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physiology

Dogma overturned

Researchers recover movements and reduce loss of neurons in rats with damaged spinal cord

In February 1996, ten months after have suffered a fall from a horse that left him immobilized in a wheelchair, unable to move his body from the neck downwards, the American actor Christopher Reeve commented, in an interview for the American television network CNN: “Anything can happen to anyone. Why should I be an exception?” The comment by the actor, known all over the world for having played Superman in the cinema, escaping unharmed from buildings that had fallen on top of him, nourished the fear of ordinary people of facing a similar fate after a car accident or a misfortunate flight in an ultra-light, resulting in a blow to the spine. Another effect was to crystallize the idea that the spinal cord does not regenerate itself ? one of the dogmas of medicine. It is not so, at least in the laboratory.

“There just has to be a favorable environment for the neurons of the central nervous system to recover successfully from the lesions”, warrants Francisco Carlos Pereira, a researcher from the University of São Paulo (USP) who helped to discover a way of contain lesions in the spinal cord, as the set of bundled fibers and nerve cells – the neurons – that runs through the inside of the vertebral column and carries the stimuli connected with the movements, feelings, and reactions of the body.

In rats submitted to controlled lesions of the spinal cord, it proved possible to recover 70% of the movements and to induce the regeneration of 50% of the neurons, with the combined use of three substances: an antidepressive medicament called rolipram, now in use with human beings; a solution with Schwann cells, which form the myelin sheath, a layer that covers the nerve fibers in a way similar to the plastic cover of telephone wires; and another solution, with cyclic adenosine monophosphate (cAMP), a compound that works as a chemical messenger, in the inside of the cells.

It was a sophisticated and difficult experiment, carried out in the Miami Project to Cure Paralysis, which occupies a six story building installed in the University of Miami, in the United States. Pereira arrived there in February 2000 as a professor of the Anatomy Department of USP’s Biomedical Sciences Institute (ICB), for a postdoctoral attachment that was to end in February 2002. In the first six months, he worked with the cultivation of cells of mice, rats, and human beings, including Schwann cells. Only then did the work start with the animals. The team coordinated by Mary Bunge, in which Australian Damien Pearse, a scholarship holder from the Christopher Reeve Paralysis Foundation also took part, used 150 rats, which were subjected to a contusion in the spinal cord at the level of the thorax. To guarantee that the degree of the lesion was similar in all the animals, an apparatus connected to computer controlled the intensity of the contusion, which caused loss of movement in the thighs, the legs, and the feet.

Multiple therapy
Next, the rodents were divided into seven groups, of which only one was not treated with any substance. The others were given one, two, or three compounds, with a subtle difference: even amongst the animals that were given the three medicaments, the rolipram was used in two ways, straight after the lesion or one week later, always released continuously by a miniature pump placed under the animal’s skin. One week after the start of the experiment, the rats were given injections with 2 million Schwann cells in the center of the lesion, and one with cAMP a few millimeters from the ends of the wound.

According to the results, published in June in Nature Medicine, eight weeks after the beginning of the study, the rodents that were given the three medicaments (rolipram straight after the lesion, and the Schwann cells with cAMP) showed the best performance: in a test to assess motor capacity, which runs from 0 (no movement) to 21 (normal movement), they earned a score of 14, equivalent n to a 70% recovery of movements. They managed stand with their feet on the ground more easily than the animals from the control group, who scored 9 and limped, without putting their weight on their paws.The study won repercussion: it was reported by about television stations and 600 radios in the United States, while in Brazil it appeared in the Jornal Hoje and Jornal Nacional news programs, both from TV Globo. All of a sudden, the prospect of walking again started to shine again in the minds of those who had suffered a serious accident and are immobilized in a wheelchair. Applying this to human beings, though, is still a rather distant possibility, which calls for more refined tests.

Of the three substances adopted in the experiment, only rolipram “may rapidly become an extra therapeutic tool, to be employed soon after the occurrence of the lesion to the spinal cord”, claims Pereira. For the time being, according to him, there is no evidence that the cAMP and the Schwann cells cultivated in the laboratory can be used without jeopardizing other functions of the organism.

