归纳和慢性复发性实验性自身免疫性脑脊髓炎的临床评分

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Summary

本视频演示诱导多发性硬化症的动物模型和临床评分标准:慢性复发DA大鼠实验性自身免疫性脑脊髓炎。这种疾病,诱发免疫大鼠包含整个大鼠脊髓和完全弗氏佐剂乳化,呈现类似人类疾病的临床体征。

Cite this Article

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Beeton, C., Garcia, A., Chandy, K. G. Induction and Clinical Scoring of Chronic-Relapsing Experimental Autoimmune Encephalomyelitis. J. Vis. Exp. (5), e224, doi:10.3791/224 (2007).

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Abstract

多发性硬化症(MS),是一种慢性中枢神经系统(CNS),常见的影响青壮年炎症性疾病。它的特点是传播领域在大脑和脊髓脱髓鞘和胶质吓唬。这些病变改变神经传导和诱导禁用在中枢神经系统的脱髓鞘斑(如截瘫,瘫痪,失明,大小便失禁)的位置不同的神经功能缺损。

实验性自身免疫性脑脊髓炎(EAE)是一个用于MS的模式。 EAE的是第一次不小心引起​​的在人类对狂犬病疫苗接种期间,利用对兔脊髓种植的病毒。脊髓注射病毒灭活残留引起的中枢神经系统疾病。根据这些意见,对EAE的第一种模式是在与中枢神经系统匀浆河流并于1935年Schwenther的免疫的非人类灵长类动物。脑脊髓炎以来一直产生多种物种,并可以按照不同的课程,取决于物种/应变和免疫抗原用于。例如,髓鞘碱性蛋白与佐剂乳化免疫Lewis大鼠诱导EAE的急性模型,而同一抗原诱导豚鼠慢性疾病。

对DA大鼠脊髓完全弗氏佐剂乳化的免疫DA大鼠EAE模型,这里描述的是诱发。大鼠免疫接种后7-14天内开发一个递增的弛缓性麻痹。临床症状,遵循了几个星期的一个复发 - 缓解的过程中。病理显示在中枢神经系统脱髓鞘斑大的免疫浸润。特别考虑照顾与EAE动物在视频结束。

Protocol

慢性复发性EAE的感应和监测

乳液是一种抗原在水溶液中添加补充完整弗氏佐剂1:1混合。添加抗原的辅助,而不是反过来,这是非常重要的!

决策和乳状液注射时,有大量的浪费。始终准备1.5或2倍,比你所需要的的更多。

使用蒸压砂浆,杵,玻璃注射器,和桥梁。所有塑料应无菌。常规实验室长凳上,可以做的准备。

1。辅以佐剂的制备

称取30毫克结核分枝杆菌 (DIFCO目录#231141)和成砂浆地。不按太硬,以避免破坏细菌成细粉末。完全弗氏佐剂H37Ra(DIFCO目录#231131)混合,并添加10毫升。现在这种补充辅助包含4 mg / ml的结核分支杆菌 。转移到管。

2。脊髓匀浆的制备

收集脊髓DA大鼠(年龄/性别无动于衷)和存储冷冻在-80 ° C。称取冷冻脊髓有足够的混合1:1(体积重量:)的补充辅助。切碎的脊髓罚款,可能用刀片,转移到在第1条中使用的砂浆,并与杵成糊状。

3。乳液的制备

  1. 广场为辅管完全弗氏佐剂。在高速的旋涡。添加一滴脊髓匀浆下降,而震荡。当所有的脊髓匀浆补充说,5分钟的旋涡。混合物变成浅粉色。

  2. 5毫升的玻璃注射器中的乳液,并链接到另一个5毫升的玻璃注射器,使用18G桥(费舍尔目录#14 - 825 - 17L)。从注射器向对方发送乳液,直到它变得坚硬(5-10分钟)。如果超过5毫升的乳液准备,用了5毫升注射器,请勿使用较大的注射器。

