Yazarlar laboratuvarda mesleki ve mükemmel teknik çalışmaları için Sayın Dalia Sela ve Bay Emi Sharon müteşekkiriz.

Hayvanlar araştırma hayvanların kullanımına ilişkin ARVO kararına uygun olarak tedavi edildi. Çalışma protokolü Laboratuar Hayvanları Araştırma kurumsal Komitesi (.; 021211 onay numarası 022-4598-2) tarafından onaylandı. Cerrahi 1. 1. Hazırlık <ol><li> Tartılır ve ketamin hidroklorür 100 mg / ml (100 mg / kg) ve ksilazin hidroklorür 25 mg / ml (12.5 mg / kg) içeren bir intramüsküler enjeksiyon ile 13 günlük bir Yeni Zelanda beyaz tavşanı anestetize. Bu dozlar nedeniyle, cerrahi prosedürün süresi için kullanılmaktadır. Bir ağrı refleksi eksikliği ile anestezi uygun seviyede sağlamak. </li><li> Steril bir pamuklu çubuk kullanarak, kurumasını kornea önlemek için sigara ameliyat gözle oftalmik merhem küçük bir miktar uygulanır. kurumasını kornealar önlemek için işlem sırasında normal tuzlu su işletilen göz kornea üzerine damla% 0.9 uygulayın. </li><li> oxybuprocain (topikal anestezi sonrası her gözün üç aksiyel uzunluk ölçümleri gerçekleştirmeksonra e hidroklorür% 0.4) bir ultrason A-tarayıcı kullanarak ve ölçümler ortalama. merkezi kornea dik probu uygulayın. </li></ol> 2. Cerrahi # 1 - Ön Çapraz Bağlama Adım <ol><li> bir göz ameliyat mikroskobu altında cerrahi göz forseps ve makas kullanılarak, cerrahi göz forseps ve makas ile 360 ​​° konjonktiva kesi yapmak. </li><li> açılı forseps veya kas kanca dört ekstraoküler rektus kasları tanımlamak ve 2-0 örgülü ipek olmayan Needled dikişlerle onları izole kullanma. NOT: ipek dikişler istenilen yönde dünyayı hareket ile yardımcı olacaktır. gözün dört rektus kaslarının (dört kadran) arasındaki kadranın ayrılmıştır. <ol><li> Bir cilt işaretleyici her çeyrekte iki bölge, ekvator sklera az bir ve posterior sklera az bir Mark. Bunlar ışınlama bölgeleri bulunmaktadır. </li></ol></li><li> % 0.1 dekstran-ücretsiz riboflavin-5-fosfat içeren 5ml şırınga 3 ml hazırlayıne. 26 G lakrimal kanül veya 25 G konik hydrodelineator şırınga bağlayın. </li><li> aşağı eğimli ısmarlama fiber optik bağlı bir UV A (370 nm) ışık kaynağı içeren ışınlama cihazını hazırlayın. Enerji gücü ve kalibrasyon ölçümü takiben (aşağıda Not bakınız), 57 mW / cm² şekilde ayarlayabilirsiniz. NOT: Kalibrasyon üretici tarafından ticari gerçekleştirildi. Bu yöntem, 57 J / cm² toplam UVA ışık dozu içerir. (0.2 cm 2 57 mW / cm2 2.2 J 0.2 cm2 nokta kümülatif yük 200 saniye boyunca 11.4 mW / 0.2 cm 2 kullanılarak 11.4 mW / 0.2 cm 2 olduğunu) </li></ol> 3. Cerrahi # 1 - Çapraz Bağlama <ol><li> Seçilen çeyreğin ters yönde ipek sütür çekerek gözün taşımak tedavi edilecek (alt temporal kadranda tedavi ederken, örneğin, yukarı çekin ve burun). 