Análisis de la actividad neuronal y cerebral mediante técnicas de redes complejas

El cerebro humano es probablemente uno de los sistemas más complejos a los que nos enfrentamos en la actualidad, si bien es también uno de los más fascinantes. Sin embargo, la compresión de cómo el cerebro organiza su actividad para llevar a cabo tareas complejas es un problema plagado de restos y obstáculos. En sus inicios la neuroimagen y la electrofisiología tenían como objetivo la identificación de regiones asociadas a activaciones relacionadas con tareas especificas, o con patrones locales que variaban en el tiempo dada cierta actividad. Sin embargo, actualmente existe un consenso acerca de que la actividad cerebral tiene un carácter temporal multiescala y espacialmente extendido, lo que lleva a considerar el cerebro como una gran red de áreas cerebrales coordinadas, cuyas conexiones funcionales son continuamente creadas y destruidas. Hasta hace poco, el énfasis de los estudios de la actividad cerebral funcional se han centrado en la identidad de los nodos particulares que forman estas redes, y en la caracterización de métricas de conectividad entre ellos: la hipótesis subyacente es que cada nodo, que es una representación mas bien aproximada de una región cerebral dada, ofrece a una única contribución al total de la red. Por tanto, la neuroimagen funcional integra los dos ingredientes básicos de la neuropsicología: la localización de la función cognitiva en módulos cerebrales especializados y el rol de las fibras de conexión en la integración de dichos módulos. Sin embargo, recientemente, la estructura y la función cerebral han empezado a ser investigadas mediante la Ciencia de la Redes, una interpretación mecánico-estadística de una antigua rama de las matemáticas: La teoría de grafos. La Ciencia de las Redes permite dotar a las redes funcionales de una gran cantidad de propiedades cuantitativas (robustez, centralidad, eficiencia, ...), y así enriquecer el conjunto de elementos que describen objetivamente la estructura y la función cerebral a disposición de los neurocientíficos. La conexión entre la Ciencia de las Redes y la Neurociencia ha aportado nuevos puntos de vista en la comprensión de la intrincada anatomía del cerebro, y de cómo las patrones de actividad cerebral se pueden sincronizar para generar las denominadas redes funcionales cerebrales, el principal objeto de estudio de esta Tesis Doctoral. Dentro de este contexto, la complejidad emerge como el puente entre las propiedades topológicas y dinámicas de los sistemas biológicos y, específicamente, en la relación entre la organización y la dinámica de las redes funcionales cerebrales. Esta Tesis Doctoral es, en términos generales, un estudio de cómo la actividad cerebral puede ser entendida como el resultado de una red de un sistema dinámico íntimamente relacionado con los procesos que ocurren en el cerebro. Con este fin, he realizado cinco estudios que tienen en cuenta ambos aspectos de dichas redes funcionales: el topológico y el dinámico. De esta manera, la Tesis está dividida en tres grandes partes: Introducción, Resultados y Discusión. En la primera parte, que comprende los Capítulos 1, 2 y 3, se hace un resumen de los conceptos más importantes de la Ciencia de las Redes relacionados al análisis de imágenes cerebrales. Concretamente, el Capitulo 1 está dedicado a introducir al lector en el mundo de la complejidad, en especial, a la complejidad topológica y dinámica de sistemas acoplados en red. El Capítulo 2 tiene como objetivo desarrollar los fundamentos biológicos, estructurales y funcionales del cerebro, cuando éste es interpretado como una red compleja. En el Capítulo 3, se resumen los objetivos esenciales y tareas que serán desarrolladas a lo largo de la segunda parte de la Tesis. La segunda parte es el núcleo de la Tesis, ya que contiene los resultados obtenidos a lo largo de los últimos cuatro años. Esta parte está dividida en cinco Capítulos, que contienen una versión detallada de las publicaciones llevadas a cabo durante esta Tesis. El Capítulo 4 está relacionado con la topología de las redes funcionales y, específicamente, con la detección y cuantificación de los nodos mas importantes: aquellos denominados “hubs” de la red. En el Capítulo 5 se muestra como las redes funcionales cerebrales pueden ser vistas no como una única red, sino más bien como una red-de-redes donde sus componentes tienen que coexistir en una situación de balance funcional. De esta forma, se investiga cómo los hemisferios cerebrales compiten para adquirir centralidad en la red-de-redes, y cómo esta interacción se mantiene (o no) cuando se introducen fallos deliberadamente en la red funcional. El Capítulo 6 va un paso mas allá al considerar las redes funcionales como sistemas vivos. En este Capítulo se muestra cómo al analizar la evolución de la topología de las redes, en vez de tratarlas como si estas fueran un sistema estático, podemos caracterizar mejor su estructura. Este hecho es especialmente relevante cuando se quiere tratar de encontrar diferencias entre grupos que desempeñan una tarea de memoria, en la que las redes funcionales tienen fuertes fluctuaciones. En el Capítulo 7 defino cómo crear redes parenclíticas a partir de bases de datos de actividad cerebral. Este nuevo tipo de redes, recientemente introducido para estudiar las anormalidades entre grupos de control y grupos anómalos, no ha sido implementado nunca en datos cerebrales y, en este Capítulo explico cómo hacerlo cuando se quiere evaluar la consistencia de la dinámica cerebral. Para concluir esta parte de la Tesis, el Capítulo 8 se centra en la relación entre las propiedades topológicas de los nodos dentro de una red y sus características dinámicas. Como mostraré más adelante, existe una relación entre ellas que revela que la posición de un