Complementary strategies
In the case of chronic patients, it is most probable that other strategies will still have to be adopted, such as the electrical stimulation of the muscles and cell replacement, alternatives that are still to be found at the experimental stage. “It won’t be an isolated strategy that will lead to the cure of lesions to the spinal cord”, claims the researcher from USP.

In scientific terms, the results of this study are noteworthy for attesting to the role of cAMP in the regeneration of neurons. One of the members of the team, biologist Marie Filbin, from Hunter College, in Nova York, had already demonstrated that this compound helped in the recovery of some kinds of neurons from the spinal nerves – sets of nervous fibers that join the central nervous system (the encephalon and the spinal cord) to the rest of the body, and are responsible for the transmission of the stimuli associated with the sensitivity and the movement of the legs, the trunk, the arms, and of the head as well.

But the spinal nerves are part of the peripheral nervous system (PNS). It remained to be proved whether the cAMP, relatively abundant in the inside of cells in general, would have the same importance in the case of neurons of the spinal cord itself, with their bundles of nerve fibers that are connected to the encephalon and are part of the central nervous system (CNS). Up until this moment, they appeared to be two worlds with very different behaviors. In the nerve fibers of the peripheral nervous system, the removal of the destroyed fragments of myelin – carried out by cells from the organism itself, and a prerequisite for the regeneration of neurons – would take about two weeks. In the nerve fibers of the central nervous system, the cleaning up of the damaged area is slower: studies carried out by other groups showed that nine months after a lesion of fibers of the CNS there were still pieces of myelin.

This degeneration leads to the accumulation of a protein known as MAG, a myelin glycoprotein, which inhibits the growth of the axons, one of the types of ramifications of the neurons. Spaniard Santiago Ramon y Cajal, regarded as one of the founders of neurology and a winner of the Nobel Prize in Medicine in 1906, together with an Italian, Camillo Golgi, had already proposed, 80 years ago, that the neurons of the central nervous system could indeed start growing again. The problem, according to him, is that something prevents this growth.As other researches have shown, besides a lot of MAG, a molecule that hampers the regeneration of the cells of the spinal cord, there was very little cAMP in the area of the lesion. So, thought the researchers, why not season the destroyed neurons with cAMP?

Taking advantage of the experiment, they added this antidepressive, which had proved to be a potent inhibitor of a molecule known as tumor necrosis factor alpha (TNF-alfa), which triggers off the death process of the cells close to the lesion. The expectation was that the lesion would not expand so much with less TNF in circulation – this property of rolipram has been put to good use in studies with human beings to hold back as well the advance of the Aids virus and the development of multiple sclerosis, a degenerative disease of the central nervous system. It was already known that this medicament prevents the destruction of the cAMP, which thus remains in action for more time.

It worked: the rolipram allowed the quantity of cAMP to remain high in the region close to the contusion, preventing the neurons from dying. The application of 2 million Schwann cells one week after the lesion caused to the animals was a reinforcement to this strategy, because it was known that these cells produce substances capable of keeping the neurons alive. These cells, though, exist only in the peripheral nerves – outside the spinal cord. “This was the best strategy (for treatment) that we have found up until now, after 15 years of hard work”, commented Mary Bunge, the coordinator of the research, in one of the interviews she granted after the publication of the results in Nature Medicine.

More recently, in another experiment also carried out with rats, Marie Filbin’s team achieved some heartening results, which reinforced the role of rolipram in assisting the recovery of lesions in the spinal cord. In a rather different approach, the researchers from Hunter College grafted a piece of the spinal cord of a rat embryo straight after causing a contusion in the spinal cord of adult rats. This time, though, they applied the rolipram only two weeks later – which is regarded as a very long time.

The animals recovered, in great measure, the capacity for controlling and moving their paws, previously paralyzed, as the study reveals, published in the June 8 issue of the Proceedings of the National Academy of Sciences. According to the authors of the work, this is a sign that rolipram may help in the regeneration of the spinal cord, even after some time had passed from the lesion.

The Project
Regeneration of the Spinal Cord (nº 99/08665-2); Modality Scholarship Abroad (Post-Doctorate); Coordinator Francisco Carlos Pereira – ICB/USP; Investment R$ 91,358.94 (FAPESP)

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