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Targeted Occlusion of Individual Pial Vessels of Mouse Cortex
小鼠皮质个别软膜血管的靶标闭塞   

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Abstract

Targeted photothrombosis is a method to occlude individual arterioles and venules that lie on the surface of the cerebral cortex. It has been used to study collateral flow patterns within the pial vascular network following occlusion of single surface vessels (Schaffer et al., 2006; Blinder et al., 2010; Nguyen et al., 2011), as well as to generate localized ischemic strokes following occlusion of single penetrating vessels (Nishimura et al., 2007; Drew et al., 2010; Shih et al., 2013). The intravascular clot is formed by irradiation of a target vessel with a focused green laser after injection of a circulating photosensitizing agent, Rose Bengal (Watson et al., 1985). We briefly describe modifications of custom-designed and commercial two-photon imaging systems required to introduce a green laser for photothrombosis. We further provide instructions on how to occlude a single penetrating arteriole within the somatosensory cortex of an anesthetized mouse.

Keywords: Two-photon imaging(双光子成像), Microinfarct(微梗死), Blood flow(血流量), Microvessel(微血管), Photothrombosis(光化学)

Materials and Reagents

  1. Mouse with cranial window implant
  2. Buprenorphine hydrochloride (Buprenex®) (Butler Schein, catalog number: 031919 )
  3. Isoflurane (Butler Schein, catalog number: 029405 )
  4. Ophthalmic ointment (Butler Schein, catalog number: 039886 )
  5. Cover Glass (no. 0 thickness) (Thomas Scientific, catalog number: 6661B40 )
  6. Filter paper (Thermo Fisher Scientific, catalog number: S47573B )
  7. 60 mm Culture dish (Thermo Fisher Scientific, catalog number: 130181 )
  8. Distilled water
  9. Glucose (Sigma-Aldrich, catalog number: G8270 )
  10. HEPES (Sigma-Aldrich, catalog number: H7006 )
  11. Artificial cerebral spinal fluid (ACSF) (see Recipes)
  12. FITC-dextran (Sigma-Aldrich, catalog number: FD2000S ) solution (see Recipes)
  13. Rose Bengal (Sigma-Aldrich, catalog number: 632-69-9 ) solution (see Recipes)

Equipment

  1. Insulin syringe, 0.3 ml volume with 29.5 gauge needle (Thermo Fisher Scientific, catalog number: 309301 )
  2. Green laser, 532 nm (Beta Laser, catalog number: MGM20 )
  3. Heating pad with feedback regulation
    1. Temperature control system (FHC Inc., catalog number: 40-90-8 )
    2. Rectal thermistor (FHC Inc., catalog number: 40-90-5D-02 )
    3. Heat pad for mouse (FHC Inc., catalog number: 40-90-2-07 )
  4. Isoflurane vaporizer (IsoTec4; Datex-Ohmeda) (GE Healthcare)
  5. Induction chamber (VetEquip, model: 941444 )
  6. Objective lens, 4x, 0.16-NA (UplanSApo) (Olympus, or equivalent for your system)
  7. Objective lens, 20x, 1.0-NA water immersion (XLUMPlanFI) (Olympus, or equivalent for your system)
  8. Two-Photon Microscope, adapted for targeted photothrombosis (custom-designed or commercial, i.e. Sutter Movable Objective Microscope)

Microscope setup

  1. With a custom-designed two-photon imaging system (Tsai et al., 2002), a green laser beam is introduced into the imaging beam path with dichroic mirror 1 (625 DRLP) (Figure 1) (Shih et al., 2011). The beam is adjusted to pass through a 3.5 mm diameter clearing etched in the dielectric coating of dichroic mirror 2 (700 DCXRU) (Figure 1). This allows transmission of the green laser to the back aperture of the objective lens, while still reflecting > 90% of emitted light from the sample toward the photomultiplier tubes (PMTs). The result is a fixed green laser beam, focused within the center of the imaging field. A shutter placed within the green laser path (LS3Z2 Uniblitz and VMM-D1 driver) will allow control over its on-off time. During occlusion of a vessel, irradiation can be periodically interrupted for brief epochs of imaging. Typically 1 frame (0.2 sec per frame) every 1 sec for an 80% duty cycle. This permits real time observation of the formation of the clot (Schaffer et al., 2006).