nodo dentro una red está íntimamente correlacionada con sus propiedades dinámicas. Finalmente, la última parte de esta Tesis Doctoral está compuesta únicamente por el Capítulo 9, el cual contiene las conclusiones y perspectivas futuras que pueden surgir de los trabajos expuestos. En vista de todo lo anterior, espero que esta Tesis aporte una perspectiva complementaria sobre uno de los más extraordinarios sistemas complejos frente a los que nos encontramos: El cerebro humano. ABSTRACT The human brain is probably one of the most complex systems we are facing, thus being a timely and fascinating object of study. Characterizing how the brain organizes its activity to carry out complex tasks is highly non-trivial. While early neuroimaging and electrophysiological studies typically aimed at identifying patches of task-specific activations or local time-varying patterns of activity, there has now been consensus that task-related brain activity has a temporally multiscale, spatially extended character, as networks of coordinated brain areas are continuously formed and destroyed. Up until recently, though, the emphasis of functional brain activity studies has been on the identity of the particular nodes forming these networks, and on the characterization of connectivity metrics between them, the underlying covert hypothesis being that each node, constituting a coarse-grained representation of a given brain region, provides a unique contribution to the whole. Thus, functional neuroimaging initially integrated the two basic ingredients of early neuropsychology: localization of cognitive function into specialized brain modules and the role of connection fibres in the integration of various modules. Lately, brain structure and function have started being investigated using Network Science, a statistical mechanics understanding of an old branch of pure mathematics: graph theory. Network Science allows endowing networks with a great number of quantitative properties, thus vastly enriching the set of objective descriptors of brain structure and function at neuroscientists’ disposal. The link between Network Science and Neuroscience has shed light about how the entangled anatomy of the brain is, and how cortical activations may synchronize to generate the so-called functional brain networks, the principal object under study along this PhD Thesis. Within this context, complexity appears to be the bridge between the topological and dynamical properties of biological systems and, more specifically, the interplay between the organization and dynamics of functional brain networks. This PhD Thesis is, in general terms, a study of how cortical activations can be understood as the output of a network of dynamical systems that are intimately related with the processes occurring in the brain. In order to do that, I performed five studies that encompass both the topological and the dynamical aspects of such functional brain networks. In this way, the Thesis is divided into three major parts: Introduction, Results and Discussion. In the first part, comprising Chapters 1, 2 and 3, I make an overview of the main concepts of Network Science related to the analysis of brain imaging. More specifically, Chapter 1 is devoted to introducing the reader to the world of complexity, specially to the topological and dynamical complexity of networked systems. Chapter 2 aims to develop the biological, topological and functional fundamentals of the brain when it is seen as a complex network. Next, Chapter 3 summarizes the main objectives and tasks that will be developed along the forthcoming Chapters. The second part of the Thesis is, in turn, its core, since it contains the results obtained along these last four years. This part is divided into five Chapters, containing a detailed version of the publications carried out during the Thesis. Chapter 4 is related to the topology of functional networks and, more specifically, to the detection and quantification of the leading nodes of the network: the hubs. In Chapter 5 I will show that functional brain networks can be viewed not as a single network, but as a network-of-networks, where its components have to co-exist in a trade-off situation. In this way, I investigate how the brain hemispheres compete for acquiring the centrality of the network-of-networks and how this interplay is maintained (or not) when failures are introduced in the functional network. Chapter 6 goes one step beyond by considering functional networks as living systems. In this Chapter I show how analyzing the evolution of the network topology instead of treating it as a static system allows to better characterize functional networks. This fact is especially relevant when trying to find differences between groups performing certain memory tasks, where functional networks have strong fluctuations. In Chapter 7 I define how to create parenclitic networks from brain imaging datasets. This new kind of networks, recently introduced to study abnormalities between control and anomalous groups, have not been implemented with brain datasets and I explain in this Chapter how to do it when evaluating the consistency of brain dynamics. To conclude with this part of the Thesis, Chapter 8 is devoted to the interplay between the topological properties of the nodes within a network and their dynamical features. As I will show, there is an interplay between them which reveals that the position of a node in a network is intimately related with its dynamical properties. Finally, the last part of this PhD Thesis is composed only by Chapter 9, which contains the conclusions and future perspectives that may arise from the exposed results. In view of all, I hope that reading this Thesis will give a complementary perspective of one of the most extraordinary complex systems: The human brain.

