Os autores reconhecem com gratidão a ajuda hábil de Sra Trine W. Mikkelsen, Sra Lise M. Fitting, e os funcionários da Påskehøjgaard. O Medical Conselho Dinamarquês de Pesquisas, a Fundação Lundbeck, ea Nordisk Fundação Novo suportado financeiramente o estudo.

A anestesia dos animais e euthanesia foi realizada em conformidade com o &quot;Princípios de cuidados de animais de laboratório&quot; (publicação NIH No. 86-23, revista 1985) e aprovado pelo Conselho dinamarquês para animal de Ética. 1. Instrumentos <ol><li> Recolher os instrumentos apresentados no vídeo e listadas na Tabela de Materiais. </li></ol> 2. decapitação NOTA: A anestesia foi induzida através de uma injecção intramuscular de 5 ml de midazolam (5 mg / ml) e 5 ml de cetamina (25 mg / mL). 5-10 minutos mais tarde, quando o animal foi profundamente sedados, uma veia da orelha foi canulada e uma sobredosagem letal (100 mg / kg de peso corporal) com pentobarbital de sódio (200 mg / mL) foi administrada por via intravenosa. Para assegurar que o animal estava completamente sacrificados, o reflexo dor interdigital foi testado como mostrado por Ettrup et al. (2011) 20. eutanásia completa foi assegurada, conforme descrito noa instrução ética acima e seguido por uma perfusão transcardial com 5 L de solução salina isotónica, tal como demonstrado por Ettrup et al. (2011) 20. Todos os procedimentos são realizados demonstraram post-mortem, excluindo a necessidade das precauções necessárias para o bem-estar anestesia longo prazo e sobrevivência pós-procedimento. <ol><li> Decapitar o porco por um elevado incisão cervical circular, usando um bisturi cirúrgico, um pouco abaixo do ângulo de mandíbula (Figura 1A). </li><li> Ainda com o bisturi cirúrgico, continuar a incisão anterior através do tecido mole do pescoço, incluindo a laringe e o esago, até que a coluna vertebral óssea é atingido, aproximadamente ao nível da junção crânio-cervical. </li><li> Avançar o corte com um bisturi cirúrgico a partir do lado anterior da junção crânio-cervical, acima do arco anterior do atlas, e através da membrana atlanto anterior, expondo desse modo o canal vertebral e da medula espinhal (A Figura 1B). Simultaneamente pedir um assistente para puxar o corpo de porco longe da cabeça de porco para facilitar o acesso entre a base do crânio ea primeira vértebra cervical. </li><li> Continue a incisão cirúrgica através do saco durai e a medula espinal (Figura 1B). Tomar cuidado especial para assegurar que o corte transversal completo da medula espinal é conseguido. NOTA: A incapacidade de realizar a etapa anterior pode resultar em tração indesejada sobre a medula espinhal e do cérebro durante as seguintes etapas do processo de decapitação. </li><li> Vigorosamente estender a junção cranocervical ao nível da secção (Figura 1C). Ao mesmo tempo, utilizar o bisturi cirúrgico para os restantes secção ligamentos atlanto para soltar a articulação entre os côndilos occipitais e o processo articular superior do atlas. Separar a cabeça do porco do corpo. </li></ol><img alt="figura 1" src="/files/ftp_upload/55511/55511fig1.jpg" style="font-size: 14px;" /> Figura 1: Minipig decapitação. Incisão (A) Pescoço (seta, o ângulo da mandíbula). (B) A incisão através dos ligamentos atlanto e da medula espinhal rodeado-dura (SC) na junção craniocervical (C1, arco anterior do atlas; OC, côndilo occipital). (C) A parte posterior da articulação atlanto é libertado por uma extensão vigorosa (setas) ao nível de secção. <a href="http://ecsource.jove.com/files/ftp_upload/55511/55511fig1large.jpg" style="font-size: 14px;" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> 3. Crânio Abertura <ol><li> Posicionar a cabeça de porco em uma mesa. </li><li> Faça uma incisão longitudinal dorsal com um bisturi cirúrgico através da pele e dos tecidos moles subjacente da parte de trás do focinho, sobre o vértice da cabeça, e para baixo através da parte posterior doa região occipital. </li><li> Expor a parte dorsal e posterior do crânio, removendo o tecido macio localizado lateral à incisão inicial com um bisturi cirúrgico. </li><li> Libertar o músculo temporal bilateral a partir do crânio (Figura 2A) com um bisturi cirúrgico. Certifique-se de que o osso occipital posterior é limpo de tecidos moles. </li><li> Utilizar a entrada posterior do forâmen magno para remover o osso occipital com um Kerrison perfurador e osso rongeurs osso e expor o cerebelo coberto-dura (Figura 2B). </li><li> Retornar para o lado anterior exposta do crânio e selecione um ponto de entrada no osso frontal, mesmo em frente dos olhos. Neste ponto, usar um cinzel osso com um martelo para penetrar o crânio e entrar no seio frontal (Figura 2C). </li><li> Utilizar a extensão do seio frontal para promover a remoção da lâmina dorsoposterior crânio exterior com um rongeur óssea ou soco e expor o interior, finalâmina crânio ósseo cobrindo o cérebro (Figura 2D). </li><li> Suavemente abrir o interior lâmina crânio ósseo anteriormente com um martelo e um cinzel