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Sep 2018
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Myelin Oligodendrocyte Glycoprotein 35-55 (MOG 35-55)-induced Experimental Autoimmune Encephalomyelitis: A Model of Chronic Multiple Sclerosis
髓鞘少突胶质细胞糖蛋白多肽( MOG35-55)诱发实验性自身免疫性脑脊髓炎:一种慢性多发性硬化模型   

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Abstract

Multiple sclerosis (MS) is the common demyelinating disease of human central nervous system. Among mouse models available to study MS, including the cuprizone application and lysolecithin-injection models, experimental autoimmune encephalomyelitis (EAE) model is widely used so that chronic EAE model of C57BL/6J can reflect the autoimmune pathogenesis of MS well. Here we introduce the EAE model based on C57BL/6J mice, which is generated by injection of myelin oligodendrocyte glycoprotein 35-55 (MOG 35-55) as an antigen. After immunization with complete Freund's adjuvant, clinical signs and changes in body weight are observed one or two weeks later. The EAE model will continue to be useful for development of therapeutics for MS.

Keywords: Experimental autoimmune encephalomyelitis (EAE) (实验性自身免疫性脑脊髓炎), Multiple sclerosis (MS) (多发性硬化), Chronic (慢性), Myelin oligodendrocyte glycoprotein 35-55 (MOG 35-55) (髓鞘少突胶质细胞糖蛋白多肽), C57BL/6J (C57BL/6J), Complete Freund's adjuvant (CFA) (完全弗氏佐剂), Autoimmune (自身免疫), Central nervous system (CNS) (中枢神经系统)

Background

Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system (CNS) and is thought to have autoimmune etiology (Thompson et al., 2018). Current therapeutic treatments mainly target lymphocytes; however, this can cause serious side effects and does not provide sufficient therapeutic efficacy for those with progressive MS. Therefore, animal models of MS are important for further elucidation of pathological mechanisms and discovery of new treatments. Available pathological mouse model of MS, including the experimental autoimmune encephalomyelitis (EAE) model, the cuprizone application model, and lysolecithin-injection models, each has its own advantages (Kipp et al., 2017). EAE is used widely to study MS (Ransohoff, 2012; Baker and Amor, 2014). All EAE models enable investigation of both the immune system and CNS, which are the targets of MS therapies (Mix et al., 2010). Although there are several animal models of EAE, two are common in MS research (Burrows et al., 2019). One is the C57BL/6 mouse, which is immunized with myelin oligodendrocyte glycoprotein (MOG) (Mendel et al., 1995), and the other is the SJL mouse, which is immunized with proteolipid protein (PLP) (Tuohy et al., 1988). The former develops chronic EAE (Mendel et al., 1995; Bittner et al., 2014) and is more popular because the C57BL/6J strain has abundant genomic resources (Gold et al., 2006). The latter model develops remitting and relapsing disease, which does not happen in the MOG-EAE model; therefore, this model is used as a model for relapsing-remitting MS (Tuohy et al., 1989).

Studies of the mechanisms underlying MS and development of suitable therapies can be conducted using EAE models; however remyelination is difficult to study in EAE model, two toxic models displaying demyelination and remyelination are widely used (Ransohoff, 2012). Cuprizone, a copper chelator, kills oligodendrocytes, resulting in demyelination and remyelination (Matsushima and Morell, 2001), whereas lysolecithin, also called lysophosphatidylcholine (LPC), is toxic to the myelin sheath (Hall, 1972). The lysolecithin-injected model has an important characteristic in that demyelination can be controlled both spatially and temporally (Keough et al., 2015). By contrast, the EAE model better reflects the autoimmune pathogenesis of MS, and represents a secondary progressive form the disease model with some modifications (Tanabe et al., 2019). Although this EAE model described in this protocol can be generated easily, disease incidence and symptoms vary according to the experimental environment. Therefore, we propose a solid method that can reliably create a C57BL/6J EAE model, which is close to human pathology and highly versatile as an animal model.

Materials and Reagents

  1. 2.5 ml Luer lock syringe (Terumo, Tokyo, Japan, catalog number: SS-02LZ)
  2. Three-way stopcock (Terumo, catalog number: TS-TR2K)
  3. 26 G Needles (Terumo, catalog number: NN-2613S)
  4. 18 G Needles (Terumo, catalog number: NN-1838S)
  5. 1 ml Syringe (Terumo, catalog number: SS-01T)
  6. 50 ml tube (Greiner, Kremsmünster, Austria, catalog number: 227261)
  7. 2 ml tube (INAOPTIKA, Osaka, Japan, catalog number: SC-0200)
  8. Mice: C57BL/6J, female, 7-12 weeks old (Japan SLC, Shizuoka, Japan)
  9. 1 mg/ml Adjuvant, Complete H37 Ra (BD Difco, Franklin Lakes, NJ, catalog number: 231131)
  10. M. Tuberculosis H37 Ra (BD Difco, catalog number: 231141)
  11. Pertussis toxin derived from Bordetella pertussis lyophilized powder (Sigma-Aldrich, St Louis, MO, catalog number: P7208)
  12. Myelin oligodendrocyte glycoprotein 35-55 (MEVGWYRSPFSRVVHLYRNGK, purity: > 95 %, Scrum, Tokyo, Japan)
  13. 5 mg/ml midazolam (Dormicum, Astellas Pharma, Tokyo, Japan)
  14. 5 mg/ml butorphanol (Vetorphale, Meiji-Seika Pharma, Tokyo, Japan)
  15. 1 mg/ml medetomidine (Domitor, Zenoaq, Fukushima, Japan)
  16. Sodium Chloride (Nakalai Tesque, Kyoto, Japan; catalog number: 31320-05)
  17. Potassium Chloride (Nakalai Tesque, catalog number: 28514-75)
  18. di-Sodium Hydrogenphosphate (Nakalai Tesque, catalog number: 31801-05)
  19. Potassium Dihydrogenphosphate (Nakalai Tesque, catalog number: 28721-55)
  20. Saline (Otsuka Pharmaceutical, Tokyo, Japan)
  21. Phosphate buffered saline (see Recipes)

