Omar, Tanya S.

Aging‐related changes in lung mechanics of human subjects are characterized by a decrease in resistance and an increase in static compliance (decrease in elastic recoil). Together, these changes result in an increase in residual volume in the absence of an increase in total lung capacity. In C57BL/6x129 mice, it was reported that lung compliance increases with aging while elastic recoil pressure does not change. However, in these studies, “old” mice at 24 months of age were compared to “young” mice at 2 months of age, but at this younger age, alveologenesis is incomplete (completion does not occur until ~6 months of age). Accordingly, it remains to be established if the same aging‐related changes in lung mechanics are observed using fully developed adult C57BL/6x129 mice for comparison. To this end, we compared lung mechanics in C57BL/6x129 mice at 6 months (100% survival, n = 6), 18 months (90% survival, n = 6), and 24 months (75% survival, n = 6) of age. Respiratory system static mechanics (Resistance, R rs ; Compliance, C rs ; Elastance, E rs ) were assessed following tracheal intubation using the FlexiVent. There was a progressive aging‐related decrease in R rs (6 month, 0.47 ± 0.02; 18 month, 0.41 ± 0.02; 24 month, 0.35 ± 0.02 cmH 2 O.s/ml), an increase in C rs (6 month, 0.046 ± 0.002; 18 month, 0.052 ± 0.002; 24 month, 0.062 ± 0.002 ml/cmH 2 O), and a decrease in E rs (6 month, 21.73 ± 1.19; 18 month, 19.30 ± 0.87; 24 month, 16.62 ± 1.25 cmH 2 O/ml). Additionally, lungs were fixed following tracheal instillation of 4% paraformaldehyde at 25 cmH 2 O. Thereafter, lungs were dehydrated, paraffin embedded, sectioned (6 μm thick), and stained (hematoxylin and eosin). Using standard stereological approaches, lung sections were analyzed in a uniform systematic but random fashion to assess alveolar morphology (measurement of average distance between alveolar walls based on mean linear intercept, L m ). There was a progressive aging‐related increase in L m (6 month, 34 ± 2; 18 month, 40 ± 2; 24 month, 45 ± 2 μm) consistent with an aging‐related emphysematous‐like change in lung morphometry. Taken together, these results in appropriately aged C57BL/6x129 mice demonstrate an aging‐related change in lung mechanics and morphometry consistent with what is observed in aging humans. Therefore, the results of the present study confirm the use of rodent models for studying aging‐related changes in lung mechanics and morphometry. Support or Funding Information National Institute of Health grants R01‐AG044615 (CBM & GCS) and T32‐HL105355 (JEE).

The risk for respiratory diseases is significantly increased in the elderly population (>65 years of age), which may be partially due to an inability to adequately clear the airways resulting from a progressive aging‐related atrophy and weakening (i.e., sarcopenia) of the diaphragm muscle (DIAm). In humans, reduced maximum inspiratory and transdiaphragmatic pressures suggest DIAm weakening, but muscle fiber atrophy has not been examined. In a National Institute on Aging (NIA) supported mouse model of aging (C57BL/6J strain), we recently showed that DIAm sarcopenia exists and affects non‐ventilatory functions (Greising et al., Exp Gerontol 48:881–887, 2013; Greising et al., Am J Physiol Lung Cell Mol Physiol 309:46–52, 2015). Importantly, rodent models such as this provide a valuable tool to explore the mechanisms underlying DIAm sarcopenia. Currently, the NIA also provides two inbred rat models for aging‐related research: the Fischer 344 (F344) and Brown Norway (BN) rat strains. The purpose of this study was to comprehensively examine whether sarcopenia also exists in the DIAm of F344 and BN rat strains provided by the NIA. Young (6 month, 100% survival) and old (24 month, 50% survival) F344 and young (6 month, 100% survival) and old (32 month, 50% survival) BN male, pathogen free, rats were obtained from the NIA rodent colony. In DIAm strips from the right midcostal region, measurements of contractility, contractile protein concentration, relative myosin heavy chain (MyHC) isoform expression, fiber type distribution and fiber cross‐sectional area (CSA) were obtained. Peak twitch specific force (P t ) was reduced by ~17% and ~14% in the aged F344 and BN rats, respectively. Additionally, maximal specific force (P o ) was reduced by ~24% and ~12% in the aged F344 and BN rats, respectively. Thus, although there was significant aging‐related DIAm weakness in both rat strains, reductions in both P t and P o were more pronounced in old F344 compared to BN rats. In F344 rats, aging also affected DIAm fiber type proportions with a significant increase in the proportion of type I fibers in old (45 ± 3%), compared to young (35 ± 3%) rats, with a concomitant decrease in the relative proportions of both type IIa fibres and type IIx and/or IIb fibers. In BN rats, DIAm fiber type proportions were not affected by aging. In both F344 and BN rats, the CSA of type I and IIa fibers were unchanged, whereas the CSA of type IIx and/or IIb DIAm fibers were reduced by ~20% in F344 rats and by ~15% is BN rats. Similar to DIAm weakness, the selective aging‐related atrophy of type IIx and/or IIb DIAm fibers was more pronounced in F344 rats. In conclusion, DIAm sarcopenia is clearly present in both F344 and BN rat strains, but more pronounced in the F344 strain. These results indicate that in addition to the C57BL/6J mouse model, the F344 rat strain is an appropriate model for research pertaining to DIAm sarcopenia. Support or Funding Information Support: National Institute of Health grants R01‐AG044615 (CBM & GCS) and T32‐HL105355 (JEE).

