Analysis of oxygen metabolism implies a neural origin for the negative BOLD response in human visual cortex

The sustained negative blood oxygenation level-dependent (BOLD) response in functional MRI is observed universally, but its interpretation is controversial. The origin of the negative response is of fundamental importance because it could provide a measurement of neural deactivation. However, a substantial component of the negative response may be due to a non-neural hemodynamic artifact. To distinguish these possibilities, we have measured evoked BOLD, cerebral blood flow (CBF), and oxygen metabolism responses to a fixed visual stimulus from two different baseline conditions. One is a normal resting baseline, and the other is a lower baseline induced by a sustained negative response. For both baseline conditions, CBF and oxygen metabolism responses reach the same peak amplitude. Consequently, evoked responses from the negative baseline are larger than those from the resting baseline. The larger metabolic response from negative baseline presumably reflects a greater neural response that is required to reach the same peak amplitude as that from resting baseline. Furthermore, the ratio of CBF to oxygen metabolism remains approximately the same from both baseline states (approximately 2:1). This tight coupling between hemodynamic and metabolic components implies that the magnitude of any hemodynamic artifact is inconsequential. We conclude that the negative response is a functionally significant index of neural deactivation in early visual cortex.     

Dynamic models of BOLD contrast

This personal recollection looks at the evolution of ideas about the dynamics of the blood oxygenation level dependent (BOLD) signal, with an emphasis on the balloon model. From the first detection of the BOLD response it has been clear that the signal exhibits interesting dynamics, such as a pronounced and long-lasting post-stimulus undershoot. The BOLD response, reflecting a change in local deoxyhemoglobin, is a combination of a hemodynamic response, related to changes in blood flow and venous blood volume, and a metabolic response related to oxygen metabolism. Modeling is potentially a way to understand the complex path from changes in neural activity to the BOLD signal. In the early days of fMRI it was hoped that the hemodynamic/metabolic response could be modeled in a unitary way, with blood flow, oxygen metabolism, and venous blood volume-the physiological factors that affect local deoxyhemoglobin-all tightly linked. The balloon model was an attempt to do this, based on the physiological ideas of limited oxygen delivery at baseline and a slow recovery of venous blood volume after the stimulus (the balloon effect), and this simple model of the physiology worked well to simulate the BOLD response. However, subsequent experiments suggest a more complicated picture of the underlying physiology, with blood flow and oxygen metabolism driven in parallel, possibly by different aspects of neural activity. In addition, it is still not clear whether the post-stimulus undershoot is a hemodynamic or a metabolic phenomenon, although the original venous balloon effect is unlikely to be the full explanation, and a flow undershoot is likely to be important. Although our understanding of the physics of the BOLD response is now reasonably solid, our understanding of the underlying physiological relationships is still relatively poor, and this is the primary hurdle for future models of BOLD dynamics.     

Attentional modulation in the human visual cortex: the time-course of the BOLD response and its implications

Throughout the visual areas of the brain, the sensory response to a stimulus is enhanced by attending to the stimulus. Neurophysiological studies in primates show that such enhancement is marked in posterior parietal cortex and some anterior occipital areas, but much more modest in the earliest processing stages, such as the primary visual cortex (V1). In contrast, human fMRI studies show large and robust attentional modulation in all visual areas, including V1. We investigate the possibility that, in the case of fMRI, the BOLD (blood oxygen level dependent) response may be increased not only by local attention-related increases in neural activity, but also by local blood-flow increases caused by remote control systems that anticipate an impending need for oxygen at the attended location. Such changes could be much more rapid than the rather slow response to oxygenation change that typifies the BOLD response. We have employed a paradigm that isolates the component of the BOLD response due to attentional modulation and the component due to the mere presence of a visual stimulus. The results show that the temporal profiles of the BOLD responses in human V1 to the onset of a stimulus and to the onset of attention are extremely similar. The time-course of the attention-related BOLD response is not consistent with the action of remote, anticipatory control mechanisms and suggests that the modulatory effect of attention seen in human V1 with fMRI probably reflects genuine changes in local neural activity that are considerably larger than in non-human primates.     

