Effects of hypoxia, hyperoxia, and hypercapnia on baseline and stimulus-evoked BOLD, CBF, and CMRO₂ in spontaneously breathing animals

Abstract

Functional magnetic resonance imaging (fMRI) was used to investigate the effects of inspired hypoxic, hyperoxic, and hypercapnic gases on baseline and stimulus-evoked changes in blood oxygenation level-dependent (BOLD) signals, cerebral blood flow (CBF), and the cerebral metabolic rate of oxygen (CMRO₂) in spontaneously breathing rats under isoflurane anesthesia. Each animal was subjected to a baseline period of six inspired gas conditions (9% O₂, 12% O₂, 21% O₂, 100% O₂, 5% CO₂, and 10% CO₂) followed by a superimposed period of forepaw stimulation. Significant stimulus-evoked fMRI responses were found in the primary somatosensory cortices. Relative fMRI responses to forepaw stimulation varied across gas conditions and were dependent on baseline physiology, whereas absolute fMRI responses were similar across moderate gas conditions (12% O₂, 21% O₂ 100% O₂, and 5% CO₂) and were relatively independent of baseline physiology. Consistent with data obtained using well-established techniques, baseline and stimulus-evoked CMRO₂ were invariant across moderate physiological perturbations thereby supporting a CMRO₂-fMRI technique for non-invasive CMRO₂ measurement. However, under 9% O₂ and 10% CO₂, stimulus-evoked CBF and BOLD were substantially reduced and the CMRO₂ formalism appeared invalid, likely due to attenuated neurovascular coupling and/or a failure of the model under extreme physiological perturbations. These findings demonstrate that absolute fMRI measurements help distinguish neural from non-neural contributions to the fMRI signals and may lend a more accurate measure of brain activity during states of altered basal physiology. Moreover, since numerous pharmacologic agents, pathophysiological states, and psychiatric conditions alter baseline physiology independent of neural activity, these results have implications for neuroimaging studies using relative fMRI changes to map brain activity.

Introduction

Functional magnetic resonance imaging (fMRI) commonly measures relative (percent) changes in blood-oxygenation-level dependent (BOLD) contrast, cerebral blood flow (CBF) and cerebral blood volume (CBV). These relative fMRI responses are highly sensitive to baseline physiological parameters such as CBF, CBV, tissue oxygenation, and oxidative metabolism (Corfield et al., 2001, Ramsay et al., 1993). Factors that affect these physiological parameters are numerous and include anesthetics, respiration rate, blood pressure, endogenous hormones, emotional states, diseases, drugs of abuse, and many commonly ingested substances such as caffeine (Dager et al., 1999), nicotine (Jacobsen et al., 2002), and alcohol (Levin et al., 1998). These observations suggest caution when comparing fMRI results across subjects whose baseline physiological parameters may differ. They also suggest that absolute fMRI signal change (i.e., change relative to a single fixed baseline state serving as a control), rather than relative fMRI signal change (i.e., change relative to its own respective baseline state), is a more accurate index of brain activity in situations where baseline physiology is markedly perturbed, such as in pharmacologic or disease-induced states. In order to unambiguously identify key baseline physiological factors that modulate fMRI responses, baseline and stimulus-evoked CBF and BOLD signals must be systematically evaluated under various perturbations of baseline cerebral blood flow and tissue oxygenation, with such perturbations being accomplished via inspiration of hypoxic, hyperoxic, or hypercapnic gases. It is hypothesized that, by determining absolute and relative stimulus-evoked changes in CBF and BOLD signals under various physiological perturbations in the same subjects and in the same experimental setting, the validity of using relative stimulus-evoked fMRI signal change as an indicator of neuronal activity under different basal physiological conditions can be tested. In addition, the cerebral metabolic rate of oxygen (CMRO₂) (Davis et al., 1998, Hoge et al., 1999, Liu et al., 2004, Mandeville et al., 1998), calculated from these measurements using Davis' CMRO₂ formalism, could be utilized to investigate metabolic changes under different baseline physiologies with and without functional stimulation.

Davis et al. (1998) introduced an eloquent formalism based on the BOLD biophysical model (Ogawa et al., 1993) to determine CMRO₂. The advantage of Davis' formalism over existing formalisms is that there are no a priori assumptions regarding resting capillary or venous oxygen saturation, blood volume fraction, blood flow, and metabolic rate of oxygen. All these parameters and other physiological quantities are lumped into the constant M which can be measured on a pixel-by-pixel basis. A modified CMRO₂ model has been proposed to take into account the arterial–venous blood volume contributions and includes a non-steady state determination of CMRO₂ (Wu et al., 2002). While Hoge et al. (1999), Mandeville et al. (1999), and others have reproduced and extended Davis' findings. However, no studies have been conducted to support the CMRO₂-MRI model under physiologically perturbed baseline states. Since it is well established that moderate and transient perturbations in arterial oxygen and carbon-dioxide partial pressures (PaO₂ and PaCO₂) per se do not change CMRO₂ (Kety and Schmidt, 1948, Novack et al., 1953), it is hypothesized that CMRO₂ values derived using Davis' formalism would be invariant under such perturbations. The same approach can be used to further test the integrity CMRO₂-MRI formalism under relatively more severe physiological perturbations. In addition, the effect of baseline conditions on stimulus-evoked neural activity can be assessed by performing somatosensory stimulation in the presence of different gas modulations.

Experiments demonstrating the self-consistency of the Davis formalism are arguably best performed in animal models under well-controlled conditions and for these reasons the established forepaw stimulation rat model is ideal. Essentially all fMRI studies of forepaw stimulation in rats use α-chloralose as the anesthetic (Duong et al., 2000, Mandeville et al., 1998, Silva et al., 1999) which has been shown to minimally perturb neural activity and hemodynamic coupling (Ueki et al., 1992). However, achieving a stable anesthesia over long durations with α-chloralose is relatively difficult and generally requires mechanical ventilation and invasive blood-gas sampling; thus, it is less suited for prolonged experiments that require repeated fMRI measurements. Our lab recently demonstrated that forepaw stimulation in rats under isoflurane anesthesia and spontaneously breathing conditions is well suited for repeated fMRI measurements because the animals can maintain stable physiology throughout the experiment (Liu et al., 2004). Of equal importance is the use of a quantitative (absolute) CBF technique based on the two-coil continuous arterial spin-labeling method in which BOLD and CBF can be simultaneously measured (Duong et al., 2000, Silva et al., 1999). This technique produces relatively high CBF contrast and eliminates inter-trial variations in CBF and BOLD responses associated with sequential measurements of these parameters, and enables calculation of CMRO₂ under different baseline physiological conditions with and without forepaw stimulation using Davis' formalism. The main goals of this study were: (i) to characterize the effects of inspired hypoxic, hyperoxic, and hypercapnic gases on baseline and forepaw-stimulation induced changes in CBF, BOLD, and CMRO₂ under spontaneously breathing conditions, and (ii) to use these findings to test the self-consistency of Davis' CMRO₂-MRI technique under relatively moderate and severe physiological perturbations.