Supplemental oxygen administration during mechanical ventilation reduces diaphragm blood flow and oxygen delivery

Abstract

During mechanical ventilation (MV), supplemental oxygen (O₂) is commonly administered to critically ill patients to combat hypoxemia. Previous studies demonstrate that hyperoxia exacerbates MV-induced diaphragm oxidative stress and contractile dysfunction. Whereas normoxic MV (i.e., 21% O₂) diminishes diaphragm perfusion and O₂ delivery in the quiescent diaphragm, the effect of MV with 100% O₂ is unknown. We tested the hypothesis that MV supplemented with hyperoxic gas (100% O₂) would increase diaphragm vascular resistance and reduce diaphragmatic blood flow and O₂ delivery to a greater extent than MV alone. Female Sprague–Dawley rats (4–6 mo) were randomly divided into two groups: 1) MV + 100% O₂ followed by MV + 21% O₂ (n = 9) or 2) MV + 21% O₂ followed by MV + 100% O₂ (n = 10). Diaphragmatic blood flow (mL/min/100 g) and vascular resistance were determined, via fluorescent microspheres, during spontaneous breathing (SB), MV + 100% O₂, and MV + 21% O₂. Compared with SB, total diaphragm vascular resistance was increased, and blood flow was decreased with both MV + 100% O₂ and MV + 21% O₂ (all P < 0.05). Medial costal diaphragmatic blood flow was lower with MV + 100% O₂ (26 ± 6 mL/min/100 g) versus MV + 21% O₂ (51 ± 15 mL/min/100 g; P < 0.05). Second, the addition of 100% O₂ during normoxic MV exacerbated the MV-induced reductions in medial costal diaphragm perfusion (23 ± 7 vs. 51 ± 15 mL/min/100 g; P < 0.05) and O₂ delivery (3.4 ± 0.2 vs. 6.4 ± 0.3 mL O₂/min/100 g; P < 0.05). These data demonstrate that administration of supplemental 100% O₂ during MV increases diaphragm vascular resistance and diminishes perfusion and O₂ delivery to a significantly greater degree than normoxic MV. This suggests that prolonged bouts of MV (i.e., 6 h) with hyperoxia may accelerate MV-induced vascular dysfunction in the quiescent diaphragm and potentially exacerbate downstream contractile dysfunction.

NEW & NOTEWORTHY This is the first study, to our knowledge, demonstrating that supplemental oxygen (i.e., 100% O₂) during mechanical ventilation (MV) augments the MV-induced reductions in diaphragmatic blood flow and O₂ delivery. The accelerated reduction in diaphragmatic blood flow with hyperoxic MV would be expected to potentiate MV-induced diaphragm vascular dysfunction and consequently, downstream contractile dysfunction. The data presented herein provide a putative mechanism for the exacerbated oxidative stress and diaphragm dysfunction reported with prolonged hyperoxic MV.

INTRODUCTION

Mechanical ventilation (MV) provides lifesaving ventilatory support in patients who are incapable of maintaining sufficient pulmonary gas exchange. Prolonged MV in animals (i.e., ≥6 h) and in humans (≥18 h) results in diaphragm contractile dysfunction, atrophy, and mitochondrial dysregulation (1–7), collectively termed ventilator-induced diaphragm dysfunction (VIDD) (8). VIDD plays a central role in weaning failure with MV, and this inability to wean from the ventilator increases patient morbidity and mortality (9). Although the pathophysiology of VIDD is clearly multifaceted, growing evidence suggests that the O₂ delivery ($\dot{\text{Q}}$O₂)-to-O₂ utilization (V̇o₂) mismatching and impaired vascular function within the diaphragm with prolonged MV are two mechanisms that portend and likely contribute to MV-induced diaphragm contractile dysfunction (10–13).

Diaphragmatic blood flow is reduced with acute MV (i.e., 10–30 min after the induction of MV) (10, 11, 14–16) and these initial reductions in flow are due primarily to diaphragm inactivity (11). Accordingly, during prolonged MV there is a further temporal reduction in diaphragm perfusion and O₂ delivery (10). Moreover, prolonged MV compromises diaphragm vasomotor function and induces resistance vessel remodeling (12, 13). Such vascular decay, and the $\dot{\text{Q}}$O₂-to- V̇o₂ mismatching following prolonged MV with room air (10) likely contributes to problematic weaning.

Arterial hyperoxemia elicits vasoconstriction in skeletal muscle (17, 18) and, in healthy vasculature, high levels of O₂ may be harmful due to this vasoconstrictor effect and the subsequent decrease in local blood flow (19). Interestingly, hyperoxia can elicit a detrimental reduction in O₂ delivery to other highly metabolic tissues such as the myocardium (20). The administration of supplemental O₂ (i.e., 1.0 $\text{F}{\text{I}}_{{\text{O}}_2}$) is common during MV (21) and is independently associated with increased mortality as well as problematic weaning (22–24). In addition, the combination of MV and hyperoxia enhances MV-induced diaphragm oxidative stress and contractile dysfunction associated with VIDD (25). Thus, the use of supplemental O₂ may exacerbate the reductions in diaphragm perfusion and O₂ delivery during MV. However, the effects of supplemental O₂ administration on diaphragm vascular resistance, blood flow, and O₂ delivery during MV are unknown.

