HHS Public Access Author manuscript Author Manuscript
Microvasc Res. Author manuscript; available in PMC 2017 May 01. Published in final edited form as: Microvasc Res. 2016 May ; 105: 15–22. doi:10.1016/j.mvr.2015.12.007.
Simultaneous Sampling of Tissue Oxygenation and Oxygen Consumption in Skeletal Muscle William H. Nugent1, Bjorn K. Song1, Roland N. Pittman1, and Aleksander S. Golub1 1Department
of Physiology and Biophysics, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298
Author Manuscript
Abstract
Author Manuscript
Under physiologic conditions, microvascular oxygen delivery appears to be well matched to oxygen consumption in respiring tissues. We present a technique to measure interstitial oxygen tension (PISFO2) and oxygen consumption (VO2) under steady-state conditions, as well as during the transitions from rest to activity and back. Phosphorescence Quenching Microscopy (PQM) was employed with pneumatic compression cycling to achieve 1 to 10 Hz sampling rates of interstitial PO2 and simultaneous recurrent sampling of VO2 (3/min) in the exteriorized rat spinotrapezius muscle. The compression pressure was optimized to 120–130 mmHg without adverse effect on the tissue preparation. A cycle of 5 s compression followed by 15 s recovery yielded a resting VO2 of 0.98 ± 0.03 ml O2/100cm3 min while preserving microvascular oxygen delivery. The measurement system was then used to assess VO2 dependence on PISFO2 at rest and further tested under conditions of isometric muscle contraction to demonstrate a robust ability to monitor the onkinetics of tissue respiration and the compensatory changes in PISFO2 during contraction and recovery. The temporal and spatial resolution of this approach is well suited to studies seeking to characterize microvascular oxygen supply and demand in thin tissues.
Keywords Oxygen Consumption (VO2); Phosphorescence Quenching Microscopy (PQM); Skeletal Muscle; Rat Spinotrapezius Muscle; Microvasculature; Oxygen Measurement; Muscle Stimulation; Tissue Compression
INTRODUCTION Author Manuscript
The oxygen (O2) supply and demand of skeletal muscle exists in a dynamic and responsive equilibrium within the interstitial fluid providing a key focal point of observation (Ferreira, Poole et al. 2005). At rest, O2 diffuses according to its partial pressure gradient from hemoglobin binding sites within red blood cells (RBCs) through the interstitium to match
Correspondence to: Roland N. Pittman, Ph.D., Department of Physiology and Biophysics, Medical College of Virginia Campus, Virginia Commonwealth University, 1101 E. Marshall Street, P. O. Box 980551, Richmond, VA 23298-0551, Tel: (804) 828-9545, Fax: (804) 828-7382, [email protected]. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Nugent et al.
Page 2
Author Manuscript
overall rates of mitochondrial oxygen consumption (VO2) within cells (Sarelius and Pohl 2010). The partial pressure of O2 in the interstitial space (PISFO2), which is dissociated from red blood cell (RBC) hemoglobin and free to diffuse to intracellular mitochondrial sinks without appreciable interference from myoglobin desaturation, is sensitive to rapid changes in the balance between supply and demand. Different approaches ranging from whole animals (Hoyt, Wickler et al. 2006, Rodrigues, Figueroa et al. 2007), isolated muscles (Goodwin, Hernandez et al. 2012); (McDonough, Behnke et al. 2005), to single fibers (Hogan 2001); (Kindig, Howlett et al. 2003); (Wust, van der Laarse et al. 2013) have sought to characterize the relationship of the partial pressure of O2 (PO2) and VO2. The description of PISFO2 supply/demand coupling for intact muscle preparations in vivo, however, remains incomplete.