Plataforma terapeútica basada en Kinect para niños con parálisis cerebral infantil

El sistema SONRIE (Sistema de terapia, basadO en KiNect, paRa nIños con parálisis cErebral), realizado como Proyecto Fin de Grado por Dña. Estefanía Sampedro Sánchez, se desarrolló con el fin de permitir el proceso de rehabilitación de los músculos faciales en niños con Parálisis Cerebral Infantil (PCI). SONRIE se compone de una plataforma de juegos cuyo objetivo es lograr una mejora terapéutica en la musculatura orofacial de niños diagnosticados de PCI con edades comprendidas entre los 4 y los 12 años. El escenario de aplicación del sistema SONRIE son las escuelas de integración que tienen escolarizados alumnos diagnosticados con este trastorno. La posibilidad de rehabilitación de los músculos faciales mediante tratamientos que se apoyan en el uso de sistemas telemáticos, junto con el empleo de tecnologías actuales (Realidad Virtual, Realidad Aumentada y Serious Games) supone una gran innovación en el entorno de la neuro-rehabilitación, entendida como el proceso de terapia que permite optimizar la participación de una persona en la sociedad, alcanzando un grado de bienestar óptimo. El trabajo realizado en este Proyecto Fin de Grado pretende escalar el sistema SONRIE, mediante el análisis, diseño y desarrollo de un Framework encargado de facilitar, ampliar y validar el uso adecuado del sistema SONRIE en entornos escolares a través de la integración de nuevas tecnologías. La plataforma desarrollada en este proyecto, permite dotar de dinamismo y persistencia a la plataforma de juegos, ofreciendo a los usuarios de SONRIE (principalmente fisioterapeutas y rehabilitadores que trabajan en entornos escolares) un sistema de terapia para niños con PCI accesible vía web. En este Proyecto Fin de Grado se describe el conjunto de componentes software desarrollados con el fin de proporcionar un entorno web que escale el sistema SONRIE, convirtiéndolo en un sistema de terapia efectivo, completo y usable. ABSTRACT. The SONRIE system (Sistema de terapia, basadO en KiNect, paRa nIños con parálisis cErebral), performed as a final project by Miss Estefanía Sampedro, was developed in order to allow the rehabilitation process of the facial muscles of children with Cerebral Palsy (CP). SONRIE consists of a gaming platform which aims to achieve a therapeutic improvement in the orofacial musculature on children diagnosed with CP aged between 4 and 12 years. The application scenario of the SONRIE system are the integration schools that have students diagnosed with this disorder. The possibility of rehabilitation of facial muscles through treatments based on the use of telematics systems, together with the use of new technologies (Virtual Reality, Augmented Reality and Serious Games) is a great innovation in the neuro-rehabilitation environment, understood as the therapy process that optimizes the participation of a person in the society, reaching an optimum level of welfare. The work done in this final project aims to scale the SONRIE system, through the analysis, design and development of a framework in charge of facilitating, extending and validating the proper use of the SONRIE system in school environments, through the integration of new technologies. The platform developed in this project, can provide dynamism and persistence to the gaming platform, offering to the SONRIE users (mainly physiotherapists and rehabilitators who work in school settings) a therapy system for children with CP accessible via web. In this final project are described the software components developed in order to provide a web environment that scales the SONRIE system, making it an effective, complete and usable therapy system.

