S och Drottning ViktoriaS Frimurarstiftelse: Bu çalışma Karolinska Enstitüsünde KID-finansman, İsveç Radyasyondan Korunma Kurumu, Karolinska Enstitüsünde Göz Araştırma Vakfı, Silah och Bertil Stohnes Stiftelse, St Erik&#39;in Göz Hastanesi Araştırma Vakfı, Ögonfonden, Konung Gustav V tarafından desteklenmiştir.

1. Ultraviyole Radyasyon Maruziyet <ol><li> 15 dakika maruz kalmadan önce, 90 mg / kg Ketalar (ketamin) ve karın zarı içinden enjeksiyon ile 10 mg / kg Rompun (ksilazin) oluşan bir karışım ile bir dişi Sprague-Dawley sıçan anestetize. </li><li> Bir sıçan süzgeç içinde hayvan yerleştirin ve bir gövde sıkmak 13 neden olmadan sıçan immobilizasyon kadar kemerleri sıkın. </li><li> Midriazis sebep olmak için her iki sıçan gözünde Mydriacyl (tropikamid), 10 mg / ml, aşılamak. </li><li> O bir gözü yarısı maksimum 10 nm tam genişliği ile 300 nm arasında 14 UVR dar bir ışın karşı konumlandırılmış böylece hayvan yerleştirin ve siyah bant ile kontralateral göz kalkan. </li><li> 15 için 300 nm dk 15 sıçan tek taraflı alt-eşik dozu 1 kJ / m 2 UVR-B Açığa. </li></ol> 2. Teşrih <ol><li> Önceden belirlenmiş bir süre maruz kalma sonrası (1, 5, 24 ve 120 saat) sonra, aktif kömür ile altı haftalık bir sıçan (ağırlık &lt;200 g) kurbann dioksit ile boğularak ve servikal vertebra dislokasyonu. </li><li> Aydınlatmak gözler. Bundan sonra, aşağı arka tarafında gözün kornea koymak. Sonra, yumruk 27 gauge kanül uygulayarak ve sklera çok yakın Merceğin zarar görmemesi için sklera teğet itin tarafından minimal bir delik. Sonra sklera arka kısmı kapalı kaldırılabilir kadar sadece limbus arkasında çevresel kesmek oftalmik cerrahi bir makas kullanın. Sonra, bir künt kavisli bir forseps ile objektif kaldırın. </li><li> 5-10 dakika daha, artık dengeli tuz çözeltisi içinde mercek tutarak, bir mikroskop altında objektif ekvatordan silier cisim kalıntıları çıkarın. </li></ol> 3. Nicel RT-PCR <ol><li> 350 ul RA1 lizis tamponu (NucleoSpin RNA II, total RNA izolasyon kiti) ve 2 ml Eppendorf tüp içinde 3.5 ul β-merkaptoetanol bir karışımı içine bir mercek yerleştirin. </li><li> Oda sıcaklığında 30 dakika boyunca bu karışıma, mercek tutun. </li><li> Lensi homojenizeObjektifin sadece sert nükleus kadar bir iğne lens (korteks ve lens kapsülü lizis tamponu ile parçalanırlar edilmiştir) gelen bırakılır. Karışımı çekirdeğini çıkarın. </li><li> Mağaza örnekleri hemen -70 ° C&#39;de </li><li> Örnekleri çözülme ve protokol &quot;kültür hücreleri ve doku total RNA saflaştırılması&quot; (NucleoSpin RNA II, total RNA izolasyon kiti) izleyin. </li><li> Mağaza RNA örnekleri -70 ° C&#39;de </li><li> 2 dakika için 95 ° C, 40 çevrim için Adım 2: Her bir numune DNA yeterli kaldırma doğrulamak için, aşağıdaki koşullar (adım 1 altında bir PCR çalıştırmak 95 ° C&#39;de 20 saniye, 55 ° C 20 sn için, 72 ° 20 saniye için C, aşama 3: 7 dakika için 72 ° C) p53 DNA spesifik primerler, ileri 