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Am J Physiol Heart Circ Physiol 285: H2064-H2071, 2003; doi:10.1152/ajpheart.00349.2003
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Microcirculatory changes during chronic adaptation to hypoxia

Enrique Saldívar,1,2 Pedro Cabrales,1 Amy G. Tsai,1 and Marcos Intaglietta1,2

1Department of Bioengineering, University of California-San Diego, La Jolla 92093; and 2La Jolla Bioengineering Institute, La Jolla, California 92037

Submitted 14 April 2003 ; accepted in final form 21 July 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Microcirculatory changes in the window chamber preparation in Syrian golden hamsters, secondary to chronic hypoxia adaptation, are presented herein. Adaptation was attained by keeping animals in a 10% oxygen environment for 1 wk and 5% the following week. The following groups were studied: group 1, adapted to chronic hypoxia and kept in a 5% oxygen environment throughout the experiment; group 2, adapted to chronic hypoxia and kept in a 21% oxygen environment 24 h before and during the experiment; and group 3, control. Adaptation caused venule enlargement and hematocrit increase (68.6 ± 2.44 in group 1, 70 ± 2.66 in group 2, and 43.27 ± 2.30 in group 3; P < 0.05). Whereas heart rate decreased in adapted animals, blood pressure remained constant. Group 1 presented alkalosis, hypocapnia, and hypoxemia. The adapted groups had decreased blood flow velocity in arterioles and veins. We found no difference in microvasculature oxygen tension between groups 2 and 3; however, the number of capillaries with flow was markedly reduced in group 1 but significantly increased in group 2. Our findings suggest that, as an adaptation to hypoxia, erythropoiesis may prove beneficial by increasing blood viscosity and shear stress, leading to vasodilatation, in addition to the increase in oxygen-carrying capacity. Calculations show that oxygen extraction in the tissue of the window chamber model was significantly lowered in adapted animals breathing 5% oxygen, but was unchanged from the control when breathing 21% oxygen, even though blood hemoglobin content was increased from 14.5 ± 0.07 g/dl at control to 21.04 ± 1.24 g/dl in the adapted animals (P < 0.05).

microcirculation; adaptation; hematocrit

CHRONIC ADAPTATION TO HYPOXIA causes hemoconcentration, i.e., polycythemia, or an increase in systemic hematocrit (Hct) above normal values (1, 6). In clinical practice, this condition is found in patients with lung disease and in individuals exposed to high altitudes for extended periods (Monge's disease) (1). Secondary polycythemia is the consequence of low blood oxygen saturation, which triggers the production of ertythropoietin and red blood cells (RBCs) (29). The functional consequence of increased Hct is presumed to be a physiological response whereby an increase in oxygen-carrying capacity compensates for the decrease in available oxygen, or lower PO2, in the case of high altitude adaptation (28). However, individuals adapted to high altitudes present a spectrum of impaired physiological conditions that is usually remedied by hemodilution or return to lower altitudes. Therefore, it is questionable whether adaptation to high altitude, via secondary polycythemia, is a normal physiological response or a pathological condition.

Polycythemia lowers cardiac output by increasing blood viscosity and, therefore, peripheral vascular resistance, as initially described by Richardson and Guyton (18). Microvascular effects, stemming from these variations in blood oxygen-carrying capacity, lead to changes in PO2 distribution in the microcirculation as well as altered oxygen delivery to the tissue. Given the direct link between polycythemia and oxygen delivery, analyzing related microvascular transport properties at the single microvessel level might provide a better understanding of whether increased Hct maintains oxygen delivery when oxygen availability decreases.

To date, microcirculatory studies (3, 4) on hemoconcentration have addressed the problem of the Fahraeus effect (decrease of Hct with reduction of vessel diameter) and the Fahraeus-Lindqvist effect (reduction of viscosity with reduction of diameter). These studies involve either acute hemoconcentration in vivo (14, 15, 22, 27) or physical modeling of artificial capillaries perfused with high Hct blood (3, 4). Acute hemoconcentration in vivo shows hysteresis (14, 27), whereby RBC velocity and flow do not return to control values after hemodilution, indicating permanent changes in the functional properties of microcirculation.

