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Photothrombotic Induction of Capillary Ischemia in the Mouse Cortex during in vivo Two-Photon Imaging
在体内双光子成像期间采用光栓法诱导小鼠皮层毛细血管缺血   

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The Journal of Neuroscience
Nov 2016

Abstract

Photothrombosis of blood vessels refers to the activation of a circulating photosensitive dye with a green light to induce clotting in vivo (Watson et al., 1985). Previous studies have described how a focused green laser could be used to noninvasively occlude pial arterioles and venules at the brain surface (Schaffer et al., 2006; Nishimura et al., 2007; Shih et al., 2013). Here we show that small regions of the capillary bed can similarly be occluded to study the ischemic response within the capillary system of the mouse cerebral cortex. The advantage of this approach is that the ischemic zone is restricted to a diameter of approximately 150-250 μm. This permits higher quality two-photon imaging of degenerative processes that would be otherwise difficult to visualize with models of large-scale stroke, due to excessive photon scattering. A consequence of capillary occlusion is leakage of the blood-brain barrier (BBB). Here, through the use of two-photon imaging data sets, we show how to quantify capillary leakage by determining the spatial extent and localization of intravenous dye extravasation.

Keywords: Blood-brain barrier (血脑屏障), Photothrombosis (光栓法), Ischemia (缺血), Two-photon imaging (双光子成像), Capillary (毛细血管), Stroke (中风)

Background

Numerous animal models exist to induce ischemia on a large scale via occlusion major cerebral arteries (Carmichael, 2005). However, there are some aspects of stroke that are not accessible to in vivo two-photon imaging. In regions experiencing more severe ischemia, cells swell due to ionic imbalance, and this edematous process contributes to increased light scattering, greatly reducing the quality and depth of in vivo two-photon imaging. A smaller zone of ischemia would reduce photon scattering, and still allow neurovascular changes associated with ischemia to be more clearly visualized over time in vivo.

We recently showed that spatially restricted regions of ischemia could be generated by focused photothrombotic irradiation of the cortical capillary bed (Underly et al., 2017). Capillary occlusions were highly reproducible, could be targeted to specific locations, and initiated at precise times through a cranial imaging window. The resulting ischemic zone occupied less than 1% of the area accessible through a typically cranial window (Figures 1D and 1E), allowing multiple strokes to be examined in one window.

Here, we describe the steps involved in inducing photothrombotic occlusion in a small region of the capillary bed during in vivo two-photon imaging. We build upon a previous protocol in which individual cortical penetrating arterioles were occluded, rather than capillaries (Taylor and Shih, 2013). We also demonstrate the analysis of BBB leakage produced as a consequence of capillary occlusion using Imaris, a 3-D visualization software.

Materials and Reagents

  1. Filter paper (GE Healthcare, catalog number: 1001-0155 )
  2. Cotton swabs (Fisher Scientific, catalog number: 23-400-119 )
  3. Cover glass (thickness: No. 0) (Thomas Scientific, catalog number: 6661B40 )
  4. 0.3 ml insulin syringes (BD, catalog number: 328438 )
  5. Petri dish
  6. An adult mouse of any common laboratory strain, ~25 to 35 g in weight
  7. Isoflurane (Patterson Veterinary Supply, catalog number: 07-806-3204 )
    Manufacturer: Zoetis, catalog number: 10015516 .
  8. Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P4417-50TAB )
  9. Agarose type 3-A (Sigma-Aldrich, catalog number: A9793 )
  10. Fluorescein-dextran (2 MDa; 5% w/v in saline) (Sigma-Aldrich, catalog number: FD2000S )
  11. Rose Bengal (1.25% w/v in saline) (Sigma-Aldrich, catalog number: 330000-1G )
  12. Buprenorphine hydrochloride (Buprenex®) (Patterson Veterinary Supply, catalog number: 07-891-9756 )
  13. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653-1KG )
  14. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333-500G )
  15. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C1016-100G )
  16. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266-100G )
  17. Glucose (Sigma-Aldrich, catalog number: G8270 )
  18. HEPES (Sigma-Aldrich, catalog number: H7006 )
  19. Artificial cerebral spinal fluid (ACSF) (see Recipes)

