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Preparation of Mosquito Salivary Gland Extract and Intradermal Inoculation of Mice
蚊子唾液腺提取物的制备与小鼠真皮内接种   

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Abstract

Mosquito-transmitted pathogens are among the leading causes of severe disease and death in humans. Components within the saliva of mosquito vectors facilitate blood feeding, modulate host responses, and allow efficient transmission of pathogens, such as Dengue, Zika, yellow fever, West Nile, Japanese encephalitis, and chikungunya viruses, as well as Plasmodium parasites, among others. Here, we describe standardized methods to assess the impact of mosquito-derived factors on immune responses and pathogenesis in mouse models of infection. This protocol includes the generation of mosquito salivary gland extracts and intradermal inoculation of mouse ears. Ultimately, the information obtained from using these techniques can help reveal fundamental mechanisms of interaction between pathogens, mosquito vectors, and the mammalian host. In addition, this protocol can help establish improved infection challenge models for pre-clinical testing of vaccines or therapeutics that take into account the natural route of transmission via mosquitoes.

Keywords: Mosquito(蚊子), Saliva(唾液), Salivary gland(唾液腺), Extract(提取物), Virus transmission(病毒传播), Intradermal(真皮内), Needle(针头), Mouse(小鼠)

Background

While probing for blood, the mosquito inoculates saliva that facilitates feeding but can also contain pathogens, if the mosquito has previously fed on an infected individual. Mosquito saliva plays an important role in establishing infection, facilitating dissemination, modulating immune responses, and exacerbating pathogenesis during West Nile virus (Schneider et al., 2006; Styer et al., 2011), Dengue virus (Cox et al., 2012; Conway et al., 2014; McCracken et al., 2014; Schmid et al., 2016), chikungunya virus (Agarwal et al., 2016), Semliki Forest virus (Pingen et al., 2016), Rift Valley Fever virus (Le Coupanec et al., 2013) and Plasmodium parasite (Schneider et al., 2011) infection. Many important questions yet remain and call for improved animal models.

Whereas inoculation via infected mosquitoes best mimics natural transmission, high variability in the inoculated dose and limited availability of insectary facilities result in restricted use of such procedures. In addition, the amount of saliva and the presence or absence of mosquito-derived components cannot be controlled when using infected mosquitoes. As an alternative, ‘spot feeding’ of uninfected female mosquitoes followed by intradermal inoculation of the pathogen via a needle mimics the natural deposition of saliva into mouse skin and delivers a defined dose of pathogen. The ‘spot feeding’ model has successfully been used to study Dengue virus (Cox et al., 2012; McCracken et al., 2014) and West Nile virus (Moser et al., 2015) infection but still requires the concomitant use of live mosquitoes and mice, and cannot control for the amount of saliva delivered. To separately control for mosquito and mouse experiments, inserting the mosquito proboscis into a sucrose solution in a capillary tube can serve to collect mosquito saliva artificially. The saliva collected during sugar feeding, however, differs qualitatively from mosquito saliva that is inoculated into the host skin during natural blood feeding (Marinotti et al., 1990; Moser et al., 2015).

Here, we describe the use of a simplified model of needle-inoculating mosquito salivary gland extract (SGE) from non-infected mosquitoes that can be delivered with a pathogen in a controlled manner at a defined dose. This method allows for independent handling of live mosquitoes and mice between collaborators and can more easily be standardized between assays and research groups. Use of SGE has proven useful to study infection with West Nile virus (Schneider et al., 2006; Moser et al., 2015), Dengue virus (Conway et al., 2014; Schmid et al., 2016), Rift Valley fever virus (Le Coupanec et al., 2013), and Sindbis virus (Schneider et al., 2004). Injection of SGE does not precisely mimic inoculation via the mosquito proboscis and likely contains non-secreted components of salivary glands. Nevertheless, this method allows collection of higher quantities of mosquito-derived factors, contains all secreted proteins, and can also be used in in vitro assays. Overall, the procedures described here should facilitate collaboration between entomologists, immunologists, and researchers studying pathogens of interest.

Materials and Reagents

Note: For the rearing of mosquitoes and general maintenance of a mosquito colony, see Bio-protocol Kauffman et al. (2017).

