Investigation of the cerebral
hemodynamic response function in single
blood vessels by functional photoacoustic
microscopy
Lun-De Liao
Chin-Teng Lin
Yen-Yu I. Shih
Hsin-Yi Lai
Wan-Ting Zhao
Timothy Q. Duong
Jyh-Yeong Chang
You-Yin Chen
Meng-Lin Li
Investigation of the cerebral hemodynamic response
function in single blood vessels by functional
photoacoustic microscopy
Lun-De Liao,a,bChin-Teng Lin,a,bYen-Yu I. Shih,cHsin-Yi Lai,aWan-Ting Zhao,dTimothy Q. Duong,c Jyh-Yeong Chang,aYou-Yin Chen,a,eand Meng-Lin Lif
aNational Chiao Tung University, Department of Electrical Engineering, Hsinchu, Taiwan bNational Chiao Tung University, Brain Research Center, Hsinchu, Taiwan
cUniversity of Texas Health Science Center at San Antonio, Research Imaging Institute, San Antonio, Texas dNational Taiwan University, Institute of Zoology, Taipei, Taiwan
eNational Yang Ming University, Department of Biomedical Engineering, Taipei, Taiwan fNational Tsing Hua University, Department of Electrical Engineering, Hsinchu, Taiwan
Abstract. The specificity of the hemodynamic response function (HRF) is determined spatially by the vascular archi-tecture and temporally by the evolution of hemodynamic changes. Here, we used functional photoacoustic micro-scopy (fPAM) to investigate single cerebral blood vessels of rats after left forepaw stimulation. In this system, we analyzed the spatiotemporal evolution of the HRFs of the total hemoglobin concentration (HbT), cerebral blood volume (CBV), and hemoglobin oxygen saturation (SO2). Changes in specific cerebral vessels corresponding to various electrical stimulation intensities and durations were bilaterally imaged with36 × 65-μm2spatial resolution. Stimulation intensities of 1, 2, 6, and 10 mA were applied for periods of 5 or 15 s. Our results show that the relative functional changes in HbT, CBV, and SO2are highly dependent not only on the intensity of the stimulation, but also on its duration. Additionally, the duration of the stimulation has a strong influence on the spatiotemporal charac-teristics of the HRF as shorter stimuli elicit responses only in the local vasculature (smaller arterioles), whereas longer stimuli lead to greater vascular supply and drainage. This study suggests that the current fPAM system is reliable for studying relative cerebral hemodynamic changes, as well as for offering new insights into the dynamics of functional cerebral hemodynamic changes in small animals.© 2012 Society of Photo-Optical Instrumentation Engineers (SPIE). [DOI:10.1117/1.JBO.17.6.061210]
Keywords: functional photoacoustic microscopy; electrical stimulation; total hemoglobin concentration; hemoglobin oxygen saturation. Paper 11507SS received Sep. 14, 2011; revised manuscript received Dec. 22, 2011; accepted for publication Jan. 16, 2012; published online May 7, 2012.
1 Introduction
To understand the hemodynamic response function (HRF) in single cerebral blood vessels, it is crucial to image changes in total hemoglobin concentration (HbT), cerebral blood volume (CBV), and hemoglobin oxygen saturationðSO2Þ.1,2The most prominent neuroimaging technique, functional magnetic resonance imaging (fMRI), maps brain function by measuring surrogate changes in cerebral blood flow (CBF), CBV, and oxygenation that accompany changes in neural activity. Although, a remarkable association exists between changes in neural activity and the hemodynamic state, a complete under-standing of the spatiotemporal evolution of the cerebral HRF is still missing. Recently, Hirano et al. investigated changes in CBF and CBV during forelimb stimulations of various durations in an anesthetized rodent model.3 However, fMRI measures only changes in selected pixels located in the activa-tion region in response to a stimulus. It is still difficult to image HRF changes in single cerebral blood vessels using fMRI with-out the use of contrast agents.4Recently, optical brain imaging
has been increasingly used to study brain functions in vivo5
because it can measure oxy-hemoglobin ðHbO2Þ and deoxy-hemoglobin (Hb) through the models’ distinctive optical absorp-tion characteristics.5–8Diffusion optical imaging (DOI)9,10and laser speckle imaging (LSI)11,12are the two most widely used
optical imaging techniques for the in vivo imaging of cerebral hemodynamic changes. The DOI technique can acquire CBV, HbO2 and Hb signals directly through the intact skull and track their changes noninvasively in response to an electrical stimulation of the rat forepaw.9Recently, Sigel et al. performed a temporal comparison of the functional changes in HbO2and Hb by DOI and fMRI during different durations of stimulation of the rat forepaw. Their results suggest that the HRF changes measured by DOI and fMRI are qualitatively similar, and that the use of optical imaging techniques to investigate changes in neurovascular coupling is becoming increasingly reliable in small animal models. However, for DOI, SO2 quantification is problematic due to tissue scattering and the nonlinear relation-ship between signal intensity and absorption coefficients.8,13,14 This problem also impairs the visualization of HRF changes in single blood vessels. In contrast, LSI can probe the hemody-namic changes in single blood vessels with high spatial resolu-tion after the skull has been removed.15Bouchard et al. proposed a novel system that, at a lower cost, enables simultaneous
Address all correspondence to: You-Yin Chen, National Yang Ming University, Department of Biomedical Engineering, Taipei, Taiwan; E-mail: irradiance@so-net.net.tw, Meng-Lin Li, National Tsing Hua University, Department of Electrical Engineering, Hsinchu, Taiwan. Tel: +886 3 516 2179; Fax: +886 3 571 5971;
E-mail:mlli@ee.nthu.edu.tw 0091-3286/2012/$25.00 © 2012 SPIE
visualization of HbT, HbO2, and Hb dynamics within single blood vessels during forepaw stimulation.15However, due to the limited penetration depth and poor depth resolution of LSI, it can access information only from single blood vessels that reflect from the cortical surface.11,12,16
Functional photoacoustic microscopy (fPAM), which can measure functional hemoglobin changes in single blood vessels without a labeling agent, is ideal for the in vivo imaging of changes in HbT, CBV, and SO2. Recently, photoacoustic (PA) imaging has been applied to subcutaneous vasculature ima-ging,17breast tumor detection,18and oxygenation monitoring in blood vessels.19,20Our recent study utilized the fPAM capability
of acquiring relative functional hemodynamic responses to forepaw electrical stimulation in exposed rodent brains.21Our
findings indicate that hemodynamic changes in specific cortical regions of the brain can be reliably imaged using fPAM.