Changes in oxygen delivery at the microscopic level have not been previously addressed. Therefore, the objective of our investigation was to study the effects of chronic hemoconcentration, secondary to low oxygen environment adaptation, on microcirculation and, specifically, on changes in oxygen delivery at the tissue level. Toward this goal, we utilized the hamster chamber window model to study the microcirculation for prolonged periods, without anesthesia, within an intact tissue isolated from the environment.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
All animal procedures were approved by the University of California, San Diego Institutional Animal Care and Use Committee and were conducted according to federal regulations regarding the care and use of laboratory animals: Public Law 99-158, the Health Research Extension Act, and Public Law 99-198, the Animal Welfare Act, regulated by USDA, APHIS, CFR, Title 9, Parts 1, 2, and 3.

Hypoxia adaptation protocol. Systemic Hct was increased by placing the animals in a normobaric chamber where air composition was controlled. The chamber was made of transparent acrylic to facilitate inspection of the animals during the adaptation period, and it accommodated a regular housing cage approved for this purpose by the University of California, San Diego (UCSD) animal subjects program. Oxygen levels were monitored with a digital system (GC-502, VICI Metronics, Santa Clara, CA). Three groups of animals were studied. One group (group 1, n = 5) was exposed to 10% oxygen for 1 wk, followed by implantation of the chamber and exposure to 5% oxygen for 1 wk (see Window chamber preparation). After having recovered from the implantation of a carotid catheter (see Catheter implantation), group 1 was investigated experimentally at 5% inspired oxygen. Another group (group 2, n = 5) was exposed to 10% oxygen for 1 wk, followed by implantation of the chamber and exposure to 5% oxygen for 1 wk. Group 2 was also investigated experimentally after recovery from catheter implantation but at 21% inspired oxygen. The last group (group 3, n = 5) was kept at 21% oxygen for 1 wk, followed by the implantation of the chamber and exposure to 21% oxygen for 1 wk. Again, after having recovered from carotid catheter implantation, group 3 was investigated experimentally at 21% inspired oxygen. Although it would be desirable to implant the chambers at the beginning of the adaptation period and follow all the experimental animals longitudinally, this is not possible with this experimental technique, because the skinfold window is a medium term preparation, having a reliable lifetime of weeks; afterward, tissue atrophy leads to a nonphysiological window. For this reason, we decided to use the schedule described in Fig. 1 and perform the experiments with the same age chambers. For the 5% and 10% gas mixtures, the balance is nitrogen. For 21%, the animals were exposed to flowing room air. The gas mixtures were obtained commercially and regulated from a pressurized tank to flow at 0.5 l/min. Oxygen levels and animal conditions were monitored twice a day. Food and water were provided ad libitum, and the cages were cleaned according to the housekeeping protocol of the vivarium at UCSD.



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  		Fig. 1. Preparation sequence for the experimental animals used in the study. Group 1 (presented as open bars in Figs. 3, 4, 5, 6) were preconditioned by exposure to a normobaric 10% oxygen environment for 1 wk, followed by implantation of the dorsal chamber (Fig. 2). This group of animals was exposed to 5% oxygen for 1 wk, followed by the implantation of a carotid catheter to monitor heart rate and blood pressure during the experiment. Animals were left to recover from the catheter surgery for 24 h in a 5% oxygen environment. The only difference between group 1 and group 2 (hatched bars in Figs. 3, 4, 5, 6) is that recovery from catheter surgery was done at 21% oxygen for group 2. Recovery at room air gives the animals the opportunity to compensate for the hypocapnia observed in group 1. Group 3 (solid bars in Figs. 3, 4, 5, 6) served as a control group. These animals were kept at room air and followed the same surgical schedule as their experimental counterparts.