Equipment

  1. Mortar and pestle (Not specified)
  2. Cooking scale (Tefal, Sarcelles, France; catalog number: BC2000J2)
  3. Freezer

Software

  1. Graph Pad Prism 5 (Graph Pad Prism Software, San Diego, CA)

Procedure

Timeline

Day 0
Step A, Preparation of anesthesia: ~15 min
Step B-D, Preparation of MOG/CFA emulsion: ~60 min
Step E, Preparation of PTx: ~10 min
Step F, Administration of MOG/CFA emulsion and PTx: ~45 min

Day 1
Step G, EAE monitoring: 1~2 min/mouse

Day 2
Step E, Preparation of PTx: ~10 min
Steps F9 and F10, Administration of PTx: ~10 min
Step G, EAE monitoring: 1~2 min/mouse

Days 3-28
Step G, EAE monitoring: 1~2 min/mouse


  1. Preparation of the cocktail of three different anesthetic agents
    Anesthesia is important to ensure subcutaneous administration of the emulsion.
    1. Prepare a cocktail of three different anesthetic agents (30 μg/ml medetomidine, 400 μg/ml midazolam, and 500 μg/ml butorphanol) in saline. This solution can be stored in a refrigerator at 4 °C, for at least 8 weeks after mixing. For non-immunized control mice, use the same anesthesia as EAE.
      1. For a 25 ml solution, combine 2 ml of midazolam with 2.5 ml of butorphanol in a 50 ml tube.
      2. Add 0.75 ml of medetomidine.
      3. Dilute to 25 ml with saline.

  2. Preparation of Complete Freund's Adjuvant (CFA)
    CFA is used to increase the immunogenicity of MOG 35-55 as an adjuvant.
    1. Calculate the required amount of M. Tuberculosis H37 Ra (MT). The required amount of MT is 500 μg per mouse. There will be some loss of MT during preparation and injection, so prepare 1.5 to 2 times more than needed amount.
    2. Calculate the required amounts of 1 mg/ml Adjuvant, Complete H37 Ra (1 mg/ml CFA). The required amount of 1 mg/ml CFA is 100 μl per mouse. There will be some loss of CFA during preparation and injection, so prepare 1.5 to 2 times more than needed amount.
    3. Place the required amount of MT into a mortar and grind with a pestle to obtain a fine powder (Figure 1A).
    4. Add the required amount of 1 mg/ml CFA into the mortar (Step B3; for example, add 5 mg MT to 1 ml of 1 mg/ml CFA) and mix to obtain a final concentration of 6 mg/ml CFA/MT mix (Figure 1B). This solution should be prepared on the day of immunization by emulsion, and can be stored at room temperature until it is used in Step D1.


      Figure 1. Preparation of Complete Freund's Adjuvant (CFA). A. Grounding MT with a mortar and a pestle. B. Mixing MT and 1 mg/ml CFA with a mortar and a pestle to obtain a final concentration of 6 mg/ml CFA/MT mix.

  3. Preparation of the MOG 35-55 peptide solution
    MOG 35-55 is used as an antigen to induce a demyelinating immune response.
    1. Dilute lyophilized MOG 35-55 in saline to obtain a 2 mg/ml stock solution in a 2 ml tube. This solution (2 mg/ml MOG 35-55 solution) can be stored in a freezer at -20 °C, for at least 8 weeks after dissolution. Avoid repeated freezing and thawing.
    2. Prior to immunization, dilute the MOG 35-55 stock solution with saline to yield a final concentration of 1 mg/ml. This solution (1 mg/ml MOG 35-55 solution) should be prepared on the day of immunization by emulsion, and can be stored in a refrigerator at 4 °C until it is used in Step D4.
    3. Use saline instead of MOG 35-55 solution for non-immunized control mice.

  4. Preparation of the MOG emulsion
    MOG 35-55/CFA emulsion induces immune response in lymph nodes.
    1. Draw 6 mg/ml CFA/MT mix from the mortar (Step B4) into a 2.5 ml Luer lock syringe fitted with an 18 G needle (Figure 2A). For non-immunized control mice, use the same 6 mg/ml CFA/MT mix as EAE.
    2. Remove the 18 G needle and connect the three-way stopcock. Release the air from the stopcock (Figure 2B).
    3. Turn the stopcock lever to close the valve connected to the CFA syringe (Figure 2C).
    4. Draw 1 mg/ml MOG 35-55 solution (Step C2) into another 2.5 ml Luer lock syringe with an 18 G needle. For non-immunized control mice, use saline instead of MOG 35-55 solution.
    5. Remove the 18 G needle and connect to the three-way stopcock. Release the air from the stopcock (Figure 2D). At this time, of either CFA or MOG solution, the liquid with the larger volume is named “solution A”, and the liquid with the smaller volume is named “solution B”.
    6. Drain “solution A” through the three-way stopcock, so that it is the same volume as “solution B”. After arranging 6 mg/ml CFA/MT mix: 1 mg/ml MOG solution to 1:1, turn the stopcock lever to close the valve not connected to a syringe (Figure 2E).
    7. Pass the solution back and forth between the two syringes for about 10 min (Figure 2F). In this step, importantly, start by pushing the syringe containing the CFA at first. This will create a white emulsion. The process is complete when the syringes suddenly begin to move easily.
    8. Drip just a drop of the emulsion into water to check the emulsion. If the droplet spreads on the water surface then the mixture is not ready (Figure 3A), so repeat Steps D7 and D8 until the emulsion is complete. It is ready when the droplet does not spread (Figure 3B). This emulsion should be prepared on the day of immunization, and can be stored in a refrigerator at 4 °C until use in Step F4.


