- Email: [email protected]
Simultaneous measurements of brain tissue pO2 and cerebral blood flow during functional stimulation
International Congress Series 1235 (2002) 155–163
Simultaneous measurements of brain tissue pO2 and cerebral blood f low during functional stimulation$ Beau M. Ances a,*, Donald G. Buerk b, Joel H. Greenberg a,c, John A. Detre a,c a
Department of Neurology, University of Pennsylvania School of Medicine, 3400 Spruce Street, Philadelphia, PA 19104, USA b Department of Physiology and Bioengineering, University of Pennsylvania School of Medicine, 3400 Spruce Street, Philadelphia, PA 19104, USA c Cerebrovascular Research Center, University of Pennsylvania School of Medicine, 3400 Spruce Street, Philadelphia, PA 19104, USA
Abstract Simultaneous partial pressure of tissue oxygen ( pO2) and laser Doppler (LD) flowmetry measurements of cerebral blood flow (CBF) were obtained from the rat somatosensory cortex during periodic electrical forepaw stimulation of either 1-min or 4-s duration. For both stimulus durations, a transient significant decrease or ‘‘initial dip’’ in tissue pO2 preceded blood flow changes, followed by a peak in blood flow and an overshoot in tissue pO2. A sustained poststimulus undershoot in tissue pO2 was observed only for the 1-min stimulus. The magnitude of this poststimulus tissue pO2 decrease was significantly greater than the ‘‘initial dip’’ observed after stimulus onset, suggesting that relative tissue hypoxia alone does not mediate blood flow changes observed with functional stimulation. Our results suggest a complex dynamic relationship between oxygen utilization and blood flow exists during functional stimulation. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Tissue oxygen electrode; Cerebral blood flow; Laser Doppler flowmetry
1. Introduction The coupling between functional stimulation and regional changes in cerebral blood flow (CBF) and cerebral metabolism has been recognized for over a century, although the Abbreviations: CBF, cerebral blood flow; CMRO2, cerebral metabolic rate of oxygen; LD, laser Doppler. $ Adapted from Ances et al., Temporal Dynamics of Brain Tissue pO2 During Functional Stimulation, Neuroscience Letters 306 (2001) 106 – 110. * Corresponding author. Tel.: +1-215-662-7341; fax: +1-215-614-1927. E-mail address: [email protected] (B.M. Ances). 0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 1 8 2 - 6
156
B.M. Ances et al. / International Congress Series 1235 (2002) 155–163
specific mediators and modulators involved have not been elucidated [21]. Controversy exists concerning the relative extents of the increases in CBF and cerebral metabolic rate of oxygen (CMRO2) during functional activation. Protracted functional activation paradigms in both humans and animals have demonstrated small or no task-induced increases in CMRO2 [7], or comparable CBF and CMRO2 changes [16,19]. With the advent of functional magnetic resonance imaging (fMRI), the blood oxygenation level dependent (BOLD) contrast technique has heightened interest in better defining the temporal dynamics of CBF and CMRO2 changes for the purpose of modeling the expected BOLD contrast responses. Early investigations of the temporal dynamics of the blood flow response were performed in the cat visual cortex using near-infrared optical spectroscopy and spectroscopic imaging techniques [8,14]. These optical imaging techniques, which are primarily sensitive to changes in the concentrations of intravascular oxyhemoglobin and deoxyhemoglobin, demonstrated an early decrease in oxyhemoglobin, an ‘‘initial dip’’, followed by a later less localized washout increase in oxyhemoglobin due to flow increases. This ‘‘initial dip’’ has subsequently been confirmed using oxygen-dependent phosphorescence quenching [20] that is sensitive to the partial pressure of intravascular oxygen. However, the extent to which intravascular oxygen changes accurately reflect tissue changes and the applicability of these observations to other brain regions and species remains uncertain as some groups using BOLD-fMRI have not observed this ‘‘initial dip’’ [15,18]. The partial pressure of tissue oxygen ( pO2) can be measured using O2 microelectrodes, which have relatively rapid response times (ms) that are accurate to low microvascular oxygen pressures (<0.1 Torr), and can be used in vivo [4]. Laser Doppler flowmetry (LD) and an O2 microelectrode previously were used simultaneously to monitor blood flow and tissue pO2 changes in the cat optic nerve during photic stimulation [3]. We have characterized the magnitude and localization of functional activation induced blood flow changes in the somatosensory cortex using a rat forepaw stimulation model [1]. To further characterize the changes in tissue O2 and blood flow during functional activation, we used an O2 microelectrode in conjunction with LD over the somatosensory cortex of rats during electrical forepaw stimulation.
2. Methods and materials Under 1– 2% halothane anesthesia, adult Sprague –Dawley rats (n=6) were tracheostomized and mechanically ventilated. A tail artery catheter was placed, the scalp retracted, and the skull thinned. A small amount of thinned skull and underlying dura was removed from an area 5-mm lateral to bregma. Anesthesia was maintained using 60 mg/kg of achloralose i.p., followed by hourly supplemental doses of 30 mg/kg. Electrical forepaw stimulation was performed using two subdermal needle electrodes inserted into the dorsal forepaw. A function generator was used to control the stimulus frequency with frequency fixed at 5 Hz while the stimulus amplitude was maintained at 2.0 mA using a constant current dense stimulus isolation device. Recessed gold microsensors [22] were fabricated from glass micropipettes with tip dimensions f3– 7 Am and recesses f20– 50 Am deep. An O2 microelectrode was inserted
B.M. Ances et al. / International Congress Series 1235 (2002) 155–163
157
into the brain and the LD probe was positioned adjacent to the microelectrode. The O2 microelectrode was inserted at various levels (200 –400 Am deep) into the somatosensory cortex. Simultaneous flow and tissue pO2 measurements were made for repetitive periodic stimulation trials of either 1 min of stimulation and 1-min interstimulus interval or 4 s of stimulation with a 20-s interstimulus interval. All data were recorded on a virtual instrument using an analog to digital converter. A single trial consisted of 10 iterations with at least two trials performed for each electrode depth. Signal averaging was performed across multiple periodic trials to increase the signal to noise. The signal averaged flow and tissue pO2 data were converted to percent changes from baseline by dividing the average baseline value obtained over the 2 s prior to stimulation. Overall averages for both blood flow and tissue pO2 for all rats were determined along with intersubject standard errors.