2-0 örgülü ipek olmayan iğneli sütür kullanarak küresi hareketini gerçekleştirmek hangi iHer rektus kas solates. </li><li> ışınlama başlamadan önce ışınlama bölgesi 20 sn üzerine% 0.1 dekstran-ücretsiz riboflavin-5-fosfat içeren foto çözümü uygulayın. </li><li> 200 sn bir ışınlama süre için aşağı eğimli özel yapılmış fiber optik kullanarak ışınlama bölgesi ışın tedavisi. 4 mm bir çapa sahip 0.2 cm² her bölgenin alanı ölçülür. </li><li> 200 sn ışınlama süresince ışınlama bölgesi üzerine her 20 saniyede% 0.1 dekstran içermeyen riboflavin-5-fosfat ihtiva eden foto-çözeltisi uygulanır. NOT: ışınlama ve iki cerrah tarafından aynı anda riboflavin düşmesini gerçekleştirin. </li><li> Tekrarlayın dört gözün kadranın her biri için 3.1-3.4 adımları. </li></ol> 4. Cerrahi # 1 - Gönder Çapraz Bağlama Adım <ol><li> Göz çevresinde kürk tıraş. Cerrahi oftalmik makas kullanılarak kapak kenar boşluklarını Trim ve daha sonra yavaşça üst ve 4-0 örgülü ipek fildişi rengi kullanarak düşük göz kapakları sütür - 3/8 daire yenidenayetin demircilik 13 mm uzunluk C-3 iğne (tarsorrhaphy). prosedür (kloramfenikol% 5) sonunda kapak kenarındaki göz merhem küçük bir miktar uygulayın. </li></ol> 5. Cerrahi # 1 - Gönder Operatif Bakım <ol><li> kurtarma sırasında bir ısıtma lambası altında hayvanların tutun. sternal yatma korumak için yeterli bilinci yerine kadar gözetimsiz bir hayvan bırakmayın. </li><li> tamamen iyileşti kadar yeni yatak ile temiz bir kafeste hayvan yerleştirin. İyileştikten sonra, hayvan, hayvan odasına iade edilecektir. sadece uyanık annesi hayvan dönün. </li><li> İlk 48 saat boyunca, genel durumunu ve hastalık veya enfeksiyon belirtileri ile ilgili hayvanları inceler. Tavşan davranış değişikliği gözlenmesi halinde gerektiğinde bölüm 1. Yönet analjezi başına analjezi yönetme. (Metamizol 50 mg / kg her 6 saat düşer). </li></ol> 6. Cerrahi 2. <ol><li> Elli beş gün yıllardan sonraurgery 1., ağırlık ve ketamin hidroklorür 100 mg / ml (100 mg / kg) ve ksilazin hidroklorür 25 mg / ml (12.5 mg / kg) içeren bir intramüsküler enjeksiyon ile anestezi tavşan. </li><li> bir göz ameliyat mikroskobu altında cerrahi göz makas kullanarak, tarsorrhaphy çıkarın. topikal anestezi (oksibuprokain hidroklorür% 0.4) bir ultrason A-tarayıcı kullandıktan sonra her gözün üç aksiyel uzunluk ölçümleri gerçekleştirmek ve sonra ölçümler ortalama. merkezi kornea dik probu uygulayın. </li><li> intraperitonal fenobarbital sodyum (200 mg / 1.5 kg vücut ağırlığı) ile tavşan öldürülür. </li></ol>

