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Functional ex-vivo Imaging of Arterial Cellular Recruitment and Lipid Extravasation
动脉细胞招募和脂质外渗的功能性离体成像   

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Circulation
Jan 2014

Abstract

The main purpose of this sophisticated and highly versatile method is to visualize and quantify structural vessel wall properties, cellular recruitment, and lipid/dextran extravasation under physiological conditions in living arteries. This will be of interest for a broad range of researchers within the field of inflammation, hypertension, atherosclerosis, and even the pharmaceutical industry. Currently, many researchers are using in vitro techniques to evaluate cellular recruitment, like transwell or flow chamber systems with cultured cells, with unclear physiological comparability. The here introduced method describes in detail the use of a sophisticated and flexible method to study arterial wall properties and leukocyte recruitment in fresh and viable murine carotid arteries ex vivo under arterial flow conditions. This model mimics the in vivo situation and allows the use of cells and arteries isolated from two different donors (for example, wildtype vs. specific knockouts) to be combined into one experiment,thereby providing information on both leukocyte and/or endothelial cell properties of both donors. As such, this model can be considered an alternative for the complicated and invasive in vivo studies, such as parabiotic experiments.

Keywords: Imaging (成像), Two-photon laser scanning microscopy (双光子激光扫描显微镜检查), Arteriograph chamber (动脉搏描计室), Cellular recruitment (细胞招募), Lipid extravasation (脂质外渗)

Background

The core of the method is the application of two-photon laser scanning microscopy (TPLSM) to visualize an ex vivo carotid artery which is mounted in an arteriograph chamber, which has been shown to mimic physiological conditions as present in the in vivo situation (Megens et al., 2007). Fresh arteries, in our case murine carotid arteries but the method is also applicable for other blood vessels of comparable size including human vessels (Bloksgaard et al., 2015), are carefully extracted and mounted in the arteriograph chamber on two glass micropipettes using thin threads. The chamber should provide sufficient space to access the artery with the microscope objective (preferably water dipping or immersion objective with a working distance of > 1 mm). After applying luminal pressure (80 mmHg) and the subsequent correction of the shortening of the length due to isolation (Megens et al., 2007), a variety of fluorescently labeled cells and/or vessel wall components of interest can be perfused into the vessel either under flow or under static conditions. This enables the user to A) count the number of adherent leukocyte subsets, B) determine molecular extravasation (dextrans, lipids) into the arterial wall, C) visualize vascular properties and structures using fluorescently conjugated (specific) antibodies or intrinsic fluorescence signals derived from extracellular matrix components, D) combine the previously described targets.

The general basis of this method was described in 2007 (Megens et al., 2007). Since then this method has been used in several scientific publications, for example to show cell recruitment under control and inflammatory conditions (Schmitt et al., 2014), chemokine presence (Soehnlein et al., 2011), detection of smooth muscle cells (Subramanian et al., 2010; Spronck et al., 2016) or proliferating endothelial cells (Schober et al., 2014), endothelial protein depositions (Ortega-Gomez et al., 2016), adhering platelets (Karshovska et al., 2015), atherosclerotic lesions in the bifurcation (Megens et al., 2007 and 2008; Weber et al., 2011), visualization of the endothelial glycocalyx (Reitsma et al., 2011), or evaluation of extracellular matrix markers (Boerboom et al., 2007; Megens et al., 2007).

TPLSM imaging can be performed prior-, during-, and/or post-perfusion. The settings of the microscope system strongly depend on the available microscope. We utilize a modern Leica SP5II MP system with a 20x WD objective and a Ti:Sa pulsed laser which allows 4 channel imaging at video rate. This is however not a requirement for application of this method as older, less well equipped TPLSM systems also suffice.

In recent years, we have further advanced the method, making it applicable to investigate recruitment of specific cell-types to the viable carotid artery (Döring et al., 2014; Schmitt et al., 2014; Karshovska et al., 2015). Not only does this method enable the user to specifically and simultaneously investigate recruitment of various cell types like monocytes, neutrophils or T-cells to highly physiological endothelium by differential fluorescent labeling (using cell trackers or equivalent), it also allows us to combine specific arteries and cells isolated (blood, bone marrow) from different (wildtype vs. knockout) mouse subsets. As a result, a system is created that can define whether effects on recruitment are mediated by the vascular and/or haematopoietic deficiency. Besides the functional readout of cell recruitment, the method further enables simultaneous or subsequent labelling and subcellular resolution imaging of vascular structures and presence of compounds in the vessel wall. As a result, altered adhesion may directly be linked to the presence or absence of specific targets.

By simultaneous application of various fluorescently labeled leukocyte subsets, the experimental conditions are equal for each cell-type, thereby limiting the experimental variation due to for example flow pattern differences (data may be presented in absolute numbers or ratios). The latter also limits the number of experiments required and, in combination with a reduced number of necessary experimental animals because inflammatory cells and arteries can be isolated from the same animals, ultimately the method reduces the number of required animals.

In addition to the cell recruitment assay we have developed an application using fluorescently labeled low-density lipoprotein particles or dextrans to visualize and quantify lipid or dextran extravasation in viable (diseased) arteries. Lastly, unlike in vivo imaging of large arteries, this ex vivo model does not suffer from unwanted motions of the arterial wall as is the case in the in vivo situation, thereby allowing imaging with subcellular resolution. Moreover, stimuli or specific dyes may be applied at any given time during the experiment giving the researcher full flexibility to tailor the methodology and achieve the required goals.

Materials and Reagents

  1. Isolation of carotid arteries
    1. Fixation tape Durapore 1.25 cm (3M, catalog number: 1538-0 )
    2. Polystyrene dishes 35 x 10 mm (2 x) (Corning, Falcon®, catalog number: 353001 )
    3. Hanks balanced saline solution (HBSS) with CaCl2 and MgCl2 (Thermo Fisher Scientific, catalog number: 1402550 ), pH 7.4

  2. Cell suspension
    1. 50 ml tubes (3 x) (SARSTEDT, catalog number: 62.547.254 )
    2. Needle 27 G x 1.5 (Grey) (BD, catalog number: 301629 )
    3. Syringe 10 ml (1 x) (BD, DiscarditTM catalog number: 309110 )
    4. Cell strainer 50 µm (Sysmex, CellTrics®, catalog number: 25004-0042-2317 )
    5. Hanks balanced saline solution (HBSS) with CaCl2 and MgCl2 (Thermo Fisher Scientific, catalog number: 1402550 ), pH 7.4
    6. Fluorescent cell markers: cell tracker green (Thermo Fisher Scientific, InvitrogenTM, catalog number: C7025 ) and Red (Thermo Fisher Scientific, InvitrogenTM, catalog number: C34565 )
    7. Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: A9434 )
    8. Potassium bicarbonate (KHCO3) (Sigma-Aldrich, catalog number: 60339 )
    9. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
    10. Lysis buffer (see Recipes)

  3. Mounting of artery
    1. Glass etching material (NH4HF2 or NH4F·HF) (Carglass, catalog number: ZB10 EI0003 )
    2. Needles 14 G x 80 mm, shortened and blunted to 40 mm and 50 mm (Braun Sterican 14 G x 80 mm) (B. Braun Medical, catalog number: 4665473 )
    3. Nylon thread Ø ~20 µm, for tying of blood vessels (Living Systems Instruments, catalog number: THR-G )
    4. Three-way tab (2 x) (B. Braun Medical, catalog number: 4095111 )
    5. Syringe 1 ml (BD, PlastipakTM, catalog number: 300026 )
    6. Silicone tubing 3 x 1 mm (Carl Roth, catalog number: 9556.1 ) cut to 0.5 m length
    7. Hanks balanced saline solution (HBSS) with CaCl2 and MgCl2 (Thermo Fisher Scientific, catalog number: 1402550 ), pH 7.4

  4. Molecular extravasation
    1. Silicone tubing 3 x 1 mm cut to 1.0 m length (Carl Roth, catalog number: 9556.1 )
    2. Needle 20 G x 1.5 (yellow) (BD, catalog number: 301300 )
    3. Safe lock tubes 1.5 ml (3 x) (Eppendorf, catalog number: 0030120086 )
    4. Syringes 1 ml (2 x) (BD, PlastipakTM, catalog number: 300026 )
    5. Three-way tab (B. Braun Medical, catalog number: 4095111 )
    6. Fixation tape Durapore 1.25 cm (3M, catalog number: 1538-0 )
    7. Human Dil-LDL (Kalen Biomedical, catalog number: 770230-9 )
    8. Hanks balanced saline solution (HBSS) with CaCl2 and MgCl2 (Thermo Fisher Scientific, catalog number: 1402550 ), pH 7.4
    9. Directly conjugated anti-CD31/eFluor450 (PECAM: Thermo Fisher Scientific, eBioscienceTM, catalog number: 48-0311-82 )
      Alternatives: Directly conjugated anti-CD54/A488 (ICAM: BioLegend, catalog number: 116112 ) or anti-CD106/A594 (VCAM: BioLegend, catalog number: 105724 )

  5. Flow assay
    1. Silicone tubing 3 x 1 mm cut to 1.5 m and 1.0 m length (Carl Roth, catalog number: 9556.1 )
    2. Needle 20 G x 1.5 (yellow) (BD, catalog number: 301300 )
    3. Syringes 1 ml (1 x) (see Reagent C5) and 10 ml (2 x) (BD, DiscarditTM, catalog number: 309110 )
    4. Three-way Tab (2 x) (B. Braun Medical, catalog number: 4095111 )
    5. 50 ml tubes (2 x) (SARSTEDT, catalog number: 62.547.254 )
    6. Fixation tape Durapore 1.25 cm (3M, catalog number: 1538-0 )
    7. Hanks balanced saline solution (HBSS) with CaCl2 and MgCl2 (Thermo Fisher Scientific, catalog number: 1402550 ), pH 7.4

Equipment

  1. Isolation of carotid arteries
    1. Pen
    2. FST student spring scissors (Fine Science Tools, catalog number: 91500-09 )
    3. Dumont forceps (2 x) (Fine Science Tools, catalog number: 91150-20 )
    4. Scissors (Fine Science Tools, catalog number: 14084-08 )
    5. Stereomicroscope Leica S8 Apo (LED light source and 0.63x objective) (Leica Microsystems, model: Leica S8 Apo )

