results similar to ours: the protein content did not change in the lung contralateral to left pneumonectomy performed in 4-week old rats. The protein content was increased in the remaining lung one week after resection and on the second week no significant statistic difference was observed among left pneumonectomy, thoracotomy and control groups.

2. Lung DNA Content

During normal growth, the lung DNA content, in male Sprague-Dawley rats, rapidly increases between day 4 and day 9 after birth³¹. Maximum amounts are reached by the 14th postnatal day. Thereafter, the rate of increase becomes slower as the animal grows older. Since only ninety-day-old animals were used in the present study, the DNA increase rate was very slow in the control group and became even slower over the second and third time intervals studied.

The lung DNA content in the cranial lobe tended to be more elevated in trilobectomized animals but no significant differences were observed.

In the left lung, a significant increase in the DNA content was observed in trilobectomized animals solely 30 days postpneumonectomy. Over the remaining time intervals studied, no difference was found among the groups.

In the right lung, the DNA content was obviously reduced in trilobectomized animals. In this group only the cranial lobe existed and it already presented the same amount of cranial lobe DNA presented by the other groups, not counting the DNA in the other three lobes of the right lung.

Although lung DNA content increased with time in trilobectomized animals, this increase was similar to that of the controls over all time intervals but the second (30 days), when an elevated DNA was observed due to the increase that took place in the left lung. Except for this, DNA was always lower in trilobectomized animals.

By comparing the lung protein content with the lung DNA content, it may be observed that compensatory growth was brought about by the lung protein content and not by the lung DNA content, which little increased or did not increase at all. This observation lead to the conclusion that compensatory growth might have been brought about by hypertrophy rather than hyperplasia of the lung tissue.

ROMANOVA et al.²² and BUHAIN & BRODY⁵ reported significant increases in the lung DNA content. In the present work, due to the methods used, no significant differences could be demonstrated but only a tendency of the lung DNA content to be larger in the remaining lobes.

3. Lung RNA Content

Both in the cranial lobe and left lung the lung RNA content was significantly more elevated in trilobectomized animals than in the other two groups, over all time intervals studied.

In the study of the right lung, over all time intervals, it was observed that the lung RNA content was significantly less in trilobectomized animals. Although a large amount was found in the cranial lobe, which underwent compensatory growth, this amount was not as large as the sum total of the four lobes of the right lung of the rats pertaining to the other groups.

With regard to the lung RNA content in the lungs, values presented by trilobectomized animals tended to be higher than those of the other groups, but this was significant over only one of the time intervals studied. Therefore, the lung RNA content which was lost with the removal of the three lobes, was totally restituted by the compensatory growth of the cranial lobe and left lung.

By comparing the lung RNA content in each lobe, or in the whole lung, with the lung protein content, it is observed that they behave very similarly demonstrating that compensatory growth was followed by a similar increase in the lung RNA content, in contrast to the lung DNA content.

As RNA is predominantly cytoplasmatic, it is believed that the compensatory growth, of the cranial lobe and the left lung as well, was brought about by an increase in cellular volume rather than an increase in cell number. Results similar to ours were found by WATKINS & RANNELS³⁰.

CONCLUSIONS

It was concluded that in our experiment with adult animals, compensatory growth after lung trilobectomy is brought about by an increase both in the lung protein content and the lung RNA content, with no significant increase in the lung DNA content, suggesting that no important cellular multiplication but an increase in the cellular volume as well as a probable increase in the intralveolar septs occurred.

Acknowledgment

The authors wish to tank FAPESP for their financial support.

RESUMO

Objetivando analisar o tipo de crescimento compensatório dos pulmões após trilobectomia no rato, foram analisados três momentos ( sete, trinta e cento e oitenta dias) após o tratamento em três grupos de animais (controle, toracotomia e trilobectomia).

Foram estudados os seguintes atributos: conteúdo protéico, conteúdo de DNA e conteúdo de RNA em cada um dos pulmões.

O crescimento compensatório no pulmão esquerdo, quando analisamos o seu conteúdo protéico, cessou aos trinta dias, enquanto que, no lobo cranial, continuou ocorrendo até os cento e oitenta dias.

O conteúdo pulmonar de DNA nos animais trilobectomizados manteve-se sempre abaixo dos outros grupos estudados, mostrando que o crescimento compensatório não ocorreu às custas de hiperplasia.

O conteúdo pulmonar de RNA nos animais trilobectomizados apresentou um incremento muito próximo ao ocorrido com o conteúdo protéico pulmonar, mostrando que as células do tecido pulmonar devem ter aumentado seu volume, uma vez que não houve acréscimo significativo de seu número, como indicou a análise do conteúdo pulmonar de DNA.

Portanto, pudemos concluir que, em nossas condições experimentais, no animal adulto, o crescimento compensatório pós-trilobectomia pulmonar ocorreu às custas de um aumento do conteúdo protéico pulmonar e de RNA, sugerindo que não ocorreu importante multiplicação celular, mas sim aumento de volume celular (Hipertrofia) e, provavelmente, aumento dos septos interalveolares.

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¹

Department of Surgery and Orthopedics, Faculty of Medicine Botucatu, São Paulo State University - UNESP

²

Department of Internal Medicine, Faculty of Medicine Botucatu, São Paulo State University - UNESP

Publication Dates

• Publication in this collection
  18 Nov 1998
• Date of issue
  Jan 1998