2.1.

Animals

Male BALB/C mice and $\mathrm{CX}3\mathrm{CR}{1}^{\mathrm{EGFP}+/-}$ (8 weeks, Wuhan University Animal Experiment Center) were housed in a specific pathogen-free environment at the Wuhan Optoelectronics National Experimental Center, Huazhong University of Science and Technology. Animal husbandry, care, and experimental procedures were conducted strictly in accordance with the Hubei Provincial Experimental Animal Regulations.

2.2.

Skull Optical Clearing Procedure

The switchable skull optical clearing window was established as reported earlier. Two solutions were used for the skull optical clearing procedure. Solution 1 (S1) was a saturated supernatant of ethanol (Sinopharm, China) and urea (Sinopharm, China) with a volume to mass ratio of 10:3 and an ethanol volume ratio of 75%. Solution 2 (S2) was a highly concentrated solution of SDBS. The key steps are as follows: (1) anesthetizing the mice with a cocktail of 2% $\alpha$-chloralose and 10% urethane ($8\mathrm{mL}/\mathrm{kg}$) via intraperitoneal injection; (2) cutting open mice scalps to expose the skull surface; (3) fixing holders to mice skulls with glue and then attaching them to custom plates; (4) adding S1 dropwise to the skull surface for about 10 min; (5) removing S1 and covering the skull with S2 for 5 min; (6) performing cortical imaging on the now transparent skull; (7) wiping off S2 after imaging and washing the skull with saline to return it to its original turbid state; and (8) removing the optical clearing agent and suturing the scalp, leaving the mice to recover from the anesthesia after 2 to 3 h. In the ex vivo experiments, the removed fresh skull was immersed in S1 for 10 min, wiped dry, and immersed in S2 for 5 min to prepare it for the optical clearing procedure. This description constitutes the entire procedure for skull optical clearing. The sham group was only anesthetized without scalp-opening operation and optical clearing treatment.

2.3.

Photothrombosis Establishment through the Skull Optical Clearing Window

We evaluated the possibility of combining the photothrombosis technique with the in vivo skull optical clearing procedure to establish a through-skull, degree controllable, targeted ischemic stroke in mice. As shown in Fig. 1(a), mice were intravenously injected with $100\mu \mathrm{L}$ of Rose Bengal ($25\mathrm{mg}/\mathrm{kg}$, Sigma-Aldrich, China) solution through the inner canthus after anesthesia before the establishment of the skull optical clearing window; therefore the subcarinal vessels were clearly visible. Cerebral blood flow distribution before stroke creation was determined using an LSCI system equipped with a He-Ne laser (632.8 nm, 3 mW), and the region of interest was irradiated with a 532-nm laser ($7\mathrm{mW}/{\mathrm{mm}}^2$, Changchun New Industry Optoelectronic Technology Co., Ltd., China) for 1, 2, or 5 min to induce cerebral infarction in the focused region at different degrees.

Fig. 1

Schematic diagram of the experimental procedure (a) and optical system (b). A pair of lenses was used to focalize the 532-nm laser onto the cortex.

2.4.

Laser Speckle Contrast Imaging of Cerebral Blood Flow

Change in cerebral blood flow velocity was monitored using a home-built LSCI system. As shown in Fig. 1(b), a He-Ne laser (632.8 nm, 3 mW) was passed through a collimated diffusion beam mirror and then uniformly irradiated to the target imaging area, and the resulting backscattered light was captured with a camera (Pixelfly, PCO GmBH©, Germany) after passing through a stereomicroscope (SZX7, Olympus, Japan) and recorded using a computer (40 frames, exposure time 20 ms). The original scattered images were processed through temporal contrast analysis to obtain blood flow distribution, which is expressed as^33,34

$1\times$ and $2.5\times$ magnifications were used to capture the bi-hemisphere and local region, respectively: the fields of view were $9\mathrm{mm}\times 6.7\mathrm{mm}$ and $3.6\mathrm{mm}\times 2.7\mathrm{mm}$, respectively.

2.5.

Immunofluorescence

The brain vasculature and neuronal nuclei were stained with Dylight 649 L.esculentum (tomato) lectin (LEL-Dylight649, DL-1178, Vector Laboratories) and an anti-NeuN antibody (Abcam, United Kingdom, ab177487), respectively. Mice were perfused with paraformaldehyde (PFA) 2 min after the intravenous injection of LEL-Dylight 649 ($0.1\mathrm{mg}/\mathrm{mL}$, $100\mu \mathrm{L}$), and their brains were removed, fixed in 4% PFA at 4°C overnight, embedded in 4% agarose (Sigma-Aldrich, St. Louis, Missouri, United States), and sliced coronally into $100\mu \mathrm{m}$-thick sections using a vibratome (Leica VT1000, Germany). These sections were subsequently washed three times (5 min each time) with PBS-Triton (PBST, 0.2% Triton X-100 in PBS), incubated in a blocking solution (5% normal goat serum/PBST) for 1 h, incubated with an anti-NeuN antibody in 5% normal goat serum/PBST overnight at 4°C and then at room temperature for 1 h, washed five times (5 min each time), incubated with 0.2% goat anti-rabbit-AF555 antibody (Invitrogen, United States, A-21428) for 5 h, washed five times (5 min each time), and imaged with a commercial confocal microscope (LSM710; Zeiss, Oberkochen, Germany).