Therefore, in the rat model of MV we tested the following hypotheses: 1) MV with hyperoxia (100% O₂) will increase diaphragm vascular resistance and decrease diaphragmatic blood flow and O₂ delivery compared with spontaneous breathing (SB), and normoxic MV (21% O₂), and 2) Following 30 min of MV + 100% O₂, during subsequent MV + 21% O₂, diaphragmatic blood flow and O₂ delivery will increase.

METHODS

Animals

Female Sprague–Dawley rats (n = 19, 4–6 mo old, ∼315 g) were obtained from Charles River Laboratories (Boston, MA) for this investigation and were randomly divided into two experimental groups: 1) MV with hyperoxic gas (100% O₂) followed by MV with room air (21% O₂) (MV + 100% O₂, n = 9; Fig. 1A) and 2) MV with room air (21% O₂) followed by MV with hyperoxic gas (100% O₂) (MV + 21% O₂, n = 10; Fig. 1B) to investigate any potential order effect. The Sprague–Dawley rat was chosen due to the similar properties (e.g., anatomical and physiological) of the rat diaphragm to the human diaphragm (26–28). 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.

Figure 1.

The random distribution of animals after spontaneous breathing (SB) and the experimental paradigm for 100% O₂ postintubation and 21% O₂ postintubation (A and B). MV, mechanical ventilation; $\dot{\text{Q}}$, injection of microspheres for blood flow determination. $\dot{\text{Q}}$1, $\dot{\text{Q}}$2, and $\dot{\text{Q}}$3 represent the three time points for fluorescent microsphere injection.

Surgical Preparation

All surgical procedures were performed using aseptic techniques. Rats were initially anesthetized with a 5% isoflurane-O₂ mixture for 5 min (isoflurane vaporizer; Harvard Apparatus, Cambridge, MA) and subsequently maintained on 3% isoflurane-O₂. Body temperature was maintained at 37 ± 1°C (via rectal thermometer) by use of a water-recirculating heating blanket. An incision was made on the ventral side of the neck and the left carotid artery was isolated and cannulated with PE-10 connected to PE-50 (Intra-Medic 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 Fluorescent Microsphere Injection). A second catheter (PE-10 connected to PE-50) was inserted into the caudal artery for the infusion of pentobarbital sodium anesthesia (50 mg/mL) and reference sampling for blood flow determination. Rats were then transitioned to pentobarbital sodium anesthesia (20 mg/kg body wt) given intra-arterially while concentrations of isoflurane were decreased and subsequently discontinued for ∼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.

Mechanical Ventilation

Once solely under pentobarbital sodium anesthesia, animals were tracheostomized and connected to a volume-cycled rodent ventilator (Kent Scientific PhysioSuite, Torrington, CT). Arterial O₂ saturation ($\text{S}{\text{a}}_{{\text{O}}_2}$%) was continuously monitored using a rodent pulse oximeter (MouseSTAT Jr., Kent Scientific) placed around the right hindlimb foot. Immediately after animals were intubated, a microcapnograph (Microstream Capnostream 20, Medtronics, Minneapolis, MN) sampling line was inserted into the expiratory tube to measure end-tidal CO₂ ($\text{P}\text{E}{\text{T}}_{\text{C}{\text{O}}_2}$). To confirm arterial hyperoxemia during hyperoxic MV, a 0.1 mL arterial blood sample was taken from the carotid artery catheter for blood gas analysis. To ensure diaphragm quiescence, $\text{P}\text{E}{\text{T}}_{\text{C}{\text{O}}_2}$ was maintained at ≤30 mmHg, and diaphragm inactivation was monitored and visually verified. In both experiments, the ventilator was maintained at an average breathing frequency of 70 ± 10 breaths/min and a tidal volume of 6–7 mL/kg body wt. The order of $\text{F}{\text{I}}_{{\text{O}}_2}$ (i.e., 100% O₂ at the onset of MV or the addition during on-going normoxic MV) follows those used clinically when a patient is presented as hypoxemic or becomes hypoxemic during MV (24).

Experiment 1: MV + 100% O₂ Followed by MV + 21% O₂

Fluorescent microspheres were infused at three different time points as outlined in the experimental paradigm (Fig. 1A). Blood flow was measured in each animal (n = 9) during SB followed by MV with hyperoxic gas (100% O₂) for 30 min. Following the second fluorescent microsphere infusion (Fig. 1A), hyperoxic gas was removed and animals were ventilated with room air for 30 min to investigate the effects of hyperoxia withdrawal on diaphragmatic blood flow during MV. Thereafter, the third and final microsphere infusion was performed (Fig. 1A). The difference in sample size between MV + 100% O₂ (n = 9) and MV + 21% O₂ (n = 8) reflects the removal of one animal that exhibited poor microsphere mixing (described under Fluorescent Microsphere Injection).

