Biz Martin Zinkernagel, MD, PhD ve Miriam Reisenhofer, doktora Federica Bisignani ve model onun mükemmel teknik yardım için kurulması, onun bilimsel giriş için teşekkür ederiz.

tüm deneylerin hayvanlar kullanım Ophthalmic ve vizyon araştırma vizyon ve Oftalmoloji (ARVO) Araştırmaları Derneği için beyannameyi yapıştırılır ve hükümet yetkilileri ilgili düzenlemelerin saygı.
 1. hayvanlar <ol><li>TgBAC korumak (gfap:gfap-GFP) Zebra balığı 167 (AB) zorlanma yaşları 6-9 ay içinde su sıcaklığını 26.5 ° C ile standart koşullar altında ve 14/10 h açık/koyu döngüsü 18 arasında.</li>
	<li>Hayvan bakımı yönergeleri hayvan deneyleri için ilgili kurumların hükümet yetkilileri tarafından onaylandıktan sonra izleyin.</li>
</ol> 2. Tersine çevrilebilir sistemik anestezi <ol><li>hazırla tricaine tozu 97,9 ml su tankı ve Tris 1 m 2.1 mL 400 mg eriterek tarafından etil 3-aminobenzoate methanesulfonate tuz (tricaine) stok çözüm tamponlu tuz (TBS). PH 7,0 1 M Tris (pH 9) ve mağaza içinde bir ay karanlık ilâ 4 ° C'de uyum. 
	Not: Tricaine gibi doğal yaşam koşulları hayvanın suda hazırlanmalı, tercihen orijinal tank su kullanılmalıdır.</li>
	<li>Tank su içinde hisse senedi çözüm 1:25 sulandırmak ve hemen kullanın.</li>
	<li>10 cm Petri kabına kadar hareketsiz hale ve dış uyaranlara karşı (yaklaşık 2-5 dk, ağırlık ve yaş bağlı olarak) yanıt vermeyen 50 mL anestezi çözeltisi içeren Zebra balığı yerleştirin.</li>
	<li>Transfer her balık el ile lazer tedavisi ( şekil 1A) için bir özel silikon pim tutucu. 
	DİKKAT! Balık tank için 10 dakikaya kadar sadece dışında imzalat kalabilir.</li>
	<li>Anestezi tedavi ve/veya görüntüleme sonra tersine çevirmek için Zebra balığı su tankı içeren kapsayıcısına getirin.</li>
	<li>Kurtarma desteklemek için bir akışı taze tank su üzerinde solungaçları Zebra balığı suyun içinde ileri geri hareket ettirerek oluşturun.</li>
</ol> 3. Lazer odak yaralanma Retina üzerinde Not: A 532 nm diode lazer Zebra balığı retina üzerinde odak hafif hasar oluşturmak için kullanılır. Lazer deneysel up yetişkin Zebra balığı tekrarlanabilir bir odak retina yaralanma kurulması sağlar.
<ol><li>Lazer çıkış gücü kadar ayarla: 70 mW; hava çapı: 50 µm; darbe süresi: 100 Bayan dikkat! Uygun kişisel koruma ve bölgesinin kalbinde etiketleme lazer ışık kullanımı gerektirir.</li>
	<li>1-2 damla % 2 hydroxypropylmethylcellulose topikal tedavi öncesi gözünde uygulayabilir ve lazer hedefleyen ışın retina üzerinde odaklanmaya 2.0 mm fundus lazer lens kullanabilirsiniz. 
	DİKKAT! hydroxypropylmethylcellulose damla viskoz ve solungaçları üzerinde giderse nefes sorunlara neden olabilir.</li>
	<li>Yer dört lazer noktalar görme sinirine soldaki çevresinde göz ve doğru tedavi edilmezse göz iç kontrol kullanın.</li>
</ol> 4. Vivo Retina morfolojisi Imaging <ol><li>gün 0, Zebra balığı retina doğrudan lazer indüksiyon sonra anesteziden canlandırıcı olmadan görselleştirmek. Tüm diğer zaman puan, istihdam genel anestezi (Bölüm 2 bkz: geri dönüşümlü sistemik anestezi). Özel yapım silikon pim tutucu ( şekil 1 B, B.1) üzerinde immobilize Zebra balığı yerleştirin.</li>
	En iyi görüntü elde etmek <li>kesmek Zebra balığı göz sığdırmak için piyasada bulunan hidrojel Kontakt lens (Ø 5.2 mm, r = 2.70 mm, Merkezi kalınlık = 0.4 mm =) aracılığıyla bir delik yumruk. Objektif içbükey yüzeyi methylcellulose ile doldurmak ve kornea yerleştirin.</li>
