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Biomedical engineering

Proven efficiency

Equipment uses birds retina to test medicines with neurological action

Automated equipment that allows one to know in seven hours if a medicine prescribed for patients is counterfeit or not, can in a short period of time become part of the hospital routine in any part of the country. Called platform tests, it evaluates the effects of neuroactive drugs, those that act on the nervous system prescribed for patients with epilepsy or migraine headaches. The device is endowed with a video microscope system that permits the monitoring, with precision, of the action of the drug on organic tissue – in this case, the retina of a bird’s eye – and to produce the test result on the computer screen. The retina of the birds, mainly hen chicks, is a material of easy manipulation and can be found in any part of the country.

The automated platform could also be very useful by the pharmaceutical industry, as suggests the project’s coordinator, the medical doctor Vera Maura Fernandes de Lima. “Let’s say that a company carries out reverse engineering in order to obtain a medicine (in reverse engineering one starts from the final product and trace back the steps in an attempt to discover how it was made). The pharmacologist could have done everything correctly and obtained a product chemically similar to the one he intended to copy, but without the same effect. This is because, in biology, the spatial distribution of the atoms in the molecule also comes into play.” The proof of the drug’s quality can easily be verified using the platform tests.

Vera Maura is a researcher at the National Sanitation Vigilance Agency (Anvisa in the Portuguese acronym), the agency responsible for checking the efficiency and security of the medicines used in Brazil. The doctor developed her work as a visiting researcher at the Polytechnic School (Poli) of the University of São Paulo (USP). Also making up the team are engineers from the Poli and from the Nuclear and Energy Research Institute – IPEN.

It was from the studies of a Brazilian that Vera Maura got her inspiration more than ten years ago to begin research using retina video microscopy that has resulted in the equipment that she has now. “Right from my second year of medicine, I fell in love with neurophysiology. Then in 1989 I began to work with a model of the retina that was created by professor Hiss Martins Ferreira, in the 60’s”. To visualize the results of her studies, Vera makes use of chicks retina, biological material that, in her opinion, bring large advantages in relation to other traditional neural tissues used in the medicines testing, such as, for example, the mice hippocampus. Furthermore, a technician with some experience can prepare the retinas in only ten minutes”, guarantees the researcher. The retina is the part of the eye responsible for image capturing. It is located at the back of the eyeball and transforms the visual stimuli into electrical signals that are taken the brain by the optical nerve.

When the retinal tissue is exposed to neuroactive compounds, some physiological properties undergo alteration. One of the most significant changes – and easily monitored – occurs in the evolution of the spreading depression waves a phenomenon first described in the 40’s by another Brazilian, the scientist Aristides Leão (see insert), spreading depression is a brisk alteration in the electrical waves of the brain that can last for various minutes and is related to some neurological situations, such as the symptoms of epilepsy and of classical migraine headache. It shows itself through outbreaks of neuronal excitation, or that is to say, excess of electrical activity, followed by depression, or inactivity of the electrical waves. These waves of extreme activity, followed by waves of inactivity, propagate themselves through the cerebral cortex, which is the uppermost chamber of the brain.

Stimulated phenomenon
“Spreading depression is a general property of the central matter of the brain, in the same way as cardiac arrhythmia is a property of the heart”, compares Vera. The probability of this state increases with the density of the synaptic terminals, a part of the nerve cells that transmit the information. What the researcher did was to stimulate the occurrence of the phenomenon in the retina in vitro without the drug to be analyzed and afterwards with it. The change in the wave pattern or of the optical signal, gives a measure of the action of the drug on the tissue.

In the time in which Vera Maura began to work with the optical signal, in 1991, was also the moment of a huge advance in video microscopy. With the help of this tool, the researcher managed to follow up with greater precision the alterations in the retinal tissue exposed to the neuroactive drugs. With this objective, she and her team created the project for the device which doesn’t do away with the use of a human operator. According to the researcher, the training in the use of the device doesn’t last more than a week. The equipment works with four retinas. First the operator has to place the retinas in a solution composed of glucose and mineral salts.

On purpose, the concentrations of these substances are higher than in a normal state, to maintain the tissue at a level close to unstable. Under these conditions, a gentle mechanical touch upon the retina is sufficient to unchain the waves. In the device developed by the team coordinated by Vera Maura, this touch is automated. “other stimuli could e used such as the addition of potassium ions or a simple flash of light”, she adds. Beginning with this stimulus, what Vera calls the “electrochemical tempest” gets going. While it is happening, the computer measures the intensity and the propagation speed of the optical signal, or that is, of the wave that now passes to be considered as the control wave. After close to twenty minutes, another control wave is generated.