Experiment 2: MV + 21% O₂ Followed by MV + 100% O₂

Blood flow was measured during SB in each animal (n = 10) followed by MV with 21% O₂ (room air) for 30 min via fluorescent microspheres. Following the second fluorescent microsphere infusion (Fig. 1B), a hyperoxic gas cylinder (100% O₂) was connected to the ventilator inlet valve, and animals were ventilated with 100% O₂ to assess the effect of supplemental oxygen administration during MV when diaphragmatic blood flow is already depressed. After 30 min of hyperoxic MV, the third and final microsphere infusion was performed (Fig. 1B). The difference in sample size between MV + 21% O₂ (n = 10) and MV + 100% O₂ (n = 8) is due to the removal of the two animals that exhibited poor microsphere mixing (described under Fluorescent Microsphere Injection).

Fluorescent Microsphere Injection

The fluorescent microsphere technique, as previously described (29, 30), was used to quantify tissue blood flow in each experimental group. Fluorescent microspheres were infused at three different time points: 1) during spontaneous breathing (SB), 2) during MV, 30 min after intubation and a stable $\text{P}\text{E}{\text{T}}_{\text{C}{\text{O}}_2}$ of ≤30 mmHg, and 3) during MV, 30 min after each gas delivery transition in both experiments (see Fig. 1 Experimental paradigm). 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 before microsphere infusion at a withdrawal rate of 0.25 mL/min and 2.0–2.5 × 10⁵ fluorescent microspheres (colors: red, scarlet, blue-green; 15.5 μm diameter, Invitrogen FluoSpheres, Carlsbad, CA) were infused into the aortic arch via the carotid artery catheter. Adequate mixing of microspheres before injection were determined by <20% difference in left and right kidney or left and right soleus muscle blood flows at each time point. Following the final microsphere infusion, rats were euthanized with pentobarbital sodium overdose (>50 mg/kg I.A.). Thereafter, tissues (diaphragm, soleus, intercostal muscles, mesentery, and kidneys) were harvested, weighed, and placed in 15-mL screw-cap polypropylene conical tubes and then placed in −80°C freezer for later blood flow analysis. The soleus, intercostal, small intestine (to assess mesentery flow), and kidneys were harvested to determine the systemic effect of hyperoxemia during MV on the distribution of blood flow to less compliant (i.e., skeletal muscle) and more compliant (i.e., splanchnic organs) vascular beds. The diaphragm was sectioned into costal (ventral, medial, and dorsal) and crural portions to determine regional distribution of diaphragmatic blood flow, whereas the sum of these portions was used to calculate total diaphragm flow.

Calculation of Blood Flow and Vascular Resistance

The fluorescent microsphere assay was performed according to Deveci and Egginton (29). 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 blood flow was calculated as follows (29):

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

where $\dot{\text{Q}}$ is blood flow (mL/min/100 g), A_t is the individual 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). Vascular resistance was calculated as:

$$\mathrm{VR=MAP}/\dot{\text{Q}}$$

where VR is vascular resistance (mmHg/mL/min/100 g), $\dot{\text{Q}}$ is blood flow (mL/min/100 g), and MAP is the mean arterial pressure (mmHg), recorded immediately before microsphere infusion.

Calculation of Arterial Oxygen Content and Oxygen Delivery

The arterial oxygen content ($\text{C}{\text{a}}_{{\text{O}}_2}$) during the SB, MV + 100% O₂, and MV + 21% O₂ conditions was calculated according to the following equation:

$$\mathrm{C}{\mathrm{a}}_{{\mathrm{O}}_{\mathrm{2}}}\mathrm{(mL/dL)=([Hb]\times 1}\mathrm{.34\times S}{\mathrm{a}}_{{\mathrm{O}}_{\mathrm{2}}}\mathrm{)+(P}{\mathrm{a}}_{\mathrm{O}}{}_{{}_{\mathrm{2}}}\mathrm{\times 0}\mathrm{.003)}$$

where [Hb] is hemoglobin concentration (g/dL), obtained by dividing the measured hematocrit values by 3, 1.34 denotes the carrying capacity of 1 g of hemoglobin (1.39 mL of O₂ less the standard methemoglobin deduction), $\text{S}{\text{a}}_{{\text{O}}_2}$ is the saturation of oxygen (%), $\text{P}{\text{a}}_{\text{O}}{}_{{}_2}$ is the arterial oxygen tension in mmHg, and 0.003 denotes the solubility coefficient of oxygen.

Medial costal diaphragm oxygen delivery was calculated according to the following equation:

$${\mathrm{O}}_{\mathrm{2}}\mathrm{delivery}\mathrm{(mL}{\mathrm{ O}}_{\mathrm{2}}\mathrm{/min/100}\mathrm{g)\hspace{0.17em}=\hspace{0.17em}\hspace{0.17em}}\dot{\text{Q}}\mathrm{\hspace{0.17em}\times \hspace{0.17em}C}{\mathrm{a}}_{{\mathrm{O}}_{\mathrm{2}}}$$

where $\dot{\text{Q}}$ is medial costal blood flow (mL/min/100 g), and $\text{C}{\text{a}}_{{\text{O}}_2}$ is the arterial oxygen content (mL/dL) described earlier. Prior to calculating O₂ delivery, $\text{C}{\text{a}}_{{\text{O}}_2}$ was divided by 100 to get oxygen content (mL) per 1 mL of blood.