  1. Cotton-tipped swab, 15 cm handle (Puritan, catalog number: 25-826 5WC )
  2. Petri dish, sterile, 60 mm (e.g., 60 mm TC-Treated Cell Culture Dish, Corning, Falcon®, catalog number: 353002 )
  3. Plastic wrap
  4. 50-ml conical centrifuge tubes (CELLTREAT Scientific Products, catalog number: 229422 )
  5. 15-ml conical centrifuge tubes (CELLTREAT Scientific Products, catalog number: 229412 )
  6. 5-ml tubes, 12 x 75 mm (Corning, Falcon®, catalog number: 352054 )
  7. 96-well plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 15041 )
  8. 5-ml serological pipets (CELLTREAT Scientific Products, catalog number: 229206B )
  9. 10-ml serological pipets (CELLTREAT Scientific Products, catalog number: 229211B )
  10. Microcentrifuge tubes, 1.5 ml (Corning , Axygen®, catalog number: MCT-150-C )
  11. Needle: 30-gauge, small RN hub, custom-made, point style 4 (10°-12° bevel), 25-mm length (Hamilton, catalog number: 7803-07 )
    Note: Alternative needles: use with disposable hypodermic needle, e.g., 30 G x 1 inch (BD, catalog number: 305128 ).
  12. Frosted microscope slides, 25 x 75 x 1.0 mm (e.g., Fisher Scientific, catalog number: 12-550-343 )
  13. Wooden applicator sticks, 15 cm (Puritan, catalog number: 807 )
  14. Insect Pins Morpho Black enameled No.000 (BioQuip, catalog number: 1208B000 )
  15. Dissecting probes fabricated from wooden applicator sticks and insect pins
    Note: Dissecting probes are fabricated from 15-cm wooden applicators and insect pins. Soak the applicator sticks in hot water for at least 30 min, and cut the heads off the pins and discard. Using plyers, hold the pin, avoiding damage to the pointy end, and push the blunt (cut) end into the stick. Let the probe dry.
  16. Transfer pipets, 1 ml, bulk, nonsterile (BioLogix, catalog number: 30-0135 )
  17. pH test strips (e.g., Sigma-Aldrich, catalog number: P4536-100EA )
  18. Female mosquitoes
  19. Mosquito holding carton (see Bio-protocol Kauffman et al., 2017 for description and construction)
  20. Mice
  21. Triethylamine (Sigma-Aldrich, catalog number: T0866 )
  22. Ethyl alcohol
  23. Phosphate-buffered saline (PBS), low endotoxin ≤ 0.25 EU/ml (i.e., Mediatech, catalog number: 21-030-CV )
  24. Micro BCA Protein Assay Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23235 )
  25. Endpoint Chromogenic Limulus Amebocyte Lysate QCL-1000 Assay (Lonza, catalog number: 50-647U )
  26. Disinfectant, such as Bleach or Umonium38 (Huckert’s Laboratoire International, catalog number: PF 12209 )
  27. Isoflurane, Iso-Vet (Chanelle, catalog number: CDS019936 ) for anesthesia of mice
  28. 70% ethanol: Combine 74 ml of ethyl alcohol with 26 ml of water. Label as flammable and store at room temperature for up to 3 months
  29. Alexa Fluor 680
  30. Monoclonal antibody 4G2

Equipment

  1. Stir plate and stir bar
  2. Floating microtube rack (i.e., Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 5974-4015 )
  3. Forceps, Superfine Tips, Swiss Style #5 (BioQuip, catalog number: 4535 )
  4. Cover slip forceps (Fisher Scientific, catalog number: S17328C )
    Manufacturer: Medco Instruments, catalog number: S17328C .
    Note: It is important that the flat parts of the forceps touch each other over the entire surface. If the opposing surfaces touch at only one point, the ear skin is more likely to generate folds when inserting the needle.
  5. Stereoscopic Zoom Microscope (e.g., Nikon Instruments, model: SMZ1500 )
  6. Branson Sonifier Model 450 (Branson 450 Analog Sonifier with 1/2” Horn, 400 W, 120 VAC) (Cole-Parmer, catalog number: EW-04715-03 )
    Manufacturer: Emerson Electric, BRANSON, model: Model 450 .
  7. Cup Horn for Ultrasonic Processors, 3” dia (Cole-Parmer, catalog number: EW-04715-39)
    Manufacturer: Emerson Electric, BRANSON, catalog number: 109-116-1760 .
  8. Branson Sound-Proof Enclosure for Ultrasonic Processors (Cole-Parmer, catalog number: EW-04715-44)
    Manufacturer: Emerson Electric, BRANSON, catalog number: 101-063-275 .
  9. Refrigerated centrifuge (e.g., Eppendorf, model: 5417 R )
  10. Reusable glass microinjection syringe type 702RN, no needle, volume 25 µl (Hamilton, catalog number: 7636-01 )
    Note: Alternative syringe: Reusable glass microinjection syringe model 702 LT (Luer tip), SYR, NDL sold separately, no needle, volume 25 µl (Hamilton, catalog number: 80401 ).
  11. Heat block capable of heating to 37 °C and 60 °C (e.g., Digital Dry Block Heater, Single Block, 240 V, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 88870004 )
  12. Plate reader capable of reading absorption at 562 nm and 405-410 nm, and temperature control capable of heating to 37 °C (e.g., BioTek Instruments, model: ELx808 )

Procedure

  1. Dissection of salivary glands of female mosquitoes (Figure 1)


    Figure 1. Diagram of Aedes mosquito showing location of salivary glands

    1. Anesthetize mosquitoes by resting a cotton swab applicator moistened with triethylamine on the mesh lid of the mosquito holding carton; cover with plastic wrap or Petri dish to contain the triethylamine (Figure 2).


      Figure 2. Mosquito holding carton used for anesthetizing mosquitoes

    2. Once the mosquitoes are anesthetized, transfer them to a Petri dish and cover. Remove a female from the dish using Superfine tip forceps and dip into a microcentrifuge tube containing 70% ethanol to sterilize the body surface (Figure 3).


