Os autores gostaria de agradecer a Yuliya Naumchuk (Loyola University Chicago) e Žiniauskaitė Agne (Experimentica Ltd.), pelo excelente apoio técnico e videográfico. Programa de pesquisa do Dr. Kaja é suportado pelo Dr. John P. e Therese E. Mulcahy dotado Professorship em oftalmologia na Loyola University Chicago.

Todos os animais foram tratados em conformidade com a instrução de ARVO para o uso de animais em oftalmologia e Vision Research e a CE Directiva 86/609/CEE para experiências em animais, usando protocolos aprovados e monitorados pelo Conselho da Finlândia experimento Animal.
1. induzida por laser rato CNV modelo 5
<ol><li>Inspecione os olhos do animal macroscopicamente para qualquer anormalidade.</li>
	<li>Pese o mouse.</li>
	<li>Calcular e preparar uma quantidade apropriada de anestésicos para utilizar, com base no peso do animal, por exemplo, uma mistura de medetomidina (1 mg/kg), ketamina (75 mg/kg) e água destilada (solução de NaCl 0,9%) na proporção de 1:1.5:2.5, ou cetamina (40-75 mg/kg), xilazina (5 mg/kg) e água destilada (solução de NaCl 0,9%) na proporção de 1:2. 5:1; para um mouse de 20g, injete 0,1 mL da mistura.</li>
	<li>Injete o anestésico intraperitonealmente.</li>
	<li>Posicione o mouse volta para a gaiola e espere até o animal é anestesiado. Confirme que o mouse é devidamente anestesiado pela falta de um pedal reflexo.</li>
	<li>Garantir o uso de equipamento de proteção pessoal da segurança do laser.</li>
	<li>Ligue a lâmpada de fenda e um laser de díodo 532 nm.</li>
	<li>Remova o rato da gaiola e coloque sobre a almofada de aquecimento.</li>
	<li>Aplique uma gota de tropicamida para dilatação da pupila. Espere 3-5 min para dilatação pupilar completo (3 mm).</li>
	<li>Posicione o mouse no palco da lâmpada de fenda.</li>
	<li>Coloque uma gota de gel líquido thoughtful sobre uma lamela de applanate da córnea.</li>
	<li>Oriente o olho do rato com a cabeça do nervo óptico no centro.</li>
	<li>Definir a potência do laser a 100 mW, a duração de 100 ms e o tamanho de ponto para 50 µm.</li>
	<li>Focar o feixe de laser o epithelium retinal do pigment (RPE).</li>
	<li>Fazer três tiros de laser em um olho, evitando os vasos da retina idealmente na 4, 8 e 12:00 posições em torno do nervo óptico, respectivamente. Inspecione o fundo do olho, depois todos tiros por ausência de hemorragia retiniana a laser. O olho contralateral serve como um controle de não-com laser.</li>
	<li>Descarte o mouse lamela e lugar volta sobre a almofada de aquecimento.</li>
	<li>Aplique uma gota de gotas de gel de PEG em ambos os olhos.</li>
</ol>2. SD-OCT 6,7
<ol><li>Coloque o mouse para a fase de alinhamento de roedores e imobilizar a cabeça.</li>
	<li>Alinhe a lente do sistema SD-OCT (por exemplo, Bioptigen/Leica Envisu R2200) para enfrentar o olho para a imagem latente com na vivo usando controladores de X - e Y-estágio.</li>
	<li>Realizar exames de SD-OCT para verificar se a quebra da membrana de Bruch: uma vez que o SD-OCT examina o olho inteiro, Mova manualmente a linha de referência nos sites lasered. Quebras de membrana de Bruch devem ser claramente visíveis em áreas lasered (ver Figura 1).</li>
</ol>3. fluoresceína angiografia 7,8,9
<ol><li>Remover o rato com o titular e coloque-o sobre o sistema de FA (por exemplo, Heidelberg Spectralis HRA2).</li>
	<li>Foco para laser queimar áreas do fundo do olho usando o modo de reflectância de infravermelho com a cabeça do nervo óptico, no meio da janela de visualização.</li>
	<li>Injete 0,1 mL de sal de sódio 5% fluoresceína para um rato de 20 g por via subcutânea ou intraperitonealmente.</li>
	<li>Concentre-se no nível da coroide.</li>
	<li>Leve uma imagem do nível de foco da coroide.</li>
	<li>Re-focar a nível da retina e uma imagem.</li>
	<li>Espere por 30 s e repita etapas 3.4-3.6.</li>
	<li>Remova o mouse do suporte e coloque-o sobre a almofada de aquecimento.</li>
	<li>Reverta anestesia por α2-antagonista de medetomidina, atipamezole (0,5 mg/kg, i.p.) ou espera para animal recuperação da anestesia.</li>
	<li>Repita em vivo SD-OCT e FA imagiologia em animais anestesiados sobre os acompanhamento dias 5, 10 e 14.</li>
</ol>4. CNV classificação
<ol><li>Classe os danos da membrana de Bruch da OCT imagens e imagens de FA da coroide tomadas imediatamente após o lasering no dia 0 como seguindo: 0 - membrana de Bruch não foi danificado 1 - sucesso dano da membrana de Bruch</li>
	<li>Classe a presença de CNV de lasered manchas que tinha vazamento, como observado, comparando a dinâmica do sinal de fluoresceína em uma série de imagens da retina de FA como seguindo: 0 - aparência normal da retina 0,5 - fraco coloração de escapamento 1.0 - áreas CNV gotejantes Nota: Use a imagem da OCT para confirmação adicional de CNV ou em FA questionável onde a presença de fluido intraretinal em imagens OCT sugeriria CNV classificação.</li>
</ol>5. medições de espessura retina
<ol><li>Use um software automatizado de segmentação para medições de espessura da retina. Certifique-se de que a espessura total da retina é considerada, como a espessura de todas as camadas da camada de fibras nervosas RPE (sites de medição saudável), ou para uma linha imaginária ligando RPE em torno do local do dano (lasered sites) (ver também Figura 7).</li>
</ol>

