Effects of aging on diaphragm hyperemia and blood flow distribution in male and female Fischer 344 rats

Abstract

Aging is associated with inspiratory muscle dysfunction; however, the impact of aging on diaphragm blood flow (BF) regulation, and whether sex differences exist, is unknown. We tested the hypotheses in young animals that diaphragm BF and vascular conductance (VC) would be greater in females and that aging would decrease the diaphragm’s ability to increase BF with contractions. Young (4–6 mo) and old (22–24 mo) Fischer 344 rats were divided into four groups: young female (YF, n = 7), young male (YM, n = 8), old female (OF, n = 9), and old male (OM, n = 9). Diaphragm BF (mL/min/100 g) and VC (mL/mmHg/min/100 g) were determined, via fluorescent microspheres, at rest and during 1 Hz contractions. In YF versus OF, aging blunted the increase in medial costal diaphragm BF (44 ± 5% vs. 16 ± 12%; P < 0.05) and VC (43 ± 7% vs. 21 ± 12%; P < 0.05). Similarly, in YM versus OM, aging blunted the increase in medial costal diaphragm BF (43 ± 6% vs. 24 ± 12%; P < 0.05) and VC (50 ± 6% vs. 34 ± 10%; P < 0.05). In female rats, age increased dorsal costal diaphragm BF, whereas in male rats, age increased crural diaphragm BF (P < 0.05). Compared with age-matched females, dorsal costal diaphragm BF was lower in YM and OM (P < 0.05). In conclusion, aging results in an inability to augment medial costal diaphragm BF and alters regional diaphragm BF distribution in response to muscular contractions. Furthermore, sex differences in regional diaphragm BF are present in young and old animals.

NEW & NOTEWORTHY This is the first study, to our knowledge, to demonstrate that old age impairs the hyperemic response and alters blood flow distribution in the diaphragm of both female and male rats. In addition, this investigation provides novel evidence of sex differences in regional diaphragm blood flow distribution with contractions. The data presented herein suggest that aging compromises diaphragm vascular function and provides a potential mechanism for the diaphragm contractile dysfunction associated with old age.

INTRODUCTION

Aging is associated with a decline in skeletal muscle function and exercise capacity (1, 2), due in part to structural and functional changes within the vasculature resulting in inadequate perfusion and decreased vascular conductance (VC) (3–11). These age-related impairments in blood flow (BF) regulation play a central role in the inability to match oxygen delivery (Q̇o₂) to the increased metabolic demand (V̇o₂) of skeletal muscle during exercise and consequently hasten fatigue (12–14). Although there are considerable data on the effect of aging on locomotory skeletal muscle perfusion, and the underlying vascular mechanisms, the impact of aging on inspiratory muscle (i.e., diaphragm) BF capacity and/or vascular control mechanisms has received scant attention.

In this regard, advanced age is associated with inspiratory muscle dysfunction, which increases morbidity and diminishes quality of life (15, 16). The diaphragm is the principal inspiratory muscle and the only skeletal muscle that is continually active throughout the mammalian lifespan. Akin to locomotory muscles, diaphragm contractile function is highly dependent on adequate perfusion, as reductions in BF, and thus Q̇o₂, promote diaphragmatic fatigue (17–20). Previous investigations demonstrate that diaphragm strength [e.g., maximal inspiratory and transdiaphragmatic pressure generation (21–23)] and contractile function [i.e., force generating capacity, fatigue resistance (24–29)] decline with old age. Although the precise mechanistic bases for aging-induced diaphragm dysfunction remain unclear, age-related changes in diaphragm hyperemia, BF distribution, and/or VC may play a central role.

