2.1.

Electrocorticography-Functional Photoacoustic Microscopy System

Figure 1 shows the experimental setup employed in this study, including the ECoG-fPAM system (i.e., the ECoG signal recording system with functional PA imaging), the photothrombotic ischemia (PTI) rat model, C-tDCS, and forepaw/hindpaw electrical stimulation (i.e., PSS). For ECoG recordings, three stainless steel epidural electrodes (including one reference electrode) were secured on the skull to acquire SSEPs and resting-state ECoG signals, which were recorded/processed via a commercial biosignal processor (RZ5D, Tucker-Davis Technologies, Florida).²² The trigger signals used to evoke neurovascular responses (e.g., functional hemodynamic changes) or induce PSS were also generated by this biosignal processor. The acoustic-resolution PAM system offers an axial resolution of $32\mu \mathrm{m}$ and a lateral resolution of $61\mu \mathrm{m}$, while the achievable penetration depth of the fPAM is estimated to be 3 mm. A frequency-tripled Nd:YAG Q-switched laser (Surelite II-10, Continuum, California) was used to pump the optical parametric oscillator (Surelite OPO Plus, Continuum, California), generating 4-ns laser pulses at a pulse repetition rate of 10 Hz. The pulsed laser light was delivered via a 1-mm multimodal fiber, which then passed through a collimation lens, an axicon, and a plexiglass mirror, forming a confocal dark-field illumination spot on the target. The laser exposure on the surface of the target was lower than the maximum permissible exposure limit of $20\mathrm{mJ}/{\mathrm{cm}}^2$ set by the American National Standards Institute. A custom-made 50-MHz ultrasonic transducer (S-Sharp, Taiwan) was used to receive the laser pulse-induced PA signals. During the PA imaging process of in vivo experiments, the ultrasonic transducer was immersed in an acrylic water tank, while the hole at the bottom of the water tank was sealed with a piece of $15\text{-}\mu \mathrm{m}$ thick polyethylene film. A thin layer of ultrasonic gel was applied to the animal’s cortex, and then the film was attached to the gel, ensuring good acoustic coupling of the generated PA signals. Afterward, the PA signals received by the ultrasonic transducer were preamplified by a low-noise amplifier (AU-3A-0110, Miteq Inc., New York) cascaded with an ultrasonic receiver (5073 PR, Olympus, Pennsylvania) and digitized by a computer-based 14-bit analog-to-digital (A/D) card (CompuScope 14200, GaGe, Illinois) at a 200-MHz sampling rate. Additionally, a photodiode (DET36A/M, Thorlabs, New Jersey) was used to monitor the fluctuations in laser light energy. The recorded signals from the photodiode were applied to compensate for variations in PA signals caused by the laser light energy instability.

Fig. 1

Schematic diagram of the ECoG-fPAM system and the C-tDCS experimental setup. The ECoG-fPAM system includes an ECoG signal recording system for neural activity evaluation and a functional PA imaging system for hemodynamic assessment. ECoG signals were acquired via two stainless steel epidural electrodes inserted into the S1FL region to evaluate evoked responses (i.e., SSEPs induced by forepaw stimulation) and resting-state neural activity. For the fPAM setup, 10-Hz laser pulses were generated and coupled to the optical path of a dark-field microscope to illuminate the region of interest and to induce a PA effect. A customized 50-MHz transducer was used to detect the generated PA waves, which were then transferred to a computer for further data analysis. Ultrasonic gel was applied to the cortex for good coupling efficiency. B-scan PA images for $\mathrm{CBV}/{\mathrm{SO}}_2$ changes in the blood vessels of the ischemic region along the AP direction were acquired using the motorized scanning stage. To induce PTI, a CW laser light was coupled to the optical path of the fPAM and was focused on the selected cerebral blood vessel within the PA imaging window. For C-tDCS intervention, the cathodal electrode was placed on the skull, while the anodal electrode was attached to the venter. For PSS intervention, forepaw/hindpaw stimulation was delivered via subdermal needle electrodes.

2.2.

