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1Department of Bioengineering, University of California-San Diego, La Jolla 92093; 2La Jolla Bioengineering Institute, La Jolla, California 92037; and 3Department of Mechanical Engineering, University of Los Andes, Bogotá, Colombia
Submitted 9 December 2003 ; accepted in final form 26 February 2004
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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hyperoxia; functional capillary density; oxygen-carrying capacity; blood substitutes; tissue oxygen delivery
Fluorocarbons have been investigated by different approaches that extended to phase 3 clinical trials (9). These materials carry oxygen, are synthetic, and, in principle, are available in very large quantities at modest costs. This has tantalized investigators, medical practitioners, and business enterprises, because it could, in principle, lead to a convenient, largely available, and pathogen-free oxygen carrier. However, fluorocarbons only become miscible with water when emulsified with phospholipids (derived from egg yolk) and therefore are not completely synthetic. Furthermore, whereas Hb carries oxygen via a reversible chemical reaction, fluorocarbons carry oxygen as a function of their solubility; therefore, at the PO2s prevailing in the microcirculation in normoxia their oxygen-carrying capacity is limited and only about twice that of plasma, or 10 times less than Hb.
The low oxygen-carrying capacity of fluorocarbons in normoxic tissue can be augmented by exposing the organism to normobaric 100% oxygen, an approach taken in all attempts to produce an oxygen-carrying blood substitute with this material (7, 9). Fluorocarbons to be used as blood substitutes would presumably be used to remedy blood losses, decreased oxygen-carrying capacity, and anemia in conditions of anemic hyperoxia. The superposition of hyperoxia on the changes of circulating fluid composition is a combination of factors whose effect on the transport of oxygen at microcirculation level is not predictable by model analysis and cannot be readily inferred from systemic measurements. Understanding of the consequences of oxygen transport to the tissue by fluorocarbons may be obtained by direct measurements and experimentation of the transport phenomena at the level of the microcirculation.
Considering that fluorocarbons are synthetic, their availability is not dependent on the transfusional blood supply, their potential ease of storage and application, and their inherent lesser cost than Hb-based oxygen-carrying plasma expanders, it would seem desirable to understand their specific mode of action in the microcirculation to determine their efficacy as a blood substitute. To accomplish this, we carried out a basic microvascular analysis of how oxygen is transported in the microcirculation when blood oxygen-carrying capacity is provided by fluorocarbons and hyperoxia in conditions of extreme hemodilution. This modality was used to evidence the underlying physiological reactions, which are not manifested and are otherwise masked by compensatory mechanisms unless the transfusion trigger is significantly exceeded.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The hamster chamber window model is widely used for microvascular studies in the unanesthetized state, and the complete surgical technique has been described in detail elsewhere (4). Briefly, the animal was prepared for chamber implantation with a 50 mg/kg ip injection of pentobarbital sodium anesthesia. After hair removal, sutures were used to lift the dorsal skin away from the animal, and one frame of the chamber was positioned on the animal's back. The chamber consists of two identical titanium frames with a 15-mm circular window. With the aid of backlighting and a stereomicroscope, one side of the skinfold was removed following the outline of the window until only a thin layer of retractor muscle and the intact subcutaneous skin of the opposing side remained. Saline and then a cover glass were placed on the exposed skin held in place by the other frame of the chamber. The intact skin of the other side was exposed to the ambient environment. The animal was allowed at least 2 days for recovery before the preparation was assessed under the microscope for any signs of edema, bleeding, or unusual neovascularization. Barring these complications, the animal was anesthetized again with pentobarbital sodium. Arterial and venous catheters were implanted in the carotid artery (polyethylene-50) and jugular vein (polyethylene-50), respectively, and filled with a heparinized saline solution (30 IU/ml) to ensure patency at the time of experiment. Catheters were tunneled under the skin and exteriorized at the dorsal side of the neck, where they were attached to the chamber frame with tape. Experiment was performed after at least 24 h but within 48 h after catheter implantation.
