# Long term direct visualization of the photothrombotic cortical infarction through the intact skull of anesthetized mice

Juliette Leclerc, Théotime Briar, Caroline Derouck, Cheima Mortier, Célia Duclos, Karelle Bénardais, Eric Verin, Jean-Paul Marie, Julien Chuquet

<figure><img src="image_1.png" /></figure>

## ► To cite this version:

Juliette Leclerc, Théotime Briar, Caroline Derouck, Cheima Mortier, Célia Duclos, et al. Long term direct visualization of the photothrombotic cortical infarction through the intact skull of anesthetized mice. Journal of Neuroscience Methods, 2026, 428, pp.110683. 10.1016/j.jneumeth.2026.110683. hal-05496947

HAL Id: hal-05496947

https://hal.science/hal-05496947v1

Submitted on 6 Feb 2026

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
# Long term direct visualization of the photothrombotic cortical infarction through the intact skull of anesthetized mice

Juliette Leclerc* , Théotime Briar, Caroline Derouck, Cheima Mortier, Célia Duclos, Karelle Bénardais, Eric Verin, Jean-Paul Marie, Julien Chuquet

Univ Rouen Normandie, Normandie Univ, GRHVN UR 3830, F-76000 Rouen, France

## ARTICLE INFO

**Keywords:**
Stroke
Ischemic lesion
Brain infarction
Photothrombotic
Rose bengal

## ABSTRACT

**Background:** To assess post-stroke functional recovery and develop new treatments, numerous preclinical models have been developed, including the photothrombotic stroke model. This reproducible and simple method induces a targeted ischemic lesion in a chosen cortical area following intravenous rose bengal injection and controlled illumination with a 532 nm laser. However, identifying the infarct's location and extent *in vivo* requires sophisticated, time-consuming, and/or expensive tools such as MRI or advanced optical imaging techniques. Thus, we introduce here a simple and low-tech method.

**New method:** Our direct method takes advantage of the long-lasting fluorescence of rose bengal remaining in the damaged cortex and detectable through the intact skull using the same 532 nm laser.

**Result:** At the lesion site, we observed an emission spot glowing through the skull for several weeks after stroke induction. *Ex vivo* immunohistochemical analysis showed that rose bengal fluorescence remains confined to the lesion, precisely delineating the infarct's boundaries.

**Comparison with existing methods:** This technique simplifies lesion localization and guides subsequent *in vivo* investigations, such as probe implantation, optogenetic fiber placement, or targeted tissue sampling in the perilesional cortex, where neuroplasticity and repair processes occur.

## 1. Introduction

Preclinical research on stroke remains crucial as this pathology still lacks effective neuroprotective or neurorepair treatments despite recent progress in neurorehabilitation (Stinear et al., 2020; Dawson et al., 2021). Numerous rodent models of stroke have been developed over the past 40 years, each exhibiting varying degrees of homology with the clinical situation. First established in 1985 (Watson et al., 1985), the photothrombotic model has gained considerable popularity in recent years, now accumulating close to 100 publications per year. Briefly, the most common version of this model consists of using a green laser or focused white light source illuminating the cerebral cortex for a few minutes through the exposed skull bone of a mouse that has received a prior systemic injection of the photosensitive dye rose bengal (4,5,6, 7-tetrachloro-2',4',5',7'-tetraiodofluorescein). This leads to a thrombosis of the cortical vessels localized under the laser beam trajectory (Labat-gest and Tomasi, 2013; Llovera et al., 2021; Knezic et al., 2022) and the development of a focal ischemic infarction. Control experiments