Data Analysis

Data were analyzed using GraphPad Prism9 (GraphPad Software, San Diego, CA). Body mass (g), diaphragm mass (g), MAP (mmHg), and heart rate (beats/min) were analyzed using a one-way ANOVA. Tissue vascular resistances, blood flows, and O₂ delivery during SB, MV + 100% O₂, and MV + 21% O₂ were analyzed using a mixed-effects one-way ANOVA. The most extreme outlier in each data set was removed after performing the Grubb’s outlier test on the data set before statistical analysis; 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–Sidak test. All data are presented as means ± SE and statistical significance was established at P < 0.05.

RESULTS

MV + 100% O₂ Postintubation Followed by MV + 21% O₂

Hyperoxemia was confirmed during hyperoxic MV as $\text{P}{\text{a}}_{\text{O}}{}_{{}_2}$ rose to 314 ± 23 mmHg from normal control values between 90 and 95 mmHg (Table 2). Compared with spontaneous breathing (SB), total and medial costal diaphragm vascular resistances were increased during MV + 100% O₂ and remained elevated during MV + 21% O₂ (P < 0.05; Figs. 2A and 3A). Accordingly, total and medial costal diaphragm blood flows were decreased during MV + 100% O₂ and MV + 21% O₂ versus SB (P < 0.05; Figs. 2C and 3C). Total and medial costal diaphragm vascular resistances and blood flows were not different between MV + 100% O₂ and MV + 21% O₂ (P > 0.05; Fig. 2, A and C; Fig. 3, A and C). Consistent with the increase in MAP (Table 1), soleus blood flow was increased during both MV + 100% O₂ and MV + 21% O₂ compared with SB (P < 0.05; Table 3), whereas soleus vascular resistance remained unchanged during SB, MV + 100% O₂, and MV + 21% O₂ (P > 0.05; Table 3). Although MAP was increased during both MV + 100% O₂ and MV + 21% O₂, (Table 1), intercostal vascular resistance and blood flow were not different across all three conditions (P > 0.05; Table 3). Mesenteric vascular resistance was increased during MV with 100% O₂ and decreased after the transition to 21% O₂ (P < 0.05). Furthermore, MV + 100% O₂ decreased mesenteric perfusion, and after the removal of 100% O₂ and transition to MV + 21% O₂, mesenteric blood flow increased versus both SB and MV + 100% O₂ (P < 0.05; Table 3). Renal vascular resistance increased with 100% O₂ (P < 0.05). After the removal of 100% O₂, renal vascular resistance decreased, and renal blood flow increased (P < 0.05; Table 3).

Table 2.

Arterial blood gases during hyperoxic mechanical ventilation

Blood Gases	MV + 100% O2	MV + 21% O2
	Postintubation, n = 9	During on-going MV, n = 8
pH	7.58 ± 0.01	7.56 ± 0.01
Hct, %	30 ± 1	30 ± 2
$\text{P}{\text{a}}_{\text{O}}{}_{{}_2}$, mmHg	314 ± 23	320 ± 19
$\text{P}{\text{a}}_{\text{C}\text{O}}{}_{{}_2}$, mmHg	20 ± 1	21 ± 2

Data are represented as means ± SE. n, number of rats. Hct, hematocrit; mmHg, millimeters of mercury; MV, mechanical ventilation; $\text{P}{\text{a}}_{\text{C}\text{O}}{}_{{}_2}$, partial pressure of carbon dioxide; $\text{P}{\text{a}}_{\text{O}}{}_{{}_2}$, partial pressure of arterial oxygen.

Figure 2.

Total diaphragm vascular resistance (VR) and blood flow (BF) during SB, MV + 100% O₂ immediately postintubation, followed by MV + 21% O₂ (A and C), and total diaphragm vascular resistance and blood flow during SB, MV + 21% O₂ immediately postintubation, followed by MV + 100% O₂ (B and D). *Significant (P < 0.05) difference vs. SB; †Significant (P < 0.05) difference vs. MV + 21% O₂. MV, mechanical ventilation; SB; spontaneous breathing.

Figure 3.

Medial costal diaphragm vascular resistance (VR) and blood flow (BF) during SB, MV + 100% O₂ immediately postintubation, followed by MV + 21% O₂ (A and C), and medial costal diaphragm vascular resistance and blood flow during SB, MV + 21% O₂ immediately postintubation, followed by MV + 100% O₂ (B and D). *Significant (P < 0.05) difference vs. SB; †Significant (P < 0.05) difference vs. MV + 21% O₂. MV, mechanical ventilation; SB; spontaneous breathing.

Table 1.