  3. 乳液可以保存在4 ° C,连续3周在注射器。我建议准备提前至少12小时的检查,它并没有打破。应该保持厚,而不是独立的两个阶段。浅黄色或米色的颜色会在一夜之间改变。

4。免疫的老鼠

  1. 在几分钟注射器混合乳化液和注射用的注射器转移。在短期麻醉下使用23G的针头和3毫升鲁尔锁注射器尾部非常基地皮下注入200μL。

  2. 收件人是7-9周岁的女DA大鼠。我们从哈伦 - 斯普拉格Dawley大鼠。

  3. 老鼠应注意观察,每天两次,重达每天。预计乳液注射后7-15天临床症状。

临床评分:

0:没病

0.5:远端跛行尾

1:跛行的尾巴

2:轻度截瘫,共济失调

3:中度截瘫,从时间的大鼠旅行时间

3.5:一个后肢瘫痪,其他动作

4:完整的后肢麻痹

5:完整的后肢麻痹和大小便失禁

6:奄奄一息,呼吸困难,没有吃或喝。安乐死立即。

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Discussion

有一个本议定书的几个重要的考虑因素。

DA大鼠的遗传学会略有不同,取决于饲养员他们都买了从。这些差异可能会增加或减少对大鼠EAE诱导的易感性。如果您的老鼠更容易受到EAE的,你会想,以减少使用非辅以完全弗氏佐剂甚至不完全弗氏佐剂免疫的强度。您也可以考虑降低乳液的量注射或用生理盐水稀释脊髓匀浆前准备的乳液。如果你的老鼠不发展EAE的临床体征,使用的全实力免疫乳液检查您的住房设施的清洁。不会发展与寄生虫(如蛲虫)感染大鼠EAE的。您可能会认为你在过滤器顶部的蒸压笼子的清洁条件下的动物的住房,并给予酸化水自由采食(准备酸化水的协议是附后)。

乳液必须使油包水型油佐剂完全大衣添加一滴一滴的抗原。准备乳液反过来(水包油型)将成为厚,但不会脑炎。

老鼠是友好的动物,如果他们已经习惯于自己的处理程序,因此它是一个好主意,他们轻轻地处理前每天注射脑炎乳液。这将减轻压力和风险咬。

严重的EAE的动物(3个或更多的得分)需要特别的照顾。老鼠不应该被拾起的尾巴,而是身体。这是减少病畜的压力,更重要的。这是非常重要,以确保所有的动物都获得食物和水,把食品和凝胶包在被褥,并提供长期吸液管上的矿泉水瓶。 EAE大鼠将继续大吃大喝,如果动物停止进食或饮水,咨询机构的兽医或安乐死的动物。生病的老鼠应该从健康的动物分开,以确保他们不断践踏,但避免独自离开老鼠,因为它们是群居动物,需要公司。

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Disclosures

CB和KGC的共同创始人和顾问Airmid公司

Materials

Name Type Company Catalog Number Comments
Complete Freund’s Adjuvant Reagent Fisher Scientific DF 311-360-5 Manufactured by Difco
Mycobacterium tuberculosis H37Ra Reagent Fisher Scientific DF3114-33-8 Manufactured by Difco
DA rat Animal Harlan Laboratories Females, 7-9 weeks old.
Glass syringes - 5 ml Tool Fisher Scientific 14-823-10 B
Metal bridge - 18G Tool Fisher Scientific 14-825-17L
Luer Lok plastic syringes - 3 ml Tool BD Biosciences 309585
Needles - 20G 1 1/2 Tool Fisher Scientific 14-826-5C

DOWNLOAD MATERIALS LIST

Comments

21 Comments

  1. Dear Dr. Beeton,
    I watched your video with great interest.
    I am a researcher at the CEA, Orsay, France, and we are interested in inducing EAE in DA rats. I would kindly like to ask you a few questions in this regard:
    1. Should the meninges be removed prior to the freezing of the spinal cord, to avoid aggregates/ clumps in the emulsion?
    ². Is it possible to reduce the spinal cord into fine powder by using a mortar and pestle in liquid nitrogen rather than using a razor and cutting it over dry ice?
    3. Can the emulsion be prepared by using a polytron or some other homogenisation system rather than the glass syringes and the connecting bridges (we seem to have a problem to obtain some of these tools).
    4. Is it possible to use rats older than 7-9 weeks or dŒs susceptibility decrease with increasing age?
    Thank you in advance.