osso para expor o cérebro coberto-dura (Figura 2E). </li><li> Continuar a remoção de osso lateralmente utilizando um cinzel osso e um rongeur osso através do osso temporal e parietal, a fim de libertar a parte dorsoposterior final do crânio, localizada entre as partes já expostas do cérebro e cerebelo coberto-dura (Figura 2F). NOTA: Muitas vezes, é possível, durante a etapa final deste processo, para usar o cinzel de abrir o restante do osso do crânio posterior de um lado, como se abre uma porta. </li></ol><img alt="Figura 2" src="/files/ftp_upload/55511/55511fig2.jpg" style="opacity: 0.9; font-size: 14px;" /> Figura 2: Minipig abertura crânio. (A) Exposição da dorsoposteriosuperfície do crânio r, incluindo a remoção do occipital e temporal do músculo. (B) A remoção do osso occipital (CB, cerebelo coberto-dura; OB, osso occipital; OC, côndilo occipital; e SC, medula espinal). (C) Um martelo e um cinzel osso são utilizadas para penetrar o crânio anteriormente e para introduzir o seio frontal ao nível dos olhos. (D) A extensão do seio frontal (FS) é utilizado para remover o osso do crânio espessa exterior (1), expondo uma lâmina óssea fina interior (2), cobrindo o cérebro. (E) Remoção da lâmina óssea fina, expondo o cérebro coberto-dura (seta). (F) Por fim, um martelo e um cinzel osso são utilizadas para ligar lateralmente o anterior e as aberturas posterior do crânio. <a href="http://ecsource.jove.com/files/ftp_upload/55511/55511fig2large.jpg" style="font-size: 14px;" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> 4. REMOV Cérebroai <ol><li> Use uma pinça cirúrgica para levantar a dura e criar uma incisão perto suave para o seio sagital superior venosa utilizando um bisturi cirúrgico fina (Figura 3A). </li><li> Use micro-tesoura ou uma dura-faca para abrir ainda mais a dura-máter que cobre a superfície dorsal do cérebro. NOTA: cuidado especial deve ser feita quando a remoção da dura-máter correspondente ao tentorium cerebelo (Figura 3B), com a preservação deste folha dural irá impedir a remoção do cérebro posterior. </li><li> Posicionar a cabeça de porco verticalmente (Figura 3C). </li><li> Utilizar o cinzel osso ou um dissecador para libertar o cérebro ventroanterior por dissecção romba do bolbo olfactivo do piso coberto de dura-máter da cavidade craniana (Figura 3D). </li><li> Use um bisturi cirúrgico multa a seção do quiasma exposta (Figura 3E). Expor e secção da haste hipofisária e os nervos oculomotores. </li><li> Solte o tronco cerebral ventral pela secção do lonervos cranianos WER (Figura 3F), com um bisturi cirúrgico fina. Certifique-se de que o tentorium cerebelar durai foi completamente incisão (Figura 3B), como esta folha da dura-máter, de outro modo atravessam o tronco cerebral durante o processo de libertação. </li></ol><img alt="Figura 3" src="/files/ftp_upload/55511/55511fig3.jpg" style="font-size: 14px;" /> Figura 3: Remoção do cérebro Minipig. Abertura (A) dural com pinça cirúrgica e uma faca de dura-máter. (B) Cuidados devem ser tomados para inciso completamente a folha de dural (seta), localizado entre o cérebro e cerebelo. (C) A cabeça de porco é posicionado verticalmente para melhor visualização das estruturas da base do crânio e em ordem para a gravidade para ajudar no deslocamento pretendida do cérebro. (D) Um de dissecção ou um cinzel osso é usado para aliviar o bolbo olfactivo por secção romba da dura-covered da base do crânio. (E) A dissecção é continuada numa direcção posterior ao longo da base do crânio para exposição e seccionamento do quiasma óptico (seta), haste infundibular, e nervos oculomotores. (F) A libertação do cérebro é completado com a secção dos nervos cranianos inferiores tal como estas se afastem da superfície ventral do tronco cerebral (III, nervo oculomotor; IV, nervo troclear; V, nervo trigeminal; e VI, abducens nervo). <a href="http://ecsource.jove.com/files/ftp_upload/55511/55511fig3large.jpg" style="font-size: 14px;" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> 5. pituitária Remoção <ol><li> Identificar o talo pituitária e a sua folha dural circundante (da sela diapragma) no andar crânio (Figura 4A). </li><li> Incisão lateral da folha da dura-máter com a haste hipofisária usando um bisturi cirúrgico fino (Figura 4B). </li><li> Use um dissector para liberar o poçouitary e levantá-lo para fora da fossa pituitária (Figura 4C). </li></ol><img alt="Figura 4" src="/files/ftp_upload/55511/55511fig4.jpg" style="opacity: 0.9; font-size: 14px;" /> Figura 4: Minipig remoção da pituitária. (A) A fossa pituitária (*) é identificado no chão crânio (1, bolbo olfactivo; 2, quiasma; e PF, fossa posterior). (B) A cobertura dural (diagphragm selar, (seta)) é incisado lateralmente. (C) A pituitária (seta) é libertado com um dissecador e elevado para fora da fossa pituitária. Escala da barra (AC) = 10 mm. <a href="http://ecsource.jove.com/files/ftp_upload/55511/55511fig4large.jpg" style="font-size: 14px;" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