Inclusion criteria. Experiments were performed between 24 and 48 h after catheter implantation if the chamber presented normal tissue, showed no bleeding or signs of infection, and had normal arteriolar flow. Systemic inclusion parameters were as follows: heart rate (HR) > 340 beats/min, mean systemic arterial blood pressure (MAP) > 80 mmHg, hematocrit (Hct) > 45%, and arterial PO2 > 50 mmHg. Animals with signs of inflammation and bleeding in the chamber were excluded from the study.
Blood chemistry and rheology. Arterial blood was collected in heparinized glass capillaries (0.05 ml) and immediately analyzed for PO2, PCO2, base excess (BE), and pH (Blood Chemistry Analyzer 248, Bayer; Norwood, MA). The comparatively low arterial PO2 and high PCO2 of these animals is a consequence of their adaptation to a fossorial environment. Blood samples for viscosity and colloid osmotic pressure measurements were quickly withdrawn from the animal with a heparinized 3-ml syringe at the end of the experiment for immediate analysis or refrigerated for next-day analysis. Blood samples were centrifuged, and colloid osmotic pressure in plasma was measured (model 420, Colloid osmometer, Wescor; Logan, UT). Calibration of the osmometer was made with a 5% albumin solution using a 30-kDa cutoff membrane (Amicon; Danvers, MA) (33). The viscosity of plasma and whole blood was determined at a shear rate of 160 s–1 and 37°C in a Dv-II+ Viscometer (Brookfield Engineering Laboratories; Middleboro, MA).
Systemic parameters. MAP and HR were continuously recorded (MP 150, Biopac Systems; Santa Barbara, CA). Hct was measured from centrifuged arterial blood samples taken in heparinized capillary tubes (Readacrit Centrifuge, Clay Adams, Division of Becton-Dickinson; Parsippany, NJ). Hb content was determined spectrophotometrically from a single fluid drop (B-Hemoglobin, Hemocue; Stockholm, Sweden) (18).
Cardiac output measurement. Cardiac output (CO) was measured by a modified thermodilution technique (3). In a group of n = 8 animals, the animals were randomly assigned to either group.
Microhemodynamic parameters. Detailed mappings were made of the chamber vasculature so that the same vessels studied in control could be followed throughout the experiment. Functional capillaries, defined as capillary segments with a RBC transit of at least a single RBC in a 30-s period (14), were assessed in 10 successive microscopic fields, totaling a region of 0.46 mm2. Observation was done systematically by displacing the microscopic field of view by a field width in 10 successive steps laterally to the observer. Each 200-µm-long displacement (when referred to the tissue) was viewed on the video monitor. The first field was chosen by a distinctive anatomic landmark (i.e., large vascular bifurcation) to easily and quickly reestablish the same fields and vessels at each phase of the experiments. Each field had between two and five capillary segments with RBC flow. Functional capillary density (FCD; in cm–1, total length of RBC-perfused capillaries divided by the area of the microscopic field of view) was evaluated by measuring and adding the length of capillaries with RBC transit in the field of view. Relative changes in FCD from baseline levels after each intervention are indicative of the extent of capillary perfusion (14).
Arteriolar and venular blood flow velocity were measured online using the photodiode/cross-correlator system (model 102B, Fiber-Optic Photo Diode Pickup and Velocity Tracker, Vista Electronics; San Diego, CA) (10). The measured centerline velocity was corrected according to vessel size to obtain the mean RBC velocity (V) (16). Vessel diameter (D) was measured online (model 908, Digital Video Image Shearing Monitor, Vista Electronics) (11). Blood flow rate (Q), equal to V times the cross-sectional area of the vessel, was calculated according to Q = V x 
(D/2)2. Wall shear stress (WSS) is defined by the following formula:
 | (1) |
is the plasma viscosity (27).
Intravascular and extravascular PO2 measurements.