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Scand. 83, 477-482 (2005).</li><li>Wollensak, G., Iomdina, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+biomechanical+properties+of+rabbit+sclera+after+collagen+crosslinking+using+riboflavin+and+ultraviolet+(UVA).">Long-term biomechanical properties of rabbit sclera after collagen crosslinking using riboflavin and ultraviolet (UVA).</a> Acta Ophthalmol. 87, 193-198 (2009).</li><li>Wildsoet, C. F., Norton, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+controlling+myopia+progression.">Toward controlling myopia progression.</a> Optom. Vis. Sci. 76, 341-342 (1999).</li><li>Avetisov, E. S., Tarutta, E. P., Iomdina, E. N., Vinetskaya, M. I., Andreyeva, M. 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Yazarlar hiçbir rakip mali çıkarları olduğunu beyan ederim.

Bu riboflavin ve UVA ışıması ile çapraz bağlama teknolojisini kullanan bir tavşan modelinde, eksenel miyopi önlenmesi vivo çalışmasında ilk sunulmaktadır. Farklı deney hayvanları bu tip çalışmalar kullanılabilir olsa da, çoğunlukla göz boyutu ve sklera yüzeyi üzerinde çapraz bağlanmasını gerçekleştirmek için ihtiyaç tavşan seçtik. Biz tavşan sklera açığa üst ve alt göz kapakları dikilmesi zorlu prosedürler olduğu bulundu. Biz kapak marjları kırpma ve göz kapakları sıkıca düzeltmeler gerek kalmadan belirli bir süre için kapalı olduğundan emin olmak için protokolde belirtilen sütür kullanmanızı öneririz. dikkate almak önemli bir şey ışınlama cihazı ve gerekli güç enerji fiber optik kalibre ihtiyacıdır. H &amp; E histolojik slaytlar çapraz bağlama prosedürü uygulandı gözleri retina hiçbir toksik bir değişiklik ortaya. İleri incelemelerde enerji ve maruz kalma süresi bir pozisyonda odaklanarak, miktarı ile, retina, koroid ve sklera üzerinde riboflavin ve UVA ışınlama potansiyel toksisitesini gidermek için yapılmalıdır. Bu çalışmada emri dikkate çeşitli sınırlamalar. Sadece ameliyat öncesi ve sonrası aksiyel uzunlukları ölçüldü. gözün aksiyel uzunluk A-scan ultrasonografi ile ölçüldü. Biz biyometrik özelliklere refraktif analiz veya sklera biyomekanik özelliklerini değerlendirmek vermedi. Birlikte ele alındığında, biz tavşan gözün eksenel uzama riboflavin ve UVA ile sklera çapraz bağlama etkisi miyopi araştırma üzerinde olumlu bir etkisi olacağını düşünüyorum.

Miyopi kırılma bozukluklarının en yaygın olanıdır. ABD&#39;de ve Avrupa&#39;da miyopi prevalansı% 30 civarında olduğu bildirilmektedir ve Asya ülkelerinde genel nüfus 1,2% 60 kadar etkiler. Miyop ilerlemesi, genellikle iki yıllık bir aralık üzerinde 3 yaklaşık -0.5 diyoptri arasında bir oranda, miyoplara% 50 kadar gerçekleşir. Miyopi dayattığı sağlık maliyetleri gözlük masrafları, kontakt lens ve refraktif cerrahi ve glokom, katarakt, retina dekolmanı ve görme bozukluğu 4-6 artmış sağlık risklerine ilişkin masraflar dahil olmak üzere, önemli bulunmaktadır. Miyopi Hayvan çalışmalarında, görme azalması göz ve kornea dövme 11 kısa mesafede gözkapağı sütür 7-10, bir tıkayıcı yerleştirme indüklenir. Yapay miyopi Bu çalışmaların gerçekleştirilmesi için, ancak, oklüzyon sürecinin hiçbir görüş yoksunluğu deneyleri Carr gibi, çok küçük hayvanlar üzerinde gerçekleştirilmelidirYetişkin numuneler üzerinde ied başarılı olmuştur. Şiddetli miyopi önemli özelliklerinden biri gibi Ehlers gibi bağlı sklera bazı metabolik bozukluk nedeniyle görsel yoksunluğu 12 veya daha sonra emmetropization bir rahatsız geri bildirim mekanizması muhtemelen sklera ilerici incelme ile sklera bir patolojik değişiklik Danlos sendromu 13. Sonuçta, her iki mekanizma nedeniyle böyle fibrillogenesis 16 bir azalma kollajen lif çapı 14,15 ve bozuklukları gibi miyop sklera yapısal anormallikler kadar uzanan ve sklera, retina ve koroid incelmesi yol açar. Çeşitli çalışmalar bozulmuş kollajen çapraz bağlama miyop sklera 17-18 zayıflaması sürecinde önemli bir faktör olduğunu göstermiştir. WOLLENSAK ve ark., 19-21 riboflavin ve ultraviyole-A (UVA) ışınlama foto uygulayarak kollajen çapraz bağlanmasını uyarılan (370 nm)ve in vitro 19 domuz ve insan sklera sertlik önemli bir,% 157 artış ve in vivo tavşan sklera sertliğinde bir 465% artış (Young modülü) 20 kaydetti. Sertliği 4 ay sonra, 3 gün sonra 320,4 göre% 277.6% arttı ve 8 ay (Young modülü) 22 sonra 502%: Çapraz bağlanma, aynı zamanda in vivo tavşan sklera üzerinde uzun süreli bir etkisi vardı. Miyop ilerlemesi tutuklama terapötik girişimler 23-26 yayınlandı ancak bu yöntemlerin başarısı tartışmalıdır. progresif miyopi önlenmesi Resim etkili bir araç bugüne kadar tespit edilmiştir. miyopi etiyolojisi hala tartışmalıdır ve tedavisi bir sorun teşkil etmektedir. Bu bulgulara dayanarak, skleral çapraz miyop ilerlemesi sklera tabanlı tedavi için bir araç olarak hizmet verebilir varsayılmaktadır. Bu çalışmanın amacı, skleral kollajen crosslinki incelemekgörsel eksen tıkanması ile uyarılan eksenel miyopi gelişmesi üzerinde etkisi ng.

