Regulation of oxygen transport during brain activation: stimulus-induced hemodynamic responses in human and animal cortices
© Seiyama et al; licensee BioMed Central Ltd. 2003
Received: 28 November 2003
Accepted: 20 December 2003
Published: 20 December 2003
The correlation between regional changes in neuronal activity and changes in hemodynamics is a major issue for noninvasive neuroimaging techniques such as functional magnetic resonance imaging (fMRI) and near-infrared optical imaging (NIOI). A tight coupling of these changes has been assumed to elucidate brain function from data obtained with those techniques. In the present study, we investigated the relationship between neuronal activity and hemodynamic responses in the occipital cortex of humans during visual stimulation and in the somatosensory cortex of rats during peripheral nerve stimulation.
The temporal frequency dependence of macroscopic hemodynamic responses on visual stimuli was investigated in the occipital cortex of humans by simultaneous measurements made using fMRI and NIOI. The stimulus-intensity dependence of both microscopic hemodynamic changes and changes in neuronal activity in response to peripheral nerve stimulation was investigated in animal models by analyzing membrane potential (fluorescence), hemodynamic parameters (visible spectra and laser-Doppler flowmetry), and vessel diameter (image analyzer).
Above a certain level of stimulus-intensity, increases in regional cerebral blood flow (rCBF) were accompanied by a decrease in regional cerebral blood volume (rCBV), i.e., dissociation of rCBF and rCBV responses occurred in both the human and animal experiments. Furthermore, the animal experiments revealed that the distribution of increased rCBF and O2 spread well beyond the area of neuronal activation, and that the increases showed saturation in the activated area.
These results suggest that above a certain level of neuronal activity, a regulatory mechanism between regional cerebral blood flow (rCBF) and rCBV acts to prevent excess O2 inflow into the focally activated area.
The existence of coupling between neuronal activity, metabolic and hemodynamic responses is a prerequisite for brain function research employing non-invasive neuroimaging techniques such as functional magnetic resonance imaging (fMRI), positron emission tomography (PET) and near-infrared optical imaging (NIOI), which can visualize stimulus-induced activation areas in the human brain (see Rev. ). Although the mechanism of the coupling between these physiological parameters remains to be elucidated despite numerous investigations conducted over past decades (see Rev. ), a tight coupling has been assumed to elucidate brain function based on the data obtained using these techniques (see Rev. ).
Within the past some dozen years, it has been reported that changes in rCBF in response to visual stimuli are accompanied by smaller changes in the regional metabolic rate of O2 (rCMRO2) in the human visual cortex (e.g., ΔrCBF: ΔrCMRO2 = 10:1  or 2:1 ). This implies that the oxygen supply is not precisely matched with the demand (referred to as "overcompensation" or "decoupling between ΔrCBF and ΔrCMRO2"). More recently, the use of optical techniques to monitor the visual cortex of animals has shown that (1) after onset of a stimulus, the concentration of deoxy-Hb increases first at a focal region in the cortex co-localized with neuronal activation and increased O2 consumption, and (2) this is followed by a decrease in deoxy-Hb and a large increase in oxy-Hb, which is caused by a delayed but large and less localized increase in rCBF [6–8].
In the course of our studies, we found a decoupling between rCBF and rCBV during visual stimulation in the human occipital cortex , although it has been empirically appreciated that an increase in rCBF accompanies an increase in rCBV. The relationship between the two was determined using whole-head measurement , which is often used for the analysis of stimulus-induced changes in rCBF and rCBV. On the basis of our results, we propose that some mechanism regulates regional blood flow (rCBF) and blood volume (rCBV) above a certain level of neuronal activity. If the mechanism works as a general rule during regional brain activation, it should occur regardless of type of stimuli, area of cortex, or animal species.
In the present study, we performed two different experiments to test the above hypothesis. First, the relationship between neuronal activity and hemodynamic responses was examined in the human occipital cortex using two types of visual stimuli (a black and white annular checkerboard and a flash-photo stimulus). Secondly, we investigated the relationship in the rat somatosensory cortex when the peripheral nerve was stimulated electrically. Here, we discuss the commonly observed regulation of oxygen transport during brain activation.
Six healthy male subjects (24–43 years old) participated in the human experiments. All subjects had normal or corrected-to-normal vision and provided written informed consent. The Communications Research Laboratory approved the experimental protocols. Four of the six subjects participated in NIOI measurements only. A flash-photo stimulator (SLS-2141, Nihon Kohden Kogyo Co. LTD, Japan) was used to provide visual stimuli (temporal frequency at 0.5, 1, 8 and 21 Hz). The time sequence of the experiments consisted of [control (30 sec) + stimulation (30 sec)]. Each of four different frequencies was shown four times in pseudo-random order. During the experiments, subjects sat still on a chair and were required to keep their eyes closed lightly. Simultaneous measurements using fMRI and NIOI were performed on the other two subjects. A black and white annular checkerboard, with a central fixation point and gray background, was used as the visual stimulus (perimacular annulus, 1.2 to 5.8 degrees; angle of each wedge, 10 degrees; number of layers, 5; temporal frequency, 0.5, 1.4, 4.7, and 14 Hz). The time sequence of the experiments consisted of [control (28 sec) + stimulation (28 sec)]. Each of four different reversal frequencies was shown four times in pseudo-random order. During the control and stimulation periods, subjects were required to fixate on a fixation cross in the middle of the checkerboard, and to lie still on his back on a patient table of MRI.