Author Manuscript Author Manuscript
When assessing the VO2 of a particular microvascular bed under physiological conditions, the difference in PO2 between the input arteries/arterioles and output veins/venules can be related to blood flow through Fick’s principle to provide a good description of O2 utilization. With regards to isolated, contracting skeletal muscle operating below the lactate threshold - an oft-studied model of PO2 and VO2 dynamics - it is acceptable to say that changes in VO2 are proportionate to energy demand (Gutierrez, Pohil et al. 1989, Poole and Richardson 1997). VO2 calculated by Fick’s principle is regarded as a metric of tissue/organ respiration and has yielded values in mammalian skeletal muscles at rest ranging from 0.054 to 3.1 ml O2/100 g*min (Edmunds and Marshall 2001); (McDonough, Behnke et al. 2005, Hoy, Peoples et al. 2009); (MacInnes and Timmons 2005). While fully practical for determining the amount of O2 consumed by a particular organ or tissue, Fick’s principle cannot directly deconstruct fiber/tissue-specific VO2 rates from these heterogeneous preparations nor indicate what spatial relations exist between VO2 and the gradients of O2 supply. Thus, for a variety of different muscle compositions a wide range of VO2 values have been reported. Additionally, there is limited temporal resolution since the hemoglobin O2 capacitance can act as a buffer between cellular respiration and changes in vascular O2 depletion.
Author Manuscript
Another approach to determining the rate of O2 extraction from a particular region of tissue is to arrest blood flow and chart the local oxygen disappearance curve (ODC). One approach uses PO2 microelectrodes (Buerk, Nair et al. 1986), but has limited spatial sensitivity and may cause tissue damage. A less invasive technique makes use of Near-Infrared Spectroscopy (NIRS) (for review see: (Ferrari, Muthalib et al. 2011). Briefly, NIRS works by illuminating the target tissue with wavelength pairs of 700–805 nm and 830–805 nm to assess hemoglobin and myoglobin oxygenation, blood volume dynamics (relative measurements of absorption at ~805 nm over time) and mitochondrial cytochrome aa3 oxygenation to assess both vascular O2 supply and tissue metabolic rate. For measurements of VO2, blood flow is arrested and the rate of oxyhemoglobin conversion to deoxyhemoglobin is related to a change in PO2 over time, thus providing indirect information on VO2 kinetics. This non-invasive approach has yielded resting VO2 values in human calf muscles around 0.20 ml O2/100 g*min (Cheatle, Potter et al. 1991); (De Blasi, Luciani et al. 2009), which fall into the lower range found using Fick’s principle, but have greater spatial and temporal resolution, as well as an assessment of mitochondrial respiration by which to compare the measured ODC. A lingering problem with these measurements is Microvasc Res. Author manuscript; available in PMC 2017 May 01.
Nugent et al.
Page 3
Author Manuscript
the muting of the ODC resulting from the buffering actions of hemoglobin and the sigmoidal shape of the ODC.
Author Manuscript
Phosphorescence Quenching Microscopy (PQM) is a well-established technique (Rumsey, Vanderkooi et al. 1988); (Smith, Golub et al. 2002) which associates the phosphorescence emission of a porphyrin dye to the PO2 of biological preparations with a high degree of spatial (~10 μm) and temporal (650nm, Newport Corp, Stratford, CT) for selective collection of phosphorescence emission. The phosphorescence signal was collected by a photomultiplier tube and sent to a modified amplifier (OP37EP, Analog Devices, Norwood, MA) which functioned as a current-to-voltage converter outfitted with a precision analog switch (ADG419BN, Norwood, MA) allowing for a gating time of 12 μs. The 12 μs delay between pulse initiation and data collection covered the 5 μs propagation delay from the plasma arc to the flash, the 4 μs light pulse, and 4–5 μs of any residual thermal tail from the flash lamp and lingering fluorescence signal from tissue or probe. Thus, the data collected were optimized to contain the highest contribution from the short phosphorescence lifetimes (i.e., highest PO2) without interference from fluorescent artifact, for reliable measurements of PISFO2. These signals were visually monitored in real-time with an oscilloscope (72-3060, Tenma, Springboro, OH) before they were passed to a 12-bit analogto-digital converter (PC-MIO-16E-4, National Instruments, Austin, TX), and saved digitally on a Dell Optiplex PC (Round Rock, TX). The tissue’s microvasculature was visualized under transillumination with a light emitting diode (Luxeon V Star white, Quadica Developments Inc., Brantford, Ontario) that was fed through the microscope’s condenser. The image, under 20× magnification, was captured in real-time by a color CCD camera (KPD20BU, Hitachi, Tokyo, Japan) and displayed on a flat-screen color video monitor (Model LN19A450C1D, Samsung, Japan). Transillumination was used for measurement site acquisition and to verify tissue flow and arrest during air bag compressions.
