2.1.

Generation and Characteristics of a Laser-Induced Shock Wave

Figure 1(a) shows the method for generation of an LISW. A laser target, which was a light-absorbing material (0.5-mm-thick natural black rubber disk) on which an optically transparent material (1.0-mm-thick polyethylene terephthalate sheet) was adhered with adhesive, was placed on the tissue. The laser target was irradiated with a high-intensity laser pulse from a 532-nm Q-switched Nd:YAG laser [pulse width, 6-ns full-width at half-maximum (FWHM); Brilliant b, Quantel, Les Ulis Cedex, France). The laser pulse was absorbed by the black rubber to induce plasma and its expansion generated a shock wave (LISW). We previously observed that an LISW generated at a laser fluence of $1.25\mathrm{J}/{\mathrm{cm}}^2$ with a spot diameter of 4 mm (peak pressure: $\sim 104\mathrm{MPa}$, impulse: $\sim 19\mathrm{Pa}·\mathrm{s}$, positive pulse duration: $\sim 0.8\mu \mathrm{s}$) caused CSD in all of the five rats examined.⁵ In this condition, transcranial application of a single LISW to a rat brain caused either no or limited hemorrhage in the cortical surface. On the basis of the histopathological analysis, we also confirmed that there was neither thermal damage nor mechanical damage (contusion) in the cortex at the same condition (data not shown). Thus, this condition can be regarded as mild or mild-to-moderate injury and was used in the present study. The peak pressure of an LISW is 2 to 3 orders of magnitude higher than that of medically relevant actual explosion shock waves, while the positive pressure duration of an LISW is 2 to 3 orders of magnitude shorter. As a result, the impulse (the time-integrated positive pressure component), which is known to be one of the most important parameters to determine tissue damage, is in the same order as that of relevant actual explosion shock waves.²⁰ Thus, we assume that the condition we used is valid to investigate the brain pathophysiology associated with bTBI in rats.

Fig. 1

Schematics showing (a) the method for generation of a LISW, (b) positions of LISW application and the cranial window for imaging, and (c) experimental setup for multispectral imaging of the rat brain.

2.2.

Surgical Preparation and LISW Application

Male Sprague–Dawley rats weighing 300 to 400 g (10 to 12 weeks of age, $n=4$, Japan SLC, Inc., Shizuoka, Japan) were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg bolus and $\sim 15\mathrm{mg}/\mathrm{kg}/\mathrm{h}$ maintenance) and placed in a stereotactic frame. After shaving the head, the scalp was incised at the midline and the parietal bone was exposed. During experiments, adequacy of anesthesia was checked by the absence of a response to tail pinch. For multispectral imaging, a cranial window with a diameter of $\sim 4.5\mathrm{mm}$ was prepared with the dura intact in the left parietal bone with a trephine (Roboz Surgical Instrument. Co., Inc., Gaithersburg, Maryland) under a surgical microscope (OPMI Pico/S100, Carl Zeiss, Germany). A disk of transparent polymethyl methacrylate with a diameter of 4.4 mm and a thickness of $\sim 0.5\mathrm{mm}$ was inserted within the window and sealed with dental cement. Figure 1(b) shows a schematic illustrating positions of the cranial window and the site of LISW application. The distance between the center of the LISW and the center of the cranial window was $\sim 7\mathrm{mm}$. After window preparation, the rat was moved to the position for imaging and placed in another stereotactic frame. The body temperature was kept constant at $37.0°\mathrm{C}\pm 0.5°\mathrm{C}$ by using a temperature-controlled body mat, and arterial oxygen saturation (${\mathrm{SpO}}_2$) was continuously monitored at the hindlimb with a pulse oximeter (8600V, Nonin Medical, Inc., Plymouth, Minnesota).

2.3.

