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INTEGRATIVE |
a11 Center for Perinatal Biology, Departments of Physiology, Obstetrics and Gynecology, School of Medicine, Loma Linda University, Loma Linda, CA 92350, USA
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Abstract |
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50% in each group (P < 0.05). For both the fetus at low altitude and that acclimatized to high altitude LTH, we present the first dose–response data on the relation of LD-CBF, cortical tPO2, and sagittal sinus blood gas values to Pa,O2. In addition, despite differences in several variables, the fetus at high altitude showed evidence of successful acclimatization, supporting the hypothesis that such fetuses demonstrate no compromise in cerebral oxygenation.
(Received 24 August 2006;
accepted after revision 21 October 2006;
first published online 26 October 2006)
Corresponding author L. D. Longo: Center for Perinatal Biology, Loma Linda University, School of Medicine, Loma Linda, CA 92350, USA. Email: llongo{at}llu.edu
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Introduction |
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Approximately 140 million people live at altitudes above 2500 m. Compared to pregnancy at low altitude, that at high altitude is associated with several problems, including intrauterine fetal growth restriction (IUGR), increased perinatal mortality and morbidity (Moore et al. 1998) with neurodevelopmental disorders (Bernstein et al. 2000; Jelliffe-Pawlowski & Hansen, 2004). Although numerous studies have explored fetal cerebrovascular responses to acute hypoxia, little information exists on cerebrovascular responses and cerebral oxygenation in response to long-term hypoxia (LTH) (Longo & Pearce, 2005). In those acclimatized fetuses, Pa,O2
= 19 ± 1 Torr, as compared with sea level control of 23 ± 1 Torr (Kamitomo et al. 1993). This long-term hypoxic preparation in pregnant sheep may prove useful, not only for pregnancy at high altitude, but also for many other disorders. For instance, pregnant women who smoke (Longo, 1977; Bureau et al. 1983), and those with certain cardiovascular (Cannell & Vernon, 1963) and pulmonary disorders (de Swiet, 1979) have somewhat similar characteristics to pregnancy at high altitude. In human fetuses at 4200 m, fetal scalp O2 tension also averaged 19 Torr (Sobrevilla et al. 1971). Several maternal and/or fetal mechanisms help to optimize fetal O2 delivery and tissue oxygenation under these circumstances (Longo, 1987). However, despite the importance of CBF and cerebral oxygenation to the high altitude acclimatized fetus, little is known of the extent to which oxygenation of its brain may be compromised.
A number of aspects of both calcium-dependent and Ca2+-independent signal transduction mechanisms in the fetal cerebral vasculature are altered in response to acclimatization to high altitude, as compared to that at sea level (Longo & Pearce, 2005). Little is known, however, regarding in vivo responses. Thus, we designed the present studies to test the hypothesis that in the fetus acclimatized to high altitude, cerebral oxygenation is not compromised in comparison to that at low altitude. For the near-term fetus at both low and high altitude, we therefore asked what are the dose–response relationships of LD-CBF to Pa,O2? To what extent are decreases in Pa,O2 associated with alterations in cortical tPO2 and sagittal sinus O2 values, as indices of cerebral oxygenation? Finally, we asked the extent to which the responses to superimposed hypoxia differ in the fetuses of the two altitude groups?
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Methods |
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For these studies, we used 14 pregnant Western ewes and their near-term singleton fetuses obtained from Nebeker Ranch (Lancaster, CA, USA). Eight were normoxic, low altitude (335 m, 1100 ft) controls, and at 30 days gestation six had been taken to high altitude (3801 m, 12 470 ft, Barcroft Laboratory, barometric pressure = 485 mmHg, White Mountain Research Station, Bishop, CA, USA) where they were maintained for 110 days. Both groups of animals were transported to our laboratory several days before the studies. In the high altitude group, within 8 h a catheter (4.0 mm o.d.) was placed in the maternal trachea, following which nitrogen was infused to maintain maternal Pa,O2 at 60 ± 3 Torr, i.e. the value we have measured at high altitude (Kamitomo et al. 1993). All surgical and experimental procedures were performed within the regulations of the Animal Welfare Act, the National Institutes of Health Guide for the Care and Use of Laboratory Animals,
‘The Guiding Principles in the Care and Use of Animals’ approved by the Council of the American Physiological Society, and the Animal Care and Use Committee of Loma Linda University.
