Rescue of cortical neurovascular functions during the

Neurobiology of Disease 75 (2015) 53–63

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Neurobiology of Disease journal homepage: www.elsevier.com/locate/ynbdi

Rescue of cortical neurovascular functions during the hyperacute phase of ischemia by peripheral sensory stimulation Lun-De Liao a,⁎, Yu-Hang Liu a,b, Hsin-Yi Lai c, Aishwarya Bandla a,d, Yen-Yu Ian Shih e, You-Yin Chen f, Nitish V. Thakor a,b,d,g a

Singapore Institute for Neurotechnology (SINAPSE), National University of Singapore, 28 Medical Drive, #05-COR 117456, Singapore Department of Electrical & Computer Engineering, National University of Singapore, 4 Engineering Drive 3 117583, Singapore Department of Physical Medicine and Rehabilitation, Chang Gung Memorial Hospital and Chang Gung University, Taoyuan 333, Taiwan, ROC d Department of Biomedical Engineering, National University of Singapore, 9 Engineering Drive 1 117575, Singapore e Department of Neurology, University of North Carolina, Chapel Hill, NC, USA f Department of Biomedical Engineering, National Yang Ming University, No. 155, Sec. 2, Linong St., Taipei 112, Taiwan, ROC g Department of Biomedical Engineering, Johns Hopkins University, Traylor 701/720 Rutland Ave, Baltimore, MD 21205, USA b c

a r t i c l e

i n f o

Article history: Received 15 July 2014 Revised 7 December 2014 Accepted 23 December 2014 Available online 5 January 2015 Keywords: Collateral circulation Electrocorticography (ECoG) Functional photoacoustic microscopy (fPAM) Ischemia Photothrombotic ischemia Peripheral somatosensory stimulation

a b s t r a c t To investigate the potential therapeutic effects of peripheral sensory stimulation during the hyperacute phase of stroke, the present study utilized electrophysiology and photoacoustic imaging techniques to evaluate neural and vascular responses of the rat cortex following ischemic insult. We employed a rat model of photothrombotic ischemia (PTI), which targeted the forelimb region of the primary somatosensory cortex (S1FL), due to its high reproducibility in creating localized ischemic injury. We also established a hybrid, dual-modality system, including six-channel electrocorticography (ECoG) and functional photoacoustic microscopy (fPAM), termed ECoG–fPAM, to image brain functional responses to peripheral sensory stimulation during the hyperacute phase of PTI. Our results showed that the evoked cerebral blood volume (CBV) and hemoglobin oxygen saturation (SO2) recovered to 84 ± 7.4% and 79 ± 6.2% of the baseline, respectively, when stimulation was delivered within 2.5 h following PTI induction. Moreover, neural activity significantly recovered, with 77 ± 8.6%, 76 ± 5.3% and 89 ± 8.2% recovery for the resting-state inter-hemispheric coherence, alpha-to-delta ratio (ADR) and somatosensory evoked potential (SSEP), respectively. Additionally, we integrated the CBV or SO2 with ADR values as a recovery indicator (RI) to assess functional recovery after PTI. The RI indicated that 80 ± 4.2% of neurovascular function was preserved when stimulation was delivered within 2.5 h. Additionally, stimulation treatment within this optimal time window resulted in a minimal infarct volume in the ischemic hemisphere (4.6 ± 2.1%). In contrast, the infarct volume comprised 13.7 ± 1.7% of the ischemic hemisphere when no stimulation treatment was applied. © 2014 Published by Elsevier Inc.

Introduction Abbreviations: ACA, anterior cerebral artery; ADR, alpha-to-delta ratio; AP, anterior– posterior; AVA, Agri-Food and Veterinary Authority of Singapore; BBB, blood–brain barrier; CBF, cerebral blood flow; CBV, cerebral blood volume; cM1, contralateral motor cortical region; cS1FL, contralateral forelimb region of the primary somatosensory cortex; CW, continuous wave; DOI, diffuse optical imaging; ECoG, electrocorticography; EEG, electroencephalography; fMRI, functional magnetic resonance imaging; fPAM, functional photoacoustic microscopy; IACUC, Institutional Animal Care and Use Committee; LSD, least significant difference; LSI, laser speckle imaging; M1, motor cortical region; ML, medial–lateral; MCA, middle cerebral artery; NACLAR, National Advisory Committee for Laboratory Animal Research; PA, photoacoustic; PBS, phosphate-buffered saline; pMCAO, permanent middle cerebral artery occlusion; PTI, photothrombotic ischemia; RI, recovery indicator; ROI, region of interest; rtPA, recombinant tissue plasminogen activator; S1FL, the forelimb region of the primary somatosensory cortex; S.D, standard deviation; SSEP, somatosensory evoked potential; SO2, hemoglobin oxygen saturation; TTC, 2,3,5-triphenyltetrazolium chloride. ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (L.-D. Liao). Available online on ScienceDirect (www.sciencedirect.com).

http://dx.doi.org/10.1016/j.nbd.2014.12.022 0969-9961/© 2014 Published by Elsevier Inc.

Stroke is a neurological deficit caused by a significant reduction in the blood supply to tissue (Lo, 2008) and is a leading cause of death and disability (Lo et al., 2003). Stroke frequently leads to irreversible tissue damage in areas where the cells are subject to necrosis (Kingwell, 2014). The timely redistribution of blood into the ischemic penumbra (salvageable tissue surrounding the ischemic core) could markedly improve the outcome (Lo, 2008; Muir et al., 2006). Currently, the most well-known therapeutic agent for stroke recovery is recombinant tissue plasminogen activator (rtPA) (Azizi et al., 2013), which breaks down clots, resulting in the reintroduction of blood into the ischemic brain region (i.e., reperfusion). Unfortunately, rtPA is viable for only a small portion (approximately 3.6%) of stroke patients (Go et al., 2013) and may cause tissue damage by weakening the blood vessel walls or disrupting the blood–brain barrier (BBB) (Abu Fanne et al., 2010). Therefore, a new

