Part of this work was supported by a Grant-in-Aid for Scientific Research (C) from the Japanese Society for the Promotion of Science (25350520, 22500401, 15K06105) and the US-ARMY ITC-PAC Research and Development Project (FA5209-15-P-0175).

cuidados com os animais, preparação e protocolos experimentais foram aprovados pelo Comitê de Pesquisa Animal da Universidade de Tóquio de Agricultura e Tecnologia. Por esta metodologia, o rato é alojado num ambiente controlado (24 ° C, 12 h ciclo de luz / escuro), com comida e água disponível ad libitum. 1. Construção de um sistema convencional Multispectral reflectância difusa imagem <ol><li> Montagem nove filtros de interferência de banda estreita ópticos com comprimentos de onda centrais de 500, 520, 540, 560, 570, 580, 600, 730, e 760 nm para os orifícios de filtro do filtro roda motorizada. </li><li> Construir um sistema de imagem multi-espectral usando uma fonte de luz branca de banda larga, uma roda de filtro motorizado com o conjunto de cima de filtros de interferência de banda estreita, uma guia de luz, uma lente de recolha, uma lente zoom de vídeo, e uma câmara CCD monocromática. A disposição de elementos ópticos, mostrado na Figura 2, pode ser referido para thé um procedimento de construção. NOTA: O ângulo de iluminação é de aproximadamente 45 ° em relação à superfície da amostra. </li><li> Ligar a fonte de luz de lâmpada de halogéneo para iluminar a superfície da amostra através de um filtro de interferência, o guia de luz, e a lente de recolha. </li><li> Abra o software de operação da câmera CCD. </li></ol> 2. Preparação de animais NOTA: Neste protocolo, o rato não foi utilizado para as futuras experiências e foi sacrificado imediatamente após as medidas de imagens multiespectrais. <ol><li> Ligar o orifício de entrada de uma câmara de indução para o orifício de saída de uma máquina de anestesia com um tubo. Conectar o orifício de saída da câmara de indução para o orifício de entrada da máquina de anestesia com um segundo tubo. </li><li> Colocar o rato para a câmara de indução e induzir a anestesia com 5,0% de isoflurano. Manter a anestesia, a uma profundidade tal que o rato não responde a pitadas dedo do pé. euower a 2,0% de isoflurano utilizando um botão rotativo na máquina de anestesia. </li><li> Fixar a cabeça do rato em um quadro estereotáxico. Anexar um bocal para anestesia à armação estereotáxica. </li><li> Ligar o orifício de entrada do bocal para a porta de saída do aparelho de anestesia com um tubo. Conectar o orifício de saída do bocal até o porto de entrada da máquina de anestesia com um tubo. </li><li> Raspar a região da cabeça além do local da incisão prospectivo usando cortadores de cabelo até a superfície da pele aparece. </li><li> Fazer uma incisão longitudinal de aproximadamente 20 mm long ao longo da linha média da cabeça, usando um bisturi cirúrgico (Figura 1 (a)) e expor os tecidos conjuntivos subcuteos (Figura 1 (b)). </li><li> Remover os tecidos conjuntivos subcuteos utilizando uma cureta afiada ou uma pinça e puxá-lo para ambos os lados da cabeça para expor o crânio de osso (Figura 1 (c)). </li><li> Escavar uma vala elipsoidal sobre o osso do crânio no interior do sutur cranianaES (sutura coronal, sagital de sutura e sutura lambdóide) utilizando uma broca de alta velocidade (Figura 1 (d)). </li><li> Lenta e homogeneamente escavar o osso do crânio dentro da vala usando a broca de alta velocidade. </li><li> Pressione levemente na superfície do crânio afinada com a ponta da pinça para estimar a espessura e força óssea depois de um vaso sanguíneo cerebral aparece. Se a região adelgaçada crânio deprime facilmente, terminar a redução do osso do crânio com a broca de alta velocidade. </li><li> Cortar a linha de fronteira elipsoidal do crânio diluído fragmentada utilizando a ponta da pinça ou pequenas tesouras cirúrgicas. </li><li> Remover o crânio diluído a partir da superfície do cérebro lenta e suavemente usando a pinça. </li><li> Suavemente banhar a janela craniana com solução salina fisiológica e cobri-lo