Fukuda Masahiro, Matsumura Takayoshi, Suda Toshio, Hirase Hajime
Kumamoto University, International Research Center for Medical Sciences, Kumamoto, Japan.
Duke-NUS Medical School, Signature Program in Neuroscience and Behavioral Disorders, Singapore.
Neurophotonics. 2022 Apr;9(2):021910. doi: 10.1117/1.NPh.9.2.021910. Epub 2022 Mar 16.
Photothrombosis is a widely used model of ischemic stroke in rodent experiments. In the photothrombosis model, the photosensitizer rose bengal (RB) is systemically introduced into the blood stream and activated by green light to induce aggregation of platelets that eventually cause vessel occlusion. Since the activation of RB 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 developed a photothrombosis protocol that can target a single vessel in the cortical parenchyma by two-photon excitation. We aim to induce a thrombotic stroke in the cortical parenchyma by two-photon activation of RB to confine photothrombosis within a vessel of a target depth. 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), RB is injected into the blood stream. The scanning wavelength is changed to 720 nm, and photothrombosis is induced by scanning the target vessel. Two-photon depth-targeted single-vessel photothrombosis was achieved with a success rate of and an irradiation duration of . Attempts without RB (i.e., only with FITC) did not result in photothrombosis at the excitation wavelength of 720 nm. 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.
光血栓形成是啮齿动物实验中广泛使用的缺血性中风模型。在光血栓形成模型中，将光敏剂孟加拉玫瑰红（RB）全身注入血流中，并通过绿光激活以诱导血小板聚集，最终导致血管闭塞。由于RB的激活是单光子现象，并且照射区域（光路）中的分子会受到激发，因此血栓形成的靶向是非特异性的，尤其是在深度维度上。我们开发了一种光血栓形成方案，该方案可以通过双光子激发靶向皮质实质中的单个血管。我们旨在通过RB的双光子激活在皮质实质中诱导血栓性中风，将光血栓形成限制在目标深度的血管内。将异硫氰酸荧光素葡聚糖（FITC-葡聚糖）注入血流中，以通过颅窗观察麻醉成年小鼠的脑血流。通过双光子成像（950nm）选择目标血管后，将RB注入血流中。将扫描波长更改为720nm，并通过扫描目标血管诱导光血栓形成。实现了双光子深度靶向单血管光血栓形成，成功率为 ，照射持续时间为 。在没有RB（即仅使用FITC）的情况下，在720nm激发波长下未产生光血栓形成。我们描述了一种通过双光子激发实现深度靶向单血管光血栓形成的方案。使用FITC葡聚糖同时对目标血管中的血流进行成像，可以确认血管闭塞并防止可能导致意外光损伤的过度照射。