5&#39;-ACCCTCTGACCTTTTTCCCA-3 &#39;ve 5&#39;-ters TGCTGGGATCTTAGGCACTC-3&#39; ve Taq DNA polimeraz (dNTPack), üretimi için uygun protokolü. Beklenen PCR ürünü 243 baz çifti olduğunu. </li><li> 243 baz çifti DNA spesifik PCR ürününü algılamak için,% 1.5 agaroz jel elektroforezi çalıştırın. Hazırlamak için:TBE tamponu% 1.5 agaroz jel agaroz 3.75 g alıp TBE tamponu 250 ml çözmek. Etidiyum bromid (0.5 mg / ml),% 1.5 agaroz jel çözeltisi, 500 ml içinde 500 ul ekle. Bir mikrodalga fırında karışımı ısıtın ve agaroz çözmek için karıştırın. </li></ol> Örneklerin hiçbiri% 1.5 agaroz jel elektroforezi üzerinde herhangi bir özel DNA PCR ürünlerinin ortaya çıkarmak için bulunur. <ol start="9"><li> RNA örnekleri (1 ul) konsantrasyonu ve bir Nanodrop ND-1000 spektrofotometre içinde 260 nm (RNA) ve 280 nm (protein) de absorbans numune oranı ölçülür. RNA örnek absorbans protein oranı RNA 2.0 ya da daha fazla ise, o zaman RNA numune saf. RNA örnek absorbans oranı 2.0 &#39;den daha düşük ise, saflaştırma adımı 3,5 RNA yinele. </li><li> 1 ug RNA, karşılık gelen örnek bir hacme al ve protokol 1. Strand cDNA sentez kiti (RT-PCR için 1. Strand cDNA sentez kiti) aşağıdaki cDNA sentezlenir. </li><li> -20 ° C&#39;de cDNA örneklerinde Mağaza(Bir dönem 1 ve daha fazla yıl için, -70 mağaza ° C). </li><li> ICycler MyiQ Tek Renk Real Time PCR sistemi kantitatif gerçek zamanlı PCR çalıştırın. Yük, 1 ul cDNA örnek, TaqMan Gen Ekspresyonu Master Mix, kaspaz 3 TaqMan Gen Ekspresyonu Testi ile 96-plaka üzerine üç kez talimatlara üretimi için uygun. Yük, 1 ul cDNA örnek, TaqMan Gen Ekspresyonu Master Mix, 18s için TaqMan Gen Ekspresyonu Assay ile başka bir 96-plaka üzerine üç kez talimatları (TaqMan Gen Ekspresyonu Assay Protokolü) üretimi için uygun. Serisi üç rastgele seçilmiş açık olmayan lensler cDNA bir kısmını seyreltin. 96 çukurlu bir plaka içinde örnekten elde edilen cDNA ile birlikte seri dilüsyonları çalıştırın. </li><li> Sonuçlarının ölçümü için standart eğri yöntemi kullanın. Eşik floresan az döngü sayısı MyiQ yazılım ölçümü olarak kullanılır. Kalibratörün göreli konsantrasyon fonksiyonu olarak eşiğinde döngü sayısı ifade standart eğri es olduğunuHer plaka seri dilüsyonları için tablished. olmayan üç kabul lens örnekleri cDNA kalibrasyonu için kullanılmıştır. CDNA ölçülen numune göreli konsantrasyonunu elde etmek için standart eğri için her bir örnek için döngü sayısı eşik ilişkilendirir. Son olarak, cDNA iç kontrol 18s için cDNA hedef göreli konsantrasyon ile ilgili olarak, hedef genlerin ekspresyon seviyesi olsun. </li></ol> 4. İmmünhistokimyasal Boyama <ol><li> Tespit <ol><li> PBS içinde göz (; pH 7.4 Fosfat Tamponlu Salin) teşrih. </li><li> 20 dakika boyunca buz gibi soğuk% 4 paraformaldehit (PFA) ile dolu bir tüp içinde göz koyun. Bir gün önce PFA% 4 hazırlayın. +4 ° C&#39;de saklayın kullanımı kadar. PFA yaklaşık bir hafta taze. </li><li> Bir mikropipet PFA ile çıkarın. Daha