Equipment

  1. Heating pad with feedback regulation (FHC, catalog number: 40-90-2-07 )
  2. Heating pad control system (FHC, catalog number: 40-90-8D )
  3. Rectal Thermistor Probe (FHC, catalog number: 40-90-5D-02 )
  4. Isoflurane vaporizer (Datex-Ohmeda, model: IsoTec4 )
  5. Induction chamber (VetEquip, catalog number: 941444 )
  6. Dental drill (Osada, model: EXL-M40 )
  7. Auxiliary equipment for two-photon microscope (Sutter Moveable Objective System [Taylor and Shih, 2013])
    1. Objective lens 4x, 0.16 NA (Olympus, model: UPLSAPO )
    2. Objective lens 20x, 1.0 NA, Water immersion (Olympus, model: XLUMPlanFI )
    3. Green laser 532 nm (Beta Electronics, model: MGM20 ). Details for how the green laser line is directed into the Sutter MOM imaging beam path was described in a separate protocol (Taylor and Shih, 2013)
  8. Computer specifications (for Imaris)
    1. 16 GB RAM
    2. 3.3 GHz CPU
    3. AMD Radeon RX 480 4GB or better
    4. 1280 x 1024 Resolution Monitor

Software

  1. Imaris (Bitplane)
    1. Imaris 7.6 (or current)
    2. Imaris Batch Module
  2. Fiji software (https://imagej.net/Fiji/Downloads)

Procedure

  1. Cranial windows
    In order to perform photothrombotic occlusion of capillaries, a cranial imaging window must be generated in the mouse skull. Both thinned skull windows (Drew et al., 2010; Yang et al., 2010) and windows involving full removal of skull can be used (Holtmaat et al., 2009). Occlusions can also be performed through windows that are acute (< 24 h) or chronic. Images shown in this protocol used polished and reinforced thinned-skull windows (PoRTs), and a video and detailed written procedure for this window type are available (Shih et al., 2012a and 2012b).

  2. Two-photon microscope and laser for photothrombosis
    Our system passes a green laser beam through to the imaging objective via the two-photon imaging beam path (Figures 1A and 1B). The microscope we use is a Sutter Moveable Objective Microscope, and details for how to introduce a green laser has been described previously (Taylor and Shih, 2013). The green laser greatly under-fills the back aperture of the 20x objective (Olympus; XLUMPlanFI), yielding a fixed laser focus with ~20-40 μm diameter at the imaging plane. The power of the green laser is ~1 mW at the sample. Prior to initiating photothrombotic occlusion, the location of the green laser focus should be determined. To do this, we place a small piece of filter paper in a Petri dish, apply 25 µl of fluorescein-dextran onto the filter paper and cover with a cover glass. The filter paper is imaged using the 20x objective, and then irradiated with the green laser. The green laser photobleaches the fluorescent dye at its focus, providing a target location relative to the imaging field (Figures 2A and 2B). This is also described in-depth in a previous protocol (Taylor and Shih, 2013). The filter set we use to detect fluorescein-dextran emission is 525/70m-2P (Chroma Corp).


    Figure 1. Photothrombotic occlusion of capillaries in mouse cortex. A. Schematic illustrating two-photon imaging laser and B. photothrombotic green laser beam paths in a typical two-photon imaging microscopy. C. The objective brings the green laser to a focus in upper layers of cortex allowing for photothrombotic irradiation of capillaries. D. Wide-field vascular map of the imaging area contained within the PoRTS window, obtained by montaging several maximally z-projected images collected using a 4x objective. E. Magnified region within the window, visualized with a 20x objective (projected over 100 µm of depth, beginning ~10 µm below the pial surface), showing ischemic damage to capillaries over 24 h.


    Figure 2. Green laser focusing for targeted photothrombosis. A. FITC-Dextran on filter paper prior to irradiation with green laser; B. Photobleached region of filter paper following irradiation with green laser light revealing the laser focus.

