Noninvasive Assessment of Cerebral Oxygenation during Acclimation to Hypobaric Hypoxia

Abstract

Factors regulating cerebral tissue Po₂ (P_tO₂) are complex. With the increased use of clinical P_tO₂ monitors, it has become important to elucidate these mechanisms. The authors are investigating a new methodology (electron paramagnetic resonance oximetry) for use in monitoring cerebral P_tO₂ in awake animals over time courses of weeks. The authors used this to study cerebral P_tO₂ in rats during chronic acclimation to hypoxia predicting that such acclimation would cause an increase in P_tO₂ because of increases that occur in capillary density and oxygen carrying capacity. The average P_tO₂ between 7 and 21 days was increased by 228% over controls.

An understanding of the factors that influence cerebral tissue Po₂ (P_tO₂) may provide insights into the control of brain oxygenation during acclimation to chronic hypoxia, and should also help us to understand regulatory mechanisms in both healthy and diseased conditions. The authors are investigating a new methodology (electron paramagnetic resonance or EPR oximetry) for use in monitoring P_tO₂ over time courses of weeks (Liu et al., 1993; Rolett et al., 2000). The authors applied EPR oximetry to the study of P_tO₂ in rats during chronic acclimation to hypoxia.

Chronic acclimation to hypoxia has been shown to increase microvascular density in brain (Boero et al., 1998; Diemer and Henn, 1965; Harik et al., 1995, 1996; LaManna et al., 1992, 1994; Miller and Hale, 1970; Opitz, 1951). Oxygen carrying capacity also increases, whereas the oxyhemoglobin dissociation curve in rats may or may not exhibit a right shift with altitude (Baumann et al., 1971; LaManna et al., 1992; Monge and Leon-Velarde, 1991; Turek et al., 1972). In addition, the sensitivity of cerebral blood flow to CO₂ is thought to increase (Severinghaus et al., 1966). These changes should result in increases in cerebral P_tO₂, unless there are homeostatic mechanisms that operate to maintain a relatively static level of P_tO₂ in awake animals.

The authors chronically adapted rats to one half an atmosphere of barometric pressure for 27 days and measured P_tO₂ when animals were breathing normobaric gases (nonanesthetized and spontaneously ventilating).

MATERIALS AND METHODS

Crystals of lithium phthalocyanine (LiPc) were used as the oxygen sensitive material (Rolett et al., 2000). The LiPc crystals were individually calibrated (Fig. 1). These data show that the calibration was linear over at least 0 to 156 mm Hg. Crystals of approximately 50 × 500 μm (0.05 mg) were implanted using a 25G needle into the frontal cortex under ketamine/xylazine anesthesia. Electron paramagnetic resonance oximetry was performed using a low frequency (1.18 GHz, L-band) spectrometer. For measurements, awake animals were placed in a plexiglas restraining tube, and the head was positioned below the EPR surface-loop resonator. The Po₂ was calculated from multiple EPR spectra (scan time 5 to 20 seconds), averaged over 5 minutes.

FIG. 1.

Calibration curve of lithium phthalocyanine (LiPc) for Po₂. (A) Example electron paramagnetic resonance (EPR) spectra from LiPc showing how the spectrum changes when LiPc is exposed to 0 mm Hg and 35.6 mm Hg O₂. (B) Calibration curve of LiPC against premixed gases of known Po₂ showing the linearity over the range of interest.

Acclimation was performed in 40-gallon drums fitted to a vacuum line to reduce pressure to 370 ± 2.1 mm Hg (range 367 to 372, mean ± SD), corresponding to an altitude of approximately 6000 m. Control subjects were placed in a drum at normobaric pressures. Ambient temperatures varied from 22.2°C to 24.4°C. Animals were housed to a maximum of 6 per drum.

The LiPc was implanted 7 days before acclimation. Each rat underwent the Po₂ measurement protocol once as a training experience at least 1 day before obtaining preacclimation data. Preacclimation P_tO₂ was measured 2 to 3 days before acclimation. Measurements were taken at 4, 7, 10, 14, 19, 23, and 27 days after onset of the acclimation period. Hypobaric animals were at normobaric pressures for at least 2 hours and for a maximum of 6 hours before measuring cerebral Po₂. Animals were out of the chambers for a maximum of 9 hours on the measurement days. Blood gases and hematocrit were not measured to minimize trauma.

Analysis of the data on the time course of P_tO₂ was performed using a general linearized model, followed by a Mann—Whitney U test (P < 0.05 was significant).

RESULTS

The LiPc was in close proximity to the surrounding tissue with no evidence of edema, red blood cell accumulation (vascular damage), or lymphocyte infiltration (Fig. 2). This was consistent with a previous report on the histologic appearance of LiPc implants in cortical tissue (Rolett et al., 2000).

FIG. 2.

Histologic appearance of the electron paramagnetic resonance (EPR) oximetry material (lithium phthalocyanine [LiPC]) in the rat cortex. Dark area is the crystal of LiPc shown in a histologic section stained with cresyl violet. Viable tissue is in close approximation to the crystal. There is no evidence of swelling, red blood cells, or local vascular damage. Arrows indicate a small blood vessel in proximity to the implant. The section was taken 7 days after implanting the material, fixed in phosphate buffered formalin, and embedded in paraffin.

