Dynamics of capillary blood flow responses to acute local changes in oxygen and carbon dioxide concentrations

Introduction

The microcirculation across all tissues is responsible for delivering nutrients, such as oxygen (O2), while simultaneously removing waste products such as carbon dioxide (CO2), produced from aerobic metabolism (Duling & Berne, 1970; Segal, 2005). Accordingly, it has been well established that blood flow within the microcirculation increases to match local oxygen demand in response to hypoxia or elevated oxidative metabolism during exercise (Hudlicka, 1985; Segal, 2005; Jackson et al., 2010). Under resting and moderate exercise conditions, energy is produced in skeletal muscle via oxidative phosphorylation that requires sufficient O2 for production of ATP, while simultaneously producing CO2 as a waste product. There is a significant positive correlation between the rate of increase in blood flow and the calculated muscle O2 uptake (V̇O2) at the onset of exercise (Hughson et al., 1996). Extensive data support the principle that increases in blood flow to working muscle during exercise are directly coupled to oxidative demand for production of ATP through vasomotor responses that promote O2 transport and delivery (Nuutinen et al., 1982). The dynamics of V̇O2 at the onset of exercise, and the accompanying bulk blood flow response, has been a major area of investigation focused on elucidating the limitations to exercise performance, and the underlying mechanisms that govern regional blood flow responses (Murias et al., 2014). At the microvascular level, local changes in arteriolar tone modulate conductance which serves as the key driver of blood flow responses during exercise. Conventional studies that logically employ exercise in humans, or stimulated contractions in animal models, to dynamically increase aerobic muscle metabolism and provoke regional blood flow responses are inherently confounded by a myriad of simultaneous microvascular vasomotor mechanisms. Indeed, multiple metabolic stimuli are integrated within the microcirculation to determine the magnitude and time course of local blood flow (Walløe & Wesche, 1988; Shoemaker and Hughson, 1999; Boushel et al., 2000; Herspring et al., 2008). In the context of exercise, it is difficult to separate the specific contributions that O2 and CO2 concentrations have on blood flow supply and demand matching from other vasoactive metabolites that regulate blood flow.

Microvascular blood flow responses at the onset of exercise result from the cumulative stimulus of multiple vasoactive stimuli and mechanical factors. Within the first 1-2 muscular contractions, blood flow increases precede changes in V̇O2 as measured at the mouth. This sudden increase in blood flow has been attributed to rapid onset vasodilation (Corondilas et al., 1964; Shoemaker and Hughson, 1999; Mihok et al., 2004; VanTeeffelen, 2006; Jackson et al., 2010). Additionally, this increase in blood flow at the arteriolar level increases the shear stress experienced by these vessels, promoting the release of nitric oxide (NO) from the endothelium which stimulates vasodilation in arterioles and feed arteries (Wray et al., 2011). Experimentally it has been shown that the onset of exercise also reduces availability of superoxide, due to an increase in oxygen consumption, leading to an increase in the concentration of NO in the interstitium, resulting in vasodilation (Golub et al., 2014).

Electrically coupled conducted responses have been implicated across multiple levels of microvessels. Changes in electrical potential in both endothelial and vascular smooth muscle cells (VSMC) have been shown to propagate upstream and ultimately modulate arteriolar tone (Cohen et al., 2000; Segal and Jacobs, 2001). Multiple transmembrane channels have been associated with blood flow responses during exercise. Arteriolar VSMC within skeletal muscle depend on Ca2+ influx through voltage-gated Ca2+ channels and release from internal stores through inositol 1,4,5-triphosphate receptors to regulate myogenic tone (Jackson & Boerman, 2018). During muscle contractions, K+ channels located in capillary endothelium are exposed to an accumulation of K+ ions and subsequently transduce a signal resulting in endothelial cell hyperpolarization that is transmitted upstream to stimulate arteriole vasodilation (Jackson, 2017).

Exercise decreases the local availability of O2 in working skeletal muscle, this increase in demand for O2 stimulates an increase in O2 supply for metabolic matching. In exercising muscle, increased aerobic metabolism and a lower capillary SO2 leads to ATP release from red blood cells (RBC), which is believed to trigger a conducted response that travels upstream causing vasodilation of arterioles and subsequently increase blood flow to areas of muscle with decreased O2 availability, thereby matching the high oxygen demand of active skeletal muscle (Jackson, 1987, 2016; Ellis et al., 2012; Ellsworth et al., 2016). An increase in lactate during exercise decreases pH in the muscle; this increase in lactate occurs in heavy exercise, yet muscle blood flow and oxygen uptake increase linearly with work loads (Andersen & Saltin, 1985; Berg et al., 1997). There have been several suggested O2 dependent mechanisms involved for local blood flow regulation in active skeletal muscle; however, mechanisms underlying the response to increased CO2 have yet to be elucidated.

Changing O2 and CO2 concentrations in skeletal muscle during exercise have been shown to elicit local blood flow responses, and several potential mechanisms responsible for these responses have been identified. The release of ATP from RBCs has been shown to elicit an increase in blood flow by dilating upstream arterioles providing support for RBCs as both a sensor and stimulus to regulate oxygen concentration in skeletal muscle. RBCs release ATP when hemoglobin desaturates in response to low oxygen environments such as that found in exercise (Jagger et al., 2001; Ellsworth et al., 2016). Arteriolar smooth muscle cells and endothelial cells have also been demonstrated, in ex vivo studies, to play a role in O2 mediated blood flow responses, however this has not been similarly observed in vivo using intravital microscopy methods (reviewed in Jackson, 2016). Furthermore, during exercise, as aerobic metabolism increases, and the rate of CO2 production increases proportionally with O2 consumption causing elevated tissue partial pressure of carbon dioxide (PCO2). Higher tissue CO2 concentration on its own, and in combination with the resulting decrease in tissue pH, has been shown to provoke vasodilation in striated muscle (Duling, 1973; Charter et al., 2018).