Animal Preparation

Fifty-eight male Sprague Dawley rats weighing 250 to 300 g (InVivos Pte. Ltd., Singapore) were randomly divided into three groups for evaluation of neurovascular functions: control PTI only ($n=16$) group, C-tDCS + PTI ($n=21$), and C-tDCS + PSS + PTI ($n=21$). Among them, 18 animals were equally divided into three groups and used for immunohistochemical studies. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the National University of Singapore and the National Health Research Institutes of Taiwan. The animals were housed at a constant temperature and humidity with free access to food and water.

During the entire surgical and experimental procedures, the body temperature of the rats was maintained at $37°\mathrm{C}\pm 0.5°\mathrm{C}$ using a rectal probe (TCAT-2 Temperature Controller, New Jersey) and a self-regulating thermal plate. The rats were anesthetized with pentobarbital (i.e., $50\mathrm{mg}/\mathrm{kg}$ bolus induction and $15\mathrm{mg}/\mathrm{kg}/\mathrm{hr}$ maintenance, i.p. injections). The anesthetized rats were mounted on a custom-made acrylic stereotaxic head holder and placed on a bed pallet during the experiment. A craniotomy was performed on the exposed skull with a high-speed drill (Micromotor drill, Stoelting, Illinois) to open a cranial window ($\sim 4\times 4{\mathrm{mm}}^2$) on the right side of the skull for B-scan PA imaging and PTI induction. Two stainless steel epidural electrodes were secured to the skull over the forelimb region of the primary somatosensory cortex (S1FL): $+0.48$ and $-0.92\mathrm{mm}$ AP, $+4.5\mathrm{mm}$ ML for ECoG recording. The reference electrode was positioned 3 mm lateral to the lambda, as shown in Fig. 1. These electrodes were connected via silver wires to the data acquisition system (RZ5D) through a connector (ZIF-Clip headstage, Tucker-Davis Technologies, Florida). For subsequent procedures, the distance between the interaural line and the bregma was used to position the head in the fPAM system.

2.3.

In Vivo Rat Photothrombotic Ischemia Model

The in vivo PTI model was established according to Watson et al.²³ Focal ischemia was induced by blocking a selected cortical blood vessel [i.e., a distal branch of the middle cerebral artery (MCA)] through the cranial window over the S1FL region of the right hemisphere.²² This effect was achieved by first injecting the photosensitizer Rose Bengal (Sigma, Missouri) diluted to $10\mathrm{mg}/\mathrm{ml}$ in HEPES-buffered saline over the course of 2 min into the tail vein at $0.2\mathrm{ml}/100\mathrm{g}$ rat body weight. The selected cortical blood vessel was then illuminated using a 5 mW, 532 nm CW laser light (AMGA-005, ONSET, Taiwan) to generate an occlusion.^23,24 The CW laser light was coupled to the dark-field optical path of the ECoG-fPAM system and focused on the selected cortical blood vessel for at least 15 min until a stable clot was formed.^22,25

2.4.

Neural Activity and Hemodynamics Assessments

As shown in Fig. 2(a), 60-s resting-state ECoG signals were recorded from the S1FL region of the right hemisphere to evaluate the effect of ischemia on neural activity, without an explicit stimulus applied to the rat. To evaluate functional changes in stimulus-evoked SSEPs and $\mathrm{CBV}/{\mathrm{SO}}_2$, forepaw electrical stimulation was delivered as a trigger via subdermal needle electrodes inserted into the left forepaw of the rat (i.e., contralateral to the ischemic hemisphere).^26,27 The electrical stimulation was delivered using a current stimulator (DS3, Digitimer, Hertfordshire, England). To synchronize this stimulator with the multichannel RZ5D biosignal processor, trigger pulses for controlling the stimulator were delivered via an output port of the processor. After the resting-state ECoG was recorded, a 5-s monophasic constant current with a 2-mA intensity and a 0.2-ms pulse width at a frequency of 3 Hz was delivered to induce SSEPs corresponding to neural activity.

