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Erythrocyte-associated transients in capillary PO₂: an isovolemic hemodilution study in the rat spinotrapezius muscle

Department of Physiology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia

Submitted 23 August 2006 ; accepted in final form 23 January 2007

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX GRANTS REFERENCES

 
Mathematical simulations of oxygen delivery to tissue from capillaries that take into account the particulate nature of blood flow predict the existence of oxygen tension (PO₂) gradients between erythrocytes (RBCs). As RBCs and plasma alternately pass an observation point, these gradients are manifested as rapid fluctuations in PO₂, also known as erythrocyte-associated transients (EATs). The impact of hemodilution on EATs and oxygen delivery at the capillary level of the microcirculation has yet to be elucidated. Therefore, in the present study, phosphorescence quenching microscopy was used to measure EATs and PO₂ in capillaries of the rat spinotrapezius muscle at the following systemic hematocrits (Hct_sys): normal (39%) and after moderate (HES1; 27%) or severe (HES2; 15%) isovolemic hemodilution using a 6% hetastarch solution. A 532-nm laser, generating 10-µs pulses concentrated onto a 0.9-µm spot, was used to obtain plasma PO₂ values 100 times/s at points along surface capillaries of the muscle. Mean capillary PO₂ (Pc; means ± SE) significantly decreased between conditions (normal: 56 ± 2 mmHg, n = 45; HES1: 47 ± 2 mmHg, n = 62; HES2: 27 ± 2 mmHg, n = 52, where n = capillary number). In addition, the magnitude of PO₂ transients (PO₂) significantly decreased with hemodilution (normal: 19 ± 1 mmHg, n = 45; HES1: 11 ± 1 mmHg, n = 62; HES2: 6 ± 1 mmHg, n = 52). Results suggest that the decrease in Pc and PO₂ with hemodilution is primarily dependent on Hct_sys and subsequent microvascular compensations.

microcirculation; oxygen transport; erythrocytes; oxygen tension gradients; phosphorescence quenching microscopy

Hellums coined the term "erythrocyte-associated transients" (EATs) (11), which refers to the variation in PO₂ caused by the alternate passage of RBCs and plasma gaps as observed at a stationary observation point along a capillary. These transients, which can also be described as spatial gradients, are a manifestation of the PO₂ heterogeneity in the plasma between RBCs (13). Therefore, rapid variations of PO₂ can be observed as a function of microcirculatory factors, such as hematocrit (Hct), blood flow, or oxygen content. The amplitude of this inter-RBC gradient has been predicted by theoretical models to vary according to cell spacing, shape, and orientation, as well as oxygen flux at the capillary wall, RBC-to-wall spatial clearance, and oxygen consumption rate of the tissue (2, 5, 6, 10, 12, 14, 21, 29).

Theoretical considerations of the discrete nature of capillary blood flow have suggested that inter-RBC PO₂ heterogeneity increases the intracapillary fraction of resistance to oxygen transport to twice that compared with previous models assuming a homogenous distribution of hemoglobin (11). This stems from the low solubility of oxygen in the plasma, which hinders diffusive oxygen movement from the RBC through the surrounding plasma sheath and plasma gaps into the tissue. Consequently, RBC separation distance plays an important role in the rate of oxygen transport from RBC to tissue (13). Under conditions of rest or maximal tissue oxygen consumption, low plasma PO₂ can become limiting for oxygen exchange, dependent on a critical RBC separation distance defined by a threshold for maintaining uniform oxygen flux at a point along a capillary (6).

Previous difficulties preventing the measurement of capillary PO₂ transients have been overcome by substantially increasing the temporal and spatial resolution of the phosphorescence quenching method with pulsed laser excitation of the phosphorescent probe. Golub and Pittman (7) previously reported measurements of EATs in rat mesenteric capillaries using rapid laser excitation to detect fast PO₂ transients. The purpose of the present study was to extend these observations to a three-dimensional tissue and to observe EATs in the capillaries of the rat spinotrapezius muscle.

Furthermore, hemodilution has been used therapeutically to increase peripheral perfusion to ischemic tissues. Rheological alterations, such as a decrease in blood viscosity (a function of hematocrit), combined with peripheral vasodilation have been credited with increased flow through vascular networks. In addition, hemodilution has been found to influence the distribution of RBC flow and Hct in microvascular networks (22). During hemodilution, the systemic hematocrit (Hct_sys) is reduced, and therefore the oxygen-carrying capacity of the blood is diminished. The role of capillaries in oxygen delivery under such conditions has yet to be investigated.