In this study, the fPAM system was used to investigate functional HRF changes in HbT, CBV, and SO2within specific cerebral blood vessels. Bilateral neurovascular changes were eli-cited by left forepaw electrical stimulation with various stimula-tion parameters. We studied the temporal dependence of the changes in specific cerebral vessels, using different stimulation intensities and durations and performed comprehensive charac-terization of vascular dynamics. This technique has the potential to advance our understanding of changes in cerebral neurovas-cular coupling and to improve the monitoring of cerebral HRF changes in small animal models.
2 Materials and Methods
2.1 Dark-Field Confocal fPAM System
A 50-MHz dark-field confocal fPAM system was used to image the functional changes in specific cortical vessels. Laser, pulses were generated by an optical parametric oscillator (Surlite OPO Plus, Continuum, Santa Clara, CA) pumped by a frequency-tripled Nd:YAG Q-switched laser (Surlite II-10, Continuum), which provides ∼4-ns laser pulses at a pulse repetition rate of 10 Hz. Laser pulses at two visible wavelengths, 560ðλ560Þ and 570 nm ðλ570Þ, were used for PA wave excitation. To probe the relative changes in HbT, CBV, and SO2 in specific blood vessels, these two wavelengths have been optimized to provide high signal-to-noise ratios (SNRs) and sensitivity.21 The 50-MHz ultrasonic transducer used in the current fPAM system was custom-made by the Resource Center for Medical Ultrasonic Transducer Technology at the University of Southern California (http://bme.usc.edu/UTRC/). It has a 6-dB fractional bandwidth of 57.5%, a focal length of 9 mm, and a 6-mm active element, offering an axial resolution of 36μm and a lateral reso-lution of 65μm.
Laser energy was delivered by a 1-mm multimode fiber. The fiber tip was coaxially aligned with a convex lens, an axicon, a plexiglass mirror, and an ultrasonic transducer on an optical bench, forming dark-field illumination that is confocal with the focal point of the ultrasonic transducer. The incident energy density on the sample surface was well within the safety limit of the American National Standards Institute (ANSI). During the imaging process, the transducer was immersed in an acrylic water tank with a hole in the bottom that was sealed with a piece of 15-μm-thick polyethylene film. A thin layer of ultraso-nic gel was applied onto the rat’s head and then attached to the thin film in order to ensure good coupling of PA waves to the tank. The PA signals received by the ultrasonic transducer were
preamplified by a low-noise amplifier (noise figure 1.2 dB, gain 55 dB, AU-3A-0110, Miteq, Hauppage, NY) and cascaded to an ultrasonic receiver (5073 PR, Olympus, Center Valley, PA). Then, the signals were digitized and sampled by a computer-based, 14-bit analog-to-digital (A/D) card (CompuScope 14200, GaGe, Lockport, ILI) at a 200-MHz sampling rate for data storage. The fluctuations in the laser energy were monitored by a photodiode (DET36A/M, Thorlabs, Newton, NJ). Finally, the recorded photodiode signals were applied to compensate for PA signal variations caused by laser energy instability before any further signal processing.
The achievable penetration depth of the fPAM is 3 mm, and the SNR (defined as the ratio of the signal peak value to the rms value of the noise) is∼18 dB. Three scanning types can be provided by this system to image the region of interest: A-line (one-dimensional images where the axis represents the imaging depth), B-scan (two-dimensional images where one axis is the lateral scanning distance and the other is the imaging depth), and C-scan (projection images from the three-dimensional images). To speed up the imaging of the rapid hemodynamic response in the subsequent functional imaging analysis, no signal averaging in time was performed. Note that the amplitude of the envelope-detected PA signal was used in the functional imaging analysis.