Fig. 2

Schematic diagram of the assessment and the therapeutic intervention protocols. (a) To evaluate neural activity, in each block, 60-s resting-state ECoG signals were recorded from the S1FL region of the right hemisphere, followed by 5 s monophasic constant current stimulation at 3 Hz to induce SSEPs of neural activity. To assess functional changes in the three different locations, three B-scan PA images were acquired in each hemodynamics assessment block. Each 1-s electrical stimulation was delivered and followed by 30 s of PA B-scan imaging at $\lambda =808$ or $\lambda =880$ for the selected cortical blood vessel. (b) Neurovascular functions were evaluated using the ECoG-fPAM system in all three groups (PTI, C-tDCS + PTI, and C-tDCS + PSS + PTI). The blue and green arrows indicate the time points of neural activity and hemodynamic assessments, respectively. The purple block shows the duration of C-tDCS administration. Current stimulation at 2 mA and 3 Hz with a 0.2-ms pulse width was delivered for 90 s every 15 min during the PSS intervention session (red block).

On the other hand, for assessing functional changes in different locations, three B-scan PA images were acquired during each hemodynamics assessment block. Thus, for evoking hemodynamic responses in these planes of the cranial window (i.e., ML: $+3.2$, $+3.7$, and $+4.2\mathrm{mm}$), three 1-s electrical stimulations were delivered, followed by 30 s of PA B-scan imaging at 808 and 880 nm wavelengths (${\lambda }_{808}$ and ${\lambda }_{880}$) for the selected cortical blood vessels in the S1FL region of the right hemisphere [Fig. 2(a)]. The time points of neural activity and hemodynamics assessments during the experiments are shown in Fig. 2(b) with blue and green arrows, respectively.

2.5.

Experimental Protocols, Cathodal-Transcranial Direct Current Stimulation, Peripheral Sensory Stimulation, and Integrated Therapeutic Intervention

Before ischemia induction, neurovascular functions were evaluated via the ECoG-fPAM system in all three groups (PTI, C-tDCS + PTI, and C-tDCS + PSS + PTI), as shown in Fig. 2(b). C-tDCS was applied using a current stimulator (Model 2100, A-M systems, Washington) to deliver a constant current of $400\mu \mathrm{A}$ for 20 min immediately after PTI. Previous studies have shown that C-tDCS has a protective effect against ischemia when applied in the very early stage.^13,14 Therefore, the duration of 20 min was selected accordingly,^12,14,18,20 and it appeared to be optimal in our preliminary studies. The cathodal electrode was attached transcranially to the rat at an anatomical location 5-mm anterior and 3-mm lateral to the bregma (Fig. 1), while the anodal electrode was attached to the venter and surrounded by gauze to avoid displacement. The contact area of the cathodal electrode was $0.196{\mathrm{cm}}^2$, whereas the area of the anodal electrode was $4\times 3.5{\mathrm{cm}}^2$. The current intensity of $400\mu \mathrm{A}$, corresponding to a current density of $20.37\mathrm{A}/{\mathrm{m}}^2$, was within the range of intensities used in other acute experiments on animals.¹⁴ For PSS, a total of four subdermal needle electrodes (two for forepaw and two for hindpaw stimulation) were inserted into the rat to deliver the PSS. Current stimulation of 3 Hz with a 0.2-ms pulse width and 2 mA was delivered for 90 s (i.e., 270 corresponding sweeps) every 15 min for 2 h after the 20-min C-tDCS [Fig. 2(b)].

2.6.