The present study attempts to understand the localized changes in capillary oxygen tension as a function of reduced Hct_sys. Isovolemic hemodilution, resulting in a dramatic reduction of Hct_sys, could affect capillary Hct and cause an increase in RBC separation distance. Therefore, defining the nature of EATs where Hct_sys is reduced to a moderate or severe level could provide further information regarding capillary oxygen exchange and microvascular compensations due to hemodilution.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX GRANTS REFERENCES

 
A total of 12 female Sprague-Dawley rats (244 ± 3.7 g; Harlan, Indianapolis, IN) were used in this investigation. One group (normal, n = 4; Hct = 38.8%) was used to assess nonhemodiluted conditions, and two other groups (HES1, n = 4; HES2, n = 4) were used to assess isovolemic hemodilution conditions at two different systemic hematocrits (26.6 and 14.8%, respectively) using Hespan (6% hetastarch, a high-molecular-weight hydroxyethyl starch; DuPont Pharma, Wilmington, DE) as the hemodiluent. Animals were initially anesthetized with a combination of ketamine-acepromazine (72 and 3 mg/kg ip, respectively). Following cannulation of the right jugular vein for administration of supplemental anesthetic, surgical preparation and measurements were conducted under a continuous intravenous infusion of alfaxalone/alfadolone (0.1 mg·kg^–1·min^–1 Saffan; Schering-Plough Animal Health, Welwyn Garden City, UK). Upon conclusion of experimentation, the animals were euthanized with administration of 0.4 ml/kg iv Euthasol (390 mg/ml pentobarbital sodium and 50 mg/ml phenytoin; Delmarva Laboratories, Midlothian, VA). All procedures and protocols were approved by the Virginia Commonwealth University Institutional Animal Care and Use Committee.

Preparation. Initially, the trachea was cannulated (PE-240; Clay Adams, Parsippany, NJ) to ensure a patent airway and spontaneous breathing of room air. Subsequent cannulation of the right jugular vein with PE-90 tubing and the left carotid artery with PE-50 tubing allowed for continuous intravenous administration of anesthetic and monitoring of mean arterial pressure (CyQ 103/301; Cyber Sense, Nicholasville, KY), respectively. The femoral artery was cannulated with PE-10 tubing and used for the withdrawal of blood during the isovolemic hemodilution procedure and blood sampling for arterial blood gas measurements, including PO₂, PCO₂, and pH (ABL 705; Radiometer America, Westlake, OH). In addition, total hemoglobin (Hb) and percent arterial hemoglobin oxygen saturation (Sa_O2) were measured using a hemoximeter (Radiometer OSM3; Radiometer, Crawley, UK). Hct was determined by capillary tube centrifugation of arterial blood. The femoral vein was cannulated with PE-20 tubing and utilized for infusion of Hespan and the albumin-bound Pd-porphyrin phosphorescence probe. The spinotrapezius muscle was partially exteriorized using a modification of the procedure originally described by Gray (9) and subsequently modified by Bailey et al. (1). Care was taken to keep the muscle moist with continuous superfusion of Ringer solution, and sutures were placed at 1-cm intervals along the lateral margins to facilitate surgical and experimental manipulation, ensuring minimal trauma to the spinotrapezius muscle. After exteriorization of the spinotrapezius, the animal was placed on its side on a thermostatic animal platform described by Golub and Pittman (8). The muscle was secured dorsal side up and spread to its physiological length by using the sutures along the lateral margins. Finally, Ringer solution was applied topically, and the muscle was covered with Saran film (Dow Chemical, Indianapolis, IN) to prevent tissue desiccation and atmospheric gas exchange.

Phosphorescence quenching microscopy. The experimental setup previously described by Golub and Pittman (7) was used to measure plasma PO₂ and maximum phosphorescence amplitude (PA) in capillaries and to measure light transmission (LT) through a capillary. The animal platform was placed on the stage of an Ortholux microscope (Leitz, Stuttgart, Germany) configured for epi-illumination through a x100/1.30 objective (Leitz). Immersion oil was placed between the objective and the Saran film covering the spinotrapezius muscle.

Phosphorescence excitation was achieved with a 532-nm laser (GMS1-038-15T; Lasermate, Pomona, CA), which delivered 60 nJ for a duration of 10 µs to a 0.9-µm-diameter region at 100 Hz. A photomultiplier tube (R3896 with high-voltage socket HC123-01; Hamamatsu, Bridgewater, NJ) detected the emitted phosphorescence collected by the x100 objective. The signal from the photomultiplier tube was directed through a gated amplifier (OP37EP; Analog Devices, Norwood, MA), designed to be a current-to-voltage converter, and then to a 12-bit analog-to-digital converter (PC-MIO-16E-4; National Instruments, Austin, TX). The digital data were stored on a Dell personal computer (Round Rock, TX).

