Este trabajo está apoyado en parte por subvenciones de los Institutos Nacionales de Salud (SR1 NS059043 y ES015988 SR1), la Sociedad Nacional de Esclerosis Múltiple, Fundación Roche para la Investigación de la Anemia, Feldstein Fundación Médica y Hospitales Shriners para niños. Nos gustaría agradecer al Dr. David Placer por sus sugerencias. Este trabajo fue apoyado en parte por subvenciones de WD de los Institutos Nacionales de Salud (SR1 NS059043 y ES015988 SR1), la Sociedad Nacional de Esclerosis Múltiple, y los Hospitales Shriners para Niños. Declaramos que no hay intereses contrapuestos en relación con este artículo.

Los procedimientos detallados de la creación de una lesión cerebral neonatal en ratones P6: <ol><li> Después del parto el día 6 (P6) de los ratones se anestesian con enfriamiento indirecto en el hielo hasta el punto de falta de respuesta a estímulos dolorosos (anestesia profunda). </li><li> Después de limpiar la piel con alcohol, una incisión en la línea media ventral se hace en la parte anterior del cuello. </li><li> Bajo un microscopio de disección, la bifurcación de la arteria carótida común derecha se acerca suavemente retracción de los músculos omohioideo y esternocleidomastoideo. </li><li> La fascia alrededor de la vaina carotídea se retira, y la rama interna proximal aislada del nervio vago y cerca ganglios simpáticos con un gancho. La arteria carótida interna se cauteriza con una punta de la cauterización. </li><li> Después de la cauterización, la incisión se sutura la piel, y el animal se mantiene caliente hasta que esté completamente despierto y luego regresó a la presa. </li><li> Una hora después de la ligadura, el animal se coloca en una cámara sellada impregnada de nitrógeno hasta un nivel del 6,0% de O 2 se alcanza. El animal se expone a 35 min de hipoxia. </li><li> Después de la exposición la hipoxia, el ratón se recupera en un cojín de la calefacción (33 ° C) durante 30 minutos y luego se devuelve a la presa. Para la creación de la lesión cerebral a la hipoxia combinada / isquemia y la infección / inflamación, el animal se inyecta por vía intraperitoneal con 0,015 ml de lipopolisacárido (LPS, 1 mg / kg). </li><li> Cuatro días post-hypoxia/ischemia, los ratones se anestesian con enfriamiento indirecto en el hielo hasta el punto de falta de respuesta a los estímulos nocivos, entonces perfundidos con paraformaldehido 4% de solución salina y después. Los cerebros se retiran y se crioprotegieron. </li></ol><img src="/files/ftp_upload/1951/1951fig1.jpg" alt="Figura 1" /> Figura 1. Caracterización de la lesión cerebral en el modelo murino de hipoxia / isquemia con tinción H &amp; E. No hay necrosis focal (flechas) en la sustancia blanca cerebral ipsilateral (A) de la ligadura de la carótida, en comparación con la sustancia blanca cerebral contralateral (B). Necrosis ipsilateral y microinfartos focales se observan en el hipocampo (C) y el tálamo (D) en el cerebro con una lesión cerebral ipsilateral materia blanca. Los resultados representativos y cifras: Modelos de ratón de PVL con hipoxia / isquemia y / o infección / inflamación se logran mediante la ligadura de carótida unilateral seguida por la exposición a la hipoxia y la inyección de LPS. Estamos definiendo los modelos de ratón nuevos modelos adecuados PVL. El examen neuropatológico de las secciones coronal teñidas con hematoxilina y eosina (H &amp; E) a través del cerebro anterior revela toda necrosis focal en la materia blanca central del