3. Results Physiological variables measured during the stimulation paradigm were (meanFS.E.M), pH=7.39F0.02, PaCO2=39.0F0.6 mm Hg, PaO2=123F2 mm Hg. The overall signal averaged time courses of changes in tissue pO2 and relative blood flow in the somatosensory cortex during 1-min forepaw stimulation with a 1-min interstimulus interval are
Fig. 1. (A) Overall time course of the signal averaged changes in cerebral blood flow (CBF) (.) and partial pressure of tissue oxygen ( pO2) (o) during 1 min of forepaw stimulation with a 1-min interstimulus interval. (B) The initial 2 s after the onset of forepaw stimulation for CBF and tissue pO2. (C) The peak responses for both CBF and tissue pO2. (D) The poststimulus time period for both CBF and tissue pO2. For all figures, the shaded area represents the 1-min stimulation period. Error bars indicate standard error for all figures (From Ances et al., Neuroscience Letters, in press).
158
B.M. Ances et al. / International Congress Series 1235 (2002) 155–163
shown in Fig. 1A. Functional stimulation led to a rapid peak in flow and tissue pO2 followed by a much lower magnitude plateau for both measurements over the final 30 s of the stimulus. Fig. 1B shows the early temporal responses of flow and tissue pO2 changes during the first 2 s of stimulation. Shortly after the stimulus onset, tissue pO2 decreased from baseline prior to blood flow changes. As shown in Fig. 1C, the maximum flow response of 155.8F5.9% of baseline blood flow was at 4.0F0.3 s, and is comparable to previous results [2]. This increase in flow was accompanied by a delayed increase in tissue pO2 with the maximum change (4.6F0.7 Torr) occurring 5.6F0.2 s after stimulus onset. A mismatch between flow changes and tissue oxygen utilization occurs during the first few seconds of stimulation. The peak increase in tissue pO2 was f11-fold larger than the ‘‘initial dip’’ in tissue pO2. At the end of protracted stimulation, tissue pO2 remained only slightly above the prestimulus baseline, while blood flow was maintained at 10F2% of baseline (Fig. 1D). These results suggest that during steady-state stimulation, blood flow increases are reasonably well matched to increases in tissue oxygen utilization. The relatively greater increase in blood flow versus tissue pO2 may be attributable to a decrease in oxygen extraction fraction [5,6]. During the poststimulus period, blood flow rapidly returned to baseline while a prominent undershoot was observed in tissue pO2, and presumably reflects an increase in tissue oxygen metabolism required to compensate for a metabolic debt incurred with protracted stimulation [9,12]. The magnitude of this
Fig. 2. (A) Overall time course of CBF (.) and tissue pO2 (o) (relative to a baseline) for 4 s of forepaw stimulation with a 20-s interstimulus interval. For both responses, a peak was observed. (B) The initial 2 s after the onset of forepaw stimulation for CBF and tissue pO2. (C) The peak responses for both CBF and tissue pO2. (D) The poststimulus time period for CBF and tissue pO2. The shaded area represents the 4-s stimulation period and error bars indicate standard error for all figures (From Ances et al., Neuroscience Letters, in press).
B.M. Ances et al. / International Congress Series 1235 (2002) 155–163
159
poststimulus tissue pO2 decrease ( 0.71F0.31 Torr at 66.4 s) was significantly greater than the initial decrease ( 0.42F0.24 Torr) observed after the stimulus onset and was not accompanied by changes in blood flow, suggesting that relative tissue hypoxia alone does not mediate flow changes observed with functional stimulation. Fig. 2A shows the overall signal averaged flow and tissue pO2 results for a shorter stimulus paradigm consisting of 4 s of stimulation with a 20-s interstimulus interval. Peaks in blood flow expressed as a percent of the baseline and changes in tissue pO2 resemble those seen for the longer stimulus. The early temporal sequence of blood flow and tissue pO2 for the 4-s stimulus was also similar to longer stimulus results, with a decrease in tissue pO2 occurring prior to flow changes (Fig. 2B). As seen in Fig. 2C, a large flow response again preceded the tissue pO2 overshoot. A lag of 1.9F0.2 s between the peak in flow and tissue pO2 could reflect the capillary transit time required for O2 to diffuse out of the blood into the tissue. For the 4-s stimulus, a prominent poststimulus undershoot was not observed in tissue pO2 (Fig. 2D). Our data suggest that the metabolic debt incurred for the 4-s stimulus may be less than that for the 1-min stimulus, and is comparable to our previous study in which both the flow response and neural activity, as measured by somatosensory evoked potentials, were shown not to be refractory for a periodic 4-s stimulus with a 20-s interstimulus interval [1].
4. Conclusion These simultaneous measurements of blood flow and tissue pO2 during functional stimulation confirm the presence of an early decrease, or ‘‘initial dip’’, in tissue pO2 in rat somatosensory cortex, indicating an early rise in CMRO2. For both stimulus durations tested, the temporal dynamics of the changes in tissue pO2 were similar to the results obtained using intravascular tracers in the cat visual cortex [8,14,20]. Our results suggest that the ‘‘initial dip’’ in tissue pO2 is generalizable to other brain regions besides the primary visual cortex, and that intravascular measures accurately reflect tissue pO2 changes. The failure to reliably observe an ‘‘initial dip’’ using BOLD-fMRI or optical imaging outside of the cat visual cortex may be attributable to richer vascularization in the visual cortex [23] and/or the increased metabolic demands of the visual cortex [17] as compared to other cortical regions. The initial decrease in tissue pO2 as measured by O2 microelectrode occurred faster than those previous measurements obtained from optical imaging and oxygen phosphorescence quenching. The maximum decrease in tissue pO2 occurred f0.7 s after the onset of stimulation compared to f1.5 s for optical imaging and oxygen phosphorescence studies [13,14,20]. This latency between the tissue and intravascular measurements is in agreement with previous results that have estimated the time required for oxygen to diffuse from the vasculature to the tissue [10]. In addition, our oxygen microelectrode measurements likely reflect local parenchymal changes at the site of increased neural activity [3,4], while optical imaging and oxygen phosphorescence measurements survey intravascular oxygen changes in vessels in as well as surrounding the activated region [8,20]. The initial increases in deoxyhemoglobin of the BOLD-fMRI may be a better localizer of neural activity as compared to the large delayed decrease in deoxyhemoglobin due to
160
B.M. Ances et al. / International Congress Series 1235 (2002) 155–163
blood flow increases [11]. Our results suggest that the relationship between blood flow and oxygen metabolism varies considerably during functional activation. Thus, while transient changes in BOLD-fMRI may be used to improve the spatial and temporal resolution of functional neuroimaging, it may be difficult to interpret these changes quantitatively in terms of neural metabolism. However, with more protracted stimulation, blood flow changes appear to be well matched to tissue oxygen utilization. 2.3.4.7. On-Site Discussion 2.3.4.7.1. Question: (Pelligrino) (1) How did you measure NO? (2) Have you examined whether neuronal NOS inhibition affects the CBF response? Answer: (Ances) (1) We measured changes in NO using a gold microsensor that was calibrated prior to experiments. Polarographic currents for the electrochemical reduction of 02 ( 650 mV relative to Ag/AgCl reference) typically ranged between 30 and 60 pA in room air equilibrated saline at 37 jC, with zero currents <1% of the room air calibration, and 90% response times <100 ms. This experimental set-up is similar to previous work by Buerk et al., Adv. Exp. Med. Bio1. 