	<li>78 D temassız slit lamba camı OCT sistemiyle teçhiz.</li>
	<li>Kızılötesi (IR) görüntünün IR içinde odak + OCT modu (fundus göz görselleştirmek ve IR almak şekil 2A) resimleri tıklayarak &#34; edinme &#34; düğmesini ( şekil 2B yerelleştirmek için) Lazer sistemiyle retina lekeler &#39; s yazılım.</li>
	<li>Visualize IR + OCT retina katmanlarda üç boyutlu bir bölümünü modu ve tıklatarak resimleri almak &#34; edinme &#34; düğmesini ( şekil 2B). Dış nükleer tabaka (yalnız) yaralanma şiddeti gözlemlemek (bkz. Bölüm 3: lazer odak yaralanma retina üzerinde) bu görüntüler.</li>
	<li>Tedavi ve/veya görüntüleme tank su içeren bir kap içinde Zebra balığı yerleştirdikten sonra anestezi tersine çevirmek için.</li>
	<li>Kurtarma desteklemek için bir akışı taze tank su üzerinde solungaçları Zebra balığı suyun içinde ileri geri hareket ettirerek oluşturun.</li>
	<li>Retina morfolojisi benzer vivo içinde görüntüleme gerçekleştirmek 1 gün, 3, 7, 14 ve hafta 6.</li>
</ol> 5. Hematoksilen &#38; Eozin (H &#38; E) Staining <ol><li>batma içine soğuk (4 ° C) tarafından Zebra balığı ötenazi anestezi çözüm için en az 10 dk ve anlatabilirsin buzda gözleri hemen tarafından anlamına gelir küçük eğri forseps.</li>
	<li>Fosfat tamponlu tuz (PBS) 20 h için 4 ° C'de % 4 paraformaldehyde (PFA) bütün gözünde düzeltin ve sonra kademeli alkol serisi örneklerinde kurutmak (Ksilen %100 5 min iki kez için etanol %100 5 min iki kez için etanol %96 3 dk iki kez ve etanol %70 3 mi n bir kez). 
	DİKKAT! İngiltere'de yılın gözler, burun ve üst solunum yolu izlemek için rahatsız edici olabilir. İngiltere'de yılın olduğu bilinen bir insan kanserojen ve şüpheli bir üreme tehlike.</li>
	<li>Parafin içinde örnekleri katıştır, optik sinir başı düzeyde 5 µm bölümleri kesilmiş ve cam slaytlarda mount.</li>
	<li>% 0.1 asit Hematoksilen çözüm 5 min için deparaffinized bölümlerle leke ve hidroklorik asit karışımı slaytları daldırma sonra iki kez distile su içindeki slaytları daldırma (250 mL HCl % 25 2 mL distile su) ve amonyak (2 mL % 25 amonyak 250 ml karışımı distile su). Musluk suyu en az 10 dakika süreyle tarafından boyama Hematoksilen gelişimi sonra Eozin G sulu çözüm %0,5 3 dk bölümlerle leke</li>
	<li>Akrilik Reçine montaj orta susuz slaytları mount ve slaytları ışık mikroskobu altında dikkat.</li>
</ol> 6. İmmünhistokimya MC harekete geçirmek için <ol><li>antijen alma arabelleği (Tris-EDTA + %0,05 non-iyonik deterjan, pH 9.0) için uygun bir vapur veya bir mikrodalga fırında 10-15 dk 3 dk deparaffinized bölümlerde ısı ve yıkama üç kez TBS ile 5 için Min her.</li>
	<li>Bir silikon bölümlerle kalem ve 100 µL eklemek daire, oda sıcaklığında 1 h. için çözüm (TBS + %10 keçi normal serum + %1 sığır serum albümin, pH 7,6) engelleme</li>
	<li>Leke anti-gliyal fibrillary asidik protein (GFAP'nin) tavşan poliklonal antikor ve anti-glutamin sentetaz (GS) fare Monoklonal antikor, 1: 200 seyreltme (örnek başına 40 µL) her ikisi de. Slayt 4 ° C'de oksijen odasında gecede kuluçkaya. Üç kez TBS + % 0.1 ile yıkama ara 20 5 min için.</li>
	<li>Bitiş görselleştirme ile uygun ikincil antikorlar. Bu iletişim kuralı bir keçi Anti-tavşan IgG H kullanılan &#38; GFAP'nin ve bir keçi Anti-fare IgG H L yeşil ikincil antikor &#38; L parlak kırmızı GS, 1 h için oda sıcaklığında 1:500 seyreltme her ikisi için</li>
	<li>Slaytlar ile montaj orta DAPI içeren dağ ve floresan mikroskop altında slaytlar gözlemlemek.</li>
</ol>