Multispectral Imaging

Through the window described above, we performed multispectral imaging of the cortex to observe CSD, morphology of vessels (changes in diameters of cortical surface vessels), ${\mathrm{rC}}_{\mathrm{Hb}}$, and ${\mathrm{StO}}_2$ before, during, and after LISW application. Figure 1(c) shows a diagram of the experimental setup. The parietal region was illuminated with the light from a halogen lamp (HL 100E, HOYA-Schott, Tokyo, Japan); the wavelength of the light was changed sequentially using a rotating filter wheel (FW102C, Thorlabs, Newton, New Jersey) with six bandpass filters. The center wavelengths of the filters were 520, 540, 560, 580, 600, and 800 nm and their bandwidths were 10 nm FWHM (520 to 600 nm) or 40 nm FWHM (800 nm); the diameters of the filters were $\sim 25\mathrm{mm}$ (Thorlabs). Diffusely reflected light from the cortex was captured with a CCD (12 bit, $1034\times 779\text{pixels}$, 1/30 s/frame, scA1000-30fm, Basler AG, Ahrensburg, Germany) equipped with a unifocal lens ($f=50\mathrm{mm}$, M118FM50, Tamron Co., Ltd., Saitama, Japan). The filter wheel was rotated every 1.5 s using accessory software (Thorlabs), and image acquisitions were synchronized with the filter rotations using an output signal from the filter wheel and LabVIEW software (National Instruments, Austin, Texas), the image acquisition rate being 1 image per 1.5 s at each wavelength. Since images at 5 of the 6 wavelengths were used for quantitative analyses as described later, the actual imaging rate was 1 image per 7.5 s. A laser target was placed on the skull in the frontal region and held with forceps; ultrasound gel was applied between the bottom of the target (rubber) and the skull surface for acoustic impedance matching. Baseline images were acquired at least for 2 min, and the target was then irradiated with a single laser pulse to produce an LISW, and imaging was continued for $\sim 60\min$. After imaging, a certified diffuse reflectance standard plate (SRS-99-020, Labsphere, Inc., North Sutton, New Hampshire) was placed at the same position and its image was acquired under the same imaging conditions. All multispectral images acquired for the cortex were divided by images acquired for the reflectance standard at the corresponding wavelengths, which were used as diffuse reflectance $r(\lambda )$ images to calculate optical and physiological parameters, as described below.

2.4.

Estimation of Cerebral Absorption (Hemodynamics) and Scattering Parameters

We performed multiple regression analysis with Monte Carlo simulation to map the reduced scattering coefficient, which was used as an optical marker for CSD,⁵ and absorption coefficient as well as ${\mathrm{rC}}_{\mathrm{Hb}}$ and ${\mathrm{StO}}_2$, using MATLAB software (Mathworks, Natick, Massachusetts). Images at 520, 540, 560, 580, and 600 nm were used for assessing the reduced scattering coefficient and concentrations of oxygenated hemoglobin ($C_{\text{HbO}}$) and deoxygenated hemoglobin ($C_{\text{HbR}}$), while images at 800 nm were used to determine the propagation velocity of CSD, as described below. The method is basically the same as that used in our previous study.^19,21 Figure 2 shows the flow diagram of the method. Absorbance spectrum $A(\lambda )$ was calculated from diffuse reflectance $r(\lambda )$ based on the following equation:

Fig. 2

Flow diagram of the process for estimating the concentration of oxygenated hemoglobin ($C_{\text{HbO}}$), the concentration of deoxygenated hemoglobin ($C_{\text{HbR}}$), and scattering amplitude $a$. (a) Preparation work for determination of the regression equations and (b) main process to estimate $C_{\text{HbO}}$ and $C_{\text{HbR}}$ from the measured reflectance spectrum $r(\lambda )$.

The total hemoglobin concentration $C_{\text{HbT}}$, also referred to as $C_{\mathrm{Hb}}$, is defined as the sum of $C_{\text{HbO}}$ and $C_{\text{HbR}}$ as follows:

The tissue oxygen saturation is determined as follows:

The reduced scattering coefficient of the brain tissue was assumed to be in the following form:^24,25