Optical signal
All of the reaction data is filmed by a video camera and stored in the micro processing unit. After a further a rest period, necessary so the retina can reestablish itself, the operator introduces a test solution. If it is a medicine that diminishes the the brain excitability – such as, for example, epilepsy medicines -, it is expected that it will diminish the propagation speed and intensity of the optical signal. The wave becomes less visible and smaller, two parameters easy to quantify”, explains the researcher. However, the operator doesn’t even have to worry about carrying this evaluation. It is the computer that will compare the results with a data bank obtained from tests with several types of drugs of proven efficiency. Once a standard of efficiency has been registered, it will be possible to see if the solution tested behaves in a similar fashion, worse or better than the standard. “The data bank has to be permanently updated. The Anvisa is going to own part of the patent and will control this archive”, explains Vera.

However, it is not only the practical application of the equipment that gets the researcher enthusiastic. Simulating lesions on the tissue and following their behavior is, for the scientist interested in models of the self-organization of the brain, one of the major applications of the equipment. Why does one patient who’s had a stroke remain with permanent damages and another doesn’t? Why do two equal lesions induce different responses? “This is a question to which I’d like to know the answer”, says Vera, capable of spending hours “playing” with the spiral evolutions of the optical signals and with the graphs that give form to the chaotic events of the brain.

The next phase in the research is to carry out experiments in the environment of micro gravity. “If the brain is an excitable world, it should be influenced by electromagnetic fields and gravity”, she deduces. In order to study gravity effects, experiments on centrifuges have already been carried out in the laboratory, and in parabolic flight, during which an aircraft quickly climbs at an angle of 40 degrees and then shut off its turbines making the nose point towards the ground to complete the parabola. Consequently, during ten to twenty seconds the plane falls freely, a situation in which everything inside the aircraft is almost free of the effects of gravity, or that is, in an environment of micro gravity. The results obtained will now be matched up against the experiments in test rockets, which allow for the reaction of micro gravity for various minutes.

Gel on the flight
In April the Brazilian rocket VS-30, developed by the Aeronautical Space Institute (IAE), of the Technical Aerospace Center of the Ministry of Aeronautics in São José dos Campos, will take off from the base of Alcântara, in Maranhão, carrying two Petri dishes (small plastic or glass receptacles) with a gel that reacts in a form very similar to that of the retina. “We want to evaluate the similarities and differences between the dynamic structures in the retina and in the gel”, Vera explains.

While this is happening, a platform for eight retinas will be prepared in Germany to take off from Sweden in 2003. These experiments make up part of a collaboration agreement into micro gravity in biology between USP and the Hohenheim University. Professor José Roberto Castilho Piqueira, of Poli, and professor Wolfgang Hanke, the director of the Physiology Division of the Membrane from the German university, are coordinating this study.

The development of a portable prototype small enough to be placed on a rocket would result in a product that could be interesting above all for hospitals distant from large urban centers. “All it takes is the location to have the minimum of infrastructure, such as good quality water and an electrical outlet”, says Vera. As well, production on a large scale could reduce the platform’s cost, which is estimated at US$ 180,000 in this phase of development of the project. Although there has been no contact with companies interested in producing the test platform yet, the important thing at this moment is that from basic research in neurology application has come a development born and bred in Brazil.

In the waves of the brain
The “Leão Wave” is a term well known by students of neurophysiology. We are talking about an excited wave that spreads through the cerebral cortex, followed by a depression, a “silencing” of the electrical waves. The phenomenon received the name of the first scientist to describe it in 1944, the Brazilian Aristides Azevedo Pacheco Leão, from the Federal University of Rio de Janeiro (UFRJ) and also the president of the Brazilian Academy of Sciences between 1967 and 1981.

On studying the electrical waves in the brain of a rabbit, an accidental touch over the exposed cortex provoked the curious phenomenon. Currently, it is known that the so called “aura” of the classical migraine, visual manifestations that proceed the headache, have a correlation with diffused depression. Today, one the greatest specialists in diffused depression is professor Hiss Martins Ferreira, a professor at UFRJ who worked with Leão and afterwards with Gustavo Oliveira e Castro, also a professor at the Biophysics Institute of the same university. In the decade of the 60s, professor Castro developed retina preparation and demonstrated that the wave that is formed in the retinal tissue is similar to the wave formed in the cortex.

The project
Self-Organization, Temporal Space (Dissipative Structures in Complex Systems Far from Equilibrium) (nº 94/03509-9); Modality Regular line of research assistance; Coordinator Vera Maura Fernandes de Lima – Anvisa and Poli/USP; Investment R$ 11,170.90 and US$ 87,566.90