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neuroprotective therapy with minimal side effects (i.e., a noninvasive, non-pharmacological method) is required for ameliorating ischemic insult (Fisher et al., 2009). Recent studies with permanent middle cerebral artery occlusion (pMCAO) animal models suggested that manipulating sensory or motor functions resulted in a neuroprotective effect during stroke recovery (Frostig et al., 2013; Lay et al., 2010; Lay et al., 2011). Although the response to peripheral sensory stimulation is likely due to remodeling of both cerebral blood flow (CBF) and electrophysiological function (Frostig et al., 1990; Lay et al., 2011), the effects of sensory stimulation on neurovascular coupling and dynamics during the hyperacute phase of ischemia are not fully understood. To address this question, measurement of hemodynamic and neural responses after ischemia induction is essential. Optical imaging techniques, such as diffuse optical imaging (DOI) and laser speckle imaging (LSI), have been used to evaluate stroke physiology (Culver et al., 2003; Luckl et al., 2010). Using these techniques, changes in hemodynamic functions, such as CBF and cerebral blood volume (CBV), can be assessed, as well as changes in oxygen concentration and cerebral oxygen metabolism in the cortical ischemic area. However, the spatial resolution of DOI reconstructed images is low because of the diffusion of light in biological tissues, thereby limiting the ability to observe changes in the fine and deep cerebral blood vessels after ischemia (Gibson and Dehghani, 2009). Compared with DOI, LSI demonstrates a higher spatial resolution of blood flow responses but limited penetration (0.5–0.8 mm only) (Liao et al., 2013). Thus, the perfusion- and metabolism-related data can only be evaluated in the superficial layers of the cortex, which is insufficient for assessing the progressive changes in the ischemic region during the entire hyperacute phase of ischemia (Dehghani et al., 2009; Liao et al., 2013; Luckl et al., 2010; Miao et al., 2010; Zhang et al., 2006). Functional magnetic resonance imaging (fMRI) provides noninvasive large-scale measurements of neural functions and can precisely distinguish both stroke mimics and small lesions in cerebral ischemia (Tatlisumak, 2002; Vymazal et al., 2012). However, this technique does not provide precise penumbra data because the estimation is based only on a difference in perfusion parameters and diffusion-weighted MRI (Vymazal et al., 2012). Furthermore, the suitability of MRI for certain stroke patients is still limited because of several factors, such as claustrophobia and the presence of pacemakers or other ferromagnetic material implants (Tatlisumak, 2002). Conversely, photoacoustic (PA) imaging is an emerging optical imaging technique that provides intrinsic optical absorption (i.e., hemoglobin), high resolution, deep tissue penetration (Zhang et al., 2006) and high compatibility with other imaging techniques (Liao et al., 2013). For instance, PA imaging technology is especially suitable for evaluating the progressive changes in the penumbra area of focal ischemia because of its attributes such as the deeper tissue penetration depth and intrinsic blood contrast. Recently, our studies have shown that functional photoacoustic microscopy (fPAM) can be used for the contrast agent-free imaging of functional CBV and hemoglobin oxygen saturation (SO2) changes in the rat cortex following peripheral sensory stimulation (Hu et al., 2009; Liao et al., 2012a; Liao et al., 2012b). In this study, we aimed to combine fPAM with electrocorticography (ECoG) recordings to investigate neurovascular function in a rodent model of photothrombotic ischemia (PTI) (Kao et al., 2014). We demonstrated that hemodynamic responses, including the CBV, SO2, and neural activity, namely, somatosensory evoked potential (SSEP) and resting-state ECoG signals, can be simultaneously measured by the proposed ECoG–fPAM system. We employed peripheral sensory stimulation as a potential treatment for PTI at multiple time points (i.e., 0, 1, or 2 h post-PTI onset) and evaluated the degree of neurovascular function recovery in the ischemic area at the forelimb region of the primary somatosensory cortex (S1FL). Inter-hemispheric coherence and alphato-delta ratio (ADR) changes calculated from the resting-state ECoG signals at the bilateral cortical regions were also evaluated. The infarct volume was also assessed for examining the efficacy of the peripheral

sensory stimulation treatment. Additionally, to investigate the recovery of neurovascular function in the PTI region, we employed the hemodynamic/ADR interaction as a recovery indicator (RI) for an in-depth assessment of functional recovery. Thus, using the ECoG–fPAM system, we demonstrated that the delivery of peripheral sensory stimulation during an appropriate time window could significantly reduce ischemic lesion severity. Materials and methods The electrocorticography-functional photoacoustic microscopy system (ECoG–fPAM) Together with the ECoG–fPAM system, we established an experimental environment capable of 1) functional PA imaging, 2) PTI induction at the cortex, 3) peripheral sensory stimulation and 4) ECoG recordings, as shown in Fig. 1. A designed 50-MHz dark-field confocal fPAM system was used to image the functional hemodynamic changes in the selected cortical blood vessels. Two visible wavelengths of laser pulses, 560 and 570 nm (λ560 and λ570), were employed for PA wave excitation. These wavelengths were used because the detected photoacoustic signals at λ560 were sensitive to changes in SO2, whereas those at λ570 were dominated by changes in CBV (Liao et al., 2010). Please refer to the supplementary material for details regarding the setup of the fPAM system and the data analysis of the relative functional changes in the CBV and SO2 in specific regions performed in this study (Liao et al., 2012a; Tsytsarev et al., 2012). For the ECoG recordings, seven stainless steel epidural electrodes (including one reference electrode) were secured on the skull to acquire SSEPs and resting-state ECoG signals, which were pre-amplified (PZ2-32, Tucker-Davis Technologies, Alachua, FL, USA) and recorded using a bio-signal processor (RZ5D, Tucker-Davis Technologies, Alachua, FL, USA). MATLAB software (MATLAB R12, MathWorks Inc., Natick, MA, USA) was used to analyze related parameters of evoked potentials in response to peripheral sensory stimulation. Please refer to the supplementary material for details concerning the data analysis of the electrophysiological recordings, including the SSEPs, inter-hemispheric coherence and ADR calculations. Animal preparation All experimental protocols used in this study were evaluated and approved by the Institutional Animal Care and Use Committee (IACUC) of the National University of Singapore. Animal care and surgical procedures were performed according to the National Advisory Committee for Laboratory Animal Research (NACLAR) guidelines for facilities licensed by the Agri-Food and Veterinary Authority of Singapore (AVA), which is the regulatory body of the Singapore Animals and Birds Act. Thirty male Wistar rats weighing 250–300 g (InVivos Pte Ltd., Singapore) were divided into four groups; six animals were included in the control group, and eight animals were included in each of the three experimental groups. The animals were anesthetized with a 50 mg/kg bolus of pentobarbital and maintained with 15 mg/kg/h pentobarbital throughout the experiment. The rats were mounted on a custom-made acrylic stereotaxic head holder. Body temperature was measured using a rectal probe and was maintained at 37 ± 0.5 °C using a self-regulating thermal plate (TCAT-2 Temperature Controller, Physitemp Instruments, Inc., Clifton, NJ, USA). The skin was subsequently removed from the skull to expose the bregma. Six stainless steel epidural electrodes were bilaterally secured to the skull over the motor cortical regions (M1: anterior–posterior (AP) = +4.2 mm, medial–lateral (ML) = ±3 mm) and the S1FL cortical regions (S1FL and S1FL* AP = +1.7 mm and −0.8 mm, respectively; ML = ±4.5 mm) for SSEPs and resting-state ECoG recordings (Fig. 1). One reference electrode was positioned at 3 mm to the right of the lambda landmark. Next, a cranial window of approximately 3 mm (AP) × 8 mm (ML), centered at the bregma, was produced for PA