As described above, the regression coefficients ${\alpha }_{\mathrm{HbO}}$, ${\alpha }_{\mathrm{HbR}}$, and ${\alpha }_0$ are related to the concentration of oxygenated hemoglobin $C_{\text{HbO}}$, that of deoxygenated hemoglobin $C_{\text{HbR}}$, and the coefficient $a$, respectively. However, ${\alpha }_{\mathrm{HbO}}$, ${\alpha }_{\mathrm{HbR}}$, and ${\alpha }_0$ are not determined by a unique regression coefficient when using only MRA1. Thus, we used further multiple regression analysis to estimate the values of $C_{\text{HbO}}$, $C_{\text{HbR}}$, and $a$ on the basis of the combination of regression coefficients ${\alpha }_{\mathrm{HbO}}$, ${\alpha }_{\mathrm{HbR}}$, and ${\alpha }_0$ that were obtained from MRA1. In this analysis, $C_{\text{HbO}}$, $C_{\text{HbR}}$, and $a$ were regarded as response variables, and the three regression coefficients ${\alpha }_{\mathrm{HbO}}$, ${\alpha }_{\text{HbR}}$, and ${\alpha }_0$ in Eq. (7) were regarded as predictor variables. Their relations are written as $C_{\text{HbO}}={\mathbf{\beta }}_{\mathbf{HbO}}\cdot \mathbf{\alpha }$, $C_{\text{HbR}}={\mathbf{\beta }}_{\mathbf{HbR}}\cdot \mathbf{\alpha }$, and $a={\mathbf{\beta }}_{\mathbf{a}}\cdot \mathbf{\alpha }$, where $\mathbf{\alpha }={[1,{\alpha }_{\mathrm{HbO}},{\alpha }_{\mathrm{HbR}},{\alpha }_0]}^{\mathrm{T}}$, ${\mathbf{\beta }}_{\mathbf{HbO}}=[{\beta }_{\mathrm{HbO},0},{\beta }_{\mathrm{HbO},1},{\beta }_{\mathrm{HbO},2},{\beta }_{\mathrm{HbO},3}]$, ${\mathbf{\beta }}_{\mathbf{HbR}}=[{\beta }_{\mathrm{HbR},0},{\beta }_{\mathrm{HbR},1},{\beta }_{\mathrm{HbR},2},{\beta }_{\mathrm{HbR},3}]$, and ${\mathbf{\beta }}_{\mathbf{a}}=[{\beta }_{a,0},{\beta }_{a,1},{\beta }_{a,2},{\beta }_{a,3}]$. The symbol ${[]}^{\mathrm{T}}$ represents the transposition of a vector. The coefficients ${\beta }_{\mathrm{HbO},i},{\beta }_{\mathrm{HbR},i}$, and ${\beta }_{a,i}(i=\mathrm{0,1},\mathrm{2,3})$ are unknown and must be determined before analysis. We adopted MCS as the foundation to establish reliable values of ${\beta }_{\mathrm{HbO},i}$, ${\beta }_{\mathrm{HbR},i}$, and ${\beta }_{a,i}$. We used the MCS source code developed by Wang et al.,²⁷ in which the Henyey–Greenstein phase function is applied to the sampling of the scattering angle of photons. The simulation model consisted of a single layer representing cortical tissue. In a single simulation of diffuse reflectance at each wavelength, 5,000,000 photons were randomly launched. The absorption coefficients of oxygenated hemoglobin ${\mu }_{a,\mathrm{HbO}}$ and deoxygenated hemoglobin ${\mu }_{a,\mathrm{HbR}}$ were obtained from the values of ${\epsilon }_{\mathrm{HbO}}(\lambda )$ and ${\epsilon }_{\mathrm{HbR}}(\lambda )$ in the literature,²⁸ where the hemoglobin concentration of blood with a 44% hematocrit is $2326\mu \mathrm{M}$ of hemoglobin. The absorption coefficient ${\mu }_a$ converted from the concentrations $C_{\text{HbO}}$ and $C_{\text{HbR}}$ and the reduced scattering coefficient ${\mu }_{\mathrm{s}}^{\prime }$ specified by the coefficient $a$ were provided as inputs to the simulation, while the diffuse reflectance was produced as output. The input concentrations and coefficient $a$ and the output reflectance are helpful as the dataset in specifying the values of ${\beta }_{\mathrm{HbO},i}$, ${\beta }_{\mathrm{HbR},i}$, and ${\beta }_{a,i}$ statistically for determining the absolute values of $C_{\text{HbO}}$, $C_{\text{HbR}}$, and $a$. The five different values of 40,172, 60,258, 80,344, 100,430, and 120,516 were calculated by multiplying the typical value²⁶ of $a$ by 0.5, 0.75, 1.0, 1.25, and 1.5, respectively, and the reduced scattering coefficients ${\mu }_{\mathrm{s}}^{\prime }(\lambda )$ of the cortical tissue with the five different values were derived from the relation of Eq. (6). The sum of the absorption coefficients of HbO and HbR, ${\mu }_{a,\mathrm{HbO}}+{\mu }_{a,\mathrm{HbR}}({\mu }_{a,\mathrm{HbT}})$, for $C_{\text{HbT}}=4.65$, 23.3, 116, 233, and $465\mu \mathrm{M}$ was used as input to the cortical tissue in the simulation. Tissue oxygen saturation ${\mathrm{StO}}_2$ was determined by ${\mu }_{a,\mathrm{HbO}}/{\mu }_{a,\mathrm{HbT}}$ and values of 0, 20, 40, 60, 80, and 100% were used for simulation. In total, 150 diffuse reflectance spectra at $\lambda =520$, 540, 560, 580, and 600 nm were simulated under the various combinations of $C_{\text{HbO}}$, $C_{\text{HbR}}$, and $a$. MRA1 for each simulated spectrum based on Eq. (7) generated the 150 sets of vector $\alpha$ and $C_{\text{HbO}}$, $C_{\text{HbR}}$, and $a$. The coefficient vectors, also referred to as conversion vector, ${\mathbf{\beta }}_{\mathbf{HbO}}$, ${\mathbf{\beta }}_{\mathbf{HbR}}$, and ${\mathbf{\beta }}_{\mathbf{a}}$ were determined statistically by performing MRA2. Once ${\mathbf{\beta }}_{\mathbf{HbO}}$, ${\mathbf{\beta }}_{\mathbf{HbR}}$, and ${\mathbf{\beta }}_{\mathbf{a}}$ were obtained, $C_{\text{HbO}}$, $C_{\text{HbR}}$, and $a$ were calculated from ${\alpha }_{\mathrm{HbO}}$, ${\alpha }_{\mathrm{HbR}}$, and ${\alpha }_0$, which were derived from MRA1 for the measured reflectance spectrum, without the Monte Carlo simulation. Therefore, the spectrum of absorption coefficient ${\mu }_a(\lambda )$ and that of reduced scattering coefficient ${\mu }_{\mathrm{s}}^{\prime }(\lambda )$ was reconstructed by Eqs. (3) and (6), respectively, from the measured reflectance spectrum. The principle of the method was previously validated using tissue phantoms;²¹ the correlation between the given value of ${\mu }_{\mathrm{s}}^{\prime }$ and estimated value of ${\mu }_{\mathrm{s}}^{\prime }$ and that between the given value of ${\mu }_a$ and estimated value of ${\mu }_a$ were shown. We also compared the results of calculation of changes in $C_{\text{HbO}}$ and $C_{\text{HbR}}$ with those obtained by the conventional modified Lambert–Beer law analysis (see Sec. 5). As described above, since the light scattering signal can be used as an optical marker of CSD, we used maps of scattering amplitude a, which is proportional to ${\mu }_{\mathrm{s}}^{\prime }$, to visualize propagation of CSD in this study. To determine the propagation velocity of CSD, reflectance images at 800 nm were used since they are also sensitive to light scattering changes due to CSD.⁵

