Name: modeling stroke in mice: focal cortical lesions by photothrombosis
Uploaded: 2021-05-06T14:00:00
Description: Watch this Scientific Journal Video about Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis at JoVE.com

We thank all our collaboration partners of the Immunostroke Consortia (FOR 2879, From immune cells to stroke recovery) for suggestions and discussions. This work was funded by the Deutsche Forschungsgemeinschaft (DFG,German Research Foundation) under Germany&#39;s Excellence Strategy within the framework of the Munich Cluster for Systems Neurology (EXC 2145 SyNergy - ID 390857198) and under the grants LI-2534/6-1, LI-2534/7-1 and LL-112/1-1.

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 &#176;C &#177; 2 &#176;C), with a 12 h light-dark cycle period and access to pelleted food and water ad libitu...

<ol>
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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...

Ischemic stroke remains a principal cause of death and acquired adult disability in developed countries in the 21st 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&#39;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 research5,6. 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 research11,12,13, 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 &#181;L/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.

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	<tbody>
		<tr>
			<td>561 nm wavelenght laser</td>
			<td>Solna</td>
			<td>Cobolt HS-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Sigma Life Science</td>
			<td>695092</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia system for isoflurane</td>
			<td>Drager</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ApopTag Peroxidase In Situ Apoptosis Detection Kit</td>
			<td>Millipore</td>
			<td>S7100</td>
			<td></td>
		</tr>
		<tr>
			<td>Bepanthen pomade</td>
			<td>Bayer</td>
			<td>1578681</td>
			<td></td>
		</tr>
		<tr>
			<td>C57Bl/6J mice</td>
			<td>Charles River</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Collimeter</td>
			<td>Thorlabs</td>
			<td>F240APC-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotons</td>
			<td>NOBA Verbondmitel Danz</td>
			<td>974116</td>
			<td></td>
		</tr>
		<tr>
			<td>Cresyl violet</td>
			<td>Sigma Life Science</td>
			<td>C5042-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Thermo Scientific CryoStarNX70</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 70%</td>
			<td>CLN Chemikalien Laborbedorf</td>
			<td>521005</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96%</td>
			<td>CLN Chemikalien Laborbedorf</td>
			<td>522078</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 99%</td>
			<td>CLN Chemikalien Laborbedorf</td>
			<td>ETO-5000-99-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Macherey-Nagel</td>
			<td>432018</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>FST</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>FST</td>
			<td>11616-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating blanket</td>
			<td>FHC DC Temperature Controller</td>
			<td>&#160;40-90-8D</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Abbot</td>
			<td>B506</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopentane</td>
			<td>Fluka</td>
			<td>59070</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Inresa Arzneimittel GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Speckle</td>
			<td>Perimed</td>
			<td>PeriCam PSI HR</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayor Scissors</td>
			<td>FST</td>
			<td>1410-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline PH: 7.4</td>
			<td>Apotheke Innestadt Uni Munchen</td>
			<td>P32799</td>
			<td></td>
		</tr>
		<tr>
			<td>Protective glasses</td>
			<td>Laser 2000</td>
			<td>NIR-ZS2-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Rose Bengal</td>
			<td>Sigma Aldrich</td>
			<td>198250-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Roti-Histokit mounting medium</td>
			<td>Roth</td>
			<td>6638.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline solution</td>
			<td>Braun</td>
			<td>131321</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomikroskop</td>
			<td>Zeiss</td>
			<td>Stemi DV4</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotactic frame</td>
			<td>Stoelting</td>
			<td>51500U</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Slides</td>
			<td>Thermo Scientific</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylacine</td>
			<td>Albrecht</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling stroke in mice: focal cortical lesions by photothrombosis

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&#160;histological analysis are demonstrated.

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...

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Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

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.

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, ...

The photothrombosis stroke model approach may by suitable for cellular and molecular studies of cortical plasticity because of the limited borders and flexibility to induce injury in different areas of the cortex. Three principle advantages distinguish the photothrombosis model from other stroke models, the possibility to direct the lesion to the desired region, it's high reproducibility, and low mortality. Proper rose bengal dosing is critical for the success and for the stability of the model.Results depend on technical specification like laser power, cross application timing, and optical shilling of the unaffected cortex. Begin by dissolving rose bengal in 0.9%saline solution for a final concentration of 10 milligrams per milliliter. Meticulously sterilize all instruments using a hot lead sterilizer and disinfect all surfaces before and after surgery with a microbial disinfectant spray.Prepare a syringe filled with saline solution to maintain the operation area hydrated and prepare the anesthesia gas. Measure the body weight of the mouse to adjust the dose of rose bengal to be injected. Set the associated feedback controlled heating pad to maintain the mouse body temperature.Once the mouse is completely anesthetized and fixed in the stereotactic frame, gently insert the rectal probe to monitor the body temperature of the mouse throughout the surgical procedure. Apply the Dex panthenol eye ointment to both eyes and clean the skin and the surrounding fur with disinfecting agent. Use scissors to make a single two to two and a half centimeter longitudinal incision and retract to expose the skull.Use cotton to gently remove the periosteum and locate the coronal sutures. Wear protective glasses while switching on the 561 nanometer laser and mark the plus three millimeters to the left, then switch off the laser and hook the sticker with four millimeter diameter hole in the marked coordinates. Inject the mouse intraparietal with rose bengal.Place the laser beam at a four to five centimeter distance from the skull. Switch on the laser and illuminate the skull for 20 minutes. Rehydrate the skull by applying two drops of 0.9%saline.After suturing the wound, place the mouse in a recovery chamber at 37 degrees Celsius to recover from anesthesia. After one hour, place the mouse back in the cage in a temperature controlled room. The crystal violet stain to serial coronal brain sections were used for the infarct volume entry analysis after 24 hours of the stroke induction.The variability of this stroke model was low, and the mean infarct volume was 29.3 cubic millimeters, representing 23%of the one brain hemisphere. The phototrombosis lesion caused a moderate and longterm sensory motor impairment indicated by the composite neuro score. Stroke animals had a significant change in the neuro score 24 hours after the surgery.Even though the differences persisted, the stroke mice improved over time. In the mice subjected to the rose bengal plus illumination, the body weight and body temperature decreased 24 hours after the surgery. However, within three days after the surgery, recovery was observed.Ischemic changes were confirmed using laser imaging. The results indicated that the rose bengal or laser illumination alone did not produce a lesion while the simultaneous application of both generated a round hypoperfused area surrounded by the narrow oligomeric zone. The crystal violet and tunnel staining for the assessment of infarct volume 24 hours after surgery revealed no tissue damage either in the rose bengal or laser illumination surgeries.On the other hand, the rose bengal plus laser illumination generated a well demarcated lesion. It's important to measure the body weight of the animal to adjust the rose bengal injection. The correct condition of coroner switchers is important to induce a consistent lesion between animals.Since this protocol leaves the skull intact and can even be performed through cranial windows, it can be combined with wide field or multi photon in vivo imaging.

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