The laser light passing through the capillary was detected with a T-5 photodiode (Intor, Socorro, NM) integrated with a 532-nm laser line filter. The LT signal collected from the diode was passed through an amplifier and directed to the second channel of the analog-to-digital converter. Both the phosphorescence and LT signals were monitored with an oscilloscope (72-3060; Tenma, Springboro, OH) for amplitude adjustments.

Capillary selection for measurement and positioning the laser spot on the vessel were carried out under transillumination using an OG-570 filter (Edmund Optics, Barrington, NJ) to prevent excitation of the phosphor. Measurements of phosphorescence excitation and LT were carried out in a darkened room, and other background light sources were minimized to reduce noise caused by stray light.

Data acquisition was performed using a program written in LabVIEW (National Instruments) at a sampling rate of 200 kHz with 400 data points per curve and 100 curves per measurement sequence. The analysis program was also written in LabVIEW and was used for the off-line determination of PO₂ for the 100 sequential curves collected. Nonlinear fitting of the phosphorescence decay curves was based on the rectangular PO₂ distribution model (30)

where t (µs) is the time from the beginning of phosphorescence decay, I(t) (V) is the magnitude of the phosphorescence signal, I(0) (V) is the amplitude of the phosphorescence signal at t = 0, M (mmHg) is the mean PO₂, (mmHg) is the half-width of the PO₂ distribution, T (µs) is the lifetime of the fast postexcitation transient, A (V) is the amplitude of the fast postexcitation transient and B (V) is the baseline offset. Stern-Volmer constants k₀ = 18.3 x 10^–4 µs^–1 and k_q = 3.06 x 10^–4 µs^–1·mmHg^–1 were determined in a separate study (30).

Capillary inclusion parameters and probe administration. Only capillaries supporting continuous RBC flow were selected for study. Before data collection, each capillary chosen for study had to meet the following additional specified selection criteria: 1) sufficient LT with evident RBC fluctuations as observed on the oscilloscope and 2) spatial separation of the capillary from other microvessels. Measurements were made in the centerline of the capillary in a region of high visual contrast.

The phosphor probe (30 mg/kg) was administered via the right femoral vein before measurements were taken, and 1 min of equilibration time was allowed before initiation of data collection to ensure a homogeneous probe distribution in the plasma. Based on a cardiac output of 100 ml/min and a circulating volume of 15 ml, the circulatory time for a 244-g rat would be 9 s (25). During a period of 1 min, probe was distributed throughout the circulation and a stable amplitude of the phosphorescence signal detected. Experimental measurements were then made within a 30-min period to exclude nonintracapillary sources from the phosphorescence signals that would arise following eventual extravasation of the albumin-bound probe.

LT, PA, and PO₂. As independent corroborative measures associated with the detection of PO₂ fluctuations, LT and maximum PA were measured. LT was measured via photodiode registration of the intensity of the laser light passing through a capillary. Maximum PA was determined by analyzing the beginning of the decay curve after the end of the excitation pulse. Upon analysis, the maximum amplitude of the phosphorescence decay curve and the light transmittance pulses were averaged using the three highest points at the peak of each signal.

The maximum amplitude of the phosphorescent signal is proportional to the amount of probe present in the detection volume. A homogeneous distribution of the dissolved probe in plasma was demonstrated by Golub and Pittman (7) using densitometric scanning and phosphorescence imaging of the Pd-porphyrin probe location, confirming that probe does not diffuse into RBCs. Therefore, PA increases when there is more probe in the detection region, which occurs when there is more plasma present (i.e., a plasma gap between RBCs). Observations of the relative minima and maxima of LT and PA are expected to be correlated with observed maxima and minima of PO₂, respectively, as RBCs and plasma gaps alternately pass through the detection region.

Isovolemic hemodilution procedure. A stepwise isovolemic blood exchange protocol was followed, using Hespan as the hemodiluent. The volume of each withdrawal/infusion step was calculated as a percentage of blood volume estimated at 6% of the body weight. The target Hcts were 25% for moderate hemodilution and 15% for severe hemodilution. Each step took 90 min to complete. An additional 15 min of equilibration time for the animal was allowed posthemodilution before systemic measurements and probe infusion. For measurements in nonhemodiluted animals, the animals were allowed a 15-min equilibration period after surgery before systemic measurements and probe infusion.