Body and diaphragm mass, heart rate, and mean arterial pressure from 100% O₂ and 21% O₂ postintubation animals

100% O2 Postintubation
Animal characteristics	SB, n = 9	MV + 100% O2, n = 9	MV + 21% O2, n = 8
Body mass, g	308 ± 11
Diaphragm mass, mg	760 ± 25
Heart rate, beats/min	360 ± 8	384 ± 5*	372 ± 4
MAP, mmHg	90 ± 3	137 ± 2*	129 ± 4*

21% O2 Postintubation
Animal characteristics	SB, n = 10	MV + 21% O2, n = 10	MV + 100% O2, n = 8
Body mass, g	316 ± 8
Diaphragm mass, mg	812 ± 39
Heart rate, beats/min	374 ± 7	400 ± 6*	366 ± 4†
MAP, mmHg	97 ± 5	139 ± 3*	142 ± 2*

Data are represented as means ± SE. n, number of rats. MAP, mean arterial pressure; MV + 100% O₂, mechanical ventilation with supplemental oxygen; MV + 21% O₂, mechanical ventilation with room air; SB, spontaneous breathing. *Significant (P < 0.05) vs. SB; †significant (P < 0.05) vs. MV + 21% O₂ (bottom).

Table 3.

Tissue vascular resistances and blood flows from 100% O₂ postintubation animals

	SB, n = 9	MV + 100% O2, n = 9	MV + 21% O2, n = 8
Vascular resistance, mmHg/mL/min/100 g
Costal diaphragm
Ventral	1.6 ± 0.2	6.4 ± 1.4*	5.4 ± 1.1*
Dorsal	1.9 ± 0.2	4.2 ± 0.7*	4.6 ± 1.0*
Crural diaphragm	2.7 ± 0.2	11.4 ± 2.1*	12.0 ± 2.4*
Soleus	4.4 ± 0.6	3.5 ± 0.8	3.5 ± 0.4
Intercostal	10.1 ± 1.9	14.2 ± 2.5	10.6 ± 2.2
Mesentery	0.9 ± 0.1	1.6 ± 0.2*	0.9 ± 0.2#
Kidney	0.15 ± 0.01	0.24 ± 0.02*	0.18 ± 0.01#
Tissue blood flow, mL/min/100 g
Costal diaphragm
Ventral	50 ± 3	20 ± 6*	27 ± 5*
Dorsal	53 ± 6	40 ± 6*	38 ± 8*
Crural diaphragm	36 ± 3	16 ± 3*	17 ± 4*
Soleus	23 ± 3	52 ± 8*	43 ± 5*
Intercostal	12 ± 2	11 ± 3	16 ± 4
Mesentery	121 ± 9	89 ± 4*	176 ± 22*#
Kidney	634 ± 38	585 ± 58	780 ± 65#

Data are represented as means ± SE. n, number of rats. MV + 100% O₂, mechanical ventilation with supplemental oxygen; MV + 21% O₂, mechanical ventilation with room air; SB, spontaneous breathing. *Significant (P < 0.05) vs. SB; #significant (P < 0.05) vs. MV + 100% O₂.

MV + 21% O₂ Postintubation Followed by MV + 100% O₂

Hyperoxemia was confirmed during hyperoxic MV ($\text{P}{\text{a}}_{\text{O}}{}_{{}_2}$ = 320 ± 19 mmHg; Table 2). As expected with MV, total and medial costal diaphragm vascular resistances were increased compared with SB (P < 0.05; Figs. 2B and 3B). When 100% O₂ was introduced during MV, total and medial costal diaphragm vascular resistance further increased versus MV + 21% O₂ (P < 0.05; Figs. 2B and 3B). Compared with SB, total and medial costal diaphragm blood flows were decreased with MV + 21% O₂ (P < 0.05; Figs. 2D and 3D). The addition of 100% O₂ during MV + 21% O₂ further reduced total and medial costal diaphragm perfusion (P < 0.05; Figs. 2D and 3D). There was a decreased vascular resistance and increased blood flow in the soleus muscle during MV + 21% O₂ versus SB (P < 0.05; Table 4). There were no differences in intercostal muscle vascular resistance or blood flow among all three conditions (P > 0.05; Table 4). In the mesentery, vascular resistance was higher during 100% O₂ versus both SB and MV + 21% O₂ (P < 0.05; Table 4). Furthermore, the addition of 100% O₂ during MV decreased mesenteric blood flow versus MV + 21% O₂ (P < 0.05; Table 4). Compared with SB, renal vascular resistance was increased with MV + 21% O₂, and further increased with the addition of 100% O₂ (P < 0.05; Table 4). Renal perfusion was decreased with 100% O₂ versus MV + 21% O₂ (P < 0.05; Table 4).

Table 4.

