O autor gostaria de agradecer a Matthew Kelley, Tiziana Cogliati, Jessica Gumerson, Uwe Wolfrum, Rivka Levron, Viola Kretschmer e Zoe Mann e para a sua avaliação crítica do manuscrito. Este trabalho foi financiado pelo Prêmio Sofja Kovalevskaya (Humbodlt Foundation) e da Universidade Johannes-Gutenberg, Mainz, Alemanha.

<ol>
	<li>Waters, A. M., Beales, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliopathies:+an+expanding+disease+spectrum.">Ciliopathies: an expanding disease spectrum.</a> Pediatr Nephrol. 26, 1039-1056 (2011).</li><li>May-Simera, H. L., Kelley, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cilia,+Wnt+signaling,+and+the+cytoskeleton.">Cilia, Wnt signaling, and the cytoskeleton.</a> Cilia. 1, 7 (2012).</li><li>Ross, A. 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=Disruption+of+Bardet-Biedl+syndrome+ciliary+proteins+perturbs+planar+cell+polarity+in+vertebrates.">Disruption of Bardet-Biedl syndrome ciliary proteins perturbs planar cell polarity in vertebrates.</a> Nat Genet. 37, 1135-1140 (2005).</li><li>Ezan, J., Montcouquiol, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+planar+cell+polarity+in+the+inner+ear.">Revisiting planar cell polarity in the inner ear.</a> Seminars in cell &amp; developmental biology. 24, 499-506 (2013).</li><li>Semenov, M. V., Habas, R., Macdonald, B. T., He, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SnapShot:+Noncanonical+Wnt+Signaling+Pathways.">SnapShot: Noncanonical Wnt Signaling Pathways.</a> Cell. 131, 1378 (2007).</li><li>Wang, 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=Regulation+of+polarized+extension+and+planar+cell+polarity+in+the+cochlea+by+the+vertebrate+PCP+pathway.">Regulation of polarized extension and planar cell polarity in the cochlea by the vertebrate PCP pathway.</a> Nat Genet. 37, 980-985 (2005).</li><li>Montcouquiol, 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=Identification+of+Vangl2+and+Scrb1+as+planar+polarity+genes+in+mammals.">Identification of Vangl2 and Scrb1 as planar polarity genes in mammals.</a> Nature. 423, 173-177 (2003).</li><li>May-Simera, H. 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=Ciliary+proteins+Bbs8+and+Ift20+promote+planar+cell+polarity+in+the+cochlea.">Ciliary proteins Bbs8 and Ift20 promote planar cell polarity in the cochlea.</a> Development. 142, 555-566 (2015).</li><li>Jones, 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=Ciliary+proteins+link+basal+body+polarization+to+planar+cell+polarity+regulation.">Ciliary proteins link basal body polarization to planar cell polarity regulation.</a> Nat Genet. 40, 69-77 (2008).</li><li>Lim, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+structure+of+the+organ+of+Corti:+a+review.">Functional structure of the organ of Corti: a review.</a> Hearing research. 22, 117-146 (1986).</li><li>Nayak, G. D., Ratnayaka, H. S., Goodyear, R. J., Richardson, G. 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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+kinocilium+of+auditory+hair+cells+and+evidence+for+its+morphogenetic+role+during+the+regeneration+of+stereocilia+and+cuticular+plates.">The kinocilium of auditory hair cells and evidence for its morphogenetic role during the regeneration of stereocilia and cuticular plates.</a> Journal of neurocytology. 24, 633-653 (1995).</li><li>Denman-Johnson, K., Forge, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+hair+bundle+polarity+and+orientation+in+the+developing+vestibular+system+of+the+mouse.">Establishment of hair bundle polarity and orientation in the developing vestibular system of the mouse.</a> Journal of neurocytology. 28, 821-835 (1999).</li><li>van Dam, T. 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+SYSCILIA+gold+standard+(SCGSv1)+of+known+ciliary+components+and+its+applications+within+a+systems+biology+consortium.">The SYSCILIA gold standard (SCGSv1) of known ciliary components and its applications within a systems biology consortium.