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Fig. 1. Illustration of the structure of our ECoG–fPAM system and the precise ECoG electrode positioning. A schematic diagram of the ECoG–fPAM system used to investigate the cortical functional changes in rats is shown in this figure. Left-forepaw electrical stimulation was delivered using a stimulator driven by a neural signal processor, which recorded the SSEPs following amplification via a front-end amplifier. For induction of PTI, a CW laser light was coupled to the dark-field optical path of the fPAM and focused on the selected cortical area. The arrows in the rat brain indicate 7 electrodes (including 6 ECoG electrodes around the PA window and 1 reference electrode on the right 3 mm from lambda). The yellow rectangular region indicates a 3 × 8 mm window created for PA imaging, and the blue area within the yellow region indicates the area targeted for PTI induction. The procedure for conducting PA imaging was as follows: (a) an optically and ultrasonically transparent, disposable polyethylene film was used to seal the window at the bottom of the water container. (b) Afterward, a commercially available ultrasound gel was applied to the rat's brain for acoustic coupling, and the brain was placed immediately below the water container and secured to the custom-made stereotaxic apparatus for imaging. (c) Laser pulses of ECoG–fPAM were generated at a 10-Hz pulse repetition rate and coupled to an optical fiber via a lens to illuminate the rat's brain. (d) PA waves were detected using a 50-MHz transducer, processed by the A/D card, and then sent to the PC for further data analysis. LNA: low noise amplifier.

imaging using a high-speed drill, and care was taken to keep the dura intact. In subsequent experiments, the interaural and bregma references were used to position the head in the fPAM system, without additional surgery. The electrodes, which were connected with silver wires, were interfaced with the data acquisition system through a ZIF-Clip headstage (Tucker-Davis Technologies, Inc., Alachua, FL, USA). Photothrombotic ischemia procedure Focal ischemia was induced using the photothrombosis method, targeting a selected cortical arteriole, which is a distal branch of the middle cerebral artery (MCA) at the right hemisphere S1FL cortical region (Liao et al., 2010). We injected the photosensitizer Rose Bengal (Na+ salt, R3877; Sigma-Aldrich, Singapore), which was diluted to 10 mg/ml in HEPES-buffered saline, into the tail vein at 0.2 ml/100 g rat body weight infused over 2 min. The cortical blood vessel selected for occlusion was subsequently illuminated with a 10 mW, 532 nm continuous wave (CW) laser light (MGM-20; Beta Electronics) (Watson et al., 1985). The CW laser light was coupled to the dark-field optical path of the ECoG–fPAM, as illustrated in Fig. 1, and focused on the selected cerebral vessel in the right S1FL region for 15 min until a stable clot was formed. The mechanism for clot formation occurs via generation of singlet oxygen (after illumination), which damages the endothelial cell membrane, resulting in subsequent platelet aggregation and thrombus formation to interrupt blood flow in the selected blood vessel

(Watson et al., 1985). An ischemic region is then formed including the irreversibly damaged infarct (ischemic core) and salvageable tissue (ischemic penumbra) (Sims and Muyderman, 2010). Peripheral sensory stimulation Subdermal needle electrodes were inserted into the rat's left forepaw (contralateral to the occlusion), and electrical stimulation was applied using a stimulator (DS3, Digitimer, Hertfordshire, UK). The trigger pulse signals controlling the stimulator were generated and delivered through the output port of a multichannel bio-signal processor (RZ5D, Tucker-Davis Technologies, Alachua, FL, USA). A monophasic constant current of 2 mA, with a 0.2 ms pulse width at a frequency of 3 Hz and 1 min stimulation duration, was used for each block, as shown in Fig. 2A. A 15 min block was employed in this study for functional signal acquisition, and each group consisted of 8 blocks after PTI induction. Each block consisted of a 1 min stimulation period, followed by a 3 min resting period to allow adequate time for the brain to return to the resting state before the subsequent measurements (Liao et al., 2010). Then, the resting-state ECoG signal was recorded for 5 min. To facilitate fPAM imaging, an additional 5 s of left forepaw electrical stimulation (i.e., 2 mA intensity, with a 0.2 ms pulse width at a frequency of 3 Hz) was applied to evoke a hemodynamic response, followed by 3 min PA imaging. A 2 min time window preceded the subsequent block.

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Fig. 2. Illustration of different stimulation (St1, St2) and recording times (resting state (RS), SSEP and photoacoustic imaging (PA)) for the control and experimental groups. (A) A schematic diagram of the experimental protocol indicates the timing of ECoG and fPAM imaging for a single 15 min block. The SSEP was recorded during the first “Stimulation-ON” phase (St1), consisting of a constant 0.2 ms electrical pulse width, a 3-Hz pulse train, and a 2 mA pulse amplitude. ECoG during the RS period was recorded beginning at the fourth minute of the block. The second 5 s stimulation (St2) evoked the hemodynamic response for PA imaging. (B) Treatment schema indicates the baseline and post-PTI recording blocks. For the control group, no treatment was delivered to the rat during the experiment, whereas groups 1–3 represent the experimental groups with stimulation treatments at different onset timings. The estimation of the infarct volume using TTC staining was performed at 48 h post-PTI induction.