Data Analysis of the Cerebral Blood Volume and Hemoglobin Oxygen Saturation Measurements

Relative functional changes in CBV and ${\mathrm{SO}}_2$ were monitored/evaluated via two optimized wavelengths (i.e., ${\lambda }_{808}$ and ${\lambda }_{880}$, respectively).²⁶ It was assumed that CBV is proportional to the cross section of a single vessel at 808-nm wavelength (${\lambda }_{808}$).²⁸ Based on the PA B-scan image at ${\lambda }_{808}$ [$I_{R(808)}$] in the selected plane of the S1FL region, the cross-sectional area $\{A[I_{R(808)}]\}$ of the selected blood vessel was calculated using the total blood vessel pixel count, while a blood vessel pixel was defined as a pixel that possessed a PA signal three times greater than the background signal.^21–23 Then, functional CBV changes ($R_{\mathrm{CBV}}$) were derived using the following equation:

PA signal changes at 880 nm wavelength (${\lambda }_{880}$) were sensitive to ${\mathrm{SO}}_2$ changes because larger absorption changes in the near-infrared spectrum occurred at approximately ${\lambda }_{880}$ when the ${\mathrm{SO}}_2$ levels changed. Images of functional ${\mathrm{SO}}_2$ changes $[\mathrm{\Delta }{I}_{F(880)}(\mathrm{t})]$ at a given time point $t$ in each block were assessed using the following equation:

2.7.

Data Analysis of Electrocorticography Recording

SSEP measurements can potentially predict functional recovery after stroke, as SSEPs correlate well with the level of disability;³⁰ therefore, SSEPs have been used as an objective way to assess the integrity of sensory and motor pathways of the central nervous system and were used as a marker of neural activity in this study.³¹ Changes in the peak-to-peak amplitude (PPA) between P1 (i.e., the first positive peak after stimulation) and N1 (i.e., the first negative peak directly following P1) were evaluated, as these values can indicate the presence of cortical dysfunction.^31–33 In this way, SSEPs were quantitatively evaluated and used to assess neural activity. The sampling rate for recording ECoG signals was 1 kHz. The recorded signals were pre-amplified through the PZ2-32 preamplifier and bandpass filtered between 0.3 and 150 Hz via an RZ5D processor. The SSEP signals evoked by left forepaw electrical stimulation were recorded pre and postischemia in the S1FL region of the right hemisphere. As mentioned in the previous section, the trigger pulses for controlling the current stimulator were generated by the biosignal processor, along with synchronous recording timestamps of each stimulus pulse onset and SSEP sweep. We administered 3 Hz stimulation for 5 s to evaluate SSEPs, and a total of 15 corresponding sweeps were extracted and averaged to generate SSEP waveforms over a 100-ms epoch poststimulus pulse. Afterward, the P1 and N1 values were extracted from the recorded SSEP waveforms to further calculate the PPA based on these two components. The percentage change in SSEPs was computed relative to the baseline value for both channels in which the baseline value was recorded before the onset of PTI for each animal and was regarded as the normal neural activity status. In this study, two-channel SSEPs from the electrodes were averaged to represent the state of neural activity at each time point, as shown in Fig. 2 (blue arrows).

Delta (i.e., 0.3 to 3 Hz) and alpha (i.e., 8 to 13 Hz) frequency bands have been reported to be strong indicators of both injury and recovery after ischemia.³⁴ Thus, the ADR [i.e., the ratio between alpha power and delta power (alpha/delta)] was used in this study to monitor functional neural changes of resting-state ECoG signals pre- and post-PTI;³⁵ a higher ratio value indicates a greater degree of neural activation, and vice versa. On the other hand, theta (i.e., 4 to 7 Hz) and beta (i.e., 14 to 20 Hz) activity may also be altered in stroke.^36–38 In comparison to the ADR, the DTABR [$(\text{delta}+\text{theta})/(\text{alpha}+\text{beta})$ ratio] additionally includes both the theta and beta bands to predict spontaneous neurological improvement after stroke.^39,40 Fast Fourier transform was used to calculate the mean power over the two channels in all frequency bands.³⁵ The percentage changes in the ADR and the DTABR were calculated with respect to the baseline value for both channels in which the baseline value was recorded before the onset of PTI and was regarded as the normal neural activity status. The two-channel ADR and DTABR values at the ischemic region were averaged to acquire a mean value representing the state of each evaluating block. More information on the detailed calculations of SSEPs, ADR, and DTABR are available in our previous studies.^22,25,39,40

2.8.

