Intestinal and Cerebral Oxygenation during Severe Isovolemic Hemodilution and Subsequent Hyperoxic Ventilation in a Pig Model

The protocol of the present study was approved by the Animal Research Committee of the Academic Medical Center at the University of Amsterdam. Animal care and handling were performed in accordance with the national guidelines for care of laboratory animals. The experiments were performed in 15 crossbred Landrace x Yorkshire pigs, 10–12 weeks old, with a mean (± SD) body weight of 25 ± 2 kg.

After an overnight fast with free access to water, the animals were sedated with an intramuscular injection of ketamine (20 mg/kg), midazolam (1 mg/kg), and atropine (0.5 mg). Anesthesia was induced with thiopental (5 mg/kg intravenously) and was maintained by intravenous infusion of midazolam (0.2 mg/kg bolus, followed by 0.2 mg · kg⁻¹· h⁻¹) and fentanyl (20 μg/kg bolus, followed by 10 μg · kg⁻¹· h⁻¹). Muscle relaxation was obtained with pancuronium bromide (0.1 mg/kg bolus, followed by 0.1 mg · kg⁻¹· h^{− 1}).

Identical anesthetic protocols have proven to be adequate for similar acute experiments in pigs. 8,14,17,18One separate time-matched sham-operated animal was anesthetized without the use of muscle relaxants to ensure that the anesthetic regimen was adequate. After tracheal intubation, ventilation was performed (AV-1; Drägerwerke, Lübeck, Germany) with oxygen in air (Fio², 0.33), maintaining normocapnia. As maintenance fluid, a crystalloid solution (Ringer's lactate) was administered (15 ml · kg⁻¹· h⁻¹). Central body temperature was maintained at approximately 37°C with a heating pad and isolation blankets.

Catheters were placed in the right brachial artery (6 French) for the measurement of arterial blood pressure and collection of arterial blood samples, the right brachial vein (14 gauge) for the administration of fluids, and the left femoral artery (8 French) for blood withdrawal during exchange transfusion. For the collection of jugular venous blood samples, a catheter (4 French) was inserted cranially into the right internal jugular vein in such a way that the tip of the catheter reached at least to the base of the skull. In this way, the internal jugular venous oxygen measurements were assumed to reflect the venous outflow of the brain in the jugular bulb, with a minimum of extracranial contamination. 19A pulmonary artery thermodilution catheter (Edwards 7 French; Baxter Healthcare Corp., Round Lake, IL) was positioned in the pulmonary artery via  an introducer in the left femoral vein for the measurement of cardiac output, right atrial pressure (RAP), pulmonary artery pressure, pulmonary capillary wedge pressure, central body temperature, and collection of mixed venous blood samples.

Following identification of the right carotid bifurcation, the external carotid artery was ligated immediately distal from the bifurcation, and an ultrasonic flow probe (3.0 mm; Transonic Systems Inc., Ithaca, NY) was placed around the right common carotid artery. If there was any artery branching from the internal carotid artery between the flow probe and the base of the skull (for instance, the occipital artery), this vessel was ligated as well.

Following midline laparotomy, an ultrasonic flow probe (4.0 mm; Transonic Systems Inc.) was placed around the superior mesenteric artery for the measurement of blood flow to the splanchnic region. After location of the terminal ileum, a mesenteric vein related to the ileum was cannulated (6 French) for the collection of mesenteric venous blood samples. An antimesenteric incision was made to expose the intestinal mucosa. The urinary bladder was cannulated to prevent distension of the bladder wall and to monitor urinary production.

A 5 × 5-cm skin flap was removed from the right side of the skull. A circular piece of bone (approximately 2 cm in diameter) was removed, exposing the dura mater of the brain. The dura was opened carefully until the cortical surface of the right hemisphere was clearly visible. Continuous irrigation with warmed saline prevented the exposed tissues from desiccation.

