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Jul 2020

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Liposomal Clodronate-mediated Macrophage Depletion in the Zebrafish Model
在斑马鱼模型中脂质体氯膦酸盐介导的巨噬细胞耗竭   

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Abstract

The ability to conduct in vivo macrophage-specific depletion remains an effective means to uncover functions of macrophages in a wide range of physiological contexts. Compared to the murine model, zebrafish offer superior imaging capabilities due to their optical transparency starting from a single-cell stage to throughout larval development. These qualities become important for in vivo cell specific depletions so that the elimination of the targeted cells can be tracked and validated in real time through microscopy. Multiple methods to deplete macrophages in zebrafish are available, including genetic (such as an irf8 knockout), chemogenetic (such as the nitroreductase/metronidazole system), and toxin-based depletions (such as using clodronate liposomes). The use of clodronate-containing liposomes to induce macrophage apoptosis after phagocytosing the liposomes is effective in depleting macrophages as well as testing their ability to phagocytose. Here we describe a detailed protocol for the systemic depletion of macrophages in zebrafish larvae by intravenous injection of liposomal clodronate supplemented with fluorescent dextran conjugates. Co-injection with the fluorescent dextran allows tracking of macrophage depletion in real time starting with verifying the successful intravenous injection to macrophage uptake of molecules and their eventual death. To verify a high degree of macrophage depletion, the level of brain macrophage (microglia) elimination can be determined by a rapid neutral red vital dye staining when clodronate injection is performed at early larval stages.


Graphical abstract:



Experimental workflow for in vivo macrophage-specific depletion by liposomal clodronate in larval zebrafish


Keywords: Liposomes (脂质体), Clodronate (氯膦酸盐), Larva injection (幼虫注射), Macrophage depletion (巨噬细胞耗竭), Zebrafish (斑马鱼), Innate Immunity (先天免疫)

Background

Macrophages are key constituents of the innate immune system and play important roles in response to infections, sterile inflammation, and environmental changes. One of the most effective ways to uncouple the functions of macrophages from a complex mix of interacting cell types in different physiological contexts is to be able to specifically eliminate macrophages and analyze the phenotypic consequence. Such depletion experiments in mice have provided much insight into the role of macrophages (Hua et al., 2018; Rosowski, 2020). However, our understanding of macrophage functions remains incomplete, and cell depletion experiments in the mouse model are difficult to track and validate in real time. For these reasons, the optical transparency and ease of manipulation of larval zebrafish offer clear advantages for highly traceable and tractable cell ablation in vivo by imaging the target cells in real time and in the whole intact organism. Zebrafish genes and immune system also share a high degree of orthology with those of the human (Yoder et al., 2002; Santoriello et al., 2012; Howe et al., 2013). Furthermore, the adaptive immune system of zebrafish does not become functionally mature until juvenile adult stages (Lam et al., 2004), making the larval zebrafish an excellent platform to study the innate immune system independent of the adaptive immune contributions.


Currently available macrophage depletion methods in zebrafish include genetic and chemogenetic manipulations, and toxin-based depletion. The development of macrophages requires early and continual function of the transcription factor Pu.1 (with the gene name spi1b), together with another transcription factor Irf8 (Li et al., 2011; Shiau et al., 2015; Tenor et al., 2015). Either disruption of pu.1 or irf8 by gene knockout or knockdown by morpholino (MO) anti-sense oligomers, makes a reliable approach for macrophage depletion, while the former ablates myeloid cells, and the latter is more specific to macrophages but also causes an increase in neutrophil numbers (Shiau et al., 2015; Yang et al., 2020). These methods are not amenable to temporal control (Rhodes et al., 2005; Li et al., 2011; Shiau et al., 2015; Rosowski, 2020), while clodronate-mediated depletion of macrophages based on localized microinjection enables some degree of spatial and temporal designation (Bernut et al., 2014).


Clodronate (also known as dichloromethylene diphosphonate) can be metabolized by cells to block mitochondrial respiration due to the formation of a non-hydrolyzable ATP analog, which then causes cell death (apoptosis) (Rosowski, 2020). Once injected as encapsulated in liposomes, clodronate is easily ingested and eliminated by macrophages as it accumulates within the cell (van Rooijen and Hendrikx, 2010). Since neither the clodronate nor the liposomal phospholipids utilized are toxic to other non-phagocytic cells (van Rooijen and Hendrikx, 2010), this approach allows specific depletion of already existing phagocytosing macrophages.