Author Manuscript
Phosphorescent Probe Pd-meso-tetra-(4-carboxyphenyl)porphyrin (Oxyphor R2) was obtained from Oxygen Enterprises (Philadelphia, PA) and conjugated to human serum albumin in PBS. The probe was then topically applied to the spinotrapezius muscle at a concentration of 10 mg/ml for 30 minutes. This allowed for deep, uniform diffusion of the probe into the muscle’s interstitium from where the phosphorescence signal exclusively emanated (Golub, Tevald et al. 2011). The preparation was then rinsed with PBS to remove unloaded probe. Probe
Microvasc Res. Author manuscript; available in PMC 2017 May 01.
Nugent et al.
Page 5
Author Manuscript
preparation, loading, and measurements were all performed under dark conditions to minimize photobleaching. Analysis of phosphorescence decay A nonlinear fitting procedure (Levenberg-Marquardt) for the individual phosphorescence decay curves was based on the rectangular PISFO2 distribution model (Golub, Popel et al. 1997) and yielded the following fitting equation for the phosphorescence time course: [1]
Author Manuscript
where t (μs) is the time from the beginning of the phosphorescence decay, I(t) (volts) is the phosphorescence decay curve, I0 (volts) is the magnitude of the phosphorescence signal at t = 0, M (mmHg) is the mean PO2 in the volume of detection, δ (mmHg) is the half-width of the PO2 distribution, and B (volts) is the baseline offset of the amplifier. The constants for Oxyphor R2 are K0 = 1.53×10−4 μs−1 and Kq = 4.3×10−4 μs−1 mmHg−1. Pneumatic Tissue Compression
Author Manuscript
Based on a previously described technique (Golub, Tevald et al. 2011), an objectivemounted air bag was inflated to produce a focal compression of the underlying tissue that resulted in immediate blood flow arrest and blood extrusion from the measurement region. Prior to compression, the air bag was pressurized to 5 mmHg to preserve gentle contact between the Krehalon barrier and the tissue to extrude any excess fluid on the surface of the tissue that might interfere with phosphorescent lifetime measurements. Next, following baseline measurements, the bag was pressurized to 120–130 mmHg. The rapid arrest of blood flow (40 mmHg were included in the analysis of resting VO2. Occasions of apparent atmospheric contamination (P0>70 mmHg) were also excluded from analysis to guard against artifactually high PO2. For the range of P0 values above 40 mmHg (Fig 3A; P0= 59.8 ± 1.1 mmHg; n= 125) where the VO2 data could be reliably considered insensitive to PISFO2, the mean VO2 was measured to be 0.98 ± 0.03 ml O2/100 cm3 min (Fig. 3B; n= 102). Muscle Contraction
Author Manuscript Author Manuscript
Electrical stimulation was applied to the spinotrapezius muscles of seven animals to assess VO2 and PISFO2 under dynamic conditions. Measurements consisted of the following series: 60 s baseline (allowing 3 measurements of VO2 and PISFO2) to provide an assessment of the resting state, 120 s of electrical stimulation, and a final 120 s of recovery without stimulation (Fig. 5). Contractile activity was isometric and confined to the period of electrical stimulation. Measurements of PISFO2 made during the 60 s baseline under resting conditions were found to be consistent with other measurements of resting skeletal muscle conditions reported here. The onset of electrical stimulation produced an immediate decline in PISFO2 (as assessed during the low pressure recovery period immediately prior to each high pressure compression), which significantly decreased from 67.7±2.0 mmHg (n=25) to 37.2±2.7 mmHg (n=25; p
Microvasc Res. Author manuscript; available in PMC 2017 May 01. Published in final edited form as: Microvasc Res. 2016 May ; 105: 15–22. doi:10.1016/j.mvr.2015.12.007.
