The authors have nothing to disclose.

Tüm işlemler &quot;Laboratuar Hayvan Bakım Prensipleri&quot; anlatılan yönergeleri izleyerek gerçekleştirilir (Sağlık yayın National Institutes hayır. 85-23, 1985 revize) ve onaylı Rosalind Franklin Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Kurulu hayvan protokol 08-19. 1.. Saç Hücre Nekroz gentamisin-indüksiyon <ol><li> % 0.004 lik bir nihai konsantrasyonda (4.32 mM) normal tuzlu su içinde gentamisin sülfat hazırlayın. </li><li> 0.004% (4.32 mM) gentamisin çözeltisi içeren bir kap içinde yetişkin balık (D. rerio, yaş 4-6 ay) yerleştirin. Herhangi bir kap kullanılabilir ancak geniş 7, yüksek 6, 7 ve uzun olan bir Pharmacal Su Sistemi Bir balık kap kullanır. 24 saat boyunca 28 ° C&#39;de ayarlanmış bir kuluçka makinesi içinde balık kap yerleştirin. 24 saatlik bir süre için uygun bir halde balık korumak için yeterli düzeyde tanktaki sıvının toplam hacmi ayarlayın. Not: gentamisin sıvının havalandırma NECE değilssary yeterli bir miktar, tedavi edilen balık sayısı için kullanılırsa. </li></ol> Saç Hücreleri 2. Vital Boyama <ol><li> 15 mg / ml &#39;lik bir işlem stok çözeltisinden floresan vital boya [4-4-diethylaminostyryl)-N-metilpiridinyum iyodit (485 nm eksitasyon ve metanol içinde λ 603 nm emisyon λ) (normal tuzlu su içinde)% 0.08 bir konsantrasyonda hazırlanması etanol. </li><li> Gentamisin ile tedavinin, tedavi araştırmak vital boya ihtiva eden bir 6 yuvalı kültür plakasının kuyudaki balık yerleştirerek hemen boyanmış kontrol ve gentamisin bir alt kümesini etkili olup olmadığını belirlemek. Bu balık 75 üzerinden dakika boyunca lekeli değildir bu nedenle, istatistiksel anlamlılık elde edilmesi için gerekli olan yeterli bir balık sayısı (ve kültür plakaları kullanın. Neuromast sayma muayene eden hızına bağlı olarak, zaman içinde karşı karşıya gelmeyen bir şekilde plakalarda balık yer Adım 2.3 &#39;de tarif edildiği gibi. </li><li> Floresan mikroskop ile bir tezgah çekmece adım 2.2 tabak yerleştirinlekeli neuromasts incelenmesi için de kullanılabilir. Oda sıcaklığında 1 saatlik bir süre boyunca boyama hayati boyanın söndürme önlemek için oda ışıkları kapatın. </li><li> Boya yıkama-out ve anestezik su depoları hem hazırlayın. Boya bir yıkama suyu normal bir balık su ve normal balık su içinde bir 1:1000 seyreltme elde edilir, böylece anestezi, su için yeterli bir 2-fenoksietanol ilave edin. </li><li> Aşırı hayati boya durulayın ve hayati boya lekeli balık gözlem için 3.1 adıma geçmek için fazla normal bir balık suda balık yerleştirin. </li><li> 28 ° C de 8-16 arasında saat için bir kuluçka makinesi normal balık su içinde yıkandı, gentamisin ile tedavi edilen balık transferi, neuromasts rejenerasyonunu incelemek </li><li> 8-16 saat arasında, çeşitli zamanlarda, balık, inkübatörden çıkarılmıştır yıkandı ve adımlar 2,1-2,4 belirtildiği gibi boyanır. Hayati boya lekeli balık gözlem için 3.1 adıma geçin. </li></ol> 3.. Anestezi Balık ve Neuromasts Floresan Sayma <ol><li&gt; Aşırı sıvı kaldırmak ve daha sonra plastik bir Petri kabı kapağının merkezli filtre kağıdı nemli bir parçası üzerine yerleştirmek için bir kağıt havlu üzerinde her balık kurutun. </li><li> Orta gövde dikiş hayati boya lekeli neuromasts bir dijital görüntü elde etmek için bir floresan stereo mikroskop sahnede kapağı yerleştirin. </li><li> Kullanın floresan stereo mikroskop üzerine yerleştirilen bir dijital kamera sonraki kantitatif analiz için görüntüleri yakalamak için 2X büyütme ayarlayın. Stereo mikroskop büyütme ayarı kullanılan mikroskop markası bağlı olabilir, ancak ayar orta gövde dikişleri içinde kolay görüntüleme ve bireysel neuromasts sayma izin vermelidir: Not. </li><li> (Bkz. Şekil 1) sağ göğüs fin için sadece yakın balık en alt ventral tarafında dört belirlenen ilmek içinde görünür neuromasts sayısını sayarak rejenerasyon miktarını belirler. İstatistiksel analiz için böyle ANOVA o gibi uygun bir testi kullanmakr Student T-testi. Deneyler, her bir zaman noktasında 5 balık az kullanmak gerekir ve bütün deneyler 3x az tekrarlanmalıdır. </li><li> Neuromast rejenerasyon zaman eğrisinin dayanarak (bkz. Şekil 3), rejenerasyon eğrisinin doğrusal faz içinde olduğu 8-16 saat sonrası gentamisin yıkama-out arasında neuromasts saymak. Not: Doğrusal zaman aşamasında kullanılması deney ve kontrol grupları arasında uygun bir kantitatif analiz için izin verir. </li></ol> Neuromast istatistiksel olarak anlamlı değildi ise Kantitatif Analiz Yüksek Çözünürlük alma için Bireysel Saç Hücreleri 4. Floresan Sayma <ol><li> Neuromasts düzeyinde kantitatif analiz önemli değilse, tek tek saç hücre düzeyinde analiz de daha yüksek bir çözünürlük derecesi elde etmek için kullanılabilir. Belirli bir zaman noktası sonrası gentamisin wash-out (zaman noktasını önceki neuromast çalışmalara dayanarak), vital boya st balıkları seçinProtokol 2 de tarif edildiği gibi balık ain ve daha sonra 1-5 dakika boyunca, bir 1:500 seyreltmede 2-fenoksietanol kullanarak balık euthanize. </li><li> Aşağıdaki gibi, bir kare flep hazırlama yapılır, böylece söndürme önlemek için, uygun ışık olarak, dört kesiler yapmak. Bu anal yüzgeçleri ile aynı hizaya gelene kadar balık üst kaburgalar boyunca bir kesi yapmak, sonra göbek arasında bir kesi yapmak, ve nihayet, bu yüzden kare flep oluşturulan bu kesiler her iki tarafında iki dikey kesiler yapmak. Not: Bu cilt hazırlama neuromast deneylerde kullanılan orta gövde dikiş dahil edecektir. </li><li> Bir cam slayt deri örneği yerleştirin ve sonra çapa yardım ve sonraki dijital görüntüleme için doku düzleştirmek için kesilip deri örneğinin üzerine bir yuvarlak cam kapak kayma yerleştirin. </li><li> Aşama 4.3 gelen deri örnekleri kullanarak, orta gövde dikişlerin her biri neuromast içindeki saç hücrelerinin dijital görüntü elde etmek. En az 60X büyütme görüntüleri almak ve sonra saç ce saymakdeney ve kontrol gruplarının karşılaştırmalı kantitatif analiz için ayrı neuromasts içinde lls (bkz. Şekil 4). </li></ol>

