Online Data Supplement
Methods
Measurement of Blood Flow
Blood flow to respiratory and selected muscles of the hindlimb, as well as the kidneys,
were determined using radiolabelled microspheres as described previously (Delp & Armstrong,
1988; Musch & Terrell, 1992). These animals received the same surgery for MV as listed above;
however, an additional catheter was placed in the caudal artery which was connected to a
Harvard infusion/withdrawal pump (model 907, Cambridge, MA). Thirty-seconds prior to
infusion of the microspheres (15 μm diameter, 46Sc, and 85Sr; DuPont/NEN, Boston, MA, USA)
blood withdrawal from the caudal artery was initiated at 0.25 ml/min and 2.5 x 105 microspheres
were infused, in random order, into the carotid artery catheter. Sequential infusions of the
quantity of microspheres utilized herein (totaling ~5.0 x 105) do not affect cardiac output, mean
arterial pressure or muscle blood flow (Ishise et al., 1980; Flaim et al., 1984; Bailey et al., 2000).
Upon completion of each experiment, the diaphragm, intercostal muscles (combined internal and
external), soleus, red portion of the gastrocnemius muscle and kidneys were removed. Tissue
radioactivity was determined via a γ-scintillation counter (Cobra II Auto Gamma Counter,
Packard, Downers Grove, IL, USA), and individual tissue flows were determined by the
reference sample method (Ishise et al., 1980) and expressed in milliliters per minute per 100
grams of tissue. To normalize for any change in arterial blood pressure vascular conductance
was calculated and expressed as milliliters per minute per 100 grams of tissue per mmHg. All
rats exhibited a < 15% difference in blood flow between the left and right kidneys at each time
point, indicating adequate mixing of microspheres during infusions.
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Phosphorescence Quenching
Phosphorescence quenching was used to measure diaphragm muscle PO2m during
spontaneous breathing and after 30 min and 6 hrs of MV. Rats were anesthetized with
pentobarbital sodium (50 mg/kg i.p., supplemented as needed) and the right carotid artery was
isolated and cannulated with a fluid-filled catheter (PE-50) to monitor arterial blood pressure
(Digi-Med BPA Model 400a; Micro Med, Inc.; Louisville, KY). This catheter was also used for
the infusion of the phosphorescent probe R2.
The diaphragm was exposed via laparotomy and the liver was reflected gently downward,
permitting access to the inferior surface of the medial and ventral regions of the diaphragm. The
phosphorimeter light guide was positioned 2-5 mm from the surface of the medial costal
diaphragm, and exposed surfaces were covered with Saran wrap to prevent moisture and heat
loss. The abdominal cavity was kept moist via superfusion of a Krebs-Henseleit bicarbonatebuffered solution equilibrated with 5% CO2/95% N2 at 37°C. A temperature probe was placed
between the liver and the diaphragm to monitor the temperature of the abdominal cavity.
The phosphor, palladium meso-tetra-(4-carboxyphenyl)-porphyrin dendrimer (R2;
Oxygen Enterprises Ltd., Philadelphia, PA), was infused ~10 min before each experiment at a
dose of 15 mg/kg and PO2m measurements were recorded every 2 s for 60 s to provide an
average steady-state PO2m during spontaneous breathing and after 30 min and 6 hrs of MV. All
PO2m measurements were performed in a dark room to avoid contamination of the signal with
ambient light. The R2 probe binds to albumin and is distributed uniformly in the plasma,
therefore providing a signal corresponding to the volume-weighed O2 pressure in the
microvascular compartment (mainly the PO2 within the capillaries, which volumetrically
represents the major intramuscular space; ref. (Poole et al., 1995)). The negative charge of the
2
R2 probe also facilitates its restriction to the muscle intravascular space (Vink & Duling, 2000).
