POSTNATAL CHANGES IN THE LUNG STRUCTUREOF ALBINO RATS

POSTNATAL CHANGES IN THE LUNG STRUCTUREOF ALBINO RATS

"HISTOLOGICAL AND MORPHOMETRICAL STUDY"

Zakya M.A.S.Alhasy^1,2 ;Ibrahim A.Maher¹;Manal M. Morsy¹

andMohammed A.S. Amin^1*

¹Anatomy & Embryology Department, Faculty of Medicine, Zagazig University, Egypt

² Anatomy&Embryology Department, Faculty of Medicine, Omar Al-Moktar University, Libya

*Corresponding author: Mohammed Ahmed Shehata Amin

E-mail: dr.mohammed.shehata2007@gmail.com

Key words: Lung- Development- Rats.

ABSTRACT

Background:The lungs represent the major part of respiratory system. It consists mainly of spongy tissue and conducting air tubules called bronchi and bronchioles. The spongy tissue at which gaseous exchange takes place between air and blood are forming alveolar duct and alveoli

The aim of this study: is to evaluate the changes that occur in the structure of the lung in the postnatal period and correlate it with clinical applications. Material and methods: 12 pregnant female rats of Wistar strain were used in this study. The study was carried out on pups of female rats after mating with male rats. Pups were given water and balanced diet by gastric intubation. Pups of pregnant rats were sacrificed at 14th and 21th days postnatal. Lung from all the age groups were processed for light microscope examination. Results:At day 14H&E stained sections revealed that the rat lung was formed of air saccules divided by large numbers of secondary septa. The wall of bronchioles consists of ciliated cuboidal epithelium. All primary and secondary septa showed the double capillary network. Masson’s trichrome stainedsections showed ordinary amount of collagen fibers in the wall of the bronchioles and bronchial vessels.At day 21H & E stained sections of lung revealed normal architecture of alveoli with many alveolar sacs.The double capillary layers inside the primary and secondary septa are transformed into a single central capillary layer. Masson’s trichrome stained sections showing average amount of collagen fibers in the wall of the bronchi.

Conclusion: There are changes occur in the structure of lung in the postnatal period at different age groups.

INTRODUCTION:

The respiratory system consists of the lungs and other airways leading to the external environment. It develops in the embryo as a ventral invagination of the foregut then grows into the thoracic mesenchyme. Subsequently, lung epithelial tubules grow and branch to give rise to the bronchial tree. This process is accompanied with development of the vascular structures, which arise by angiogenesis (Wei et al., 2009). The lung development has been divided into 5 stages: embryonicstage, pseudo glandular stage, canalicular stage, saccular stage, and finally the alveolar stage. However, more recently the alveolar stage has been split in two and a sixth stage has been defined as the stage of microvascular maturation. Each of these stages is defined by a specific developmental milestone and each requires a unique set of developmental factors to accomplish its specific end goal (Kotecha, 2000).The Embryonic Stage started in rat at day 11-13 of gestation. It’s the start of organogenesis and formation of major airways (Schittny&Burri, 2007). The bronchial buds then give rise to unilobar left and quadrilobed right lungs (Warburton et al., 2000). The double origin of the lung tissue is important. Many processes of lung development are dependent on the interaction between epithelium and mesenchyme. Classical transplantation experiments have shown that a cross-talk between the endodermal epithelium and the mesodermal mesenchyme is needed for the control of branching morphogenesis and cytodifferentiation (Schittny&Burri, 2007). Pseudo glandular Stage started in rat at day 13-18.5 of gestation. At this stage the lungs are already subdivided into definitive lobes (Kotecha, 2000). Canalicular stage started in rat at day 18.5–20 of gestation, which comprises important steps in the development of the fetal lung. The Saccular (or Terminal sac) stage started in Rat from day 21 of gestation to day 4 postnatal. At the beginning of this stage, the peripheral airways form typical clusters of widened air spaces termed saccules or terminal sacs (Rutter, 2008). The Alveolar Stage started in rat at day 4-14 postnatal days, while the micro-vascular maturation stagestarted in rat at day 14- 21 postnatal .The essence of this stage is the restructuring of the double capillary networks in the parenchymal septa to the mature aspect with a single capillary system. In addition, a large overlap with the stage of alveolarization exists (Joshi &Kotecha, 2007). Inorder to demonstrate these different stages we perform this study.