2.2 Experimental Animals
Six male Wistar rats (National Laboratory Animal Center, Taiwan), weighing 250 to 300 g each, were used. The animals were housed at a constant temperature and humidity with free access to food and water. Before the imaging experiments, the rats fasted for 24 h but were given water ad libitum. All animal experiments were conducted in accordance with the guidelines of the Animal Research Committee of National Chiao Tung University and National Tsing Hua University. The animals were initially anesthetized with 3% isoflurane. Supplemental α-chloralose anesthesia (70 mg∕kg) was injected intraperitone-ally as needed. The anesthetized rats were mounted on a custom-made acrylic stereotaxic head holder to reduce motion artifacts during the experiment, and the skin and muscle were cut away from the skull to expose the bregma landmark. The anteropos-terior (AP) distance between the bregma and the interaural line22was surveyed directly. The bregma was 9.3 0.12 mm [mean standard deviationðSDÞ] anterior to the interaural line.23 Next, a craniotomy was performed on each animal, and a bilateral cranial window of approximately 8ðhorizontalÞ × 6ðverticalÞ mm2was made with a high-speed drill. After the rat was secured to the stereotaxic frame and placed on the bed pal-let, the pallet was moved into position at the bregma, which was 9 mm anterior to an imaginary line drawn between the center of each ear bar (the interaural line).21 In the subsequent
experi-ments, the interaural and bregma references were then used to position the head in the fPAM system without additional surgery.
After bregma positioning, a PA C-scan (i.e., a projection image from the three-dimensional images) was performed to acquire reference images of the cortical vasculature. The cortical blood vessels under the open-skull window of the rat cortical surface, indicated by solid red arrows in Fig.1(a), were imaged in vivo by fPAM atλ570, as shown in Fig.1(b). In addition, arter-iolar branches from the anterior cerebral artery (ACA) vessel system can be seen in the projected C-scan image.24,25These
both Fig.1(a) and 1(b), along with the superior sagittal sinus (SSS). The SSS is the largest vein in the rodent brain cortex.25 The MI and MII arterioles are two different-sized arterioles that branch from the ACA blood vessel system and extend from the bottom center of the brain to the cortical surface. The bilateral MI arterioles cross through the anatomical borders of the pri-mary motor cortex and secondary motor cortex.25 However, the bilateral MII arterioles are located in the anatomical borders of the primary somatosensory cortex that innervates the forepaw (S1FL).25,26Functional changes in HbT, CBV, and SO
2 in the SSS and the bilateral MI and MII arterioles were imaged by fPAM scanning during stimulation. These images were acquired along the black solid and dashed lines in the bilateral regions shown in Fig.1(b). Moreover, some branches of these vessels were also visualized in the projected C-scan image. Note that the SSS was not imaged well by the current fPAM system, which is similar to previous reports.21,27The geometric focusing and the finite detection bandwidth of the ultrasound transducer that was used may have accounted for the weakness of the PA signals detected from the SSS.28 Sensing with spherical geometric
focusing inherently preferred point-like PA sources, which was not applicable to the PA signals of the SSS because of its large size. In addition, the frequency of the PA signal gen-erated by the SSS might be out of the detection bandwidth of the transducer, thus weakening the detected PA signals.
2.3 Electrical Forepaw Stimulation
Forepaw stimulation was applied to evoke functional hemody-namic changes in the cerebral vessels, and it was achieved by insertion of thin-needle stainless electrodes under the skin of the rat’s left forepaw. Electrical stimulation was then applied using a stimulator (Model 2100, A-M Systems, Sequim, WA). A mono-phasic constant current of four pulses of different intensities (1, 2, 6, and 10 mA) with a 0.2-ms pulse width at a frequency of 3 Hz was used. Various stimulation intensities were employed to evoke hemodynamic changes of different strengths during the stimulation onset. In addition, two stimulation durations of 5 and 15 s were employed. A block-design paradigm was employed in this study for functional signal acquisition. The short stimulation duration (5 s) was originally selected to pro-vide enough time for the hemodynamic response to reach a max-imum value in order to enable the large amount of averaging necessary.29The longer stimulation duration (15 s) was selected to enable the cerebral volume response to reach a nearly max-imal response while avoiding the progressive attenuation that is seen for stimuli of longer durations.30 Each trial consisted of three blocks, and each block began with a 20-s baseline that was followed by a 5-s Stimulation-ON and a 75-s Stimulation-OFF period for the shorter stimulation or a 15-s Stimulation-ON and a 65-s Stimulation-OFF period for the longer stimulations, as illustrated in Fig.2(a) and2(b). There was a 5-s lapse between each block, and the PA signals [Fig.2(c)] atλ560orλ570were acquired in each block to assess stimulation-induced hemodynamic changes in specific single cortical vessels (the SSS and bilateral MI and MII arterioles). Note that the timing used in our block-design paradigm is ade-quate for the rat’s brain to recover to the resting state before the next stimulus.31
2.4 Data Analysis of the Functional Changes in CBV, HbT, and SO2
Two optimized wavelengths (i.e.,λ560andλ570) were employed to monitor functional HbT, CBV, and SO2changes with a high SNR and high sensitivity.21It was assumed that the CBV is pro-portional to the cross-sectional area of a single blood vessel.32
Functional CBV changesðRCBVÞ in a single vessel can be mon-itored by assessing the changes in the cross-section of the vessel during each block against a cross-section baseline acquired immediately before the electrical stimulation onset (i.e., ∼20 s in each block). PA cross-sectional B-scan images of
Fig. 1 Comparison of an open-skull photograph with a PA-projected C-scan image acquired atλ570. (a) An open-skull photograph of the cerebral
surface showing an unobstructed view of the cortex vessels. Some branches of the blood vessels, which are indicated by the red solid arrows, are imaged by fPAM in (b). The green dashed line indicates the position of the bregma. The red dashed arrow indicates the section of the bregma þ0.98 mm activated during left forelimb stimulation. (b) In vivo PA-projected C-scan image of the blood vessels in the superficial layer of the cortex acquired atλ570. The green dashed arrow indicates the position of the bregma. The SSS and some cortical vessels on the cortical surface can be
identified. Functional B-scan images of the SSS and bilateral MI and MII arterioles were acquired by scanning along the black solid and dashed lines in the bilateral region.
