Age-Related Changes in Respiratory System Mechanics and Lung Morphometry

Abstract

Aging leads to degenerative changes in lung structural and mechanical properties that may impact breathing. Relatively little is known about how age-related alterations in lung mechanical properties impact expiratory duration (T_E) and residual volume (RV). The present study tested three hypotheses: 1) alveolar airspace increases with age due to alveolar wall breakdown, 2) lung elastic recoil decreases, slowing expiratory flow (E-flow), and 3) airway compliance (Caw) decreases, promoting air trapping reflected by increased RV. To test these hypotheses, we evaluated changes in alveolar size using histological assessments, mechanical properties using forced oscillation perturbations with a programmable mechanical ventilator and breathing parameters using plethysmography in young (6 months) and old (24 months) Fischer 344 rats. Mean linear intercept (L_m) was higher in old (65 ± 10 μm) compared to young (55 ± 5 μm) rats, indicating alveolar airspace increase. In old rats, decreased elastic recoil was indicated by increased parameter K (0.178 ± 0.010 vs. 0.167 ± 0.013 in young) and higher V₁₀/TLC (80.7 ± 1.2% vs. 78.5 ± 2.5% in young). In addition, RV was higher in old rats (0.41 ± 0.10 mL vs. 0.27 ± 0.05 mL in young), consistent with air trapping. These mechanical changes were associated with increased E-flow (~30%) in old rats, attributable to increased tidal volume (2.08 ± 0.41 mL vs. 1.53 ± 0.36 mL in young) as T_E was unchanged. These results indicate important changes in the lung parenchyma, causing alteration in passive and active properties of the respiratory system in aging rats.

New & Noteworthy:

The present study provides novel physiological insights into aging by measuring airway compliance and residual volume via forced oscillation perturbations using a programmable mechanical ventilator (flexiVent—currently considered a gold standard for in vivo lung function measurements). In addition, dynamic measurements of lung inflation reported herein may serve as early indicators of altered elastic properties. Taken together, the findings offer a comprehensive assessment of age-related changes in lung structure and function.

Graphical Abstract

Introduction

Aging leads to progressive changes in lung structure, respiratory muscle function, and respiratory mechanics in both humans (1–3) and rodent models (4–7). The global population is aging, and by 2050 an estimated 21% of the world’s population will be 60 years of age or older (1, 8). This demographic shift is accompanied by an increased incidence of chronic obstructive pulmonary disease (COPD) and higher mortality from pneumonia in older adults (2, 3, 9). These age-related differences in respiratory function may be understood in the context of underlying structural and functional alterations in lungs.

There are age-related changes in lung structure that affect lung mechanics and ventilation. Structural changes in the lungs with aging include irreversible alveolar airspace enlargement and alveolar wall breakdown (4, 10–14). These age-related structural changes in the lung result in increased compliance with a decrease in elastic recoil in humans (15, 16) and mice (4, 7, 17). In contrast, reports of lung compliance in aging rat models are more inconsistent, possibly reflecting differences in strain (e.g., Sprague-Dawley vs Fischer 344) (11, 18, 19). An age-related decrease in lung elastic recoil impairs proper lung emptying, leading to air trapping and hyperinflation, which subsequently increases residual volume (RV) and/or functional residual capacity (FRC) as seen in humans (2, 20, 21) and rats (18). Furthermore, the conducting airway resistance decreases in old mice (4, 7, 22) and old Fischer 344 rats (23).

Changes in lung and chest wall mechanics with aging impact both inspiratory and expiratory patterns (2, 21, 24). A previous study reported increased respiratory rate (RR) in old rats (25), whereas in mice RR decreased with age when comparing young (26) and old (27) animals within the same laboratory. Age-related changes in inspiratory (T_I) and expiratory durations (T_E) in rats are unclear, but by comparing results from young (26) and old (27) mice the duty cycle of breathing decreases with age due to a prolongation of expiration.

The present study tested the hypotheses that in older Fischer 344 rats: 1) alveolar airspace increases, 2) lung elastic recoil decreases slowing expiratory flow rate, 3) airway compliance decreases, leading to air trapping reflected by an increase in residual volume. Accordingly, we characterized histological, mechanical, and functional parameters in young (6-month-old) and old (24-month-old) Fischer 344 rats.

MATERIALS AND METHODS

All protocols were approved by the Institutional Animal Care and Use Committee (A00003068–17-R23) at the Mayo Clinic and followed the American Physiological Society and National Institute of Health Guidelines. A total of 33 Fischer 344 rats (19 females, 14 males) were used in the study, comprising 21 young (6-month) and 12 old (24-month) animals. Animals were assigned to three experimental procedures and whenever feasible, the same animals were used across multiple procedures to minimize animal use and enable cross-comparison of the data.