Systolic and diastolic arterial blood pressures (millimeters of mercury), heart rate (beats per minute), RAP (millimeters of mercury), systolic and diastolic pulmonary pressures (millimeters of mercury), and pulmonary capillary wedge pressure (millimeters of mercury) were monitored. Cardiac output was measured by thermodilution and a cardiac output computer (Vigilance; Baxter Edwards Critical Care, Round Lake, IL). The average of 3 consecutive bolus injections of 5 ml of room-temperature saline was considered representative for the cardiac output at each measurement point. Blood flows in the internal carotid artery (Q̇^ICA) and superior mesenteric artery (Q̇^SMA) were measured continuously (Flow meter T206; Transonic Systems Inc.). By distal occlusion of the vessels, zero flow values were obtained, which were compared with the values measured at the end of the experiment after the animal had been killed. Hemodynamic values were indexed according to body weight. Systemic vascular resistance index, mesenteric vascular resistance index, and internal carotid vascular resistance index (all millimeters of mercury per milliliter per minute per kilogram) were calculated as follows: (MAP − RAP)/CI, (MAP − RAP)/Q̇^SMA, and (MAP − RAP)/Q̇^ICA, respectively, where MAP = mean arterial pressure.

At each measurement point, simultaneously an arterial sample was taken from the brachial artery, a mixed venous sample from the pulmonary artery catheter, a mesenteric venous sample from the mesenteric venous catheter, and a jugular venous sample from the internal jugular venous catheter. The samples were used to determine blood gas values (ABL505; Radiometer, Copenhagen, Denmark), as well as hematocrit, hemoglobin concentration, and hemoglobin oxygen saturation (So²) (OSM 3; Radiometer), corrected for species.

Systemic Do2 (Do^2SYS; milliliters per minute per kilogram body weight) was calculated as:

where CI is expressed as milliliters per minute per kilogram, and arterial oxygen content is expressed as milliliters oxygen per milliliter blood, which was calculated as:

where Pao²is arterial oxygen partial pressure and Sao²is arterial oxygen saturation. Systemic oxygen consumption (V̇o^2SYS; milliliters per minute per kilogram bodyweight) was calculated as:

The systemic O²ER (O²ER^SYS; percent) was calculated as:

In a similar way, the intestinal Do²(DO^2SMA), V̇o²(V̇o^2SMA), and O²ER (O²ER^SMA) were calculated. Measurement of the internal jugular venous oxygen content allowed the calculation of the arteriovenous oxygen content difference, and thereby the O²ER of the brain 20–22(O²ER^ICA) was calculated as:

The μPo²was measured in the cerebral cortex and the serosa and mucosa of the ileum, using the oxygen-dependent quenching of Pd-porphyrin phosphorescence. Excitation of Pd-porphyrin by a pulse of light causes emission of phosphorescence with a decay in time, which is quantitatively related to the oxygen concentration. 23–25Pd-meso-tetra(4 carboxy-phenyl)porphine (Porphyrin Products, Logan, UT) is coupled to human serum albumin to form a large molecular complex that, when injected intravenously, is confined mainly to the vascular compartment. 25,26Fifty milliliters of a 4-mm Pd-porphyrin solution was administered, corresponding with a dosage of 12 mg/kg bodyweight. The μ Po²measurements were made with optical fibers for the transmission of excitation and emission light, attached to a phosphorimeter. To determine which microvascular compartment is measured by fiber phosphorimetry, the Pd-porphyrin phosphorescence fiber technique has been compared with a microscopic phosphorimeter: simultaneous Po²measurements with the fiberoptic technique showed excellent correlation with microscopically measured Po²in capillaries and first-order venules, but not with arteriolar or venous Po²values, at different Fio²levels. 27Thus, this result allowed us to term the fiberoptic measurement of Po²as the measurement of μPo². Fiberoptic measurements of μPo²incorporate blood vessels over an area of approximately 1 cm²with a penetration depth of approximately 0.5 mm. 17,25As the calibration constants in the calculation of the μPo²from the phosphorescence decay time are temperature dependent, intestinal surface temperature measurements were used for correction of these constants. In the present study, a multifiber phosphorimeter was used, with three separately operated optical fibers. The fibers were placed near the surfaces of the cerebral cortex and the serosa and mucosa of the ileum. In this way, the μ Po²was measured in three different regions simultaneously.

After preparation and a stabilization period of at least 30 min, two baseline measurements were made during a 1-h period. The animals were randomized between a hemodilution group (n = 10) and a time-matched control group (n = 5). Stepwise isovolemic hemodilution was accomplished by withdrawal of blood from the femoral artery and simultaneous administration of an equal volume of HAES-steril 6% (6% hydroxyethylstarch, degree of substitution 0.5, in 0.9% NaCl solution, Mw 200,000; Fresenius, Germany) through the femoral vein at the same rate. Four dilution steps were made: three steps of 20 ml/kg bodyweight and a final step of 30 ml/kg, resulting in a total volume exchange of 90 ml/kg. Twenty minutes after each hemodilution step, all hemodynamic, blood gas, and μPo²measurements were repeated. Lactate measurements were performed at baseline and after a total volume exchange of 40 and 90 ml/kg. Following the measurements after the final hemodilution step, Fio²was increased to 1.0, and after 20 min stabilization, all measurements were repeated. In the control group, identical measurements were made at corresponding time intervals, but no hemodilution was performed. In the final stage of the experiment, the Fio²was increased to 1.0. All experiments were terminated by administration of 30 mmol of potassium chloride.