2.5.

Evaluation of Cortical Vessel Diameters

To assess changes in diameters of cortical arterioles and venules, we used reflectance images at 520 nm, which is an isosbestic point of HbO and HbR. For the cortical image, we placed a linear line that was perpendicularly across the centerline of the vessel of interest on LabVIEW software (see white and red lines on the top image in Fig. 5), creating an image intensity profile of the vessel. Vessel diameter was defined as the FWHM value of the profile.^29,30,31 Arterioles and venules were distinguished on the basis of their diameters, positions, orientations, and values of ${\mathrm{StO}}_2$ before LISW application. In addition, we carefully distinguished cortical surface arterioles from the vessels in the dura matter on the basis of their diameters; the diameter of cortical surface arterioles is about two to three times larger than that of dural vessels ($\sim 25\mu \mathrm{m}$) in their proximal portion in rats.²³

2.6.

Statistical Analysis

Statistical methods for data obtained in this study were selected on the basis of analysis of data normality and uniformity of variance using the Ekuseru–Toukei 2012 statistical software package (Social Survey Research Information Co., Ltd., Tokyo, Japan). With regard to changes in diameters of pial arterioles and venules, results were compared using the nonparametric Dunn’s multiple comparisons test. Changes in ${\mathrm{rC}}_{\mathrm{Hb}}$ and ${\mathrm{StO}}_2$ were tested by the parametric Dunnett’s multiple comparisons test or the nonparametric Dunn’s multiple comparisons test. Changes in OEF were tested by the parametric Tukey’s multiple comparisons test. The multiple comparison tests were performed using GraphPad Prism software (Prism 6 for Windows, GraphPad software, Inc., La Jolla, California); a difference was considered statistically significant when $p<0.05$. Data are presented as means ± standard deviation (SD) when the parametric method was used, and data are presented as medians with ranges (in box plots) when the nonparametric method was selected.