    Figure 1. Schematic of custom-designed two-photon system modified to introduce a continuous wave green laser beam. (CW) continuous wave, (PMT) photomultiplier tube. The beam control module for the green laser refers to a neutral density filter to attenuate the laser intensity to a level suitable for photothrombosis. A ~ 3.5 mm diameter hole is etched in the coating of dichroic mirror 2 to allow the green laser to pass while the PMT assembly is in place, thereby allowing visualization of clot formation in real-time. Components and instructions to adapt a custom-designed two-photon system (Tsai et al., 2002) for targeted photothrombosis are provided in Shih et al. (2011).

  2. For the enclosed design of commercial two-photon imaging systems, there is often limited space within the microscope for additional optomechanical components. Here we illustrate how the green laser may be introduced through the camera port of a Sutter Movable Objective Microscope (MOM), using a custom green laser module described by Sigler et al. 2008. The port is located above the ocular lenses, as is the case for most commercial microscope systems (Figure 2a). This path leads to a movable mirror (mirror 2) already located within the Ti-Sapphire imaging beam path between the scan and tube lenses. The green laser is then deflected toward the back aperture of the objective. When transitioning from two-photon to wide-field imaging mode with the MOM system, built-in servo-motors move mirror 2 into the beam path and move the primary dichroic above the objective out of the path synchronously (Figure 2a). This allows the green laser to pass to the objective. In this configuration, however, one will be not be able to visualize the formation of the clot in real-time. Rather, the extent of clot is monitored in between epochs of continuous irradiation (30 sec), by transitioning back to two-photon imaging mode when the green laser is off (Figure 2b). Finally, while not discussed here, another entry point for the green laser is through an epifluorescence filter slot, as described in detail by Sigler et al. (2008).


    Figure 2. Schematic of commercial two-photon system (Sutter Movable Objective Microscope) modified to introduce a continuous wave green laser beam through the camera port. The beam control module refers to a neutral density filter to attenuate the laser intensity to a level suitable for photothrombosis, and a polarizing filter on a rotation mount for finer adjustments of intensity (see Sigler et al., 2008 for detailed instruction for green laser assembly for camera port). A single plano convex lens, rather than the lens doublet described by Sigler et al. 2008, is added to the optical cage assembly to ensure that the beam is collimated after it passes through the tube lens of the microscope. The beam control module and lens are housed in an optical cage generated from Newport parts, which is bolted to a C-mount adaptor that fits into the camera port. The PMT assembly is moved out of the imaging beam path to allow the green laser to pass.

  3. With both custom and commercial two-photon imaging systems, additional optics may be necessary to adjust the power of the green laser and modify the diameter of the beam, depending upon the system. Power control is typically achieved by adding a neutral density filter and/or polarizer filter on a rotation mount (Sigler et al., 2008; Shih et al., 2011). The power of the green laser at its focus after the microscope objective should range between 0.5–1 mW. The green laser beam should under fill the back aperture of the microscope objective, in order to generate a relatively large 3 to 5 μm diameter region of photoactivation within the center of the imaging plane. The beam should also be collimated to ensure that the green laser is focused at the imaging plane. It may therefore be necessary to add lenses to alter the beam diameter and/or adjust for convergence/divergence of the green laser caused by other lenses within the beam path. For example, with the Sutter MOM system an additional plano convex lens (LA1978-A) was added to reduce convergence caused by the tube lens (Figure 2a; located within optical cage system). All necessary optics can be housed together with the green laser using cage systems available from ThorLabs or Newport, and then mounted on the camera port as a single unit, similar to that described by Sigler et al. (Figure 2a) (Sigler et al., 2008).
    We now describe the procedures involved in occluding a single penetrating arteriole in mouse cortex using a green laser beam coupled through the camera port of a Sutter MOM.

