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Freeze-fracture: probing the myelin depths.

Freeze-Fracture: Probing the Myelin Depths

In an effort to learn what goes awry in multiple sclerosis, many scientists are intensively studying the effects of myelin deterioration on nerve fiber membranes.

Traditional methods of studying nerve membranes are useful but provide limited information. Now, a comparatively new technique called freeze-fracturing is offering dramatic new clues as to how nerve fibers are organized and what mechanisms may be responsible for producing the neurological changes seen when the myelin sheath surrounding them is damaged.

Dr. Jack Rosenbluth, a neurocytologist at New York University School of Medicine, is using a freeze-fracture method to study nerve fibers in normal and in myelin-deficient animals. In an interview with INSIDE MS, the Society grantee explained how he does the procedure.

"We first freeze a sample of tissue between two flat metal discs," he explains. "Then we mount it in a vacuum chamber on a platform cooled by liquid nitrogen. We fracture the tissue by pulling the discs apart so that a layer of tissue sticks to each disc, like two halves of a sandwich. Next, we apply an electrical current to a platinum-carbon wire that also sits inside the chamber. The wire glows, giving off platinum atoms which literally "rain" on the exposed tissues. This forms a thin platinun coating which becomes an exquisitely detailed metallic replica of the surface of the tissue. When the coating procedure is complete, we take the specimen out, warm it to room temperature, and remove the tissue chemically. We now have ready for study a platinum mirror image of the tissue we had initially prepared."

This method, Dr. Rosenbluth points out, is particularly valuable because the tissue tends to fracture selectively along membranes, providing unique surface views of their molecular structure. Such studies at NYU have demonstrated that at the interruptions in the myelin sheath called the nodes of Ranvier, the nerve fiber membrane has high concentrations of protein molecules corresponding to the sodium channels essential for the conduction of nerve impulses that jump from node to node. (See INSIDE MS, Fall '88, "Sodium Channels.") In unmyelinated nerve fibers, on the other hand, these molecules are distributed randomly and at much lower concentrations throughout the membrane. When myelin is damaged, the normal high concentration of sodium channels at the node appears to revert gradually to the random distribution. characteristic of unmyelinated fibers.

These laboratory studies have raised several questions in Dr. Rosenbluth's mind about the mechanism of interaction between myelin and nerve fibers and about how loss of myelin affects nerve function. Answers may come from a very special animal he and other scientists are studying.

"This is a mutant rat whose central nervous system is virtually devoid of myelin," he says. "Half the males will be born without myelin, though the whole litter looks perfectly normal. About two weeks after birth the mutants develop a tremor, mostly in the hind legs. Later, seizures appear; the life span is only four weeks.

"The central nervous system in these animals is normal except for the lack of myelin. It's a very dramatic loss: The animals don't just have a slight diminution or patchy loss of myelin; they have practically no myelin anywhere in their brain and spinal cord." Nonetheless, the scientist points out, these rats can eat, suckle, move about, make noises, and react to stimuli during their brief lives. Even without myelin their central nervous system seems to function rather well.

Freeze-fracture studies of these animals show clearly that the sodium

channel pattern in their peripheral nervous system, where myelin is intact, is normal, and the sodium channels are, as expected, concentrated at the nodes of Ranvier, while in the central system the channels are not localized at the nodes but randomly scattered. "We can see this structural difference when we look at a mutant's nerve fiber that passes all the way from the peripheral to the central nervous system," he says. "When the fiber is in the peripheral system, we find the sodium channels closely packed at the nodes; but if we follow this same fiber into the central nervous system, which is unmyeliminated, there we find them randomly scattered. It's likely to be the absence of myelin that makes the difference in the sodium channel pattern."

So how can this mutant animal function without myelin? "The answer seems to be that you don't have to have myelin to conduct an impulse," Dr. Rosenbluth says "We see now that an unmyelinated fiber will conduct. Only instead of the impulse jumping from node to node, it moves continuously all along the length of the fiber membrane with the help of the scattered sodium channels. This is a far less efficient way of conducting signals, and it does result in eventual neurological abnormalities in the mutants. Nevertheless, it can work."

The NYU scientist conjectures that something similar may be happening in multiple sclerosis. "A patient will have an acute attack and may lose function very suddenly. Then, over the course of a few weeks, a considerable amount of function may be restored. This usually happens too quickly for myelin to be re-forming. What's probably happening is that the sodium channels are redistributing and new ones are being added, until the point is reached where the nerve fiber -- even without myelin -- is able to conduct slowly along the segment. We know it's slow because we can measure its speed to conduction. This is one theory to explain the partial remission that often follows exacerbations in MS."

Along with several other scientists, Dr. Rosenbluth has been attempting to remyelinate his mutant rats to prolong their lives. He is using transplantation methods pioneered by NMSS grantee Madeleine Gumpel at Salpetriere Hospital in Paris (INSIDE MS, Fall '90, "Transplanting Oligos"). Dr. Rosenbluth's technique differs in that he transplants healthy fetal cells -- precursors of myelin-making cells called oligodendrocytes --into juvenile rats' spinal cords.

"We recently reported that these fetal cells do in fact survive the granting, differentiate into oligos, and begin to form myelin sheaths in the mutant rats," he says. "However, the myelin forms only in focal patches at the site of the fetal cell injections. It doesn't extend to other areas. We haven't been able to remyelinate the animals completely. But we're trying to work out strategies to increase the spread of new myelin formation after transplanting."

Would it be possible to use fetal transplants to reverse demyelinating disease in humans? "All we can say is that if the technique works, it may some day be possible to apply it in treating MS lessions," Dr. Rosenbluth observes. "Our first task is to see whether is succeeds on a long-term basis in animal models and to determine from our freeze-fracture studies whether the transplants restore the normal structure of the nerve fiber membrane."
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Author:Shaw, Phyllis
Publication:Inside MS
Date:Jan 1, 1991
Words:1127
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