Although it has previously been demonstrated that R2 does not extravasate in skeletal muscle
(Poole et al., 2004), we measured diaphragm PO2m obtained only after 6 hrs of MV in a separate
group of animals (n=5) and compared PO2m values from these animals to those of animals that
had the R2 circulating for the entire 6 hrs period. In this group MV was initiated as described
above, however, the surgery to expose the diaphragm began after 5.5 hrs of MV. The infusion of
R2 and measurement of diaphragm PO2m was not initiated until 6 hrs of MV. We did not
observe any difference in PO2m between the 6 hrs groups (i.e., those with R2 circulating for the
entire 6 hrs or those with the R2 circulating for <5 min (Supplemental Figure 1)), confirming R2
probe compartmentalization to the intravascular as previously demonstrated (Poole et al., 2004).
Upon completion of the experiment each rat was euthanized with an overdose of anesthesia
(pentobarbital sodium, >100 mg/kg, i.a.) and a thoracotomy was performed to visually verify
cardiac arrest.
PO2m Measurements and Calculations
The principles of the phosphorescence quenching technique have been discussed
previously (Poole et al., 1995; Behnke et al., 2001). The Stern-Volmer relationship allows the
calculation of PO2m through the direct measurement of a phosphorescence lifetime via the
following equation (Rumsey et al., 1988):
PO2m = [(to/ t)] ― 1/(kq*to)
where kQ is the quenching constant (expressed in mmHg/s) and τ° and τ are the phosphorescence
lifetimes in the absence of O2 and the ambient O2 concentration, respectively. For R2, kQ is 409
mmHg/s and τ° is 601 μs (Lo et al., 1997) and the effects of pH and temperature are negligible
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within the physiological range in vivo (Pawlowski & Wilson, 1992). The light guide contained
within the probe focused excitation light (524 nm) on the medial region of the exposed
diaphragm (~2.0 mm diameter, to ~500 μm deep). The PO2m was determined with a PMOD
5000 Frequency Domain Phosphorometer (Oxygen Enterprises Ltd., Philadelphia, PA) at
frequencies between 100 Hz and 20 kHz, which allows phosphorescence lifetime measurements
from 10 μs to ~2.5 ms. In the single frequency mode, 10 scans (100 ms) were used to acquire the
resultant lifetime of the phosphorescence signal (700 nm) (Rev. see (Vinogradov, 2001)) and
phosphorescence lifetimes were computationally obtained based on the decomposition of data
vectors to a linearly independent set of exponentials (Vinogradov & Wilson, 1994).
．
Calculation of Diaphragm VO2
．
Muscle oxygen uptake (VO2) of the medial costal diapragm (i.e., region of PO2m
measures) was calculated from blood flow and PO2m measurements in the diaphragm during the
resting and contracting (see below) conditions as described by Behnke et al. (Behnke et al.,
．
2002). VO2 was calculated using the Fick equation using PO2m as an analogue for venous PO2
(Wagner, 1991; Roca et al., 1992) and, from the O2 dissociation curve, microvascular blood O2
content (CmO2)(McDonough et al., 2001). As there was no observable change in muscle
temperature or blood pH, no significant shift in the O2 dissociation curve would be expected.
．
From data collected on arterial blood samples, PO2m and muscle blood flow (Qm), VO2 was
calculated using:
．
VO2 = Qm ˟ (CaO2-CmO2)
．
where VO2 is the oxygen uptake of the diaphragm muscle, Qm is diaphragm muscle blood flow,
and CaO2 and CmO2 are the oxygen contents of the arterial and microvascular blood,
．
respectively. Muscle VO2 was expressed as ml/min/per 100 g tissue.
4
Diaphragm Contractions
The diaphragm was exposed as described above and stainless steel electrodes were
sutured (6-0 silk; Ethicon, Somerville, NJ) to the right ventral costal (cathode) and the right
dorsal costal (anode) diaphragm according to the methods of Geer et al. (Geer et al., 2002).