  3. Targeted photothrombotic occlusion
    1. Anesthetize the mouse and affix the animal’s head to a stable imaging apparatus under the two-photon microscope. For anesthesia, the data shown here is collected from a C57BL/6 mouse under 0.75 to 1.5% isoflurane supplied in medical air. However, other types of anesthesia can also be used. Typically, a custom machined aluminum flange is attached to the head using dental cement during a pre-surgical procedure. Later, the metal flange can be secured to a custom holder using screws. There are many variations of head fixation that are compatible to this protocol. See methods in Shih et al. (2012a) for one method for head fixation of mice.
    2. Administer 25 µl of 5% w/v fluorescein-dextran (2 MDa) into the infraorbital vein. Using a 0.3 ml insulin syringe, carefully insert the needle tip 3 mm into the corner of the eye closest to the animal’s midline. To ensure that the needle does not penetrate too deeply, place a mark on the needle 3 mm from the tip using a black marker. The needle should go behind the eye, slightly pushing the eye laterally. Inject the contents of the syringe over a period of 20 sec. None of the solution should accumulate outside the eye, when correctly administered. Withdraw the syringe and carefully clean the eye with a moistened cotton swab. Ophthalmic ointment should then be applied to both eyes to avoid desiccation. A video for this procedure is also available (Shih et al., 2012b). This procedure should be done under anesthesia, and with care to avoid damage to the eye. The vasculature should be clearly visible with two-photon imaging immediately after injection. Too deep insertion of the needle will result in fluorescein-dextran within the cranium and increased subdural fluorescence during two-photon imaging.
    3. Starting at a low magnification with the 4x objective, obtain z-stacks of the vasculature over the entire cortical area accessible through the cranial window. These images, when montaged, provide a map of the cortical vasculature for navigation at higher magnification (Figure 1D).
    4. Change to the 20x objective and focus on the larger vessels at the pial surface. Use the map collected at lower magnification to determine your location within the imaging window.
    5. Locate a region of capillaries without larger vessels overlying them (Figure 1E). When finding a capillary location of interest, target a region at least 20 µm away from larger penetrating vessels, which may lead to larger strokes if inadvertently occluded.
    6. Place the target of the green laser over the region of the capillary bed that is to be irradiated (Figures 1D and 1E; white dot).
    7. Inject 25-50 µl of 1.25% Rose Bengal into the infraorbital vein, using the same method described above for fluorescein-dextran.
      IMPORTANT: Check to be sure that the field of view and green laser focus has not shifted prior to beginning laser irradiation.
    8. Initiate photothrombosis by turning on the green laser light (Figure 1B) (1 mW at the sample) and allowing irradiation for 25 sec.
      IMPORTANT: The Rose Bengal dye will only remain in the blood stream for ~5-10 min, with rapid removal from the blood plasma over this period. Thus, it is important to perform irradiation as quickly as possible after administering Rose Bengal.

  4. Imaging the evolution of capillary ischemia
    One can expect to observe complete cessation of blood flow in ~10 capillary segments within a 211 x 211 x 150 µm cube of tissue immediately after the 25 sec irradiation (Figure 3A; yellow arrowheads). A capillary segment is defined as a length of capillary between branch-points. The integrity of the capillary wall is typically maintained in for the first 0.5-1 h after which localized sites of dye leakage begin to appear from non-flowing capillaries (Figure 3A; white arrows). The number of these leakage sites increases gradually over a period of 3 h. For each imaging time-point, we collect image z-stacks of the irradiated region. While our studies have characterized changes occurring in the first 3 h post-occlusion (Underly et al., 2017), the precise experimental time-line and imaging interval can be adapted based on the experimental question.


    Figure 3. In vivo observation and post-hoc quantification of capillary leakage. A. In vivo tracking of capillary leakage over 3 h following photothrombotic irradiation of cortical capillaries; B. Two-dimensional (left) and 3-dimensional (right) representation of capillaries before irradiation. Sixteen ROIs (purple) are strung along a capillary segment in the 3-dimensional panel, with ROI 8 positioned at the central point of leakage. C. The same vessels are shown 1 h after irradiation, when fluorescein-dextran leakage is evident. D. Graph of fluorescein-dextran volumes pre (dotted line) and post (solid line) irradiation, corresponding to the capillary leakage site. The post-irradiation dye volume trace for data of panel C is shown as the solid red line.