As part of the design for our protocol, we co-injected fluorescently labeled dextran with the liposomal clodronate to allow us to verify a precise and accurate injection and track the effect of the clodronate on macrophages in the entire larvae. To this end, after intravenous co-injection of clodronate liposome with fluorescently tagged dextran, we visually verified the successful injection of the substances into circulation and monitored the uptake of fluorescent dextran by macrophages and their ultimate demise over time. We designed the protocol to include a 48-hour post injection period to allow the effect of clodronate induced apoptosis in macrophages to materialize in light of previous work in chicken and mice showing that the efficacy of clodronate can take a few days depending on the tissue (Kameka et al., 2014; Ponzoni et al., 2018). We confirmed the efficacy of clodronate-mediated macrophage depletion at 48 hours post injection by assessing the remaining number of brain-resident macrophages (microglia), because a rapid analysis of microglia in the living larvae is available by the neutral red vital dye staining. We have chosen to inject at the early larval stage at 3 dpf (days post fertilization) because this is prior to maturation of the blood-brain-barrier (Jeong et al., 2008; O’Brown et al., 2019) at which time we found that our injected substances easily reach macrophages throughout the body including the brain. Using the 48-hour window for clodronate to take effect, we were able to achieve full ablation of microglia in most injected larval zebrafish (Yang et al., 2020). Overall, we found that intravenous microinjection of liposomal clodronate at 3 dpf with a 48-hour incubation time is effective in eliminating macrophages.


Materials and Reagents

  1. 1.5 ml microfuge tubes (Eppendorf, SafeLock, catalog number: 0030120086)

  2. Polystyrene Petri dish (VWR, catalog number: 25384-342)

  3. Thin wall borosilicate glass capillaries, 4 inches, OD 1.5 mm with filament (World Precision Instruments, catalog number: TW150F-4)

  4. Glass bottle

  5. 7.5 ml transfer pipettes (VWR, catalog number: 414004-005)

  6. Low melt agarose (Fisher Scientific, IBI Scientific, catalog number: 50-550-455), store at room temperature

  7. PTU (N-Phenylthiourea) (Sigma-Aldrich, catalog number: P7629), store at room temperature; made into PTU solution, store at -20 °C

  8. Clodronate Liposomes (Liposoma, catalog number: C41J0781, https://clodronateliposomes.com), store at 4-7 °C

  9. Control Liposomes (Liposoma, catalog number: B#P37J0718, https://clodronateliposomes.com), store at 4-7 °C

  10. Dextran, Alexa FluorTM 568: 10,000 MW (Invitrogen, catalog number: D22912), store in freezer and protect from light

  11. Neutral Red Dye (Sigma-Aldrich, catalog number: N4638), store at room temperature

  12. Tricaine (3-amino benzoic acidethylester) (Sigma-Aldrich, catalog number: A-5040) made into tricaine solution, store at -20 °C

  13. 50× PTU stock (see Recipes)

  14. 25× Tricaine stock solution (100 ml) (see Recipes)

  15. 3% Methyl cellulose (see Recipes)

  16. 1,000× neutral red solution (see Recipes)

  17. 1.5% low melt agarose (see Recipes)

Equipment

  1. Stereomicroscope with LED illuminated base and articulating mirror (World Precision Instruments, model: 504941, PZMIII-MI)

  2. Fluorescent stereomicroscope fully apochromatic corrected with 16:5:1 zoom optics (Leica, model: M165 MC)

  3. Manual micromanipulator (World Precision Instruments, catalog number: M3301)

  4. PicoNozzle Kit v2 (World Precision Instruments, catalog number: 5430-ALL)

  5. Pneumatic PicoPump PV 820 (World Precision Instruments, catalog number: SYS-PV820)

  6. Incubator (Benchmark Scientific, model: H2200-H)

  7. Microelectrode Puller (World Precision Instruments, catalog number: PUL-1000)

  8. Microwave

Software

  1. Fiji (Schindelin et al., 2012, https://fiji.sc/)

Procedure

  1. Larval zebrafish growth

    1. Incubate zebrafish embryos in fish water in a 28.5 °C incubator.

    2. Conduct a daily water change, and starting at 24 h post-fertilization (hpf), the fish water is also supplemented with PTU at a final concentration of 0.003% to inhibit pigmentation.

      PTU (N-Phenylthiourea) inhibits melanogenesis (pigmentation) by blocking all tyrosinase-dependent steps in the melanin pathway (Karlsson et al., 2001).

    3. Check the health status of the zebrafish larvae before injection. By 3 dpf healthy larvae will have a consistent rhythmic heartbeat with apparent blood flow, intermittent bursts of movement, and a straight body.


  2. Prepare liposome mix with fluorescent dextran

    1. Use clodronate liposomes and control liposomes directly at 5 mg/ml at the concentration they were delivered in liquid form from the manufacturer. We recommend making aliquots of the liposomes for storage at 4 °C to eliminate possible contamination from multiple usage; we use the aliquots within 12 months of storage. Supplement the liposome with Alexa 568 labeled dextran at 1:100 dilution of a 5 ng/nl stock for visualization of the injection.

      For example: add 0.5 μl of a 1:10 dilution of Alexa 568 labeled dextran at 5 ng/nl to 4.5 μl of clodronate liposomes at its original concentration of 5 mg/ml.

    2. Vortex to mix until a homogenous mixture for about 30-60 s.


  3. Needle and microinjector set-up

    Review the manufacturer’s recommendations and instructions for the micro-needle puller and the microinjector before operating.