 



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  		Fig. 3. Systemic values for the three groups studied. A: heart rate of all studied animals. This variable was independent of the level of oxygen in the inspired air but depended on hematocrit value. BPM, beats/min. B: blood pressure presented no significant difference between the groups. See Fig. 1 for bar descriptions.

 


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  		Fig. 4. Experimental results of arterial blood gases. The pH was appreciably increased in group 1 (A) secondary to hypocapnia (B). There was a marked decrease in arterial oxygen in adapted animals breathing 5% oxygen compared with animals breathing room air (C). See Fig. 1 for bar descriptions.

 


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  		Fig. 5. Vessel diameter and blood velocity. A: arterioles summarized show a noticeable reduction in the diameters of group 1. There was an increase in the diameters measured in group 2. Venular diameters (B) were increased in both adapted groups. Blood flow velocity (C and D) was markedly reduced in the adapted groups for both arterioles (C) and venules (D). See Fig. 1 for bar descriptions.

 


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  		Fig. 6. Microvascular oxygen tension and functional capillary density. Oxygen tension (A and B) was markedly reduced in group 1, but virtually the same in group 2 and group 3 controls, for both arterioles (A) and venules (B). C: summary result for functional capillary density. Adapted animals breathing 5% oxygen had a noticeable reduction in the number of flowing capillaries (FCD) per microscopic field, whereas adapted animals, breathing 21% oxygen, had an increase in FCD compared with control group 3.

 


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  		Fig. 2. Macroscopic comparison between a control animal (left) and an animal adapted to hypoxia (right). The two chambers correspond to the same region of the skin. As detailed in the text, adapted animals were exposed to a 10% oxygen environment for 1 wk and 5% oxygen the following week. Venular diameters of medium size veins (VM) increased, whereas large veins (VL) and arteries (A) remained unchanged.

 
Window chamber preparation. While under general anesthesia (injection of 50 mg/kg ip pentobarbital sodium), the hamster's dorsum was shaved with an electric razor. Hair remover cream was applied for 2 min to remove any remaining hair. The area was rinsed first with water and then with a 70% alcohol solution. To create a skinfold, the disinfected area was lifted from the animal and gently pulled back by sutures between the edge of the skinfold and a frame that outlines both the animal and the skinfold. Two titanium frames, one of which is sutured to one side of the skinfold, support the skinfold and provide a 12-mm circular opening. An incision was made to separate tissue layers from the opposite side of the skinfold. These were removed according to the outline of the window, leaving only a thin layer of retractor muscle, adipose, connective tissue, and intact skin, which was sealed with a glass cover slip held by the other frame (2, 5). This other frame has an identical aperture that remains open, exposing the skin to its natural environment. The frames were held together by three bolts and nuts; a spacer maintained a gap between the chambers to prevent excessive compression of the tissue. After surgery, the animals were left to recover for a period of 24–48 h, and the chambers were checked under the microscope for hemorrhagic spots or signs of inflammation, such as edema or slow flow.

Catheter implantation. At the end of the second week, after an animal implanted with the chamber was verified to be in good, stable condition, a carotid catheter was implanted while the animal was under general anesthesia with 50 mg/kg ip pentobarbital sodium. Catheters were flushed with a heparin solution of 30 IU/ml to keep them unobstructed.

RBC velocity. Arteriolar and venular blood flow velocity were measured on-line using the photodiode cross-correlation technique (Fiber Optic Photo Diode Pickup and Velocity Tracker model 102B; Vista Electronics, San Diego, CA). The video image-shearing technique was used to measure vessel diameter (D) online. The measured centerline velocity v was corrected, according to vessel size, to obtain the RBC velocity (V). Blood flow was calculated from the measured parameters as Q = V x {pi}(D/2)2 (10). V, D, and Q were measured while the animals breathed 5% (group 1) or 21% oxygen (groups 2 and 3).