The PA signals at λ560 or λ570 were acquired in the block to assess stimulation-induced hemodynamic changes in the S1FL region. PA B-scan images acquired in the S1FL region were used to assess stimulation-induced relative hemodynamic changes. In total, 4 groups were used in this study, including one control and three experimental groups (Fig. 2B). The control group received PTI induction but no peripheral sensory stimulation. In the three experimental groups, the peripheral sensory stimulation was applied at different time points as follows: immediately following PTI induction (group 1), 1 h post-PTI induction (group 2), and 2 h post-PTI induction (group 3). Neurovascular function as an indicator of ischemia recovery In this study, we developed a recovery indicator (RI) that comprehensively evaluates the neurovascular coupling function by integrating information on the neural activity and hemodynamic changes into a single diagram/measure. Thus, using this developed RI, we were able to assess the efficacy of our stimulation therapy and the degree of recovery after ischemia based on the deduced value of the neurovascular coupling function. The ADR and CBV/SO2 were used as indicators of neural activity and hemodynamics, respectively, for the RI. The main concept of the RI is that the trends in the ADR and CBV/SO2 values after 2 h of treatment could merge at a certain unique point in the RI diagram, which would represent the neurovascular function. Based on the position of this intersection point, we could rapidly and intuitively determine the recovery efficacy. For instance, if the intersection point is far from the origin (i.e., the worst case), the recovery degree is better. Furthermore,

an intersection point in the upper triangular region of the RI diagram would indicate that the recovery level of neural activity (ADR) is better than that of hemodynamics (CBV/SO2), and vice versa for any intersection point in the lower triangular region. The following is a brief introduction on how to calculate the RI value: In the RI diagram, the bottom x-axis (normalized CBV/SO2 value) corresponds to the right y-axis (time), while the top x-axis (time) corresponds to the left y-axis (normalized ADR value). The baseline values of the relative CBV, SO2 and ADR changes were used to normalize the curves from 0 (i.e., the worst case after PTI) to 1 (i.e., the baseline value). The values of normalized CBV/SO2 with the ADR at the end of each treatment (i.e., 2 h) constituted a “coupling line” of neurovascular function. The perpendicular distance from the origin (x0, y0) to this coupling line was calculated. Additionally, the distance between (x0, y0) and (xM, yM) (i.e., the diagonal vertex relative to the origin of coordinates) was calculated for comparison. The percentage difference between the above-mentioned perpendicular distance and the maximum distance of the index (from (x0, y0) to (xM, yM)) was regarded as the RI of neurovascular function. This RI of neurovascular function, which was based on the integration of the CBV/SO2 with the ADR results, indicated the level of recovery following PTI and was expressed as the following equation: 20

1

3

jax0 þ by0 þ cj 6B 7 qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiC RI ða; b; cÞ ¼ 2 4@ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi A 100%5 ‐ 1; a2 þ b2 ðxM ‐ x0 Þ2 þ ðyM ‐ y0 Þ2

ð1Þ


where ax + by + c = 0 is the coupling line equation formed by normalizing the CBV/SO2 with the ADR values at the end of each treatment; jax0 þ by0 þ cj pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi is the distance from (x0, y0) (the origin of the coordinates a2 þ b 2 representing the worst case of the CBV/SO2 with the ADR values following PTI) to the coupling line; and (xM, yM) is the diagonal vertex relative to the origin of coordinates denoting the best case of the CBV/SO2 with ADR values (i.e., baseline values). According to the RI value, we can evaluate the effects of each treatment group and determine the recovery of neurovascular function using ECoG–fPAM. Please refer to Fig. S1 of the supplementary material for details regarding RI calculations. Measurement of infarct volume The extent of infarct was measured using 2,3,5-triphenyl-tetrazolium chloride (TTC). At 48 h following ischemia onset, the rats were deeply anesthetized using 10% chloral hydrate, and their brains were rapidly removed, washed in phosphate-buffered saline (PBS) at room temperature (25 ± 1 °C), and then frozen at −20 °C for 10 min. Brain tissue from an area 4 mm anterior and 6 mm posterior to the bregma was cut into 10 serial 1 mm coronal sections. The sliced brain tissues were stained with 2% TTC (AMRESCO, Solon, OH, USA) for 20 min at 37 °C in the dark, followed by overnight immersion in 4% paraformaldehyde in 0.1 M PBS, pH 7.4, at 4 °C. The infarcted tissue remained unstained (white), whereas normal tissue was stained red. The extent of ischemic infarct was traced, and the integrated volume was calculated using ImageJ software (NIH Image). The infarct volume was calculated by adding the infarct areas of all sections and multiplying by the slice thickness. To compensate for the effect of brain edema, the corrected infarct volume was calculated using the following equation: percentage of corrected infarct volume = {[total lesion volume − (non-ischemic hemisphere volume − ischemic hemisphere volume)] / ischemic hemisphere volume} × 100. Statistical analysis The experiment was designed to quantitatively measure PA signals (i.e., IR(570)) and the corresponding SSEP changes, as well as changes in the bilateral S1FL regions following peripheral sensory stimulation treatment for PTI. The baseline values of the relative CBV, SO2, SSEPs, inter-hemispheric coherence and ADR were used to normalize the changes pre- and post-PTI (i.e., the baseline value was 1, and the worst outcome after PTI was 0). Statistical significance was assessed using a paired t-test, with significance defined as a probability (p) value of less than 0.05. Case-to-case differences in averaged PA signals of the studied areas and changes in cross-sectional areas (i.e., CBV changes) were examined using paired t-tests (p b 0.05; n = 8 for groups 1–3; n = 6 for the control group). The significance of the changes observed in the averaged PA signals of the studied areas in response to peripheral sensory stimulation was analyzed using the Wilcoxon matched-pairs signed-rank test (two-tailed; p b 0.05; n = 8 for groups 1–3; n = 6 for the control group) (Liao et al., 2010). An ANOVA was performed to assess the changes in SSEPs and inter-hemispheric coherence in different brain areas and between cases using a repeated-measures ANOVA with Fisher's least significant difference (LSD) post-hoc analysis (Lai et al., 2012). All statistical analyses were performed using SPSS software (version 10.0, IBM®, Armonk, New York, USA). The data are presented as the mean ± standard deviation (S.D.). Results Comparison of CBV and SO2 changes with peripheral sensory stimulation at 0 h, 1 h, and 2 h post-PTI The diagram of the PA imaging and ECoG recording sites is shown in Fig. 1. Following PTI induction at the distal MCA branch, the effect of

