1.

Introduction

It is well known that there is a tight coupling between changes in electrical activity in the brain and responses of the microcirculation.^{1 2} Although this coupling is used by neuroimaging techniques to visualize the neuronal electrical activity indirectly in living brain, such as positron emission tomography (PET), optical imaging based on intrinsic signal, and functional magnetic resonance imaging (fMRI), the dynamics of neurovascular coupling and the underlying mechanism of vascular regulation are not yet fully understood. There are two possible locations in the vascular compartment where the initial activity-evoked microcirculatory regulation cause take place: arterioles and capillaries.³

By tracking pial arteriolar diameter during 20 s of sciatic nerve stimulation in the rat, Ngai et al.;⁴ observed a dilation of pial arterioles in the hindlimb sensory cortex.⁴ Using laser Doppler flowmetry (LDF), they subsequently showed that dilation of surface arterioles was accompanied by a local increase in cerebral perfusion.⁵ Matsuura et al.;³ used LDF to measure the changes in red blood cell (RBC) velocity and concentration independently during activation of rat somatosensory cortex by direct microelectrial stimulation, and no significant time lag in the latency between RBC velocity and concentration was observed, which suggests that both arteriole dilation and capillary volume change contribute simultaneously to the initial regulation of local cerebral blood flow (CBF).⁶

Malonek et al.;⁶ reported their results with LDF and imaging spectroscopy studies in the cat visual cortex, which showed that the stimulation-evoked changes in cerebral blood volume (CBV) displayed an apparent similarity with the CBF-LDF curves but preceded the CBF increase by about 1 s. They thus supposed that the capillary change might occur before arteriolar dilation. However, the LDF used in these experiments had low spatial resolution (the sample volume was estimated to be approximately 1 mm³) and only an integrated CBF change was measured.^{3 6} Although the improved LDF monitors the RBC velocity and concentration independently, no detailed behavioral image of different microvascular compartments, such as venules, arterioles, and capillaries in parenchyma, during cortical activation evoked by the sensory stimulation was provided separately.³

Nuran and Lauritzen⁷ examined the contribution of single vascular elements to the local increase of CBF accompanying the increased neuron activity in response to electrical stimulation of parallel and climbing fibers by using scanning laser Doppler flowmetry (SLDF). The high spatial resolution (∼5 μm) applied in their studies enabled them to investigate the flow changes in arterioles, venules, and small vessels separately. But the 2-s temporal resolution of SLDF limited its application for observing the initial phase of neurovascular coupling.

To explore the possible location of the vasculature where microcirculation is initially regulated, it is necessary to obtain spatiotemporal activity-dependent vascular-response images with high resolution around the sites where the electrical recordings are targeted. The intrinsic optical imaging technique has achieved a two-dimensional spatial resolution of 20 to 100 μm and a temporal resolution of milliseconds, which has been used to approximate local blood volume changes at the hemoglobin-isosbestic wavelength (570 nm) and reveal Hb changes at 605 nm during sensory stimulation.⁸ Therefore optical imaging may shed light on the spatiotemporal dynamics of microvascular regulation and mechanisms controlling this local interaction. However, based on the finding that the early increase in the deoxyhemoglobin concentration starting less than 100 ms after the onset of stimulation is highly collocated with electrical activity at the columnar level whereas the delayed (300 ms after the onset of stimulation) increase in blood volume is not,⁹ studies using optical imaging of an intrinsic signal mainly focused on visualizing the functional architecture at the level of individual cortical columns such as the orientation columns of the visual cortex in cat and monkey^{9 10} and the single whisker functional representation in the rat barrel cortex,^{11 12} and regarded the blood vessel response observed as an artifact.⁸ There have been a few attempts to give the spatiotemporal characteristics of the vascular response evoked by peripheral nerve stimulation.^{10 11 13 14} However, details on the statistical estimation of the timing of CBV changes in different microvascular compartments have not been fully provided. In the present study, optical reflectance recording at 570 nm was performed obtain a detailed estimate of the spatiotemporal characteristics of blood volume changes, such as the start latency, peak latency, termination time, and peak amplitude, in different microcirculation compartments (cortical parenchyma, where capillaries are embedded, and in arteries and veins), in response to 2-s electrical stimulation of the sciatic nerve, and to help in understanding the dynamics of neurovascular coupling.

2.

Materials and Methods

2.4.

