Radial alveolar count assessment in the aging

Marta Ortega-Martínez1, Abel Gutiérrez-Marín1, Idalia Coronado-Hernández1, Ricardo M. Cerda-Flores2, Adriana Ancer-Arellano1, Carlos de-la-Garza-González3, Laura E. Rodríguez-Flores1, Jesús Ancer-Rodríguez1 and Gilberto Jaramillo-Rangel1 1 Departamento de Patología, Facultad de Medicina, Universidad Autónoma de Nuevo León, Av. Madero s/n, 64460

Monterrey, Mexico 2 Facultad de Enfermería, Universidad Autónoma de Nuevo León, Av. Gonzalitos 1500 Norte, 64460 Monterrey, Mexico 3 Departamento de Embriología, Facultad de Medicina, Universidad Autónoma de Nuevo León, Av. Madero s/n, 64460

Monterrey, Mexico

There are several parameters employed to evaluate lung growth or development in biopsies. The simplest method is the radial alveolar count (RAC), which is the number of alveoli transected by a perpendicular line drawn from the center of a respiratory bronchiole to the nearest septal division or pleural margin. The RAC method provides a reliable index of lung growth in intrauterine and early postnatal development, as well as in childhood. The method has been also applied in assessment of lung development in diverse pathological conditions. Up to date there are no data regarding RAC in healthy adulthood and its relation with aging. Lung specimens from CD1 mice at the age of 2, 12 or 24 months were fixed in 10% neutral-buffered formalin and paraffin-embedded. After staining of 5-µm sections with hematoxylin and eosin, RAC was determined. Results showed a statistically significant difference between the RAC means at 2 months (6.2±3.5) and 12 and 24 months (4.3±3.5 and 4.8±2.6, respectively); there was no significant difference between the means at 12 and 24 months (F=8.61, df1=2, df2=259, p=0.000; Student-Newman-Keuls test: M2≠M12=M24). Thus, our findings show that the RAC decreases during aging until it plateau. Aging is associated with morphometric changes in the lung that lead to decreased lung function. The chronicity of this process is poorly understood with respect to time of onset or progression. This work augments our understanding of this phenomenon and demonstrates that the RAC provides a simple and accurate method to analyze aging-related changes in the lung.

Keywords: radial alveolar count (RAC); mouse; aging

1. Introduction

Morphometry is the term applied to measurement of form or structure. Morphometric methods are particularly effective in studying and evaluating the organization of the biological tissues as they are sensitive enough to reveal remarkably small structural and developmental changes which otherwise would go undetected by qualitative observations. Furthermore, they do have the advantage of simplicity [1]. There are several morphometric data that give insight into structure-functional correlations in the lung [2, 3]. In 1960 Emery and Mithal introduced the radial alveolar count (RAC), which is the number of alveoli transected by a perpendicular line drawn from the center of a respiratory bronchiole to the nearest septal division or pleural margin [4, 5]. This method was later improved by Cooney and Thurlbeck for their application in intrauterine and early postnatal lung [6] and in late postnatal lung [7]. The RAC method provides a reliable index of lung growth in intrauterine and early postnatal development, as well as in childhood [4, 6, 7]. The method has been also applied in assessment of lung development in diverse pathological conditions and in experimental studies [8, 9]. On the other hand, the respiratory system undergoes various anatomical, physiological and immunological changes with age. These changes cause pulmonary disease which has significant consequences for the aging population. Chronic lower respiratory tract disease, defined as asthma, emphysema, chronic bronchitis, bronchiectasis, and chronic obstructive pulmonary disease (COPD), is the third leading cause of death in people aged 65 years and older. As the elderly population is increasing globally, it is necessary to understand the effect of the aging process in the lung in order to provide better care to this population [10, 11]. A stereotyped pattern of structural changes which occur in the lung as it ages is characterized by airspace enlargement [12]. Previous studies have shown that other senescent changes in lung, including the number of alveoli, vary among species and strains [13, 14]. Up to date there are no data regarding RAC in healthy adulthood and its relation with aging. In this study, we analyzed RAC in the aging mouse lung.