Procedure

  1. Targeting the green laser
    1. Before imaging, locate the focus of the green laser within the imaging plane by bleaching a piece of filter paper soaked with FITC-dextran solution.
    2. Cut a 1 x 1 cm piece of filter paper and place it in a culture dish. Cover the paper with 50 μl of FITC-dextran and overlay it with a coverslip. Place a drop of distilled water onto the surface of the cover glass and bring the filter paper into focus at the eyepiece.
    3. Use the same objective lens that you would use for photothrombosis (i.e., 20x, 1.0-NA). Use the translation stage to find an area free of bubbles. Turn off the PMTs and transition to wide-field mode. Ensure the cameral port is open and that the green laser passes through the objective.
    4. Irradiate the sample for 30 sec and then deactivate the beam. Transition back to two-photon imaging mode. An area of the filter paper should now be bleached, marking the focus of the green laser beam.
    5. Place a clear piece of tape on the computer screen over the bleached area. Use a pen to mark the location of where the laser is focused. Repeat the bleaching procedure in a different location to ensure that the location of irradiation is consistent.

  2. Photothrombosis
    1. This procedure requires optical access to the brain either through a skull-removed, which has been described in detail in past publications (Mostany and Portera-Cailliau, 2008; Drew et al., 2010; Shih et al., 2012a; Shih et al., 2012b) or thinned skull cranial window (Holtmaat, 2009). A method of fixing the head of the mouse for imaging is also required (Shih et al., 2012b).
    2. Anesthetize the mouse with isoflurane and affix the animal’s head in the optical imaging apparatus. The apparatus should have a method of delivering isoflurane continuously to provide anesthesia throughout the procedure.
    3. Administer 25 μl of FITC-dextran through the tail vein or retro-orbital vein to label the blood serum.
    4. Using a low magnification objective (i.e., 4x, 0.16-NA), take an image stack (200 μm deep, 5 μm steps) of the pial vasculature through the cranial window. A maximally projected image of this stack is used as a map to provide guidance when using higher magnification objectives (Figure 3a).


      Figure 3. Generation of cortical microinfarct by targeted photothrombosis in mouse cortex. a. low magnification, maximally projected image of pial vasculature visualized through a thinned skull PoRTS window (Drew, 2010). The vasculature is labeled with intravenous FITC-dextran. A single penetrating arteriole within the window is identified for photothrombosis (inset). b. Green laser irradiation of a single penetrating arteriole (left panel) immediately following intravenous administration of Rose Bengal leads to localized clotting (right panel). After successful photothrombosis, a dark clot is seen at the site of irradiation and the vessel becomes brighter upstream due to stagnation of red blood cell flow. c. Post-mortem immunohistochemistry with the pan-neuronal marker NeuN demarcates the boundaries of the resulting microinfarct (yellow-dotted line), as observed 48 hours following occlusion.

    5. Exchange the low magnification lens for a high magnification objective (i.e., 20x, 1.0-NA). Place a drop of distilled water over the cranial window and bring the pial surface into focus. Navigate within the cranial window using the image made with the low magnification objective. Locate the neck of a penetrating arteriole just before it descends into the cortex. Penetrating arterioles branch from the surface arteriolar network and descend into the brain (Blinder, 2010; Shih, 2013). To confirm the identity of the target, use two-photon microscopy to image deeper layers of cortex and ensure that it penetrates into the brain. Once verified, return to the pial surface and maneuver the location of the green laser focus into the lumen of the target vessel by moving the microscope stage.
    6. Inject 50 μl of Rose Bengal solution through the tail vein or retro-orbital vein. Immediately irradiate the vessel with the green laser focus for 30 sec. Return to two-photon imaging mode. The vessel should now be occluded. A successfully occluded vessel will show no dark streaks caused by the movement of red blood cells. There will typically be a dark thrombus with a bright region of stagnant serum directly upstream (Figure 3b, right panel). If the vessel fails to occlude, the photothrombotic procedure may be repeated. Irradiation must be performed within 5 to 10 min after intravenous injection of Rose Bengal as the dye is quickly extravasated from circulation (Zhang and Murphy, 2007). The vessel should be re-examined after 1 h to ensure the vessel has not de-occluded. De-occlusion can sometimes occur with large penetrating arterioles.
    7. The occlusion of a single penetrating arteriole will result in a columnar region of ischemia in mouse cortex ranging from 300 to 500 μm in diameter and can often span the entire depth of cortex (Drew et al., 2010). This region of ischemia will eventually become infarcted and the discrete boundary between viable and infarcted tissue can be delineated with post-hoc immunohistology (Figure 3c).