    1. Use a micropipette needle puller to make a fine microinjection needle (refer to Table 1 and Figure 1).

      Microelectrode puller program (see Table 1)


      Table 1. 4-step protocol for glass capillary tube pulling
      Step Heat Force Distance Delay
      1 690 260 7.3 0
      2 500 240 0.5 4
      3 500 230 0.5 10
      4 380 240 0.5 20


      Figure 1. Pulling microinjection needle. A. Capillary tube loaded into micro-needle puller. B. Magnified view of heated filament surrounding glass capillary. C. Creation of 2 microinjection needles from pulling (double-sided white arrow).


    2. Load 3 μl of injection material into the wide end of the microcapillary glass tube.

    3. Shake the capillary tube to bring the injection material to the tip of the needle and to remove any air bubbles.

    4. Turn on the air source and the microinjector, and transfer the needle into the microinjector.

    5. Break the tip of the microinjection needle using forceps to an extent that allows the injection materials to release consistently and minimize the needle puncture on the target tissue.

    6. Adjust the pressure on the pneumatic pump and the location of microinjection syringe pump to ensure consistent 0.5 nl per foot-pedal kick of injection so each injection consists of two kicks per larval fish for a total of 1 nl.

      Size of each injection kick can be estimated by injecting into air which creates a spherical drop at the needle tip. The diameter of the liquid sphere of about 100 μm is estimated using a stage micrometer to give 0.5 nl volume.

    7. Adjust the micromanipulator so that the needle can reach the center of the stage and touch the base so that the needle can reach any mounted larvae in your field of view under microscope.


  4. Mounting zebrafish for microinjections

    1. Use a microwave to melt 1.5% low-melt agarose (for a short time, 10-20 s). See Recipes for low-melt agarose preparation.

    2. Use a plastic transfer pipette to transfer multiple larvae at 3 dpf to the center of a 10 cm Petri dish lid. Many larvae can be transferred at once upwards of 10-15 larvae.

    3. Remove as much fish water as possible to leave only minimal water around fish as to not dehydrate them, then slowly pipette in lukewarm low melt agarose and gently swirl the larvae to mix them into the agarose (or by pipetting them up and down 1-2 times).

      1. The agarose will be very hot immediately after heating. To prevent scalding and killing the larvae, wait until the agarose is not hot to the touch, but still warm and fluid. The low-melt agarose will remain fluid at 37 °C and set rapidly below 25 °C. So you want to aim to use the agarose at the temperature between 30 °C and 40 °C. You can also keep the agarose on a heat block at 60 °C so it remains in solution for a longer period of time as you work on mounting the larvae. You can expedite the cooling process by pipetting hot agarose up and down on a clean surface (such as in another dish).

      2. The goal is to have the larvae embedded in a thin layer of agarose so that when it comes to using the fine glass needle for injection, the needle does not need to penetrate through much gel to reach your desired tissue target (see Figure 2). Too much agarose will make it hard to direct the needle to your target site without having it bend or swerve in the agarose.



      Figure 2. Set-up of zebrafish larval microinjections. A. Mounted larvae are placed under a fluorescent stereomicroscope adjacent to a microinjection needle attached to a micro-manipulator. B. Higher magnification of mounted larvae. Dotted circle shows solidified thin layer of low-melt agarose covered with a small pool of system water supplemented with PTU. Black arrows point to larvae. Red arrow points to tip of needle (Note: This arrow is not pointing to the injection site.)


    4. The most time-sensitive step is in orienting all the zebrafish larvae quickly in the agarose before it begins to re-solidify. You can expect to have less than 1-2 min for this step. Use fine forceps to quickly orient each one on their sides, but without concern of the exact orientation or body alignment, only that the tail region containing the caudal vein plexus is accessible to your needle. Take caution to not poke the larvae to cause injury but to use the agarose around the larvae to move them.

    5. Wait for agarose around the larvae to cool down and solidify before starting injections.

    6. Add a small amount of fish system water to cover the agarose to prevent dehydration and keep the larvae healthy during the operation (see Figure 2).


  5. Intravenous microinjections

    1. Use the foot pedal to inject 1 nl of clodronate liposomes or control liposomes into caudal vein plexus, which is located in the tail tissue right past the yolk extension (see Graphical abstract).

    2. Validate each injection by seeing an immediate distribution of the injected fluorescent dextran mix into the heart and subsequent circulation throughout the body vasculature using a fluorescent stereomicroscope. Any larvae after injection that do not show the correct distribution of the fluorescent marker in the body vasculature is removed from the dish using forceps.

    3. Gently recover the injected fish from the agarose into fresh fish water supplemented with 0.003% PTU and transfer them back into the 28.5 °C incubator.

      Use the side of the forceps to create a break in the agarose beginning underneath the head and dragging the forceps along the body until the tip of the tail is reached. Larvae should be able to squirm out easily and swim into the system water surrounding the agarose. Pipette released larval zebrafish into the dish with fresh fish water.

    4. After microinjection, monitor all the fish for normal health and behavior. Remove any sickly or abnormal fish, and only continue processing the healthy and normal larvae.


  6. Neutral red staining

    1. In a Petri dish, stain a subset of your injected larvae at 48 hpi (hours post injection) with neutral red at 2.5 μg/ml in fish water supplemented with 0.003% PTU at room temperature for 45 min to 1 h (see Figure 3A).

