Este trabalho é, em parte suportado por concessões do National Institutes of Health (RO1 NS059043 e RO1 ES015988), Multiple Sclerosis Society Nacional, Fundação Roche para Anemia Research, Feldstein Fundação Médica e Hospitais Shriners para Crianças. Gostaríamos de agradecer ao Dr. David Prazer para suas sugestões. Este trabalho foi em parte suportado por concessões para WD de National Institutes of Health (RO1 NS059043 e RO1 ES015988), Sociedade Nacional de Esclerose Múltipla, e Hospitais Shriners para Crianças. Declaramos a inexistência de interesses concorrentes relacionados para este artigo.

Procedimentos detalhados de criação de lesão cerebral neonatal em ratos P6: <ol><li> Pós-natal do dia 6 (P6) ratos são anestesiados com refrigeração indireta sobre o gelo ao ponto de insensibilidade ao estímulo nocivo (anestesia profunda). </li><li> Após a limpeza da pele com álcool, uma incisão mediana ventral é feita no pescoço anterior. </li><li> Sob um microscópio de dissecação, a bifurcação da artéria carótida comum direita é abordado por gentilmente retrair os músculos omo-hióideo e esternocleidomastóideo. </li><li> A fascia em torno da bainha carotídea é removido, eo ramo proximal interna isolada do nervo vago nas proximidades e gânglios simpáticos com um gancho. A artéria carótida interna é então cauterizados com uma ponta de cauterização. </li><li> Após a cauterização, a incisão na pele é suturada, eo animal é mantido quente até que esteja completamente desperto e depois voltou para a represa. </li><li> Uma hora após a ligadura, o animal é colocado em uma câmara selada infundido com nitrogênio, até um nível de 6,0% O 2 é atingido. O animal é então exposto a 35 min de hipóxia. </li><li> Após a exposição a hipóxia, o mouse se recupera em uma almofada de aquecimento (33 ° C) por 30 min e depois é devolvido à represa. Para a criação de lesão cerebral com hipóxia combinado / isquemia e infecção / inflamação, o animal é então injetado intraperitonealmente com 0,015 ml de lipopolissacarídeo (LPS, 1 mg / kg). </li><li> Quatro dias post-hypoxia/ischemia, os ratos são anestesiados com refrigeração indireta sobre o gelo até o ponto de indiferença a estímulos nocivos, então perfundidos com solução salina e, em seguida, paraformaldeído 4%. Cérebros são removidos e cryoprotected. </li></ol><img src="/files/ftp_upload/1951/1951fig1.jpg" alt="Figura 1" /> Figura 1. Caracterização de lesão cerebral no modelo do rato hipóxica / isquêmica com coloração H &amp; E. Há necrose focal (setas) na substância branca cerebral ipsilateral (A) para a ligadura de carótida, em comparação com a matéria cerebral contralateral branco (B). Necrose focal ipsilateral e microinfartos são observadas no hipocampo (C) eo tálamo (D) no cérebro com lesão da substância branca cerebral ipsilateral. Resultados representante e números: Modelos do rato do PVL com hipóxia / isquemia e / ou infecção / inflamação são alcançados pela ligadura de carótida unilateral seguido pela exposição à hipóxia e injeção de LPS. Estamos definindo os modelos novo mouse como adequados modelos LPV. O estudo neuropatológico de cortes coronais corados com hematoxilina e eosina (H &amp; E) através da parte frontal do cérebro inteiro revela necrose focal na questão central branca do hemisfério cerebral ipsilateral à ligadura de carótida (Fig. 1A), com relativa preservação dos neurônios no o córtex cerebral sobrejacente comparação com a matéria cerebral contralateral branco (Figura 1B). Em matéria de necrose focal branco, existe um marcado aumento da celularidade composto de macrófagos e astrócitos reativos, como é característico de LPV focal humana. Na questão central contralateral branca, os núcleos são organizados em oligodendroglial fascicular-como feixes, essa arquitetura é completamente interrompido pela necrose focal no lado ipsilateral. De nota, também observamos necrose ipsilateral no hipocampo (Fig. 1C) eo tálamo (microinfartos focal) (Figura 1D) no cérebro com lesão da substância branca cerebral ipsilateral. PVL humana também não ocorre em completo isolamento que acompanham lesões massa cinzenta. A marca da PVL é o relativo, embora não seja completa, poupando do córtex cerebral que cobre o assunto feridos branco - um recurso que nós acreditamos que está presente em nossos modelos de mouse. <img src="/files/ftp_upload/1951/1951fig2.jpg" alt="Figura 2" /> Figura 2. Escala de pontuação de 0-5 para avaliar lesão da substância branca pelo PAM ou O1 em modelos do rato de leucomalácia periventricular. Nossos modelos de rato mostram características de PVL, tanto a nível fenotípico e molecular. Temos usado a detecção imunocitoquímica da proteína básica da mielina (MBP) ou O1 antígeno para avaliar lesão, criaram uma escala padrão 0-5 para marcar a lesão da substância branca (Fig. 2), e caracterizaram a substância branca patologia (Fig . 3). Mais recentemente, um ensaio não-tendenciosa estereológica foi implementado para substituir o método semi-quantitativo inicial. Além disso, para testar efeitos correlatos de lesão da substância branca nos resultados funcionais, temos realizado testes comportamentais para a função motora. Temos demonstrado que os ratos apresentam déficits motores atacando P10-21 hipóxia seguintes / isquemia mais LPS em P6. Nós estabelecemos critérios de pontuação com uma escala de 0-4 para definir disfunção membro contralateral em ratos feridos no P10-21. Normalmente, os ratos normais pode scamper uma inclinação de 30 º (nota 4) e subir a 45 ° inclinação sem deslizar (escore de 3). Camundongos feridos têm notas mais baixas em correlação com os graus de lesão da substância branca. <img src="/files/ftp_upload/1951/1951fig3.jpg" alt="Figura 3" /> Figura 3. Caracterização de lesão da substância branca pelo PAM ou O1 coloração e estrutura da mielina por microscopia eletrônica (EM). MBP e coloração O1 mostrar a perda de mielina na substância branca cerebral ipsilateral à ligadura, em comparação com substância branca contralateral. EM exame mostra axônios mielinizados na massa branca cerebral contralateral à ligadura e degenerou tecido na substância branca ipsilateral. Prevemos que o estabelecimento dos modelos novo mouse da LPV nos permitirá estudar mecanismos específicos de PVL-like lesão no cérebro imaturo. Iremos abordar patogênese usando linhagens de camundongos transgênicos para examinar o papel de moléculas individuais de interesse e avaliar as metas de neuroproteção.