Prospects for quantitative fMRI: investigating the effects of caffeine on baseline oxygen metabolism and the response to a visual stimulus in humans

Functional magnetic resonance imaging (fMRI) provides an indirect reflection of neural activity change in the working brain through detection of blood oxygenation level dependent (BOLD) signal changes. Although widely used to map patterns of brain activation, fMRI has not yet met its potential for clinical and pharmacological studies due to difficulties in quantitatively interpreting the BOLD signal. This difficulty is due to the BOLD response being strongly modulated by two physiological factors in addition to the level of neural activity: the amount of deoxyhemoglobin present in the baseline state and the coupling ratio, n, of evoked changes in blood flow and oxygen metabolism. In this study, we used a quantitative fMRI approach with dual measurement of blood flow and BOLD responses to overcome these limitations and show that these two sources of modulation work in opposite directions following caffeine administration in healthy human subjects. A strong 27% reduction in baseline blood flow and a 22% increase in baseline oxygen metabolism after caffeine consumption led to a decrease in baseline blood oxygenation and were expected to increase the subsequent BOLD response to the visual stimulus. Opposing this, caffeine reduced n through a strong 61% increase in the evoked oxygen metabolism response to the visual stimulus. The combined effect was that BOLD responses pre- and post-caffeine were similar despite large underlying physiological changes, indicating that the magnitude of the BOLD response alone should not be interpreted as a direct measure of underlying neurophysiological changes. Instead, a quantitative methodology based on dual-echo measurement of blood flow and BOLD responses is a promising tool for applying fMRI to disease and drug studies in which both baseline conditions and the coupling of blood flow and oxygen metabolism responses to a stimulus may be altered.     

A three-compartment model of the hemodynamic response and oxygen delivery to brain

We describe a mathematical model linking changes in cerebral blood flow, blood volume and the blood oxygenation state in response to stimulation. The model has three compartments to take into account the fact that the cerebral blood flow and volume as measured concurrently using laser Doppler flowmetry and optical imaging spectroscopy have contributions from the arterial, capillary as well as the venous compartments of the vasculature. It is an extension to previous one-compartment hemodynamic models which assume that the measured blood volume changes are from the venous compartment only. An important assumption of the model is that the tissue oxygen concentration is a time varying state variable of the system and is driven by the changes in metabolic demand resulting from changes in neural activity. The model takes into account the pre-capillary oxygen diffusion by flexibly allowing the saturation of the arterial compartment to be less than unity. Simulations are used to explore the sensitivity of the model and to optimise the parameters for experimental data. We conclude that the three-compartment model was better than the one-compartment model at capturing the hemodynamics of the response to changes in neural activation following stimulation.     

Calibrated fMRI during a cognitive Stroop task reveals reduced metabolic response with increasing age

fMRI studies of aging have revealed increased blood oxygenation level dependent (BOLD) response to tasks of executive function with advancing age, which is generally interpreted as increased neural activity. However, changes in the cerebrovascular system with age can alter the BOLD signal, complicating this interpretation. Arterial spin labeling (ASL) allows simultaneous acquisition of BOLD and cerebral blood flow (CBF) information and can be used to quantify the component parts of the BOLD signal. We used this calibrated BOLD approach in 58 healthy participants over an age range of 18-71 years to determine the relative vascular and neuronal contributions to the age-related BOLD changes in response to a Stroop task. The percentage BOLD response increased significantly with increasing age but the percentage CBF response did not alter, such that the BOLD increase is attributed to a significant reduction in the oxygen metabolism response with increasing age. Hence, in this study, the BOLD increase with age should be interpreted as a reduction in neural activity. The greatest percentage BOLD increases with age were found in the left and right medial frontal gyri and the primary motor cortex and were again linked to a reduction in oxygen metabolism. On separating the participants into three groups (young, old high performers and old low performers), age-related differences in percentage BOLD response and oxygen metabolism response could be attributed to the low performing old group. This study demonstrates the need to take into account alterations in vascular-metabolic coupling and resting blood volume when interpreting changes in the BOLD response with aging.     