Functional capillary density. Functional capillary density (FCD) was tabulated from capillary lengths with RBC flow in an area composed of 10 successive microscopic fields (420 x 320 µm). Functional capillary density (cm–1) is the number of RBC-perfused capillary segments divided by the area of the microscopic field of view. Capillary segments, i.e., capillaries between bifurcations, are considered functional if RBCs are observed to transit over a 30-s period. FCD was measured while the animals breathed 5% oxygen (group 1)or 21% (groups 2 and 3).

Microvascular PO2 distribution. High-resolution microvascular PO2 measurements were made using phosphorescence quenching microscopy, which has been described in greater detail elsewhere (12). Oxygen measurements are based on the oxygen-dependent quenching of phosphorescence emitted by an albumin-bound metalloporphyrin complex after light excitation. It is independent of tissue dye concentration and is optimal for detecting hypoxia because the decay time is inversely proportional to the PO2 level. This technique was used to measure both intravascular and extravascular PO2 because the albumin-dye complex continuously extravasates the circulation into the interstitium (9, 13, 23, 25). Animals received a slow intravenous injection of 15 mg/kg body wt of 10.0 mg/ml palladium-meso-tetra(4-carboxyphenyl)porphyrin (Porphyrin Products, Logan, UT). The dye was allowed to circulate for 20 min before PO2 measurements. The image of the measuring window (10 x 30 µm) was projected optically onto the tissue being measured to precisely locate the PO2 measurement sites. Microvascular PO2 distribution was measured while the animals breathed 5% (group 1) or 21% oxygen (groups 2 and 3).

Systemic parameters. Mean arterial blood pressure was monitored throughout the experimental procedures. Arterial blood gases and systemic Hct were determined by collecting blood samples in heparinized capillaries. Samples were centrifuged for 4 min using an IEC Micro-MB centrifuge (International Equipment) and an Adams Micro-Hct reader (Clay Adams; Parsippany, NJ). Hemoglobin level was determined by using a B-Hemoglobin photometer (Hemocue; Ängelholm, Sweden). Arterial gases and blood pH were measured with a Blood Chemistry Analyzer 248 (Chiron Diagnostics; New York, NY). Blood viscosity was measured with a DV-II+Viscometer (Brookfield Engineering Laboratories; Midleboro, MA).

Data analysis. Results are presented as means ± SE. Before statistical analysis, we determined whether the data were normally distributed, using a normality test, and whether the sets had equal variances, using a variance equality test. A one-way analysis of variance test was used when both conditions were satisfied. Differences between groups were found by using a t-test applying the Bonferroni correction. In case the normality or equal variance conditions were not satisfied, a Kruskal-Wallis one-way analysis of variance on the ranks was used. Differences between groups were investigated by using either the Tukey test, for those tests where the number of samples were equal, or Dunn's method, for groups with different number of samples (SigmaStat 2.0, Windows 98; SPSS). Differences were considered statistically significant if P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
All adapted animals showed a pronounced increase in systemic Hct and hemoglobin concentration. The adapted groups showed a decrease in physical activity, more evident during the second week of adaptation, but remained responsive after stimulation. All the adapted animals presented hyperemic paws, and the macroscopic observation of the chambers revealed an increase in the diameter of the vessels when compared with animals not exposed to low oxygen environments (Fig. 2). The adapted animals presented an increase in systemic Hct to a level of 68.6 ± 2.44 (group 1) and 70 ± 2.66 (group 2) compared with 43.27 ± 2.30 (group 3, control) and a concomitant increase in hemoglobin concentration to 19.8 ± 0.5 g/dl (group 1) and 21.09 ± 1.24 g/dl (group 2) compared with the control (group 3) 14.57 ± 0.07 g/dl (Table 1). Both the increase in Hct and hemoglobin were statistically significant (P < 0.05).