      Figure 2. Preparation of the MOG emulsion. A. Drawing 6 mg/ml CFA/MT mix from a mortar into a 2.5 ml leur lock syringe. B. Connecting CFA syringe to a three-way stopcock and releasing air from the stopcock. C. Closing the valve connected to the CFA syringe. D. Connecting MOG 35-55 syringe to the stopcock and releasing air. E. Arranging amount of MOG 35-55:CFA = 1:1 and closing the valve without syringe. F. Mixing MOG 35-55 and CFA to get white emulsion.


      Figure 3. Checking the condition of the emulsion. A. Unfinished emulsion spread like this picture. B. Completed emulsion does not spread.

  5. Preparation of Pertussis toxin (PTx)
    PTx is used to disrupt the blood brain barrier and allow immune cells to invade the CNS.
    1. Dilute one vial of Pertussis toxin derived from Bordetella pertussis lyophilized powder (50 μg) in 1 ml of phosphate buffered saline (PBS) to obtain a 50 μg/ml PTx stock solution. This solution (50 μg/ml PTx stock solution) can be stored in a refrigerator at 4 °C, for at least 6 months after dissolution.
    2. Calculate the required amount of PTx. The required amount of PTx for each mouse is at a dose of 10 μg/kg. Some Ptx will be lost during preparation and injection, so prepare 1.1 to 1.5 times more than the needed amount.
    3. Dilute the PTx stock solution 1:50 in PBS to yield a final concentration of 1 μg/ml in a 2 ml tube. This solution (1 μg/ml Ptx) should be prepared on the day of injection (Day 0 and Day 2), and can be stored in a refrigerator at 4 °C until it is used in Steps F7 and F10. For non-immunized control mice, use the same PTx solution as EAE.
    4. Prepare a 1 ml syringe and a 26 G needle for intraperitoneal injection.

  6. Animal immunization
    This immunization step is preferably performed during the light phase. Anesthesia, emulsion and PTx administration day is set to Day 0, and PTx-only injection day is Day 2.
    1. Ensure that all mice can be identified easily to enable daily evaluation, e.g., by color marking the tail base.
    2. Put each mouse on the cooking scale and measure the body weight of each mouse. 
    3. Anesthetize with the cocktail of three different anesthetic agents. Each mouse is anesthetized by intraperitoneal (i.p.) injection (10 ml/kg). Use a 1 ml syringe fitted with a 26 G needle. For non-immunized control mice, use the same anesthesia as EAE. The mouse is fully anesthetized about 10 min after administration, and the anesthetic lasts about 1 h (Kirihara et al., 2013).
    4. Remix the complete emulsion, which is prepared in Step D, and transfer all of the emulsion to a single syringe.
    5. Remove the stopcock and connect a 26 G needle (Figure 4A).
    6. Inject 100 μl of the MOG 35-55/CFA emulsion subcutaneously (s.c.) at two different sites (upper back (neck) (Figure 4B) and lower back (root of the right hind limb) (Figure 4C). For non-immunized control mice, use the control emulsion made in Step D, which excludes only MOG 35-55.
    7. Inject 10 ml/kg PTx solution i.p. (10 μg/kg PTx). For non-immunized control mice, inject the same PTx solution as EAE.
    8. Check the following points of the mice which are injected the emulsion: Whether the mouse has awakened from anesthesia, is not dead, or has an emulsion leaked from the administration site. Mice with any problems are excluded from the experiment.
    9. Prepare the PTx solution as in Step E on Day 2.
    10. Measure the body weight and inject a second dose of PTx on Day 2 post-immunization as in Step F7.


      Figure 4. Animal immunization. A. Connecting a 26 G needle to the emulsion syringe. B and C. Injecting emulsion subcutaneously to the neck (B) and base of the right hindlimb (C).

  7. EAE monitoring
    This monitoring step is preferably performed during the light phase. On Day 0 (the day of emulsion administration), the mice are evaluated after recovering from anesthesia. On Day 2 (the day of only PTx injection), the evaluation is performed immediately after administration.
    1. Weigh each mouse (EAE and non-immunized control) daily.
    2. On a daily basis, evaluate the clinical signs of each mouse (EAE and non-immunized control) and score as follows:
      1. 0: no clinical deficit.
      2. 1: partial tail paralysis (Figure 5A, Video 1).
      3. 2: full tail paralysis (Figure 5B, Video 2).
      4. 3: partial hindlimb paralysis (Figure 5C, Video 3).
      5. 4: full hindlimb paralysis (Figure 5D, Video 4).
      6. 5: forelimb paresis (Figure 5E, Video 5).
      7. 6: dead.
      Details of clinical score evaluation
      First, when mouse is lifted by the center of the tail, give a score of 1 or higher if the tip of the tail hangs down and a score of 0 if not. Second, when mouse is lifted by the base of the tail, give a score of 2 or higher if the all of the tail hangs down without tension. Next, when mouse is hanged on the lid of the cage with the only forelimbs, give a score of 2 if lower body can be lifted to grab the lid not only on the forelimbs but also on the hind limbs, and a score of 3 or higher if not. Then, when mouse is put on the flat desk, give a score of 4 or higher if it drags with both hind limbs facing down, and a score of 3 if not. After that, when the lower body of a mouse is lifted, give a score of 5 if the mouse could move by the forelimbs only in one direction, either left or right, and a score of 4 if it could move in both directions. Note that non-immunized control mice do not develop such clinical symptoms at all.


