Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

Summary

Described here is the photothrombotic stroke model, where a stroke is produced through the intact skull by inducing permanent microvascular occlusion using laser illumination after administration of a photosensitive dye.

Abstract

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, there remain limited therapeutic options for stroke patients. Therefore, more research is needed for pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Given the inability of in vitro models to reproduce the complexity of the brain, experimental stroke models are essential for the analysis and subsequent evaluation of novel drug targets for these mechanisms. In addition, detailed standardized models for all procedures are urgently needed to overcome the so-called replication crisis. As an effort within the ImmunoStroke research consortium, a standardized photothrombotic mouse model using an intraperitoneal injection of Rose Bengal and the illumination of the intact skull with a 561 nm laser is described. This model allows the performance of stroke in mice with allocation to any cortical region of the brain without invasive surgery; thus, enabling the study of stroke in various areas of the brain. In this video, the surgical methods of stroke induction in the photothrombotic model along with histological analysis are demonstrated.

Introduction

Ischemic stroke remains a principal cause of death and acquired adult disability in developed countries in the 21^st century accounting for approximately 2.7 million deaths in 2017 worldwide¹. Even with the immense efforts of the scientific community, few treatments are available. Furthermore, with such high exclusion criteria, these already limited options are not accessible to many patients, resulting in an urgent need for novel treatments to improve functional recovery after stroke.

Considering the incapability of in vitro models to replicate the complex interactions of the brain, animal models are essential for preclinical stroke research. Mice are the most frequently used animal model in the stroke research field. The majority of these mouse models aim to induce infarctions by blocking the blood flow within the middle cerebral artery (MCA) since the majority of human stroke lesions are located in the MCA territory². Although these models better recapitulate human stroke lesions, they involve convulated surgeries with high infarct volume variability.

Since Rosenblum and El-Sabban's proposal of the photothrombotic model in 1977³, and later the application of this model to rats Watson et al.⁴, it has become widely used in ischemic stroke research^5,6. The photothrombotic stroke model induces a local and defined cortical infarct as a result of the photoactivation of a light-sensitive dye previously injected into the blood flow. This causes local thrombosis of the vessels in the areas exposed to light. Briefly, upon exposure to light from the injected photosensitive dye, localized oxidative injury of the endothelial cell membrane is induced, leading to platelet aggregation and thrombus formation, followed by local disruption of cerebral blood flow⁷.

The principal advantage of this technique resides in its simplicity of execution and the possibility to direct the lesion to the desired region. Unlike other experimental stroke models, minor surgical expertise is needed to perform the photothrombotic stroke model as the lesion is induced through illumination of the intact skull. Moreover, the well-delimited borders (Figure 2A and Figure 5B) and the flexibility to induce the lesion to a specific brain region can facilitate the study of cellular responses within the ischemic or intact cortical area⁸. For these reasons, this approach is suitable for the study of cellular and molecular mechanisms of cortical plasticity.

Over the past few decades, the growing concern regarding the lack of reproducibility between research groups has been coined the so-called replication crisis⁹. After the coordination of the first preclinical randomized controlled multicenter trial study in 2015¹⁰, a proposed tool to improve preclinical research^11,12,13, it was confirmed that one cause for failing reproducibility between preclinical studies from independent laboratories was the lack of sufficient standardization of experimental stroke models and outcome parameters¹⁴. Accordingly, when the ImmunoStroke consortium was established (https://immunostroke.de/), a collaboration which aims to understand brain-immune interactions underlying the mechanistic principles of stroke recovery, the standardization of all the experimental stroke models among each research group was essential.

Described here is the standardized procedure for the induction of the photothrombotic model as used in the above-mentioned research consortium. Briefly, an animal underwent anesthetics, received a Rose Bengal injection (10 µL/g) intraperitonally, and the intact skull, 3 mm left from bregma, was immediately illuminated by a 561 nm laser for 20 min (Figure 1). Additionally, a related histological and behavioral method to analyze the stroke outcome in this model is reported. All methods are based on standard operating procedures developed and used in the laboratory.

Protocol

The experiments reported in this video were conducted according to the national guidelines for the use of experimental animals, and the protocols were approved by the German governmental committees (Regierung von Oberbayern, Munich, Germany). The mice used in this study were male C57Bl/6J mice, 10-12 weeks old, and dispatched by Charles River Germany. The animals were housed under controlled temperatures (22 °C ± 2 °C), with a 12 h light-dark cycle period and access to pelleted food and water ad libitum.

1. Preparation of the material and instruments

1. Dissolve Rose Bengal in 0.9% saline solution to reach a final concentration of 10 mg/mL. Connect the heat blanket to keep the operation area warm and maintain the mouse body temperature during anesthesia at 37 °C.
2. Prepare scissors, forceps, pieces of cotton, dexpanthenol eye ointment, and suture material. Prepare a syringe with saline solution (without needle) to maintain the operation area hydrated. Prepare the anesthesia gas (100% O₂ + isoflurane).

