Supported by NIH award R01 DK093525 (GFG). We greatly appreciate the scientific review and grammatical editing of the manuscript by Dr. Amber Shaffer (University of Pittsburgh) and thank Michael Burcham for assistance in preparation of figures.

NOT: Bu protokol gözden ve Pittsburgh Kurumsal Hayvan Bakım ve Kullanım Kurulu Üniversitesi tarafından onaylanmıştır. Modifiye Krebs Çözüm ve Test İlaç Alikot 1. Hazırlık <ol><li> 117,9 NaCl, 4,7 KCİ, 25 NaHCO 3, 1.3, NaH 2 PO 4, MgSO 4 1.2, 2.5 CaCl2, 11.1, D-glukoz, 2 sodyum bütirat, 20 sodyum asetat (mM olarak) içeren değiştirilmiş bir Krebs çözeltisi içinde 6 L yapmak 0.004 nifedipin (spontan kas kasılmaları bloke etmek için), ve 0.003 indometasin (endojen prostaglandin sentezini bloke etmek için). Sırasıyla doku diseksiyonu ve tek lif kayıt için buz-soğuk ve sıcak Krebs çözümleri kullanın. </li><li> Herhangi bir kimyasal çözümleri hazırlayın (örn, IS, kapsaisin, ATP) istenen konsantrasyonlarda alikotlarda. </li></ol> Kolorektumun-sinir Doku 2. Diseksiyon <ol><li> Uyutmak ve erkek fareler euthanize (6 - 8 haftalık, 20-30 g)10 yerine geçen bir akış oranında bir CO2 odası - farelerde kadar dakika başına bölme hacminin% 30 göğüs hareketinin olmaması ile gösterildiği gibi tamamen nefes durdurun. </li><li> Hemen sonra, torasik bölme kesmenin sağ atriyum delme ve buz soğukluğunda Krebs geniş bir hacmi (~ 500 mi) fare karkas içine daldırarak ötenazi, asıl bulbus olfaktoryusları aşağıdaki gibidir (4 ° C) bir karbojen (% 95 O2 ile kabarcıklar halinde % 5 CO2). </li><li> Dikkatle tüm iç organları ama kolon ve pelvik organları çıkarın. Biraz göğüs diyafram üzerinde T12 spinal segment boyunca yarısında fare transect ve buz soğuk içeren bir diseksiyon odasına kuyruk yarısını aktarmak, Krebs solüsyonu kabarmış. </li><li> Steromikroskopla, üretraya kavşaklarda transecting ile mesane ve üreme organları kaldırmak ve ortak iliak arterlere üzere iki kola ayrılır kadar inen / abdominal aorta çıkarın. PN veya surroundin gelen LSN Freeg künt diseksiyon ile dokular ve L6 ve S1 vertebra (PN için) sütun veya T13 ve (LSN) L1 vertebra sütuna ventral giriş noktasına kadar iliak dışından sinir izleyin. </li><li> Pubis eklemi ve sağ ve sol asetabuler eklemleri kesin ve iliyak kemiği kaldırmak. Sinir vertebra sütun girdiği yere kadar kolorektal yakın ekli kas ve bağ dokusundan ya PN veya LSN dikkatlice ücretsiz. </li><li> Dikkatle uzak kolorektal maruz iliak kemik rezeksiyonu. Sürekliliğinde bağlı PN veya LSN distal kolorektal parçalara ayır. </li><li> Doku odasının banyo bölmesine bağlı sinir kolorektal aktarın. Daha fazla diseksiyon ile aşırı bağ dokusu çıkarın, ve anti-mezenterik sınırı boyunca uzunlamasına kolorektal açın. </li><li> Yukarı bakacak şekilde mukozal tarafı, Chambe silikon üssü haline kayıt bölmesine bitişik kolorektumun mezenterik kenarını pinR ve bir zorlamalı tahrik düzenine bağlı kanca kesintiye kolorektal antimezenterik uzunluğu bağlamak (Şekil 1 &#39;de gösterilen ve Şekil 2A&#39;da fotoğraflandı). </li><li> Bir fare deliği