Surgery and instrumentation
We have described this previously (Tomimatsu et al. 2006a,b). Briefly, the pregnant ewes and their fetuses were instrumented at 122 ± 3 days of gestation (term 145 days). Following induction of anaesthesia with thiopental sodium (10 mg kg–1
I.V.), the ewe was placed in the supine position and intubated. Anaesthesia was maintained throughout the surgical procedure with mechanical ventilation of 1.0% halothane in O2. All surgical procedures were carried out under aseptic conditions. The maternal abdominal wall and uterus were incised, and the fetal head and one forelimb were delivered. We inserted a polyvinyl catheter (2.3 mm o.d.) into the brachial artery and advanced it into the aortic arch for arterial blood sampling and recording of blood pressure and heart rate. We also placed a catheter into the brachial vein, and advanced it into the superior vena cava. Following replacement of the forelimb into the uterus, this was repeated in the other forelimb, which then was replaced into the uterus. Each catheter was anchored subcutaneously and exteriorized through the fetal skin, uterus and maternal abdominal wall to the ewe's left flank.
We then incised the scalp rostral to the coronal suture, exposing the right and left parietal bones. By use of a 15 gauge syringe needle to make an opening in the skull and dura, we placed a polyvinyl catheter (2.3 mm o.d.) 1.5 cm into the sagittal sinus, to sample mixed venous blood from the brain, including the tissue containing the LDF probe. This catheter was secured to the fetal scalp with tissue glue and latex dental dam. Through an 18 gauge needle on the right side 5 mm lateral to the sagittal suture and 15 mm caudal to the coronal suture, we inserted the tip of the composite tPO2 laser Doppler flow probe with thermocouple (Oxford Optronix, Ltd, Oxford, UK) into the parasagittal parietal lobe cortex to a depth of 5 mm below the dura mater, and fixed this to the skull with tissue glue. We repeated the same steps on the left side, and then closed the scalp incision.
Next we placed a polyvinyl catheter (3.5 mm o.d.) in the amniotic fluid for measurement of amniotic fluid pressure and administration of antibiotics. The uterine wall was closed in two layers. Catheters and probe connections were exteriorized to the ewe's left flank, and stored in a pouch attached to the maternal skin. Lastly, in the ewe's right femoral artery and vein we inserted Tygon polyvinyl catheters (2.8 mm o.d.) filled with heparinized saline for blood sampling and infusion of fluids, respectively. Postoperatively, the ewe was given 900 000 U penicillin intramuscularly for three days, and the fetus was given claforan (50 mg day–1, I.V.). We also administered ampicillin (500 mg) and gentamicin (40 mg) into the amniotic fluid daily until the experiments were completed. During the postoperative period (0–24 h) the ewes were monitored for signs of discomfort such as restlessness, teeth grinding, abnormal posture or respiratory rate, and changes in mental alertness. Any animal that displayed these signs was treated with buprenorphine hydrochloride (Buprenex, Reckitt & Colman Products Ltd, Hull, UK, 0.005 mg kg–1 I.M. every 4–6 h). Typically, ewes resumed normal feeding 6–12 h following surgery. We monitored arterial blood gases daily for 4–5 days of postoperative recovery before experiments were commenced.
Experimental design
The protocol was designed to measure LD-CBF during a 40 min normoxic control period, and then during 40 min of superimposed isocapnic, hypoxic hypoxia. The studies examined the relation of LD-CBF to Pa,O2, and their relationship to cortical tPO2, sagittal sinus PO2, and [HbO2], and cerebral metabolic rate for O2 (CMRO2). For each group, while maintaining maternal and fetal arterial PCO2 (Pa,CO2) values at their baseline levels (i.e. 35 ± 1 and 44 ± 1 Torr, and 33 ± 3 and 44 ± 1 Torr, respectively), we induced isocapnic hypoxia by having the ewe breathe 10.5% O2 with 3–5% CO2 to achieve a fetal Pa,O2 of 11 ± 1 Torr. This was administered by passing the gas mixture at 30 l min–1 through an opaque plastic bag over the ewe's head, and was tolerated by the ewes with minimal evidence of distress. In general, for each animal a second hypoxic exposure was repeated 48–72 h following the first. We saw no evidence that responses to the second exposure differed significantly from that of the first.