<ol>
	<li>Dobi, E. T., Puliafito, C. A., Destro, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+experimental+choroidal+neovascularization+in+the+rat.">A new model of experimental choroidal neovascularization in the rat.</a> Arch. Ophthalmol. Chic. Ill 1960. 107, 264-269 (1989).</li><li>Tobe, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+neovascularization+in+mice+with+overexpression+of+vascular+endothelial+growth+factor+in+photoreceptors.">Evolution of neovascularization in mice with overexpression of vascular endothelial growth factor in photoreceptors.</a> Invest. Ophthalmol. Vis. Sci. 39, 180-188 (1998).</li><li>Seo, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dramatic+inhibition+of+retinal+and+choroidal+neovascularization+by+oral+administration+of+a+kinase+inhibitor.">Dramatic inhibition of retinal and choroidal neovascularization by oral administration of a kinase inhibitor.</a> Am. J. Pathol. 154, 1743-1753 (1999).</li><li>Grossniklaus, H. E., Kang, S. J., Berglin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+choroidal+and+retinal+neovascularization.">Animal models of choroidal and retinal neovascularization.</a> Prog. Retin. Eye Res. 29, 500-519 (2010).</li><li>Shah, R. S., Soetikno, B. T., Lajko, M., Fawzi, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mouse+Model+for+Laser-induced+Choroidal+Neovascularization.">A Mouse Model for Laser-induced Choroidal Neovascularization.</a> J Vis Exp. (106), e53502 (2015).</li><li>Giani, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+evaluation+of+laser-induced+choroidal+neovascularization+using+spectral-domain+optical+coherence+tomography.">In vivo evaluation of laser-induced choroidal neovascularization using spectral-domain optical coherence tomography.</a> Invest. Ophthalmol. Vis. Sci. 52, 3880-3887 (2011).</li><li>Gong, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+an+Image-Guided+Laser-Induced+Choroidal+Neovascularization+Model+in+Mice.">Optimization of an Image-Guided Laser-Induced Choroidal Neovascularization Model in Mice.</a> PloS One. 10, e0132643 (2015).</li><li>Sheets, K. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotectin+D1+attenuates+laser-induced+choroidal+neovascularization+in+mouse.">Neuroprotectin D1 attenuates laser-induced choroidal neovascularization in mouse.</a> Mol. Vis. 16, 320-329 (2010).</li><li>Hoerster, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-vivo+and+ex-vivo+characterization+of+laser-induced+choroidal+neovascularization+variability+in+mice.">In-vivo and ex-vivo characterization of laser-induced choroidal neovascularization variability in mice.</a> Graefes Arch. Clin. Exp. Ophthalmol. Albrecht Von Graefes Arch. Klin. Exp. Ophthalmol. 250, 1579-1586 (2012).</li><li>Sulaiman, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Optical+Coherence+Tomography+Quantification+Method+for+Choroidal+Neovascularization.">A Simple Optical Coherence Tomography Quantification Method for Choroidal Neovascularization.</a> J. Ocul. Pharmacol. Ther. Off. J. Assoc. Ocul. Pharmacol. Ther. Off. J. Assoc. Ocul. Pharmacol. 31, 447-454 (2015).</li></ol>