MATERIAL AND METHODS:

• Animals:

This study was carried out on 12 virgin female albino rats and 6 male albino rats of 160-200 gm were obtained from the animal house Faculty of veterinarymedicine, Zagazig University. All animals were housed in environmentally controlled rooms, in spacious wire mesh cages. Temperature was kept at room temperature (25~30 ͦ c). A cycle of twelve hours of Light and twelve hours of darkness was maintained throughout this study. All animals received food and tap water ad libtium.

Determination of pregnancy

Animals were mated overnight and after that separated and examined in the following morning for the presence of sperm in the vaginal smear. Vaginal smears stained with Papanicolaou stain (Kiernan, 2000) were taken every day. When smear was positive, the first day of pregnancy was considered. The females with negative vaginal smear were also isolated and re kept again with males. The process was repeated until all females became pregnant.

• Study design

The length of gestation for animals averaged 22days. 12 pregnant female rats of Wistar strain were used. Pups were sacrificed at 14th and 21th days postnatal. The study contained 12 pregnant rats which were not received any treatment and were given water and balanced diet by gastric intubation. The offspring of these pregnant rats are subdivided into 2 subgroups:

• Subgroup Ia14PND: contains 6 mothers with their pups.
• Subgroup Ib21PND; contains 6 mothers with their pups.

The day of birth was referred to as postnatal day 0. Within 24hr after birth, pups were kept with their mothers to be sacrificed on day 14and 21 after parturition. Pups were anesthetized to avoid stress. Histological examination of lung tissue was done. The rats were decapitated and the lung removed by sagittal section. Specimens from all the age groups were processed for light microscope examination.

Histological Examination:

The lungs of albino rats were collected at the end of experiment. The samples were fixed in Bouin’s solution, then dehydrated in ascending grades of alcohols, cleared in xylene and embedded in paraffin. The samples were casted, then sliced into 5 µm in thickness and placed onto glass slides. The slides were stained by hematoxylin and Eosin (H&E) and Masson's Trichrome stains (Bancroft and Gamble, 2008).

Immunohistochemical study:

Immunohistochemical reactions were carried out on sections of the lung using an antibody against alpha smooth muscle actin antigen (αSMA) marker for smooth muscle specific protein around the developing epithelial tubes (Yamada et al., 2002). A mouse anti alpha smooth muscle actin antibody (1A4, Sigma, ST Louis, USA) diluted as supplied (1:800) and designed for the specific localization of αSMA in paraffin sections. They were delivered from DAKO life trade Egypt. A biotinylated secondary anti-immunoglobulin capable of binding to both the primary antibody and the streptavidinbiotin enzyme complex since both primary antibody and antibody of the label are produced in the same animal species. Paraffin blocks were cut by a microtome at 4 micron thickness. Sections were mounted on positively charged glass slides. The slides were incubated at 37°C overnight for accurate adhesion of the sections to the slides. Slides were placed in 60 Coven for 13 to 18 hours. Slides were transferred into three changes of fresh xylene for 10 minutes each change. Slides were transferred into graded ethanol (two changes of 100%, two changes of 95%, two changes of 70% and two changes of 50%), 3 minutes for each change. Then washed in phosphate buffer saline of PH 7.2 for minutes. The PH of the buffer was checked with each run. The primary antibody replaced by buffer was included for each specimen, stained with Mayer's haematoxylin and was first noted to determine the amount of specific staining seen in the examined sections.

RESULTS:

Histological examination:

Light microscopic examination

Subgroup Ia: age 14^th postnatal

1. 1.      Hematoxylin and eosin(H&E):

Examination of H&E stained sections from rat lung at 14th post natal day revealed that the rat lung was formed of air saccules divided by large numbers of developing crests (secondary septa) which recognized along the primary septa into smaller units (primitive alveoli) (Fig 1.).The wall of bronchioles consists of ciliated cuboidal epithelium (Fig 2). All primary and secondary septa showed the double capillary network which bulge into the lumen so that they were considered immature septa. Primitive alveoli were lined by flattened type I pneumocytes and cuboidal type II pneumocytes (Fig3).