Blood sampling
Maternal and fetal blood samples (0.3 ml) were collected and analysed for blood gases corrected to the body temperature (ABL3, Radiometer, Copenhagen, Denmark) every 10 min throughout the control period. To standardize the degree of hypoxia, additional blood samples were taken every 5 min during this period, and the inspired gas mixture was adjusted accordingly. We also measured spectrophotometrically haemoglobin concentration [Hgb] and oxyhaemoglobin saturation (OSM2 Hemoximeter, Radiometer), and calculated O2 content (Ca,O2) as the product [HbO2] x [Hgb] x 1.34. Blood gas values were corrected to fetal body temperature (Lotgering et al. 1983). As previously reported, we calculated relative cerebral O2 delivery (LD-CBF x Ca,O2), cerebral fractional O2 extraction, i.e. O2 consumed as a fraction of that delivered (CMRO2/cerebral O2 delivery) (Jones et al. 1982; Jones & Traystman, 1984), and the cerebral metabolic rate for O2 (LD-CBF x arterial to sagittal sinus O2 content difference) (Tomimatsu et al. 2006a,b).
Measurements of laser doppler cerebral blood flow and intracerebral oxygen tension
In each group of fetuses, we continuously recorded both right and left relative cortical blood flow by use of a laser Doppler flowmeter (Oxford Optronix). We also measured cortical tPO2 by use of a fluorescent oxygen probe (Oxylite, Oxford Optronix), temperature, mean arterial blood pressure (MABP), and fetal heart rate (FHR). Analog outputs were digitized (sampling rate 100 Hz) and stored using an analog to digital converter (Powerlab 16/SP, ADInstruments, Colorado Springs, CO, USA), and data acquisition software (Chart v 4, ADInstruments). In the fetal lamb, the sagittal sinus drains blood from the anterior one-third of the brain (Grant et al. 1995), and its blood gas values are considered to represent global cerebral oxygenation.
Data acquisition and statistical analyses
We recorded laser Doppler CBF and tPO2 signals from both right and left hemispheres of the brain. Analysis of variance showed no systematic differences between hemispheres. Thus, the two results were averaged to provide a mean estimate of changes in relative blood flow and tPO2 values. We measured mean arterial blood pressure and heart rate continuously by use of a pressure transducer (Argon Medical, Athens, TX, USA) and the data acquisition software. For the LD-CBF, tPO2, mean arterial blood pressure, and heart rate data we took 1 min averages, and exported the values to a spreadsheet for analysis. Because LD-CBF provides a relative measure of cerebral blood flow, these values as well as those for relative vascular resistance (e.g. mean arterial pressure/relative LD-CBF), O2 delivery, and CMRO2 for each animal were calculated as a percentage of the mean values during the baseline control period (Tomimatsu et al. 2006a,b). We expressed results as means ± S.E.M., and used two-way analysis of variance (ANOVA) to compare control and hypoxic periods in both altitude groups. We performed both linear and nonlinear regressions (GraphPad Prism, GraphPad Software, San Diego, CA, USA); P < 0.05 was considered statistically significant.
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Results |
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Following acclimatization to high-altitude, maternal Pa,O2 was 60 ± 3 Torr, compared with a sea-level value of 100 ± 5 Torr. In the low altitude fetuses, acute hypoxia decreased Pa,O2 from a baseline value of 23 ± 1 Torr to 11 ± 1 Torr (P < 0.01), while in the high altitude acclimatized fetuses this decreased from the baseline value of 19 ± 1 Torr to 11 ± 1 Torr (P < 0.01) (Table 1). Following the hypoxic period, Pa,O2 returned rapidly to the baseline value in both altitude groups. Also in both altitude groups, fetal arterial PCO2 and pH were similar during the baseline control period, and did not change significantly during isocapnic hypoxia (Table 1). Figure 1 shows the mean values of LD-CBF and cortical tissue PO2 during the 40 min baseline control period, 40 min of superimposed hypoxia, and 40 min recovery. Figure 1A shows LD-CBF for both groups of fetuses as a percentage change from the baseline value. During the initial 20 min of hypoxia in the low altitude fetus, LD-CBF increased 31 ± 2% above the control value, then increased further to 44 ± 2% above baseline from 20 to 40 min (P < 0.01). In contrast, in the high altitude fetuses, during the initial 20 min period of superimposed hypoxia, LD-CBF increased 10 ± 2% above the control value, then increased to 19 ± 2% (P < 0.01) above baseline during 20–40 min continued hypoxia (Table 1). In both altitude groups, following superimposed hypoxia LD-CBF returned to the baseline control value within 5–10 min.