Immunohistochemistry

Rats were overdosed with pentobarbital ($300\mathrm{mg}/\mathrm{ml}$) and perfused with $0.1\mathrm{mol}/\mathrm{L}$ phosphate buffer through the heart. The brains were then removed and snap frozen in crushed dry ice. Coronal sections ($30\mu \mathrm{m}$) in the regions of interests ($+1$, $+0.7$, $+0.4$, $+0.1$, $-0.2$, $-0.5$, and $-0.8\mathrm{mm}$) were cut using a cryostat and mounted on glass slides. Antibodies against NeuN (1:200, Millipore, Massachusetts) and ED-1 (1:200, Millipore, Massachusetts) were used, and slides were incubated at 4°C overnight. Sections were then washed and incubated with Alexa Fluor 555-conjugated goat antimouse (Thermo Fisher Scientific, Massachusetts) secondary antibody for 1 h at room temperature. Brain slices were counterstained by 4’,6-diamidino-2-phenylindole (Thermo Fisher Scientific, Massachusetts) and finally mounted with ProLong Gold (Thermo Fisher Scientific, Massachusetts) mounting media. As a negative control, sections were probed with phosphate-buffered saline (PBS) with 0.1% Triton instead of antibodies overnight. All fluorescent images were captured with an Olympus IX71 (Olympus, Japan) laser scanning confocal microscope, and images captured were processed by Image-Pro Insight software (Media Cybernetics, Maryland). Cell counts were performed by quantifying the number of immunopositive cells in a selected $1{\mathrm{mm}}^2$ site at the infarcted region in a blinded manner.

2.9.

Measurement of the Infarct Volume and Behavioral Assessment

The infarct volume was measured using 2,3,5-triphenyl-tetrazolium chloride (TTC).⁴¹ At 48 h following the onset of PTI, the rats were overdosed with pentobarbital ($300\mathrm{mg}/\mathrm{ml}$), and the brains were rapidly removed, washed in $1\times$ PBS at room temperature, and snap frozen in dry ice until use. Six serial 1.5-mm coronal sections were cut from each brain (from 4-mm anterior to 5-mm posterior to the bregma). The sections were stained with 2% TTC (chloride, T84859, Sigma, Missouri) for 12 min at 37°C in the dark, followed by overnight immersion in 4% paraformaldehyde in $0.1\mathrm{mol}/\mathrm{L}$ PBS, pH 7.4, at 4°C. The infarcted tissue remained unstained (i.e., white), whereas normal tissue was stained red. The volume of the ischemic infarct was traced and 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. The true infarct volume was obtained by correction for brain edema as follows:²²

Wood et al.⁴² demonstrated that photothrombotic cortical lesions produce a deficit in grip strength, which could be observed at 24 h post-PTI. For behavioral assessment, the grip strength test was conducted in a blinded manner 2 days post-PTI. A total of five grip strength trials were performed for each animal in 5 min intervals. Each trial included three pulls, and the average of all 15 pulls was used for data analysis.

2.10.

Statistical Analysis

Baseline values of CBV, ${\mathrm{SO}}_2$, SSEPs, ADR, and DTABR were used to normalize the observed post-PTI changes (i.e., the most extreme value post-PTI was scaled to 0, and the baseline value was scaled to 1). The changes in SSEPs, ADR, and DTABR in the ischemic hemisphere between cases were assessed using repeated-measures analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) posthoc analysis.⁴³ Case-to-case differences in averaged PA signals of the studied regions and changes in cross-sectional areas (i.e., CBV and ${\mathrm{SO}}_2$ changes) were examined using paired $t$-tests (i.e., $p<0.05$, $n=40$). The significance of the relative changes observed in the averaged PA signals of the studied areas in response to electrical stimulation was analyzed using the Wilcoxon matched-pairs signed-rank test (i.e., two-tailed, $p<0.05$, $n=40$).²⁶ Immunofluorescence and behavioral data were assessed by a one-way ANOVA followed by a posthoc (Bonferroni) test (i.e., $p<0.05$, $n=6$). All statistical analyses were performed using SPSS (version 10.0, SPSS^®). Data are presented as the $\mathrm{mean}\pm \text{standard deviation}$ (S.D.). Statistical significance was reached for $p<0.05$.