Values are reported as mean ± SD. Because the consecutive baseline measurements were not significantly different for any parameter, they were averaged and presented as a single data point. Intragroup differences were analyzed using analysis of variance for repeated measurements. When appropriate, post hoc  analyses were performed with the Student-Newman-Keuls test. Intergroup differences for each measurement point were analyzed with the unpaired t  test; Bonferroni correction for multiple comparisons was used. P  values < 0.05 were considered significant. A critical level of hemodilution was determined for the whole body V̇o²and the μPo²of the intestinal serosa and mucosa and the cerebral cortex. From plots of hematocrit against V̇o^2SYSand μ Po², it was possible to determine the points at which V̇o²or μPo²became dependent on the hematocrit with further hemodilution. These points were determined for each animal separately by the intersection of the two best-fit regression lines, as determined by a least sum of squares technique.

Because of the type of measurements, muscle relaxation was necessary throughout the experimental protocol. To ensure that the anesthetic regimen was sound, one separate time-matched sham-operated animal was anesthetized without the use of muscle relaxants. Throughout the experiment, the animal did not try to escape or move purposefully. Only slight shivering was observed 150 min after start of anesthesia. All hemodynamic parameters remained stable as in all control group animals. All hemodynamics of the study animals are summarized in table 1.

Baseline measurements in the hemodilution and the control groups were not significantly different, except for the heart rate, which was lower in the control group. In the control animals, all systemic hemodynamic parameters remained constant throughout the experiment. In the hemodilution group, the decrease in hematocrit, from 25.3 ± 3.0 at baseline to 7.6 ± 1.2% after exchange of 90 ml/kg, was accompanied by an increase in cardiac index (CI;fig. 1). Heart rate increased significantly, whereas stroke volume did not change from baseline values. MAP remained constant during hemodilution, although the systemic vascular resistance index decreased significantly compared with baseline and control group values. The pulmonary artery pressure, vascular resistance index, and pulmonary capillary wedge pressure did not change significantly during hemodilution.

Hyperoxia decreased the heart rate to the range of baseline values, resulting in a slight but not significant decrease in CI. In addition, the pulmonary capillary wedge pressure and the systemic vascular resistance index increased after hyperoxia.

Data are summarized in table 2and figure 1. Despite the increase in CI, Do^2SYSdecreased with the onset of hemodilution. The O²ER^SYSincreased, and as a result the V̇o^2SYSwas preserved through a hematocrit of 9.9% but decreased significantly after the final hemodilution step at a hematocrit of 7.6%. Consequently, the mixed venous Po²and So²decreased with progressive hemodilution.

Following the final dilution step, hyperoxia significantly increased the arterial oxygen content in the hemodilution group from 3.4 ± 0.3 to 4.7 ± 0.5 ml/dl. This did not change Do^2SYS, despite the decrease in CI. The O²ER^SYSdecreased significantly following hyperoxia, and V̇o^2SYSwas not restored. Mixed venous Po²and So²increased above baseline values (P < 0.05). In the control group, the arterial oxygen content was increased by hyperoxia as well. However, this did not change the Do^2SYSand V̇o^2SYS, although mixed venous Po²and So²exceeded baseline values.

Although all animals underwent the same surgical and anesthetic protocols and were randomly assigned to the hemodilution or the control group, mesenteric blood flow (Q̇^SMA) in the control group was lower (fig. 2) compared with the hemodilution group. All control group parameters remained constant. Hemodilution did not change Q̇^SMA, resulting in a progressive decrease in Do^2SMA. After exchange of 60 ml/kg (corresponding hematocrit, 9.9%), V̇o^2SMAwas decreased from baseline, whereas O²ER^SMAincreased from 21 ± 7% at baseline to 46 ± 9% after the final hemodilution step (fig. 2).