Whole Body Plethysmography

Ventilatory parameters were measured using a single-chamber whole-body plethysmograph (DSI Buxco FinePointe Series WBP - Rat). In this system, conscious and unrestrained animals breathe naturally while enclosed in a sealed chamber. Breathing causes small pressure fluctuations in the chamber referred to as box flow. The system software applies Boyle’s Law to convert measured box flow into respiratory flow and then applies the Drorbaugh & Fenn correction to correct tidal volume (V_T), for temperature and humidity (28). The corrected signal is then analyzed by the FinePointe software to calculate V_T, RR, minute ventilation (V_E), T_I, and related ventilatory parameters. This approach stemmed from early whole-body plethysmography methods (28, 29) that was later adapted to rodents (30). Prior to each experiment, the system was calibrated according to manufacturer guidelines. Animals were allowed to acclimate to the chamber for 10–15 min before data collection. After the acclimatization period baseline measurements were recorded for 5 min, eupnea for 7 min, and recovery for 7 min while a constant flow of fresh air (3 L/min), referred to as bias flow was maintained to prevent CO₂ accumulation. V_T, respiratory frequency (f), V_E, T_I and T_E were recorded every 2 s for at least 30 min.

Assessment of Respiratory System Mechanics

Rats were anesthetized by intraperitoneal (IP) injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). Anesthetic depth was confirmed at regular 15-minute intervals by absence of toe pinch and palpebral reflexes. Supplementary doses of ~1/3 the initial dose were given if either reflex was present. A tracheostomy was performed, and the trachea cannulated using a 14 G cannula (XS-C14, SCIREQ, Montréal, Canada) and an airtight seal was secured with silk sutures. The animals were then placed in a supine position and connected to a programmable mechanical ventilator (flexiVent FX system with FX4 module, flexiWare v8.3, SCIREQ Scientific Respiratory Equipment Inc., Montréal, Canada). Muscles were paralyzed by IP injection of pancuronium (0.2 mg/kg) and rats were then ventilated for 3 min under controlled parameters (90 breaths/min) to ensure drug efficacy. Three distinct mechanical perturbations were applied, each performed three times and always preceded by a deep inflation of the lungs to 30 cm H₂O over 3 s. The peak pressure was held for an additional 3 s to allow alveolar pressure to equilibrate. This process expands the lungs from FRC to total lung capacity (TLC), providing a measure of inspiratory capacity (IC) (31, 32).

Single Frequency Forced-Oscillation Maneuver - Snapshot 90

The first mechanical perturbation applied was the “SnapShot 90”, a brief (0.72 s), single-frequency (1.5 Hz, equivalent to 90 breaths per min) forced-oscillation maneuver performed according to the manufacturer’s guidelines (flexiVent FX, SCIREQ). The amplitude (1 mL/100 g) of the imposed oscillation approximated the animal’s V_T. During each measurement, volume and pressure signals were recorded, and the flow signal was derived from the volume trace. These signals—volume, pressure, and flow—were fit to the single-compartment model using linear regression. The single-compartment method treats the entire respiratory system (airways + lung tissue + chest wall) as one compartment with a single resistance (Rrs), elastance (Ers), and compliance (Crs). This perturbation returns values for total Rrs, Ers, and Crs. SnapShot 90 provides a detailed characterization of the resistive and elastic properties of the respiratory system during normal tidal breathing. These measurements include contributions from the conducting and peripheral airways, lung tissue, and chest wall mechanics.

Broadband Forced-Oscillation Maneuver - Quick Prime 3

Following the SnapShot 90 maneuver, the “Quick Prime 3” perturbation was performed (3 mL/kg, 3 s) with oscillatory frequencies ranging from 1 to 20.5 Hz—spanning both above and below the animal’s natural ventilatory frequency—and was conducted according to the manufacturer’s guidelines (flexiVent FX, SCIREQ). During this procedure, volume and pressure signals were recorded, and flow was derived from the volume signal at each frequency. Respiratory input impedance was then calculated at every frequency and fit to the Constant Phase Model introduced by Hantos and colleagues (33), which is implemented in the analysis software. This model mathematically represents the mechanical properties of the respiratory system, allowing interpretation of the impedance data. From it, the following parameters are extracted: Newtonian resistance (Rn), tissue damping (G), tissue elastance (H), and hysteresivity (η). The Constant Phase Model separates the contributions of the central conducting airways and the peripheral alveolar tissues. It enables frequency-dependent analysis of how the respiratory system resists the imposed oscillatory flow. Quick Prime 3 thus provides a comprehensive profile of the respiratory system’s mechanical response across a wide frequency range.

Pressure-Volume Loop

Pressure-volume (P-V) loop were obtained by gradually increasing airway pressure in discrete steps, pausing airflow briefly after each increment to allow the lungs to equilibrate and was performed according to the manufacturer’s guidelines (flexiVent FX, SCIREQ). This process continued until a pressure of 30 cm H₂O was reached, representing TLC. Static compliance was calculated by evaluating the volume at 5 cm H₂O during the deflation limb. When the chest cavity is closed, Cst reflects both lung and chest wall mechanics. Lastly, the hysteresis area of the P-V loop corresponds to energy loss due to tissue resistance and surfactant-related surface tension changes during breathing. The lung deflation decay constant (K) derived from the deflation limb using the Salazar–Knowles equation (V=A−Be^−KP) provides insight into lung recoil or compliance, independent of TLC (19, 32, 34, 35). P-V loop measure the distensibility of the lungs and chest from the end of exhalation to full inflation. Typically, sigmoidal shape, P-V loops display mechanical properties of lung tissue and energy loss known as hysteresis (4).