3.1.

Effect of Interventions on Neural Activity During Hyperacute Phase of Photothrombotic Ischemia

Figure 3 shows the PPA of SSEP waveforms and the ADR and DTABR of resting-state ECoG signal recordings pre- and post-PTI, which were used to indicate neural activity in this study.^33,35,40 As shown in Fig. 3(a), the PPA of all three groups (i.e., PTI, C-tDCS + PTI, and C-tDCS + PSS + PTI) decreased by $\sim 70\%$ from the baseline value immediately after the induction of PTI ($p<0.05$). There was no spontaneous recovery during the hyperacute phase (within 4 h) as observed in the PTI-only group. In contrast, C-tDCS treatment caused the PPA to increase, reaching $\sim 290\%$ above the baseline level within 4 h ($p<0.05$). The combined treatment of C-tDCS + PSS yielded similar patterns of change except that the increase was more modest, reaching only approximately 100% above the baseline level ($p<0.05$). The observed changes in ADR were similar to those of PPA [Fig. 3(b)], recovering from a decreased level of $\sim 40\%$ of baseline level to $\sim 140\%$ and 30% above baseline in the C-tDCS-treated and C-tDCS + PSS treated groups, respectively, within 4-h post-PTI.

Fig. 3

Effect of interventions on neural activity during the hyperacute phase of PTI. (a) Changes in the normalized PPA of SSEPs are shown. (b and c) The ADR and DTABR values of resting-state ECoG signal recordings were also used as indicators to evaluate neural functions pre- and post-PTI in this study. Both ADR and DTABR significantly recovered and exceeded the baseline values for the C-tDCS + PTI and C-tDCS + PSS + PTI groups, while the values of the PTI group worsened, consistent with the PPA results. The results showed that therapeutic interventions including C-tDCS were able to restore neural activity in the ischemic region during the hyperacute phase of PTI. The values of all three indicators for each time point were averaged over the two electrode channels and plotted with standard deviations, as these two channels were both located in the S1FL region and within the ischemic area. These results represented the overall state of each evaluation block and were used to compare the values obtained at various time points. A posthoc analysis comparing the indicators at each time point was also performed.

DTABR, unlike PPA and ADR, increased to $\sim 100\%$ above the baseline value in all three groups [Fig. 3(c)] ($p<0.05$). Here, both treatment groups showed similar changes and DTABR returned to baseline within 1 h. Over the next 3 h, DTABR decreased to $\sim 50\%$ and 30% of the baseline value for the C-tDCS and C-tDCS + PSS groups, respectively ($p<0.05$), but, overall, the two treatment groups did not significantly differ. Again, no recovery of DTABR was observed in the PTI-only group. The above results showed that C-tDCS treatment was able to restore neural activity in the ischemic region during the hyperacute phase of PTI. Moreover, PSS modulated the effects of C-tDCS toward baseline levels for at least two of the three parameters measured.

3.2.

Effect of Interventions on Hemodynamics During the Hyperacute Phase of Photothrombotic Ischemia

PA imaging was employed to evaluate the effect of interventions on hemodynamics as measured by CBV and ${\mathrm{SO}}_2$ following PTI. To assess different locations near the ischemic induction region, three planes of the cranial window were scanned to evaluate the hemodynamic changes at the PTI induction region ($+3.7\mathrm{mm}$ from the bregma) and its downstream ($+3.2\mathrm{mm}$) and upstream ($+4.2\mathrm{mm}$) regions, as shown in Fig. 4(a). The PA-measured CBV significantly changed in the C-tDCS + PSS + PTI group at different locations pre- and post-PTI [Fig. 4(b)]. The results showed that the CBV of the PTI induction region significantly recovered to $\sim 50\%$ of the baseline value during the hyperacute phase of ischemia following C-tDCS + PSS treatment ($p<0.05$). On the other hand, the B-scan images of upstream and downstream regions also showed similar recovery of CBV after applying the intervention.