  2. Cell suspension
    1. Timer
    2. Pen (see Equipment A1)
    3. Scissors (Fine Science Tools, catalog number: 91401-12 )
    4. 1 ml pipette (Eppendorf, catalog number: 3120000062 )
    5. Centrifuge (Eppendorf, model: 5430 )
    6. Cell counting chamber (Neubauer)
    7. Cell culture microscope (Leica DMi1 with phase contrast and 10x NA0.3 objective) (Leica Microsystems, model: Leica DMi1 )
    8. Flow cytometer (BD, model: BD FACSCANTO SYSTEM )

  3. Mounting of artery
    1. Arteriograph chamber (2 x, IDEE©, Maastricht University, the Netherlands)
    2. Glass pipettes 1.5 x 0.86 mm (2 x, Harvard Apparatus, catalog number: 30-0057 )
    3. Pipette puller (NARISHIGE, catalog number: PP-830 )
    4. Pipette tip grinder (IDEE©, Maastricht University, the Netherlands)
      Alternative: Glass pipettes readymade (Living Systems Instruments, catalog number: GCP-300-325 )
    5. Silicone kit, 73 clear (Farnell, catalog number: 101705 )
    6. Sphygmomanometer (Riester, Big Ben®, catalog number: 1453-100 ) adapted with a 500 ml air chamber (Schott) and Luer-connectors to fit 1 x 3 mm silicone tubing (IDEE©, Maastricht University, the Netherlands)
    7. Luer-coupling adapter female (1x) (Carl Roth, catalog number: CT62.1 )
    8. Stereomicroscope Leica S8 Apo (with LED light source and 0.63x objective) (Leica Microsystems, model: Leica S8 Apo)

  4. Microscope
    1. Commercially available Leica SP5IIMP laser scanning microscope system based on a DM6000FS microscope stand (for more info we refer to the manufacturer’s website:
      https://www.leica-microsystems.com/products/confocal-microscopes/details/product/leica-tcs-sp5-ii/downloads/)
      Alternative: Any functional TPLSM enabling spectral specificity in ≥ 2 detectors and a water dipping objective with sufficient working distance (≥ 1 mm) can be used in combination with the here described assays.
    2. Spectra physics MaiTai DeepSee Ti:Sa laser source
    3. Leica Hybrid detectors (4 x)
    4. Luigs and Neumann SM7 motorized microscope stage
    5. Leica 20x NA1.00WD objective
    6. Ludin Climate control /laser safety box

  5. Molecular extravasation
    1. Timer (see Equipment B1)
    2. Pen (see Equipment A1)
    3. Luer-coupling adapters: male (1 x) (Carl Roth, catalog number: CT58.1 ) and female (1 x) (Carl Roth, catalog number: CT62.1 )
    4. Syringe infusion pump (Harvard Apparatus pump11 elite) (Harvard Apparatus, catalog number: 70-4504 )

  6. Flow assay
    1. Luer-coupling adapters: male (2 x) (Carl Roth, catalog number: CT58.1 ) and female (1 x) (Carl Roth, catalog number: CT62.1 )
    2. Syringe infusion pump (Harvard Apparatus pump11 elite) (Harvard Apparatus, catalog number: 70-4504 )
    3. Column stand with clamp (unknown brand, length 100 cm, footprint 15 x 25 cm)
    4. Pc with excel and/or paper for cell count

Software

  1. Leica LAS AF 2.6 acquisition software
  2. Leica LAS X 3.11 image processing software (including 3D analyses package); offline
    Note: Any processing software package that allows the user to handle multidimensional data can be used.

Procedure

  1. Isolation of carotid arteries
    Isolate the mouse carotid arteries (length after isolation preferably 3-6 mm) following the local ethical committee guidelines using a stereo microscope (see Equipment A5) and place them in HBBS (pH 7.4) in a polystyrene dish (Ø 35 mm) (see Figure 1 for detailed information).


    Figure 1. Isolation of carotid arteries. Fix euthanised mouse on the stereomicroscope stage (A) and remove the skin in the neck region using scissors (14084-08) and clean the area using a wet tissue (B). The submaxillary glands (I) become visible. C. Pull one side of the thyrotic gland (I) to the outside and remove musculus sternocleidomastoidius (II) and musculus XXXX (III) by using Dumont forceps. D. Underneath some thin see-through layers, the common carotid artery becomes visible (IV). Carefully remove tissue over the artery and check whether there are atypical sidebranches present. E. Moreover carefully remove tissue from the bifurcation (green arrow) and isolate the cleaned common carotid artery by dissecting it at the blue lines (using spring scissors). Hold the dissected artery by one of both outer end (Dumont forceps) and place it into a small dish filled with HBSS to keep it moist. After isolation the artery must be kept cool (fridge or ice) until further processing.

    There are some prerequisites to be met which are crucial for maintaining arterial viability and TPLSM imaging:
    1. Mechanical damage either by pinching or overstretching the vessel must be avoided at all cost as this will immediately result in altered structural and functional behaviour.
    2. Contact of air with the artery should be minimized. Isolate the artery while keeping the wound moist at all time, after dissection quickly transfer the artery into medium.
    3. The artery must be very clean for imaging. Any ‘dirt’, hair, or tissue on the outside of the artery will result in loss of visibility of the parts of the artery underneath it. So work meticulously and also remove thin sheets of connective tissue which are covering the artery.
      Note: Flushing of the circulation of the mouse, as often used for harvesting of aortas, is not recommended as it potentially introduces air bubbles in the lumen of the arteries which impact the endothelial cells. Flushing can be performed in a controlled way directly after isolation by temporarily mounting (carefully holding it in place with the forceps thus without threads) the common part on an air-free single glass pipette in the perfusion chamber and gently flushing HBSS through it. This however has to be done quickly (within 30 min) after carotid isolation in order to avoid blood clots! (See also procedure in section ‘mounting of artery’.)

  2. Isolation of bone marrow derived leukocytes
    1. Isolate the femur and/or tibia of the mice following the local ethical committee guidelines and place them in HBBS (pH 7.4).
      Note: The most convenient method of isolation is to first remove the skin from the hind limbs, subsequently pull on the hind limbs to dislocate the hip. Afterwards it is easy to remove the hind limb by cutting in the hip area, without the risk of cutting the bone. After removal, remaining muscles and tissue can be safely removed.
    2. Cut off the epiphyses (heads) of the bones with scissors so that the marrow cavity is open.
    3. Flush the bone with HBSS into a 50 ml tube by using a 27 G needle and 10 ml syringe.
      Note: It is important that all bone marrow cells are removed from the bones. Use as much volume as needed for flushing until practically all cells (red colour) are removed.
    4. Prepare a single cell solution by resuspending the cells using a 1 ml pipette.
    5. Filter the resuspended bone marrow cells with a 50 µm cell strainer to reduce tissue in the final cell suspension.
    6. Centrifuge the cells for 5 min at 300 x g.
    7. Resuspend the pellet with 5 ml of lysis buffer (in order to lyse the red blood cells) and incubate for 1 min at room temperature.
    8. Centrifuge the cells for 5 min at 300 x g.
      Note: If required, different leukocyte subsets (e.g., monocytes, neutrophils and lymphocytes) can be isolated according to lab specific protocols.
    9. Prepare cell tracker solution by diluting the cell tracker 1:1,000 in HBSS.
      Note: Different colours of cell tracker can be used to distinguish between leukocyte subsets (e.g., monocytes, neutrophils and lymphocytes) or between different leukocyte sources (e.g., wild-type vs. knock-out)
    10. Resuspend the cells in prepared cell tracker solution (total volume 3 ml per leukocyte subset/source).
    11. Incubate for 15 min at room temperature.
      Notes:
      1. Alternate cell tracker green and cell tracker red between the different experimental runs to avoid any impact of the dyes on the results as well as to keep the observers blinded.
      2. Other cell marker combinations such as Calcein (eBioscience) and Rhodamin (Sigma-Aldrich) or other cell tracker combinations may also be used.
    12. Wash the cells in suspension twice after the staining procedure, resuspend in 3 ml HBSS, use 50 ml tubes, centrifuge at 300 x g for 5 min.
      Note: Washing of the cells is critical in order to avoid overall staining of the vessel wall by unbound cell trackers in the prepared cell suspension which causes loss of contrast!
    13. Count total numbers of cells per leukocyte subset/source using a Neubauer chamber and a microscope (with phase contrast).
    14. Mix both types of leukocytes (2 different subsets or sources, labelled with different colours to distinguish them from each other) to achieve a 1:1 ratio of both resuspended in a total of 6 ml HBSS.
    15. Cell density for each leukocyte group should be 1.5 x 106 leukocytes (thus, the total number of cells in suspension for each flow assay is 3 x 106 in 6 ml HBSS).
    16. Analyse the resulting leukocyte suspension by flow cytometry in order to confirm the 1:1 ratio.

  3. Preparation of arteriography chamber
    1. Pull and grind pipettes from 0.58 x 1 x 80 mm glass tubes by using a pipette puller (heater = 60; weight= 3 x large block) and a grinder setup. The goal is to generate pipettes with a length of 40 mm and 50 mm and a tip diameter of 250-350 µm with a 45° tip angle. Tip size and angle can be checked using any bright field microscope (≈ 100x magnification) and a scale bar slide (see Figure 2).


      Figure 2. Micropipette construct. A. Overview picture micropipette as used for home-built perfusions chamber. The glass micropipette is glued into a blunted and shortened 14 G needle (green, total length approximately 50 mm). B. The glass pipette sticks approximately 0.8-10 mm out of the blunted needle. C. Close up of the tip of the pipette which has a tip diameter of 250-300 µm and is grinded to obtain a 45° angle. After gluing and grinding the tip is etched in order to create a roughened glass surface (not visible) giving better friction for the mounting of the artery.