<ol>
	<li>Lind, M. N., Moustgaard, A., Jelsing, J., Vajta, G., Cumming, P., Hansen, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+pigs+in+neuroscience:+Modeling+brain+disorders.">The use of pigs in neuroscience: Modeling brain disorders.</a> Neurosci Biobehav Rev. 31, 728-751 (2007).</li><li>Bjarkam, C. 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=Neuromodulation+in+a+minipig+model+of+Parkinson+disease.">Neuromodulation in a minipig model of Parkinson disease.</a> British J Neurosurg. 22 (Suppl. 1), S9-S12 (2008).</li><li>Bjarkam, C. R., Cancian, G., Glud, A. N., Ettrup, K. S., &#216;stergaard, L., S&#248;rensen, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI-guided+stereotaxic+targeting+in+pigs+based+on+a+stereotaxic+localizer+box+fitted+with+an+isocentric+frame+and+use+of+SurgiPlan+computer-planning+software.">MRI-guided stereotaxic targeting in pigs based on a stereotaxic localizer box fitted with an isocentric frame and use of SurgiPlan computer-planning software.</a> J Neurosci Methods. 183 (2), 119-126 (2009).</li><li>Sauleau, P., Lapouble, E., Val-Laillet, D., Malbert, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pig+model+in+brain+imaging+and+neurosurgery.">The pig model in brain imaging and neurosurgery.</a> Animal. 3 (8), 1138-1151 (2009).</li><li>Bjarkam, C. 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=Safety+and+function+of+a+new+clinical+intracerebral+microinjection+instrument+for+stem+cells+and+therapeutics+examined+in+the+Göttingen+minipig.">Safety and function of a new clinical intracerebral microinjection instrument for stem cells and therapeutics examined in the Göttingen minipig.</a> Stereotact Funct Neurosurg. 88 (1), 56-63 (2010).</li><li>Fjord-Larsen, L., 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=Long-term+delivery+of+nerve+growth+factor+by+encapsulated+cell+biodelivery+in+the+minipig+basal+forebrain.">Long-term delivery of nerve growth factor by encapsulated cell biodelivery in the minipig basal forebrain.</a> Mol Therapy. 18 (12), 2164-2172 (2010).</li><li>S&#248;rensen, J. C., 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=Development+of+neuromodulation+treatments+in+a+large+animal+model+-+Do+neurosurgeons+dream+of+electric+pigs?.">Development of neuromodulation treatments in a large animal model - Do neurosurgeons dream of electric pigs?.</a> Prog Brain Res. 194, 97-103 (2011).</li><li>Dolezalova, D., 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=Pig+models+of+neurodegenerative+disorders:+utilization+in+cell+replacement-based+preclinical+safety+and+efficacy+studies.">Pig models of neurodegenerative disorders: utilization in cell replacement-based preclinical safety and efficacy studies.</a> J Comp Neurol. 522 (12), 2784-2801 (2014).</li><li>Mikkelsen, M., Moller, A., Jensen, L. H., Pedersen, A., Harajehi, J. B., Pakkenberg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MPTP-induced+Parkinsonism+in+minipigs:+A+behavioral,+biochemical,+and+histological+study.">MPTP-induced Parkinsonism in minipigs: A behavioral, biochemical, and histological study.</a> Neurotoxicol Teratol. 21, 169-175 (1999).</li><li>Danielsen, E. H., 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+DaNEX+study+of+embryonic+mesencephalic,+dopaminergic+tissue+grafted+to+a+minipig+model+of+Parkinson's+disease:+Preliminary+findings+of+effect+of+MPTP+poisoning+on+striatal+dopaminergic+markers.">The DaNEX study of embryonic mesencephalic, dopaminergic tissue grafted to a minipig model of Parkinson's disease: Preliminary findings of effect of MPTP poisoning on striatal dopaminergic markers.</a> Cell Transplant. 9 (2), 247-259 (2000).</li><li>Dalmose, A., Bjarkam, C. R., S&#248;rensen, J. C., J&#248;rgensen, T. M., Djurhuus, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+high+frequency+deep+brain+stimulation+on+urine+storage+and+voiding+function+in+conscious+minipigs.">Effects of high frequency deep brain stimulation on urine storage and voiding function in conscious minipigs.</a> Neurourol Urodyn. 23 (3), 265-272 (2004).