com uma placa de vidro transparente, aproximadamente, 0,1 mm de espessura. </li></ol> 3. Regulamentação da fração inspirada de oxigênio NOTA: O condi respiratóriação pode ser alterada através do controlo da fracção de oxigénio inspirada (FiO 2). <ol><li> Usando um tubo, ligar o primeiro porto de um conector de tubo em forma de Y (conector 1) para a primeira porta de outro conector de tubo em forma de Y (conector 2). </li><li> Ligar o orifício de entrada do bocal para a segunda porta de conector do tubo 1. </li><li> Usando um tubo, ligar a terceira porta de conector do tubo 1 para um dispositivo de oxigénio monitor de concentração. </li><li> Com um tubo, ligar a segunda porta de ligação de tubo 2 para a porta de saída de um aparelho de anestesia. </li><li> Usando um tubo, ligar a terceira porta de ligação de tubo 2 para a porta de saída de um dispositivo de mistura de gás. </li><li> Conectar um orifício de entrada do dispositivo de mistura de gás a uma alta pressão de 95% de O2 - 5% de CO 2 do cilindro de gás, utilizando um tubo. </li><li> Ligue a outra porta de entrada do dispositivo de mistura de gás a uma alta pressão de 95% de N 2 - 5% cilindro de gás de CO 2 usando um tubo. </li><li> Alterar as taxas de fluxo de gás Sf O 2 e N 2, utilizando os botões rotativos no dispositivo de mistura de gás. </li><li> Verificar e regular a FiO 2, utilizando o dispositivo de oxigénio monitor de concentração. </li></ol> 4. Aquisição das Multispectral de reflectância difusa Imagens <ol><li> Aquisição de imagens de referência NOTA: Os componentes ópticos utilizados nesta experiência, tais como a fonte luminosa, de fibra óptica, e os detectores têm as suas próprias características espectrais. Portanto, a intensidade da luz que passou através destes componentes deve ser gravado como uma imagem de referência. A imagem de referência é uma imagem tirada com um difusor branco padrão iluminado com a luz da fonte de luz. <ol><li> Coloque o difusor branco padrão no palco horizontalmente. </li><li> O foco da lente da câmara sobre a superfície do difusor branco, rodando o anel de zoom no barril. </li><li> Ajustar o tempo de integração da câmera selecionando o valor apropriado nadrop-down lista de tempos de integração no software de operação da câmara para que a maior quantidade de luz produz um sinal que é aproximadamente 75% das contagens máximas. Enquanto observa o histograma de valores de pixel, ajustar o tempo de integração até que o nível de intensidade de sinal é de aproximadamente 75% das contagens máximas. </li><li> Selecione o comando &quot;salvar&quot; no menu arquivo para salvar uma imagem em um arquivo. </li><li> Alterar a localização do filtro, rodando a roda de filtro. </li><li> Guardar uma imagem aos outros comprimentos de onda de acordo com o processo descrito acima. O nome do arquivo deve identificar a amostra e o comprimento de onda utilizado (por exemplo, W500, W520, W540 ... W760). </li></ol></li><li> Aquisição de imagens de exemplo Nota: Imagens da intensidade da luz difusa refletida de cérebro de rato expostos em nove comprimentos de onda são capturados e salvos no disco rígido de um computador pessoal utilizando as mesmas condições de aquisição. <ol><li> Delicadamente, coloque o rna no palco e, lentamente, ajustar o nível do palco para que a câmara pode focar na superfície do cérebro de rato. </li><li> Selecione o comando &quot;salvar&quot; no menu arquivo para salvar uma imagem em um arquivo. </li><li> Alterar a localização do filtro, rodando a roda de filtro. </li><li> Guardar uma imagem aos outros comprimentos de onda de acordo com o processo descrito acima. O nome do arquivo deve identificar a amostra e o comprimento de onda (por exemplo, R500, R520, R540 ... R760). </li></ol></li><li> Aquisição de imagens escuras NOTA: A câmara CCD pode gerar uma intensidade de luz em resposta a um sinal eléctrico. No entanto, existe alguma saída menor devido ao ruído nos circuitos elétricos e detectores, mesmo se a luz não entra no detector; isso é chamado de ruído atual escuro. Para