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.

1. Pre-surgery 
<ol>
 <li> 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 &mu;m filter and store it in the dark at room temperature up to two months. 
 </li>
 <li>Sterilize all surgical instruments by autoclaving. The surgical area should be sanitized less than one hour before initiating the surgery.</li>
 <li>Record the mouse body weight to adjust the dose of Rose Bengal to be injected. We injected 10 &mu;...

<ol>
	<li>Lopez, A. D., Mathers, C. D., Ezzati, M., Jamison, D. T., Murray, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+and+regional+burden+of+disease+and+risk+factors.">Global and regional burden of disease and risk factors.</a> Lancet. 367, 1747-1757 (2001).</li><li>Mathers, C. D., Boerma, T., Ma Fat, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+and+regional+causes+of+death.">Global and regional causes of death.</a> Br. Med. Bull. 92, 7-32 (2009).</li><li>Rosenblum, W. I., El-Sabban, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+aggregation+in+the+cerebral+microcirculation:+effect+of+aspirin+and+other+agents.">Platelet aggregation in the cerebral microcirculation: effect of aspirin and other agents.</a> Circ. Res. 40, 320-328 (1977).</li><li>Watson, B. D., Dietrich, W. D., Busto, R., Wachtel, M. S., Ginsberg, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+reproducible+brain+infarction+by+photochemically+initiated+thrombosis.">Induction of reproducible brain infarction by photochemically initiated thrombosis.</a> Ann. Neurol. 17, 497-504 (1985).</li><li>Bergeron, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducing+photochemical+cortical+lesions+in+rat+brain.">Inducing photochemical cortical lesions in rat brain.</a> Curr. Protoc. Neurosci. Chapter 9, Unit 9 16 (2003).</li><li>Lee, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochemically+induced+cerebral+ischemia+in+a+mouse+model.">Photochemically induced cerebral ischemia in a mouse model.</a> Surg. Neurol. 67, 620-625 (2007).</li><li>Dietrich, W. D., Watson, B. D., Busto, R., Ginsberg, M. D., Bethea, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochemically+induced+cerebral+infarction.+I.+Early+microvascular+alterations.">Photochemically induced cerebral infarction. I. Early microvascular alterations.</a> Acta Neuropathol. 72, 315-325 (1987).</li><li>Schroeter, M., Jander, S., Stoll, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+induction+of+focal+cerebral+ischemia+in+mice+by+photothrombosis+of+cortical+microvessels:+characterization+of+inflammatory+responses.">Non-invasive induction of focal cerebral ischemia in mice by photothrombosis of cortical microvessels: characterization of inflammatory responses.</a> J. Neurosci. Methods. 117, 43-49 (2002).</li><li>Kitagawa, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+ischemia+after+bilateral+carotid+artery+occlusion+and+intraluminal+suture+occlusion+in+mice:+evaluation+of+the+patency+of+the+posterior+communicating+artery.">Cerebral ischemia after bilateral carotid artery occlusion and intraluminal suture occlusion in mice: evaluation of the patency of the posterior communicating artery.</a> J. Cereb. Blood Flow Metab. 18, 570-579 (1998).</li><li>Sigler, A., Goroshkov, A., Murphy, T. 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Korean Neurosurg. Soc. 46, 479-483 (2009).</li><li>Silva, V. M., Corson, N., Elder, A., Oberdorster, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rat+ear+vein+model+for+investigating+in+vivo+thrombogenicity+of+ultrafine+particles+(UFP).">The rat ear vein model for investigating in vivo thrombogenicity of ultrafine particles (UFP).