</li><li>Dalmose, A., Bjarkam, C. R., Djurhuus, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotactic+electrical+stimulation+of+the+pontine+micturition+center+in+the+pig.">Stereotactic electrical stimulation of the pontine micturition center in the pig.</a> Br J Urol. 95, 886-889 (2005).</li><li>Andersen, F., Watanabe, H., Bjarkam, C. R., Danielsen, E. H., Cumming, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pig+brain+stereotaxic+standard+space:+Mapping+of+cerebral+blood+flow+normative+values+and+effect+of+MPTP-lesioning.">Pig brain stereotaxic standard space: Mapping of cerebral blood flow normative values and effect of MPTP-lesioning.</a> Brain Res Bull. 66 (1), 17-29 (2005).</li><li>Glud, A. 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=Direct+gene+transfer+in+the+minipig+CNS+using+stereotaxic+lentiviral+microinjections.">Direct gene transfer in the minipig CNS using stereotaxic lentiviral microinjections.</a> Acta Neurobiol Exp. 70 (3), 1-8 (2010).</li><li>Glud, A. 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=Direct+MRI-guided+stereotaxic+viral+mediated+gene+transfer+of+alpha-synuclein+in+the+minipig+CNS.">Direct MRI-guided stereotaxic viral mediated gene transfer of alpha-synuclein in the minipig CNS.</a> Acta Neurobiol Exp. 71 (4), 508-518 (2011).</li><li>Ettrup, K. S., S&#248;rensen, J. C., Rodell, A., Alstrup, A. K. O., Bjarkam, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothalamic+deep+brain+stimulation+influences+autonomic+and+limbic+circuitry+involved+in+the+regulation+of+aggression+and+cardiocerebrovascular+control+in+the+minipig.">Hypothalamic deep brain stimulation influences autonomic and limbic circuitry involved in the regulation of aggression and cardiocerebrovascular control in the minipig.</a> Stereotact Funct Neurosurg. 90 (5), 281-291 (2012).</li><li>Nielsen, 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=Continuous+MPTP+intoxication+in+the+minipig+results+in+chronic+parkinsonian+deficits.">Continuous MPTP intoxication in the minipig results in chronic parkinsonian deficits.</a> Acta Neurobiol Exp. 76, 198-210 (2016).</li><li>Bjarkam, C. 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=A+MRI-compatible+stereotaxic+localizer+box+enables+high-precision+stereotaxic+procedures+in+pigs.">A MRI-compatible stereotaxic localizer box enables high-precision stereotaxic procedures in pigs.</a> J Neurosci Methods. 139 (2), 293-298 (2004).</li><li>Bjarkam, C. R., Jorgensen, R. L., Jensen, K. N., Sunde, N. A. A., S&#248;rensen, J. C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+brain+stimulation+electrode+anchoring+using+BioGlue&#174;,+a+protective+electrode+covering,+and+a+titanium+microplate.">Deep brain stimulation electrode anchoring using BioGlue&#174;, a protective electrode covering, and a titanium microplate.</a> J Neurosci Methods. 168, 151-155 (2008).</li><li>Ettrup, K. 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=Basic+Surgical+Techniques+in+the+Minipig:+Intubation,+Transurethral+Bladder+Catheterization,+Femoral+Vessel+Catheterization,+and+Transcardial+Perfusion.">Basic Surgical Techniques in the Minipig: Intubation, Transurethral Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion.</a> J Vis Exp. (52), e2652 (2011).</li><li>Ettrup, K. S., Torn&#248;e, J., S&#248;rensen, J. C., Bjarkam, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+surgical+device+for+minimally+invasive+implantation+of+experimental+deep+brain+stimulation+leads+in+large+research+animals.">A surgical device for minimally invasive implantation of experimental deep brain stimulation leads in large research animals.</a> J Neurosci Methods. 200 (1), 41-46 (2011).</li><li>Danielsen, E. H., 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=Positron+emission+tomography+of+living+brain+in+minipigs+and+domestic+pigs.">Positron emission tomography of living brain in minipigs and domestic pigs.</a> Scand J Lab Anim Sci Suppl. 25 (1), 127-135 (1998).</li><li>R&#248;hl, L., 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=Time+evolution+of+cerebral+perfusion+and+ADC+measured+by+MRI+in+a+porcine+stroke+model.">Time evolution of cerebral perfusion and ADC measured by MRI in a porcine stroke model.