Yazarlar görüntülemede ona cömert yardım için, Ruth Zelkha, MS teşekkür ederim. Bu araştırma ME Klinik Scientist Kalkınma Programı Ödülü K12EY021475 tarafından desteklenen, ARD, Ulusal Göz Enstitüsü, NIH ve Körlük Önlemek için Araştırma sınırsız bağışı çekirdek hibe EY01792 için R01 EY024349-01A1 verin. ARD Araştırma engelle Körlük bir Kariyer Geliştirme Ödülü almıştır.

Hayvanlar üzerinde yapılan tüm işlemler vizyon araştırma hayvan kullanım için Vizyon ve Oftalmoloji tablolarında Araştırma Derneği ile uyumludur. Altı aylık erkek / kadın C57BL / 6J vahşi tip farelere Ondört dört kullanıldı: on yaralanma modeli için fareler ve kontrol hayvanlarına olarak dört (yaralı olmayan korneası). 1. Hayvan hazırlanması <ol><li> Bir 100 mg / kg ketamin intraperitonal enjeksiyon ve 5 mg / kg ksilazin karışımı ile bir fare anestezisi. Anestezi etkisi almıştır sonra, anestezi derinliği onaylamak için ayak tutam; Hayvan tepkimeye girmemelidir. </li><li> herhangi bir önceki kornea hasarı yokluğunu doğrulamak için deneylerini önce bir yarık lamba altında göz kontrol edin. </li><li> gözün üzerine% 1 tetracaine hidroklorür bir damla aşılamak. 60 Sonra - 90 saniye, duyu yokluğunu doğrulamak için kornea refleksi test edin. Proparakain hidroklorür% 0.5 yerine kullanılabilir. </li><li> Ne zaman kornea duyu kaybıBelirli nazikçe göz ve göz çevresinde saç% 5 povidon-iyot bir damla yerleştirin. 1 dakika sonra damla silin ya da araç ipucu müdahale saç önlemek için göz çevresinde saç üzerine yayılır. </li></ol> 2. Kornea epiteli zımpara <ol><li> cerrahi eldivenleri ve bir önlük giyin. Steril dönen çapak ve steril bir cımbız: gerekli araçları hazırlayın. Daha iyi kontrol için, bükülmüş cımbız kullanın. İşlem sırasında steril bir kağıda araçları yerleştirin. </li><li> bükülmüş cımbız kullanarak, burun tarafında kapaklarını kapma ve yavaşça göz proptosing ile göz stabilize. Çok fazla baskı kullanmaktan kaçının. </li><li> Cerrahi mikroskop altında, iki kez steril dönen çapak kullanarak limbal alanı çevresinde 360 ​​° tıraş. Tur başına 6 sn - 5 hızında limbusta etrafında gidin. NOT: çapak bir sabit hıza sahip. Ancak, kurulan hızda döndürmek için, &quot;son fındık&quot; tamamen sıkılmalıdır. </li><li> Dönen çapak ile limbusa limbustan bütün kornea epiteli çıkarın. Daha iyi sonuçlar için, dairesel bir şekilde aracı hareket ettirmek ve kornea yüzeyinin biraz paralel olarak ucunun yan tarafına çalışmak tutun. re-fırçalama tarafından değil epiteli zaten kaldırılmış alanları stroma kesmemeye dikkat edin. göz üzerinde herhangi bir baskı uygulamayın. gerektiği gibi fırça ucu temizleyin. </li><li> korneada steril fosfat tamponlu bir damla serum fizyolojik aşılamak ve yumuşak bir temizlik dokusu ile herhangi bir müstakil epitel sayfaları silin. </li><li> varsa kalan epitel tıraş. </li><li> Ameliyat sırasında fare kuyruğu dikkat edin. NOT: herhangi bir hareket oluşursa, anestezi derinliği soluk ve daha fazla enjeksiyon gereklidir. Birincil enjekte hacminin üçte ikisine üçte biri yeterli olmalıdır. anestetik ilaç ile hayvan aşırı doz etmeyin. </li><li> Başka bir göz bunları kullanmadan önce tüm araçları sterilize edin. </li></ol> 3. Onayla Komple epitel Kaldırma <ol><li> kornea olarak 1 mg / ml floresein sodyum bir damla uygulanır. 30 saniye sonra kornea temizleyin ve epitel tümüyle kaldırılmasını doğrulamak için bir kobalt mavisi filtreli bir mikroskop altında incelemek. NOT: epitel defekti ile Kornea alanlar yeşil görünür hale gelir. </li></ol> 4. Post-işlem Bakımı <ol><li> Bir ısıtma battaniye hayvan koyun ve kurtarma altında iken (kendi kafeste, örneğin) güvenli bir yerde saklayın. bilinçli olanların şirket bilinçsiz bir hayvan tutmayın. </li><li> % 1 eritromisin oftalmik merhem sürün. Bu aynı zamanda göz kuruluğu engelleyecektir. Uygulamayı bir hafta boyunca antibiyotik gazetesine devam edin. </li><li> kg buprenorfin subkutan işlemden sonra tekrar 12 saat sonra / 0.1 mg enjekte edilir. İki gün süre ile günde iki defa enjeksiyon devam edin. </li><li> anesteziden tam iyileşme kadar, 45 dakika - 30 hayvan gözlemleyin. NOT: Ventral yüzeyinde solunum kaslarının hafif hareket kurtarma altında iken hayvan welness gösterir. </li></ol>

