Este trabalho foi financiado pela Fundação de ciência Natural de Beijing (No.7162124) e Xin-ao Fundação para a Universidade de Beijing de medicina chinesa.

Todos os animais foram mantidos em conformidade com as diretrizes da legislação chinesa sobre o uso ético e cuidar de animais de laboratório.
1. pré-modelagem preparação
<ol><li>
Preparação do laboratório
	<ol>
<li>Execute a lâmpada de UV durante pelo menos 30 min antes do experimento.</li>
		<li>Controle a temperatura do laboratório a 25 ± 3 ° C e umidade relativa em torno de 30%.</li>
		<li>Ligue o laboratório luz às 06:00 e desligar às 18:00 para estabelecer um ciclo claro/escuro de 12 h/12 h.</li>
	</ol>
</li>
	<li>
Construção de caixa de plataforma múltipla
	<ol>
<li>Construa um depósito de plástico opaco sem uma cobertura de 110 × 60 × 40 cm3.</li>
		<li>Corrigir a quinze plataformas circulares (h = 8 cm, d = 6,5 cm) na parte inferior do tanque, o enfermeiro que distribuir em três linhas e cinco colunas. Deixe espaço suficiente entre cada plataforma, mais ou menos 10 cm entre colunas e entre linhas de 13 centímetros.</li>
		<li>Definir uma saída de água no lado lateral do tanque e instalar uma torneira.</li>
		<li>Fazer uma cobertura de rede de fio de ferro para o tanque com uma caixa de comida pendurado nela.</li>
	</ol>
</li>
	<li>
Agrupamento e ratos de habitação Nota: Ratos machos Wistar de 8 semanas de idade, pesando aproximadamente 200-210 g, são utilizados no experimento. Os ratos vivem em grupos durante o processo de modelagem.<ol>
<li>Número de raízes de cauda dos ratos com uma caneta.</li>
		<li>Pesar os ratos, excluir o extremamente leve ou mais pesada e dividir o resto aleatoriamente em grupos de controle e modelo.</li>
		<li>Delicadamente colocou os ratos as gaiolas limpas e permitir-lhes para se aclimatar ao laboratório pelo menos 3 dias. Fornece água suficiente e fornecimento de alimentos.</li>
	</ol>
</li>
</ol>2. modelagem com MMPM
Nota: O processo começa às 18:00 e termina às 08:00 no dia seguinte, para um total de 14 h por dia, durante 21 dias. Para evitar fatores de interferência, a mesma pessoa é necessária para conduzir todo o experimento, enquanto usava o mesmo casaco de laboratório. 10 ratos Wistar são usados no experimento.
<ol><li>Coloque o reservatório sobre uma superfície plana, por exemplo, no chão. Em seguida, encha o reservatório com aproximadamente 7 cm de água morna (25 ± 3 ° C), cerca de 1 cm abaixo da plataforma plana.</li>
	<li>Prepare comida e bebida suficiente para todos os ratos no tanque para 1 dia. Forragem e água colocaram na caixa de comida e pendurá-lo na capa. Nota: Alguns ratos inteligentes aprendem a descansar na caixa de comida. Em caso afirmativo, conduzi-los a volta para o tanque.</li>
	<li>Levar os ratos do grupo de modelo fora da gaiola, agarrá-los pelo rabo e colocá-los suavemente para o tanque. Lançar todos os ratos na água em vez das plataformas para motivar o seu medo de água. Certifique-se de que todos os ratos Obtém uma plataforma para se sustentar, enquanto ratos do grupo controle permanecer em suas gaiolas originais com comida e água suficientes.</li>
	<li>Tampa do tanque. Monitore os ratos para evitar ferimento acidental. Se um rato permanece na água por mais de 1h sem subir na plataforma, buscá-lo fora do tanque e removê-lo do teste.</li>
	<li>Após 14 h, tirar os ratos do modelo do tanque e seque os cabelos com um secador. Re-marca as caudas dos ratos se desvanece-se. Retornar os ratos para suas gaiolas originais e fornecer-lhes comida e água suficientes.</li>
	<li>Limpe todos os cantos do aquário. Elevar um lado do tanque e abra a torneira para a água de esgoto de saída.</li>
	<li>Esterilizar o tanque com um spray de etanol 75% e expô-lo à luz UV.</li>
</ol>3. modelo de avaliação: Teste comportamental
Nota: Todos os testes são realizados no laboratório comportamental. Ruído e luz extra não são permitidas durante o teste para evitar a perturbação. Se possível, use o mesmo pessoa (s) para realizar cada teste. Um casaco escuro e luvas são necessárias para o reconhecimento de escala de cinza no processamento de imagem. Executar o OFT primeiro como tem o menor efeito sobre o comportamento do rato.