    2. Remove the majority of the neutral red supplemented fish water by exchanging it for fresh new fish water supplemented with PTU (see Figure 3B).

    3. Leave the larvae in incubator for another 2-3 h before analyzing under a stereomicroscope.

    4. Before analyzing, anesthetize larvae with tricaine at 0.5× strength, which the larvae can stay in for up to 45 min with the ability to fully recover back to normal and moving again. For the anesthetics to be working, larvae should not be moving so you can use them for your analysis.

      Note: Please follow your institutional guidelines on the appropriate protocol for anesthetizing zebrafish.

    5. Mount larvae in 3% methyl cellulose to position the larvae for analyzing and imaging under a stereomicroscope with a color camera. The larvae should be mounted brain side up to image the microglia under an upright stereomicroscope (see Figure 3C-3E).

      Notes:

      1. You can mount larvae on a dish or any flat platform to visualize them under a stereoscope.

      2. Because methyl cellulose is highly viscous and these larvae were temporarily anesthetized, they remain sufficiently still for quick imaging without further anesthesia in only methyl cellulose for at least up to 10 min.

      3. Depending on the endpoint of the experiment, these larvae can be directly processed after imaging for genotyping, or recovered by washing them 1-2 times in clean fish water to remove the methyl cellulose at which point they can return to their growing chamber (an incubator) or fixed for further analysis. Fish larvae should remain well and alive during this entire period up to the endpoint processing.



    Figure 3. Neutral red analysis. A. Color of fish water after addition of neutral red. B. Color of fish and water after removal of neutral red. C. Full body image of control uninjected 4 dpf larvae after neutral red staining. D and E. Lateral (D) and dorsal (E) view of neutral red staining. C-E. Larvae imaged in 3% methyl cellulose. Black arrows point to individual microglial cells.

Data analysis

The intravenous administration of clodronate liposomes offer the ability to deplete macrophages throughout the body, including microglia and peripheral macrophages, without the maintenance of a transgenic line. Analyze the efficiency of clodronate-mediated macrophage depletion by comparing the number of microglia in the brains of liposomal clodronate injected group with the control liposomal vehicle or control uninjected group using the neutral red vital dye assay at 48 hpi (see Figure 4–Figure Supplement 2 in Yang et al., 2020).

Recipes

  1. 50× PTU stock (1 L)

    1. Dissolve 1.5 g N-Phenylthiourea (PTU) in 1 L of distilled water by stirring overnight at room temperature.

    2. Aliquot and store concentrated stock at -20 °C.

    3. To inhibit pigmentation in developing embryos,use at a 1× concentration in fish water and replace daily.

    4. 1× is 0.003% [weight (g) by volume (per 100 ml)] in fish water.

  2. 25× Tricaine stock solution (100 ml)

    Add 400 mg of Tricaine powder (3-amino benzoic acid ethyl ester) to 100 ml of distilled water. Store concentrated stock at -20 °C. To use tricaine as an anesthetic,use at a 0.5-1.0× concentration.

  3. 3% Methyl cellulose

    Note: You will need 3 g of methyl cellulose powder in 100 ml of distilled water.

    1. To dissolve methyl cellulose powder in water, bring half of the distilled water to 60 °C. The remaining volume of water should be stored at 4 °C and left to cool for 30 min.

    2. Add 3 g of methyl cellulose powder to boiling water and stir. The mixture will become extremely viscous quickly.

    3. Remove the solution from the heat as soon as stirring becomes difficult.

    4. Add the chilled water and continue to stir for 30 s.

    5. To fully dissolve particles and remove bubbles the solution must be placed at -20 °C overnight. To prevent glass from breaking, gradually lower the temperature by placing glass at 4 °C for 1-2 h before placing at -20 °C overnight.

  4. 1,000× neutral red dye solution

    Dissolve neutral red powder in distilled water at a concentration of 2.5 mg/ml for a 1,000× stock. For live staining of microglia in zebrafish, use neutral red at 1× strength.

  5. 1.5% low melt agarose

    1. Dissolve 1.5 g of low melt agarose in 100 ml distilled water using the microwave for less than 30 s.

    2. Monitor the heating process as low melt agarose can easily overheat and bubble over.

    3. Once this solution is made, it can be stored in a glass bottle with a lid at room temperature and be repeatedly re-used by re-melting the agarose in the microwave.

Acknowledgments

The graphical abstract was created with BioRender.com. This protocol accompanies the publication (Yang et al., 2020). The work was funded by NIH NIGMS grant 1R35GM124719 to C.E.S.

Competing interests

The authors declare no competing interests.

Ethics

Animal experimentation: This study was performed in strict accordance with the approved institutional animal care and use committee (IACUC) protocols (#16-160 and #19-132) of the UNC Chapel Hill.