      Figure 3. Tools for salivary gland removal

    3. Place a drop (approximately 50 µl) of PBS on a microscope slide using a transfer pipet and spread the drop around.
    4. Place the slide on the stage of the dissecting microscope.
    5. Remove the legs of the mosquito, then place the body on its side in the PBS (see Video 1).

      Video 1. Dissection of mosquito salivary glands

    6. Remove the tip of the abdomen with a sharp cut from the dissecting probe; do not pull.
    7. Place one dissecting probe on the thorax of the mosquito and the other between the head and the thorax. Apply gentle pressure to the thorax, then slowly remove the head by gently pulling away from the thorax (Figure 4 and Video 1).


      Figure 4. Mosquito diagram: positioning for salivary gland removal

    8. The salivary glands will be apparent, if they remain with the head and may be removed by lifting them up and away from the head with the probe, while holding one probe between the glands and the head (Figure 5A and Video 1). If the glands remain within the thorax, apply gentle pressure with one probe held flat against the thorax and use the other probe to locate and gently tease out the glands (Figure 5B and Video 1).


      Figure 5. Mosquito diagram: removal of salivary glands. A. Lift salivary glands up & away from head; B. Tease salivary glands from thorax.

    9. Once the glands are removed, place an additional two drops of fresh PBS separately on the slide, near but not touching the initial drop, and carefully rinse the glands by drawing them sequentially through the PBS drops (Figure 6).


      Figure 6. Rinse salivary glands after removal

    10. Place one pair of salivary glands in 10 µl of PBS, pooling up to 50 pairs in a volume of 500 µl.
      Because mosquitoes inoculate volumes < 5 µl while probing for blood vessels, one may choose to prepare SGE at a higher concentration (using lower volumes of PBS) for later being able to reduce the volume of intradermal inoculation.
    11. Store at -20 °C until ready to prepare the SGE.

  2. Preparation of SGE
    1. Remove frozen glands and thaw quickly at 37 °C.
    2. Add water and ice chips to the horn of the sonicator.
    3. Place a microfuge tube containing salivary glands into the floating microtube rack.
    4. Program the sonicator with the following parameters: 100 mV, 3 bursts of 20 sec, 1 min cooling between bursts.
    5. Centrifuge at 5,000 x g at 4 °C for 10 min.
    6. Remove the supernatant to a fresh microcentrifuge tube.
    7. Determine the protein concentration of the SGE using the Micro BCA Protein Assay Kit, following the manufacturer’s microplate procedure. The typical amount of protein obtained per one pair of salivary gland is 0.8-1.2 µg, which we obtained in a volume of 10 µl PBS.
    8. Use pH strips to test pH of SGE stocks, which should be neutral pH of 7.0 due to the buffering capacity of PBS. In the case that the pH is not neutral, interpretation of the data should take this into account. On the one hand, pH may be an intrinsic property of saliva from the used vector species. On the other hand, differences in pH may change infectivity and transmission of a virus or another pathogen used in the experiments.
    9. Confirm that SGE stock is free of endotoxin or contains very low levels that are well below the FDA-approved limit for injection solutions (< 5 endotoxin units per kg per hour), as tested using the endpoint chromogenic Limulus Amebocyte Lysate kit (Lonza) and 10 µl SGE stock, which corresponds to approximately 1 µg of protein. If endotoxin levels are high, the SGE preparation should be discarded because observed effects cannot clearly be attributed to components of the SGE or the presence of endotoxin.
    10. Store at -80 °C.

  3. Intradermal inoculation
    1. Dilute the SGE in sterile PBS in a 1.5-ml tube. Adjust concentration to contain the extract of 0.2 to 1 salivary gland (each mosquito has one pair of salivary glands) per 20 µl PBS. If testing the effect of SGE on arbovirus infection, mix SGE with a concentrated virus stock of e.g., DENV (104-106 plaque forming units, see [Schmid and Harris, 2014; Schmid et al., 2016]). Use virus diluted in PBS as control for the absence of mosquito saliva. As further controls, use non-injected (untouched) ears or intradermal inoculation of PBS alone.
      If titers of the virus and SGE stock solutions are high enough, it may be beneficial to inoculate a total volume of less than 20 µl because mosquitoes typically inoculate < 5 µl.
    2. Assemble and sterilize the reusable glass syringe and needle (Figure 7) by flushing at least three times with 70% ethanol. Rinse three times with sterile PBS


      Figure 7. Hamilton glass syringe and needle

    3. For a 20 µl injection, take up a total of 25 µl of the salivary gland extract into the glass syringe, while excluding air bubbles. Avoid touching the surface of the tube to avoid blunting of the tip of the reusable needle.
    4. In the meantime, anesthetize mice via inhalation of 2-4% isoflurane in a stream of 2 L/min oxygen. Adjust inhalation time and percent isoflurane until the mouse breathes approximately once per second. After removing from the anesthesia, the mouse will still be anesthetized for approximately one minute, to allow the intradermal inoculation.
      Note: Anesthesia of mice via isoflurane inhalation must be performed by trained laboratory personnel.
    5. Immobilize one ear of an anesthetized mouse using cover slip forceps so that the ventral part of the ear faces up. The bottom of the forceps should end approximately in the center of the ear at the site of injection. Tilt the ear backwards and remove folds within the ear skin (Figure 8A).
      Note: Make sure that the ear skin is dry prior to injection in order to avoid sticking of the ear to the forceps. If necessary, dry the skin with tissue or gauze. See also Video 2, Intradermal Inoculation.