    Reply
    Posted by: Anonymous
    February 5, 2008 - 11:20 AM
  2. We use whole spinal cords, without removing any parts.
    Since we published this video and following advice from Dr. Holmdahl (Lund, Sweden), we have started using fresh spinal cords from ~1² weeks old DA rats. The emulsions give more homogeneous diseases when working with a large group of animals (> 40 recipients). We have not tried using a mortar and pestle. I don't think it would work for fresh cords but might on frozen cords.
    I haven't tried any other methods for preparing the emulsion. As a graduate student in France I was able to buy glass syringes but we had to ask facilities to make metal bridges to connect the syringes by using two metal needles.
    Recipients should be young adults for best susceptibility to EAE. We have never tried using older DA rats but in other rat models of EAE (using Lewis rats) we have found that the older the recipients, the less susceptible to EAE they were. Note that not all DA rats are identical, there are small variations from vendor to vendor so you may want to try rats from several vendors in your area to find which are more susceptible to EAE.
    If you have any more questions you are welcome to contact me by email at beeton@bcm.edu.

    Reply
    Posted by: Christine B.
    February 5, 2008 - 4:02 PM
  3. Dear Dr. Beeton, thank you for very interesting video. I would like to ask you , if you have any experience with the EAE induction in mice using this protocol. Thank You for the answer. Sincerely Jan Pazour

    Reply
    Posted by: Anonymous
    June 30, 2008 - 9:31 AM
  4. I have never used this protocol in mice. For mice I have only used the MOG 35-55 peptide to induce EAE [in C57BL/6 mice]. Using whole spinal cord should work in mice but the protocol would have to be adjusted. You would first have to ensure you are working with the right strain and then you would have to work out the doses and type of adjuvant. Most protocols in mice also call for an additional injection of pertussis toxin and very often mice need a booster injection to develop EAE. This can be a rather tricky protocol to adapt to mice. Sorry that I could not give you a more helpful answer.

    Reply
    Posted by: Christine B.
    June 30, 2008 - 10:38 AM
  5. Dear all, could there be any difference in using M.butyricum instead of M. tuberculosis H37RA containing adjuvant in inducing EAE in wistar rats (mylein basic protein- MP bio-vendor). I plan to use the former.Please suggest.

    raghul
    toxicologist
    SGS -LSS india

    Reply
    Posted by: Anonymous
    August 25, 2008 - 1:24 PM
  6. Dear Raghul, I have no experience inducing EAE in Wistar rats so am unsure of the results. When inducing acute EAE in Lewis rats with MBP we used CFA made with M. butyricum and supplemented it with 3 mg/ml M. tuberculosis and this worked well with 100% EAE incidence and a homogeneous disease. Best wishes, Christine

    Reply
    Posted by: Christine B.
    August 25, 2008 - 5:46 PM
  7. what is the concentration of the HCl for drink wather?

    Reply
    Posted by: Andres Q.
    September 15, 2009 - 2:40 PM
  8. To prepare acidified drinking water, fill a clean carboy with 10 liters of water. Add 5 ml of 1²N (concentrated) hydrochloric acid (HCl) to the water and stir. The pH of the solution should be in the range of ².4-3.0.
    For more safety, ensure that your stock of concentrated acid is not used for anything else but preparing acidified drinking water. This will reduce the risk of accidental contamination with toxic chemicals.
    Do not check the pH directly in your carboy but rather in a sample taken into a beaker.
    This acidified drinking water is not quite as acidic as common sodas many people drink every day. It will not damage the lining of the Œsophagus of the rats.