in the literature have robustly demonstrated that neither laser illumination alone (up to 150 mW, 10 min, 532 nm) nor rose bengal injection alone is sufficient to induce a lesion or any measurable neuronal damage, confirming that the photothrombotic effect is strictly dependent on the interaction of the photosensitizer and the light source (Shabani et al., 2023). The major advantage of this approach is that lesion localisation does not depend on vascular arborisation, enabling the precise induction of a targeted ischemic lesion in a chosen cortical area (e.g., the forelimb motor cortex). Surgically, this model is therefore relatively simple to implement, requiring only minimal invasive procedures since the skull bone remains intact. Nevertheless, the occlusion is permanent, thus it does not allow for simulating the clinical pathophysiological complexity, for example, by incorporating the effects of reperfusion (but see Conti et al., 2021). Nonetheless, the model is highly favored by laboratories focusing on neurorepair and post-stroke neurorehabilitation, since it permits the study of functional recovery related to the targeted cortical region (Bandet and Winship, 2024; Knezic et al., 2022; Zeiger et al., 2021). As with all stroke models, identifying the

<footnote>* Corresponding author.
E-mail address: juliette.leclerc12@univ-rouen.fr (J. Leclerc).</footnote>
location and extent of the infarct *in vivo* while keeping the mouse alive for further exploration requires the use of sophisticated, time consuming and/or expensive tools such as MRI or advanced optical imaging techniques like wide-field fluorescence microscopy (Conti et al., 2021; Bandet and Winship, 2024).

Besides its use as a photosensitive dye for stroke research, rose bengal has found applications in medicine and biology, for example in ophthalmology, cancer treatment and photoablation (Díaz et al., 2011; Kurosu et al., 2022; Murube, 2014; Patil et al., 2018). Rose bengal belongs to the xanthene family of dyes, such as the widely used fluorescein. It is therefore a fluorescent compound with an excitation peak at 559 nm and an emission peak at 571 nm. However, this property has never been exploited in the context of its use for stroke research. Here we report that the fluorescence signal of rose bengal remains stable and strictly localized into the necrotic area for several weeks following the induction of photothrombotic stroke. The fluorescence can be easily detected through the intact skull, allowing for the precise 2D localization and assessment of the infarct extent at the cortical surface, in order to guide further investigations, for instance probe or electrode positioning at controlled distances.

# 2. Material and methods

## 2.1. Animals and approvals

Eight to twelve-week-old (20–25 g) male and female C57BL/6 J mice were purchased from Janvier Laboratories. Experiments, approved by the Ethics Committee for Animal Research of Normandy (European Commission identifier: NTS-FR-536122) were conducted by authorized investigators in accordance with the recommendations of the European Communities 86/609/EEC. All procedures were undertaken, and reporting was done in accordance with the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines.

## 2.2. Photothrombotic stroke model

Anesthesia was induced with 5 % isoflurane delivered by a 2 L/min air flow in an induction chamber until the complete sedation of the animal. The mouse was then placed prone in a stereotaxic frame (RWD Life Science) in front of a face mask diffusing isoflurane (2 ± 0.5 %, 0.8 L/min). Body temperature was maintained at 37.5 ± 0.5°C and the eyes were protected with ophthalmic gel (Lubrital). General analgesia was provided by subcutaneous injection of Buprenorphine (0.1 mg/kg). The scalp was then shaved, and local asepsis was performed twice with a solution of Betadine and alcohol. After local analgesia with EMLA® (Lidocaine 2.5 % + Prilocaine 2.5 %) and loss of the paw retraction reflex, the skin was incised over 1.5 cm to expose the skull.

The periosteum was delicately scratched off and a piece of opaque black tape presenting a 1 mm pinhole was placed according to the stereotaxic coordinates of the right caudal forelimb area (AP: 0; ML: 1.5 mm). After application of eye ointment, a green laser (532 nm, 3 mm in diameter, 50 mW, Besram Technology Inc) was positioned 1 cm above the skull, in vertical alignment with the pinhole. Rose bengal (Sigma-Aldrich 330000; 3.75 mg/mouse diluted in 250 µL of saline) was administered intraperitoneally, then anesthesia was adjusted to 1.5 % isoflurane. Five minutes after injection, the animal's skull was illuminated by laser for ten minutes, before the pinholed tape was removed. The wound was then sutured (6/0 silk thread) and sanitized, and the animal was placed in a heated chamber until it woke up. Buprenorphine (0.1 mg/kg, sc.) was administered subcutaneously daily for the following 3 days.