Aging alters BF distribution within active skeletal muscle and compromises the matching of BF to metabolic demand (13, 30). Similar to large locomotory muscles [e.g., gastrocnemius (31, 32)], there is considerable regional heterogeneity in diaphragm BF distribution at rest and during maximal exercise (19, 33–39). Specifically, within the costal diaphragm, the medial costal region sustains the highest relative perfusion and performs the greatest proportion of inspiratory work (19, 33–39). Although resting bulk diaphragm BF is not different between young and aged rats (40), the distribution of BF within the aged diaphragm is not known. Moreover, in skeletal muscles that exhibit a fiber-type composition (i.e., spinotrapezius) and oxidative capacity (i.e., red gastrocnemius) akin to the diaphragm, aging blunts the hyperemic response to contractions (30, 41–43). Thus, it is plausible that old age alters regional BF distribution and/or diminishes the intrinsic capacity of the diaphragm to augment BF with increased metabolic demand. If such an age-related hyperemic impairment exists in the diaphragm, it would mechanistically underpin the diaphragmatic fatigue associated with aging.

Differences in skeletal muscle BF and vascular function may play a key role in the dichotomy of skeletal muscle fatigue between biological sexes (12). In young adults, previous investigations suggest that the diaphragm is more fatigue resistant in females compared with male counterparts (44). Intriguingly, these sex differences in diaphragm performance are absent in aged animals (45). These data suggest that in young animals, there may be sex differences in the hyperemic response and/or BF distribution within the diaphragm that are abolished with advanced age. Currently, the effect of biological sex on diaphragm BF control in young and old animals is unknown.

Therefore, the overall objective of this study was to investigate the effects of aging and biological sex on diaphragmatic hyperemia and BF distribution during electrically induced contractions in an established animal model. Specifically, we tested the following hypotheses in young and old rats of both sexes: 1) aging will compromise the ability to increase diaphragm BF and VC, 2) aging will alter the regional distribution of diaphragm BF, 3) diaphragm BF and VC will be greater in young females versus young males, and 4) there will be no sex differences in diaphragm BF or VC in old rats.

METHODS

Animals

A total of 42 young (4–6 mo) and old (22–24 mo) Fischer 344 rats (National Institutes on Aging Colony) were used for this investigation. Of the 42 animals used, 9 were excluded due to poor microsphere mixing (described in Fluorescent Microsphere Injection). Animals were divided into two experimental groups (n = 33): 1) young (n = 15) and 2) old (n = 18). For sex comparisons, young and old groups were subdivided into four experimental groups: 1) young female (YF; n = 7), 2) young male (YM; n = 8), 3) old female (OF; n = 9), and 4) old male (OM; n = 9). For age comparisons, power analyses revealed that a minimum of 11 rats per group would be needed for adequate statistical power, and the sample sizes for young (n = 15) and old (n = 18) yielded a power of 0.94. All procedures were approved by the Kansas State University Institutional Animal Care and Use Committee and complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Upon arrival, animals were housed and maintained in a temperature-controlled (23 ± 2°C) room with a 12:12-h light-dark cycle with water and rat chow provided ad libitum.

Surgical Preparation

All surgical procedures were performed using aseptic techniques. Rats were initially anesthetized with a 5% isoflurane-O₂ mixture (isoflurane vaporizer; Harvard Apparatus, Cambridge, MA) and subsequently maintained on 2%–3% isoflurane-O₂. Body temperature was maintained at 37 ± 1°C (measured via rectal thermometer) by use of a water-recirculating heating blanket. An incision was made on the ventral side of the neck, and the right carotid artery was isolated and cannulated with PE-10 connected to PE-50 (Intramedic Polyethylene Tubing; Clay Adams Brand; Becton, Dickinson, Sparks, MD) for measurements of mean arterial pressure (MAP; Digi-Med BPA; Micro-Med Inc., Louisville, KY) and infusion of fluorescent microspheres (see later). A second catheter (PE-10 connected to PE-50) was inserted into the caudal artery for the infusion of pentobarbital sodium anesthesia and reference sampling for BF determination. Rats were then transitioned to pentobarbital sodium anesthesia (20 mg/kg body wt) given intra-arterially (total volume per injection was ∼0.2 mL of fluid) while concentrations of isoflurane were decreased and subsequently discontinued over ∼30 min. The level of anesthesia was regularly monitored via toe pinch and palpebral reflex, with pentobarbital anesthesia supplemented (3.5–7.0 mg/kg) as necessary.

