1. Gimm Colon Doku hazırlanması <ol><li> Gastrointestinal Motilite Monitör (Gimm), ilk etapta izole kolon (121 mM NaCl, 5.9 mM KCl, 2.5 mM CaCl 2, 1.2 mM MgCl 2, 25 mM NaHCO 3 buz Krebs çözümü için distal kolon segmenti hazırlamak için 1.2 mM NaH 2 PO 4 ve 8 mM glukoz,% 95 O 2 /% 5 CO 2 ile gazlı). Kalan mezenter dış duvardan uzak temizleyin ve organ banyosuna yerleştirilir zaman ayırt edilir, böylece oral sonunda küçük bir kesi yapmak. Not: doku 2 saat önce deney için buzlu Krebs çözüm kalabilirler. </li><li> Sonra, görüntü elde etme girişimi önlemek için kamera alanının dışında organ banyosuna giriş ve çıkış kanallar yerleştirin. Sürekli prewarmed organ banyosu serpmek (37 ° C) 10 ml / dk akış hızında Krebs çözeltisi oksijenli (% 95,% 5 CO 2). </li><li> Anal vs oral biter takip etmek, orta segmentte 1 cm kadar serbestçe hareket ettiğinden, küçük bir gevşeklik derecesi sağlayan organ banyosunda iki ucundaki pin distal kolon segment (en az 5 cm) . Fekal pelet yerleştirme kolaylığı için araştırmacı doğru sözlü sonuna yerleştirilmelidir. Segment uzunluğu ve gerginlik boylamasına streç ile transit (Dickson ve ark oranını azaltarak, itici motilite oranı etkiler çünkü Kolon bölümleri belirli bir deney seti içindeki her deney için aynı araştırmacı tarafından aynı şekilde tutturulmuş olmalıdır. , 2007). </li><li> Hazırlanması, en az 30 dakika boyunca gelmesini sağlayınız. </li></ol> 2. Gimm ve Veri Toplama ayarlama <ol><li> Gimm sisteminde, perfüzyon odasında kolonik segment altından aydınlatılır. Bir bilgisayar ile arabirim bir dijital video kamera odasının üstünde bir konuma sahiptir. Işık aydınlatma kaynağı ve Gimm yazılım hem de açık olduğundan emin olun. </li><li> Gimm yazılım uygulaması yeni bir deneme kurduktan sonra, epoksi kaplı bir fekal pelet peristaltizm başlatmak için kolon segmentinin oral ucunu takarak ilk deneme başlayacak. Kamerayı açmak ve Kayda başlamak için kayıt düğmesine tıklayın bilgisayardaki kamera düğme üzerine tıklayın. Pelet anal yönde hareketi, video kamera tarafından kaydedilen ve dijital filmlerinizi daha sonra analiz için bir PC üzerinde saklanır. Pelet kolon segmentinin sonuna ulaştığında, kaydı durdurmak için kayıt düğmesini tıklatın. </li><li> Sevk oranı için bir kontrol değeri elde etmek için, sağlıklı bir hayvanın bir kolon segmenti ve ilaç uygulamadan başlar. Her çalışma arasında 5 dakikalık bir iyileşme süresi, tek bir hazırlık 3-5 deneyler. </li><li> Kolonik motilite belirli koşullar veya ilaçların etkilerini belirlemek için, her deneysel durum için 3-5 denemeler / hazırlık yaparlar. Buna ek olarak, en az beş farklı hayvanların en az beş farklı iki nokta üst üste her bir deney gerçekleştirir. </li><li> Dijital film analizi, fekal pelet sevk oranı kolonik segment X cm çapraz bir pelet için gereken süre olarak hesaplanır. </li></ol> 3. Uzaysal Haritalar İnşaatı <ol><li> Tek tek Gimm çalışır elde edilen dijital video, özel Gimm yazılımı kullanılarak Spatiotemporal haritalar dönüştürülebilir. </li><li> Yatay eksen, kolon çapı değişiklikleri videonun her karesini bir siluet kolon görüntü dönüştürme ve hesaplanması ve gri-skala içine boydan boya çapı dönüştürme zamanla çizilir. Sonuçta daralma alanlarda beyaz görünmesini pelet ve dinlenme alanları, siyah görünür. </li><li> Kolon segment üzerinden pelet tarafından katedilen mesafe dikey ekseni temsil edilmektedir. </li></ol> 4. Temsilci Spatiotemporal Haritalar <ol><li> Çeşitli deneysel koşullar altında kolon segmentlerinde pelet motilite gösteren temsili Spatiotemporal haritalar burada gösterilir. X (yatay) eksen pelet kolon segment üzerinden ilerlediğini mesafe temsil ederken, y (dikey) eksen, zamanla pelet konumu temsil eder. <img src="/files/ftp_upload/2435/2435fig1.jpg" alt="Şekil 1" /> </li><li> Uzaysal harita bir kontrol kolon (normal, işlenmemiş), pelet ~ 2 mm / sn hızında doğrusal ilerleme olacaktır. Buna karşılık, bazı ilaçlar ya da kolonda enflamasyon idaresi durduruldu motilite ve tıkalı motilite gibi kesintiye motilite desenler neden olabilir. </li><li> Y ekseni fekal pelet ve x-ekseni saniye cinsinden zamanı temsil tarafından katedilen mesafe milimetre olarak temsil eden, Gimm gelen sonuçları da grafik olarak gösterilen olabilir. Örneğin, DAMGO (D-ALA 2, N-Me-Phe4, Gly-ol5) yönetimi, biropiat μ-reseptör agonisti, kolon itici hareketliliğinde azalmaya neden olur. </li></ol>