</a> Toxicol. Sci. 85, 983-989 (2005).</li><li>Watson, B. D., Prado, R., Dietrich, W. D., Ginsberg, M. D., Green, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochemically+induced+spinal+cord+injury+in+the+rat.">Photochemically induced spinal cord injury in the rat.</a> Brain Res. 367, 296-300 (1986).</li><li>Van Reempts, J., Van Deuren, B., Van de Ven, M., Cornelissen, F., Borgers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flunarizine+reduces+cerebral+infarct+size+after+photochemically+induced+thrombosis+in+spontaneously+hypertensive+rats.">Flunarizine reduces cerebral infarct size after photochemically induced thrombosis in spontaneously hypertensive rats.</a> Stroke. 18, 1113-1119 (1987).</li><li>Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+focal+stroke:+size,+mechanism,+and+purpose.">Rodent models of focal stroke: size, mechanism, and purpose.</a> NeuroRx. 2, 396-409 (2005).</li><li>Kleinschnitz, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+of+platelets+or+intrinsic+coagulation+pathway-driven+thrombosis+does+not+prevent+cerebral+infarctions+induced+by+photothrombosis.">Blocking of platelets or intrinsic coagulation pathway-driven thrombosis does not prevent cerebral infarctions induced by photothrombosis.</a> Stroke. 39, 1262-1268 (2008).</li><li>Porritt, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photothrombosis-induced+infarction+of+the+mouse+cerebral+cortex+is+not+affected+by+the+Nrf2-activator+sulforaphane.">Photothrombosis-induced infarction of the mouse cerebral cortex is not affected by the Nrf2-activator sulforaphane.</a> PLoS One. 7, e41090 (2012).</li><li>Baskin, Y. K., Dietrich, W. D., Green, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+effective+behavioral+tasks+for+evaluating+sensorimotor+dysfunction+following+traumatic+brain+injury+in+mice.">Two effective behavioral tasks for evaluating sensorimotor dysfunction following traumatic brain injury in mice.</a> J. Neurosci Methods. 129, 87-93 (2003).</li><li>Markgraf, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+histopathologic+consequences+of+photothrombotic+occlusion+of+the+distal+middle+cerebral+artery+in+Sprague-Dawley+and+Wistar+rats.">Comparative histopathologic consequences of photothrombotic occlusion of the distal middle cerebral artery in Sprague-Dawley and Wistar rats.</a> Stroke. 24, 286-292 (1993).</li><li>Wester, P., Watson, B. D., Prado, R., Dietrich, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photothrombotic+'ring'+model+of+rat+stroke-in-evolution+displaying+putative+penumbral+inversion.">A photothrombotic 'ring' model of rat stroke-in-evolution displaying putative penumbral inversion.</a> Stroke. 26, 444-450 (1995).</li><li>Hu, X., Wester, P., Brannstrom, T., Watson, B. D., Gu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+and+reproducible+focal+cortical+ischemia+with+or+without+late+spontaneous+reperfusion+generated+by+a+ring-shaped,+laser-driven+photothrombotic+lesion+in+rats.">Progressive and reproducible focal cortical ischemia with or without late spontaneous reperfusion generated by a ring-shaped, laser-driven photothrombotic lesion in rats.</a> Brain Res. Brain Res. Protoc. 7, 76-85 (2001).</li><li>Maxwell, K. A., Dyck, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+reproducible+focal+ischemic+lesions+in+neonatal+mice+by+photothrombosis.">Induction of reproducible focal ischemic lesions in neonatal mice by photothrombosis.</a> Dev. 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No conflicts of interest declared.