Targeted manipulation of [O2] and [CO2] while simultaneously measuring the resulting capillary blood flow response is possible when coupled with intravital video microscopy and precisely controlled gas conditions. The microcirculation is essential in the transport of O2 from the blood delivering it into tissue while simultaneously removing CO2. Direct manipulation of local O2 and CO2 concentrations has been previously achieved by employing microfluidic gas exchange chambers that can maintain a constant gas partial pressure in the tissue while simultaneously measuring capillary blood flow in skeletal muscle (Ghonaim, 2011; Ghonaim, 2013; Sové 2021). The use of a gas exchange chamber allows dynamic manipulation of [O2] and [CO2] both individually and in combination with one another. Previous work manipulating [O2] and [CO2] have largely focused on steady state flow conditions and as a result, dynamic characterization of capillary level blood flow in response to acute changes in [O2] and [CO2] have yet to be thoroughly described. Characterizing the time course of [O2] and [CO2] mediated microvascular blood flow responses is essential to understanding the broader dynamics of conduit artery flow at the onset of exercise. Furthermore, examining the dynamics of microvascular responses under various conditions can provide mechanistic insights which may be masked by overlapping or redundant mechanisms. In this study, we aim to quantify the magnitude and time transient of capillary blood flow responses to acute changes in local [O2] and [CO2] in skeletal muscle. Additionally, we sought to quantify the additive response to both [O2] and [CO2] to mimic muscle microenvironment conditions similar to those found following the onset of moderate exercise.

Materials and methodsAnimal surgery

13 male Sprague Dawley rats (170 g–211 g) were obtained from Charles River Laboratories and were allowed to acclimatize in animal care for 1 week before use. Rats were fed Teklad 2018 (Envigo, Indianapolis, IND, United States) standard rodent chow and allowed water ad libitum. All animal protocols were approved by Memorial University Animal Care Committee.

On the day of testing, animals were anesthetized with a 65 mg/kg intraperitoneal injection of sodium pentobarbital (Euthanyl, Bimeda, Cambridge, ON, Canada). Following induction, depth of anesthesia was assessed with palpebral reflex and toe pinch prior to the start of surgery to verify the animal was sufficiently anesthetized. Once in the surgical plane, a rectal temperature probe was inserted to monitor the body temperature of the animal throughout the experiment. Physiological temperatures were maintained between 36–37°C using a heated mat and/or heat lamp as necessary.

The common carotid artery was cannulated to allow continuous monitoring of blood pressure and heart rate (400a Blood Pressure Analyzer, Micro-Med, Louisville, KY, United States). The right jugular vein was cannulated to provide fluid resuscitation (0.5 ml/kg/hr) and maintenance anaesthetic as required. The animal’s heart rate and blood pressure were monitored continuously for variability as well as regular testing the palpebral reflexes and toe pinch to ensure acceptable depth of anaesthesia. Maintenance doses of sodium pentobarbital (22 mg/kg) were administered intravenously when the animal’s mean arterial pressure exceeded 110 mmHg or if the animal responded to adverse stimuli. Animals were tracheotomized and mechanically ventilated (Inspira ASV, Harvard Apparatus, Holliston, MA, United States) with an initial gas mixture of ∼30% O2 and 70% N2. Respiratory rates and volumes were automatically determined by the ventilator’s built in software based on the animal’s weight. The right extensor digitorum longus (EDL), a muscle of the lower hind limb, was blunt dissected and isolated as previously described (Tyml and Budreau, 1991; Fraser et al., 2012). The distal tendon was cut, the muscle was lifted and cleared from the remaining tissue without damaging the feed artery and vein. The EDL muscle was reflected over the gas exchange chamber on the stage of an inverted microscope (IX73, Olympus, Tokyo, Japan). The EDL was fixed under slight tension at approximately in situ length, covered with a polyvinylidene chloride film, bathed in warm saline, and gently compressed with a glass coverslip and microscope slide to isolate the EDL from room air, and to aid in establishing a uniform optical interface that is orthogonal to the incident light path. The animal was allowed to acclimatize on the stage for 30 min following positioning. Following the acclimatization, and with the animal’s body temperature between 36–37°C, an arterial blood sample was collected to measure blood gases (VetScan iSTAT, Abbott Point of Care Inc. Princeton, NJ, United States). Arterial PCO2 and PO2 were maintained within normal physiological range by adjusting ventilation rate and volume as needed prior to data collection.

The microscopy imaging setup was composed of an Olympus IX73 microscope (Olympus, Tokyo, Japan) fitted for transillumination with a 300 W Xenon light source (Lambda LS-30, Sutter Instruments, Novato, CA, United States). A parfocal beam splitter (Optosplit II Bypass, Cairn Research Ltd. Faversham, United Kingdom) directed light through 420 nm (isosbestic wavelength) and 438 nm (oxygen-sensitive) bandpass filters. Simultaneous and parfocal capture of video sequences were recorded for both wavelengths, with each wavelength on separate halves of the camera chip. Video recordings were made at 16 bit depth 2048 × 2048 resolution using a 10× objective (NA 0.40, Olympus, Tokyo, Japan) using an Orca Flash 4.0 v3 scientific digital camera (Hamamatsu, Hamamatsu City, Japan) and controlled by HCImage Live software (Hamamatsu, Hamamatsu City, Japan) on a desktop computer.