hemisferio cerebral ipsilateral a la ligadura de la carótida (Fig. 1A), con preservación relativa de las neuronas en la corteza que recubre cerebral en comparación con la sustancia blanca cerebral contralateral (Fig. 1B). En el centro de sustancia blanca necrótico, hay un marcado aumento de la celularidad que consiste en macrófagos y astrocitos reactivos, como es característico de PVL humanos focal. En el contralateral sustancia blanca central, el núcleo oligodendroglial están dispuestos en fascicular, como haces, esta arquitectura es completamente alterada por la necrosis focal en el lado ipsilateral. Es de destacar que también se observa necrosis ipsilateral en el hipocampo (Fig. 1C) y el tálamo (microinfartos focales) (Fig. 1D) en el cerebro con una lesión cerebral ipsilateral materia blanca. PVL humanos tampoco se produce en un aislamiento total de las lesiones que acompañan la materia gris. El sello distintivo de PVL es la relación, aunque no completa, ahorradores de la corteza cerebral que recubre el asunto heridos blanco - una característica que creemos que está presente en nuestros modelos de ratón. <img src="/files/ftp_upload/1951/1951fig2.jpg" alt="Figura 2" /> Figura 2. Escala de puntuación de 0-5 para la evaluación de lesiones de sustancia blanca por MBP o O1 en modelos de ratón de la leucomalacia periventricular. Nuestros modelos de ratón muestran las características de la FPV, tanto a nivel fenotípico y molecular. Hemos utilizado la detección inmunohistoquímica de la proteína básica de mielina (MBP) o antígeno O1 para evaluar una lesión, han establecido una escala estándar de 0 a 5 de la puntuación de la lesión de sustancia blanca (Fig. 2), y han caracterizado a la patología de la sustancia blanca (Fig. . 3). Más recientemente, un ensayo de no sesgada estereológicos se ha implementado para sustituir a la primera semi-cuantitativos método. Además, para probar los efectos correlativos de la lesión de sustancia blanca en los resultados funcionales, se han realizado pruebas de comportamiento de la función motora. Hemos demostrado que los ratones presentan déficit motor impresionante a la hipoxia siguientes P10-21 / isquemia más LPS en P6. Hemos establecido los criterios de puntuación con una escala de 0-4 para definir la disfunción extremidad contralateral en ratones heridos en P10-21. Por lo general, los ratones normales se pueden corretear de un 30 ° de inclinación (puntuación de 4) y sube a 45 ° de inclinación, sin deslizamiento (puntuación de 3). Los ratones lesionados tienen puntuaciones más bajas en correlación con los grados de lesión de sustancia blanca. <img src="/files/ftp_upload/1951/1951fig3.jpg" alt="Figura 3" /> Figura 3. Caracterización de lesiones de sustancia blanca por MBP o O1 coloración y la estructura de la mielina por microscopía electrónica (EM). MBP y tinción O1 mostrar la mielina perdida en la sustancia blanca cerebral ipsilateral a la ligadura, en comparación con la sustancia blanca contralateral. Examen EM muestra axones mielinizados en la sustancia blanca cerebral contralateral a la ligadura y el tejido degenerado en la materia blanca ipsilateral. Anticipamos que el establecimiento de los modelos de ratón de nuevo PVL nos permitirá estudiar los mecanismos específicos de PVL-como la lesión del cerebro inmaduro. Nos ocuparemos de la patogénesis utilizando cepas de ratones transgénicos para examinar el papel de las moléculas individuales de interés y evaluar los objetivos de la neuroprotección.