      Figure 5. Clinical score evaluation of EAE mice. Representative mouse images of each clinical score. A. Clinical score 1: partial tail paralysis, B. Clinical score 2: full tail paralysis, C. Clinical score 3: partial hindlimb paralysis, D. Clinical score 4: full hindlimb paralysis, E. Clinical score 5: forelimb paresis.

      Video 1. EAE mouse with clinical score 1. (All experiments were conducted in accordance with the ethical guidelines of the Kyoto University Animal Experimentation Committee and with the guidelines of the Japanese Pharmacological Society. The approval ID of the animal experiment in this protocol is 14-42 and the validity period is from 2014 to 2019.)

      Video 2. EAE mouse with clinical score 2. (All experiments were conducted in accordance with the ethical guidelines of the Kyoto University Animal Experimentation Committee and with the guidelines of the Japanese Pharmacological Society. The approval ID of the animal experiment in this protocol is 14-42 and the validity period is from 2014 to 2019.)

      Video 3. EAE mouse with clinical score 3. (All experiments were conducted in accordance with the ethical guidelines of the Kyoto University Animal Experimentation Committee and with the guidelines of the Japanese Pharmacological Society. The approval ID of the animal experiment in this protocol is 14-42 and the validity period is from 2014 to 2019.)

      Video 4. EAE mouse with clinical score 4. (All experiments were conducted in accordance with the ethical guidelines of the Kyoto University Animal Experimentation Committee and with the guidelines of the Japanese Pharmacological Society. The approval ID of the animal experiment in this protocol is 14-42 and the validity period is from 2014 to 2019.)

      Video 5. EAE mouse with clinical score 5. (All experiments were conducted in accordance with the ethical guidelines of the Kyoto University Animal Experimentation Committee and with the guidelines of the Japanese Pharmacological Society. The approval ID of the animal experiment in this protocol is 14-42 and the validity period is from 2014 to 2019.)

      These videos were made at Kyoto University in accordance with the ethical guidelines of the Kyoto University Animal Experimentation Committee and the guidelines of the Japanese Pharmacological Society, and approved by the Kyoto University Animal Experimentation Committee (Protocol Number: 19-36).

Data analysis

  1. Display the daily body weight measurement data as an XY plot, where Y is “Body weight” and X is “Days post-immunization” (Figure 6A) by using GraphPad Prism (table format: grouped).
  2. Display the daily clinical score evaluation data in an XY plot, where Y is the “Clinical score” and X is “Days post-immunization” (Figure 6B) by using GraphPad Prism (table format: grouped).
  3. Analyze the data using two-way ANOVA, with Tukey’s multiple-comparisons test.


    Figure 6. Changes in weight and clinical symptoms of EAE. Development of EAE in MOG-immunized C57BL/6 mice. Body weight (A) and clinical score (B) were monitored for 28 days. Note that mice began to lose body weight at the onset of clinical signs during EAE. n = 4, **P < 0.01, ***P < 0.001.

Notes

  1. Importance of grounding the MT into a fine powder (Step B3)
    This step is important because the EAE incidence was higher when this operation was firmly performed compared with the careless operation. The fine powder of MT can be easily mixed with CFA, so that the EAE incidence should be increased.
  2. Reasons for using a three-way stopcock to get the emulsion (Step D)
    It is very important to make a strong emulsion. Therefore, we can judge the completeness of the emulsion by the slipping of syringe operation when we use a 3-way stop-cock.
  3. Importance of removing the air from the stopcock (Steps D2 and D5)
    If the air is not removed, the content of MOG 35-55 and CFA in the emulsion administered to one mouse could decrease, resulting in variability from experiment to experiment. Thus, the air in the three-way stopcock and the syringes should be removed.
  4. Importance of starting with the CFA syringe first (Step D7)
    We have tried both pushing from the CFA syringe and pushing from the MOG syringe in the step of mixing them to make an emulsion. As a result, we found that the former was easy to push when mixing by pushing the syringe, so we recommend pushing the CFA syringe first.
  5. Necessity of the emulsion injection to two different sites (Step F6)
    The location of each emulsion administration targets a different lymph node; the upper back (neck) is an axillary lymph node, and the lower back (root of the hind limb) is an inguinal lymph node. We think that the EAE incidence increases by causing immune responses in various lymph nodes. When the incidence is low, one of the causes could be that the administration position may be far from the lymph nodes.
  6. Importance of daily body weight measurement (Step G1)
    Since worsening of symptoms and weight loss correlate, it is considered that recording weight together with the clinical score leads to proper evaluation of EAE. Moreover, weight loss often occurs before the appearance of EAE clinical scores, which helps to avoid overlooking the clinical score of 1.
  7. Importance of daily clinical score evaluation (Step G2)
    EAE pathological condition can be evaluated by confirming daily changing symptoms with clinical score. This symptom evaluation with drug administration or genetically modified mice leads to elucidation of the disease state and evaluation of the therapeutic effect. Thus, daily scoring is important. Clinical symptoms become apparent about 10-14 days after antigen immunization, and then reach the maximum score within a few days to a week, and often become chronic as it is. The incidence is 90-100%.
  8. Importance of the genetic background
    Genetic background is very important because MOG 35-55 EAE model does not work in SJL mice, and PLP 139-151 EAE model does not work in C57BL/6J mice. However, NOD (non-obese diabetes) mouse is also usable for MOG EAE model. For details, see the citation of Stromnes and Goverman, 2006.