Full-Range Pressure-Volume Curves

Prior to the full-range pressure–volume (P-V) measurements performed according to the manufacturer’s guidelines (flexiVent FX, SCIREQ) animals were ventilated with 100% oxygen under closed chest wall for approximately 5 min, after which the ventilator valves were closed for 10 min. During this interval, pulmonary oxygen uptake caused progressive collapse of the alveoli. The piston delivered a constant airflow to inflate the lungs up to 35 cm H₂O, followed by withdrawal to −10 cm H₂O resulting in a total of three inflation–deflation loops. The first cycle was performed in degassed lungs, whereas subsequent cycles were carried out after alveolar recruitment, with the final deflation returning to baseline pressure (0 cm H₂O).

Static compliance (C) was calculated as the slope of the linear segment of the deflation limb between 3 and 8 cm H₂O. The deflation P-V relationship was characterized by the V₁₀/TLC, which is defined as the volume at 10 cm H₂O (V₁₀) normalized to TLC. Lung opening pressure (Pₒₚ) was determined from the initial portion of the first inflation limb by identifying the characteristic elbow shape associated with a fast opening of the lung parenchyma. Compliance of collapsible airways (Caw, μL/cm H₂O) was calculated by linear regression of the early inflation segment before parenchymal opening, reflecting airway tree distension (36). All mechanical measurements were obtained with the chest wall intact to obtain residual volume measurements.

Lung Histology and Morphometry

After mechanical ventilation, animals were detached, and lungs were inflated via a tracheal cannula at 25 cm H₂O with 4% paraformaldehyde. After ≥15 min equilibration, the trachea was securely tied off, the cannula withdrawn, and the lungs excised. The reference lung volume (LV) was determined using a water displacement method. In this procedure, a lung that was tied off at a pressure of 25 cm H₂O was fully submerged without touching the sides of the beaker in a pre-weighed beaker filled with saline. The change in the water level and the weight of the saline was then measured, which indicated the initial LV. This method is described in detail in the study by (37).

Lungs were fixed in 4% paraformaldehyde for ≥24 h before identical tissue processing consisting of buffered formalin fixation, graded ethanol dehydration, xylene clearing, wax infiltration (Leica Biosystems, ASP300s, Buffalo Grove, IL). Paraffin embedding was performed in multiple orientations (horizontal and vertical) to achieve isotropy (Leica EG1150 H-C). Paraffin embedded lung tissues were sectioned at 5 μm thickness using a Leica RM2165 microtome, mounted on slides, and dried at 56 °C for 2 h. Slides were then stained with hematoxylin and eosin, and imaged with the Motic EasyScan system at 20x magnification. (4, 38). Importantly, paraffin embedding is associated with tissue shrinkage that may affect stereological measurements (39). However, because all samples were processed identically, this limitation is expected to influence absolute values rather than comparisons between experimental groups (40).

Our morphometric sampling followed the stereological methods described previously, which outline mathematical approaches to infer the properties of irregular 3D structures from 2D tissue section (41). After imaging, lung sections were digitized using Aperio ImageScope (v12.4.6.5003; 20x magnification) and analyzed with Fiji (ImageJ). Systematic random sampling was used for field selection: approximately every eighth lung section was chosen, moving systematically from left to right, resulting in ~20 test fields per lung. Each test field was overlaid with 5 evenly spaced horizontal test lines (38).

The framework for measuring mean linear intercept (L_m) originates from Campbell and Tomkeieff, who introduced an indirect method in which L_m is derived from the volume-to-surface (V/S) ratio of alveolar airspaces. In this approach, endpoints of test lines landing within airspace provide an estimate of volume, while intersections of test lines with alveolar walls provide an estimate of surface area; their ratio allows L_m to be calculated (42). The L_m represents the average free distance across alveolar airspaces and can be estimated either indirectly from the volume-to-surface ratio or directly by measuring chord lengths along test lines (43).

Knudsen and colleagues (43) describe the direct intercept method in detail which measures the actual distances (chords) between alveolar walls along test lines. To avoid sampling bias, we followed the test line + guard-zone design of Knudsen and colleagues (43). Each test line consisted of a solid initial test line segment (~¼ of the entire line) and a dashed extension line segment called the guard line (~¾ of the line). Measurements always began at the left end of the solid line and proceeded along to the right. The first intersection of the test line with an alveolar wall marked the start of an intercept; measurement continued along the line until the next alveolar wall intersection, defining one alveolar airspace (intersect) length (Lx). If an intersect started along the solid test line but extended into the guard line, it was measured to avoid excluding large alveoli or ducts. However, intersections that began entirely outside of the test line were excluded. The guard-zone design ensured unbiased sampling and prevented under sampling of long intercepts like in emphysematous regions. All intercepts were measured in micrometers, and L_m was calculated as follows:

$$L_m=\frac{\sum _{i=1}^{N}L{x}_i}{N},$$

where N is the total number of intercepts measured across all fields and Lx represents each intercept/chord length (43). For each lung, we analyzed approximately 20 test fields (dimensions/test field: 1396 × 1054 pixels, with a resolution of 0.514 μm/pixel), with 5 tests lines per field, yielding a total of about 160–260 intercepts/lung. This intercept number is considered sufficient as detailed by (43). From the measured intercepts (μm), we calculated L_m shown in Figure 1A.