PO2 measurements were made using palladium-porphyrin phosphorescence quenching microscopy (21) based on the relationship between the decay rate of excited palladium-mesotetra(4-carboxyphenyl)porphyrin (Porphyrin Products; Logan, UT) bound to albumin and the PO2 according to the Stern-Volmer equation (35). Animals received a slow intravenous injection of 15 mg/kg body wt (10.1 mg/ml) of the phosphorescence dye 
10 min before PO2 measurements, which provided for an adequate signal-to-noise ratio for interstitial PO2 measurements (21). Simultaneous tissue PO2 measurements using this system and the microelectrode technique have shown nearly identical values (2). In our system (21), intravascular measurements were made by placing a rectangular optical window within the vessel of interest, the longest side of the rectangle positioned parallel to the vessel wall. Intravascular PO2 measurements were made in large feeding arterioles, smaller arcading arterioles, large venules, and smaller collecting venules. Tissue PO2 measurements were made in interstitial regions void of large vessels with a 10 x 10-µm optical window to estimate the lowest oxygen level within the chamber (24). The decay curves were analyzed offline using a standard single-exponential least-squares numerical fitting technique. Resultant time constants were applied to the Stern-Volmer equation to calculate PO2, where the quenching constant and the phosphorescence lifetime in the absence of oxygen measured are 325 mmHg/s and 600 µs, respectively. The phosphorescence decay due to quenching at a specific PO2 yields a single decay constant, and in vitro calibration is valid for in vivo measurements (13).
Acute isovolemic hemodilution and experimental groups. Progressive hemodilution to a final systemic Hct level of 25% of baseline was accomplished with three isovolemic exchange steps according to previously described protocol (22). Briefly, the volume of each exchange-transfusion step was calculated as a percentage of blood volume, estimated as 7% of body weight. An acute anemic state was induced by lowering the systemic Hct by 60% with two steps (exchange levels 1 and 2) of progressive isovolemic hemodilution using the colloid solution [10% 200-kDa hydroxyethyl starch (HES 200)]. Level 1 exchange was 40% of blood volume, and level 2 and 3 exchanges were each 35% of blood volume.
After level 2 exchange, animals were randomly divided into three experimental groups by sorting on a set of random numbers produced in a random ordering scheme (1), and the level 3 exchange was performed: 1) control group, with colloid solution to Hct of 25% of baseline; and 2) perfluorocarbon (PFC) group, with PCF emulsion.
One hour after level 3 exchange (in both groups), the animal's fraction of inspired oxygen (FIO2) was increased from normal (21%) to 100%. At each level of dilution and after FIO2 was changed, the animal was given a 10-min recovery period before data acquisition. Details of fluid administration and oxygen inspired levels are shown in Fig. 1.
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Fields of observations and vessels were chosen for study at locations in the tissue where vessels were in sharp focus. The same fields and vessels were used throughout the experiment, allowing for direct comparisons with baseline levels.
In the experimental exchange groups, successive hemodilution was performed after baseline systemic, microvascular, and hemodynamic characterization. After each exchange and ensuing stabilization period, measurements were performed following the schedule shown in Fig. 1, where exchanges began every hour. Second exchange was begun exactly 1 h after the first exchange. Blood samples were withdrawn from level 3 exchange animals at the end of the experiment for subsequent analysis of viscosity and colloid osmotic pressure. The duration of the experiments was 5 h.
Fluorocrit measurement. Fluorocrit was assessed in the same capillaries used for Hct measurements by centrifugation (Readacrit Centrifuge, Clay Adams) and is given as a percentage, representing the length of PCF emulsion (indicated by the white color) in the centrifuged capillary tube relative to the entire length of the centrifuged whole blood (consisting of subsequent layers of plasma, cells, and PCF emulsion) (7).
Tissue oxygen supply from microvascular data.