Diaphragm Contractions

Following carotid and caudal artery catheterization, animals were tracheotomized and mechanically ventilated with positive end-expiratory pressure (PEEP) set at 1 cmH₂O (Kent Scientific PhysioSuite, Torrington, CT) for the duration of the experimental protocol (≤30 min) to enhance survival and render the diaphragm inactive to minimize the interference of spontaneous diaphragm contractions. All experiments were performed with the animal in the supine position, which has been demonstrated to have no effect on the distribution of costal diaphragm blood flow (33). The diaphragm was exposed as previously described (36, 39, 46, 47) and kept moist with warm saline. Following diaphragm exposure, stainless steel electrodes were sutured to the left ventral costal (cathode) and the left dorsal costal (anode) diaphragm. After a 10-min recovery period, and prior to contractions, fluorescent microspheres were infused (described in Fluorescent Microsphere Injection) to assess baseline diaphragmatic BF. Thereafter, electrically stimulated twitch contractions were induced at 1 Hz (3–6 V, 2-ms pulse duration) with a Grass S88 stimulator (Quincy, MA) for 180 s. At 180 s of stimulation, fluorescent microspheres were infused to assess the contracting steady-state diaphragm hyperemia.

Fluorescent Microsphere Injection

The fluorescent microsphere technique, as previously described (37–39, 48), was used to quantify tissue BF in each experimental group. Fluorescent microspheres were infused at two different time points: 1) rest (baseline; 10 min postelectrode placement and prior to contractions) and 2) 180 s of electrical stimulation. For each measure, a reference blood sample was taken from the caudal artery catheter, using a Harvard withdrawal pump (model 907; Cambridge, MA) that was initiated 30 s prior to microsphere infusion at a withdrawal rate of 0.25 mL/min, and 2.0–2.5 × 10⁵ fluorescent microspheres (randomized colors: red, scarlet, or blue-green; 15.5 μm diameter; Invitrogen FluoSpheres, Carlsbad, CA) were infused into the aortic arch via the carotid artery catheter. In each animal, adequate mixing of microspheres prior to injection was determined by <20% difference in left and right kidney or left and right soleus muscle BFs at each time point. Following the final microsphere infusion, rats were euthanized with pentobarbital sodium overdose (>50 mg/kg ia) and the thorax was opened to confirm proper placement of the carotid artery catheter into the aortic arch before tissues were identified and excised. Thereafter, tissues (diaphragm, soleus, and kidneys) were harvested, weighed, and placed in 15-mL screw-cap polypropylene conical tubes and then placed in a –80°C freezer for later BF analysis. The diaphragm was sectioned into costal (ventral, medial, and dorsal) and crural portions (Fig. 1) to determine regional distribution of diaphragmatic BF, whereas the sum of these portions was used to calculate total diaphragm flow (37–39).

Figure 1.

Inferior aspect of the rodent diaphragm (left) and the regional sections of the costal and crural diaphragm with black lines denoting the left and right phrenic feed arteries (right). CR, crural; DC, dorsal costal; MC, medial costal; VC, ventral costal.

Calculation of BF and VC

The fluorescent microsphere assay was performed according to Deveci and Egginton (48). Chemically digested tissue samples were placed into a 96-well plate, with each sample analyzed in quadruplicate. After measuring the fluorescence intensity of each tissue and reference blood sample, using a Spectramax i3 plate reader (Molecular Devices, Sunnyvale, CA), tissue BF was calculated as follows (48):

$$\dot{\text{Q}}=[\left({\text{A}}_{\text{t}}{/\text{A}}_{\text{b}}\right)\times \left(\text{s/w}\right)]\times 100$$

where Q̇ is BF (mL/min/100 g), A_t is the individual tissue sample intensity, A_b is the reference blood sample intensity, s is the withdrawal rate (0.25 mL/min) of the reference blood sample, and w is the tissue weight (g). VC was calculated as follows:

$$\text{VC}=\dot{\text{Q}}/\text{MAP}$$

where VC is vascular conductance (mL/mmHg/min/100 g), Q̇ is BF (mL/min/100 g), and MAP is the mean arterial pressure (mmHg), recorded immediately before microsphere infusion.