References

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简介

[摘要]为了进行能力在体内巨噬细胞比耗尽仍然是有效的手段,在广泛的生理环境的巨噬细胞的揭开功能。与鼠模型相比,斑马鱼具有良好的成像能力,因为它们的光学透明性从单细胞阶段到整个幼体发育期开始。这些素质成为重要的体内细胞特异的枯竭,使靶细胞的消除可以跟踪并通过显微镜进行实时验证。多种方法以耗尽巨噬细胞在斑马鱼中是可用的,包括遗传(如IRF8敲除),chemogenetic(如在 硝基还原酶/甲硝唑系统)和基于毒素的消耗(例如使用氯膦酸盐脂质体)。在吞噬脂质体后,使用含氯膦酸盐的脂质体诱导巨噬细胞凋亡可有效地消耗巨噬细胞并测试其吞噬能力。在这里,我们描述了通过静脉注射补充有荧光右旋糖酐共轭物的脂质体氯膦酸盐对斑马鱼幼虫体内巨噬细胞进行系统耗竭的详细方案。与荧光右旋糖酐共注射可以实时跟踪巨噬细胞耗竭情况,从验证静脉内注射成功到摄取巨噬细胞分子及其最终死亡开始。为了验证高度的巨噬细胞耗竭,可以在幼虫早期进行氯膦酸盐注射时通过快速中性红色活体染料染色来确定脑巨噬细胞(小胶质细胞)的清除水平。

图形概要:

幼虫斑马鱼脂质体氯膦酸盐体内巨噬细胞特异性清除的实验工作流程


[背景]巨噬细胞是先天免疫系统的关键组成部分,并响应于感染,无菌性炎症,与环境变化中发挥重要作用。使巨噬细胞功能与不同生理环境中相互作用细胞类型的复杂混合物脱钩的最有效方法之一是能够特异性消除巨噬细胞并分析表型结果。小鼠中的此类耗竭实验为巨噬细胞的作用提供了很多见识(Hua等人,2018; Rosowski,2020)。但是,我们对巨噬细胞功能的理解仍然不完整,并且小鼠模型中的细胞耗竭实验难以实时跟踪和验证。由于这些原因,幼虫斑马鱼的光学透明性和易于操作性,通过对靶细胞进行实时成像并在整个完整生物体内成像,为体内高度可追溯和可处理的细胞消融提供了明显的优势。斑马鱼的基因和免疫系统也与人类有着高度的同源性(Yoder等,2002; Santoriello等,2012; Howe等,2013)。此外,斑马鱼的适应性免疫系统直到成年幼体阶段才在功能上成熟(Lam等,2004),这使得幼体斑马鱼成为了研究先天免疫系统而与适应性免疫作用无关的优秀平台。

斑马鱼中目前可用的巨噬细胞耗竭方法包括遗传和化学遗传学操作,以及基于毒素的耗竭。巨噬细胞的发育需要转录因子Pu.1(基因名称为spi1b )以及另一种转录因子Irf8的早期和持续功能(Li等,2011; Shiau等,2015; Tenor等, 2015)。通过基因敲除或吗啉代(MO)反义寡聚体的敲低来破坏pu.1或irf8 ,这是一种可靠的巨噬细胞耗竭方法,而前者消灭了髓样细胞,后者消融了巨噬细胞,但也导致了巨噬细胞的凋亡。嗜中性白血球数目增加(Shiau等人,2015; Yang等人,2020)。这些方法不适用于时间控制(Rhodes等,2005; Li等,2011; Shiau等,2015; Rosowski,2020),而基于局部显微注射的氯膦酸盐介导的巨噬细胞耗竭则可在一定程度上缓解癌症。时空指定(Bernut et al 。,2014)。

氯膦酸盐(也称为二氯亚甲基二膦酸盐)可由于不可水解的ATP类似物的形成而被细胞代谢,从而阻止线粒体呼吸,进而导致细胞死亡(细胞凋亡)(Rosowski,2020年)。一旦注射入脂质体中,氯膦酸盐就很容易被巨噬细胞摄取和清除,因为它会在细胞内积聚(van Rooijen和Hendrikx,2010年)。由于所用的氯膦酸盐和脂质体磷脂均对其他非吞噬细胞无毒(van Rooijen和Hendrikx,2010年),因此该方法可特异性清除已经存在的吞噬巨噬细胞。

作为方案设计的一部分,我们将荧光标记的葡聚糖与脂质体氯膦酸盐共注射,以使我们能够验证准确而准确的注射方式,并跟踪氯膦酸盐对整个幼虫中巨噬细胞的作用。为此,在静脉注射氯膦酸盐脂质体和荧光标记的葡聚糖后,我们目测验证了该物质是否已成功注入循环系统并监测了巨噬细胞对荧光葡聚糖的吸收及其随时间的最终消亡。我们设计的方案包括注射后48小时,以使氯膦酸盐在巨噬细胞中诱导的凋亡效应得以实现,这是根据先前在鸡和小鼠中的工作表明,氯膦酸盐的疗效可能需要几天的时间,具体取决于组织( Kameka et al。,2014; Ponzoni et al。,2018)。我们通过评估剩余的大脑驻留巨噬细胞(小胶质细胞)的数量,证实了注射后48小时氯膦酸盐介导的巨噬细胞耗竭的功效,因为通过中性红色生命染料染色可对活体幼虫中的小胶质细胞进行快速分析。我们选择在幼虫早期以3 dpf (受精后的天数)注射,因为这是在血脑屏障成熟之前进行的(Jeong等,2008; O'Brown等,2019)。一次,我们发现我们注射的物质很容易到达包括大脑在内的整个人体的巨噬细胞。使用氯膦酸盐生效的48小时窗口,我们能够在大多数注射的幼虫斑马鱼中实现小胶质细胞的完全消融(Yang等人,2020年)。总体而言,我们发现静脉滴注3 dpf的氯膦酸氯膦酸盐具有48小时的孵育时间,可有效消除巨噬细胞。