Recipes

  1. Artificial cerebral spinal fluid (ACSF)
    Prepared from:
    125 mM NaCl
    5 mM KCl
    10 mM glucose
    3.1 mM CaCl2
    1.3 mM MgCl2
    10 mM HEPES (pH 7.4)
    Sterile filter and maintain as aliquots at 4 °C (Kleinfeld and Delaney, 1996)
  2. FITC-dextran solution
    Prepare a 5% solution (w/v) in sterile PBS
    Maintain as aliquots at -20 °C
  3. Rose Bengal solution
    Prepare a 1.25% solution (w/v) in PBS
    Maintain as aliquots at -20 °C

Acknowledgments

Our work is generously supported by grants to A.Y.S. from the NINDS (NS085402), the Dana Foundation, and South Carolina Clinical and Translational Institute (UL1TR000062).

References

  1. Blinder, P., Shih, A. Y., Rafie, C. and Kleinfeld, D. (2010). Topological basis for the robust distribution of blood to rodent neocortex. Proc Natl Acad Sci U S A 107(28): 12670-12675.
  2. Drew, P. J., Shih, A. Y., Driscoll, J. D., Knutsen, P. M., Blinder, P., Davalos, D., Akassoglou, K., Tsai, P. S. and Kleinfeld, D. (2010). Chronic optical access through a polished and reinforced thinned skull. Nat Methods 7(12): 981-984.
  3. Kleinfeld, D. and Delaney, K. R. (1996). Distributed representation of vibrissa movement in the upper layers of somatosensory cortex revealed with voltage-sensitive dyes. J Comp Neurol 375(1): 89-108.
  4. Mostany, R. and Portera-Cailliau, C. (2008). A method for 2-photon imaging of blood flow in the neocortex through a cranial window. J Vis Exp(12).
  5. Nguyen, J., Nishimura, N., Fetcho, R. N., Iadecola, C. and Schaffer, C. B. (2011). Occlusion of cortical ascending venules causes blood flow decreases, reversals in flow direction, and vessel dilation in upstream capillaries. J Cereb Blood Flow Metab 31(11): 2243-2254.
  6. Nishimura, N., Schaffer, C. B., Friedman, B., Lyden, P. D. and Kleinfeld, D. (2007). Penetrating arterioles are a bottleneck in the perfusion of neocortex. Proc Natl Acad Sci U S A 104(1): 365-370.
  7. Schaffer, C. B., Friedman, B., Nishimura, N., Schroeder, L. F., Tsai, P. S., Ebner, F. F., Lyden, P. D. and Kleinfeld, D. (2006). Two-photon imaging of cortical surface microvessels reveals a robust redistribution in blood flow after vascular occlusion. PLoS Biol 4(2): e22.
  8. Shih, A. Y., Mateo, C., Drew, P. J., Tsai, P. S. and Kleinfeld, D. (2012). A polished and reinforced thinned-skull window for long-term imaging of the mouse brain. J Vis Exp (61).
  9. Shih, A. Y., Driscoll, J. D., Drew, P. J., Nishimura, N., Schaffer, C. B. and Kleinfeld, D. (2012). Two-photon microscopy as a tool to study blood flow and neurovascular coupling in the rodent brain. J Cereb Blood Flow Metab 32(7): 1277-1309.
  10. Shih. A.Y., Nishimura. N., Nguyen. J., Friedman. B., Lyden. P.D., B. S.C., Kleinfeld, D. (2011). Optically Induced Occlusion of Single Blood Vessels in Neocortex. In: Imaging in Neuroscience: A Laboratory Manual (Helmchen F, Konnerth A, Yuste R, eds), p. New York: Cold Spring Harbor Laboratory Press, Chapter 85, 939-948.
  11. Shih, A. Y., Blinder, P., Tsai, P. S., Friedman, B., Stanley, G., Lyden, P. D. and Kleinfeld, D. (2013). The smallest stroke: occlusion of one penetrating vessel leads to infarction and a cognitive deficit. Nat Neurosci 16(1): 55-63.
  12. Sigler, A., Goroshkov, A. and Murphy, T. H. (2008). Hardware and methodology for targeting single brain arterioles for photothrombotic stroke on an upright microscope. J Neurosci Methods 170(1): 35-44.
  13. Tsai, P.S., Nishimura, N., Yoder, E.J., Dolnick, E.M., White, G.A., Kleinfeld, D. (2002). Principles, design, and construction of a two photon laser scanning microscope for in vitro and in vivo brain imaging. In: In Vivo Optical Imaging of Brain Function (Frostig RD, ed), pp 113-171. Boca Raton: CRC Press.
  14. Watson, B. D., Dietrich, W. D., Busto, R., Wachtel, M. S. and Ginsberg, M. D. (1985). Induction of reproducible brain infarction by photochemically initiated thrombosis. Ann Neurol 17(5): 497-504.
  15. Zhang, S. and Murphy, T. H. (2007). Imaging the impact of cortical microcirculation on synaptic structure and sensory-evoked hemodynamic responses in vivo. PLoS Biol 5(5): e119.

