Analysis of the area and the number of pulmonary alveoli through the normal aging process in CD1 mouse

During the aging process, the lung exhibits structural changes accompanied by a decline in its function. The related information currently available is still scarce and contradictory. In addition, changes in some pulmonary parameters through aging process are species- and strain-dependent. The aim of this study was the assessment of the area and the number of pulmonary alveoli through the normal aging process in CD1 mouse. Paraffin-embedded sections of lungs from CD1 mice at age of 2, 6, 12, 18, or 24 months were stained with hematoxylin and eosin and examined using a light microscope. Images were captured using a camera linked to an image analysis software to measure areas and count alveoli. There was a significant difference in the alveolar area among the ages analyzed (F=87.53, Sig.=0.000). The alveolar area of the 6-, 12-, 18-, and 24-month-old mice was significantly greater (all p values < 0.001) than in mice at 2 months of age. Also, the alveolar number was significantly different among the ages tested (F=3.21, Sig.=0.023). The number of alveoli in mice at 2 months of age was greater than in mice at all other age groups, reaching statistical significance when compared with the 6-, 12-, and 18-month-old mice (p values of 0.044, 0.014, and 0.002, respectively). Thus, we observed an increase in alveolar area and a decrease in alveolar number through the aging process. This information might be useful to understand pathologic changes underlying susceptibility of elderly individuals to chronic lower respiratory tract diseases.


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23 Abstract 24 During the aging process, the lung exhibits structural changes accompanied by a decline in its 25 function. The related information currently available is still scarce and contradictory. In 26 addition, changes in some pulmonary parameters through aging process are species-and 27 strain-dependent. The aim of this study was the assessment of the area and the number of 28 pulmonary alveoli through the normal aging process in CD1 mouse. Paraffin-embedded 29 sections of lungs from CD1 mice at age of 2, 6, 12, 18, or 24 months were stained with 30 hematoxylin and eosin and examined using a light microscope. Images were captured using a 31 camera linked to an image analysis software to measure areas and count alveoli. There was a 32 significant difference in the alveolar area among the ages analyzed (F=87.53, Sig.=0.000). 33 The alveolar area of the 6-, 12-, 18-, and 24-month-old mice was significantly greater (all p 34 values < 0.001) than in mice at 2 months of age. Also, the alveolar number was significantly Aging process is associated with structural changes in the lung that lead to a decrease in its 47 function. These alterations may increase susceptibility of elderly individuals to chronic lower 48 respiratory tract diseases, such as asthma and chronic obstructive pulmonary disease (COPD) 49 [1,2]. Lung aging-related research has been devoted mainly to the analysis of alveolar 50 structure. There is almost a consensus that the principal feature of the senile lung is the 51 homogeneous enlargement of alveolar airspaces [3][4][5] show alterations in this parameter [10]. Also, it has been suggested that BALB/cNNia mice 58 may be resistant to an aging-related increase in alveolar size [11], whereas DBA/2 and 59 C57BL/6 mice display an air space enlargement during late life stages [12,13]. These events 60 could be related to innate pulmonary variations between mouse strains; for example, it has 61 been described that C3H/HeJ, C57BL/6J and A/J mice differ naturally in alveolar size [14].

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Although the mouse strain CD1 has been widely used to examine multiple aspects of the 63 general aging process [15][16][17], its alveolar morphometric analysis has not been reported yet. 64 We previously evaluated the cell turnover of the bronchiolar epithelium and analyzed the    The results are presented as means ± 1 standard error (SE). One-way ANOVA test was used 109 to determine statistical significance (p < 0.05), followed by post hoc analysis using the least 6 / 15 110 significant difference (LSD) test when significant differences were found between groups.

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The data were analyzed using the SPSS for Windows software (SPSS, Inc., Chicago, IL, 112 USA), release 21.0.

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The results are summarized in Table 1 that the AA of the 6-, 12-, 18-, and 24-month-old mice (598±12, 668±18, 881±20, and 119 743±21 m 2 , respectively) was significantly greater (all p values < 0.001) than in mice at 2 120 months of age (488±11 m 2 ). The AA in mice at 24 months of age was significantly higher 121 than in the 6-and 12-month-old mice (p values < 0.001, and 0.002, respectively). Mice at 18 122 months of age had a significantly higher AA than mice at 6 and 12 months of age (both p 123 values < 0.001). Also, mice at 12 months of age had a significantly higher AA than in mice at 124 6 months of age (p value of 0.002). Finally, the AA in mice at 24 months of age was 125 significantly smaller than in the 18-month-old mice (p value < 0.001) (Fig 2). The alveolar respectively) than in mice at 2 months of age (105.3±14.6) (Fig 3).      than in mice at 2 months of age (*). The AA in mice at 24 months of age was significantly 145 greater (at most p = 0.002) than in the 6-( †), and 12-month-old mice ( §). Mice at 18 months 146 of age had a significantly higher AA than mice at 6 ( †) and 12 ( §) months of age (both p 147 values < 0.001). Also, mice at 12 months of age had a significantly higher AA than in mice at 148 6 months of age ( †; p = 0.002). Finally, the AA in mice at 24 months of age was significantly 149 smaller than in the 18-month-old mice ( ‡; p < 0.001). Values are expressed as means ± 1 150 standard error. Data were analyzed by a one-way ANOVA followed by a least significant 151 difference test. Results were considered statistically significant at p < 0.05.  3. Alveolar number (AN) from healthy 2-,6-, 12-, 18-, and 24-month-old CD1 mice. 154 The AN of the 6-, 12-, and 18-month-old mice was significantly smaller than in mice at 2  Our findings revealed that alveolar area increased and number of alveoli decreased with 167 age. Alveolar area increased significantly among all ages between 2 and 18 months (Table 1   168 and Fig 2), while the 2-month-old mice had a significantly higher number of alveoli when 169 compared to mice at 6, 12, and 18 months of age (Table 1 and Fig 3).

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The results obtained in most of the previous studies coincide with ours (Table 2)   Interestingly, we found a significant decrease in alveolar area between the 18-and 24-188 month-old mice (Fig 2) and an increase in alveolar number between the same ages, although 189 it was not statistically significant (Fig 3). We and other authors previously reported changes