Recipes

  1. Phosphate buffered saline (PBS)
    1 L Ultra-pure water
    8 g Sodium chloride (NaCl)
    0.2 g Potassium chloride (KCl)
    1.15 g di-Sodium hydrogenphosphate (Na2HPO4)
    0.2 g Potassium dihydrogenphosphate (KH2PO4)

Acknowledgments

This study was supported by MEXT/JSPS KAKENHI Grant Numbers 17K19486 and 19K03377 (to H.S.). The procedure was adapted from that described in a previous study (Tsutsui et al., 2018).

Competing interests

The authors declare no conflicts of interest associated with this manuscript.

Ethics

All experiments were conducted in accordance with the ethical guidelines of the Kyoto University Animal Experimentation Committee and with the guidelines of the Japanese Pharmacological Society. The approval ID of the animal experiment in this protocol is 14-42 and the validity period is from 2014 to 2019.

References

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简介

多发性硬化症(MS)是人类中枢神经系统常见的脱髓鞘疾病。在可用于研究MS的小鼠模型中,包括铜酮应用和溶血卵磷脂注射模型中,实验性自身免疫性脑脊髓炎(EAE)模型被广泛使用,因此C57BL / 6J的慢性EAE模型可以很好地反映MS的自身免疫性发病机理。在这里,我们介绍基于C57BL / 6J小鼠的EAE模型,该模型是通过注射髓鞘少突胶质细胞糖蛋白35-55(MOG 35-55)作为抗原而生成的。用完全的弗氏佐剂免疫后,一到两周后观察到临床体征和体重变化。EAE模型将继续用于开发MS疗法。

【背景】多发性硬化症(MS)是中枢神经系统(CNS)的一种慢性炎症性疾病,被认为具有自身免疫病因(Thompson et al。,2018)。目前的治疗方法主要是针对淋巴细胞。但是,这可能会导致严重的副作用,并且不能为进行性MS的患者提供足够的治疗效果。因此,MS的动物模型对于进一步阐明病理机制和发现新的治疗方法很重要。MS可用的病理小鼠模型,包括实验性自身免疫性脑脊髓炎(EAE)模型,cuprizone应用模型和溶血卵磷脂注射模型,各有其优势(Kipp et al。,2017)。EAE被广泛用于研究MS(Ransohoff,2012; Baker和Amor,2014)。所有EAE模型都可以研究免疫系统和中枢神经系统,这是MS治疗的目标(Mix et al。,2010)。尽管有几种EAE动物模型,但在MS研究中有两种很常见(Burrows et al。,2019)。一种是用髓鞘少突胶质细胞糖蛋白(MOG)免疫的C57BL / 6小鼠(Mendel等),1995),另一种是用蛋白脂蛋白(PLP)免疫的SJL小鼠。 )(Tuohy等,1988)。前者发展为慢性EAE(Mendel等人,1995; Bittner等人,2014),并且由于C57BL / 6J菌株具有丰富的基因组资源而广受欢迎(金 et al。,2006)。后一种模型会引起疾病的复发和复发,而在MOG-EAE模型中则不会发生。因此,该模型被用作复发缓解型MS的模型(Tuohy等,1989)。



可以使用EAE模型对MS潜在机制和合适疗法的发展进行研究。然而,在EAE模型中很难研究髓鞘再生,显示脱髓鞘和髓鞘再生的两种毒性模型被广泛使用(Ransohoff,2012)。铜螯合剂Cuprizone杀死少突胶质细胞,导致脱髓鞘和再髓鞘化(Matsushima and Morell,2001),而溶血卵磷脂,也称为溶血磷脂酰胆碱(LPC),对髓鞘具有毒性(Hall,1972)。溶血卵磷脂注射模型具有一个重要特征,即可以在空间和时间上控制脱髓鞘作用(Keough等,2015)。相比之下,EAE模型更好地反映了MS的自身免疫性发病机制,并代表了经过某些修改的疾病模型的继发性进行性疾病(Tanabe et al。,2019)。尽管可以轻松生成此协议中描述的EAE模型,但疾病发生率和症状会根据实验环境而有所不同。因此,我们提出了一种可靠的方法,该方法可以可靠地创建C57BL / 6J EAE模型,该模型接近于人类病理学并且作为动物模型具有很高的通用性。

关键字:实验性自身免疫性脑脊髓炎, 多发性硬化, 慢性, 髓鞘少突胶质细胞糖蛋白多肽, C57BL/6J, 完全弗氏佐剂, 自身免疫, 中枢神经系统

材料和试剂

  1. 2.5 ml鲁尔锁注射器(Terumo,Tokyo,Japan,货号:SS-02LZ)
  2. 三通旋塞阀(Terumo,货号:TS-TR2K)
  3. 26 G针(Terumo,货号:NN-2613S)
  4. 18 G针(Terumo,货号:NN-1838S)
  5. 1 ml注射器(Terumo,目录号:SS-01T)
  6. 50 ml管(Greiner,Kremsmünster,Austria,目录号227261)
  7. 2 ml管(INAOPTIKA,日本大阪,目录号:SC-0200)
  8. 小鼠:C57BL / 6J,雌性,7-12周大(日本SLC,静冈,日本)
  9. 1 mg / ml佐剂,完整的H37 Ra(BD Difco,Franklin Lakes,NJ,目录号231131)
  10. M。结核 H37 Ra(BD Difco,目录号:231141)
  11. 百日咳毒素来源于百日咳博德特氏菌冻干粉(密苏里州圣路易斯的西格玛奥德里奇公司,目录号:P7208)
  12. 髓磷脂少突胶质细胞糖蛋白35-55(MEVGWYRSPFSRVVHLYRNGK,纯度:> 95%,Scrum,东京,日本)
  13. 5 mg / ml咪达唑仑(Dormicum,Astellas Pharma,Tokyo,Japan)
  14. 5 mg / ml布托啡诺(Vetorphale,明治制衣制药公司,东京,日本)
  15. 1 mg / ml美托咪定(Domitor,Zenoaq,Fukushima,Japan)
  16. 氯化钠(Nakalai Tesque,京都,日本;目录号:31320-05)
  17. 氯化钾(Nakalai Tesque,目录号:28514-75)
  18. 磷酸氢二钠(Nakalai Tesque,目录号:31801-05)
  19. 磷酸二氢钾(Nakalai Tesque,目录号:28721-55)
  20. 盐水(大冢制药,日本东京)
  21. 磷酸盐缓冲盐水(请参阅食谱)