1998; 454: 159) for measurement of tissue NO that was performed in the cat retina. (2) We have not attempted nNOS inhibition with these experiments of simultaneous measurements of NO and CBF using the NO electrode, and laser Doppler (LD). We do have previous data concerning the effect of nNOS inhibition on the CBF using the neuronal NOS inhibitor 7-Nitroindazole (7-NI). In these experiments, we have shown that 7-NI leads significantly reduced the CBF response while neuronal activity remained unaffected (Ances et al., J. Cereb. Blood Flow Metab. 2001; 21: P124). These results are also similar to work that has previously been performed using 7-NI (Lindauer et al., Am. J. Physiol. 1999; 277(2 Pt 2): H799). 2.3.4.7.2. Question: (Harder) The ‘‘Dip’’ is very small, for it to be relevant physiologically in needs to be amplified. Answer: (Ances) The dip is quite small in our measurements with this initial dip approximately 1/10 in magnitude compared to later increases in tissue pO2 as seen with an increases in CBF. Our results are different then those previously seen with oxygen phosphorescence quenching where 1/4 ratio was observed. As shown in the last scene of slides in this presentation, a potential mechanism that could relatively increase the magnitude of this initial dip would be stronger variations in the inter-stimulus interval (ISI). As we have previously shown with shorter ISIs, the relative magnitude of the initial dip increases (Ances et al., 2000; J. Cereb. Blood Flow Metab. 20: 290). Further pharmacological interventions using NOS inhibitors (as suggested in above question) will be investigated to magnify or minimize the dip. 2.3.4.7.3. Question: (Pearce) Did you measure and correct for differences in the time constants of measurements of both CBF and tissue pO2? How confident are you that the relative registrations of the CBF and pO2 signals were not out of phase? Answer: (Ances) In these experiments, the temporal resolution of the experiments were limited by laser Doppler (LD) collection. If we had only obtained tissue pO2 values with stimulation, we could have had a temporal resolution of milliseconds. However, since
B.M. Ances et al. / International Congress Series 1235 (2002) 155–163
161
these experiments investigated the simultaneous measurements of CBF and tissue pO2 we were limited by the temporal resolution of LD. We are quite confident that the CBF and pO2 signals that we obtained were not out of phase. 2.3.4.7.4. Question: (Gjedde) In your conclusions, you mentioned that tissue pO2 might serve as a buffer for increased CMRO2 without affecting the mitochondrial pO2. As stated in your conclusion, these results would be in agreement with calculations offered by Dr. Buxton in his opening presentation. It is of interest to know how the tissue pO2 might be able to fall without affecting mitochondrial pO2? Answer: (Ances) Mitochondrial O2 may remain elevated in the face of decreasing tissue pO2 by a siphoning of tissue pO2. An increase in neuronal activity will lead to an increase in metabolic requirements. In order to maintain mitochondrial O2 that is required for metabolic processes, tissue pO2 may decrease with O2 flowing from the tissue into the mitochondria. Therefore, the initial dip in tissue pO2 we observed may occur due to this siphoning of tissue pO2 by the mitochondria. Once blood flow increases, a corresponding increase in tissue pO2 will occur. These results are entirely consistent with the model proposed by Buxton at this meeting. 2.3.4.7.5. Question: (Golanov) (1) What are the reasons to choose the particular paradigm of stimulation used in experiments? Have you tried single pulse stimulation? (2) Did you try other anesthetics? Answer: (Ances) (1) We used the paradigm of either 1-min stimulation and 1 min of interstimulus interval or 4 s of stimulation with a 20-s of interstimulus interval (ISI) as we have previous experience using these paradigm (Detre et al., Brain Research 1998; 726 (1– 2): 91 and Ances et al., Neurosci. Lett. 1999; 257 (1): 25) Furthermore, we were interested at seeing if the initial dip could be seen for both short and long stimulus durations. Our paradigm allowed us to signal average our results and is similar to paradigms that we used in functional neuroimaging studies. We have not tried a single pulse experiment. However, even with one iteration (i.e. one stimulation of 4-s duration and a 20-s interstimulus interval) we were able to see thus initial dip. In the future, we plan to investigate the minimum stimulus duration required for producing the ‘‘initial dip’’. (2) In other experiments, we initially anesthetized the rat with halothane and then maintained anesthesia with a-chloralose. This anesthetic was chosen as a-chloralose has previously been shown to maintain the coupling of CBF and neuronal activity with functional stimulation (Ueki et al., J. Cereb. Blood Flow Metab. 1988; 8: 486). We have not attempted to use other anesthetic agents. 2.3.4.7.6. Comment: (Bandettini) Use of 4-s ISI is difficult to interpret. Even a predicted response is sensitive to what type of function you use as a model. So any difference between predicted and revealed may not have much meaning. 2.3.4.7.7. Question: (Jones) Was your data controlled for the initial brain tissue pO2? My fear is that your results might be influenced by the wide variations possible in brain tissue. pO2 have been noted previously by many workers (Wilson et al., J. Appl. Physiol. 1993; 74: 580; Kozniewska et al., J. Cereb. Blood Flow Metab. 1987; 7: 464; Silver. Med. Electron Biol. Eng. 1965; 3: 377; Smith et al., Microvasc. Res. 1977; 13: 233). Your dip
162
B.M. Ances et al. / International Congress Series 1235 (2002) 155–163
results might be different if your probe was placed in a region with a brain pO2 of 4 mm Hg vs. a region with a pO2 of 40 mm Hg. Answer: (Ances) We attempted to control for variations in the tissue pO2 by sampling over a depth of a few microns. In these experiments we advanced the O2 electrode from depths of 200– 400 mm in order to ensure that we sampled from similar regions that were measured using the LD probe (1 mm3). In our experiments, we did not see significant variations in the tissue pO2 with advancement of the tissue pO2 probe. Furthermore, tissue pO2 values were also similar across all rats. 2.3.4.7.8. Question: (Gaehtgens) My question is related to the lack of synchrony between the signals for pO2, NO and LDF. While the observations are very interesting, I think the interpretation depends on the measurements being made at identical locations in the tissue. (1) Therefore, do you have information about the precise location of your electrode, e.g. in relation to neighbouring blood vessels, etc.? (2) Since NO- and CBF-signals are clearly not ‘‘in phase’’ but CBF increase with a delay relative to NO, how can one conclude that NO is initiating the flow increase? Answer: (Ances) (1) In these experiments, we placed the LD probe with a few microns of either the O2 or NO microelectrode. Both prior to and after removal of the microelectrodes we obtained flow responses from the region from which the O2 probe was inserted. We did not do post-mortem studies looking at the exact site of the microelectrode tip placement. We therefore cannot be sure that electrodes were not next to blood vessels. However, since we never recorded extremely elevated NO or pO2 measurements, our results strongly suggest that we were within the tissue and not in blood vessels. (2) All of our tissue measurements had the same temporal resolution. In our results, we have shown that with stimulation there is an almost immediate increase in tissue NO after the stimulus onset. These results suggest that an increase in tissue NO occurs prior to the onset of CBF changes. With an increase in CBF, we observe a decrease in NO and our data show very similar shape curves with increases in CBF leading to decrease in NO due to a washout effect by CBF. However, the NO still remains above baseline until the end of stimulation. Once the stimulus is terminated NO decreases with an undershoot observed before returning to baseline during the understimulus interval. In the future, we plan to perform mathematical modeling concerning the relationship between the changes in NO and CBF.