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			<td height="21" style="height:21px;width:244px;">2-0 braided silk non-needled sutures&#160;</td>
			<td style="width:137px;">ETHICON</td>
			<td style="width:119px;">W193</td>
			<td style="width:167px;"></td>
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			<td height="25" style="height:25px;">4-0 braided silk ivory color&#160;</td>
			<td style="width:137px;">ETHICON</td>
			<td style="width:119px;">W816</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">0.1% dextran-free riboflavin-5-phosphate 1mg:1ml</td>
			<td style="width:137px;">Concept for Pharmacy Ltd</td>
			<td style="width:119px;">D2-5025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV A (370nm) light source&#160;</td>
			<td>O/E LAND Inc</td>
			<td style="width:119px;">NCSU033B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Beveled down custom made fiber optic&#160;</td>
			<td>Prizmatix Ltd</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:244px;">26G lacrimal cannula&#160;</td>
			<td style="width:137px;">Beaver-visitec International Ltd.&#160;</td>
			<td style="width:119px;">REF581276</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:244px;">25G tapered hydrodelineator [Blumenthal]&#160;</td>
			<td style="width:137px;">Beaver-visitec International Ltd.&#160;</td>
			<td style="width:119px;">REF585107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">13 days old rabbits</td>
			<td style="width:137px;">Harlan&#160;</td>
			<td style="width:119px;">1NZWR40</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:244px;">Ultrasonic biometer</td>
			<td>Allergan-Humphrey</td>
			<td style="width:119px;">820-519</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Skin marker</td>
			<td style="width:137px;">Devon</td>
			<td style="width:119px;">4237101664X</td>
			<td></td>
		</tr>
	</tbody>
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scleral cross-linking using riboflavin and ultraviolet-a radiation for prevention of axial myopia in a rabbit model

Miyop kişiler, şiddetli miyopi ile özellikle, katarakt, glokom, retina dekolmanı ve korioretinal anormalliklerin yüksek normalden risk altındadır. Buna ek olarak, patolojik miyopi görme bozukluğu ve körlük 1-3 ortak bir geri dönüşü olmayan bir nedenidir. Çalışmamız bir tavşan modelinde eksenel miyopi gelişimine riboflavin ve ultraviyole-A ışınları kullanılarak skleral çapraz bağlanma etkisini gösterir. gözün aksiyel uzunluk 13 gün (erkek ve dişi) yaş Yeni Zelanda beyaz tavşanlarda A-tarama ultrason ile ölçüldü. Sonra göz tarsorrhaphy tarafından takip sklera çapraz bağlanma, 360 ° konjonktiva kesi uygulandı. Eksenel uzama onların sağ göz kapaklarını (tarsorrhaphy) dikilmesi ile 13 gün eski Yeni Zelanda tavşanlarda oluşturuldu. gözler çeyrek ayrıldı ve her kadran 0.2 cm² bir alanı ve 4 mm çapa sahip, iki sklera ışınlama bölgeleri, her biri vardı. Çapraz bağlanma,% 0.1 bırakarak gerçekleştirildidekstran içermeyen morötesi A radyasyondan önce radyasyon bölgeleri 20 saniye ve 200 saniye ışınlama süresi boyunca her 20 saniyede üzerine riboflavin-5-fosfat. UVA radyasyonu (370 nm) 57 mW / cm² (toplam UVA ışık dozu, 57 J / cm²) de sklera dik uygulandı. Tarsorrhaphies tekrar eksenel uzunlukta ölçümleri, ardından günde 55 ayrılmıştır. Bu çalışma, riboflavin ve ultraviyole-Radyasyonla skleral çapraz bağlama etkin bir tavşan modelinde oklüzyon kaynaklı eksenel uzamaya önlediğini göstermektedir.

