SN e MDD receberam apoio do NIH K08 NS083740 e do Departamento de Neurologia UCLA. AJG reconhece o apoio do Dr. Miriam e Sheldon G. Adelson Fundação de Pesquisa Médica e da L. Hillblom Fundação Larry. KLN agradece o apoio da Stroke Center American Heart Association 14BFSC17760005 ASA-Bugher. ILL, EGS e STC foram apoiados pelo NIH R01 NS071481. JDH reconhece o apoio do NIH K08 NS083740.

O uso de animais neste protocolo foi realizado de acordo com os procedimentos aprovados pela University of California Los Angeles Animal Care e do Comitê Use. Nota: Comece identificando a população murina alvo. Em estudos anteriores, do tipo selvagem ratinhos C57 / Bl6 machos única têm sido utilizadas, no entanto vários ratinhos transgénicos ou knockout também pode ser usado. Note-se que as coordenadas estereotáxicas baseiam-se em murganhos C57 / BL6 anatomia. Recomenda-se que cada usuário inicialmente verificar a localização do curso de substância branca. 1. Branco Matéria curso de indução - Medial Abordagem angular <ol><li> Comece a preparação de uma pipeta de vidro removido utilizando tubo capilar 0,5 milímetros de tal modo que o diâmetro distai é entre 15-25 uM 11. </li><li> Prepara-se uma alíquota de 10 ul estéril de L-NIO (N (5) - de HCl (1) -iminoethyl-L-ornitina) a 27,4 mg / ml (130 ^ M) em solução salina normal a 0,9% estéril. </li><li> Pré-encher a pipeta de vidro puxadocom um pequeno volume de L-NIO (2-5 ul) por meio da fixação do pipeta de vidro para a tubagem ligada a uma linha de vácuo. Deite o pipeta sobre a bancada de trabalho e inserir a extremidade puxado para dentro da solução de L-Nio. <ol><li> Aplicar o vácuo até pelo menos 2 mm da porção de 0,5 mm da pipeta é preenchido. Desligue o vácuo e retirar a pipeta. Colocá-lo de lado até Passo 1,12. </li></ol></li><li> Colocar o rato numa câmara de indução e induzir anestesia do rato utilizando isoflurano padrão de 32% fluiu através de um vaporizador (5 L / min inalado com 5 L / min de oxigénio e 0,5 L / min de N2) durante 1 minuto ou até profundamente anestesiados. Transferir o rato a um aparelho de estereotaxia equipado com um microscópio estereotáxica. Proporcionar a manutenção da anestesia utilizando 32% de isoflurano fluiu através de um vaporizador (2 L / min inalado com 5 L / min de oxigénio e 0,5 L / min de N2) e um cone de nariz. Verifique a profundidade da anestesia com uma pitada dedo do pé. </li><li> Ajustar o braço de injecção para 36 °. </li><li> AffiXA puxado titular pipeta de vidro para a extremidade distal de um sistema de injecção de baixa pressão de volume e anexar ao braço de injecção da configuração estereotáxica. </li><li> Brasão bigodes do animal anestesiado com vaselina e colocar pomada lágrima artificial sobre ambos os olhos. Prepare um campo cirúrgico estéril, colocando uma cortina estéril sobre a cabeça do animal com um 5-10 cm de abertura sobre a cabeça. Prepare uma superfície cirúrgica aspetic raspando o pêlo que cobre o crânio. Limpar o couro cabeludo com a alternância de betadine e 70% compressas com álcool. </li><li> Adicione uma 1,5 centímetros incisão na linha média do couro cabeludo com uma tesoura fina estéreis para expor a superfície do crânio. Seca-se o crânio com uma mecha de algodão estéril e usando um microscópio de ampliação estereotáxica em 1-3X, remover qualquer tecido periostal sobrejacente usando uma ferramenta de micro ponto de estéril. </li><li> Marcar o bregma como ponto de