Fig. 2 Schematic diagram of the experimental protocol, including the two different stimulations and the fPAM imaging timing. (a) Block design utilized for the short electrical forepaw stimulation task (5 s). (b) Block design utilized for the long electrical forepaw stimulation task (15 s). (c) PA data were acquired in three active blocks under dif-ferent stimulation intensities. Multiple PA B-scan images of specific sin-gle cortical vessels were acquired in each active block. Note that in (b) and (c), the task began at the baseline “Stimulation-OFF” state; the active“Stimulation-ON” state consisted of a constant 0.2-ms elec-trical pulse width, a 3-Hz pulse train and a pulse amplitude of 1, 2, 6, or 10 mA. There was a 5-s lapse between the PA scan and the block-design paradigm to the next block.
each specific single vessel at λ570 [i.e., IRð570Þ] were used. RCBVðtÞ was constructed according to the following equation:
RCBVðtÞ ¼
A½IRð570ÞðtÞ
A½IRð570Þ;baseline
; (1)
where t is the time in each block, A½IRð570ÞðtÞ represents the cross-sectional area of a single vessel at a given time in each block, and A½IRð570Þ;baseline is the cross-section baseline esti-mated from the baseline image acquired immediately before electrical stimulation onset in each block. A½IRð570Þ was calcu-lated from the total vessel pixel count of a single vessel image [i.e., IRð570Þ]. A vessel pixel was defined as any pixel with a PA signal three times greater than the background signal.21,33
The optical absorption of blood at λ570 is insensitive to the SO2level because λ570 is an isosbestic point of the molar extinction spectra of HbO2 and Hb. That is, changes in the PA signal atλ570largely result from changes in the total amount of hemoglobin (i.e., HbT) within the PA resolution cell (HbT¼ ½HbO2 þ ½Hb).27Therefore, PA B-scan images atλ570½IRð570Þ were used to probe the changes in HbT. Because the PA signal at a given pixel in IRð570Þis proportional to the HbT within the PA resolution cell centered at that pixel, the mean functional HbT changes [RHbTðtÞ] in a single vessel during the stimulation per-iod could be assessed as follows:
RHbTðtÞ ¼ X ðx;yÞ∈vessel pixel ½IRð570Þðx; z; tÞ∕A½IRð570ÞðtÞ − X ðx;yÞ∈vessel pixel ½IRð570Þ;baselineðx; z; tÞ∕ A½IRð570Þ;baselineðtÞ; (2)
where (x, z) is the pixel position and IRð570Þ;baselineis the baseline image atλ570acquired immediately before electrical stimulation onset in each block.
In contrast to those atλ570, PA signal changes atλ560 were sensitive to SO2changes because the largest absorption changes in the visible spectrum occur at approximately λ560, when the SO2 level changes. To exclude the effects of changes in HbT on PA signals and to observe only the SO2level changes,21 func-tional images of SO2 changes [ΔIFð560ÞðtÞ] at the given time point t in each block were then assessed according to the fol-lowing equation: ΔIFð560ÞðtÞ ¼ Ið560ÞðtÞ IRð570ÞðtÞ − Ið560Þ;baseline IRð570Þ;baseline ¼ IFð560ÞðtÞ − IFð560Þ;baseline; (3)
where Ið560Þ(i.e., the PA image acquired atλ560) was normalized to IRð570Þon a pixel-by-pixel basis, and Ið560Þ;baselinewas the base-line image atλ560acquired immediately before electrical stimu-lation onset in each block. In this equation, negative values in ΔIFð560Þ(i.e., a positive−ΔIFð560Þ) indicate increases in the SO2 level and vice versa.21 The mean functional SO2 changes [RSO2ðtÞ] in a single vessel during the stimulation period
were probed as follows:
RSO2ðtÞ ¼ X ðx;zÞ∈vessel pixel ½IFð560Þðx; z; tÞ∕A½IRð570ÞðtÞ − X ðx;zÞ∈vessel pixel ½IFð560Þ;baselineðx; z; tÞ∕ A½IRð570Þ;baselineðtÞ: (4)
When IRð570Þis used as a marker for HbT and IFð560Þis used as a marker for SO2, the fPAM system can be used to probe the changes in HbT and SO2independently, as shown in Eqs.(2)and(4). 2.5 Statistical Analyses
The experiments were designed to quantitatively measure PA signal½IRð570Þ changes, as well as changes in the SSS and bilat-eral MI and MII arterioles, following electrical stimulation. Sta-tistical significance was assessed using a paired t-test, with the significance defined as a probabilityðpÞ value of <0.05. Side-to-side differences in PA signals [IRð570ÞandΔIFð560Þ] of the stu-died areas and CBV changes were both examined using paired t-tests (p < 0.05, n¼ 6). The significance of changes observed in the fPAM signals [ΔIFð560Þ] of the studied areas in response to electrical stimulation was evaluated using the Wilcoxon matched-pairs signed-rank test (two-tailed, p < 0.05, n¼ 6). All statistical analyses were performed using SPSS (version 10.0, SPSS, IBM, Armonk, NY).