O autor Symantas Ragauskas, pH.d. é um funcionário (cientista) e acionista do Ltd. de Experimentica que oferece contrato de serviços de pesquisa empregando o modelo CNV pré-clínicos usado neste artigo.
O autor Eva Kielczewski é um empregado (engenheiro de aplicações de pesquisa, OCT) da Leica Microsystems que produz sistemas de SD-OCT usados neste artigo.
O autor Joseph Vance é um empregado (at OCT Sales Director) da Leica Microsystems que produz sistemas de SD-OCT usados neste artigo. Joseph Vance também é presidente e diretor executivo da leção, LLC.
O autor Simon Kaja, pH.d. é consultor Chief Scientific Officer e acionista da Experimentica Ltd., uma contrato pré-clínicos pesquisa organização que oferece contrato de serviços de pesquisa, incl. modelo de CNV pré-clínicos usado neste artigo. Simon Kaja, pH.d. é também CEO da K & P científica, LLC, das Ciências da vida, consultoria e atua como o Dr. John P. e Therese E. Mulcahy dotado Professor em oftalmologia na Loyola University Chicago Stritch School of Medicine. Os termos desse acordo foram revistos e aprovados pela Loyola University Chicago, em conformidade com sua política de conflito de interesses.
O autor Giedrius Kalesnykas, pH.d. é um funcionário (CEO) e acionista do Ltd. de Experimentica que oferece contrato de serviços de pesquisa empregando o modelo CNV pré-clínicos usado neste artigo.

Imagiologia multimodal oferece valiosas ferramentas para avaliação de patologia CNV. Aqui apresentamos um protocolo de imagem consistindo de FA, SD-OCT e segmentação automática para a avaliação rápida, reprodutível e confiável de patologia CNV. Uma ruptura da membrana de Bruch após administração do laser foi confirmada. Além disso, o uso de SD-OCT nesta fase também permitiu a visualização imediata dos possíveis hemorragias intraretinal e subretinal, que pode confundir a interpretação dos resultados. V...

A primeira tentativa bem sucedida para imitar a patologia humana CNV em roedores foi demonstrada há quase três décadas com um laser de krypton em Long-Evans ratos1. Depois disso, um laser de krypton foi usado para invadir a cepa de rato mais popular, C57BL/6J2,3,4a membrana de Bruch. A taxa de sucesso da indução CNV foi verificada com FA e manchas histológicas. Um rápido desenvolvimento de modalidades de imagem não-invasivos, como OCT, estimulou o crescimento do campo de roedores modelos pré-clínicos. A capacidade de monitorar mudanças morfológicas na retina em múltiplos pontos de tempo no mesmo olho significativamente contribui para a redução do uso de animais e aumenta a eficiência em estudos experimentais. Avaliação histológica das lesões CNV é bastante simples e exige a rotulagem de crescimento vascular anormal ao redor do local de administração do laser, aquisição de imagem e estimativa de área/volume usando um software de análise de imagem. Em contraste, na vivo modalidades de imagem apresentar análises mais complexas de patologia CNV e sua interpretação.
Aqui nós apresentamos um método simples e relativamente rápido para indução de grau, progressão e regressão de CNV usando FA, SD-OCT, e o método de segmentação automatizada no mouse laser-induzida CNV modelo.

<table border="0" cellpadding="0" cellspacing="0" width="917">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:275px;">Medetomidine (commercial name Domitor)</td>
			<td style="width:123px;">Orion</td>
			<td style="width:344px;">Vnr 01 56 02</td>
			<td style="width:177px;">Anesthesia</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ketamine</td>
			<td>Intervet</td>
			<td>Vnr 51 14 85</td>
			<td>Anesthesia</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">0,9% NaCl</td>
			<td>B Braun</td>
			<td>357 0340</td>
			<td>Anesthesia</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Xylazine (commercial name Rompun vet)</td>
			<td>Bayer</td>
			<td>vnr 14 89 99</td>
			<td>Anesthesia</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tropicamide</td>
			<td>Santen</td>
			<td>Vnr 04 12 36</td>
			<td>Mydriatic agent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Viscotears</td>
			<td>Alcon</td>
			<td>Vnr 44 54 81</td>
			<td>Lubricant</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Systane</td>
			<td>Alcon</td>
			<td>&#160;-</td>
			<td>Lubricant</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">5% Fluorescein sodium salt</td>
			<td>Sigma Aldrich</td>
			<td>F6377-100G</td>
			<td>Fluoresent agent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Atipamezole (commercial name Antisedane)</td>
			<td>Orion</td>
			<td>Vnr 47 19 53</td>
			<td>Anesthesia</td>
		</tr>
	</tbody>
</table>