<ol>
	<li>Haddad, S., Chen, C. A., Santangelo, S. L., Seddon, 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=The+genetics+of+age-related+macular+degeneration:+a+review+of+progress+to+date.">The genetics of age-related macular degeneration: a review of progress to date.</a> Surv. Ophthalmol. 51 (4), 316-363 (2006).</li><li>Stefano Ferrari, S., Di Iorio, E., Barbaro, V., Ponzin, D., Sorrentino, F. S., Parmeggiani, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinitis+Pigmentosa:+Genes+and+Disease+Mechanisms.">Retinitis Pigmentosa: Genes and Disease Mechanisms.</a> Curr Genomics. 12 (4), 238-249 (2011).</li><li>Berson, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinitis+pigmentosa.+The+Friedenwald+Lecture.">Retinitis pigmentosa. The Friedenwald Lecture.</a> Invest Ophthalmol Vis Sci. 34 (5), 1659-1676 (1993).</li><li>Strettoi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Survey+of+Retinal+Remodeling.">A Survey of Retinal Remodeling.</a> Front Cell Neurosci. 9, 494 (2015).</li><li>Hartong, D. T., Berson, E. L., Dryja, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinitis+pigmentosa.">Retinitis pigmentosa.</a> Lancet. 368, 1795-1809 (2006).</li><li>Wang, D. Y., Chan, W. M., Tam, P. O., Baum, L., Lam, D. S., Chong, K. K., Fan, B. J., Pang, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+mutations+in+retinitis+pigmentosa+and+their+clinical+implications.">Gene mutations in retinitis pigmentosa and their clinical implications.</a> Clin Chim Acta. 351 (1-2), 5-16 (2005).</li><li>Pierce, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathways+to+photoreceptor+cell+death+in+inherited+retinal+degenerations.">Pathways to photoreceptor cell death in inherited retinal degenerations.</a> BioEssays. 23, 605-618 (2001).</li><li>Tackenberg, M. A., Tucker, B. A., Swift, J. S., Jiang, C., Redenti, S., Greenberg, K. P., Flannery, J. G., Reichenbach, A., Young, 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=Muller+cell+activation,+proliferation+and+migration+following+laser+injury.">Muller cell activation, proliferation and migration following laser injury.</a> Mol. Vis. , 1886-1896 (2009).</li><li>Newman, E., Reichenbach, 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+M&#252;ller+cell:+a+functional+element+of+the+retina.">The M&#252;ller cell: a functional element of the retina.</a> Trends Neurosci. 19 (8), 307-312 (1996).</li><li>Kubota, R., Hokoc, J. N., Moshiri, A., McGuire, C., Reh, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+of+neurogenesis+in+the+retinal+ciliary+marginal+zone+of+homeothermic+vertebrates.">A comparative study of neurogenesis in the retinal ciliary marginal zone of homeothermic vertebrates.</a> Brain Res Dev Brain Res. 134, 31-41 (2002).</li><li>Zhao, T. T., Tian, C. Y., Yin, Z. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+M&#252;ller+cells+occurs+during+retinal+degeneration+in+RCS+rats.">Activation of M&#252;ller cells occurs during retinal degeneration in RCS rats.</a> Adv Exp Med Biol. 664, 575-583 (2010).</li><li>DiCicco, R. M., Bell, B. A., Kaul, C., Hollyfield, J. G., Anand-Apte, B., Perkins, B. D., Tao, Y. K., Yuan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+Regeneration+Following+OCT-Guided+Laser+Injury+in+Zebrafish.">Retinal Regeneration Following OCT-Guided Laser Injury in Zebrafish.</a> Invest Ophthalmol Vis Sci. 55 (10), 6281-6288 (2014).</li><li>Bilotta, J., Saszik, 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+visual+system.">The zebrafish as a model visual system.</a> Int. J. Dev. Neurosci. , 621-629 (2001).</li><li>Fausett, B. V., Goldman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+alpha1+tubulin-expressing+M&#252;ller+glia+in+regeneration+of+the+injured+zebrafish+retina.">A role for alpha1 tubulin-expressing M&#252;ller glia in regeneration of the injured zebrafish retina.</a> J Neurosci. 26 (23), 6303-6313 (2006).</li><li>Yurco, P., Cameron, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+M&#252;ller+glia+to+retinal+injury+in+adult+zebrafish.">Responses of M&#252;ller glia to retinal injury in adult zebrafish.</a> Vision Res. 45, 991-1002 (2005).</li><li>Ashutosh, P. J., Roesch, K., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+neurogenic+potential+of+M&#252;ller+gial+cells+in+the+vertebrate+retina.">Development and neurogenic potential of M&#252;ller gial cells in the vertebrate retina.