Abstract Significance Photothrombosis is a widely used model of ischemic stroke in rodent experiments. In the photothromboris model, the photosensitizer Rose Bengal is systemically introduced to the blood stream and activated by green light to induce aggregation of platelets that eventually cause vessel occlusion. Since the activation of Rose Bengal is a one-photon phenomenon and the molecules in the illuminated area (light path) are subject to excitation, targeting of thrombosis is unspecific especially in the depth dimension. We have developed a photothrombosis protocol that can target a single vessel in the cortical parenchyma by two-photon excitation. Aim We aim to induce a thrombotic stroke in the cortical parenchyma by two-photon activation of Rose Bengal so that we confine photothrombosis within a vessel of a target depth. Approach FITC-dextran is injected into the blood stream to visualize the cerebral blood flow in anesthetized adult mice with a cranial window. After a target vessel is chosen by two-photon imaging (950 nm), Rose Bengal is injected into the blood stream. The scanning wavelength is changed to 720 nm and photothrombosis was induced by scanning the target vessel. Results Two-photon depth-targeted single vessel photothrombosis was achieved with a success rate of 84.9±1.7% within 80 s. Attempts without Rose Bengal (i.e., only with FITC) did not result in photothrombosis at the excitation wavelength of 720 nm. Conclusions We described a protocol that achieves depth-targeted single vessel photothrombosis by two-photon excitation. Simultaneous imaging of blood flow in the targeted vessel using FITC dextran enabled the confirmation of vessel occlusion and prevention of excess irradiation that possibly induces unintended photodamage.

Depth-targeted intracortical microstroke by two-photon photothrombosis in rodent brain