At this point, another lively collaborator appeared on the scene: Margaret (Margo) DeMerritt. Margo not only knew about blood cells but also had the enthusiasm and sparkle that are important in any research activity, particularly on the many routine days when everything else in the laboratory either plods along or turns out wrong. In the course of these blood studies, and particularly when it came to interpreting the kinds of cells in bone marrow, I became increasingly dependent on Margo's skill and enthusiasm. She started in the lab as a part-time technician, then became more and more a full-time collaborator. Finally, growing all the while in scientific stature, she entered our medical school at Oregon and graduated with distinction.
Our research problem was this: we could see abnormal deposits in white blood cells of the two MLD children, and some of these deposits were metachromatic with toluidine blue. But no good histochemical method existed which could clearly tell us whether sulfatase activity was deficient in white cells. We had to devise our own.
There are three different groups of sulfatases: A, B, and C. Our original method measured all at once and did not distinguish among them. Therefore, if any single sulfatase were absent (A, for example), the evidence might be obscured because the two other sulfatases were still active in the cell. For this reason, even though MLD white blood cells showed no consistent decrease in sulfatase activity, we could still not conclude they were not deficient in a single sulfatase. It was discouraging to have invested a great deal of time perfecting the histochemical method only to have ended with nothing conclusive.
Let me now move ahead in the story briefly to emphasize a point. Years later, despite the initial setback, the histochemical method did help us after all. All we finally had to do was make a minor change-run the test in an alkaline solution instead of an acid one. In doing so, we finally had a test for the one sulfatase (sulfatase C) which is active under more alkaline conditions. This modified test finally showed us that sulfatase C was deficient in tissues from the MLD patients in the McLean family who had abnormal granules in their white blood cells. The vignette illustrates how unpredictable research can be: first, the hypothesis is formulated, then a method is developed to test the hypothesis. The method fails at first, but later on, it turns out to be most helpful if carried out in just a slightly different way.
Back in 1957, our research in one area spun off into another. For example, I was still curious to learn more about metachromasia and wondered whether sulfated metachromatic molecules like sulfatides could be harmful in high concentration. Two questions arose: (1) Could one create an experimental metachromatic disorder by injecting animals with an excess of sulfated molecules? (2) Would the response of the tissue to an excess of these metachromatic molecules help us understand any of the changes seen in hypertrophic neuritis? Here, of course, my thoughts were looping back to the starting point-to my old interest in hypertrophic neuritis. I was still trying to understand why metachromatic areas occurred inside the peripheral nerves in that disease. Writing this years later, I still am not certain.
From John Harris, an ophthalmologist, I learned that a new sulfated molecule had been produced by a drug company. It was a complex sugar (polysaccharide) called hyaluronic acid sulfate. I decided to use this soluble metachromatic molecule in an experiment that was designed to answer several questions. The questions could all be formulated quite consciously.
Too consciously. The plan was to inject large amounts of the compound into experimental animals. I would then look at the white blood cells under the microscope to see if some of the material had entered them and at the kidney to find out if it had entered the tubule cells. Moreover, by injecting the compound near the sciatic nerve, there was the remote possibility that some of it might penetrate inside the nerve. This might create a local excess of metachromatic material as occurs in hypertrophic neuritis.
I chose young rabbits in order to create a research situation analogous to that of the MLD children we were studying, reasoning that when a young animal is growing rapidly, it might be most vulnerable to a metabolic error. In approach, the new experiment was rather simpleminded, but from it there evolved an astonishing variety of unexpected observations.
We started by injecting hyaluronic acid sulfate under the skin of the back of young rabbits. A few days later, the rabbits dragged both hind legs. Our first thought was that their peripheral nerves or spinal cord might have been damaged. I discovered, however, that being a rabbit doctor required more diagnostic skill than I had anticipated. In fact, the physical examination showed that the rabbits had fractured both thigh bones. X rays and subsequent microscopic examinations showed that the bones had broken because they were abnormally thinned and softened. Then, a few days later, each rabbit began to develop a large lower abdominal hernia. Microscopic examination later showed that these hernias were caused by weak abdominal muscles and weak connective tissue. The hernias were filled with intestines which had spilled out from the abdominal cavity and entered the hernial sac. Not only were bones, muscles, and connective tissues weakened, but the skin and subcutaneous tissues were easily torn.
These major results were far removed from MLD and from hypertrophic neuritis. However, they were reminiscent of some abnormalities found in other diseases affecting connective tissue. We had stumbled onto a new kind of experimental model. The animal model was of considerable interest, because genetic diseases of connective tissue do exist in man in which bones fracture, hernias develop, and skin becomes thin and weak.
But what was the real explanation for these disorders? What was going on down at the submicroscopic level-at the level of the molecules themselves? We decided to explore the situation. Briefly, what we found was that hyaluronic acid alone (which lacks sulfate groups) did not produce the abnormalities in our rabbits, nor did other similar polysaccharide molecules which had only a few sulfate groups. Essentially, two things were required to produce the experimental lesions: (1) The molecule had to be a special kind containing many negatively charged sulfate groups. (2) This sulfated compound had to be concentrated in the tissues.
