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Neuroscience

Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

Published: May 6th, 2021

DOI:

10.3791/62536

1Institute for Stroke and Dementia Research, LMU Munich, 2Munich Cluster for Systems Neurology (SyNergy)

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, 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.

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 ....

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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 libitu.......

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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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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.......

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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'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.

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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

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