Tissue vascular resistances and blood flows from 21% O₂ postintubation animals

	SB, n = 10	MV + 21% O2, n = 10	MV + 100% O2, n = 8
Vascular resistance, mmHg/mL/min/100 g
Costal diaphragm
Ventral	2.2 ± 0.3	5.4 ± 1.0*	8.0 ± 0.6*#
Dorsal	2.2 ± 0.2	4.3 ± 0.8*	6.2 ± 1.1*#
Crural diaphragm	3.7 ± 0.6	11.2 ± 2.5*	12.8 ± 2.0*
Soleus	4.5 ± 0.6	2.7 ± 0.4*	3.9 ± 0.8
Intercostal	8.4 ± 1.8	15.4 ± 4.0 (P = 0.13)	15.3 ± 1.5 (P = 0.07)
Mesentery	1.0 ± 0.1	1.1 ± 0.1	1.6 ± 0.1*#
Kidney	0.15 ± 0.01	0.20 ± 0.02*	0.28 ± 0.03*#
Tissue blood flow, mL/min/100 g
Costal diaphragm
Ventral	53 ± 8	41 ± 14	17 ± 2*#
Dorsal	52 ± 10	38 ± 4*	29 ± 5*#
Crural diaphragm	31 ± 4	17 ± 3*	13 ± 2*
Soleus	25 ± 3	69 ± 10*	53 ± 12*
Intercostal	15 ± 3	14 ± 4	10 ± 1
Mesentery	115 ± 14	132 ± 13	89 ± 5#
Kidney	663 ± 61	739 ± 63	498 ± 39#

Data are represented as means ± SE. n, number of rats. MV + 100% O₂, mechanical ventilation with supplemental oxygen; MV + 21% O₂, mechanical ventilation with room air; SB, spontaneous breathing. *Significant (P < 0.05) vs. SB; #significant (P < 0.05) vs. MV + 21% O₂.

Arterial Oxygen Content and Oxygen Delivery during MV + 100% O₂ Postintubation

Administration of supplemental 100% O₂ postintubation significantly increased arterial O₂ content ($\text{C}{\text{a}}_{{\text{O}}_2}$) versus SB (P < 0.05), and upon transition from 100% to 21% O₂, $\text{C}{\text{a}}_{{\text{O}}_2}$ during MV + 21% O₂ was decreased compared with MV + 100% O₂ (P < 0.05; Fig. 4A). Although supplemental O₂ increased $\text{C}{\text{a}}_{{\text{O}}_2}$, medial costal diaphragm O₂ delivery was decreased during MV + 100% O₂ versus SB (P < 0.05; Fig. 4C). After the removal of 100% O₂, medial costal diaphragm O₂ delivery was not different during MV + 21% O₂ (P > 0.05; Fig. 4C).

Figure 4.

Arterial oxygen content and medial costal diaphragm oxygen delivery during SB, MV + 100% O₂ immediately postintubation, followed by MV + 21% O₂ (A and C), and arterial oxygen content and medial costal diaphragm oxygen delivery during SB, MV + 21% O₂ immediately postintubation, followed by MV + 100% O₂ (B and D). *Significant (P < 0.05) difference vs. SB; #Significant (P < 0.05) difference vs. MV + 100% O₂; †Significant (P < 0.05) difference vs. MV + 21% O₂. SB; spontaneous breathing; MV, mechanical ventilation.

Arterial Oxygen Content and Oxygen Delivery during MV + 21% O₂ Postintubation

$\text{C}{\text{a}}_{{\text{O}}_2}$ was not different between SB and MV + 21% O₂ (P > 0.05), and was significantly elevated during MV + 100% O₂ (P < 0.05; Fig. 4B). However, the addition of 100% O₂ during on-going MV further decreased medial costal O₂ delivery versus MV + 21% O₂ (P < 0.05; Fig. 4D). Compared with MV + 21% O₂ postintubation, the reductions in medial costal blood flow and O₂ delivery were greater during MV + 100% O₂ postintubation (P < 0.05; Fig. 5, A and B).

Figure 5.

Medial costal diaphragmatic blood flow (BF; A) and oxygen delivery (B) postintubation during MV + 100% O₂ and MV + 21% O₂. *Significant (P < 0.05) difference vs. MV + 100% O₂. MV, mechanical ventilation.

DISCUSSION

The present results demonstrate that administration of supplemental oxygen (i.e., 100% O₂) amplifies the MV-induced increases in diaphragm vascular resistance and reductions in bulk and regional diaphragm perfusion. Thus, concomitant arterial hyperoxemia and MV compromised diaphragm O₂ delivery: An effect that was especially marked in the medial costal region. These effects were specific to the diaphragm muscle, as blood flow was unchanged (e.g., intercostals) or even increased to other skeletal muscles (likely due to increased arterial pressure) (e.g., soleus; Tables 3 and 4). Interestingly, MV + 100% O₂ increased splanchnic vascular resistances such that blood flow was reduced (e.g., kidneys, mesentery; Tables 3 and 4). These data provide novel support for the notion that use of supplemental O₂ during prolonged MV may exacerbate diaphragm vascular dysfunction and, consequently, contractile dysfunction.

Total and Regional Diaphragmatic Blood Flow and O₂ Delivery

The reductions in bulk diaphragmatic blood flow with hyperoxic MV, regardless of when supplemental O₂ was administered (i.e., immediately postintubation vs. on-going normoxic MV) (Fig. 2, A–D), suggests an increased tonic vasoconstriction of the diaphragm vasculature that is not seen in other skeletal muscles (Tables 3 and 4). Contrary to our hypothesis, total diaphragm vascular resistance remained elevated and blood flow did not increase after the removal of supplemental O₂ during MV (i.e., transition from 100% O₂ to room air MV) (Fig. 2, A and C), suggesting that hyperoxia elicits a powerful local vasoconstriction in the inactive diaphragm that does not subside even after removal of 100% O₂.