<ol><li>
OFT
	<ol>
<li>Verifique se o gravador sobre a caixa de campo aberto para se certificar que está ligado à estação de trabalho e cobre cada canto da caixa. Ajuste a iluminação para eliminar sombras na caixa.</li>
		<li>Mova os ratos em laboratório comportamental em suas gaiolas originais. Permita-lhes para se aclimatar pelo menos 1 h antes do teste.</li>
		<li>Limpar e desinfetar a caixa com 75% de etanol para garantir que não há nenhum excremento ou cheiro do experimento anterior.</li>
		<li>Remover um rato da gaiola por suas costas e colocá-lo suavemente na área central da caixa. Rapidamente os braços retiro da caixa para não bloquear o tiro.</li>
		<li>Entrada de número de ratos e iniciar a gravação. Contagem e registro da frequência de atividades verticais, como o rato, incluindo a criação e a escalada.</li>
		<li>Depois de 5 min, parar a gravação, tira da caixa com o rato e devolvê-lo para a gaiola.</li>
		<li>Repita as etapas 3.1.3 - 3.1.6 até que todos os ratos terminaram o teste.</li>
	</ol>
</li>
	<li>
EPM
	<ol>
<li>Execute as etapas de pré-seleção e aclimatação quanto o OFT (etapas 3.1.1 - 3.1.2).</li>
		<li>Remover um rato da gaiola por suas costas e colocá-lo suavemente sobre a parte de junção dos dois braços. Aterrar o rato para o braço esquerdo aberto e sair rapidamente para não bloquear o tiro.</li>
		<li>Entrada de número de ratos e iniciar a gravação. Contagem e registo a frequência de entradas diferentes de braço. Se o rato cai fora do labirinto no teste, buscá-lo e enviá-lo para o labirinto. Gravar informações detalhadas para análise de dados.</li>
		<li>Depois de 5 min, parar a gravação, pegue o rato fora e devolvê-lo para a gaiola.</li>
		<li>Remover o excremento e limpe o labirinto com 75% de etanol para eliminar cheiro do antigo rato.</li>
		<li>Repita as etapas 3.2.2 - 3.2.5 até que todos os ratos terminaram o teste.</li>
	</ol>
</li>
	<li>
Teste de ES
	<ol>
<li>Encha o tanque de natação (70 × 30 × 110 cm3) com 80 cm de água morna (25 ± 3 ° C). Nota: Se houver um termostato no tanque, temperatura da água deve ser definida em 37 ° C, que é semelhante à temperatura do corpo do rato. Caso contrário, defina-o como a temperatura para mantê-la constante.</li>
		<li>Faça uma carga para cada rato com pino cachos e amarrá-lo suavemente na sua raiz da cauda. A carga pesa 10% do peso do rato.</li>
		<li>Pegar um rato pela cauda e jogá-lo no tanque de natação. Se os ratos huddle ou agarrar-se à parede, os distinguem e conduzi-los de volta na água.</li>
		<li>Iniciar a cronometragem no momento em que o rato é colocado em água e parar o tempo quando ele está esgotado, que é demonstrada como a incapacidade de lutar fora da água com a boca e o nariz por baixo da água por mais de 10 s. Nota: Às vezes, exaustão e afogamento ocorrem de repente. Certifique-se de ter bastante experimentadores para gravar e salvar o animal ao mesmo tempo.</li>
		<li>Remova os ratos exaustos fora da água sem interromper os outros. Seque os cabelos, re-marcar seus números e enviá-los de volta para a gaiola.</li>
		<li>Mude a água no tanque após conclusão de um grupo. Depois de fazem todos os ratos, esvaziar o tanque de natação e limpar e esterilizá-lo com etanol e luz UV.</li>
	</ol>
</li>
</ol>4. modelo de avaliação: Deteção de neurotransmissor Central
<ol><li>Anestesia o rato com injeção intra-peritoneal de hidrato de cloral 10% (3ml/kg) até que esteja inconsciente.</li>
	<li>Decapitar o rato.</li>
	<li>Faça uma incisão longitudinal ao longo da linha pós-medial, abrir o crânio para ambos os lados e expor o cérebro. Vire o crânio, remover o cérebro e colocar o cérebro em um saco de gelo.</li>
	<li>Separar e remover o hipotálamo, que é a área em forma de diamante na parte central da base do cérebro que tem uma fronteira clara com tecidos circundantes. Coloque-o em um tubo estéril e congelá-lo com nitrogênio líquido. Armazene todas as amostras em um frigorífico de-80 ° C.</li>
	<li>Detecta o conteúdo da DA e 5-HT no hipotálamo usando cromatografia líquida de alto desempenho (HPLC)8.</li>
</ol>

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Os autores não têm nada para divulgar.