Detailed analysis of tissue oxygen supply was made by calculating oxygen consumption using Eq. 2 when PFCs are not present and Eq. 3 when they are
 | (2) |
 | (3) |
is the oxygen-carrying capacity of Hb at 100% saturation or 1.34 ml O2/g Hb, SA% is the arteriolar oxygen saturation of RBCs, (1 – Hct) is the fractional plasma volume and converts the equation from per deciliter of plasma to per deciliter of blood, (1 – Hct – Fct) is the fractional plasma volume and converts the equation from per deciliter of plasma to per deciliter of blood when PFCs are present, 
is the solubility of oxygen in plasma and equal to 3.14 x 10–3 ml O2·dl plasma–1·mmHg–1, pfc is the solubility of oxygen in Oxycyte and equal to 1.7 x 10–2 ml O2·dl plasma–1·mmHg–1, and Q is microvascular flow for each microvessel. The oxygen dissociation curve for hamster RBCs was determined from freshly collected blood (31). Data analysis. Results are presented as means ± SD. Data within each group were analyzed using ANOVA for repeated measurements. When appropriate, post hoc analyses were performed with the Tukey multiple-comparison test. For repeated measurements, time-related changes were assessed by ANOVA (SigmaStat 3.0 for Windows 2000, SPSS). Changes were considered statistically significant if P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Systemic and blood gas parameters. Systemic and blood gas parameters for each experimental group are presented in Tables 2 and 3. There were no statistically significant differences between experimental groups before the exchange protocol. Systemic Hcts after level 1 and 2 exchange were 29.4 ± 1.7% (28.8 ± 1.0% in control and 30.0 ± 2.2% with PFC) and 19.1 ± 1.1% (18.9 ± 0.9% in control and 19.4 ± 1.4% with PFC, P < 0.05 relative to baseline), respectively. Level 3 control and PFC exchange reduced systemic Hct to 11.1 ± 0.6% and 10.6 ± 0.5%, respectively, which were significantly reduced from baseline (P < 0.05) but not statistically different from each other. There was no statistically significant difference in Hct or Hb 1 h after level 3 exchange was achieved and after FIO2 was increased to 100%.
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Systemic arterial blood gas analysis showed (see Table 3) a statistically significant (P < 0.05) rise in PO2 from baseline with an increasing degree of hemodilution in levels 2 and 3 [76 ± 1 and 89 ± 2 mmHg for the control group and 79 ± 12 and 101 ± 12 mmHg for the PFC group, significantly higher than baseline (P < 0.05)]. Increasing FIO2 significantly increased PO2 [412 ± 12 mmHg in control and 594 ± 18 mmHg with PFC (P < 0.05 relative to baseline and level 3 exchange)].
PCO2 was not significantly changed relative to baseline by the hemodilution protocols, whereas the increase of FIO2 showed a significant rise of PCO2 in the control group compared with level 3 exchange of the same group (48 ± 3 mmHg, level 3 FIO2 100%, control, P < 0.05).
Blood pH was not statistically changed from baseline among all experimental groups except in the control group, which showed a statistically significant decrease when the FIO2 was increased during level 3 exchange (7.340 ± 0.021, level 3 FIO2 100%, control, P < 0.05).
Blood BE was significantly decreased after level 3 hemodilution in both groups compared with baseline (–2.0 ± 2.7 mmol/l in control and –3.0 ± 1.5 mmol/l with PFC, P < 0.05). Increasing FIO2 significantly increased BE in the PFC group to 5.5 ± 2.5 mmol/l (P < 0.05).
Cardiac output. Eight animals (n = 8, 67.7 ± 5.5 g) entered the CO protocol and tolerated the entire procedure without visible signs of discomfort. The animals were assigned randomly to the following experimental groups: control (n = 4, 68.1 ± 8.9 g) and PFC (n = 4, 66.8 ± 7.4 g). CO increased after level 1 and 2 exchange in both groups, 123 ± 6% (22.1 ± 1.9 ml/min, control level 1, P < 0.05) and 159 ± 10% (28.6 ± 1.8 ml/min, control level 1, P < 0.05) for the control group and 151 ± 8% (24.7 ± 1.7 ml/min, control level 1, P < 0.05) and 169 ± 16% (27.5 ± 1.8 ml/min, control level 1, P < 0.05) for the PFC group, respectively (Fig. 2C). CO decreased significantly after level 3 exchange in the control group relative to baseline, 89 ± 4% (16.2 ± 1.9 ml/min in control, P < 0.05).