2. Masson's trichrome stain:

Masson’strichrome stainedsections showed ordinary amount of collagen fibers in the wall of the bronchioles and bronchial vessels. In addition, the surrounding lung tissue demonstrates minimal amount of collagen fibers in the interalveolar septa (Fig4).

3.α SMA immunostaining:

Immunoperoxidase technique for α SMA showed a population of round α SMA –positive cells appeared at the tips of the developing secondary septa in addition to the slender α SMA –positive cells in primitive septa (Fig5).

Subgroup Ib: age 21^th day postnatal

1. 1.      Hematoxylin and eosin(H&E):

Examination of H & E stained sections of lung at 21 ^Th postnatal day revealed normal architecture of alveoli with many alveolar sacs. Alveoli were separated by apparently thin primary septa. (Fig6). Alveoli were lined by squamous pneumocytes type I cell and cuboidal pneumocytes type II cell. Alveolar walls were less cellular with thin interstitium but groups of interstitial cells were observed where several septa were joined. The double capillary layers inside the primary and secondary septa are transformed into a single central capillary layer (Fig7). The terminal bronchiole is lined by simple columnar epithelium and surrounded by thin layer of smooth muscle (Fig8).

1. 2.      Masson's trichrome stain:

Masson’s trichrome stained sections of 21 ^Th DPN showing average amount of collagen fibers in the wall of the bronchi and bronchial vessels. In addition, the surrounding lung tissue demonstrates scattered amount of collagen fibers in the interalveolar septa. (Fig8).

1. 3.      α SMA immunostaining:

Immunoperoxidase technique for α SMA showed a round α SMA –positive cells at the tips of the developing secondary septa, whereas the slender α SMA –positive interstitial cells in the primary septa are disappeared (Fig9).

Figure (1): A photomicrograph of a section in the lung of the control group at 14 DPN showing medium sized bronchiolar wall (B) with the lining respiratory epithelium and bronchial vessels (BV), air saccules (S), secondary septa (arrow) and primitive alveoli (A).  (H&E X 100)

Figure (2): A photomicrograph of a section in the lung of the control group at 14 DPN showing medium sized bronchiolar wall which consists of respiratory epithelium, ciliated cells (arrow) and Clara cells (arrow heads).    (H&E X 100)

Figure (3): A photomicrograph of a section in the lung of the control group at 14 DPN showing the surrounding lung tissue with developing alveoli (DA), secondary septa(arrow), primary septa (*), capillary network (double arrow),flattened type I pneumocytes (curved arrow) and cuboidal type II pneumocytes(arrow heads) (H&E X 400).

Figure (4): A photomicrograph of a section in the lung of the control group at 14 DPN showing ordinary amount of collagen fibers (Green) in the wall of the bronchioles and bronchial vessels (arrows). In addition, the surrounding lung tissue demonstrates minimal amount of collagen fibers (Green) in the interalveolar septa (arrow heads). (Masson’s trichrome X 100)

Figure (5): A photomicrograph of a section in the lung of the control group at 14 DPN showing prominent expression of α-SMA–positive cells in the primitive alveolar septa (*) and other α-SMA–positive cells in the developing secondary septa (arrow heads).  (α-SMA X 400)

Figure (6): A photomicrograph of a section in the lung of the control group at 21 DPN showing medium sized bronchiolar wall (B) with the lining respiratory epithelium and bronchial vessels (BV). In addition, air saccules (S), secondary septa (arrow) and primitive alveoli (A) with thin interalveolar septa are seen. (H&E X 100)

Figure (7): A photomicrograph of a section in the lung of the control group at 21 DPN showing the surrounding lung tissue where Alveoli were lined by squamous pneumocytes type I cell (arrow heads), and cuboidal pneumocytes type II cell (arrows). Secondary septa with single central capillary layer are also seen (curved arrow).       (H&E X 400)

Figure (8): A photomicrograph of a section in the lung of the control group at 21 DPN showing average amount of collagen fibers (Green) in the wall of the bronchi and bronchial vessels (arrows). In addition, the surrounding lung tissue demonstrates scarce amount of collagen fibers (Green) in the interalveolar septa (arrow heads). (Masson’s trichrome X 100)

Figure (9): A photomicrograph of a section in the lung of the control group at 21 DPN showing prominent expression of α-SMA–positive cells in the interstitial tissue of immature alveoli (*) and other α-SMA–positive cells in the developing secondary septa (arrow heads) are visible.      (α-SMA X 400)

Morphometrical Results:

There was a highly significant difference (p<0.001) between 14PND group and 21 PND group regarding Interalveolar septa thickness, ATETAS; area percentage (%) occupied by both terminal segments of epithelial tubes & by alveolar sacs  and area percentage(%) of α-SMA. There was a highly significant reduction in 21PND group in all parameters (Table 1 and Fig 10,11&12).