2. Preparation of the animal

1. Inject analgesia 30 min before surgery (4 mg/kg Carprofen and 0.1 mg/kg Buprenorphine).
2. Record the mouse body weight to adjust the dose of Rose Bengal to be injected (10 µL/g i.e., 100 µg/g).
3. Place the mouse in the induction chamber with an isoflurane flow rate of 4% to anesthetize it until the spontaneous movement of the body and vibrissae stops.
4. Transfer the mouse into the stereotactic frame and place it in a prone position with its nose into the anesthesia mask. Fix the animal and maintain the isoflurane concentration at 4% for 1 min. Then reduce and maintain the isoflurane concentration at 2%.
5. Gently insert the rectal probe to monitor the temperature throughout the surgical procedures. Set the associated feedback-controlled heating pad to maintain the mouse body temperature at 37 °C.
6. Apply dexpanthenol eye ointment to both eyes and clean the skin and surrounding fur with a disinfectant agent.

3. Photothrombosis model

1. Make a 2.0-2.5 cm longitudinal incision and retract to expose the skull. Perform the skull exposure with a single cut to avoid wound complications.
2. Remove the periosteum gently with cotton and identify the coronal sutures.
3. Put on the protective glasses, switch on the 561 nm laser and mark the bregma +3 mm left.
4. Switch off the laser, attach a sticker with a 4 mm diameter hole placed at the marked coordinates mentioned above.
5. Inject the mouse with Bengal Rose (10 µL/g), intraperitoneally. Place the laser beam at 4-5 cm from the skull, switch on the 561 nm laser and illuminate the skull for 20 min.
6. Apply two drops of 0.9% saline on the skull to rehydrate, suture the wound, and place the animal in a recovery chamber at 37 °C to recover from anesthesia. After 1 h, return the mice to their cages in a temperature-controlled room.
7. Inject analgesia every 12 h for 3 days after surgery (4 mg/kg Carprofen and 0.1 mg/kg Buprenorphine).

4. Sham operation

1. Carry out two different procedures of Sham operations as described in steps 4.1.1 and 4.1.2.
   1. Perform all the procedures identically to the operation described above. Inject Rose Bengal without switching on the laser. After 20 min under anesthesia, allow the animals to stay in the recovery chamber for 1 h to recover, before being returned to their cages.
   2. Perform all the procedures identically to the operation described above, switching on the laser. Do not inject Rose Bengal. After 20 min of laser illumination, allow the animals to stay in the recovery chamber for 1 h to recover from anesthesia, before being returned to their cages.

5. Laser speckle

1. Connect the heated blanket to keep the operation area warm and maintain the mouse body temperature during anesthesia at 37 °C.
2. Place the mouse into the induction chamber with an isoflurane flow rate of 4% to anesthetize it until the spontaneous movement of the body and vibrissae stops and then transfer the mouse into the stereotactic frame.
3. Place the mouse in a prone position with its nose into the anesthesia mask. Fix the animal and maintain the isoflurane concentration at 4% for 1 min. T hen reduce and maintain it at 2%.
4. Gently insert the rectal probe to monitor the temperature throughout the surgical procedures. Set the associated feedback-controlled heating pad to maintain the mouse body temperature at 37 °C and apply dexpanthenol eye ointment to both eyes. Clean the skin and the surrounding fur with a disinfectant agent.
5. Make a 2.0-2.5 cm longitudinal incision and retract to expose the skull. Perform the skull exposure with a single cut to avoid wound complications.
6. Place the sterotactic frame under the laser speckle and adjust the height to obtain a sharp image. Focus the laser speckle perfusion imaging (LSI) camera on the cranial window. Configure the high resolution laser speckle imaging (LSI) camera system as previously described¹⁵.
7. Acquire data from a 1 cm x 1 cm field of view using a 785 nm wavelength and 80 mW lasers with a frame rate of 21 images/s at a working distance of 1 cm for 1 min.
8. After imaging, apply two drops of 0.9% saline to the skull to rehydrate, suture the wound and place the animal in a recovery chamber at 37 °C to recover from anesthesia for 1 h. After 1 h, return the mice to their cages in a temperature-controlled room.

6. Neuroscore

NOTE: For the neurological deficit analysis, a modified neurological scale published by Eckenstein et al. in 1997 is used¹⁵.

1. Score the animals for general (Table 1) and focal deficits (Table 2). This composite scale ranges from 0 (no deficits) to 46 (severe impairments).
2. Perform the neuroscore at the same time each day and use surgical clothes to keep a neutral smell.
3. Habituate the mice for 30 min in the room with an open cage prior to the testing and allow them to observe each item for 30 s.

7. Perfusion

1. Prepare a 20 mL syringe containing PBS-heparin (2 U/mL) and place it 1 m above the bench to facilitate/ensure gravity-driven perfusion.
2. Inject intraperitoneally 100 µL of ketamine and xylazine (120/16 mg/kg body weight, respectively). Wait for 5 min and corroborate the cessation of spontaneous body movement and vibrissae.
3. Fix the animal in a supine position and disinfect the abdominal body surface with 100% ethanol. Make a 3 cm long incision in the abdomen; cut the diaphragm and the ribs to completely visualize the heart.
4. Make a small incision in the right atrium and insert the perfusion cannula into the left ventricle and perfuse with 20 mL PBS-heparin.
5. After perfusion, decapitate the animal and remove the brain, freeze it using dry ice and store them at -80 °C until further use.