设备

  1. 研钵和杵(未指定)
  2. 烹饪秤(特法尔,萨尔塞勒斯,法国;目录号:BC2000J2)
  3. 冰柜

软件

  1. Graph Pad Prism 5(Graph Pad Prism软件,加利福尼亚州圣地亚哥)

程序

时间轴

第0天
步骤A,麻醉准备:〜15分钟
BD步骤,MOG / CFA乳液的制备:〜60分钟
步骤E,准备PTx:〜10分钟
步骤F,MOG / CFA乳液和PTx的给药:约45分钟

第1天
步骤G,EAE监控:1〜2分钟/鼠标

第2天
步骤E,准备PTx:〜10分钟
管理PTx的步骤F9和F10:〜10分钟
步骤G,EAE监控:1〜2分钟/鼠标

第3-28天
步骤G,EAE监控:每只鼠标1-2分钟


  1. 三种不同麻醉剂鸡尾酒的制备
    麻醉对于确保乳剂的皮下给药是重要的。
    1. 准备在盐水中的三种不同麻醉剂(30μg/ ml美托咪定,400μg/ ml咪达唑仑和500μg/ ml丁烷醇)的混合物。混合后,该溶液可以在4°C的冰箱中保存至少8周。对于未免疫的对照小鼠,请使用与EAE相同的麻醉剂。
      1. 对于25毫升的溶液,在50毫升的试管中将2毫升的咪达唑仑与2.5毫升的丁烷酚合并。
      2. 加入0.75毫升美托咪定。
      3. 用盐水稀释至25毫升。

  2. 弗氏完全佐剂(CFA)的制备
    CFA用于增加佐剂MOG 35-55的免疫原性。
    1. 计算所需的 M量。结核 H37 Ra(MT)。每只小鼠所需的MT量为500μg。在准备和注射过程中会损失MT,因此准备的量要比所需量多1.5至2倍。
    2. 计算所需量的1 mg / ml佐剂,完全H37 Ra(1 mg / ml CFA)。1 mg / ml CFA的所需量为每只小鼠100μl。在准备和注射过程中会损失CFA,因此准备量要比所需量多1.5至2倍。
    3. 将所需量的MT放入研钵中,并用杵研磨以获得细粉(图1A)。
    4. 将所需量的1 mg / ml CFA添加到研钵中(步骤B3;例如,将5 mg MT添加到1 ml 1 mg / ml CFA中)并混合,以获得6 mg / ml CFA / MT混合物的最终浓度(图1B)。此溶液应在通过乳剂免疫的当天制备,并可以在室温下保存,直到用于步骤D1。


      图1.弗氏完全佐剂(CFA)的制备。 A.用研钵和研棒将MT接地。B.用研钵和研棒混合MT和1 mg / ml CFA,使最终浓度为6 mg / ml CFA / MT。

  3. MOG 35-55肽溶液的制备
    MOG 35-55用作诱导脱髓鞘免疫反应的抗原。
    1. 在盐水中稀释冻干的MOG 35-55,在2 ml试管中获得2 mg / ml储备液。该溶液(2 mg / ml MOG 35-55溶液)可以在溶解后于-20°C的冰箱中保存至少8周。避免反复冻融。
    2. 免疫前,用盐水稀释MOG 35-55储备溶液,使最终浓度为1 mg / ml。该溶液(1 mg / ml MOG 35-55溶液)应在通过乳剂免疫的当天制备,并可以在4°C的冰箱中保存,直到用于步骤D4。
    3. 对于未免疫的对照小鼠,使用生理盐水代替MOG 35-55溶液。

  4. MOG乳液的制备
    MOG 35-55 / CFA乳液可诱导淋巴结免疫反应。
    1. 从研钵(步骤B4)中将6 mg / ml CFA / MT混合物吸入装有18 G针头的2.5 ml鲁尔锁注射器中(图2A)。对于未免疫的对照小鼠,使用与EAE相同的6 mg / ml CFA / MT混合物。
    2. 取下18 G针并连接三通旋塞阀。从旋塞阀释放空气(图2B)。
    3. 转动旋塞阀杆以关闭连接到CFA注射器的阀(图2C)。
    4. 将1 mg / ml MOG 35-55溶液(步骤C2)吸入另一个带有18 G针头的2.5 ml鲁尔锁注射器中。对于未免疫的对照小鼠,请使用生理盐水代替MOG 35-55溶液。
    5. 卸下18 G针并连接到三通旋塞阀。从旋塞阀释放空气(图2D)。此时,对于CFA溶液或MOG溶液,体积较大的液体称为“溶液A”,体积较小的液体称为“溶液B”。
    6. 通过三通旋塞阀排放“溶液A”,使其体积与“溶液B”相同。安排6 mg / ml CFA / MT混合物:1 mg / ml MOG溶液至1:1后,转动旋塞阀杆以关闭未连接到注射器的阀(图2E)。
    7. 将溶液在两个注射器之间来回传递约10分钟(图2F)。在这一步中,重要的是,首先要推入装有CFA的注射器。这将产生白色乳液。当注射器突然开始容易移动时,该过程完成。
    8. 只需将一滴乳液滴入水中即可检查乳液。如果液滴散布在水表面,则表明混合物尚未准备就绪(图3A),因此重复步骤D7和D8,直到乳液完成。当液滴不扩散时就准备就绪(图3B)。该乳液应在免疫当天制备,并可以在4°C的冰箱中保存,直到在步骤F4中使用为止。