Data analysis. The "peak detection" method in Origin version 6.1 (OriginLab, Northampton, MA) was used to determine peak values on the unfiltered data, corresponding to the near-RBC PO₂, and troughs, corresponding to the lowest PO₂ in the plasma gap. PO₂ peak values, as well as the corresponding times these peaks occurred during the 1-s measurement period, were determined using this time series analysis method. In addition, the same method was applied to detect the times when peak minima and maxima occurred in the LT and PA time tracings corresponding to the PO₂ time tracings for a given capillary. This information was used to help substantiate the presence or absence of RBCs in the detection volume.

The Origin analysis tool "pick peaks" uses a search rectangle in which width and height (the percentage of the total number of points in the data range, i.e., time) and the total amplitude of the data in the range (i.e., LT, PA, or PO₂, respectively) can be specified. A peak is rejected if it does not meet the minimum height criterion, the percentage of the total amplitude of the data in the range relative to the minimum determined within the specified width. The test point is at the top center of the rectangle, so any peak found may not correspond to an absolute local maximum. This situation can occur when data are asymmetrically distributed around a relative maximum (Origin). A 5 x 5 search rectangle with a minimum height of 5 was used to detect both positive and negative peaks simultaneously.

For the 100 consecutive PO₂ measurements collected over the 1-s measurement period, all positive peaks detected were averaged, giving a mean peak PO₂ (PO₂^Max), and all negative peaks detected were averaged, resulting in the mean trough PO₂ (PO₂^Min). This was done for each capillary PO₂ time course for all three conditions (total of 159 capillaries). The individual mean capillary PO₂ (Pc) is an average PO₂ of the 100 sequential curves collected (unfiltered) over the acquisition period of 1 s. The difference between PO₂^Maxand PO₂^Min is termed the PO₂. In addition, RBC flux was estimated, based on the number of positive peaks detected by the time series analysis (i.e., peak detection method) using the unfiltered PO₂ data from each capillary.

Oxygen loss. A mass balance approach was used in the determination of oxygen loss between the systemic arterial blood and the site of capillary PO₂ measurements for each capillary (see APPENDIX). Sa_O2 was measured from arterial blood obtained from the carotid artery. Capillary oxygen saturation (Sc) was estimated from the Hill equation, using capillary PO₂ values and P₅₀, recalculated due to differences in pH and PCO₂ under the different conditions (i.e., normal, HES1, HES2). To account for the shift in the oxygen dissociation curve, the Severinghaus equation (24) was used as follows:

where P is the standard P₅₀ of Sprague-Dawley rats (36 mmHg; Ref. 3), pH is the experimental value determined by the blood gas analyzer, T is the stabilized temperature of the rat spinotrapezius, and BE refers to the base excess, also determined by the blood gas analyzer. Changes in blood pH and BE between the arterial and capillary measurement sites were neglected. Sc was then estimated from the Hill equation,

where PO₂^Max is the maximum capillary PO₂ associated with the presence of a RBC, P₅₀ is the shifted value determined as described above, and n_H is 2.7, the Hill coefficient (15). Equation A10 (see APPENDIX) was then used to calculate the oxygen loss per RBC (L_R) as follows:

where SO₂ is Sa_O2 – Sc, [Hb]_RBC is the average hemoglobin concentration per RBC (see Table 1), and C_Hb is 1.34 ml O₂/g Hb, the oxygen binding capacity of hemoglobin.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX GRANTS REFERENCES

 
Systemic data, capillary PO₂, and Hct. A total of 159 capillaries were evaluated in the 12 female Sprague-Dawley rats. There were no significant differences in mean arterial pressure (MAP) among normal (99.8 ± 7.4 mmHg), HES1 (114.7 ± 9.9 mmHg), and HES2 conditions (96.2 ± 8.9 mmHg). Sa_O2 also was not significantly different among the three conditions (93.6 ± 2.1, 92.8 ± 0.7, and 94.0 ± 1.5% for normal, HES1, and HES2, respectively).

Arterial PO₂ (Pa_O2) varied significantly between the normal (97.0 ± 6.1 mmHg) and HES1 conditions (76.1 ± 1.7 mmHg), whereas Pa_O2 for the HES2 condition (82.6 ± 6.0 mmHg) was not significantly different from either the normal or HES1 value. Following hemodilution, the Hct_sys was reduced significantly from 38.8 ± 0.9% (normal condition) to 26.6 ± 1.3% (HES1, moderate hemodilution) and then to 14.8 ± 1.3% (HES2, severe hemodilution). As the Hct_sys was reduced, Pc also decreased. Pc was 56.5 ± 2.2 mmHg (n = 45) for the normal condition, 47.2 ± 1.7 mmHg (n = 62) after moderate hemodilution, and 26.8 ± 1.5 mmHg (n = 52) after severe hemodilution, with a significant difference evident among all conditions. Table 1 summarizes the systemic variables for the three conditions, and comparisons of Pa_O2, Pc, and Hct_sys among the three conditions are shown in Fig. 1.