Baseline values for the carotid blood flow (Q̇^ICA;fig. 3) and the corresponding vascular resistance (table 1) were comparable for both groups. Q̇^ICAincreased from 2.5 ± 0.5 ml · min⁻¹· kg⁻¹at baseline to 4.2 ± 0.6 ml · min⁻¹· kg^{− 1}after exchange of 90 ml/kg (fig. 3). Internal jugular venous Po²(fig. 4) and So²(table 3) decreased during hemodilution, reflecting the increase in O²ER^ICA(from 30 ± 6% at baseline to 47 ± 6% at 90-ml/kg exchange;fig. 3). Mesenteric venous Po²(fig. 4) and So²values (table 3) decreased in a similar way.

Hyperoxia did not influence Q̇^SMAafter hemodilution. The Do^2SMAincreased slightly but not significantly, and V̇o^2SMAdid not improve. Mesenteric venous Po²and So²increased substantially following hyperoxia; the Po²even exceeded baseline values. In the control group, however, the mesenteric vascular resistance index increased after hyperoxia and the Q̇^SMAdecreased, resulting in a decrease in Do^2SMAbut not in V̇o^2SMA. The change in O²ER^SMAwas not statistically significant, and the mesenteric venous Po²and So²did not significantly increase after hyperoxia in the control group.

Hyperoxia decreased the Q̇^ICAsignificantly in the hemodilution and the control groups. Po²and So²in the internal jugular vein in the hemodilution group exceeded baseline values, whereas they did not change in the control group.

Microvascular and regional venous Po²measurements are shown in figure 4. Baseline measurements were similar for the hemodilution and the control groups. The μPo²of the serosa (59 ± 7 mmHg at baseline) decreased at the second hemodilution step and fell to 21 ± 7 mmHg after the last step (hematocrit, 7.6 ± 1.2%). The mucosal μPo²(27 ± 4 mmHg at baseline) remained unaffected for a longer period of time and was decreased after the third hemodilution step (hematocrit, 9.9 ± 1.1%). The μ Po²of the cerebral cortex (26 ± 3 mmHg at baseline) demonstrated a similar behavior as the mucosal μPo²and was also decreased after the third hemodilution step. μPo²measurements in the control groups did not change in time.

In the hemodilution group, hyperoxia increased both the serosal and the mucosal μPo²; the mucosal μ Po²was even restored to baseline values. The cerebral cortex μPo²was also significantly increased but did not return to baseline values. Hyperoxia had no significant effect in the control group.

The critical hematocrit levels for the systemic V̇o²and intestinal and cerebral μPo²measurements are shown in figure 5. V̇o^2SYS(fig. 5A) started to decrease with hemodilution at an average hematocrit of 13.7 ± 3.5%. In the intestines, the critical hematocrit for the intestinal mucosa (fig. 5B) was 11.4 ± 2.6%, whereas the serosal μPo²(fig. 5C) started to decrease with hematocrit already at an average value of 16.9 ± 4.2. The μPo²in the cerebral cortex (fig. 5D) displayed a similar behavior as the intestinal mucosa and V̇o^2SYSand started to decrease with hematocrit at an average value of 12.1 ± 3.1%. The critical hematocrit values for the mucosal and cerebral μPo²and V̇o^2SYSwere not significantly different; compared with the serosal μPo², the critical hematocrit of the latter was found to be significantly higher.

Systemic and regional parameters are summarized in tables 2 and 3, respectively. Arterial carbon dioxide partial pressure did not change during the entire experiment in the hemodilution and the control groups. The systemic and regional venous p  H measurements did not change in the control group. In the hemodilution group, the arterial p  H decreased significantly after the final hemodilution step. The mixed venous p  H did not change, but the mesenteric venous p  H decreased after the second step and the jugular venous p  H after the third step. There was no significant change in systemic and regional lactate levels during hemodilution.

The main finding of this study is that the systemic V̇o², the cerebral μPo², and the intestinal mucosal μPo²became impaired at the same stage during hemodilution, whereas the intestinal serosal μ Po²became impaired at an earlier stage. The systemic response to the decreased arterial oxygen content consisted of an increase in CI and an increase in O²ER^SYS. At a regional level, the increased CI was redistributed in favor of other organ systems than the intestines, as Q̇^SMAremained constant. Despite the redistribution of blood flow, systemic as well as intestinal V̇o²became impaired by the diminished oxygen supply at the same level of hemodilution. Although systemic redistribution may have favored the oxygenation of, for instance, the brain, the intestines successfully compensated for the diminished Do²by a larger increase in the oxygen extraction from the blood: 130% increase for the intestinal versus  52% for the cerebral O²ER from baseline to the final hemodilution step. An increase in O²ER as the predominant mechanism for the preservation of intestinal V̇o²has been reported in previous studies as well. 1,12,28Finally, a level of hemodilution was reached below which all compensatory mechanisms became insufficient, resulting in a general critical level of hemodilution for the whole body, the intestinal mucosa, and the cerebral cortex.