    Reply
    Posted by: Anonymous
    September 15, 2009 - 3:03 PM
  9. Dear Dr Beeton,
    I found and watched with great interest you movie about induction and clinical
    scoring of chronic-relapsing EAE in rats and i would like to ask you if
    you have some hystological or/and MRI findings to prove that the major
    pathophisiologycal changes, induced by the immunization, occur in the brain
    and not in the spinal cord? How much this animal model mimics what is
    happening in human brain in MS? I am interested more by demyelination
    which i can observe using DT-MRI.

    Thank you in advance

    Reply
    Posted by: Anonymous
    October 5, 2009 - 8:32 AM
  10. We have not done any histology the brain of these animals as our focus was on the spinal cord.
    We used the brains to isolate mononuclear cells and found more with a CCR7- differentiated phenotype in EAE rats than controls. This suggest brain-infiltration and therefore a potential for demyelinating lesions, but I can't guaranty it.
    This model is definitely demyelinating in the spinal cord (see figure S6 in the online supplement of the paper Matheu, Beeton et al. Immunity ²008, ²9:60²).
    Christine

    Reply
    Posted by: Anonymous
    October 5, 2009 - 1:54 PM
  11. Dear Dr. Beeton, thank you for very interesting video. Do you have the protocol to induce EAE in mice? Can you please provide injecting protocol for mice. Thank you.

    Reply
    Posted by: Anonymous
    July 12, 2010 - 1:24 PM
  12. The protocol will depend on the mouse strain you are interested in. Some strains are more susceptible to EAE than others. A good place to start is the following protocol: http://www.penningerlab.com/uploads/media/EAE_induction_01.pdf

    Reply
    Posted by: Anonymous
    September 30, 2011 - 11:34 PM
  13. Dear Dr Beeton
    Thanks for the vieo, it is very helpful. I have just one question regarding the part you use as "SPINAL CORD". Is it seperated from spinal column or you just use spinal colum containing spinal cord?

    Reply
    Posted by: Anonymous
    November 12, 2011 - 5:01 AM
  14. You want to use the spinal cord itself, removed from the bone. It is pretty easy to collect since you don't need to keep it in a single piece (as you would for histology for example). You can simply scrape it out.
    Hope this helps!
    Christine

    Reply
    Posted by: Anonymous
    November 14, 2011 - 4:36 PM
  15. Dear Dr Beeton
    Thanks for the video. Please let me know how to make acidified water and HOW LONG THE ANIMAL SHOUL DRINK SUCH A WATER BEFORE INDUCTION OF EAE?
    Regards

    Reply
    Posted by: Anonymous
    November 27, 2011 - 3:58 AM
  16. For preparation of the acidified water, please refer to my answer to a similar question from Andres Quintanar, posted 09/15/²009.
    We put our rats on acidified water as soon as we receive them, which is 4-7 days before we induce EAE.

    Reply
    Posted by: Anonymous
    November 27, 2011 - 4:40 PM
  17. Dear Dr Beeton
    Thanks for the video. It is really helpful. Can we use Sprague-Dawley and Wistar Rats. Do we need in your model to use pertussis toxin.

    Reply
    Posted by: Anonymous
    December 5, 2011 - 6:32 AM
  18. Hello Dear Christine Beeton
    Thanks for your useful video. I would be grateful if you response my question: In this video when you work with Mycobacterium tuberculosis H37RA for making it thin powder, you didn't wear mask, is it safe for you?

    Regards

    Reply
    Posted by: Anonymous
    January 22, 2012 - 5:34 AM
  19. Hello Dear Professor Beeton
    I have another question dear Dr Beeton: Should we perfuse the rat before spinal cord isolation (for EAE induction) or no the perfusion is just necessary when we want to isolate the cells from spinal cord or brain? I want to isolate the guina pig spinal cord for EAE induction and I don't know the perfusion is necessary or no.
    Best Regards

    Reply
    Posted by: Anonymous
    March 10, 2012 - 7:46 AM

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