View larger version (30K): [in this window] [in a new window]   Fig. 1. Comparison of general declining trend of arterial PO2 (PaO2), capillary PO2 (Pc), and systemic hematocrit (Hct) in the normal condition and conditions of moderate (HES1) and severe isovolemic hemodilution (HES2). Values are means ± SE. *P < 0.001, significantly different from other 2 conditions. P < 0.05, significantly different from normal. P < 0.0001, significantly different from other 2 conditions.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Example of PO2 tracing containing 100 consecutive data points for a HES2 capillary (Hct = 15%). The solid line connects the unfiltered data collected over the 1-s measurement period. The overlying dashed line is the data filtered with a 20-Hz cutoff frequency low-pass filter. Using a "peak detection" method of analysis, we determined positive and negative peaks for all capillaries representing PO2 values (either PO2Max or PO2Min) and time points (either t or t). Peaks are marked by the time points at which they occur on the unfiltered data. In addition, temporal changes in PO2 are easily recognizable over the 1-s data collection period. The PO2Max and PO2Min for this capillary are 28.9 and 23.9 mmHg, respectively. The Pc and PO2 are 26.7 and 5.0 mmHg, respectively.

View larger version (12K): [in this window] [in a new window]   Fig. 3. Correlation analysis between t and t, t and t, and t and t was performed to determine whether the maximum and minimum peak values detected from the individual PO2, light transmission (LT), and phosphorescence amplitude (PA) signals would correspond. All relationships in peak time for the three signals in this HES2 capillary demonstrate high correlation coefficients of 0.99 (P < 0.0001). The linear fit line (solid) lies almost directly on the line of identity (dashed), indicating negligible shift between the different signals and high temporal and spatial resolution of the measurement system.

EAT amplitude. PO₂^Max, corresponding to the PO₂ value associated with the RBCs, decreased as the Hct_sys was reduced by isovolemic hemodilution with Hespan. The difference between PO₂^Max and PO₂^Min (PO₂) was found to decrease from 18.5 ± 1.1 mmHg under the normal condition to 10.9 ± 0.6 mmHg after moderate hemodilution and then to 6.5 ± 0.5 mmHg following severe hemodilution (Table 2). Thus PO₂ fluctuations were reduced by 41 and 65% for moderate and severe hemodilution, respectively, compared with the normal condition. Figure 4 shows PO₂ plotted against PO₂^Max. This graph indicates that during isovolemic hemodilution with Hespan, PO₂ is related to Hct and RBC PO₂ (Fig. 5).

View larger version (8K): [in this window] [in a new window]   Fig. 4. PO2 is plotted against PO2Max. Values are shown as means ± SE in mmHg. *P < 0.001, significantly different from other 2 conditions.

View larger version (8K): [in this window] [in a new window]   Fig. 5. PO2Max, PO2Min, and PO2 are plotted against Hct, demonstrating a more drastic decrease in the near-red blood cell (RBC) PO2 (PO2Max) than in the plasma PO2 (PO2Min). Values are shown as means ± SE. *P < 0.05, significantly different from normal. P < 0.05, significantly different from HES1.

View larger version (8K): [in this window] [in a new window]   Fig. 6. Arterial (SaO2) and capillary oxygen saturation (ScO2) plotted against Hct. A greater change in Sc occurred between the Hct of 26.6% (HES1) and 14.8% (HES2) than between the nonhemodiluted animals with a Hct of 38.8% and the HES1 condition. This finding indicates that significant derangements in oxygen delivery begin to occur after 35% of the normal Hct is reduced.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX GRANTS REFERENCES

Laser excitation of the Pd-porphyrin phosphorescent probe confined to the plasma allowed the measurement of plasma PO₂ transients and PA, proportional to the amount of plasma in the detection region. The particulate nature of blood flow was also revealed by the simultaneous measurement of LT through the capillary. As a result of the intrinsic nature of RBCs to absorb light, the signal would decrease in the presence of a RBC and increase once the RBC moved out of the detection region. The signal for PA would correspondingly increase, reflecting the presence of plasma measured by the amount of light emitted by the excited phosphor probe in the detection region, and decrease when an RBC was present (i.e., less plasma).