Figure 1:

Panel A summarizes mean linear intercept (L_m) for each animal, with young (n = 9, blue) and old (n = 8, orange) plotted as individual points (females = circles, males = triangles), group means ± SD (black lines). Old rats had significantly larger alveolar airspaces than young (63 ± 9 vs. 55 ± 6 μm; p = 0.049; Cohen’s d = 1.06), with no sex differences. Panel B shows representative histological images of lung parenchyma from a young (left) and an old (right) animal. Solid black lines indicate test lines used for mean linear intercept (L_m) measurement, with corresponding dashed guard lines overlaid to guide systematic sampling during analysis.

Statistical analysis

A priori power analyses were conducted based on the variance measured in previous studies (4, 26, 27) with α = 0.05, β = 0.20 (Power = 0.80), and a detectable effect size of 20% in each experiment (i.e., lung histology, mechanics, plethysmography). Two-way ANOVAs with sex and age as independent variables were first performed to evaluate potential impacts of sex. It should be noted that while sex was included as a biological variable the present study was not powered to detect sex differences. If no significant effects of sex or sex × age interactions were detected, then Welch’s unpaired t-tests were used for independent age group comparisons to avoid assumptions of equal variance. Effect sizes were calculated for each independent variable as Cohen’s d, using the mean values of the independent young and old age groups and the pooled standard deviation. An effect size of 0.2 or less was interpreted as small, between 0.3 to 0.7 as moderate, and 0.8 or greater as large (44).

RESULTS

Animals

A total of 33 Fischer 344 rats (19 females, 14 males) were used in the study, comprising 21 young and 12 old animals. On the day of recording, the mean ± SD body masses for young females and males were 207 ± 31 g and 323 ± 38 g, respectively, while old females and males weighed 241 ± 11 g and 391 ± 28 g. No significant age-related weight differences were observed. The histology cohort included 9 young and 8 old rats. Measurements from the programmable mechanical ventilator were obtained from 13 young and 10 old rats but full-range P-V curves, part of the mechanical ventilator measurements, involved 9 young and 9 old rats. Whole-body plethysmography measurements were performed on 12 young and 10 old rats.

Lung Histology and Morphometry

For the characterization of alveolar airspace length, linear intercept measurements were calculated from young (n = 9) and old rats (n = 8). Histological measurements showed significantly larger alveolar airspaces in old rats compared to young (L_m; Young: 55 ± 6 μm, Old: 63 ± 9 μm; p = 0.049; Cohen’s d: 1.06) a difference demonstrated both quantitatively and in the representative photomicrographs (Figure 1). Lung volume measurements from the water displacement procedure were not significantly different between young (n = 8) and old (n = 6) rats (LV; Young: 7.3 ± 1.4 mL, Old: 8.8 ± 1.8 mL; p = 0.127).

Respiratory System Mechanics

Representative full-range P-V curves for one young and one old animal are shown in Figure 2 and were used to calculate parameters associated with respiratory system mechanics. Lung volumes and capacities measured with a programmable mechanical ventilator were similar between young (n = 9) and old (n = 9) rats as shown in Table 1, with no significant differences in TLC, vital capacity (VC), IC, FRC, or LV. This indicates that the overall size and capacity of the lungs did not change with age. However, as shown in Table 1, RV was significantly higher in old rats compared to young rats (RV; Young: 0.27 ± 0.05 mL, Old: 0.41 ± 0.10 mL; p = 0.003; Cohen’s d: 1.81), consistent with age-related changes in gas trapping or lung emptying efficiency.

Figure 2:

Representative full-range pressure–volume (P–V) curves from a young (blue) and an old (orange) animal are shown. These curves were used to derive parameters associated with respiratory system mechanics. Filled circles (orange for the old animal and blue for the young animal) indicate the discrete pressure steps applied during mechanical ventilation, illustrating the incremental inflation of the lungs at each pressure level.

Table 1.

Lung volumes (LV) and capacities in young (n = 9) and old (n = 9) rats, presented as mean ± SD.

Parameters	Young 6-month-old	Old 24-month-old
RV (mL)	0.27 ± 0.05	0.40 ± 0.10*
FRC (mL)	3.60 ± 0.80	3.53 ± 1.02
FRC/g (mL/g)	0.014 ± 0.003	0.012 ± 0.002
IC (mL)	7.8 ± 1.5	6.6 ± 1.2
TLC (mL)	11.4 ± 2.0	10.1 ± 2.0
TLC/g (mL/g)	0.043 ± 0.006	0.035 ± 0.003

No significant differences were observed in total lung capacity (TLC) inspiratory capacity (IC), functional residual capacity (FRC) indicating similar overall lung size and capacity between groups. However, residual volume (RV) significantly increased in old rats compared to young (RV; Young: 0.27 ± 0.05 mL, Old: 0.41 ± 0.10 mL; p = 0.003; Cohen’s d: 1.81).