<ol>
	<li>Dambly-Chaudire, C., Sapde, D., Soubiran, F., Decorde, K., Gompel, N., Ghysen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Lateral+Line+of+Zebrafish:+a+Model+System+for+the+Analysis+of+Morphogenesis+and+Neural+Development+in+Vertebrates.">The Lateral Line of Zebrafish: a Model System for the Analysis of Morphogenesis and Neural Development in Vertebrates.</a> Biol. Cell. 95 (9), 579-587 (2003).</li><li>Montgomery, J., Carton, G., Voigt, R., Baker, C., Diebel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+Processing+of+Water+Currents+by+Fishes.">Sensory Processing of Water Currents by Fishes.</a> Phil. Trans. Royal Soc. London B Biol. Sci. 355 (1401), 1325-1327 (2000).</li><li>Buck, L. M., Winter, M. J., Redfern, W., Whitfield, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ototoxin-Induced+Cellular+Damage+in+Neuromasts+Disrupts+Lateral+Line+Function+in+Larval+Zebrafish.">Ototoxin-Induced Cellular Damage in Neuromasts Disrupts Lateral Line Function in Larval Zebrafish.</a> Hearing Res. 284 (1-2), 1-2 (2012).</li><li>Engelmann, J., Hanke, W., Mogdans, J., Bleckmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+Stimuli+and+the+Fish+Lateral+Line.">Hydrodynamic Stimuli and the Fish Lateral Line.</a> Nature. 408 (6808), 51-52 (2000).</li><li>Olszewski, J., Haehnel, M., Taguch, M., Liao, 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=Zebrafish+Larvae+Exhibit+Rheotaxis+and+Can+Escape+a+Continuous+Suction+Source+Using+Their+Lateral+Line.">Zebrafish Larvae Exhibit Rheotaxis and Can Escape a Continuous Suction Source Using Their Lateral Line.</a> PloS One. 7 (5), e36661 (2012).</li><li>Raible, D. W., Kruse, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+the+Lateral+Line+System+in+Embryonic+Zebrafish.">Organization of the Lateral Line System in Embryonic Zebrafish.</a> J. Comp. Neurol. 421 (2), 189-198 (2000).</li><li>Coffin, A. B., Reinhart, K. E., Owens, K. N., Raible, D. W., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Extracellular Divalent Cations Modulate Aminoglycoside-Induced Hair Cell Death in the Zebrafish Lateral. 253 (1-2), 1-2 (2009).</li><li>Harris, J. A., Cheng, A. G., Cunningham, L. L., MacDonald, G., Raible, D. W., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neomycin-Induced Hair Cell Death and Rapid Regeneration in the Lateral Line of Zebrafish (Danio. 4 (2), 219-234 (2003).</li><li>Ma, E. Y., Rubel, E. W., Raible, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+Signaling+Regulates+the+Extent+of+Hair+Cell+Regeneration+in+the+Zebrafish+Lateral+Line).">Notch Signaling Regulates the Extent of Hair Cell Regeneration in the Zebrafish Lateral Line).</a> J. Neurosci. 28 (9), 2261-2273 (2008).</li><li>Brignull, H. R., Raible, D. W., Stone, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feathers+and+Fins:+Non-Mammalian+Models+for+Hair+Cell+Regeneration.">Feathers and Fins: Non-Mammalian Models for Hair Cell Regeneration.</a> Brain Res. 1277, 12-23 (2009).</li><li>Bibliowicz, J., Tittle, R. K., Gross, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+Better+Understanding+of+Human+Eye+Disease+Insights+From+the+Zebrafish,+Danio+Rerio.">Toward a Better Understanding of Human Eye Disease Insights From the Zebrafish, Danio Rerio.</a> Prog. Mol. Biol. Transl. Sci. 100, 287-330 (2011).</li><li>Mione, M. C., Trede, N. 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+Zebrafish+As+a+Model+for+Cancer.">The Zebrafish As a Model for Cancer.</a> Dis. Model. Mech. 3 (9-10), 9-10 (2010).</li><li>Norton, W., Bally-Cuif, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+Zebrafish+As+a+Model+Organism+for+Behavioural+Genetics.">Adult Zebrafish As a Model Organism for Behavioural Genetics.</a> BMC. Neurosci. 11, (2010).</li><li>Mathur, P., Guo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Zebrafish+As+a+Model+to+Understand+Mechanisms+of+Addiction+and.">Use of Zebrafish As a Model to Understand Mechanisms of Addiction and.</a> Complex Neurobehavioral Phenotypes. Neurobiol. Dis. 40 (1), 66-72 (2010).</li><li>Ignatius, M. S., Langenau, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+As+a+Model+for+Cancer+Self-Renewal.">Zebrafish As a Model for Cancer Self-Renewal.</a> Zebrafish. 6 (4), 377-387 (2009).