</a> Cilia. 2, 7 (2013).</li><li>Blacque, O. E., Sanders, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartments+within+a+compartment:+what+C.+elegans+can+tell+us+about+ciliary+subdomain+composition,+biogenesis,+function,+and+disease.">Compartments within a compartment: what C. elegans can tell us about ciliary subdomain composition, biogenesis, function, and disease.</a> Organogenesis. 10, 126-137 (2014).</li><li>Wallingford, J. B., Mitchell, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strange+as+it+may+seem:+the+many+links+between+Wnt+signaling,+planar+cell+polarity,+and+cilia.">Strange as it may seem: the many links between Wnt signaling, planar cell polarity, and cilia.</a> Genes &amp; development. 25, 201-213 (2011).</li><li>Borovina, A., Ciruna, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFT88+plays+a+cilia-+and+PCP-independent+role+in+controlling+oriented+cell+divisions+during+vertebrate+embryonic+development.">IFT88 plays a cilia- and PCP-independent role in controlling oriented cell divisions during vertebrate embryonic development.</a> Cell reports. 5, 37-43 (2013).</li><li>Huang, P., Schier, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dampened+Hedgehog+signaling+but+normal+Wnt+signaling+in+zebrafish+without+cilia.">Dampened Hedgehog signaling but normal Wnt signaling in zebrafish without cilia.</a> Development. 136, 3089-3098 (2009).</li><li>Ocbina, P. J., Tuson, M., Anderson, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+cilia+are+not+required+for+normal+canonical+Wnt+signaling+in+the+mouse+embryo.">Primary cilia are not required for normal canonical Wnt signaling in the mouse embryo.</a> PloS one. 4, e6839 (2009).</li><li>Jones, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+electron+microscopy:+preparation+and+imaging+for+SEM.">Scanning electron microscopy: preparation and imaging for SEM.</a> Methods Mol Biol. 915, 1-20 (2012).</li><li>Curtin, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+of+Celsr1+disrupts+planar+polarity+of+inner+ear+hair+cells+and+causes+severe+neural+tube+defects+in+the+mouse.">Mutation of Celsr1 disrupts planar polarity of inner ear hair cells and causes severe neural tube defects in the mouse.</a> Current biology : CB. 13, 1129-1133 (2003).</li><li>Wang, Y., Guo, N., Nathans, J. <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+Frizzled3+and+Frizzled6+in+neural+tube+closure+and+in+the+planar+polarity+of+inner-ear+sensory+hair+cells.">The role of Frizzled3 and Frizzled6 in neural tube closure and in the planar polarity of inner-ear sensory hair cells.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 26, 2147-2156 (2006).</li><li>Montcouquiol, M., Jones, J. M., Sans, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+planar+polarity+proteins+in+mammalian+cochlea.">Detection of planar polarity proteins in mammalian cochlea.</a> Methods Mol Biol. 468, 207-219 (2008).</li><li>May-Simera, H., Kelley, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examining+planar+cell+polarity+in+the+mammalian+cochlea.">Examining planar cell polarity in the mammalian cochlea.</a> Methods Mol Biol. 839, 157-171 (2012).</li><li>Yin, H., Copley, C. O., Goodrich, L. V., Deans, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+phenotypes+between+different+vangl2+mutants+demonstrates+dominant+effects+of+the+Looptail+mutation+during+hair+cell+development.">Comparison of phenotypes between different vangl2 mutants demonstrates dominant effects of the Looptail mutation during hair cell development.</a> PloS one. 7, e31988 (2012).</li><li>Rachel, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+Cep290+and+Mkks+ciliopathy+alleles+in+mice+rescues+sensory+defects+and+restores+ciliogenesis.">Combining Cep290 and Mkks ciliopathy alleles in mice rescues sensory defects and restores ciliogenesis.</a> J Clin Invest. 122, 1233-1245 (2012).