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occlusion could be visualized in the right S1FL area (Fig. 3A). A postPTI PA B-scan image at λ570 (i.e., IR(570)) at 0.2 mm anterior to the bregma is shown in Fig. 3B for group 1. Fig. 3C shows the PAM-measured relative CBV (i.e., RCBV) changes pre- and post-PTI for each group in the region of interest (ROI). The ROI includes a 1.5 × 1 mm region on the ischemic hemisphere (shown in Fig. 3B by the red box on the right hemisphere). The PA images/values in Figs. 3C-E were normalized to the maximum change in IR(570) among all groups. Note that IR(570) is proportional to the CBV (Liao et al., 2010). Peripheral sensory stimulation was delivered at different time points in different groups, as follows: 1) immediately following PTI induction, 2) 1 h post-PTI induction, and 3) 2 h post-PTI induction. The average functional CBV (i.e., RCBV) and SO2 (i.e., −RSO2 ) changes in bilateral S1FL areas of the experimental groups are shown as a function of time in Figs. 3D–E, respectively. After PTI induction in groups 1, 2, and 3, the relative CBV over the S1FL region in the ischemic hemisphere significantly decreased by 70 ± 4.6%, 68 ± 4.3%, and 71 ± 4.8% and SO2 decreased by 73 ± 5.9%, 72 ± 1.8% and 71 ± 4.7%, respectively, compared with the baseline value (p b 0.05; n = 8 per group). Following peripheral sensory stimulation, the relative CBV and SO2 significantly recovered to 84 ± 7.4% and 79 ± 6.2%, respectively, compared with the baseline in group 1 (p b 0.05; n = 8). In contrast, the relative CBV and SO2 only recovered to 38 ± 1.4% and 53 ± 4.3% of the baseline in group 2 and to 52 ± 3.2% and 44 ± 4.3%, respectively, of the baseline in group 3 (p b 0.05; n = 8). The results of the CBV and SO2 changes after stimulation in each group showed that treatment initiated immediately after PTI (group 1) showed the best rescue effect among all groups and helped recover the oxygen saturation level close to the baseline. Comparison of SSEPs with peripheral sensory stimulation at 0 h, 1 h, and 2 h post-PTI The SSEP waveforms (Sakatani et al., 1990) and extracted parameters, including the P1 amplitude, N1 amplitude, P1 latency, and N1 latency, were used to evaluate neural activity changes pre- and post-PTI (Fig. 4A). The averaged peak-to-peak amplitudes of SSEPs continually recovered toward the baseline in group 1 (Fig. 4B). The peak-to-peak amplitudes showed a trend of increasing values at the beginning of the treatment in group 2 and group 3; however, the peak-to-peak amplitudes decreased again when the stimulation continued to be delivered 2.5 h after PTI induction. These results indicated that stimulation delivered within 2.5 h following PTI induction is beneficial for SSEPs recovery. The changes in SSEP amplitude and latency at multiple time points are shown in Fig. 4C. The repeated-measures ANOVA indicated that the P1 (F = 9.742, p b 0.05) and N1 (F = 13.218, p b 0.05) amplitudes differed among the three groups. The post-hoc analysis comparing the P1 amplitude at each time point showed that the baseline P1 and N1 amplitudes were not significantly different across groups. The P1 amplitude decreased by 38 ± 11.3% (p b 0.05) after PTI induction, indicating that neural activity was immediately affected by ischemia. In group 1, the P1 amplitude recovered to 89 ± 8.2% 2 h after PTI induction. Increases in the P1 amplitude were also observed in groups 2 and 3 within the initial 2.5 and 3 h following PTI induction, respectively. However, the P1 amplitude decreased when the peripheral sensory stimulation was continuously delivered beyond 2.5 h (group 2, 42 ± 14.2%) and 3 h (group 3, 12 ± 9.6%) after PTI induction. In contrast, in group 1, the N1 amplitude recovered to 59 ± 10.8% of the baseline after treatment, whereas the N1 amplitudes in group 2 and group 3 largely decreased, as shown in Fig. 4C. Collectively, these data demonstrated that SSEP amplitude recovered when peripheral sensory stimulation was delivered within the initial 2.5 h following PTI induction, whereas sustained administration of the stimulation beyond the appropriate time window was less effective in rescuing neural activity. The repeated-measures ANOVA indicated that the SSEP latency differed between these three groups (interaction effect, P1 latency: F = 17.542, p b 0.05 and N1 latency: F = 18.265, p b 0.05). The P1

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Fig. 3. The results of PA imaging in the hyperacute phase of PTI. (A) Open-skull photograph of the surface of the ischemic hemisphere rat brain taken prior to (baseline) and following PTI (group 1, at 2 h). The red dashed line indicates the location of the ROI, which is 1.5 mm in width and 1 mm in depth for PA B-scans at the bregma +0.2 mm. The red scale bar indicates 1 mm. (B) An in vivo IR(570) PA B-scan image at the position of the bregma +0.2 mm, which is proportional to the CBV changes. The window for bilateral PA B-scan images is 8 mm wide and 3 mm deep. The solid red arrows in both (A) and (B) indicate the location of PTI induction (coordinates from the bregma: AP, +0.2 mm; ML, +4 mm). The solid yellow lines in both (A) and (B) indicate the S1FL regions. The solid red line in (B) indicates the ROI window for PA imaging, which is also indicated by the red dashed line in (A). (C) The maximum in vivo PA B-scan images IR(570) of three groups at the selected contralateral ROI window (1.5 × 1 mm) over the S1FL region. The solid yellow arrows in (C) indicate that a significant recovery of the changes in CBV was observed in groups 1 and 2 following peripheral sensory stimulation treatment. (D-E) The average functional CBV (i.e., RCBV) and SO2 (i.e., −RSO2 ) changes in the bilateral S1FL regions in groups 1–3 as a function of time. The results for groups 1–3 show that the relative CBV values after 2 h of peripheral sensory stimulation recovered better than the SO2 values in the ischemic region (p b 0.05), indicating that the relative CBV changes may be more correlated with the region of PTI than the SO2 changes and are therefore more suitable for evaluating PTI recovery. Standard deviations are indicated by the error bars.