Acknowledgements This work was supported by MH12078, NS02079, NS337859, and EY09260.
References [1] B.M. Ances, J.H. Greenberg, J.A. Detre, Effects of variations in interstimulus interval on activation-flow coupling response and somatosensory evoked potentials with forepaw stimulation in the rat, J. Cereb. Blood Flow Metab. 20 (2000) 290 – 297. [2] B.M. Ances, D.F. Wilson, J.H. Greenberg, J.A. Detre, Dynamic changes in cerebral blood flow, O2 tension,
B.M. Ances et al. / International Congress Series 1235 (2002) 155–163
[3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
[14] [15] [16]
[17] [18]
[19] [20] [21] [22] [23]
163
and calculated cerebral metabolic rate of O2 during functional activation using oxygen phosphorescence quenching, J. Cereb. Blood Flow Metab. 21 (5) (2001) 511 – 516. D.G. Buerk, D.N. Atochin, C.E. Riva, Simultaneous tissue pO2, nitric oxide, and laser Doppler blood flow measurements during neuronal activation of optic nerve, Adv. Exp. Med. Biol. 454 (1998) 159 – 164. D.G. Buerk, P. Nair, PtiO2 and CMRO2 changes in cortex and hippocampus of aging gerbil brain, J. Appl. Physiol. 74 (1993) 1723 – 1728. R.B. Buxton, L.R. Frank, A model for the coupling between cerebral blood flow and oxygen metabolism during neural stimulation, J. Cereb. Blood Flow Metab. 17 (1997) 64 – 72. R.B. Buxton, E.C. Wong, L.R. Frank, Dynamics of blood flow and oxygenation changes during brain activation: the balloon model, Magn. Reson. Med. 39 (1998) 855 – 864. P.T. Fox, M.E. Raichle, Focal physiological uncoupling of cerebral blood flow and oxidative metabolism during somatosensory stimulation in human subjects, Proc. Natl. Acad. Sci. U. S. A. 83 (1986) 1140 – 1144. A. Grinvald, E. Leike, R.D. Frostig, C.D. Gilbert, T.N. Weisel, Functional architecture of the cortex revealed by optical imaging of intrinsic signals, Nature 324 (1986) 269 – 271. J. Hamer, K. Wiedemann, H. Berlet, F. Weinhardt, S. Hoyer, Cerebral glucose and energy metabolism, cerebral oxygen consumption, and blood flow in arterial hypoxaemia, Acta Neurochir. 44 (1978) 151 – 160. A.G. Hudetz, Mathematical model of oxygen transport in the cerebral cortex, Brain Res. 817 (1999) 75 – 83. D.S. Kim, T.Q. Duong, S.G. Kim, High-resolution mapping of iso-orientation columns by fMRI [see comments], Nat. Neurosci. 3 (2000) 164 – 169. D. Kintner, J.H. Fitzpatrick, J.A. Louie Jr., D.D. Gilboe, Cerebral oxygen and energy metabolism during and after 30 minutes of moderate hypoxia, Am. J. Physiol. 247 (1984) E475 – E482. D. Malonek, U. Dirnagl, U. Lindauer, K. Yamada, I. Kanno, A. Grinvald, Vascular imprints of neuronal activity: relationships between the dynamics of cortical blood flow, oxygenation, and volume changes following sensory stimulation, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 14826 – 14831. D. Malonek, A. Grinvald, Interactions between electrical activity and cortical microcirculation revealed by imaging spectroscopy; implications for functional brain mapping, Science 272 (1996) 551 – 554. J.J. Marota, C. Ayata, M.A. Moskowitz, R.M. Weisskoff, B.R. Rosen, J.B. Mandeville, Investigation of the early response to rat forepaw stimulation, Magn. Reson. Med. 41 (1999) 247 – 252. S. Marrett, H. Fujita, E. Meyer, L. Ribeiro, A. Evans, H. Kuwabara, A. Gjedde, Stimulus specific increases of oxidative metabolism in human visual cortex, in: K. Uemura, N.A. Lassen, T. Jones, I. Kanno (Eds.), Quantification of Brain Function. Tracer Kinetics and Image Analysis in Brain PET, Elsevier, New York, 1993, pp. 217 – 228. T.M. Preuss, J.H. Kaas, Cytochrome oxidase ‘blobs’ and other characteristics of primary visual cortex in a lemuroid primate, Cheirogaleus medius, Brain Behav. Evol. 47 (1996) 103 – 112. A.C. Silva, S.P. Lee, C. Iadecola, S.G. Kim, Early temporal characteristics of cerebral blood flow and deoxyhemoglobin changes during somatosensory stimulation, J. Cereb. Blood Flow Metab. 20 (2000) 201 – 206. L. Sokoloff, R. Mangold, R.L. Wechsler, C. Kennedy, S. Kety, The effects of mental activation cerebral circulation and metabolism, J. Clin. Invest. 34 (1955) 1101 – 1108. I. Vanzetta, A. Grinvald, Increased cortical oxidative metabolism due to sensory stimulation: implications for functional brain imaging, Science 286 (1999) 1555 – 1558. A. Villringer, U. Dirnagl, Coupling of brain activity and cerebral blood flow: basis of functional neuroimaging, Cereb. Brain Metab. Rev. 7 (1995) 240 – 276. W.J. Whalen, J. Riley, P. Nair, A microelectrode for measuring intracellular pO2, J. Appl. Physiol. 23 (1967) 798 – 801. D. Zheng, A.S. LaMantia, D. Purves, Specialized vasculaturization of the primate visual cortex, J. Neurosci. 91 (1991) 2622 – 2629.