</li><li>Milan, D. J., MacRae, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+Genetic+Models+for+Arrhythmia.">Zebrafish Genetic Models for Arrhythmia.</a> Prog. Biophys. Mol. Biol. 98 (2-3), 2-3 (2008).</li><li>Van Trump, W. J., Coombs, S., Duncan, K., McHenry, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gentamicin+Is+Ototoxic+to+All+Hair+Cells+in+the+Fish+Lateral+Line+System.">Gentamicin Is Ototoxic to All Hair Cells in the Fish Lateral Line System.</a> Hear. Res. 261 (1-2), 1-2 (2010).</li><li>Littleton, R. M., Hove, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+a+Nontraditional+Model+of+Traditional+Medicine.">Zebrafish: a Nontraditional Model of Traditional Medicine.</a> J. Ethnopharmacol. 145 (3), 677-685 (2013).</li><li>Harris, J. A., Cheng, A. G., Cunningham, L. L., MacDonald, G., Raible, D. W., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neomycin-Induced Hair Cell Death and Rapid Regeneration in the Lateral Line of Zebrafish (Danio. 4 (2), 219-234 (2003).</li><li>Olszewski, J., Haehnel, M., Taguchi, M., Liao, 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=Zebrafish+Larvae+Exhibit+Rheotaxis+and+Can+Escape+a+Continuous+Suction+Source+Using+Their+Lateral+Line).">Zebrafish Larvae Exhibit Rheotaxis and Can Escape a Continuous Suction Source Using Their Lateral Line).</a> PLoS One. 7 (5), 36661-36 (2012).</li><li>Liang, J., Wang, D., Renaud, G., Wolfsberg, T. G., Wilson, A. F., Burgess, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Stat3/Socs3a+Pathway+Is+a+Key+Regulator+of+Hair+Cell+Regeneration+in+Zebrafish+[Corrected.">The Stat3/Socs3a Pathway Is a Key Regulator of Hair Cell Regeneration in Zebrafish [Corrected.</a> J. Neurosci. 32 (31), 10662-10673 (2012).</li><li>Nakae, M., Asaoka, R., Wada, H., Sasaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Dye+Staining+of+Neuromasts+in+Live+Fishes:+An+Aid+to+Systematic+Studies.">Fluorescent Dye Staining of Neuromasts in Live Fishes: An Aid to Systematic Studies.</a> Ichthyol Res. , 286-290 (2012).</li><li>Magrassi, L., Purves, D., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Probes+That+Stain+Living+Nerve+Terminals.">Fluorescent Probes That Stain Living Nerve Terminals.</a> The J. Neurosci. 7 (4), 1207-1214 (1987).</li><li>Owens, K. N., Coffin, A. B., Hong, L. S., Bennett, K. O., Rubel, E. W., Raible, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+Mechanosensory+Hair+Cells+of+the+Zebrafish+Lateral+Line+to+Aminoglycosides+Reveals+Distinct+Cell+Death+Pathways.">Response of Mechanosensory Hair Cells of the Zebrafish Lateral Line to Aminoglycosides Reveals Distinct Cell Death Pathways.</a> Hear. Res. 253 (1-2), 1-2 (2009).</li><li>Namdaran, P., Reinhart, K. E., Owens, K. N., Raible, D. W., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Identification of Modulators of Hair Cell Regeneration in the Zebrafish Lateral. 32 (10), 3516-3528 (2012).</li><li>Herrera, A. A., Banner, L. 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+Use+and+Effects+of+Vital+Fluorescent+Dyes:+Observation+of+Motor+Nerve+Terminals+and+Satellite+Cells+in+Living+Frog+Muscles.">The Use and Effects of Vital Fluorescent Dyes: Observation of Motor Nerve Terminals and Satellite Cells in Living Frog Muscles.</a> J. Neurocytol. 19 (1), 67-83 (1990).</li><li>Hickey, P. C., Jacobson, D., Read, N. D., Louise Glass, ., L, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-Cell+Imaging+of+Vegetative+Hyphal+Fusion+in+Neurospora+Crassa.">Live-Cell Imaging of Vegetative Hyphal Fusion in Neurospora Crassa.</a> Fungal. Genet. Biol. 37 (1), 109-119 (2002).</li><li>Olsen, A. S., Sarras, M. P., Intine, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limb+Regeneration+Is+Impaired+in+an+Adult+Zebrafish+Model+of+Diabetes+Mellitus.">Limb Regeneration Is Impaired in an Adult Zebrafish Model of Diabetes Mellitus.</a> Wound Repair Regen. 18 (5), 532-542 (2010).</li><li>Olsen, A. S., Sarras, M. P., Leontovich, A., Intine, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heritable+Transmission+of+Diabetic+Metabolic+Memory+in+Zebrafish+Correlates+With+DNA+Hypomethylation+and+Aberrant+Gene+Expression.">Heritable Transmission of Diabetic Metabolic Memory in Zebrafish Correlates With DNA Hypomethylation and Aberrant Gene Expression.</a> Diabetes. 61 (2), 485-491 (2012).</li></ol>