and N1 latencies in the three groups increased by 9 ± 4.3% after PTI induction, indicating that neural activity was affected. After treatment, both the P1 and N1 latencies recovered to approximately the baseline value in group 1. In contrast, the group 2 and group 3 treatments increased both the P1 and N1 latencies when continually delivered beyond 2.5 h (group 2) and 3 h (group 3) following PTI induction, which was consistent with the results of the SSEP amplitude changes. In group 3, the stimulation efficacy was particularly low because the P1 (47 ± 8.7%, p b 0.05) and N1 (43 ± 9.4%, p b 0.05) latencies were significantly greater than the baseline value, demonstrating that stimulation delivered more than 2.5 h after PTI induction may not rescue neural function. Comparison of bilateral S1FL connectivity with peripheral sensory stimulation at 0 h, 1 h, and 2 h post-PTI Inter-hemispheric coherence, which is a measurement of the linear relationship between two signals (i.e., the signals of ischemic and nonischemic hemispheres) at a specific frequency, was calculated in this study. Because delta (0.1–4 Hz) and alpha (8–12 Hz) frequency bands have been reported to be strong indicators for both injury and recovery after ischemia (Zhang et al., 2013), these two frequency bands were used to estimate inter-hemispheric coherence in our study. Specifically,

the changes in inter-hemispheric coherence in the bilateral S1FL regions (Ch3–Ch4) were used to evaluate the effects of peripheral sensory stimulation treatment before and after PTI induction (Figs. 5A and B). The repeated-measures ANOVA indicated that the interaction among the groups, time points, and frequency bands was significant (F = 19.564, p b 0.05). In the control group, the changes in interhemispheric coherence over bilateral S1FL regions in the alpha band significantly decreased by 72 ± 10.2% (p b 0.05) after 2 h of PTI induction, indicating decreased inter-hemispheric coherence when no treatment was delivered. After treatment in group 1, the inter-hemispheric coherence between the bilateral S1FL regions in the alpha band steadily recovered to 77 ± 8.6% of the baseline. In groups 2 and 3, the interhemispheric coherence values increased at the beginning of the treatments; however, the inter-hemispheric coherence of group 2 decreased from 2.5 h post-PTI induction (decreased to 62 ± 11.4% of the baseline after treatment), and the inter-hemispheric alpha coherence of group 3 decreased to 54 ± 14.9% of the baseline after treatment. The changes in inter-hemispheric coherence in the delta band pre- and post-PTI were similar to the alpha band. Only group 1 exhibited sustained recovery (increased to 82 ± 6.2% of the baseline, F = 18.871, p b 0.05). Collectively, after treatment, group 1 exhibited maximal recovery of interhemispheric coherence in bilateral S1FL regions, whereas group 2 and group 3 also showed increased inter-hemispheric coherence values


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Fig. 4. Illustration of SSEP-related parameters and experimental results. The four parameters of SSEPs are indicated in (A), including the P1 amplitude, P1 latency, N1 amplitude, and N1 latency. (B) The SSEP waveform changes of different groups pre- and post-PTI; group 1, group 2, and group 3 denote treatments applied after 0, 1, and 2 h following PTI induction, respectively. Only group 1 exhibited continual recovery of the SSEP peak-to-peak amplitude during treatment. (C) The corresponding parameter changes in SSEP components over the cS1FL region following PTI induction, with different applied treatments. The results of groups 1–3 are shown; when treatment was applied within 2.5 h following PTI induction, neural activity significantly recovered from ischemic damage, whereas treatment applied after 2.5 h was not beneficial for neural activity restoration.

with stimulation delivered within 2.5 h following the PTI induction. Changes in the inter-hemispheric coherence of bilateral M1 (Ch1– Ch2) and bilateral S1FL* (Ch5–Ch6) regions pre- and post-PTI are shown in Figs. S2A and B in the supplementary material, respectively. ADR changes with peripheral sensory stimulation at 0 h, 1 h, and 2 h post-PTI ADR was calculated to evaluate the neural activity in the ischemic hemisphere. ADR has been reported to be an accurate indicator of

recovery/injury when monitoring cerebral ischemia; a lower ADR indicates a slower ECoG, which indicates worse or no recovery, and vice versa. Therefore, ADR was used as a relevant measure of ischemic recovery in this study (Claassen et al., 2004). Compared with the baseline, the ADR changes in the contralateral S1FL (cS1FL) (i.e., Ch4) region decreased by 62 ± 14.4% following PTI induction (Fig. 5C). The control group exhibited a sustained decrease in the ADR at cS1FL. The treatment in group 1 produced an increase in the ADR values at the cS1FL region, which recovered to 76 ± 5.3% of the baseline after 2 h following stimulation onset. Group 2 exhibited recovery in the ADR values at the

Fig. 5. Sequential changes in inter-hemispheric connectivity between bilateral S1FL regions before and following PTI, and functional recovery in the damaged S1FL region. (A) and (B) indicate inter-hemispheric coherence changes in bilateral S1FL regions (Ch3 and Ch4), and (C) indicates ADR changes in the cS1FL region. The locations of Ch3 and Ch4 are shown in Fig. 1. Corresponding bilateral coherence changes in the S1FL regions before and following PTI are shown in (A) the alpha band and (B) the delta band. The results indicate that inter-hemispheric coherence between the bilateral S1FL regions in both the alpha and the delta bands could recover when timely treatment was administered (group 1). In contrast, treatment delivered after 2.5 h of PTI induction only decreased the coherence value. A similar result can be observed in (C). ADR is a suitable indicator to evaluate the degree of recovery following PTI (in general, increased delta band power and decreased alpha band power indicate brain injury); therefore, ADR changes in the cS1FL also demonstrate the recovery of the injured brain region when treatment was delivered within the optimal time window.

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cS1FL region; however, the ADR values decreased again after 2.5 h following PTI induction. The ADR of group 3 exhibited a trend of recovery from 2 to 2.5 h following PTI induction and then began to deteriorate toward the end of the treatment (p b 0.05). Supplementary Figs. S2A and B show the ADR changes in contralateral M1 (cM1; Ch2) and contralateral S1FL* (cS1FL*; Ch6) regions of the ischemic hemisphere preand post-PTI. Because no significant differences were observed in the ADR values of the non-ischemic hemisphere (i.e., Ch1, Ch3 and Ch5) pre- and post-PTI induction, only the ADR values of the ischemic hemisphere are later discussed in the Discussion section.