</li><li>Jagger, 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=Alstrom+Syndrome+protein+ALMS1+localizes+to+basal+bodies+of+cochlear+hair+cells+and+regulates+cilium-dependent+planar+cell+polarity.">Alstrom Syndrome protein ALMS1 localizes to basal bodies of cochlear hair cells and regulates cilium-dependent planar cell polarity.</a> Human molecular genetics. 20, 466-481 (2011).</li><li>Collin, G. B., 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+Alstrom+Syndrome+Protein,+ALMS1,+Interacts+with+alpha-Actinin+and+Components+of+the+Endosome+Recycling+Pathway.">The Alstrom Syndrome Protein, ALMS1, Interacts with alpha-Actinin and Components of the Endosome Recycling Pathway.</a> PloS one. 7, e37925 (2012).</li><li>Copley, C. O., Duncan, J. S., Liu, C., Cheng, H., Deans, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+refinement+of+auditory+hair+cell+planar+polarity+deficits+occurs+in+the+absence+of+Vangl2.">Postnatal refinement of auditory hair cell planar polarity deficits occurs in the absence of Vangl2.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 33, 14001-14016 (2013).</li><li>Sorusch, N., Wunderlich, K., Bauss, K., Nagel-Wolfrum, K., Wolfrum, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Usher+syndrome+protein+network+functions+in+the+retina+and+their+relation+to+other+retinal+ciliopathies.">Usher syndrome protein network functions in the retina and their relation to other retinal ciliopathies.</a> Advances in experimental medicine and biology. 801, 527-533 (2014).</li><li>Hebert, J. M., McConnell, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+cre+to+the+Foxg1+(BF-1)+locus+mediates+loxP+recombination+in+the+telencephalon+and+other+developing+head+structures.">Targeting of cre to the Foxg1 (BF-1) locus mediates loxP recombination in the telencephalon and other developing head structures.</a> Dev Biol. 222, 296-306 (2000).</li><li>Willott, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+8,+Measurement+of+the+auditory+brainstem+response+(ABR)+to+study+auditory+sensitivity+in+mice.">Chapter 8, Measurement of the auditory brainstem response (ABR) to study auditory sensitivity in mice.</a> Current protocols in neuroscience. , Unit8 21B (2006).</li><li>Martin, G. K., Stagner, B. B., Lonsbury-Martin, B. 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=Chapter+8,+Assessment+of+cochlear+function+in+mice:+distortion-product+otoacoustic+emissions.">Chapter 8, Assessment of cochlear function in mice: distortion-product otoacoustic emissions.</a> Current protocols in neuroscience. , Unit8 21C (2006).</li><li>Finetti, 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=Intraflagellar+transport+is+required+for+polarized+recycling+of+the+TCR/CD3+complex+to+the+immune+synapse.">Intraflagellar transport is required for polarized recycling of the TCR/CD3 complex to the immune synapse.</a> Nature cell biology. 11, 1332-1339 (2009).</li><li>Sedmak, T., Wolfrum, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraflagellar+transport+molecules+in+ciliary+and+nonciliary+cells+of+the+retina.">Intraflagellar transport molecules in ciliary and nonciliary cells of the retina.</a> J Cell Biol. 189, 171-186 (2010).</li><li>Yuan, S., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+horizons:+ciliary+proteins+reach+beyond+cilia.">Expanding horizons: ciliary proteins reach beyond cilia.</a> Annual review of genetics. 47, 353-376 (2013).</li><li>Tarchini, B., Jolicoeur, C., Cayouette, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+blueprint+at+the+apical+surface+establishes+planar+asymmetry+in+cochlear+hair+cells.">A molecular blueprint at the apical surface establishes planar asymmetry in cochlear hair cells.</a> Developmental cell. 27, 88-102 (2013).</li><li>Ezan, 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=Primary+cilium+migration+depends+on+G-protein+signalling+control+of+subapical+cytoskeleton.">Primary cilium migration depends on G-protein signalling control of subapical cytoskeleton.</a> Nature cell biology. 15, 1107-1115 (2013).</li></ol>