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Table 1 also presents the fetal responses of mean arterial blood pressure, heart rate, and other variables in the low and high altitude groups. In the low altitude fetuses, in response to acute hypoxia, fetal MABP rose from a baseline value of 43 ± 2 mmHg to 48 ± 3 mmHg. Similarly in the high altitude acclimatized fetuses, with superimposed hypoxia MAPB increased from 41 ± 2 to 48 ± 3 mmHg. During the hypoxic periods in each altitude group these changes were associated with a moderate but significant decrease in relative cerebral vascular resistance (Table 1). During acute hypoxia in low altitude animals, FHR decreased 18% from the baseline value of 158 ± 3 to 130 ± 3 beats per minute (bpm) at 40 min (P < 0.01). For the fetuses at high altitude, in response to superimposed hypoxia FHR decreased 25% from a baseline value of 175 ± 3 to 131 ± 2 bpm (P < 0.01; Table 1). Again for each group, during the recovery period FHR returned to values slightly greater than that during the prehypoxic period. As seen in Table 1, the difference in baseline FHR between fetuses of the two altitude groups was significant (P < 0.05).
As also noted in Table 1, in low altitude fetuses baseline haemoglobin concentration increased from a control value of 9.2 ± 0.1 g dl–1 to 10.1 ± 0.1 g dl–1 in response to acute hypoxia (P < 0.05). In fetuses at high altitude, [Hb] increased from 11.1 ± 0.1 g dl–1 during control period to 12.0 ± 0.1 g dl–1 during superimposed hypoxia. The differences between these [Hb] values in the two altitude groups were highly significant (P < 0.05).
LD-CBF and cortical tissue PO2 dose–response relations to arterial PO2
To examine CBF-Pa,O2 dose–responses, Fig. 2A illustrates the relation of fetal LD-CBF to arterial PO2 values for both the fetuses at low altitude and those at high altitude. With superimposed hypoxia, the increase in fetal LD-CBF showed a linear relationship to Pa,O2 (Fig. 2A). This relationship was similar for both [HbO2] and O2 content. As observed in Fig. 1A, in the high altitude fetus the slope of the acute hypoxic-induced increase was significantly less than that of low altitude controls (P < 0.01; regressions equations are given in the figure legends; Table 1). Note: data for this and other figures do not include the recovery period.
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2 ± 1 Torr (Table 1). Sagittal sinus blood gas values
To explore further the relation of fetal cerebral oxygenation to Pa,O2 and LD-CBF in both altitude groups, we measured several functions. Strikingly, for fetuses at both altitudes baseline sagittal sinus PO2 and [HbO2] were 16 Torr and 43%, respectively. Also in both altitude groups, the decreases in these values during superimposed hypoxia were similar (Table 1). Figure 3 presents the data on sagittal sinus PO2 values as a function of both Pa,O2 and cortical tissue PO2 in these fetuses. As seen in Fig. 3A, for both altitude groups this relationship was linear and did not differ significantly between the groups (regression equations are given in the figure legend). Also as shown in Fig. 3B, sagittal sinus PO2 showed a curvilinear relation to cortical tPO2, plateauing above 4 Torr; and this relationship did not differ significantly between the two altitude groups. Figure 3C shows that the relation of sagittal sinus [HbO2] to cortical tPO2 did not differ significantly from that for sagittal sinus PO2
versus tPO2.
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To examine the relations of fetal cerebral O2 delivery, fractional O2 extraction, and CMRO2 to Pa,O2 in the two altitude groups, Fig. 4 presents plots of these variables. During superimposed hypoxia in fetuses at both altitudes, fetal relative O2 delivery (arterial O2 content x LD-CBF) (Fig. 4A) and fetal arterial to sagittal sinus O2 content difference (Fig. 4B) decreased significantly as a function of Pa,O2. Importantly, the lower relative O2 delivery in the high altitude fetuses was less than that seen in the low altitude controls, which probably reflects the lower hypoxic-induced increase in CBF (regression equations are given in the legends to figures). Figure 4C shows that during acute hypoxia in the low altitude fetus's cerebral fractional O2 extraction (1 – (venous O2 content/arterial O2 content)) remained constant at 0.36 ± 0.02. In contrast, in the high altitude fetuses under baseline conditions this value was significantly lower (0.28 ± 0.02) (P < 0.05). However, with superimposed hypoxia fractional O2 extraction increased significantly to a value similar to that of the fetuses at low altitude (0.35 ± 0.03; Table 1). We calculated the relationship of relative cerebral metabolic rate for O2 to Pa,O2 values for fetuses in each altitude group from the product of LD-CBF (% baseline) and the arterial to sagittal sinus O2 content difference. For fetuses at both low and high altitude, Fig. 4D shows the CMRO2 over a range of hypoxic-induced Pa,O2 values. CMRO2 decreased with Pa,O2, so that at Pa,O2 of 10 Torr CMRO2 had decreased 50% in both altitude groups (Fig. 4D and Table 1). When we plotted CMRO2 as a function of sagittal sinus PO2, the relationship was quite similar, although the venous PO2 values were lower (data not shown).