Figure 3 shows the time course of changes in cerebral P_tO₂, measured in awake, restrained animals, at normobaric pressures. The P_tO₂ before acclimation was not significantly different (27.1 ± 7.5 vs. 26.0 ± 15, n = 6, mean ± SD in the acclimated and normobaric groups). The P_tO₂ in the acclimated versus control groups were significantly different by day 4 (the first EPR measurement during acclimation), with values of 49 ± 11 mm Hg versus 24 ± 6 mm Hg, respectively. The P_tO₂ in acclimated animals increased rapidly in the first 7 days, then stabilized for the duration of the study. The average P_tO₂ in the acclimated group (from 7 days on) was 62 mm Hg versus 26 mm Hg in the control groups, an increase of 238%.

FIG. 3.

The P_tO₂ in brains of animals being acclimated to chronic hypoxia. Cortical P_tO₂ was measured when both groups were breathing normoxic FiO₂ at normobaric pressure. Closed circles represent rats living at one-half atmospheric pressures. Open circles designate rats living at normobaric pressures.

DISCUSSION

Electron paramagnetic resonance oximetry is a relatively new method for monitoring cerebral P_tO₂ (Liu et al., 1993; Rolett et al., 2000). Although trauma can occur during implant, the damage is minimal and would be similar to that which would occur when using a micro-electrode of similar size to the needle. However, because the measurements were obtained more than 5 days after the implant, there was time for the tissue to recover (Hoopes et al., 1997; Rolett et al., 2000). This feature, and the capability to measure P_tO₂ in awake animals, makes EPR an attractive alternative.

Because the implant is large, with respect to cells and capillary units, the P_tO₂ will likely reflect an average value and may be more comparable to mean Po₂ values obtained with microelectrodes of smaller size (Goda et al., 1995; Swartz et al., 1996). The current study's control values of 27 mm Hg were similar to the average rat cortical value of 29 ± 5 mm Hg obtained under isoflurane anesthesia using microelectrodes (Seyde and Longnecker, 1986), and 31 ± 5 mm Hg when using urethane (Metzger et al., 1980). The authors' values were also similar to the value of 33 mm Hg measured in human parenchyma during surgery (Charbel et al., 1997).

Studies with animal models show that there is vascular remodeling in response to chronic hypoxia. Early histologic studies show increased capillary density in neural tissue after chronic exposure to hypoxia (Diemer and Henn, 1965; Opitz, 1951). More recent studies of microvessel density confirm that acclimation increases density and segment length in studies on rats, mice, and cats (Boero et al., 1998; Cervos-Navarro et al., 1991; Harik et al., 1995; LaManna et al., 1992; Mironov et al., 1994).

Interestingly, the time course of the increase in vascularity does not match the changes in the regulation of cerebral oxygenation observed in this study. Rats acclimated to a similar pressure did not have an increased microvessel density after 4 days of chronic hypoxia (Harik et al., 1996). However, in the current study, there was a significant increase in P_tO₂ after 4 days and the maximum level was reached by 7 days.

Elevated P_tO₂ values would be expected if perfusion was higher than was required for oxygen utilization. A possible cause of an elevation in cerebral blood flow (CBF) would be a relative hypoventilation on returning to normobaric high oxygen environment, which would result in an increase in Paco₂ and stimulation of CBF. Calculations indicate that in humans acclimated to altitude, if arterial Paco₂ is elevated to preacclimation levels, the CBF would be approximately 60% higher than if their Paco₂ was allowed to remain low (Severinghaus et al., 1966). Because cerebral metabolic rate for oxygen appears to change little (Severinghaus et al., 1966), the change in co₂ reactivity could result in an increase in CBF and P_tO₂ in the acclimated rats when they are breathing normobaric oxygen. When Pco₂ was measured in a similar rat model to that used in the current study, the values increased considerably from the acclimated value of 18 mm Hg to 28 mm Hg (LaManna et al., 1992) when animals were exposed to normobaric conditions. In a similar study, rats acclimated to 380 mm Hg showed a marked reduction of Paco₂ relative to normoxic controls, followed by an increase to 31 mm Hg when exposed to normobaric conditions (Aaron and Powell, 1993).

However, Lamanna et al. (1992) measured CBF and found it to be similar to the normoxic controls, and similar to or less than in rats acclimated to hypoxia and measured during hypoxia. Their flow data do not indicate that CBF is high in the brains of rats acclimated to hypoxia but breathing normobaric gasses. These CBF data indicate that rats do not show the increased sensitivity to CO₂ that has been reported in humans.

Even if there was a marked increase in CBF, the reported changes in P_tO₂ indicate that this variable could not account for the magnitude of the change observed in the current study. In rats anesthetized with sodium pentobarbital, exposed to inspired values of Pco₂ as high as 10%, P_tO₂ increased by 150% to 180% (Metzger et al., 1971), whereas the authors observed an increase of 238% in the current study.

Conclusions

Electron paramagnetic resonance oximetry was an effective and sensitive method for measuring cerebral P_tO₂ over a period of weeks in awake animals. These data show that the model of chronic hypoxia exhibits a large elevation in cerebral P_tO₂. The model would allow for studying factors that influence P_tO₂, such as the relative impact of changes in vessel density, oxygen carrying capacity, and CBF. A host of questions could be asked with respect to chronic adaptation to hypoxia: What are the values for P_tO₂ at the acclimated oxygen levels? Would chronic exposure to high oxygen result in a relative hypoxia on returning to normal oxygen? What oxygen levels would cause the acclimation observed in this study? Could they be achieved by duty cycles such as those encountered by flight crews? Also, Does the change in P_tO₂ indicate a time course of angiogenesis that may relate to the onset of high altitude cerebral edema?