<ol>
	<li>Bayliss, W. M., Starling, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+movements+and+innervation+of+the+small+intestine.">The movements and innervation of the small intestine.</a> J of Physiol (Lond). 24, 99-143 .</li><li>Dickson, E. J., Spencer, N. J., Hennig, G. W., Bayguinov, P. O., Ren, J., Heredia, D. J., Smith, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enteric+occult+reflex+underlies+accommodation+and+slow+transit+in+the+distal+large+bowel.">An enteric occult reflex underlies accommodation and slow transit in the distal large bowel.</a> Gastroenterology. 132, 1912-1924 (2007).</li><li>Foxx-Orenstein, A. E., Jin, J. G., Grider, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-HT4+receptor+agonists+and+delta-opioid+receptor+antagonists+act+synergistically+to+stimulate+colonic+propulsion.">5-HT4 receptor agonists and delta-opioid receptor antagonists act synergistically to stimulate colonic propulsion.</a> Am J Physiol. 275, 979-983 (1998).</li><li>Grider, J. R., Foxx-Orenstein, A. E., Jin, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Hydroxytryptamine4+receptor+agonists+initiate+the+peristaltic+reflex+in+human,+rat,+and+guinea+pig+intestine.">5-Hydroxytryptamine4 receptor agonists initiate the peristaltic reflex in human, rat, and guinea pig intestine.</a> Gastroenterology. 115, 370-380 (1998).</li><li>Kadowaki, M., Wade, P. R., Gershon, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Participation+of+5-HT3,+5-HT4,+and+nicotinic+receptors+in+the+peristaltic+reflex+of+guinea+pig+distal+colon.">Participation of 5-HT3, 5-HT4, and nicotinic receptors in the peristaltic reflex of guinea pig distal colon.</a> Am J Physiol. 271, 849-857 (1996).</li><li>Krauter, E. M., Strong, D. S., Brooks, E. M., Linden, D. R., Sharkey, K. A., Mawe, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+colonic+motility+and+the+electrophysiological+properties+of+myenteric+neurons+persist+following+recovery+from+trinitrobenzene+sulfonic+acid+colitis+in+the+guinea+pig.">Changes in colonic motility and the electrophysiological properties of myenteric neurons persist following recovery from trinitrobenzene sulfonic acid colitis in the guinea pig.</a> Neurogastroenterol Motil. 19, 990-1000 (2007).</li><li>Langley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Autonomic Nervous System. , (1921).</li><li>Linden, D. R., Sharkey, K. A., Mawe, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+excitability+of+myenteric+AH+neurones+in+the+inflamed+guinea-pig+distal+colon.">Enhanced excitability of myenteric AH neurones in the inflamed guinea-pig distal colon.</a> J Physiol. 547, 589-601 (2003).</li><li>Linden, D. R., Sharkey, K. A., Ho, W., Mawe, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclooxygenase-2+contributes+to+dysmotility+and+enhanced+excitability+of+myenteric+AH+neurones+in+the+inflamed+guinea+pig+distal+colon.">Cyclooxygenase-2 contributes to dysmotility and enhanced excitability of myenteric AH neurones in the inflamed guinea pig distal colon.</a> J Physiol. 557, 191-205 (2004).</li><li>Linden, D. R., Chen, J. X., Gershon, M. D., Sharkey, K. A., Mawe, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+availability+is+increased+in+mucosa+of+guinea+pigs+with+TNBS-induced+colitis.">Serotonin availability is increased in mucosa of guinea pigs with TNBS-induced colitis.</a> Am J Physiol Gastrointest Liver Physiol. 285, 207-216 (2003).</li><li>Linden, D. R., Couvrette, J. M., Ciolino, A., McQuoid, C., Blaszyk, H., Sharkey, K. A., Mawe, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indiscriminate+loss+of+myenteric+neurones+in+the+TNBS-inflamed+guinea-pig+distal+colon.">Indiscriminate loss of myenteric neurones in the TNBS-inflamed guinea-pig distal colon.</a> Neurogastroenterol Motil. 17, 751-760 (2005).</li><li>Lomax, A. E., Mawe, G. M., Sharkey, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+facilitation+and+enhanced+neuronal+excitability+in+the+submucosal+plexus+during+experimental+colitis+in+guinea-pig.">Synaptic facilitation and enhanced neuronal excitability in the submucosal plexus during experimental colitis in guinea-pig.</a> J Physiol. 564, 863-875 (2005).</li><li>Lomax, A. E., O'Hara, J. R., Hyland, N. P., Mawe, G. M., Sharkey, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+alterations+to+enteric+neural+signaling+in+the+guinea+pig+colon+following+the+resolution+of+colitis.">Persistent alterations to enteric neural signaling in the guinea pig colon following the resolution of colitis.</a> Am J Physiol Gastrointest Liver Physiol. 292, 482-491 (2007).</li><li>Strong, D. S., Cornbrooks, C. F., Roberts, J. A., Hoffman, J. M., Sharkey, K. A., Mawe, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purinergic+neuromuscular+transmission+is+selectively+attenuated+in+ulcerated+regions+of+inflamed+guinea+pig+distal+colon.">Purinergic neuromuscular transmission is selectively attenuated in ulcerated regions of inflamed guinea pig distal colon.</a> J Physiol. 588, 847-859 (2010).</li><li>Wade, P. R., Chen, J., Jaffe, B., Kassem, I. S., Blakely, R. D., Gershon, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+and+function+of+a+5-HT+transporter+in+crypt+epithelia+of+the+gastrointestinal+tract.">Localization and function of a 5-HT transporter in crypt epithelia of the gastrointestinal tract.</a> J Neurosci. 16, 2352-2364 (1996).</li><li>Wood, M. J., Hyman, N. H., Mawe, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effects+of+Daikenchuto+(DKT)+on+Propulsive+Motility+in+the+Colon.">The Effects of Daikenchuto (DKT) on Propulsive Motility in the Colon.</a> J Surg Res. , (2009).</li></ol>