## 2.3. Trans-skull visualization of the lesion and image acquisition

Sedation and analgesia were induced as described above. The mouse was then placed prone in a stereotaxic frame (RWD Life Science) in front

of a face mask diffusing isoflurane (2 ± 0.5 %, 0.8 L/min). The scalp was then shaved, and local asepsis was performed as detailed above.

After the periosteum was opened, a larger green laser (532 nm, 2 cm in diameter, 50 mW, Besram Technology Inc) was used to illuminate the mouse exposed skull around the lesion. Using red protective glasses (filtering between 200 and 540 nm), the rose bengal emission peaking at 572 nm could be observed without being overlapped by its excitation laser (532 nm). A regular digital reflex camera (Sony SLT-A65V; SAL1855 lens) was used to image the cortex illuminated with the laser (in order to see rose bengal fluorescence) and with regular white light.

## 2.4. Infarct surface location determination

Just after trans-skull visualization of the lesion, mice were euthanized with an intracardiac injection of Euthoxin (100 mg/kg). Rapidly after extraction, whole brains were stained by submerging them in a 2,3,5-Triphenyltetrazolium chloride (TTC) solution [13.3 mg/mL, diluted in phosphate-buffered saline (PBS)]. For comparison between rose bengal and TTC staining, pictures were taken under white light and then overlaid with the fluorescent one.

## 2.5. Immunofluorescent labeling and image acquisition

Anesthesia was induced with 5 % isoflurane delivered by a 2 L/min air flow in an induction chamber until the complete sedation of the animal. Fifteen minutes after general analgesia by Buprenorphine (s.c. 0.1 mg/kg), Euthoxin (100 mg/kg) was injected intraperitoneally. The animal was then perfused transcardially with saline solution (50 mL) followed by 4 % paraformaldehyde (PFA) in 1X PBS (50 mL). Whole brains were extracted, fixed in 4 % PFA for 24 h and cryoprotected in 20 % sucrose solution before being soaked in isopentane and stored at -80°C.

Using a cryostat (Leica CM1860), 20 µm-thick slices were taken from each tenth section and mounted on slides. Samples were rinsed in PBS and incubated for 15 min in 4 % PFA before washing in PBS with Tween. Immunohistochemical labeling was then performed with a mix of primary antibodies including rat anti-glial fibrillary acidic protein (GFAP; 1:250; Thermo Fischer 13-0300) and rabbit anti-NeuN (1:200, Novius Bio NBP1-7768AF350) in blocking solution (0.3 % Triton-X, 3 % donkey serum and 5 % Bovine Serum Albumin in 1X PBS) in a humid chamber for 12 h. Sections were rinsed twice in PBS and 2 times in PBS-T and incubated with the corresponding fluorophore-coupled secondary antibodies (Fluorescein Isothiocyanate (FITC; 1:500; Interchim 712-545-150) and Cyanin 5 (Cy5; 1:500; Interchim 711-605-152) for 1 h, then rinsed again in PBS and PBS-T. Finally, the slides were mounted with mowiol and dried.

Immunohistochemical sections were acquired using a widefield fluorescence microscope (Leica Thunder Imager), and deconvolution was systematically applied to reduce background noise. While fluorescent secondary antibodies were imaged using filters specifically designed for their coupled fluorophores, rose bengal was imaged with Cyanin 3 (Cy3) filters (532-558 nm excitation filter; 570-640 nm emission filter).

## 2.6. Image analysis

All collected images were processed with the FIJI program and CLIP STUDIO Paint. Windowing/leveling and luminosity were adapted for each fluorophore and conserved for every condition. For transcranial and whole-brain rose bengal imaging, a thresholding, established from the pre-injection image, was used to eliminate the autofluorescence signal of the brain.