ve kapısı banyo bölmesi bağlanır kayıt bölmesine, içine PN veya LSN uzatın. Yavaşça sinir uymak için bir hidrofil yüzey sağlar kayıt bölmesi, bir küçük cam ayna üzerine sinir gövde yatıyordu. Sıcak ile banyo bölmesini Superfuse (30-32 ° C), Krebs solüsyonu oksijenli ve mineral yağ ile kayıt bölmesini doldurun. </li></ol> 3. Tek-lif Kayıt ve Alıcı Field lokalizasyonu <ol><li> Dikkatle yüksek büyütme (- 60X 50) de stereomicroscope altında PN veya LSN&#39;den epinöriyuma (sinir kılıfı) geri soyma. ~ 100 mikron kalınlığında 8 sinir demetleri - ince forseps kullanarak, 5 içine sinir gövde alay. </li><li> Platin-iridyum başvuru yerleştirindoku bölmesi içinde Krebs çözeltisi ile temas halindeki elektrot. Sıralı aynı malzemeden yapılmış kayıt elektrot üzerine bireysel sinir demetleri yerleştirin. </li><li> Yavaşça yukarı okşayarak tarafından ve kolorektal yüzeye aşağı kolorektal afferentleri gelen AP uyandırmak için yumuşak bir boya fırçası kullanın. Saptanabilir AP (aksiyon potansiyeli) kayıtları ile kolon innerve sinir demeti (ler) bulun. NOT: PN ve SSN da idrar kesesi ve diğer pelvik organlar innerve. </li><li> Ayrıca 10 mikron kalınlığında ~ ince fasikül filamentler içine sinir demeti bölmek ve kayıt elektrot üzerine bireysel bir filamanın yerleştirmek için 30 G iğne uçları bir çift kullanın. </li><li> Elektriksel akım yayılması ~ 2 mm çapındaki üreten suprathreshold uyaran yoğunluğu (10 mA büyüklüğü, 0,3 Hz @ 0,5 msn süre), en afferent sonlar heyecanlandırmak için mukozal yüzeye dik yuvarlak uçlu eş elektrot yerleştirin. Sistematik (~ 1.5 mm adımlarını elektrot Taşı) Düzleşmiş kolorektumun uzunluğu ve genişliği boyunca açık sonlar yerelleştirilmesine. </li><li> Bir afferent biten heyecanlı olduğunda, asgari uyaran yoğunluğunu (uyaran eşik) gerektirir aktivasyon (açık alan, RF) siteyi kesin olarak belirlemek için elektrot konumunu ayarlamak. &gt; 3 mA 19 bir uyarıcı eşik sonlar atın. </li><li> Uyaran parazit ve aksiyon potansiyeli başlangıcı arasındaki iletim hızı 1 (CV)) alıcı alan (RF) en uyarıcı elektrot ve kayıt sitesi ve 2) iletim gecikmesi (örneğin, Şekil 2B arasındaki mesafe) hesaplayın . Özgeçmiş (m / sn) = uzaklık (mm) / iletim gecikmesi (msn). </li></ol> Mechanosensitive Kolorektal afferentlerin 4. Fonksiyonel Sınıflandırılması <ol><li> Elektriksel stimülasyon ile bir RF bulduktan sonra, RF aşağıdaki üç mekanik uyaranlara geçerlidir: <ol><li> Bir Calibra ucu basarak tarama uyaran Davranışted von Frey gibi naylon monofilament (0.4 ve 1 gr gücü) dik düzleştirilmiş kolorektumun üzerinde RF doğru. </li><li> Yavaşça RF küçük bir yüzey kayma gerilmesi oluşturmak için ince bir naylon iplik telinin (10 mg kuvveti) ile kolorektal mukozayı okşayarak tarafından okşayarak uyaran Davranış. </li><li> Rampalı bir germe kuvveti sağlayan bir bilgisayar kontrollü kuvvet aktüatörü kullanılarak çevresel streç Davranış - aşama 2.8 &#39;de tarif edilen kanca tırmık ile kolorektal anti-mezenterik kenarı boyunca çevresel yönde (0 5 mN / sn 170 mN) . </li></ol></li><li> Serozal