referência usando um marcador de ponta fina. </li><li> Perfurar uma craniotomia ellipitical 2 mm, utilizando uma fina broca cirúrgica stip estéril0; começando posteriormente ao bregma e estendendo anteriormente apenas à esquerda da linha média. Retirar os fragmentos de osso e tecido mole sobrejacente de modo que o córtex cerebral pode ser visualizado. </li><li> Manter o campo cirúrgico e superfície cortical úmida, aplicando de forma intermitente gotas de solução salina estéril. </li><li> Juntar uma pipeta de vidro puxado para o braço do injector do aparelho estereotáxico. Alinhar a extremidade distal da pipeta com a bregma e zero as coordenadas estereotáxicas. </li><li> Avançar a pipeta para o primeiro anterior / posterior (A / P) e medial / lateral (M / L) coordenadas fornecidas na Tabela 1. </li><li> Avançar a pipeta para a superfície cortical e zerar o ventral (A / V D) Medição dorsal /. </li><li> Lentamente passar a pipeta no cérebro até atingir o primeiro D / V de coordenadas na Tabela 1. </li><li> Usando um sistema de injecção de baixa pressão de volume ajustado a 20 psi durante 20 pulsos ms, injectar 100 nl de L-NIO no cérebro e esperar cinco minutos para evitar o refluxoa pista pipeta. <ol><li> Use um retículo calibrado na ocular do microscópio estereotáxica e uma ampliação de 3X. </li><li> Por conseguinte, deslocar um total de 0.100 mm 3 (comprimento com 0,5 mm de diâmetro de uma pipeta de 0,5 mm, correspondendo a 100 nl) a partir da pipeta de vidro puxado para cada conjunto de coordenadas. Usando uma bolsa, medir e padronizar uma vez que cada configurar varia dependendo da ampliação e escalas usadas. </li><li> Para a medição do volume precisas durante cada injecção, a abordagem da pipeta em ângulo com o microscópio do lado de modo que o menisco hidroaéreo tem uma vista sagital. O menisco deve aparecer no mesmo plano focal de tanto a parede interior e exterior da pipeta. </li></ol></li><li> Lentamente, retirar a pipeta e repita os passos 1.13-1.16.3 no segundo e terceiro conjunto de coordenadas fornecidas na Tabela 1. </li><li> Após a injecção final, remover a pipeta e colocar cera de osso suficiente para encher o local craniotomia. UMApproximate as bordas da ferida couro cabeludo e ligam-se com adesivo dérmico. </li><li> Injectar 0,1 mL de 0,5% Marcaine nas margens da ferida, usando uma agulha 30 G estéril para evitar a prevenir a dor associada com o local de incisão no couro cabeludo. </li><li> Devolver o animal para habitação e abastecimento de pós-operatório de antibióticos (0,48 mg / ml sulfametoxazoltrimetoprim, ou 0,5 mg / ml levofloxacina) na água de beber durante 5 dias. </li></ol> 2. Branco Matéria curso de indução - Posterior Abordagem angular <ol><li> Execute os passos 1,1-1,12 como no protocolo de abordagem em ângulo medial, exceto ajustar o braço para injeções da configuração estereotáxica a 45 graus orientados anterior para posterior. </li><li> Avançar a pipeta para o primeiro A / P e M / L coordenadas fornecidas na Tabela 2. </li><li> Conclua as etapas restantes 1.14-1.20 como no protocolo de abordagem em ângulo lateral. </li></ol> 3. neuronal retrógrada Labeling <ol><li> Prepare uma estéril 10aliquota ul de L-NIO a 54,8 mg / ml em 0,9% de solução salina normal. </li><li> Prepara-se uma aliquota de 20% estéril Fluororuby (ou 20% de amina dextrano biotinilado ou 2% de Fluorogold) em 0,9% de solução salina normal. </li><li> Dilui-se em conjunto 1: 1, para