Redistribution of a diminished oxygen supply in favor of organ systems with a lower oxygen extraction capacity can increase the efficiency of Do². 29However, the mechanisms behind the redistribution of blood flow during hemodilution are not clear. The results of the present study demonstrated the functional consequences of redistribution and only allow for speculations to be made regarding regulatory mechanisms. Possible mechanisms that could account for systemic or local redistribution could include increased sympathetic activity, 30–32although the level of circulating catecholamines does not increase during hemodilution. 8On the other hand, activity of nitric oxide might play an important role in the systemic and splanchnic response to hemodilution. 33–35An increase in cerebral blood flow during hemodilution has been attributed to decreases in arterial oxygen concentration and blood viscosity 36–38; increased nitric oxide activity should not be involved in this process. 39 

Although the V̇o^2SMAand the mucosal μPo²were preserved until a hematocrit of ±11.0%, the serosal μPo²started to decline at a hematocrit of 17.0% (P < 0.05). This finding implies that the mucosa contains the predominant oxygen-consuming part of the intestinal wall and is in agreement with the oxygen electrode measurements of Haisjackl et al.  14during hemodilution. Being the site for absorption and secretion in the gastrointestinal tract and the barrier to microbial invasion from the intestinal lumen, the gut mucosa can be expected to have a greater oxygen demand than the serosa. To preserve these important functions during conditions of diminished oxygen supply, it is not unlikely that during hemodilution the diminished intestinal oxygen supply was redistributed in favor of the mucosa. 14,35A redistribution of intraorgan blood flow during hemodilution has been shown to occur in the heart 8,40,41and the kidney. 8 

In the present study, systemic and regional oxygenation became impaired in the hematocrit range of 10–15%, which is in agreement with critical values in previous studies. 2,4,6In addition, the constant regional and systemic lactate concentrations in the present study point at adequate tissue oxygenation until this level of hemodilution, 42to which is contributed by the values of the regional intestinal carbon dioxide partial pressure. At the lowest hematocrit levels, only the decreased p  H values indicated that tissue oxygenation was impaired, although systemic or regional acidosis did not occur. Considering critical levels of hemodilution in general, it must be noted that anesthetized animals respond differently to hemodilution as compared with conscious animals. Although not supported by the results of the present study, in anesthetized animals cardiac output increased mainly because of an increase in stroke volume, whereas in conscious animals the increased cardiac output was attributable to an increment in heart rate. 43The influence of anesthesia is emphasized by studies in which a critical level of Do²could not be demonstrated during hemodilution in conscious animals and humans. 44–46 

The increase in Fio²did not improve systemic or intestinal V̇o²despite an increase in arterial oxygen content in the hemodilution and the control groups. Furthermore, it was found that only at low hematocrit levels did hyperoxia increase the regional venous Po²values far more than the μPo²values, indicating that in combination with severe hemodilution, the increased amount of physically dissolved oxygen was being shunted to the venous side of the organ vascular beds. It can be hypothesized that an interaction between the physiologic responses to hemodilution (e.g. , increased capillary density and blood flow) on the one hand and hyperoxia (reduced capillary density and blood flow 47) on the other hand resulted in at least a partial diversion of the dissolved oxygen away from the microcirculation.

In conclusion, similar critical levels of hemodilution were found for V̇o^2SYSand the cerebral and intestinal mucosal μPo², whereas the intestinal O²ER increased to a greater extent than the cerebral O²ER, indicating that during hemodilution the diminished oxygen supply was efficiently redistributed in favor of the organs with a lower capacity to increase oxygen extraction. The serosal μPo²decreased at an earlier stage than the mucosal μPo², suggesting that the decreased intestinal oxygen supply was redistributed within the intestinal wall during hemodilution. During oxygen supply dependency, hyperoxic ventilation did not improve systemic or regional V̇o². The increased regional venous Po²values in combination with the decreased O²ER indicate that the increased amount of physically dissolved oxygen was shunted to the venous side of the organ vascular beds during severe hemodilution.