    2. Etch the tips of the grinded pipettes by NH4HF2 or NH4F·HF compound to the tip of the grinded pipettes (one can simply hold the tip several mm in the compound in the opened storage flask). (10-20 min or when the etching process was successful), rinse carefully with distilled water after the procedure, and let them dry (RT). Etched tips enable more stable tying of the artery to the glass.
      Note: Because of differences in arteriography chamber design the preparation and properties of the pipettes are different for the various chambers. The most important property of the pipette for this method is the tip opening diameter and the 45° angular shape of the tip since both directly influence the flow pattern within the artery.
    3. Cut the 14 G needles by making a circular edge using a small file and then breaking the tip off using small pincers and remove any sharp edges using sandpaper. The needles should have a length of 40 mm and 50 mm (50 mm for the adjuster side of the chamber).
    4. Glue the glass pipettes into the shortened and blunt 20 G needles using silicone kit. Make sure the space between the glass and the steel needle is fully closed to ensure air tightness of the construct. Moreover, make sure the non-sharpened outer edge of the glass pipette just reaches the Luer-lock connector of the 20 G needle in order to avoid accumulation of debris in the non-filled space within the construct.
    5. Carefully rinse the created constructs with distilled water after the procedure and store them safely or mount them in the arteriography chambers.

  4. Mounting of artery
    1. Prepare a thread dish by applying some tape (5 cm) upside down (sticky side up) in a polystyrene Petri dish (Ø 100 mm). Tape the outer ends of the upside down tape to the polystyrene dish with some short pieces of tape.
    2. Prepare threads (Ø ~20 µm; living systems instrumentation) in the thread dish (Figure 3) using a stereomicroscope (see Equipment A5). The threads are generated by 1) curve the straight thread and make a circle by crossing the outer ends with each other, 2) leashing one outer end twice through the circle.


      Figure 3. Preparation of the nylon threads as used for mounting of an artery. A. Overview of materials needed for the procedure; 10 cm polystyrine Petri dish (I) with Durapore tape and 8-10 mm long nylon threads (Ø ≈ 20 µm: arrow); 2 Dumont forceps (II), and the nylon thread (III). B-F. Close ups of Durapore tape with nylon threads showing the stepwise procedure of thread preparation; B. Single thread next to a cluster of threads (green arrow); C. Curve the straight thread and make a circle by crossing the outer ends with each other; D. Placing one outer end in the circle (diameter 500-700 µm) using the sticky properties of the tape to keep it in place; E. Leashing one outer end through the circle; F. By repeating Procedure D and Procedure E the thread is ready. Place the finished thread with its circle half on the sticky Durapore and half over the tape for improved handling (threads are visible and gradable without the use of a stereomicroscope).

    3. Clean the interior and the pipettes of the home built (but also commercially available) arterial perfusion chamber (Figure 4A) carefully with 70% ethanol (1x flush) and HBSS (2x flush), attach a 3-way tab on both pipettes with the female Luer-couplers and fill with HBSS medium with CaCl2 and MgCl2 (pH 7.4). Make sure that both pipettes are completely air-free and leave a 1 ml syringe filled with medium connected to the 3-way tab at the side of the length adjuster of the chamber.
    4. Transfer the freshly isolated and cleaned carotid artery to the perfusion chamber and place it at the stereomicroscope (see Equipment A5) (see Figure 4).


      Figure 4. Mounting of murine carotid artery in home-built perfusion chamber. A. Example of a perfusion chamber with longitudinal length adjuster (green arrow); B. Fill the clean perfusion chamber with HBSS and attach 3-way tabs (I) on both pipettes. Remove any air from both pipettes using a HBSS filled 1 ml syringe (II). C. View of the interior of the perfusion chamber using a stereomicroscope; D. Close up of a glass pipette with 2 pre-prepared nylon threads slid over it; E. Overview of mounted but unpressurized artery. Two threads are used on each pipette. The bifurcation (III) helps determining the correct flow direction. F. The mounted artery is then closed at the flow outlet (bifurcation side) and connected to a sphygmomanometer (IV) at the flow entrance side to reveal any leakage of the mounted artery. G. Stepwise increase pressure to and correct the distance between both pipettes (using the adjuster, green arrow) until the artery is straight at 80 mmHg. Note the length difference between the artery in Procedure E and Procedure G, where a pressurized artery at 80 mmHg is approximately 30% longer.

    5. Place two or three threads over the pipette using the stereomicroscope (see Equipment A5).
    6. Mount the common part of the carotid by pulling it over the pipette at the length adjuster side of the chamber (Not too far!).
    7. Tighten the two or three threads so that the artery is fixed in between the glass tip and the threads.
    8. Gently flush some medium through the carotid in order to get rid of possible blood leftovers.
      Note: It is preferred to flush the artery inside the chamber and not flush the mouse as a total in order to limit air in the lumen. This however has to be done quickly (within 30 min) after carotid isolation in order to avoid blood clots!
    9. Adjust the distance between both pipettes using the adjuster on the outside of the chamber (Figure 4B; green).
    10. Place two or three threads over the 2nd pipette.
    11. Mount the bifurcation side of the carotid by pulling it over the pipette (Figure 4B). Make sure you have tightened all branches that are not cannulated to create a closed circuit.
      Notes: Make sure you mount the artery in its physiological direction for the flow assay, i.e., flow goes from common to bifurcation. A failure of the flow direction will result in false-positive or negative results. Also make sure the artery is not mounted under tension due to twisting of the artery!
    12. Open the 3-way tab on the exit side (the non-length adjustment side of the chamber) and gently flush some medium to see whether pipettes are open (HBSS runs through the artery and comes out on the exit side) and artery is mounted stably.
    13. Connect the sphygmomanometer with 3 x 1 mm silicone tubing and a female Luer-connector to the length adjustment side of the chamber and fill the 3 x 1 mm tubing of the manometer with some HBSS.
    14. Adjust the 3-way connectors to create a closed circuit that can be pressure controlled using the manometer and stepwise increase the average intraluminal pressure while adjusting the length of the unfolding vessel using the external adjuster until you reach 100 mmHg. Be aware of leaks of the mounted artery by tracking the medium level in the 3 x 1 mm silicone tube connected to the sphygmomanometer.
      Note: A leak somewhere in the total system will result in alteration of the buffer level in the 3 x 1 mm silicone tubing!
    15. Make sure the length is corrected properly at 100 mmHg and then lower the pressure to 80 mmHg (mimicking physiological stretch of the artery).
    16. When all is correctly mounted, proceed to the next step.

  5. Two-photon laser scanning microscopy (TPLSM)
    1. Set the climate box to 37 °C. Make sure the temperature sensor inside the climate box is positioned close to the objective. To reach a stable temperature the climate control box must be started approx. 1 h prior to the first measurement. No additional CO2/O2 gas control is required for these experiments.
    2. Turn on the system according to protocol (different for every microscope).
    3. Place the perfusion chamber onto the microscope stage and focus on the carotid artery using the 20x 1.00 WD objective, the fluorescent light source and the RGB-filter.
      Note: Be very careful not to focus too deep as this will result in destruction of the sample, chamber and potentially the objective!
    4. Select the spectral channels required (channel one: 400-430 nm for collagen; 440-500 nm for elastin; 510-540 nm for green cell tracker; 620-670 nm for cell tracker deep red).
    5. Tune the excitation laser (Ti:Sa) to 800 nm.
    6. Select the imaging mode (XYZ), scan speed (200-400 Hz), and xyz resolution (XY-pixel format according to Nyquist criterion, Z-step 0.8-1.5 µm). For creating movies of the artery during flow/cell adhesion, choose XYT or XYZT scans, select ‘minimize’ time interval and maximize overall recording time. Moreover, reduce the number of pixels and increase scan speed to 600 Hz in order to have a reasonable refreshment rate of the pictures.
    7. Select the area of interest in the mounted carotid artery in ‘live’ mode; avoid the first field of view directly next to the glass pipettes as this area suffers from mechanical damage due to the mounting protocol!
      Note: The easiest way to find the vessel is to focus on the threads that tighten the artery to the pipette. The threads will appear very bright (autofluorescence) and by looking at the loop one can derive whether focus is on the top or the lower part of the artery.
    8. Select the Z-range (total thickness of the imaged volume).
    9. Start the selected scan using the ‘record’ function.
    10. Record as many datasets as required.
    11. When ready with imaging make sure to rename and save the generated data.
    12. Copy the data to one of the processing computers.
    13. Shut down according to the TPLSM protocol. Don’t forget the fluorescent light bulb and the climate chamber!
      Note: The here described TPLSM settings should be considered as an example only. Settings of the TPLSM system strongly depend on the layout of the system and as such should be optimized specifically for every different system! Try to achieve maximum detection efficiency with lowest bleed through for the required channels (minimum of 2 channels, preferably 3-4 channels).

  6. Static experiment for LDL permeability (Figure 5)


    Figure 5. Schematic overview of experimental setup of flow experiment and static experiment

    1. After the mounting procedure, reduce the luminal pressure again and open the 3-way tab on the exit side.
    2. Remove the 3-way connector on the inlet side.
    3. Remove any air from the inlet Luer-connector (using a 1 ml syringe filled with HBSS with a 14 G needle) of the pipette.
    4. Slowly inject mounted vessels with 50 µg/ml Dil-LDL, diluted in HBSS using a 1 ml syringe.
      Note: DiI is a fluorescent lipophilic tracer attached to LDL with an excitation of 549 nm and emission of 565 nm.
    5. Re-pressurize the artery to 80 mmHg using the sphygmomanometer.
    6. Place the sphygmomanometer outside on top of the climate box and keep an eye on it while imaging. A drop in pressure means that the system is not completely closed and therefore leaky.
    7. Place the mounted artery on the microscope stage inside the climate box.
    8. Incubate for 90 min at 37 °C.
    9. Perfuse the vessel with HBSS to wash away non-bound LDL (similar procedure as previously described injection of Dil-LDL).
    10. Inject anti-CD31 eFluor450 antibody (labeling endothelial cells, more specifically endothelial junctions): same procedure as previously described injection of Dil-LDL.
    11. Incubate for 10 min at 37 °C.
    12. Perfuse the vessel with HBSS to wash away non-bound antibody (similar procedure as previously described injection of Dil-LDL), re-pressurize and place it again on the microscope stage.
      Note: Flushing or loading of the vessel is best performed outside the climate chamber with help of a stereomicroscope.
    13. Start recording (see image acquisition section above).
    14. When analyzing permeability, one needs to scan the whole artery systematically! Keep the field-of-view (f.o.v.) size and scanned volume equal and move field by field through the artery until you have reached the other pipette.
      Note: Discard the last f.o.v. closest to the pipettes because of potential damage due to the mounting procedure.
    15. Quantification of LDL uptake is performed after the experiments in recorded xyz-scans using processing software (Figure 6).
      Alternative: Any image processing software that enables working with multidimensional datasets will suffice.
      Note: Approximately 25% of the overall artery can be visualized from one side when using f.o.v. of 450 x 450 µm and when scanning from tunica adventitia to the lumen until both vessel wall borders disappear from the field of view (see Figure 6C for an impression of the scanned volume). In case a total overview of the artery is required, one can turn the carotid artery mechanically with 90°, 180° and 270° by simultaneous turning of both pipettes (careful!). It is not advised to focus directly through the artery to reach the ‘bottom’ because this will strongly reduce the image quality and increase risk of focusing too deep/destroying the sample.