Although the time points of maxima and minima were highly correlated among the three independent variables (P < 0.0001 in all cases), some noise is still present in the signals. Golub and Pittman (7) found that, due to the complex scattering of light (i.e., reflection and refraction) by a RBC with random size, shape, and orientation, PA was a better indicator of the presence of plasma or RBC. In the present study we could not conclude whether LT or PA was a better independent corroborative measure, perhaps because of other factors that might have contributed to variability between signals in skeletal muscle, including muscle thickness, refraction of transmitted light by muscle tissue, and the presence of multiple microvessels along the incident light beam.

Mathematical predictions of oxygen tension associated with RBCs and plasma in capillaries suggest that significant PO₂ gradients can exist in the plasma between RBCs (11, 13). In early modeling studies, PO₂ was predicted to be no greater than 6 mmHg for conditions of rest, anemia, or exercise (13, 16). Using RBC shape as a determinant of oxygen transport, Wang and Popel (29) calculated PO₂ amplitudes as great as 20 mmHg under simulated conditions of maximal tissue oxygen demand. Assuming normal Hct under resting conditions, a maximum PO₂ oscillation of 10 mmHg has also been predicted (14). Homer et al. (13) calculated that the maximum PO₂ difference between RBCs and plasma would be more pronounced under conditions of severe acute anemia, reasoning that the distance between red cells would increase and therefore cause the PO₂ to be depressed in the plasma gap between cells. In the current study this expectation was not observed; rather, the largest difference in oxygen tension was found to occur at normal Hct_sys. A conclusion reached in several mathematical models analyzing oxygen tension between RBCs and plasma is that cell spacing is a critical determinant of the difference observed between the near-red cell and plasma gap PO₂ (5, 6, 13, 16, 17). Therefore, many models predict that with hemodilution, the spatial distribution of RBCs changes so that they become spaced further apart as they flow through the capillaries. This prediction assumes that in the process of hemodilution, where the Hct_sys is reduced, there is a concomitant decrease in capillary hematocrit whereby RBC spacing increases. This assumption implies that the smaller number of RBCs is distributed among the same capillaries, thereby increasing the average RBC spacing. Results in the current study show that with isovolemic hemodilution, the difference between the near-RBC and plasma gap PO₂ instead decreases, suggesting that the distribution of RBCs among capillary networks is significantly altered.

Tsai et al. (28) studied hemodynamic parameters in resting rabbit skeletal muscle as the Hct_sys was gradually reduced to 26% of normal by isovolemic hemodilution with a 6% dextran solution. Their results suggest that the spacing between RBCs during capillary transit is independent of the reduction in Hct_sys. They also reported that RBC flux was maintained by an increase in RBC velocity. Similar results have been reported by Sarelius (23), who measured capillary blood flow variables in the hamster cremaster muscle after isovolemic hemodilution. In this case, no change was found in capillary RBC flux or velocity at Hct_sys values 32 and 49% of normal. This study also concluded that capillary RBC flow and capillary RBC content (cells/100 µm) were not directly proportional to the changes in Hct_sys. In the present study, capillary RBC flux was estimated by counting the number of peaks in the PO₂ signal over the 1-s measurement period, and the results are consistent with previous studies indicating no significant change in capillary RBC flux with hemodilution in skeletal muscle (18, 23, 28).

Because EATs were the focus of the current investigation, only capillaries perfused by RBCs were selected for study. This raises the issue of how systemic hemodilution affects the distribution of RBCs within the microcirculation, especially in capillary networks. From previous studies (22), it appears that hemodilution results in higher RBC velocity and increased axial migration of RBCs in arterioles. Side branches from the proximal parts of the arteriolar network are more likely to contain blood that is much lower in Hct due to plasma skimming. The capillary networks derived from those branches will hence be perfused mostly with plasma and a paucity of RBCs. Accordingly, the capillary networks derived from the more distal parts of the arteriolar network will be the recipients of most of the RBCs, consistent with the finding that in those capillaries perfused by RBCs, capillary flux remains relatively unchanged following hemodilution. This overall increase in the heterogeneity of RBC flow among capillary networks results in a maldistribution of oxygen delivery, where some capillary networks receive an inadequate supply of RBCs and therefore oxygen, whereas others are adequately supplied with RBCs with reduced SO₂ under hemodiluted conditions. Also, as a result of redistribution of RBCs, cell spacing in the smaller number of RBC-perfused capillaries will not vary much from that at normal Hct. This could be part of the explanation as to why the magnitude of the EATs (i.e., PO₂) does not increase with reduced Hct_sys, and it appears that the distribution of RBCs within capillary networks with hemodilution may result in maintained RBC spacing in fewer networks (i.e., functional capillary density decreases roughly in proportion to the degree of hemodilution).