3 Results
3.1 HbT Changes in the Bilateral Cerebral Vessels During Stimulations of Different Intensities and Durations
The first rows of Fig.3(a)and3(b)show RHbTðtÞ in the SSS and bilateral MI and MII arterioles during 1 stimulation block and using the stimulation intensities of 1, 2, 6, and 10 mA, with a dura-tion of 5 s. The yellow zone indicates the 5-s Stimuladura-tion-ON per-iod. Significant HbT changes were observed in the SSS and MI and MII arterioles contralateral to the electrically stimulated left fore-paw. In contrast, no significant HbT changes were found in the ipsilateral MI or MII arteriole. The time to peak RHbT in the SSS and the contralateral MI and MII arterioles after stimulation onset using a stimulation intensity of 2 mA was 16.24, 9.68, and 19.75 s, respectively. There was no significant difference among the times to peak RHbTin the SSS and contralateral MI and MII arterioles using any of these stimulation intensities. However, the peak RHbTvalues were significantly higher than the baseline when these stimulation intensities were used. The average RHbT response time was 26.56, 23.42, and 25.84 s for the SSS and the contralateral MI and MII arterioles, respectively. The response time is defined as the difference between the time of stimulation onset (i.e., 20 s in each stimulation block) and the time when the RHbTvalue return to the baseline. Here the baseline is the RHbT value obtained immediately before the stimulation onset. Note that the same definition is also applied to RCBVand−RSO2.
Figure4(a)shows the peak RHbTvalues in the SSS and con-tralateral MI and MII arterioles as a function of stimulation intensity. Using a stimulation intensity of 2 mA, the peak RHbT value was 0.98 0.002 (mean SD) (p < 0.001) in the SSS, 2.940 0.003 (mean SD) (p < 0.001) in the contralat-eral MI arteriole and 3.510 0.004 (mean SD) (p < 0.001) in the MII arteriole during stimulation (Wilcoxon matched-pairs signed-rank test, n¼ 6). Three additional statistically
Fig. 3 Functional changes in the HbT, CBV, and SO2(i.e.,RHbT,RCBV,−RSO2) in the SSS and bilateral MI and MII arterioles during one stimulation block,
shown as a function of time under different stimulation intensities (1, 2, 6, and 10 mA) and with a duration of 5 s. (a) Ipsilateral. (b) Contralateral. The error bars represent the standard deviations of the data from six rats. The yellow zone indicates the 5-s Stimulation-ON period. Note that the ipsilateral SSS is the SSS during the ipsilateral-side measurement, and the contralateral SSS is the same SSS during the contralateral-side measurement.
significant peak RHbTvalues using stimulation intensities of 1, 6, and 10 mA are plotted in Fig.4(a). There was a slight trend of increasing RHbT peak amplitude with increasing stimulation intensity. No significant peak RHbT values were observed in the ipsilateral MI or MII arterioles from Stimulation-ON to Stimulation-OFFðp > 0.05Þ.
The first rows of Fig.5show RHbTðtÞ in the SSS and bilateral MI and MII arterioles in one 15-s stimulation block using a sti-mulation intensity of 2 mA. In this long stisti-mulation, the time to peak RHbTafter stimulation onset in the SSS and the contralat-eral MI and MII arterioles was 18.41, 17.12, and 21.08 s, respec-tively. The times to peak RHbTin the SSS and the contralateral MI and MII arterioles were longer than those using the 5-s sti-mulation. During stimulation, the peak RHbTvalue using a 15-s stimulation was 1.16 0.001 (mean SD) (p < 0.001) in the SSS, 3.240 0.003 (mean SD) (p < 0.001) in the contralat-eral MI arteriole and 3.890 0.004 (mean SD) (p < 0.001) in the MII arteriole (Wilcoxon matched-pairs signed-rank test, n¼ 6). The peak RHbT values were significantly higher when the 15-s stimulation was used than when the values from the 5-s stimulation were used.
Under the long stimulation condition, the response time of RHbT from the RHbT to baseline was 28.42, 23.75, and 26.14 s for the SSS and the contralateral MI and MII arterioles, respectively. Only the SSS and the contralateral MII showed longer response times when the 15-s stimulation was used. The response time of RHbT in the contralateral MI vessel was only slightly increased compared with the shorter stimulation. The time to peak RHbTof the contralateral MI arteriole increased significantly under the 15-s stimulation compared with 5 s; how-ever, the times to peak RHbTof the SSS and contralateral MII arteriole were not increased significantly.