The author declares no competing financial interests.

Ao preparar o tecido coclear para análise, existem alguns pontos-chave a ter em conta. Em primeiro lugar, as diferenças de fundo genético pode modificar o fenótipo coclear, tornando-se necessário analisar e comparar apenas os controlos da mesma ninhada. Em segundo lugar, a remoção completa da membrana tectória é necessário para obter as melhores imagens com imuno-histoquímica e é essencial para SEM. A membrana tectória é uma estrutura opaca e pode obscurecer as células do epitélio sensorial directamente ...

cílios são apêndices longos à base de microtúbulos que se estendem a partir da superfície da maioria das células de mamífero. cílios são muitas vezes confundidos com cílios móveis, dos quais há sempre múltiplas por célula, e cuja finalidade é mover o líquido através de superfícies de membranas. cílios, ao contrário, adotar papéis sensoriais e são, consequentemente, também referida como cílios sensorial. Uma vez que há muito esquecido, esta organela foi recentemente &#39;redescoberto&#39; como resultado de sua associação com uma infinidade de doenças genéticas humanas 1. Idealmente posicionado como um organelo de sinalização, o cílio primário tem sido demonstrado que regulam numerosas vias de sinalização, muitas das quais são importantes, não só na homeostasia dos tecidos e doença, como também durante o desenvolvimento 2. Uma das primeiras vias de sinalização mostrado estar associada com disfunção cílios foi a via de sinalização Wnt não canónicas, também conhecida como a polaridade celular planar (PCP) via <sup<html> &gt; 3. Esta cascata de sinalização inicialmente identificado em Drosophila, é crítico para a embriogénese; em particular para os processos de convergência e de extensão e para a orientação correcta de células no plano de epitélios 4. A sinalização sequencial de um núcleo de um conjunto de proteínas reguladoras traduz sinais direccionais que em última análise levam a rearranjos do citoesqueleto e resultam na polarização coordenada de células epiteliais num plano 5. O processo de convergência e de extensão é absolutamente necessária para o ducto coclear para alongar e para a correcta modelação celular 6. Como este é regulado através da activação da via de PCP, um dos mais marcantes de fenótipos mutantes PCP cóclea é um ducto coclear encurtado com desorganizado epitélio sensorial 7. Da mesma forma, ratos mutantes, que não têm cílios, também apresentam como uma convergência e extensão fenótipo 8,9, embora precisamente como isso é regulado continua a ser elucidado. ve_content &quot;&gt; Como os processos de convergência e de extensão são críticas para o crescimento do ducto coclear, e padronização celular do epitélio sensorial dentro do ducto coclear, na cóclea em desenvolvimento é um órgão ideal para analisar a sinalização PCP durante o desenvolvimento dos vertebrados. O órgão de Corti, o termo dado para o epitélio sensorial especializado que as linhas do ducto coclear, é constituído por células de suporte não-sensoriais e células ciliadas mecanosensorial que devem ser uniformemente orientada para a cóclea para funcionar 10. as células ciliadas mecanosensorial são assim chamados por causa da feixes stereociliary que se estendem a partir da placa cuticular (superfície apical) de cada uma das células pilosas sensitivas 11. Estes agem como transdutores primárias de mechanosensation e apesar da sua nomenclatura como estereocílios, são na verdade, composto de microvilosidades à base de filamentos de actina modificado. Dentro de cada cabelo em forma de viga pacote, três fileiras de estereocílios são organizados em uma pa altamente ordenada e regular,ttern em um caso semelhante a forma de escada. Cílios à base de microtúbulos real, kinocilia chamados, são necessários para o desenvolvimento e orientação dos feixes stereociliary 12. Em cima de cada célula de cabelo, um único cinocílio está fisicamente ligado ao pacote stereocilia, com localização central adjacente à linha mais alta de estereocílios. A função precisa da cinocílio não é clara, e uma hipótese é que a cinocílio &#39;puxa&#39; estereocílios em forma à medida que amadurecem a partir microvilosidades 12. Nos vertebrados, kinocilia na cóclea estão presentes apenas transitoriamente e retrair a partir das células ciliadas em ratos antes do início da audição 11,13,14. Perda completa dos cílios na cóclea em desenvolvimento resulta em ductos cocleares severamente encurtado, mis-formados e mal-orientada feixes stereociliary, bem como corpos basais mis-posicionado 8,9. Um cílio funcional não é apenas composto pelo axonema ciliar. Muitas proteínas associadas com cíliosfunção ocorrem em complexos localizados para subdomínios cílios-relacionadas, como o corpo basal, zona de transição, ou axonema ciliar 15. O corpo basal, derivado do centriole mãe do centrossoma, é também um centro-organização de microtúbulos de microtúbulos que se estendem para longe do cílio para o corpo celular e pode regular o tráfico intracelular, bem como o tráfico ciliar. A zona de transição ciliar é outra região onde a função ciliar é regulada em termos de organização de importação e exportação de compostos ciliares 16. Vários estudos têm identificado uma ligação entre os cílios e Wnt não canónicas (sinalização PCP), embora o mecanismo exacto é incerto 17. Redundância dos genes ciliares e do PCP e da sensibilidade da polaridade celular para anormalidades celulares generalizadas, tornam difícil ligar diretamente uma mutação a déficits específicos do PCP. Uma das saídas de leitura de sinalização PCP é o posicionamento do corpo basal e Primary cílio, portanto, segregando o principal dos defeitos secundários é um desafio. Alguns estudos em peixes-zebra e mouse mutantes sugeriram nenhuma conexão entre os cílios e sinalização Wnt 18-20. Discrepâncias nos dados podem refletir espécies, tecidos ou diferenças temporais dependentes das contribuições ciliares no sentido de sinalização Wnt. Além disso, a capacidade de resposta Wnt normal, pode ser mantido se os corpos basais permanecem funcionais. Uma visão mais profunda sobre o papel das proteínas ciliares em vias de sinalização celular e outros fenômenos biológicos é crucial para a nossa compreensão da biologia celular e de desenvolvimento, bem como para o desenvolvimento de estratégias de tratamento direcionados.