Şekiller 1 ve 2, grafiksel iki grup eksenel uzunluğu ölçümleri göstermektedir. Sol göz (Şekil 1) ameliyat değil iken Grup 1 tavşan sağ göz skleral çapraz bağlanma ve tarsorrhaphy uygulandı. Sol göz (Şekil 2) ameliyat değil iken Grup 2 tavşan sağ gözüne sadece kesi ve tarsorrhaphy uygulandı. skleral çapraz bağlanma ve tarsorrhaphy uygulanan grupta 1, olarak, sağ göz eksenel uzunluk ortalama 3.61 ± 0.76 mm, ortalama farkın, daha sonra gözkapağı sütür önce 10.68 ± 0.74 mm ve 14.29 ± 0.3 mm 55 gün ölçtük. Unsutured / Opere sol gözünde gelen değerler 4.44 ± 0.81 mm (Şekil 1) ortalama farkı için, 10.70 ± 0.79 mm ve 15.14 ± 0.32 mm idi. dikilir eksenel uzunluklarının karşılaştırılmasıve tıkanma fazının sonunda unsutured gözler dikildi gözünde daha az net artış göstermiştir. Sadece keratektomi ve tarsorrhaphies uygulanan grupta 2, olarak, sağ göz eksenel uzunluk ortalama 5.19 ± 0.85 mm, ortalama farkın, daha sonra gözkapağı sütür önce 10.50 ± 0.67 mm ve 15.69 ± 0.39 mm 55 gün ölçtük. Unsutured / Opere sol gözünde gelen değerler 4.20 ± 0.67 mm (Şekil 2) ortalama farkı için, 10.54 ± 0.71 mm ve 14.74 ± 0.38 mm idi. tıkanıklığı fazının sonunda dikilerek ve unsutured gözlerin eksenel uzunluklarının karşılaştırılması dikilir gözünde daha büyük bir net artış saptandı. Grup 2 (5.19 ± 0.85 mm) ve grup 1 (3.61 ± 0.76 mm) arasında doğru gözlerinin eksenel uzunluğu ortalama değişiklik karşılaştırılması tıkanıklığı p sonunda önemli ölçüde daha düşük bir değer vermiştirhase çapraz bağlama işlemi (p &lt;0.001, Mann-Whitney testi) yapılan gözlerde (55 gün). Sol gözünde ortalama eksenel uzunluğu grup arası fark (4.20 ± 0.67 mm vs 4.44 ± 0.81 mm) (p = 0.39, Mann-Whitney parametrik olmayan testi) istatistiksel olarak anlamlı değildi. <img alt="Şekil 1" src="/files/ftp_upload/53201/53201fig1.jpg" /> Şekil 1. Öncesi ve skleral çapraz bağlama ve sol göz eksenel ölçümlerin vs tarsorrhaphy sonra sağ göz eksenel ölçümler. Skleral çapraz bağlama ve tarsorrhaphy (RE başlangıcı) ve 55 gün sonra tarsorrhaphy kaldırılması (RE sona) sonra önce sağ gözünde eksenel uzunluğu anlamına gelir. Başlangıçta sol göz ortalama aksiyel uzunluk (LE başlangıç) ve 55 gün sonra (LE sonu). sol göz ameliyat değildi ve açık bırakılmıştır. Hata çubukları, ortalamanın standart hatasını gösterir. (Permissio ile yeniden basılmışn referans 27) elde edilmiştir. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53201/53201fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="şekil 2" src="/files/ftp_upload/53201/53201fig2.jpg" /> Şekil 2. Öncesi ve sol göz eksenel ölçümlerin vs tarsorrhaphy sonra sağ göz eksenel ölçümler. Eksenel tarsorrhaphy (RE başlamadan önce) sağ gözünün uzunluğunu ve 55 gün sonra tarsorrhaphy kaldırılması (RE sona) sonra gelir. Başlangıçta sol gözünün eksenel uzunluğunu ortalama hayranlarıyla sol göz ameliyat değildi ve açık bırakılmıştır (LE başlangıç) ve 55 gün sonra (LE sonu). Hata çubukları, ortalamanın standart hatasını gösterir. (Referans 27 izni ile yeniden basılmıştır). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53201/53201fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için tıklayınız</a>.