关键字:脂质体, 氯膦酸盐, 幼虫注射, 巨噬细胞耗竭, 斑马鱼, 先天免疫

材料和试剂
1.5 ml微量离心管(Eppendorf,SafeLock,目录号:0030120086)
聚苯乙烯陪替氏培养皿(VWR,目录号:25384-342)
薄壁硼硅酸盐玻璃毛细管,4英寸,外径1.5 mm,带细丝(World Precision Instruments,目录号:TW150F-4)
玻璃瓶
7.5 m升移液器(VWR,目录号:414004-005)
低熔点琼脂糖(Fisher Scientific,IBI Scientific,目录号:50-550-455),在室温下保存
PTU (N-苯基硫脲)(Sigma-Aldrich,目录号:P7629),在室温下保存;制成PTU溶液,在-20 °C下保存
氯膦酸盐脂质体(Lip o soma,目录号:C41J0781,https : //clodronateliposomes.com ),在4-7 °C下储存
对照脂质体(Lip o soma,目录号:B#P37J0718,https : //clodronateliposomes.com ),储存在4-7 °C
Dextran,Alexa Fluor TM 568:10,000 MW(Invitrogen,目录号:D22912),存储在冰箱中并避光
中性红染料(Sigma-Aldrich,目录号:N4638),在室温下保存
将三卡因(3-氨基苯甲酸乙酯)(Sigma-Aldrich,目录号:A-5040)制成三卡因溶液,储存在-20 °C
50 × PTU库存(请参阅食谱)
25 × Tricaine储备溶液(100 ml)(请参阅食谱)
3%甲基纤维素(请参阅食谱)
1,000 ×中性红溶液(请参阅配方)
              1.5%低熔点琼脂糖(请参阅食谱)


设备

带有LED照明底座和铰接镜的立体显微镜(World Precision Instruments,型号:504941,PZMIII-MI)
荧光立体显微镜,通过16:5:1变焦光学元件完全复消色差校正(Leica,型号:M165 MC)
手动微操作器(World Precision Instruments,目录号:M3301)
PicoNozzle套件v2(World Precision仪器,目录号:5430-ALL)
气动PicoPump PV 820 (World Precision Instruments ,目录号:SYS-PV820 )
孵化器(Benchmark Scientific,型号:H2200-H )
微电极拉拔器(世界精密仪器公司,目录号:PUL-1000)
微波


软件

斐济(Schindelin et al。,2012 ,https://fiji.sc/)


程序

幼虫斑马鱼生长
在28.5 °C的孵化器中将斑马鱼胚胎在鱼水中孵化。
进行日常换水,从施肥后24小时(hpf)开始,鱼水中还添加了PTU,其最终浓度为0.003%,以抑制色素沉着。
PTU(N-苯基硫脲)通过阻断黑色素途径中所有酪氨酸酶依赖性步骤来抑制黑色素生成(色素沉着)(Karlsson等,2001)。


注射前检查斑马鱼幼虫的健康状况。由3旦健康幼虫将具有明显的血流量,运动的间歇脉冲串,并且提供一致的有节奏的心跳一个直BOD ÿ 。


用荧光右旋糖酐制备脂质体混合物
使用氯膦酸盐脂质体和对照脂质体直接以5mg / ml的在它们被输送的浓度以液体形式从制造商。我们建议将脂质体等分试样保存在4 °C,以消除多次使用可能造成的污染;我们在储存12个月内使用等份试样。在Alexa 568标记的葡聚糖上补充脂质体,以5 ng / nl的储备液以1:100的比例稀释,以观察注射液。
例如:添加0.5 μ升以5ng / NL 1:10稀释的Alexa 568标记的葡聚糖的4.5 μ升在其原始的5毫克/毫升浓度氯膦酸盐脂质体。


涡旋混合直至均匀混合物约30-60 s 。


针头和微型注射器的设置
在操作之前,请阅读制造商对微针拔针器和微注射器的建议和说明。


使用微量移液器拔针器制成精细的微量注射针(请参阅表1和图1)。


微电极拉拔器程序(请参阅表1)