Modeling the hemodynamic response to brain activation

Neural activity in the brain is accompanied by changes in cerebral blood flow (CBF) and blood oxygenation that are detectable with functional magnetic resonance imaging (fMRI) techniques. In this paper, recent mathematical models of this hemodynamic response are reviewed and integrated. Models are described for: (1) the blood oxygenation level dependent (BOLD) signal as a function of changes in cerebral oxygen extraction fraction (E) and cerebral blood volume (CBV); (2) the balloon model, proposed to describe the transient dynamics of CBV and deoxy-hemoglobin (Hb) and how they affect the BOLD signal; (3) neurovascular coupling, relating the responses in CBF and cerebral metabolic rate of oxygen (CMRO(2)) to the neural activity response; and (4) a simple model for the temporal nonlinearity of the neural response itself. These models are integrated into a mathematical framework describing the steps linking a stimulus to the measured BOLD and CBF responses. Experimental results examining transient features of the BOLD response (post-stimulus undershoot and initial dip), nonlinearities of the hemodynamic response, and the role of the physiologic baseline state in altering the BOLD signal are discussed in the context of the proposed models. Quantitative modeling of the hemodynamic response, when combined with experimental data measuring both the BOLD and CBF responses, makes possible a more specific and quantitative assessment of brain physiology than is possible with standard BOLD imaging alone. This approach has the potential to enhance numerous studies of brain function in development, health, and disease.     

Simultaneous recording of task-induced changes in blood oxygenation, volume, and flow using diffuse optical imaging and arterial spin-labeling MRI

Increased neural activity in brain tissue is accompanied by an array of supporting physiological processes, including increases in blood flow and the rates at which glucose and oxygen are consumed. These responses lead to secondary effects such as alterations in blood oxygenation and blood volume, and are ultimately the primary determinants of the amplitude and temporal signature of the blood oxygenation level-dependent (BOLD) signal used prevalently to map brain function. We have performed experiments using a combination of optical and MRI-based imaging methods to develop a more comprehensive picture of the physiological events accompanying activation of primary motor cortex during a finger apposition task. Temporal profiles for changes in tissue hemoglobin concentrations were qualitatively similar to those observed for MRI-based flow and oxygenation signals. Quantitative analysis of these signals revealed peak changes of +16 +/- 2% for HbO, -13 +/- 2% for HbR, +8 +/- 3% for total Hb, +83 +/- 9% for cerebral blood flow, and +1.4 +/- 0.1% for the BOLD MRI signal. A mass balance model was used to estimate the change in rate of oxidative metabolism implied by the optical and flow measurements, leading to a computed value of +47 +/- 5%. It should be noted that the optical and MRI observations may in general reflect changes over different volumes of tissue. The ratio of fractional changes in oxidative metabolism to fractional change in blood flow was found to be 0.56 +/- 0.08, in general agreement with previous studies of flow-metabolism coupling.     

Retinotopic maps and hemodynamic delays in the human visual cortex measured using arterial spin labeling

Cortical representations of the visual field are organized retinotopically, such that nearby neurons have receptive fields at nearby locations in the image. Many studies have used blood oxygenation level-dependent (BOLD) fMRI to non-invasively construct retinotopic maps in humans. The accuracy of the maps depends on the spatial extent of the metabolic and hemodynamic changes induced by the neural activity. Several studies using gradient-echo MRI at 1.5 T and 3 T showed that most of the BOLD signal originates from veins, which might lead to a spatial displacement from the actual site of neuronal activation, thus reducing the specificity of the functional localization. In contrast to BOLD signal, cerebral blood flow (CBF) as measured using arterial spin labeling (ASL) is less or not at all affected by remote draining veins, and therefore spatially and temporally more closely linked to the underlying neural activity. In the present study, we determined retinotopic maps in the human brain using CBF as well as using BOLD signal in order to compare their spatial relationship and the temporal delays of each imaging modality for visual areas V1, V2, V3, hV4 and V3AB. We tested the robustness and reproducibility of the maps across different sessions, calculated the overlap as well as signal delay times across visual areas. While area boundaries were relatively well preserved, we found systematic differences of response latencies between CBF and the BOLD signal between areas. In summary, CBF data obtained using ASL allows reliable retinotopic maps to be constructed; this approach is, therefore, suitable for studying visual areas especially in close proximity to large veins where the BOLD signal is spatially inaccurate.     

Regional differences in the coupling of cerebral blood flow and oxygen metabolism changes in response to activation: implications for BOLD-fMRI

Functional magnetic resonance imaging (fMRI) based on blood oxygenation level dependent (BOLD) signal changes is a sensitive tool for mapping brain activation, but quantitative interpretation of the BOLD response is problematic. The BOLD response is primarily driven by cerebral blood flow (CBF) changes, but is moderated by M, a scaling parameter reflecting baseline deoxyhemoglobin, and n, the ratio of fractional changes in CBF to cerebral metabolic rate of oxygen consumption (CMRO(2)). We compared M and n between cortical (visual cortex, VC) and subcortical (lentiform nuclei, LN) regions using a quantitative approach based on calibrating the BOLD response with a hypercapnia experiment. Although M was similar in both regions (~5.8%), differences in n (2.21+/-0.03 in VC and 1.58+/-0.03 in LN; Cohen d=1.71) produced substantially weaker (~3.7x) subcortical than cortical BOLD responses relative to CMRO(2) changes. Because of this strong sensitivity to n, BOLD response amplitudes cannot be interpreted as a quantitative reflection of underlying metabolic changes, particularly when comparing cortical and subcortical regions.     