2.6.

Infarct Volume Measurement

Anti-NeuN-stained brain sections were imaged with a confocal microscope, and the maximum projection was obtained for each brain section. Areas that remained unstained were considered infarcts. The infarct area $S_{\text{infarct}}$ of each anti-NeuN-stained coronal surface brain slice with a thickness of $h$ ($100\mu \mathrm{m}$) was measured with ImageJ (Bethesda, Maryland, United States). The infarct volume was obtained by determining the sum of the volumes of each infarcted brain slice, that is $V_{\text{infarct}}=\mathrm{SUM}(S_{\text{infarct}}\times h)$.

2.7.

Statistical Analysis

Data are expressed as the mean ± standard error and were analyzed using Statistical Product Service Solutions software (SPSS, IBM, United States). One-way analysis of variance (ANOVA) was used to compare more than two groups. Values were considered significant at $P<0.05$ (*$P<0.05$, **$P<0.01$, and ***$P<0.001$).

3.1.

Establishment of Cortical Photothrombosis through the Skull Optical Clearing Window

To assess the turbid skull-caused limitation of the photothrombotic model and the skull optical clearing-induced improvement, both in vivo and ex vivo experiments were performed. First, we passed focused light (the radius of the light spot was 0.5 mm) through a mouse skull sample and imaged the result with a charge-coupled device (CCD). As shown in Fig. 2(a), the energy of light passing through the optical clearing treated skull was highly concentrated than that through the original cloudy skull. Figure 2(b) depicts the examination of all the detected signals through the skull under different conditions. Quantitative analysis demonstrated that the distance from the central position to the site where intensity drops by half when the laser passed through the turbid skull was 0.58 mm; it was 0.52 mm after optical clearing treatment [Fig. 2(b)], suggesting that the optical clearing treatment widened the 0.5-mm radius spot 12% less as the light beam passed through the original skull. Figure 2(b) indicated that the total laser power through the optical clearing treated skull was five times stronger as it went through the original skull.

Fig. 2

Enhancement of light transmission after skull optical clearing treatment. (a) Representative normalized images of 532-nm laser spots through skull samples before and after optical clearing treatment. The color represents the recorded intensity, which was normalized against the highest intensity. (b) Light intensity distribution from the center in (a). (c) Representative in vivo photographs of a skull-covered brain’s subjection to the 532-nm laser irradiation. It was captured using the stereomicroscope under a bright field, with the 532-nm laser focused on the cortex. (d) LSCI-assisted CBF images after Rose Bengal injection and 532-nm laser irradiation for 5 min. The irradiation location was the same ($\mathrm{AP}=-2\mathrm{mm}$, $\mathrm{ML}=2\mathrm{mm}$) for each mouse. For the skull optical clearing group, irradiation was introduced after skull optical clearing window establishment, followed by LSCI detection. For the control group, irradiation was performed first, followed by skull optical clearing window establishment and LSCI detection. The color represents relative blood flow velocity. The red circles represent the laser irradiation area. Mean ± SD; $n=3$ per group.

In vivo experiments were used to evaluate the enhancement of skull optical clearing in establishing the photothrombotic model. As shown in Fig. 2(c), before skull optical clearing, we only observed the 532-nm light spot on the skull and were not aware of the cerebrovascular distribution under it. In contrast, the skull optical clearing window allowed for easy selection of areas or vessels to be occluded without opening the skull. As Fig. 2(d) shows, blood flow in the exposed regions decreased remarkably after the 532-nm laser irradiation through the skull optical clearing window, but the same irradiation dosage yielded no observable infarction through the original turbid skull, pointing to enhancement in the skull optical clearing window-induced photothrombotic efficacy.