<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>

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Prejuízo para o desenvolvimento do cérebro leva a consequências devastadoras neurológicos. Surpreendentemente, o padrão de lesão cerebral perinatal é altamente dependente da idade. Em bebês nascidos a termo, que afeta predominantemente o córtex cerebral com perda neuronal característica. No entanto, em bebês prematuros, que afeta seletivamente substância branca cerebral com lesão importante para o desenvolvimento oligodendrócitos, um distúrbio chamado de leucomalácia periventricular (LPV). A magnitude do problema de lesão cerebral em bebês prematuros é extraordinário. Parto prematuro e de cuidados intensivos neonatal melhorou levaram a quase 90% de sobrevivência dos 13 milhões de bebês prematuros em todo o mundo (cerca de 56.000 nos Estados Unidos), nascido anual com um peso menor que 1500 gramas, ou seja, de muito baixo peso ao nascer. Aproximadamente 10% dos sobreviventes posteriormente apresentam paralisia cerebral e cerca de 50% têm déficit cognitivo e comportamental. PVL é o mais importante patologia cerebral paralisia cerebral subjacente em prematuros. Nenhuma terapia específica para PVL existe presentemente, em parte porque a patogênese não foi bem compreendida 1,2. Embora a etiologia da PVL é multifatorial, perinatal hipóxia / isquemia com infecção intra-uterina materna é pensado para ser uma causa primária de LPV. Um modelo de hipóxia / isquemia da LPV no rato em P7 foi previamente estabelecido 3. Este modelo é o único que se assemelha a PVL em prematuros. A principal característica deste modelo é seletiva substância branca patologia, em contraste com a maioria dos modelos de acidente vascular cerebral que são caracterizados por infarto massa cinzenta. No entanto, as limitações do modelo hipóxico-isquêmica no rato não deve ser menosprezada. Primeiro, a etiologia da PVL é frequentemente multifatorial. Além da inflamação hipóxia / isquemia, infecção materno-fetal / está fortemente associada a LPV. Assim, a inflamação hipóxia / isquemia e infecção / combinado provavelmente criar modelos mais PVL-like. A idade de roedores que melhor se correlaciona com o período de desenvolvimento humano de maior risco para lesões PVL (24-32 semanas de gestação) é pós-natal, porque a diferenciação oligodendroglial é predominantemente um evento pós-natal em roedores. Como é impossível produzir uma infecção materno-fetal / inflamação nas idades pós-natal, seria razoável para simular uma infecção numa fase pós-natal relevantes do desenvolvimento para criar um modelo do roedor da LPV. Temos procurado combinar a injeção de LPS com o modelo hipóxica / isquêmica em ratos em P6, para produzir mais clinicamente relevantes lesões LPV. Em segundo lugar, a abordagem de prova de conceito do ideal experimental para um papel causal de uma molécula requer o uso de camundongos transgênicos; manipulações genéticas são muito mais difíceis no rato do que no mouse. Assim, procuramos converter o nosso modelo experimental de PVL do rato para o mouse. Modelos do rato do PVL irá facilitar grandemente 1) o estudo da biologia da doença e patogênese disponíveis usando linhagens de camundongos transgênicos, 2) realização de testes de drogas para descobrir agentes candidato com potencial terapêutico; 3) testar a utilidade de células-tronco usando linhagens de camundongos imunodeficientes.