Modelado y Evaluación de Entornos Virtuales Interactivos para Rehabilitación Cognitiva en Daño Cerebral

El Daño Cerebral (DC) se refiere a cualquier lesión producida en el cerebro y que afecta a su funcionalidad. Se ha convertido en una de las principales causas de discapacidad neurológica de las sociedades desarrolladas. Hasta la más sencilla de las actividades y acciones que realizamos en nuestro día a día involucran a los procesos cognitivos. Por ello, la alteración de las funciones cognitivas como consecuencia del DC, limita no sólo la calidad de vida del paciente sino también la de las persona de su entorno. La rehabilitación cognitiva trata de aumentar la autonomía y calidad de vida del paciente minimizando o compensando los desórdenes funciones causados por el episodio de DC. La plasticidad cerebral es una propiedad intrínseca al sistema nervioso humano por la que en función a la experiencia se crean nuevos patrones de conectividad. El propósito de la neurorrehabilitación es precisamente modular esta propiedad intrínseca a partir de ejercicios específicos, los cuales podrían derivar en la recuperación parcial o total de las funciones afectadas. La incorporación de la tecnología a las terapias de rehabilitación ha permitido desarrollar nuevas metodologías de trabajo. Esto ha ayudado a hacer frente a las dificultades de la rehabilitación que los procesos tradicionales no logran abarcar. A pesar del gran avance realizado en los Ãoltimos años, todavía existen debilidades en el proceso de rehabilitación; por ejemplo, la trasferencia a la vida real de las habilidades logradas durante la terapia de rehabilitación, así como su generalización a otras actividades cotidianas. Los entornos virtuales pueden reproducir situaciones cotidianas. Permiten simular, de forma controlada, los requisitos conductuales que encontramos en la vida real. En un contexto terapéutico, puede ser utilizado por el neuropsicólogo para corregir en el paciente comportamientos patológicos no deseados, realizar intervenciones terapéuticas sobre Actividades de Vida Diaria que estimulen conductas adaptativas. A pesar de que las tecnologías actuales tienen potencial suficiente para aportar nuevos beneficios al proceso de rehabilitación, existe cierta reticencia a su incorporación a la clínica diaria. A día de hoy, no se ha podido demostrar que su uso aporte una mejorar significativa con respecto a otro tipo de intervención; en otras palabras, no existe evidencia científica de la eficacia del uso de entornos virtuales interactivos en rehabilitación. En este contexto, la presente Tesis Doctoral trata de abordar los aspectos que mantienen a los entornos virtuales interactivos al margen de la rutina clínica diaria. Se estudian las diferentes etapas del proceso de rehabilitación cognitiva relacionado con la integración y uso de estos entornos: diseño de las actividades, su implementación en el entorno virtual, y finalmente la ejecución por el paciente y análisis de los respectivos datos. Por tanto, los bloques en los que queda dividido el trabajo de investigación expuesto en esta memoria son: 1. Diseño de las AVD. La definición y configuración de los elementos que componen la AVD permite al terapeuta diseñar estrategias de intervención terapéutica para actuar sobre el comportamiento del paciente durante la ejecución de la actividad. En esta parte de la tesis se pretende formalizar el diseño de las AVD de tal forma que el terapeuta pueda explotar el potencial tecnológico de los entornos virtuales interactivos abstrayéndose de la complejidad implícita a la tecnología. Para hacer viable este planteamiento se propone una metodología que permita modelar la definición de las AVD, representar el conocimiento implícito en ellas, y asistir al neuropsicólogo durante el proceso de diseño de la intervención clínica. 2. Entorno virtual interactivo. El gran avance tecnológico producido durante los Ãoltimos años permite reproducir AVD interactivas en un contexto de uso clínico. El objetivo perseguido en esta parte de la Tesis es el de extraer las características potenciales de esta solución tecnológica y aplicarla a las necesidades y requisitos de la rehabilitación cognitiva. Se propone el uso de la tecnología de Vídeo Interactivo para el desarrollo de estos entornos virtuales. Para la evaluación de la misma se realiza un estudio experimental dividido en dos fases con la participación de sujetos sanos y pacientes, donde se valora su idoneidad para ser utilizado en terapias de rehabilitación cognitiva. 3. Monitorización de las AVD. El uso de estos entornos virtuales interactivos expone al paciente ante una gran cantidad de estímulos e interacciones. Este hecho requiere de instrumentos de monitorización avanzado que aporten al terapeuta información objetiva sobre el comportamiento del paciente, lo que le podría permitir por ejemplo evaluar la eficacia del tratamiento. En este apartado se propone el uso de métricas basadas en la atención visual y la interacción con el entorno para conocer datos sobre el comportamiento del paciente durante la AVD. Se desarrolla un sistema de monitorización integrado con el entorno virtual que ofrece los instrumentos necesarios para la evaluación de estas métricas para su uso clínico. La metodología propuesta ha permitido diseñar una AVD basada en la definición de intervenciones terapéuticas. Posteriormente esta AVD has sido implementada mediante la tecnología de vídeo interactivo, creando así el prototipo de un entorno virtual para ser utilizado por pacientes con déficit cognitivo. Los resultados del estudio experimental mediante el cual ha sido evaluado demuestran la robustez y usabilidad del sistema, así como su capacidad para intervenir sobre el comportamiento del paciente. El sistema monitorización que ha