I'd like to perform an experiment on mice inducing EAE with whole myelin as the immunogen. The most important reference seems to be "Oct;21(4):749-57.
Myelination in rat brain: method of myelin isolation. Norton WT, Poduslo SE.J. of Neurochemistry 1973" that obviously is about rats.. Thank you in advance for your help.
Christian, you will find a method described in:
Anderton S.M. & Wraith D.C. Hierarchy in the ability of T cell epitopes to induce peripheral tolerance to antigens from myelin. Eur. J. Immunol. (1998) 28, 1251-1261
You will find that spinal cord is predominantly white matter and therefore can be used as a crude myelin preparation. We have used this to induce EAE in a range of models. The dose required depends on the strain but we have been successful with doses between 100 ug and 1 mg. Let me know if you need further advice.
Just a few simple pratical suggestions.
For an optimal immunization animals must receive an intradermal injection of the antigen in a depot form that will release it slowly. For this purpose any lyophilized preparation must be reconstituted in water or in saline solution up to the appropriate concentration and the suspension must be emusioned with Complete or Incomplete Freund's Adjuvant until a thick compound similar to a cream will be obtained. This is usually carried out connecting two opposed syringes with a two/three way connector one containing the Freund's adjuvant and one the water/saline suspension (a connector can also be built in house, connecting a large caliber needle with the cap of a secon identical needle). Pumping grdually the water solution in the oil a nice thick emulsion will be easily obtained. The thickness of the emulsion is critical for a efficacy of the depot.
Animals can be injected in the foot paths or in the skin around shoulder or hind limb joints, with insulin syringe and needle. There isn't much room for large volumes of emulsion in these sites, therefore the concentration of the antigen in the emulsion must be carefully planned in advance.
Purification of Myelin from brain or spinal cord is simple, all the methods are based on gradient centriugations. In addition I believe that at least bovine and mouse purified myelin prparations can be purchased from the market.
However, as already suggested by others, fresh brain white matter or spinal cord, whatever the source, will work nicely as well. In this case fresh tissue can be lyophilized and stored or can be used as a fresh preparation. If used fresh it may not be necessary to dissoleve it in saline solution
Best regards, and good luck with your experiments.
Yes, the method described by Norton and Poduslo for Myelin purificationis still the gold standard and one of the most effcient.
For immunization with whole myelin:
In Lewis rats, myelin will induce acute EAE.
Massacesi L et al. Suppression of experimental allergic encephalomyelitis by retinoic acid.J Neurol Sci. 1987, 80:55-64.
Each rat must receive in each hind foot pads 0.25 ml of an emulsion containing 1 mg guinea pig myelin in 0.5 ml Complete Freund's Adjuvant (this containig 3 mg /ml Mycobacterium tuberculosis H37RA).
In new wolrd primates whole myelin will induce chronic EAE.
Massacesi L, et al. Active and passively induced experimental autoimmune encephalomyelitis in common marmosets: A new model for multiple sclerosis. Ann Neurol 1995; 37: 519–530.
0.2 mg fresh-frozen WMH, emulsified in complete
Freund’s adjuvant containing 2 mg/ml of killed
Mycobacterium tuberculosis.
Best regards,
Luca Massacesi
PS: in UCSF the Neurology Dept has all the expertise needed
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