in vivo multimodal imaging and analysis of mouse laser-induced choroidal neovascularization model

Induzida por laser a neovascularização de coroide (CNV) é um modelo bem estabelecido para imitar a forma úmida da degeneração macular relacionada à idade (DMRI). Neste protocolo, nosso objetivo é orientar o leitor não simplesmente através das considerações técnicas de gerar lesões induzida por laser para desencadear processos neovascular, mas prefiro focar a informação poderosa que pode ser obtida multimodal longitudinal vivo em de imagem durante todo o período de seguimento.
O mouse laser-induzida modelo CNV foi gerado por uma administração de laser de diodo. Multimodal no vivo técnicas de imagem foram utilizadas para monitorar CNV indução, progressão e regressão. Primeiro, a tomografia de coerência óptica de domínio espectral (SD-OCT) foi realizada imediatamente após o lasering para verificar uma ruptura da membrana de Bruch. Subsequentes na vivo imagens usando angiografia fluoresceína (FA) confirmaram sucesso dano da membrana de Bruch da série imagens adquiridas a nível da coroide. Acompanhamento longitudinal da proliferação de CNV e regressão nos dias 5, 10 e 14 após o lasering foi realizado usando SD-OCT e FA. Simples e confiável de classificação de leasions de CNV com vazamento de imagens de FA é apresentado. Segmentação automatizada para medição da espessura total da retina, combinada com a aplicação manual calibre para medição da espessura da retina em sites CNV, permitir uma avaliação imparcial da presença de edema. Finalmente, verificação histológica da CNV é executada usando isolectin GS-IB4 mancha na flatmounts da coroide. A coloração é thresholded, e a área de isolectin-positivo é calculada com o ImageJ.
Este protocolo é especialmente útil em estudos de terapêutica que exigem alta taxa de transferência-como triagem de patologia CNV como rápido, permite multimodal e classificação confiável de edema de patologia e retinal CNV. Além disso, a alta resolução SD-OCT permite a gravação de outras características patológicas, tais como o acúmulo de fluido subretinal ou intraretinal. No entanto, este método não oferece a possibilidade de automatizar a análise de volume CNV de imagens SD-OCT, que tem de ser executada manualmente.

Uma bolha ou apresentam sangramento imediatamente após lasering nem sempre é visível. Portanto, o SD-OCT é particularmente importante para verificar os danos da membrana de Bruch. A Figura 1 mostra um exemplo de imagem OCT em pontos diferentes de tempo após a administração do laser.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56173/56173fig1.jpg"> Figura...

Watch this Scientific Journal Video about In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model at JoVE.com

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Na Vivo Imagiologia multimodal e análise do modelo de neovascularização coroide de induzida por Laser de Mouse

Aqui, apresentamos a utilidade da imagem longitudinal na vivo na sequência de alterações morfológicas da neovascularização de coroide do laser-induzida em camundongos.

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

in-vivo-multimodal-imaging-analysis-mouse-laser-induced-choroidal

Research

JoVE Journal

Neuroscience

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

9.0K visualizações.  Experimentica Ltd.. Aqui, apresentamos a utilidade da imagem longitudinal na vivo na sequência de alterações morfológicas da neovascularização de coroide do laser-induzida em camundongos.

Vídeo: In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

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 ...