<ol>
	<li>Volpe, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neurology of newborn. , 217-276 (2001).</li><li>Deng, W., Pleasure, J., Pleasure, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+periventricular+leukomalacia.">Progress in periventricular leukomalacia.</a> Arch. Neurol. 65, 1291-1295 (2008).</li><li>Follett, P. L., Deng, W., Dai, W., Talos, D. M., Massillon, L. J., Rosenberg, P. A., Volpe, J. J., Jensen, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+receptor-mediated+oligodendrocyte+toxicity+in+periventricular+leukomalacia:+a+protective+role+for+topiramate.">Glutamate receptor-mediated oligodendrocyte toxicity in periventricular leukomalacia: a protective role for topiramate.</a> J. Neurosci. 24, 4412-4420 (2004).</li></ol>

La lesión del cerebro en desarrollo lleva a devastadoras consecuencias neurológicas. Sorprendentemente, el patrón de la lesión cerebral perinatal es altamente dependiente de la edad. En recién nacidos a término, que afecta principalmente a la corteza cerebral con pérdida neuronal característica. Sin embargo, en los bebés prematuros, que afecta selectivamente la materia blanca cerebral con daño importante a los oligodendrocitos en desarrollo, un trastorno denominado leucomalacia periventricular (LPV). La magnitud del problema de las lesiones cerebrales en el bebé prematuro es extraordinario. Parto prematuro y la mejora de los cuidados intensivos neonatales han llevado a casi el 90% de supervivencia de los 13 millones de niños prematuros en todo el mundo (aproximadamente 56.000 en los Estados Unidos), nacido el año con un peso al nacer inferior a 1500 gramos, es decir, de muy bajo peso al nacer. Aproximadamente el 10% de los sobrevivientes posteriormente presentan parálisis cerebral y aproximadamente el 50% tienen déficits cognitivos y de comportamiento. PVL es el cerebro más importante de la patología subyacente parálisis cerebral en recién nacidos prematuros. No hay tratamiento específico para PVL existe en la actualidad, en parte debido a la patogenia no ha sido bien entendido 1,2. Aunque la etiología de la CVP es multifactorial, hipoxia perinatal / isquemia con infección intrauterina materna se cree que es una causa primaria de la FPV. Un modelo de hipoxia / isquemia del PVL en la rata a P7 ha sido establecido con anterioridad 3. Este modelo es único en el que se asemeja a LPV en recién nacidos prematuros. El sello distintivo de este modelo es selectiva de la materia blanca patología, a diferencia de la mayoría de los modelos de carrera que se caracterizan por un infarto de la materia gris. Sin embargo, las limitaciones del modelo hipóxico-isquémica en la rata no debe pasarse por alto. En primer lugar, la etiología de la CVP suele ser multifactorial. Además de la hipoxia / isquemia, materno-fetal infección / inflamación se asocia fuertemente con la FPV. Por lo tanto, la inflamación combinado hipoxia / isquemia y la infección / probable que crear modelos más PVL-como. La edad de roedores que mejor se correlaciona con el período de desarrollo humano de mayor riesgo de lesiones PVL (24-32 semanas de gestación) es después del parto porque la diferenciación oligodendroglial es predominantemente un evento post-natal en los roedores. Ya que es imposible producir una infección materno-fetal / inflamación de edad posnatal, que sería razonable para simular una infección en una fase de desarrollo postnatal relevante para crear un modelo de roedores de la FPV. Hemos tratado de combinar la inyección de LPS con el modelo de hipoxia / isquemia en ratones a P6, para producir lesiones PVL más clínicamente relevante. En segundo lugar, el óptimo de prueba de concepto de enfoque experimental para un papel causal de una molécula hace necesario el uso de ratones transgénicos, la manipulación genética son mucho más difíciles en la rata que en el ratón. Por lo tanto, hemos tratado de convertir nuestro modelo experimental de PVL de la rata en el ratón. Modelos de ratón de PVL facilitará en gran medida 1) el estudio de la biología de la enfermedad y la patogénesis utilizando disponibles cepas de ratones transgénicos, 2) la realización de ensayos de medicamentos para descubrir posibles agentes con potencial terapéutico, y 3) las pruebas de la utilidad de las células madre utilizando cepas inmunodeficientes ratón.

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mouse models of periventricular leukomalacia

Se describe un protocolo para el establecimiento de modelos de ratón de la leucomalacia periventricular (PVL). PVL es la forma predominante de la lesión cerebral en los bebés prematuros y el antecedente más común de parálisis cerebral. PVL se caracteriza por daño la sustancia blanca periventricular, con lesiones oligodendroglial prominentes. Hipoxia / isquemia, con o sin infección sistémica / inflamación son las principales causas de la FPV. Nosotros usamos ratones P6 para crear modelos de lesión cerebral neonatal mediante la inducción de hipoxia / isquemia, con o sin infección sistémica / inflamación con la ligadura de carótida unilateral seguida por la exposición a la hipoxia, con o sin la inyección de la endotoxina lipopolisacárido (LPS). Inmunohistoquímica de la proteína básica de mielina (MBP) o O1 y el examen microscópico electrónico muestran la pérdida de mielina prominentes en la sustancia blanca cerebral con daño adicional en el hipocampo y el tálamo. Establecimiento de modelos de ratón de PVL facilitará en gran medida el estudio de la patogénesis de la enfermedad utilizando disponibles cepas de ratones transgénicos, la conducción de los ensayos de medicamentos de una manera relativamente alto rendimiento para identificar posibles agentes terapéuticos, y el ensayo de transplante de células madre utilizando cepas de inmunodeficiencia ratón.