Watch this Scientific Journal Video about Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model at JoVE.com

Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model

Bu eksenel uzama tavşan göz riboflavin ve UVA sklera çapraz bağlanma etkisini göstermektedir. Eksenel uzama onların sağ göz kapaklarını (tarsorrhaphy) dikilmesi yoluyla (erkek ve dişi) 13 gün eski Yeni Zelanda tavşanlarda oluşturuldu.

Skleral bir Tavşan Modelinde Aksiyel Miyopide Önleme Riboflavin ve Ultraviyole-A Radyasyon Kullanımı Çapraz bağlama

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal ...

scleral-cross-linking-using-riboflavin-ultraviolet-radiation-for

Nanyang Technological University

Research

JoVE Journal

Medicine

8.8K Görüntüleme.  Rabin Medical Center, Beilinson Campus.  Bu eksenel uzama tavşan göz riboflavin ve UVA sklera çapraz bağlanma etkisini göstermektedir. Eksenel uzama onların sağ göz kapaklarını (tarsorrhaphy) dikilmesi yoluyla (erkek ve dişi) 13 gün eski Yeni Zelanda tavşanlarda oluşturuldu. 

Video: Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

Meme Kanseri Beyin metastaz tümör Hipoksi Dynamics in vivo Biyoparlaklık Görüntüleme Fare Modeli

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Tıp

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

Stereotaktik intrakranial implantasyon ve<em&gt; In vivo</emGlioblastoma Multiforme bir Mouse Model Sistemde Tümör Xenografts arasında&gt; Bioluminescent Görüntüleme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated cartilage lesions lead to ongoing pain, negatively affect the quality of life and predispose for osteoarthritis. During the last decades, several surgical techniques have been developed to treat such lesions. However, until now it was not possible to achieve a full repair in terms of covering the defect with hyaline articular cartilage or of providing satisfactory long-term recovery 2-4. Therefore, articular cartilage injuries remain a prime target for regenerative techniques such as Tissue Engineering. In contrast to other surgical techniques, which often lead to the formation of fibrous or fibrocartilaginous tissue, Tissue Engineering aims at fully restoring the complex structure and properties of the original articular cartilage by using the chondrogenic potential of transplanted cells. Recent developments opened up promising possibilities for regenerative cartilage therapies.
The first cell based approach for the treatment of full-thickness cartilage or osteochondral lesions was performed in 1994 by Lars Peterson and Mats Brittberg who pioneered clinical autologous chondrocyte implantation (ACI) 5. Today, the technique is clinically well-established for the treatment of large hyaline cartilage defects of the knee, maintaining good clinical results even 10 to 20 years after implantation 6. In recent years, the implantation of autologous chondrocytes underwent a rapid progression. The use of an artificial three-dimensional collagen-matrix on which cells are subsequently replanted became more and more popular 7-9.
MACT comprises of two surgical procedures: First, in order to collect chondrocytes, a cartilage biopsy needs to be performed from a non weight-bearing cartilage area of the knee joint. Then, chondrocytes are being extracted, purified and expanded to a sufficient cell number in vitro. Chondrocytes are then seeded onto a three-dimensional matrix and can subsequently be re-implanted. When preparing a tissue-engineered implant, proliferation rate and differentiation capacity are crucial for a successful tissue regeneration 10. The use of a three-dimensional matrix as a cell carrier is thought to support these cellular characteristics 11.
The following protocol will summarize and demonstrate a technique for the isolation of chondrocytes from cartilage biopsies, their proliferation in vitro and their seeding onto a 3D-matrix (Chondro-Gide, Geistlich Biomaterials, Wollhusen, Switzerland). Finally, the implantation of the cell-matrix-constructs into artificially created chondral defects of a rabbit's knee joint will be described. This technique can be used as an experimental setting for further experiments of cartilage repair.