Results of the recovery indicator for neurovascular coupling with peripheral sensory stimulation Fig. 6 showed a comparison of the recovery indicator for neurovascular function among the experimental groups. Both changes in the normalized functional hemodynamics and in the ADR were included in this indicator. According to the calculated RIs from the curves of normalized CBV and ADR changes in the cS1FL region, the percentages of recovery in groups 1, 2, and 3 were 80 ± 4.2%, 38 ± 3.5%, and 35 ± 3.8% of the baseline, respectively (Fig. 6A). The percentages of recovery of RIs (integrating normalized SO2 and ADR changes) in the cS1FL region for groups 1, 2, and 3 were 79 ± 5.9%, 35 ± 4.8%, and 28 ± 5.7% of the baseline, respectively (Fig. 6B).

Evaluation of the infarct volume with peripheral sensory stimulation intervention We compared the infarct volumes in coronal sections from each group using TTC staining (Fig. 7A) and expressed the lesion volume as a percentage of the ischemic hemispheric volume (Fig. 7B). The infarcts were pale (unstained) regions in the somatosensory cortex. Group 1 exhibited a significant reduction in the volume of the infarct, whereas group 2 showed a slightly reduced infarct volume (% contralateral hemisphere) (p b 0.05; 4.6 ± 2.1% for group 1 and 10.1 ± 1.6% for group 2) compared with the results without stimulation (control group: 13.7 ± 1.7%). In contrast, a reduction of the infarct was not observed in group 3, indicating that stimulation was less effective when delivered beyond the optimal time window.

Discussion In the present study, the fPAM technique was integrated with ECoG recordings, for the first time, to measure functional cerebral hemodynamics, SSEPs, and resting-state ECoG changes over the bilateral S1FL regions in rats. The study was designed to evaluate the efficacy of peripheral sensory stimulation in ameliorating PTI-induced lesions during the hyperacute phase of ischemia. Our major finding is that peripheral sensory stimulation delivered within 2.5 h following PTI induction could facilitate the recovery of cortical functions. Neural activity and hemodynamic changes in the hyperacute phase of PTI, which were measured using the ECoG–fPAM system When the peripheral sensory stimulation was delivered within 2.5 h of PTI induction, a significant recovery of neural activity was observed, as shown by the restoration of ADR values and P1 (N1) peak amplitudes compared with the baseline level, as well as the recovery of functional CBV and SO2. The mechanism by which functional recovery is promoted may involve lepto-meningeal anastomoses, which interconnect the distal branches of the cortical vessels and can be divided into two types, intra-arterial and inter-arterial anastomoses (Luo et al., 2008). A previous study demonstrated that intra-arterial anastomosis helps to immediately reintroduce blood flow into the ischemic region following ischemia. Blood perfusion is rapidly reestablished at the first downstream branch through a reversal of flow in a blood vessel (Schaffer et al., 2006). An alternative route of blood supply could be provided by the collateral vasculature between the anterior cerebral artery (ACA) and the MCA (Armitage et al., 2010). The infarct size is highly correlated with cerebral collateral circulation. For instance, animals with extensive collateral vessels between the ACA and MCA exhibited significantly smaller cortical infarcts than animals with fewer collaterals (Zhang et al., 2010). In contrast, blood vessels exhibit robust vasodilation upon stimulation, which enhances blood redistribution in the vessels surrounding ischemic regions and contributes to the maintenance of uninterrupted flow in the proximal penetrating arterioles, preserving the reactivity of neurons (Shih et al., 2009). Therefore, together with the above findings, we postulate that peripheral sensory stimulation treatment may increase the blood flow in the penumbra region via collateral circulation, which has a neuroprotective effect for ischemia. In addition, according to our group 1 results (Fig. 3C), the relative CBV changes in

Fig. 6. The results of the RI for neurovascular function following PTI in groups 1–3. Please refer to Fig. S1 in the supplementary material for details regarding the RI concept. (A–B) The solid and dashed lines (i.e., red, blue and black) indicate the normalized CBV or SO2 changes correlated to the ADR changes at the cS1FL region in groups 1–3, respectively. The solid lines with arrows in red, blue and black indicate the coupling lines of groups 1–3, respectively. The perpendicular distance from the origin to the coupling line was calculated, and the distance of the diagonal line (between the origin and the diagonal vertex relative to the origin) was calculated for comparison. The percentage difference between the perpendicular distance and the diagonal line is the RI of neurovascular function, and indicates the level of recovery following PTI based on integration of the CBV (SO2) with the ADR results. The recovery indicators based on the relative CBV or SO2 changes with ADR in (A–B) exhibited the maximal 80 ± 4.2% and 79 ± 5.9% of RI values for the recovery of neurovascular function following treatment in group 1, respectively.


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Fig. 7. Infarct volume detected using TTC staining and cerebral infarct volume analysis determined in S1FL areas (denoted by the black arrow) based on a rat brain atlas (Paxinos and Watson, 2007). (A) Representative ischemic brain coronal sections from the untreated group (Control) and treated groups that received peripheral sensory stimulation immediately (Group 1), 1 h (Group 2), and 2 h (Group 3) post-PTI. (B) The percentage of infarct volume in all groups. Significant reductions of infarct volumes were found in groups 1 and 2 compared with the control group (p b 0.05), where these two groups received peripheral sensory stimulation treatment within 3 h following PTI induction. The data are presented as the mean ± S.D. n = 6 for the control group, and n = 8 for each experimental group.

the superficial blood vessel in the ischemic region (from the superficial to deeper region of the brain) largely decreased immediately after PTI induction. After delivering the peripheral sensory stimulation in group 1, the relative CBV changes in deeper blood vessels primarily recovered, whereas the superficial blood vessels showed only partial recovery. The above evidence indicated that the core area of the PTI infarct cannot be recovered and that the penumbra tissue surrounding the core region might be salvageable via peripheral sensory stimulation treatment. Our findings indicate that peripheral sensory stimulation treatment could accelerate the reintroduction of blood flow to the aforementioned penumbra, thus preventing infarct expansion. Under certain experimental conditions, peripheral sensory stimulation can exacerbate stroke damage when the treatment is applied at an inappropriate time following stroke (Lay et al., 2010). According to our results, cortical functions cannot be restored and may decline even further when we deliver the stimulation after the first 2.5-h period following PTI onset. Nevertheless, another study also indicated that peripheral sensory stimulation is beneficial for preserving cortical functions because it increases neural stem/progenitor cell proliferation during the chronic phase post-ischemia (Liu et al., 2013). Thus, although the period for delivering peripheral sensory stimulation to recover brain functions during the hyperacute phase of PTI is limited (i.e., within 2.5 h following PTI onset), this stimulation treatment remained favorable for long-term ischemic rehabilitation (Liu et al., 2013). Interestingly, we noticed the following specific phenomena related to the two essential components of SSEP, P1 and N1: 1) In some cases, P1 components completely disappeared following PTI induction; however, N1 components were only slightly affected (i.e., group 2 of Fig. 4B). 2) In some other cases, the N1 amplitude recovered and increased to greater than the baseline after our treatment was applied (i.e., group 1 of Fig. 4B). A difference in the neural transmission pathways for P1 and N1 may explain the former phenomenon. The depolarization of proximal apical dendrites of supragranular pyramidal cells results in the P1 component, whereas the N1 component results from the distal depolarization of apical dendrites of both supragranular and infragranular pyramidal cells (Di and Barth, 1991). The P1 component is created by a deep current sink in layers IV–V and by a superficial