Simultaneous measurements of brain tissue pO2 and cerebral blood f low during functional stimulation$ Beau M. Ances a,*, Donald G. Buerk b, Joel H. Greenberg a,c, John A. Detre a,c a
Department of Neurology, University of Pennsylvania School of Medicine, 3400 Spruce Street, Philadelphia, PA 19104, USA b Department of Physiology and Bioengineering, University of Pennsylvania School of Medicine, 3400 Spruce Street, Philadelphia, PA 19104, USA c Cerebrovascular Research Center, University of Pennsylvania School of Medicine, 3400 Spruce Street, Philadelphia, PA 19104, USA
Abstract Simultaneous partial pressure of tissue oxygen ( pO2) and laser Doppler (LD) flowmetry measurements of cerebral blood flow (CBF) were obtained from the rat somatosensory cortex during periodic electrical forepaw stimulation of either 1-min or 4-s duration. For both stimulus durations, a transient significant decrease or ‘‘initial dip’’ in tissue pO2 preceded blood flow changes, followed by a peak in blood flow and an overshoot in tissue pO2. A sustained poststimulus undershoot in tissue pO2 was observed only for the 1-min stimulus. The magnitude of this poststimulus tissue pO2 decrease was significantly greater than the ‘‘initial dip’’ observed after stimulus onset, suggesting that relative tissue hypoxia alone does not mediate blood flow changes observed with functional stimulation. Our results suggest a complex dynamic relationship between oxygen utilization and blood flow exists during functional stimulation. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Tissue oxygen electrode; Cerebral blood flow; Laser Doppler flowmetry
1. Introduction The coupling between functional stimulation and regional changes in cerebral blood flow (CBF) and cerebral metabolism has been recognized for over a century, although the Abbreviations: CBF, cerebral blood flow; CMRO2, cerebral metabolic rate of oxygen; LD, laser Doppler. $ Adapted from Ances et al., Temporal Dynamics of Brain Tissue pO2 During Functional Stimulation, Neuroscience Letters 306 (2001) 106 – 110. * Corresponding author. Tel.: +1-215-662-7341; fax: +1-215-614-1927. E-mail address: [email protected] (B.M. Ances). 0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 1 8 2 - 6
156
B.M. Ances et al. / International Congress Series 1235 (2002) 155–163
specific mediators and modulators involved have not been elucidated [21]. Controversy exists concerning the relative extents of the increases in CBF and cerebral metabolic rate of oxygen (CMRO2) during functional activation. Protracted functional activation paradigms in both humans and animals have demonstrated small or no task-induced increases in CMRO2 [7], or comparable CBF and CMRO2 changes [16,19]. With the advent of functional magnetic resonance imaging (fMRI), the blood oxygenation level dependent (BOLD) contrast technique has heightened interest in better defining the temporal dynamics of CBF and CMRO2 changes for the purpose of modeling the expected BOLD contrast responses. Early investigations of the temporal dynamics of the blood flow response were performed in the cat visual cortex using near-infrared optical spectroscopy and spectroscopic imaging techniques [8,14]. These optical imaging techniques, which are primarily sensitive to changes in the concentrations of intravascular oxyhemoglobin and deoxyhemoglobin, demonstrated an early decrease in oxyhemoglobin, an ‘‘initial dip’’, followed by a later less localized washout increase in oxyhemoglobin due to flow increases. This ‘‘initial dip’’ has subsequently been confirmed using oxygen-dependent phosphorescence quenching [20] that is sensitive to the partial pressure of intravascular oxygen. However, the extent to which intravascular oxygen changes accurately reflect tissue changes and the applicability of these observations to other brain regions and species remains uncertain as some groups using BOLD-fMRI have not observed this ‘‘initial dip’’ [15,18]. The partial pressure of tissue oxygen ( pO2) can be measured using O2 microelectrodes, which have relatively rapid response times (ms) that are accurate to low microvascular oxygen pressures (<0.1 Torr), and can be used in vivo [4]. Laser Doppler flowmetry (LD) and an O2 microelectrode previously were used simultaneously to monitor blood flow and tissue pO2 changes in the cat optic nerve during photic stimulation [3]. We have characterized the magnitude and localization of functional activation induced blood flow changes in the somatosensory cortex using a rat forepaw stimulation model [1]. To further characterize the changes in tissue O2 and blood flow during functional activation, we used an O2 microelectrode in conjunction with LD over the somatosensory cortex of rats during electrical forepaw stimulation.
2. Methods and materials Under 1– 2% halothane anesthesia, adult Sprague –Dawley rats (n=6) were tracheostomized and mechanically ventilated. A tail artery catheter was placed, the scalp retracted, and the skull thinned. A small amount of thinned skull and underlying dura was removed from an area 5-mm lateral to bregma. Anesthesia was maintained using 60 mg/kg of achloralose i.p., followed by hourly supplemental doses of 30 mg/kg. Electrical forepaw stimulation was performed using two subdermal needle electrodes inserted into the dorsal forepaw. A function generator was used to control the stimulus frequency with frequency fixed at 5 Hz while the stimulus amplitude was maintained at 2.0 mA using a constant current dense stimulus isolation device. Recessed gold microsensors [22] were fabricated from glass micropipettes with tip dimensions f3– 7 Am and recesses f20– 50 Am deep. An O2 microelectrode was inserted
B.M. Ances et al. / International Congress Series 1235 (2002) 155–163
157
into the brain and the LD probe was positioned adjacent to the microelectrode. The O2 microelectrode was inserted at various levels (200 –400 Am deep) into the somatosensory cortex. Simultaneous flow and tissue pO2 measurements were made for repetitive periodic stimulation trials of either 1 min of stimulation and 1-min interstimulus interval or 4 s of stimulation with a 20-s interstimulus interval. All data were recorded on a virtual instrument using an analog to digital converter. A single trial consisted of 10 iterations with at least two trials performed for each electrode depth. Signal averaging was performed across multiple periodic trials to increase the signal to noise. The signal averaged flow and tissue pO2 data were converted to percent changes from baseline by dividing the average baseline value obtained over the 2 s prior to stimulation. Overall averages for both blood flow and tissue pO2 for all rats were determined along with intersubject standard errors.