</a> Prog Retin Eye Res. 28 (4), 249-262 (2009).</li><li>Xia, X., Ahmad, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unlocking+the+Neurogenic+Potential+of+Mammalian+M&#252;ller+Glia.">Unlocking the Neurogenic Potential of Mammalian M&#252;ller Glia.</a> Int J Stem Cells. 9 (2), 169-175 (2016).</li><li>Brand, M., Granato, M., N&#252;sslein-Volhard, C., N&#252;sslein-Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+and+raising+zebrafish.">Keeping and raising zebrafish.</a> Zebrafish: A Practical Approach. , 7-38 (2002).</li><li>Riepe, R. E., Norenburg, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M&#252;ller+cell+localisation+of+glutamine+synthetase+in+rat+retina.">M&#252;ller cell localisation of glutamine synthetase in rat retina.</a> Nature. 268 (5621), 654-655 (1977).</li><li>Derouiche, A., Rauen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coincidence+of+L-glutamate/L-aspartate+transporter+(GLAST)+and+glutamine+synthetase+(GS)+immunoreactions+in+retinal+glia:+evidence+for+coupling+of+GLAST+and+GS+in+transmitter+clearance.">Coincidence of L-glutamate/L-aspartate transporter (GLAST) and glutamine synthetase (GS) immunoreactions in retinal glia: evidence for coupling of GLAST and GS in transmitter clearance.</a> J Neurosci Res. 42 (1), 131-143 (1995).</li><li>Bignami, A., Dahl, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+radial+glia+of+M&#252;ller+in+the+rat+retina+and+their+response+to+injury.+An+immunofluorescence+study+with+antibodies+to+the+glial+fibrillary+acidic+(GFA)+protein.">The radial glia of M&#252;ller in the rat retina and their response to injury. An immunofluorescence study with antibodies to the glial fibrillary acidic (GFA) protein.</a> Exp Eye Res. 28 (1), 63-69 (1979).</li><li>Sherpa, T., Fimbel, S. M., Mallory, D. E., Maaswinkel, H., Spritzer, S. D., Sand, J. A., Li, L., Hyde, D. R., Stenkamp, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ganglion+cell+regeneration+following+whole-retina+destruction+in+zebrafish.">Ganglion cell regeneration following whole-retina destruction in zebrafish.</a> Dev Neurobiol. 68 (2), 166-181 (2008).</li><li>Cameron, D. A., Carney, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+mosaic+patterns+in+the+native+and+regenerated+inner+retina+of+zebrafish:+implications+for+retinal+assembly.">Cell mosaic patterns in the native and regenerated inner retina of zebrafish: implications for retinal assembly.</a> J Comp Neurol. 416 (3), 356-367 (2000).</li><li>Raymond, P. A., Barthel, L. K., Bernardos, R. L., Perkowski, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+retinal+stem+cells+and+their+niches+in+adult+zebrafish.">Molecular characterization of retinal stem cells and their niches in adult zebrafish.</a> BMC Dev Biol. 6, 36 (2006).</li><li>Bailey, T. J., Davis, D. H., Vance, J. E., Hyde, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral-domain+optical+coherence+tomography+as+a+noninvasive+method+to+assess+damaged+and+regenerating+adult+zebrafish+retinas.">Spectral-domain optical coherence tomography as a noninvasive method to assess damaged and regenerating adult zebrafish retinas.</a> Invest Ophthalmol Vis Sci. 53 (6), 3126-3138 (2012).</li><li>Koinzer, S., Saeger, M., Hesse, C., Portz, L., Kleemann, S., Schlott, K., Brinkmann, R., Roider, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+with+OCT+and+histology+of+photocoagulation+lesions+in+patients+and+rabbits.">Correlation with OCT and histology of photocoagulation lesions in patients and rabbits.</a> Acta Ophthalmol. 91 (8), e603-e611 (2013).</li><li>Wan, J., Zheng, H., Chen, Z. L., Xiao, H. L., Shen, Z. J., Zhou, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+regeneration+of+photoreceptor+from+M&#252;ller+glia+after+retinal+degeneration+in+adult+rat.">Preferential regeneration of photoreceptor from M&#252;ller glia after retinal degeneration in adult rat.</a> Vision Res. (2), 223-234 (2008).</li><li>Thomas, J. L., Thummel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+light+damage+paradigm+for+use+in+retinal+regeneration+studies+in+adult+zebrafish.">A novel light damage paradigm for use in retinal regeneration studies in adult zebrafish.</a> J Vis Exp. (80), e51017 (2013).</li></ol>