Watch this Scientific Journal Video about Mouse Models of Periventricular Leukomalacia at JoVE.com

Mouse Models of Periventricular Leukomalacia

Se establecieron modelos de ratón de leucomalacia periventricular (LPV), la lesión cerebral predominante en los bebés prematuros se caracteriza por lesiones de sustancia blanca periventricular. Hipoxia / isquemia con / sin infección sistémica son las causas principales de la FPV. La ligadura de carótida unilateral y la exposición hipoxia con / sin inyección de lipopolisacárido crea PVL-como lesiones en los ratones P6.

Los modelos de ratón de la Retinopatía

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature ...

mouse-models-of-periventricular-leukomalacia

Research

JoVE Journal

Neuroscience

29.7K vistas.  University of California, Davis. Se establecieron modelos de ratón de leucomalacia periventricular (LPV), la lesión cerebral predominante en los bebés prematuros se caracteriza por lesiones de sustancia blanca periventricular. Hipoxia / isquemia con / sin infección sistémica son las causas principales de la FPV. La ligadura de carótida unilateral y la exposición hipoxia con / sin inyección de lipopolisacárido crea PVL-como lesiones en los ratones P6.

Video: Mouse Models of Periventricular Leukomalacia

A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebella ataxia. It is derived from previously published phenotype assessments in several disease models, including spinocerebellar ataxias, Huntington s disease and spinobulbar muscular atrophy. Measures include hind limb clasping, ledge test, gait and kyphosis. Each measure is recorded on a scale of 0-3, with a combined total of 0-12 for all four measures. The results effectively discriminate between affected and non-affected individuals, while also quantifying the temporal progression of neurodegenerative disease phenotypes. Measures may be analyzed individually or combined into a composite phenotype score for greater statistical power. The ideal combination of the four described measures will depend upon the disorder in question. We present an example of the protocol used to assess disease severity in a transgenic mouse model of spinocerebellar ataxia type 7 (SCA7).
Albert R. La Spada and Gwenn A. Garden contributed to this manuscript equally.

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebellar ataxia. Measures include hind limb clasping, ledge test, gait and kyphosis. This protocol effectively discriminates between affected and non-affected individuals, and detects the progression of affected individuals over time.

A simple fenotipo compuesto el sistema de puntaje para la evaluación de modelos de ratón de Ataxia cerebelosa

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebella ataxia. It is derived from previously ...

Investigación

Neurociencias

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

Múltiples imágenes del ratón neuroanatómicos de Resonancia Magnética

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Olfactory Assays for Mouse Models of Neurodegenerative Disease

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process and may be a useful behavioral marker for early detection. Earlier detection in neurodegenerative disease is a major goal in the field because this is when neuroprotective therapies have the best potential to be effective. Therefore, in preclinical studies testing novel neuroprotective strategies in rodent models of neurodegenerative disease, olfactory assessment could be highly useful in determining therapeutic potential of compounds and translation to the clinic. In the present study we describe a battery of olfactory assays that are useful in measuring olfactory function in mice. The tests presented in this study were chosen because they measure olfaction abilities in mice related to food odors, social odors, and non-social odors. These tests have proven useful in characterizing novel genetic mouse models of Parkinson&#8217;s disease as well as in testing potential disease-modifying therapies.

Impairment in olfactory function is a common feature in many neurodegenerative disorders including Parkinson, Alzheimer, and Huntington diseases. In the present article, we describe a set of tests for assessing olfaction discrimination and detection in mice that can be used to measure olfactory abilities in mouse models of neurodegenerative diseases.

Los ensayos olfativos para modelos de ratón de Enfermedades Neurodegenerativas

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process ...