3. Results Physiological variables measured during the stimulation paradigm were (meanFS.E.M), pH=7.39F0.02, PaCO2=39.0F0.6 mm Hg, PaO2=123F2 mm Hg. The overall signal averaged time courses of changes in tissue pO2 and relative blood flow in the somatosensory cortex during 1-min forepaw stimulation with a 1-min interstimulus interval are
Fig. 1. (A) Overall time course of the signal averaged changes in cerebral blood flow (CBF) (.) and partial pressure of tissue oxygen ( pO2) (o) during 1 min of forepaw stimulation with a 1-min interstimulus interval. (B) The initial 2 s after the onset of forepaw stimulation for CBF and tissue pO2. (C) The peak responses for both CBF and tissue pO2. (D) The poststimulus time period for both CBF and tissue pO2. For all figures, the shaded area represents the 1-min stimulation period. Error bars indicate standard error for all figures (From Ances et al., Neuroscience Letters, in press).
158
B.M. Ances et al. / International Congress Series 1235 (2002) 155–163
shown in Fig. 1A. Functional stimulation led to a rapid peak in flow and tissue pO2 followed by a much lower magnitude plateau for both measurements over the final 30 s of the stimulus. Fig. 1B shows the early temporal responses of flow and tissue pO2 changes during the first 2 s of stimulation. Shortly after the stimulus onset, tissue pO2 decreased from baseline prior to blood flow changes. As shown in Fig. 1C, the maximum flow response of 155.8F5.9% of baseline blood flow was at 4.0F0.3 s, and is comparable to previous results [2]. This increase in flow was accompanied by a delayed increase in tissue pO2 with the maximum change (4.6F0.7 Torr) occurring 5.6F0.2 s after stimulus onset. A mismatch between flow changes and tissue oxygen utilization occurs during the first few seconds of stimulation. The peak increase in tissue pO2 was f11-fold larger than the ‘‘initial dip’’ in tissue pO2. At the end of protracted stimulation, tissue pO2 remained only slightly above the prestimulus baseline, while blood flow was maintained at 10F2% of baseline (Fig. 1D). These results suggest that during steady-state stimulation, blood flow increases are reasonably well matched to increases in tissue oxygen utilization. The relatively greater increase in blood flow versus tissue pO2 may be attributable to a decrease in oxygen extraction fraction [5,6]. During the poststimulus period, blood flow rapidly returned to baseline while a prominent undershoot was observed in tissue pO2, and presumably reflects an increase in tissue oxygen metabolism required to compensate for a metabolic debt incurred with protracted stimulation [9,12]. The magnitude of this
Fig. 2. (A) Overall time course of CBF (.) and tissue pO2 (o) (relative to a baseline) for 4 s of forepaw stimulation with a 20-s interstimulus interval. For both responses, a peak was observed. (B) The initial 2 s after the onset of forepaw stimulation for CBF and tissue pO2. (C) The peak responses for both CBF and tissue pO2. (D) The poststimulus time period for CBF and tissue pO2. The shaded area represents the 4-s stimulation period and error bars indicate standard error for all figures (From Ances et al., Neuroscience Letters, in press).
B.M. Ances et al. / International Congress Series 1235 (2002) 155–163
159
poststimulus tissue pO2 decrease ( 0.71F0.31 Torr at 66.4 s) was significantly greater than the initial decrease ( 0.42F0.24 Torr) observed after the stimulus onset and was not accompanied by changes in blood flow, suggesting that relative tissue hypoxia alone does not mediate flow changes observed with functional stimulation. Fig. 2A shows the overall signal averaged flow and tissue pO2 results for a shorter stimulus paradigm consisting of 4 s of stimulation with a 20-s interstimulus interval. Peaks in blood flow expressed as a percent of the baseline and changes in tissue pO2 resemble those seen for the longer stimulus. The early temporal sequence of blood flow and tissue pO2 for the 4-s stimulus was also similar to longer stimulus results, with a decrease in tissue pO2 occurring prior to flow changes (Fig. 2B). As seen in Fig. 2C, a large flow response again preceded the tissue pO2 overshoot. A lag of 1.9F0.2 s between the peak in flow and tissue pO2 could reflect the capillary transit time required for O2 to diffuse out of the blood into the tissue. For the 4-s stimulus, a prominent poststimulus undershoot was not observed in tissue pO2 (Fig. 2D). Our data suggest that the metabolic debt incurred for the 4-s stimulus may be less than that for the 1-min stimulus, and is comparable to our previous study in which both the flow response and neural activity, as measured by somatosensory evoked potentials, were shown not to be refractory for a periodic 4-s stimulus with a 20-s interstimulus interval [1].
4. Conclusion These simultaneous measurements of blood flow and tissue pO2 during functional stimulation confirm the presence of an early decrease, or ‘‘initial dip’’, in tissue pO2 in rat somatosensory cortex, indicating an early rise in CMRO2. For both stimulus durations tested, the temporal dynamics of the changes in tissue pO2 were similar to the results obtained using intravascular tracers in the cat visual cortex [8,14,20]. Our results suggest that the ‘‘initial dip’’ in tissue pO2 is generalizable to other brain regions besides the primary visual cortex, and that intravascular measures accurately reflect tissue pO2 changes. The failure to reliably observe an ‘‘initial dip’’ using BOLD-fMRI or optical imaging outside of the cat visual cortex may be attributable to richer vascularization in the visual cortex [23] and/or the increased metabolic demands of the visual cortex [17] as compared to other cortical regions. The initial decrease in tissue pO2 as measured by O2 microelectrode occurred faster than those previous measurements obtained from optical imaging and oxygen phosphorescence quenching. The maximum decrease in tissue pO2 occurred f0.7 s after the onset of stimulation compared to f1.5 s for optical imaging and oxygen phosphorescence studies [13,14,20]. This latency between the tissue and intravascular measurements is in agreement with previous results that have estimated the time required for oxygen to diffuse from the vasculature to the tissue [10]. In addition, our oxygen microelectrode measurements likely reflect local parenchymal changes at the site of increased neural activity [3,4], while optical imaging and oxygen phosphorescence measurements survey intravascular oxygen changes in vessels in as well as surrounding the activated region [8,20]. The initial increases in deoxyhemoglobin of the BOLD-fMRI may be a better localizer of neural activity as compared to the large delayed decrease in deoxyhemoglobin due to
160
B.M. Ances et al. / International Congress Series 1235 (2002) 155–163
blood flow increases [11]. Our results suggest that the relationship between blood flow and oxygen metabolism varies considerably during functional activation. Thus, while transient changes in BOLD-fMRI may be used to improve the spatial and temporal resolution of functional neuroimaging, it may be difficult to interpret these changes quantitatively in terms of neural metabolism. However, with more protracted stimulation, blood flow changes appear to be well matched to tissue oxygen utilization. 2.3.4.7. On-Site Discussion 2.3.4.7.1. Question: (Pelligrino) (1) How did you measure NO? (2) Have you examined whether neuronal NOS inhibition affects the CBF response? Answer: (Ances) (1) We measured changes in NO using a gold microsensor that was calibrated prior to experiments. Polarographic currents for the electrochemical reduction of 02 ( 650 mV relative to Ag/AgCl reference) typically ranged between 30 and 60 pA in room air equilibrated saline at 37 jC, with zero currents <1% of the room air calibration, and 90% response times <100 ms. This experimental set-up is similar to previous work by Buerk et al., Adv. Exp. Med. Bio1. 