Yazarlar ifşa gerek yok.

Zebra balığı retina rejenerasyon/dejenerasyon Sitotoksin aracılı hücre ölüm22, mekanik yaralanma23ve termal yaralanma24gibi farklı yaklaşımlarla incelemeye gittim. Zebra balığı retina hasar için 532 nm diode lazer çalıştırmaya başladık. Böylece, bizim modeli birçok avantaj sunar. Örneğin, biz hızla PRs katmanındaki özellikle dış retina lokalize yaralanma iyi tanımlanmış bir alan oluşturdu. Ayrıca, bu deneysel kurulu...

Vizyon muhtemelen insanın en temel anlamda ve onun bozulma bir yüksek sosyo-ekonomik etkisi vardır. Sanayileşmiş dünyada retina dejeneratif hastalıkları görme kaybı ve körlük arasında1yetişkin nüfusun çoğunluğu için hesap. Retinitis pigmentosa (RP) körlüğü yaklaşık 1.5 milyon kişi dünya çapında2,3etkileyen 60 ve 20 yaşları arasındaki kişilerde en sık devralınan nedenidir. Kalıtsal retina bozuklukları dejenerasyonu retina pigment epiteli ve daha sonra gliosis tarafından takip ve iç nöronlar4remodeling ilerici kaybı photoreceptors (PRs) tarafından karakterize heterojen bir ailedir. Hastalığın seyrini genellikle çubuklar, loş ışık ve renk görme ve görme keskinliği5için gerekli olan, koniler akromatik vizyon için sorumlu olan ile başlayan iki PR hücre tipleri, artımlı kaybı açıklanabilir. RP neden için yeterli bir tek gen kusurdur. Defa 130'dan fazla 45'in üzerinde genlerdeki mutasyonlar hastalığı6ile ilişkili bulunmuştur. Bu değişen hastalığı fenotipleri yol açar ve gen tedavisi olmayan genelleştirilebilir bir neden ve böylece karmaşık bir tedavi yaklaşımı. Bu nedenle, hastalıklar kör olarak retina dejenerasyonlar tedavisinde yeni genel tedavi yaklaşımları geliştirmek için acil bir ihtiyaç vardır.
Retina dejenerasyonu kez PR kaybı içerir; Bu nedenle, PR hücre ölümü dejeneratif süreçleri retina7özelliğidir. Zaten PR hücre ölümü Müller glia hücre (MC)8harekete geçirmek ve nükleer silahların yayılmasına karşı uyarır gösterilmiştir. MCs, omurgalı retina büyük gliyal hücre tipinde bir kez kabul "yapıştırıcı" başka bir şey olmak arasında retina sinir hücreleri. Son yıllarda, birçok çalışma daha fazla sadece yapısal9çekmek gibi MCs hareket göstermiştir. Farklı fonksiyonları arasında MCs da neurogenesis içinde katılmak ve10onarmak. Nitekim, karşılık diffusible faktörler degenerating retina, MCs önemli ölçüde gliyal fibrillary asidik protein (GFAP'nin) ifade artırın. Bu nedenle, retina hasarı ve dejenerasyon11ikincil bir tepki olarak MC harekete geçirmek için bir işaret olarak GFAP'nin etiketleme kullanılabilir.
Son zamanlarda, Zebra balığı (Danio rerio) retinal dejenerasyon ikna etmek için bir lazer kullanarak odak yaralanma bir roman uyarlaması geliştirdik. Odak yaralanma hücre yaralı siteye geçiş ve retina rejenerasyon12sırasında gerçekleşen olayların hassas zamanlama gibi bazı biyolojik süreçlerin eğitimi için avantajlıdır. Ayrıca, Zebra balığı onun görsel sistem ve bu diğer omurgalıların arasındaki benzerlikleri nedeniyle görsel araştırma önemli hale gelmiştir. İnsan ve teleost retinae brüt morfolojik ve histolojik özellikleri bazı farklılıklar görüntüler. Buna göre insan ve Zebra balığı retinae retina projeksiyon nöronlar, ganglion hücrelerinin en içteki içinde ikamet ederken nerede ışık algılama photoreceptors en dış katmanı işgal aynı katmanlı model düzenlenen aynı büyük hücre sınıfları içerir nöronal katmanı, lens proksimal. Retina interneurons, amacrine, bipolar ve yatay hücreler, photoreceptor ve ganglion hücre katmanları13arasında yerelleştirilmesine. Ayrıca, Zebra balığı retina koni egemen olduğu ve bu nedenle daha, örneğin, çalışılmış kemirgen retina insan retina için yakın olduğunu. Büyüleyici bir fark teleost ve memeliler arasında balık retina ve retina yeniden oluşturma tamamlandıktan sonra hasar kalıcı neurogenesis vardır. Zebra balığı, MCs dedifferentiate ve rejenerasyon yaralı retina14,15dakika sonra arabuluculuk. Tavuk, MCs aynı zamanda hücre döngüsü yeniden girmek için ve dedifferentiate için bazı kapasitesine sahiptir. Retina yaralanma yetişkin balık MCs Dede ve kök hücre belirli özellikleri kabul, hasarlı retina dokusu ile göç ve yeni nöronlar16üretmek. Gen ifadesi memeli MCs profil oluşturma retina ataları beklenmeyen benzerlikler ortaya ve MCs içsel Nörojenik potansiyelini tavuk, kemirgen ve insan bile retina için kanıt17büyüyor. Yine de, neden kuşlar ve memeliler rejeneratif yanıtta düşük güçlü yanıt olarak balık ile karşılaştırıldığında henüz anlaşılamamıştır. Bu nedenle, Zebra balığı endojen tamir mekanizmaları anlama stratejileri uyarıcı retina rejenerasyon memeliler ve insanlar için önerebilir miyim? MCs retina dejenerasyonu olan hastaların tedavisi için endojen onarım mekanizması bir tedavi aracı olarak istihdam bizim toplum için olağanüstü bir etkisi olurdu.
Burada, biz göze ait araştırma dejenerasyon/rejenerasyon modelinde istihdam için gerekli adımları sağlar. Biz ilk neurosensory retina görüntüleme yaralanma site ve son olarak görüntülenmesi bitişik MCs katılımı gelişen olayların ardından üzerinde odak hasar inducing üzerinde duruldu. Genel iletişim kuralı gerçekleştirmek nispeten kolay ve çok çeşitli retina daha sonra değerlendirmek için olanaklar açar.