      Figure 6. Examples of TPLSM imaging of bone marrow derived leukocyte adhesion of two classes (green; wildtype leukocytes, red; knock out leukocytes) in carotid artery after the flow assay. A. XZ projection; B. XY projection; C. 3D reconstruction, and D. YZ projection of a single field-of-view. The arterial wall extracellular matrix components collagen (bright blue, tunica adventitia) and elastin (blue, tunica media) are visualized using the intrinsic signals of autofluorescence and second harmonics generation. Scale bars = 100 µm.

  7. Dynamic (flow) experiment for cell recruitment (Figure 5)
    1. Connect the flow chamber with the common carotid artery side to the syringe pump using a 10 ml syringe, a male Luer-connector, a 3-way tab, 3 x 1 mm silicone tubing (1 m), and a female Luer-connector (from the syringe to the 3-way connector attached to the chamber). The pump can be placed on top of or next to the climate chamber and should be tilted slightly to make sure the syringe empties completely. Use some extra length of 3 x 1 mm silicone tubing (1 m) and make a loop inside the climate chamber (with durapore tape) in order to preheat the medium or cell suspension to 37 °C before it reaches the artery.
    2. Make sure the whole system is air-free and filled with HBSS before attaching it to the artery!
    3. Connect the exit side of the vessel/chamber to the water column using another 3-way tab and 3 x 1 mm silicone tubing. Make sure the water column is filled with HBSS and positioned at the correct height (black line). Use a 50 ml tube attached to the clamp and the column at 80 cm height above the position of the artery to collect the ‘flushed’ fluids. Tape the 3 x 1 mm silicone tubing to the falcon to stabilize the position of the silicone tubing going into the 50 ml tube.
    4. Select the appropriate flow rate on the pump (see manual). Make sure you select the correct syringe (size and brand), flow direction (push) and total volume (5.5 ml, dependent on the experiment).
    5. For mimicking arterial flow rate set the pump to 0.54 ml/min (calculated based on flow rates and average carotid diameters in vivo).
    6. Open the 3-way tabs in the appropriate way and perform a test run with medium only to check the open flow system. Fluid should slowly drip out of the 3 x 1 mm silicone tubing into the 50 ml tube on the water column.
    7. Change the syringe with a similar one containing the pre-prepared cell suspension mix. Make the Luer-connector and 3-way tab air free using a 14 G needle and an HBSS filled 1 ml syringe. Apply a total volume of 6 ml of cell suspension mix, which allows for a flow time of 10 min.
      Note: Make sure that every time you change syringes there are no air bubbles introduced to the system as air bubbles will alter or destroy the endothelial cells of the arteries! Air bubbles may be avoided by carefully removing air/filling the outer end of Leur-connectors with HBSS (using a 1 ml syringe with HBSS and a 20 G needle) before (re)connecting syringes or 3-way tabs. Also make sure that the silicone tubing is properly filled with HBSS before attaching it to the chamber.
    8. When image acquisition settings are set, start the flow procedure.
    9. When a live recording of the flow assay is needed, start the xyzt scan.
      Note: Adhesive cell will be round-shaped and flowing cells in the lumen will appear as stripes due to the laser scanning methodology of the TPLSM.
    10. Once the cell suspension has run out, replace the cell suspension syringe with the HBSS containing syringe and start the flow again for 3 min (or approximately 2 ml). The latter is to avoid cell loss in the dead volume of the silicone tubing on the pump side and to reduce the number of non-adhesive cells in the luminal space.
      Alternative: One could add specific antibodies to the washing flush of HBSS to visualize additional targets or structures of interest.
    11. For still xyz-images (Figure 6), stop the pump.
    12. When analyzing cell recruitment, one needs to scan the whole artery systematically! Keep the field of view (f.o.v.) size and scanned volume equal and move field by field through the artery until you’ve reached the other pipette (discard the f.o.v.’s closest to the pipettes).
    13. Counting of adhesive cells can be performed live (while scanning through the artery; 2 observers are needed and always record some reference xyz-scans) or after the experiments in recorded xyz-scans (Figure 6) using processing software (Leica LasX)
      Notes:
      1. Approximately 25% of the overall artery can be visualized from one side when using f.o.v. of 450 x 450 µm and when scanning from tunica adventitia to the lumen until both vessel wall borders disappear from the field of view (see Figure 6C for an impression of the scanned volume). In case a total overview of the artery is required, one can turn the carotid artery mechanically with 90°, 180° and 270° by simultaneous turning of both pipettes (careful!). It is not advised to focus directly through the artery to reach the ‘bottom’ because this will strongly reduce the image quality and increase risk of focusing too deep/destroying the sample.
      2. Optimally, the cell count is performed by two blinded observers.
      3. Required time is approximately 2.5 h per artery: from isolation of the artery and preparation of the cell suspension, up to counting of adhesive cells on 50% of the endothelial surface. In case detailed imaging of additional targets is required, another 30-60 min should be added to the total time. As a result, one may realistically perform 4 flow experiments per working day. Arteries can be stored on ice or in a fridge for a while (up to 4 h did not change the live/dead staining pattern; Megens et al., 2007) but in order to improve reproducibility of the experiments, it is advisable to process them as quickly as possible after isolation.
    14. Repeat the whole procedure for the carotid artery derived from the 2nd donor (either wildtype or knock-out mouse) mounted in a 2nd arteriography chamber.
      Note: To reduce the influence of experimental timing and vessel chamber properties it is best to set up the next round of experiments with alternating frequency (i.e., use the 2nd mouse type as the first one in the initially used chamber) and alternate the applied cell markers for staining the isolated leukocytes subsets (i.e., green becomes red in the next series of experiments).

Data analysis

  1. Analyses of Dil-LDL extravasation
    1. Quantification of the number and size of junctional Dil-LDL particles per cell was performed using Leica LASX 3.11 software.
      Note: Any image processing software that enables multidimensional analyses will be useable for this method.
    2. Recorded xyz-stacks were recombined into maximum intensity projections, transforming the 3D information into a resultant 2D image showing all cell junction in the xy-plane (Figure 7A).


      Figure 7. Example of TPLSM imaging of Dil-LDL extravasation in the carotid artery. A. Max. image showing XY projection (Dil-LDL in Red and CD31 in Blue). The arterial wall extracellular matrix components collagen (white, tunica adventitia) and elastin (green, tunica media) are visualized using the intrinsic signals of autofluorescence and second harmonics generation. Image also includes analysis method showing selection of ROIs (red arrows). Scale bar = 50 µm. B. Example of intensity scheme showing individual peaks representing the Dil-LDL particles.

    3. A further reduction of back ground noise was achieved using a blur filter (kernel size 3).
    4. Ten individual endothelial cells were chosen based on the junctional CD31 signal and blinded from the LDL signal by temporarily excluding the spectral channel representing the Dil-LDL particles.
    5. A polyline was drawn around each selected cell based on its CD31 junctional staining, resulting in specific regions of interest for each individual cell (Figure 7A, red arrows).
    6. The polyline selection was used to generate intensity profiles (Figure 7B).
    7. A threshold of the Dil-LDL channel was set just above the background intensity to exclude background noise.
      Note: The specific threshold level will vary depending on the experimental background level and has to be determined individually.
    8. The total number of individual peaks in the intensity profile represented the number of particles, the full width of each peak was used to estimate the size of junctional Dil-LDL particles.
      Note: Dead cells were excluded and the cells used for the quantification did not share any cellular junctions.

Recipes

  1. Lysis buffer
    155 mM NH4Cl
    10 mM KHCO3
    0.1mM EDTA
    Dissolved in 1 L water (MilliQ), pH 7.2

Acknowledgments

The study was financially supported by the DFG (SFB1123 TB Z01, A01 and B03 and INST409/97-1 FUGG) and the Alexander von Humboldt Foundation. Christian Weber is supported by the ERC.