      Video 2. Intradermal inoculation of the mouse ear


      Figure 8. Intradermal inoculation of the mouse ear
       
    6. Force the part of the forceps that faces up towards the top and the part at the dorsal side of the ear (facing away) towards the bottom. Thus forming a ‘bench’ or ‘ledge’ will provide a solid support for the injection site (Figure 8A).
    7. Hold the needle with the bevel pointing up. Approach the ‘ledge’ with the need held at a ~45° angle and gently penetrate the surface of the skin (Figure 8B).
    8. Drop the 45° angle of the needle until it is parallel to the forceps and skin, while precisely maintaining the position of the tip of the needle. Being then parallel to the skin, one should feel the support of the forceps along the length of the needle (Figure 8C).
    9. Insert the needle ~3 mm while moving parallel to the surface within the skin tissue. One should feel substantial resistance and continuously see the bevel of the needle through the surface of the skin (Figure 8D). If the resistance suddenly decreases or the bevel can no longer be seen, the needle may have poked through the back of the skin (if this occurs, start a second attempt after removing the forceps and needle).
    10. Carefully let go of the forceps without moving the ear skin relative to the needle (Figure 8E).
    11. Move the hand that held the forceps over the other hand to reach the plunger of the syringe.
    12. Slowly inoculate 20 µl of solution and monitor the appearance of a small blister underneath the skin (Figure 8F). Successful injection leads to a transient stiffening of the ear (induced by the small blister), and some resistance should be felt while injecting. If no blister develops or no stiffening occurs, the inoculum may have leaked through the back of the ear. If this occurs, remove the needle and reinsert at a slightly different site of the ear.
    13. If a small amount of inoculum leaks through the front or back of the skin, compensate this volume with the extra 5 µl that have been taken up into the syringe to ensure injection of exactly 20 µl. If the inoculated volume is still < 20 µl, inoculate more at a different site of the same ear skin.
    14. After inoculation, hold the side of the ear (not where the blister has formed) with the forceps and slowly remove the needle.
      Note: Start using a new needle once initial puncture of the skin becomes more difficult (every 6-10 experiments).
    15. Throughout the procedure, closely monitor the breathing of the anesthetized mouse. Also, monitor the mice regularly during and after waking up from anesthesia.
    16. After use, clean the glass syringe by flushing several times with Bleach or other disinfectant, followed by PBS and then 70% ethanol. For reusing the glass syringes, designate one syringe per reagent (e.g., SGE alone, virus alone, SGE + virus, PBS alone) to avoid cross-contamination. The glass syringes and needles can also be autoclaved.

Data analysis

For details on animal experiments and ethical approvals, methods used, data processing and analysis, and statistical tests that were used to generate the example data in Figure 9, see our original publication (under the open access Creative Commons Attribution license [Schmid et al., 2016]), from which the representative example of data (below) was extracted:


Figure 9. Intradermal inoculation of SGE induces vascular leak into the skin and exacerbates Dengue disease in a mouse model of infection. A. Wild-type mice were inoculated intravenously with dextran that was labeled with Alexa Fluor 680. Immediately thereafter, 15 µl SGE or PBS was inoculated intradermally. After 30 min, mice were euthanized, and ears were scanned using the Odyssey CLx Infrared Imaging System (Licor). Representative scan showing fluorescence that leaked with plasma into the skin at sites of intradermal inoculation or corresponding areas in steady-state ears (white circles). B. Mice deficient in the interferon-α/β receptor (Ifnar-/-) were inoculated intradermally with 105 plaque forming units of Dengue virus-2 strain D220 in the absence or presence of enhancing antibodies (5 µg of monoclonal antibody 4G2, targeting Dengue virus E protein) to model antibody-dependent enhancement (ADE). Dengue virus was inoculated alone or after mixing with Ae. aegypti SGE (+SGE). Kaplan-Meier curves showing survival of mice. Data were pooled from three experiments. Statistically significant differences were tested between the presence and absence of SGE using the Log-rank (Mantel-Cox) test and are marked as ** for P < 0.01.

Acknowledgments

All experiments using mice were performed with approval of and strictly followed the guidelines of the Animal Care and Use Committee of the University of California, Berkeley, USA (AUP-2014-08-6638) and the Ethical Committee of the University of Leuven, Belgium (P140-2016). The development and use of this protocol at University of California, Berkeley was supported by the US National Institute of Allergy and Infectious Diseases, National Institutes of Health grant R01 AI085607 (EH) and the German Research Foundation (Deutsche Forschungsgemeinschaft), research fellowship SCHM 3011/1-1 (MAS). We would like to thank Johan Neyts and Kai Dallmeier for support, Ruben Pholien for technical assistance, and the KU Leuven Rega Foundation for a postdoctoral fellowship (MAS). The original work of the laboratory at KU Leuven was supported by the IVAP-project of the Belgian Society of Virology (BELVIR) from the Belgian Federal Science Policy Office (Belspo). This protocol was originally established to generate the primary data in Schmid et al., 2016 and was briefly described therein.