sonra, 20 dakika süreyle buz gibi soğuk PBS ile yıkayın. </li><li> ° C gece boyunca +4 sakaroz% 30 göz koy. </li><li> Optimal Kesme Sıcaklık (OCT) orta ile dolu bir kap içine göz koyun (Doku-Tek). Kesme işlemi için uygun bir konuma göz koyun. </li><li> Kuru buz üzerinde OCT-orta dondurun. </li><li> -70 ° C&#39;de saklayın kullanımı kadar. </li></ol></li><li> Kesit <ol><li> Uygun bir düzlemde kriyo-kesit için göz yerleştirin. </li><li> Her bir lensin merkezi bir kısmı üç 5 um kalınlıkta orta sagital bölümleri kesin. Farklı bölümlerde aynı çekirdekleri boyama önlemek için sıralı bölümler arasında en az 6 bölümden atın. </li><li> Bir bölümü kesip edildikten sonra, hafifçe üstüne bir slayt yerleştirin. Bölümü daha sonra slayt ayrılmamak gerekir. </li><li> Kullanmadan önce kurumasını bekleyin slaytlar bırakın. Slaytlar kullanılıncaya kadar -20 ° C&#39;de muhafaza edilebilir. </li></ol></li><li> Floresans immünhistokimya <ol><li> Numuneler oda sıcaklığında (5-10 dk) elde edelim. </li><li> Bir PAP-kalem (Invitrogen) ile örnekler etrafında kenarları çizin. </li><li> Bir süre (10 dk) için sınır kurumasını bekleyin. </li><li> Mikroskop slaytlar etiketleyin. </li><li> 1 × P rehidrate15 dakika boyunca BS. </li><li> 30 dakika için standart blok çözelti içinde geçirgen. </li><li> Standart blok çözüm 1:3,000 seyreltilmiş; (Cep Sinyalizasyon Teknolojisi, Inc tavşan poliklonal, bölünmüş kaspaz-3 antikor Asp175 9661) primer antikor ekleyin. </li><li> Gece boyunca +4 ° C&#39;de nemlendirilmiş bir odasında saklayın. </li><li> (3 × 5 dk) pipetleme veya büyük bir banyo (&gt; 15 dk, 1-2 değişiklikleri) daldırarak PBS içinde yıkayın. </li><li> Standart blok çözelti içinde 1:300 oranında seyreltilmiş sekonder antikor (belirli bir emilim / emisyon spektrumunu ile anti-tavşan ikincil antikoru) ekleyin. </li><li> (3 × 5 dk) pipetleme veya büyük bir banyo (&gt; 15 dk, 1-2 değişiklikleri) daldırarak PBS içinde yıkayın. </li><li> PAP-kalem lekeleri silin. </li><li> 3 saat boyunca oda sıcaklığında nemlendirilmiş bir oda içinde muhafaza edin. Işığa maruz kalmaktan kaçının. </li><li> Vectashield bir damla ekleyin ve slayt üzerinde bir kapak kayma yerleştirin. Kabarcıklar yapmaktan kaçının. </li><li> Vectashield sertleşmeye (~ 20 dk) olsun. </li><li> Bölümler i tutunanalizi kadar karanlık n. </li><li> Kontrol grubundaki bütün bölümler primer antikorun yokluğunda işlenir. </li><li> Bir floresan mikroskop altında birkaç saat içinde sonuç arayın. </li><li> Bir taraftan nükleer yay her merceğin diğer tarafında nükleer yay epitel hücre &#39;çekirdekler sayın. Mavi tüm lens epitel çekirdekler ve yeşil kaspaz-3 pozitif boyanan görmek için ikincil antikor emisyon uyan bir standart filtre görmek için standart mavi filtre uygulayın. </li><li> Tüm çekirdekler mercek epitelyal ve pozitif çekirdek sayısı numarasını kaydedin. Her bölüm için hücreleri üç kez sayın. </li></ol></li></ol>