Experimental protocol

To evaluate O2 and CO2 dependence on blood flow in the microcirculation both independently and simultaneously, a series of gas perturbations were imposed on the surface of the EDL muscle in contact with the gas exchange chamber membrane similar to those described previously (Sové et al., 2021). The series of 9 perturbations were conducted on multiple fields of view across the muscle at a focal depth within ∼60 µm of the surface. Each field was recorded during the following four O2 perturbations: 1) 1 min 7% O2 followed by 2 min 12% O2, 2) 1 min 7% O2 followed by 2 min 2% O2, 3) 1 min 12% O2 followed by 2 min of 7% O2, 4) 1 min 2% O2 followed by 2 min of 7% O2. CO2 was maintained at 5% throughout the O2 challenges, N2 made up the balance of the gasses delivered via the exchange chamber. Following completion of O2 challenges, recordings were made during the following four CO2 challenges: 5) 1 min of 5% CO2 then 2 min of 10% CO2, 6) 1 min of 5% CO2 then 2 min of 0% CO2, 7) 1 min of 10% CO2 then 2 min of 5% CO2, and 8) 1 min of 0% CO2 then 2 min of 5% CO2. [O2] was maintained at 7% throughout the duration of the CO2 challenges. The combined perturbation was as follows: 1 min of 7% O2 and 5% CO2, then 1 min of 2% O2 and 5% CO2, and lastly 2 min of 2% O2 and 10% CO2 with N2 being the balance of gasses delivered. Prior to the start of each perturbation the muscle was allowed to equilibrate for 1–2 min at the baseline O2 and CO2 concentrations for the next perturbation. Following the full series of 9 challenges the muscle was allowed to re-equilibrate at 7% [O2] and 5% [CO2] for 10 min prior to repeating the sequence in the next field of view.

Gas perturbations were imposed on the surface of the muscle using a three-dimensionally (3D) printed gas exchange chamber as described previously (Sové et al., 2021). The device in the present study employed an exchange window (5 mm × 3.5 mm) fitted with a 50 µm thick gas permeable membrane fabricated by spin coating polydimethylsiloxane onto a standard glass slide. The assembled device was connected by plastic tubing to a triple-inlet manifold supplied by three computer-controlled mass flow meters (SmartTrak100, Sierra Instruments, Monterey, CA, United States) for each gas channel, with a frequency response of <300 ms. The EDL muscle was placed over the exchange window of the device and isolated from room air as described above. Gas concentrations from the mass flow meters were dynamically controlled by custom MATLAB software allowing changes in O2 and CO2 concentrations to be triggered automatically at the appropriate time and sequence.

Offline analysis and statistics

Offline analysis was conducted using custom software written in MATLAB (Mathworks, Natick, MA, United States). The software generated mp4 videos of captured sequences and functional images including variance and sum of absolute difference images used to facilitate identification of in-focus vessel segments for analysis (Japee et al., 2004). In focus capillaries with single file RBC flow were semi-automatically selected for analysis and space time images (STIs) were generated at each wavelength as described previously (Ellis et al., 1990, 1992, 2002, 2012; Japee et al., 2004; Ghonaim et al., 2011; Fraser et al., 2012). STIs were analyzed using the custom software package written in MATLAB and 1 second means were calculated from frame-by-frame measurements of velocity, lineal density, RBC supply rate (SR), and RBC oxygen saturation (SO2). Resulting second-by-second means were used for statistical comparisons.

Statistical analysis of the resulting capillary hemodynamic data was completed using Prism (Graphpad, California, United States). Time constants and associated parameters were determined using a mono-exponential non-linear least-squared curve fitting implemented in Prism for O2, CO2, and combined O2 and CO2 challenges, similar to approaches used to quantify V̇O2 kinetics (Tschakovsky et al., 2006). O2 and CO2 responses were fit to the following mono-exponential model:

where Y(t) is the response over time, Yb is the magnitude at baseline, X0 is the time delay, Y0 is the asymptotic amplitude of the response, and τ is the time constant representing the time to reach 63% of the full response. Combined O2 and CO2 responses were fit to a multi-exponential model as follows:

Yt=Yb+Y11−e−t−X1τ1+Y21−e−t−X2τ2

where Y(t) is the response over time, Yb is the magnitude at baseline, X1 is the time delay, Y1 is the amplitude of the response to O2, τ1 is the time constant for the O2 response, Y2 is the amplitude of the CO2 response, X2 is the time delay, and τ2 is the time constant for the CO2 response. Repeated measures one-way ANOVA (using a mixed effects model to account for missing values) and Dunnett’s multiple comparisons were used to evaluate differences in the baseline period and the second-by-second mean responses following gas changes in the 3- and 4-min challenges. A p-value of <0.05 was considered significant. Mean and standard deviation are reported unless otherwise noted.

ResultsSystemic animal data

Animal weight and systemic physiological animal monitoring data are shown in Table 1. Animal weights were measured immediately prior to the experiment. Reported mean arterial, systolic, and diastolic blood pressures represent the values recorded from the start of the capturing protocol and therefore include periods immediately following administration of anaesthetic. Mechanical ventilation respiratory rate and stroke volume are reported in Table 1. Arterial blood gasses are listed in Table 1.

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TABLE 1. Systemic physiological and arterial blood gas measurements.

Oxygen challenges

On-transient O2 mediated flow responses were measured during the 1-min baseline period at 7% [O2] followed by 12% high and 2% low O2 challenges for the remaining 2 min with a constant 5% [CO2] throughout the sequence. Off-transient flow responses were measured over 1-min periods of 12% (high), and 2% (low) chamber [O2] followed by 2 min of baseline 7% [O2]. Modeled parameters determined by non-linear least-squared fitting to mono-exponential curves for hemodynamic and capillary RBC SO2 following oxygen perturbation curves are listed in Table 2, stable exponential fits were determined for each hemodynamic and saturation measure in response to the O2 challenges.