References

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简介

蚊子传播的病原体是人类严重疾病和死亡的主要原因之一。蚊子唾液中的组分促进血液供体,调节宿主反应,并允许有效传播病原体,如登革热,紫草,黄热病,西尼罗河,日本脑炎和基孔肯雅病毒,以及疟原虫寄生虫等。在这里,我们描述了评估蚊子衍生因子对小鼠感染模型中免疫反应和发病机制的影响的标准化方法。该方案包括产生蚊子唾液腺提取物和皮内接种小鼠耳朵。最终,使用这些技术获得的信息可以帮助揭示病原体,蚊子和哺乳动物宿主之间的相互作用的基本机制。此外,该协议还可以帮助建立疫苗或治疗剂的临床前检测改进的感染挑战模型,以考虑通过蚊子传播的自然途径。
【背景】在探测血液的同时,蚊子接种有助于喂养的唾液,但如果蚊子曾经感染过感染个体,也可能含有病原体。蚊子唾液在建立感染,促进传播,调节免疫应答和加剧西尼罗河病毒发病过程中发挥重要作用(Schneider等人,2006; Styer等人)。 ,2011),登革热病毒(Cox等人,2012; Conway等人,2014; McCracken等人,2014; Schmid ,2016),基孔肯雅病毒(Agarwal等人,2016),Semliki Forest病毒(Pingen等人,2016) ,裂谷热病毒(Le Coupanec等人,2013)和疟原虫寄生虫(Schneider等人,2011)感染。许多重要问题仍然存在,需要改进动物模型。
&NBSP;而通过感染的蚊子接种最好地模仿自然传播,接种剂量的高度变异性和有限的昆虫设施的可用性导致这种程序的有限使用。此外,使用感染的蚊子时,不能控制唾液的含量和蚊子组分的存在或不存在。作为替代方案,未感染的雌性蚊子的“斑点喂养”,然后通过针对病原体进行真皮内接种,模拟了唾液在小鼠皮肤中的自然沉积并递送了一定剂量的病原体。 “斑点喂养”模型已经成功地用于研究登革病毒(Cox等人,2012; McCracken等人,2014)和西尼罗病毒(Moser 等人,2015年)感染,但仍然需要同时使用活的蚊子和小鼠,并且不能控制输送的唾液量。为了单独控制蚊子和小鼠实验,将蚊子长鼻虫插入毛细管中的蔗糖溶液可以人工收集蚊子唾液。然而,在喂食期间收集的唾液与天然供血期间接种到宿主皮肤中的蚊子唾液有差异(Marinotti等人,1990; Moser等人, ,,2015)。
这里,我们描述使用来自非感染蚊子的针接种蚊子唾液腺提取物(SGE)的简化模型,其可以以受控的方式以定义的剂量与病原体一起递送。这种方法允许在合作者之间独立处理活的蚊子和小鼠,并且可以更容易地在测定和研究组之间进行标准化。 SGE的使用已被证明可用于研究西尼罗河病毒感染(Schneider等,2006; Moser等人,2015),登革热病毒(Conway 裂谷热病毒(Le Coupanec et al。,2013)和辛德毕斯病毒(Sindbis virus,et al。,2014; Schmid等人,2016) Schneider等人,2004)。注射SGE不能通过蚊子长鼻精确地模拟接种,并且可能含有唾液腺的非分泌成分。然而,这种方法允许收集更多量的蚊子来源的因子,含有所有分泌的蛋白质,并且也可以用于体外实验。总之,这里描述的程序应该促进昆虫学家,免疫学家和研究人员研究感兴趣的病原体之间的协作。