Data Analysis

Data were analyzed using GraphPad Prism10 (GraphPad Software, San Diego, CA). Body mass (g), diaphragm mass (mg), diaphragm-to-body mass ratio (mg/g), and MAP (mmHg) were analyzed using a one-way ANOVA. Tissue VC and BF at rest and during 1 Hz contractions were analyzed using a mixed-effects two-way RM ANOVA. Age-related and biological sex comparisons were analyzed with separate mixed-effects two-way RM ANOVAs. A Grubb’s outlier test was performed on the dataset before statistical analysis and resulted in one outlier and subsequent removal from the dataset; this procedure did not alter the statistical inferences or resultant conclusions, thereby supporting the robustness of the data presented herein. Post hoc analyses were performed using a Holm–Šidák test. All data are presented as means ± SE, and statistical significance was established at P < 0.05.

RESULTS

Body mass and diaphragm mass were greater in old animals compared with their respective young counterparts (body mass: P = 0.0181; diaphragm mass: P = 0.0133; Table 1). In young and old animals, body and diaphragm mass were greater in males versus females (all P < 0.0001; Table 2). However, diaphragm-to-body mass ratio was not different between young and old or female and male animals (P > 0.05; Tables 1 and 2), suggesting diaphragm atrophy was not present in aged animals. MAP was lower during 1 Hz contractions compared with rest in both young and old animals (young: P = 0.0016; old: P = 0.0229; Table 1). There were no age-related or sex differences in MAP at rest or during 1 Hz contractions (P > 0.05; Tables 1 and 2).

Table 1.

Body mass, diaphragm mass, diaphragm-to-body mass ratio, and mean arterial pressure in young and old animals

Animal Characteristics	Young (n = 15)	Old (n = 18)
Body mass, g	265 ± 22	340 ± 21†
Diaphragm mass, mg	745 ± 59	952 ± 53#
Diaphragm/body mass, mg/g	2.85 ± 0.06	2.89 ± 0.07
MAP, mmHg, rest	117 ± 2	122 ± 4
MAP, mmHg, 1 Hz	105 ± 3**	112 ± 3*

Data are means ± SE. Data were analyzed using a one-way ANOVA, and post hoc analyses were performed using a Holm–Šidák test. n, number of rats; MAP, mean arterial pressure.

†Significant (P = 0.0181) vs. young;

#significant (P = 0.0133) difference vs. young;

*significant (P = 0.0229) difference vs. MAP at rest;

**significant (P = 0.0017) difference vs. MAP at rest.

Table 2.

Body mass, diaphragm mass, diaphragm-to-body mass ratio, and mean arterial pressure in young and old animals of both sexes

Animal Characteristics	YF (n = 7)	OF (n = 9)	YM (n = 8)	OM (n = 9)
Body mass, g	179 ± 2**	250 ± 5*#	339 ± 10	421 ± 5**
Diaphragm mass, mg	528 ± 9**	747 ± 21*#	935 ± 40	1148 ± 28¥
Diaphragm/body mass, mg/g	2.95 ± 0.05	3.00 ± 0.12	2.76 ± 0.09	2.77 ± 0.04
MAP, mmHg, rest	116 ± 2	124 ± 5	119 ± 4	121 ± 6
MAP, mmHg, 1 Hz	106 ± 3a	114 ± 4b	105 ± 6c	109 ± 5d

Data are means ± SE. Data were analyzed using a one-way ANOVA, and post hoc analyses were performed using a Holm–Šidák test. MAP, mean arterial pressure; n, number of rats; OF, old female; OM, old male; YF, young female; YM, young male.

*Significant (P < 0.0001) difference vs. YF;

**significant (P < 0.0001) difference vs. YM;

#significant (P < 0.0001) difference vs. OM;

¥significant (P = 0.0005) difference vs. YM;

^asignificant (P = 0.0011) difference vs. MAP at rest;

^bsignificant (P = 0.0023) difference vs. MAP at rest;

^csignificant (P = 0.0109) difference vs. MAP at rest;

^dsignificant (P = 0.0051) difference vs. MAP at rest.