sido integrado con el entorno virtual aporta datos objetivos sobre el comportamiento del paciente durante la ejecución de la actividad. Los resultados obtenidos permiten contrastar las hipótesis de investigación planteadas en la Tesis Doctoral, aportando soluciones que pueden ayudar a la integración de los entornos virtuales interactivos en la rutina clínica. Esto abre una nueva vía de investigación y desarrollo que podría suponer un gran progreso y mejora en los procesos de neurorrehabilitación cognitiva en daño cerebral. ABSTRACT Brain injury (BI) refers to medical conditions that occur in the brain, altering its function. It becomes one of the main neurological disabilities in the developed society. Cognitive processes determine individual performance in Activities of Daily Living (ADL), thus, the cognitive disorders after BI result in a loss of autonomy and independence, affecting the patient’s quality of life. Cognitive rehabilitation seeks to increase patients’ autonomy and quality of life minimizing or compensating functional disorders showed by BI patients. Brain plasticity is an intrinsic property of the human nervous system whereby its structure is changed depending on experience. Neurorehabilitation pursuits a precise modulation of this intrinsic property, based on specific exercises to induce functional changes, which could result in partial or total recovery of the affected functions. The new methodologies that can be approached by applying technologies to the rehabilitation process, permit to deal with the difficulties which are out of the scope of the traditional rehabilitation. Despite this huge breakthrough, there are still weaknesses in the rehabilitation process, such as the transferring to the real life those skills reached along the therapy, and its generalization to others daily activities. Virtual environments reproduce daily situations. Behavioural requirements which are similar to those we perceive in real life, are simulated in a controlled way. In these virtual environments the therapist is allowed to interact with patients without even being present, inhibiting unsuitable behaviour patterns, stimulating correct answers throughout the simulation and enhancing stimuli with supplementary information when necessary. Despite the benefits which could be brought to the cognitive rehabilitation by applying the potential of the current technologies, there are barriers for widespread use of interactive virtual environments in clinical routine. At present, the evidence that these technologies bring a significant improvement to the cognitive therapies is limited. In other words, there is no evidence about the efficacy of using virtual environments in rehabilitation. In this context, this work aims to address those issues which keep the virtual environments out of the clinical routine. The stages of the cognitive rehabilitation process, which are related with the use and integration of these environments, are analysed: activities design, its implementation in the virtual environment, and the patient’s performance and the data analysis. Hence, the thesis is comprised of the main chapters that are listed below: 1. ADL Design.Definition and configuration of the elements which comprise the ADL allow the therapist to design intervention strategies to influence over the patient behaviour along the activity performance. This chapter aims to formalise the AVD design in order to help neuropsychologists to make use of the interactive virtual environments’ potential but isolating them from the complexity of the technology. With this purpose a new methodology is proposed as an instrument to model the ADL definition, to manage its implied knowledge and to assist the clinician along the design process of the therapeutic intervention. 2. Interactive virtual environment. Continuous advancements make the technology feasible for re-creating rehabilitation therapies based on ADL. The goal of this stage is to analyse the main features of virtual environments in order to apply them according to the cognitive rehabilitation’s requirements. The interactive video is proposed as the technology to develop virtual environments. Experimental study is carried out to assess the suitability of the interactive video to be used by cognitive rehabilitation. 3. ADL monitoring system. This kind of virtual environments bring patients in front lots of stimuli and interactions. Thus, advanced monitoring instruments are needed to provide therapist with objective information about patient’s behaviour. This thesis chapter propose the use of metrics rely on visual patients’ visual attention and their interactions with the environment. A monitoring system has been developed and integrated with the interactive video-based virtual environment, providing neuropsychologist with the instruments to evaluate the clinical force of this metrics. Therapeutic interventions-based ADL has been designed by using the proposed methodology. Interactive video technology has been used to develop the ADL, resulting in a virtual environment prototype to be use by patients who suffer a cognitive deficits. An experimental study has been performed to evaluate the virtual environment, whose overcomes show the usability and solidity of the system, and also its capacity to have influence over patient’s behaviour. The monitoring system, which has been embedded in the virtual environment, provides objective information about patients’ behaviour along their activity performance. Research hypothesis of the Thesis are proven by the obtained results. They could help to incorporate the interactive virtual environments in the clinical routine. This may be a significant step forward to enhance the cognitive neurorehabilitation processes in brain injury.