  5. Image processing
    Imaris supports numerous imaging formats, but Fiji software can be used to convert formats not compatible with Imaris to .tif files. Ensure that the pixel/micron ratios for x-y and z planes are correctly entered into Imaris. The ratios can be modified in Image Properties within the Imaris software. It is also important to consider your file names as Imaris will combine similar file names not separated into different folders.
    1. Begin by identifying the initial regions of dye leakage by utilizing the two-dimensional .tif stacks in Fiji. These are regions in which the intravascular dye has begun to expand beyond the initial (pre-image) border of the blood vessels, and are associated with a region of high intensity fluorescence (Figure 3A; white arrows).
    2. Using Imaris, open the image stacks containing the fluorescein channel, captured during or after dye leakage has begun.
    3. With an image rendered in 3-dimensions in Imaris, string regions of interest (ROIs) along the vasculature where leakage is occurring (Figures 3B and 3C; purple, right column).
      1. These ROIs are created by using the ‘Surface’ feature in Imaris and selecting the ‘region of interest’ option.
      2. Start by placing an initial region identified at the site of leakage. Then string ROIs outward from this central point, without overlap, to sample surrounding regions of the capillary segment. These ROIs should be appropriately sized so that the depth of the ROI is never completely filled to avoid a volume ceiling effect which would prevent finding the region of peak dye extravasation.

Data analysis

With ROIs volumetrically rendered (the concluding step in Surface creation) a graphical representation of the volumes can be produced. This can be done by graphing the dye leakage volumes within each ROI (found in the ‘statistics tab’ of Imaris). By having several ROIs strung along the capillary, areas with a significant increase in leakage can be differentiated by showing a significant change in volume between ROIs from images prior to, and following, capillary occlusion (Figure 3D). This difference can be found by performing t-tests comparing each of the corresponding ROIs (ROI 1 pre vs. ROI 1 post, etc.). An ANOVA may be necessary instead of a t-test if the number of groups or times used during experimentation fit the criteria for an ANOVA.

Notes

In regard to the capillary occlusions and dye leakage, there is a lot of variability in the volume of dye depending on the time of sampling. It is important to image at time points (every 15 min or more) frequent enough to capture the formation of the event.

Recipes

  1. Fluorescein-dextran
    5% (w/v) in sterile PBS
    Preloaded as aliquots in 0.3 ml insulin syringes
    Stored at -20 °C
  2. Rose Bengal
    1.25% solution (w/v) in sterile PBS
    Preloaded as aliquots in 0.3 ml insulin syringes
    Stored at -20 °C
  3. Artificial cerebral spinal fluid (ACSF)
    125 mM NaCl
    5 mM KCl
    10 mM glucose
    3.1 mM CaCl2
    1.3 mM MgCl2
    10 mM HEPES (pH 7.4)
    Maintain at 4 °C

Acknowledgments

Our work is supported by grants to A.Y.S. from the NINDS (NS085402, NS096997), National Science Foundation (1539034), the Dana Foundation, the American Heart Association (14GRNT20480366), Alzheimer’s Association NIRG award, South Carolina Clinical and Translational Institute (UL1TR000062), Charleston Conference on Alzheimer’s Disease New Vision Award, and an Institutional Development Award (IDeA) from the NIGMS under grant number P20GM12345.