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  		Table 1. Hemoglobin and hematocrit values of the three groups studied

 

The adapted animals presented a decrease in heart rate but maintained the same blood pressure as the control group (Fig. 3). The heart rate showed a nonsignificant decrease in both adapted groups: 336.6 ± 22.1 beats/min (group 1), with a P = 0.06 compared with the control, and 320.2 ± 25.3 beats/min (group 2), with a P = 0.05 compared with the control. The control group presented a value of 412.4 ± 11.6 beats/min. Blood pressure showed no significant difference among the studied groups: 96.5 ± 9.4 mmHg for group 1, 110.9 ± 7.5 mmHg for group 2, and 97.6 ± 1.6 mmHg for group 3 (P = 0.306).

In group 1, arterial pH increased, while PCO2 and PO2 decreased, compared with both groups 2 and 3 (Fig. 4). Arterial pH of group 1 was 7.43 ± 0.02, a difference not significantly higher than the values found in groups 2 and 3: 7.34 ± 0.01 (P = 0.05) and 7.37 ± 0.01, respectively. No other differences in pH were found when other comparisons were performed between groups. PCO2 in group 1 was 18.2 ± 4.2 mmHg, significantly lower than the values found in groups 2 and 3: 62.5 ± 4.3 and 58.7 ± 1.9 mmHg, respectively. Arterial PO2 was also found to be significantly lower in group 1 compared with the other two groups: 16.4 ± 1.2 mmHg for group 1, 48.9 ± 5.4 mmHg for group 2, and 54.0 ± 1.9 mmHg for group 3. This suggests the increase in pH, observed in the adapted hypoxic animals (group 1), is due to hypocapnia, secondary to hyperventilation.

Arterial diameters were reduced in group 1 and increased in group 2 compared with control group 3. Vein diameters were increased in group 1, and even more in group 2, compared with the control group (Fig. 5). Arterial diameters in group 1 had a value of 51.2 ± 5.1 µm (significantly lower than the other two groups, P = 0.003), whereas group 2 had a value of 93.1 ± 9.8 µm and group 3 presented a value of 74.6 ± 6.6 µm. Vein diameters were 140.6 ± 19.3 µm in group 1, 185.1 ± 31.1 µmin group 2, and 99.1 ± 8.7 µmin group 3. Values in group 3 were significantly lower than the other two groups (P = 0.016).

Arterial and vein blood velocities were significantly reduced in the adapted animals (groups 1 and 2) compared with the control group, P < 0.05 (Fig. 5). Arterial blood velocity was 0.94 ± 0.13 mm/s in group 1, 1.17 ± 0.24 mm/s in group 2, and 3.62 ± 0.33 mm/s in group 3. Vein blood velocity was 0.86 ± 0.12 mm/s in group 1, 0.57 ± 0.10 mm/s in group 2, and 2.08 ± 0.17 mm/s in group 3. These results suggest a significant increase in the peripheral resistance of the adapted groups, presumably from an increase in blood viscosity due to high Hct.

In situ oxygen tension in the microvasculature depends on the percentage of oxygen in the inspired air, not the Hct level (Fig. 6). Arteriolar oxygen tension in group 1 was 11.3 ± 0.7 mmHg (significantly lower than the other two groups, P < 0.05), 26.7 ± 0.7 mmHg for group 2, and 26.0 ± 0.9 mmHg for the control group. Vein oxygen tension in group 1 was 7.8 ± 0.9 mmHg (again, significantly lower than the other two groups, P < 0.05), 21.6 ± 0.6 mmHg for group 2, and 20.3 ± 0.7 mmHg for group 3.