# 3. Results

After intraperitoneal injection of rose bengal, targeted illumination
of the mouse skull for 10 min with a 532 nm laser through a 1 mm pin-hole induced a localized and permanent photothrombosis of the caudal forelimb area (Fig. 1A). Several days later, the mouse was anesthetized, and its skull was reilluminated with a wider 532 nm laser for a few seconds to capture a digital image of the entire exposed skull through a red filter (band-stop 200–540 nm) (Fig. 1B). Three (Fig. 1D) and seven

days (Fig. 1E) after ischemia induction, a focal fluorescent spot (≈ 571 nm) was revealed at the presumed lesion site, which was not seen beforehand (Fig. 1C). To find out whether the observed fluorescence actually corresponds to the lesion, the mouse brain was then extracted and stained with TTC to reveal the infarction (unstained white zone). Illuminated with the laser, the cortical fluorescent spot visible at 3 days

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Induction and visualization of the focal ischemic lesion through the intact mouse skull. (A) Photothrombosis induction: five minutes after intraperitoneal injection of rose bengal, the skull is illuminated for 10 min with a 50 mW laser (λ<sub>ex</sub> = 532 nm) focused on the right caudal forelimb area to induce its permanent thrombosis (B) Fluorescent detection of the cortical lesion. Later (days/weeks), the mouse skull is illuminated with a larger 50 mW laser (λ<sub>ex</sub>= 532 nm) (left) and imaged through a red band-stop filter (λ<sub>filt</sub> = 200–540 nm) revealing a fluorescent spot (peak λ<sub>em</sub>= 571 nm) which corresponds to the lesion (right). (C-E) Fluorescence visualization through the skull before and after focal thrombosis induction. Skull of an anesthetized mouse illuminated with white light (top) or the green laser (λ = 532 nm, 50 mW) (bottom), captured prior to ischemia induction (C, pre injection), and at 3 days (D3) and 7 days (D7) following ischemia induction (D and E). The sagittal, coronal, and frontonasal skull sutures are drawn. Laser illumination reveals a shiny spot at 3 days that coincides with the lesion site and decreases in size by day 7, also depicted by dashed lines in the top pictures. (F-J) Colocalization and matching area of the rose bengal fluorescent signal and the lesion. Ex-vivo extracted brain with photothrombotic lesion visualized under white light in F and with the laser (λ = 532 nm, 50 mW) in G, compared to subsequent TTC staining in H. A coronal section of the same sample further confirms the similar 3D extent of the TTC staining in I and the fluorescent rose bengal staining in J. (K) Long-term detection of the fluorescent lesion: 50 days post-ischemia induction (D50), laser illumination still reveals the lesion (extracted, PFA-fixed brain).</figcaption></figure>
exactly colocalized the TTC-unstained cortex (Fig. 1F-G-H). A coronal section of the same sample further confirmed the similar 3D extent of the fluorescent signal and the TTC negative signal (Fig. 1I-J). The fluorescence remained detectable on the cortical surface at least until 50 days after stroke (Fig. 1K).

We assumed that the observed fluorescence might originate from the fluorescent rose bengal itself at the lesion site. To test this hypothesis, we used immunolabeling of surviving neurons (NeuN⁺) and reactive astrocytes (GFAP⁺) to delineate the lesion boundaries. Seven days after ischemia induction, fluorescent rose bengal was strongly accumulated within the lesion area, confirming our prediction (Fig. 2A-B-C). Rose bengal fluorescence signal remained localized at the lesion site on the long term (50 days after ischemia induction) (Fig. 2D-E).

## 4. Discussion

The method reported here presents for the first time a very simple, low-cost and low-carbon way to highlight the location and precise extent of a cortical photothrombotic ischemic lesion in mice. The originality lies in the possibility of visualizing the cortical infarct through the skull of an anesthetized mouse, several days or even weeks after the photo-thrombotic procedure.