concentrações finais de 27,4 mg / ml de L-NIO e 10% Fluororuby. </li><li> Execute o protocolo de acidente vascular cerebral como acima nas etapas 1.3-1.23. </li><li> Visualizar o marcador nativamente fluorescente na secção de tecido por meio da fixação de perfusão, cryosectioning e microscopia, como descrito anteriormente 8. </li></ol> 4. Processamento de tecido por imunofluorescência <ol><li> Em um intervalo pós-AVC apropriada variando de 3 horas a 14 dias após o acidente vascular cerebral, eutanásia ratos através de overdose isoflurano ou IACUC locais procedimento aprovado. </li><li> Abrir a cavidade torácica com uma tesoura em ângulo e inserir uma agulha G 23 borboleta para o ventrículo esquerdo. </li><li> Coloque um pequeno corte no átrio direito com uma tesoura fina para permitir uma via de saída para o fluido de perfusão. </li><li> Transcardialmente perfundir com 30-40 ml de solução salina tamponada com fosfato fria seguido de 30-40 ml de paraformaldeído a 4% frio, a uma taxa de 10 ml / min à temperatura ambiente. </li><li> Decapitar o mouse e remover o cérebro usando uma tesoura esterilizada para abrir o crânio posteriormente e em seguida, remover suavemente a caveira sobreposta com uma espátula, coloque o cérebro em frio PFA 4% durante 24 h, e depois transferir para 30% de sacarose em PBS durante 48 horas . </li><li> Prepare secções de quarenta micron flutuante usando um criostato e executa o processamento de anticorpo, tal como anteriormente descrito 6-8. Neste estudo, utilizar os seguintes anticorpos: (: diluição 1 500); de coelho anti-neurofilamento 200 de coelho anti-vimentina (1: 500); de cabra anti-GFAP (1: 500); de coelho anti-Iba-1 (1: 1,000). </li></ol> 5. Processamento de Tecidos para a proteína ou RNA Análise <ol><li> Em um intervalo pós-AVC apropriada variando de 3 horas a 14 dias após o acidente vascular cerebral, eutanásia através de overdose isoflurano ou IACUC locais procedimento aprovado. </li><li> decapitar orato e remover o cérebro usando uma tesoura esterilizada para abrir o crânio posteriormente e em seguida, remover suavemente a caveira sobreposta com uma espátula. </li><li> Inserir uma espátula estéril 4 milímetros na parte frontal do cérebro para cortar o bolbo olfactivo e os nervos ópticos. Suavemente levantar o cérebro para fora da calvária e colocar em tampão de dissecção gelado (Solução Salina Equilibrada de 1x Hank, 25 mM de HEPES-KOH, pH 7,4, glicose 35 mM, bicarbonato de sódio 4 mM, e 0,01 mg / ml de ciclo-hexamida). </li><li> Usando um bloco cérebro e novas lâminas de barbear estéreis, prepare lajes 2-3 mm contendo o curso e colocar em buffer de dissecção fria. </li><li> Sob um microscópio de dissecação, identificar o assunto córtex motor subjacente branca no hemisfério injetado. Em intervalos mais longos de pós-acidente vascular cerebral, a região pode ser visualmente identificado por necrose focal e palidez mielina. Nota: Em intervalos pós-AVC anteriores, a injecção de L-NIO misturado com 1 ul de 10% Fast Green pode permitir a identificação visual do acidente vascular cerebral ( <strong&gt; Figura 4A). </li><li> Sob a orientação de um microscópio de dissecação e utilizando uma nova bisturi, dissecar cuidadosamente a região da substância branca que contém o acidente vascular cerebral, identificado por qualquer rotulagem Verde Rápido ou perda de tecido. Remover córtex sobrejacente e subjacente estriado como desejado. </li></ol>