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...

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 extensive infarctions by interrupting (temporarily or permanently) the blood flow within the middle cerebral artery, whereas other models were developed to study lesions of small size in specific areas, typically the motor and somatosensory cortices. However, several factors may contribute to generate a certain degree of variability in experimental stroke studies, including the mouse strain used, the age and sex of animals included in the study and, above all, the technique adopted to induce the ischemic damage. With regard to the latter point, the duration and invasiveness of the surgery (i.e. the need for a craniotomy) as well as the surgical skill required to the operator to reliably induce an ischemic lesion are critical determinants for a successful and unbiased in vivo stroke study. 
The concept of photothrombosis was initially proposed by Rosenblum and El-Sabban in 19773 and became renowned by its application in rat brain by Watson et al. in 19854 in which the technique was largely improved and set the basis of the current model3-6. The photothrombotic approach aims to induce a cortical infarction through the photo-activation of a light-sensitive dye previously delivered into blood system, which results in local vessel thrombosis in the areas exposed to the light. When the circulating dye is illuminated at the appropriate wavelength by a cold light source, it releases energy to oxygen molecules, which in turn generate a large amount of highly reactive singlet oxygen products. These oxygen intermediates induce endothelial cell membrane peroxidation, leading to platelet adhesion and aggregation, and eventually to the formation of thrombi which determine local cerebral flow interruption7.
Photothrombosis is a non-canonical ischemic model that does not occlude or break only one artery as it usually happens in human stroke, but induces lesions in more superficial vessels, which results in selective interruption of blood flow in the areas exposed to light. For this reason, this approach may be suitable for cellular and molecular studies of cortical plasticity. The principal advantage of this technique resides in its simplicity of execution. Moreover, photothrombosis can be easily performed in approximately forty minutes per animal, including twenty-minute wait (3 min for anesthesia; 1 min to shave the scalp; 3 to 5 min to place the animal on the stereotaxic apparatus; 2 min to scrub the scalp with antiseptic solution, make an incision and clean the skull; 2 to 4 min to place the cold light fiber; 1 min to inject the rose Bengal solution; 5 min-wait for intraperitoneal diffusion; 15 min of illumination; and 5 min to clean the wound and suture the animal). Furthermore, no surgical expertise is needed to perform this technique as the lesion is induced through simple illumination of the intact skull. Unlike classical arterial occlusion, this method determines selective occlusions of pial and intraparenchymal microvessels within the irradiated zone and reduces the variability among lesions as no collateral vessel is left to supply oxygen in the targeted area.
In spite of its particular nature, the photothrombotic damage shares essential mechanisms occurring in brain stroke. Similarly to artery occlusion in human stroke, platelet aggregation and clot formation determine interruption of blood flow in the irradiated area7. Likewise, this model also shares essential inflammatory responses as in middle cerebral artery occlusion8. However, because of the well-delimitated boundaries, the penumbral zone, which corresponds to an area of partially preserved metabolism, is very reduced or inexistent after a photothrombotic lesion. This clear border can facilitate the study of cellular responses within ischemic or intact cortical area. Photothrombosis mouse model is particularly suitable for stroke studies in a variety of transgenic animals. Indeed classical models cannot fit to all strains and long period studies in C57BL/6 mouse strain reported a high mortality ratio that can cause bias9.

<table border="1">
<tbody>
 <tr>
 <td>MATERIAL NAME</td>
 <td>COMPANY </td>
 <td>CATALOGUE NUMBER</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Solutions and chemicals</td>
 </tr>
 <tr>
 <td>Rose Bengal </td>
 <td>Sigma, Italy </td>
 <td>330000 </td>
 <td></td>
 </tr>
 <tr>
 <td>Isoflurane Vet </td>
 <td>Merial </td>
 <td> 103120022 </td>
 <td></td>
 </tr>
 <tr>
 <td>Betadine </td>
 <td>Asta Medica </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Paraformaldehyde </td>
 <td>Sigma-Aldrich </td>
 <td> 158127 </td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Surgical material and equipment</td>
 </tr>
 <tr>
 <td>Fluosorber Filter </td>
 <td>Havard apparatus </td>
 <td> 340415 </td>
 <td></td>
 </tr>
 <tr>
 <td>150W fiber optic illuminator</td>
 <td>Photonic</td>
 <td>PL3000</td>
 <td></td>
 </tr>
 <tr>
 <td>Temperature Controller for Plate TCAT-2DF </td>
 <td>Havard apparatus</td>
 <td>727561 </td>
 <td></td>
 </tr>
 <tr>
 <td>Stereotaxic Instrument </td>
 <td>Stoelting</td>
 <td> 51950 </td>
 <td></td>
 </tr>
 <tr>
 <td>Operating microscope </td>
 <td>Takagi </td>
 <td> OM8 </td>
 <td></td>
 </tr>
 <tr>
 <td>Heating pad </td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Oxygen and nitrogen gas</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Surgery Tools</td>
 <td>World precision instrument </td>
 <td></td>
 <td>Optic fiber taps and mask are custom-made </td>
 </tr>
 </tbody>
</table>

photothrombotic ischemia: a minimally invasive and reproducible photochemical cortical lesion model for mouse stroke studies

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.

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...

Watch this Scientific Journal Video about Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies at JoVE.com

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

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 ...

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34.2K Views.  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.