<ol>
	<li>Afsharkhamseh, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+limbal+stem+cell+deficiency+after+mechanical+and+thermal+injuries+in+mice.">Stability of limbal stem cell deficiency after mechanical and thermal injuries in mice.</a> Exp. Eye Res. 145, 88-92 (2015).</li><li>Dor&#224;, N. J., Hill, R. E., Collinson, J. M., West, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+tracing+in+the+adult+mouse+corneal+epithelium+supports+the+limbal+epithelial+stem+cell+hypothesis+with+intermittent+periods+of+stem+cell+quiescence.">Lineage tracing in the adult mouse corneal epithelium supports the limbal epithelial stem cell hypothesis with intermittent periods of stem cell quiescence.</a> Stem Cell Res. 15 (3), 665-677 (2015).</li><li>Ahmad, S., Osei-Bempong, C., Dana, R., Jurkunas, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+culture+and+transplantation+of+human+limbal+stem+cells.">The culture and transplantation of human limbal stem cells.</a> J. Cell Physiol. 225 (1), 15-19 (2010).</li><li>Kolli, S., Ahmad, S., Lako, M., Figueiredo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+clinical+implementation+of+corneal+epithelial+stem+cell+therapy+for+treatment+of+unilateral+limbal+stem+cell+deficiency.">Successful clinical implementation of corneal epithelial stem cell therapy for treatment of unilateral limbal stem cell deficiency.</a> Stem Cells. 28 (3), 597-610 (2010).</li><li>Ahmad, S., Figueiredo, F., Lako, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+epithelial+stem+cells:+characterization,+culture+and+transplantation.">Corneal epithelial stem cells: characterization, culture and transplantation.</a> Regen. Med. 1 (1), 29-44 (2006).</li><li>He, H., Yiu, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-based+therapy+for+treating+limbal+stem+cells+deficiency:+A+review+of+different+strategies.">Stem cell-based therapy for treating limbal stem cells deficiency: A review of different strategies.</a> Saudi J. Ophthalmol. 28 (3), 188-194 (2014).</li><li>Pal-Ghosh, S., Pajoohesh-Ganji, A., Tadvalkar, G., Stepp, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+the+basement+membrane+enhances+corneal+wound+healing.">Removal of the basement membrane enhances corneal wound healing.</a> Exp Eye Res. 93 (6), 927-936 (2011).</li><li>Stepp, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wounding+the+cornea+to+learn+how+it+heals.">Wounding the cornea to learn how it heals.</a> Exp. Eye Res. 121, 178-193 (2014).</li><li>Amirjamshidi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limbal+fibroblast+conditioned+media:+a+non-invasive+treatment+for+limbal+stem+cell+deficiency.">Limbal fibroblast conditioned media: a non-invasive treatment for limbal stem cell deficiency.</a> Mol. Vis. 17, 658-666 (2011).</li><li>Li, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+AlgerBrush+II+rotating+burr+as+a+tool+for+inducing+ocular+surface+failure+in+the+New+Zealand+White+rabbit.">Evaluation of the AlgerBrush II rotating burr as a tool for inducing ocular surface failure in the New Zealand White rabbit.</a> Exp. Eye Res. 147, 1-11 (2016).