</a> J Magn Reson Imaging. 15 (2), 123-129 (2002).</li><li>Cumming, P., Gillings, N. M., Jensen, S. B., Bjarkam, C. R., Gjedde, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+the+uptake+and+distribution+of+the+dopamine+D2/3+agonist+(R)-N-[1-11C]n-propylnorapomorphine+in+brain+of+healthy+and+MPTP-poisoned+Gottingen+miniature+pigs.">Kinetics of the uptake and distribution of the dopamine D2/3 agonist (R)-N-[1-11C]n-propylnorapomorphine in brain of healthy and MPTP-poisoned Gottingen miniature pigs.</a> Nucl Med Biol. 30 (5), 547-553 (2003).</li><li>Jensen, K. N., Deding, D., S&#248;rensen, J. C., Bjarkam, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+implantation+of+deep+brain+stimulation+electrodes+in+the+pontine+micturition+centre+of+the+minipig.">Long-term implantation of deep brain stimulation electrodes in the pontine micturition centre of the minipig.</a> Acta Neurochir. 151 (7), 785-794 (2009).</li><li>Rosendal, F., 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=Does+chronic+low+dose+treatment+with+ciclosporin+influence+the+brain?+A+histopathological+study+in+pigs.">Does chronic low dose treatment with ciclosporin influence the brain? A histopathological study in pigs.</a> Transplantation Proc. 37 (8), 3305-3308 (2005).</li><li>Nielsen, M. S., S&#248;rensen, J. C., Bjarkam, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+substantia+nigra+pars+compacta+of+the+minipig:+An+anatomical+and+stereological+study.">The substantia nigra pars compacta of the minipig: An anatomical and stereological study.</a> Brain Struct Funct. (4-5), 481-488 (2009).</li><li>S&#248;rensen, J. C., Bjarkam, C. R., Simonsen, C. Z., Danielsen, E., Geneser, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oriented+sectioning+of+irregular+tissue+blocks+in+relation+to+computerized+scanning+modalities.+Results+from+the+domestic+pig+brain.">Oriented sectioning of irregular tissue blocks in relation to computerized scanning modalities. Results from the domestic pig brain.</a> J Neurosci Methods. 104, 93-98 (2000).</li><li>Bjarkam, C. R., Pedersen, M., S&#248;rensen, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+strategies+for+embedding,+orientation+and+sectioning+of+small+brain+specimens+enable+direct+correlation+to+MR-images,+brain+atlases,+or+use+of+unbiased+stereology.">New strategies for embedding, orientation and sectioning of small brain specimens enable direct correlation to MR-images, brain atlases, or use of unbiased stereology.</a> J Neurosci Methods. 108, 153-159 (2001).</li><li>Bjarkam, C. R., S&#248;rensen, J. C., Geneser, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+and+morphology+of+serotonin-immunoreactive+axons+in+the+hippocampal+region+of+the+New+Zealand+white+rabbit.+I.+Area+dentata+and+hippocampus+proper.">Distribution and morphology of serotonin-immunoreactive axons in the hippocampal region of the New Zealand white rabbit. I. Area dentata and hippocampus proper.</a> Hippocampus. 13 (1), 21-37 (2003).</li><li>Bjarkam, C. R., Glud, A. N., Orlowski, D., S&#248;rensen, J. C., Palomero-Gallagher, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+telencephalon+of+the+minipig,+cytoarchitecture+and+cortical+surface+anatomy.">The telencephalon of the minipig, cytoarchitecture and cortical surface anatomy.</a> Brain Struct Funct. , (2016).</li><li>Boltze, J., Nitzsche, B., Geiger, K. D., Schoon, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathological+investigation+of+different+MCAO+modalities+and+impact+of+autologous+bone+marrow+mononuclear+cell+administration+in+an+ovine+stroke+model.">Histopathological investigation of different MCAO modalities and impact of autologous bone marrow mononuclear cell administration in an ovine stroke model.</a> Transl Stroke Res. 2, 279-293 (2011).</li><li>Jortner, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+return+of+the+dark+neuron.+A+histological+artifact+complicating+contemporary+neurotoxicologic+evaluation.">The return of the dark neuron. A histological artifact complicating contemporary neurotoxicologic evaluation.</a> Neurotoxicology. 27, 628-634 (2006).</li></ol>