Bu makalede kullanılan alet üreten Catamount Araştırma ve Geliştirme, Inc, bu video üretim sponsor oldu.

Gastrointestinal sistem, sinir aracılı itici motilite bir asır önce ilk defa Bayliss ve Starling (Bayliss ve Starling, 1899) tarafından tarif edilmiştir. Bu gözlem, enterik sinir sistemi (ENS), otonom sinir sisteminin farklı bir bölümü (Langley, 1921) gibi bağırsak sinirler atama yol açtı. Nörojenik bağırsak peristaltizm belirli bir noktaya ve refleks aracılı kasılmaları şeyden önce, ya da sözlü, aboral yönünde uyaran ve rahatlama düzeyine yayma ve / veya mukozal stimülasyonu gerektirir. Sonuç aboral yönde bir sözlü luminal içeriğini propels bir basınç gradiyenti nesil. Türlerin çeşitli ince bağırsak ve kalın bağırsak sıçan, kasılmaların peristaltik dalgalar barsak segmentlerinin lümen içine sıvı infüzyonu tarafından aktif hale getirilebilir. Kobay distal kolonda kolon oral ucunu, doğal ya da yapay fekal pelet eklenecek ve kolon segment boyunca ilerleme kolayca takip edilebilir. Böylece, kobay distal kolon, bağırsak itici motilite araştırmak için basit ve kullanışlı bir çalışma sağlar. Peristaltizm nöroaktif bileşikleri ve reseptörlerin sayısı içeren karmaşık bir sinir refleks. Sonuç olarak, ENS de nörotransmisyon etkileyen bileşikler itici motilite oranını etkilemez. Serotonin, bağırsak mukozasının enterochromaffın hücreleri öncelikle piyasaya peristaltik refleks başlanması yer almaktadır. Itici motilite 4 5-HT reseptör agonistleri (Foxx-Orenstein ve ark, 1998; Grider ve ark, 1998) intralumenal idaresi tarafından geliştirilmiş olduğunu göstermiştir kobay distal kolon, Grider ve arkadaşları kullanarak, uygulama, 5 banyo -HT 3 ve 4, 5-HT reseptör antagonistleri sevk oranı (. Linden ve ark, 2003b. Kadowaki ve ark, 1996) azalır. Ayrıca, serotonin seçici gerialım inhibitörlerinin (SSRI) ile serotonin taşıyıcı inhibisyonu henüz 5-HT reseptörleri (Kadowaki ve ark, 1996, duyarsızlaştırma nedeniyle muhtemelen daha yüksek konsantrasyonlarda peristaltizm, azaltan, düşük konsantrasyonlarda peristaltizm artırır. Wade ve ark , 1996). Kobay itici motilite katkıda bulunan diğer sinyal molekülleri bağırsaklarda internöronal sinyal birincil arabulucu nikotinik reseptörleri, oyunculuk asetilkolin içerir. Sevk oranı (Kadowaki ve ark, 1996) daha az konsantrasyonlarda etkilemez Engelleme nikotinik reseptör antagonisti hexamethonium sinaptik iletim, yüksek konsantrasyonlarda pelet tahrik azalır. Uzun gastrointestinal motilite üzerine inhibitör etkileri olduğu bilinmektedir Opiatlar ve opioid reseptör agonistleri, kobay itici motilite modeli pelet tahrik azaltmak (; Wood ve ark, 2009 Foxx-Orenstein ve ark, 