<table>
	<tbody>
		<tr>
			<td>Tools/Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone elastomere - Sylgard 184</td>
			<td>Sigma-Aldrich</td>
			<td>761028-5EA</td>
			<td>See Note 2</td>
		</tr>
		<tr>
			<td>Micro dissecting scissors-straight blade</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps (no. 5 and 55) and blunt forceps</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope.</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Uncoated glass microscope slides</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover slips (22 mm &#215; 40 mm &#215; 0.15 mm)</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipettes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Minutien pins</td>
			<td>&#160;Fine Science Tools</td>
			<td>26002-10</td>
			<td></td>
		</tr>
		<tr>
			<td>SEM sample holder</td>
			<td>tousimis</td>
			<td>8762</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning electron microscopy studs</td>
			<td>TED PELLA</td>
			<td>16111</td>
			<td></td>
		</tr>
		<tr>
			<td>PELCO Tabs: Carbon adhesive</td>
			<td>TED PELLA</td>
			<td>16084-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent Microscope</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Critical Point Dryer</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass microscope slides</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipe Tissue&#160;</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Paint Brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1&#215; Phosphate buffered saline (PBS)</td>
			<td>Gibco/Life Technologies</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde&#160; (PFA) (EM Grade Required for EM)</td>
			<td>Various</td>
			<td></td>
			<td>Prepare a 4% solution in 1&#215; PBS made fresh each time. EM Grade Required for EM.</td>
		</tr>
		<tr>
			<td>2.5% Glutaraldehyde Grade1</td>
			<td>Sigma-Aldrich</td>
			<td>G5882</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl (pH 7.5)</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl 2</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AffiniPure Fab Fragment Donkey Anti-Mouse IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>715-007-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G Mounting media</td>
			<td>SouthernBiotech</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>10&#215; Hanks&#8217; Balanced Salt Solution (HBSS)</td>
			<td>Gibco/Life Technologies</td>
			<td>14065</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Gibco/Life Technologies</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetroxide (OsO4 )</td>
			<td>Sigma-Aldrich/Fluka Analytical</td>
			<td>75632</td>
			<td></td>
		</tr>
		<tr>
			<td>Tannic acid</td>
			<td>Sigma-Aldrich</td>
			<td>403040</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti Arl13b</td>
			<td>Protein Tech</td>
			<td>17711-1-AP</td>
			<td>Suggested concentration 1:1000</td>
		</tr>
		<tr>
			<td>anti acetylated tubulin (611-B1)</td>
			<td>Sigma-Aldrich</td>
			<td>T6793</td>
			<td>Suggested concentration 1:800</td>
		</tr>
		<tr>
			<td>anti gamma tubulin (GTU-88)</td>
			<td>Sigma-Aldrich</td>
			<td>T6557</td>
			<td>Suggested concentration 1:200</td>
		</tr>
		<tr>
			<td>anti Zo_1&#160;</td>
			<td>Invitrogen</td>
			<td>40-2300</td>
			<td>Suggested concentration 1:500</td>
		</tr>
		<tr>
			<td>Myosin VI</td>
			<td>Proteus Biosciences</td>
			<td>25-6791</td>
			<td>Suggested concentration 1:1000</td>
		</tr>
		<tr>
			<td>Myosin VIIa</td>
			<td>Proteus Biosciences</td>
			<td>25-6790</td>
			<td>Suggested concentration 1:1000</td>
		</tr>
		<tr>
			<td>anti Vangl2</td>
			<td>Merk Millipore</td>
			<td>ABN373</td>
			<td>Suggested concentration 1:250</td>
		</tr>
		<tr>
			<td>anti G&#945;i3</td>
			<td>Sigma-Aldrich</td>
			<td>G4040</td>
			<td>Suggested concentration 1:250</td>
		</tr>
		<tr>
			<td>Alexa Fluor&#174; 488 Phalloidin</td>
			<td>Invitrogen/Life Technologies</td>
			<td>A12379</td>
			<td>Suggested concentration 1:300-1000</td>
		</tr>
		<tr>
			<td>Alexa Fluor&#174; 568 Phalloidin</td>
			<td>Invitrogen/Life Technologies</td>
			<td>A12380</td>
			<td>Suggested concentration 1:300-1000</td>
		</tr>
	</tbody>
</table>

