Effects of hypoxia, hyperoxia, and hypercapnia on baseline and stimulus-evoked BOLD, CBF, and CMRO2 in spontaneously breathing animals
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.
Section snippets
Animal preparation
Nine male Sprague–Dawley rats (300–340 g) were initially anesthetized with the vaporizer set to 2.0% isoflurane. Needle electrodes were inserted subcutaneously into the forepaws. In 5 out of 9 rats, the femoral artery was catheterized for continuous recording of heart rate (HR) and mean arterial blood pressure (MABP). Blood gases were sampled once for each gas condition. Respiration rate (RR) was derived from the slow modulations on top of the cardiac waveforms. Animals were secured in an
Physiological measurements
Physiological parameters are summarized in Table 1. HR, MABP, and RR during 21% O2 were consistent with those reported previously under similar experimental conditions (Liu et al., 2004) and were stable during the breaks between gas challenges. All of the following changes are relative to 21% O2 and are statistically significant (P < 0.05) unless otherwise noted. Inhalation of hypoxic gas decreased HR, MABP, PaCO2, PaO2, and arterial oxygen saturation (SaO2), and increased RR and arterial pH.
Potential drawbacks of the isoflurane-anesthetized forepaw-stimulation model
The use of isoflurane anesthesia has some drawbacks. First, isoflurane suppresses neural activity which could explain the higher stimulation current needed relative to that used under α-chloralose (1.5–2.0 mA; Duong et al., 2000, Silva et al., 1999) which could potentially cause pain-induced changes in MABP and HR, as well as activation outside the somatosensory pathway. However, the applied current was previously optimized to elicit robust fMRI responses without producing sustained changes in
Acknowledgments
We thank Drs. K. Uludag and J.B. Mandeville for their critical and helpful comments. This work was supported by grants from the American Heart Association (SDG-0430020N) and the National Institute of Health (NEI R01-EY014211, NINDS R01-NS45879).
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