2. Material and Methods

Male CD1 mice were examined. Mice were maintained on a 12 h: 12h light-dark cycle with food and water ad libitum. Three animals were sacrificed at the age of 2, 12 or 24 months by cervical dislocation. To more consistently control the selection of sample sections, only the right lungs were processed and analyzed. Lungs were fixed in 10% neutral-buffered formalin and paraffin-embedded. Serial 5-µm sections were cut, deparaffinized in xylene and hydrated in a

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graded series of alcohol. Sections were stained with hematoxylin and eosin according to standard techniques [15]. RAC analysis was conducted using three tissue sections per animal taken from the middle of each lung, so that a portion of each lung lobe was included in each section. Identifying information on each slide was temporarily masked, until after RAC was completed. All respiratory bronchioles for each of the three sections were used for the counts. The RAC method was strictly performed as previously described [7]. Briefly, a line was dropped from each respiratory bronchiole to the nearest connective tissue septum. The number of alveoli cut by this line was then counted. Only closed alveoli were evaluated; alveoli that opened to an alveolar duct or alveolar sacs were neglected. According to this method, bronchioles partly lined by epithelium were selected (Fig. 1). The optical microscope images were taking using a Primo Star light microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) equipped with a Axio-Cam ICc1camera (Carl Zeiss Microscopy GmbH, Oberkochen, Germany). Images were edited and labeled using the software Zen lite 2011 (Carl Zeiss Microscopy GmbH, Oberkochen, Germany). All the information was captured in SPSS (IBM v.22). Values are presented as means (SD). The data was statistically analyzed using a one-way analysis of variance (ANOVA) followed by a Student-Newman-Keuls test. A p value less than 0.05 was considered significant.

Fig. 1 Radial alveolar count (RAC) in a mouse lung. A line was dropped from a respiratory bronchiole to the nearest connective tissue septum. The number of alveoli cut by this line was then counted. When a respiratory bronchiole was symmetrical, the line began at its geometric center (A). If the respiratory bronchiole was irregular, the line began at the midpoint between the conducting epithelium and the first branch point (B). AS= alveolar sac. Bars= 100 µm.

3. Results

Evaluation of tissue sections from 2-month-old mice revealed normal lung microanatomy. In contrast, light microscopic sections from 12- and 24-month-old mice showed an increase of alveolar size and a reduction in the alveoli number (Fig. 2). There was a statistically significant difference between the RAC means at 2 months (6.2 ± 3.5) and 12 and 24 months (4.3 ± 3.5 and 4.8 ± 2.6, respectively); there was no significant difference between the means at 12 and 24 months (F=8.61, df1=2, df2=259, p=0.000; Student-Newman-Keuls test: M2≠M12=M24) (Table 1). Table 1 Mean and standard deviation (SD) values of radial alveolar count stratified by age group.

Age (months) n* Mean SD

2 110 6.2 3.5 12 24

85 67

4.3 4.8

3.5 2.6

*n= number of respiratory bronchioles evaluated.

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Fig. 2 Representative histological sections of hematoxylin and eosin-stained lungs from 2-, 12-, and 24-month-old mice. Compared with the 2-month-old mice (A), an increase of alveolar size and a reduction in the alveoli number were evident in the 12- (B) and 24- (C) month-old mice. Bars= 50 µm.

4. Discussion

Morphometric techniques are effective in evaluating the organization of the biological tissues and could reveal interspecimen differences that reflect functional changes. Aging lungs exhibit both structural and functional alterations. In this study, we examined the RAC technique in CD1 mouse lungs during normal aging. Airspace enlargement may be a general characteristic of aging lung [10, 11]. Lungs undergo changes that lead to an increase in alveolar size. This lowers the alveolar surface tension and so reduces the elastic recoil of the lungs, which in turn causes a reduction in maximum achievable flow in the airways during the breathing cycle [16]. In concordance with this fact, we qualitatively observed an increase of alveolar size in the 12- and 24-month-old mice in comparison to the 2-month-old mice (Fig. 2). More controversial is if alveoli number diminishes with age. While Thurlbeck and Angus found that alveoli are not lost with age in humans [13], Herring et al. in rhesus monkeys [14] and Hyde et al. in dogs [17] found that the number of alveoli declined significantly with age. Our results revealed that in aging CD1 mice RAC initially diminishes and then plateau (Table 1). To the best of our knowledge, this is the first time that RAC is evaluated in aging. Thus, similar analyses are necessary in other species and in other mouse strains. Calvi et al. found that aging associated airspace enlargement in mouse develops during middle age (8-12 months approximately) and is accompanied by early oxidative stress, cell death and elastase activation [12]. Assessment of RAC is necessary at other time points during mouse adult life in order to establish the exact age at which the RAC diminution commences. Also, other studies are required to determine the molecular basis of this diminution. In conclusion, our findings show that the RAC decreases during aging until it plateau. Aging is associated with morphometric changes in the lung that lead to decreased lung function. This work augments our understanding of this phenomenon and demonstrates that the RAC provides a simple and accurate method to analyze aging-related changes in the lung.

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Acknowledgements We are grateful to Martín García-Guerrero and Ruth Alvarez-Cantú for technical assistance.

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