Os autores não têm nada a revelar.

A maior parte dos estudos experimentais neuroscience são realizados em espécies animais pequenos, tais como ratinhos e ratos, em que o acesso ao SNC é facilitado por um de espessura da dura-máter e o crânio fina. No entanto, em animais experimentais maiores, como porcos 1, 4, 8, ovinos 32, e primatas não-humanos, a espessura considerável destas estruturas que requer a utilização de instrumentos ...

<html> estudos de neurociência de translação em suínos têm se tornado cada vez mais popular durante as últimas duas décadas. O grande tamanho do cérebro de porco permite o uso de geradores de imagens cerebrais clínica convencionais e a utilização directa e os ensaios de procedimentos neurocirúrgicos e equipamento de clínica humana 1, 2, 3, 4, 5, 6, 7, 8. Nos últimos 20 anos, porcos, especialmente porcos chineses (por exemplo, Gottingen minipig), foram usadas para examinar as modalidades de tratamento neuromoduladores, tais como transplante de células estaminais; transfecção de vector viral; e estimulação cerebral profunda dirigida para a doença de Parkinson, obesidade, depressão, doença de Alzheimer e 2, 6,= &quot;refex&quot;&gt; 9, 10, 11, 12, 13, 14, 15, 16, 17. Esta foi seguida pelo desenvolvimento de abordagens cirúrgicas estereotáxicas e para manipular o minipig SNC 3, 18, 19, 20, 21. As alterações do sistema nervoso central instituídos foram avaliadas em animais vivos utilizando imagiologia cerebral (PET 10, 13, 22, 24 e 23 MR), cistometria 11, 12, 25, a análise da marcha17, a avaliação neurológica 9, 17, e exame pós-morte com base na histologia e análise estereologia 14, 15, 17, 26, 27, 31. No entanto, a análise post-mortem requer a exposição e a remoção do cérebro de porco, que não é uma tarefa fácil, como uma cabeça dura, ósseo e uma dura-máter fibrosa que cobre rodeiam o cérebro de porco. O objectivo deste trabalho e vídeo de instruções é descrever como o cérebro de porco post-mortem e na pituitária podem ser expostos e removido em estado intacto em 15-20 min, utilizando instrumentos cirúrgicos não motorizados. O vídeo de instruções e ilustrações fotográficas mostram miniporcos do sexo masculino (idade: 6 meses, peso corporal: 20-25 kg) utilizados para um estudo anatômico na glândula pituitária minipig.

<table border="0" cellpadding="0" cellspacing="0" width="1036">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Heavy Scalpel Handle #4</td>
			<td style="width:191px;">FST (Fine Science Tools)</td>
			<td style="width:149px;">10008-13</td>
			<td style="width:481px;">Good for skin incision and soft tissue removal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-Sterile Scalpel Blades #23</td>
			<td style="width:191px;">FST&#160;</td>
			<td>10023-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel Handle #7</td>
			<td style="width:191px;">FST&#160;</td>
			<td>10007-12</td>
			<td>Optimal for dural incision and precision work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-Sterile Scalpel Blades #11</td>
			<td style="width:191px;">FST&#160;</td>
			<td>10011-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical Forceps</td>
			<td style="width:191px;">FST&#160;</td>
			<td>11024-18</td>
			<td>The tip of the surgical forceps ensure a firm grip&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kerrison Bone Punch</td>
			<td style="width:191px;">Aesculap Neurosurgery</td>
			<td style="width:149px;">FF713R</td>
			<td>Must be robust, bite size 3-5 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bone Rongeur</td>
			<td>Aesculap Neurosurgery</td>
			<td style="width:149px;">MD615</td>
			<td>Must be robust, bite size 15 x 5 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bone Rongeur</td>
			<td>Aesculap Neurosurgery</td>
			<td style="width:149px;">FO551R</td>
			<td>Must be robust, bite size 25 x 15 mm&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bone Chisel</td>
			<td>Lawton</td>
			<td>67-0335</td>
			<td>The size of the chisel head should not exceed 20 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mallet (Hammer)</td>
			<td>Millarco</td>
			<td>5624108</td>
			<td>Weigth 300 g, length 30 cm, head hit area size 2 x 2 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-Scissor</td>
			<td>FST&#160;</td>
			<td>14002-14&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissector</td>
			<td>Aesculap Neurosurgery</td>
			<td style="width:149px;">OL165R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">&#160;G&#246;ttingen minipigs</td>
			<td style="width:191px;">&#160;Ellegaard G&#246;ttingen Minipigs A/S, Denmark</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Euthanimal</td>
			<td style="width:191px;"></td>
			<td style="width:149px;"></td>
			<td>pentobarbital</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamine</td>
			<td style="width:191px;">Pfizer</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Midazolam&#160;</td>
			<td style="width:191px;">Hameln Pharmaceuticals</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

exposure of the pig cns for histological analysis: a manual for decapitation, skull opening, and brain removal

Os porcos têm se tornado cada vez mais popular em grande animal investigação translacional neurociência como um substituto economicamente e eticamente viável para primatas não-humanos. O tamanho grande do cérebro do porco permite o uso de geradores de imagens cerebrais clínica convencionais e a utilização directa e testes de procedimentos e equipamentos neurocirúrgicos de clínica humana. Uma análise mais aprofundada macroscópico e histológico, no entanto, requer exposição post-mortem do sistema de porco nervoso central (SNC) e subsequente remoção do cérebro. Esta não é uma tarefa fácil, como o porco SNC é encapsulado por uma cabeça dura, ósseo e da coluna vertebral. O objectivo deste trabalho e vídeo de instruções é descrever como a expor e remover o cérebro de porco post-mortem e a glândula pituitária num estado intacto, adequado para análise macroscópica e histológica subsequente.