An experimental technique for the treatment of chondral defects in the rabbit's knee joint is described. The implantation of autologous chondrocytes seeded on a matrix is a well-accepted method for the remodeling and repair of articular cartilage lesions providing satisfying long-term results. Matrix-assisted autologous chondrocyte transplantation (MACT) offers a standardized and clinically established implantation method.

Bir Tavşan Modelinde Kondral Kusurların Tadilat ve Onarım için matris destekli Otolog Kondrosit Nakli

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Bir Zebra balığı Otokton Tümör Modeli kullanılarak Melanom Değiştiricileri için Tarama

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

<em&gt; Ovo</emSarkoma için bir Xenograftmodel olarak&gt; CAM-testi

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.

Subaraknoid Kanama Akut ve Geç sekeli Çalışmaları Tavşan Kan şant Modeli: Teknik Yönleri

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and ...

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and negatively impact patient quality of life and long term survival. Advances in the understanding of the biological pathways associated with normal tissue toxicities have allowed for the development of interventional drugs, however, current response studies are limited by a lack of quantitative metrics for assessing the severity of skin reactions. Here we present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe the instrumentation design of the DOS system as well as the inversion algorithm for extracting the optical parameters. Finally, to demonstrate clinical utility, we present representative data from a pre-clinical mouse model of radiation induced erythema and compare the results with a commonly employed visual scoring. The described DOS method offers an objective, high through-put evaluation of skin toxicity via functional response that is translatable to the clinical setting.

We present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe DOS instrumentation design, optical parameters extraction algorithms and the animal handling procedures required to yield representative data from a pre-clinical mouse model of radiation induced erythema.

Bir fare Modeli Kullanılarak Akut Radyasyondan Bağlı Cilt zehirlilik Kantitatif Değerlendirme optik tayf Diffüz

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and ...

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon&#39;s space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using&#160;second harmonic generation imaging and differential scanning calorimetry.

Tavşan sklera bir araç olarak ikinci harmonik üretimi sinyalleri tedavi doku (TXL) için miyopi Cross-linking değerlendirilmesi için

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

İzometrik Contractility ölçüm tel Myography kullanarak fare mezenterik arter

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the safety of stem cell transplantation. Although the transplantation of differentiated rather than undifferentiated cells is common practice, teratomas can still arise from the presence of residual undifferentiated stem cells at the time of transplant or from spontaneous mutations in differentiated cells. Because stem cell therapies are often delivered into anatomically sensitive sites, even small tumors can be clinically devastating, resulting in blindness, paralysis, cognitive abnormalities, and cardiovascular dysfunction. Surgical access to these sites may also be limited, leaving patients with few therapeutic options. Controlling stem cell misbehavior is, therefore, critical for the clinical translation of stem cell therapy.
External beam radiation offers an effective means of delivering targeted therapy to decrease the teratoma burden while minimizing injury to surrounding organs. Additionally, this method avoids genetic manipulation or viral transduction of stem cells-which are associated with additional clinical safety and efficacy concerns. Here, we describe a protocol to create pluripotent stem cell-derived teratomas in mice and to apply external beam radiation therapy to selectively ablate these tumors in vivo.

Research on treatment strategies for pluripotent stem cell-derived teratomas is important for the clinical translation of stem cell therapy. Here, we describe a protocol to, first, generate stem cell-derived teratomas in mice and, then, to selectively target and treat these tumors in vivo using a small-animal irradiator.

Pluripotent kök hücre kaynaklı Teratomas Dış Beam radyasyon bir küçük-hayvan modelinde kullanarak hedeflenen ve seçici tedavisinde

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the ...