References

  1. Carmichael, S. T. (2005). Rodent models of focal stroke: size, mechanism, and purpose. NeuroRx 2(3): 396-409.
  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. Holtmaat, A., Bonhoeffer, T., Chow, D. K., Chuckowree, J., De Paola, V., Hofer, S. B., Hubener, M., Keck, T., Knott, G., Lee, W. C., Mostany, R., Mrsic-Flogel, T. D., Nedivi, E., Portera-Cailliau, C., Svoboda, K., Trachtenberg, J. T. and Wilbrecht, L. (2009). Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window. Nat Protoc 4(8): 1128-1144.
  4. 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.
  5. 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.
  6. 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.
  7. Shih, A. Y., Driscoll, J. D., Drew, P. J., Nishimura, N., Schaffer, C. B. and Kleinfeld, D. (2012a). 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.
  8. Shih, A. Y., Mateo, C., Drew, P. J., Tsai, P. S. and Kleinfeld, D. (2012b). A polished and reinforced thinned-skull window for long-term imaging of the mouse brain. J Vis Exp (61).
  9. Taylor, Z. J. and Shih, A. Y. (2013). Targeted occlusion of individual pial vessels of mouse cortex. Bio Protoc 3(17).
  10. Underly, R. G., Levy, M., Hartmann, D. A., Grant, R. I., Watson, A. N. and Shih, A. Y. (2017). Pericytes as inducers of rapid, matrix metalloproteinase-9 dependent capillary damage during ischemia. J Neurosci 37:129-140.
  11. 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.
  12. Yang, G., Pan, F., Parkhurst, C. N., Grutzendler, J. and Gan, W. B. (2010). Thinned-skull cranial window technique for long-term imaging of the cortex in live mice. Nat Protoc 5(2): 201-208.

简介

血管的血栓形成是指用绿光激活循环的光敏染料,以在体内诱发凝血(Watson等人,1985)。以前的研究已经描述了如何将聚焦的绿色激光器用于非侵入性地封闭脑表面的小动脉和小静脉(Schaffer等人,2006; Nishimura等人, 2007; Shih等人,2013)。这里我们显示毛细血管床的小区域可以类似地闭塞,以研究小鼠大脑皮质毛细管系统内的缺血反应。这种方法的优点是缺血区被限制在约150-250μm的直径。这允许退化过程的更高质量的双光子成像,否则由于过度的光子散射,否则难以用大规模中风的模型来可视化。毛细血管闭塞的结果是血脑屏障(BBB)的泄漏。在这里,通过使用双光子成像数据集,我们展示了如何通过确定静脉内染料外渗的空间范围和定位来量化毛细血管渗漏。
【背景】存在许多动物模型通过闭塞主要大脑动脉大规模诱发缺血(Carmichael,2005)。然而,在体内双光子成像中不能获得中风的一些方面。在经历更严重缺血的区域,由于离子不平衡而导致细胞膨胀,这种水肿过程有助于增加光散射,大大降低了体内双光子成像的质量和深度。较小的局部缺血区域将减少光子散射,并且仍然允许与局部缺血相关的神经血管变化随着时间的推移在体内更清晰地显现。
我们最近表明,空间受限区域的缺血可以通过聚焦的血栓形成的皮质毛细血管床照射产生(Underly等人,2017)。毛细血管闭塞是高度可重复的,可以针对特定的位置,并通过颅骨成像窗口在精确的时间开始。所得到的缺血区域占据通过典型颅窗可接近的面积的1%(图1D和1E),允许在一个窗口中检查多个笔触。
&NBSP;这里,我们描述了在体内双光子成像期间在毛细血管床的小区域中诱导光血栓闭塞所涉及的步骤。我们建立在以前的方案,其中个体皮质穿透小动脉闭塞,而不是毛细血管(Taylor和Shih,2013)。我们还展示了使用Imaris(一种3-D可视化软件)作为毛细血管闭塞产生的BBB泄漏的分析。

