Ômega-conotoxina MVIIC no trauma experimental da medula espinal.

RESUMO

O trauma medular é uma afecção comum na medicina veterinária. Ela pode acarretar graves consequências tais como defecit na função motora sensorial e autonimica para o animal acometido e pode determinar a eutanásia do mesmo. As lesões medulares causam danos no tecido nervoso por mecanismos primário e secundário. A lesão primaria e de tipo irreversivel, ja no mecanismo secundário um influxo exacerbado de cálcio é produzido, principalmente devido à ativação de canais para cálcio voltagem-dependentes (CCVD). Esse evento é considerado crítico na fisiopatogenia da lesão medular. A Ômega-conotoxina MVIIC obtida da purificação do veneno de Conus magus é capaz de bloquear CCVD e, assim, reduzir o influxo de cálcio. O presente estudo avaliou o efeito da aplicação intratecal da Ômega-conotoxina MVIIC nas doses 15 e 30 pmol e nos tempos 5 minutos e uma hora apos o trauma medular experimental em ratos. Foram utilizados 36 ratos machos adultos, variedade Wistar, aleatoriamente divididos em seis grupos. Os animais do grupo Controle Negativo (CN) foram submetidos à laminectomia dorsal. Nos demais grupos, além da laminectomia, os animais foram submetidos ao trauma medular agudo contusivo pelo aparelho MASCIS impactor. Realizou-se aplicação intratecal de placebo nos animais dos grupos Controle Positivo (CP). Nos Grupos G3 e G5 foram aplicadas doses de 15 e 30 pmol, respectivamente, da toxina Ômega-conotoxina MVIIC, nos animais tratados 5 minutos após o trauma. Nos Grupos G4 e G6 foram aplicadas as doses de 15 e 30, respectivamente, uma hora após o trauma. 48 horas após a intervenção cirúrgica, os animais foram eutanasiados. Por fim, coletaram-se amostras de sangue para avaliar os efeitos secundários, e segmentos de medula espinhal, para quantificação de espécies reativas de oxigênio e peroxidação lipídica e para a avaliação da expressão gênica de fatores relacionados à apoptose por meio de técnica de qRTPCR. Observou-se redução de peroxidação lípica no grupo tratado com 15 pmol/5 minutos após o trauma, o que sugere ação neuroprotetora desta toxina.

ABSTRACT

Spinal cord trauma is a common affection in veterinary medicine. It can have serious consequences for the affected animal and can end with euthanasia. The spinal cord lesions cause damage to the nervous tissue by two mechanisms, the primary and the secondary. In the secondary mechanism, there is an exacerbated influx of calcium, mainly due to the activation of voltagedependent calcium channels (CCVD). This secondary event is considered critical in the pathophysiology of the spinal cord injury. The Omega-conotoxin MVIIC obtained from the purification of the venom of Conus magus is able to block CCVD and reduce the influx of calcium. The present study evaluated the effect of intrathecal application of diverse doses at different Omega-conotoxin MVIIC times in rats submitted to experimental acute medullary trauma. Thirty-six adult male Wistar rats were randomly divided into six groups. The animals of the Negative Control (NC) group underwent dorsal laminectomy without causing trauma by the MASCIS impactor device. In the other groups, animals were submitted to the laminectomy, as well as acute medullary trauma. Intrathecal application of placebo was performed in the animals of the control and 15 and 30 pmol doses of the Omega-conotoxin toxin MVIIC in treated animals 5 minutes and one hour post trauma. 48 hours after surgery, the animals were euthanized and samples of blood, urine and spinal cord segments were collected for the quantification of reactive oxygen species and lipid peroxidation and evaluation of the gene expression of factors related to apoptosis through the qRT technique -PCR. Spinal cord trauma is a common affection in veterinary medicine. It can have serious consequences for the affected animal and can end with the euthanasia of the same. The medullary lesions cause damage to the nervous tissue by two mechanisms, the primary and secondary, being the exacerbated influx of calcium, mainly due to the activation of voltage-dependent calcium channels (CCVD), a secondary event considered critical in the pathophysiology of the spinal cord injury. The Omega-conotoxin MVIIC obtained from the purification of the venom of Conus magus is able to block CCVD and thus reduce the influx of calcium. The present study evaluated the effect of intrathecal application of different doses at different Omega-conotoxin MVIIC times in rats submitted to experimental acute medullary trauma. Thirty-six adult male Wistar rats were randomly divided into six groups. The animals of the CN group underwent dorsal laminectomy without causing trauma by the MASCIS impactor device. In the other groups, in addition to laminectomy, the animals were submitted to acute medullary trauma. Besides, doses of 15 and 30 pmol of the Omega-conotoxin toxin MVIIC were applied in the treated animals for 5 minutes and one-hour post trauma. 48 hours after surgery, the animals were euthanized. Finally, samples of blood, urine and spinal cord segments were collected for the quantification of reactive oxygen species and lipid peroxidation and for the evaluation of the gene expression of factors related to apoptosis through the qRT-PCR technique.