Electrically stimulated twitch contractions were induced a 1 Hz (3-6 V, 2-ms pulse duration)
with a Grass S88 stimulator (Quincy, MA) for 3 min. This contraction protocol elicits
contractions at a similar frequency as during with spontaneous breathing. However, electrical
stimulation would be expected to contract all muscle fibers and consequently it might be
．
supposed that the resultant VO2 would be higher than that observed during spontaneous
breathing. For example, in vivo during normal breathing there is a heterogeneous pattern of
blood flow distribution suggesting a similar heterogeneous distribution of metabolic activity
(Brancatisano et al., 1991; Sexton & Poole, 1995). Electrical stimulation of the muscle may alter
the heterogeneity of motor unit activation due to the concurrent activation of the majority of
fibers. The stimulation protocol was initiated after 30 min and 6 hrs of MV and PO2m was
measured at 2-s intervals across the rest-to-contractions transition. Blood flow was measured
．
after 180 s of stimulation to assess the contracting steady-state muscle hyperemia. VO2 was then
calculated as described above for resting and steady-state contracting conditions.
Statistics
A statistical power analysis (Sigma Stat 4.0, Systat Software Inc.) was performed to
estimate the sample size needed for a given set of experiments. KaleidaGraph 4.0 (Synergy
Software, Reading, PA) was used for data analysis by fitting a user-defined function to the PO2m
data by using an iterative least squares error method. A monoexponential function with a time
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delay was used to assess PO2m dynamics across the rest-to-contractions transition according to
the methods of Geer et. al. (Geer et al., 2002). Goodness of fit was determined by the coefficient
of determination. Differences between parameters (i.e., time delay (TD), time constant (tau) and
the mean response time (TD + tau)) were determined by a repeated measures analysis of variance
(ANOVA). Resting and steady-state contracting PO2m, blood pressure, arterial blood gases, pH
from the individual animals across time were analyzed with repeated measures ANOVA. Blood
flow, vascular conductance, O2 delivery, and VO2 were analyzed with a one-way ANOVA.
．
Linear regression analysis was performed to compare MAP and arterial PO2 to PO2m, VO2 and
the PO2m mean response time. Individual significant differences were examined post hoc using
Tukey’s test. Data are presented as means ± SE. Significance was accepted at P ≤ 0.05.
Reference
Bailey JK, Kindig CA, Behnke BJ, Musch TI, Schmid-Schoenbein GW & Poole DC. (2000).
Spinotrapezius muscle microcirculatory function: effects of surgical exteriorization. Am J
Physiol Heart Circ Physiol 279, H3131-3137.
Behnke BJ, Barstow TJ, Kindig CA, McDonough P, Musch TI & Poole DC. (2002). Dynamics
of oxygen uptake following exercise onset in rat skeletal muscle. Respir Physiolo
Neurobiol 133, 229-239.
Behnke BJ, Kindig CA, Musch TI, Koga S & Poole DC. (2001). Dynamics of microvascular
oxygen pressure across the rest-exercise transition in rat skeletal muscle. Respir Physiol
126, 53-63.
Brancatisano A, Amis TC, Tully A, Kelly WT & Engel LA. (1991). Regional distribution of
blood flow within the diaphragm. J Appl Physiol 71, 583-589.
Delp MD & Armstrong RB. (1988). Blood flow in normal and denervated muscle during
exercise in conscious rats. Am J Physiol 255, H1509-1515.
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Flaim SF, Nellis SH, Toggart EJ, Drexler H, Kanda K & Newman ED. (1984). Multiple
simultaneous determinations of hemodynamics and flow distribution in conscious rat. J
Pharmacol Methods 11, 1-39.
Geer CM, Behnke BJ, McDonough P & Poole DC. (2002). Dynamics of microvascular oxygen
pressure in the rat diaphragm. J Appl Physiol 93, 227-232.
Ishise S, Pegram BL, Yamamoto J, Kitamura Y & Frohlich ED. (1980). Reference sample
microsphere method: cardiac output and blood flows in conscious rat. Am J Physiol 239,
H443-H449.