Age-Related Differences in Diaphragm Hemodynamics

In both young and old animals, total diaphragm VC and BF increased from rest to contractions (young VC and BF, old VC: P < 0.0001; old BF: P = 0.0003; Fig. 2, A and B). Total diaphragm VC and BF at rest and during 1 Hz contractions were not different between young and old animals (P > 0.05; Fig. 2, A and B). In both young and old animals, medial costal diaphragm VC and BF increased from rest to contractions (young VC and BF: P < 0.0001; old VC: P = 0.0002; old BF: P = 0.0300; Fig. 3, A and B). However, medial costal diaphragm VC and BF were higher during 1 Hz contractions in young versus old (VC: P < 0.0001; BF: P = 0.001; Fig. 3, A and B). There were no age-related differences in kidney BF (439 ± 45 vs. 413 ± 40 mL/min/100 g; P > 0.05) or soleus BF (22 ± 2 vs. 25 ± 3 mL/min/100 g; P > 0.05).

Figure 2.

Total diaphragm vascular conductance (VC) (A) and blood flow (BF) (B) in young (n = 15) and old (n = 17) rats at rest and during 1 Hz contractions. Data were analyzed using a mixed-effects two-way RM ANOVA, and post hoc analyses were performed using a Holm–Šidák test. *Significant (P = 0.0003) difference vs. rest. **Significant (P < 0.0001) difference vs. rest.

Figure 3.

Medial costal diaphragm vascular conductance (VC) (A) and blood flow (BF) (B) in young (n = 15) and old (n = 18) rats at rest and during 1 Hz contractions. Data were analyzed using a mixed-effects two-way RM ANOVA, and post hoc analyses were performed using a Holm Šidá test. ***Significant (P < 0.0001) difference vs. rest. **Significant (P = 0.0028) difference vs. rest. *Significant (P = 0.0100) vs. rest. #Significant (P < 0.0001) vs. young. †Significant (P = 0.0010) difference vs. young.

Differences in Diaphragm Hemodynamics between Biological Sexes: Young and Old

In young and old groups, there were no sex differences in resting or contracting total diaphragm VC nor BF (P > 0.05; Fig. 4, A–D). Medial costal diaphragm VC and BF were not different between sexes in either young or old animals (P > 0.05; Fig. 5, A–D). There were no differences between biological sexes in ventral costal or crural diaphragm BF during 1 Hz contractions in either young or old animals (P > 0.05; Fig. 6). However, contracting dorsal costal diaphragm BF was significantly higher in YF and OF compared with their age-matched male counterparts (YF vs. YM: P = 0.0418; OF vs. OM: P = 0.0050; Fig. 6).

Figure 4.

Total diaphragm vascular conductance (VC) and blood flow (BF) in young rats of both sexes (A, C) and old rats of both sexes (B, D) at rest and during 1 Hz contractions. Data were analyzed using a mixed-effects two-way RM ANOVA, and post hoc analyses were performed using a Holm–Šidá test. ***Significant (P < 0.0001) difference vs. rest. **Significant (P = 0.0002) difference vs. rest. *Significant (P = 0.0022) difference vs. rest. #Significant (P = 0.0055) difference vs. rest. ¥Significant (P = 0.0381) difference vs. rest. †Significant (P = 0.0300) difference vs. rest. OF, old female (n = 8); OM, old male (n = 8); YF, young female (n = 7); YM, young male (n = 8).

Figure 5.

Medial costal diaphragm vascular conductance (VC) and blood flow (BF) in young rats of both sexes (A, C) and old rats of both sexes (B, D) at rest and during 1 Hz contractions. Data were analyzed using a mixed-effects two-way RM ANOVA, and post hoc analyses were performed using a Holm–Šidák test. ***Significant (P < 0.0001) difference vs. rest. **Significant (P = 0.0002) vs. rest. *Significant (P = 0.0019) difference vs. rest. †Significant (P = 0.0123) difference vs. rest. #Significant (P = 0.0424) vs. rest. OF, old female (n = 8); OM, old male (n = 9); YF, young female (n = 7); YM, young male (n = 8).

Figure 6.