We next selected four regions on the bi-hemispheres of the mice and established a targeted photothrombotic stroke, as shown in Fig. 3. The four regions correspond to the upper left, upper right, lower left, and lower right regions of the bregma [Fig. 3(b)]. The diameter of the light spot was 1 mm, and blood flow after stroke establishment with 5-min laser irradiation is demonstrated in Fig. 3(c). Blood flow in groups region of interest (ROI) 1, ROI 2, ROI 3, and ROI 4 decreased to $0.44\pm 0.07$, $0.49\pm 0.09$, $0.45\pm 0.08$, and $0.45\pm 0.03$, respectively, relative to baseline blood flow. Such results suggest that the turbid skull can limit the targeting ability and consistency of the photothrombotic model, whereas the skull optical clearing technique provides a craniotomy-free window for a stable model of ischemic strokes in regions of interest.

Fig. 3

Evaluation of blood flow changes in selected areas of interest in the large field of view of the cerebral cortex for stroke. (a) Four regions based on the relative position of bregma in the cerebral cortex. The diameter of the ROI is 1 mm. (b) Diagram of the four regions. (c) Changes in blood flow in the four regions after stroke. Mean ± SD; ns—$P>0.05$, $n=3$ per group. ROI, region of interest.

3.2.

Continuous Monitoring of CBF Post-Skull Optical Clearing Window-Induced Stroke

Using the skull optical clearing technique, we not only established a targeted photothrombotic model but also tracked the changes in CBF distribution after stroke. Figure 4(a) shows CBF levels after Rose Bengal injection and laser irradiation for 6 h. Blood flow changes in the irradiated region and in the vessels inside and outside the region were measured as in Fig. 4(b). CBF in the laser-irradiated area gradually decreased during the 6 h of irradiation, dropping to 50% at 2 h post-stroke and eventually to about 30%. Similarly, blood flow in the relatively larger vessels in the region (indicated as number 1) dropped to about 50% within 3 h after the stroke but remained constant for the next 3 h. The drop in relatively smaller vessels in the region (indicated as number 2) was more rapid, reaching 50% after 1.5 h and about 20% after 6 h post-stroke. On the contrary, blood flow in the vessels outside the irradiated region (indicated as number 3) showed a slow and small decrease after 6 h. In addition to the in vivo observation of blood flow changes in the superficial layer, an ex vivo evaluation of the vascular blockage in the deep cortex was performed 6 h post-stroke [Figs. 4(c) and 4(d)]. The results display no significant difference in vessel density between the two sides of the cerebral cortex ($0.12\pm 0.03$ versus $0.10\pm 0.01$) right after the 2-min laser irradiation. After 6 h, there was a significant infarction on the ipsilateral side, and the vessel density decreased to $0.01\pm 0.00$, while the density on the contralateral side remained $0.11\pm 0.03$. These results suggest that the extent of vascular damage after 2 min of irradiation is progressively amplified over 6 h.

Fig. 4

The 6-h observation after skull optical clearing window-assisted targeted photothrombosis. (a) Blood flow monitoring after the 2-min laser exposure. The diameter of the laser spot was 1 mm and is denoted by the green circle. Vessel-1 and vessel-2 represent relatively large and small vessels, respectively, within the laser-irradiated area, while vessel-3 is the vessel outside the area. (b) Changes in CBF in the green circle, vessel-1, vessel-2, and vessel-3 for 6 h after PT. CBF values were normalized against the baseline value (CBF value before PT). (c) Representative ex vivo coronal brain sections of mice (where the photothrombosis center was located) at 0 min and 6 h post-stroke, labeled with LEL-649 and imaged with a commercial confocal microscope (LSM710, Zeiss, Germany). (d) The density of blood vessels in the ischemic region. $1\mathrm{mm}\times 1\mathrm{mm}$ ROIs in the ischemic region and contralateral side were selected [white frames in (c)], where vasculature was binarized. Then, the ratio of vascular area to total ROI was calculated as the vascular density. Mean ± SD; ns—$P>0.05$, ***$P<0.001$; **$P<0.01$, $n=3$ per group. PT, photothrombosis.

3.3.

Long-Term Monitoring of Skull Optical Clearing Window-Assisted Photothrombotic Model at Different Light Doses