Estimating sequestered parasite population dynamics in cerebral malaria

Clinical investigation of malaria is hampered by the lack of a method for estimating the number of parasites that are sequestered in the tissues, for it is these parasites that are thought to be crucial to the pathogenesis of life-threatening complications such as cerebral malaria. We present a method of estimating this hidden population by using clinical observations of peripheral parasitemia combined with an age-structured mathematical model of the parasite erythrocyte cycle. Applying the model to data from 217 Gambian children undergoing treatment for cerebral malaria we conclude that although artemether clears parasitemia more rapidly than quinine, the clearance of sequestered parasites is similar for the two drugs. The estimated sequestered mass was found to be a more direct predictor of fatal outcome than clinically observed parasitemia. This method allows a sequential analysis of sequestered parasite population dynamics in children suffering from cerebral malaria, and the results offer a possible explanation for why artemether provides less advantage than might have been expected over quinine in reducing mortality despite its rapid effect on circulating parasites.

MEK1 protein kinase inhibition protects against damage resulting from focal cerebral ischemia

The MEK1 (MAP kinase/ERK kinase)/ERK (extracellular-signal-responsive kinase) pathway has been implicated in cell growth and differentiation [Seger, R. & Krebs, E. G. (1995) FASEB J. 9, 726–735]. Here we show that the MEK/ERK pathway is activated during focal cerebral ischemia and may play a role in inducing damage. Treatment of mice 30 min before ischemia with the MEK1-specific inhibitor PD98059 [Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T. & Saltiel, A. R. (1995) J. Biol. Chem. 270, 27489–27494] reduces focal infarct volume at 22 hr after ischemia by 55% after transient occlusion of the middle cerebral artery. This is accompanied by a reduction in phospho-ERK1/2 immunohistochemical staining. MEK1 inhibition also results in reduced brain damage 72 hr after ischemia, with focal infarct volume reduced by 36%. This study indicates that the MEK1/ERK pathway contributes to brain injury during focal cerebral ischemia and that PD98059, a MEK1-specific antagonist, is a potent neuroprotective agent.