Optical Imaging

On each trial, the images of backscattered and reflected light were collected and stored in the random-access memory (RAM) of a computer over a 9-s period at 40 Hz using a 12-bit 640×480 pixels video camera (PixelFly VGA, Germany) attached to a microscope (Olympus SZ6045TRCTV, Japan). After the acquisition of all 360 frame images was completed, the images were transferred from RAM to the hard disk. Frames were recorded 1 s before the start of stimulation. The stimulations were generated with a stimulator (STG1004, Germany) and the stimulator was triggered by the CCD busy output signal of the CCD camera. The rat hindlimb somatosensory cortex was illuminated with light at 570 nm through a dual light guide (Olympus LG-DI, Japan) (for optical imaging setup, see Fig. 1). No dark-field method was used in our optical imaging studies. The two-dimensional optical spatial resolution and time resolution applied in these optical imaging studies were 5 μm/pixel (over a 3.2×2.4-mm field) and 25 ms, respectively.

Figure 1

Schematic drawing of optical imaging setup.

3.

Results

The results shown here are based on experiments performed in ten male Wistar rats. The mean arterial blood pressure (MABP) and blood gas values of all ten animals were as follows: MABP=115.2±9.3 mm Hg, pO ₂=125.5±14 mm Hg, pCO ₂=37.2±3.6 mm Hg, and pH=7.4±0.06. The values were maintained within a stable range throughout each experiment. The arterial blood pressure did not change significantly during the stimulation.

3.1.

Spatial Pattern of Evoked CBV Changes: Cortical Parenchyma, Arterioles, and Veins

In Fig. 2(a) the raw CCD image of the exposed cortex illuminated with 570-nm light clearly shows the anatomical cerebral vascular pattern through a thinned skull. In each rat, electrical stimulation of the sciatic nerve evoked a monophasic decrease in the optical reflectance of 570 nm at a location centered 2 mm laterally and caudally to the bregma, which was consistent with the hindlimb projection area of the somatosensory cortex. A decrease in the optical reflectance with 570-nm light indicated an increase in evoked local blood volume (under the assumption that the hematocrit remains constant) at the activated cortex. This increase in blood volume peaked at about 3 s and returned to the baseline about 7 s after the stimulus onset [see Fig. 2(d)]. As shown in Fig. 2(b) the vascular response evoked by sciatic nerve stimulation was found only in the arteries and the cortical parenchyma compartments where capillaries are embedded. No such stimulus-dependent blood volume increase during sciatic nerve stimulation was observed in the large cortical vein sites in our studies. All ten experimental animals exhibited a similar spatial response.

Figure 2

Spatial pattern of stimulus-induced optical reflectance changes (ΔR/R₀) at 570 nm. (a) Raw image of exposed rat cortex through a thinned skull at 570 nm illumination. The parietal branches of the superior cerebral vein and arteries are clearly distinguishable. (b) The spatial pattern of a stimulation-evoked vascular response. White circles indicate arteries, black dots indicate cortical parenchyma. The image was obtained by averaging the activation maps from 2.5 to 3 s after the onset of stimulation. The activation map was a visualization of the optical reflectance difference between an individual frame after stimulus onset and the mean intensity of frames prior to the stimulation. The color bar indicates the amplitude of signal change ΔR/R₀, where R=optical reflectance collected during an individual image and ΔR=R_i−R₀ denotes the reflectance difference between frame i’th and the baseline level. (c) Time course of activation maps in one experimental animal. Among the top images, the left, middle, and right images correspond, respectively, to mean activation maps during 0.5 s (averaged from 0 to 0.5 s), 1.5 s (averaged from 1 to 1.5 s), and 2.5 s (averaged from 2 to 2.5 s). The left, middle, and right images shown at bottom correspond, respectively, to mean activation maps during 3.5 s (averaged from 3 to 3.5 s), 4.5 s (averaged from 4 to 4.5 s) and 5.5 s (averaged from 5 to 5.5 s). (d) Mean temporal response of optical reflectance changes over the entire activated region across animals (n=10). The horizontal bar in the chart indicates the duration of stimulation.

3.2.

Temporal Pattern of Evoked CBV Changes

3.2.1.

Mean temporal pattern over entire activated region

The evoked change in reflectance started to decrease rapidly after the onset of stimulus and kept decreasing until it peaked at 2.85±0.66 s and returned to the baseline level 6.60±0.45 s after the onset of stimulation [see Fig. 2(d)]. The mean amplitude of the peak of optical signal changes evoked by the 5-Hz, 2-s electrical sciatic nerve stimulus was 0.38±0.18 over the activated region, including both cortical parenchyma and arteries.

3.2.2.