This work was supported by a research grant from the Iacocca Family Foundation, National Institutes of Health Grant DK092721 (to R.V.I.), and Rosalind Franklin University start-up funds. No potential conflicts of interest relevant to this article were reported. G.C.P., S.M.M, and N.D. researched data. M.P.S. Jr. and RI oversaw the project, contributed to the discussion of the data, and oversaw the writing and editing of the manuscript.

Embriyonik ve larva zebrabalıkları 8,24,25 lateral hat (LL) rejenerasyon analizi için kurulmuştur edebiyat geniş gövdesine dayanarak, çalışmamızın amacı zebrabalıkları yanal hat rejenerasyon için niceliksel bir test geliştirmek olduğunu olabilir en iyi yetişkin balık incelenir hastalık modelleri uygulanabilir. Biz yetişkin balık embriyonik / larva balık için geliştirilen yöntemler uygulanarak zaman belli kritik noktalarda önemli olduğunu bulundu. Bu noktaların en önemli kabul edi...

Yanal çizgi (LL) sistemi işitme, denge, rheotaxis ve bu tür eğitim ve yırtıcı kaçınma 1-5 olarak aracılık davranışları sorumludur hem de balık ve amfibiler bulunan bir mechanosensory organdır. Bu destek hücreleri ile çevrili saç hücre kümeleri oluşur, her ikisi de neuromasts 6&#39;ya yapılar konumlandırılmıştır. Bu neuromasts tipik olarak balık baş görülen yatay bir dikiş ile gövde ve kuyruk uzunlamasına ekseni boyunca (dikişler adlandırılır) dik çizgiler halinde düzenlenir. Yetişkinde, neuromasts embriyonik ya da larva balık 6 ile karşılaştırıldığında, dikiş içinde sayısında önemli ölçüde daha fazladır. Zebrafish Biyomedikal çalışmalar antibiyotik tedavisi, gürültü kaynaklı travma, kronik enfeksiyon, vb etkisi üzerinde odaklanmıştır. saç hücrelerinin çabasıyla 7,8 daha iyi insanlarda etkilerini anlamak için. Pek çok omurgalılarda aksine, teBöyle Zebra balığı (Br.rerio) gibi leosts, kayıp saç hücrelerinin yenilenmesine yeteneği var. Zebrafish nedeniyle hızlı bir gelişme süresi ve yüksek rejeneratif kapasitesinin özellikle yararlıdır. Ancak bugüne kadar; yanal hat geliştirme ve / veya rejenerasyon Zebrafish çalışmalar kaynaklanmaktadır kolay sayma ve analiz 6,9,10 sağlayan yanal çizgi neuromasts azaltılmış sayısına embriyonik ve larva balık kullanmıştır. Ancak, nörolojik ve non-nörolojik hastalıklar 11-16 gibi birçok zebrabalıkları modelleri yetişkin balık değil larva incelenir, biz gentamisini kullanılarak yetişkin zebrabalıkları bir yanal çizgi rejeneratif tahlili (önce Zebra balığı larvaları kullanılan bir aminoglikozid geliştirilmesi üzerinde duruldu ve bir deney kullanılabilir, böylece daha yakın) yetişkin balık 17, kullanılan mevcut yetişkin zebrabalıkları hastalık modellerinde de uygulanabilir. Daha önce Van Trump e prosedürleri yayınlandığı süret al. 17 yetişkin balıklarda saç hücre ablasyonu için kurulmuş koşullar, bu kontrol ve bu transgenik çizgiler zebra balığı veya farmakolojik-kaynaklı hastalık durumlarını kullanırken olduğu gibi Deney grupları arasında Kantitatif karşılaştırma için gereklidir neuromast rejenerasyon için bir standart eğri oluşturmak yoktu Zebra balığı 18. Bu nedenle kontrol ve deney grupları karşılaştırırken böyle yetişkin zebrabalıkları hastalık modellerinde olduğu gibi bizim verilerini kullanmak müfettişleri sağlamak için neuromast rejenerasyon standart bir eğri oluşturmak için saç hücre ablasyon için Van Trump ve ark. 17 prosedürleri takip etti, ama onların çalışmaları üzerine inşa . Tahlil, aynı zamanda çözünürlüğü daha yüksek bir düzeyde gerekli olduğunda tek tek saç hücreye analiz uzantısı izin verecek şekilde tasarlanmıştır.