Optical Measurements and Analysis
A 16-channel near-infrared optical imaging system, OPTIM_A, was used to obtain optical images of changes in concentration of hemoglobin (Hb) species in the occipital cortex for simultaneous measurement with fMRI . The system consisted of six optical source units, each having three laser diodes (780, 805, and 830 nm) and six photomultiplier tubes. The source units and detector tubes were connected to glass-fiber bundles for the illumination of incident light and for the collection of reflected light from the head. Combinations of 16 nearest-neighbor pairs of input and output fibers were used to obtain a topographical image covering a 76 × 76 mm area in the occipital region of the head. The pixel size of the NIOI was estimated to be 20 × 20 mm at a source-detector distance of 27 mm. A single-channel near-infrared spectroscopy (OM-100A, Shimadzu Co., Japan) was used to monitor changes in concentrations of Hb species during the flash-photo stimulation. The system consisted of one optical source unit, with three laser diodes (780, 805, and 830 nm), and one photomultiplier tube. Sampling interval of near-infrared optical measurements was 1 sec. Changes in the Hb species concentration, expressed in an arbitrary unit, (Δ[oxy-Hb], Δ[deoxy-Hb], and Δ[total-Hb] (= Δ[oxy-Hb] + Δ[deoxy-Hb])) from the control conditions were calculated based on a modified Lambert-Beer law , using the extinction coefficient of chromophores reported by Matcher et al.  as follows.
Δ[oxy-Hb] = -1.489ΔAbs780 + 0.597ΔAbs805 + 1.485ΔAbs830
Δ[deoxy-Hb] = 1.855ΔAbs780 - 0.239ΔAbs805 - 1.095ΔAbs830
fMRI Measurements and Analysis
A 1.5 T MRI scanner (Magnetom Vision; Siemens, Germany) was used to obtain blood oxygen level-dependent contrast functional images. Functional images weighted with the apparent transverse relaxation time (T2*) were obtained with an echo planar imaging (EPI) sequence (repetition time (TR), 4000 msec; echo time (TE), 55.24 msec; flip angle (FA), 90°; field of view (FoV), 256 × 256 mm2; matrix size, 64 × 64; slice thickness, 4 mm). Areas of significant activation were determined using SPM99 http://www.fil.ion.ucl.ac.uk. Motion correction and spatial smoothing (three-dimensional Gaussian kernel, 11 mm full width at half maximum) were successively performed for each subject. Areas of activation were determined by a statistical threshold of P < 0.0001 (voxel level) corrected for multiple comparison for the entire search volume). To enable the fMRI and optical signals to be compared, the time series of the fMRI signals were processed as follows. After removing motion artifacts from all the T2*-weighted images using Automated Image Registration (AIR, http://bishopw.loni.ucla.edu/AIR3 version 3.0, the time courses of the fMRI signals from the region of interest were obtained using AVS/Express version 3.2 (Advanced Visual Systems Inc., USA).
All animal experiments were conducted in accordance with our institutional guidelines for the care and use of laboratory animals. Male Wistar rats weighing 190–220 g were purchased from SLC (Shizuoka, Japan) and allowed free access to food and water. The rats were initially anesthetized with urethane (0.8 g/kg body wt. i.p.). They were then tracheotomized, immobilized with pancuronium bromide (2 mg/kg/h), and artificially ventilated with room air. The ventilation was adjusted to maintain arterial blood gas tension in the physiological range. A craniotomy (4 × 5 mm) was performed on the left hemisphere and the dura mater was removed to expose the somatosensory cortex. A pair of needle electrodes was inserted underneath the skin of the plantar and ankle region in the contralateral hindlimb. Except as otherwise noted, the posterior tibial nerve (in part, the peroneal nerve) was electrically stimulated with a rectangular pulse of 3.8 mA intensity and 0.5 msec duration at 5 Hz. No major changes were detected in the mean arterial blood pressure (110 mmHg ± 5 S.D.) during and after posterior tibial (PT) nerve stimulation.
Measurement of Hemodynamic Parameters
Regional changes in red-blood-cell flow (rRBCFlow), velocity (rRBCVeloc), and content (rRBCMass) were measured in 12 rats using a laser-Doppler tissue flowmeter (LDF) (FLO-CE1, Omega Flow Inc., Japan) combined with a microscopy system . Signal changes from the surface of the cortex (semiglobular, 500 μm in diameter) were collected at a time constant of 0.5 sec. In separate experiments (5 rats), changes in the diameters (D) of second- and third- branches of the middle cerebral artery (MCA) and RBC velocity (v) in these single pial arterioles were measured using a fiber-optic laser-Doppler anemometer microscope (FLDAM) . The blood-flow rate in individual microvessels was calculated as v* π(D/2)2.
Measurements of Activation Area and Blood-Flow Distribution
Another five rats were used to acquire digitized images through transmission filters at 577.3 (± 1.2) nm with a charge-coupled device (CCD) camera. The magnitude of absorption change at 577 nm (ΔA577) was calculated in each pixel and color-coded to produce a false-color map. The same rats were then used for the measurement of membrane potential. The exposed cortex was stained with a voltage-sensitive dye JPW-1114 (Molecular Probes, USA) (0.5 mg/ml for 90 min). The hindlimb was electrically stimulated 21 times with a rectangular pulse of 3.8 mA intensity and 0.5 msec duration at a frequency of 0.33 Hz. The fluorescence associated with membrane potential changes was measured with a high-speed CCD imaging system, MiCAM01 (Brain Vision Inc., Japan). For each stimulation, 84 images were acquired at 2 msec intervals, and 21 series of time-course images were averaged.
Results and Discussion
This research was supported by the Breakthrough 21 Project of the Ministry of Post and Telecommunications of Japan, and in part by Grants-in-Aid from the Nissan Science Foundation of Japan.
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