3D Modeling of the Lateral Ventricles and Histological Characterization of Periventricular Tissue in Humans and Mouse

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional units of the ventricles are ciliated epithelial cells termed ependymal cells, which line the ventricles and through ciliary action are capable of generating laminar flow of CSF at the ventricle surface. This monolayer of ependymal cells also provides barrier and filtration functions that promote exchange between brain interstitial fluids (ISF) and circulating CSF. Biochemical changes in the brain are thereby reflected in the composition of the CSF and destruction of the ependyma can disrupt the delicate balance of CSF and ISF exchange. In humans there is a strong correlation between lateral ventricle expansion and aging. Age-associated ventriculomegaly can occur even in the absence of dementia or obstruction of CSF flow. The exact cause and progression of ventriculomegaly is often unknown; however, enlarged ventricles can show regional and, often, extensive loss of ependymal cell coverage with ventricle surface astrogliosis and associated periventricular edema replacing the functional ependymal cell monolayer. Using MRI scans together with postmortem human brain tissue, we describe how to prepare, image and compile 3D renderings of lateral ventricle volumes, calculate lateral ventricle volumes, and characterize periventricular tissue through immunohistochemical analysis of en face lateral ventricle wall tissue preparations. Corresponding analyses of mouse brain tissue are also presented supporting the use of mouse models as a means to evaluate changes to the lateral ventricles and periventricular tissue found in human aging and disease. Together, these protocols allow investigations into the cause and effect of ventriculomegaly and highlight techniques to study ventricular system health and its important barrier and filtration functions within the brain.

Using MRI scans (human), 3D imaging software, and immunohistological analysis, we document changes to the brain&#8217;s lateral ventricles. Longitudinal 3D mapping of lateral ventricle volume changes and characterization of periventricular cellular changes that occur in the human brain due to aging or disease are then modeled in mice.

Modelado 3D del lateral Ventrículos y histológico Caracterización de Tejidos periventricular en los seres humanos y de ratón

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional ...

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer&#039;s Mouse Models

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

El uso de biosensores basados ​​en enzimas para medir tónica y fásica glutamato en modelos de ratones con Alzheimer

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

Exploring Deep Space - Uncovering the Anatomy of Periventricular Structures to Reveal the Lateral Ventricles of the Human Brain

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this challenging. Because the ventricles are negative spaces located deep within the brain, the only way to understand their anatomy is by appreciating their boundaries formed by related structures. Looking at a 2D representation of these spaces, in any of the cardinal planes, will not enable visualisation of all of the structures that form the boundaries of the ventricles. Thus, using 2D sections alone requires students to compute their own mental image of the 3D ventricular spaces. The aim of this study was to develop a reproducible method for dissecting the human brain to create an educational resource to enhance student understanding of the intricate relationships between the ventricles and periventricular structures. To achieve this, we created a video resource that features a step-by-step guide using a fiber dissection method to reveal the lateral and third ventricles together with the closely related limbic system and basal ganglia structures. One of the advantages of this method is that it enables delineation of the white matter tracts that are difficult to distinguish using other dissection techniques. This video is accompanied by a written protocol that provides a systematic description of the process to aid in the reproduction of the brain dissection. This package offers a valuable anatomy teaching resource for educators and students alike. By following these instructions educators can create teaching resources and students can be guided to produce their own brain dissection as a hands-on practical activity. We recommend that this video guide be incorporated into neuroanatomy teaching to enhance student understanding of the morphology and clinical relevance of the ventricles.

This paper demonstrates the effective use of a fiber dissection method to reveal the superficial white matter tracts and periventricular structures of the human brain, in three-dimensional space, to aid student comprehension of ventricular morphology.

Explorar el espacio profundo - descubrimiento de la anatomía de las estructuras periventriculares para revelar los ventrículos laterales del cerebro humano

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this ...

Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly and additional brain abnormalities. However, the mechanism underlying the pathogenesis of ZIKV in the developing brain remains unclear. Intracerebral surgical methods are frequently used in neuroscience research to address questions about both normal and abnormal brain development and brain function. This protocol utilizes classical surgical techniques and describes methods that allow one to model ZIKV-associated human neurological disease in the mouse nervous system. While direct brain inoculation does not model the normal mode of virus transmission, the method allows investigators to ask targeted questions concerning the consequence after ZIKV infection of the developing brain. This protocol describes embryonic, neonatal, and adult stages of intraventricular inoculation of ZIKV. Once mastered, this method can become a straightforward and reproducible technique that only takes a few hours to perform.