<ol>
	<li>Go, A. 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=Heart+disease+and+stroke+statistics--2014+update:+a+report+from+the+American+Heart+Association.">Heart disease and stroke statistics--2014 update: a report from the American Heart Association.</a> Circulation. 129, 28-292 (2014).</li><li>Saini, M., 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=Silent+stroke:+not+listened+to+rather+than+silent.">Silent stroke: not listened to rather than silent.</a> Stroke. 43, 3102-3104 (2012).</li><li>Koton, 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=Burden+and+outcome+of+prevalent+ischemic+brain+disease+in+a+national+acute+stroke+registry.">Burden and outcome of prevalent ischemic brain disease in a national acute stroke registry.</a> Stroke. 44, 3293-3297 (2013).</li><li>Jiwa, N. S., Garrard, P., Hainsworth, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+of+vascular+dementia+and+vascular+cognitive+impairment:+a+systematic+review.">Experimental models of vascular dementia and vascular cognitive impairment: a systematic review.</a> J Neurochem. 115, 814-828 (2010).</li><li>Sozmen, E. G., Hinman, J. D., Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+that+matter:+white+matter+stroke+models.">Models that matter: white matter stroke models.</a> Neurotherapeutics. 9, 349-358 (2012).</li><li>Sozmen, E. G., Kolekar, A., Havton, L. A., Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+white+matter+stroke+model+in+the+mouse:+axonal+damage,+progenitor+responses+and+MRI+correlates.">A white matter stroke model in the mouse: axonal damage, progenitor responses and MRI correlates.</a> J Neurosci Methods. 180, 261-272 (2009).</li><li>Rosenzweig, S., Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+exacerbation+of+white+matter+stroke+outcomes:+a+role+for+oxidative+damage+and+inflammatory+mediators.">Age-dependent exacerbation of white matter stroke outcomes: a role for oxidative damage and inflammatory mediators.</a> Stroke. 44, 2579-2586 (2013).</li><li>Hinman, J. D., Rasband, M. N., Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodeling+of+the+axon+initial+segment+after+focal+cortical+and+white+matter+stroke.">Remodeling of the axon initial segment after focal cortical and white matter stroke.</a> Stroke. 44, 182-189 (2013).</li><li>McCall, T. B., Feelisch, M., Palmer, R. M., Moncada, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+N-iminoethyl-L-ornithine+as+an+irreversible+inhibitor+of+nitric+oxide+synthase+in+phagocytic+cells.">Identification of N-iminoethyl-L-ornithine as an irreversible inhibitor of nitric oxide synthase in phagocytic cells.</a> Brit j pharmacol. 102, 234-238 (1991).</li><li>Gadea, A., Aguirre, A., Haydar, T. F., Gallo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelin-1+regulates+oligodendrocyte+development.">Endothelin-1 regulates oligodendrocyte development.</a> J Neurosci. 29, 10047-10062 (2009).</li><li>Dean, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+(pulling)+of+needles+for+gene+delivery+by+microinjection.">Preparation (pulling) of needles for gene delivery by microinjection.</a> CSH prot. , (2006).</li><li>Hughes, P. M., 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=Focal+lesions+in+the+rat+central+nervous+system+induced+by+endothelin-1.">Focal lesions in the rat central nervous system induced by endothelin-1.</a> J. Neuropathol. Exp. Neurol. 62, 1276-1286 (2003).</li><li>Whitehead, S. N., Hachinski, V. C., Cechetto, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+a+rat+model+of+cerebral+ischemia+and+beta-amyloid+toxicity:+inflammatory+responses.">Interaction between a rat model of cerebral ischemia and beta-amyloid toxicity: inflammatory responses.</a> Stroke. 36, 107-112 (2005).</li><li>Frost, S. B., Barbay, S., Mumert, M. L., Stowe, A. M., Nudo, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+animal+model+of+capsular+infarct:+endothelin-1+injections+in+the+rat.">An animal model of capsular infarct: endothelin-1 injections in the rat.</a> Behav Brain Res. 169, 206-211 (2006).</li><li>Horie, N., 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=Mouse+model+of+focal+cerebral+ischemia+using+endothelin-1.">Mouse model of focal cerebral ischemia using endothelin-1.</a> J Neurosci Methods. 173, 286-290 (2008).</li><li>Wang, M., 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=Cognitive+deficits+and+delayed+neuronal+loss+in+a+mouse+model+of+multiple+microinfarcts.">Cognitive deficits and delayed neuronal loss in a mouse model of multiple microinfarcts.</a> Neuroscience. 32, 17948-17960 (2012).</li><li>Shih, A. 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=The+smallest+stroke:+occlusion+of+one+penetrating+vessel+leads+to+infarction+and+a+cognitive+deficit.">The smallest stroke: occlusion of one penetrating vessel leads to infarction and a cognitive deficit.</a> Nat Neurosci. 16, 55-63 (2013).</li><li>Jung, K. J., 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=The+role+of+endothelin+receptor+A+during+myelination+of+developing+oligodendrocytes.">The role of endothelin receptor A during myelination of developing oligodendrocytes.</a> J Korean Med Sci. 26, 92-99 (2011).</li></ol>