3.1.

Spatiotemporal Changes in Scattering Amplitude Associated with Cortical Spreading Depolarization

In our previous studies, we showed that light scattering signals can be used as an optical marker of CSD.⁵ Figure 3 shows difference images of scattering amplitude in the rat cortex after exposure to an LISW, where the image before LISW application is subtracted from the image at each timepoint (rat no. 3). After LISW application, a large venule showed an increase in scattering amplitude, and the region with increased scattering amplitude appeared in the parenchyma and in the pial arterioles in the right edge of the region of interest (RoI) (anterior side of the cortex) at 1 min 21 s after LISW application. The region with increased scattering amplitude in the parenchyma and arterioles was propagating like a wave to the left (posterior side), indicating a propagation of CSD. Such CSD-related scattering change was observed in all four rats examined, and only in one rat (rat no. 4), the second consecutive wave of CSD was seen at 5 min 45 s after the start of the first wave. The propagation velocity of the wavefront of the first CSD wave in the parenchyma was $2.96\pm 0.55\mathrm{mm}/\min$ ($\text{mean}\pm \mathrm{SD}$) ($n=4$), being consistent with the results of our previous study⁵ and other studies using KCl injection or pinprick to induce CSD.^15,16,32

Fig. 3

Difference images of scattering amplitude during CSD propagation for rat no. 3. Pixel sizes in the images are $455\times 418$; the imaging field of view corresponds to $\sim 2.6\times 2.4\mathrm{mm}$. Each time indicates the time after LISW application.

3.2.

Vasculature Morphological Changes

Figure 4 shows images at 520 nm, an isosbestic wavelength of HbO and HbR, of pial arterioles and venules before and after exposure to an LISW in the same rat, as that shown in Fig. 3 (rat no. 3). Vessel type-dependent morphological changes were seen. A pial arteriole marked “A” that is running in the anterograde direction to the CSD propagation direction showed almost no change in diameter just after LISW application but showed distinct vasodilatation during the CSD propagation and then recovered soon after the CSD propagation [Fig. 4(a)]. Such vasodilatation during CSD was not observed in the pial arteriole marked “B” that is running perpendicular to the CSD propagation direction [Fig. 4(b)]. Pial venules observed through the cranial window were all anatomically perpendicular to or retrograde to the CSD propagation direction, while their initial diameters were greatly different. Thus, we divided them into two groups: smaller-diameter (less than $80\mu \mathrm{m}$) pial venules such as that marked “C” and larger-diameter ($80\mu \mathrm{m}$ or more) pial venules such as that marked “D.” Both types of venules showed distinct constriction immediately after LISW application regardless of their orientations and distances from the site of LISW application [Figs. 4(c) and 4(d)]; smaller-diameter venule C appeared to be more prominently constricted. The constrictions in both types of venules tended to recover during CSD propagation, but both types of venules reconstricted after the CSD propagation.

Fig. 4

Representative reflectance images at 520 nm of four different regions before and after LISW application in rat no. 3. Region (a) shows a pial arteriole (A), which is anterograde to the direction of CSD propagation, with red arrows. Region (b) shows another pial arteriole (B), which is perpendicular to the direction of CSD propagation, with red arrows. Region (c) and region (d) show pial venules (C) and (D), respectively, with blue arrows; region (c) shows a relatively small venule and region (d) shows a large venule. Pixel sizes in the images (a-d) are $165\times 121$. Each bar in the image before LSIW application represents $100\mu \mathrm{m}$. Each position of (a) to (d) and direction of CSD propagation are shown at the top.