Tissue oxygen consumption was preserved in group 2 compared with the control (group 3) but markedly reduced in group 1 (Table 2). The microvascular methodology used in our studies permits a detailed analysis of oxygen consumption in the tissue. Calculations of oxygen consumption, normalized relative to control, are made using the equation

(1)
where RBCHb is the hemoglobin in RBCs expressed as grams per deciliter of blood, {Delta}A-V% is the difference in arteriolar-venular oxygen saturation of the RBCs, and Q is the averaged microvascular flow for all microvessels as a percentage of baseline. The oxygen dissociation curve for hamster RBCs was determined from freshly collected hamster RBCs, as previously described (21). Determination of oxygen saturation requires information on the Bohr effect, because adaptation to low oxygen causes a significant change in pH. Whereas these data are not available for hamster blood, the change of blood oxygen during 5% oxygen inspiration is small, and the correction should minimally affect the results. In this quantitative comparison, we use the averaged microvascular flow for all the microvessels because of the heterogenous responses in the network. This lack of uniformity is due to the presence of interconnecting (arcading) arterioles within a given vessel order.


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  		Table 2. Oxygen delivery and consumption in the microcirculation of the hamster window model

 

FCD was markedly decreased in group 1, but significantly increased in group 2, with respect to the control group (Fig. 6). Group 1 presented an average of 2.41 ± 0.45 capillaries/field with 14.42 ± 0.80 capillaries/field for group 2 and 10.63 ± 0.33 capillaries/field for the control group. Each group showed a significant difference compared with the other groups (P < 0.05).

In a subgroup of group 1 (n = 3 animals), we measured the viscosity of blood. The Hct in this subgroup presented an average Hct of 71.7 ± 1.7% and a blood viscosity of 9.0 ± 0.32 cP compared with 4.0 cP for control. Thus, whereas Hct increased by 1.43-fold, blood viscosity increased 2.24-fold.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
The principal finding of this study is that an increase in Hct, consistent with adaptation to chronic hypoxia, is accompanied by an expansion of the venular network that is observable to the naked eye. As depicted in Fig. 2, this increase in venular diameter may be responsible for the hyperemia observed in the paws of the adapted groups and, presumably, corresponds to the conjunctival congestion (16) and reddish color (1) observed in patients with chronic mountain sickness (Monge's disease). This finding suggests that oxygen delivery in the adapted animals respiring a low oxygen atmosphere may be shifted to the venular compartment, because this portion of the microcirculation presents a significantly increased oxygen-carrying capacity.

Whereas heart rate in adapted animals decreased marginally leading a nonsignificant difference, blood pressure remained the same as in the control group (Fig. 3). To analyze this finding, it is useful to consider a simplistic model for blood pressure; namely, blood pressure = cardiac output x peripheral resistance (19). A major limitation of current technology is that cardiac output cannot be measured in these animals due to their small size. However, we can consider changes in peripheral resistance due to changes in blood viscosity. In the adapted animals, viscosity was 9.0 cP; thus the 1.4-fold increase in Hct caused a 2.2-fold increase in viscosity. Because peripheral vascular resistance is a linear function of blood viscosity, without changes in the vascular diameters of resistance arteries and arterioles, cardiac output must decrease to prevent hypertension in the adapted animals. Therefore, the marginally lowered heart rate found in adapted animals, indicating decreased cardiac output, can be related to systemic blood pressure regulation, an effect that is additionally reenforced by the increased arteriolar diameter also found in the adapted animals.

Our study shows that adapted animals respiring 5% O2 exhibit hypocapnia and hypoxemia. These conditions are indicative of respiratory alkalosis due to hyperventilation in response to hypoxia. Presumably, animals continuously hyperventilate during hypoxia and are in respiratory alkalosis during the early stages of adaptation. In a later stage, the animals develop a compensatory metabolic acidosis, which normalizes pH. When the animals are exposed to a normal concentration of oxygen, hyperventilation is corrected, and the animals develop metabolic acidosis. Given that pH in group 2 is normal, the 24-h recovery period before experiments in room air appears sufficient to compensate for acidosis due to normocapnia in adapted animals.