O MMPM é projetado originalmente por privação de sono9. Ratos são lançados em um tanque de água com plataformas fixadas na parte inferior. Impulsionado pelo medo instintivo de água, ratos permanecem em pé nas plataformas e ocorre sem dormir. O estudo mostra que diferentes horas de privação de sono levam a várias alterações no comportamento do rato e humor, incluindo reconhecimento de imparidade10en11e fadiga central. Alguns pesquisadores...

A fadiga é um dos principais fatores que ameaçam a saúde humana1. Nas últimas décadas, várias pesquisas provaram que fadiga é acionada por perifericamente mas centralmente orientada e sempre acompanhado com distúrbios emocionais e cognitivos. Fisiologista italiano Mosso r. proposto pela primeira vez a palavra fadiga Central2. Geralmente é definida como a actividade voluntária limitada e comprometimento da cognição devido à disfunção da transmissão do impulso no sistema nervoso central (SNC)3. Comparado com fadiga muscular periférica, fadiga central enfatiza alterações no SNC, bem como as consequentes perturbações emocionais/comportamentais, incluindo depressão, ansiedade, deficiência de cognição e perda de memória. Um estudo mostra que muitos fatores podem induzir a fadiga central, entre os quais excessiva atividade física e estresse mental, são imprescindíveis4. Quanto a patogênese, teorias como o triptofano-Quinurenina via hipótese5 explicar alterações em determinadas vias; no entanto, estudos mais aprofundados ainda são necessários para revelar as correlações central-periférico da fadiga central.
Como o mecanismo subjacente de fadiga central ainda não está claro, um modelo animal eficaz é muito importante para futuras pesquisas. Os modelos existentes de fadiga são principalmente induzidos por exercício excessivo, como escadas rolantes6 e natação peso carregado7, com pouca preocupação sobre fatores mentais. Para melhor simular o desenvolvimento da fadiga central, nosso grupo desenvolveu um modelo do rato com o MMPM. Durante o processo de modelagem, ratos permanecem em pé nas plataformas estreitas na caixa múltipla plataforma por longas horas, incluindo a parte do tempo de sono. Diferente dos modelos de exercício excessivo, o modelo MMPM usa a privação do sono parcial como um fator mental tendo em consideração a complexa patogênese da fadiga central.
Para avaliação do modelo, usamos os OFT e EPM testes para determinar o clima de ansiedade e voluntariado. O teste de ES é realizado para medir o desempenho muscular periférica. Além disso, tomamos o cérebro do rato e detectar a dopamina (DA) / conteúdo de serotonina (5-HT) em ambos os hypothalamuses para observar as diferenças de neurotransmissor central.
O protocolo apresentado abaixo destina-se à fadiga central de modelo induzida pela atividade física repetida e falta de sono, imitando uma condição comum na vida humana. No entanto, ao ajustar a duração do modelo, pode ser usada em muitos outros campos, como em estudos de observação e estresse do sono. No futuro, pesquisa, esperamos que este modelo vai ajudar a descobrir mais mudanças do CNS e sua conexão com o sistema periférico, para revelar o mecanismo de patogênese da fadiga central.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>multiple platform sleep deprivation water tank</td>
			<td>Customization,it is provided by the neuroimmunological laboratory of Beijing University of Chinese Medicine</td>
			<td></td>
			<td>110cm x 60cm x 40cm. There are 15 plastic small platforms at the bottom. The small platform is 6.5cm in diameter and 8cm high</td>
		</tr>
		<tr>
			<td>Wistar rats</td>
			<td>Beijing Weitong Lihua Experimental Animal Technology Company</td>
			<td>license number SYXK (Beijing) 2016-0011</td>
			<td>Use 32 Wistar healthy male rats ,8 week old (200-210 g)</td>
		</tr>
		<tr>
			<td>Agilent 1100LC high performance liquid chromatograph&#160;</td>
			<td>Agilent</td>
			<td>&#160;G1379A, G1311A, G1313A , G1316A&#160;</td>
			<td>&#160;G1379A, G1311A type chromatographic pump, G1313A automatic sampler, G1316A column temperature box</td>
		</tr>
		<tr>
			<td>DECADE II SDC electrochemical detector</td>
			<td>Dutch ANTEC company</td>
			<td></td>
			<td>glassy carbon electrode, Ag/AgCl reference electrode, workstations (Clarity CHS)</td>
		</tr>
		<tr>
			<td>Biofuge Stratos high-speed refrigeration centrifuge</td>
			<td>HERAEUS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>VCX130 ultrasonic fracturing instrument</td>
			<td>SONICS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ACS-ZEAS electronic scale</td>
			<td>Phos technology development, Beijing.</td>
			<td></td>
			<td>The weight of the weighing rats can be accurate to 0.1g.</td>
		</tr>
		<tr>
			<td>Open Field Box</td>
			<td>Customization,it is provided by the neuroimmunological laboratory of Beijing University of Chinese Medicine</td>
			<td></td>
			<td>wooden box of open field&#160; 100 cm by 100 cm x 40 cm, inside wall and bottom as the gray.The bottom is divided into 25 equal area squares, each of which is 20cm x 20cm, and the 16 grids along the outer wall are the external ones, and the other 9 grids are central.The camera is mounted above the median.</td>
		</tr>
		<tr>
			<td>Elevated Plus-maze</td>
			<td>Beijing zhongshi dechuang technology development co. LTD.</td>
			<td></td>
			<td>The open arms and close&#160; arms of the cross are composed of 30cm x 5cm x 15cm, and the central area is 5cm x 5cm, with a camera mounted above the center and 45cm high.</td>
		</tr>
		<tr>
			<td>rat swimming bucket.</td>
			<td>Zhenhua biological instrument equipment co., LTD. Anhui,China.</td>
			<td></td>
			<td>The volume of plastic drum is 70cm x 30cm x 110cm, which is used for swimming in rats.</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td>Shiya instrument co., LTD., changzhou,China.</td>
			<td></td>
			<td>Control water temperature</td>
		</tr>
		<tr>
			<td>Small water pump</td>
			<td>Xincheng technology co., LTD., chengdu,China.</td>
			<td></td>
			<td>Used for water tank and swimming behavior.</td>
		</tr>
		<tr>
			<td>Ethovition3.0 behavioral software.</td>
			<td>Nuldus,Netherlands</td>
			<td></td>
			<td>Measurement analysis of rat behavior videos.</td>
		</tr>
	</tbody>
</table>