The modeling of stroke using rose bengal through photoreaction exploits the photosensitivity property of this molecule but does not make use of its fluorescence property. We discovered that this fluorescence remains stable enough and powerful over time to be detectable through the relatively translucent mouse skull, whereas its applicability to rats, whose skull and dura mater is thicker, remains to be tested (Liu et al.,

2017; Wiersma and Winship, 2018). Moreover, the fluorescence remains confined to the limits of the necrotic tissue. Rose bengal can be uptaken in blood flow when injected intraperitoneally and doesn't appear to cross the blood barrier, likely due to its binding with proteins in the blood (Alarcon et al., 2017). Its detection within necrotic tissue may be due to the breakdown of the blood-brain barrier caused by the rose bengal photochemical reaction (Knezic et al., 2022). Specifically, the immunohistochemistry analyses show that rose bengal is highly concentrated in compartments compatible with the size of cells that are neither neurons nor astrocytes. Given their high density within the necrotic mass, we suggest that these are macrophages internalizing rose bengal, although further studies are needed to confirm their identity. It is therefore primarily the extravascular fluorescence of rose bengal that enables the transcranial imaging of the lesion. The strong enrichment of rose bengal within the necrotic lesion creates a sharp contrast, clearly delineating the lesion's boundary. We also confirmed that this fluorescence is indeed due to the presence of rose bengal and not to an acquired autofluorescence of the necrotic tissue. Of note, to prevent misinterpretation of immunohistofluorescence studies, it has to be kept in mind that rose bengal shares the same range of emission and excitation wavelengths than commonly used fluorophore-coupled antibodies (e.g. Cy3, Alexa Fluor 532–661).

State of the art methods for non-invasively mapping the lesion area *in vivo* use sophisticated imaging tools: MRI is the tool of choice for longitudinally tracking the evolution of lesion volume and other parameters such as edema with high spatial resolution (Freret et al., 2009; Marteau et al., 2013). However, most laboratories can't afford or easily use MRI for *in vivo* anatomopathology of the mouse brain. Clearly, our

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Accumulation of rose bengal within the boundaries of the necrotic cortex. (A-C) Immunohistology of the cortex 7 days after photothrombotic ischemia, showing the fluorescence signal of rose bengal (magenta in A) alone, then with NeuN immunoreactivity labeling surviving neurons (cyan in B), and GFAP immunoreactivity labeling of reactive astrocytes (green) surrounding the lesion in C. Left: entire cortex, center: close-up of the lesion, right: close-up of the lesion border (white dashed lines). (D-E) Rose bengal fluorescence signal and GFAP immunoreactivity of the damaged cortex 50 days after photothrombotic ischemia.</figcaption></figure>
method does not replace such an approach: the *in vivo* mapping achievable with rose bengal precisely marks the pial surface of the lesion, but not its 3D volume. Furthermore, our method is limited to visualizing the part of the lesion located under the parietal skull, while the lateral part of the lesion, as observed after photothrombotic occlusion of the middle cerebral artery, requires additional surgical exposure. Thus, this method does not substitute for other methods, invasive or non-invasive, aimed at quantifying the lesion volume and its evolution. Interestingly, small photothrombotic lesions (1 mm) are not detectable at 14 days post-stroke with most used MRI (Summers et al., 2017), while infra-millimeter lesions are still visible at 50 days with rose bengal fluorescence detection.

It is therefore a sensitive, practical and very easy alternative for locating the lesion and, conversely, the surviving peripheral cortex, known as the perilesional cortex. The perilesional cortex is a focus of great interest in stroke research, to study the plasticity mechanisms involved in the neurorepair phase that underpins the relearning of lost functions (Krakauer and Carmichael, 2017; Campos et al., 2023; Bandet and Winship, 2024; Alasoadura et al., 2024). Numerous studies focus on the neuronal, vascular, and glial activity of this cortex, which represents a fertile territory spanning a few millimeters around the lesion. Our visualization approach thus allows targeting a precise position relative to the lesion for implanting an electrode, a probe, a pipette, or an optical fiber for optogenetics.