<table border="0" cellpadding="0" cellspacing="0" width="431">
	<tbody>
		<tr>
			<td>Gentamicin sulfate solution (50mg/ml)</td>
			<td>Sigma Aldrich</td>
			<td>G1397</td>
			<td></td>
		</tr>
		<tr>
			<td>2 Phenoxyethanol</td>
			<td>Sigma Aldrich</td>
			<td>P1126</td>
			<td></td>
		</tr>
		<tr>
			<td>4-4-diethylaminostryryl-N-methylpyridinium iodide (4-Di-2-Asp)</td>
			<td>Aldrich</td>
			<td>D-3418</td>
			<td>485 nm excitation &#955; and 603 nm emission &#955;</td>
		</tr>
		<tr>
			<td>in methanol</td>
		</tr>
		<tr>
			<td>6 well plates</td>
			<td>Mid Sci</td>
			<td>TP92006</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>08-757-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom Microwell Dishes</td>
			<td>Matek Corporation</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting&#160; Microscope</td>
			<td>Nikon</td>
			<td>TMZ-1500</td>
			<td>Any dissecting microscope is fine.</td>
		</tr>
		<tr>
			<td>Camera for Imaging</td>
			<td>Nikon</td>
			<td>Q imaging</td>
			<td>Any camera is suitable.</td>
		</tr>
		<tr>
			<td>Image J software</td>
			<td>National Institutes of Health</td>
			<td>NIH Image</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elements</td>
			<td>Nikon</td>
			<td></td>
			<td>Any imaging software is suitable.</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus</td>
			<td>FV10i</td>
			<td>Any high resolution fluorescent microscope is suitable</td>
		</tr>
		<tr>
			<td>Aquatic System</td>
			<td>&#160;KG Aquatics ZFS Rack System.</td>
			<td></td>
			<td>Any aquatic system can be used</td>
		</tr>
	</tbody>
</table>