Age-related and sex differences in regional diaphragm blood flow (BF) during 1 Hz contractions in young and old rats. Data comparisons for YF vs. OF, YM vs. OM, YF vs. YM, and OF vs. OM were analyzed separately using a mixed-effects two-way RM ANOVA, and post hoc analyses were performed using a Holm–Šidák test. **Significant (P = 0.0096) difference vs. YF dorsal. *Significant (P = 0.0070) difference vs. YF medial. #Significant (P = 0.0170) difference vs. YM medial. †Significant (P = 0.0050) vs. OM dorsal. ¥Significant (P = 0.0418) vs. YM dorsal. $Significant (P = 0.0313) vs. YM crural. OF, old female (n = 8); OM, old male (n = 9); YF, young female (n = 7); YM, young male (n = 8).

Sex-Specific Differences in Diaphragm Hemodynamics with Aging

Compared with YF, contracting dorsal costal diaphragm BF was greater in OF (P = 0.0096; Fig. 6). In the ventral costal and crural diaphragm, there were no significant differences in BF during 1 Hz contractions between YF and OF (P > 0.05; Fig. 6). In males, contracting crural diaphragm BF was greater in OM versus YM (P = 0.0313; Fig. 6). There were no age-related differences in contracting BF in the ventral or dorsal costal diaphragm in males (P > 0.05; Fig. 6).

DISCUSSION

Several novel findings emerge from this investigation in young and old rats: 1) regional diaphragm BF distribution was altered with advanced age, 2) aging decreased the ability to augment BF and VC in the medial costal diaphragm, and 3) sex differences in regional diaphragm perfusion are present in both young and old rats. These data support the notion that, similar to locomotory skeletal muscle, the diaphragm vasculature undergoes age-related changes that compromise diaphragm BF regulation and likely contribute to inspiratory muscle dysfunction in older individuals.

Aging and Diaphragm BF Regulation

In spontaneously breathing rats, bulk diaphragm BF is not different between young and old rats (40). The diaphragm contraction protocol utilized herein produces a contractile stimulus analogous to spontaneous breathing (36, 39, 47) and has been used previously to study the hyperemic response in nonrespiratory skeletal muscle(s) of young and old animals (41, 49, 50). Furthermore, this contractile stimulus elicited diaphragm blood flows that are similar to the conscious, spontaneously breathing rat diaphragm (35). Accordingly, the present data agree with that of Delp et al. (40), as total diaphragm BF and VC were not different between young and old rats during contractions (Fig. 2, A and B). These data alone would suggest that aging has no impact on the local control of diaphragm BF; however, within active skeletal muscle, aging induces a redistribution of BF away from highly oxidative (e.g., soleus, red gastrocnemius) to highly glycolytic skeletal muscle (e.g., white gastrocnemius) (30). Given the heterogenous oxidative capacity (51) and BF pattern within the diaphragm (19, 33–39), measurement of total diaphragm hemodynamics alone may belie any age-related alterations in regional diaphragm perfusion that would represent significant changes in diaphragm BF regulation. Therefore, investigating regional diaphragm hemodynamics is requisite to delineate the effects of aging on the local control of diaphragm BF.

The diaphragm comprises two distinct muscle regions: the costal (ventral, medial, dorsal) and crural diaphragm (Fig. 1). Importantly, the medial costal diaphragm sustains the greatest proportion of inspiratory work (19), demanding the highest relative perfusion within the costal diaphragm (34–39) and therefore may be more susceptible to aging-induced aberrations in vascular function. In the present investigation, young animals demonstrated a robust increase in medial costal diaphragm BF and VC with contractions; however, these increases in BF and VC were blunted in old animals (Fig. 3, A and B). In addition, there was an age-related redistribution of regional diaphragm BF during increased contractile activity (Fig. 6). Specifically, in old males, BF was increased in the crural diaphragm, which possesses the lowest oxidative capacity within the diaphragm (51). On the contrary, in old females, the redistribution of diaphragm BF was confined to the dorsal costal diaphragm. These data support the notion that aging 1) impairs diaphragmatic hyperemia, specifically in the medial costal diaphragm, and 2) alters the distribution of diaphragm BF during contractions. Although aging results in alterations in chest wall compliance and pulmonary mechanics (52), it is unknown whether these changes significantly impact regional diaphragm mechanical work and/or perfusion. Notwithstanding this, the altered diaphragm BF distribution and diminished medial costal hyperemic response in the aged diaphragm would result in a poor matching of Q̇o₂-to-V̇o₂, via suboptimal O₂ delivery, and promote diaphragmatic fatigue (13, 18, 20).