evaluation of planar-cell-polarity phenotypes in ciliopathy mouse mutant cochlea

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest signaling pathways shown to be associated with ciliary function was the non-canonical Wnt signaling pathway, also referred to as planar cell polarity (PCP) signaling. One of the best places in which to study the effects of planar cell polarity (PCP) signaling during vertebrate development is the mammalian cochlea. PCP signaling disruption in the mouse cochlea disrupts cochlear outgrowth, cellular patterning and hair cell orientation, all of which are affected by cilia dysfunction. The goal of this protocol is to describe the analysis of PCP signaling in the developing mammalian cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy. Defects in convergence and extension are manifested as a shortening of the cochlear duct and/or changes in cellular patterning, which can be quantified following dissection from developing mouse mutants. Changes in stereociliary bundle orientation and kinocilia length or positioning can be observed and quantitated using either immunofluorescence or scanning electron microscopy (SEM). A deeper insight into the role of ciliary proteins in cellular signaling pathways and other biological phenomena is crucial for our understanding of cellular and developmental biology, as well as for the development of targeted treatment strategies.

Cóclea Dissecção e Preparação de tecidos Após remoção do cérebro, pós-linha média dissecção sagital de uma cabeça de rato P0, o labirinto ósseo, visto de trás, pode ser visualizado (Figura 1A, seta branca) e removido. A Figura 1B mostra os ósseas labirintos isolado com o cabelo coclear virados para cima, ventral, (à esquerda) e por trás, dorsal (direita). A ...