1998; 454: 159) for measurement of tissue NO that was performed in the cat retina. (2) We have not attempted nNOS inhibition with these experiments of simultaneous measurements of NO and CBF using the NO electrode, and laser Doppler (LD). We do have previous data concerning the effect of nNOS inhibition on the CBF using the neuronal NOS inhibitor 7-Nitroindazole (7-NI). In these experiments, we have shown that 7-NI leads significantly reduced the CBF response while neuronal activity remained unaffected (Ances et al., J. Cereb. Blood Flow Metab. 2001; 21: P124). These results are also similar to work that has previously been performed using 7-NI (Lindauer et al., Am. J. Physiol. 1999; 277(2 Pt 2): H799). 2.3.4.7.2. Question: (Harder) The ‘‘Dip’’ is very small, for it to be relevant physiologically in needs to be amplified. Answer: (Ances) The dip is quite small in our measurements with this initial dip approximately 1/10 in magnitude compared to later increases in tissue pO2 as seen with an increases in CBF. Our results are different then those previously seen with oxygen phosphorescence quenching where 1/4 ratio was observed. As shown in the last scene of slides in this presentation, a potential mechanism that could relatively increase the magnitude of this initial dip would be stronger variations in the inter-stimulus interval (ISI). As we have previously shown with shorter ISIs, the relative magnitude of the initial dip increases (Ances et al., 2000; J. Cereb. Blood Flow Metab. 20: 290). Further pharmacological interventions using NOS inhibitors (as suggested in above question) will be investigated to magnify or minimize the dip. 2.3.4.7.3. Question: (Pearce) Did you measure and correct for differences in the time constants of measurements of both CBF and tissue pO2? How confident are you that the relative registrations of the CBF and pO2 signals were not out of phase? Answer: (Ances) In these experiments, the temporal resolution of the experiments were limited by laser Doppler (LD) collection. If we had only obtained tissue pO2 values with stimulation, we could have had a temporal resolution of milliseconds. However, since
B.M. Ances et al. / International Congress Series 1235 (2002) 155–163
161
these experiments investigated the simultaneous measurements of CBF and tissue pO2 we were limited by the temporal resolution of LD. We are quite confident that the CBF and pO2 signals that we obtained were not out of phase. 2.3.4.7.4. Question: (Gjedde) In your conclusions, you mentioned that tissue pO2 might serve as a buffer for increased CMRO2 without affecting the mitochondrial pO2. As stated in your conclusion, these results would be in agreement with calculations offered by Dr. Buxton in his opening presentation. It is of interest to know how the tissue pO2 might be able to fall without affecting mitochondrial pO2? Answer: (Ances) Mitochondrial O2 may remain elevated in the face of decreasing tissue pO2 by a siphoning of tissue pO2. An increase in neuronal activity will lead to an increase in metabolic requirements. In order to maintain mitochondrial O2 that is required for metabolic processes, tissue pO2 may decrease with O2 flowing from the tissue into the mitochondria. Therefore, the initial dip in tissue pO2 we observed may occur due to this siphoning of tissue pO2 by the mitochondria. Once blood flow increases, a corresponding increase in tissue pO2 will occur. These results are entirely consistent with the model proposed by Buxton at this meeting. 2.3.4.7.5. Question: (Golanov) (1) What are the reasons to choose the particular paradigm of stimulation used in experiments? Have you tried single pulse stimulation? (2) Did you try other anesthetics? Answer: (Ances) (1) We used the paradigm of either 1-min stimulation and 1 min of interstimulus interval or 4 s of stimulation with a 20-s of interstimulus interval (ISI) as we have previous experience using these paradigm (Detre et al., Brain Research 1998; 726 (1– 2): 91 and Ances et al., Neurosci. Lett. 1999; 257 (1): 25) Furthermore, we were interested at seeing if the initial dip could be seen for both short and long stimulus durations. Our paradigm allowed us to signal average our results and is similar to paradigms that we used in functional neuroimaging studies. We have not tried a single pulse experiment. However, even with one iteration (i.e. one stimulation of 4-s duration and a 20-s interstimulus interval) we were able to see thus initial dip. In the future, we plan to investigate the minimum stimulus duration required for producing the ‘‘initial dip’’. (2) In other experiments, we initially anesthetized the rat with halothane and then maintained anesthesia with a-chloralose. This anesthetic was chosen as a-chloralose has previously been shown to maintain the coupling of CBF and neuronal activity with functional stimulation (Ueki et al., J. Cereb. Blood Flow Metab. 1988; 8: 486). We have not attempted to use other anesthetic agents. 2.3.4.7.6. Comment: (Bandettini) Use of 4-s ISI is difficult to interpret. Even a predicted response is sensitive to what type of function you use as a model. So any difference between predicted and revealed may not have much meaning. 2.3.4.7.7. Question: (Jones) Was your data controlled for the initial brain tissue pO2? My fear is that your results might be influenced by the wide variations possible in brain tissue. pO2 have been noted previously by many workers (Wilson et al., J. Appl. Physiol. 1993; 74: 580; Kozniewska et al., J. Cereb. Blood Flow Metab. 1987; 7: 464; Silver. Med. Electron Biol. Eng. 1965; 3: 377; Smith et al., Microvasc. Res. 1977; 13: 233). Your dip
162
B.M. Ances et al. / International Congress Series 1235 (2002) 155–163
results might be different if your probe was placed in a region with a brain pO2 of 4 mm Hg vs. a region with a pO2 of 40 mm Hg. Answer: (Ances) We attempted to control for variations in the tissue pO2 by sampling over a depth of a few microns. In these experiments we advanced the O2 electrode from depths of 200– 400 mm in order to ensure that we sampled from similar regions that were measured using the LD probe (1 mm3). In our experiments, we did not see significant variations in the tissue pO2 with advancement of the tissue pO2 probe. Furthermore, tissue pO2 values were also similar across all rats. 2.3.4.7.8. Question: (Gaehtgens) My question is related to the lack of synchrony between the signals for pO2, NO and LDF. While the observations are very interesting, I think the interpretation depends on the measurements being made at identical locations in the tissue. (1) Therefore, do you have information about the precise location of your electrode, e.g. in relation to neighbouring blood vessels, etc.? (2) Since NO- and CBF-signals are clearly not ‘‘in phase’’ but CBF increase with a delay relative to NO, how can one conclude that NO is initiating the flow increase? Answer: (Ances) (1) In these experiments, we placed the LD probe with a few microns of either the O2 or NO microelectrode. Both prior to and after removal of the microelectrodes we obtained flow responses from the region from which the O2 probe was inserted. We did not do post-mortem studies looking at the exact site of the microelectrode tip placement. We therefore cannot be sure that electrodes were not next to blood vessels. However, since we never recorded extremely elevated NO or pO2 measurements, our results strongly suggest that we were within the tissue and not in blood vessels. (2) All of our tissue measurements had the same temporal resolution. In our results, we have shown that with stimulation there is an almost immediate increase in tissue NO after the stimulus onset. These results suggest that an increase in tissue NO occurs prior to the onset of CBF changes. With an increase in CBF, we observe a decrease in NO and our data show very similar shape curves with increases in CBF leading to decrease in NO due to a washout effect by CBF. However, the NO still remains above baseline until the end of stimulation. Once the stimulus is terminated NO decreases with an undershoot observed before returning to baseline during the understimulus interval. In the future, we plan to perform mathematical modeling concerning the relationship between the changes in NO and CBF.
