#142 from R&D Innovator Volume 4, Number 2          February 1995

Paying Attention to a Sick Rabbit
by Lorise C. Gahring, Ph.D.

Dr. Gahring is assistant professor of medicine at University of Utah.

I want to describe my involvement in a basic research project which unexpectedly led to a possible treatment for a devastating disease.  The fact that I’d never heard of the disease did nothing to diminish the intellectual and personal fascination of the work. 

The story began in 1988, when Scott Rogers and I were postdocs in San Diego (at the Salk and Scripps Institutes, respectively).  We were interested in glutamate receptors--proteins that bind to glutamate, an amino acid which acts as a messenger in the brain. For many of our studies, we needed antibodies that specifically recognize and bind to glutamate receptors. These antibodies would help us locate these receptors in microscopic slices of mouse brain.

It was relatively easy to obtain and purify the receptors with molecular biology techniques.  We made antibodies by injecting glutamate receptors into rabbits, which would produce antibodies specific for glutamate receptors.  (This is the normal immune response --the rabbit sees the injected material as “foreign,” and makes antibodies to rid the body of it.)  After a few weeks, we collected sera containing the desired antibody.  As a graduate student and post-doc in immunology, I had done this routine hundreds of times.  The rabbits, while not happy to have a needle stuck in their ears during serum collection, seem content afterwards and remain healthy.

One Sick Rabbit

In 30 months, we injected different types of glutamate receptors into about 50 rabbits, and collected their sera.  Toward the end, the animal caretaker called Scott with some "bad" news: one of our rabbits was sick.    Sick was an understatement for whatever was ailing this poor rabbit--during the night it lost a substantial amount of blood from a laceration on its tongue, in a syndrome that was reminiscent of tongue-biting in epileptics.  We’d never seen this in other rabbits in this project (or in any other research project, either, for that matter). 

Was this interesting phenomenon also significant? We sacrificed this rabbit, collected its remaining blood, and removed its brain.  James O. McNamara, an MD on sabbatical at Salk, suggested we send the brain to Duke University pathologist Dr. Barbara Crain, an expert on brain pathology, for analysis. 

In the meantime, we kept a close eye on the other rabbit which we'd  injected with the same glutamate receptor subunit, and used video to document its behavior.  Sure enough, as we taped all night long, this rabbit started having what looked like seizures.

Had we induced seizures by immunizing the rabbit with a brain receptor?  This was exciting, and possibly significant.  After Dr. Crain reported that the brain had a pathology very reminiscent of Rasmussen’s encephalitis, a rare, severe form of childhood epilepsy, Dr. Ian Andrews, a pediatric neurologist at Duke University, sent us human blood samples.  He wanted to know if we could distinguish children with Rasmussen’s from controls, merely by examining glutamate-receptor antibodies in their blood.  Over one long night, we tested 25 samples, and found two that carried antibodies against the same glutamate-receptor we'd injected into those rabbits! 

To our relief and satisfaction, these were the samples from children with active Rasmussen’s encephalitis.  Using a different assay for the same antibody, we confirmed our correlation.  In other words, Rasmussen's suddenly looked like an autoimmune disease, in which the immune system creates antibodies which attack glutamate receptors in the brain.  The logical conclusion was that this attack caused interference in the glutamate communication system, and the seizures. 

Putting it to the Test

Rasmussen’s encephalitis causes a particularly devastating form of seizure, which cannot be controlled by any medication, so the recommended treatment for its unfortunate patients is to remove the affected side of the brain.  In fact, one of the patients who had tested positive for glutamate-receptor antibody was scheduled for such a hemispherectomy. 

Ian suggested that we try to remove the antibody from her system by a dialysis-like procedure (in which plasma is removed from the blood, cleansed, and returned).  With approval from the National Institutes of Health, we began this unconventional therapy, even though skepticism was running high. One physician in our group even said he’d “take his shingle down” if it worked.

In 1992, within three days of initiating the procedure, the nine-year old girl began speaking her first words in almost two years, and her seizure rate fell dramatically!  And at the same time, we found a drop in the amount of the glutamate-receptor antibody in her plasma. 

Sadly, as with any autoimmune disease, we still can't specifically eliminate the harmful antibody production, so the benefits of treatment start to wane as the immune system continues to make more destructive antibodies. We had to watch her glutamate-binding site antibodies rise, and along with it, her rate of seizures.  In an effort to prevent the dangerous hemispherectomy, Ian is trying a number of autoimmune therapies with her.

As we push forward with this work, it's easy to forget--but essential to remember--that this entire line of investigation started with a rabbit with an unexpected seizure.  That event gave us two choices: 1) to ignore the unexpected and start over, or 2)  to follow up on the observation, even though it could have led to a dead end. Fortunately, we chose the second action.  (Luck was with us: we’ve since found that not all rabbits immunized with this glutamate-receptor site have seizures.)

Now, after investing several years of work and substantial resources in the basic biology of glutamate receptors and the clinical treatment of this tragic illness, I'm gratified we made the correct choice--to follow up on the unexpected.  Science is not just about proving hypotheses and being logical--it's also about being creative and remaining alert. 

This experience has also shown me the power of synergism between basic and clinical researchers, something I’ll continue to facilitate.