Watch this Scientific Journal Video about Evaluation of Planar-Cell-Polarity Phenotypes in Ciliopathy Mouse Mutant Cochlea at JoVE.com

Evaluation of Planar-Cell-Polarity Phenotypes in Ciliopathy Mouse Mutant Cochlea

Avaliação da Planar-Cell-Polaridade fenótipos em Ciliopathy rato mutante cóclea

Primary cilia influence various signaling pathways. The mammalian cochlea is ideal for examining planar cell polarity (PCP) signaling. Cilia dysfunction affects cochlear outgrowth, cellular patterning and hair cell orientation, readouts of PCP. Our goal is to analyze PCP signaling in mouse cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy.

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest ...

evaluation-planar-cell-polarity-phenotypes-ciliopathy-mouse-mutant

Research

JoVE Journal

Medicine

10.3K visualizações.  Johannes Gutenberg-Universität Mainz. Primary cilia influence various signaling pathways. The mammalian cochlea is ideal for examining planar cell polarity (PCP) signaling. Cilia dysfunction affects cochlear outgrowth, cellular patterning and hair cell orientation, readouts of PCP. Our goal is to analyze PCP signaling in mouse cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy.

Vídeo: Evaluation of Planar-Cell-Polarity Phenotypes in Ciliopathy Mouse Mutant Cochlea

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

Hiperinsulinêmico euglicêmico-Clamps em Consciente, Ratos Unrestrained

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

Medicina

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

Avaliação longitudinal do rato Perda Hind Limb óssea após lesão medular usando Novel, In vivo Metodologia,

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

Stroke is the third cause of mortality and the leading cause of disability in the World. Ischemic stroke accounts for approximately 80% of all strokes. However, the thrombolytic tissue plasminogen activator (tPA) is the only treatment of acute ischemic stroke that exists. This led researchers to develop several ischemic stroke models in a variety of species. Two major types of rodent models have been developed: models of global cerebral ischemia or focal cerebral ischemia. To mimic ischemic stroke in patients, in whom approximately 80% thrombotic or embolic strokes occur in the territory of the middle cerebral artery (MCA), the intraluminal middle cerebral artery occlusion (MCAO) model is quite relevant for stroke studies. This model was first developed in rats by Koizumi et al. in 1986 1. Because of the ease of genetic manipulation in mice, these models have also been developed in this species 2-3.
	Herein, we present the transient MCA occlusion procedure in C57/Bl6 mice. Previous studies have reported that physical properties of the occluder such as tip diameter, length, shape, and flexibility are critical for the reproducibility of the infarct volume 4. Herein, a commercial silicon coated monofilaments (Doccol Corporation) have been used. Another great advantage is that this monofilament reduces the risk to induce subarachnoid hemorrhages. Using the Zeiss stereo-microscope Stemi 2000, the silicon coated monofilament was introduced into the internal carotid artery (ICA) via a cut in the external carotid artery (ECA) until the monofilament occludes the base of the MCA. Blood flow was restored 1 hour later by removal of the monofilament to mimic the restoration of blood flow after lysis of a thromboembolic clot in humans. The extent of cerebral infarct may be evaluated first by a neurologic score and by the measurement of the infarct volume. Ischemic mice were thus analyzed for their neurologic score at different post-reperfusion times. To evaluate the infarct volume, staining with 2,3,5-triphenyltetrazolium chloride (TTC) was usually performed. Herein, we used cresyl violet staining since it offers the opportunity to test many critical markers by immunohistochemistry. In this video, we report the MCAO procedure; neurological scores and the evaluation of the infarct volume by cresyl violet staining.

The intraluminal middle cerebral occlusion model in mice is herein presented. The extent of cerebral infarct is evaluated by a neurologic score and cresyl violet staining, an alternative staining to TTC, offering the great advantage to test in parallel many interest markers.

Modelo de Rato OACM Intraluminal: Avaliação infarto cerebral por coloração violeta Cresyl

Stroke is the third  cause of mortality and the leading cause of disability in the World. Ischemic  stroke accounts for approximately 80% of all strokes. ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

Um método simples de intubação Lung Rato

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Avaliação de metástases pulmonares em Rato mamárias tumorais Models por Quantitative PCR em tempo real

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

Avaliação histomorfométrica padronizada da osteoartrite em um modelo de rato cirúrgico

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...

Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining endocochlear potential, ion transport, and fluid balance. Different forms of hearing disorders are reported to involve abnormal microcirculation in the cochlea. Investigation of how cochlear blood flow (CoBF) pathology affects hearing function is challenging due to the lack of feasible interrogation methods and the difficulty in accessing the inner ear. An open vessel-window in the lateral cochlear wall, combined with fluorescence intravital microscopy, has been used for studying CoBF changes in vivo, but mostly in guinea pig and only recently in the mouse. This paper and the associated video describe the open vessel-window method for visualizing blood flow in the mouse cochlea. Details include 1) preparation of the fluorescent-labeled blood cell suspension from mice; 2) construction of an open vessel-window for intravital microscopy in an anesthetized mouse, and 3) measurement of blood flow velocity and volume using an offline recording of the imaging. The method is presented in video format to show how to use the open window approach in mouse to investigate structural and functional changes in the cochlear microcirculation under normal and pathological conditions.

An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological conditions.

Medição do fluxo sanguíneo strial em Cochlea de rato utilizando uma janela de vaso aberto e microscopia de fluorescência intravital

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining ...

Evaluation of Hepatic Glucose Production in a Polycystic Ovary Syndrome Mouse Model

Polycystic ovary syndrome (PCOS) is a common disease that results in disorders of glucose metabolism, such as insulin resistance and glucose intolerance. Dysregulated glucose metabolism is an important manifestation of the disease and is the key to its pathogenesis. Therefore, studies involving evaluation of glucose metabolism in PCOS are of utmost importance. Very few studies have quantified hepatic glucose production directly in PCOS models using non-radioactive glucose tracers. In this study, we discuss step-by-step instructions for the quantification of the rate of hepatic glucose production in a PCOS mouse model by measuring M+2 enrichment of [6,6-2H2]glucose, a stable isotopic glucose tracer, via gas chromatography - mass spectrometry (GCMS). This procedure involves creation of stable isotopic glucose tracer solution, use of tail vein catheter placement and infusion of the glucose tracer in both fasting and glucose-rich states in the same mouse in tandem. The enrichment of [6,6-2H2]glucose is measured using pentaacetate derivative in GCMS. This technique can be applied to a wide variety of studies involving direct measurement of the rate of hepatic glucose production.

This study describes the direct measurement of hepatic glucose production in a polycystic ovary syndrome mouse model by using a stable isotopic glucose tracer via tail vein in both fasting and glucose-rich states in tandem.

Avaliação da Produção de Glicose Hepática em um Modelo de Rato da Síndrome do Ovário Policístico

Polycystic ovary syndrome (PCOS) is a common disease that results in disorders of glucose metabolism, such as insulin resistance and glucose intolerance. ...

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.

Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.

Avaliação fisiofisiológica da retina em um modelo de rato

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal ...

Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to detect drug exposure. The rat blood sampling method determines the quality of the plasma and further affects the precision of the test results. The subclavian vein blood collection method described in this protocol collects blood samples repeatedly in the conscious state of animals to meet the needs of PK and TK tests. The skills of restraint handling and appropriate procedure of needle incision ensure the success rate of blood collection. It is easy to operate while ensuring the quality of plasma and at the same time catering to animal welfare. However, this method requires skilled operation, and an improper one may cause animal weakness, pain, lameness, and even mortality. The current method has been used in the testing facility for a 4-week oral toxicity study in Sprague Dawley (SD) rats with TK. The maximum amount of blood collected within 24 h did not exceed 20% of the animal's total blood. The animals' body weight was more than 200 g for males and females. The data showed that the animals' bodyweight increased steadily every week, and the clinical observation was normal after repetitive sample collection.

The present protocol describes a simple and efficient method for collecting blood from the subclavian vein in rats. It enables rapid, timely, and easily identifiable sampling without anesthesia and obtains high-quality blood through repetitive sample collection.

Amostragem Repetitiva de Sangue da Veia Subclávia de Rato Consciente

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to ...