To the world at large, the hyaluronic acid sulfate experiments could only be judged a failure. They produced nothing resembling hypertrophic neuritis, nothing like the white cell changes in MLD, and led to no published results. The editorial boards of three different journals rejected the manuscripts. Yet, on the other hand, the experiments were not merely a diversion, nor were they a waste of time. They reinforced my keen interest in determining exactly what it was about the configuration of a molecule that caused tissues to undergo a pathological change. The studies also showed us that an excess of sulfated molecules could cause a tissue to break down slowly. This might be one of several potential mechanisms by which myelin sheaths could break down in metachromatic leukodystrophy. During these studies we also learned how properly to control an animal experiment, and how to think of disease in terms of the disintegration of large molecules in a tissue. I also got into the habit of injecting a certain molecule and then looking at the injection site under the microscope. Each hard-won lesson turned out to be prerequisite for the next experiments.
7
Controls and the Experimental
Globoid Response, 1960
Scientific investigations and experimental ideas may be born as a result of fortuitous and almost involuntary chance observations which present themselves either spontaneously or in an experiment designed with quite a different purpose.
Claude Bernard
In 1890, the German bacteriologist Robert Koch formulated the scientific rules for proving that a certain microorganism was, in fact, the cause of a given disease. The microbe must first be isolated from the infected patient. Then, after being cultured, it must cause the same disease in a normal experimental animal. And finally, the organism isolated from the diseased animal and grown in culture should be the same as the one cultured earlier from the sick patient. These rules, termed Koch's postulates, still form the foundation for our contemporary research in
infectious diseases.
While in Oregon, I decided to use a somewhat similar approach in my chemical and histochemical research. I chose the sulfatide molecule instead of a microorganism, and a metabolic disease, MLD, rather than an infectious one. In these experiments, I teamed up with Darwin Lehfeldt, a hard-working, tenacious medical student from Montana, who has since gone on to specialize in pathology. First, we devised a system for injecting human sulfatide (obtained from the brain of a patient with MLD) into the white matter of rat brain. We next waited two weeks or more for the tissue reaction to evolve, then made frozen sections of the injection site and stained them with toluidine blue. The results were striking. Within a few days the sulfated molecules were engulfed by phagocytes, scavenger cells that engulf debris, digest it, and thus serve a cleanup function.
The phagocytes in the rat brain became enlarged and gave a strong red-purple metachromatic stain. Moreover, after working over the sulfatide molecules for a few more days, some scavenger cells turned the deposits into the golden-brown color associated with human MLD. It seemed to take at least three weeks before the cell could reorganize the sulfatide molecules it had ingested. These experiments in rats finally suggested an explanation for the old mystery of why some sulfatide deposits in humans stained a golden-brown color: it took time for the appropriate arrangement of sulfatide molecules to evolve in the human deposits. The important finding, in any event, was that sulfatides caused the metachromatic phagocytes of MLD. This was now clear.
But once again, the most striking finding was completely unexpected. The surprise awaited me when I examined sections of brain from a rat injected with one of the appropriate control molecules. The following narrative illustrates an important principle-that the unexpected yield from a control experiment may be more fruitful than that from the main experiment.
First, an explanation about what the word "control" means when used in this context. Suppose you wish to test the theory that molecule X, and it alone, will cause a given abnormality in tissue. A basic philosophical principle governs the way you set up the experiment: you design the experiment as if to prove yourself wrong. Indeed, the principle is so fundamental that you become operationally defined as an investigator only to the degree that you adhere to it. This means doing more than injecting molecule X. It means you must test out at least three other groups of animals to cover (and thus to control) the unforseen pitfalls in the experiment. For example, your control groups will include: (1) no injection-this to make certain that your animals are free from a disease that might itself cause the abnormality; (2) sham injection-this to find out what changes the stress and the needle alone make; (3) injections of closely related molecules (K, Y, and Z, for instance, which resemble X)these injections will determine whether molecule X is really unique in its effects.
In our experiment, I included cerebroside into the design of the experiment as one of the control molecules. The idea was simply to see how cells in the brain would react to injections of a relevant molecule closely resembling sulfatide but lacking a sulfate group. In fact, the only difference between the two molecules is that sulfatide has a sulfate group and cerebroside doesn't (figures 2 and 6).
The results were startling in their implications. Around the edge of the cerebroside injections, the rats had developed a most unusual tissue response. Indeed, at the injection site, sections of the rat brains resembled the tissue reaction found in yet another human disease! The human illness was called globoid leukodystrophy (GLD) (figure 6). Quite by accident, we had produced one of its manifestations in rats. At the moment this dawned on me, I felt a sober, puzzled amazement, a feeling unlike anything I had experienced during the MLD research and which remains with me to this day.