an assay for lateral line regeneration in adult zebrafish

Nedeniyle insanda işitme ve denge bozuklukları, klinik önemi, bu tür Zebra balığı gibi model organizmalar yanal hat geliştirme ve rejenerasyon çalışması için kullanılmıştır. Zebra balığı nedeniyle hızlı gelişimi zaman ve yüksek rejeneratif kapasitesinin bu tür çalışmalar için özellikle çekici. Bugüne kadar, yanal çizgi rejenerasyon Zebrafish çalışmalar esas olarak, çünkü bu aşamada neuromasts alt sayısının embriyonik ve larva evrelerinin balık kullanmıştır. Bu yanal çizgi rejenerasyon ve / veya daha önceki gelişim evrelerinde daha kolay gelişme kantitatif analizini yapmıştır. Nörolojik ve non-nörolojik hastalıkların çok zebra balığı model yetişkin balık olup embriyo / larva incelenir için bir deney kullanılabilir, böylece o akım tatbik edilebilir, yetişkin Zebrafish kantitatif bir yan hat rejeneratif tahlili geliştirilmesi üzerine odaklanmıştır yetişkin zebrabalıkları hastalık modelleri. Van TRUM tarafından daha önceki çalışmaları üzerine inşap vd. 17. yetişkin Meksika kör mağara balık ve Zebra balığı (Br.rerio) saç hücrelerinin ablasyonu için prosedürleri tarif, bizim tahlil deney ve kontrol grupları arasında nicel bir karşılaştırmasını sağlamak üzere tasarlanmıştır. Bu yanal doğrultusunda belirli bir bölgede saç hücrelerinin gentamisin kaynaklı nekroz izlenerek ve 24 saatlik bir zaman periyodu boyunca neuromast yeniden ortaya çıkma yüzdesi göre bir rejeneratif neuromast standart eğri geliştirerek gerçekleştirildi. Tahlil, aynı zamanda çözünürlüğü daha yüksek bir düzeyde gerekli olan tek tek saç hücre seviyesine analiz uzantısı izin verecek şekilde tasarlanmıştır.

Yetişkin zebrabalıkları yanal çizginin neuromast rejenerasyon miktarının belirlenmesi için prosedürlerin optimizasyonu. Larva zebrabalıkları neuromasts kolaylıkla ölçülebilir vardır; Bununla birlikte, yetişkin zebrafish yan hat 6,17,19,20 kantitatif analiz daha zor hale dikiş başına neuromasts çok daha büyük bir sayı vardır. Şekil 1A&#39;da görüldüğü gibi, baş orta bölümü ya da kuyruk ya kıyasla neuromasts bir be...

Watch this Scientific Journal Video about An Assay for Lateral Line Regeneration in Adult Zebrafish at JoVE.com

An Assay for Lateral Line Regeneration in Adult Zebrafish

Nörolojik ve non-nörolojik hastalıkların çok zebra balığı model yetişkin balık ziyade embriyo / larva incelenir için, erişkin zebrabalıkları hastalık modellerinde uygulanabilen bir nicel yan hat rejeneratif deneyi geliştirilmiştir. Deney 1) neuromast ve 2) tek tek saç hücre düzeyde çözünürlüğü dahil.