^*Parameters with p < 0.05 are marked with an.

From the SnapShot 90 maneuver, respiratory resistance (Rrs; Young: 0.04 ± 0.04 cm H₂O·s/mL, Old: 0.03 ± 0.01 cm H₂O·s/mL; p = 0.452), respiratory system elastance (Ers; Young: 2.05 ± 0.57 cm H₂O·s/mL, Old: 2.04 ± 0.40 cm H₂O/mL; p = 0.993), and dynamic compliance (Crs; Young: 0.51 ± 0.11 mL/cm H₂O, Old: 0.52 ± 0.11 mL/cm H₂O; p = 0.933) did not differ significantly between young and old animals. Similarly, static compliance (C) obtained from the full-range P-V curve (Young: 0.85 ± 0.17 mL/cm H₂O, Old: 0.77 ± 0.19 mL/cm H₂O; p = 0.380) and static compliance derived from the Salazar–Knowles equation (Cst) using the P-V loop (Young: 0.76 ± 0.16 mL/cm H₂O, Old: 0.65 ± 0.13 mL/cm H₂O; p = 0.094) were not significantly different between groups.

However, additional and less commonly reported measurements from the programmable mechanical ventilator (flexiVent) provided further insight into respiratory mechanics. One such parameter, the deflation decay constant (K) from the Salazar–Knowles equation, relates volume and pressure. A higher parameter K reflects decreased elastic recoil or increased compliance. Parameter K as shown in Figure 3A, was significantly higher in old rats compared to young (Young: 0.168 ± 0.013, Old: 0.179 ± 0.010; p = 0.038; Cohen’s d: 0.90). The deflation P-V relationship can be partly characterized by the V₁₀/TLC ratio, defined as the volume at 10 cm H₂O normalized to TLC. V₁₀/TLC as shown in Figure 3B, was significantly higher in old rats compared to young rats (Young: 78.5 ± 2.5, Old: 80.7 ± 1.2; p = 0.042; Cohen’s d: 1.08). Both measures are consistent with decreased lung elastic recoil with aging.

Figure 3.

Panel A shows the lung deflation decay constant (K) in young (blue, n = 13) and old (orange, n = 10) rats, and Panel B the volume at 10 cm H₂O normalized to total lung capacity (V₁₀/TLC) in young (blue, n = 9) and old (orange, n = 9) rats. Individual values are plotted (females = circles, males = triangles) with group means ± SD (black lines). Significant differences were found for K (p = 0.038; Cohen’s d = 0.90) and V₁₀/TLC (p = 0.042; Cohen’s d = 1.08), marked with (*). No sex differences were observed.

As shown in Figure 4, RV was significantly higher in old rats compared to young rats (RV; Young: 0.27 ± 0.05 mL, Old: 0.41 ± 0.10 mL; p = 0.003; Cohen’s d: 1.81).

Figure 4.

Residual volume (RV) was significantly higher in old (orange, n = 9) compared to young (blue, n = 9) rats (young: 0.27 ± 0.05 mL; old: 0.41 ± 0.10 mL; p = 0.003; Cohen’s d = 1.81). Individual values are shown (females = circles, males = triangles) with group means ± SD (black lines). Significant differences (p < 0.05) are marked with (*). No sex differences were observed.

Airway compliance (Caw), measured from the full-range P-V curves immediately before the alveolar opening pressure, provides information about the compliance of collapsible airways prior to alveolar opening due to large pressure gradients. As shown in Figure 5, Caw was significantly lower in old rats compared to young rats (Young: 19.2 ± 4.2 μL/cm H₂O, Old:13.4 ± 1.6 μL/cm H₂O; p = 0.003; Cohen’s d: 1.82). No significant sex differences were observed.

Figure 5.

Airway compliance (Caw), measured from the full-range P-V curve immediately before alveolar opening pressure, decreased significantly in old (orange, n = 9) compared to young (blue, n = 9) rats (young: 19.2 ± 4.2 μL/cm H₂O; old: 13.4 ± 1.6 μL/cm H₂O; p = 0.003; Cohen’s d = 1.82). Individual values are shown (females = circles, males = triangles) with group means ± SD (black lines). Significant differences (p < 0.05) are marked with (*). No sex differences were observed. Values are mean ± SD.

Newtonian resistance (Rn), representing the resistance of the central/conducting airways, did not differ significantly between young and old rats (Young: 0.038 ± 0.015 cm H₂O•s/mL, Old: 0.033 ± 0.007 cm H₂O•s/mL; p = 0.327). Peripheral airway properties, assessed through tissue damping (G) and tissue elastance (H), were also not significantly different between the two age groups. Tissue damping (G), reflecting energy loss to heat within tissues due to friction, was not significantly different between young and old rats (Young: 0.376 ± 0.100 cm H₂O/mL, Old: 0.351 ± 0.083 cm H₂O/mL; p = 0.527). Tissue elastance (H), representing the elastic energy stored in the tissues and their ability to recoil, was also not significantly different between the two age groups (Young: 1.966 ± 0.545 cm H₂O/mL, Old: 1.783 ± 0.280 cm H₂O/mL; p = 0.290). The hysteresivity (η), calculated as the ratio of G/H, was 0.192 ± 0.030 in young and 0.195 ± 0.023 in old rats, with no significant differences observed (p = 0.894).