medir com precisão a intensidade espectral da luz, a componente de corrente escura deve ser gravado como uma imagem escura e então subtraído a partir do sinal medido. A imagem escura é uma tomada de imagemn com o percurso da luz bloqueada. <ol><li> Desligue a fonte de luz lâmpada de halogéneo. </li><li> Bloquear o caminho da luz ao sistema de câmera CCD usando uma placa de blindagem. </li><li> Selecione o comando &quot;salvar&quot; no menu arquivo para salvar uma imagem em um arquivo. O nome do arquivo deve identificar a amostra (por exemplo, Escuro). </li></ol></li></ol> 5. Visualizando o conteúdo de hemoglobina eo espalhamento de luz Parâmetro NOTA: Um conjunto de imagens por reflexão multiespectrais é salva no disco rígido de um computador pessoal e analisados ​​offline. Uma análise de regressão múltipla auxiliado por uma simulação de Monte Carlo 19 das imagens de reflectância difusa multi-espectrais em nove comprimentos de onda (500, 520, 540, 560, 570, 580, 600, 730, e 760 nm) é então realizada para visualizar o bidimensional mapas de concentração de hemoglobina oxigenada, a concentração de hemoglobina desoxigenada, a concentração total de hemoglobina, saturação de oxigénio cerebral regional, e fezestering poder. O algoritmo detalhada foi publicada nas literaturas 17, 18. <ol><li> Subtrair a imagem escura de ambas a imagem de referência e a imagem da amostra em cada comprimento de onda. </li><li> Normalizar a imagem da amostra pela imagem de referência em cada comprimento de onda λ. Tratar a imagem normalizada, tal como a imagem de reflectância difusa R. </li><li> Calcular a absorvância (ou densidade óptica) uma imagem, tendo o logaritmo do recíproco da imagem reflectância difusa R em cada comprimento de onda λ: <img alt="equação 1" src="/files/ftp_upload/55399/55399eq1.jpg" /> (1) </li><li> Gerar uma matriz tridimensional por empilhamento das imagens de absorvância na ordem dos seus comprimentos de onda, onde o x - plano y mostra a informação estrutural obtida para a superfície do cérebro e o z -axis mostra a informação espectral. </li><li> PERuma análise de regressão múltipla para o espectro de absorvância A ( λ ) em cada coordenada xy. </li><li> Utilize o espectro de absorvância A ( λ ) como variável dependente e os espectros de coeficiente de extinção molar da hemoglobina oxigenada ε HbO ( λ ) e da hemoglobina desoxigenada ε HbR ( λ ) como variáveis ​​independentes para o passo 5.5 (valores publicados para ε HbO ( λ ) E ε HbR ( λ ) são fornecidos na Tabela 1 ). </li><li> Verifique os mapas bidimensionais (imagens) dos três coeficientes de regressão múltipla a HbO , a HbR e a 0 . </li><li> Gere uma matriz tridimensional empilhando as imagens dos coeficientes de regressão múltipla na ordem de um HbO , um HbR , e um 0 , onde o <em&gt; x - y plano mostra a informação estrutural obtida para a superfície do cérebro e o z -axis mostra os vários coeficientes de regressão. </li><li> Calcule a concentração de hemoglobina oxigenada C HbO, a hemoglobina desoxigenada concentração C HBr, e o poder de dispersão b a partir do conjunto de regressão múltipla coeficientes a HbO, um HBr, e um 0 em cada coordenada xy usando as seguintes fórmulas empíricas (ele valores de β HbO, i, β HBr, i, e β 0, i (i = 0,1,2,3) são fornecidos na Tabela 2): <img alt="equação 2" src="/files/ftp_upload/55399/55399eq2.jpg" /> (2) <img alt="equação 3" src="/files/ftp_upload/55399/55399eq3.jpg" /> (3) <img alt="equação 4" src="/files/ftp_upload/55399/55399eq4.jpg" /> (4) </li><li> Verificar os mapas bidimensionais (imagens) da concentração de hemoglobina oxigenada C HbO, a concentração de hemoglobina desoxigenada C HBr, e o poder de dispersão b. </li><li> Calcular um mapa bidimensional do C HBT concentração total de hemoglobina pela soma C e HbO C HBr em cada x - y de coordenadas. </li><li> Calcular um mapa bidimensional da saturação de oxigénio cerebral regional RSO 2 dividindo a concentração de hemoglobina oxigenada C HbO pelo C HBT concentração total de hemoglobina em cada x - y de coordenadas. </li></ol>