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Çıkar çatışması ilan etti.

<html> Bu kağıt UVR-B ile indüklenen katarakt sırasında meydana gelen moleküler olayları inceleyerek sağlayan yöntemler açıklanır. Vivo UVR indüklenmiş katarakt kullanılabilir bilgilerin çoğu albino Sprague-Dawley sıçanlara 7, 16, 17, 18, ​​19, deneylerden türetilen olduğu göz önüne alındığında, biz bu çalışmada albino Sprague-Dawley sıçan kullanmaya karar verdi. Sıçanların Yaş altı hafta eski oldu. Erkeklerde aksine, kad?...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> Ketalar </td><td> Pfizer </td><td> 150086 </td><td> 50 mg / ml </td></tr><tr><td> Rompun </td><td> Bayer </td><td> 022545 </td><td> 20 mg / ml </td></tr><tr><td> Oculentum Simplex </td><td> Apoteket, İsveç </td><td> 336164 </td><td> 5 g </td></tr><tr><td> Mydriacyl </td><td> Alcon </td><td> 00352 </td><td> 10 mg / ml </td></tr><tr><td> Dengelenmiş tuz çözeltisi </td><td> Alcon </td><td> 0007950055 </td><td></td></tr><tr><td> 3.5 ul β-merkaptoetanol </td><td></td><td></td><td></td></tr><tr><td> NucleoSpin RNA II, total RNA izolasyon kiti </td><td> Macherey-Nagel GmbH &amp; Co, Düren, Almanya </td><td> 740955.50 </td><td></td></tr><tr><td> p53 DNA spesifik primerler </td><td> biomers.net GmbH </td><td> Özel yapılmış </td><td></td></tr><tr><td> Taq DNA polimeraz, dNTPack </td><td> Roche </td><td> 04 728 866 001 </td><td></td></tr><tr><td> Agaroz </td><td> Sigma </td><td> A5093 </td><td></td></tr><tr><td> Etidyum bromür çözeltisi 0.5 mg / ml &#39; </td><td> Sigma </td><td> E1385 </td><td></td></tr><tr><td> Nano-Bırak ND-1000 spektrofotometre </td><td> Nanodrop Ürünler </td><td></td><td></td></tr><tr><td> RT-PCR için 1. Strand cDNA sentez kiti (AMV) </td><td> Roche Diagnostics GmbH </td><td> 11 483 188 001 </td><td></td></tr><tr><td> iCycler MyiQ Tek Renk Real Time PCR algılama sistemi </td><td> Bio-Rad Laboratories </td><td></td><td></td></tr> &lt;tr&gt; <td> 96-kuyulu plakalı </td><td> Sarstedt </td><td> 72.1979.202 </td><td></td></tr><tr><td> TaqMan Gen Ekspresyonu Master Mix </td><td> Applied Biosystems </td><td> 4369016 </td><td></td></tr><tr><td> Kaspaz 3 TaqMan Gen Ekspresyonu Assay </td><td> Applied Biosystems </td><td> Rn00563902_m1 </td><td></td></tr><tr><td> 18s için TaqMan Gen Ekspresyonu Assay </td><td> Applied Biosystems </td><td> Hs99999901_s1 </td><td></td></tr><tr><td> MyiQ yazılım </td><td> Bio-Rad Laboratories </td><td></td><td></td></tr><tr><td> Cleaved kaspaz-3 (Asp175) </td><td> Hücre sinyalizasyon teknolojisi </td><td> 9661 </td><td></td></tr><tr><td> Anti-tavşan IgG </td><td> Abcam </td><td> Ab6798 </td><td></td></tr><tr><td> Sakaroz </td><td> Sigma Aldrich </td><td> 84097 </td><td></td></tr&gt; <tr><td> Vectashield </td><td> Vektör Laboratuvarları </td><td></td><td></td></tr><tr><td> Evrensel Mikroskop Axioplan 2 Görüntüleme </td><td> Carl Zeiss </td><td></td><td></td></tr><tr><td> Paraformaldehyde </td><td> Sigma Aldrich </td><td> 441244 </td><td></td></tr><tr><td> 10x TBE tamponu </td><td> Promega </td><td> V4251 </td><td></td></tr></tbody></table>