Breathing Parameter Measurements (Whole Body Plethysmography)

As shown in Figure 6A, plethysmograph measurements in unrestrained, awake rats revealed a significantly larger V_T in old rats compared to young rats (Young: 1.5 ± 0.4 mL, Old: 2.0 ± 0.4 mL; p = 0.004; Cohen’s d: 1.43). RR did not differ significantly between the two age groups (Young: 94 ± 21 breaths/min, Old: 91 ± 15 breaths/min; p = 0.798; Cohen’s d: 0.11). However as shown in Figure 6B, V_E, calculated as the product of V_T and RR, was significantly higher in old rats compared to young rats (Young: 129 ± 29 mL/min, Old: 168 ± 29 mL/min; p = 0.005; Cohen’s d: 1.35).

Figure 6.

Point plots show significant whole-body plethysmography results in young (blue, n = 12) and old (orange, n = 10) rats. Panels display tidal volume (V_T) (A), minute ventilation (V_E ) (B), inspiratory flow (C), and expiratory flow (D). Individual values are shown (females = circles, males = triangles) with group means ± SD (black lines). Significant differences (p < 0.05) are marked with (*). No sex differences were observed.

Inspiratory (T_I) and expiratory time (T_E) durations were not significantly different between the two age groups. T_I was 232 ± 43 ms in young rats and 244 ± 28 ms in old rats (p = 0.460; Cohen’s d: 0.30), while T_E was 511 ± 95 ms and 525 ± 85 ms, respectively (p = 0.78; Cohen’s d: 0.15). Dividing V_T by T_I and T_E yielded mean inspiratory (I-flow) and expiratory flow (E-flow) rates. I-flow as shown in Figure 6C was significantly higher in old rats compared to young rats (Young: 6.59 mL/s, Old: 8.54 mL/s; p = 0.007; Cohen’s d: 0.30). E-flow, shown in Figure 6D was also significantly higher in old rats compared to young (Young: 3.10 mL/s, Old: 3.96 mL/s; p = 0.007; Cohen’s d: 0.16).

DISCUSSION

The present study confirmed the hypothesis that alveolar airspace increases with age as reflected by increased L_m in old compared to young rats. Although no overall age-related changes were observed in Ers, Crs, or C, measures quantifying the deflation P-V relationship revealed that aging decreases elastic recoil, specifically increasing the parameter K and V₁₀/TLC. Contrary to our hypothesis, E-flow increased in old rats, driven primarily by increased V_T rather than changes in T_E. In addition, data from the present study show that Caw decreases, with a concomitant increase in RV, consistent with air trapping. Collectively, these outcomes provide a comprehensive assessment of age-related structural and functional changes in the lungs of Fischer 344 rats. Notably, The present study provides novel physiological insights into aging by measuring airway compliance (Caw) and residual volume (RV) via forced oscillation perturbations using a programmable mechanical ventilator (flexiVent—currently considered a gold standard for in vivo lung function measurements) (45).

Fischer 344 rats are a widely used model in aging physiology (6, 11, 18, 46–49) and are an aging model approved by the National Institute of Aging (NIA). Although both Fischer 344 and Brown Norway rats are NIA-approved models of aging, Fischer 344 rats were selected because they are more extensively used across aging studies, facilitating comparison with existing literature (50). By comparison, male Sprague-Dawley rats display excessive weight gain. By 24 months of age, Fischer 344 rats develop phrenic motor neuron loss (47, 51, 52), deficits in neuromuscular transmission (48, 51, 53) and changes in muscle cross sectional area (54), including sarcopenia (6, 46, 55, 56). The genetic uniformity of Fischer 344 rats and their extensive use across organ systems facilitate comparison with existing literature and provide the potential for a more holistic understanding of the physiology of aging. The selection of age groups in Fischer 344 rats was based on survival information provided by the National Institute of Aging (NIA) (46, 48, 54). Importantly, Fischer 344 rats maintain relatively stable body weight after reaching adulthood, making them well suited for studying age-related lung physiology (6, 23, 49). Additionally, rats may offer advantages over mice due to their larger size and metabolic demands (26, 57). Several previous studies included only males (4, 7, 11, 19, 22, 46, 58), with relatively few including both sexes (13, 18). Importantly, similar to previous studies (13, 18) the present study was powered to detect aging-related effect, rather than sex-specific differences. In the present study, based on the limited sample, none of the outcome measures were impacted by sex.