8. Infarct volumetry

1. Cryosectioning: Cut the brain serially on a cryostat to 20 µm thick sections every 120 µm and mount on slides. Store the slides at -80 °C until further processing.
2. Cresyl violet (CV) staining
   1. To prepare the staining solution, mix 0.5 g of CV acetate in 500 mL of H₂O. Stir and heat (60 °C) until the crystals are dissolved. Allow the solution to cool and store it in a dark bottle. Reheat to 60 °C and filter (paper filter) before every use.
   2. Dry the slides at room temperature for 30 min. Place them in 95% ethanol for 15 min, followed by 70% ethanol for 1 min, and afterwards in 50% ethanol for 1 min.
   3. Place the slides in distilled water for 2 min, refresh the distilled water, and place the slides in water again for 1 min. Then, place the slides in the pre-heated staining solution for 10 min at 60 °C. Wash the slides twice in distilled water for 1 min.
   4. Place the slides in 95% ethanol for 2 min. Then place them into 100% ethanol for 5 min, refresh the 100% ethanol and place the slides in 100% ethanol again for 2 min. Afterwards, cover the slides with a mounting medium.
   5. Analysis: Scan the slides and analyze the indirect infarct volume by the Swanson method¹⁶ to correct for edema: Ischemic area = (ischemic region)-((ipsilateral hemisphere) - (contralateral hemisphere)).

9. Tunel staining (in situ apoptosis detection kit)

1. Dry the slides, post-fix in 4% paraformaldehyde in PBS (ph 7.4) for 10-20 min at RT. Wash in PBS, post-fix in precooled ethanol: acetic acid 2:1 for 5 min at -20 °C.
2. Wash in PBS and apply equilibration buffer (10 s to a maximum of 60 min at RT) and apply working strength TdT enzyme (1 h at 37 °C in humidified chamber)
3. Apply working strength stop/wash enzyme (10 min at RT), wash in PBS and apply warmed (RT) working strength anti-digoxigenin conjugate (30 min at RT in dark)
4. Wash in PBS, incubate with DAPI for 5 min at RT and mount the slides with fluoromount media.

Results

The model described here is a photothrombotic stroke model by Rose Bengal injection and intact skull illumination for 20 min, at a constant 561 nm wavelength and 25 mW output power at the fiber. Although the complete photothrombotic surgery lasts 30 min, the animal is kept under low anesthesia and the brain damage is moderate. Approximately 10 min after transfer to their cages, all the animals were awake, freely moving in the cage, and interacting with littermates.

Infarct volumetry was perfor...

Discussion

The presented protocol describes the experimental stroke model of photothrombosis by illuminating the intact skull with a 561 nm laser, with a previous intraperitoneal injection of Rose Bengal. Until recently, the use of this model has been low but is steadily increasing.

Mortality during stroke induction in this model is absent. The overall mortality of less than 5% arises during operation due to anesthesiological complications or sacrifice after meeting the exclusion criteria. To warrant the...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
561 nm wavelenght laser	Solna	Cobolt HS-03
Acetic Acid	Sigma Life Science	695092
Anesthesia system for isoflurane	Drager
ApopTag Peroxidase In Situ Apoptosis Detection Kit	Millipore	S7100
Bepanthen pomade	Bayer	1578681
C57Bl/6J mice	Charles River	000664
Collimeter	Thorlabs	F240APC-A
Cotons	NOBA Verbondmitel Danz	974116
Cresyl violet	Sigma Life Science	C5042-10G
Cryostat	Thermo Scientific CryoStarNX70
Ethanol 70%	CLN Chemikalien Laborbedorf	521005
Ethanol 96%	CLN Chemikalien Laborbedorf	522078
Ethanol 99%	CLN Chemikalien Laborbedorf	ETO-5000-99-1
Filter paper	Macherey-Nagel	432018
Fine Scissors	FST	15000-00
Forceps	FST	11616-15
Heating blanket	FHC DC Temperature Controller	40-90-8D
Isoflurane	Abbot	B506
Isopentane	Fluka	59070
Ketamine	Inresa Arzneimittel GmbH
Laser Speckle	Perimed	PeriCam PSI HR
Mayor Scissors	FST	1410-15
Phosphate Buffered Saline PH: 7.4	Apotheke Innestadt Uni Munchen	P32799
Protective glasses	Laser 2000	NIR-ZS2-38
Rose Bengal	Sigma Aldrich	198250-5G
Roti-Histokit mounting medium	Roth	6638.1
Saline solution	Braun	131321
Stereomikroskop	Zeiss	Stemi DV4
Stereotactic frame	Stoelting	51500U
Superfrost Plus Slides	Thermo Scientific	J1800AMNZ
Xylacine	Albrecht