Table (1): Showing the mean values of morphometric parameters in studied groups

Fig. (10): Bar chart showing the mean of inter-alveolar septa thickness in studied groups

Fig. (11): Bar chart showing the mean of area percentage (%) of ATETAS in studied groups

Fig. (12): Bar chart showing the mean of area percentage (%) of α-SMA in studied groups

DISCUSSION

The current study was designed to detect the changes in the lung structure during the postnatal period at different age groups. The rat was chosen as it is considered to be the first animal to be domesticated for strictly scientific purposes and become a species of choice because of metabolic similarities, as well as their small size and short life span (Gad, 2007). In this research, we studied pups from the 14th day of pregnancy until day21 after delivery. This age corresponds to lung development which has been divided into 5 stages: embryonic stage, pseudo glandular stage, canalicular stage, saccular stage, and finally the alveolar stage (Laudy&Wladimiraoff, 2002). In the current study, the control group at age 14^th day postnatal shows air saccules and secondary septa that appear as finger like projection which divided air space to primitive alveoli this was similar to the findings ofBolle et al., (2007) and Joshi &Kotecha (2007) said. In addition there were several characteristic features shown in the lung at this age group as respiratory epithelium, ciliated cells and clara cells. This description was in accordance with (Ross &Pawlina, 2009).Also the surrounding lung tissue show developing alveoli, secondary septa, primary septa, capillary network, flattened type I pneumocytes and cuboidal type II pneumocytes.  This description was in accordance with (Tomashefski&Farver, 2008). There were large numbers of developing crests (secondary septa) along the primary septa giving them acrenated appearance. The secondary septa increased in height and subdivided the air saccules into smaller units (primitive alveoli) .These septa still contain double capillary layer, this was in agreement with many authors Prodhan&Kinane (2002), Roth-

Kleiner& Post (2005) and Joshi &Kotecha (2007), who stated that the formation of the pulmonary alveoli as shown in the present study was initiated by developing of secondary septa from the pre- existed primary septa. These secondary septa subdivided the air saccules into smaller units, the primitive alveoli. All the primary and secondary septa showed a double capillary network. In the other side contrast the interalveolar walls of the adult lung contain only a single capillary layer. Masson's trichrome stained sections show ordinary amount of collagen fibers in the wall of the bronchioles and bronchial vessels, the surrounding lung tissue demonstrates minimal amount of collagen fibers in the interalveolar septa. This description was in accordance with (Ahmed and Mazher 2009) who found that minimal amount of collagen fibers were concentrated mainly around bronchus and blood vessels.α-SMA immunohistochemical stained sections showed prominent expression of α-SMA in the wall of the bronchi as well as, α-SMA–positive cells in the primitive alveolar septa  and other α-SMA–positive cells in the developing secondary septa  are clearly demonstrated. This description was in accordance with (Ibáñez-Vea et al., 2018) who demonstrated the lung compartment specific expression of α-SMA positive cells, which are apparently myofibroblasts, in alveoli, bronchioles and bronchi in lung tissue. Lastly α-SMA is a useful marker for smooth muscle cells in the developing lung because of its consistent appearance in vascular and bronchiolar smooth muscle cells from the initial stage of their differentiation. By the use of this marker, we could succeed in clarifying the early differentiation and later distribution of the bronchiolar and vascular smooth muscle cells (Mitchell et al., 1990). In the current study, the control group at age 21th day postnatal shows bronchiolar wall with the lining respiratory epithelium and bronchial vessels, air saccules, secondary septa and primitive alveoli with thin interalveolar septa. This description was in accordance with Ross &Pawlina (2009) who said that  the surrounding lung tissue show alveoli lined by squamous pneumocytes type I cell, cuboidal pneumocytes type II cell and secondary septa with single central capillary layer. The lung was formed of alveoli with thin interalveolar septa. The double capillary layers inside the primary and secondary septa are transformed into a single central capillary layer this agreed with Schittny&Burri (2007) who stated that the essence of this stage is the restructuring of the double capillary networks in the parenchymal septa to the mature aspect with a single capillary system. This process results in a reduction of tissue mass and alveolar septal thickness, as well as the total number of fibroblasts and epithelial type II cells. With finalization of septal restructuring at around day 21, lung structural change is considered complete and the lung enters a period of equilibrated growth. Masson's trichrome stained sections show average amount of collagen fibers in the wall of the bronchi and bronchial vessels. In addition, the surrounding lung tissue demonstrates scarce amount of collagen fibers in the interalveolar septa. This description was in accordance with Prokopakis et al., (2013) who noticed the presence of minimal amount of collagen fibers in alveolar wall, bronchi, bronchial vessels, the interstitial tissue of immature alveoli and the developing secondary septa. This description was in accordance with Warburton et al., (2005) who stated that the round α-SMA–positive cells persist in the tips of the secondary septa whereas the slender α-SMA–positive in the primary septa disappeared in mature alveoli but persisted in immature alveoli.