简介

目标光血栓形成是阻塞位于大脑皮质表面上的个体小动脉和小静脉的方法。它已经用于在封闭单表面血管后研究在Pial血管网内的侧支血流模式(Schaffer等人,2006; Blinder等人,2010; Nguyen 2011),以及在单一穿透血管闭塞后产生局部缺血性中风(Nishimura et al。,2007; Drew et al。 ,2010; Shih et al。,2013)。在注射循环的光敏剂,玫瑰红(Watson等人,1985)后,通过用聚焦的绿色激光器照射目标血管形成血管内凝块。我们简要介绍了定制设计和商业双光子成像系统的修改,引入绿色激光用于光血栓形成。我们进一步提供如何闭塞麻醉鼠标的体感皮层内的单个穿透小动脉的说明。

关键字:双光子成像, 微梗死, 血流量, 微血管, 光化学

材料和试剂

  1. 鼠标与颅窗植入
  2. 盐酸丁丙诺非(Buprenex)(Butler Schein,目录号:031919)
  3. 异氟醚(Butler Schein,目录号:029405)
  4. 眼用软膏(Butler Schein,目录号:039886)
  5. 盖玻璃(no.0厚度)(Thomas Scientific,目录号:6661B40)
  6. 滤纸(Thermo Fisher Scientific,目录号:S47573B)
  7. 60mm培养皿(Thermo Fisher Scientific,目录号:130181)
  8. 蒸馏水
  9. 葡萄糖(Sigma-Aldrich,目录号:G8270)
  10. HEPES(Sigma-Aldrich,目录号:H7006)
  11. 人工脑脊髓液(ACSF)(见配方)
  12. FITC-葡聚糖(Sigma-Aldrich,目录号:FD2000S)溶液(参见Recipes)
  13. 玫瑰红(Sigma-Aldrich,目录号:632-69-9)溶液(参见Recipes)

设备

  1. 胰岛素注射器,使用29.5号针(Thermo Fisher Scientific,目录号:309301),0.3ml体积
  2. 绿色激光,532nm(Beta Laser,目录号:MGM20)
  3. 带反馈调节的加热垫
    1. 温度控制系统(FHC Inc.,目录号:40-90-8)
    2. 直流热敏电阻(FHC Inc.,目录号:40-90-5D-02)
    3. 用于鼠标的热垫(FHC Inc.,目录号:40-90-2-07)
  4. 异氟烷蒸发器(IsoTec4; Datex-Ohmeda)(GE Healthcare)
  5. 感应室(VetEquip,型号:941444)
  6. 物镜,4x,0.16-NA(UplanSApo)(Olympus或您的系统的等效物)
  7. 物镜,20x,1.0-NA浸水(XLUMPlanFI)(Olympus或您的系统的等效物)
  8. 双光子显微镜,适用于目标光血栓症(定制设计或商业化,即 Sutter可移动物镜显微镜)

显微镜设置

  1. 使用定制设计的双光子成像系统(Tsai等人,2002),用分色镜1(625DRLP)将绿色激光束引入成像光束路径(图1)(图1) Shih等人,2011)。调节光束以通过在分色镜2(700DCXRU)(图1)的电介质涂层中蚀刻的3.5mm直径的透明。这允许绿色激光器传输到物镜的后孔径,同时仍然反射> 90%的从样品朝向光电倍增管(PMT)的发射光。结果是固定的绿色激光束,聚焦在成像场的中心内。放置在绿色激光路径(LS3Z2 Uniblitz和VMM-D1驱动器)内的快门将允许控制其开关时间。在血管闭塞期间,可以针对短暂的成像时期周期性地中断照射。对于80%的占空比,通常为每1秒1帧(每帧0.2秒)。这允许实时观察凝块的形成(Schaffer等人,2006)。