Neurociência

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

Imagem Multimodal de implante de células-tronco no sistema nervoso central de camundongos

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

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 ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

Ex vivo imagens ao vivo de divisões celulares simples no mouse neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Um<em&gt; Ex Vivo</em&gt; Induzida por laser Lesão Medular modelo para avaliar mecanismos de axonal Degeneration em tempo real

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma establishes a lifelong persistent infection in the brain. While this brain infection is asymptomatic in most immunocompetent people, in the developing fetus or immunocompromised individuals such as acquired immune deficiency syndrome (AIDS) patients, this predilection for and persistence in the brain can lead to devastating neurologic disease. Thus, it is clear that the brain-Toxoplasma interaction is critical to the symptomatic disease produced by Toxoplasma, yet we have little understanding of the cellular or molecular interaction between cells of the central nervous system (CNS) and the parasite. In the mouse model of CNS toxoplasmosis it has been known for over 30 years that neurons are the cells in which the parasite persists, but little information is available about which part of the neuron is generally infected (soma, dendrite, axon) and if this cellular relationship changes between strains. In part, this lack is secondary to the difficulty of imaging and visualizing whole infected neurons from an animal. Such images would typically require serial sectioning and stitching of tissue imaged by electron microscopy or confocal microscopy after immunostaining. By combining several techniques, the method described here enables the use of thick sections (160 &#181;m) to identify and image whole cells that contain cysts, allowing three-dimensional visualization and analysis of individual, chronically infected neurons without the need for immunostaining, electron microscopy, or serial sectioning and stitching. Using this technique, we can begin to understand the cellular relationship between the parasite and the infected neuron.

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Using this protocol, we were able to image 160 &#181;m thick brain sections from mice infected with the parasite Toxoplasma gondii, which enables visualization and analysis of the spatial relationship between the encysting parasite and the infected neuron.

Imagem 3-D e Análise de Neurônios Infected<em&gt; In Vivo</em&gt; Com<em&gt; Toxoplasma gondii</em

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

Ativação de células da Müller Glia em um modelo de regeneração no Zebrafish e degeneração retiniana induzida por Laser

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

Laser-Induced Brain Injury in the Motor Cortex of Rats

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) occlusion of the middle cerebral artery (MCA) using a catheter. This generally accepted technique, however, has some limitations, thereby limiting its extensive use. Stroke induction by this method is often characterized by high variability in the localization and size of the ischemic area, periodical occurrences of hemorrhage, and high death rates. Also, the successful completion of any of the transient or permanent procedures requires expertise and often lasts for about 30 minutes. In this protocol, a laser irradiation technique is presented that can serve as an alternative method for inducing and studying brain injury in rodent models.
When compared to rats in the control and MCAO groups, the brain injury by laser induction showed reduced variability in body temperature, infarct volume, brain edema, intracranial hemorrhage, and mortality. Furthermore, the use of a laser-induced injury caused damage to the brain tissues only in the motor cortex unlike in the MCAO experiments where destruction of both the motor cortex and striatal tissues is observed.
Findings from this investigation suggest that laser irradiation could serve as an alternative and effective technique for inducing brain injury in the motor cortex. The method also shortens the time for completing the procedure and does not require expert handlers.

The protocol presented here shows a technique to create a rodent model of brain injury. The method described here uses laser irradiation and targets motor cortex.

Lesão cerebral induzida por laser no córtex motor de ratos

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) ...

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study of this movement in situ was only possible using radioisotopic pulse-labeling, which permits analysis of axonal transport in whole nerves with a temporal resolution of days and a spatial resolution of millimeters. To study neurofilament transport in situ with higher temporal and spatial resolution, we developed a hThy1-paGFP-NFM transgenic mouse that expresses neurofilament protein M tagged with photoactivatable GFP in neurons. Here we describe fluorescence photoactivation pulse-escape and pulse-spread methods to analyze neurofilament transport in single myelinated axons of tibial nerves from these mice ex vivo. Isolated nerve segments are maintained on the microscope stage by perfusion with oxygenated saline and imaged by spinning disk confocal fluorescence microscopy. Violet light is used to activate the fluorescence in a short axonal window. The fluorescence in the activated and flanking regions is analyzed over time, permitting the study of neurofilament transport with temporal and spatial resolution on the order of minutes&#160;and microns, respectively. Mathematical modeling can be used to extract kinetic parameters of neurofilament transport including the velocity, directional bias and pausing behavior from the resulting data. The pulse-escape and pulse-spread methods can also be adapted to visualize neurofilament transport in other nerves. With the development of additional transgenic mice, these methods could also be used to image and analyze the axonal transport of other cytoskeletal and cytosolic proteins in axons.

We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein.

Imagem e Análise do Transporte de Neurofilamento em Nervo Tibial de Camundongo Excised

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

Imagem transpupilar de dois fótons in vivo da retina do rato

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...