CONCLUSION:

This study revealed the histological and immunohistochemical changes in the lung structure of postnatal albino rats. This could explain pathogenesis of many respiratory diseases found in clinical practice.

REFERENCE:

Ahmed, R. R. andK. Mazher (2009): Histological, histochemical and biochemical changes in the liver, kidney, lung and spleen under the effect of repetitive hyperthermia in rat neonates. Iranian Journal of Cancer Prevention, 2(2): 91-101

Bancroft, J. and A.Gamble (2008):Theory and Practice of Histological Techniques. Churchill Livingstone, New York, London.

Bolle, I. ;G. Eder ; S. Takenaka ; K. Ganguly ; S. Karrasch ; C. Zeller andH. Schulz (2007):Postnatal lung function in the developing rat. Journal of Applied Physiology, 104(4):1167-1176.

Gadbarbora, O. ;H.D.J. Eric and R. Stein (2007): Textbook of anxiety disorders. Second edition, britishlaibrary cataloguing in publication data, Chapter 8, Pp: 201-204.

Ibáñez-Vea, M. ;M.Zuazo ; M.Gato ; H.Arasanz ; G.Fernández- Hinoja ; D. EscorsandG. Kochan (2018): Myeloid-derived suppressor cells in the tumor microenvironment: current knowledge and future perspectives. Archivumimmunologiaeettherapiaeexperimentalis, 66(2): 113-123.

Joshi, S. and S.Kotecha (2007): Lung Growth And Development. Early Human; 83: 789–794.

Kiernan, J.A. (2000): Histological and Histochemical Methods. Theory and Practice, 3rd ed., Butterworth Heinemann, Oxford, Boston., Pp: 23-29.

Kotecha, S. (2000): Lung growth for beginners. Paediatr Respir Rev; 1: 308–313.

Laudy, J.A.M. and J.W. Wladimiraff (2002): Fetal lung Developmental Aspect. Ultrasound Obstet Gynacol; 16: 284-290.

Mitchell, J.J. ;SusanE. Reynolds ; KevinO. Leslie; Robert B. Low and W.M. Janet (1990): Smooth Muscle Cell Markers in Developing Rat Lung. American Journal of Respiratory Cell and Molecular Biology; 3(6):515-523.

Prodhan, P. and T.B. Kinane (2002): Developmental paradigms in terminal lung development. Bio Essays; 24: 1052–1059.

Prokopakis, E. ; A.Vardouniotis ; H.Kawauchi ; G.Scadding,; C.Georgalas ; P.HellingsandL.Kalogjera (2013): The pathophysiology of the hygiene hypothesis. International journal of pediatric otorhinolaryngology, 77(7): 1065-1071.

Ross, M.H. and W.Pawlina (2009): Histology A text and Atlas. 5th ed., Lippincott Williams and Wilkins. Baltimore and Philadelphia.; pp: 670-678.