1998). İlginçtir, opioid antagonisti nalokson ile birlikte uygulandığında daikenchuto, kusma, karın ağrısı ve düzensiz motilite tedavisi için klinik olarak kullanılan geleneksel Japon bitkisel ilaç, pelet sevk oranı azalır ve tek başına verildiğinde ters peristaltizm sonuçları (Wood ve ark. 2009). Bu bulgular, çeşitli bileşikler ve bunların reseptörleri kolonik motilite desen katkıları değerlendirmek için kobay itici motilite modeli yararlılığını göstermektedir. Bir dizi çalışmada, inflamasyon kolon nöron (. Linden ve ark, 2003a; Lomax ve ark, 2005) Elektrik ve sinaptik özelliklerinde değişikliklere neden olduğunu göstermiştir ve bu değişiklikler (Kräuter inflamasyon kurtarma aşağıdaki hafta devam edebilir et al, 2007; Lomax ve ark, 2007). Kolit indüklenen nöroplastik değişikliklerin itici motilite Gimm sistemini kullanarak kobay distal kolon etkisi araştırılmıştır. Bu sistem, sadece bir fekal pelet belli bir mesafe yolculuk zamanı ölçmek için itici motilite değerlendirir kolonik motilite, izleme geleneksel yöntem üzerinde birçok avantajı vardır. Gimm sistemi kullanarak daha fazla son araştırmalar, henüz daha ortaya koymuştur; kolonda enflamasyon kaynaklı nöral plastisite Önceki çalışmalarda pelet tahrik (Linden ve ark, 2005 Linden ve ark, 2003a) hızında bir azalma göstermiştir karmaşık değişiklikler (Güçlü ve ark, 2010). Örneğin, ülserli bölgelerde iltihaplı distal kolon pelet sevk oranı azalır, henüz motilite bitişik bölgeler, daha önceki &quot;kronometre&quot; yöntemi tarafından tanınan bir fenomen ivme. Diğer çalışmalarda aktif inflamasyon aşamasında dismotilite COX-2 (Linden ve ark, 2004), henüz ötesinde devam değişmiş motilite inhibe ile restore edilebilir olduğunu göstermiştirinflamasyon çözünürlük COX-2 büyüklüğüne duyarsız (Kräuter ve ark, 2007). Ayrıca, bu daha sonraki çalışmalarda, Gimm sistem tarafından üretilen Spatiotemporal haritalar tahrik azalmış oranları katkıda post-inflamatuar hayvanlarda gözlenmiştir kolonik motilite adım adım bir model ortaya koymuştur. Sonuç olarak, yüksek verim kobay distal kolon itici motilite tahlil kolonik motilite test bileşikleri ve patolojik durumların etkilerini ölçmek anlamına gelir temsil eder, Ayrıca, Gimm sistemi in vitro kolonik itici motilite değerlendirmek için basit ve anlaşılır bir yaklaşım sağlar. Pelet tahrik sürekli ölçümlerin yanı sıra spontan aktivite desen çalışması için Spatiotemporal haritaların üretimi için izin verir. Geleneksel yöntemlerle karşılaştırıldığında, aynı zamanda dijital dosyaları sisteme kaydedilir ve kolayca erişilebilir quantitated ve tekrar analiz birden çok yolu olabilir daha karmaşık bir olgu verebilmesidir.