Lo LW, Vinogradov SA, Koch CJ & Wilson DF. (1997). A new, water soluble, phosphor for
oxygen measurements in vivo. Adv Exp Med Biol 428, 651-656.
McDonough P, Behnke BJ, Kindig CA & Poole DC. (2001). Rat muscle microvascular PO2
kinetics during the exercise off-transient. Exp Physiol 86, 349-356.
Musch TI & Terrell JA. (1992). Skeletal muscle blood flow abnormalities in rats with a chronic
myocardial infarction: rest and exercise. Am J Physiol 262, H411-419.
Pawlowski M & Wilson DF. (1992). Monitoring of the oxygen pressure in the blood of live
animals using the oxygen dependent quenching of phosphorescence. Adv Exp Med Biol
316, 179-185.
Poole DC, Behnke BJ, McDonough P, McAllister RM & Wilson DF. (2004). Measurement of
muscle microvascular oxygen pressures: compartmentalization of phosphorescent probe.
Microcirculation 11, 317-326.
Poole DC, Wagner PD & Wilson DF. (1995). Diaphragm microvascular plasma PO2 measured
in vivo. J Appl Physiol 79, 2050-2057.
Roca J, Agusti AG, Alonso A, Poole DC, Viegas C, Barbera JA, Rodriguez-Roisin R, Ferrer A
& Wagner PD. (1992). Effects of training on muscle O2 transport at VO2max. J Appl
Physiol 73, 1067-1076.
Rumsey WL, Vanderkooi JM & Wilson DF. (1988). Imaging of phosphorescence: a novel
method for measuring oxygen distribution in perfused tissue. Science 241, 1649-1651.
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Sexton WL & Poole DC. (1995). Costal diaphragm blood flow heterogeneity at rest and during
exercise. Respir Physiol 101, 171-182.
Vink H & Duling BR. (2000). Capillary endothelial surface layer selectively reduces plasma
solute distribution volume. Am J Physiol Heart Circ Physiol 278, H285-289.
Vinogradov S, M.A. Fernandez-Searra, B.W. Dugan, and D.F. Wilson. (2001). Frequency
domain instrument for measuring phosphorescence lifetime distributions in
heterogeneous samples. Review of Scientific Instruments 72, 3396-3406.
Vinogradov SA & Wilson DF. (1994). Phosphorescence lifetime analysis with a quadratic
programming algorithm for determining quencher distributions in heterogeneous systems.
Biophys J 67, 2048-2059.
Wagner PD. (1991). Central and peripheral aspects of oxygen transport and adaptations with
exercise. Sports Med 11, 133-142.
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Microvascular PO2 (torr)
25
6 Hrs MV with R2 circulating entire duration
6 Hrs MV with R2 infused directly before PO2 measured
20
15
10
5
0
Supplemental Figure 1. Mean diaphragm microvascular PO2 measured after 6 hrs of
MV with the R2 probe in the circulation for the entire period (n=8) and immediately
after the R2 probe was infused (i.e., within 1 minute) in a separate group of animals
(n=5) subjected to 6 hrs of MV. There was no difference in PO2m between groups
confirming the PO2 values obtained in the 6 hrs MV group with the R2 probe
circulating for the entire period originated from the vascular compartment. For further
information see “Phosphorescence quenching” in the online data supplement.
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Vascular Conductance
(ml/min/100 g/mmHg)
0.6
¥
#
¥
#
¥
#
0.4
¥
#
*
0.2
0.0
Medial Costal
Ventral
Dorsal
Crural
Spontaneous
Breathing
30 min
MV
* *
* * †
†
† †
6 hr
MV
Supplemental Figure 2. Vascular conductance to regionally delineated
portions of the diaphragm muscle during spontaneous breathing and after 30
min and 6 hrs of MV. *P<0.05 versus same muscle during spontaneous
breathing; †P<0.05 versus same muscle after 30 min MV. ¥P<0.05 versus
ventral diaphragm. #P<0.05 versus medial costal diaphragm.
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