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To examine the relation of blood lactate levels to isocapnic, hypoxic hypoxia, we measured these in fetuses in both altitude groups. In the low altitude fetus, arterial lactate levels increased significantly from the baseline level of 0.9 ± 0.1 mM to 1.6 ± 0.2 mM at 20 min hypoxia and 3.2 ± 0.3 mM at 40 min isocapnic hypoxia (P < 0.05 at both time points). In concert with this increase, arterial pH decreased as a function of hypoxia duration from a baseline value of 7.36 ± 0.1 to 7.30 ± 0.1 at 40 min (Table 1). Similarly in the high altitude fetus, baseline lactate was 1.0 ± 0.1 mM during the control period increasing to 1.5 ± 0.2 and 3.3 ± 0.3 mM at 20 and 40 min hypoxia, respectively. Again, the arterial pH values decreased significantly (Table 1). These values did not differ significantly between the two altitude groups. These hypoxic-induced pH changes represented systemic lactate production, as the arterial to sagittal sinus lactate difference remained relatively unchanged. Importantly in the low altitude fetuses, when superimposed hypoxia extended beyond 40 min, metabolic acidosis became more severe (data not shown). In the high altitude animals, we did not extend the hypoxia period so as not to take a chance of compromising them further.
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Discussion |
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In the fetus at both low and high altitude, superimposed acute hypoxia was associated with significant increases in LD-CBF, and decreases in cortical tissue PO2, sagittal sinus PO2, and relative cerebral metabolic rate for O2. It was striking that although the hypoxic-induced increase in LD-CBF in the high altitude fetus was only one-half that of that at low altitude, cortical tPO2 and sagittal sinus O2 values were similar in the two groups. This suggests that despite the seemingly hostile environment, the fetus at moderately high altitude is successfully acclimatized, and cerebral oxygenation is not compromised. This study also suggests that sagittal sinus O2 values can serve as an indicator of cortical tissue oxygenation.
Many aspects of fetal cerebrovascular responses in response to acute hypoxia have been documented to change. However, little is known about the responses to superimposed hypoxia in those subjected to long-term hypoxia. At high altitude, despite the reduction in fetal Pa,O2 there was no reduction in total body weight or that of other organs, the exception being the brain, the weight of which decreased 23% from 64 ± 2 to 47 ± 3 g (P < 0.01, so that the brain to body weight ratio decreased from 1.4 ± 0.1 to 1.1 ± 0.1; P < 0.01) (Penninga & Longo, 1998). This raised the question of the extent to which compromised oxygenation might have resulted in the smaller brains of these animals. These findings in sheep contrast with human studies in which newborn birth weight is reduced at high altitude (Howard et al. 1957; Lichty et al. 1957; McClung, 1969; Moore et al. 1982). Nor in the LTH fetus were there major changes in the levels of many circulating hormones (Kitanaka et al. 1989; Kamitomo et al. 1992; Longo & Pearce, 1998). Thus, in many respects the ovine fetus appears to represent an example of successful acclimatization. In the present study, we tested the hypothesis that responses to superimposed hypoxia, in terms of brain oxygenation, did not differ significantly in the high altitude acclimatized fetus, as compared to low altitude controls.
In the developing fetus, acute hypoxia is associated with a significant increase in cerebral blood flow (Peeters et al. 1979; Ashwal et al. 1980). In contrast, the fetus acclimatized to high altitude long-term hypoxia demonstrates near-normal CBF, despite a significant 27 ± 4% decrease in cardiac output, and a 49 ± 6% decrease in blood flow to the carcass and most other organs (Kamitomo et al. 1993). Also in adult human subjects acclimatized to high altitude, despite changes in respiration and function of many organs, CBF remains at relatively normal levels with little change in cerebrovascular resistance (Huang et al. 1987; Xu & LaManna, 2006). These findings raise questions regarding the mechanisms whereby fetal cerebrovascular homeostasis is maintained during LTH, as well as during superimposed hypoxia.