表1 。玻璃毛细管拉制的4步方案








力量


距离


延迟


1个


690


260


7.3


0


2个


500


240


0.5


4


3


500


230


0.5


10


4


380


240


0.5


20



图1.拉动微注射针。A.将毛细管装入微针拔轮器中。B.加热的灯丝围绕玻璃毛细管的放大图。C. 2的创作显微注射针š从拉(双面白色箭头)。

将3μl注射材料装入微毛细管玻璃管的宽端。
摇动毛细管,将注射材料带到针尖,并清除气泡。
打开气源和微型注射器,然后将针头转移到微型注射器中。
使用镊子将微注射针的尖端折断到一定程度,以使注射材料始终释放,并最大程度地减少目标组织上的针刺。
调整气动泵上的压力和微注射注射器泵的位置,以确保每英尺脚踏进水0.5 nl一致,因此每次进样由每条幼虫鱼两次进水组成,总计1 nl。
可以通过将空气注入到会在针尖产生球形液滴的空气中来估算每个喷射行程的大小。液球直径约100μ m的使用阶段微米至得到0.5 NL体积估算。


调整微操纵器,使针头可以到达平台的中心并接触基座,从而使针头可以到达显微镜下视场中任何已安装的幼虫。
安装斑马鱼以进行显微注射
我们Ë微波炉融化1.5%低熔点琼脂糖(短时间,10-20或多个)。有关低熔点琼脂糖的制备方法,请参见食谱。
使用塑料移液管以3 dpf的速度将多个幼体移至10厘米培养皿盖的中心。许多幼虫可以一次转移到10-15个以上的幼虫中。
去除尽可能多的鱼水,使鱼周围只剩下极少量的水,以免使它们脱水,然后缓慢吸取温热的低熔点琼脂糖,然后轻轻旋动幼虫将它们混合到琼脂糖中(或通过上下吸打1-2)次)。
加热后,琼脂糖会非常热。为防止烫伤和杀死幼虫,请等到琼脂糖不烫手,但仍要温暖而流动。低熔点琼脂糖在37 °C时仍会保持液态,并在25 °C以下迅速凝固。因此,您希望在30 °C至40 °C的温度下使用琼脂糖。您也可以将琼脂糖放在60 °C的加热块上,以便在安装幼体时将其在溶液中保留更长的时间。您可以通过在干净的表面上(例如在另一个盘子中)上下吸热琼脂糖来加快冷却过程。
目的是将幼虫包埋在薄薄的琼脂糖层中,以便在使用细玻璃针进行注射时,该针不需要穿透太多的凝胶即可达到所需的组织目标(见图2)。琼脂糖过多会导致难以将针引导到目标部位而又不会使其在琼脂糖中弯曲或弯曲。




图2.斑马鱼幼虫微量注射的样品。A.将已安装的幼虫放置在荧光立体显微镜下,靠近连接到微操纵器的微注射针。B.幼虫的放大倍数更高。虚线圆圈显示低熔点琼脂糖凝固的薄层,覆盖有少量补充PTU的系统水。黑色箭头指向幼虫。红色箭头指向针尖(注意:此箭头未指向注射部位。)

对时间最敏感的步骤是将所有斑马鱼幼虫在琼脂糖中快速定向,然后再开始重新固化。您可以期望此步骤少于1-2分钟。使用细镊子使每个人的侧面快速定向,但不必担心确切的方向或身体对准,只需要针刺触及包含尾静脉丛的尾巴区域即可。小心不要戳伤幼虫,以免造成伤害,而应使用幼虫周围的琼脂糖移动它们。
等待幼虫周围的琼脂糖冷却并固化,然后再开始注射。
在手术过程中,添加少量鱼系统水以覆盖琼脂糖,以防止脱水并保持幼虫健康(参见图2)。


静脉注射
用脚踏板向尾静脉丛注射1毫升氯膦酸盐脂质体或对照脂质体,尾静脉丛位于卵黄延伸区正后的尾巴组织中(请参见图形摘要)。
通过观察荧光右旋糖酐混合物在心脏中的即时分布以及随后使用荧光体视显微镜在整个身体脉管系统中的循环,来验证每次注射。注射后,使用镊子将任何未显示荧光标记在人体脉管系统中正确分布的幼虫从培养皿中取出。
从琼脂糖中轻轻注入注入的鱼到补充有0.003%PTU的新鲜鱼水中,然后将它们转移回28.5 °C的培养箱中。
使用镊子的侧面在琼脂糖中产生一个断裂,从头下方开始,沿着身体拖动镊子,直到到达尾巴的尖端。幼虫应该能够轻松地蠕动并进入琼脂糖周围的系统水中。移液器用新鲜的鱼水将幼虫斑马鱼放到盘中。


显微注射后,监测所有鱼的健康状况和行为。除去任何有病或异常的鱼,然后仅继续处理健康和正常的幼虫。


中性红染色
在P etri皿中,在48 hpi (注射后数小时)的部分注射幼虫中,于室温下以2.5μg/ ml补充有0.003%PTU的鱼水中中性红染色45分钟至1 h (见图3A) )。
通过将补充了PTU的新鲜新鱼水更换为新鲜的补充了中性红鱼水,以除去其中的大部分(见图3B)。
在立体显微镜下进行分析之前,将幼虫在培养箱中再放置2-3小时。
在分析之前,用0.5 ×强度的三卡因麻醉幼虫,该幼虫可以在长达45分钟的时间内停留,并能够完全恢复正常并再次移动。为了使麻醉剂起作用,幼虫不应移动,因此可以将其用于分析。
注意:请按照您的机构指南对麻醉斑马鱼的适当方案进行操作。