Figure 5 shows time courses of changes in diameters of each type of arteriole and venule for $\sim 60\min$ after LISW application (rat no. 3), for which we randomly selected three positions on each arteriole/venule shown in Fig. 4. The diameter of arteriole A slightly increased and then decreased by 10% to 15% over a period of 1 min after LISW application and then suddenly increased by a maximum of $\sim 80\%$ concomitantly with the propagation of CSD. Soon after CSD propagation, the diameter recovered and thereafter it remained at the baseline level or decreased by 5% to 30% until the end of the measurement. In arteriole B, the diameter was limitedly changed at two positions (B-1 and B-2) over a period of $\sim 50\min$ after LISW application [Fig. 5(b)]. At the other position (B-3), however, the diameter decreased by $\sim 20\%$ immediately after LISW application but transiently recovered. During CSD propagation, the diameter decreased again by $\sim 30\%$, after which the diameter gradually recovered to the baseline. In pial venules C and D, on the other hand, the diameters decreased by $\sim 45\%$ and $\sim 25\%$, respectively, over a period of $\sim 1\min$ soon after LISW application and then both increased almost concomitantly with the propagation of CSD over a period of $\sim 1\min$. Immediately after the CSD propagation, they decreased again by 20% to 30% from the baseline over a period of $\sim 1\min$ and then started to recover [Figs. 5(c) and 5)d)]. Interestingly, the diameters started to increase from the baseline at $\sim 7\min$ after LISW application and reached 120% to 155% at $\sim 20\min$; the smaller venules showed larger variations in their diameters. Thereafter, the diameters gradually decreased in smaller venule C but showed little changes in larger venule D.

Fig. 5

Time courses of changes in diameters of pial arterioles and venules after LISW application for rat no. 3; diameters were normalized by average diameters before LISW application. (a) Arteriole A anterograde to the direction of CSD propagation (A-1 to 3). (b) Arteriole B perpendicular to the direction of CSD propagation (B-1 to 3). (c) Venule C with a small diameter (C-1 to 3). (d) Venule D with a large diameter (D-1 to 3). Each position to measure the diameter is shown in the top image. Each graph on the left side is a magnified view of the bold-lined square of the graph on the right side. Arrowheads from $t_1$ to $t_5$ show timepoints for analysis in Figs. 6, 9, and 10.

On the basis of the time courses of vessel diameters shown in Fig. 5, six characteristic time points were determined: $t_0$, before LISW application; $t_1$, immediately after LISW application; $t_2$, during CSD propagation; $t_3$, immediately after CSD propagation; $t_4$, $\sim 20\min$ post-CSD; and $t_5$, $\sim 50\min$ post-CSD [see arrowheads at the top of Fig. 5(a)]. Figure 6 shows changes in diameters of pial arterioles and venules relative to the baseline diameters (values before LISW application, $t_0$) at these time points for all four rats examined. For this analysis, we randomly selected a total of 14 positions from 5 arterioles for type A (running anterograde to the CSD propagation direction), 8 positions from 3 arterioles for type B (running perpendicular to or retrograde to the CSD propagation direction), 17 positions from 8 venules for type C (with a diameter less than $80\mu \mathrm{m}$), and 10 positions from 7 venules for type D (with a diameter of $80\mu \mathrm{m}$ or more); all of the observable arterioles and venules were evaluated. The mean diameters before LISW application were $48\pm 4\mu \mathrm{m}$ ($n=14$, $\text{mean}\pm \mathrm{SEM}$) and $44\pm 5\mu \mathrm{m}$ ($n=8$) for type A and type B arterioles and $53\pm 4\mu \mathrm{m}$ ($n=17$) and $150\pm 13\mu \mathrm{m}$ ($n=12$) for type C and type D venules, respectively. Unique responses depending on the vascular type were seen in all rats examined. The diameter of type A arterioles showed a significant increase at $t_2$ and the percent increase was $59\pm 9\%$ ($\text{mean}\pm \mathrm{SEM}$). For type B arterioles, however, the diameter did not show a significant change at $t_2$ but was significantly decreased at $t_3$ ($-10\pm 3\%$). For both C and D types of venules, the diameters were significantly decreased at $t_1$ ($-22\pm 4\%$ and $-20\pm 2\%$, respectively). After the propagation of CSD, both C and D types of venules showed significant increases at $t_4$ ($34\pm 4\%$ and $25\pm 3\%$, respectively). Vascular responses to the second consecutive CSD, which were observed only for rat no. 4, were excluded from analysis in this study.