</li><li>Ti, S. E., Anderson, D., Touhami, A., Kim, C., Tseng, S. C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+outcome+following+transplantation+of+ex+vivo+expanded+limbal+epithelium+on+amniotic+membrane+for+total+limbal+deficiency+in+rabbits.">Factors affecting outcome following transplantation of ex vivo expanded limbal epithelium on amniotic membrane for total limbal deficiency in rabbits.</a> Invest. Ophthalmol. Vis. Sci. 43 (8), 2584-2592 (2002).</li><li>Ma, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+chemically+burned+rat+corneal+surface+by+bone+marrow-derived+human+mesenchymal+stem+cells.">Reconstruction of chemically burned rat corneal surface by bone marrow-derived human mesenchymal stem cells.</a> Stem Cells. 24 (2), 315-321 (2006).</li><li>Bu, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+activated+omental+cells+on+rat+limbal+corneal+alkali+injury.">Effects of activated omental cells on rat limbal corneal alkali injury.</a> Exp. Eye Res. 121, 143-146 (2014).</li><li>Luengo Gimeno, F., Lavigne, V., Gatto, S., Croxatto, J. O., Correa, L., Gallo, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+corneal+stem-cell+transplantation+in+rabbits+with+severe+ocular+alkali+burns.">Advances in corneal stem-cell transplantation in rabbits with severe ocular alkali burns.</a> J. Cataract Refract. Surg. 33 (11), 1958-1965 (2007).</li><li>Lin, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+limbal+stem+cell+deficiency+induced+by+topical+medication+with+the+preservative+benzalkonium+chloride.">A mouse model of limbal stem cell deficiency induced by topical medication with the preservative benzalkonium chloride.</a> Invest. Ophthalmol. Vis. Sci. 54 (9), 6314-6325 (2013).</li><li>Huang, A. J., Tseng, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+epithelial+wound+healing+in+the+absence+of+limbal+epithelium.">Corneal epithelial wound healing in the absence of limbal epithelium.</a> Invest. Ophthalmol. Vis. Sci. 32 (1), 96-105 (1991).</li><li>Liu, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cornea-specific+expression+of+K12+keratin+during+mouse+development.">Cornea-specific expression of K12 keratin during mouse development.</a> Curr. Eye Res. 12 (11), 963-974 (1993).</li><li>Nakatsu, M. N., Gonz&#225;lez, S., Mei, H., Deng, S. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+limbal+mesenchymal+cells+support+the+growth+of+human+corneal+epithelial+stem/progenitor+cells.">Human limbal mesenchymal cells support the growth of human corneal epithelial stem/progenitor cells.</a> Invest. Ophthalmol. Vis. Sci. 55 (10), 6953-6959 (2014).</li><li>Pajoohesh-Ganji, A., Pal-Ghosh, S., Tadvalkar, G., Stepp, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+goblet+cells+and+their+niche:+implications+for+corneal+stem+cell+deficiency.">Corneal goblet cells and their niche: implications for corneal stem cell deficiency.</a> Stem Cells. 30 (9), 2032-2043 (2012).</li><li>McCloy, R. A., Rogers, S., Caldon, C. E., Lorca, T., Castro, A., Burgess, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+inhibition+of+Cdk1+in+G+2+phase+overrides+the+SAC+and+decouples+mitotic+events.">Partial inhibition of Cdk1 in G 2 phase overrides the SAC and decouples mitotic events.</a> Cell Cycle. 13, 1400-1412 (2014).</li></ol>