Evidence for a protective role of metallothionein-1 in focal cerebral ischemia

Metallothioneins (MTs) are a family of metal binding proteins that have been proposed to participate in a cellular defense against zinc toxicity and free radicals. In the present study, we investigated whether increased expression of MT in MT-1 isoform-overexpressing transgenic mice (MT-TG) affords protection against mild focal cerebral ischemia and reperfusion. Transient focal ischemia was induced in control (wild type) and MT-TG mice by occluding the right middle cerebral artery for 45 min. Upon reperfusion, cerebral edema slowly developed and peaked at 24 hr as shown by T2-weighted MRI. The volume of affected tissue was on the average 42% smaller in MT-TG mice compared with control mice at 6, 9, 24, and 72 hr and 14 days postreperfusion (P < 0.01). In addition, functional studies showed that 3 weeks after reperfusion MT-TG mice showed a significantly better motor performance compared with control mice (P = 0.011). Although cortical baseline levels of MT-1 mRNA were similar in control and MT-TG mice, there was an increase in MT-1 mRNA levels in the ischemic cortex of MT-TG mice to 7.5 times baseline levels compared with an increase to 2.3 times baseline levels in control mice 24 hr after reperfusion. In addition, MT-TG mice showed an increased MT immunoreactivity in astrocytes, vascular endothelial cells, and neurons 24 hr after reperfusion whereas in control mice MT immunoreactivity was restricted mainly to astrocytes and decreased in the infarcted tissue. These results provide evidence that increased expression of MT-1 protects against focal cerebral ischemia and reperfusion.

Immunologic tolerance to myelin basic protein decreases stroke size after transient focal cerebral ischemia

Immune mechanisms contribute to cerebral ischemic injury. Therapeutic immunosuppressive options are limited due to systemic side effects. We attempted to achieve immunosuppression in the brain through oral tolerance to myelin basic protein (MBP). Lewis rats were fed low-dose bovine MBP or ovalbumin (1 mg, five times) before 3 h of middle cerebral artery occlusion (MCAO). A third group of animals was sensitized to MBP but did not survive the post-stroke period. Infarct size at 24 and 96 h after ischemia was significantly less in tolerized animals. Tolerance to MBP was confirmed in vivo by a decrease in delayed-type hypersensitivity to MBP. Systemic immune responses, characterized in vitro by spleen cell proliferation to Con A, lipopolysaccharide, and MBP, again confirmed antigen-specific immunologic tolerance. Immunohistochemistry revealed transforming growth factor β1 production by T cells in the brains of tolerized but not control animals. Systemic transforming growth factor β1 levels were equivalent in both groups. Corticosterone levels 24 h after surgery were elevated in all sham-operated animals and ischemic control animals but not in ischemic tolerized animals. These results demonstrate that antigen-specific modulation of the immune response decreases infarct size after focal cerebral ischemia and that sensitization to the same antigen may actually worsen outcome.

Functional impact of cerebral connections

Cerebral networks are complex sets of connections that resemble a ladder-like web of multiple parallel feedforward, lateral, and feedback connections. This static anatomical description has been pivotal in guiding our understanding of signal processing within cerebral networks. However, measures on both magnitude and functional significance of connections are extremely limited. Here, we compare the anatomically defined strengths of a set of cerebral pathways emerging from the visual middle suprasylvian (MS) cortex of the cat with measures of the functional impact the same region has over distant sites. These functional measures were obtained by analyzing the local and distant effects of MS cooling deactivation on deoxyglucose uptake. Relative to major efferent projections from MS cortex that have a strong influence, projections to early visual processing stages have weaker functional influences than predicted from the anatomy. For higher processing stages, the converse holds: projections from MS cortex have stronger functional influence than predicted from the anatomy. We conclude that these and future functional measures, obtained using the same combination of techniques, will furnish fundamental, new information that complements and extends current models of static cerebral networks, and lead to more realistic models of cerebral network function and component interactions.