The present imaging technique can't be done through the skin. Longitudinal imaging is therefore clearly limited: reopening the sutured scalp for repeated imaging must respect the strict technical and ethical standards. Interesting developments remain to be tested and expand the applications of rose bengal fluorescence. For example, injecting this rose bengal ahead of brain lesion induction to specifically label lesions not caused by photoreaction, such as permanent middle cerebral artery occlusion (MCAO model) or traumatic lesions of the cerebral cortex.

### CRediT authorship contribution statement

**Julien Chuquet:** Writing – review & editing, Writing – original draft, Supervision, Project administration, Funding acquisition. **Juliette Leclerc:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization. **Caroline Derouck:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis. **Théotime Briar:** Writing – review & editing, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. **Célia Duclos:** Writing – review & editing, Investigation. **Mortier Cheïma:** Writing – review & editing, Investigation. **Eric Verin:** Writing – review & editing, Funding acquisition. **Karelle Bénardais:** Writing – review & editing, Visualization, Formal analysis. **Jean-Paul Marie:** Writing – review & editing, Resources, Funding acquisition.

### Funding

This work was supported by the University of Rouen Normandy, the Fondation AVC sous égide de la Fondation pour la Recherche Médicale et ses partenaires (AVC20230600558) to JC and La Fondation des "Gueules Cassées" (45–2024) to JL.

### Declaration of Competing Interest

The authors declare no competing financial interests.

### Acknowledgements

Immunohistofluorescence images were acquired at Plateforme de Recherche en Imagerie Cellulaire de Normandie (PRIMACEN). During the preparation of this work the authors used ChatGPT in order to assist

with the readability and language. After using this tool/service, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

### Data availability

Data will be made available on request.

### References

Alarcon, E.I., Poblete, H., Roh, H., Couture, J.-F., Comer, J., Kochevar, I.E., 2017. Rose bengal binding to collagen and tissue photobonding. ACS Omega 2, 6646–6657.

Alasoadura, M., Leclerc, J., Hazime, M., Leprince, J., Vaudry, D., Chuquet, J., 2024. The Excessive tonic inhibition of the peri-infarct cortex depresses low gamma rhythm power during poststroke recovery. J. Neurosci. 44.

Bandet, M.V., Winship, I.R., 2024. Aberrant cortical activity, functional connectivity, and neural assembly architecture after photothrombotic stroke in mice. eLife 12, RP90080.

Campos, B., Choi, H., DeMarco, A.T., Seydell-Greenwald, A., Hussain, S.J., Joy, M.T., Turkeltaub, P.E., Zeiger, W., 2023. Rethinking remapping: circuit mechanisms of recovery after stroke. J. Neurosci. 43, 7489–7500.

Conti, E., Piccardi, B., Sodero, A., Tudisco, L., Lombardo, I., Fainardi, E., Nencini, P., Sarti, C., Allegra Mascaro, A.L., Baldereschi, M., 2021. Translational stroke research review: using the mouse to model human futile recanalization and reperfusion injury in ischemic brain tissue. Cells 10, 3308.

Dawson, J., Liu, C.Y., Francisco, G.E., Cramer, S.C., Wolf, S.L., Dixit, A., Alexander, J., Ali, R., Brown, B.L., Feng, W., DeMark, L., Hochberg, L.R., Kautz, S.A., Majid, A., O'Dell, M.W., Pierce, D., Prudente, C.N., Redgrave, J., Turner, D.L., Engineer, N.D., Kimberley, T.J., 2021. Vagus nerve stimulation paired with rehabilitation for upper limb motor function after ischaemic stroke (VNS-REHAB): a randomised, blinded, pivotal, device trial. Lancet 397, 1545–1553.

Díaz, R., Casanova, A., Ariza, J., Moriyón, I., 2011. The rose bengal test in human brucellosis: a neglected test for the diagnosis of a neglected disease. PLOS Negl. Trop. Dis. 5, e950.

Freret, T., Bouet, V., Leconte, C., Roussel, S., Chazalviel, L., Divoux, D., Schumann-Bard, P., Boulouard, M., 2009. Behavioral deficits after distal focal cerebral ischemia in mice: usefulness of adhesive removal test. Behav. Neurosci. 123, 224–230.