Fig. 6

Relative changes in diameters of pial arterioles and venules at five time points after LISW application from the baseline for four rats examined; description for each time point is shown at the bottom of the graphs. (a) Arteriole A anterograde to the direction of CSD propagation. (b) Arteriole B perpendicular to the direction of CSD propagation. (c) Venule C with a diameter less than $80\mu \mathrm{m}$. (d) Venule D with a diameter of $80\mu \mathrm{m}$ or more. The horizontal dashed line in each graph shows the baseline before LISW application. Numbers of positions for vessel diameter measurements in rat no. 1, no. 2, no. 3, and no. 4 are as follows: (a) arteriole A, 3, 6, 3, and 2; (b) arteriole B, 2, 0, 3, and 3; (c) venules C, 5, 4, 4, and 4; (d) venule D, 4, 2, 4, and 2, respectively. Data are presented as medians with ranges. Asterisks ($*p<0.05$, $**p<0.01$) indicate a statistically significant difference (Dunn’s multiple comparisons test).

3.3.

Regional Hemoglobin Concentration and Tissue Oxygen Saturation

Figure 7 shows false-color maps of the distributions of ${\mathrm{rC}}_{\mathrm{Hb}}$ and ${\mathrm{StO}}_2$ before and after LISW application (rat no. 3; see also Fig. 8 for ${\mathrm{StO}}_2$). The maps clearly show site-dependent variations in ${\mathrm{rC}}_{\mathrm{Hb}}$ and ${\mathrm{StO}}_2$ among the arterioles, venules, and parenchyma. Immediately after LISW application ($t=1$ min 11 s), both ${\mathrm{rC}}_{\mathrm{Hb}}$ and ${\mathrm{StO}}_2$ decreased in almost all regions; their decreases in the parenchyma were remarkable and pial venules showed distinct constriction. Thereafter, ${\mathrm{rC}}_{\mathrm{Hb}}$ and ${\mathrm{StO}}_2$ started to increase in the right side region ($t=1$ min 21 s), and the regions with increased ${\mathrm{rC}}_{\mathrm{Hb}}$ and ${\mathrm{StO}}_2$ were moving to the left side, corresponding to the propagation of CSD (until $t=2\min$ 29 s), indicating hyperemic responses to CSD (see Fig. 3). After the CSD propagation, ${\mathrm{rC}}_{\mathrm{Hb}}$ and ${\mathrm{StO}}_2$ started to drastically decrease ($t=3$ min 26 s). Thereafter, ${\mathrm{rC}}_{\mathrm{Hb}}$ tended to gradually recover but still continued to be lower than the values before LISW application in the parenchymal region ($t=66\min$ 37 s). On the other hand, ${\mathrm{StO}}_2$ remained very low in all regions; the values in the parenchyma and venules were especially low until $\sim 40\min$ post-CSD and those in the parenchyma at $\sim 1\mathrm{h}$ post-CSD were still lower than the values before LISW application.

Fig. 7

Maps of (a) regional hemoglobin concentration (${\mathrm{rC}}_{\mathrm{Hb}}$) and (b) tissue oxygen saturation (${\mathrm{StO}}_2$) before and after LISW application for rat no. 3. The bottom left images in (a) and (b) are corresponding reflectance images at 520 nm before LISW application, showing identification of arterioles, venules, and parenchyma as “a,” “v,” and “p,” respectively. Pixel sizes in the images are $455\times 418$.

Fig. 8

Tissue oxygen saturation (${\mathrm{StO}}_2$) map before and after LISW application for rat no. 3 corresponding to Fig. 7. Time (mm:ss) in the bottom right indicates the time after LISW application; minus means before LISW application [MPEG, 6.0 MB (URL: https://doi.org/10.1117/1.JBO.24.3.035005.1)].