a rat model of central fatigue using a modified multiple platform method

Neste artigo, apresentamos um modelo do rato de fadiga central usando o modificado método de plataforma múltipla (MMPM). A caixa de plataforma múltipla foi concebida como um tanque de água com plataformas estreitas na parte inferior. Os ratos do modelo foram colocados no tanque e ficou nas plataformas para 14 h (18:00-08:00) por dia por 21 dias consecutivos, com um grupo de controle em branco para contraste. No final da modelagem, ratos no grupo modelo mostraram uma aparência cansada óbvia. Para avaliar o modelo, realizamos vários testes comportamentais, incluindo o campo aberto (OFT), teste da elevada além de labirinto (EPM) e o teste de natação exaustiva (ES). Os resultados mostraram que essa ansiedade, comprometimento da cognição espacial, desempenho pobre do músculo e recusada atividade voluntária apresentado em ratos modelo confirmam o diagnóstico da fadiga central. Alterações de neurotransmissores a central também verificado o resultado. Em conclusão, o modelo simulado com êxito a fadiga central, e estudos futuros com o modelo podem ajudar a revelar o mecanismo patológico da doença.

Descrevemos um modelo do rato de fadiga central usando MMPM. 24 ratos Wistar são aleatoriamente divididos em grupo controle e o grupo modelo, com 12 ratos em cada grupo. O aparelho modelo é projetado como um tanque de água com plataformas estreitas na parte inferior (Figura 1). Ratos do modelo fique nas plataformas para 14 h por dia, incluindo o tempo de sono parcial, durante 21 dias (Figura 2).
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Watch this Scientific Journal Video about A Rat Model of Central Fatigue Using a Modified Multiple Platform Method at JoVE.com

A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

Aqui, apresentamos um protocolo para apresentar um modelo do rato de fadiga central usando o modificado método de plataforma múltipla (MMPM).