3.2 CBV Changes in Bilateral Cerebral Vessels During Stimulations of Different Intensities and Durations The second rows of Fig.3(a)and3(b)show RCBVðtÞ in the SSS and bilateral MI and MII arterioles in 1 stimulation block under stimulation intensities of 1, 2, 6, and 10 mA and a stimulation duration of 5 s. Using a stimulation intensity of 2 mA, the time to peak RCBV was 8.34 s in the contralateral MI arteriole and 16.13 s in the contralateral MII arteriole after stimulation onset. There was no significant difference in the time to peak RCBV between the contralateral MI and MII arterioles using any of the four stimulation intensities; however, the peak RCBV values were significantly different between the MI and MII arterioles. The average RCBVresponse time from the stimulation
onset to the return of RCBV to baseline was 14.12 s in the contralateral MI arteriole and 20.78 s in the contralateral MII arteriole under a stimulation intensity of 2 mA [Fig.3(b)].
Figure4(b)shows the peak RCBVvalues in the contralateral MI and MII arterioles as a function of stimulation intensity. Using a stimulation intensity of 2 mA, the peak RCBVvalues in the con-tralateral MI and MII arterioles were 1.160 0.003 (mean SD) (p < 0.0001) and 1.270 0.002 (mean SD) (p < 0.001), respectively (paired t-test; n¼ 6). In contrast, neither the MI nor the MII arterioles displayed significant CBV changes (p> 0.05) in the hemispheres ipsilateral to the forepaw response region. Additionally, no significant RCBVwas found in the SSS during stimulation (p> 0.05). Another three significant peak RCBV values using stimulation intensities of 1, 6, and 10 mA are plotted in Fig.4(b). The results indicate that the peak RCBV values were sensitive to the increasing stimulation intensities.
The second row of Fig.5shows RCBVðtÞ in the SSS and bilat-eral MI and MII arterioles in one stimulation block under 15-s stimulation. Using the long stimulation condition (Fig.5), the RCBV response times from the stimulation onset to the return of RCBVto baseline were 17.85 and 22.38 s for the contralateral MI and MII arterioles, respectively.
3.3 SO2 Changes in Bilateral Cerebral Vessels During Stimulations of Different Intensities and Durations The third rows of Fig.3(a)and3(b)illustrate time course mea-surements of−RSO2in the SSS and bilateral MI and MII
arter-ioles in one stimulation block using stimulation intensities of 1, 2, 6, and 10 mA and a duration of 5 s. After stimulation onset at 2 mA, the time to peak−RSO2was 12.14, 9.87, and 17.82 s in the
SSS and contralateral MI and MII arterioles, respectively. There was no significant difference among the times to peak−RSO2in
the SSS and the contralateral MI and MII arterioles when any of the four stimulation intensities were being used. However, the peak−RSO2values were significantly higher than baseline under
these stimulation intensities. The peak−RSO2values changed in
proportion to the stimulation intensity. Using a 5-s stimulation, the average response times of−RSO2from stimulation onset to
the return of−RSO2to baseline were 26.12, 22.87, and 24.96 s
for the SSS and the contralateral MI and MII arterioles, respectively.
Figure4(c) shows the peak−RSO2 values in the SSS and
the contralateral MI and MII arterioles as a function of the stimulation intensity. The peak−RSO2values during stimulation
were 0.96 0.002 (mean SD) (p < 0.001) in the SSS, 2.490 0.003 (mean SD) (p < 0.001) in the contralateral
Fig. 4 PeakRHbT,RCBV, and−RSO2 values in the SSS and contralateral MI and MII arterioles during one stimulation block under a 5-s stimulation,
plotted as a function of stimulation intensity. The error bars represent the standard deviations of the data from six rats. Note that the ipsilateral SSS is the SSS during the ipsilateral-side measurement, and the contralateral SSS is the same SSS during the contralateral-side measurement.
MI arteriole and 3.520 0.004 (mean SD) (p < 0.001) in the MII arteriole (Wilcoxon matched-pairs signed-rank test, n¼ 6) using a stimulation intensity of 2 mA. Slight peak −RSO2
increases accompanied the increasing stimulation intensities. No significant−RSO2 changes were observed in the ipsilateral
MI or MII arterioles from Stimulation-ON to Stimulation-OFF (p> 0.05). Another three significant peak −RSO2 values using
stimulation intensities of 1, 6, and 10 mA are plotted in Fig. 4(c). These results indicate that the peak −RSO2 values
were sensitive to the increasing stimulation intensity.
