Estudio de los marcadores del metabolismo energético cerebral en el líquido cefalorraquídeo del perro

Resumen

1. introducción o motivación de la tesis En la actualidad, tanto en medicina humana como en medicina veterinaria, se investiga constantemente con el fin de encontrar nuevas técnicas diagnósticas y nuevos marcadores que sirvan como indicador pronóstico de la evolución de una enfermedad. De esta manera, conociendo en profundidad la fisiopatología de una enfermedad, se podrá conseguir un diagnóstico más precoz y en consiguiente, un tratamiento más efectivo para la misma. En el caso concreto de las enfermedades que afectan al sistema nervioso central (SNC), la obtención de muestras para el diagnóstico es complicada. El SNC se compone del parénquima nervioso, las meninges y el líquido cefalorraquídeo (LCR). Este último es un líquido resultante del filtrado del plasma y de la secreción membranosa, por lo que su composición difiere de la del plasma. En condiciones fisiológicas se trata de un fluido transparente con muy poca celularidad y baja concentración de proteínas. El LCR mantiene un contacto estrecho con el parénquima nervioso, por lo que las alteraciones que se producen en el tejido neuronal pueden verse reflejadas en la composición del LCR. Su obtención se lleva a cabo mediante punción en cisterna cerebelomedular o en espacio lumbosacro, para lo cual es necesaria la anestesia general del paciente. Una de las tendencias actuales en medicina humana se centra en buscar biomarcadores en LCR que reflejen cambios en el ratio metabólico cerebral que puedan ser justificados por determinadas patologías. Estudios como el de Parnetti y col. (2000) o el de Liguori y col. (2015) encuentran niveles anormalmente altos de lactato y piruvato en personas con Alzheimer, demencia vascular, meningitis infecciosa y linfoma. Alteraciones similares han sido descritas por Pugliese y col. (2005) en perros con un grado severo de disfunción cognitiva. En medicina veterinaria exiten relativamente pocos estudios que relacionen las alteraciones en las concentraciones de neurotransmisores, enzimas y sustratos metabólicos neuronales con diferentes patologías. Además, los estudios que establecen rangos fisiológicos de referencia en el perro son aún más escasos y variables. En base a ello, resulta necesario llevar a cabo estudios basales en pacientes sanos. Dichos estudios deberían tener en cuenta parámetros como la raza, edad, sexo, lugar de punción y protocolo anestésico utilizado para la toma de muestras, ya que los agentes anestésicos pueden interferir en el ratio metabólico celular. El pilar esencial del estudio de la fisiología del metabolismo energético cerebral (MEC) se fundamenta en el hecho de que la actividad neuronal está íntimamente ligada al flujo sanguíneo cerebral y al metabolismo energético. La reducción en el aporte de sangre y nutrientes al parénquima encefálico se traduce en una alteración del metabolismo glucídico oxidativo, obligando a las neuronas a emplear rutas metabólicas alternativas que van a producir metabolitos detectables en el LCR. Como ocurre en el síndrome de disfunción cognitiva canina (SDCC), la alteración del metabolismo oxidativo provoca una cascada metabólica que conlleva una menor producción de ATP. Esto comporta una reducción de la actividad de la bomba Na+-K+-ATPasa, provocando el aumento del K+ extracelular, el bloqueo de la despolarización neuronal y el deterioro de la función cognitiva. Hasta la fecha se conoce que tanto personas con demencia como perros con SDCC se benefician de la ingesta de dietas enriquecidas con antioxidantes, vitaminas, ácidos grasos omega 3 y triglicéridos de cadena media. Sin embargo, en el perro, no se ha determinado el impacto real de este tratamiento nutricional en el metabolismo cerebral. Con el presente estudio se pretende profundizar en el conocimiento de la composición del LCR en perros sanos atendiendo a los siguientes factores: (i) la obtención de datos que contribuyan a establecer unos valores de referencia de determinados biomarcadores del MEC, (ii) la observación de las fluctuaciones de dichos marcadores en pacientes sometidos a suplemento dietético con nutracéuticos, (iii) la detección de posibles variaciones de estos parámetros en función del protocolo anestésico utilizado, o (iv) en función del lugar de extracción de LCR. 2.contenido de la investigación Para el desarrollo de esta tesis doctoral se escogieron perros de raza Beagle sanos, a los que se sometió a una primera anestesia general para extraer líquido cefalorraquídeo con el