关键字:蚊子, 唾液, 唾液腺, 提取物, 病毒传播, 真皮内, 针头, 小鼠

材料和试剂

注意:对于蚊子的饲养和蚊子殖民地的一般维护,参见Bio-protocol Kauffman等人(2017)。

  1. 棉针织棉签,15厘米手柄(清教徒,目录号:25-826 5WC)
  2. 培养皿,无菌,60mm(例如,60mm TC处理的细胞培养皿,Corning,Falcon,目录号:353002)
  3. 塑料包装
  4. 50ml锥形离心管(CELLTREAT Scientific Products,目录号:229422)
  5. 15毫升锥形离心管(CELLTREAT Scientific Products,目录号:229412)
  6. 5 ml管,12 x 75 mm(Corning,Falcon ®,目录号:352054)
  7. 96孔板(Thermo Fisher Scientific,Thermo Scientific TM,目录号:15041)
  8. 5 ml血清移液管(CELLTREAT Scientific Products,目录号:229206B)
  9. 10 ml血清移液管(CELLTREAT Scientific Products,目录号:229211B)
  10. 微量离心管,1.5ml(Corning,Axygen ,目录号:MCT-150-C)
  11. 针头:30尺,小RN轮毂,定制,点式4(10°-12°斜面),25毫米长度(汉密尔顿,目录号:7803-07)
    注意:替代针头:使用一次性皮下注射针,例如30 G x 1英寸(BD,目录号:305128)。
  12. 25 x 75 x 1.0 mm的磨砂显微镜载玻片(例如,Fisher Scientific,目录号:12-550-343)
  13. 木制施药棒,15厘米(清教徒,目录号:807)
  14. 昆虫针头Morpho Black搪瓷No.000(BioQuip,目录号:1208B000)
  15. 用木制的涂抹棒和昆虫针制成的探针探针 注意:解剖探针由15厘米的木制施药器和昆虫针制成。将涂抹棒浸泡在热水中至少30分钟,并将头部从针脚上切下并丢弃。使用洗涤器,握住针脚,避免损坏尖端,并将钝头(切割)端部推入棒中。让探针干燥。
  16. 转移移液管,1毫升,散装,无毒(BioLogix,目录号:30-0135)
  17. pH测试条(例如,Sigma-Aldrich,目录号:P4536-100EA)
  18. 女性蚊子
  19. 蚊子手提箱(参见Bio-protocol Kauffman等人,2017,用于描述和建造)
  20. 小鼠
  21. 三乙胺(Sigma-Aldrich,目录号:T0866)
  22. 乙醇
  23. 磷酸盐缓冲盐水(PBS),低内毒素≤0.25EU/ ml(即Mediatech,目录号:21-030-CV)
  24. 微量BCA蛋白测定试剂盒(Thermo Fisher Scientific,Thermo Scientific TM,目录号:23235)
  25. 端点显色鲎Amebocyte裂解液QCL-1000测定(Lonza,目录号:50-647U)
  26. 消毒剂,如漂白剂或Umonium 38(Huckert's Laboratoire International,目录号:PF 12209)
  27. 异氟烷,Iso-Vet(Chanelle,目录号:CDS019936)用于麻醉小鼠
  28. 70%乙醇:将74ml乙醇与26ml水混合。标记为易燃并在室温下储存长达3个月
  29. Alexa Fluor 680
  30. 单克隆抗体4G2

设备

  1. 搅拌棒和搅拌棒
  2. 浮动微管架(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:5974-4015)
  3. 镊子,超细窍门,瑞士风格#5(BioQuip,目录号:4535)
  4. 盖滑镊子(Fisher Scientific,目录号:S17328C)
    制造商:Medco Instruments,目录号:S17328C。
    注意:镊子的平坦部分在整个表面上彼此接触很重要。如果相对的表面仅接触一点,则在插入针时,耳廓皮肤更有可能产生褶皱。
  5. 立体放大显微镜(例如,尼康仪器,型号:SMZ1500)
  6. Branson Sonifier 450型(Branson 450模拟超声波器,1/2“喇叭,400 W,120 VAC)(Cole-Parmer,目录号:EW-04715-03)
    制造商:Emerson Electric,BRANSON,型号:Model 450。
  7. 用于超音波处理器的杯形喇叭,3“直径(Cole-Parmer,目录号:EW-04715-39)
    制造商:Emerson Electric,BRANSON,目录号:109-116-1760。
  8. Branson防爆外壳超声波处理器(Cole-Parmer,目录号:EW-04715-44)
    制造商:Emerson Electric,BRANSON,目录号:101-063-275。
  9. 冷冻离心机(例如,Eppendorf,型号:5417R)
  10. 可重复使用的玻璃显微注射器型号702RN,无针,体积25μl(Hamilton,目录号:7636-01)
    注意:替代注射器:可重复使用的玻璃显微注射器型号702 LT(Luer tip),SYR,NDL单独出售,无针,体积25μl(Hamilton,目录号:80401)。
  11. 能够加热到37℃和60℃的热块(例如,数字干块加热器,单块240V,Thermo Fisher Scientific,Thermo Scientific TM ,目录号:88870004)
  12. 能够读取562nm和405-410nm吸收的读板器,以及能够加热到37℃的温度控制(例如,BioTek Instruments,型号:ELx808)

程序

  1. 女性唾液腺的解剖(图1)


    图1.伊蚊的图示显示唾液腺的位置的蚊子

    1. 通过将蚊子沾上三乙胺的棉签放在蚊帐的网眼盖上来麻醉蚊子;用塑料包装或培养皿盖住三乙胺(图2)

      图2.用于麻醉蚊子的蚊子夹持

    2. 一旦蚊子被麻醉,将它们转移到培养皿和盖子上。使用超细尖端镊子从盘中取出雌性,并浸入含70%乙醇的微量离心管中以对体表进行灭菌(图3)。


      图3.唾液腺去除工具

    3. 使用转移移液管在显微镜载玻片上放一滴(约50μl)的PBS,并将其滴落
    4. 将幻灯片放置在解剖显微镜的舞台上。
    5. 取出蚊子的腿,然后将身体放在PBS的侧面(见视频1)。

      Video 1. Dissection of mosquito salivary glands

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


    6. 从解剖探头上切开腹部,取出腹部尖端;不要拉。
    7. 将一个解剖探头放在蚊子的胸部,另一头在头部和胸部之间。对胸部施加温和的压力,然后轻轻地从胸部拉出头部(图4和视频1)。


      图4.蚊子图:唾液腺移除定位

    8. 如果唾液腺保持头部,并且可以用探头将其抬起并离开头部,同时在腺体和头部之间握住一个探针(图5A和视频1)可以清除唾液腺。如果腺体保留在胸腔内,应用温和的压力将一个探头平放在胸部上,并使用另一个探头定位并轻轻挑出腺体(图5B和视频1)。