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TABLE 2. Parameters and constraints for mono-exponential non-linear least squared fit modeling of oxygen challenge responses.

Increases in [O2] from 2–7% and 7–12% caused significant increases in RBC SO2 within 2 s of the step change, compared to the respective baseline periods (Table 3; Figures 1A,D). Significant decreases in RBC velocity, lineal density, capillary hematocrit, and RBC supply rate, were observed in response to increased [O2] (Table 3; Figures 25). Capillary RBC velocity decreased by 66 s following the 2–7% change in [O2]; whereas lineal density did not show significantly decreases until 79 s (Table 3; Figures 25). Decreasing [O2] from 12 to 7% and 7 to 2% resulted in significant decreases in RBC SO2 by 62 s after the step change in [O2] (Table 3; Figures 1B,C). Significant increases in all measured hemodynamic parameters were observed within 18 s following the decrease in [O2] (Table 3; Figures 25). Changes in RBC SO2 in response to increased [O2] from 7 to 12%, yielded the fastest time transient (τ = 1.009 s), while the slowest time constant was observed with capillary hematocrit in response to increases in [O2] from 2—7% (τ = 74.75 s). Time constants were fastest for saturation changes and slowest for lineal density and capillary hematocrit responses.

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TABLE 3. Mean capillary blood flow responses to oxygen challenges.

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FIGURE 1. Mean capillary red blood cell oxygen saturation in response to O2 challenges. Second-by-second capillary red blood cell (RBC) oxygen saturation (SO2) measurements were made from intravital video microscopy of rat skeletal muscle microcirculation, recorded during four different stepwise O2 challenges. Each oxygen challenge began with a 1-min baseline period where chamber O2 concentration ([O2]) was set to a low (2%), normal (7%), or high (12%) concentration as the initial [O2]. Following the baseline period, the chamber [O2] was abruptly changed to the concentration of interest for the remaining 2-min so that hemodynamic responses could be quantified. The regime of [O2] challenges were 7–12% (A), 12–7% (B), 7–2% (C), and 2–7% (D) with a steady 5% [CO2] and the balance of the gas mixture being composed of N2. The mean SO2 at each time point consists of all capillaries measured during a given challenge, with the shaded region representing the standard error of the mean. Resulting mean responses were modeled to a mono-exponential using established non-linear least squared fitting methods to aid in describing the dynamics of responses. Time constants (τ) for each SO2 response were 1.01 s (Panel A, n = 167), 1.44 s (Panel B, n = 168), 1.64 s (Panel C, n = 221) and 1.27 s (Panel D, n = 214). A repeated measures one-way ANOVA (using a mixed effects model to account for missing values) was used to compare the last 10 s of the baseline period to the second-by-second mean responses following the step-change, the Dunnett’s multiple comparisons test was used. p < 0.05 (*) were considered to be significant.

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FIGURE 2. Mean capillary red blood cell velocity in response to O2 challenges. Second-by-second capillary red blood cell (RBC) velocity measurements were made from intravital video microscopy of rat skeletal muscle microcirculation, recorded during four different stepwise O2 challenges. Each oxygen challenge began with a 1-min baseline period where chamber O2 concentration ([O2]) was set to a low (2%), normal (7%), or high (12%) concentration as the initial [O2]. Following the baseline period, the chamber [O2] was abruptly changed to the concentration of interest for the remaining 2-min so that hemodynamic responses could be quantified. The regime of [O2] challenges were 7–12% (A), 12–7% (B), 7–2% (C), and 2–7% (D) with a steady 5% [CO2] and the balance of the gas mixture being composed of N2. The mean velocity at each time point consists of all capillaries measured for a given challenge, with the shaded region representing the standard error of the mean. Resulting mean responses were modeled to a mono-exponential using established non-linear least squared fitting methods to aid in describing the dynamics of responses. Time constants (τ) for each velocity response were 6.96 s (Panel A, n = 272 capillaries), 35.54 s (Panel B, n = 294), 38.54 s (Panel C, n = 300) and 13.36 s (Panel D, n = 293). A repeated measures one-way ANOVA (using a mixed effects model to account for missing values) was used to compare the last 10s of the baseline period to the second-by-second mean responses following the step-change, the Dunnett’s multiple comparisons test was used. p < 0.05 (*) were considered to be significant.

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FIGURE 3. Mean capillary red blood cell lineal density in response to O2 challenges. Second-by-second capillary red blood cell (RBC) lineal density (LD) measurements were made from intravital video microscopy of rat skeletal muscle microcirculation, recorded during four different stepwise O2 challenges. Each oxygen challenge began with a 1-min baseline period where chamber O2 concentration ([O2]) was set to a low (2%), normal (7%), or high (12%) concentration as the initial [O2]. Following the baseline period, the chamber [O2] was abruptly changed to the concentration of interest for the remaining 2-min so that hemodynamic responses could be quantified. The regime of [O2] challenges were 7–12% (A), 12–7% (B), 7–2% (C), and 2–7% (D) with a steady 5% [CO2] and the balance of the gas mixture being composed of N2. The mean LD at each time point consists of all capillaries measured during a given challenge, with the shaded region representing the standard error of the mean. Resulting mean responses were modeled to a mono-exponential using established non-linear least squared fitting methods to aid in describing the dynamics of responses. Time constants (τ) for each LD response were 24.03 s (Panel A, n = 279), 26.93 s (Panel B, n = 298), 35.93 s (Panel C, n = 305) and 68.24 s (Panel D, n = 294). A repeated measures one-way ANOVA (using a mixed effects model to account for missing values) was used to compare the last 10 s of the baseline period to the second-by-second mean responses following the step-change, the Dunnett’s multiple comparisons test was used. p < 0.05 (*) were considered to be significant.