Let us observe, parenthetically, how little I knew about this other myelin disease. The few fragments of information I possessed formed no coherent picture. They were like a small handful of jigsaw puzzle pieces scattered widely about on the floor. I did recall that the disease was also termed Krabbe's disease, after the late Danish neuropathologist who described it. I reflected back to nine years before, in New York City, where I had seen my only living patient with GLD. As I examined this affected child, D.W., his clinical abnormalities did resemble those of the MLD children, but his symptoms began much earlier, when he was only four months of age. First he became abnormally fretful and cried seemingly without cause. Gradually, he developed stiffness of the neck, back, arms, and legs, accompanied by blindness and loss of mental function. Finally, he died from an extensive paralysis only ten months after his symptoms began. I obtained permission for the autopsy, looked under the microscope at the havoc the disease had caused in his brain, and saw that GLD had damaged his white matter in a very distinctive way.
Now, after a gap of many years, I was astonished to see that our rat brains had developed a replica of some of the same microscopic features of this dread human disease. Peering though the microscope, I saw many similar distended cells (figure 6). Several of these large globular cells in rat brain had more than one nucleus, and they stained the very same way as did cells in human GLD when special chemical methods were used.
Could cerebrosides also cause the globoid response in human GLD?
Naturally, we went on to test many other control compounds. After many such rat experiments, it finally seemed clear that, yes, cerebroside molecules in particular could cause the special globoidlike response of globoid leukodystrophy.' What made cerebroside molecules so distinctive? Perhaps it was that they were just soluble enough to get into the cell, yet insoluble enough so that even the normal cell could only digest them and dispose of them slowly.
But the findings in rats explained only part of the problem. For the next question was: did In.nnan globoid bodies, in fact, also contain cerebroside? Soon I encountered more children with GLD in our medical school hospital, and all too soon they succumbed to the harsh momentum of their disease. I also wrote to other physicians who kindly sent autopsy material from their cases for us to analyze. Looking through the microscope at the broken-down human white matter, I could see many scattered clusters of the now familiar globular cells and bodies. What remained, therefore, was to take this devastated white matter, analyze it for various lipids, and see whether cerebrosides were increased. This we did.
The cerebroside results, looked at alone, were disappointing. The data did not support the hypothesis. In fact, our chemical studies soon showed that cerebrosides and all other lipids were reduced in GLD. Still, we could rationalize away this unexpected result, because the myelin sheaths of white matter that normally contain the lipids were themselves destroyed in the course of the disease (see figure 7). Such a process would, of itself, reduce all the lipids. Perhaps, then, there was no absolute increase in cerebrosides, but only a relative one that was largely confined to globoid bodies. This turned out to be the case: cerebrosides were relatively elevated when compared with the low levels of all the other myelin lipids. The analyses of human tissue were therefore consistent with our experimental results in rats, for the human data too suggested that a local increase in cerebrosides could be the critical factor in. GLD. Still, the evidence was not conclusive.
Figure 6
GLD; the globoid body and its chemical background. Under the microscope, the globoid body is a distended phagocytic cell that has more than one nucleus. It contains an excess of cerebroside molecules because the cell is deficient in the enzyme cerebroside galactosidase. Normally, this enzyme splits galactose from cerebroside. By this means, the enzyme normally keeps down the level of this lipid molecule. The phagocytic cell reacts to this particular undigested molecule in a very distinctive way. It becomes globular ("globoid") and develops more than one nucleus.
At this time, Saul Korey himself played a stimulating role in our studies. He visited our Oregon laboratory one day, and I brought him up to date with the findings in our GLD research. Saul's supple mind probed to the root of the problem, and his question was characteristically cogent: "Why analyze
all that white matter; why don't you just get out the globoid bodies and look at them?" I protested: it would be too formidable a task to get globoid bodies out of the potpourri of other material in the diseased white matter. He agreed; it would be difficult. He also observed that progress wouldn't be made otherwise. That seemed to settle the topic; we then moved on to the next one.
Saul's challenge mobilized my efforts. I had known all along that if I wanted to understand what was inside human globoid bodies I first had to isolate them in reasonably pure form. But that seemed incredibly difficult, and I had not faced up to the issue. Furthermore, I knew I would have to learn the techniques of thin-layer chromatography. This new method of separating and identifying lipids was not simple, but it seemed much superior to the older methods we had used.
Soon, we were launched on two new fronts: isolating human globoid bodies and studying them by thin-layer chromatography. In terms of personal enjoyment, I remember the whole globoid body isolation project as one of the most fun-filled and adventurous. It involved problem solving all the way. The techniques needed had to be more sophisticated than those used for detecting metachromatic granular bodies in MLD urine. Other requirements were also more rigorous. First, I had to find places in white matter where globoid elements were concentrated and dissect these pieces out. Next I had to free the bodies from surrounding brain tissue without destroying them in the process. This meant grinding the tissue in a loosely fitting homogenizer. Then it was essential to filter out contaminating bits of blood vessels by using a sieve. The next step was to separate out the heavier globoid bodies by centrifuging them down to the bottom of a tube filled with concentrated solutions of sucrose. Because I had very little GLD tissue, I had to make a special, small test tube for this purpose. Finally, I had to devise a new method for taking globoid bodies out from the bottom of this test tube.
Chase, Chance, and Creativity Page 5