    (CW)连续波,(PMT)光电倍增管。图1.定制设计的双光子系统的示意图,其被修改以引入连续波绿色激光束。用于绿色激光的光束控制模块是指中性密度滤光器,以将激光强度衰减到适合于光血栓形成的水平。在二向色镜2的涂层中蚀刻直径为〜3.5mm的孔,以允许绿色激光器在PMT组件就位时通过,从而允许实时观察凝块形成。 Shih等人(2011)提供了用于适应针对性光致血栓形成的定制设计的双光子系统(Tsai等人,2002)的组件和说明书。 br />
  2. 对于商业双光子成像系统的封闭设计,显微镜内的附加光机械部件通常有有限的空间。这里我们说明如何使用由Sigler等人描述的定制绿色激光器模块通过Sutter可移动物镜显微镜(MOM)的相机端口引入绿色激光器。端口位于目镜上方,如大多数商业显微镜系统的情况(图2a)。该路径通向已经位于扫描透镜和管透镜之间的Ti-Sapphire成像光束路径内的可移动反射镜(反射镜2)。然后绿色激光器朝向物镜的后孔偏转。当使用MOM系统从双光子成像模式转换到广视场成像模式时,内置伺服电机将反射镜2移动到光束路径中,并且将物镜上方的主二向色同步地移出路径(图2a)。这允许绿色激光器传递到物镜。然而,在这种构造中,人们将不能实时地可视化凝块的形成。相反,当绿色激光器关闭时(图2b),通过转变回到双光子成像模式,在连续照射的时期(30秒)之间监测凝块的程度。最后,虽然这里没有讨论,但是绿色激光器的另一个入口点是通过落射荧光滤光器槽,如Sigler等人( 2008) />

    图2.修改为通过相机端口引入连续波绿色激光束的商用双光子系统(Sutter可移动物镜显微镜)的示意图。光束控制模块是指中性密度滤光器,激光强度到适合于光血栓形成的水平,以及在旋转支架上的偏振滤光片,用于更精细地调节强度(参见Sigler等人,2008,关于绿色的详细说明 相机端口激光组件)。单个平凸透镜而不是Sigler等人描述的透镜双合透镜2008被添加到光学笼组件以确保光束在穿过显微镜的管透镜之后被准直。光束控制模块和透镜容纳在从Newport部件产生的光学笼中,该光学笼被螺栓连接到适配到相机端口中的C-mount适配器。 PMT组件移出成像光束路径,以允许绿色激光通过。

  3. 对于定制和商业双光子成像系统,根据系统,可能需要附加的光学器件来调节绿色激光器的功率并修改光束的直径。功率控制通常通过在旋转座架上添加中性密度滤波器和/或偏振器滤波器来实现(Sigler等人,2008; Shih等人,2011)。绿色激光在显微镜物镜后的焦点处的功率应在0.5-1mW之间。绿色激光束应当填充显微镜物镜的后孔径,以便在成像平面的中心内产生相对大的3至5μm直径的光活化区域。光束还应该被准直,以确保绿色激光在成像平面处聚焦。因此,可能需要增加透镜以改变光束直径和/或调整由光束路径内的其它透镜引起的绿色激光器的会聚/发散。例如,对于Sutter MOM系统,添加了附加的平凸透镜(LA1978-A)以减少由管透镜(图2a;位于光学笼系统内)引起的会聚。所有必要的光学器件可以使用可从ThorLabs或Newport获得的笼系统与绿色激光器一起容纳,然后作为单个单元安装在照相机端口上,类似于Sigler等人 。 (图2a)(Sigler等人,2008)。
    我们现在描述涉及使用通过Sutter MOM的相机端口耦合的绿色激光束在小鼠皮质中阻塞单个穿透小动脉的过程。