关键字:血脑屏障, 光栓法, 缺血, 双光子成像, 毛细血管, 中风

材料和试剂

  1. 滤纸(GE Healthcare,目录号:1001-0155)
  2. 棉签(Fisher Scientific,目录号:23-400-119)
  3. 盖玻璃(厚度:0号)(Thomas Scientific,目录号:6661B40)
  4. 0.3ml胰岛素注射器(BD,目录号:328438)
  5. 培养皿
  6. 任何常见实验室菌株的成年小鼠,体重25〜35g
  7. 异氟烷(Patterson Veterinary Supply,目录号:07-806-3204)
    制造商:Zoetis,目录号码:10015516。
  8. 磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:P4417-50TAB)
  9. 琼脂糖3-A型(Sigma-Aldrich,目录号:A9793)
  10. 荧光素 - 葡聚糖(2 MDa;盐水中5%w / v)(Sigma-Aldrich,目录号:FD2000S)
  11. 玫瑰红(1.25%w / v盐水)(Sigma-Aldrich,目录号:330000-1G)
  12. 丁丙诺啡盐酸盐(Buprenex ®)(Patterson Veterinary Supply,目录号:07-891-9756)
  13. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653-1KG)
  14. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333-500G)
  15. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C1016-100G)
  16. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266-100G)
  17. 葡萄糖(Sigma-Aldrich,目录号:G8270)
  18. HEPES(Sigma-Aldrich,目录号:H7006)
  19. 人造脑脊髓液(ACSF)(见食谱)

设备

  1. 加热垫带反馈调节(FHC,目录号:40-90-2-07)
  2. 加热垫控制系统(FHC,目录号:40-90-8D)
  3. 直热电阻探头(FHC,目录号:40-90-5D-02)
  4. 异氟烷蒸发器(Datex-Ohmeda,型号:IsoTec4)
  5. 感应室(VetEquip,目录号:941444)
  6. 牙科钻(Osada,型号:EXL-M40)
  7. 双光子显微镜辅助设备(Sutter可移动目标系统[Taylor and Shih,2013])
    1. 物镜4x,0.16 NA(Olympus,型号:UPLSAPO)
    2. 物镜20x,1.0 NA,水浸(Olympus,型号:XLUMPlanFI)
    3. 绿色激光532 nm(Beta电子,型号:MGM20)。关于如何将绿色激光线引导到Sutter MOM成像光束路径的细节在单独的协议中描述(Taylor和Shih,2013)
  8. 计算机规格(Imaris)
    1. 16 GB RAM
    2. 3.3 GHz CPU
    3. AMD Radeon RX 480 4GB或更高版本
    4. 1280 x 1024分辨率监视器

软件

  1. Imaris(Bitplane)
    1. Imaris 7.6(或当前)
    2. Imaris批量模块
  2. 斐济软件( https://imagej.net/Fiji/Downloads )< br />

程序

  1. 颅窗
    为了进行毛细血管的光血栓闭塞,必须在小鼠头骨中产生颅骨成像窗口。可以使用两个变薄的颅骨窗(Drew等人,2010; Yang等人,2010),并且可以使用涉及完全去除颅骨的窗口(Holtmaat et al。,et al。 。,2009)。也可以通过急性(<24小时)或慢性的窗口进行闭塞。该协议中所示的图像使用抛光和加固的薄型颅骨窗(PoRT),并且可以使用该窗口类型的视频和详细的书面程序(Shih等人,2012a和2012b)。

  2. 双光子显微镜和激光治疗血栓形成
    我们的系统通过双光子成像光束路径将绿色激光束通过成像物镜(图1A和1B)。我们使用的显微镜是Sutter可移动物镜显微镜,以前介绍了如何引入绿色激光的细节(Taylor和Shih,2013)。绿色激光大大弥补了20x物镜(Olympus; XLUMPlanFI)的后孔,在成像平面上产生直径约20-40μm的固定激光对焦。绿色激光的功率在样品约为1 mW。在引发光血栓闭塞之前,应确定绿色激光焦点的位置。为了做到这一点,我们将一小片滤纸放在培养皿中,将25μl荧光素 - 葡聚糖涂于滤纸上,并用盖玻片盖住。使用20x物镜对滤纸进行成像,然后用绿色激光照射。绿色激光使荧光染料在其焦点处漂白,提供相对于成像场的目标位置(图2A和2B)。这也在以前的协议中深入描述(Taylor和Shih,2013)。我们用于检测荧光素 - 葡聚糖发射的过滤器组是525 / 70m-2P(Chroma Corp)。