Figure 9 shows changes in ${\mathrm{rC}}_{\mathrm{Hb}}$ and ${\mathrm{StO}}_2$ in the parenchyma, arterioles, and venules at the six characteristic time points shown in Figs. 5 and 6 for all four rats examined, for which a total of 68 RoIs, including 24 RoIs in the parenchyma, 20 RoIs in arterioles running anterograde to the CSD propagation direction, and 24 RoIs in venules, were sampled. Immediately after LISW application (at $t_1$), ${\mathrm{rC}}_{\mathrm{Hb}}$ was significantly decreased in all of the regions, by $\sim 8\%$ in the parenchyma, by $\sim 6\%$ in arterioles, and by $\sim 8\%$ in venules from the baseline levels. During CSD propagation (at $t_2$, light blue shadow), ${\mathrm{rC}}_{\mathrm{Hb}}$ was significantly increased in the parenchyma by $\sim 21\%$ and in arterioles by $\sim 22\%$, and then immediately after CSD propagation (at $t_3$), ${\mathrm{rC}}_{\mathrm{Hb}}$ was significantly decreased in the parenchyma, arterioles, and venules by $\sim 6\%$, by $\sim 4\%$, and by $\sim 8\%$, respectively. At $t_4$ and $t_5$ (more than $\sim 20\min$ post-CSD), while ${\mathrm{rC}}_{\mathrm{Hb}}$ in the arterioles at $t_5$ was significantly decreased, ${\mathrm{rC}}_{\mathrm{Hb}}$ had recovered to the baseline levels in the parenchyma and venules and did not change significantly. Corresponding to these ${\mathrm{rC}}_{\mathrm{Hb}}$ changes, ${\mathrm{StO}}_2$ also changed, but its behavior was more complex. At $t_1$, ${\mathrm{StO}}_2$ was significantly decreased in all of the regions by $\sim 29\%$ in the parenchyma, by $\sim 4\%$ in arterioles, and by $\sim 14\%$ in venules from the baseline levels, but at $t_2$, it was significantly increased in these regions by $\sim 64\%$, $\sim 14\%$, and $\sim 19\%$ from the baseline levels, respectively. At $t_3$, ${\mathrm{StO}}_2$ was significantly decreased by $\sim 46\%$ in the parenchyma, by $\sim 10\%$ in arterioles, and by $\sim 16\%$ in venules from the baseline levels. After the CSD propagation (at $t_4$ and $t_5$), ${\mathrm{rC}}_{\mathrm{Hb}}$ recovered to the baseline levels in almost all of the regions as described above, but ${\mathrm{StO}}_2$ still remained at significantly low levels, indicating a unique abnormal hemodynamic condition. Figure 9(g) shows arterial oxygen saturation (${\mathrm{SpO}}_2$) measured at the hindlimb simultaneously with ${\mathrm{rC}}_{\mathrm{Hb}}$ and ${\mathrm{StO}}_2$ for the cortex. It should be noted that even after LISW application, ${\mathrm{SpO}}_2$ showed no significant changes at any time point, suggesting that the above-mentioned drastic changes in ${\mathrm{rC}}_{\mathrm{Hb}}$ and ${\mathrm{StO}}_2$ were local phenomena in the brain.

Fig. 9

Regional hemoglobin concentration (${\mathrm{rC}}_{\mathrm{Hb}}$), tissue oxygen saturation (${\mathrm{StO}}_2$), and arterial oxygen saturation measured at the hindlimb before and after LISW application for four rats examined; description for each time point is shown at the bottom of the graphs. (a–c) ${\mathrm{rC}}_{\mathrm{Hb}}$ in the parenchyma (a), pial arterioles (b), and pial venules (c). (d–f) Corresponding ${\mathrm{StO}}_2$ in the parenchyma (d), pial arterioles (e), and pial venules (f). (g) Arterial oxygen saturation (${\mathrm{SpO}}_2$) simultaneously measured at the hindlimb. The horizontal dashed line in each graph shows the baseline before LISW application. Numbers of RoIs used for analysis in rat no. 1, no. 2, no. 3, and no. 4 are as follows: 6, 6, 6, and 6 for parenchyma; 4, 6, 4, and 6 for arterioles; 6, 6, 6, and 6 for venules, respectively. ${\mathrm{rC}}_{\mathrm{Hb}}$ levels in the parenchyma (a) are presented as medians with ranges; Dunn’s multiple comparisons test was performed. Other data are presented as means ± SD; Dunnett’s multiple comparisons test was performed. Asterisks ($*p<0.05$, $**p<0.01$, $****p<0.0001$) indicate a statistically significant difference.

On the basis of the data of the arteriolar and venular ${\mathrm{StO}}_2$ described above, we evaluated OEF by the equation:³³

Fig. 10

OEF at the same time points as in Figs. 6 and 9 after LISW application for four rats examined: $t_0$, before LISW application; $t_1$, immediately after LISW application; $t_2$, during CSD propagation (with blue shadow); $t_3$, immediately after CSD propagation; $t_4$, $\sim 20\min$ post-CSD; $t_5$, $\sim 50\min$ post-CSD. The horizontal dashed line shows the baseline before LISW application. Data are presented as $\text{means}\pm \mathrm{SD}$ ($n=4$); Tukey’s multiple comparisons test was performed. Asterisks ($**p<0.01$) indicate a statistically significant difference.