A tetracycline derivative, minocycline, reduces inflammation and protects against focal cerebral ischemia with a wide therapeutic window

The only treatment of patients with acute ischemic stroke is thrombolytic therapy, which benefits only a fraction of stroke patients. Both human and experimental studies indicate that ischemic stroke involves secondary inflammation that significantly contributes to the outcome after ischemic insult. Minocycline is a semisynthetic second-generation tetracycline that exerts antiinflammatory effects that are completely separate from its antimicrobial action. Because tetracycline treatment is clinically well tolerated, we investigated whether minocycline protects against focal brain ischemia with a wide therapeutic window. Using a rat model of transient middle cerebral artery occlusion, we show that daily treatment with minocycline reduces cortical infarction volume by 76 ± 22% when the treatment is started 12 h before ischemia and by 63 ± 35% when started even 4 h after the onset of ischemia. The treatment inhibits morphological activation of microglia in the area adjacent to the infarction, inhibits induction of IL-1β-converting enzyme, and reduces cyclooxygenase-2 expression and prostaglandin E2 production. Minocycline had no effect on astrogliosis or spreading depression, a wave of ionic transients thought to contribute to enlargement of cortical infarction. Treatment with minocycline may act directly on brain cells, because cultured primary neurons were also salvaged from glutamate toxicity. Minocycline may represent a prototype of an antiinflammatory compound that provides protection against ischemic stroke and has a clinically relevant therapeutic window.

Effects of in vivo adventitial expression of recombinant endothelial nitric oxide synthase gene in cerebral arteries

Nitric oxide (NO), synthesized from l-arginine by NO synthases (NOS), plays an essential role in the regulation of cerebrovascular tone. Adenoviral vectors have been widely used to transfer recombinant genes to different vascular beds. To determine whether the recombinant endothelial NOS (eNOS) gene can be delivered in vivo to the adventitia of cerebral arteries and functionally expressed, a replication-incompetent adenoviral vector encoding eNOS gene (AdCMVNOS) or β-galactosidase reporter gene (AdCMVLacZ) was injected into canine cerebrospinal fluid (CSF) via the cisterna magna (final viral titer in CSF, 109 pfu/ml). Adventitial transgene expression was demonstrated 24 h later by β-galactosidase histochemistry and quantification, eNOS immunohistochemistry, and Western blot analysis of recombinant eNOS. Electron microscopy immunogold labeling indicated that recombinant eNOS protein was expressed in adventitial fibroblasts. In AdCMVNOS-transduced arteries, basal cGMP production and bradykinin-induced relaxations were significantly augmented when compared with AdCMVLacZ-transduced vessels (P < 0.05). The increased receptor-mediated relaxations and cGMP production were inhibited by eNOS inhibitors. In addition, the increase in cGMP production was reversed in the absence of calcium, suggesting that the increased NO production did not result from inducible NOS expression. The present study demonstrates the successful in vivo transfer and functional expression of recombinant eNOS gene in large cerebral arteries. It also suggests that perivascular eNOS gene delivery via the CSF is a feasible approach that does not require interruption of cerebral blood flow.

Cerebral cortical astroglia from the trisomy 16 mouse, a model for Down syndrome, produce neuronal cholinergic deficits in cell culture

Trisomy 21 (Down syndrome) is associated with a high incidence of Alzheimer disease and with deficits in cholinergic function in humans. We used the trisomy 16 (Ts16) mouse model for Down syndrome to identify the cellular basis for the cholinergic dysfunction. Cholinergic neurons and cerebral cortical astroglia, obtained separately from Ts16 mouse fetuses and their euploid littermates, were cultured in various combinations. Choline acetyltransferase activity and cholinergic neuron number were both depressed in cultures in which both neurons and glia were derived from Ts16 fetuses. Cholinergic function of normal neurons was significantly down-regulated by coculture with Ts16 glia. Conversely, neurons from Ts16 animals could express normal cholinergic function when grown with normal glia. These observations indicate that astroglia may contribute strongly to the abnormal cholinergic function in the mouse Ts16 model for Down syndrome. The Ts16 glia could lack a cholinergic supporting factor present in normal glia or contain a factor that down-regulates cholinergic function.