Simultaneous Sampling of Tissue Oxygenation and Oxygen Consumption in Skeletal Muscle William H. Nugent1, Bjorn K. Song1, Roland N. Pittman1, and Aleksander S. Golub1 1Department
of Physiology and Biophysics, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298
Author Manuscript
Abstract
Author Manuscript
Under physiologic conditions, microvascular oxygen delivery appears to be well matched to oxygen consumption in respiring tissues. We present a technique to measure interstitial oxygen tension (PISFO2) and oxygen consumption (VO2) under steady-state conditions, as well as during the transitions from rest to activity and back. Phosphorescence Quenching Microscopy (PQM) was employed with pneumatic compression cycling to achieve 1 to 10 Hz sampling rates of interstitial PO2 and simultaneous recurrent sampling of VO2 (3/min) in the exteriorized rat spinotrapezius muscle. The compression pressure was optimized to 120–130 mmHg without adverse effect on the tissue preparation. A cycle of 5 s compression followed by 15 s recovery yielded a resting VO2 of 0.98 ± 0.03 ml O2/100cm3 min while preserving microvascular oxygen delivery. The measurement system was then used to assess VO2 dependence on PISFO2 at rest and further tested under conditions of isometric muscle contraction to demonstrate a robust ability to monitor the onkinetics of tissue respiration and the compensatory changes in PISFO2 during contraction and recovery. The temporal and spatial resolution of this approach is well suited to studies seeking to characterize microvascular oxygen supply and demand in thin tissues.
Keywords Oxygen Consumption (VO2); Phosphorescence Quenching Microscopy (PQM); Skeletal Muscle; Rat Spinotrapezius Muscle; Microvasculature; Oxygen Measurement; Muscle Stimulation; Tissue Compression
INTRODUCTION Author Manuscript
The oxygen (O2) supply and demand of skeletal muscle exists in a dynamic and responsive equilibrium within the interstitial fluid providing a key focal point of observation (Ferreira, Poole et al. 2005). At rest, O2 diffuses according to its partial pressure gradient from hemoglobin binding sites within red blood cells (RBCs) through the interstitium to match
Correspondence to: Roland N. Pittman, Ph.D., Department of Physiology and Biophysics, Medical College of Virginia Campus, Virginia Commonwealth University, 1101 E. Marshall Street, P. O. Box 980551, Richmond, VA 23298-0551, Tel: (804) 828-9545, Fax: (804) 828-7382, [email protected]. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Nugent et al.
Page 2
Author Manuscript
overall rates of mitochondrial oxygen consumption (VO2) within cells (Sarelius and Pohl 2010). The partial pressure of O2 in the interstitial space (PISFO2), which is dissociated from red blood cell (RBC) hemoglobin and free to diffuse to intracellular mitochondrial sinks without appreciable interference from myoglobin desaturation, is sensitive to rapid changes in the balance between supply and demand. Different approaches ranging from whole animals (Hoyt, Wickler et al. 2006, Rodrigues, Figueroa et al. 2007), isolated muscles (Goodwin, Hernandez et al. 2012); (McDonough, Behnke et al. 2005), to single fibers (Hogan 2001); (Kindig, Howlett et al. 2003); (Wust, van der Laarse et al. 2013) have sought to characterize the relationship of the partial pressure of O2 (PO2) and VO2. The description of PISFO2 supply/demand coupling for intact muscle preparations in vivo, however, remains incomplete.