Given the heterogeneous distribution of blood flow in the diaphragm (10, 11, 14, 15, 28, 31), measurements of total diaphragm hemodynamics alone do not capture the regional effects of hyperoxia during MV on blood flow regulation. Within the diaphragm, the medial costal portion sustains the greatest proportion of inspiratory work and commands the highest relative blood flow (28), and therefore appears more susceptible to inactivity-mediated tissue and vascular injury with MV (12, 13, 32). Similar to previous MV investigations (10, 11), the reductions in diaphragmatic blood flow herein, with both normoxic and hyperoxic MV, were most apparent within the medial costal portion. In the present investigation, during MV + 100% O₂, medial costal vascular resistance was increased, whereas blood flow and O₂ delivery were decreased to a greater extent than MV alone (Fig. 4C). Interestingly, the addition of 100% O₂ during on-going normoxic MV further increased vascular resistance and diminished medial costal perfusion and O₂ delivery (Fig. 3, B and D). The initial vasoconstriction of the medial costal diaphragm vasculature during hyperoxic MV is undoubtedly due to the vasoconstrictive nature of hyperoxia (19, 33, 34). On the other hand, its failure to reverse upon restoration of normoxic MV implicates other mechanisms (discussed under Hyperoxic MV and Exacerbated VIDD: Potential Mechanisms) in the maintenance of increased tonic vasoconstriction following hyperoxia + MV.

Systemic Hemodynamic Effects of Hyperoxic MV

Although the skeletal muscle perfusion decrements determined herein with normoxic MV are specific to the diaphragm, the additive and systemic effects of hyperoxia on peripheral skeletal muscle (i.e., soleus and intercostals) and splanchnic (i.e., mesentery and kidney) hemodynamics during MV are not well characterized. Recently, we demonstrated that soleus vascular resistance is reduced, and blood flow is increased, whereas intercostal muscle vascular resistance and perfusion were unchanged with acute normoxic MV (i.e., 10 min) (11). In the present investigation, the same hemodynamic changes occurred in the soleus and intercostal muscles during both MV + 21% O₂ and MV + 100% O₂ with the exception of intercostal vascular resistance, which increased with the addition of 100% O₂ during on-going normoxic MV (Table 4). Hyperoxia has a systemic vasoconstrictor effect, however, the data herein suggest that peripheral skeletal muscle vasculature may be relatively insensitive to hyperoxia’s effect during MV, as blood flow to the intercostal and soleus muscles was not different between MV with 21% and 100% O₂. This may be due to the changes in sympathetic nerve activity and its effect on the redistribution of cardiac output during acute MV.

Splanchnic organs receive a substantial portion of the cardiac output (∼25%) at rest and nearly 50% of total sympathetic outflow (35, 36), and thus represent an important aspect of blood flow regulation and cardiovascular homeostasis. In animals and humans, MV elicits reflex sympathoexcitation and increases renal sympathetic activation (37, 38). Previously, we have shown that renal vascular resistance is elevated, and renal perfusion is significantly decreased with 10 min of MV (11). In the present investigation, during MV with 21% and 100% O₂ immediately postintubation, renal vascular resistance was increased with no change in blood flow (Tables 3 and 4). However, with the MV protocol herein, animals were ventilated for 30 min in each condition before measuring tissue blood flow, supporting that the hemodynamic effect of increased renal sympathetic activation wanes at some point beyond 10 min of MV.

Previous literature regarding the effects of 100% O₂ on splanchnic hemodynamics is conflicting. Specifically, Waisman et al. (39) showed that 100% O₂ elevated mesenteric vascular resistance with no change in perfusion. Other investigations have demonstrated that 100% O₂ increased renal and mesentery blood flow, with either no change or a decrease in vascular resistance (40, 41). Furthermore, Van den Bos et al. (42) showed that elevating tissue Po₂ had no effect on mesenteric arteriolar or venular tone. Herein, MV + 100% O₂ postintubation elevated mesenteric vascular resistance, diminishing blood flow (Table 3). After removal of 100% O₂ during MV, mesenteric and renal vascular resistances decreased, driving an increase in mesenteric and renal perfusion. Interestingly, the addition of 100% O₂ during on-going MV increased both renal and mesenteric vascular resistance with concomitant reductions in renal and mesenteric perfusion (Table 4). This suggests that during MV, the addition of supplemental oxygen and subsequent hyperoxemia elicits a vasoconstriction in these splanchnic organs that diminishes splanchnic perfusion. Moreover, peripheral skeletal muscles appear to be resistant to the systemic vasoconstrictive effects of hyperoxemia during MV.