Difference of temporal dynamics of CBV changes evoked at different vascular compartments

In order to investigate the temporal dynamics of the local blood volume changes evoked at different vascular compartments, six small regions (each 0.1×0.1 mm) were selected for analysis. The region of interest was determined in the following manner: (1) three 0.01-mm² regions were selected in the parenchyma compartment and in the arterial compartment, denoted by black and white circles, respectively [see Fig. 2(b)]; (2) all the regions of interest were discrete and at least 0.2 mm apart. The temporal responses of the changes in optical reflectance in the cortical parenchyma and arteries are summarized in Table 1. As shown in Fig. 3(a), the local changes in optical reflectance at both parenchymal and arterial sites exhibited a similar shape of a monophasic decrease. The normalized ΔCBV started to increase rapidly after the stimulus onset and reached 10 of the peak response 0.70±0.32 s after the onset of stimulation. There was no significant time lag in this 10 start latency time between parenchyma (capillaries) and arteries at the site of the activated cortex [see Fig. 3(b)]. However, there were significant differences in peak amplitude, peak latency, and termination time of the changes in optical signal between the parenchyma and the arterial compartment. At the parenchyma site, the stimulus-induced signal changes (ΔR/R₀, where ΔR is the reflectance difference compared with that of the first frame and R₀ is the optical reflection intensity of the first frame) reached their peak value of 0.25±0.047 at 2.66±0.61 s after the start of sciatic nerve stimulation and returned to baseline 5.90±1.20 s after stimulus onset. At arterial sites, the changes in optical signal reached their peak value of 0.504±0.068 at 3.06±0.70 s after stimulus onset and returned to the baseline 6.70±1.30 s after the onset. Further ANOVA statistical analysis showed that both peak amplitude and peak latency were significantly different in parenchymal and arterial sites [peak amplitude: F(1,58)=17.30, P<0.01; peak latency: F(1,58)=5.41, P<0.05] . The termination time of the optical signal changes in the two vascular compartments was also significantly different [F(1,58)=5.595, P<0.05] . However, varying the location within the parenchymal and arterial compartments themselves (the three regions sampled for parenchymal and artery sites, respectively) did not affect the peak amplitude, peak latency, and termination time significantly [see Fig. 3(c)].

Figure 3

Time course of stimulus-evoked optical reflectance changes at 570 nm in different microvascular compartments across animals (n=10) for the cortical parenchyma and arteries. The horizontal bars indicate the duration of stimulation. (a) Mean temporal dynamics over the regions indicated in Fig. 2(b). Both the parenchyma and artery plots are the average result of the time courses of their three 0.01-mm² “sampling” regions. (b) Normalized changes in blood volume regions indicated in Fig. 2(b). The blood volume changes were normalized to the peak amplitude of the signal changes. (c) Time courses of changes in optical reflectance in the six selected regions. Each plot results from averaging the intensity changes of all the pixels within the region of interest across ten experimental animals.

Table 1

Temporal characteristics of optical reflectance changes in somatosensory cortex evoked by sciatic nerve stimulation.
	Peak Amplitude ()	Start Latency (seconds)	Peak Latency (seconds)	Termination Time (seconds)
Cortical parenchyma	0.25 ± 0.047	0.70 ± 0.32	2.66 ± 0.61	5.90 ± 1.20
Arteries	0.50 ± 0.068		3.06 ± 0.70	6.70 ± 1.30

4.

Discussion

Neuron activity initiates microcirculatory regulation. Using optical imaging spectroscopy^{18 19} and laser-Doppler flowmetry.^{3 6 7 18} the relationships between cerebral blood flow, oxygenation, and volume have been investigated on the rodent barrel cortex, the anesthetized cat, and the awake monkey. Focused on the initial phase of activation, some investigators observed a significant lag (∼1 s) between blood volume and blood flow changes in cats and suggested that the increase in local blood volume prior to the increase in flow may result from an early functional recruitment of capillaries before the arteriolar segment dilates.⁶ Myles et al.;¹⁸ obtained similar results in rodent studies but with a slight lag (∼0.2 s) and the lag was just detectable with temporal resolution. Others found that there was no significant time lag in the latency between red blood cell velocity and concentration upon neuronal activation and suggested that both arteriole dilation and capillary volume changes contribute simultaneously to the initial regulation of CBF.³

In the present study, changes in local blood volume started to increase rapidly after the onset of the stimulus, which is consistent with the result of previous studies in cat visual cortex⁶ and rat barrel cortex¹⁸ using optical imaging spectroscopy. The signal change reached 10 of the peak response 0.7±0.32 s after the stimulus. There was no significant time lag in this 10 start latency time between parenchymal (capillaries) and arterial compartments at the activation site [see Fig. 3(b)]. This result suggests that the stimulus evokes a rapid increase in local blood volume in both capillaries and arterioles. The initial increase in capillaries may result from their functional recruitment, as suggested in Refs. 6 and 7, but the mechanism controlling the increase in blood volume (HbT concentration) in arterioles during this early phase is unclear. One possibility is that an activity-induced dilation of arterioles occurs, together with an independent increase in blood volume in capillaries. Although it has been reported that a prolonged (20 s) electrical stimulation of the sciatic nerve evoked a pial arteriole dilation after an initial brief latent period (<2 s)²⁰ and the prolonged (20 s) stimulation of parallel fibers also produced arteriolar dilation,¹⁶ the diameter changes in pial arterioles in response to short (2 s) stimulations requires further investigation.