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The authors have nothing to disclose.

O passo mais importante neste protocolo é a remoção da região do crânio diluído para fazer a janela craniana; esta deve ser realizada com cuidado para evitar hemorragias inesperadas. Este passo é importante para a obtenção de alta qualidade multiespectrais difundir imagens de reflectância com alta precisão. O uso de um microscópio estereoscópico é recomendado para o procedimento cirúrgico, se possível. Pequenos pedaços de esponja de gelatina são úteis para hemostasia. O si...

A técnica de reflexão difusa multicêntrica é a técnica mais comum para obtenção de um mapa espacial de sinais ópticos intrínsecos (IOSs) no tecido cortical. Os IOS observados no cérebro in vivo são atribuídos principalmente a três fenômenos: variações na absorção de luz e propriedades de dispersão devido à hemodinâmica cortical, variação na absorção dependendo da redução ou oxidação de citocromos nas mitocôndrias e variações nas propriedades de dispersão da luz induzidas por alterações morfológicas 1 . A luz na gama espectral visível (VIS) para infravermelho próximo (NIR) é efetivamente absorvida e dispersa pelo tecido biológico. O espectro de reflectância difusa do cérebro in vivo é caracterizado por espectros de absorção e dispersão. Os coeficientes de dispersão reduzidos μ s &#39; do tecido cerebral na faixa de comprimento de onda VIS-para-NIR resultam numa exposição de espectro de dispersão monótonaMagnitudes menores em comprimentos de onda mais longos. O espectro do coeficiente de dispersão reduzido μ s &#39;(λ) pode ser aproximado para estar na forma da função de lei de potência 2 , 3 como μ s &#39; (λ) = a × λ -b . O poder de dispersão b está relacionado com o tamanho dos dispersores biológicos no tecido vivo 2 , 3 . Alterações morfológicas do tecido e redução da viabilidade do tecido cortical vivo podem afetar o tamanho dos dispersores biológicos 4 , 5 , 6 , 7 , 8 , 9 . Um sistema ótico para a imagem de reflexão difusa multiespectral pode ser facilmente construído a partir de uma lâmpada incandescenteFonte óptica, componentes ópticos simples e um dispositivo monocromático carregado acoplado (CCD). Portanto, vários algoritmos e sistemas óticos para a imagem de reflexão difusa multiespectral foram utilizados para avaliar a hemodinâmica cortical e / ou a morfologia do tecido 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 . O método descrito neste artigo é utilizado para visualizar tanto a hemodinâmica como as propriedades de dispersão da luz do tecido cerebral de rato in vivo utilizando um sistema de imagem de reflexão difusa multiespectral convencional. As vantagens deste método em relação às técnicas alternativas são a capacidade de avaliar alterações espaciotemporais tanto na hemodinâmica cerebral como no tecido corticalmorfologia, assim como a sua aplicabilidade para vários modelos animais para a disfunção cerebral. Portanto, o método será adequado para investigações de lesão traumática cerebral, ataque epiléptico, acidente vascular cerebral e isquemia.

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			<td>Andover Corporation, NH, USA</td>
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simultaneous evaluation of cerebral hemodynamics and light scattering properties of the in vivo rat brain using multispectral diffuse reflectance imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of hemodynamics and the light scattering properties of the in vivo rat brain.

Imagens espectrais representativas de reflectância difusa adquirido a partir de cérebros de rato in vivo são apresentados na Figura 3. As imagens em 500, 520, 540, 560, 570, e 580 nm visualizar claramente uma rede densa de vasos sanguíneos no córtex cerebral. A deterioração do contraste entre os vasos sanguíneos e o tecido circundante observadas nas imagens em 600, 730, e 760 nm reflecte a diminuição da absorção de luz pela hemoglobina no mais longo ...

Watch this Scientific Journal Video about Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging at J

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

A avaliação simultânea da hemodinâmica cerebral e das propriedades de dispersão da luz do tecido in vivo do cérebro de rato é demonstrada utilizando um sistema de imagem de reflexão difusa multiespectral convencional.

Avaliação simultânea de Cerebral Hemodinâmica e de espalhamento de luz Propriedades do<em&gt; In Vivo</em&gt; Cérebro de Rato Utilizando Imagem de Reflexão Difusa Multispectral

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...

simultaneous-evaluation-cerebral-hemodynamics-light-scattering

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Neuroscience

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

7.6K visualizações.  Tokyo University of Agriculture & Technology.  A avaliação simultânea da hemodinâmica cerebral e das propriedades de dispersão da luz do tecido in vivo do cérebro de rato é demonstrada utilizando um sistema de imagem de reflexão difusa multiespectral convencional. 