Hyperoxic MV and Exacerbated VIDD: Potential Mechanisms

In skeletal muscle, deep tissue Po₂ falls when muscle surface Po₂ is elevated (43, 44). Given the close apposition of the diaphragm to the pulmonary parenchyma, the Po₂ at the superior surface of the diaphragm may be increased during MV + 100% O₂. In an aged rodent model, the combination of 100% O₂ and 3 h of MV compromises diaphragm contractile function (45), suggesting that hyperoxia may accelerate MV-induced diaphragm contractile dysfunction. Prolonged hyperoxic MV decreases diaphragm contractility and increases MV-induced oxidative stress (25), potentially exacerbating VIDD. It is plausible that the exaggerated diaphragm dysfunction with hyperoxic MV may be related to prolonged hyperoxia-mediated vasoconstriction of the diaphragm resistance vasculature, and the subsequent reductions in blood flow and O₂ delivery; all of which would negatively impact diaphragm vascular and contractile function.

In addition to eliciting vasoconstriction, hyperoxia has been shown to increase vascular reactive oxygen species (ROS) (e.g., superoxide anion) production and lead to endothelial damage (46, 47). Herein, 100% O₂ at MV onset elevated diaphragm vascular resistance, which persisted throughout subsequent MV + 21% O₂ (Figs. 2A and 3A), evidencing a prolonged hyperoxia-induced vasoconstriction. This effect may be consequent to a hyperoxia-mediated inhibition of endothelial prostaglandins (48), increased vascular reactive oxygen species (ROS) formation (46, 49, 50), diminished nitric oxide (NO) bioavailability (51, 52), and/or ROS-mediated endothelial damage (46, 47, 53); all acting in concert to limit diaphragm perfusion. Furthermore, in humans, hyperoxic vasoconstriction impairs endothelium-dependent vasodilation via increased oxidative stress (47). Thus, the augmented vasoconstriction and changes in vasoactive mediators (e.g., NO, ROS) with hyperoxia, if present during prolonged MV (i.e., 6 h), may promote vascular entrenchment (54, 55) and accelerate and/or exacerbate MV-induced diaphragm vascular dysfunction (12, 13).

Experimental Considerations

Diaphragm quiescence was verified visually in our investigation, but not through electromyography; however, the diaphragm is rendered inactive (verified via electromyography) during prolonged controlled MV in the anesthetized rat (4, 5). Given that end-tidal CO₂ was maintained at or below 30 mmHg during the MV protocol herein, it was unlikely that there was any meaningful chemoreceptor-induced drive to breathe. Since the diaphragm was inactivated during MV in the same manner in each experiment, it is unlikely that hypocapnia influenced the results herein. However, we cannot rule out that some diaphragm motor units were not inactivated during MV. Similar to previous investigations (10–13), adult female Sprague–Dawley rats (∼6 mo) were used for this investigation. This age range, healthy status, and the slower growth rate of females allowed for experimental investigation in the absence of underlying pathologies and body mass differences among groups. Moreover, VIDD occurs equally in males and females (3), and it is acknowledged that VIDD can be studied in both male and female rats. Finally, the estrous cycle was not tracked in these animals, however, it has been demonstrated that diaphragm blood flow is not impacted by the stage of the estrous cycle (56).

Ramifications and Conclusions

The data herein demonstrate that increasing $\text{C}{\text{a}}_{{\text{O}}_2}$ during MV (via supplemental 100% O₂) diminishes medial costal diaphragmatic blood flow and O₂ delivery. These hyperoxia-mediated reductions in perfusion and O₂ delivery may expedite the time course of MV-induced vasomotor dysfunction with prolonged MV (12), providing a potential mechanism for the downstream diaphragm contractile dysfunction with prolonged hyperoxic MV (25). VIDD is primarily initiated by diaphragm inactivity during MV (57) and it has been demonstrated that hyperoxic MV exacerbates VIDD (25). Given that reductions in diaphragm perfusion promote contractile fatigue (58) and that diaphragm inactivity is not different between normoxic MV and hyperoxic MV, the more severe reductions in diaphragmatic blood flow and O₂ delivery during hyperoxic MV would potentiate diaphragm vascular dysfunction, and thus, exacerbate diaphragm contractile dysfunction. Therefore, the reduced diaphragm perfusion and O₂ delivery during hyperoxic MV may predispose patients to prolonged MV, VIDD, and problematic weaning. We predict that the use of hyperoxia during prolonged MV may exacerbate MV-induced diaphragm vascular dysfunction and argue for the careful titration of inspired hyperoxia to balance patient needs while avoiding pernicious hyperoxemia.

GRANTS

This work was supported by the National Heart, Lung, and Blood Institute Grant 1 R15 HL-137156-01A1, the National Institute of Aging Grant 1 R15 AG 078060, an award from the Johnson Cancer Research Center A 21-0645, and a K-State College of Veterinary Medicine Sustained Momentum for Investigators with Laboratories Established (S.M.I.L.E.) Award (to D. C. Poole).

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., O.N.K., K.M.S., D.R.B., and R.E.W. performed experiments; A.G.H. analyzed data; A.G.H., D.C.P., and B.J.B. interpreted results of experiments; A.G.H. prepared figures; A.G.H. drafted manuscript; A.G.H., O.N.K., K.M.S., D.R.B., R.E.W., D.C.P., and B.J.B. edited and revised manuscript; A.G.H., O.N.K., K.M.S., D.R.B., R.E.W., D.C.P., and B.J.B. approved final version of manuscript.