Exposure to low oxygen environments occurs naturally at high altitudes. Therefore, it is relevant to establish the equivalent altitude above sea level where 10% and 5% oxygen compositions are encountered. This can be obtained from the formula PO2 (mmHg) = (barometric pressure – partial pressure of water vapor at body temperature) x %O2 in air. Usually, lowered partial pressure of oxygen at high altitude is due to lowered barometric pressure. In our case, however, it is due to the change in the percentage of oxygen of the inspired air. Because barometric pressure at sea level is 760 mmHg, and, at body temperature, the partial pressure of water vapor is 47 mmHg, the oxygen content is 20.93%, and its partial pressure is 159 mmHg. Therefore, at sea level with 10% of oxygen content, the PO2 corresponds to 71.3 mmHg. As a reference, PO2 at the highest permanent habitation (altitude 5,300 m) is 72 mmHg; at the summit of Everest, PO2 is 43 mmHg; and a PO2 of 35 mmHg corresponds to 5% oxygen. Our experimental system is set at 760 mmHg total (atmospheric) pressure, whereas at the summit of the Everest, pressure is 253 mmHg.

The effect of absolute pressure on blood oxygenation is determined by Henry's law, which states that the volume of gas dissolved in a liquid is proportional to its partial pressure (C = KPx, where C is the concentration of gas dissolved in the liquid, K is the solubility constant, and Px is the partial pressure of the gas on the liquid). Because the concentration of oxygen in blood is determined by its partial pressure, there is an infinite set of absolute pressures and gas mixture ratios that result in the same concentration of dissolved oxygen. An example is oxygen breathing by scuba divers. In this case, the partial pressure of air oxygen increases proportionally to the absolute pressure. Therefore, to maintain constant the amount of oxygen dissolved in the blood, the percentage of oxygen must be reduced. To avoid oxygen toxicity during deep dives, the amount of oxygen inspired has to be reduced to <1% (28).

In our experiments, analysis shows the amount of oxygen dissolved in blood, even under normobaric conditions, corresponds to the same amount of oxygen dissolved in blood at high altitude. Thus our model is representative of adaptation to high altitude. However, it is important to consider the effects of hypobaria per se. Although acute nomoxic hypobaria has no effect on blood pressure, urinary output, heart rate, or other physiological variables, it has been reported that chronic exposure to low pressure enhances the sodium and water retention in hypoxia (24).

In the present study, we determined the microvascular changes in animals secondary to adaptation to chronic extreme hypoxia. To properly evaluate the nature of these changes we designed three study groups. Group 1 was adapted to hypoxia and was maintained throughout the experiment in hypoxia conditions, to mimic the conditions of animals under hypoxic stress after adaptation. However, changes observed in this group (i.e., increased Hct and viscosity) may be due not only to the adaptation itself but also to extreme hypoxia. For this reason, we incorporated another group (group 2), which was previously adapted to hypoxia but conditioned to breathe room air (at 21% oxygen). This group allowed us to study the changes due only to adaptation and to distinguish them from those caused by hypoxia. The last group (group 3) was used a control group.

A common explanation of the role of erythropoiesis in adaptation to hypoxia is that increased oxygen-carrying capacity, due to increased circulating hemoglobin, compensates for the diminished PO2. This explanation assumes that, although PO2 and oxygen saturation are low, the oxygen content of the arterial blood may be normal or elevated. For example, inhabitants of the Peruvian Andes, living at altitudes of 4,500 m, with an arterial PO2 of 45 mmHg and an arterial oxygen saturation of 81%, have an arterial oxygen content of 22 ml/100 ml, which is above the sea level value due to the increased hemoglobin concentration (28). However, the oxygen tension in vessels of the adapted animals at room air (21% O2) yielded the same value as the control group. These paradoxical results may indicate that adaptation to hypoxia occurs independently of adjustments in oxygen-carrying capacity.