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		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Krebs&#x2019; Solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane</td>
<td>Anesthesia</td>
<td>Webster Veterinary</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Epoxy-coated fecal pellet </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>native guinea pig pellet dried and epoxy (black nail polish) coated</td>
</tr>
<tr>
<td>Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micro Scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Stainless Steel Pins</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Beakers</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>500 mL</td>
</tr>
<tr>
<td>Gas Tank</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>95% O2/5% CO2</td>
</tr>
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<td>Timer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Gastrointestinal Motility Monitor</td>
<td>&nbsp;</td>
<td>Catamount Research and Development</td>
<td>&nbsp;</td>
<td><a href="http://www.catamountresearch.com/products/gimm.htm">http://www.catamountresearch.com/products/gimm.htm</a></td>
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gastrointestinal motility monitor (gimm)

Gastrointestinal Motilite Monitör (Gimm Catamount Araştırma ve Geliştirme; St Albans, VT), in vitro sistemde izole kobay distal kolon segmentlerinde itici motilite izler . Komple bir sistem, bir bilgisayar, video kamera, ışıklı organ banyosu, pompalar dolaşan peristaltik ve ısıtmalı su banyosu, ve veri kaydı ve analiz için özel Gimm yazılım oluşur. Kolonik peristaltizm izleme geleneksel yöntemlerle karşılaştırıldığında, Gimm sistemi motilite sürekli, kantitatif değerlendirme sağlar. Kobay distal kolon ısındı, oksijenli Krebs çözüm yıkandığı ve oral sonuna eklenen fekal pelet yaklaşık 2 mm / sn hızında kolon segment boyunca hareketli. Filmler segment boyunca fekal pelet geçmeden yakalanır ve Gimm yazılım fekal pelet ilerlemeyi izlemek kullanılabilir. Itici motilite oranları tüm segment için veya herhangi bir özel ilgi bölge için elde edilebilir. Bolus indüklenen motilite desen analizine ek olarak, Spatiotemporal haritalar, spontan motor aktivite kalıplarını değerlendirmek için çekilen video segmentleri inşa edilecek. Bu sistem uygulamaları farmakolojik itici motilite reseptör agonistleri ve antagonistleri etkilerinin değerlendirilmesi yanı sıra, enflamasyon ya da stres gibi fizyopatolojik koşullardaki kaynaklanan değişikliklerin değerlendirilmesi. Gimm sistemini kullanarak kobay distal kolon itici motilite testi, öğrenmesi kolay ve basittir ve bu itici motilite değerlendirilmesinde güvenilir ve tekrarlanabilir bir yöntem sağlar.