<table>
	<tbody>
		<tr>
			<td>Acid hematoxylin solution</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>2852</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>A07030</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>5470</td>
			<td></td>
		</tr>
		<tr>
			<td>Dako Pen</td>
			<td>Dako, Glostrup, Danmark</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI mounting medium</td>
			<td>Vector Labs, Burlingame, CA, USA</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin G aqueous solution 0.5%</td>
			<td>Carl Roth, Arlesheim, Switzerland</td>
			<td>X883.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>2860</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene diamine tetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>ED</td>
			<td></td>
		</tr>
		<tr>
			<td>Eukitt</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>3989</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG H&#38;L Alexa Fluor&#174; 488</td>
			<td>Life Technologies, Zug, Switzerland</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG H&#38;L Alexa Fluor&#174; 594</td>
			<td>Life Technologies, Zug, Switzerland</td>
			<td>A11020</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat normal serum</td>
			<td>Dako, Glostrup, Danmark</td>
			<td>X0907</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogel contact lens</td>
			<td>Johnson &#38; Johnson AG, Zug, Switzerland</td>
			<td>n.a.</td>
			<td>1-Day Acuvue Moist</td>
		</tr>
		<tr>
			<td>Hydroxypropylmethylcellulose 2%</td>
			<td>OmniVision, Neuhausen, Switzerland</td>
			<td>n.a.</td>
			<td>Methocel 2%</td>
		</tr>
		<tr>
			<td>Ethyl 3-aminobenzoate methanesulfonate</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>A5040</td>
			<td>Tricaine, MS-222</td>
		</tr>
		<tr>
			<td>Visulas 532s</td>
			<td>Carl Zeiss Meditec AG, Oberkochen, Germany</td>
			<td>n.a.</td>
			<td>532 nm laser</td>
		</tr>
		<tr>
			<td>Mouse anti-GS monoclonal antibody</td>
			<td>Millipore, Billerica, MA, USA</td>
			<td>MAB302</td>
			<td></td>
		</tr>
		<tr>
			<td>HRA + OCT Imaging System</td>
			<td>Heidelberg Engineering, Heidelberg, Germany</td>
			<td>n.a.</td>
			<td>Spectralis</td>
		</tr>
		<tr>
			<td>Heidelberg Eye Explorer</td>
			<td>Heidelberg Engineering, Heidelberg, Germany</td>
			<td>n.a.</td>
			<td>Version 1.9.10.0</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>P5368</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>P5368</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-GFAP polyclonal antibody</td>
			<td>Invitrogen, Waltham, MA, USA</td>
			<td>180063</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone pin holder</td>
			<td>Huco Vision AG Switzerland</td>
			<td>n.a.</td>
			<td>Cut by hand from silicone pin mat of the sterilization tray accordingly.</td>
		</tr>
		<tr>
			<td>Slit lamp BM900</td>
			<td>Haag-Streit AG, Koeniz, Switzerland</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Slit lamp adapter</td>
			<td>Iridex Corp., Mountain View, CA, USA</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus glass slides</td>
			<td>Gehard Menzel GmbH, Braunschweig, Germany</td>
			<td>10149870</td>
			<td></td>
		</tr>
		<tr>
			<td>TgBAC (gfap:gfap-GFP) zf167 (AB) strain</td>
			<td>KIT, Karlsruhe, Germany</td>
			<td>15204</td>
			<td>http://zfin.org/ZDB-ALT-100308-3</td>
		</tr>
		<tr>
			<td>Tris buffered saline (TBS)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>P5912</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>78D non-contact slit lamp lens</td>
			<td>Volk Optical, Mentor, OH, USA</td>
			<td>V78C</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>534056</td>
			<td></td>
		</tr>
		<tr>
			<td>Ocular fundus laser lens</td>
			<td>Ocular Instruments, Bellevue, WA, USA</td>
			<td>OFA2-0</td>
			<td></td>
		</tr>
		<tr>
			<td>2100 Retriever</td>
			<td>Aptum Biologics Ltd., Southampton, United Kingdom</td>
			<td>R2100-EU</td>
			<td>Steamer</td>
		</tr>
	</tbody>
</table>

m&#252;ller glia cell activation in a laser-induced retinal degeneration and regeneration model in zebrafish