Author Manuscript Author Manuscript
When assessing the VO2 of a particular microvascular bed under physiological conditions, the difference in PO2 between the input arteries/arterioles and output veins/venules can be related to blood flow through Fick’s principle to provide a good description of O2 utilization. With regards to isolated, contracting skeletal muscle operating below the lactate threshold - an oft-studied model of PO2 and VO2 dynamics - it is acceptable to say that changes in VO2 are proportionate to energy demand (Gutierrez, Pohil et al. 1989, Poole and Richardson 1997). VO2 calculated by Fick’s principle is regarded as a metric of tissue/organ respiration and has yielded values in mammalian skeletal muscles at rest ranging from 0.054 to 3.1 ml O2/100 g*min (Edmunds and Marshall 2001); (McDonough, Behnke et al. 2005, Hoy, Peoples et al. 2009); (MacInnes and Timmons 2005). While fully practical for determining the amount of O2 consumed by a particular organ or tissue, Fick’s principle cannot directly deconstruct fiber/tissue-specific VO2 rates from these heterogeneous preparations nor indicate what spatial relations exist between VO2 and the gradients of O2 supply. Thus, for a variety of different muscle compositions a wide range of VO2 values have been reported. Additionally, there is limited temporal resolution since the hemoglobin O2 capacitance can act as a buffer between cellular respiration and changes in vascular O2 depletion.
Author Manuscript
Another approach to determining the rate of O2 extraction from a particular region of tissue is to arrest blood flow and chart the local oxygen disappearance curve (ODC). One approach uses PO2 microelectrodes (Buerk, Nair et al. 1986), but has limited spatial sensitivity and may cause tissue damage. A less invasive technique makes use of Near-Infrared Spectroscopy (NIRS) (for review see: (Ferrari, Muthalib et al. 2011). Briefly, NIRS works by illuminating the target tissue with wavelength pairs of 700–805 nm and 830–805 nm to assess hemoglobin and myoglobin oxygenation, blood volume dynamics (relative measurements of absorption at ~805 nm over time) and mitochondrial cytochrome aa3 oxygenation to assess both vascular O2 supply and tissue metabolic rate. For measurements of VO2, blood flow is arrested and the rate of oxyhemoglobin conversion to deoxyhemoglobin is related to a change in PO2 over time, thus providing indirect information on VO2 kinetics. This non-invasive approach has yielded resting VO2 values in human calf muscles around 0.20 ml O2/100 g*min (Cheatle, Potter et al. 1991); (De Blasi, Luciani et al. 2009), which fall into the lower range found using Fick’s principle, but have greater spatial and temporal resolution, as well as an assessment of mitochondrial respiration by which to compare the measured ODC. A lingering problem with these measurements is Microvasc Res. Author manuscript; available in PMC 2017 May 01.
Nugent et al.
Page 3
Author Manuscript
the muting of the ODC resulting from the buffering actions of hemoglobin and the sigmoidal shape of the ODC.
Author Manuscript
Phosphorescence Quenching Microscopy (PQM) is a well-established technique (Rumsey, Vanderkooi et al. 1988); (Smith, Golub et al. 2002) which associates the phosphorescence emission of a porphyrin dye to the PO2 of biological preparations with a high degree of spatial (~10 μm) and temporal (650nm, Newport Corp, Stratford, CT) for selective collection of phosphorescence emission. The phosphorescence signal was collected by a photomultiplier tube and sent to a modified amplifier (OP37EP, Analog Devices, Norwood, MA) which functioned as a current-to-voltage converter outfitted with a precision analog switch (ADG419BN, Norwood, MA) allowing for a gating time of 12 μs. The 12 μs delay between pulse initiation and data collection covered the 5 μs propagation delay from the plasma arc to the flash, the 4 μs light pulse, and 4–5 μs of any residual thermal tail from the flash lamp and lingering fluorescence signal from tissue or probe. Thus, the data collected were optimized to contain the highest contribution from the short phosphorescence lifetimes (i.e., highest PO2) without interference from fluorescent artifact, for reliable measurements of PISFO2. These signals were visually monitored in real-time with an oscilloscope (72-3060, Tenma, Springboro, OH) before they were passed to a 12-bit analogto-digital converter (PC-MIO-16E-4, National Instruments, Austin, TX), and saved digitally on a Dell Optiplex PC (Round Rock, TX). The tissue’s microvasculature was visualized under transillumination with a light emitting diode (Luxeon V Star white, Quadica Developments Inc., Brantford, Ontario) that was fed through the microscope’s condenser. The image, under 20× magnification, was captured in real-time by a color CCD camera (KPD20BU, Hitachi, Tokyo, Japan) and displayed on a flat-screen color video monitor (Model LN19A450C1D, Samsung, Japan). Transillumination was used for measurement site acquisition and to verify tissue flow and arrest during air bag compressions.