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FIGURE 4. Mean capillary red blood cell hematocrit in response to O2 challenges. Second-by-second capillary red blood cell (RBC) hematocrit measurements were made from intravital video microscopy of rat skeletal muscle microcirculation, recorded during four different stepwise O2 challenges. Each oxygen challenge began with a 1-min baseline period where chamber O2 concentration ([O2]) was set to a low (2%), normal (7%), or high (12%) concentration as the initial [O2]. Following the baseline period, the chamber [O2] was abruptly changed to the concentration of interest for the remaining 2-min so that hemodynamic responses could be quantified. The regime of [O2] challenges were 7–12% (A), 12–7% (B), 7–2% (C), and 2–7% (D) with a steady 5% [CO2] and the balance of the gas mixture being composed of N2. The mean hematocrit at each time point consists of all capillaries measured during a given challenge, with the shaded region representing the standard error of the mean. Resulting mean responses were modeled to a mono-exponential using established non-linear least squared fitting methods to aid in describing the dynamics of responses. Time constants (τ) for each hematocrit response were 27.19 s (Panel A, n = 278), 32.42 s (Panel B, n = 298), 35.21 s (Panel C, n = 305) and 74.75 s (Panel D, n = 294). A repeated measures one-way ANOVA (using a mixed effects model to account for missing values) was used to compare the last 10 s of the baseline period to the second-by-second mean responses following the step-change, the Dunnett’s multiple comparisons test was used. p < 0.05 (*) were considered to be significant.

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FIGURE 5. Mean capillary red blood cell supply rate in response to O2 challenges. Second-by-second capillary red blood cell (RBC) supply rate (SR) measurements were made from intravital video microscopy of rat skeletal muscle microcirculation, recorded during four different stepwise O2 challenges. Each oxygen challenge began with a 1-min baseline period where chamber O2 concentration ([O2]) was set to a low (2%), normal (7%), or high (12%) concentration as the initial [O2]. Following the baseline period, the chamber [O2] was abruptly changed to the concentration of interest for the remaining 2-min so that hemodynamic responses could be quantified. The regime of [O2] challenges were 7–12% (A), 12–7% (B), 7–2% (C), and 2–7% (D) with a steady 5% [CO2] and the balance of the gas mixture being composed of N2. The mean SR at each time point consists of all capillaries measured during a given challenge, with the shaded region representing the standard error of the mean. Resulting mean responses were modeled to a mono-exponential using established non-linear least squared fitting methods to aid in describing the dynamics of responses. Time constants (τ) for each SR response were 20.61 s (Panel A, n = 278), 41.87 s (Panel B, n = 298), 36.06 s (Panel C, n = 304) and 23.07 s (Panel D, n = 294). A repeated measures one-way ANOVA (using a mixed effects model to account for missing values) was used to compare the last 10s of the baseline period to the second-by-second mean responses following the step-change, the Dunnett’s multiple comparisons test was used. p < 0.05 (*) were considered to be significant.

Carbon dioxide challenges

On-transient CO2 mediated flow responses were measured during the 1-min baseline period at 5% followed by 0% low and 10% high [CO2] challenge for the remaining 2 min with stable 7% [O2] throughout. Off-transient challenges were measured by 1-min periods of 0% low and 10% high CO2 with sequential change to 5% CO2. Parameters defined for mono-exponential non-linear least squared fitting of CO2 perturbation curves are listed in Table 4.

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TABLE 4. Parameters and constraints for mono-exponential non-linear least squared fit modeling of carbon dioxide challenge responses.

Reducing [CO2] from 5 to 0% and 10 to 5% lead to significant increases in RBC SO2 within 2 s compared to 51–60 s baseline period in both challenges (Table 5; Figures 6A,D). RBC velocity and RBC supply rate significantly decreased within 17 s of the step decrease in [CO2] (Figures 7A,D, 10A,D, respectively). Lineal density and capillary hematocrit were significantly decreased by 6 s after [CO2] was decreased from 5 to 0% (Figures 8A, 9A); however, lineal density and hematocrit did not significantly change in response to the 10–5% challenge (Figures 8D, 9D). Red blood cell SO2 significantly decreased in response to increased [CO2] both from 0 to 5% and 5–10% within 17 s following the change in [CO2] (Table 5; Figures 6B,C). There were significant increases in RBC velocity, lineal density, hematocrit, and RBC supply rate in response to increased [CO2] (Table 5; Figures 710). Increases in hemodynamic measurements occurred between 62 and 80 s following the change in gas conditions. The time constants for SO2 changes were among the fastest with tau ranging from 0.38 s to 5.3 s. The increased [CO2] from 5—10% resulted in the longest time constants for hemodynamic changes (τ = 30.3 s–88.1 s).

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TABLE 5. Mean capillary blood flow responses to carbon dioxide challenges.