Here we describe a method for establishing a model of Zika virus-induced microcephaly in mouse. This protocol includes methods for embryonic, neonatal, and adult-stage intracerebral inoculation of the Zika virus.

Establecimiento de modelos de ratón para trastornos neurológicos inducidos por el Virus Zika utilizando estrategias de inyección Intracerebral: embrionario, Neonatal y adulto

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly ...

Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection

This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory neurons and glial satellite cells. These are useful and versatile models to investigate a variety of biological responses associated with physiological and pathological conditions of the peripheral nervous system (PNS) ranging from neuron-glial interaction, neuroplasticity, neuroinflammation, and viral infection. The usage of DRG explant is scientifically advantageous compared to simplistic single cells models for multiple reasons. For instance, as an organotypic culture, the DRG explant allows ex vivo transfer of an entire neuronal network including the extracellular microenvironment that play a significant role in all the neuronal and glial functions. Further, DRG explants can also be maintained ex vivo for several days and the culture conditions can be perturbed as desired. In addition, the harvested DRG can be further dissociated into an in vitro co-culture of primary sensory neurons and satellite glial cells to investigate neuronal-glial interaction, neuritogenesis, axonal cone interaction with the extracellular microenvironment, and more general, any aspect associated with the neuronal metabolism. Therefore, the DRG-explant system offers a great deal of flexibility to study a wide array of events related to biological, physiological, and pathological conditions in a cost-effective manner.

In this report, the advantages of organotypic cultures and dissociated primary cultures of mouse-derived dorsal root ganglia are highlighted to investigate a wide range of mechanisms associated with neuron-glial interaction, neuroplasticity, neuroinflammation, and response to viral infection.

Ratón adulto DRG explante y disociado modelos celulares para investigar respuestas a insultos ambientales incluyendo infección Viral y la neuroplasticidad

This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory ...

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

En Vivo Medición electrofisiológica de potencial de acción compuesto del músculo de las extremidades anteriores en modelos de ratón de la degeneración de la motoneurona

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

A Behavioral Screen for Heat-Induced Seizures in Mouse Models of Epilepsy

Transgenic mouse models have proved to be powerful tools in studying various aspects of human neurological disorders, including epilepsy. The SCN1A-associated genetic epilepsies comprise a wide spectrum of seizure disorders with incomplete penetrance and clinical variability. SCN1A mutations can result in a large variety of seizure phenotype ranging from simple, self-limited fever-associated febrile seizures (FS), moderate-level genetic epilepsy with febrile seizures plus (GEFS+) to more severe Dravet Syndrome (DS). Although FS are commonly seen in children below 6-7 years of age who do not have genetic epilepsy, FS in GEFS+ patients continue to occur into adulthood. Traditionally, experimental FS have been induced in mice by exposing the animal to a stream of dry air or heating lamps, and the rate of change in body temperature is often not well controlled. Here, we describe a custom-built heating chamber, with a plexiglass front, that is fitted with a digital temperature controller and a heater-equipped electric fan, which can send heated forced air into the test arena in a temperature-controlled manner. The body temperature of a mouse placed in the chamber, monitored through a rectal probe, can be increased to 40-42 &#176;C in a reproducible manner by increasing the temperature inside the chamber. Continual visual monitoring of the animals during the heating period demonstrates induction of heat-induced seizures in mice carrying an FS mutation at a body temperature that does not elicit behavioral seizures in wild-type litter mates. Animals can be easily removed from the chamber and placed on a cooling pad to rapidly return body temperature to normal. This method provides for a simple, rapid, and reproducible screening protocol for the occurrence of heat-induced seizures in epilepsy mouse models.

The goal of the method is to screen for hyperthermia or heat-induced seizures in mouse models. The protocol describes the use of a custom-built chamber with continuous monitoring of the body temperature to determine whether elevated body temperature leads to seizures.