程序

  1. 瞄准绿色激光
    1. 在成像之前,通过漂白一块用FITC-葡聚糖溶液浸泡的滤纸来定位成像平面内的绿色激光的焦点。
    2. 切一个1×1厘米的滤纸,并将其放在培养皿中。用50μl的FITC-葡聚糖覆盖纸并用盖玻片覆盖。将一滴蒸馏水滴在盖玻片的表面上,使滤纸在目镜处聚焦。
    3. 使用与用于光血栓形成(即,20x,1.0-NA)相同的物镜。使用翻译平台找到没有气泡的区域。关闭PMT并转换到宽视场模式。确保摄像头端口打开,绿色激光通过物镜。
    4. 照射样品30秒,然后停用光束。转换回双光子成像模式。滤纸的一个区域现在应该被漂白,标记绿色激光束的焦点。
    5. 在计算机屏幕上的漂白区域放置一条清晰的磁带。使用笔标记激光聚焦的位置。在不同的位置重复漂白程序,以确保辐照位置是一致的。

  2. 光血栓
    1. 该过程需要通过去除颅骨的光学接入,其已经在过去的出版物中详细描述(Mostany和Portera-Cailliau,2008; Drew等人,2010; Shih >等人,2012a; Shih等人,2012b)或变薄的颅骨窗口(Holtmaat,2009)。还需要固定小鼠头部用于成像的方法(Shih等人,2012b)。
    2. 用异氟烷麻醉小鼠并将动物的头部固定在光学成像装置中。该装置应当具有连续递送异氟烷以在整个程序中提供麻醉的方法
    3. 管理25微升的FITC葡聚糖通过尾静脉或眶后静脉标记血清。
    4. 使用低放大率物镜(,即4x,0.16-NA),取通过颅窗的软膜脉管系统的图像堆叠(200μm深,5μm步长)。此堆叠的最大投影图像用作地图,以在使用较高放大倍数的物镜时提供指导(图3a)

      图3.通过小鼠皮质中的靶向光血栓形成产生皮层微梗塞。 a。低放大率,通过减薄的头骨PoRTS窗口可视化的血管脉管系统的最大投影图像(Drew,2010)。脉管系统用静脉内FITC-葡聚糖标记。识别窗内的单个穿透性小动脉用于光血栓形成(插图)。 b。在静脉内施用玫瑰红后立即单个穿透性小动脉(左图)的绿色激光照射导致局部凝血(右图)。成功的光血栓形成后,在照射部位看到黑色凝块,并且由于红细胞流动的停滞,血管在上游变得更亮。 C。使用泛神经元标记物NeuN的尸体后的免疫组织化学分析在闭塞后48小时观察到的所得微梗死(黄点线)的边界。

    5. 更换用于高放大率物镜的低倍率镜头(,即,20x,1.0-NA)。将一滴蒸馏水放在颅窗上,并使唾液表面聚焦。使用低放大倍数物镜的图像在颅窗内导航。在下降到皮层之前,找到穿透性小动脉的颈部。穿透性小动脉从表面小动脉网络分支并下降到大脑中(Blinder,2010; Shih,2013)。要确认目标的身份,使用双光子显微镜成像更深层的皮质,并确保它渗透到大脑。一旦验证,返回到表面并通过移动显微镜载物台将绿色激光焦点的位置操纵到目标血管的管腔中。
    6. 通过尾静脉或眶后静脉注射50μl玫瑰红溶液。立即用绿色激光焦点照射容器30秒。返回双光子成像模式。船舶现在应该堵塞。成功阻塞的血管将不显示由红细胞运动引起的暗条纹。通常会有黑色血栓,明亮区域的血清停滞在直接上游(图3b,右图)。如果血管未闭塞,可以重复光血栓形成程序。辐照必须在静脉注射玫瑰红后5至10分钟内进行,因为染料从循环中迅速渗出(Zhang和Murphy,2007)。该容器应在1小时后重新检查,以确保容器尚未脱出。有时可能会发生大穿透性小动脉的闭塞
    7. 单个穿透性小动脉的闭塞将导致直径在300至500μm范围内的小鼠皮质中的缺血的柱状区域,并且通常可跨越皮质的整个深度(Drew等人,2010) 。这种局部缺血区域最终将梗塞,并且可以用事后免疫组织学(图3c)描绘活的和梗死的组织之间的离散边界。

食谱

  1. 人工脑脊液(ACSF)
    准备:
    125 mM NaCl 5 mM KCl
    10mM葡萄糖 3.1mM CaCl 2。 1.3mM MgCl 2
    10mM HEPES(pH 7.4)
    无菌过滤并保持在4°C等分试样(Kleinfeld和Delaney,1996)
  2. FITC-葡聚糖溶液 制备5%溶液(w/v)的无菌PBS
    保持等分于-20°C
  3. 玫瑰红溶液
    制备1.25%溶液(w/v)的PBS
    保持等分于-20°C