Also, in contrast to the predictions of mathematical models, as the animals were hemodiluted with a 6% hetastarch solution, the PO₂ in the capillaries became more homogeneous as reflected by the significant decrease in the ratio PO₂/Pc(proportional to the coefficient of variation) from normal (see Table 2). The exchange of blood with a non-oxygen-carrying fluid caused a decrease in the overall systemic oxygen-carrying capacity of the blood via a reduction in Hct_sys, which also contributed to the decrease in plasma PO₂. From the data collected, it can be concluded that there was a greater change in near-RBC PO₂ than there was in plasma gap PO₂ as the animals were hemodiluted, indicating that homogeneity on a local level is more a function of the RBC PO₂ decrease than the plasma gap PO₂ decrease (see Fig. 5). This can be explained by considering oxygen exchange between discrete regions of relatively high PO₂ (i.e., RBCs) or relatively low PO₂ (i.e., plasma gaps) and the even lower PO₂ in the tissue. As a result of the oxygen diffusion gradient between these respective regions and the tissue, the higher oxygen flux from the RBC region would lead to a larger PO₂ drop for the RBCs than for the plasma gaps.

In acutely anemic animals with significantly reduced Hct and total Hb concentration, normal oxygen consumption is maintained by parallel increases of cardiac output and oxygen extraction (19). Although cardiac output was not measured in this study, we report that SO₂ increased significantly at 15% Hct, indicating that oxygen extraction increased significantly between the large arterioles and the capillary measurement sites. Swain and Pittman (27) reported that about two-thirds of the oxygen loss occurring between arterioles and venules actually takes place in precapillary arterioles. The current study also demonstrates that a substantial amount of oxygen loss must occur before the blood reaches the center of capillaries (where, on average, measurements were made) and that the degree of loss is augmented by hemodilution. Although the systemic arterial PO₂ stayed relatively steady, the mean capillary PO₂ showed a significant decreasing trend (Tables 1 and 2), reflecting greater oxygen loss from the arteries to the middle of capillaries under such conditions. The finding of the oxygen saturation difference above shows that as the oxygen-carrying capacity is compromised, the burden of tissue supply of oxygen from each erythrocyte increases. This oxygen loss per RBC is also represented in Table 3 as L_R, demonstrating that the largest oxygen loss occurs under severe hemodilution conditions, three times the loss per RBC compared with normal. Therefore, isovolemic hemodilution results in the depression of RBC PO₂ with a lesser depression of plasma PO₂ contributing to dampened EATs (shown in graphic form in Figs. 5 and 6).

In addition, on the basis of theoretical modeling, we hypothesized that PO₂ would increase with isovolemic hemodilution. Results demonstrate that this does not occur. Rather, the analysis of PO₂ data revealed that as the Hct_sys was reduced, the PO₂ decreased from roughly 18 mmHg (normal) to 11 mmHg after moderate hemodilution (HES1) and then to 6 mmHg after severe hemodilution (HES2). Figure 7 shows the relationship between PO₂ from the different conditions and L_R. As the hemodilution becomes more severe, the L_R increases and PO₂ decreases. This is consistent with the explanation above concerning the greater burden, with decreasing Hct_sys, of each erythrocyte to supply oxygen to the tissue under unchanged conditions of demand, despite an increase in cardiac output.

View larger version (11K): [in this window] [in a new window]   Fig. 7. The capillary PO2 shows a decreasing trend as more oxygen is lost per RBC (LR) due to diffusive shunting to tissue or other vessels in the vicinity of the capillaries. A reduction of the relative spread of PO2 values occurs as the hematocrit is dropped to either the moderate (HES1) or severe (HES2) level. When the LR is high, the PO2 is generally low despite the condition, which becomes increasingly the case as capillary PO2 becomes more homogenous with hemodilution.

Although this study used the most current method of phosphorescence quenching microscopy, difficulties still exist in making such measurements in muscle. Initially, the rat mesentery was used to study EATs, where the two-dimensional microvascular network of the mesentery assured a limited contribution of extravasated albumin-bound probe to the phosphorescence signal from the interstitial space or from any microvessels in the laser light path. The three-dimensional anatomy and high vascular spatial density of the spinotrapezius muscle increased the probability of signal artifact from other compartments, including microvessels and the interstitial space, along the incident light path. To minimize the effects on LT and PA from extraneous microvessels and the tissue, we employed a 30-min time limit for capillary measurements based on published observations that extravasation of the phosphorescent probe over 35 min of perfusion was insufficient to yield a detectable phosphorescence signal in skeletal muscle (20). Each capillary chosen for the present study also met strict inclusion criteria to ensure that no other microvessels were in the incident laser light path.