Um número de modelos anteriores de acidente vascular cerebral subcorticais tenham sido descritos incluindo injecções focais de endotelina-1 para a cápsula interna, matéria branca subcortical e corpo estriado do rato 6,15 12-14 e rato. Modelos mais recentes de pequenos acidentes vasculares cerebrais focais têm utilizado a injeção microembolias colesterol na artéria carótida 16 e oclusão photothrombotic de um único arteríolas penetrantes 17. Cada um destes modelos t...

Stroke affecting the subcortical white matter is a common clinical entity, accounting for up to 25% of clinical strokes annually in the US 1. Ischemic damage to white matter also occurs silently at a significantly higher rate and contributes to the development of vascular dementia 2,3. Presently, patients with this form of cerebral ischemia have few, if any treatment choices. Despite the clinical importance of this disease, few clinically relevant animal models exist 4,5. 
The goal of this protocol is to produce a focal ischemic lesion within the murine white matter. This murine model of human disease allows the specific study of axonal injury response to stroke and how the cellular elements of white matter, namely oligodendrocytes and astrocytes along with axons, respond to and repair after stroke.
Previous reports have described a model of subcortical white matter stroke using endothelin-1 (ET-1) 6 that is similar to the one described here. Several key changes to the experimental protocol have been made thereby the potential uses of this model have expanded 7,8. This protocol provides a reliable and modifiable strategy to produce a focal stroke within mouse brain white matter. 
The major advantages of this model are the use of a chemical endothelial nitric oxide synthase (eNOS) inhibitor N(5)-(1)-iminoethyl-L-ornithine HCl (L-Nio) 9 with no known paracrine effects on cellular elements of white matter which had been a complication of models using endothelin-1 10. In addition, the stereotactic targeting of white matter in the mouse allows the use of any variety of transgenic or knockout strains, greatly expanding the available tools to determine the effect of stroke on brain white matter. Here, two variations on this technique are described and demonstrate some of the additional variations that can be utilized to enhance the understanding of axonal and white matter damage and repair after stroke.

<table border="0" cellpadding="0" cellspacing="0" width="725">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">L-N5-(1-Iminoethyl)ornithine, Dihydrochloride</td>
			<td style="width:204px;">Calbiochem</td>
			<td style="width:133px;">400600-20MG</td>
			<td style="width:81px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane</td>
			<td>Phoenix Pharmaceutical, Inc.</td>
			<td>NDC 57319-559-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Capillary tubes</td>
			<td>World Precision Instruments</td>
			<td>50-821-807</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Picospritzer</td>
			<td>Parker Instrumentation</td>
			<td>Picospritzer II</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereotactic setup</td>
			<td>Kent Scientific</td>
			<td>KSC51725</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipette puller</td>
			<td>KOPF</td>
			<td>Model 720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereomicroscope SZ51</td>
			<td>Olympus</td>
			<td>88-124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine scissors</td>
			<td>Fine Scientific Tools</td>
			<td>14084-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps</td>
			<td>Harvard Apparatus</td>
			<td>PY2 72-8547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved Forceps</td>
			<td>Harvard Apparatus</td>
			<td>PY2 72-8598</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blunt dissection tool</td>
			<td>Fine Scientific Tools</td>
			<td>10066-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drill</td>
			<td>Dremel</td>
			<td>8220-1/28</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drill bits</td>
			<td>Fine Scientific Tools</td>
			<td>19007-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vetbond</td>
			<td>3M</td>
			<td>1469SB&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Marcaine</td>
			<td>HOSPIRA</td>
			<td>NDC 0409-1610-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trimethoprim-Sulfamethaxole</td>
			<td>STI Pharmacy</td>
			<td>NDC 54879-007-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluororuby</td>
			<td>Fluorochrome Inc</td>
			<td>30mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Fisher</td>
			<td>O4042-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td>Fisher</td>
			<td>BP220-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostat</td>
			<td>Leica CM3050 S</td>
			<td>14047033518</td>
			<td></td>
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			<td height="21" style="height:21px;">Glass slides</td>
			<td>Fisher</td>
			<td>12-544-7</td>
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			<td height="21" style="height:21px;">Fast Green&#160;</td>
			<td>Sigma</td>
			<td>F7252-5G</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Dissection microscope</td>
			<td>Nikon</td>
			<td>SMZ1500</td>
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			<td height="21" style="height:21px;">23 gauge butterfly needle</td>
			<td>Fisher</td>
			<td>14-840-35</td>
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			<td height="21" style="height:21px;">10X Hank&#39;s Balanced Salt Solution</td>
			<td>Life Technologies</td>
			<td>14065056</td>
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			<td height="21" style="height:21px;">1M HEPES-KOH, pH 7.4</td>
			<td>Affymetrix</td>
			<td>16924</td>
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			<td height="21" style="height:21px;">D-Glucose</td>
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			<td>G8270</td>
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			<td height="21" style="height:21px;">Sodium bicarbonate</td>
			<td>Sigma</td>
			<td>S5761</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Cyclohexamide</td>
			<td>Sigma</td>
			<td>01810</td>
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a versatile murine model of subcortical white matter stroke for the study of axonal degeneration and white matter neurobiology