Acknowledgements This work was supported by MH12078, NS02079, NS337859, and EY09260.
References [1] B.M. Ances, J.H. Greenberg, J.A. Detre, Effects of variations in interstimulus interval on activation-flow coupling response and somatosensory evoked potentials with forepaw stimulation in the rat, J. Cereb. Blood Flow Metab. 20 (2000) 290 – 297. [2] B.M. Ances, D.F. Wilson, J.H. Greenberg, J.A. Detre, Dynamic changes in cerebral blood flow, O2 tension,
B.M. Ances et al. / International Congress Series 1235 (2002) 155–163
[3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
[14] [15] [16]
[17] [18]
[19] [20] [21] [22] [23]
163
and calculated cerebral metabolic rate of O2 during functional activation using oxygen phosphorescence quenching, J. Cereb. Blood Flow Metab. 21 (5) (2001) 511 – 516. D.G. Buerk, D.N. Atochin, C.E. Riva, Simultaneous tissue pO2, nitric oxide, and laser Doppler blood flow measurements during neuronal activation of optic nerve, Adv. Exp. Med. Biol. 454 (1998) 159 – 164. D.G. Buerk, P. Nair, PtiO2 and CMRO2 changes in cortex and hippocampus of aging gerbil brain, J. Appl. Physiol. 74 (1993) 1723 – 1728. R.B. Buxton, L.R. Frank, A model for the coupling between cerebral blood flow and oxygen metabolism during neural stimulation, J. Cereb. Blood Flow Metab. 17 (1997) 64 – 72. R.B. Buxton, E.C. Wong, L.R. Frank, Dynamics of blood flow and oxygenation changes during brain activation: the balloon model, Magn. Reson. Med. 39 (1998) 855 – 864. P.T. Fox, M.E. Raichle, Focal physiological uncoupling of cerebral blood flow and oxidative metabolism during somatosensory stimulation in human subjects, Proc. Natl. Acad. Sci. U. S. A. 83 (1986) 1140 – 1144. A. Grinvald, E. Leike, R.D. Frostig, C.D. Gilbert, T.N. Weisel, Functional architecture of the cortex revealed by optical imaging of intrinsic signals, Nature 324 (1986) 269 – 271. J. Hamer, K. Wiedemann, H. Berlet, F. Weinhardt, S. Hoyer, Cerebral glucose and energy metabolism, cerebral oxygen consumption, and blood flow in arterial hypoxaemia, Acta Neurochir. 44 (1978) 151 – 160. A.G. Hudetz, Mathematical model of oxygen transport in the cerebral cortex, Brain Res. 817 (1999) 75 – 83. D.S. Kim, T.Q. Duong, S.G. Kim, High-resolution mapping of iso-orientation columns by fMRI [see comments], Nat. Neurosci. 3 (2000) 164 – 169. D. Kintner, J.H. Fitzpatrick, J.A. Louie Jr., D.D. Gilboe, Cerebral oxygen and energy metabolism during and after 30 minutes of moderate hypoxia, Am. J. Physiol. 247 (1984) E475 – E482. D. Malonek, U. Dirnagl, U. Lindauer, K. Yamada, I. Kanno, A. Grinvald, Vascular imprints of neuronal activity: relationships between the dynamics of cortical blood flow, oxygenation, and volume changes following sensory stimulation, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 14826 – 14831. D. Malonek, A. Grinvald, Interactions between electrical activity and cortical microcirculation revealed by imaging spectroscopy; implications for functional brain mapping, Science 272 (1996) 551 – 554. J.J. Marota, C. Ayata, M.A. Moskowitz, R.M. Weisskoff, B.R. Rosen, J.B. Mandeville, Investigation of the early response to rat forepaw stimulation, Magn. Reson. Med. 41 (1999) 247 – 252. S. Marrett, H. Fujita, E. Meyer, L. Ribeiro, A. Evans, H. Kuwabara, A. Gjedde, Stimulus specific increases of oxidative metabolism in human visual cortex, in: K. Uemura, N.A. Lassen, T. Jones, I. Kanno (Eds.), Quantification of Brain Function. Tracer Kinetics and Image Analysis in Brain PET, Elsevier, New York, 1993, pp. 217 – 228. T.M. Preuss, J.H. Kaas, Cytochrome oxidase ‘blobs’ and other characteristics of primary visual cortex in a lemuroid primate, Cheirogaleus medius, Brain Behav. Evol. 47 (1996) 103 – 112. A.C. Silva, S.P. Lee, C. Iadecola, S.G. Kim, Early temporal characteristics of cerebral blood flow and deoxyhemoglobin changes during somatosensory stimulation, J. Cereb. Blood Flow Metab. 20 (2000) 201 – 206. L. Sokoloff, R. Mangold, R.L. Wechsler, C. Kennedy, S. Kety, The effects of mental activation cerebral circulation and metabolism, J. Clin. Invest. 34 (1955) 1101 – 1108. I. Vanzetta, A. Grinvald, Increased cortical oxidative metabolism due to sensory stimulation: implications for functional brain imaging, Science 286 (1999) 1555 – 1558. A. Villringer, U. Dirnagl, Coupling of brain activity and cerebral blood flow: basis of functional neuroimaging, Cereb. Brain Metab. Rev. 7 (1995) 240 – 276. W.J. Whalen, J. Riley, P. Nair, A microelectrode for measuring intracellular pO2, J. Appl. Physiol. 23 (1967) 798 – 801. D. Zheng, A.S. LaMantia, D. Purves, Specialized vasculaturization of the primate visual cortex, J. Neurosci. 91 (1991) 2622 – 2629.