      图2. MOG乳液的制备。 A.从研钵中将6 mg / ml CFA / MT混合物吸入2.5 ml leur lock注射器中。B.将CFA注射器连接到三通旋塞阀,然后从旋塞阀释放空气。C.关闭连接到CFA注射器的阀。D.将MOG 35-55注射器连接到旋塞阀并释放空气。E.安排MOG 35-55:CFA = 1:1的量,并在没有注射器的情况下关闭阀门。F.将MOG 35-55和CFA混合,得到白色乳液。


      图3.检查乳液的状态。 A.未完成的乳液如上图所示铺开。B.完整的乳液不扩散。

  5. 百日咳毒素(PTx)的制备
    PTx用于破坏血脑屏障,并使免疫细胞侵入CNS。
    1. 在1 ml磷酸盐缓冲盐水(PBS)中稀释一小瓶百日咳杆菌毒素(百日咳杆菌)冻干粉(50μg),以得到50μg/ ml PTx储备液。该溶液(50μg/ ml PTx储备溶液)可以在溶解后于4°C的冰箱中保存至少6个月。
    2. 计算所需的PTx量。每只小鼠所需的PTx量为10μg/ kg。在准备和注射过程中会损失一些Ptx,因此准备的量要比所需量多1.1至1.5倍。
    3. 将PTx储备溶液在PBS中按1:50稀释,在2 ml试管中的终浓度为1μg/ ml。该溶液(1μg/ ml Ptx)应在注射当天(第0天和第2天)制备,并可以在4°C的冰箱中保存,直到在步骤F7和F10中使用为止。对于未免疫的对照小鼠,请使用与EAE相同的PTx溶液。
    4. 准备1毫升注射器和26 G针进行腹膜内注射。

  6. 动物免疫
    该免疫步骤优选在光照阶段进行。麻醉,乳剂和PTx的给药日设为第0天,仅PTx的注射日设为第2天。
    1. 确保可以通过对尾巴进行颜色标记来轻松识别所有小鼠,以便进行日常评估,例如 eg 。
    2. 将每只老鼠放在烹饪秤上,并测量每只老鼠的体重。
    3. 用三种不同麻醉剂的混合物进行麻醉。通过腹膜内(ip)注射(10 ml / kg)麻醉每只小鼠。使用装有26 G针的1 ml注射器。对于未免疫的对照小鼠,请使用与EAE相同的麻醉剂。给药后约10分钟将小鼠完全麻醉,麻醉持续约1小时(Kirihara et al。,2013)。
    4. 重新混合在步骤D中制备的完整乳液,并将所有乳液转移到单个注射器中。
    5. 卸下旋塞阀并连接26 G针(图4A)。
    6. 在两个不同部位(上背部(颈部)(图4B)和下背部(右后肢的根部))(图4C)皮下注射(sc)100μlMOG 35-55 / CFA乳液。对照小鼠,使用步骤D中制得的对照乳剂,该乳剂仅不含MOG 35-55。
    7. ip注射10 ml / kg PTx溶液(10μg/ kg PTx)。对于未免疫的对照小鼠,注射与EAE相同的PTx溶液。
    8. 检查注射乳剂的小鼠的以下几点:小鼠是否已从麻醉中醒来,未死亡或乳剂从给药部位漏出。有任何问题的小鼠从实验中排除。
    9. 按照第2步的步骤E,准备PTx解决方案。
    10. 如步骤F7所述,在第二天免疫后测量体重并注射第二剂PTx。


      图4.动物免疫。 A.将26 G针头连接到乳液注射器。B和C。在颈部(B)和右后肢(C)的底部皮下注射乳液。

  7. EAE监控
    该监视步骤优选地在光阶段期间执行。在第0天(乳液当日
    给药),从麻醉中恢复后评估小鼠。第2天(只有
    PTx注射),给药后立即进行评估。
    1. 每天称重每只小鼠(EAE和非免疫对照)。
    2. 每天评估每只小鼠(EAE和未免疫对照)的临床体征,并按以下方式评分:
      1. 0:无临床缺陷。
      2. 1:部分尾巴麻痹(图5A,视频1)。
      3. 2:全尾巴麻痹(图5B,视频2)。
      4. 3:后肢部分麻痹(图5C,视频3)。
      5. 4:后肢完全瘫痪(图5D,视频4)。
      6. 5:前肢轻瘫(图5E,视频5)。
      7. 6:死了。
      临床评分评估的详细信息
      首先,将鼠标举到尾巴的中心时,如果尾巴的尖端垂下,则得分为1或更高;否则,得分为0。其次,当鼠标被尾巴的底部抬起时,如果所有的尾巴都垂下来而没有拉力,则得分为2或更高。接下来,将鼠标悬吊在只有前肢的笼子盖上时,如果可以抬起下半身抓住前盖和后肢的盖子,则得分为2,得分为3或2。如果没有更高。然后,将鼠标放在平坦的桌子上时,如果其后肢都朝下拖动,则得分为4分或更高;如果不是,则得分为3分。此后,抬起鼠标的下半身时,如果鼠标只能沿一个方向(左右方向)移动前肢,则得分为5;如果鼠标可以沿两个方向移动,则得分为4。请注意,未免疫的对照小鼠根本不会出现这种临床症状。