References

  1. Bloksgaard, M., Leurgans, T. M., Nissen, I., Jensen, P. S., Hansen, M. L., Brewer, J. R., Bagatolli, L. A., Marcussen, N., Irmukhamedov, A., Rasmussen, L. M. and De Mey, J. G. (2015). Elastin organization in pig and cardiovascular disease patients' pericardial resistance arteries. J Vasc Res 52(1): 1-11.
  2. Boerboom, R. A., Krahn, K. N., Megens, R. T., van Zandvoort, M. A., Merkx, M. and Bouten, C. V. (2007). High resolution imaging of collagen organisation and synthesis using a versatile collagen specific probe. J Struct Biol 159(3): 392-399.
  3. Döring, Y., Noels, H., Mandl, M., Kramp, B., Neideck, C., Lievens, D., Drechsler, M., Megens, R. T., Tilstam, P. V., Langer, M., Hartwig, H., Theelen, W., Marth, J. D., Sperandio, M., Soehnlein, O. and Weber, C. (2014). Deficiency of the sialyltransferase St3Gal4 reduces Ccl5-mediated myeloid cell recruitment and arrest: short communication. Circ Res 114(6): 976-981.
  4. Karshovska, E., Zhao, Z., Blanchet, X., Schmitt, M. M., Bidzhekov, K., Soehnlein, O., von Hundelshausen, P., Mattheij, N. J., Cosemans, J. M., Megens, R. T., Koeppel, T. A., Schober, A., Hackeng, T. M., Weber, C. and Koenen, R. R. (2015). Hyperreactivity of junctional adhesion molecule A-deficient platelets accelerates atherosclerosis in hyperlipidemic mice. Circ Res 116(4): 587-599.
  5. Megens, R. T., Oude Egbrink, M. G., Cleutjens, J. P., Kuijpers, M. J., Schiffers, P. H., Merkx, M., Slaaf, D. W. and van Zandvoort, M. A. (2007). Imaging collagen in intact viable healthy and atherosclerotic arteries using fluorescently labeled CNA35 and two-photon laser scanning microscopy. Mol Imaging 6(4): 247-260.
  6. Megens, R. T., Oude Egbrink, M. G., Merkx, M., Slaaf, D. W. and van Zandvoort, M. A. (2008). Two-photon microscopy on vital carotid arteries: imaging the relationship between collagen and inflammatory cells in atherosclerotic plaques. J Biomed Opt 13(4): 044022.
  7. Megens, R. T., Reitsma, S., Schiffers, P. H., Hilgers, R. H., De Mey, J. G., Slaaf, D. W., oude Egbrink, M. G. and van Zandvoort, M. A. (2007). Two-photon microscopy of vital murine elastic and muscular arteries. Combined structural and functional imaging with subcellular resolution. J Vasc Res 44(2): 87-98.
  8. Ortega-Gomez, A., Salvermoser, M., Rossaint, J., Pick, R., Brauner, J., Lemnitzer, P., Tilgner, J., de Jong, R. J., Megens, R. T., Jamasbi, J., Doring, Y., Pham, C. T., Scheiermann, C., Siess, W., Drechsler, M., Weber, C., Grommes, J., Zarbock, A., Walzog, B. and Soehnlein, O. (2016). Cathepsin G controls arterial but not venular myeloid cell recruitment. Circulation 134(16): 1176-88.
  9. Reitsma, S., Oude Egbrink, M. G., Heijnen, V. V., Megens, R. T., Engels, W., Vink, H., Slaaf, D. W. and van Zandvoort, M. A. (2011). Endothelial glycocalyx thickness and platelet-vessel wall interactions during atherogenesis. Thromb Haemost 106(5): 939-946.
  10. Schmitt, M. M., Megens, R. T., Zernecke, A., Bidzhekov, K., van den Akker, M. N., Rademakers, T., van Zandvoort, M. A., Hackeng, T. M., Koenen, R. R. and Weber, C. (2014). Endothelial JAM-A guides monocytes into flow-dependent predilection sites of atherosclerosis. Circulation 129(1): 66-76.
  11. Schober, A., Nazari-Jahantigh, M., Wei, Y., Zhe, Z., Gremse, F., Grommes, J., Megens, R. T. A., Heyll, K., Thiemann, A., Iruela-Arispe, M. L., Wang, S., Kiessling, F., Olson, E. N. and Weber, C. (2014). MicroRNA-126-5p promotes endothelial proliferation and limits atherosclerosis by suppressing Dlk1. Nat Med 20(4): 368-376.
  12. Soehnlein, O., Wantha, S., Simsekyilmaz, S., Doring, Y., Megens, R. T., Mause, S. F., Drechsler, M., Smeets, R., Weinandy, S., Schreiber, F., Gries, T., Jockenhoevel, S., Moller, M., Vijayan, S., van Zandvoort, M. A., Agerberth, B., Pham, C. T., Gallo, R. L., Hackeng, T. M., Liehn, E. A., Zernecke, A., Klee, D. and Weber, C. (2011). Neutrophil-derived cathelicidin protects from neointimal hyperplasia. Sci Transl Med 3(103): 103ra198.
  13. Spronck, B., Megens, R. T., Reesink, K. D. and Delhaas, T. (2016). A method for three-dimensional quantification of vascular smooth muscle orientation: application in viable murine carotid arteries. Biomech Model Mechanobiol 15(2): 419-432.
  14. Subramanian, P., Karshovska, E., Reinhard, P., Megens, R. T., Zhou, Z., Akhtar, S., Schumann, U., Li, X., van Zandvoort, M., Ludin, C., Weber, C. and Schober, A. (2010). Lysophosphatidic acid receptors LPA1 and LPA3 promote CXCL12-mediated smooth muscle progenitor cell recruitment in neointima formation. Circ Res 107(1): 96-105.
  15. Weber, C., Meiler, S., Doring, Y., Koch, M., Drechsler, M., Megens, R. T., Rowinska, Z., Bidzhekov, K., Fecher, C., Ribechini, E., van Zandvoort, M. A., Binder, C. J., Jelinek, I., Hristov, M., Boon, L., Jung, S., Korn, T., Lutz, M. B., Forster, I., Zenke, M., Hieronymus, T., Junt, T. and Zernecke, A. (2011). CCL17-expressing dendritic cells drive atherosclerosis by restraining regulatory T cell homeostasis in mice. J Clin Invest 121(7): 2898-2910.

简介

这种复杂和多功能方法的主要目的是在活体动物的生理条件下可视化和量化结构血管壁性质,细胞募集和脂质/葡聚糖外渗。这对于广泛的炎症,高血压,动脉粥样硬化甚至制药行业的研究人员都是有兴趣的。目前,许多研究人员正在使用体外技术来评估细胞募集,如具有培养细胞的transwell或流动室系统,具有不清楚的生理学可比性。这里介绍的方法详细描述了使用复杂而灵活的方法来研究在动脉血流条件下体外新鲜和活的鼠颈动脉中的动脉壁特性和白细胞募集。该模型模拟体内情况,并允许使用从两个不同供体分离的细胞和动脉(例如,野生型与特异性敲除)组合成一个实验,从而提供关于白细胞和/或内皮细胞特性的信息两个捐助者。因此,该模型可以被认为是复杂和侵入性体内研究的替代方案,例如对比实验。
【背景】该方法的核心是应用双光子激光扫描显微镜(TPLSM)来显示安装在动脉造影腔室中的体外颈动脉,其已显示模拟体内情况下存在的生理条件(Megens et al。,2007)。新鲜的动脉,在我们这种情况下是小鼠颈动脉,但该方法也适用于其他类似大小的血管(包括人类血管)(Bloksgaard等,2015),仔细提取并安装在使用细线的两个玻璃微量移液管的动脉造影室。该腔室应提供足够的空间以用显微镜物镜(优选为具有> 1mm的工作距离的浸水或浸没物镜)进入动脉。在应用腔内压力(80mmHg)和随后校正由于分离引起的长度缩短(Megens等人,2007)之后,可以将多种感兴趣的荧光标记的细胞和/或血管壁组分灌注到血管无论是在流动下还是在静态条件下。这使得用户能够计算贴壁白细胞亚群的数量,B)确定分子外渗(葡聚糖,脂质)进入动脉壁,C)使用荧光共轭(特异性)抗体或源自细胞外基质成分,D)结合前述目标。
该方法的一般基础在2007年描述(Megens et al。,2007)。从那时起,这种方法已被用于几个科学出版物中,例如在控制和炎症条件下显示细胞募集(Schmitt等,2014),趋化因子存在(Soehnlein等,2011),平滑肌细胞检测(Subramanian内皮细胞沉积(Ortega-Gomez等,2016),粘附血小板(Karshovska等,2015) ,分叉中的动脉粥样硬化病变(Megens等人,2007和2008; Weber等,2011),内皮糖萼的可视化(Reitsma等人,2011)或细胞外基质标记物的评估(Boerboom等人, 2007; Megens et al。,2007)。
TPLSM成像可以在灌注前,灌注和/或灌注后进行。显微镜系统的设置强烈依赖于可用的显微镜​​。我们利用现代Leica SP5II MP系统与20x WD物镜和Ti:Sa脉冲激光器,允许以视频速率进行4通道成像。然而,这不是要求应用这种方法,因为较旧的,装备不善的TPLSM系统也足够了。
近年来,我们进一步推进了该方法,适用于研究颈动脉颈动脉特异性细胞类型的募集(Döring等,2014; Schmitt等,2014; Karshovska等,2015)。这种方法不仅可以使用户能够通过差异荧光标记(使用细胞跟踪器或等同物)来特异性地并且同时调查各种细胞类型如单核细胞,嗜中性粒细胞或T细胞的募集到高度生理的内皮,还可以使特定的动脉和来自不同(野生型与敲除)小鼠亚群的细胞分离(血液,骨髓)。因此,创建了一个系统,可以定义对招募的影响是否由血管和/或造血缺陷介导。除了细胞募集的功能读出之外,该方法还能够同时或随后的血管结构的标记和亚细胞分辨率成像以及化合物在血管壁中的存在。结果,改变的粘附性可以直接与特定靶的存在或不存在相关联。
通过同时应用各种荧光标记的白细胞亚群,实验条件对于每种细胞类型是相等的,从而限制了由于例如流动模式差异的实验变化(数据可以以绝对数或比率表示)。后者还限制了所需实验的数量,并结合减少数量的必需实验动物,因为炎症细胞和动脉可以从相同的动物中分离,最终该方法减少了所需动物的数量。
除了细胞募集实验,我们开发了使用荧光标记的低密度脂蛋白颗粒或葡聚糖的应用来可视化和量化可行(患病)动脉中的脂质或葡聚糖外渗。最后,与大动脉的体内成像不同,该离体模型不像体内情况那样遭受动脉壁的不想要的运动,从而允许用亚细胞分辨率进行成像。此外,可以在实验期间的任何给定时间施加刺激物或特定染料,使研究人员能够充分灵活地定制方法并达到所需目标。

关键字:成像, 双光子激光扫描显微镜检查, 动脉搏描计室, 细胞招募, 脂质外渗

材料和试剂

  1. 分离颈动脉
    1. 固定胶带Durapore 1.25厘米(3M,目录号:1538-0)
    2. 35 x 10 mm(2 x)(Corning,Falcon ®,目录号:353001)的聚苯乙烯盘
    3. Hanks平衡盐水溶液(HBSS)与CaCl 2和MgCl 2(Thermo Fisher Scientific,目录号:1402550),pH 7.4

  2. 细胞悬浮液
    1. 50ml管(3×)(SARSTEDT,目录号:62.547.254)
    2. 针27 G x 1.5(灰色)(BD,目录号:301629)
    3. 注射器10 ml(1 x)(BD,Discardit TM 目录号:309110)
    4. 细胞过滤器50μm(Sysmex,CellTrics ®,目录号:25004-0042-2317)
    5. Hanks平衡盐水溶液(HBSS)与CaCl 2和MgCl 2(Thermo Fisher Scientific,目录号:1402550),pH 7.4
    6. 荧光细胞标记物:细胞追踪器绿(Thermo Fisher Scientific,Invitrogen公司,目录号:C7025)和Red(Thermo Fisher Scientific,Invitrogen公司,目录号:C34565) br />
    7. 氯化铵(NH 4 Cl)(Sigma-Aldrich,目录号:A9434)
    8. 碳酸氢钾(KHCO 3)(Sigma-Aldrich,目录号:60339)
    9. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E6758)
    10. 裂解缓冲液(见配方)

