The postnatal development and growth of the cardio-respiratory system in sprague-dawley rats Ancuta Apreutese 1,2, Cedric Gordon 1, Roy Forster 3, Andrew Graham 1, Bernard Palate 3, Julius Haruna 1 and Marie-Odile Benoit-Biancamano 2 1 CiToxLAB in North America, Laval, Qc, Canada - 2 Faculté de Médecine Vétérinaire, Université de Montréal, St-Hyacinthe, Qc, Canada - 3 CiToxLAB in France, Evreux CEDEX, France Abstract The purpose of this study was to investigate the histomorphological changes of the cardiorespiratory system in rat pups over the first month of life. The heart weight and the heart weight relative to body weight ratio were calculated for 51 rats over 13 timepoints, ranging from postnatal day 1 (PND1) to PND30. For each timepoint, whenever possible, equal number of females and males were used. The aortic wall progressively increased in thickness throughout the duration of our study as a result of an accumulation of extracellular matrix (collagen and elastic fibers). Postnatally, the cardiac volume essentially increased by cardiomyocyte hypertrophy, which showed a more mature appearance around PND21. In the newborn myocardium, mitotic figures and apoptotic bodies were frequently seen, increased on PND4 and showed a diffuse pattern. At birth, the trachea was lined by immature columnar ciliated epithelial cells and submucosal glands became visible around one week after birth. The newborn rat has no alveoli and breathes with smooth, large gas exchange units termed “primary saccules”. An intense interstitial cellular proliferation was observed in the lungs between PND6 and PND14, when the majority of alveoli are separated by new secondary septa, formed from the primary walls. These changes were accompanied by few mitoses and/ or individual apoptotic cells. The findings described herein suggest that the cardio-respiratory system of rats is immature at birth. The data generated will serve as background historical database that will be valuable when performing postnatal developmental toxicity studies. Introduction In recent years juvenile toxicity studies have been an area of significant concern in preclinical development of drugs and have been target of academic interest as well. Added to other issues of testing strategies (e.g. route of administration, target organs, age of pups), monitoring of landmarks of postnatal histomorphological development in rodents is a critical component in study design and a fundamental element of such studies. In the work presented here we have investigated the histomorphological changes of cardio-respiratory system in rat pups over the first month of life. Materials and methods The 51 pups in this study were the progeny of 6 time-mated Sprague-Dawley rats dams Crl:CD (SD) purchased from Charles River Laboratories Canada Inc. (St-Constant, QC) at Day 0 of pregnancy. The tissues were obtained from pups at post-natal day (PND) 1, PND2, PND4, PND6, PND, PND10, PND14, PND17, PND21, PND24, PND26, PND28 and PND30. When possible, equal number of females and males were used for each occasion. The body weight and heart weight were recorded for each time point and the organ weight to body weight ratio was calculated; the results were statistically evaluated using a dispersion diagram and a polynomial regression. The aorta, heart, trachea and lungs were collected and placed in 10% buffered formalin. After 24hrs, the tissues were rinsed and stored in 70% ethanol until histological processing. The fixed samples were trimmed, processed and paraffin-embedded. Sections were cut at 4µm thickness and stained with hematoxylin and eosin (H&E). The immunochemical demonstration of vimentin within lung samples was performed using the kit Dako Envision HRP(AEC) (a rabbit monoclonal antibody diluted at 1:400) and used according to manufacturer’s instructions. In order to clarify the distinction between collagen and elastin fibers within the aortic wall, Masson’s trichrome stain was employed. third postnatal week. The neonatal cardiomyocytes are elliptical to elongate, scant and short, with a single, central nucleus. The intercalated discs are poorly developed, the cross striations are absent and the myofibrils are scant and mainly located peripherally (figure 4). An increase in the myocardial contractile function appears to be associated with enlarged cellular size by addition of new, orderly sarcomeres leading to the apparition of cross-striations, visible around PND10. Through the first month of life, the tracheal lumen increased approximately 4-5 folds. At birth, the trachea was lined by immature columnar, poorly ciliated epithelium; the first submucosal glands became visible around one week after birth. The newborn rat has no alveoli and breathes with large gas exchange units termed “primary saccules”. These structures were lined by a smooth and thick, highly cellular wall, with a central sheet of connective tissue flanked on both sides by flattened cells (primary septa) (figure 5). The respiratory bronchioles were lined by a simple non ciliated cuboidal epithelium while the mucosa of the large bronchi was composed of pseudostratified ciliated epithelium. Perinatally, the mucosal glands