Teleost ve memeliler arasında büyüleyici bir fark teleost retina retina neurogenesis ve yeniden oluşturma tamamlandıktan sonra ciddi hasar için ömür boyu potansiyelidir. Zebra balığı rejenerasyon yollar soruşturma memelilerde retina dejeneratif hastalıkları tedavisi için yenilikçi stratejiler geliştirmek için yeni anlayışlar getirebilir. Burada, biz Fokal lezyon yetişkin Zebra balığı dış retinada için indüksiyon yoluyla 532 nm diode lazer odaklı. Yerelleştirilmiş bir yaralanma retina dejenerasyonu ve hasar alan, doğrudan rejenerasyon sırasında gerçekleşecek biyolojik süreçlerin soruşturma sağlar. Non-invaziv optik Koherens tomografi (OCT) kullanarak, biz were güçlü-e doğru bozuk alan ve monitör sonraki rejenerasyon konumunu tanımlamak içinde vivo. Gerçekten de, OCT görüntüleme histolojik analizler ile daha önce yalnızca bilgi veren Zebra balığı retina, yüksek çözünürlüklü, kesitsel görüntüler üretir. Gerçek zamanlı OCT verilerden onaylamak için histolojik kesitler gerçekleştirilen ve retina yaralanma indüksiyon sonra rejeneratif yanıt immünhistokimya tarafından araştırıldı.

Gerçek zamanlı OCT: MCs rol retina tamir amacıyla, biz iyi arasındadır bölgenin Zebra balığı retina hasar inducing bir lazer yaralanma modeli kullanılır. Hasar sitenin içinde vivo OCT yaralanma (şekil 3) takip 60 dakika içinde ilk kez (0 gün) aracılığıyla görüntüsü. Balık gözü optik için telafi etmek için özel yapım bir kontakt lens kornea üzerinde yerleştirildi. Hemen sonra lazer tedavisi, diffüz bir...

Watch this Scientific Journal Video about MÃ¼ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish at JoVE.com

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

Zebra balığı retina dejenerasyonu/rejenerasyon omurgalılarda mekanizmaları incelemek için popüler bir hayvan modelidir. Bu iletişim kuralı yerelleştirilmiş yaralanma dış retina iç retina en az hasar ile kesintiye neden yöntemi açıklanır. Daha sonra in vivo retina morfoloji ve Müller glia yanıt retina yeniden oluşturma işlemi boyunca izliyoruz.

Bir lazer kaynaklı retina dejenerasyonu, Zebra balığı rejenerasyon modelini Müller Glia hücre aktivasyonu

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

mller-glia-cell-activation-laser-induced-retinal-degeneration

Research

JoVE Journal

Neuroscience

9.7K Görüntüleme.  University of Bern. Zebra balığı retina dejenerasyonu/rejenerasyon omurgalılarda mekanizmaları incelemek için popüler bir hayvan modelidir. Bu iletişim kuralı yerelleştirilmiş yaralanma dış retina iç retina en az hasar ile kesintiye neden yöntemi açıklanır. Daha sonra in vivo retina morfoloji ve Müller glia yanıt retina yeniden oluşturma işlemi boyunca izliyoruz.

Video: Müller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Fare Retina Ganglion Hücreleri Transfeksiyon tarafından In vivo Elektroporasyon

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Nörobilim

Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato

Larval zebrafish are emerging as a model for describing the development and function of simple neural circuits. Due to their external fertilization, rapid development, and translucency, zebrafish are particularly well suited for optogenetic approaches to investigate neural circuit function. In this approach, light-sensitive ion channels are expressed in specific neurons, enabling the experimenter to activate or inhibit them at will and thus assess their contribution to specific behaviors. Applying these methods in larval zebrafish is conceptually simple but requires the optimization of technical details. Here we demonstrate a procedure for expressing a channelrhodopsin variant in larval zebrafish somatosensory neurons, photo-activating single cells, and recording the resulting behaviors. By introducing a few modifications to previously established methods, this approach could be used to elicit behavioral responses from single neurons activated up to at least 4 days post-fertilization (dpf). Specifically, we created a transgene using a somatosensory neuron enhancer, CREST3, to drive the expression of the tagged channelrhodopsin variant, ChEF-tdTomato. Injecting this transgene into 1-cell stage embryos results in mosaic expression in somatosensory neurons, which can be imaged with confocal microscopy. Illuminating identified cells in these animals with light from a 473 nm DPSS laser, guided through a fiber optic cable, elicits behaviors that can be recorded with a high-speed video camera and analyzed quantitatively. This technique could be adapted to study behaviors elicited by activating any zebrafish neuron. Combining this approach with genetic or pharmacological perturbations will be a powerful way to investigate circuit formation and function.

