043 - The Antibody Response of Rabbits to Injections of Emulsions and Extracts of Homologous Brain

Along the lines of 040, here is an update (in relative time) about researchers' attempts to produce encephalomyelitis in lab animals. To recap: some people a while ago, when given a vaccine against rabies containing brain material, came down with a serious brain disorder that may have been due to an immune reaction against their own nervous tissue. Thomas Rivers and colleagues managed to induce a similar condition in a few monkeys by injecting them with rabbit brain tissue.

In the above-named study, Rivers and his colleague Francis Schwentker attempted to learn more about this process.¹

Considering the difficulty they had before in inducing the reaction in monkeys (dozens of injections over a long period), they tried to find ways to increase the ability of the brain tissue to cause a reaction. The methods: adding pig serum, letting the brain tissue sit around until it broke down on its own, and infecting it with vaccinia. The brains were dissolved either in water or in alcohol.

They tested a total of 79 rabbits in groups of four or five. After injecting them with the brain tissue, they tested their blood to see what kinds of antibodies they had.

Fresh rabbit brain with nothing added didn't induce any antibody responses, at least not in this study. In contrast, autolyzed brain (the one that sat around until it broke itself down) induced a lot. The other mixtures were intermediate. So apparently the additions could help form antigen somewhat, but the brain could form its own antigen too.

Schwentker and Rivers wanted to see if the antibodies formed were specific to the brain, so they tested their ability to bind to the brain and some other organs both of rabbits and of guinea pigs. Turned out that the antibodies formed against brain dissolved in water also bound to liver, kidney, and spleen, and all the same organs in guinea pigs, though the activity was less than against the brain itself. Antibodies from brain dissolved in alcohol only targeted the brain though.

It seemed that there were at least two kinds of antibody: one specific for a component of the brain that is soluble in alcohol, and one that was present in many different organs of the body.

To confirm this, the scientists removed each type of antibody by mixing the antibody-containing serum with organs. First they mixed the serum with kidney emulsions, and then tested what remained in the same way as before. The remaining antibodies bound only to the brain, not to any other organs. So antibodies to the non-specific component had been removed.

Next they mixed serum with brain dissolved in water. The remaining antibodies, if there were any, did not bind to any of the organs they tested.

Finally they mixed serum with brain dissolved in alcohol. The remaining antibodies only bound to the non-brain organs, not to the brain. This was somewhat surprising (if the antibodies bind to a common component that is also present in the brain, shouldn't they bind just as much to the brain?), but the authors explain that it is because the solution of brain tested was so dilute (so as not to overwhelm the other tests) and the antibodies present didn't bind to it very much, so it didn't show activity. Not sure why they didn't just make a less dilute solution of brain to test that, but oh well.

To figure out which component of the brain was inducing the most antibodies, Schwentker and Rivers tested the rabbit serum with antibodies against brain tissue using different proportions of white matter and grey matter from the brain. They found that as the relative amount of white matter increased, the antibodies bound to it better and better, so it seemed that something in the white matter was the antibodies' target.

One thing that's more common in white matter than in grey matter is myelin, which surrounds the sheaths of neurons. To confirm that myelin is the component of interest, they tested the anti-brain antibodies on emulsions of brain from different ages of rabbits. Newborn rabbits have hardly any myelin in their brains, and the amount increases with age, so if the antibodies primarily target myelin, they would expect more and more to bind to older and older rabbits, and that is indeed what they found.

So it seemed that injecting rabbits with solutions of rabbit brain could, in some circumstances, induce the rabbits to form antibodies against their own myelin. This could indeed be the mechanism by which encephalomyelitis occurs (in which myelin is broken down) after rabies vaccination.

Finally, the authors analyzed the health of the rabbits they used in their experiments. Most of them were fine, but a number did develop fatal paralysis. Looking at their brain tissue after they died, they noticed increased immune cell activity and inflammation in these animals, but they didn't see any breakdown of the myelin, so they were unwilling to conclude that there was a direct relationship between the antibodies against myelin and the paralysis they observed. It was interesting that the highest number of paralyzed animals (32%) was found in the group injected with autolyzed brain, which was also the group with the most anti-myelin antibodies.

This study gives some good hints at the problem, though it isn't entirely conclusive. A later study speculated that the antibody response might be protective somehow, rather than damaging:

"It is conceivable that a component of brain which is both toxic and antigenic could be released under conditions favoring autolysis, such as might exist locally in the injected brain-adjuvant mass. In this case, the antibody might represent a protective reaction...It might also account for the observation by Schwentker and Rivers that autolyzed brain extracts produced paralysis and antibody formation in rabbits, while fresh brain had no effect. Although no direct evidence for a toxin has been obtained in the present study, the matter warrants further exploration."²

And another cautioned about the interpretation of these results:

"While these studies have indeed confirmed the existence of antibodies specific for nervous tissue, the use of placenta has shown that certain 'antibodies to brain' may be antibodies to some element of the brain other than specifically nervous tissue, possibly vascular endothelium. Caution is therefore needed in interpreting results obtained with crude brain tissue or tissue extracts as due to antibodies specific for brain tissue."³

And I must reiterate that, while encephalomyelitis seemed to be an unfortunate reaction for some recipients of the rabies vaccine, it was the best they had at the time (we have better and safer now) and the alternative was often almost certainly dying of rabies, compared to a very rare side effect. So this story is, at worst, an interesting lesson from the past to inform medical efforts in the future.

Citations:
1. Schwentker, F. F. & Rivers, T. M. The Antibody Response of Rabbits to Injections of Emulsions and Extracts of Homologous Brain. J Exp Med 60, 559–574 (1934). 2. Thomas, L., Paterson, P. Y. & Smithwick, B. Acute Disseminated Encephalomyelitis Following Immunization with Homologous Brain Extracts I. Studies on the Role of a Circulating Antibody in the Production of the Condition in Dogs. J Exp Med 92, 133–152 (1950). 3. Lumsden, C. E., Kabat, E. A., Wolf, A. & Bezer, A. E. Studies on Acute Disseminated Encephalomyelitis Produced Experimentally in Rhesus Monkeys V. Complement-Fixing Antibodies. J Exp Med 92, 253–270 (1950).