Author Manuscript
Phosphorescent Probe Pd-meso-tetra-(4-carboxyphenyl)porphyrin (Oxyphor R2) was obtained from Oxygen Enterprises (Philadelphia, PA) and conjugated to human serum albumin in PBS. The probe was then topically applied to the spinotrapezius muscle at a concentration of 10 mg/ml for 30 minutes. This allowed for deep, uniform diffusion of the probe into the muscle’s interstitium from where the phosphorescence signal exclusively emanated (Golub, Tevald et al. 2011). The preparation was then rinsed with PBS to remove unloaded probe. Probe
Microvasc Res. Author manuscript; available in PMC 2017 May 01.
Nugent et al.
Page 5
Author Manuscript
preparation, loading, and measurements were all performed under dark conditions to minimize photobleaching. Analysis of phosphorescence decay A nonlinear fitting procedure (Levenberg-Marquardt) for the individual phosphorescence decay curves was based on the rectangular PISFO2 distribution model (Golub, Popel et al. 1997) and yielded the following fitting equation for the phosphorescence time course: [1]
Author Manuscript
where t (μs) is the time from the beginning of the phosphorescence decay, I(t) (volts) is the phosphorescence decay curve, I0 (volts) is the magnitude of the phosphorescence signal at t = 0, M (mmHg) is the mean PO2 in the volume of detection, δ (mmHg) is the half-width of the PO2 distribution, and B (volts) is the baseline offset of the amplifier. The constants for Oxyphor R2 are K0 = 1.53×10−4 μs−1 and Kq = 4.3×10−4 μs−1 mmHg−1. Pneumatic Tissue Compression
Author Manuscript
Based on a previously described technique (Golub, Tevald et al. 2011), an objectivemounted air bag was inflated to produce a focal compression of the underlying tissue that resulted in immediate blood flow arrest and blood extrusion from the measurement region. Prior to compression, the air bag was pressurized to 5 mmHg to preserve gentle contact between the Krehalon barrier and the tissue to extrude any excess fluid on the surface of the tissue that might interfere with phosphorescent lifetime measurements. Next, following baseline measurements, the bag was pressurized to 120–130 mmHg. The rapid arrest of blood flow (40 mmHg were included in the analysis of resting VO2. Occasions of apparent atmospheric contamination (P0>70 mmHg) were also excluded from analysis to guard against artifactually high PO2. For the range of P0 values above 40 mmHg (Fig 3A; P0= 59.8 ± 1.1 mmHg; n= 125) where the VO2 data could be reliably considered insensitive to PISFO2, the mean VO2 was measured to be 0.98 ± 0.03 ml O2/100 cm3 min (Fig. 3B; n= 102). Muscle Contraction
Author Manuscript Author Manuscript
Electrical stimulation was applied to the spinotrapezius muscles of seven animals to assess VO2 and PISFO2 under dynamic conditions. Measurements consisted of the following series: 60 s baseline (allowing 3 measurements of VO2 and PISFO2) to provide an assessment of the resting state, 120 s of electrical stimulation, and a final 120 s of recovery without stimulation (Fig. 5). Contractile activity was isometric and confined to the period of electrical stimulation. Measurements of PISFO2 made during the 60 s baseline under resting conditions were found to be consistent with other measurements of resting skeletal muscle conditions reported here. The onset of electrical stimulation produced an immediate decline in PISFO2 (as assessed during the low pressure recovery period immediately prior to each high pressure compression), which significantly decreased from 67.7±2.0 mmHg (n=25) to 37.2±2.7 mmHg (n=25; p