Yetişkin Zebra balığı Lateral Hat Rejenerasyon için bir Deneyi

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

an-assay-for-lateral-line-regeneration-in-adult-zebrafish

Research

JoVE Journal

Biology

12.5K Görüntüleme.  Dr. William M Scholl College of Podiatric Medicine, Rosalind Franklin University of Medicine and Science.  Nörolojik ve non-nörolojik hastalıkların çok zebra balığı model yetişkin balık ziyade embriyo / larva incelenir için, erişkin zebrabalıkları hastalık modellerinde uygulanabilen bir nicel yan hat rejeneratif deneyi geliştirilmiştir. Deney 1) neuromast ve 2) tek tek saç hücre düzeyde çözünürlüğü dahil. 

Video: An Assay for Lateral Line Regeneration in Adult Zebrafish

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

Yetişkin Zebra balığı Tüm Böbrek İliği Nakli

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

Biyoloji

Cellular Toxicity of Nanogenomedicine in MCF-7 Cell Line: MTT assay

Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based medicine and/or its carrier may elicit cellular toxicity. For assessment of such cytotoxicity, a common methodology is largely dependent upon utilization of the 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay which has been widely used as a colorimetric approach based on the activity of mitochondrial dehydrogenase enzymes in cells. In this current investigation, MCF-7 cells were inoculated in 96-well plate and at 50% confluency they were treated with different nanopolyplexes and subjected to MTT assay after 24 hours. Water soluble yellow MTT is metabolized by the metabolically active cells to the water insoluble purple formazan, which is further dissolved in dimethylsulfoxide and Sornson s buffer pH 10.5. The resultant product can be quantified by spectrophotometry using a plate reader at 570 nm.

The MTT assay is an easy and reproducible colorimetric assay for evaluation of cell viability based on reduction of yellow MTT and production of water insoluble purple formazan. Here, the viability of MCF-7 cells upon treatment of nanogenomedicine has been evaluated.

MCF-7 Hücre Line Nanogenomedicine Hücresel Toksisite: MTT assay

Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based ...

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

Yetişkin Zebra balığı retro-orbital Enjeksiyon

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

Yetişkin Zebra balığı içine intraperitoneal enjeksiyon

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

Morpholinos in vivo Elektroporasyon Yetişkin Zebra balığı Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

Yenileyici Yetişkin Zebra balığı Tail Fin içine Morpholinos in vivo olarak Elektroporasyon

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

Yetişkin Zebra balığı Biyokimyasal Analiz için Kan Alma

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Yetişkin Zebra balığı Gavaging

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel &#8220;repeated&#8221; blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be&#160;&#8804;0.4% of body weight every week or &#8804;1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

Yetişkin Zebra balığı Kan Testleri için Tekrarlanan Kan Alma

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

In Vivo Surface Electrocardiography for Adult Zebrafish

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of zebrafish not only as a research model for human cardiac electrophysiology and myopathies but also as a surrogate model in high throughput pharmaceutical screening for potential cardiotoxicities to humans, such as QT prolongation. As such, in vivo electrocardiography for adult zebrafish is an electrical phenotyping tool that is necessary, if not indispensable, for cross-sectional or longitudinal in vivo electrophysiological characterizations. However, too often, the lack of a reliable, practical, and cost-effective recording method remains a major challenge preventing this in vivo diagnostic tool from becoming more readily accessible. Here, we describe a practical, straightforward approach to in vivo electrocardiography for adult zebrafish using a low-maintenance, cost-effective, and comprehensive system that yields consistent, reliable recordings. We illustrate our protocol using healthy adult male zebrafish of 12-18 months of age. We also introduce a rapid real-time interpretation strategy for quality validation to ensure data accuracy and robustness early in the electrocardiogram recording process.

Here, we present a reliable, minimally invasive, and cost-effective method to record and interpret electrocardiograms in live anesthetized adult zebrafish.

Yetişkin zebrafish için In vivo yüzey elektrokardiyografi

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of ...