Physical properties of blood. Table 4 compares rheological properties and the colloid osmotic pressure of blood after level 3 exchange. Blood and plasma viscosities were significantly different from baseline in the control and PFC groups (P < 0.05). Blood viscosity was reduced to 2.21 ± 0.34 and 2.67 ± 0.41 cP, and plasma viscosity was increased to 1.29 ± 0.21 and 1.49 ± 0.27 cP, respectively. Colloid osmotic pressure was statistically unchanged, indicating that the control group might be hypervolemic relative to the PFB group, because according to calculations this group received a total 0.52 g of HES 200 versus 0.38 g for the PFB group. However, a major shift of blood volume was not evidenced by the change of Hct, a relatively good indicator of volume shifts in these conditions of extreme hemodilution, which was virtually identical for the two groups.
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RBC velocity and blood flow. Figure 3 shows the change in RBC velocity in arterioles (B) and venules (E) as a function of the hemodilution level. Arteriolar velocity increased after level 1 exchange to 139 ± 8% for the control group (6.5 ± 2.7 mm/s, P < 0.05) and 147 ± 14% for the PFC group (6.9 ± 1.5 mm/s, P < 0.05) of baseline, respectively. Venular RBC velocity also increased in both groups after level 1 exchange to 142 ± 9% in the control group (1.6 ± 0.8 mm/s, P < 0.05) and 130 ± 13% for the PFC group (1.8 ± 0.7 mm/s, P < 0.05) of baseline in the PFC group.
After level 2 exchange, arteriolar RBC velocity remained elevated relative to baseline for both the control and PFC groups [130 ± 8% for the control group (6.1 ± 2.7 mm/s, P < 0.05) and 145 ± 13% for the PFC group (6.8 ± 1.7 mm/s, P < 0.05), respectively]. Conversely, venular RBC velocity returned to baseline levels. Level 3 exchange reduced the arteriolar RBC velocity versus baseline in both groups, being 66 ± 7% for the control group (3.1 ± 1.3 mm/s, P < 0.05) and 76 ± 15% for the PFC group (3.6 ± 1.0 mm/s, P < 0.05), respectively. Venular RBC velocity was significantly decreased relative to baseline in the control group [69 ± 8% (0.8 ± 0.5 mm/s, P < 0.05)].
Increasing FIO2 reduced arteriolar RBC velocity relative to baseline in both groups [69 ± 5% for the control group (3.2 ± 1.4 mm/s, P < 0.05) and 85 ± 15% for the PFC group (4.0 ± 1.3 mm/s, P < 0.05), respectively]. Venular RBC velocity was significantly increased compared with baseline in the PFC group [112 ± 20% (1.6 ± 0.7 mm/s with PFC, P < 0.05)].
Arteriolar and venular blood flows after the hemodilution protocols are presented in Fig. 3. The results are given as means ± SE to show the trend of this parameter calculated from vessel diameter and RBC velocity. Arteriolar blood flow (Fig. 3C) was statistically increased from baseline after level 1 and 2 exchange to 141 ± 21% (24.6 ± 17.4 µl/s, control level 1, P < 0.05) and 147 ± 27% (24.2 ± 15.7 µl/s, control level 2, P < 0.05) in the control group and 146 ± 30% (28.9 ± 12.1 µl/s, PFC level 1, P < 0.05) and 157 ± 39% (31.0 ± 13.5 µl/s, PFC level 2, P < 0.05) in the PFC group. Venular blood flow (Fig. 3F) was statistically decreased from baseline after level 2 exchange to 77 ± 25% (2.3 ± 1.5 µl/s, P < 0.05) in the control group and 78 ± 19% (3.0 ± 1.5 µl/s, P < 0.05) in the PFC group. After level 3 exchange, control and PFC groups decreased arteriolar blood flow from baseline to 57 ± 10% (9.6 ± 6.4 µl/s in control, P < 0.05) and 67 ± 23% (12.9 ± 6.0 µl/s with PFC, P < 0.05), respectively. Venular blood flow was significantly decreased from baseline after level 3 exchange and after FIO2 was increased in the control group to 71 ± 16% (2.1 ± 1.5 µl/s, level 3, P < 0.05) and 71 ± 19% (2.0 ± 1.3 µl/s, FIO2 100%, P < 0.05), respectively.