将幼虫安装在3%的甲基纤维素中,以放置幼虫,以便在带有彩色相机的立体显微镜下进行分析和成像。幼虫应安装在大脑一侧,以在直立的立体显微镜下对小胶质细胞成像(见图3C-3 E)。
笔记:


您可以将幼虫安装在盘子或任何平坦的平台上,以在立体镜下对其进行可视化。
因为甲基纤维素是高度粘性的,并且这些幼虫暂时麻醉,它们保持足够的仍然为快速成像,而不只在甲基纤维素进一步麻醉至少高达1 0分钟。
根据实验的终点,这些幼虫可以在成像后直接进行基因分型,也可以通过在干净的鱼水中清洗1-2次以去除甲基纤维素的方式回收,然后再返回到生长室(孵化箱)。 )或固定以进行进一步分析。鱼的幼虫在整个过程中应保持良好的生命力,直至最终加工。




图3.中性红色分析。A.添加中性红色后鱼水的颜色。B.去除中性红色后鱼和水的颜色。C.中性红色染色后未注射的4dpf幼虫的对照的全身图像。D和E。中性红色染色的侧面(D)和背面(E)视图。CE。幼虫在3%的甲基纤维素中成像。黑色箭头指向单个小胶质细胞。

数据分析

氯膦酸盐脂质体的静脉内给药提供了消耗整个体内巨噬细胞(包括小胶质细胞和外周巨噬细胞)的能力,而无需维持转基因系。通过在48 hpi处使用中性红色活体染料分析比较脂质体氯膦酸盐注射组与对照脂质体媒介物或对照未注射组脑中小胶质细胞的数量,从而分析氯膦酸盐介导的巨噬细胞耗竭的效率(参见图4–图附录2)在杨等人。,2020)。

菜谱

1. 50 × PTU库存(1 L)     

通过在室温下搅拌过夜,将1.5 g N-苯基硫脲(PTU)溶解在1 L蒸馏水中。
分装和集中库存在-20 ℃。
为抑制发育中的胚胎中的色素沉着,请以1倍的浓度在鱼水中使用并每日更换。
1 ×是[体积(每100m重(g)0.003%升)]在鱼水。
2. 25 × Tricaine储备溶液(100 ml)     

将400毫克的三卡因粉末(3-氨基苯甲酸乙酯)添加到100毫升的蒸馏水中。将浓缩液储存在-20 °C下。要将曲卡因用作麻醉剂,请以0.5-1.0 ×的浓度使用。


3. 3%甲基纤维素     

注意:您则需要3克甲基纤维素粉末在百米升蒸馏水。


要将甲基纤维素粉末溶解在水中,请将一半的蒸馏水升至60 °C 。剩余的水量应储存在4 °C并冷却30分钟。
将3克甲基纤维素粉末加到沸水中,搅拌。混合物将很快变得非常粘稠。
搅拌变得困难时,将溶液从热源中移开。
加入冷水,继续搅拌30秒钟。
为了完全溶解颗粒并去除气泡,必须将溶液在-20 °C下放置过夜。为防止玻璃破裂,可将玻璃于4 °C放置1-2 h逐渐降低温度,然后于-20 °C放置过夜。
4. 1,000 ×中性红色染料溶液     

以2.5毫克/毫升,对于1的浓度溶解在蒸馏水中,中性红粉末,000 ×库存。要对斑马鱼中的小胶质细胞进行实时染色,请使用强度为1 ×的中性红。


5. 1.5%低熔点琼脂糖     

用微波将1.5 g低熔点琼脂糖溶解在100 ml蒸馏水中少于30 s。
监视加热过程,因为低熔点琼脂糖很容易过热并起泡。
制成此溶液后,可以将其保存在室温下带盖的玻璃瓶中,并通过在微波中重新融化琼脂糖来重复使用。


致谢

图形摘要是使用BioRender.com创建的。该协议随出版物一起发布(Yang等,2020)。这项工作由NIH NIGMS授予CES的1R35GM124719资助

利益争夺

作者宣称没有利益冲突。

伦理

动物实验:严格按照UNC教堂山批准的机构动物护理和使用委员会(IACUC)规程(#16-160和#19-132)进行这项研究。

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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Yang, L., Rojas, A. M. and Shiau, C. E. (2021). Liposomal Clodronate-mediated Macrophage Depletion in the Zebrafish Model. Bio-protocol 11(6): e3951. DOI: 10.21769/BioProtoc.3951.
  2. Yang, L., Jimenez, J. A., Earley, A. M., Hamlin, V., Kwon, V., Dixon, C. T. and Shiau, C. E. (2020). Drainage of inflammatory macromolecules from the brain to periphery targets the liver for macrophage infiltration. Elife 9: e58191.
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