Acidente vascular cerebral que afecte contas de matéria branca para até 25% das apresentações clínicas acidente vascular cerebral, ocorre silenciosamente em taxas que podem ser 5-10 vezes maior, e contribui significativamente para o desenvolvimento de demência vascular. Alguns modelos de acidente vascular cerebral focal substância branca existir e esta falta de modelos apropriados tem dificultado a compreensão dos mecanismos neurobiológicos envolvidos na resposta a uma lesão e reparação após este tipo de acidente vascular cerebral. A principal limitação de outros modelos de acidente vascular cerebral subcorticais é que eles não focalmente restringir o enfarte e a substância branca ou foram validados principalmente em espécies não murinas. Isto limita a capacidade de aplicar a grande variedade de ferramentas de investigação para estudar o murino de acidente vascular cerebral neurobiologia substância branca. Aqui apresenta-se uma metodologia para a produção fiável de um acidente vascular cerebral focal em matéria branca murino utilizando uma injecção local de um inibidor de eNOS irreversível. Apresentamos também diversas variações no protocolo geral, incluindo dois estereotáxica únicavariações, traçando retrógrada neuronal, bem como de rotulagem tecido fresco e dissecação que expandir muito as potenciais aplicações desta técnica. Estas variações permitem múltiplas abordagens para analisar os efeitos neurobiológicos desta forma comum e pouco estudado de acidente vascular cerebral.

Usando o modelo apresentado, a matéria branca forelimb subjacente córtex sensório-motor confiável pode ser alvo. Este modelo de acidente vascular cerebral induzida quimicamente produz axonal focal e a perda de mielina, astrocitose e microgliose (Figura 1), como é tipicamente visto nos enfartes lacunares humanos. Usando três injeções, um modelo clinicamente útil é estabelecida com comprometimento precoce em tarefas motoras membro anterior 7 e uma pequ...

Watch this Scientific Journal Video about A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology at JoVE.com

A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.

A Stroke Versátil Modelo murino de Subcortical Branco Matéria para o Estudo da axonal Degeneration e branco Matéria Neurobiology

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and ...

a-versatile-murine-model-subcortical-white-matter-stroke-for-study

Research

JoVE Journal

Medicine

8.0K visualizações.  University of California, Los Angeles. Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.

Vídeo: A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Um método para isolamento de ilhotas murino e transplante renal subcapsular

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

Medicina

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarcadores em um modelo animal para Revelando correlatos neurais, hematológica e Comportamental do TEPT

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Um modelo murino de implante de stent na artéria carótida para o Estudo da reestenose

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

Um modelo murino de hemorragia subaracnóide

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Excitotóxico Estimulação do Microslices cérebro como um<em&gt; In vitro</em&gt; Modelo de AVC