Vídeo: Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

FMRI simultânea e Eletrofisiologia no cérebro de roedores

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

Neurociência

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Neuroimagem Funcional Usando rompimento da barreira hemato-encefálica ultra-sônico e Manganês-RM

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

Imagem Funcional do Cortex Auditivo em gatos adultos usando de alta campo fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

Estimulação Cerebral Profunda com fMRI simultâneo em Roedores

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Simultâneo<em&gt; Ex vivo</em&gt; Teste Funcional de Dois retinas por<em&gt; In vivo</em&gt; Sistema Eletrorretinograma

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

Simultânea de dois fótons<em&gt; In Vivo</em&gt; Imagem de Synaptic Entradas e metas pós-sinápticos no córtex do rato retrosplï¿½ico

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types that comprise the BBB; these cells have a very high-energy demand, which requires optimal mitochondrial function. In the case of disease or injury, the mitochondrial function in these cells can be altered, resulting in disease or&#160;the opening of the BBB. In this manuscript, we introduce a method to measure mitochondrial function in CVE cells by using whole, intact cells and a bioanalyzer. A mito-stress assay is used to challenge the cells that have been perturbed, either physically or chemically, and evaluate their bioenergetic function. Additionally, this method also provides a useful way to screen new therapeutics that have direct effects on mitochondrial function. We have optimized the cell density necessary to yield oxygen consumption rates that allow for the calculation of a variety of mitochondrial parameters, including ATP production, maximal respiration, and spare capacity. We also show the sensitivity of the assay by demonstrating that the introduction of the&#160;microRNA, miR-34a, leads to a pronounced and detectable decrease in mitochondrial activity. While the data shown in this paper is optimized for the bEnd.3 cell line, we have also optimized the protocol for primary CVE cells, further suggesting the utility in preclinical and clinical models.

Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells.

Avaliação da função bioenergética em células endoteliais Vascular Cerebral

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

Viver a imagem das células do córtex Cerebral primário usando um sistema de cultura 2D

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Diffuse Reflectance Spectroscopy: Getting the Capillary Refill Test Under One's Thumb

The capillary refill test was introduced in 1947 to help estimate circulatory status in critically ill patients. Guidelines commonly state that refill should occur within 2 s after releasing 5 s of firm pressure (e.g., by the physician&#39;s finger) in the normal healthy supine patient. A slower refill time indicates poor skin perfusion, which can be caused by conditions including sepsis, blood loss, hypoperfusion, and hypothermia. Since its introduction, the clinical usefulness of the test has been debated. Advocates point out its feasibility and simplicity and claim that it can indicate changes in vascular status earlier than changes in vital signs such as heart rate. Critics, on the other hand, stress that the lack of standardization in how the test is performed and the highly subjective nature of the naked eye assessment, as well as the test&#39;s susceptibility to ambient factors, markedly lowers the clinical value. The aim of the present work is to describe in detail the course of the refill event and to suggest potentially more objective and exact endpoint values for the capillary refill test using diffuse polarization spectroscopy.

This protocol describes how the use of diffuse polarization spectroscopy can improve the clinical usefulness of the capillary refill test. We suggest a more detailed analysis of the course of the capillary refill in healthy volunteers using diffuse reflectance spectroscopy videos and new informatic endpoints.

Espectroscopia de reflectância difusa: Ficando o teste capilar sob o polegar

The capillary refill test was introduced in 1947 to help estimate circulatory status in critically ill patients. Guidelines commonly state that refill ...

Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large percentage of TBI patients requiring hospitalization. DAI involves widespread axonal damage from shaking, rotation or blast injury, leading to rapid axonal stretch injury and secondary axonal changes that are associated with a long-lasting impact on functional recovery. Historically, experimental models of DAI without focal injury have been difficult to design. Here we validate a simple, reproducible and reliable rodent model of DAI that causes widespread white matter damage without skull fractures or contusions.

This protocol validates a reliable, easy-to-perform and reproducible rodent model of brain diffuse axonal injury (DAI) that induces widespread white matter damage without skull fractures or contusions.

Indução de lesão cerebral axonal difusa em ratos com base na aceleração rotacional

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large ...

Avaliação simultânea de Cerebral Hemodinâmica e de espalhamento de luz Propriedades do

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