WSS. WSS was calculated for each vessel studied after level 3 exchange in control and PFC groups and when FIO2 was increased (see Fig. 4). The calculated values of WSS are presented as means ± SE to show the trend rather than the distribution of the parameter. Before the exchange protocol, there were no statistically significant differences in WSS among the arterioles (control: 7.30 ± 0.86 dyn/cm2; PFC: 6.39 ± 0.42 dyn/cm2) and venules (control: 2.01 ± 0.29 dyn/cm2; PFC: 2.35 ± 0.15 dyn/cm2). After the level 3 exchange, the control group arterial WSS decreased to 77 ± 3% (5.63 ± 0.66 dyn/cm2, P < 0.05) of baseline, and increasing FIO2 did not change the arterial WSS.
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There was no significant difference in arteriolar WSS between control and PFC groups after level 3 exchange; however, the level 3 PFC group had a significant increase in WSS relative to the control group in venules (P < 0.05), and the increase of FIO2 caused a significant difference in arteriolar and venular WSSs (P < 0.05).
Functional capillary density. Figure 5 shows the effect of hemodilution on the length of RBC-perfused capillaries per unit area. FCD after level 1 exchange was reduced relative to baseline to 92 ± 5% (P < 0.05) in the control group and 93 ± 3% in the PFC group (not statistically different from baseline). Level 2 exchange reduced FCD to 84 ± 4% of baseline (P < 0.05) for the control group and 88 ± 3% of baseline (P < 0.05) for the PFC group. FCD was further reduced after level 3 exchange in control and PFC groups to 66 ± 4% and 63 ± 10% of baseline, respectively (P < 0.05). FCD was significantly lower than baseline after the increase of FIO2, FCD being 66 ± 5% and 66 ± 7% of baseline in control and PFC groups, respectively (P < 0.05). No statistical difference was found as a consequence of increase of FIO2 between groups.
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Microvascular oxygen distribution. The PO2 distribution in arterioles, venules, and tissue is shown in Fig. 6. Arteriolar PO2 was not significantly different between groups after the level 3 exchange, being 19.5 ± 4.6 mmHg for the control group and 16.5 ± 4.2 mmHg for the PFC group. Venules had a significantly higher PO2 in the control group (13.3 ± 3.5 mmHg, P < 0.05) than in the PFC group (7.7 ± 2.5 mmHg). After the increase of FIO2, the arterioles of the PFC group had a significant increase in PO2 (42.7 ± 9.7 mmHg, P < 0.05) compared with the control group. Venules did not change significantly in either group, but the difference between the control (14.1 ± 2.5 mmHg, P < 0.05) and PFC groups (7.8 ± 1.4 mmHg) was significant with 100% FIO2.
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Oxygen delivery. Oxygen delivery was measured directly by combining systemic data of CO, carotid artery PO2, and blood composition (Eqs. 2 and 3). This parameter was significantly reduced after level 1 exchange relative to baseline in the control group to 75 ± 4% (2.4 ± 0.5 ml O2/min in control, P < 0.05). Level 2 and 3 exchange oxygen delivery was also reduced significantly relative to baseline in the control and PFC groups to 64 ± 2% (2.0 ± 0.1 ml O2/min in control, P < 0.05) and 24 ± 1% (0.8 ± 0.1 ml O2/min in control, P < 0.05) and 73 ± 16% (2.0 ± 0.2 ml O2/min with PFC, P < 0.05) and 26 ± 5% (0.7 ± 0.1 ml O2/min with PFC, P < 0.05), respectively. Increasing FIO2 significantly lowered oxygen delivery compared with baseline in both groups to 32 ± 1% (1.0 ± 0.1 ml O2/min in control, P < 0.05) in the control group and 51 ± 16% (1.4 ± 0.2 ml O2/min with PFC, P < 0.05) in the PFC group, respectively (Fig. 7).