Structural Changes in the Lung

Morphometric analysis followed stereological methods described previously (41). Changes in lung tissue structure were analyzed using L_m, a widely applied index of alveolar airspace size (4, 38, 41–43, 59). In old compared to young Fischer 344 rats, L_m increased by ~15% (Figure 1A), consistent with irreversible age-related alveolar enlargement (4, 11–13). This effect was large (Cohen’s d = 1.06) and aligned with prior studies in Fischer 344 rats reporting an ~12% increase from 12 to 24 months (11) and ~25% increase from 9 to 24 months (13). Alveolar diameters in the young Fischer 344 rat group also matched baseline values reported previously (11, 13, 60, 61). Compared with other strains, Fischer 344 rats exhibit smaller age-related lung structural changes (11, 49), which may explain why age-related differences in the present study, while statistically significant, were only modest. Similar age-related increase in L_m has been described in mice (4, 7, 62). As a previous study noted, changes in L_m reflect alveolar airspace enlargement but cannot, by themselves, distinguish between alveolar wall breakdown and differences in lung inflation or elastic recoil during fixation (43). Given the relatively modest difference in L_m in the present study, we cannot exclude the possibility that differences in lung inflation partly influenced L_m.

The literature emphasizes the importance of post-fixation LV when interpreting morphometric data (4, 43). LV measured by water displacement following fixation at a constant inflation pressure of 25 cm H₂O was not significantly different between young and old rats (Young: 7.3 ± 1.4 mL; Old: 8.8 ± 1.8 mL; p = 0.127), indicating comparable lung inflation across groups. As shown in Table 1 lung inflation performed in vivo using the mechanical ventilator reached 35 cm H₂O defining TLC. Post-fixation LV were substantially greater than functional residual capacity (FRC; Young: 3.60 ± 0.80 mL, Old: 3.53 ± 1.02 mL) and closely approximated inspiratory capacity (IC; Young: 7.8 ± 1.5 mL, Old: 6.6 ± 1.2 mL). As expected, post-fixation LV determined by water displacement were lower than TLC (Young: 11.4 ± 2.0 mL, Old: 10.1 ± 2.0 mL) determined by flexiVent pressurization to 35 cm H₂O. A regression analysis demonstrated a strong correlation (R² = 0.98) between TLC and post-fixation LV, with TLC values consistently ~25–30% higher, reflecting the difference in inflation pressures (~35 vs 25 cm H₂O) Figure 7. These findings suggest that the lungs from both young and old rats were adequately inflated to near IC at 25 cm H₂O, which minimizes the risk of parenchymal overdistension (4, 11, 18, 58). Consequently, the difference in L_m observed in the present study is likely due to age-related effects rather than methodological issues.

Figure 7:

Regression analysis of post-fixation displacement lung volume (LV) at 25 cm H₂O versus total lung capacity (TLC) at 35 cm H₂O in young (blue, n = 9) and old (orange, n = 8) animals. Individual values are shown (females = circles, males = triangles) with the linear regression line (solid black) and line of identity (dashed). A strong correlation is observed (R² = 0.9818), with TLC values consistently ~25–30% higher than post-fixation lung volume, consistent the pressure difference during inflation.

Changes in Lung Mechanics

While numerous studies have examined lung mechanics in old age, measurements that directly quantify the deflation P-V relationship have generally received less scrutiny. Prior work has focused mainly on respiratory system or lung compliance and elastance in mice (4, 7, 17) and rats (11, 18, 19). Contemporary applications of programmable mechanical ventilators allow assessment of parameter K (decay constant of exponential defining the deflation P-V relationship), V₁₀/TLC, Caw, and RV, providing deeper insight into lung mechanics (19, 32, 35, 36). In the present study, we used a terminal degassing procedure to measure RV, which has not been previously reported in young vs. old rats. Because measurement of RV required an intact chest wall, our measurements of mechanical properties reflect respiratory system level changes rather than isolated lung mechanics. However, a prior study from our laboratory compared open and closed chest wall compliance and found no difference between the two indicating that chest-wall intactness likely had minimal impact on our results (4).

Consistent with earlier reports, we observed no age-related changes in overall respiratory system compliance (4, 7, 11, 17). Given that aging decreases chest-wall compliance while increasing lung compliance, lung-specific changes may be offset at whole-system level (21, 63). By contrast, parameters from the deflation limb of the P-V relationship revealed clear age-related changes. Nagase and colleagues (19) reported ~11% increase in parameter K in 29-month vs. 14-month Sprague-Dawley rats, indicating slower lung deflation. Similarly, we found ~6% increase in parameter K in old vs. young Fischer 344 rats, with a large effect size (Cohen’s d = 0.91; Figure 3A). Parameter K is obtained from the deflation limb of the P-V loop by fitting the Salazar–Knowles equation (V = A − Be^−KP). K describes the curvature of the deflation limb and increased values indicate decreased lung elastic recoil and greater volume retention for a given pressure. Unlike conventional compliance measures, K is independent of absolute LV and captures recoil over the entire P-V deflation curve, rather than at a single pressure point (19, 32, 34). Recent work in mice further emphasized these advantages, showing that K is sensitive to intrinsic tissue mechanics such as airway smooth muscle contraction but not to inflammation, possibly making it a sensitive marker of lung tissue compliance (35). Beyond rodent models, the parameter K has been applied in human studies; ~50% of older asthmatic patients showed increased K, consistent with age-related loss of recoil even in an otherwise hypercontractile environment (64).