The authors declare that they have nothing to disclose.

Bu makalede, LSCD olan bir fare modeli oluşturmak için yeniden üretilebilir ve nispeten basit bir teknik açıklanmaktadır. fazlalaştı olan bu modelin bazı önemli yönleri vardır. İlk olarak, kimyasal madde 11, 12 kullanımı farklı modeller, yaralanma esas olarak altta yatan kornea stroma veya başka göz içi yapıların en az zararla yüzey epiteli (ve bazal zar) içerir. Böylece, prosedürü komplike ve daha zor limbal kök hücrelerin yönelik herhangi bir müdahalelerin sonucunu değerlendir...

kornea epitel kornea netlik ve bütünlüğünü korumak için gereklidir. Sürekli kornea ve konjonktiva kavşağında Limbustan-dar bir bölgede yaşayan epitel kök hücreleri tarafından yaşamı boyunca yenilenir. Bunlar kendini sürekli yenileyen limbal kök hücreler hem normal ve yaralanma sonrası, kornea epitel rejenere önemli bir rol oynamaktadır. Bu kök hücrelerin kısmen ya da tamamen tükenmesi tekrarlayan kornea erozyonlar, ağrı, kornea yara ve neovaskülarizasyon, goblet hücre görünümü neden olacak ve tedavi edilmezse, korneal körlük bırakılırsa. Bu durum limbal kök hücre eksikliği (LSCD) olarak da bilinir ve idiyopatik olabilir; kalıtsal; veya bağlı kimyasal veya termal yaralanmalar, uzun süreli kontak lens takmak ve kronik inflamasyon 1-6 kazandı. LSCD üzerine yapılan araştırmalar, uygun bir hayvan modeli gerektirir, sadece taklit insanlarda hastalık, ancak en az olan, tekrarlanabilir ve sürdürülebilirdiğer kornea ve oküler yapıların yaralanma montaj. Bu model tedavileri değerlendirmek ve moleküler ve hücresel düzeyde hastalık mekanizmalarını açıklamak gereklidir. Dönen bir çapak kullanımı ile, 1 Daha önce anlatıldığı gibi, kolayca bahsedilen avantajlara sahiptir ve en az üç ay boyunca devam LSCD bir fare modeli geliştirebilir. Bu çalışmanın amacı LSCD basit, tekrarlanabilir ve sürdürülebilir fare modeli sunmaktır. Dönen çapak eşit altta yatan stroma 1 zarar vermeden epitel kaldıran bir araçtır. Yara iyileştirici çalışmalarda kornea erozyon 7, 8 uyarılması için kullanılmıştır. Bir fare LSCD oluşturmak için vesile-sunulan teknik daha önce rapor edilmemiştir. Daha önce daha fazla restorasyon o ile, daha az homojen yaralanma-özellikle limbus-ve daha değişken bir fenotip 1, 9 bir künt spatula sonucu epitel kazıma yöntemleri tanıttıf Normal kornea epitel 1. Kör kazıyıcı farklı olarak, döner çapak oyuk 7, 8, 10 gibi epitelyal bazal membran kaldırır. kök hücreleri ayırmak için bildirilen diğer metotlar, altta yatan göz yapılarına istenmeyen yaralanmalara neden olabilir, aynı zamanda önemli enflamasyon ve daha sonra kornea opasitesi ya yol açabilir, sadece sodyum hidroksit, n-heptanol, ve benzalkonyum klorür gibi kimyasal kullanılmasını içerir epitel skuamöz metaplazi 11-15. Dönen çapak bu ciddi komplikasyonlar ile ilişkili değildir. Cerrahi, tek başına ya da kimyasal maddelerin 10, 11, 16 kullanımı ile, limbal epitel kaldırma gerçekleştirmek için daha zordur ve küçük gözler ve (fare gibi) ince limbal epitel hayvanlarda en iyi seçenek değildir. Buna ek olarak, şaşırtıcı, limbectomy hala 10 gerisinde limbal epitel bazı bırakabilir. Aşağıda tarif edilen teknik, neovaskülarizasyon AN LSCD neden olurhastada tam LSCD sunumunu taklit ve en az üç ay 1 sürer D konjonktiva. Bu farelerde LSCD veya yara iyileşmesi patofizyolojisi, immünoloji ve potansiyel tedavi çalışma amacı olanlar için uygundur. Muhtemelen, bazı değişikliklerle, bu prosedür sıçan ya da tavşan 10 gerçekleştirilebilir. Dönen çapak verimli epitel kaldırır gibi, korneal epitel aşınma / yaralama ve herhangi bir ilgili tedavi ya da moleküler biyoloji çalışmalarında ilgili herhangi bir araştırma kullanılabilir.