      图5.蚊子图:去除唾液腺。 A.将唾液腺升起&amp;远离头B.从胸部抽出唾液腺。

    9. 一旦取出腺体后,将另外两滴新鲜PBS分别放在载玻片上,但不接触初始液滴,并通过PBS液滴依次绘制,仔细冲洗腺体(图6)。


      图6.清除后冲洗唾液腺

    10. 将一对唾液腺置于10μlPBS中,最多放入50对,体积为500μl。
      因为蚊子接种体积<在探查血管时为5μl,可以选择以较高浓度(使用较低体积的PBS)制备SGE,以便以后能够减少皮内接种的体积。
    11. 储存于-20°C,直至准备好SGE。

  2. SGE的准备
    1. 去除冷冻的腺体并在37°C迅速解冻。
    2. 将水和冰芯片添加到超声波仪器的喇叭。
    3. 将含有唾液腺的微量离心管放入浮动微管架中。
    4. 使用以下参数对超声波仪器进行编程:100 mV,3秒20秒,突发之间1分钟冷却。
    5. 在4℃下以5,000×g离心10分钟。
    6. 将上清液移至新鲜的微量离心管。
    7. 按照制造商的微孔板程序,使用Micro BCA Protein Assay Kit确定SGE的蛋白质浓度。每一对唾液腺获得的蛋白质的典型量为0.8-1.2μg,我们以10μlPBS的体积获得。
    8. 使用pH条测试SGE储备液的pH值,由于PBS的缓冲能力,其应为中性pH 7.0。在pH不是中性的情况下,数据的解释应该考虑到这一点。一方面,pH可能是来自所用载体物种的唾液的固有特性。另一方面,pH的差异可能改变实验中使用的病毒或其他病原体的感染性和传播。
    9. 确认SGE库存没有内毒素,或含有非常低的水平,远低于FDA批准的注射液限制(<5内毒素单位/ kg /小时),如使用终点显色鲎Amebocyte裂解液试剂盒(Lonza)测试的,和10μlSGE原料,其对应于约1μg蛋白质。如果内毒素水平高,则应该丢弃SGE制剂,因为观察到的效果不能清楚地归因于SGE的组分或内毒素的存在。
    10. 储存于-80°C。

  3. 皮内接种
    1. 在无菌PBS中的1.5ml管中稀释 SGE。调整浓度,每20μlPBS含有0.2至1个唾液腺(每只蚊子有一对唾液腺)的提取物。如果测试SGE对蛛网膜病毒感染的影响,则将SGE与浓缩的病毒储备物例如混合,将DENV(10μg/ ml)参见[Schmid和Harris,2014; Schmid等人,2016])。使用在PBS中稀释的病毒作为无蚊子唾液的对照。作为进一步的对照,使用非注射(未触动)耳朵或单独的PBS的皮内接种。
      如果病毒和SGE储备溶液的滴度足够高,则接种总体积小于20μl可能是有益的,因为蚊子通常接种< 5μl。
    2. <70>通过用70%乙醇冲洗至少三次,将可重复使用的玻璃注射器和针头(图7)组装和消毒。用无菌PBS冲洗三次

      图7.汉密尔顿玻璃注射器和针头

    3. 对于20μl注射液,将总共25μl的唾液腺提取物吸收到玻璃注射器中,同时排除气泡。避免接触管的表面,以避免可重复使用的针头尖端变钝。
    4. 同时,通过在2L / min氧气流中吸入2-4%异氟烷来麻醉小鼠。调整吸入时间和异氟烷百分比,直到鼠标每秒钟大约呼吸一次。从麻醉中取出后,小鼠仍然麻醉约1分钟,以进行皮内接种。
      注意:经过异氟烷吸入的小鼠麻醉必须由经过培训的实验室人员进行。
    5. 使用盖滑动镊子使麻醉的小鼠的一只耳朵固定,使得耳朵的腹侧面朝上。镊子的底部应该在注射部位大致在耳朵的中心。向后倾斜耳朵并清除耳部皮肤中的褶皱(图8A)。
      注意:为了避免将耳朵粘到镊子上,请确保注射前的耳朵皮肤干燥。必要时,用纸巾或纱布干燥皮肤。另见视频2,真皮内接种。