characterization of molecular mechanisms of in vivo uvr induced cataract

Katarakt dünyada 1 körlüğün önde gelen nedenidir. Dünya Sağlık Örgütü ışık transferi engellediğini göz merceğinden bir karaltı olarak katarakt tanımlar. Katarakt diyabet, sigara, ultraviyole radyasyon (UVR), alkol, iyonize radyasyon, steroidler ve hipertansiyon ile ilişkili bir çok faktörlü bir hastalıktır. UVR katarakt neden olduğu güçlü 2-4 deneysel ve epidemiyolojik kanıtlar 5,6 yoktur. Biz anestezi 7 ve olmayan anestezi altındaki hayvanlarda 8 hem UVR B indüklenmiş katarakt için bir hayvan modeli geliştirdi. Katarakt için tek çare cerrahidir ancak bu tedavinin herkes için erişilebilir değildir. Bu 10 yıl boyunca katarakt başlangıcı bir gecikme 50% 9 ile katarakt ameliyatı gereksinimini azaltabilir tahmin edilmiştir. Katarakt insidansı geciktirmek için, katarakt oluşumu mekanizmasını anlamak ve etkin önleme stratejilerini bulmak için gereklidiregies. Katarakt gelişimi için mekanizmalar arasında, apoptoz insanlar ve hayvanlar 10 katarakt başlamasında önemli bir rol oynar. Bizim odak son zamanlarda katarakt gelişimi 8,11,12 için mekanizma olarak objektif apoptoz olmuştur. Bu apoptoz yolu üzerinde UVR etkisi daha iyi anlaşılmasını katarakt önlemek için yeni ilaç keşfi için imkanlar sağlayacağı tahmin edilmektedir. Bu yazıda, deneysel UVR-B vivo maruz kalınmasına neden olabilir nasıl katarakt açıklar. Ayrıca RT-PCR ve immünohistokimyasal araçları UVR-B ile indüklenen katarakt moleküler mekanizmaları çalışmak olarak sunulmaktadır.

Ölçümlerinde varyasyon çeşitli kaynaklardan varyans analizi ile tahmin edildi ve hayvan başına üç ölçümleri dikkate ölçümler için varyans hayvanlar için bunun% 15 mertebesinde olduğu görülmüştür. Bu nedenle, bütün lens analiz göz önüne alındığında, bu hassasiyetini artırmak için mümkün değildir. Ortogonal test 120 saat gecikme aralığının karşılık daha kısa gecikme aralıkları arasında kaspaz-3 mesajı için istatistiksel olarak anlamlı bir kontrast aydınlatılmıştır. <...

Watch this Scientific Journal Video about Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract at JoVE.com

Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

Katarakt dünyada körlüğün en önde gelen nedenidir. Güneş ultraviyole radyasyon (UVR) katarakt gelişimi için en önemli risk faktörüdür. Kadar UVR-B ile indüklenen katarakt bir hayvan modeli geliştirilmiştir. UVR, kantitatif RT-PCR ve immünohistokimyasal maruziyet: Bu yazıda katarakt oluşumu soruşturma yöntemleri açıklanmaktadır.