Differential sympathetic neural control of oxygenation in resting and exercising human skeletal muscle.

Metabolic products of skeletal muscle contraction activate metaboreceptor muscle afferents that reflexively increase sympathetic nerve activity (SNA) targeted to both resting and exercising skeletal muscle. To determine effects of the increased sympathetic vasoconstrictor drive on muscle oxygenation, we measured changes in tissue oxygen stores and mitochondrial cytochrome a,a3 redox state in rhythmically contracting human forearm muscles with near infrared spectroscopy while simultaneously measuring muscle SNA with microelectrodes. The major new finding is that the ability of reflex-sympathetic activation to decrease muscle oxygenation is abolished when the muscle is exercised at an intensity > 10% of maximal voluntary contraction (MVC). During high intensity handgrip, (45% MVC), contraction-induced decreases in muscle oxygenation remained stable despite progressive metaboreceptor-mediated reflex increases in SNA. During mild to moderate handgrips (20-33% MVC) that do not evoke reflex-sympathetic activation, experimentally induced increases in muscle SNA had no effect on oxygenation in exercising muscles but produced robust decreases in oxygenation in resting muscles. The latter decreases were evident even during maximal metabolic vasodilation accompanying reactive hyperemia. We conclude that in humans sympathetic neural control of skeletal muscle oxygenation is sensitive to modulation by metabolic events in the contracting muscles. These events are different from those involved in either metaboreceptor muscle afferent activation or reactive hyperemia.     

BOLD response during uncoupling of neuronal activity and CBF

The widely used technique of functional magnetic resonance imaging (fMRI) based on the blood oxygenation level-dependent (BOLD) effect is a tool for the investigation of changes in local brain activity upon stimulation. The principle of measurement is based on the assumption that there is a strong coupling between changes in neural activity, metabolism, vascular response and oxygen extraction in the area under investigation. As fMRI is on the way to become a routine tool in clinical examinations, we wanted to investigate whether, generally and under a variety of conditions, there is a strong link between the BOLD signal and neural activity. For clinical and experimental application of the method, it is crucial, whether the absence of changes in BOLD signal intensity upon stimulation can always be interpreted as an absence of changes in brain activity. We approached this question by inhibiting the nitric oxide mediated 'neurovascular coupling' via application of 7 nitroindazole. Before and after inhibition of this neurovascular coupling, we acquired evoked potentials and performed fMRI during somatosensory stimulation in rats. Cerebral blood flow response as well as BOLD signal intensity changes following electrical stimulation were abolished within 10 min after application of 7 nitroindazole, whereas somatosensory-evoked potentials were only slightly affected but still clearly detectable. Even 1 h after injection of 7 nitroindazole, there was still remaining electrical activity. Thus, we observed an uncoupling between electrical, i.e., neural activity and the BOLD signal. According to our results, the absence of BOLD signal changes did not permit the conclusion that there was no neural activity in the area under investigation. Our findings are especially relevant for the clinical application of fMRI in patients suffering from cerebrovascular and other brain diseases.     

Photoacoustic microscopy of vascular adaptation and tissue oxygen metabolism during cutaneous wound healing

Cutaneous wounds affect millions of people every year. Vascularization and blood oxygen delivery are critical bottlenecks in wound healing, and understanding the spatiotemporal dynamics of these processes may lead to more effective therapeutic strategies to accelerate wound healing. In this work, we applied multi-parametric photoacoustic microscopy (PAM) to study vascular adaptation and the associated changes in blood oxygen delivery and tissue oxygen metabolism throughout the hemostasis, inflammatory, proliferation, and early remodeling phases of wound healing in mice with skin puncture wounds. Multifaceted changes in the vascular structure, function, and tissue oxygen metabolism were observed during the 14-day monitoring of wound healing. On the entire wound area, significant elevations of the arterial blood flow and tissue oxygen metabolism were observed right after wounding and remained well above the baseline over the 14-day period. On the healing front, biphasic changes in the vascular density and blood flow were observed, both of which peaked on day 1, remained elevated in the first week, and returned to the baselines by day 14. Along with the wound closure and thickening, tissue oxygen metabolism in the healing front remained elevated even after structural and functional changes in the vasculature were stabilized. On the newly formed tissue, significantly higher blood oxygenation, flow, and tissue metabolism were observed compared to those before wounding. Blood oxygenation and flow in the new tissue appeared to be independent of when it was formed, but instead showed noticeable dependence on the phase of wound healing. This PAM study provides new insights into the structural, functional, and metabolic changes associated with vascular adaptation during wound healing and suggests that the timing and target of vascular treatments for wound healing may affect the outcomes.     