olarak, mukozal (sondalama künt sadece cevap) afferentler sınıflandırır kas (çevresel streç cevap ve sondalama künt) kas / mukozal (çevresel streç, mukozal okşayarak cevap ve sondalama künt), ya da, (okşayarak mukoza ve sondalama künt cevap) MIA (üç mekanik uyarıcılara herhangi birine yanıt yok). </li><li> Sadece (mezenterik afferentleri içinZor LSN innervasyon), elektrik stimülasyonu ile seçici aktive mezenter sondalama / mekanik okşayarak kendi alıcı sonlar bulmak için. </li><li> Streç duyarlı afferentleri (kas ve kas-mukozal) için, rampalı streç sırasında ilk AP çağrıştırıyor kuvvet olarak tanımlanan tepki eşiğini belirlemek. </li><li> Serozal afferentlerin için, bilgisayar kontrollü kuvvet aktüatör tarafından yönlendirilen açık alan tarama punktat düzeylerini artan onların yanıtlarını kaydedin. </li></ol> Alıcı Endings 5. Kimyasal Uygulama / Modülasyon <ol><li> Mekanik uyarana bir temel yanıtı kaydedin (yani, rampalı streç, sondalama punktat veya mukozal okşayarak yanıt). </li><li> Coat alt boru (10 mm yüksekliğinde ve 4 x 4 mm 2 kare veya 4 pirinç veya paslanmaz çelikten, - 5 mm çapında) bir parçasının kenar vazelin ve kolorektal üzerinde açık alana üzerine yerleştirin. </li><li> KaldırKimyasal (lar), test edilecek içeren çözeltinin 5 dakika 170 ul - Krebs boru içine çözeltisi ve 3 için açık bir son maruz kalmaktadır. </li><li> Kimyasal uygulama sırasında aferentinin yanıtı Monitör (bazı afferentler kemosensitif vardır). </li><li> Kimyasal işlem sona erdirmek için kimyasal çözeltisi ve boru çıkarın. 4 içinde - 6 dakika, bazal yanıt aynı mekanik uyarana afferent cevabı test. </li><li> Yıkama-out yeterli bir süre (&gt; 15 dk) sonra yeniden mekanik uyaran yeniden uygulayın. </li></ol> 6. Kayıt ve Ayırıcı AP sivri <ol><li> 20 kHz akson kaydedilen elektrik sinyallerini dijital ortama aktarmak ve bir bilgisayara verileri kaydetmek. Bir ses monitör ile on-line sinyali izleyin. </li><li> Off-line AP ani analiz ve bireysel başak dalga formları 25 temel bileşen analizi dayalı tek birimleri ayırt. NOT: Bir rekor inci artık içermelidirbir, iki, kolayca discriminable aktif birimleri. </li></ol>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+visceral+hypersensitivity+by+glial+cell+line-derived+neurotrophic+factor+family+receptor+&#945;-3+in+colorectal+afferents.">Modulation of visceral hypersensitivity by glial cell line-derived neurotrophic factor family receptor &#945;-3 in colorectal afferents.</a> Am J Physiol Gastrointest Liver Physiol. 300, G418-G424 (2011).</li></ol>

The authors have nothing to disclose.

Burada tarif edilen in vitro kolorektum-sinir hazırlık güzel visseral duyusal nöronlar diğer fonksiyonel olmayan yaklaşımlar (örn, hücresel, moleküler ve histolojik çalışmalar) tamamlar bireysel kolorektal aferentlerin, nöral kodlama işlevlerini (çalışmak için güçlü bir yaklaşım olduğu kanıtlanmıştır ) Detaylar için gözden 27. bkz. Nosisepsiyon ve uzun vadeli kolorektal aşırı duyarlılık katkıda Nöronal mekanizmalar ortaya konmuştur ve farmakolojik manip...