objetivo de medir niveles de lactato (1,189 mM/L), piruvato (0,0577 mM/L) y ratio lactato/piruvato (44,247) en muestras extraídas en cisterna cerebelomedular. Tras obtener los niveles basales de estos biomarcadores, evaluamos el efecto de la suplementación de la dieta con nutracéuticos. Para ello, los animales fueron anestesiados en dos ocasiones, con una separación de 50 días, tiempo durante el cual estuvieron tomando el suplemento nutricional. Obtenemos muestras de líquido cefalorraquídeo antes y después del tratamiento para medir proteínas totales (21 g/dL), recuento de células nucleadas (< 5 células/µL), glucosa (59 mg/dL), sodio (151 mM/L), cloro (132 mM/L), potasio (2,96 mM/L), lactato (1,53 mM/L), piruvato (0,028 mM/L) y su ratio (16,2). Tras el tratamiento observamos que los niveles de sodio (160 mM/L) y glucosa (73 mg/dL) aumentaron significativamente a la vez que disminuyeron los valores de lactato (1,21 mM/L) y el ratio lactato/piruvato (9,9), poniendo de manifiesto la mejoría en el estado oxidativo del encéfalo. Para valorar el efecto de los agentes anestésicos y el tiempo de duración de la anestesia en los biomarcadores del metabolismo energético cerebral, sometimos a todos los animales a dos anestesias, una exclusivamente con isofluorano y otra con propofol. Medimos valores de lactato, piruvato, glutamato, glucosa, creatin kinasa, proteínas totales y electrolitos en dos tiempos diferentes, una vez a los 15 minutos de la inducción anestésica (T0) y por segunda vez a las 3 horas de la inducción (T3). Observamos que los valores de lactato en el grupo anestesiado con propofol fueron significativamente más bajos en T3 (1,02 mM/L) que en T0 (1,4 mM/L). Sin embargo, en el grupo de perros anestesiados exclusivamente con isofluorano, los valores de lactato en T3 (1,58 mM/L) mostraron una tendencia a aumentar con el tiempo anestésico (T0 = 1,43 mM/L). Finalmente, para valorar el efecto del lugar de extracción del líquido cefalorraquídeo, llevamos a cabo la medición de lactato en cisterna cerebelomedular y cisterna lumbar. Para la extracción de líquido cefalorraquídeo empleamos el mismo procedieminto anestésico descrito en el párrafo anterior. Observamos resultados significativamente mayores de lactato en cisterna lumbar (1,58 mM/L) en comparación con los obtenidos en cisterna magna (1,44 mM/L). De esta forma se corrobora el flujo rostrocaudal del líquido cefalorraquídeo en el sistema nervioso central del perro, al igual que en humanos y en el caballo. 3.conclusión Las mediciones de determinados marcadores del MEC en LCR en el perro de raza Beagle van a sufrir modificaciones asociadas a factores como el momento y el punto de extracción de la muestra, el protocolo anestésico utilizado y el tratamiento previo. El veterinario debe conocer los posibles cambios que pueden sufrir los diferentes biomarcadores con el objetivo de evitar errores en el proceso diagnóstico. Por lo tanto, las conclusiones de esta tesis son las siguientes: 1. La determinación de lactato, piruvato y ratio L/P en LCR de 18 perros sanos de raza Beagle nos permite establecer las bases para la elaboración de un rango fisiológico de estos biomarcadores del MEC en cisterna magna. El empleo de un mismo protocolo anestésico en todos los perros y la utilización de la misma cohorte de edad reduce el sesgo de los datos observado en estudios previos. 2. Tras 50 días de tratamiento con nutracéuticos (Aktivait®), los valores de glucosa y sodio en LCR aumentaron de forma significativa, mientras que los niveles de lactato y ratio L/P descendieron. Estos datos sugieren una mejoría en el estado oxidativo del MEC tras el tratamiento en estos perros. Las proteínas totales, recuento celular, cloro, potasio y piruvato permanecieron sin cambios. 3. Tanto el protocolo anestésico como el momento de extracción del LCR influyen en la concentración de biomarcadores del MEC. En T0 no se observaron diferencias significativas; sin embargo, en T3, en el grupo-Propo se observa una disminución significativa de los niveles de lactato, mientras que en el grupo-Iso el lactato tiende a aumentar. La necesidad de anestesiar a los perros para la extracción de LCR y las diferencias observadas en nuestro estudio condicionan que se deba tener en cuenta las variaciones de biomarcadores del MEC generadas por el agente anestésico empleado. 4. La concentración de lactato en LCR fue mayor en las muestras obtenidas en cisterna lumbar en comparación con las obtenidas en cisterna cerebelomedular en perros de raza Beagle. 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