致谢

我们的工作慷慨支持A.Y.S.赠款。 来自NINDS(NS085402),Dana基金会和南卡罗来纳州临床和转化研究所(UL1TR000062)。

参考文献

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  2. Drew,P.J.,Shih,A.Y.,Driscoll,J.D.,Knutsen,P.M.,Blinder,P.,Davalos,D.,Akassoglou,K.,Tsai,P.S.and Kleinfeld,D。(2010)。 慢性 通过抛光和加强的薄颅骨进行光学访问。 7(12):981-984。
  3. Kleinfeld,D。和Delaney,K.R。(1996)。 在电压敏感染料显示的体感皮层上层中的vibrissa运动的分布式表示。 a> J Comp Neurol 375(1):89-108。
  4. Mostany,R。和Portera-Cailliau,C。(2008)。 通过颅窗在新皮层中进行双光子成像的方法。 J Vis Exp (12)。
  5. Nguyen,J.,Nishimura,N.,Fetcho,R.N.,Iadecola,C.and Schaffer,C.B。(2011)。 皮质上行小静脉的阻塞导致血流量减少,血流方向逆转,上游毛细血管扩张。 J Cereb Blood Flow Metab 31(11):2243-2254。
  6. Nishimura,N.,Schaffer,C.B.,Friedman,B.,Lyden,P.D.and Kleinfeld,D。(2007)。 穿透小动脉是新皮层灌注的瓶颈。美国国家科学院Sci USA 104(1):365-370。
  7. Schaffer,C.B.,Friedman,B.,Nishimura,N.,Schroeder,L.F.,Tsai,P.S.,Ebner,F.F.,Lyden,P.D.and Kleinfeld,D。(2006)。 皮质表面微血管的双光子成像显示血管闭塞后血流中的强烈再分布。 a> PLoS Biol 4(2):e22。
  8. Shih,A.Y.,Mateo,C.,Drew,P.J.,Tsai,P.S.and Kleinfeld,D。(2012)。 抛光和加固的稀疏颅骨窗口,用于长期成像小鼠大脑。 J Vis Exp (61)。
  9. Shih,A.Y.,Driscoll,J.D.,Drew,P.J.,Nishimura,N.,Schaffer,C.B.and Kleinfeld,D。(2012)。 双光子显微镜作为研究啮齿动物脑中血流和神经血管偶联的工具。 a> J Cereb Blood Flow Metab 32(7):1277-1309。
  10. Shih。 A.Y.,Nishimura。 N.,Nguyen。 J.,Friedman。 B.,Lyden。 P.D.,B. S.C.,Kleinfeld,D。(2011)。单层血管在大脑皮层中的光诱导闭塞。 In:Imaging in Neuroscience:A Laboratory Manual(Helmchen F,Konnerth A,Yuste R,eds),p。 New York:Cold Spring Harbor Laboratory Press,第85章,939-948
  11. Shih,A.Y.,Blinder,P.,Tsai,P.S.,Friedman,B.,Stanley,G.,Lyden,P.D.and Kleinfeld,D.(2013)。 最小中风:一个穿透血管的闭塞导致梗死和认知缺陷。 Nat Neurosci 16(1):55-63。
  12. Sigler,A.,Goroshkov,A。和Murphy,T.H。(2008)。 在直立显微镜上针对单个脑动脉进行光血栓形成中风的硬件和方法。 em> J Neurosci Methods 170(1):35-44
  13. Tsai,P.S.,Nishimura,N.,Yoder,E.J.,Dolnick,E.M.,White,G.A.,Kleinfeld,D。(2002)。用于体外和体内脑成像的双光子激光扫描显微镜的原理,设计和构造。 In:In Vivo Optical Imaging of Brain Function(Frostig RD,ed),第113-171页。 Boca Raton:CRC Press
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Taylor, Z. J. and Shih, A. Y. (2013). Targeted Occlusion of Individual Pial Vessels of Mouse Cortex. Bio-protocol 3(17): e897. DOI: 10.21769/BioProtoc.897.
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