  3. 动脉的安装
    1. 玻璃蚀刻材料(NH 4 HF 2或NH 4 F·HF)(Carglass,目录号:ZB10 EI0003)
    2. 针14 G x 80 mm,缩短并钝化至40 mm和50 mm(Braun Sterican 14 G x 80 mm)(B.Braun Medical,目录号:4665473)
    3. 尼龙线Ø〜20μm,用于绑扎血管(Living Systems Instruments,目录号:THR-G)
    4. 三路标签(2 x)(B.Braun Medical,目录号:4095111)
    5. 注射器1ml(BD,Plastipak TM,目录号:300026)
    6. 硅胶管3 x 1毫米(Carl Roth,目录号:9556.1)切成0.5米长度
    7. Hanks平衡盐水溶液(HBSS)与CaCl 2和MgCl 2(Thermo Fisher Scientific,目录号:1402550),pH 7.4

  4. 分子外渗
    1. 硅胶管3 x 1 mm切割至1.0米长(Carl Roth,目录号:9556.1)
    2. 针20 G x 1.5(黄色)(BD,目录号:301300)
    3. 安全锁管1.5 ml(3 x)(Eppendorf,目录号:0030120086)
    4. 注射器1ml(2×)(BD,Plastipak TM,目录号:300026)
    5. 三向标签(B.Braun Medical,目录号:4095111)
    6. 固定胶带Durapore 1.25厘米(3M,目录号:1538-0)
    7. 人类Dil-LDL(Kalen Biomedical,目录号:770230-9)
    8. Hanks平衡盐水溶液(HBSS)与CaCl 2和MgCl 2(Thermo Fisher Scientific,目录号:1402550),pH 7.4
    9. 直接缀合的抗CD31/eFluor450(PECAM:Thermo Fisher Scientific,eBioscience TM,目录号:48-0311-82)
      替代方案:直接缀合的抗CD54/A488(ICAM:BioLegend,目录号:116112)或抗CD106/A594(VCAM:BioLegend,目录号:105724)

  5. 流量分析
    1. 硅胶管3 x 1 mm切割至1.5 m和1.0 m长度(Carl Roth,目录号:9556.1)
    2. 针20 G x 1.5(黄色)(BD,目录号:301300)
    3. 注射器1 ml(1 x)(见试剂C5)和10ml(2 x)(BD,Discardit TM,目录号:309110)
    4. 三路标签(2 x)(B.Braun Medical,目录号:4095111)
    5. 50ml管(2×)(SARSTEDT,目录号:62.547.254)
    6. 固定胶带Durapore 1.25厘米(3M,目录号:1538-0)
    7. Hanks平衡盐水溶液(HBSS)与CaCl 2和MgCl 2(Thermo Fisher Scientific,目录号:1402550),pH 7.4

设备

  1. 分离颈动脉

    1. FST学生弹簧剪(精细科学工具,目录号:91500-09)
    2. 杜邦镊子(2 x)(精细科学工具,目录号:91150-20)
    3. 剪刀(精细科学工具,目录号:14084-08)
    4. 立体显微镜Leica S8 Apo(LED光源和0.63x物镜)(Leica Microsystems,型号:Leica S8 Apo)

  2. 细胞悬浮液
    1. 计时器
    2. 笔(见设备A1)
    3. 剪刀(精细科学工具,目录号:91401-12)
    4. 1 ml移液器(Eppendorf,目录号:3120000062)
    5. 离心机(Eppendorf,型号:5430)
    6. 细胞计数室(Neubauer)
    7. 细胞培养显微镜(Leica DMi1,相位对比度和10倍NA0.3目标)(Leica Microsystems,型号:Leica DMi1)
    8. 流式细胞仪(BD,型号:BD FACSCANTO SYSTEM)

  3. 动脉的安装
    1. 动脉造影室(2 x,荷兰马斯特里赫特大学IDEE ©
    2. 玻璃移液管1.5 x 0.86 mm(2 x,哈佛仪器,目录号:30-0057)
    3. 移液器拉杆(NARISHIGE,目录号:PP-830)
    4. 移液器研磨机(荷兰马斯特里赫特大学IDEE ©
      替代方案:现成的玻璃移液器(Living Systems Instruments,目录号:GCP-300-325)
    5. 硅胶套件,73清除(Farnell,目录号:101705)
    6. 血压计(Riester,Big Ben ®,目录号:1453-100)适应于500ml空气室(Schott)和Luer连接器,以安装1 x 3 mm硅胶管(IDEE © ; ,马斯特里赫特大学,荷兰)
    7. Luer耦合适配器母头(1x)(Carl Roth,目录号:CT62.1)
    8. 立体显微镜Leica S8 Apo(带有LED光源和0.63x物镜)(Leica Microsystems,型号:Leica S8 Apo)

  4. 显微镜
    1. 基于DM6000FS显微镜支架的市售徕卡SP5IIMP激光扫描显微镜系统(有关更多信息,请参阅制造商网站:
      https://www.leica-microsystems.com/products/confocal-microscopes/details/product/leica-tcs-sp5-ii/downloads/
      替代方案:使用≥2个检测器中的光谱特异性的任何功能性TPLSM和具有足够工作距离(≥1mm)的水浸物体可以与本文所述的测定结合使用。
    2. 光谱物理MaiTai DeepSee Ti:Sa激光源
    3. 徕卡混合探测器(4 x)
    4. Luigs和Neumann SM7电动显微镜舞台
    5. 徕卡20x NA1.00WD目标
    6. 鲁丁气候控制/激光安全箱

  5. 分子外渗
    1. 定时器(见设备B1)
    2. 笔(见设备A1)
    3. Luer耦合适配器:男性(1 x)(Carl Roth,目录号:CT58.1)和女性(1 x)(Carl Roth,目录号:CT62.1)
    4. 注射器输液泵(Harvard Apparatus pump11 elite)(Harvard Apparatus,catalog number:70-4504)

  6. 流量分析
    1. Luer耦合适配器:男性(2 x)(Carl Roth,目录号:CT58.1)和女性(1 x)(Carl Roth,目录号:CT62.1)
    2. 注射器输液泵(Harvard Apparatus pump11 elite)(Harvard Apparatus,catalog number:70-4504)
    3. 立柱与夹具(未知品牌,长度100厘米,占地面积15 x 25厘米)
    4. 具有excel和/或用于细胞计数的纸的Pc

软件

  1. Leica LAS AF 2.6采集软件
  2. 徕卡LAS X 3.11图像处理软件(含3D分析包);离线
    注意:可以使用允许用户处理多维数据的任何处理软件包。

程序

  1. 颈动脉分离
    使用立体显微镜(见设备A5),按照当地的伦理委员会指南,分离小鼠颈动脉(分离后的长度,优选3-6mm),并将它们放置在聚苯乙烯盘(Ø35mm)中的HBBS(pH 7.4)中(见图1详细信息)。


    图1.颈动脉的分离将安乐死的小鼠固定在立体显微镜(A)上,并使用剪刀(14084-08)去除颈部皮肤,并使用湿纸巾(B )。下颌腺(I)变得可见。 C.将甲状腺(I)的一侧拉到外面,用Dumont镊子去除肌肉胸锁乳突肌(II)和肌肉XXXX(III)。 D.在一些细透明层下面,颈总动脉变得可见(IV)。仔细清除动脉上的组织,检查是否存在非典型侧分支。 E.此外,仔细地从分叉(绿色箭头)移除组织,并用蓝色线解剖清洁的颈总动脉(使用弹簧剪刀)。通过外端(杜蒙镊子)之一握住解剖的动脉,并将其放入填充有HBSS的小盘中以保持其潮湿。隔离后,动脉必须保持凉爽(冰箱或冰)直至进一步处理
    有一些必须满足的先决条件,对于维持动脉生存能力和TPLSM成像至关重要:
    1. 必须不惜一切代价避免容器受到挤压或过度拉伸造成的机械损伤,因为这将立即导致结构和功能的变化。
    2. 空气与动脉的接触应尽量减少。隔开动脉同时保持伤口潮湿,解剖后迅速将动脉转移到培养基中。
    3. 动脉必须非常干净才能进行成像。在动脉外侧的任何"污垢",头发或组织将导致其下面的动脉部分的可视性丧失。所以工作细致,也清除覆盖动脉的薄片结缔组织 注意:不推荐像通常用于收获主动脉的小鼠的循环冲洗,因为它可能会在影响内皮细胞的动脉内腔中引入气泡。冲洗可以通过临时安装(使用镊子,因此没有螺丝将其固定在适当位置)直接在隔离后进行,将灌注腔中的无空气单个玻璃移液管上的公共部分轻轻地冲洗通过其进行HBSS。然而,这必须在颈动脉分离后迅速(30分钟内)完成,以避免血块堵塞! (另见"动脉安装"一节中的程序。)

  2. 分离骨髓来源的白细胞
    1. 按照当地的伦理委员会指南,分离小鼠的股骨和/或胫骨,将其置于HBBS(pH 7.4)中。
      注意:最方便的隔离方法是首先从后肢去除皮肤,然后拉扯后肢脱位髋关节。之后,通过在臀部区域切割,很容易去除后肢,没有切割骨的危险。去除后,剩下的肌肉和组织可以安全地取出。
    2. 用剪刀切断骨骼的骨骼(头),使骨髓腔开放。
    3. 用27 G针和10ml注射器将HBSS的骨头冲洗成50ml管 注意:所有骨髓细胞从骨骼中移除是很重要的。使用所需的体积,直到几乎所有的细胞(红色)被清除。
    4. 通过使用1 ml移液管重悬细胞来制备单细胞溶液。
    5. 用50μm细胞过滤器过滤重悬的骨髓细胞,以减少最终细胞悬浮液中的组织
    6. 以300×g离心细胞5分钟。
    7. 用5ml裂解缓冲液(为了裂解红细胞)重悬细胞沉淀,并在室温下孵育1分钟。
    8. 以300×g离心细胞5分钟。
      注意:如果需要,可以根据实验室特异性方案分离不同的白细胞亚群(例如单核细胞,嗜中性粒细胞和淋巴细胞)。
    9. 通过在HBSS中稀释细胞跟踪器1:1,000来准备细胞追踪器解决方案。
      注意:细胞跟踪器的不同颜色可用于区分白细胞亚群(例如,单核细胞,嗜中性粒细胞和淋巴细胞)或不同白细胞来源之间(例如野生型与淘汰型) />
    10. 将细胞重悬于准备的细胞追踪器溶液中(总体积为3ml /白细胞亚群/来源)
    11. 在室温下孵育15分钟。
      注意:



