As shown in Fig. 3(b), after reaching 10 of the peak response, the blood volume increases at two microvascular compartments diverged. The changes in the parenchyma compartment peaked 2.66±0.61 s after the onset of the stimulus, which is, 0.4±0.2 s before the change at an arterial site (P<0.05). This difference in the time-lag of peak latency indicates a faster increase in blood volume in capillaries than in arterioles, which suggests that the stimulus-induced increase in capillaries cannot be entirely accounted for by the dilation of arterioles.

In the present study, a decrease in optical reflectance induced by stimulation of the sciatic nerve occurred only at the arterial and parenchymal sites of the activated cortex, whereas no such response was observed in large cortical veins. The spatial pattern of vascular activities we observed is similar to that found by Nemoto et al.;²¹ at a similar region of the rat somatosensory cortex under 586-nm illumination during electrical stimulation of the hindlimb. Since both 570 nm and 586 nm are the isosbestic points of oxyhemoglobin and deoxyhemoglobin, the decreases in optical signal evoked by stimulation of the peripheral nerve are likely to indicate an activity-related increase in the concentration of total hemoglobin in arteries and capillaries.

However, at vein sites, although Narayan et al.;¹³ reported a functional venous optical signal with the stimulation of forelimb and whisker under both 550-nm (which is also a hemoglobin-isosbestic wavelength) and 605-nm light, our results are consistent with the findings of Nemoto et al.;²¹ and are supported by the observation of changes in the diameter of pial vessels during sciatic nerve stimulation in rats by Ngai et al.;^{4 5} In their studies, a dilation of pial arterioles in the hindlimb sensory cortex⁴ and an accompanying increase in local cerebral blood flow⁵ were detected during sciatic nerve stimulation, but no alteration in vein diameter was observed.⁴ The absence of a decrease in optical reflectance under 570 nm in a large cortical vein during neuron activation indicates that no net change in the total hemoglobin concentration was evoked in veins, although the blood oxygenation in veins changes during cortex activation according to the results of optical imaging using 605 nm.

As shown in previous studies in the rat hindlimb (Nemoto et al.;²¹), forelimb (Narayan et al.;¹³), and barrel (Hess et al.;,²² Masino et al.;,¹⁴ Narayan et al.;¹³) somatosensory cortex, an activity-related optical signal using 605 nm occurred at the cortical parenchyma and the draining cortical vein sites. Furthermore, the optical responses at this wavelength at the veins and parenchymal sites initially exhibited a biphasic pattern of decrease and then an increase in the optical reflectance. It is interesting that using another wavelength (577 nm), in which the extinction coefficient for oxyhemoglobin is only 1.67 times more than that for deoxyhemoglobin, Nemoto et al.;²¹ and Hess et al.;²² also reported the absence of a functional venous signal.

Based on the findings obtained with all these wavelengths, we arrived at the following picture of the dynamics of neurovascular coupling at veins: During the initial phase of neuron activation, there is an increase in deoxyhemoglobin and a decrease in oxyhemoglobin, but no net change in total hemoglobin concentration, whereas during the later phase, oxyhemoglobin content increases and deoxyhemoglobin content decreases, with a roughly constant total hemoglobin content. The changes in blood oxygenation occurring at cortical vein sites could be a result of the venous drainage effect when the blood travels down the vascular tree from the site of activation.²³ Nemoto et al.;²¹ suggested that the absence of an optical signal decrease in veins during activation may be explained by the nonlinear relationship between the changes in concentrations of hemoglobin derivatives and the absorbance changes in the high absorbance range. However, this assumption may be questionable because of the existence of the optical signal at 605 nm.

5.

Conclusion

The spatiotemporal characteristics of changes in local cerebral blood volume in the rat hindlimb somatosensory cortex evoked by electrical stimulation of the sciatic nerve were investigated by optical imaging at 570 nm. We statistically estimated the spatiotemporal characteristics of blood volume changes, such as the start latency, peak latency, termination time, and peak amplitude, in different microcirculation compartments (cortical parenchyma, arteries, and veins). Our results suggest that sciatic nerve stimulation evokes an increase in local blood volume in both arteries and cortical parenchyma sites soon after the onset of the stimulus, but that the blood volume increase in capillaries could not be entirely accounted for by the dilation of arterioles. However, we found no activity-related blood volume increase in large cortical veins.