The third row of Fig.5shows−RSO2in the SSS and bilateral
MI and MII arterioles in 1 15-s block using a 2-mA stimulation. For long stimulations, the time to peak−RSO2in the SSS and the
contralateral MI and MII arterioles was 15.51, 15.14, and 18.89 s, respectively, after stimulation onset. The peak−RSO2
values using the 15-s stimulation were 1.12 0.001 (mean SD) (p < 0.001) in the SSS, 3.140 0.002 (mean SD) (p < 0.001) in the contralateral MI arteriole, and
3.840 0.005 (mean SD) (p < 0.001) in the MII arteriole (Wilcoxon matched-pairs signed-rank test, n¼ 6). Using the long stimulation condition, the−RSO2response times from
sti-mulation onset to the return of−RSO2 to baseline were 27.62,
24.95, and 26.27 s in the SSS and the contralateral MI and MII arterioles, respectively. The time to peak−RSO2in the
con-tralateral MI arteriole was longer under the 15-s stimulation than the 5-s stimulation. However, the times to peak−RSO2in the SSS
and contralateral MII arterioles did not differ between the 15- and 5-s stimulations.
4 Discussion
The present study used fPAM to investigate the HRFs of the HbT, CBV, and SO2in the SSS and bilateral MI and MII arter-ioles in response to forepaw stimulations of various intensities and durations. Our main findings were as follows: (1) the HRFs of the HbT, CBV, and SO2 in specific cerebral blood vessels can be investigated reliably using fPAM; (2) functional HbT
Fig. 5 Functional HbT, CBV, and SO2changes (i.e.,RHbT,RCBV,−RSO2) in the SSS and bilateral MI and MII arterioles in one stimulation block as a
function of time using 15-s stimulations of 2 mA. The error bars represent the standard deviations of the data from six rats. The yellow zone indicates the Stimulation-ON period. Note that the ipsilateral SSS is the SSS during the ipsilateral-side measurement, and the contralateral SSS is the same SSS during the contralateral-side measurement.
and SO2increased sublinearly (i.e., compressed) with increasing stimulation intensities; (3) the CBV changes, within a limited dynamic range of stimulation intensities and durations, were correlated more linearly than the HbT and SO2 with the arterioles; (4) the times to peak HbT and CBV were lower in the MI arterioles than in the MII arterioles; and (5) the peak values of the HbT, CBV and SO2 responses were higher in the MII arterioles than in the MI arterioles.
4.1 Functional Changes in the HbT, CBV, and SO2 in Response to Various Stimulation Intensities Our experiments in which various electrical stimulation intensities were applied to the left rat forepaw showed significant increases in contralateral HbT (i.e., increases in IRð570Þ), CBV (i.e., increases in vascular cross-sectional area in IRð570Þ) and SO2 (i.e., negative ΔIFð560Þor decreases in IFð560Þ) in the MI and MII arterioles. In the SSS, significant increases in HbT and SO2 were observed under various stimulation intensities (1, 2, 6, and 10 mA) of fixed duration (5 s), whereas the CBV in the SSS did not increase under any stimulation. With increasing stimulation intensities, the maximum functional HRF changes in the HbTand SO2were sub-linear (i.e., compressed) phenomena.10The peak values of the CBV were more linearly correlated with increasing stimulation intensities in arterioles than the peak HbT and SO2. This result implies that saturation in neural activation occurs at a stimulation intensity larger than 2 mA. According to a previous study,34a simi-lar phenomenon has been found in response to an amplitude of about 1.5 mA. On the other hand, our results suggest that the larger CBV changes were more significantly correlated to the local acti-vation region. An explanation for this is that with increasing sti-mulation intensity, the local activation region demands more oxygenated blood, while more deoxygenated blood drains out of the activation region, resulting in the CBV changing signifi-cantly and linearly with the stimulation intensity, especially in the arterioles.2,3The oxygenated and deoxygenated blood will afflux into the smaller arterioles and induce more significant CBV changes than the changes in HbT and SO2. Nemoto et al. also showed that the CBV-related signals are more highly corre-lated than oxygenation-recorre-lated signals with the activated region.35
In addition, the phenomena of HbT and SO2onset overshoots may be due to blood flow increases near the local activation sites, which would lead to a fresh blood supply and greater HbT and SO2increases in the surrounding arterioles.15The oxy-genated blood that subsequently flows into the veins may lead to HbT and SO2increases.2,36The current fPAM system found that the times to peak HbT and SO2were shorter in the MI arterioles than in veins under different stimulation intensities. To transfer a significant amount of HbT and SO2into the activation region, shorter times are needed to reach peak HbT and SO2in MI arter-ioles than in veins.15,36,37Moreover, the greater increases in the
HbT and SO2levels also indicate increased CBF.32This finding of dissociation of the HbT and CBV responses strongly suggests that Grubb’s relationship between CBF and CBV [CBV¼ 0.8 × CBF (Ref. 38)] does not always apply and requires revision.2Additionally, a significant lateralization
phe-nomenon during different unilateral forepaw electrical stimula-tion intensities was observed, in agreement with previous studies.15,39,40
With changing stimulation intensities, HRF changes in HbT, CBV, and SO2were all faster in the contralateral MI arteriole than in the contralateral MII arteriole. This occurred because the HRF results in these increases in large blood vessels before
smaller blood vessels.41Conversely, the peak values of HbT,
CBV, and SO2 were larger in the contralateral MII arteriole than in the contralateral MI arteriole. A potential explanation for this is that the contralateral MII arteriole is closer to the acti-vated cerebral regions than the contralateral MI arteriole.2,42To