    图1.小鼠皮层毛细血管的光血栓闭塞。A.在典型的双光子成像显微镜中示意性说明双光子成像激光和B.光血红蛋白激光束路径。 C.目标使绿色激光成为皮层上层的焦点,允许毛细血管的光血栓形成照射。 D.包含在PoRTS窗口内的成像区域的宽场血管图,通过使用4x物镜收集的几个最大z投影图像进行蒙皮获得。 E.窗口内的放大区域,可视化为20x物镜(投影超过100μm的深度,从pial表面开始〜10μm),显示24小时内毛细血管的缺血性损伤。

    “”src
    图2.绿色激光聚焦目标光血栓形成。 :一种。用绿色激光照射前的滤纸上的FITC-葡聚糖; B.用绿色激光照射后的滤纸的漂白区域显示激光焦点
  3. 靶向光血栓闭塞
    1. 在双光子显微镜下麻醉鼠标并将动物的头部固定在稳定的成像装置上。对于麻醉,这里显示的数据是从C57BL / 6小鼠收集在0.75-1.5%在医用空气中供应的异氟醚。然而,也可以使用其他类型的麻醉。通常,在手术前的过程中,使用牙科粘固剂将定制的加工铝法兰连接到头部。后来,金属法兰可以使用螺丝固定到定制的支架上。头部固定的许多变体与本协议兼容。请参阅Shih等人(2012a)中针对小鼠头部固定的一种方法的方法。
    2. 将25μl5%w / v荧光素 - 葡聚糖(2 MDa)管理到眶下静脉。使用0.3ml胰岛素注射器,将针尖小心地插入距离动物中线最近的眼角。为了确保针头不能太深地穿透,请使用黑色标记将尖端上的针标距3毫米。针头应该在眼后,稍微推动眼睛。在20秒内注射注射器的内容物。当正确给药时,没有任何溶液应积聚在眼睛外部。取出注射器,并用湿润的棉签仔细清洁眼睛。应将眼科软膏施用于双眼以避免干燥。此程序的视频也可用(Shih等人,2012b)。该程序应在麻醉下进行,并小心避免损伤眼睛。注射后立即用双光子成像清楚可见脉管系统。针头的深度插入将导致头颅内的荧光素 - 葡聚糖,并在双光子成像期间增加硬膜下荧光。
    3. 从4倍目标的低放大倍数开始,通过颅窗可以通过整个皮质区获得脉管系统的z-叠层。这些图像在蒙皮时提供了用于在较高放大倍数下导航的皮质脉管系统图(图1D)。
    4. 改变为20倍的目标,并将重点放在较大的船体表面。使用以较低放大倍率收集的地图来确定您在成像窗口内的位置。
    5. 找到一个毛细血管区,没有较大的血管覆盖(图1E)。当找到感兴趣的毛细血管位置时,瞄准距离较大穿透血管至少20微米的区域,如果无意中闭塞可能会导致较大的中风。
    6. 将绿色激光的目标放在待照射的毛细管床的区域(图1D和1E;白点)。
    7. 使用上述荧光素 - 葡聚糖相同的方法将25-50μl的1.25%玫瑰红注入眶下静脉。
      重要事项:检查以确保在开始激光照射之前,视野和绿色激光对焦没有移动。
    8. 通过打开绿色激光(图1B)(样品1 mW)并允许照射25秒,开始光血栓形成。
      重要事项:玫瑰红色染料只能在血液中保留约5-10分钟,在此期间迅速从血浆中清除。因此,在施用罗伯孟加拉之后尽可能快地进行照射是重要的。

  4. 成像毛细血管缺血的进展
    在25秒照射后立即可以观察到在211×211×150微米立方体的组织内〜10个毛细血管段的血流完全停止(图3A;黄色箭头)。毛细管段被定义为分支点之间的毛细管长度。毛细管壁的完整性通常保持在第一个0.5-1小时,此后染色剂渗漏的局部位点从非流动毛细管开始出现(图3A;白色箭头)。这些泄漏点的数量在3小时内逐渐增加。对于每个成像时间点,我们收集照射区域的图像z-叠层。虽然我们的研究表明在闭塞后3小时内发生的变化(Underly等人,2017),但是精确的实验时间线和成像间隔可以基于实验问题进行调整。