Moleküler Mekanizmalarının Karakterizasyonu<em&gt; In vivo</em&gt; UVR İsteyerek Katarakt

Cataract is the leading cause of blindness in the world 1. The World Health Organization defines cataract as a clouding of the lens of the eye which ...

characterization-molecular-mechanisms-vivo-uvr-induced

Research

JoVE Journal

Medicine

Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

9.8K Görüntüleme.  Karolinska Institutet. Katarakt dünyada körlüğün en önde gelen nedenidir. Güneş ultraviyole radyasyon (UVR) katarakt gelişimi için en önemli risk faktörüdür. Kadar UVR-B ile indüklenen katarakt bir hayvan modeli geliştirilmiştir. UVR, kantitatif RT-PCR ve immünohistokimyasal maruziyet: Bu yazıda katarakt oluşumu soruşturma yöntemleri açıklanmaktadır.

Video: Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

İnflamatuar Plak Kızılötesi Floresans (NIRF) İntravasküler Moleküler Görüntüleme, Multimodal Yaklaşım civarında in vivo Ateroskleroz Görüntüleme

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

Tıp

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

Bir In vivo</emDaralma indüklenen Sakatlık ve Kurtarma Non-invaziv İzleme> Kemirgen Model

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

Hücre Takip için vivo 19 F MRG

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

Moleküler Kolorektal Kanser Hücrelerinin bir 3-Boyutlu Modeli İnvaziv tümör mikro Profili ve<em&gt; Ex vivo</em&gt; Fibroblastlar

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Kemoterapi kaynaklı Vasküler Toksisite - Gerçek zamanlı<em&gt; In vivo</em&gt; Gemi Düşüklüğü Görüntüleme

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Fare Modelinde Kuruluş ve İYE ve CAUTI Karakterizasyonu

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

İnsan Tümör ksenogratflarının gelen Fare Hücrelerinin tükenmesi Anlamlı Hedef Hücre Alt Analizi geliştirir

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

Vitro ve In Vivo insan hücreleri, C. Elegansve fareler Mitophagy tespiti

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac performance in the absence of hypertension, valvular, congenital, or ischemic heart diseases, and which may be related to infection, autoimmune or metabolic abnormalities, or family inheritance. It can progress into congestive heart failure with a poor prognosis. Doxorubicin (Dox) is widely employed as a chemotherapeutic drug, but its use is limited because it causes DCM-like changes of the myocardium. Its myocardial toxicity is attributed to oxidative stress, chronic inflammation, and cardiomyocyte apoptosis. A model of DCM exploiting these Dox-induced DCM symptoms has not been established.

Described is a protocol to establish a Doxorubicin-induced dilated cardiomyopathy (DCM) model in mice via long-term intraperitoneal injection of Doxorubicin.

Vivo'da Genişlemiş Kardiyomiyopatinin Doxorubicin Kaynaklı Murine Modeli

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac ...

Generation and Characterization of Right Ventricular Myocardial Infarction Induced by Permanent Ligation of the Right Coronary Artery in Mice

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In contrast to the extensively used mouse myocardial infarction (MI) model generated by left coronary artery ligation, the RVI mouse model is rarely employed due to the difficulty associated with model generation. Research on the mechanisms and treatment of RVI-induced RV remodeling and dysfunction requires animal models to mimic the pathophysiology of RVI in patients. This study introduces a feasible procedure for RVI model generation in C57BL/6J mice. Further, this model was characterized based on the following: infarct size evaluation at 24 h after MI, assessment of cardiac remodeling and function with echocardiography, RV hemodynamics assessment, and histology of the infarct zone at 4 weeks after RVI. In addition, a coronary vasculature cast was performed to observe the coronary arterial arrangement in RV. This mouse model of RVI would facilitate the research on mechanisms of right heart failure and seek new therapeutic targets of RV remodeling.

There are several differences between the right and left ventricles. However, the pathophysiology of right ventricular infarction (RVI) has not been clarified. In the present protocol, a reproducible method for RVI mouse model generation is introduced, which may provide a means to explain the mechanism of RVI.

Farelerde Sağ Koroner Arterin Kalıcı Ligasyonu ile İndüklenen Sağ Ventrikül Miyokard İnfarktüsünün Oluşumu ve Karakterizasyonu

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In ...