transfer a significant quantity of oxygenated blood into the acti-vated region during the stimulation period, the peak changes in HbT, CBV, and SO2 in the contralateral MII arteriole must be larger than those in the contralateral MI arteriole.42These results indicate that the HRF is affected by the stimulation intensity.2
4.2 Functional HbT, CBV, and SO2 Changes in Response to Various Stimulation Durations Our findings suggest that the response times of CBV, HbT, and SO2in single blood vessels are independent of the stimulation duration. Therefore, the response times of CBV, HbT, and SO2 in those blood vessels will not be affected significantly by sti-mulation duration. Rather, only the degrees of CBV, HbT, and SO2 changes are affected by stimulation duration.2,3DOI can measure hemodynamic changes from only a selected region of interest,9,10but not the changes in single vessels, as presented in this study. This fact may explain the previously reported pla-teau response phenomenon after stimulation onset, as well as the longer response time compared to the shorter stimulation dura-tion when measuring changes in HbT, CBV, and SO2.3,10We were only interested in the HRF changes in single blood vessels, not the total changes from the selected region of interests. The same problems also exist when using fMRI, which senses only the changes in response to stimulation from selected pixels, not a single cerebral vessel. Thus, the stimulation-induced HRF changes in single vessels cannot be measured directly by fMRI.4 In addition, the results under different stimulation durations (5 and 15 s) indicate that relative functional HbT, CBV, and SO2 changes were more strongly correlated in the contralateral MI arteriole than in the contralateral MII arteriole. It is possible that the contralateral MII arteriole is closer to the activation region and that its cross-section is also smaller than that of the contralateral MI arteriole, which would lead the MII arterioles to reach their maximum blood volume soon after sti-mulation onset.2 Therefore, using longer stimulation, smaller functional hemodynamic changes in the contralateral MII arter-iole were found. However, our data suggest that changes in HbT, CBV, and SO2 are more prominently associated with smaller arterioles using stimulations of different intensities and dura-tions.2This finding, consistent with fMRI data of previous
stu-dies,42 can be attributed to the immediate proximity of the neuronal activity to the smaller arterioles.
Our data also suggest that HbT, CBV, and SO2 responses remained elevated for a few seconds after the onset of the sti-mulation in the contralateral MI and MII arterioles under differ-ent stimulation intensities (1, 2, 6, and 10 mA) and durations (5 and 15 s). In delivering blood flow and oxygenation to locally activated tissues, the total response times of HbT, CBV, and SO2 are longer in smaller arteries than in large ones,2,43likely due to the locations of the smaller arteries.
In addition, our findings demonstrate that HbT changes in the SSS were significant, but there were no significant CBV changes. This finding appears contradictory because HbT and CBV, despite being calculated using different techniques, should be relatively similar. There are two potential reasons for this dis-crepancy: (1) because the SSS is the largest vein in the rat brain, small changes in its cross-section are difficult to detect, whereas
changes in CBV could cause large changes in HbT; and (2) the CBV calculation requires an accurate image of a specific blood vessel to calculate the cross-sectional area. Although the SSS could be seen in the PA B-scan images, the PA signals from the SSS were much weaker than those from other cortical ves-sels.21,27This difficulty may have resulted from the spherical geometric focusing and the finite detection bandwidth of the 50-MHz ultrasound transducer that was used in our current fPAM setup. Concordant with our results, a previous study has indicated significant SO2 changes in veins and arterioles, with significant CBV changes seen only in arterioles during somatosensory stimulation.43
5 Conclusions
The present study demonstrates the unique capabilities of an fPAM system for characterizing the HRF changes in single cer-ebral blood vessels. Local relative functional HRFs (i.e., HbT, CBV, and SO2) in single blood vessels using different stimula-tion intensities (1, 2, 6, and 10 mA) and durastimula-tions (5 and 15 s) were analyzed and discussed comprehensively. Our results indi-cate that both the times to reach the peak values and the mag-nitudes of the peak values of the HbT, CBV, and SO2responses were faster and higher, respectively, in the MII arterioles than in the MI arterioles. This may have occurred because the MII arter-ioles, which are smaller, are much closer to the local activated region and, therefore, require greater metabolism. We also found that integrated CBV increased more linearly with stimulation intensity and duration than did the HbT or SO2, which indicates that the significant CBV changes were more colocalized with the neuronal activation and corresponding blood consumption. Our findings are concordant with those reported in the literature, indicating that the regulation of hemodynamic changes in single blood vessels can be studied reliably by fPAM without the use of contrast agents. The current fPAM system will be useful as a complementary modality to other neurovascular imaging tech-niques for label-free investigations of HRFs to assess neuronal activity in single cerebral blood vessels.
Acknowledgments
We acknowledge the National Science Council, R.O.C., for its support of this research (NSC Nos. 97-2221-E-007-084-MY3, NSC 100-2221-E-007-010-MY2, 96-2220-E-009-029, 97-2220-E-009-029, 99-2221-E-009-154, and 99-2911-I-009-101), as well as funding support from the National Tsing Hua University (Boost program 98N2531E1) and the Ministry of Education, Taiwan. The authors would also like to thank Po-Hsun Wang for his help in developing the fPAM system.
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