FCD was found to be directly related to Hct and the percentage of oxygen in the inspired air. Comparison of groups 2 and 3 shows that, at the same oxygen level, the group with the highest Hct had a significantly greater FCD. Maintained or increased FCD has been shown to be as or more important than tissue oxygenation in ensuring tissue survival (11), and this variable was the only one in our study that appeared to confer to the adapted animals a physiological advantage over the control group. The increase in functional capillary density may be related to the increased Hct found in extreme hemodilution performed with a highly viscous plasma expander, where the augmented shear stress causes vasodilation of the arterioles and the viscous plasma pressurizes and distends the capillaries (26). There was a marked decrease in FCD in group 1 compared with groups 2 and 3. Because groups 1 and 2 have a similar increase in Hct, these differences are due to the oxygen level breathed by the animals. However, hypoxia causes a twofold decrease in arteriolar diameter, leading to an increase of peripheral resistance that may be as much as 16-fold. It should be noted that adapted animals that are returned to respiring 21% oxygen show a FCD that is significantly higher than controls. Because histological analysis of adapted versus nonadapted tissues shows that there is no change in capillary density (17, 20), the observed difference indicates the recruitment of nonperfused capillaries in the adapted animals that respire 21% O2. This finding suggests that FCD observed in adapted animals at 5%, albeit significantly smaller than in the other group, is related to the process of adaptation. Accordingly, in the absence of this adaptation, FCD at 5% O2 would be near zero, a condition that imperils survival and which might explain why animals that are suddenly exposed to 5% O2 succumb in a short time.

Our findings suggest that the increased Hct found in the adaptation to extreme chronic hypoxia may not be solely for the purpose of augmenting oxygen-carrying capacity. Higher Hct significantly increases blood viscosity, and therefore shear tress, which leads to the production of vasodilators such as nitric oxide and prostacyclin by the endothelium (7, 8). Peripheral vascular resistance falls and perfusion increases. This mechanism, however, does not result in improved tissue perfusion because arterioles constrict during conditions of extreme hypoxia and return to their original diameter or dilate during conditions of normoxia.

Our oxygen consumption calculation shows that a significant increase in Hct, in the hypoxic condition, is not beneficial because oxygen delivery is only 10% of normal. These results also show that, for this tissue model, oxygen consumption remains constant during a significant increase in Hct at normal inspired air, a result that may have implications regarding the presumed beneficial effects of increasing Hct in athletes. The point should be made, however, that we cannot deduce what is the effect of increased Hct in conditions of exercise.

In summary, our experiments show that adaptation to extremely low oxygen concentration in a normobaric environment leads to increased Hct. Functionally, this process is mainly venular, because most of the extra RBCs occupy the distended venules acting as a erythrocyte reservoir in an environment where flow is below that of control. Contrary to the commonly accepted idea, the increase in Hct does not compensate for low oxygen tensions, potentially causing unfavorable conditions due to the increase in peripheral resistance. Restoration of normal oxygen partial pressure returns the organism to normal physiological conditions, with the additional improvement that high viscosity appears to have a direct effect on improving functional capillary density, ultimately ameliorating peripheral perfusion. Our findings show that adapted animals returned to 21% O2 have significantly greater FCD than control, suggesting that the process of adaptation leads to the maintenance of threshold level of FCD that otherwise is eliminated in nonadapted animals suddenly exposed to hypoxia (5% O2). Our results also show that the significant increase of systemic Hct does not correct for the oxygen deficit but may be a contributive factor in maintaining the threshold of peripheral perfusion. Because adaptation to 5% oxygen may be on the limit of organism survival, the beneficial effects of an increased Hct will require more extended investigations, where intermediate levels of adaptation are studied.


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute (NHLBI) Grant HL-57430 (to E. Saldívar, Principal Investigator) and the NHLBI Bioengineering Partnership Grant R24-HL-64395 and Grants R01-HL-62318 and R01-HL-62354 (to M. Intaglietta, Principal Investigator).


   ACKNOWLEDGMENTS
 
The authors are indebted to Froilan P. Barra for the surgical preparation of the animals and to Stanley Block for the manufacturing of the hypoxic chambers.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. Saldívar, La Jolla Bioengineering Institute, 505 Coast Boulevard South, La Jolla, CA 92037 (E-mail: enrique{at}ljbi.org).

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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