      1. 也可以使用其他细胞标记物组合,例如钙黄绿素(eBioscience)和罗丹明(Sigma-Aldrich)或其他细胞跟踪剂组合。
    12. 在染色程序后洗涤细胞悬浮液两次,重悬于3ml HBSS中,使用50ml管,以300×g离心5分钟。
      注意:洗涤细胞是至关重要的,以避免造成造成对比度损失的准备的细胞悬浮液中未结合的细胞追踪器导致血管壁的整体染色!
    13. 使用Neubauer室和显微镜(相位对比)计数每个白细胞亚群/来源的细胞总数。
    14. 混合两种类型的白细胞(2种不同的亚类或来源,用不同的颜色标记以区分它们)以达到在总共6ml HBSS中重新悬浮的1:1比例。
    15. 每个白细胞组的细胞密度应为1.5×10 6个白细胞(因此,每个流式测定的悬浮液中的细胞总数在6ml HBSS中为3×10 6) 。
    16. 通过流式细胞术分析所得到的白细胞悬浮液,以确认1:1的比例。

  3. 动脉造影室的准备
    1. 通过使用移液器拉拔器(加热器= 60;重量= 3×大块)和研磨机设置,从0.58 x 1 x 80 mm玻璃管中拉出并研磨移液器。目标是产生长度为40mm和50mm的移液管,并且尖端直径为250-350μm,具有45°的尖角。可以使用任何明视野显微镜(≈100x放大倍率)和比例尺幻灯片检查尖端尺寸和角度(见图2)。


      图2.微量移液器构造 A.概述用于自制灌注腔的图像微量移液器。将玻璃微量移液管胶合成钝化并缩短的14 G针(绿色,总长约50 mm)。 B.玻璃移液管从钝头针上伸出大约0.8-10毫米。 C.关闭尖端直径为250-300微米的移液管尖端,并研磨以获得45度角。在胶合和研磨之后,蚀刻尖端以产生粗糙的玻璃表面(不可见),为安装动脉提供更好的摩擦。

    2. 将研磨的移液管的尖端通过NH 4或HF 4 HF/HF或HF 4 H 3 F·HF化合物吸到研磨的移液管的尖端(一个罐简单地将开头的容器中的化合物中的尖端保持几毫米)。 (10-20分钟或蚀刻工艺成功时),手术后用蒸馏水小心冲洗干燥(RT)。蚀刻的技巧可以使动脉更稳定地绑在玻璃上。
      注意:由于动脉造影室设计的差异,各种腔室的移液器的制备和性质不同。这种方法的移液器最重要的特性是尖端开口直径和尖端的45°角形状,因为两者直接影响动脉内的流动模式。
    3. 通过使用小文件使圆形边缘切割14 G针,然后使用小的钳子打破尖端,并使用砂纸去除任何锋利的边缘。针应该有40毫米和50毫米的长度(50毫米为腔室的调节器一侧)
    4. 使用硅胶套将玻璃移液器胶入缩短且钝的20 G针中。确保玻璃和钢针之间的空间完全关闭,以确保结构的气密性。此外,确保玻璃移液管的非磨削外边缘刚到达20 G针的Luer-lock连接器,以避免碎屑堆积在构件内的未填充空间中。
    5. 在手术后用蒸馏水小心冲洗造成的结构,并将其安全存放在动脉造影室。

  4. 动脉的安装
    1. 通过在聚苯乙烯培养皿(Ø100 mm)中将一些胶带(5厘米)上下颠倒(粘稠的一面)准备好。将上下带的外端粘贴到聚苯乙烯盘上,并放上一些短片。
    2. 使用立体显微镜在线盘(图3)中准备螺纹(Ø〜20μm;活体系统仪器)(见设备A5)。螺纹由1)弯曲直线,并通过将外端彼此交叉而形成一个圆,2)将一个外端两次穿过圆圈。


      Figur e 3。用于安装 a n r t的尼龙线的制备 e r y A.程序所需材料概述10厘米聚苯乙烯培养皿(I),带有Durapore带和8-10毫米长的尼龙线(Ø≈20微米:箭头); 2杜蒙钳(II)和尼龙线(III)。 B-F。近距离的带有尼龙线的Durapore胶带展示了螺纹准备的逐步程序; B.线程旁边的单线程(绿色箭头);曲线直线,并通过相互穿过外端做一个圆; D.使用胶带的粘性将一个外端放在圆(直径500-700μm)内,使其保持在适当的位置;一个外端穿过圆圈; F.通过重复步骤D和程序E线程就绪。将完成的线圈放在粘性Durapore上的一半,并将其一半放在胶带上以改进处理(螺纹是可见的并且不使用立体显微镜可分级)。

    3. 用70%乙醇(1x冲洗)和HBSS(2x冲洗)小心清洁家用(也可商购)的动脉灌注腔(图4A)的内部和移液管,将两个移液管上的三向标签连接到雌性Luer-Couplers,并用CaCl 2和MgCl 2(pH 7.4)填充HBSS培养基。确保两个移液器完全无空气,并留下1毫升注射器,填充介质,连接到腔室长度调节器侧面的3向接头。
    4. 将新鲜分离和清洁的颈动脉转移到灌注室,并将其置于立体显微镜(见设备A5)(见图4)。


      图 ure 4。 小鼠颈动脉 动脉 。 A.具有纵向长度调节器的灌注室(绿色箭头)的示例; B.使用HBSS填充干净的灌注腔,并在两个移液管上连接3路标签(I)。使用装有HBSS的1ml注射器(II)从移液管中取出任何空气。 C.使用立体显微镜观察灌注室的内部; D.用2个预先制备好的尼龙线将玻璃移液管关上,滑过它; E.安装但未加压动脉的概况。每个移液器使用两个螺纹。分岔(III)有助于确定正确的流向。 F.然后将安装的动脉在流出口(分叉侧)关闭并连接到流入口侧的血压计(IV),以显示所安装的动脉的任何泄漏。 G.逐步增加压力并校正两个移液管之间的距离(使用调节器,绿色箭头),直到动脉在80 mmHg处直线。注意步骤E中的动脉与程序G之间的长度差异,其中80mmHg的加压动脉长约30%。

    5. 使用立体显微镜将两个或三个线放在移液管上(见设备A5)。
    6. 将颈动脉的共同部分通过将其移动到腔室长度调节器侧的移液管上(不要太远!)。
    7. 拧紧两个或三个螺纹,使得动脉固定在玻璃尖端和螺纹之间。
    8. 通过颈动脉轻轻冲洗一些介质,以消除可能的血液残留物。
      注意:优选冲洗腔内的动脉,而不是总共冲洗鼠标,以限制内腔中的空气。
    9. 使用室外调节器调节两个移液器之间的距离(图4B;绿色)。
    10. 在2 nd 移液器上放置两条或三条线。
    11. 将其拉出移液管(图4B)将颈动脉的分叉侧安装。确保您已收紧所有未插管的分支,以创建闭路电路。
      注意:确保将动脉沿其生理方向安装用于流量测定,即流动从普通到分叉。流动方向的失败将导致假阳性或阴性结果。还要确保由于动脉扭曲而紧张地安装动脉!
    12. 打开出口侧(室的非长度调整侧)的3向接头,轻轻冲洗一些介质,以确定移液器是否打开(HBSS穿过动脉并从出口侧出来)并且动脉被安装稳定
    13. 将血压计与3 x 1毫米硅胶管和一个母Luer连接器连接到腔室的长度调节侧,并用一些HBSS填充压力计的3 x 1 mm管道。
    14. 调整3路连接器以创建可以使用压力计进行压力控制的闭路电路,并使用外部调节器调节展开容器的长度,逐步增加平均管腔内压力,直到达到100 mmHg。通过跟踪连接到血压计的3 x 1 mm硅胶管中的介质水平,注意安装的动脉的泄漏。
      注意:在整个系统中的某处泄漏会导致3 x 1 mm硅胶管中的缓冲液水平发生变化!
    15. 确保长度在100 mmHg下正确校正,然后将压力降低至80 mmHg(模拟动脉的生理伸展)。
    16. 全部安装正确后,请继续下一步。

  5. 双光子激光扫描显微镜(TPLSM)
    1. 将气候箱设置为37°C。确保气候箱内的温度传感器靠近物镜。要达到稳定的温度,气温控制箱必须大约启动。在第一次测量前1小时。这些实验不需要额外的CO 2/O 2/2气体控制。
    2. 按照协议开启系统(每台显微镜不同)。
    3. 将灌注室放置在显微镜平台上,并使用20x 1.00 WD物镜,荧光光源和RGB滤镜对焦于颈动脉。
      注意:注意不要过于深入,因为这样会导致样品,室的破坏和潜在的目标!
    4. 选择所需的光谱通道(通道1:胶原为400-430nm;弹性蛋白为440-500nm;绿色细胞追踪器为510-540nm;细胞跟踪器深红色为620-670nm)。
    5. 将激发激光(Ti:Sa)调谐至800 nm。
    6. 选择成像模式(XYZ),扫描速度(200-400Hz)和xyz分辨率(XY-像素格式根据奈奎斯特标准,Z步0.8-1.5μm)。要在流动/细胞粘附过程中创建动脉影像,请选择XYT或XYZT扫描,选择"最小化"时间间隔并最大化总体记录时间。此外,减少像素数量并将扫描速度提高到600Hz,以便具有合理的图像刷新率。
    7. 在"活"模式下选择颈动脉颈部感兴趣的区域;避免玻璃移液器旁边的第一个视野,因为这个区域由于安装协议而受到机械损坏!
      注意:找到血管的最简单的方法是集中在将动脉收紧到移液管上的螺纹上。线程将 显示非常明亮(自动荧光),并且通过查看循环,可以得出焦点是否位于动脉的顶部或底部 。
    8. 选择Z范围(成像体积的总厚度)。
    9. 使用'record'功能启动所选扫描。
    10. 记录所需的数据集数量。
    11. 当准备好成像时,请确保重命名并保存生成的数据。
    12. 将数据复制到其中一个处理计算机。
    13. 根据TPLSM协议关闭。不要忘记荧光灯泡和气候室!
      他在这里描述的TPLSM设置应该仅仅作为一个例子被认为是 。 TPLSM系统的设置在系统的布局上强烈地 专门为每一个不同的系统!尝试实现 > 频道)。