Watch this Scientific Journal Video about Gastrointestinal Motility Monitor GIMM at JoVE.com

Gastrointestinal Motility Monitor GIMM

Gastrointestinal Motilite izleyicisi ile kobay distal kolonda kolonik motilite değerlendirilmesi (Gimm), gastrointestinal sistem itici motilite kantitatif olarak değerlendirmek için öğrenmek için basit ve basit bir yaklaşımdır.

Gastrointestinal Motilite Monitör (Gimm)

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility ...

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Unit 1

Gastrointestinal Motility Monitor (GIMM)

Drugs Affecting Gastrointestinal Motility

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30.5K Görüntüleme.  The University of Vermont. Gastrointestinal Motilite izleyicisi ile kobay distal kolonda kolonik motilite değerlendirilmesi (Gimm), gastrointestinal sistem itici motilite kantitatif olarak değerlendirmek için öğrenmek için basit ve basit bir yaklaşımdır.

Video: Gastrointestinal Motility Monitor GIMM

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

Nöral Kök Hücre Motilite değerlendirilmesi Agaroz Jel tabanlı mikroakışkan Cihaz Kullanımı

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Biyoloji

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

Kemirgen Havayolu Epiteli içinde Cilia Motilite Yüksek Çözünürlüklü Görüntüleme için ex vivo Yöntemi

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more &#8220;temperate swarmers&#8221; that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. &#8220;Wettability&#8221;, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.

Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques.

Bakteriyel Yüzey Motilite Tahliller hazırlanması, Görüntüleme ve miktarının belirlenmesi

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

Larvaların Faaliyet Ölçümü<em&gt; Drosophila</em&gt; Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, and membrane potential. All of these approaches depend on measurement of activity at one or multiple locations along the gut simultaneously which are then interpreted to provide a sense of overall motility patterns. Recently, the development of video recording and spatiotemporal mapping (STmap) techniques have made it possible to observe and analyze complex patterns in ex vivo whole segments of colon and intestine. Once recorded and digitized, video records can be converted to STmaps in which the luminal diameter is converted to grayscale or color [called diameter maps (Dmaps)]. STmaps can provide data on motility direction (i.e., stationary, peristaltic, antiperistaltic), velocity, duration, frequency and strength of contractile motility patterns. Advantages of this approach include: analysis of interaction or simultaneous development of different motility patterns in different regions of the same segment, visualization of motility pattern changes over time, and analysis of how activity in one region influences activity in another region. Video recordings can be replayed with different timescales and analysis parameters so that separate STmaps and motility patterns can be analyzed in more detail. This protocol specifically details the effects of intraluminal fluid distension and intraluminal stimuli that affect motility generation. The use of luminal receptor agonists and antagonists provides mechanistic information on how specific patterns are initiated and how one pattern can be converted into another pattern. The technique is limited by the ability to only measure motility that causes changes in luminal diameter, without providing data on intraluminal pressure changes or muscle tension, and by the generation of artifacts based upon experimental setup; although, analysis methods can account for these issues. When compared to previous techniques the video recording and STmap approach provides a more comprehensive understanding of gastrointestinal motility.

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Recently available video recording and spatiotemporal mapping (STmap) techniques make it possible to visualize and quantify both propagating and mixing patterns of intestinal motility. The goal of this protocol is to explain the generation and analysis of STmaps using the GastroIntestinal Motility Monitoring (GIMM) system.