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mouse models of periventricular leukomalacia

Nós descrevemos um protocolo para o estabelecimento de modelos de ratos de leucomalácia periventricular (LPV). PVL é a forma predominante de lesão cerebral em bebês prematuros e de o antecedente mais comum de paralisia cerebral. PVL é caracterizada por lesão da substância branca periventricular com lesão oligodendroglial proeminente. Hipóxia / isquemia, com ou sem infecção sistêmica / inflamação são as principais causas de LPV. Nós usamos ratos P6 para criar modelos de lesão cerebral neonatal pela indução de hipóxia / isquemia, com ou sem infecção sistêmica / inflamação com ligadura de carótida unilateral seguido pela exposição à hipóxia, com ou sem injeção de lipopolissacarídeo endotoxina (LPS). Imunohistoquímica da proteína básica da mielina (MBP) ou O1 e exame de microscopia eletrônica mostram perda de mielina proeminente na matéria branca cerebral com dano adicional para o hipocampo eo tálamo. Estabelecimento de modelos do rato do PVL vai facilitar muito o estudo da patogênese da doença usando linhagens de camundongos transgênicos disponíveis, realização de testes de drogas de uma forma relativamente alto rendimento para identificar agentes terapêuticos candidato, e testes do transplante de células-tronco usando linhagens de camundongos imunodeficiência.

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

Mouse Models of Periventricular Leukomalacia

Nós estabelecemos modelos do rato de leucomalácia periventricular (LPV), a lesão cerebral predominante em prematuros caracterizada por lesões na substância branca periventricular. Hipóxia / isquemia com / sem infecção sistêmica são as principais causas de LPV. Ligadura da carótida unilateral e exposição a hipóxia com / sem injeção de lipopolissacarídeo cria PVL-como lesões em camundongos P6.

Modelos de mouse de leucomalácia periventricular

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 visualizações.  University of California, Davis. Nós estabelecemos modelos do rato de leucomalácia periventricular (LPV), a lesão cerebral predominante em prematuros caracterizada por lesões na substância branca periventricular. Hipóxia / isquemia com / sem infecção sistêmica são as principais causas de LPV. Ligadura da carótida unilateral e exposição a hipóxia com / sem injeção de lipopolissacarídeo cria PVL-como lesões em camundongos P6.

Vídeo: 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.

Um sistema simples de pontuação Composite Fenótipo para avaliar modelos de ratos de ataxia cerebelar

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

Neurociência

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.

Múltiplas mouse-Imagem por Ressonância Magnética neuroanatômicos

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.

Ensaios olfativas para o Rato modelos de doenças 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.

Modelagem 3D do lateral ventrículos e histológica Caracterização do Tecido Periventricular em humanos e mouse

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).

Utilizando biossensores baseados em Enzyme medir Tonic e Phasic glutamato na doença de Alzheimer rato Models

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.

Explorando o espaço profundo - descobrindo a anatomia das estruturas periventriculares para revelar os ventrículos laterais do cérebro 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.

Estabelecer modelos de Mouse para doenças neurológicas induzida por vírus Zika usando estratégias de injeção Intracerebral: embrionárias, Neonatal e 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.

Rato adulto DRG explantes e dissociado de modelos de célula para investigar neuroplasticidade e respostas aos insultos ambientais incluindo infecção Viral

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.

Na Vivo Eletrofisiológica medição do potencial de ação muscular composto de membros dianteiros em modelos de Mouse de degeneração do neurônio Motor

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.