LD-CBF responses to hypoxic hypoxia
In association with advances in laser Doppler (LD) technology is the ability to measure continuously cerebral blood flow and cortical tPO2 (Bishai et al. 2003) and, by sampling blood from the sagittal sinus, to estimate cerebral oxygenation. Despite several studies using this LDF technology (Bishai et al. 2003; Hunter et al. 2003a,b; Tomimatsu et al. 2006a), in neither the fetus at low altitude nor that acclimatized to high altitude is the relation of LD-CBF to Pa,O2 values, nor their relationship to cortical tissue oxygenation (tPO2 and sagittal sinus PO2) known.
In response to 40 min of acute isocapnic hypoxia, in low altitude animals cerebral cortical blood flow increased to 44 ± 2% above baseline (similar to that of earlier studies; Ashwal et al. 1980; Blood et al. 2002; Hunter et al. 2003b). In the high altitude acclimatized fetus, in contrast, in response to superimposed hypoxia LD-CBF increased only one-half that value. At first, this attenuated response may appear counterintuitive, as one might predict a more robust response in the chronically hypoxic fetus. However, in view of the significant increase in haemoglobin concentration and arterial O2 content, this may be quite reasonable. If one assumes a normal CMRO2 in the high altitude fetus, then with a given degree of hypoxaemia in the presence of significantly elevated blood O2 capacity, the requirement for a smaller increase in CBF could be anticipated. Additional factors may account for the blunted CBF response to superimposed hypoxia. Chronic hypoxia alters the composition and reactivity of cerebral arteries and depresses the magnitude of depolarization-induced contraction (Longo et al. 1993), and decreases densities of several receptors (Ueno et al. 1997; Zhou et al. 1997). In addition, chronic hypoxia appears to attenuate the ability of the ATP-sensitive potassium channel to promote relaxation (Long et al. 2002; Longo & Pearce, 2005). Other studies have shown that chronic hypoxia depresses the function of nitric oxide releasing nerves in the cerebral arteries, due to decreased expression of neuronal nitric oxide synthase in the perivascular nerves (Mbaku et al. 2003). Additionally, chronic hypoxia depresses endothelial vasodilatory capacity, attributed to depression of endothelial nitric oxide synthesis (Aguan et al. 1998). In the fetuses of both altitude groups, with a curvilinear analysis the threshold for hypoxia-induced increase in CBF appeared to be at or near a Pa,O2 value of 16 ± 1 Torr, only a few Torr less than the baseline value of the high altitude group.
Cortical tissue PO2
Cerebral cortical tPO2 represents local tissue oxygenation, and reflects Pa,O2, Pa,CO2, CBF, haemoglobin O2 affinity, diffusion distance from the capillaries, O2 delivery and CMRO2 (Kreuzer, 1982; Jones & Traystman, 1984; Wagerle & Jones, 1998), and is a rather dynamic entity (Duling et al. 1979; Kreuzer, 1982; Doppenberg et al. 1998; Ereciñska & Silver, 2001; Tsai et al. 2003). In the fetuses at low and high altitude, baseline cortical tPO2 was 8 ± 1 and 6 ± 1 Torr, respectively, decreasing to 2 ± 1 Torr (P < 0.01) (Table 1) in each group in response to acute hypoxia. Neither control nor hypoxic-induced values differed significantly between the two altitude groups, probably reflecting the compensatory mechanisms of successful acclimatization.