      图5. EAE小鼠的临床评分评估。每个临床评分的代表性小鼠图像。A.临床评分1:部分尾巴麻痹B.临床评分2:完全尾巴麻痹C.临床评分3:部分后肢麻痹D.临床评分4:完全后肢麻痹E.临床评分5:前肢麻痹。 br />

      视频1.临床得分为1的EAE小鼠



      视频2.临床评分为2的EAE鼠标


      视频3.临床评分为3的EAE鼠标


      视频4.临床得分为4的EAE鼠标


      视频5.临床评分为5的EAE鼠标

      这些视频是根据京都大学动物实验委员会的道德准则和日本药理学会的准则在京都大学制作的,并经京都大学动物实验委员会批准(协议编号:19-36)。

数据分析

  1. 通过使用GraphPad Prism(表格式:分组),将每日体重测量数据显示为XY图,其中Y为“体重”,X为“免疫后天数”(图6A)。
  2. 通过使用GraphPad Prism(表格式:分组),在XY图中显示每日临床评分评估数据,其中Y为“临床评分”,X为“免疫后天数”(图6B)。
  3. 使用Tukey的多重比较测试,使用双向方差分析分析数据。


    图6. EAE的体重和临床症状的变化。在MOG免疫的C57BL / 6小鼠中EAE的发展。监测体重(A)和临床评分(B)28天。请注意,小鼠在EAE期间开始出现临床体征时开始体重减轻。n = 4,** P &lt; 0.01,*** P &lt; 0.001。

笔记

  1. 将MT磨成细粉的重要性(步骤B3)
    此步骤很重要,因为与粗心操作相比,牢固执行此操作时EAE发生率更高。MT的细粉易于与CFA混合,因此应增加EAE的发生率。
  2. 使用三通旋塞阀获取乳液的原因(步骤D)
    制备强乳液非常重要。因此,当我们使用三通旋塞阀时,我们可以通过注射器操作的打滑来判断乳液的完整性。
  3. 清除旋塞阀中空气的重要性(步骤D2和D5)
    如果不除去空气,则给予一只小鼠的乳剂中MOG 35-55和CFA的含量可能会降低,从而导致实验之间存在差异。因此,应清除三通旋塞阀和注射器中的空气。
  4. 首先从CFA注射器开始的重要性(步骤D7)
    在将它们混合制成乳液的步骤中,我们尝试了从CFA注射器推动和从MOG注射器推动。结果,我们发现前者在推动注射器混合时很容易推动,因此我们建议先推动CFA注射器。
  5. 必须将乳液注入两个不同的位置(步骤F6)
    每次乳剂给药的位置都针对不同的淋巴结。上背部(颈部)是腋窝淋巴结,下背部(后肢的根部)是腹股沟淋巴结。我们认为,EAE发病率会通过在各种淋巴结中引起免疫反应而增加。当发生率低时,原因之一可能是给药位置可能距离淋巴结较远。
  6. 每日体重测量的重要性(步骤G1)
    由于症状的恶化和体重减轻相关,因此认为记录体重和临床评分可以正确评估EAE。此外,体重减轻经常发生在EAE临床评分出现之前,这有助于避免忽视临床评分1。
  7. 日常临床评分评估的重要性(步骤G2)
    EAE病理状况可以通过用临床评分确认每日变化的症状来评估。用药物给药或转基因小鼠进行的症状评估可阐明疾病状态并评估治疗效果。因此,每日评分很重要。抗原免疫后约10-14天,临床症状变得明显,然后在几天至一周内达到最高评分,并通常变得慢性。发生率为90-100%。
  8. 遗传背景的重要性
    遗传背景非常重要,因为MOG 35-55 EAE模型不适用于SJL小鼠,而PLP 139-151 EAE模型不适用于C57BL / 6J小鼠。但是,NOD(非肥胖糖尿病)小鼠也可用于MOG EAE模型。有关详细信息,请参见Stromnes和Goverman的引用,2006。

菜谱

  1. 磷酸盐缓冲盐水(PBS)
    1升超纯水
    8克氯化钠(NaCl)
    0.2克氯化钾(KCl)
    1.15 g磷酸氢二钠(Na 2 HPO 4
    0.2克磷酸二氢钾(KH 2 PO 4

致谢

这项研究得到了MEXT / JSPS KAKENHI资助号17K19486和19K03377(HS资助)的支持。该程序改编自先前研究中描述的程序(Tsutsui et al。,2018)。

利益争夺

作者声明与此手稿无关的利益冲突。

伦理

所有实验均根据京都大学动物实验委员会的道德准则和日本药理学会的准则进行。该协议中动物实验的批准ID为14-42,有效期为2014年至2019年。

参考文献

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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Miyamura, S., Matsuo, N., Nagayasu, K., Shirakawa, H. and Kaneko, S. (2019). Myelin Oligodendrocyte Glycoprotein 35-55 (MOG 35-55)-induced Experimental Autoimmune Encephalomyelitis: A Model of Chronic Multiple Sclerosis. Bio-protocol 9(24): e3453. DOI: 10.21769/BioProtoc.3453.
  2. Tsutsui, M., Hirase, R., Miyamura, S., Nagayasu, K., Nakagawa, T., Mori, Y., Shirakawa, H., Kaneko, S. (2018). TRPM2 exacerbates central nervous system inflammation in experimental autoimmune encephalomyelitis by increasing production of CXCL2 chemokines. J Neurosci. 38(39):8484-8495.
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