In addition to scrutinizing blood flow changes in a short period, we tracked blood flow for a long period of up to 14 days to understand the occurrence of injury after stroke. And the light time was adjusted as a control to obtain different degrees of damage to the vascular injury. As shown in Fig. 5(a), 5 min of laser irradiation without Rose Bengal injection did not affect blood flow, indicating that such a light dose itself was safe for animals. On the contrary, alongside the Rose Bengal injection, the lengthier the exposure to light, the more significant the post-stroke damage induced. To make this clearer, we examined blood flow changes in the 1-mm light spot region [green circles in Fig. 5(a)] and a circle of 2 mm diameter centered on the light spot [reds circle in Fig. 5(a)]. Figures 5(b) and 5(c) show that blood flow fluctuated above and below the baseline when irradiated for 5 min in the absence of Rose Bengal injection. However, combinedly administering Rose Bengal and irradiation at different exposure times resulted in the fall and then rise of blood flow during the 14 days of scrutiny, with blood flow in the light spot region recovering slowly. To be specific, there were no vascular flow signals within the red circles in the 5-min-irradiation group, whose relative baseline blood flow average was $0.00\pm 0.00$, on day 1 post-stroke. On the other hand, we noted significant flow in the blood vessels in the 2-min-irradiation group. Besides, blood flow signals outside distinguishable vessels were also higher than zero, and the mean value was $0.27\pm 0.12$. In the 1-min group, the mean blood flow value was $0.39\pm 0.05$. On day 4 post-stroke, CBF in the 5-min irradiation group was still $0.00\pm 0.00$, but CBF in the 2- and 1-min irradiation groups increased to $0.69\pm 0.20$ and $0.84\pm 0.16$, respectively: they were not significantly different from the control group values ($1.00\pm 0.06$, $p=0.11$ versus group 2-min, $p=0.27$ versus group 1-min). On day 7 post-stroke, CBF in the 5-min irradiation group increased to $0.38\pm 0.02$; CBF in the 2- and 1-min irradiation groups were $0.84\pm 0.15$ and $1.07\pm 0.13$, respectively. By day 14, CBF had recovered to $0.64\pm 0.14$ in the 5-min group, $1.00\pm 0.10$ in the 2-min group, and $0.94\pm 0.15$ in the 1-min group, with no significant difference between each injury group and the control group ($0.94\pm 0.06$). Such results indicate that mice experienced different degrees of damage after exposure to different light doses in the skull optical clearing window procedure, and the two correlated positively. Blood flow further spontaneously recovered over the next 2 weeks, with the recovery ability correlating negatively with the light dose.

Fig. 5

Cerebral blood flow over 14 days after photothrombosis establishment with various light doses. (a) Representative LSCI images of skull optical clearing window-engendered cortical blood flow for 14 days after photothrombosis. Green circles represent the positions of the 532-nm laser spots. (b) Changes in blood flow intensity with various light doses for 14 days in the green and red circles in (a). The values were normalized against the baseline. (c) CBF in the red circle in (a) on days 1, 4, 7, and 14 post-stroke at different irradiation doses. Mean ± SD; ns—$P>0.05$, ***$P<0.001$; **$P<0.01$, $n=3$ per group. RB, Rose Bengal; PT, photothrombosis.

To verify the aforementioned conclusions, we also counted the infarction volumes under different light doses (Fig. 6). The neurons were labeled with an anti-NeuN antibody and the vessels with LEL-649 (Fig. S1 in the Supplemental Material), and both were merged together. The results post-stroke day 1 with 5-, 2-, and 1-min laser irradiation are shown in Fig. 6(a), and those after 14 days of stroke are presented in Fig. 6(b). Blood flow in the LEL-649 channel recovered remarkably 14 days after stroke, which was consistent with in vivo findings (Fig. S1 in the Supplemental Material). Infarct volume statistics are based on neuronal staining. As shown in Figs. 6(c) and 6(d), the infarct volume in the 5-min irradiation group was the largest, reaching $15.29\pm 4.49{\mathrm{mm}}^3$ post-stroke day 1. The infarct volume in the 2-min irradiation group was $7.06\pm 1.23{\mathrm{mm}}^3$, only half of that of the 5-min irradiation group. The infarct volume in the 1-min irradiation group was the smallest, only $3.31\pm 1.50{\mathrm{mm}}^3$. After 14 days, the infarct volume in each group dropped considerably: $2.41\pm 0.36{\mathrm{mm}}^3$, $1.20\pm 0.50{\mathrm{mm}}^3$, and $0.58\pm 0.15{\mathrm{mm}}^3$ in the 5-, 2-, and 1-min irradiation groups, respectively.

Fig. 6

Infarct volumes on days 1 and 14 after photothrombosis establishment with various light doses. (a) Coronal sections of the brain on day 1 post-stroke under the 5-, 2-, and 1-min irradiation, all spaced $500\mu \mathrm{m}$ apart, where neurons were labeled with an anti-NeuN antibody. (b) Coronal sections of the brain on day 14 post-stroke under the 5-, 2-, and 1-min irradiation. The white arrows in (a) and (b) indicate stroke regions. (c) Infarct volumes 1 day after photothrombosis establishment. (d) Infarct volumes 14 days after photothrombosis establishment. Mean ± SD; ns—$P>0.05$, *$P<0.05$, $n=3$ per group.

Herein, the degree of injury in our stroke model is controllable: the longer the duration of exposure to light, the more severe the injury. CBF in mice post-stroke fell and then rose, and the infarct volume diminished, indicating that the mice recovered by themselves.