    图3. 体内 毛细管渗漏的观察和事后定量。 A.体内跟踪皮质毛细血管的光凝照射3小时后的毛细血管渗漏; B.辐射前毛细管的二维(左)和三维(右)表示。六维ROI(紫色)沿着三维面板中的毛细管段串联,ROI 8位于泄漏的中心点。 C.照射后1小时显示相同的血管,当荧光素 - 葡聚糖渗漏明显时。 D.荧光素 - 葡聚糖体积前(虚线)和后(实线)照射的图,对应于毛细管渗漏部位。面板C数据的后照射染料体积迹线显示为实线红线。

  5. 图像处理
    Imaris支持多种成像格式,但斐济软件可用于将与Imaris不兼容的格式转换为.tif文件。确保x-y和z平面的像素/微米比正确输入到Imaris中。可以在Imaris软件中的图像属性中修改这些比率。同样重要的是考虑您的文件名,因为Imaris将组合类似的文件名不会分成不同的文件夹。
    1. 首先通过利用斐济的二维.tif栈识别染料泄漏的初始区域。这些是血管内染料开始扩张超出血管初始(前图像)边界的区域,并且与高强度荧光区域相关联(图3A;白色箭头)。
    2. 使用Imaris打开包含荧光素通道的图像堆,在染料泄漏开始之前或之后捕获。
    3. 在Imaris中以3维呈现的图像,沿着发生泄漏的脉管系统的感兴趣的字符串区域(ROI)(图3B和3C;紫色右列)。
      1. 这些ROI是通过使用Imaris中的“Surface”功能创建的,并选择“感兴趣的区域”选项。
      2. 首先放置在泄漏现场识别的初始区域。然后从该中心点向外输出ROI,而不重叠,以对毛细血管段的周围区域进行采样。这些投资回报率应该适当地确定大小,以便ROI的深度从未被完全填满,以避免体积上限效应,从而阻止发现峰染色外渗的区域。

数据分析

随着ROI(体积)渲染(Surface创建中的最后一步),可以生成卷的图形表示。这可以通过绘制每个ROI内的染料泄漏体积(在Imaris的“统计”选项卡中找到)来完成。通过沿着毛细管串联几个ROI,可以通过在毛细血管闭塞之前和之后的图像的ROI之间显示体积的显着变化来区分泄漏显着增加的区域(图3D)。通过执行比较每个相应的ROI(ROI 1 pre与ROI 1 post,等等)之间的差异,可以找到这种差异。如果实验过程中使用的组数或次数符合方差分析的标准,则可能需要ANOVA而不是 t 测试。

笔记

关于毛细血管闭塞和染料渗漏,根据取样时间,染料体积的变化很大。重要的是在时间点(每15分钟或更长时间)拍摄频率足以捕获事件的形成。

食谱

  1. 荧光素 - 葡聚糖
    5%(w / v)无菌PBS
    预先加入0.3ml胰岛素注射器等分试样 储存于-20°C
  2. 玫瑰孟加拉
    1.25%溶液(w / v)在无菌PBS中 预先加入0.3ml胰岛素注射器等分试样 储存于-20°C
  3. 人造脑脊髓液(ACSF)
    125 mM NaCl
    5 mM KCl
    10 mM葡萄糖
    3.1mM CaCl 2
    1.3mM MgCl 2
    10 mM HEPES(pH 7.4)
    维持在4°C

致谢

我们的工作得到了A.Y.S.的资助。来自NINDS(NS085402,NS096997),国家科学基金会(1539034),达纳基金会,美国心脏协会(14GRNT20480366),阿尔茨海默氏症协会NIRG奖,南卡罗来纳临床和转化研究所(UL1TR000062),查尔斯顿阿尔茨海默病新视力会议颁奖典礼,颁奖典礼,以及授权号码为P20GM12345的NIGMS颁发的机构发展奖(IDeA)。

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Underly, R. G. and Shih, A. Y. (2017). Photothrombotic Induction of Capillary Ischemia in the Mouse Cortex during in vivo Two-Photon Imaging. Bio-protocol 7(13): e2378. DOI: 10.21769/BioProtoc.2378.
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