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FIGURE 6. Mean capillary red blood cell oxygen saturation in response to CO2 challenges. Second-by-second capillary red blood cell (RBC) oxygen saturation (SO2) measurements were made from intravital video microscopy of rat skeletal muscle microcirculation, recorded during four different stepwise CO2 challenges. Each CO2 challenge began with a 1-min baseline period where chamber CO2 concentration ([CO2]) was set to a low (0%), normal (5%), or high (10%) concentration as the initial [CO2]. Following the baseline period, the chamber [CO2] was abruptly changed to the concentration of interest for the remaining 2-min so that hemodynamic responses could be quantified. The regime of [CO2] challenges were 5–0% (A), 0–5% (B), 5–10% (C), and 10–5% (D) with a steady 7% [O2] and the balance of the gas mixture being composed of N2. The mean SO2 at each time point consists of all capillaries measured during a given challenge, with the shaded region representing the standard error of the mean. Resulting mean responses were modeled to a mono-exponential using established non-linear least squared fitting methods to aid in describing the dynamics of responses. Time constants (τ) for each SO2 response were 0.84 s (Panel A, n = 82), 0.38 s (Panel B, n = 90), 2.887 s (Panel C, n = 197) and 5.339 s (Panel D, n = 183). A repeated measures one-way ANOVA (using a mixed effects model to account for missing values) was used to compare the last 10s of the baseline period to the second-by-second mean responses following the step-change, the Dunnett’s multiple comparisons test was used. p < 0.05 (*) were considered to be significant.

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FIGURE 7. Mean capillary red blood cell velocity in response to CO2 challenges. Second-by-second capillary red blood cell (RBC) velocity measurements were made from intravital video microscopy of rat skeletal muscle microcirculation, recorded during four different stepwise CO2 challenges. Each CO2 challenge began with a 1-min baseline period where chamber CO2 concentration ([CO2]) was set to a low (0%), normal (5%), or high (10%) concentration as the initial [CO2]. Following the baseline period, the chamber [CO2] was abruptly changed to the concentration of interest for the remaining 2-min so that hemodynamic responses could be quantified. The regime of [CO2] challenges were 5–0% (A), 0–5% (B), 5–10% (C), and 10–5% (D) with a steady 7% [O2] and the balance of the gas mixture being composed of N2. The mean velocity at each time point consists of all capillaries measured during a given challenge, with the shaded region representing the standard error of the mean. Resulting mean responses were modeled to a mono-exponential using established non-linear least squared fitting methods to aid in describing the dynamics of responses. Time constants (τ) for each velocity response were 18.88 s (Panel A, n = 245), 21.79 s (Panel B, n = 242), 79.34 s (Panel C, n = 288) and 20.66 s (Panel D, n = 285). A repeated measures one-way ANOVA (using a mixed effects model to account for missing values) was used to compare the last 10s of the baseline period to the second-by-second mean responses following the step-change, the Dunnett’s multiple comparisons test was used. p < 0.05 (*) were considered to be significant.

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FIGURE 8. Mean capillary red blood cell lineal density in response to CO2 challenges. Second-by-second capillary red blood cell (RBC) lineal density (LD) measurements were made from intravital video microscopy of rat skeletal muscle microcirculation, recorded during four different stepwise CO2 challenges. Each CO2 challenge began with a 1-min baseline period where chamber CO2 concentration ([CO2]) was set to a low (0%), normal (5%), or high (10%) concentration as the initial ([CO2]). Following the baseline period, the chamber [CO2] was abruptly changed to the concentration of interest for the remaining 2-min so that hemodynamic responses could be quantified. The regime of [CO2] challenges were 5–0% (A), 0–5% (B), 5–10% (C), and 10–5% (D) with a steady 7% [O2] and the balance of the gas mixture being composed of N2. The mean LD at each time point consists of all capillaries measured during a given challenge, with the shaded region representing the standard error of the mean. Resulting mean responses were modeled to a mono-exponential using established non-linear least squared fitting methods to aid in describing the dynamics of responses. Time constants (τ) for each LD response were 15.89 s (Panel A, n = 255), 30.63 s (Panel B, n = 246) and 30.31 s (Panel C, n = 292); there was no time constant for 10–5% (Panel D, n = 289). A repeated measures one-way ANOVA (using a mixed effects model to account for missing values) was used to compare the last 10 s of the baseline period to the second-by-second mean responses following the step-change, the Dunnett’s multiple comparisons test was used. p < 0.05 (*) were considered to be significant.

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FIGURE 9. Mean capillary red blood cell hematocrit in response to CO2 challenges. Second-by-second capillary red blood cell (RBC) hematocrit measurements were made from intravital video microscopy of rat skeletal muscle microcirculation, recorded during four different stepwise CO2 challenges. Each CO2 challenge began with a 1-min baseline period where chamber CO2 concentration ([CO2]) was set to a low (0%), normal (5%), or high (10%) concentration as the initial ([CO2]). Following the baseline period, the chamber ([CO2]) was abruptly changed to the concentration of interest for the remaining 2-min so that hemodynamic responses could be quantified. The regime of ([CO2]) challenges were 5–0% (A), 0–5% (B), 5–10% (C), and 10–5% (D) with a steady 7% [O2] and the balance of the gas mixture being composed of N2. The mean hematocrit at each time point consists of all capillaries measured during a given challenge, with the shaded region representing the standard error of the mean. Resulting mean responses were modeled to a mono-exponential using established non-linear least squared fitting methods to aid in describing the dynamics of responses. Time constants (τ) for each hematocrit response were 15.27 s (Panel A, n = 250), 38.50 s (Panel B, n = 248) and 65.94 s (Panel C, n = 292); there was no time constant for 10–5% ([CO2]) (Panel D, n = 289). A repeated measures one-way ANOVA (using a mixed effects model to account for missing values) was used to compare the last 10s of the baseline period to the second-by-second mean responses following the step-change, the Dunnett’s multiple comparisons test was used. p < 0.05 (*) were considered to be significant.