Consistent with increased K, V₁₀/TLC was ~2% higher in old vs. young rats (81% vs. 79%). Although this change was modest in absolute terms, the effect size was large (Cohen’s d = 1.10; Fig. 3B), underscoring its potential physiological significance. A higher V₁₀/TLC indicates greater volume retention at 10 cm H₂O, consistent with age-related loss of lung elastic recoil. Taken together, these measures show that while conventional compliance measurements were unaffected by age, measures quantifying the deflation P-V relationship revealed decreased elastic recoil/increased lung compliance in old Fischer 344 rats. The results of the present study suggest that these measures may serve as early indicators of changes in lung elastic properties.

The resistance of upper, cartilage-supported airways decreases in old Fischer 344 rats (23). These upper airways are larger and less prone to collapse (65). In the present study, we observed no age-related changes in lung, tissue, or airway resistance in Fischer 344 rats, consistent with a previous study comparing 14- and 29-month-old Sprague-Dawley rats (19). Although airway resistance was unchanged, compliance of collapsible airways (Caw) was ~43% lower in old vs. young rats, with a large effect size (Cohen’s d = 1.82). This decrease in Caw may arise from age-related changes in airway smooth muscle tone, extracellular matrix remodeling, or low-grade inflammation. Regardless of the mechanism, the impact of lower Caw is likely to be a greater likelihood for air trapping.

Prior studies have emphasized the importance of starting LV in determining airway resistance (22), with apparent decreases in airway resistance attributed to increases in FRC (19, 22)––a phenomenon perhaps more pronounced in mice than rats (4, 22). In Fischer 344 rats, FRC was unchanged in older animals, whereas RV increased, indicating air trapping. Notably, previous studies reporting age-related changes in airway resistance did not measure RV (4, 7, 19, 22), and thus did not make the direct link between the collapsibility of small airways (reflected by decreased Caw) and air trapping (reflected by increased RV) that we do in the present study. Although some previous studies document relatively high RV values, methodological differences may explain the lower values we observed (18, 49). In the present study, a considerable number of quality control steps were taken (detailed in the Methods) to ensure accurate assessments of RV. Indeed, 5 animals were excluded due to quality control failures. Notably, RV in young Fischer 344 rats closely matched the RV value reported in Sprague-Dawley rats (32) using the same mechanical ventilator (flexiVent). In the present study, RV increased by ~48% in old compared with young rats, reflecting a large age-related effect size (Cohen’s d = 1.81), consistent with previous studies in humans (2, 20, 21, 66) and rats (18). This increase in RV is concordant with the increase in parameter K, V₁₀/TLC, and decrease in Caw, together indicating incomplete lung emptying and air trapping.

Whole Body Plethysmography

The breathing parameters of young Fischer 344 rats aligned with prior work (25, 26, 57, 67, 68) and were consistent with earlier Fischer 344 rat data (18), as RR did not differ between age groups (~90 breaths/min). A previous study reported increased RR in old Sprague-Dawley rats (25), whereas in mice, RR decreased with age when comparing young (26) and old (27) animals within the same laboratory. By contrast, V_T increased by ~33% in old rats, resulting in ~30% increase in V_E as shown in Figure 6A & 6B, respectively. This volume-driven rise in V_E may reflect compensation for alveolar enlargement, decreased elastic recoil, and possible increases in physiological dead space (e.g., increased RV). While we did not measure dead space directly, human studies have reported age-related increases in alveolar dead space (21). In addition, decreased lung recoil forces may favor increased V_T rather than RR as a strategy to increase E-flow. Despite these structural and mechanical changes, T_I and T_E were not different between young vs old rats. However, increased V_T and unchanged T_E increased the average I-flow (~30%, Fig. 6C) and E-flow (~28%, Fig. 6D), though effect sizes were small (Cohen’s d = 0.30 and 0.16). Together, this pattern suggests that aging increases flow without altering timing, which may reflect age-related changes in descending ventilatory drive as well as post-inspiratory diaphragm activity. A more detailed, breath-by-breath assessment using plethysmography may further unveil subtle impacts of age on ventilatory parameters (69), but it is beyond the scope of the present study.

Conclusion

In the present study, awake unrestrained Fischer 344 rats showed no age-related differences in timing parameters (RR, T_I, T_E), whereas tidal volume (V_T), minute ventilation (V_E), inspiratory flow (I-flow), and expiratory flow (E-flow) were significantly increased in the old compared to young rats. These age-related changes in breathing parameters occurred alongside increased alveolar size (L_m), decreased lung elastic recoil (K and V₁₀/TLC), Caw and increased RV. Importantly, more conventional measurements of respiratory system compliance/elastance remained unchanged with age. Thus, parameters derived from the deflation P-V relationship (e.g., parameter K and V₁₀/TLC) revealed subtle but physiologically meaningful decreases in elastic recoil, consistent with the modest increase in L_m. The present study extends prior work by integrating measurements of Caw and RV with structural and ventilatory outcomes in Fischer 344 rats. The study also provides a comprehensive overview of age-related changes that show modest but physiologically meaningful structural, mechanical, and functional alterations in Fischer 344 rats.