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

DTI of the Visual Pathway - White Matter Tracts and Cerebral Lesions

DTI is a technique that identifies white matter tracts (WMT) non-invasively in healthy and non-healthy patients using diffusion measurements. Similar to visual pathways (VP), WMT are not visible with classical MRI or intra-operatively with microscope. DTI will help neurosurgeons to prevent destruction of the VP while removing lesions adjacent to this WMT. We have performed DTI on fifty patients before and after surgery between March 2012 to January 2014. To navigate we used a 3DT1-weighted sequence. Additionally, we performed a T2-weighted and DTI-sequences. The parameters used were, FOV: 200 x 200 mm, slice thickness: 2 mm, and acquisition matrix: 96 x 96 yielding nearly isotropic voxels of 2 x 2 x 2 mm. Axial MRI was carried out using a 32 gradient direction and one b0-image. We used Echo-Planar-Imaging (EPI) and ASSET parallel imaging with an acceleration factor of 2 and b-value of 800 s/mm&#178;. The scanning time was less than 9 min.
The DTI-data obtained were processed using a FDA approved surgical navigation system program which uses a straightforward fiber-tracking approach known as fiber assignment by continuous tracking (FACT). This is based on the propagation of lines between regions of interest (ROI) which is defined by a physician. A maximum angle of 50, FA start value of 0.10 and ADC stop value of 0.20 mm&#178;/s were the parameters used for tractography.
There are some limitations to this technique. The limited acquisition time frame enforces trade-offs in the image quality. Another important point not to be neglected is the brain shift during surgery. As for the latter intra-operative MRI might be helpful. Furthermore the risk of false positive or false negative tracts needs to be taken into account which might compromise the final results.

Diffusion tensor imaging (DTI) was performed to try to depict the major parts of the visual pathway. The goal was to use an FDA approved standard commercial workstation which could be used for everyday routine in order to try to reduce postoperative damage of the visual pathway in the patients.

DTI do Caminho Visual - substância branca panfletos e Cerebral Lesões

DTI is a technique that identifies white matter tracts (WMT) non-invasively in healthy and non-healthy patients using diffusion measurements. Similar to ...

Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated blood is the aorta. The aorta may become affected in diseases such as atherosclerosis and aneurysm. Researchers investigating these diseases would benefit from direct observation of the aorta to characterize disease progression as well as to evaluate efficacy of potential therapeutics. The goal of this protocol is to describe proper isolation and excision of the aorta to aid investigators researching cardiovascular disease. Isolation and excision of the aorta allows investigators to look at gross morphometric changes as wells as allowing them to preserve and stain the tissue to look at histologic changes if desired. The aorta may be used for molecular studies to evaluate protein and gene expression to discover targets of interest and mechanisms of action. This technique is superior to imaging modalities as they have inherent limitations in technology and cost. Additionally, primary isolated cells from a freshly isolated and excised aorta can allowing researchers to perform further in situ and in vitro assays. The isolation and excision of the aorta has the limitation of having to sacrifice the animal however, in this case the benefits outweigh the harm as it is the most versatile technique in the study of aortic disease.

Pathology of the aorta can lead to severe morbidity and mortality, therefore research of disease progression and potential therapies is warranted. Here, we present a protocol to isolate and excise the murine aorta to aid researchers in their investigation of cardiovascular disease.

Isolamento e excisão de Murino Aorta; A técnica versátil no Estudo de Doenças Cardiovasculares

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

Um Defeito modelo simples Critical porte Femoral em Ratos

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Oxigênio-Glucose Privação e reoxigenação como um<em&gt; In Vitro</em&gt; Isquemia e reperfusão Lesão modelo para estudar Disfunção Barreira Hematoencefálica

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model

Pedicle screw fixation is the gold standard for the treatment of spinal diseases. However, many studies have reported the issue of loosening pedicle screws after spinal surgery, which is a serious concern. To address this problem, diverse types of pedicle screws have been examined to identify those with good fixation strength and osseointegration in spine bone. The porcine spine is a good alternative for the human spine in the evaluation of pedicle screws due to the anatomical size, mechanical characteristics, and cost. Although several studies have reported that pedicle screws are efficient in the porcine model, no study has described detailed protocols for the evaluation of a pedicle screw using the porcine model. Here, we describe a detailed method for evaluating transpedicular screws using an in vivo porcine lumbar spine model. The technical details for anesthesia, spine surgery, and harvest provided here will facilitate with the evaluation of the transpedicular screw fixation model.

An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model

Here, we introduce a method to evaluate transpedicular screws by using an in vivo porcine lumbar spine model.

Método de anestesia, cirurgia e colheita para avaliação de parafusos transpediculares usando um In Vivo Modelo de espinha lombar porcina

Pedicle screw fixation is the gold standard for the treatment of spinal diseases. However, many studies have reported the issue of loosening pedicle ...