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Tissue oxygen supply. The supply of oxygen to the tissue was measured after level 3 exchange in both groups and after the increase of FIO2, as shown in Fig. 8 in terms of means ± SE. The PFC group showed a statistically significant (P < 0.05) rise in tissue oxygen supply after the increase of FIO2 compared with the same group at level 3 exchange, from 1.0 ± 0.2 x 10–7 to 4.5 ± 0.7 x 10–7 ml O2/s. During level 3 hemodilution with HES 200, the calculated tissue oxygen consumption was 6.1 x 10–8 ml O2/s and with the increase of FIO2 it was 6.6 x 10–8 ml O2/s, unlike in the PFC group, which changed from 6.7 x 10–8 to 42.6 x 10–8 ml O2/s when FIO2 was increased.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The present results also show that there is no significant difference between animals with and without PFC in the circulation, in terms of both systemic and microvascular parameters, in extreme hemodilution when breathing normobaric air. Thus it is appropriate to conclude that the extra oxygen delivery observed in the microcirculation during 100% oxygen inspiration is due to the presence of the PFCs in the circulation.
Our investigations were carried out using an extreme hemodilution protocol already tested in previous studies aimed at determining the efficacy of oxygen-carrying plasma expanders [Tsai et al. (30)]. In our protocol, Hct was reduced to 11% (Hb 3.5 g/dl), which is a value close to the critical Hct for this species, i.e., the Hct (or Hb concentration) beyond which further hemodilution places the animal in an oxygen supply-dependent condition. At this level of hemodilution, in our experiments, the acid-base balance was negative and remained negative in control animals that were hemodiluted only with HES 200, even when they inspired 100% oxygen. Conversely, the acid-base balance returned to normal values in the animals treated with PFC breathing 100% oxygen. Furthermore, determinations of systemic oxygen delivery show that fluorocarbon caused a significant increase in the amount of oxygen transported and consumed (20) compared with the minimal increase due to the oxygen carried by plasma in the HES 200-treated group during 100% oxygen inspiration.
The effect of fluorocarbons on oxygen transport at 100% oxygen inspiration is also evident in the differences in CO because control (no PFC) animals did not increase CO upon inspiring 100% oxygen, whereas animals treated with PFC increased CO by 50% with the increased FIO2. Considering that a similar increase in oxygen delivery was found using microvascular data from the window chamber, it is plausible to assume that these microvascular observations may reflect changes occurring in other tissues.
This study shows that infusion of a 4.2 g/kg dose of PFC emulsion combined with 100% oxygen ventilation is well tolerated in all animals and has no negative effects on microvascular perfusion, a result also reported by Nolte et al. (17). The increased oxygen delivery in the microcirculation found in this study is due to the increased arteriolar PO2. Venular PO2 did not change, causing the arteriolar-venous difference across the microcirculation to increase. These two events, coupled with the maintenance of blood flow in the microcirculation and the increased oxygen-carrying capacity, caused oxygen delivery to increase substantially, and thus in this tissue and microvascular network the presence of fluorocarbon increases arteriolar PO2 at 100% FIO2, increasing the amount of oxygen delivered by RBCs.
Exposure to hyperoxia has been shown to cause vasoconstriction (23); however, there was no evidence for this effect in our investigations. This may be in part due to the high level of hemodilution to which the animals were subjected before breathing 100% oxygen. The negative acid-base balance suggests that the animals were borderline hypoxic, which is a stimulus for dilatation. Notably, the combination of circulating PFC and 100% inspired oxygen inspiration did restore the acid-base balance, suggesting that hypoxia was reversed and that hyperoxic inspiration did not cause vasoconstriction. As shown in several studies recently reviewed by Tsai et al. (29), there is a continous exit of oxygen from both the circulation and microcirculation driven by the oxygen gradient between blood and tissue. In normal conditions for this species, this oxygen efflux determines that arteriolar PO2 is in the range of 50 mmHg. In hemodilution, the initial arterial oxygen gradients are the same, and thus the magnitude of the efflux of oxygen, which solely depends on the gradient, is the same. However, the progressive depletion of oxygen from blood lowers its saturation, and therefore blood arriving to the microcirculation in extreme hemodilution has a significantly lower PO2.