Roth-Kleiner, M. and M. Post (2005): Similarities and dissimilarities of branching and septation during lung development. Pediatric Pulmonology; 40: 113–134.

Rutter, M. (2008): Institutional effects on Children: Design Issues and Substantive Findings. Monographs of the Society for Research in Child Development, 73(3): 271-278.‏

Schittny, J. and P.H. Burri (2007): Development and growth of the lung; Annual review of cell and developmental biology ,139: 111-124

Tomashefski, J.F. and C.F. Farver (2008): Dail and Hammer’s Pulmonary Pathology. 3th edition.; pp: 20-28

Warburton, D. ;S. Bellusci ; S. De Langhe ; P.M. Del Moral ; V. Fleury ; A.MailleuxandW. Shi (2005): Molecular mechanisms of early lung specification and branching morphogenesis. Pediatric research, 57(5 Part 2), 26R.‏

Wei, G. W. (2009):Uncertain linguistic hybrid geometric mean operator and its application to group decision making under uncertain linguistic environment. International Journal of Uncertainty, Fuzziness and Knowledge-Based Systems, 17(02): 251-267.‏

Yamada, T.; E.Suzuki ;F. Gejyo and T. Ushiki (2002): Developmental changes in the structure of the rat fetal lung, with special reference to the airway smooth muscle and vasculature. Archives of histology and cytology, 65(1): 55-69.‏

التغیرات فی بنیة الرئة بعد الولادة لدى الفئران البیضاء

)دراسة نسیجیة(

زکیه محمد عابد سلیمان الحاسی^{1 2} ،  ابراهیم امین ماهر¹، منال محمد مرسی ¹

ومحمد احمد شحاته¹

¹ قسم التشریح الآدمی وعلم الأجنة - کلیة الطب البشری - جامعة الزقازیق- مصر

²قسم التشریح الآدمی وعلم الأجنة - کلیة الطب والجراحة - جامعة عمر المختار -  لیبیا

یقوم الجهاز التنفسی بوظائف حیویة فی الجسم. بالإضافة إلى کونه مسؤولاً عن توصیل وتبادل الغازات ، فإنه یلعب أیضًا وظائف مهمة مثل إصدار الأصوات ، والشم ، وإنتاج وإزالة العدید من المواد مثل الهستامین والبروستاجلاندین.

یتکون الجهاز التنفسی من الرئتین والممرات الهوائیة الأخرى المؤدیة إلى البیئة الخارجیة.

تم تقسیم تطور الرئة إلى خمس مراحل: المرحلة الجنینیة ، المرحلة الغدیة الزائفة ، المرحلة القنیة ، المرحلة التکیسیة، وأخیرًا مرحلة الحوصلة.

الهدف من هذه الدراسة هو تقییم التغیرات التی تحدث فی بنیة الرئة فی فترة ما بعد الولادة وربطها بالتطبیقات الإکلینیکیة.

أجریت هذه الدراسة على اثنتی عشرة أنثى و ست ذکور من الفئران البیضاء تم الحصول علیهم من بیت الحیوانات بکلیة الطب البیطری جامعة الزقازیق بمصر.

أظهرت الدراسة انه فی الیوم الرابع عشر للحمل ان رئة أجنة الفئران قد تشکلت من تکیسات هوائیة مقسمة بأعداد کبیرة من الحواجز الثانویة التی تحتوی علی شعیرات دمویة مزدوجة  ویبطن جدار القصیبات الهوائیة نسیج طلائی من الخلایا المکعبة ذات الأهداب. و قد تشکلت فی الیوم الحادی والعشرون بنیة الحویصلات الهوائیة الطبیعیة. وتتحول الشعیرات المزدوجة داخل الحواجز إلى شعیرات مرکزیة واحدة.  وتترکز ​​الکمیة الطبیعیة لألیاف الکولاجین فی جدار القصبات الهوائیة.

خلصت هذه الدراسة الی الکشف عن التغیرات النسیجیة والنسیجیة الکیمیائیة فی بنیة الرئة فی الجرذان البیضاء بعد الولادة. حیث یمکن أن تستخدم هذه المشاهدات فی تفسیر التطور المرضی للعدید من أمراض الجهاز التنفسی الموجودة فی الممارسة الإکلینیکیة.