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Medicine

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

Published: June 9th, 2013

DOI:

10.3791/50370

1Department of Neuroscience, University of Turin , 2Neuroscience Institute of Turin (NIT), University of Turin , 3Neuroscience Institute Cavalieri-Ottolenghi (NICO), University of Turin , 4Department of Anatomy, Pharmacology and Forensic Medicine, University of Turin

Photothrombosis is a quick, minimally-invasive technique for inducing small and well-delimited infarction in areas of interest in highly reproducible manner. It is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected light-sensitive dye. Following illumination, the dye is activated and produces singlet oxygen that damages components of endothelial cell membranes, with subsequent platelet aggregation and thrombi formation, which eventually determines the interruption of local blood flow. This approach, initially proposed by Rosenblum and El-Sabban in 1977, was later improved by Watson in 1985 in rat brain and set the basis of the current model. Also, the increased availability of transgenic mouse lines further contributed to raise the interest on the photothrombosis model. Briefly, a photosensitive dye (Rose Bengal) is injected intraperitoneally and enters the blood stream. When illuminated by a cold light source, the dye becomes activated and induces endothelial damage with platelet activation and thrombosis, resulting in local blood flow interruption. The light source can be applied on the intact skull with no need of craniotomy, which allows targeting of any cortical area of interest in a reproducible and non-invasive way. The mouse is then sutured and allowed to wake up. The evaluation of ischemic damage can be quickly accomplished by triphenyl-tetrazolium chloride or cresyl violet staining. This technique produces infarction of small size and well-delimited boundaries, which is highly advantageous for precise cell characterization or functional studies. Furthermore, it is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

At the beginning of the 21th century, ischemic stroke is a devastating disorder that represents the second cause of long term disability1 and the second cause of mortality worldwide, in which stroke accounted for approximately 5,7 million deaths in 20042. In spite of the many efforts that were put in, there is still no effective treatment available to improve functional recovery after stroke. Animal models of stroke are widely used in the field of stroke research as they allow modeling of the pathophysiology of ischemic damage and test the efficacy of different neuroprotective strategies in vivo. Most of these models aim to induce extens....

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1. Pre-surgery

  1. Weigh Rose Bengal in an 1.5 ml tube and dissolve in sterile saline solution until reaching a final concentration of 15 mg/ml. Filter sterilize through a 0.2 μm filter and store it in the dark at room temperature up to two months.
  2. Sterilize all surgical instruments by autoclaving. The surgical area should be sanitized less than one hour before initiating the surgery.
  3. Record the mouse body weight to adjust the dose of Rose Bengal to be injected. We injected 10 μ.......

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This protocol will produce a cortical lesion that is already visible upon dissection of the cortex to the unaided eye (Figures 1A-1C). The photothrombotic lesion develops in superficial and deep cortical layers in which the tissue is sufficiently translucent to allow photo-activation of the Rose Bengal. Measurement of the extent of cerebral infarction can be performed quickly by histological staining with triphenyl-tetrazolium chloride (TTC) on fresh tissue or by cresyl violet after fixation in 4% parafo.......

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Modifications and substitutions

Because of its absorption peak at 562 nm, a green light laser from a filtered xenon arc lamp was originally chosen to irradiate the photosensitive Rose Bengal. Although laser-mediated excitation was still used recently5, it can be replaced by cold light lamp that also ensure dye excitation10,15. Cold light optic fibers are easier to manipulate and less expensive than laser sources. However, it should be noticed that lasers are commonly used to target ind.......

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We thank Annalisa Buffo for insightful suggestions and comments, and Maurizio Grassano, Marina Boido and Ermira Pajaj for the shooting. This work was funded by FP7-MC-214003-2 (Marie Curie Initial Training Network AXREGEN) and the Compagnia di San Paolo, gliarep project.

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Name Company Catalog Number Comments
MATERIAL NAME COMPANY CATALOGUE NUMBER
Solutions and chemicals
Rose Bengal Sigma, Italy 330000
Isoflurane Vet Merial 103120022
Betadine Asta Medica
Paraformaldehyde Sigma-Aldrich 158127
Surgical material and equipment
Fluosorber Filter Havard apparatus 340415
150W fiber optic illuminator Photonic PL3000
Temperature Controller for Plate TCAT-2DF Havard apparatus 727561
Stereotaxic Instrument Stoelting 51950
Operating microscope Takagi OM8
Heating pad
Oxygen and nitrogen gas
Surgery Tools World precision instrument Optic fiber taps and mask are custom-made

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