Sarcopenia is the age-related reduction of muscle mass and specific force. In previous studies, we found that sarcopenia of the diaphragm muscle (DIAm) is evident by 24 mo of age in both rats and mice and is associated with selective atrophy of type IIx and IIb muscle fibers and a decrease in maximum specific force. These fiber type-specific effects of sarcopenia resemble those induced by DIAm denervation, leading us to hypothesize that sarcopenia is due to an age-related loss of phrenic motor neurons (PhMNs). To address this hypothesis, we determined the number of PhMNs in young (6 mo old) and old (24 mo old) Fischer 344 rats. Moreover, we determined age-related changes in the size of PhMNs, since larger PhMNs innervate type IIx and IIb DIAm fibers. The PhMN pool was retrogradely labeled and imaged with confocal microscopy to assess the number of PhMNs and the morphometry of PhMN soma and proximal dendrites. In older animals, there were 22% fewer PhMNs, a 19% decrease in somal surface area, and a 21% decrease in dendritic surface area compared with young Fischer 344 rats. The age-associated loss of PhMNs involved predominantly larger PhMNs. These results are consistent with an age-related denervation of larger, more fatigable DIAm motor units, which are required primarily for high-force airway clearance behaviors. NEW & NOTEWORTHY Diaphragm muscle sarcopenia in rodent models is well described in the literature; however, the relationship between sarcopenia and frank phrenic motor neuron (MN) loss is unexplored in these models. We quantify a 22% loss of phrenic MNs in old (24 mo) compared with young (6 mo) Fischer 344 rats. We also report reductions in phrenic MN somal and proximal dendritic morphology that relate to decreased MN heterogeneity in old compared with young Fischer 344 rats.

New FindingsWhat is the central question of this study?Several rat models are commonly used to study the physiology of ageing (e.g. Fischer 344 and Brown Norway rats are recommended by the USA National Institute of Ageing). Diaphragm muscle sarcopenia (ageing‐related muscle weakness and atrophy) remains incompletely described in these rat models.What is the main finding and its importance?Diaphragm muscle sarcopenia is present in both the Fischer 344 and Brown Norway rat strains, but appears more pronounced in Fischer 344 rats.The risk for respiratory diseases increases in adults >65 years of age, which may be attributable in part to ageing‐related weakening and atrophy (i.e. sarcopenia) of the diaphragm muscle (DIAm). The mechanisms underlying DIAm sarcopenia remain unknown. Based on existing evidence, we hypothesized that sarcopenia is most evident in type IIx and/or IIb DIAm fibres, i.e. more fatigable motor units. Currently, the USA National Institute on Aging supports Fischer 344 (F344) and Brown Norway (BN) rat strains for ageing‐related research, yet DIAm sarcopenia has not been evaluated comprehensively in either strain. Thus, the present study examined DIAm sarcopenia in older adult F344 (24 months old, 50% survival) and BN rats (32 months old, 50% survival), compared with young adult (6‐month‐old) F344 and BN rats. Measurements of contractility, contractile protein concentration, fibre type distribution and fibre cross‐sectional area were obtained from midcostal DIAm strips. Maximal specific force was reduced by ∼24 and ∼13% in older F344 and BN rats, respectively. Additionally, although the cross‐sectional area of type I and IIa DIAm fibres was unchanged in both F344 and BN rats, the cross‐sectional area of type IIx and/or IIb DIAm fibres was reduced by ∼20 and ∼15% in F344 and BN rats, respectively. Thus, although there was ageing‐related DIAm weakness and atrophy, selective to type IIx and/or IIb DIAm fibres, in both F344 and BN rats, the sarcopenic phenotype was more pronounced in F344 rats.