In conclusion, the present study shows that EATs can be identified in the rat spinotrapezius muscle as independently corroborated by the PO₂, LT, and PA signals under normal conditions and after isovolemic hemodilution. Furthermore, the largest PO₂ occurs under normal conditions, and the PO₂ gradients in plasma, as well as mean capillary PO₂, decrease as the Hct_sys is reduced via isovolemic hemodilution. These results differ from some theoretical predictions, in part because of assumptions in the models that were required to simplify the complex conditions of the microcirculation. Several theoretical predictions have emphasized the importance of RBC spacing on plasma PO₂ gradients. The current study found no evidence of significant changes in RBC spacing (near constant RBC flux for different Hct values) so that capillary EATs under the conditions of study appear to be mostly dependent on the PO₂ and SO₂ of RBCs entering the capillary. The decreased oxygen-carrying capacity caused by reduced hematocrit increases the burden of each erythrocyte to supply oxygen to the tissue, especially under conditions of severe hemodilution. In addition, the effect of systemic hematocrit on the distribution of RBC flow in microcirculatory networks due to rheological alterations (i.e., reduced viscosity and increased axial migration) has a profound influence on the magnitude of EATs. These conclusions strongly suggest that during isovolemic hemodilution, passive compensatory mechanisms of oxygen delivery exist in the microvasculature that alter EATs in contrast to what might be hypothesized on the basis of theoretical works.

	APPENDIX

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX GRANTS REFERENCES

 
To interpret capillary PO₂ measurements under conditions of hemodilution where some variables could not be measured, we employed the following scenario. One of two cases is assumed to exist in a given capillary: 1) it is perfused only by plasma, or 2) it is perfused by RBCs and plasma. In the case of the plasma-perfused capillaries, the number of total plasma-perfused capillaries is N_P, flow is Q_c,P, and capillary hematocrit is by definition Hct_c,P = 0. For RBC-perfused capillaries, the total number of capillaries is N_R, flow is Q_c,R, and capillary discharge hematocrit is Hct_c,R.

Note that functional capillary density can be defined as FCD = N_R/(N_P + N_R). The input artery, in this case the spinotrapezius feed arteriole, is assumed to have the same values for oxygen tension (Pa_O2), oxygen saturation (Sa_O2), and hematocrit (Hct_a) as systemic arterial values (i.e., negligible loss of oxygen and flow separation, respectively, between the systemic artery and feed arteriole). All blood flow entering through this artery (Q_a) is assumed to go through either the plasma- or RBC-perfused capillaries. Therefore, conservation of blood flow gives

(A1)

Assuming that all RBCs entering the artery go through the RBC-perfused capillaries, then conservation of RBC flow gives the following relationship, since the plasma-perfused capillaries do not contain any RBCs:

(A2)

The convective oxygen flow for the upstream inflow vessel (neglecting oxygen carried by the plasma) is

(A3)

where [O₂]_a is arterial oxygen content, Sa_O2 is arterial oxygen saturation, [Hb]_RBC is the average Hb concentration in a single RBC, and C_Hb is the oxygen-binding capacity of Hb. For the capillaries, Q_c, is

(A4)

where the term pertaining to plasma flow can be neglected because [O₂]_c,P is very small compared with [O₂]_c,R. Therefore, the equation can be simplified as

(A5)

where Sc_R is calculated from the Hill equation using the PO₂ of the RBCs.

The rate at which oxygen diffuses from the arteriolar network between the arterial inflow and the middle of the RBC-perfused capillaries ("middle" reflects that the capillary sites are chosen at random positions along a capillary) is given by the following:

(A6)

The rate of oxygen loss from the network between the inflow artery and middle of the capillaries relative to the oxygen inflow to the network is

(A7)

From conservation of RBC flow (Eq. A2), we have

(A8)

where SO₂ = Sa_O2 – Sc_R. The average oxygen loss per RBC is then

(A9)

Again, using Eq. A2, we have

(A10)

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX GRANTS REFERENCES

 
This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-18292, American Heart Association (Mid-Atlantic Affiliate) Grant 065449U, and the A. D. Williams Foundation.

	FOOTNOTES

 

Address for reprint requests and other correspondence: R. N. Pittman, Dept. of Physiology, Medical College of Virginia Campus, Virginia Commonwealth Univ., 1101 E. Marshall St., PO Box 980551, Richmond, VA 23298-0551 (e-mail: pittman{at}vcu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX GRANTS REFERENCES

 

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