Um rato do modelo de fadiga Central usando um modificado método de plataforma múltipla

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed ...

a-rat-model-central-fatigue-using-modified-multiple-platform

Research

JoVE Journal

Neuroscience

8.9K visualizações.  Beijing University of Chinese Medicine. Aqui, apresentamos um protocolo para apresentar um modelo do rato de fadiga central usando o modificado método de plataforma múltipla (MMPM).

Vídeo: A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.

In this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.

Aplicação de uma condutância do receptor NMDA em neurônios dopaminérgicos Rat Midbrain Usando a Técnica Clamp dinâmico

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

Neurociência

Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various heterologous genes could thus be investigated easily while maintaining many aspects of in vivo biology. There has been increasing interest to transfect terminally differentiated neurons for which conventional transfection methods have been fraught with difficulties such as low yields and significant losses in viability. Biolistic transfection can circumvent many of these difficulties yet only recently has this technique been modified so that it is amenable for use in mammalian tissues.
New modifications to the accelerator chamber have enhanced the gene gun&#39;s firing accuracy and increased its depths of penetration while also allowing the use of lower gas pressure (50 psi) without loss of transfection efficiency as well as permitting a focused regioselective spread of the particles to within 3 mm. In addition, this technique is straight forward and faster to perform than tedious microinjections. Both transient and stable expression are possible with nanoparticle bombardment where episomal expression can be detected within 24 hr and the cell survival was shown to be better than, or at least equal to, conventional methods. This technique has however one crucial advantage: it permits the transfection to be localized within a single restrained radius thus enabling the user to anatomically isolate the heterologous gene&#39;s effects. Here we present an in-depth protocol to prepare viable adult organotypic slices and submit them to regioselective transfection using an improved gene gun.

Recent improvements in organotypic brain slice preparations have permitted their exploitation for biotechnological applications. Organotypic slices maintain local structural characteristics of in vivo biology, including functional synaptic connections. Here we present a regioselective biolistic delivery method to label and genetically manipulate these slices.

Regiosseletiva Biolistic Segmentação em Organotípicas fatias do cérebro do Usando um Modificado Gene Gun

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various ...

A Method of Nodose Ganglia Injection in Sprague-Dawley Rat

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located in the organs of the abdomen and thorax. The vagus nerve communicates information from stimuli such as heart rate, blood pressure, bronchopulmonary irritation, and gastrointestinal distension to the nucleus of solitary tract of the medulla. The cell bodies of the vagus nerve are located in the nodose and petrosal ganglia, of which the majority are located in the former. The nodose ganglia contain a wealth of receptors for amino acids, monoamines, neuropeptides, and other neurochemicals that can modify afferent vagus nerve activity. Modifying vagal afferents through systemic peripheral drug treatments targeted at the receptors on nodose ganglia has the potential of treating diseases such as sleep apnea, gastroesophageal reflux disease, or chronic cough. The protocol here describes a method of injection neurochemicals directly into the nodose ganglion. Injecting neurochemicals directly into the nodose ganglia allows study of effects solely on cell bodies that modulate afferent nerve activity, and prevents the complication of involving the central nervous system as seen in systemic neurochemical treatment. Using readily available and inexpensive equipment, intranodose ganglia injections are easily done in anesthetized Sprague-Dawley rats.

Afferent vagal signaling transmits important information to central nervous system from receptors located in organs of the abdomen and thorax. The nodose ganglia of vagus nerves contain many types of receptors that modulate vagal activity. This protocol describes a method of local injections of neurochemicals into the nodose ganglia.

Um método de Nodoso Gânglios Injecção em ratos Sprague-Dawley

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located ...

Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic applications. This protocol describes a fabrication technique of a modified cylindrical nerve cuff electrode to meet these criteria. Minimum computer-aided design and manufacturing (CAD and CAM) skills are necessary to consistently produce cuffs with high precision (contact placement 0.51 &#177; 0.04 mm) and various cuff sizes. The precision in spatially distributing the contacts and the ability to retain a predefined geometry accomplished with this design are two criteria essential to optimize the cuff&#39;s interface for selective recording and stimulation. The presented design also maximizes the flexibility in the longitudinal direction while maintaining sufficient rigidity in the transverse direction to reshape the nerve by using materials with different elasticities. The expansion of the cuff&#39;s cross sectional area as a result of increasing the pressure inside the cuff was observed to be 25% at 67 mm Hg. This test demonstrates the flexibility of the cuff and its response to nerve swelling post-implant. The stability of the contacts&#39; interface and recording quality were also examined with contacts&#39; impedance and signal-to-noise ratio metrics from a chronically implanted cuff (7.5 months), and observed to be 2.55 &#177; 0.25 k&#8486; and 5.10 &#177; 0.81 dB respectively.