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FIGURE 10. Mean capillary red blood cell supply rate in response to CO2 challenges. Second-by-second capillary red blood cell (RBC) supply rate (SR) measurements were made from intravital video microscopy of rat skeletal muscle microcirculation, recorded during four different stepwise CO2 challenges. Each CO2 challenge began with a 1-min baseline period where chamber CO2 concentration ([CO2]) was set to a low (0%), normal (5%), or high (10%) concentration as the initial ([CO2]). Following the baseline period, the chamber ([CO2]) was abruptly changed to the concentration of interest for the remaining 2-min so that hemodynamic responses could be quantified. The regime of ([CO2]) challenges were 5–0% (A), 0–5% (B), 5–10% (C), and 10–5% (D) with a steady 7% [O2] and the balance of the gas mixture being composed of N2. The mean SR at each time point consists of all capillaries measured during a given challenge, with the shaded region representing the standard error of the mean. Resulting mean responses were modeled to a mono-exponential using established non-linear least squared fitting methods to aid in describing the dynamics of responses. Time constants (τ) for each SR response were 13.84 s (Panel A, n = 250), 32.96 s (Panel B, n = 246), 88.08 s (Panel C, n = 290) and 20.18 s (Panel D, n = 287). A repeated measures one-way ANOVA (using a mixed effects model to account for missing values) was used to compare the last 10 s of the baseline period to the second-by-second mean responses following the step-change, the Dunnett’s multiple comparisons test was used. p < 0.05 (*) were considered to be significant.

Combined O2 and CO2 challenges

Combined O2 and CO2 mediated flow responses were measured during a 4-min challenge. The 4-min sequence consisted of a 1-min baseline period with 7% [O2] and 5% [CO2], followed by a 1-min period with 2% [O2] and 5% [CO2], and lastly a 2-min period with 2% [O2] and 10% [CO2]. Parameters defined by mono-exponential non-linear least squared fitting hemodynamic and oxygen saturation responses to the combined O2 and CO2 perturbation are provided in Table 6. Stable exponential fits were achieved for all hemodynamic and saturation measures in response to the combined challenge.

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TABLE 6. Parameters and constraints for multi-exponential non-linear least squared fit modeling of combined oxygen and carbon dioxide responses.

A significant decrease in RBC SO2 was observed by 62 s in response to a decrease in gas exchange chamber [O2] from 7–2%; however, this decrease in SO2 was followed by an increase in RBC SO2 at 142 s in response to increased [CO2] from 5–10% compared to the last 10 s of the low O2 period (111–120 s) (Table 7; Figure 11A). The combined challenge caused significant increases in RBC velocity by 67 s following the step change to 2% [O2] compared to the mean of the baseline period between 51–60 s, and subsequently velocity significantly increased by 123 s following the change from 5 to 10% [CO2] when compared to the low O2 period (111–120 s) (Figure 11B). The step change in gas exchange chamber [O2] from 7 to 2% caused significant increases in lineal density and mean capillary hematocrit by 83 s (Figures 11C,D respectively). The [CO2] step change from 5 to 10% during the combined challenge caused significant increases in lineal density by 162 s compared to the 111–120 s time period of the initial [O2] step change (Figure 11C). Similarly, hematocrit significantly increased by 161 s following the change to 10% [CO2] (Figure 11D). Mean capillary RBC SR significantly increased by 68 and 124 s in response to the combined step change in [O2] and [CO2] respectively (Figure 11E).

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TABLE 7. Mean capillary blood flow responses to combined oxygen and carbon dioxide challenges.

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FIGURE 11. Mean hemodynamic measures in responses to combined O2 and CO2 challenges. Second-by-second capillary hemodynamic measurements were made from intravital video microscopy of rat skeletal muscle microcirculation, recorded during a 4-min combined O2 and CO2 stepwise challenge. For O2, the chamber O2 concentration ([O2]) was set to baseline 7% for 1-min, then decreased to 2% [O2] for the remaining 3 min. For CO2 concentrations ([CO2]), a 5% baseline period was set for 2 min, then [CO2] increased to 10% for the second 2 min period. During the 4-min sequence, the balance of the gas mixture was composed of N2. The hemodynamic parameters quantified were red blood cell (RBC) oxygen saturation (SO2) (A), RBC velocity (B), lineal density (LD) (C), hematocrit (D), and RBC supply rate (SR) (E). Each time point consists of all capillaries measured during the combined challenge, with the shaded region representing the standard error of the mean. Resulting mean responses were modeled to a mono-exponential using established non-linear least squared fitting methods to aid in describing the dynamics of responses. Time constants (τ) for each O2 response were 23.30 s (Panel A, n = 286 capillaries), 22.38 s (Panel B, n = 292), 21.52 s (Panel C, n = 292), 20.94 s (Panel D, n = 291) and 0.68 s (Panel E, n = 193). Time constants (τ) for each CO2 response were 85.78 s (Panel A), 23.63 s (Panel B), 20.73 s (Panel C), 49.87 s (Panel D) and 92.32 s (Panel E). A repeated measures one-way ANOVA (using a mixed effects model to account for missing values) was used to compare the 51–60 s of the baseline period and the 111–120 s period to the second-by-second mean responses following the O2 and CO2 step-change, respectively, and the Dunnett’s multiple comparisons test was used. *: p < 0.05, compared to 51–60 s #: p < 0.05, compared to 111–120 s.