Knezic, A., Broughton, B.R.S., Widdop, R.E., McCarthy, C.A., 2022. Optimising the photothrombotic model of stroke in the C57BI/6 and FVB/N strains of mouse. Sci. Rep. 12, 7598.

Krakauer, J.W., Carmichael, S.T., 2017. Broken Movement. The Neurobiology of Motor Recovery after Stroke. MIT Press.

Kurosu, M., Mitachi, K., Yang, J., Pershing, E.V., Horowitz, B.D., Wachter, E.A., Lacey, John W., Ji, I.I.I., Rodrigues, Y., D.J., 2022. Antibacterial activity of pharmaceutical-grade rose bengal: an application of a synthetic dye in antibacterial therapies. Molecules 27, 322.

Labat-gest, V., Tomasi, S., 2013. Photothrombotic ischemia: a minimally invasive and reproducible photochemical cortical lesion model for mouse stroke studies. J. Vis. Exp. 50370.

Liu, N.-W., Ke, C.-C., Zhao, Y., Chen, Y.-A., Chan, K.-C., Tan, D.T.-W., Lee, J.-S., Chen, Y.-Y., Hsu, T.-W., Hsieh, Y.-J., Chang, C.-W., Yang, B.-H., Huang, W.-S., Liu, R.-S., 2017. Evolutional characterization of photochemically induced stroke in rats: a multimodality imaging and molecular biological study. Transl. Stroke Res. 8, 244–256.

Llovera, G., Pinkham, K., Liesz, A., 2021. Modeling stroke in mice: focal cortical lesions by photothrombosis. J. Vis. Exp., e62536

Marteau, L., Valable, S., Divoux, D., Roussel, S.A., Touzani, O., MacKenzie, E.T., Bernaudin, M., Petit, E., 2013. Angiopoietin-2 is vasoprotective in the acute phase of cerebral ischemia. J. Cereb. Blood Flow. Metab. 33, 389–395.

Murube, J., 2014. Rose bengal: the second most commonly used surfocular vital stain. Ocul. Surf. 12, 14–22.

Patil, N., Truong, V., Holmberg, M.H., Lavoie, N.S., McCoy, M.R., Dutton, J.R., Holmberg, E.G., Parr, A.M., 2018. Safety and efficacy of rose bengal derivatives for glial scar ablation in chronic spinal cord injury. J. Neurotrauma 35, 1745–1754.

Shabani, Z., Farhoudi, M., Rahbarghazi, R., Karimipour, M., Mehrad, H., 2023. Cellular, histological, and behavioral pathological alterations associated with the mouse model of photothrombotic ischemic stroke. J. Chem. Neuroanat. 130, 102261.

Stinear, C.M., Lang, C.E., Zeiler, S., Byblow, W.D., 2020. Advances and challenges in stroke rehabilitation. Lancet Neurol. 19, 348–360.

Summers, P.M., Hartmann, D.A., Hui, E.S., Nie, X., Deardorff, R.L., McKinnon, E.T., Helpern, J.A., Jensen, J.H., Shih, A.Y., 2017. Functional deficits induced by cortical microinfarcts. J. Cereb. Blood Flow. Metab. 37, 3599–3614.

Watson, B.D., Dietrich, W.D., Busto, R., Wachtel, M.S., Ginsberg, M.D., 1985. Induction of reproducible brain infarction by photochemically initiated thrombosis. Ann. Neurol. 17, 497–504.

Wiersma, A.M., Winship, I.R., 2018. Induction of photothrombotic stroke in the sensorimotor cortex of rats and preparation of tissue for analysis of stroke volume and topographical cortical localization of ischemic infarct. Biol. Protoc. 8, e2861.

Zeiger, W.A., Marosi, M., Saggi, S., Noble, N., Samad, I., Portera-Cailliau, C., 2021. Barrel cortex plasticity after photothrombotic stroke involves potentiating responses of pre-existing circuits but not functional remapping to new circuits. Nat. Commun. 12, 3972.