Optogenetic techniques have made it possible to study the contribution of specific neurons to behavior. We describe a method in larval zebrafish for activating single somatosensory neurons expressing a channelrhodopsin variant (ChEF) with a diode-pumped solid state (DPSS) laser and recording the elicited behaviors with a high-speed video camera.

Chef-tdTomato kullanarak Zebrafish Somatosensoriyel Nöron Optogenetic Etkinleştirme

Larval  zebrafish are emerging as a model for describing the development and function  of simple neural circuits. Due to their  external fertilization, ...

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, photoreceptor degeneration triggers M&#252;ller glial cells to re-enter the cell cycle and produce transient-amplifying progenitors. These progenitors continue to proliferate as they migrate to the damaged area, where they ultimately give rise to new photoreceptors. Currently, there are two widely-used LIRD paradigms, each of which results in varying degrees of photoreceptor loss and corresponding differences in the regeneration response. As more genetic and pharmacological tools are available to test the role of individual genes of interest during regeneration, there is a need to develop a robust LIRD paradigm. Here we describe a LIRD protocol that results in widespread and consistent loss of both rod and cone photoreceptors in which we have combined the use of two previously established LIRD techniques. Furthermore, this protocol can be extended for use in pigmented animals, which eliminates the need to maintain transgenic lines of interest on the albino background for LIRD studies.

Multiple light damage protocols have been described to damage photoreceptors and consequently induce a retinal regeneration response in adult zebrafish. This protocol describes an improved method that can be used in pigmented animals and that damages the vast majority of rod and cone photoreceptors across the entire retina.

Yetişkin Zebra balığı Retina Rejenerasyon Çalışmaları Kullanım için Yeni Bir Işık Hasar Paradigma

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, ...

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

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

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

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

Bir<em&gt; Ex vivo</em&gt; Lazer kaynaklı Omurilik Felçlileri Modeli Gerçek zamanlı aksonal Dejenerasyon mekanizmaları değerlendirmek için

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

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Modelleme nöronal ölüm ve fare birincil serebellar granül nöronlar dejenerasyon

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

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

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.
The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch&#39;s membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch&#39;s membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is&#160;presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.
This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

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

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

Vivo Multimodal görüntüleme ve fare lazer kaynaklı Choroidal neovaskülarizasyon Model analizi

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

Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection is considered a useful and reproducible model. Compared with other optic nerve injury models used commonly for assessing secondary degeneration, e.g. complete optic nerve transection and optic nerve crush models, the partial optic nerve transection model is superior as it distinguishes primary from secondary degeneration in situ. Therefore, it serves as an excellent tool for evaluating secondary degeneration. This study describes a novel operative approach of partial optic nerve transection by directly accessing the area of the retrobulbar optic nerve through the orbital lateral wall of the eyeball. Moreover, we present a newly designed, low cost surgical instrument to assist with transection. As demonstrated by the representative results in distinguishing the boundary of primary and secondary injury areas, the new approach and instrument ensures high efficiency and stability of the model by providing adequate space for surgical operation. This in turn makes it easy to separate the meningeal sheath and ophthalmic vessels from the optic nerve before transection. An additional benefit is that this space-saving operative approach improves the investigators&#39; ability to administer drugs, carriers, or selective RGC tracers to the stump of the partially transected optic nerve, allowing the exploration of mechanisms behind secondary injury in RGCs, in a new way.

Secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. This study describes an innovative operative approach for partial optic nerve transection. The use of this space-saving operative approach extends the model's application range, and allows exploration of secondary injury mechanisms in RGCs in a new way.

Kısmi optik sinir transeksiyon Sıçanlarda: ikincil dejenerasyonu retina Ganglion hücrelerinin değerlendirmek için yeni bir operatif yaklaşım ile kurulan bir Model

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection ...

Laser-Induced Brain Injury in the Motor Cortex of Rats

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

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

Farelerin Motor Korteksinde LazerE Bağlı Beyin Hasarı

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

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

Beyin Dilimlerinde ve Birincil Hücre Kültüründe Piramidal Nöronların Balistik Etiketlemesi

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

İskemide Nöron-Glia Etkileşimlerinin Rolünü Nörade İncelemek İçin Hücre Kültürü Modeli

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...