Para evitar que o material tecido de secar, recomenda-se a armazenar o cérebro removido e pituitária em um frasco cheio com o fixador ou solução salina isotónica imediatamente após análise macroscópica foi realizada. O material de tecido pode ser armazenado no fixador por ano, ao passo que o armazenamento em solução salina isotónica, mesmo no frigorífico, irá conduzir à deterioração do tecido com o tempo. A pit...

Watch this Scientific Journal Video about Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal at JoVE.com

Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal

O objectivo deste trabalho e vídeo de instruções é descrever como a expor e remover o cérebro de porco e glândula pituitária pós-morte num estado intacto, adequado para análise macroscópica e histológica subsequente.

Exposição do SNC porco para análise histológica: A manual por decapitação, crânio de abertura, e Cérebro Remoção

Pigs have become increasingly popular in large-animal translational neuroscience research as an economically and ethically feasible substitute to ...

exposure-pig-cns-for-histological-analysis-manual-for-decapitation

Research

Education

JoVE Core

JoVE Journal

Anatomy and Physiology

Neuroscience

Unit 1

The Axial Skeleton

SCIENTISTS IN ACTION

15.3K visualizações.  Aalborg University Hospital.  O objectivo deste trabalho e vídeo de instruções é descrever como a expor e remover o cérebro de porco e glândula pituitária pós-morte num estado intacto, adequado para análise macroscópica e histológica subsequente. 

Vídeo: Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

A polido e reforçado Janela afinado crânio para longo prazo de imagens do cérebro de camundongo

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Neurociência

Co-analysis of Brain Structure and Function using fMRI and Diffusion-weighted Imaging

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when studying the brain, as the functional role that a brain area fulfills may be largely defined by its connections to other brain areas. In this report, we describe a novel, non-invasive approach for relating brain structure and function using magnetic resonance imaging (MRI). This approach, a combination of structural imaging of long-range fiber connections and functional imaging data, is illustrated in two distinct cognitive domains, visual attention and face perception. Structural imaging is performed with diffusion-weighted imaging (DWI) and fiber tractography, which track the diffusion of water molecules along white-matter fiber tracts in the brain (Figure 1). By visualizing these fiber tracts, we are able to investigate the long-range connective architecture of the brain. The results compare favorably with one of the most widely-used techniques in DWI, diffusion tensor imaging (DTI). DTI is unable to resolve complex configurations of fiber tracts, limiting its utility for constructing detailed, anatomically-informed models of brain function. In contrast, our analyses reproduce known neuroanatomy with precision and accuracy. This advantage is partly due to data acquisition procedures: while many DTI protocols measure diffusion in a small number of directions (e.g., 6 or 12), we employ a diffusion spectrum imaging (DSI)1, 2 protocol which assesses diffusion in 257 directions and at a range of magnetic gradient strengths. Moreover, DSI data allow us to use more sophisticated methods for reconstructing acquired data. In two experiments (visual attention and face perception), tractography reveals that co-active areas of the human brain are anatomically connected, supporting extant hypotheses that they form functional networks. DWI allows us to create a "circuit diagram" and reproduce it on an individual-subject basis, for the purpose of monitoring task-relevant brain activity in networks of interest.

We describe a novel approach for simultaneous analysis of brain function and structure using magnetic resonance imaging (MRI). We assess brain structure with high-resolution diffusion-weighted imaging and white-matter fiber tractography. Unlike standard structural MRI, these techniques allow us to directly relate anatomical connectivity to functional properties of brain networks.

Co-análise de estrutura e função cerebral usando ressonância magnética funcional e imagem ponderada em difusão

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when ...

A Comprehensive Protocol for Manual Segmentation of the Medial Temporal Lobe Structures

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, hippocampus, and the associated parahippocampal regions (perirhinal, entorhinal,&#160;and parahippocampal proper). Unlike most other tracing protocols available, typically focusing on certain MTL areas (e.g., amygdala and/or hippocampus), the integrative perspective adopted by the present tracing guidelines allows for clear localization of all MTL subregions. By integrating information from a variety of sources, including extant tracing protocols separately targeting various MTL structures, histological reports, and brain atlases, and with the complement of illustrative visual materials, the present protocol provides an accurate, intuitive, and convenient guide for understanding the MTL anatomy. The need for such tracing guidelines is also emphasized by illustrating possible differences between automatic and manual segmentation protocols. This knowledge can be applied toward research involving not only structural MRI investigations but also structural-functional colocalization and fMRI signal extraction from anatomically defined ROIs, in healthy and clinical groups alike.

The present work provides a comprehensive set of guidelines for manually tracing the medial temporal lobe (MTL) structures. This protocol can be applied to research involving structural and/or combined structural-functional magnetic resonance imaging (MRI) investigations of the MTL, in both healthy and clinical groups.