The presence of PFC at 100% FIO2 significantly increases the oxygen transported from the lungs to the major blood vessels. The intrinsically higher blood tissue oxygen gradient in these conditions could increase premicrocirculatory oxygen exit; however, this efflux is not sufficient to deplete the oxygen in blood, and in the present experiments arteriolar blood attains an approximately normal PO2. We found that both the control and PFC groups reached the same pre-100% FIO2 inhalation venular PO2, suggesting that oxygen extraction in the PFC group is enhanced, an effect that may be due to the lowering of the diffusional resistance in the plasma layer by the presence of PFC particles, thus enhancing oxygen-unloading capacity.
Tissue oxygen supply with PFC was 0.7 x 10–7 and 4.2 x 10–7 ml O2/s for normoxia and hyperoxia, respectively, and was significantly lower in both groups compared with baseline on an identical animal preparation (23), where it was 33.7 x 10–7 ml O2/s for normoxia and 34.1 x 10–7 ml O2/s for hyperoxia.
A critical parameter that is a determinant of microvascular function is FCD, which was not different between control and PFC-infused animals nor between normoxic and hyperoxic ventilation. The drop of FCD in extreme hemodilution in both hypoxia and hyperoxic conditions is probably a consequence of the low blood viscosity, as proposed by Tsai and Intaglietta (27), who indicated that there is a need for a threshold capillary pressure to maintain capillaries sufficiently distended to allow for the passage of RBCs. However, in all conditions, FCD ranged to 60–65% of control, a value that is low, but not pathological, according to the findings of Kerger et al. (14) and Tsai et al. (26), who determined that microvascular function is compromised when FCD drops below 40% of control.
Fluorocarbons have been tested for their ability to transport oxygen in various experimental conditions and even clinical trials, and analysis of their effectiveness at the cellular level was usually based on determining whether tissue PO2 is improved by the presence of PFCs in the circulation and 100% FIO2 (7). In our study, tissue PO2 increased significantly from a statistical view point; however, the increase is in all probability not physiologically important for tissue oxygenation. The low values found in our experiments, even with 100% FIO2, may be a characteristic of the tissue studied and that we could measure tissue PO2 in areas between microvessels. In this situation, measurements are not affected by the diffusion field from arterioles, an effect likely to be present when using larger electrodes that prevent identifying the location of measurement.
The lack of evidence of vasoconstriction with PFC perfusion and 100% FIO2 may in part be due to the increased blood and plasma viscosity caused by PFC relative to that attained with HES 200 hemodilution. Increased plasma viscosity in hemodilution produces a vasodilatory stimulus and the enhancement of microvascular function (26), an effect that may be due to the increased production of vasodilators such as nitric oxide and prostacyclin by the endothelium (6).
In conclusion, this study shows that the presence of a 4.2 g/kg dose of PFC emulsion and ventilation with 100% oxygen in animals that have undergone extreme hemodilution to a Hct of 11% reverses the consequences of anoxia present when PFC is not present in the circulation. Our findings demonstrate that PFCs are able to deliver oxygen to the tissue when combined with hyperoxic ventilation. Furthermore, in the extreme hemodilution protocol followed in this study, hyperoxic ventilation and PFC administration gave no evidence of impaired microvascular function. Our results show that PFCs and hyperoxia restore microvascular and systemic functionality in extreme hemodilution, a finding that may not necessarily be obtained with lesser degrees of Hct reductions, which were not explored in this study. However, systemic and microvascular improvements found in the conditions of the present experiments exceed those attainable with hyperoxia and conventional plasma expanders.
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Significantly different from baseline (BL) (P < 0.05); 
significantly different with change in FIO2 (P < 0.05); 
significantly different between hemodilution fluids (P < 0.05).