      Video 2. Intradermal inoculation of the mouse ear

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player



      图8.小鼠耳朵的真皮接种
      &nbsp;
    6. 强制镊子的朝向顶部的部分,以及耳朵背侧部分(朝外)朝向底部。因此,形成“台阶”或“壁架”将为注射部位提供坚实的支持(图8A)
    7. 用斜面朝上的方式握住针头。接近需要保持在〜45°角度的“凸缘”,并轻轻地穿透皮肤表面(图8B)。
    8. 降低针的45°角,直到其平行于镊子和皮肤,同时精确地保持针尖的位置。然后平行于皮肤,应该感觉到镊子沿着针的长度的支撑(图8C)。
    9. 插入针〜3毫米,同时平移于皮肤组织内的表面。应该感觉到很大的阻力,并通过皮肤表面不断地看到针的斜面(图8D)。如果阻力突然降低或不能再看到斜面,针头可能会穿过皮肤背面(如果发生这种情况,请在拆下镊子和针头后再次尝试)。
    10. 仔细放开镊子,而不会相对于针头移动耳朵皮肤(图8E)。
    11. 移动握住镊子的手,另一只手到达注射器的柱塞。
    12. 慢慢接种 20μl溶液,并监测皮肤下面的小水泡的外观(图8F)。成功的注射导致耳朵的瞬时僵硬(由小水泡引起),并且在注射时应该感觉到一些阻力。如果不发生水疱发生或不发生僵化,接种物可能已经通过耳朵后面渗漏。如果发生这种情况,请取下针头并重新插入耳朵稍微不同的位置。
    13. 如果少量的接种物通过皮肤前部或背部渗漏,请用额外的5μl补充该体积,将其吸入注射器,以确保注射完全20μl。如果接种体积仍然< 20μl,在同一耳朵皮肤的不同部位接种更多。
    14. 接种后,用镊子握住耳朵的一侧(不是水泡形成的地方),并慢慢移除针头。
      注意:一旦初次穿刺皮肤变得更加困难(每6-10次实验),开始使用新的针头。
    15. 在整个程序中,仔细观察麻醉鼠标的呼吸。此外,在从麻醉中醒来期间和之后定期监测小鼠。
    16. 使用后,使用漂白剂或其他消毒剂冲洗几次,然后用PBS然后70%乙醇清洗玻璃注射器。为了重复使用玻璃注射器,每个试剂(例如,单独的SGE,单独的病毒,单独的SGE +病毒,PBS单独)指定一个注射器,以避免交叉污染。玻璃注射器和针头也可以被高压灭菌。

数据分析

有关动物实验和道德审批的详细信息,使用的方法,数据处理和分析以及用于生成图9中的示例数据的统计测试,请参阅我们的原始出版物(根据开放获取知识共享署名许可[Schmid 等等,2016]),从中提取了数据的代表性例子(下面):


图9. SGE的皮内接种诱导血管渗漏到皮肤中,并加重感染小鼠模型中的登革热病。A.用Alexa Fluor 680标记的葡聚糖静脉内接种野生型小鼠。之后立即将15μlSGE或PBS皮内接种。 30分钟后,将小鼠安乐死,并使用Odyssey CLx红外成像系统(Licor)扫描耳朵。代表性的扫描显示在皮肤接种的位置或稳态耳朵中的相应区域(白色圆圈)等离子体泄漏到皮肤中的荧光。 B.用干酪素-α/β受体缺陷的小鼠( - / - )用登革热病毒-2菌株D220在不存在或存在增强抗体(5μg单克隆抗体4G2,靶向登革病毒E蛋白))以模拟抗体依赖性增强(ADE)。登革热病毒单独或与Ae混合后接种。 aegypti SGE(+ SGE)。 Kaplan-Meier曲线显示小鼠的生存。从三个实验汇总数据。使用对数秩(Mantel-Cox)测试在SGE的存在和不存在之间测试统计学上的显着性差异,并将其标记为**用于P < 0.01。

致谢

所有使用小鼠的实验均经过美国加利福尼亚大学伯克利分校(AUP-2014-08-6638)和比利时鲁汶大学伦理委员会的批准并严格遵守。 (P140-2016)。加利福尼亚大学伯克利分校的这一协议的开发和使用得到了美国国家过敏和传染病研究所,国家卫生研究院授予R01 AI085607(EH)和德国研究基金会(Deutsche Forschungsgemeinschaft)的支持。 >),SCHM 3011 / 1-1(MAS)研究奖学金。我们要感谢Johan Neyts和Kai Dallmeier的支持,Ruben Pholien的技术援助和KU Leuven Rega基金会的博士后研究金(MAS)。来自比利时联邦科学政策办公室(Belspo)的比利时病毒学协会(BELVIR)的IVAP项目支持了鲁汶大学实验室的原始工作。该协议最初被建立以在Schmid等人,2016中生成主要数据,并在其中简要描述。

参考

  1. Agarwal,A.,Joshi,G.,Nagar,D.P.,Sharma,A.K.,Sukumaran,D.,Pant,S.C.,Parida,M.M.and Dash,P.K。(2016)。 蚊子唾液诱导的皮肤事件增加基孔肯雅病毒复制和疾病进展。 / a> &nbsp; Infect Genet Evol 40:126-135。
  2. Conway,MJ,Watson,AM,Colpitts,TM,Dragovic,SM,Li,Z.,Wang,P.,Feitosa,F.,Shepherd,DT,Ryman,KD,Klimstra,WB,Anderson,JF和Fikrig,E 。(2014)。蚊子唾液丝氨酸蛋白酶增强传播登革热病毒进入哺乳动物宿主。 J Virol 88(1):164-175。
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引用:Schmid, M. A., Kauffman, E., Payne, A., Harris, E. and Kramer, L. D. (2017). Preparation of Mosquito Salivary Gland Extract and Intradermal Inoculation of Mice. Bio-protocol 7(14): e2407. DOI: 10.21769/BioProtoc.2407.
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