<table border="0" cellpadding="0" cellspacing="0" width="1091">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;Rotating burr: AlgerBrush II rust ring remover&#160;</td>
			<td style="width:277px;">Rumex International Co, Clearwater, FL</td>
			<td style="width:144px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 16-141</td>
			<td style="width:351px;">0.5 mm burr. Also available from other companies.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;Surgical microscope</td>
			<td style="width:277px;">Wild Heerbrugg, Switzerland</td>
			<td style="width:144px;">&#160;&#160;&#160;&#160;&#160;&#160; Wild M691</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;Digital camera</td>
			<td style="width:277px;">Nikon, Thailand</td>
			<td style="width:144px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;Nikon FS-2 slit lamp</td>
			<td style="width:277px;">Nikon, Japan</td>
			<td style="width:144px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;Polyclonal anti-CK12 antibody</td>
			<td style="width:277px;">Santa Cruz Blotechnology, CA,</td>
			<td style="width:144px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160; SC-17101</td>
			<td>1:100 concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;Monoclonal anti-CK8 antibody</td>
			<td style="width:277px;">TROMA-I-s, Iowa City, IA</td>
			<td style="width:144px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160; AB_531826</td>
			<td>1:50 concentration</td>
		</tr>
	</tbody>
</table>

a simple mechanical procedure to create limbal stem cell deficiency in mouse

Limbal kök hücre eksikliği (LSCD) arıza veya kornea epitel konjonktiva ile değiştirilir sonra limbal epitel kök hücrelerin, kaybı durumudur. Hastalar tekrarlayan kornea defektleri, ağrı, enflamasyon ve görme kaybı muzdarip. Daha önce, LSCD bir fare modeli tarif ve diğer iki model ile karşılaştırılmıştır. amaç hem taklit insanlarda fenotip ve hastalık patofizyolojisini incelemek için ve yeni tedaviler değerlendirmek mümkün kılmak için yeterince uzun sürer LSCD tutarlı bir fare modeli üretmek oldu. Burada, tekniğin daha ayrıntılı olarak tarif edilmektedir. Dönen bir çapak ile bir motorlu araç kornea yüzeyinden pas halkaları çıkarmak için ya da hastaların pterjium yatak düzeltmek için tasarlanmıştır. İstenen LSCD model oluşturmak için uygun bir cihazdır. Sanki, küçük gözleri üzerinde çalışmak için uygun kılan bir ince ucu ile bir hazır, kolay kullanımlı bir araçtırFarelerde. Bu uygulama, göze gereksiz travma önler ve genellikle kimyasal yaralanma modellerinde olduğu gibi, istenmeyen yaralanmalara neden olmaz. künt kazıyıcı aksine, bu bazal membran ile epitel kaldırır. Bu protokolde, limbal alan iki kez aşınmış ve daha sonra bütün kornea epitel limbustan limbusa tıraş edildi. Stroma yaralanmasını önlemek için, bakım epiteli zaten kaldırıldı kez kornea yüzeyi fırça değil alındı.

Bu tekniği gerçekleştirdikten sonra bir ay içinde, kornealar% 100 yüzeyel neovaskülarizasyon gelişti (Şekil 1A. Fotoğraflar parlak alan ve kobalt mavisi filtrenin altına bir yarık lamba bağlı bir kamera ile çekildi). Gözün 18/20 (% 90) &#39;de, yeni damar oluşumu, kornea bütün yüzeyi (Şekil 1A) içeriyordu. 2/20 (% 10), neovaskülarizasyon üç dört kornea kadranlarda gözlendi, ama yine de kornea merkezine ulaştı. <p class="j...

Watch this Scientific Journal Video about A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse at JoVE.com

A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse

aşağıdaki makale etkin bir limbal kök hücre yetmezliği (LSCD) sürdürülebilir bir fare modeli oluşturmak için kolay, tekrarlanabilir teknik sağlar. Bu hayvan modeli test ve limbal kök hücre hastalıkları için tedavi etkinliğini karşılaştıran yararlıdır.

Basit mekanik Prosedür Mouse Limbal Kök Hücre Eksikliği oluşturma

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with ...

a-simple-mechanical-procedure-to-create-limbal-stem-cell-deficiency

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

8.3K Görüntüleme.  University of Illinois at Chicago.  aşağıdaki makale etkin bir limbal kök hücre yetmezliği (LSCD) sürdürülebilir bir fare modeli oluşturmak için kolay, tekrarlanabilir teknik sağlar. Bu hayvan modeli test ve limbal kök hücre hastalıkları için tedavi etkinliğini karşılaştıran yararlıdır. 