To act or not to act. Neural correlates of executive control of learned motor behavior

Successful behavior requires contextual modulation of learned "programs", that is, the retrieval or nonretrieval (inhibition) of behavioral elements depending on situative context. Here we report neural correlates of these elementary aspects of behavior as identified with functional magnetic resonance imaging (fMRI). Inhibition of a "ready-to-go" behavioral program was represented in the brain by reduction of net synaptic activity in the cerebro-cerebellar pathway. The metabolic correlate of inhibition was a multifocal (premotor, primary sensorimotor, superior parietal, cingulate cortex, and cerebellum) decrease of the blood oxygenation level-dependent (BOLD) signal to below the resting state (negative BOLD) with a concomitant decrease of motor cortical excitability. The reverse was true for retrieval. We propose that contextual modulation of learned behavioral programs depends on an interplay of focal increases and decreases of neural activity and that the inhibitory changes are reflected by negative BOLD responses in an extended cerebro-cerebellar network of sensorimotor structures.     

The BOLD response and vascular reactivity during visual stimulation in the presence of hypoxic hypoxia

A disproportionate increase in cerebral blood flow (CBF) relative to the cerebral metabolic rate of oxygen (CMRO(2)), in response to neuronal activation, results in a decreased oxygen extraction fraction (OEF) and hence local 'hyperoxygenation'. The mismatch is the key 'physiological substrate' for blood oxygenation level dependent (BOLD) fMRI. The mismatch may reflect inefficient O(2) diffusion in the brain tissue, a factor requiring maintenance of a steep [O(2)] gradient between capillary bed and neural cell mitochondria. The aim of this study was to assess vascular responsiveness to reduced blood oxygen saturation, using both BOLD fMRI and the CBV-weighted vascular space occupancy (VASO)-dependent fMRI technique, during visual activation in hypoxic hypoxia. Our fMRI results show decreased amplitude and absence of initial sharp overshoot in the BOLD response, while VASO signal was not influenced by decreasing oxygen saturation down to 0.85. The results suggest that the OEF during visual activation may be different in hypoxia relative to normoxia, due to a more efficient oxygen extraction under compromised oxygen availability. The data also indicate that vascular reactivity to brain activation is not affected by mild hypoxia.     

早期应用高压氧对脑梗死患者日常生活活动能力的影响

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Source of nonlinearity of the BOLD response revealed by simultaneous fMRI and NIRS

The nonlinearity of the blood oxygenation level-dependent (BOLD) response to stimuli of different duration, particularly those of short duration, has been well studied by functional magnetic resonance imaging (fMRI). This nonlinearity is assumed to be due to neural adaptation and the nonlinearity of the response in the oxygen extraction fraction (OEF); the latter has not been examined quantitatively in humans. To evaluate how the OEF response contributes to the nonlinearity of the BOLD response to neural activity, we used simultaneous fMRI and near-infrared spectroscopy (NIRS). The responses to visual stimuli of four different durations were measured as changes in the BOLD signal and the NIRS-derived hemoglobin concentrations. The hemodynamic response nonlinearity was quantified using an impulse response function model with saturation nonlinearity scaling in the response amplitude, assuming that the unknown neural adaptation parameters varied within a physiologically feasible range. Independent of the degree of neural adaptation, the BOLD response consistently showed saturation nonlinearity similar to that of the OEF response estimated from the NIRS measures, the nonlinearity of which was greater than that of the response in the total hemoglobin concentration representing the cerebral blood volume (CBV). We also found that the contribution of the OEF response to the BOLD response was four to seven times greater than the contribution of the CBV response. Thus, we conclude that the nonlinearity of the BOLD response to neural activity originates mainly from that of the OEF response.     