and the bronchus-associated lymphoid tissue (BALT) were absent. Between PND6 and PND14, the primary septa became modified by the elevation of multiple, low secondary crests, appearing as narrow ridges projecting into the airspace and subdividing it into many, incompletely closed smaller units (figure 6). These new septa were thickened by an intense cellular proliferation of interstitial cells which have indistinct borders, scant cytoplasm and large oval nuclei. The cells were positive for vimentin immunolabeling, consistent with fibroblasts (figure 7). By the end of the third postnatal week, the septa underwent noticeable thinning and the lung parenchyma progressively matured, resulting in increased gas exchange surfaces (figure 8). Organs development and growth are driven by concurrent processes of programmed cell death and increased mitoses. Those histological features are present in specific cell types and their proportions vary in developing animals. Within the myocardium, apoptotic bodies and increased mitoses were more obvious, peaked at PND4 and showed a diffuse pattern; while the lung, trachea and aorta were more discreetly remodeled by apoptosis and/or mitoses. Figure 4 : Histological aspect of immature myocardium (Sprague-Dawley rat PND1). The myocardium is hypercellular, cardiomyocytes have a primitive appearance, are pale with high nuclear to cytoplasmic ratio, have less prominent cross-striations and high mitotic index (arrows). H&E 400x. Figure 5 : Histological aspect of immature lung parenchyma (SpragueDawley rat PND1). The “primary saccules” are lined by a thick and highly cellular wall, with a central sheet of connective tissue flanked on both sides by flattened cells (arrows). H&E 200x. Figure 6 : Light micrograph of lung parenchyma (Sprague-Dawley rat PND10). Note the intense cellular proliferation within the alveolar wall forming secondary septa projecting into the air space (arrows). H&E. 200x. Figure 7 : Histological aspect of lung parenchyma (Sprague-Dawley rat PND21). The alveoli are lined by slender walls and the air spaces are much more subdivided into smaller gas units creating larger gas exchange surface areas. H&E 200x. Figure 1: Diagram of the polynomial regression of the ratio of heart weight relative to body weight in Sprague-Dawley rats. Note that, at birth, the ratio of heart weight relative to body weight was higher than that on PND30 suggesting that by the end of the first month of life, the heart weight increased at a less dramatic rate than body weight. Figure 8 : Photomicrograph of immature lung parenchyma (SpragueDawley rat PND1). Note the scattered vimentin-positive cells with a brown-colored cytoplasm. Vimentin immunolabelling 200x. Figure 9 : Photomicrograph of immature lung parenchyma (Sprague-Dawley rat PND10). Note the increased signal for vimentin immunolabelling within the alveolar septa. Vimentin immunolabelling 200x. Results and Discussion During the first month of life, both body and heart weight increased with age, and most significantly after PND21. The heart weight relative to body weight was higher at birth, and progressively decreased until PND30, suggesting that by the end of first month of life the heart weight increased at a less steep rate than body weight (figure 1). As a broad generalization, at birth, most organs showed increased cellularity, the interstitium was edematous and contained loosely arranged mesenchymal cells. Qualitative changes in the aortic wall from PND1 to PND30 were indicative of the adaptive growth response of the thoracic aorta to the rapidly increasing size of the whole body. When compared with the perinatal period, the thoracic aorta showed an approximately 1.5 fold increase in luminal diameter after the first week of life and around 3-fold increase at the end of the first month. The wall appears hypercellular and elastic fibers are less pronounced (figure 2); subsequently the proportion between components changed in favor of elastic fibers addition (figure 3). The rat heart is morphologically immature at birth and grows by cell hyperplasia over the perinatal period followed by cell hypertrophy around the Figure 3 : Photomicrograph of thoracic aorta (Sprague-Dawley rat PND30). Note the pronounced amount of elastic fibers within the media (orange) with concurrent increase in wall thickness. Masson’s trichrome 400x. Conclusion As in human, the cardio-respiratory system of Sprague-Dawley rats is immature at birth. The lungs and airways reached maturity 3 weeks after birth while cardiomyocyte hypertrophy was noticed until PND30. Tissue remodeling by apoptosis and/or mitosis was minimal in the lung, trachea and aorta and marked within the heart, especially at PND4. These results will serve as database of background age-related changes of neonatal and juvenile rats in preclinical toxicologic studies. Figure 2 : Histological aspect of immature thoracic aorta (SpragueDawley rat PND1). At birth, the aortic wall appears hypercellular and elastic fibers are less pronounced than in mature aorta. Masson’s trichrome 400x. www.citoxlab.com references 1. Kok Wah Hew and Kit A. 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