Video: A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

Bir Fare Modeli Utero Organ Nakli

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Tıp

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

İzole İskelet Kası Myo-mekanik Analizi

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

A Simple Method of Mouse Lung Intubation

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

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

Fare Akciğer Entübasyonun Basit Bir Yöntem

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

Parabiosis in Mice: A Detailed Protocol

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of microvasculature at the site of inflammation. Parabiotic partners share their circulating antigens and thus are free of adverse immune reaction. First described by Paul Bert in 18641, the parabiosis surgery was refined by Bunster and Meyer in 1933 to improve animal survival2. In the current protocol, two mice are surgically joined following a modification of the Bunster and Meyer technique. Animals are connected through the elbow and knee joints followed by attachment of the skin allowing firm support that prevents strain on the sutured skin. Herein, we describe in detail the parabiotic joining of a ubiquitous GFP expressing mouse to a wild type (WT) mouse. Two weeks after the procedure, the pair is separated and GFP positive cells can be detected by flow cytometric analysis in the blood circulation of the WT mouse. The blood chimerism allows one to examine the contribution of the circulating cells from one animal in the other.

Parabiotic joining of two organisms leads to the development of a shared circulatory system. In this protocol, we describe the surgical steps to form a parabiotic connection between a wild-type mouse and a constitutive GFP-expressing mouse.

Farelerde parabiyoz: Detaylı Bir Protokol

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of ...

A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation of the animal for surgery, opening the thoracic body wall, exposing the desired rib from the surrounding intercostal muscles, excising the desired section of rib without inducing a pneumothorax, and closing the incisions. Compared to the bones of the appendicular skeleton, the ribs are highly accessible. In addition, no internal or external fixator is necessary since the adjacent ribs provide a natural fixation. The surgery uses commercially available supplies, is straightforward to learn, and well-tolerated by the animal. The procedure can be carried out with or without removing the surrounding periosteum, and therefore the contribution of the periosteum to repair can be assessed. Results indicate that if the periosteum is retained, robust repair occurs in 1 - 2 months. We expect that use of this protocol will stimulate research into rib repair and that the findings will facilitate the development of new ways to stimulate bone repair in other locations around the body.

The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

Fare kaburga Rezeksiyonu için Cerrahi Prosedür: Büyük Ölçekli Uzun Kemik Onarım İçin Bir Model

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pankreas Kanseri Yeni Tedavi Stratejileri Geliştirme Hücre Özellikleri Kök incelenmesi

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe the use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations of superior and uniform quality to that which can be achieved by hand. The device comprises a base that holds 4 stainless steel rods and a top, which acts a cutting guide. The rods are inserted into the lumen of the small intestine [divided into thirds] and the colon. The rods and samples are then placed over a piece of filter paper or card into the holding slots in the base of the device. The top of the device is then positioned and serves as a cutting guide. The two angled sections in the center of the top piece are used to guide a knife or scalpel and cut the intestines longitudinally on the top of the rods. Once the intestinal sections have been cut, the top is removed and the card,&#160;tissue and rods gently removed from the device and placed on the bench. The rods are then gently rolled sideways to flatten and stick the intestinal segments onto the underlying piece of filter paper or card. The final preparation can then be examined or fixed and stored for later analysis. The preparations are invaluable for the study of intestinal changes in normal or genetically modified mouse models. The preparations have been used for the study and quantification of the effects of inflammation (colitis), damage, pre-cancerous lesions (aberrant crypt foci (ACFs) and mucin depleted foci (MDFs)) and polyps or tumors.

The use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations is described.

Basit Aygıt Hızla Fare Bağırsak Tüm Mounts Hazırlanır

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Bir Yenidoğan Fare Omurilik Sıkıştırma Yaralanması Modeli

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

Ven interpozisyonu Model: baypas açıklığının Eğitim için uygun bir model

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of therapeutic stem cells via the intranasal route is a recently discovered alternative strategy, with strong potential for clinical translation, due to its non-invasive nature compared to intracranial implantation or delivery via systemic routes. The lack of blood brain barrier further strengthens the therapeutic potential of stem cells undergoing intranasal brain entry. This article summarizes the essential techniques utilized in our studies and outlines the basic principles of intranasal strategy for stem cell delivery using a mouse model of intracranial glioma xenografts. We demonstrate the optimized procedures that generate consistent and reproducible results with specific predetermined experimental parameters and offer guidelines for streamlined work flow that ensure efficient execution and reliable experimental outcome. The article is designed to serve as a baseline for further experimental customization based on hypothesis, stem cell types, or tumor specifics.

Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma.

Bir Fare Modelinde Tedavi Edici Kök Hücrelerin Glioblastomaya İntranazal Dağıtımı

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of ...