Mechanistic Bases for the Reduced Medial Costal Diaphragm Hyperemic Response in Old Age

The inability to augment medial costal diaphragm BF and VC in old rats suggests that aging compromises vasomotor function and/or induces structural alterations in the diaphragm vasculature. Although there is a preponderance of data regarding age-related vascular dysfunction in peripheral skeletal muscle [for review, see Muller-Delp (53)], to our knowledge, there are no studies investigating the effects of aging on diaphragm vasomotor function. Based on the evidence from locomotory skeletal muscle, diaphragm vascular dysfunction with advanced age may be related to diminished endothelial-mediated vasodilation (i.e., decreased nitric oxide bioavailability, endothelial nitric oxide synthase uncoupling) (5, 8, 10, 54, 55), reduced myogenic responsiveness (6), and/or structural modifications (9) (i.e., vascular wall remodeling), all of which would act in concert to limit medial costal diaphragm hyperemia. It is important to note that in locomotory skeletal muscle, vascular smooth muscle relaxation is preserved in old age (5). However, due to the unique physiological characteristics [e.g., high BF capacity, counter-current capillary flow, differential vasomotor function (35, 56, 57)] and lifelong activity of the diaphragm, aging may differentially impact the diaphragm vasculature. Therefore, identifying the vasomotor pathways (i.e., endothelial-dependent, endothelial-independent) and the associated mechanisms involved in age-related diaphragm vascular dysfunction is worthy of further investigation.

Diaphragm BF Distribution and Biological Sex

Sex differences in skeletal muscle fatigue may be related to differences in muscle perfusion (12, 58). Previous work in young adults indicates that in females, the diaphragm may be more fatigue resistant compared with males (44). Contrary to our hypotheses, there were no sex differences in total or medial costal diaphragm BF or VC during contractions in young animals (Fig. 4, A–D and Fig. 5, A–D). However, dorsal costal BF was greater during contractions in young females versus young males (Fig. 6), and this sex difference in regional diaphragm perfusion persisted in old age (Fig. 6). This may be related to differences in diaphragm recruitment between females and males. Specifically, the higher contracting dorsal costal blood flow may reflect a greater reliance on the dorsal costal diaphragm, and thus a higher metabolic demand, than males to sustain the same ventilatory workload. Given that the medial costal diaphragm and dorsal costal diaphragm have similar oxidative capacities (51), the higher dorsal costal BF in young females may explain, in part, the greater diaphragm fatigue resistance reported in females (44). In support of this notion, sex differences in peripheral skeletal muscle fatigue are abolished during ischemia (12). Although dorsal costal BF was greater in old females than in old males, aging reduced medial costal diaphragm perfusion to a similar degree in old animals (Fig. 5D and Fig. 6). Furthermore, age-related decrements in diaphragm function are not different between male and female rats (45). This suggests that the higher dorsal costal BF in old females may not be sufficient to compensate for the blunted medial costal hyperemic response. Therefore, despite the higher dorsal costal diaphragm BF in old females, the diminished medial costal hyperemia in old animals of both sexes could mechanistically contribute to the absence of sex differences in diaphragm dysfunction with old age.