This article provides a detailed description on the fabrication process of a high contact-density flat interface nerve electrode (FINE). This electrode is optimized for recording and stimulating neural activity selectively within peripheral nerves.

Fabricação de alta Contact densidade, eletrodos nervosas Plano de Interface para gravação e estimulação Applications

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic ...

Dynamic Quantitative Sensory Testing to Characterize Central Pain Processing

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain processing is present in many chronic pain conditions and can influence responses to specific therapies. Thus, the ability to precisely describe the state of central pain processing has profound clinical significance in both prognosis and prediction. Because it is not practical to record neuronal firings directly in the human spinal cord, surrogate behavior tests become an important tool to assess the state of central pain processing. Dynamic QST is one such test, and can probe both the ascending facilitation and descending modulation of incoming nociceptive signals via TS and CPM, respectively. Due to the large between-individual variability in the sensitivity to noxious signals, standardized TS and CPM tests may not yield any meaningful data in up to 50% of the population due to floor or ceiling effects. We present methodologies to individualize TS and CPM so we can capture these measures in a broader range of individuals than previously possible. We have used these methods successfully in several studies at the lab, and data from one ongoing study will be presented to demonstrate feasibility and potential applications of the methods.

By assessing pain in response to repetitive or different types of standardized stimuli, dynamic quantitative sensory testing (QST) can reveal changes in the central processing of pain. We present methods to optimize and individualize two dynamic QST measures: temporal summation (TS) and conditioned pain modulation (CPM).

Dinâmica Quantitative Sensory Testing Caracterizar Central Dor Processing

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain ...

Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating disease, with clinical features similar to the progressive forms of multiple sclerosis (MS). The mice show progressive disability with loss of motor and sensory functions, which can be assessed with multiple apparatuses and protocols. Among them, the Rotarod performance test is a very common behavioral test, its advantage being that it provides objective measurements, but it is often used assuming that it is straightforward and simple. In contrast to visual scoring systems used in some models of MS, which are highly subjective, the Rotarod test generates an objective, measurable, continuous variable (i.e., length of time), allowing almost perfect inter-rater concordances. However, inter-laboratory reliability is only achieved if the various testing parameters are replicated. In this manuscript, recommendations of specific testing parameters, such as size, speed, and acceleration of the rod; amount of training given to the animals; and data processing, are presented for the Rotarod test.

An optimized testing protocol is presented in this paper for the Rotarod performance test, used for measuring progressive neurological disability in TMEV-infected mice.

Medindo Progressive Neurological Disability em um modelo de rato com esclerose múltipla

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating ...

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor determining patient outcome and is therefore frequently taken as a study endpoint. However, in small animal studies on SAH, quantification of cerebral vasospasm is a major challenge. Here, an ex vivo method is presented that allows quantification of volumes of entire vessel segments, which can be used as an objective measure to quantify cerebral vasospasm. In a first step, endovascular casting of the cerebral vasculature is performed using a radiopaque casting agent. Then, cross-sectional imaging data are acquired by micro computed tomography. The final step involves 3-dimensional reconstruction of the virtual vascular tree, followed by an algorithm to calculate center lines and volumes of the selected vessel segments. The method resulted in a highly accurate virtual reconstruction of the cerebrovascular tree shown by a diameter-based comparison of anatomical samples with their virtual reconstructions. Compared with vessel diameters alone, the vessel volumes highlight the differences between vasospastic and non-vasospastic vessels shown in a series of SAH and sham-operated mice.

The goal of this article is to present a method that allows a 3-dimensional reconstruction of the cerebrovascular tree in mice after micro computed tomography and determination of volumes of entire vessel segments that can be used to quantify cerebral vasospasm in murine models of subarachnoid hemorrhage.

Um método volumétrico para quantificação de vasoespasmo Cerebral em um modelo murino de hemorragia subaracnoide

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

Injeção direta de um vetor de Lentivirus destaca várias vias motoras na medula espinhal de ratos

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

Dissecção e isolamento da glia murina de múltiplas regiões do sistema nervoso central

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

Um método de limpeza de tecido hidrofóbico para tecido cerebral de rato

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...