Discussion

The dynamics of V̇O2 uptake at the onset of exercise is coupled to the increased oxidative requirements of working skeletal muscle and the resulting time course of blood flow responses necessary to match oxygen demand (Andersen & Saltin, 1985). Measurements of conduit vessel blood flow during moderate exercise have demonstrated a two-phase blood flow response composed of a fast component attributed to mechanical factors, and a slower second phase driven by multiple agents produced or diminished during elevated aerobic metabolism (Shoemaker and Hughson, 1999; VanTeeffelen and Segal, 2006). While the full contribution of metabolic products to blood flow responses and their underlying mechanisms have not been fully elucidated, oxygen and carbon dioxide have long been understood to have independent vasoactive properties which act in a concentration dependent manner to increase blood supply during exercise. Indeed, there is evidence for multiple mechanisms governing vasoactive responses for both oxygen and carbon dioxide, though there is little data in the literature that describes the dynamics of these mechanisms or the overall time course of the resulting change in blood flow (reviewed in Jackson, 2016). To address this gap in our understanding, we quantified the microvascular blood flow responses to direct step changes in skeletal muscle PO2 and PCO2 in the absence of muscular contractions or other changes in aerobic metabolism. For the purposes of this discussion, we focus primarily on salient responses that are comparable with the decrease in skeletal muscle [O2] and increase in [CO2] seen at the onset of exercise and electrically stimulated contractions.

Oxygen challenges

In this study we manipulated muscle oxygen concentration using a microfluidic gas exchange chamber that was directly interfaced with the EDL muscle via a gas permeable membrane. As expected, step changes in gas exchange chamber [O2] provoked rapid and profound alterations in capillary RBC SO2 that provides essential insight into the dynamics of our experimental method and important context for the resulting capillary blood flow responses (Figure 1). In each of the 4 oxygen challenges employed, significant changes in RBC SO2 were observed within 1–2 s of the step-change within the chamber with times to new steady state SO2 conditions ranging from 5—9 s. It is important to note that the dynamics of this imposed change in SO2 [τ = 1.4 s, for 7–2% (O2) challenge] is much faster than reported fast component decreases in microvascular (τ = 9.0 s) and interstitial (τ = 12.8 s) PO2 at the onset of electrically stimulated contractions using similar rat models (Behnke et al., 2001; Hirai et al., 2018). Step changes in chamber [O2] from 7–2% provoked a 52% peak increase in capillary RBC velocity, an 83% increase in RBC supply rate, and a 30% increase in capillary hematocrit over the course of the 2 min challenge, each of which are remarkably similar to previous observations of capillary hemodynamics during 1 Hz stimulated contractions in rat muscle (Kindig et al., 2002). The determined time transients of capillary RBC velocity (τ = 35.5 s), hematocrit (τ = 32.4 s), and supply rate (τ = 42.0 s) responses to 7–2% [O2] in our model were all of similar time scales and notably slower than responses during muscle contraction, particularly with respect to reported contraction induced supply rate response in animals (τ = 16 s) (Kindig et al., 2002; Poole et al., 2021) and early phase flow response in humans (τ < 7 s) (Shoemaker and Hughson, 1999). The discrepancy in dynamics with our model is almost certainly due to the absence of contraction that drives the early phase of exercise hyperemia via mechanical factors and rapid onset vasodilation (Tschakovsky et al., 1996; Laughlin et al., 1999; Mihok and Murrant, 2004; VanTeeffelen and Segal, 2006). Transient changes in capillary hematocrit in the present study suggest involvement of higher order arterioles that would be capable of affecting downstream hematocrit based on the Fåhræus-Lindqvist effect as also suggested by Kindig et al. (Fåhræus and Lindqvist, 1931; Barbee and Cokelet, 1971; Pries et al., 1986; Kindig et al., 2002). Further, in our model it is also likely that hematocrit and lineal density changes are driven, at least in part, by asymmetries in blood flow distribution between upstream arteriolar branches that supply the muscle volume influenced by our imposed gas perturbations, compared to other regions of the muscle which remain at basal conditions (Pries et al., 1989). Indeed, the hemodynamic changes observed during the 7–2% [O2] challenge likely integrate dilation of multiple levels of the arteriolar tree from terminal arterioles to higher order vessels via conducted signaling (reviewed in Bagher and Segal, 2011).

It is interesting to note that the dynamics of on and off transient responses to 12% and 2% oxygen challenges were not symmetrical, with faster capillary RBC velocity kinetics determined for 7–12% [O2] (τ = 7.0 s) and 2–7% [O2] (τ = 13.4 s) challenges (Table 2). However, the slower velocity dynamics based on the exponential fit to the 7–2% challenge does not describe the apparent multi-exponential nature of the response that includes a fast component evident over the first 20 s of the step change, and followed by a slower component that persists over the remainder of the 2 min challenge (Figure 2C). Similarly, this fast component is visible in the first 20 s of the velocity response to the 2–7% [O2] challenge (Figure 2D) and over the same time period in the 7–2% [O2] portion of the combined [O2] and [CO2] challenge (Figure 11B). The fast component of the velocity response to these challenges supports the role of rapid conducted signaling in oxygen mediated blood flow regulation, and provides further evidence of multiple mechanisms with distinct kinetics that overlap in time to produce the observed response. In contrast, the kinetics of hematocrit and lineal density responses were well represented by mono-exponential fits, and in the case of the 2–7% [O2] challenge showed profoundly slower dynamics (τ = 74.75 s) compared to the velocity response, with hematocrit changes likely driven by higher order arterioles as explained above.

Carbon dioxide challenges

Step changes in the gas exchange chamber [CO2] provoked robust blood flow responses in each of the 4 challenge sequences studied. Increasing [CO2] from the putative resting concentration in the 5–10% challenge resulted in a 58% peak increase in capillary RBC velocity, a 16% increase in capillary hematocrit, and a 63% increase in capillary RBC supply rate which were similar in proportion to the increases seen over 2 min during the 7–2% [O2] challenges. The dynamics of the 5–10% [CO2] capillary hemodynamic responses were markedly slower than other CO2 challenges studied. Capillary RBC velocity (τ = 79.3 s), hematocrit (τ = 65.9), and supply rate (τ = 88.1 s) demonstrated distinctly slower kinetics that were two-fold longer than the oxygen mediated responses in the 7

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