Fig. 4

Changes in the normalized CBV and ${\mathrm{SO}}_2$ values pre- and postischemia. (a) To assess different locations near the ischemic induction region, three planes of the cranial window (i.e., ML: $+3.7\mathrm{mm}$, $+3.2\mathrm{mm}$, and $+4.2\mathrm{mm}$) were scanned to evaluate the hemodynamic changes at the PTI induction region and its downstream and upstream regions, respectively. Note that the photo shown here is only for illustration [not related with PA signals in (b)]. (b) The PA-measured CBV ($R_{\mathrm{CBV}}$) changes of the C-tDCS + PSS + PTI group at three locations pre- and post-PTI. The results showed that CBV of the PTI induction region could not be recovered to the baseline value, which might be because this region had become the ischemic core due to C-tDCS (decreasing the CBF over the stimulated region). On the other hand, the B-scan images of downstream and upstream regions showed that more CBV was restored to the surrounding regions (i.e., reperfusion), which may protect the ischemic area. (c) The normalized CBV ($R_{\mathrm{CBV}}$) and ${\mathrm{SO}}_2$ ($-R_{{\mathrm{SO}}_2}$) changes of all groups at three locations. The results indicated that C-tDCS + PSS intervention led to the greatest improvement in ischemic outcome according to hemodynamics ($p<0.05$).

The normalized CBV ($R_{\mathrm{CBV}}$) and ${\mathrm{SO}}_2$ ($-R_{{\mathrm{SO}}_2}$) changes of the three groups at the three different locations over time are shown in Fig. 4(c). Following PTI induction, the relative ${\mathrm{SO}}_2$ of all three groups decreased initially by $\sim 66\%$ relative to baseline at the PTI induction region. Both treatment groups recovered to $\sim 50\%$ of the baseline value after 240 min, while the untreated group further deteriorated to 80% below baseline. On the other hand, the results indicated that the CBV value of the C-tDCS + PSS + PTI group was the highest among all groups 4 h postischemia at the three B-scan locations. The CBV at the PTI induction region initially decreased significantly to $\sim 36\%$ of the baseline level after PTI induction in all three groups ($p<0.05$). This was followed by a significant recovery to 47% of the baseline value in the C-tDCS + PSS-treated group at 240 min ($p<0.05$). In the upstream and downstream regions, the initial decreases in ${\mathrm{SO}}_2$ and CBV following PTI were markedly less than those at the induction region. However, consistent observations were made in which both ${\mathrm{SO}}_2$ and CBV recovered after the initial decreases in both C-tDCS-treated groups but not in the untreated group. In summary, the results demonstrated that the C-tDCS + PSS intervention resulted in substantial improvement in stroke outcome during the hyperacute phase of ischemia. We also noted that the downstream ${\mathrm{SO}}_2$ levels were higher in the untreated group versus all other groups. A potential reason for this unexpected ${\mathrm{SO}}_2$ value might be due to the instant collateral circulation of the deeper microvessels of this batch of rats.^44,45 Since the 880-nm wavelength of light penetrates to a deeper tissue region, the effect of the potential deep collateral circulation could be observed by investigating ${\mathrm{SO}}_2$, while CBV (808 nm) could only examine the blood vessels at a surface region.

3.3.

Evaluations of Infarct Volume and Behavior

The infarct volume obtained in rats treated with C-tDCS + PSS was significantly reduced by 50% and 40% compared with the no treatment group and the C-tDCS-treated group, respectively ($p<0.05$). The PTI- and C-tDCS-only groups were not significantly different [Fig. 5(a)], showing that only C-tDCS + PSS is an effective intervention in reducing the infarct size following the ischemic insult. This result is also supported by our previous study,²² as we have shown that infarct volume can be reduced if PSS is delivered during the hyperacute phase of ischemia (i.e., 5%, 10%, and 14% if applied immediately, 1 and 2 h postischemia, respectively; in this study, PSS was applied 30 min postischemia).²² Similarly, as shown in Fig. 5(b), the grip strength of the C-tDCS + PSS-treated group was well preserved and significantly stronger ($634\pm 56\mathrm{g}$ force) than the grip strength of both the untreated ($495\pm 39\mathrm{g}$) and the C-tDCS-treated ($534\pm 68\mathrm{g}$) groups ($p<0.05$). In addition, the grip strength of the C-tDCS + PSS group was also the closest to the baseline value of healthy animals ($783\pm 79\mathrm{g}$). Thus, C-tDCS + PSS was able to induce the greatest restoration in the grip strength of the rats after ischemia.