Sagittal sinus blood gas values
In critically ill newborn infants and adults, cerebral venous PO2 and/or [HbO2] are used to assess oxygenation status (Siggaard-Andersen et al. 1995; Ahmad et al. 2000; Macmillan & Andrews, 2000; Dunn et al. 2006). At both altitudes, the present study demonstrates the significant decreases in fetal sagittal sinus PO2 and [HbO2] in concert with decreases of cortical tPO2, in response to superimposed hypoxia (Fig. 3). The observation that sagittal sinus PO2 and [HbO2] values were identical in fetuses of both altitude groups (Table 1) strongly suggests successful acclimatization, and adequacy of cerebral oxygenation. For the fetuses at high altitude this may have been a consequence of greater O2 extraction, as compared with baseline, and the greater blood O2 capacity. In both altitude groups, fetal sagittal sinus PO2 varied linearly with Pa,O2 (Fig. 3A), while the relation of both sagittal sinus PO2 to cortical tPO2 (Fig. 3B), and of sagittal sinus [HbO2] to cortical tPO2 (Fig. 3C) was flat above a tPO2 of 4 Torr. This fits with the observation that the relation of cortical tPO2 to Pa,O2 did not differ significantly in the two altitude groups (Fig. 2B). It also reinforces the idea that sagittal sinus blood gases may provide a reasonable assessment of brain tissue oxygenation.
Cerebral metabolic rate for O2
The cerebral metabolic rate for O2 is one of the important factors in cerebral tissue oxygenation. Nonetheless, the relation of CMRO2 to CBF remains poorly documented, and the mechanisms that regulate this linkage even more so (Vannucci & Vannucci, 2004). Also of note, by use of radioactive microsphere measurements, and obtaining the cerebral arterial–venous O2 difference in fetuses at sea level, several reports have demonstrated a moderate increase in CBF with the low voltage, high frequency EEG state, as compared with the high voltage, low frequency state (Richardson et al. 1989; Morrison et al. 2005). The present study did not include EEG state.
The relation of fetal CMRO2 to CBF is an important consideration. In the low altitude fetus, cerebral fractional O2 extraction remained constant during acute hypoxia (Fig. 4C). In contrast, in the high altitude fetus, baseline fractional O2 extraction was significantly lower (Table 1), increasing in response to superimposed hypoxia to a value similar to that of the low altitude fetus. The mechanisms of the lower fractional O2 extraction under control conditions in the high altitude fetus is unknown, but may be a consequence of its increase in haemoglobin concentration and arterial O2 content, or perhaps a decreased diffusion distance of O2.
In the fetuses at both altitudes, relative CMRO2 decreased 50% in response to superimposed hypoxia (Fig. 4D). This finding agrees with several studies in adult animals that have demonstrated significantly reduced CMRO2 in response to hypoxia (Haggendal & Norback, 1966; Gardiner, 1980; Gautier & Murariu, 1999). However, a caveat must be noted. One group has suggested that fetal CMRO2 remains relatively constant in response to acute hypoxia (Jones & Traystman, 1984). As we have shown, LD-CBF may underestimate the CBF increase as compared to the microsphere method by 9% (Bishai et al. 2003). If, in fact the hypoxic-induced increase in cerebral blood flow was greater than that we recorded with laser-Doppler, the decrease in CMRO2 would not be as great, 40% perhaps. Thus, although we believe the present results to be valid, the effect of superimposed hypoxia on fetal CMRO2 requires further study. The degree to which fetal Pa,O2 can fall and fractional O2 extraction can increase before CMRO2 decreases, that is the ‘oxygen margin of safety’, may be an indication of the oxygen reserve available to the fetus (Richardson & Bocking, 1998).
Perspective
For the fetus at either low or high altitude, the present study gives the first measurements of isocapnic hypoxic-induced changes in cerebral blood flow, cortical tissue PO2, and sagittal sinus blood gas values, and their relation to the Pa,O2 and to cerebral metabolic rate for O2. In the fetus at high altitude, in response to 40 min isocapnic hypoxia, LD-CBF increased by only one-half that of low altitude controls. Concurrently, fetal cortical tPO2 decreased to a similar extent in both altitude groups. Also in both groups, sagittal sinus PO2 and [HbO2] values were similar, and importantly they reflected cortical tPO2. In addition, the arterial lactate and pH values were similar. As noted above, acidosis increased as a function of duration of hypoxia. In light of the role of acidosis in neuronal injury, and the significant association of acidosis at birth with newborn and childhood encephalopathy and neurological deficits (Vannucci & Yager, 1992), these observations are of considerable relevance.
Overall, these data suggest that in response to high altitude long-term hypoxia, the fetus successfully acclimatized or ‘adapted’, despite having a significantly lower Pa,O2 value (in association with a considerably decreased maternal Pa,O2). In conclusion, we suggest that the present study serves as a reference point for further in vivo and in vitro studies of cellular and subcellular mechanisms of the regulation of CBF and tissue oxygenation in the fetus, and mechanisms of successful acclimatization to long-term hypoxaemia.
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