current source in layers I-II, with activity in supragranular cells (Di et al., 1990). Following the initial positivity of SSEP, the N1 component is induced by the activity of infragranular cells, with a superficial sink developed in layers I-IV and with current sources in the deep layers (V–VI) (Di et al., 1990). Because our PTI model induced ischemia on the cortical surface, the P1 components were more likely to be affected than the N1 components. Thus, in some cases, P1 components were largely affected following PTI induction, whereas only minor variations occurred for N1 components. A potential explanation for the second phenomenon is the increased extracellular K+ concentrations following PTI (Sakatani et al., 1990), which may depolarize and bring the cells closer to the activation threshold (Sakatani et al., 1990). Therefore, after the stimulation was delivered, the N1 amplitude of SSEP was greater than the baseline. ADR, inter-hemispheric coherence, and recovery indicators of neurovascular functions in the hyperacute phase of PTI Quantitative ECoG measures, which have been extensively studied, possess a strong correlation with sensorimotor recovery (Finnigan et al., 2004). Both positive indicators (i.e., alpha power) and negative indicators (i.e., delta power) predict stroke recovery (Finnigan et al., 2004). For instance, after PTI induction, the ADR values of all groups largely decreased over the ischemic S1FL region, indicating that neural activity was affected. Without stimulation, the ADR values in the cS1FL region continually decreased following PTI induction, representing sustained injury of the cortex. In contrast, ADR values increased when the stimulation was delivered within an appropriate time window, suggesting better recovery of neural activity following PTI in this group (i.e., group 1) (Tolonen and Sulg, 1981). These results support our hypothesis that peripheral sensory stimulation may be beneficial for functional recovery. A previous study indicated that acute ischemic injury is followed by the loss of inter-hemispheric coherence between the non-ischemic and ischemic hemispheres (Molnár et al., 1988); our coherence analysis of PTI is consistent with these observations. The loss of interhemispheric connectivity could be attributed to post-PTI diaschisis, which caused interrupted synchronization by widespread cerebral

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metabolic changes extending from the affected hemisphere to the unaffected hemisphere (Jablonka et al., 2010). The inter-hemispheric coherence appeared to recover within 2.5 h post-PTI induction by treatment with peripheral sensory stimulation, as shown in Fig. 5. Based on the results of a previous study, the recovered inter-hemispheric coherence may be due to anatomical and functional reorganization during recovery, recruiting healthy, remote, and secondary cortical areas to compensate for the injured region's function (Knecht et al., 1996). Thus, the manifestation of post-PTI cortical plasticity plays a critical role in sensorimotor recovery following injury. In contrast, the recovery of inter-hemispheric connectivity after the onset of PTI and the notable efferent cortico-cortical connections from the ischemic hemisphere could be attributed to reduced trans-collosal inhibition following hyperacute ischemia. Therefore, the ischemic hemisphere plays a crucial role in early motor recovery following hyperacute stroke (Liepert et al., 2000), and delivering stimulation contralateral to the ischemic hemisphere during the hyperacute phase of ischemia may largely restore neurovascular function. Although the quantitative ECoG measurement of the ADR is an effective indicator to evaluate recovery from cerebral ischemia, this measurement can only provide information related to changes in neural activity. To further observe the recovery of neurovascular function following PTI using ECoG–fPAM, we proposed a reliable recovery indicator that combined CBV or SO2 with ADR changes in this study. Based on the RI, we were able to assess the degree of neurovascular functional recovery. Because ADR changes in the cS1FL (Ch4) were more sensitive to PTI than changes in cS1FL* (Ch6) (due to the proximity of cS1FL to the PTI location), we present RIs of normalized CBV or SO2 with ADR changes in the cS1FL region only (Fig. 6). The RI results of both CBV–ADR and SO2–ADR combinations (calculated by Eq. (1)) indicate that the group 1 treatment, with immediate peripheral sensory stimulation after PTI, can facilitate the recovery of both neural activity and hemodynamics. In contrast, the relative CBV and SO2 values were compared to determine which parameter is more suitable for the RI calculation. According to our data, the RI value of CBV–ADR changes was slightly higher than that of the SO2–ADR changes, indicating that the relative CBV changes had higher correlation to the region of PTI (Liao et al., 2010). Nemoto et al. also demonstrated that CBV-related signals had higher correlation with activated/damaged regions than oxygenation-related signals (Nemoto et al., 2004), which supports our conclusion that CBV–ADR combination RI values are efficient measures for assessing the recovery of neurovascular function. In summary, the present study demonstrates that peripheral sensory stimulation is an effective method to facilitate PTI recovery within the first 2.5 h optimal window following PTI onset in rats. Using the novel ECoG–fPAM system, we evaluated the evoked functional CBV and SO2 changes, SSEP changes and resting-state ECoG changes in the bilateral S1FL regions pre- and post-PTI. Our results indicate that neurovascular functions could be rescued and recovered in the PTI region when peripheral sensory stimulation intervention was delivered within an appropriate time window and that treatment delivered at an inappropriate time can exacerbate ischemic damage. Additionally, a comprehensive RI value (either CBV or SO2 combined with ADR) was proposed in this study. Using this indicator in a single setting, the recovery of neurovascular function can be quantified conveniently, and the RI results correlated well with other measurements, such as SSEPs, inter-hemispheric coherence and infarct volume. Our future studies will investigate peripheral sensory stimulation effects in the chronic phase of ischemia and further utilize this ECoG–fPAM technique to probe neurovascular function in different disorders in small animal models.

Disclosure/conflict of interest The authors declare no conflicts of interest.

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