Fig. 5

C-tDCS + PSS protected against ischemic injuries in vivo. (a) Infarct volume was significantly reduced by C-tDCS + PSS compared with no treatment (PTI alone), while C-tDCS was not effective. Representative TTC-stained coronal brain sections show infarct areas (white) on the right. The lesion volumes are expressed as a percentage of the ischemic hemispheric volume after correction for edema. (b) Functional recovery was assessed by measuring the grip strength. Similarly, grip strength was significantly preserved in rats treated with C-tDCS + PSS compared with animals with no treatment or C-tDCS treatment. *$p<0.05$ and **$p<0.01$.

3.4.

Neuroprotective Effect of Therapeutic Interventions

NeuN immunohistochemical staining in the infarcted area was observed to be markedly decreased following PTI compared with the noninfarcted contralateral side (Fig. 6). Upon treatment, the number of NeuN-positive cells was markedly preserved (2-fold higher) in the C-tDCS + PSS-treated group compared with the no treatment group ($p<0.05$). However, there was no significant difference in the C-tDCS-treated group compared with the PTI group.

Fig. 6

Neuroprotective effects of C-tDCS + PSS. (a and b) NeuN staining (a) was used to visualize neurons in selected areas I and II, as shown in (b). The results showed that PTI markedly reduced the number of neurons compared with the contralateral side and that C-tDCS + PSS treatment protected neurons in the infarcted region following PTI. $\text{Scale bar}=250\mu \mathrm{m}$. (c) Cell counts of NeuN-positive cells revealed significant preservation by more than 2-fold in the C-tDCS + PSS + PTI group compared with the untreated group in the ischemic region $I$. $n=5$ to 6. *$p<0.05$ and **$p<0.01$.

In contrast, as shown in Fig. 7, ED-1-immunopositive cells significantly increased in the infarcted area following PTI in the untreated group ($2809\pm 555\mathrm{cells}\mathrm{per}{\mathrm{mm}}^2$). Similar cell counts were obtained in the C-tDCS-treated group ($2739\pm 307\mathrm{cells}\mathrm{per}{\mathrm{mm}}^2$), indicating microglia activation in both groups. In the C-tDCS + PSS-treated group, however, the obtained cell counts ($440\pm 104\mathrm{cells}\mathrm{per}{\mathrm{mm}}^2$) were only $\sim 15\%$ of those in the other groups, indicating a drastic suppression of microglial activation by C-tDCS + PSS treatment. The above results showed that the C-tDCS + PSS intervention may decrease infarct volume and improve functions such as grip strength by protecting against neuronal loss and suppressing inflammatory responses.

Fig. 7

C-tDCS + PSS suppressed microglia activation. (a and b) ED-1 staining was used to visualize activated microglia in selected areas I and II, as shown in (b). The results showed that PTI markedly increased the number of activated microglia compared with the contralateral side and that C-tDCS + PSS treatment prevented microglia activation in the infarcted region following PTI. a(II) lower panel represents the magnification of images taken from the respective ED-1 staining of infarcted regions, indicating the amoeboid reactive microglia. a(II) top panel $\text{scale bar}=250\mu \mathrm{m}$. a(II) bottom panel $\text{scale bar}=125\mu \mathrm{m}$. a(I) $\text{scale bar}=250\mu \mathrm{m}$. (c) Cell counts of ED-1-positive cells showed a significant reduction by more than 2-fold in the C-tDCS + PSS + PTI group in ischemic region $I$. $n=5$ to 6. **$p<0.01$.