  6. LDL渗透性的静态实验(图5)

    图 ure 5.流程实验和静态实验的实验设置示意图

    1. 安装步骤后,再次降低管腔压力,并打开出口侧的3路标签。
    2. 卸下入口侧的3路连接器。
    3. 从移液器的入口Luer连接器(使用填充有HBSS的1 ml注射器,14 G针)取出任何空气。
    4. 缓慢注射装有50μg/ml Dil-LDL的血管,使用1ml注射器在HBSS中稀释。
      注意:DiI是连接到LDL的荧光亲脂性示踪剂,具有549nm的激发和565nm的发射。
    5. 使用血压计将动脉重新加压至80 mmHg。
    6. 将血压计放在气候箱的顶部,并在成像时保持眼睛。压力下降意味着系统不完全关闭,因此泄漏。
    7. 将安装的动脉放置在气候箱内的显微镜平台上。
    8. 在37°C孵育90分钟。
    9. 用HBSS清洗血管以清洗无限制的LDL(与前述Dil-LDL注射相似的程序)。
    10. 注射抗CD31 eFluor450抗体(标记内皮细胞,更具体地说是内皮结):与以前描述的Dil-LDL注射相同的程序。
    11. 在37°C孵育10分钟。
    12. 用HBSS清洗血清以洗去未结合的抗体(与前述Dil-LDL注射相似的步骤),再次加压并将其置于显微镜载物台上。
      注意:在立体显微镜的帮助下,冲洗或装载容器最好在气候室外进行。
    13. 开始录音(见上文图像采集部分)。
    14. 分析渗透性时,需要系统地扫描整个动脉!保持视野(f.o.v.)尺寸和扫描体积相等,并通过动脉逐场移动,直到达到其他移液管。
      注意:丢弃最后一个f.o.v.最靠近移液器由于安装程序而导致潜在的损坏。
    15. 在使用处理软件记录的xyz扫描实验后进行LDL摄取的定量(图6) 替代方案:任何能够处理多维数据集的图像处理软件就足够了。
      注意:当使用f.o.v时,可以从一侧可视化整个动脉的大约25%。 450×450μm,当从外膜到内腔扫描时,直到两个血管壁边缘从视野消失(参见图6C为扫描体积的印象)。如果需要对动脉的总体概况,则可以通过同时转动移液管(小心!)以90°,180°和270°的方式将颈动脉机械地转动。不建议通过动脉直接聚焦到"底部",因为这将大大降低图像质量,并增加深度/摧毁样本的聚焦风险。


      图 ure 6。来自两种类型的 (绿色 ;野生型白细胞,红色;敲击)的骨髓TPLSM成像实例 衍生白细胞粘附在流动测定之后,颈动脉中出现白细胞)。 B. XY投影; C.三维重建和D.YZ投影的单一视野。使用自发荧光和二次谐波产生的固有信号可视化动脉壁细胞外基质组分胶原(亮蓝色,外膜)和弹性蛋白(蓝色,中膜)。比例尺=100μm
  7. 细胞募集的动态(流)实验(图5)
    1. 使用10ml注射器,公Luer连接器,3向接头,3 x 1毫米硅胶管(1米)和母Luer连接器(1毫米)将流动室与颈总动脉侧连接到注射器泵从注射器到连接到腔室的3路连接器)。泵可以放置在气候室的顶部或旁边,并应稍微倾斜以确保注射器完全清空。使用一些额外长度的3 x 1毫米硅胶管(1米),并在气候室内(带有新加坡胶带)进行回路,以便将介质或细胞悬浮液预热至37°C,然后到达动脉。
    2. 确保整个系统无空气充满HBSS,然后再将其连接到动脉!
    3. 使用另一个三向接头和3 x 1毫米硅胶管将容器/腔室的出口侧连接到水柱。确保水柱充满HBSS并且位于正确的高度(黑线)。使用连接到夹具上的50ml管和位于动脉位置80厘米高处的柱以收集"冲洗"的液体。将3 x 1毫米硅胶管粘贴到猎鹰,以稳定硅胶管进入50ml管的位置。
    4. 在泵上选择合适的流量(参见手册)。确保选择正确的注射器(尺寸和品牌),流动方向(推动)和总体积(5.5 ml,取决于实验)。
    5. 为了模拟动脉血流速率,将泵设定为0.54ml/min(基于流速和平均颈动脉直径计算 我 v o )
    6. 以适当的方式打开三向选项卡,并用介质执行测试运行,以检查打开的流量系统。液体应慢慢从3×1毫米硅胶管中滴入水柱上的50ml管中
    7. 更换注射器与类似的包含预先准备好的细胞悬液混合物。使用14 G针和HBSS填充的1 ml注射器,使Luer连接器和3向接头空气畅通。使用总体积为6ml的细胞悬浮液混合物,其允许10分钟的流动时间。
      确保每次更换注射器时,气泡将不会引入到系统中,因为气泡会 或破坏动脉内皮细胞!在重新连接注射器或3号注射器之前,请仔细取出空气/填充Leur连接器的外端,使用HBSS(使用1 ml HBSS注射器和20 G针头)方式 标签。同时确保硅胶管在将其连接到室之前已正确填充HBSS。
    8. 设置图像采集设置时,启动流程。
    9. 当需要流式测定的实时记录时,启动xyzt扫描。
      ote:粘合细胞将是圆形的,由于TPLSM的激光扫描方法,流明的细胞将显示为条纹。
    10. 一旦细胞悬浮液用尽,用含有HBSS的注射器代替细胞悬液注射器,再次开始流动3分钟(或约2毫升)。后者是为了避免在泵侧的硅胶管的死体积中的细胞损失,并减少腔内空间中的非粘性细胞的数量。

    11. 对于静止的xyz图像(图6),停止泵。
    12. 分析细胞募集时,需要系统扫描整个动脉!保持视野(f.o.v.)大小和扫描体积相等,并通过动脉逐场移动,直到您到达其他移液器(丢弃最接近移液器的f.o.v.)。
    13. 粘附细胞的计数可以直接进行(扫描通过动脉;需要2名观察者,总是记录一些参考的xyz扫描),或者在使用处理软件(Leica LasX)的记录的xyz扫描中进行实验(图6) />
      1. 当使用f.o.v时,可以从一侧可视化整个动脉的大约25%。 450×450μm,当从外膜到内腔扫描时,直到两个血管壁边缘从视野消失(参见图6C ,以获得扫描体积的印象)。如果需要对动脉的总体概况,则可以通过同时转动移液管(小心!)以90°,180°和270°的方式将颈动脉机械地转动。不建议通过动脉直接集中到达"底部",因为这将大大降低图像质量,并增加对焦 的风险太深/破坏样本。
      2. 最佳的情况是,细胞计数由两名盲人观察员进行。
      3. 每个动脉需要时间约2.5h:从分离动脉和细胞悬浮液的制备,直到50%内皮表面上的粘附细胞计数。如果需要额外目标的详细成像,则应在总时间内添加另外30-60分钟。因此,每个工作日可以实际执行4个流程实验。动物可以储存在冰上或冰箱中一段时间(最多4小时不改变活/死染色模式; Megens等人,2007),但是为了提高重现性实验之后,隔离后尽可能快地处理它们。
    14. 重复来自安装在2号动物造影腔室中的2只雄性供体(野生型或敲除小鼠)的颈动脉的整个过程。
      减少实验时间和容器室属性的影响,最好设置下一轮 e 具有交替频率(即,使用2 nd 鼠标类型作为最初使用的房间中的第一个)的实例 替代应用的细胞标记用于染色分离的白细胞亚群(即,在下一系列实验中绿色变为红色)。 />

数据分析

  1. Dil-LDL外渗分析
    1. 使用Leica LASX 3.11软件对每个细胞的连接Dil-LDL颗粒的数量和大小进行定量。
      注意:使用多维分析的任何图像处理软件都可用于此方法。
    2. 将记录的xyz堆叠重新组合成最大强度投影,将3D信息转换为显示xy平面中的所有细胞结的所得2D图像(图7A)。


      Figu re 7。 STRONG>。 A.最大图像显示XY投影(Dil-LDL为红色,CD31为蓝色)。使用自发荧光和二次谐波产生的固有信号可视化动脉壁细胞外基质组分胶原(白色,外膜)和弹性蛋白(绿色,中膜)。图像还包括显示ROI选择(红色箭头)的分析方法。刻度棒=50μm。 B.强度方案的示例,显示表示Dil-LDL颗粒的各个峰。

    3. 使用模糊滤波器(内核大小3)实现了背景噪声的进一步降低。
    4. 通过临时排除代表Dil-LDL颗粒的光谱通道,基于连接的CD31信号选择十个单独的内皮细胞,并从LDL信号中盲目。
    5. 基于其CD31连接染色,围绕每个所选择的细胞绘制折线,导致每个单独细胞的特定的感兴趣区域(图7A,红色箭头)。
    6. 折线选择用于产生强度分布图(图7B)
    7. 将Dil-LDL通道的阈值设置为高于背景强度以排除背景噪声。
      注意:具体的阈值水平将根据实验背景水平而有所不同,必须单独确定 。
    8. 强度分布中单个峰的总数表示颗粒数,每个峰的全宽用于估计连接的Dil-LDL颗粒的大小。
      任何细胞结。

食谱

  1. 裂解缓冲液
    155mM NH 4 Cl
    10mM KHCO 3
    0.1mM EDTA
    溶于1L水(MilliQ),pH7.2

致谢

研究由DFG(SFB1123 TB Z01,A01和B03以及INST409/97-1 FUGG)和亚历山大·冯·洪堡基金会提供财务支持。

参考

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:van der Vorst, E. P., Maas, S. L., Ortega-Gomez, A., Hameleers, J. M., Bianchini, M., Asare, Y., Soehnlein, O., Döring, Y., Weber, C. and Megens, R. T. (2017). Functional ex-vivo Imaging of Arterial Cellular Recruitment and Lipid Extravasation. Bio-protocol 7(12): e2344. DOI: 10.21769/BioProtoc.2344.
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