Experimental Considerations

It is possible that the redistribution of diaphragm BF in aged rats could be related to increases in sympathetic activation or changes in local sympathetic activity, such as impaired sympatholysis, associated with old age (59, 60). In the present study, muscle sympathetic activity was not measured; however, we found no differences in MAP or renal BF between young and old rats, which suggests any potential age-related changes in local sympathetic activation had little impact on the results herein. Furthermore, age-related reductions in VC and muscle BF are independent of sympathetic influence on local vascular tone (61). It is important to note that compared with rest, MAP was lower during contractions in each experimental group. It has been demonstrated previously that phrenic artery blood flow is well regulated at arterial pressures of 90–120 mmHg (62), and given that MAP was not different between groups, and in all instances above 90 mmHg, the lower MAP during contractions likely had little influence on the results herein. In addition, the estrous cycle was not tracked in these animals; however, in rats, diaphragm BF is not affected by estrous cycle stage (63), and previous literature demonstrates that 100% of female rats have irregular cycling at 18 mo of age and 75% of female rats are acyclic at 24 mo of age (64). Given the vasodilatory influence and vasoprotective role of estrogen (65, 66), it is possible that sex differences exist in the specific mechanisms that regulate diaphragm vasomotor function; however, this remains to be determined. Regardless, the data herein are novel, as sex differences in diaphragm BF distribution in both young and aged animals have not been reported in the literature.

Implications

Adequate perfusion, and thus resistance vessel function, is a major determinate of diaphragm contractile function (17, 18). The compromised diaphragm hemodynamics during increased contractile activity in old animals suggests that aging induces functional and/or structural deficits in the diaphragm vasculature. Interestingly, in old mice, 14 days of dietary nitrate (NaNO₃) supplementation, which enhances skeletal muscle vascular control and O₂ delivery (67), improves in vitro diaphragm contractile function (68). These data, in addition to the present investigation, support the notion that age-related deficits in diaphragm BF and vasomotor control may contribute to the diaphragm dysfunction associated with old age. Furthermore, elderly patients are at a greater risk of weaning complications with prolonged mechanical ventilation (69, 70), which in and of itself diminishes diaphragm BF and vasomotor function (36–38, 71, 72). Therefore, in pathologies that compromise diaphragm function, age-related decrements in diaphragm blood flow regulation may exacerbate diaphragm dysfunction and contribute to poor clinical outcomes in elderly patients.

Perspectives and Significance

To date, diaphragm vascular control is an area of limited research (73). Despite being chronically active, the diaphragm vasculature and hyperemic capacity are surprisingly vulnerable (36, 39, 71, 72, 74). The present findings suggest that aging may impair diaphragm vascular control within the medial costal diaphragm vasculature. Furthermore, the data herein are the first evidence, to our knowledge, of sex differences in regional diaphragm blood flow distribution in young and old rats. This investigation builds upon our previous work on diaphragm BF regulation in health and disease (36, 39, 71, 72, 74) and represents a key advancement in our understanding of the impact of aging on inspiratory muscle function and cardiovascular control. The next steps in this line of work would be to identify the specific vascular mechanisms contributing to age-related alterations in diaphragm BF control to develop therapeutic approaches that may serve as viable strategies to improve diaphragm perfusion and potentially mitigate aging-induced diaphragm dysfunction.

Conclusions

Old age, independent of biological sex, impaired the hyperemic response in the medial costal diaphragm and altered regional diaphragm BF distribution with contractions. Furthermore, sex differences in regional diaphragm BF are present in young and old animals. Specifically, contracting dorsal costal diaphragm BF was greater in young and old females compared with age-matched males. These data support the notion that age-related changes in diaphragm BF regulation may contribute to the diaphragm contractile dysfunction associated with old age. Future studies are needed to identify the underlying vasomotor pathways involved in the diminished medial costal diaphragm BF response in advanced age.

DATA AVAILABILITY

Data will be made available upon reasonable request.

GRANTS

This work was supported by the National Institute on Aging awarded to Bradley J. Behnke and David C. Poole (1R15AG078060) and Ruth L. Kirschstein National Research Service Awards from the National Heart, Lung, and Blood Institute awarded to Andrew G. Horn (1F31HL167618-02) and Kiana M. Schulze (1F31HL170643-02).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

A.G.H., D.C.P., and B.J.B. conceived and designed research; A.G.H. and K.M.S. performed experiments; A.G.H. and K.M.S. analyzed data; A.G.H., K.M.S., J.M.-D., D.C.P., and B.J.B. interpreted results of experiments; A.G.H. prepared figures; A.G.H. drafted manuscript; A.G.H., K.M.S., J.M.-D., D.C.P., and B.J.B. edited and revised manuscript; A.G.H., K.M.S., J.M.-D., D.C.P., and B.J.B. approved final version of manuscript.