Hareketliliği zamanmekansal Haritalama içinde<em&gt; Ex Vivo</emBağırsakların&gt; Hazırlıklar

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, ...

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. A detailed analysis of IRF3 activation is essential to understand how pathogens induce or evade the innate antiviral response. Distinct activated forms of IRF3 can be distinguished based on their phosphorylation and monomer vs dimer status. In vivo discrimination between the different activated species of IRF3 can be achieved through the separation of IRF3 phosphorylated forms based on their mobility shifts on SDS-PAGE. Additionally, the levels of IRF3 monomer and dimer can be monitored using non-denaturing electrophoresis. Here, we detail a procedure to reach the highest resolution to gain the most information regarding IRF3 activation status. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple total and phosphospecific antibodies. This experimental strategy constitutes an affordable and sensitive approach to acquire all the necessary information for a complete analysis of the phosphorylation-mediated activation of IRF3.

Here we describe a procedure allowing a detailed analysis of the phosphorylation-dependent activation of the IRF3 transcription factor. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple phosphospecific antibodies.

Bir Yüksek Çözünürlüklü Yöntem IRF3 fosforilasyonu bağımlı Aktivasyon Monitör

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

pHluorin etiketli Reseptör kullanarak Subselüler Yerelleşme ve Ticareti Monitör

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.

Ex-Vivo Konfokal Mikroskopi ile Lenf Nodal Yapısı nın Ve Hücresel Lokalizasyonun Görselleştirilmesi

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are ...

Measuring Crop Motility and Food Passaging in Drosophila

Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. Disordered GI motility and gastric emptying cause multiple diseases and symptoms. As a powerful genetic model organism, Drosophila can be used in GI motility research. The Drosophila crop is an organ that contracts and moves food into the midgut for further digestion, functionally similar to a mammalian stomach. Presented is a protocol to study Drosophila crop motility using simple measurement tools. A method for counting crop contractions to evaluate crop motility and a method for detecting the distribution of food dyed blue between the crop and gut using a spectrophotometer to investigate the effect of the crop on food passaging is described. The method was used to detect the difference in crop motility between control and nprl2 mutant flies. This protocol is both cost-efficient and highly sensitive to crop motility.

Measuring Crop Motility and Food Passaging in Drosophila

The goal of this protocol is to measure crop contraction and quantify food distribution in the Drosophila gut.

Drosophila'da Bitki Hareketliliği ve Gıda Geçişinin Ölçülmesi

Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. ...

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, prevent fracture. In the interest to both optimize peak bone mass and combat various musculoskeletal diseases (i.e., post-menopausal osteoporosis, anorexia nervosa, type 1 and 2 diabetes mellitus), incredible efforts have been made in the field of bone biology to fully characterize osteoblasts throughout their differentiation process. Given the primary role of mature osteoblasts to secrete matrix proteins and mineralization vesicles, it has been noted that these processes take an incredible amount of cellular energy, or adenosine triphosphate (ATP). The overall cellular energy status is often referred to as cellular bioenergetics, and it includes a series of metabolic reactions that sense substrate availability to derive ATP to meet cellular needs. Therefore, the current method details the process of isolating primary, murine bone marrow stromal cells (BMSCs) and monitoring their bioenergetic status using the Real-time cell metabolic flux analyzer at various stages in osteoblast differentiation. Importantly, these data have demonstrated that the metabolic profile changes dramatically throughout osteoblast differentiation. Thus, using this physiologically relevant cell type is required to fully appreciate how a cell's bioenergetic status can regulate the overall function.

Real-time cell metabolic flux assay measures the oxygen consumption rate and extracellular acidification rate, which corresponds to mitochondrial and glycolytic adenosine triphosphate production, using pH and oxygen sensors. The manuscript explains a method to understand the energy status of osteoblasts and the characterization and interpretation of the cellular bioenergetic status.

Osteoblast Biyoenerjetik Teknolojilerini İzlemek için Gerçek Zamanlı Hücre Metabolik Akı Analizörünü Kullanma

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, ...