Um protocolo abrangente para segmentação manual das estruturas do lobo temporal medial

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

Analisando Synaptic Modulação da<em&gt; Drosophila melanogaster</em&gt; Fotorreceptores após exposição à luz prolongada

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

Análise do elevado-throughput de comportamento Locomotor no ensaio de ilha de Drosophila

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Gravação de plasticidade sináptica em fatias Hippocampal agudas, mantidas em um pequeno volume reciclagem - perfusão- e sistema de câmara de submersão-tipo

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, a multitude of novel clearing methodologies including CUBIC (clear, unobstructed brain imaging cocktails and computational analysis), SWITCH (system-wide control of interaction time and kinetics of chemicals), MAP (magnified analysis of the proteome), and PACT (passive clarity technique), have been established to further expand the existing toolkit for the microscopic analysis of biological tissues. The present study aims to improve upon and optimize the original PACT procedure for an array of intact rodent tissues, including the whole central nervous system (CNS), kidneys, spleen, and whole mouse embryos. Termed psPACT (process-separate PACT) and mPACT (modified PACT), these novel techniques provide highly efficacious means of mapping cell circuitry and visualizing subcellular structures in intact normal and pathological tissues. In the following protocol, we provide a detailed, step-by-step outline on how to achieve maximal tissue clearance with minimal invasion of their structural integrity via psPACT and mPACT.

Here, we present two novel methodologies, psPACT and mPACT, for achieving maximal optical transparency and subsequent microscopic analysis of tissue vasculature in the intact rodent whole CNS.

Passivo novo métodos de compensação para a produção rápida de transparência óptica no tecido inteiro do CNS

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Crônica implantação de matriz Electrocorticographic todo-cortical no sagui comum

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Extraction and Dissection of the Domesticated Pig Brain

Use of the pig as a preclinical and translatable animal model has been well-documented and accepted by research fields investigating cardiovascular systems, gastrointestinal systems, and nutrition, and the pig is increasingly being used as a large animal model in neuroscience. Furthermore, the pig is an accepted model to study neurodevelopment as it displays brain growth and development patterns similar to what occurs in humans. As a less common animal model in neuroscience, surgical and dissection procedures on pigs may not be as familiar or well-practiced among researchers. Therefore, a standardized visual protocol detailing consistent extraction and dissection methods may prove valuable for researchers working with the pig. The following video showcases a technique to remove the pig brain while keeping the cortex and brainstem intact and reviews methods to dissect several commonly investigated brain regions including the brainstem, cerebellum, midbrain, hippocampus, striatum, thalamus, and medial prefrontal cortex. The purpose of this video is to provide researchers with the tools and knowledge necessary to consistently perform a brain extraction and dissection on the four-week-old pig.

This protocol details the technique for removal of the pig brain in its entirety and dissection of several brain regions commonly studied in neuroscience.

Extração e Dissecção do Cérebro de Suínos Domesticados

Use of the pig as a preclinical and translatable animal model has been well-documented and accepted by research fields investigating cardiovascular ...

In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at cellular and subcellular resolution. Advances in molecular and imaging technologies have allowed us to gain numerous detailed insights into cellular and molecular mechanisms of brain development in the transparent zebrafish embryo. Recently, processes of refinement of neuronal connectivity that occur at later larval stages several weeks after fertilization, which are for example control of social behavior, decision making or motivation-driven behavior, have moved into focus of research. At these stages, pigmentation of the zebrafish skin interferes with light penetration into brain tissue, and solutions for embryonic stages, e.g., pharmacological inhibition of pigmentation, are not feasible anymore.
Therefore, a minimally invasive surgical solution for microscopy access to the brain of awake zebrafish is provided that is derived from electrophysiological&#160;approaches. In teleosts, skin and soft skull cartilage can be carefully removed by micro-peeling these layers, exposing underlying neurons and axonal tracts without damage. This allows for recording neuronal morphology, including synaptic structures and their molecular contents, and the observation of physiological changes such as Ca2+ transients or intracellular transport events. In addition, interrogation of these processes by means of pharmacological inhibition or optogenetic manipulation is feasible. This brain exposure approach provides information about structural and physiological changes in neurons as well as the correlation and interdependence of these events in live brain tissue in the range of minutes or hours. The technique is suitable for in vivo brain imaging of zebrafish larvae up to 30 days post fertilization, the latest developmental stage tested so far. It, thus, provides access to such important questions as synaptic refinement and scaling, axonal and dendritic transport, synaptic targeting of cytoskeletal cargo or local activity-dependent expression. Therefore, a broad use for this mounting and imaging approach can be anticipated.

Here we present a method to image the zebrafish embryonic brain in vivo upto larval and juvenile stages. This microinvasive procedure, adapted from electrophysiological approaches, provides access to cellular and subcellular details of mature neuron and can be combined with optogenetics and neuropharmacological studies for characterizing brain function and drug intervention.

In vivo Imagem de Tecido Cerebral Totalmente Ativo em Larvas de Zebrafish Acordados e Juvenis por Remoção de Crânio e Pele

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at ...