Brain activity relating to the contingent negative variation: an fMRI investigation

The contingent negative variation (CNV) is a long-latency electroencephalography (EEG) surface negative potential with cognitive and motor components, observed during response anticipation. CNV is an index of cortical arousal during orienting and attention, yet its functional neuroanatomical basis is poorly understood. We used functional magnetic resonance imaging (fMRI) with simultaneous EEG and recording of galvanic skin response (GSR) to investigate CNV-related central neural activity and its relationship to peripheral autonomic arousal. In a group analysis, blood oxygenation level dependent (BOLD) activity during the period of CNV generation was enhanced in thalamus, somatomotor cortex, bilateral midcingulate, supplementary motor, and insular cortices. Enhancement of CNV-related activity in anterior and midcingulate, SMA, and insular cortices was associated with decreases in peripheral sympathetic arousal. In a subset of subjects in whom we acquired simultaneous EEG and fMRI data, we observed activity in bilateral thalamus, anterior cingulate, and supplementary motor cortex that was modulated by trial-by-trial amplitude of CNV. These findings provide a likely functional neuroanatomical substrate for the CNV and demonstrate modulation of components of this neural circuitry by peripheral autonomic arousal. Moreover, these data suggest a mechanistic model whereby thalamocortical interactions regulate CNV amplitude.     

Cerebral blood flow and BOLD responses to a memory encoding task: a comparison between healthy young and elderly adults

Functional magnetic resonance imaging (fMRI) studies of the medial temporal lobe have primarily made use of the blood oxygenation level dependent (BOLD) response to neural activity. The interpretation of the BOLD signal as a measure of medial temporal lobe function can be complicated, however, by changes in the cerebrovascular system that can occur with both normal aging and age-related diseases, such as Alzheimer's disease. Quantitative measures of the functional cerebral blood flow (CBF) response offer a useful complement to BOLD measures and have been shown to aid in the interpretation of fMRI studies. Despite these potential advantages, the application of ASL to fMRI studies of cognitive tasks and at-risk populations has been limited. In this study, we demonstrate the application of ASL fMRI to obtain measures of the CBF and BOLD responses to the encoding of natural scenes in healthy young (mean 25 years) and elderly (mean 74 years) adults. The percent CBF increase in the medial temporal lobe was significantly higher in the older adults, whereas the CBF levels during baseline and task conditions and during a separate resting-state scan were significantly lower in the older group. The older adults also showed slightly higher values for the BOLD response amplitude and the absolute change in CBF, but the age group differences were not significant. The percent CBF and BOLD responses are consistent with an age-related increase in the cerebral metabolic rate of oxygen metabolism (CMRO(2)) response to memory encoding.     

Blood bank conditions and RBCs: the progressive loss of metabolic modulation

BACKGROUND: Human RBC metabolism is modulated by the cell oxygenation state. Among other mechanisms, competition of deoxyhemoglobin and some glycolytic enzymes for the cytoplasmic domain of band 3 is probably involved in modulation. This metabolic modulation is connected to variations in intracellular NADPH and ATP levels as a function of the oxygenation state of the cell, and, consequently, it should have physiologic relevance. The present study investigates the effect of storage on this metabolic modulation and its relationship with the alteration of membrane protein composition. STUDY DESIGN AND METHODS: RBCs stored in CPD-saline-adenine-glucose-mannitol were assayed for glucose uptake and partition between glycolysis and the pentose phosphate pathway at high and low oxygen saturation by nuclear magnetic resonance spectroscopy after 1, 14, 21, 35, and 42 days of storage. Membrane protein composition was determined by SDS-PAGE on Days 1, 14, 35, and 42. Metabolic values and 2,3 DPG concentration were also measured after rejuvenation for 1 hour at 37 degrees C with pyruvate-inosine-phosphate-adenine solution on Day 21. RESULTS: Metabolic differences between RBCs incubated at high and low oxygen saturation decreased during storage, and, on Day 35, the two groups did not have significant differences (p = 0.111). SDS-PAGE showed that membrane protein composition was concurrently modified. The percentage of unmodified band 3 decreased during storage, principally between Days 14 and 35. In rejuvenated RBCs, oxygen-dependent modulation was not restored. CONCLUSIONS: RBCs stored in CPD-saline-adenine-glucose-mannitol do show a progressive loss of oxygen-dependent metabolic modulation, which is not restored after rejuvenation and which seems partly related to modifications in membrane proteins, mainly band 3.