Ağrı ve aşırı duyarlılık belirgin biyopatolojik neden veya doku hasarının olmaması durumunda, irritabl bağırsak sendromu (IBS) gibi mide-bağırsak işlev bozuklukları, mustarip hastalarda baskın şikayetleri bulunmaktadır. Örneğin, IBS hastaları (karın alan palpasyonla yani, hassasiyet) normal bağırsak fonksiyonu sırasında rektal balon şişkinlik ve artan duyarlılığı artan tepkiler de dahil olmak üzere aşırı duyarlılık, yanı sıra somatik sevk aşırı duyarlılık gösterirler 1. Arasında, daha iyi anlaşılması, kolorektal ileticileri hedefleme IBS hastalarında (guanilat siklaza Cı agonisti linaclotide 4-6 oral yoldan, örneğin, lokal anestezik 2,3 intra-rektal instilasyon) ağrı ve aşırı duyarlılık azaltmada etkili olduğu kanıtlanmıştır çünkü kolorektumun afferent innervasyon önemlidir. Kolorektal afferentlerin dahil viseral afferentleri,/ Kimyasal besleyici- ve termal modaliteleri (örneğin, 7-9) yanıt yeteneğine sahiptir. Ancak, mekanik uyaranlara (yani, mechanosensitive afferentleri) yanıt visseral afferentlerin olmuştur mekanik uyaranlara (örn, lümen distansiyonu, streç), genellikle rahatsızlık ve ağrı 10-16 olmak üzere bilinçli duyumlar, yol olanlardır çünkü çoğu iyice okudu. Buna ek olarak, organlar da yaygın, mekanik duyarsız afferentleri (MIAS) tarafından innerve sessiz ya da uyku nosiseptörleri 17 adlandırılır. Normal fizyolojik şartlar altında, MIAS mekanik uyarıya yanıt ya da çok yüksek bir tepki 18 eşikleri, ancak aktif hale gelir ve patofizyolojik koşullarda mechanosensitivity kazanmak ve aşırı duyarlılık katkıda bulunabilir yoktur. Burada tarif edilen in vitro hazırlık ve protokolü kullanarak, biz geliştirdik ve denize bir elektrik uyaran stratejisi istihdam<html> kolorektal 19 hem mechanosensitive ve MIA sonlar tarafsız kimlik izin alıcı sonlar için rch. kolorektal innervasyon lomber splanknik (SSN) ve pelvik sinir (PN) yollar türetilen ve beş mechanosensitive sınıflar (serozal, mukozal, kas, kas-mukozal, mezenterik) ve bir MIA sınıfına 20 olarak kategorize edilebilir kolorektal afferentler içerir. Bu in vitro hazırlanmasını kullanarak, kolorektal MIAS elde edilen mechanosensitivity LSN yolunun PN yolunda MIAS% 71 ve MIAS% 23 duyarlı enflamatuar bir çorba (IS), kendi açık alanları arasında kısa maruz kaldıktan sonra (duyarlı) bulundu 19. Ayrıca, uzun süreli davranış viseral aşırı duyarlılık bağlamında MIAS uzun süreli hassasiyet (en fazla 28 gün) belgelenmiştir (zimosan 21 ya da 2,4,6-trinitrobenzen sülfonik asit içinin tedavi uygulanan farelerde, örneğin, (TNBS) 22) . mechanosensitive afferentlerin, kas ve kas-mukozal afferentleri arasında jove_content &quot;&gt; (yani, streç duyarlı) ve zararlı kolorektal distansiyon 23,24 kodlamasını işine yaramak tonik kolorektumun çevresel streç kodlamak tek sınıflardır. Bir bilgisayar-kontrollü kullanma Zorlamalı tahrik düzeni, biz düzleştirilmiş kolorektal doku çevresel yönde bir standart, homojen ve yeniden üretilebilir bir rampalı streç uygulanan ve streç duyarlı ileticilerin, düşük eşik değeri ve yüksek eşik 23 kategorize. Buna ek olarak, germe sensitizasyon zaman sürecini intrakolonik zimosan 21 veya TNBS 22 tedavisinden sonra -duyarlı afferentleri visseral ağrı ve aşırı duyarlılık streç duyarlı kolorektal aferentlerin bir rolü olduğunu düşündürmektedir, davranışsal visseral aşırı duyarlılık başlangıcı, sebat ve / veya kurtarma karşılık gelir.

<table border="0" cellpadding="0" cellspacing="0" width="1173">
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			<td height="21" style="height:21px;width:375px;">Name of Material/ Equipment</td>
			<td style="width:291px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td style="width:372px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica MZ16 stereo microscope&#160;</td>
			<td>Leica Microsystems Inc.</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica IC D camera</td>
			<td>Leica Microsystems Inc.</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Amplifier</td>
			<td>World Precision instruments, Inc.</td>
			<td>SYS-DAM80&#160;</td>
			<td>Low-noise differential amplifier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Two-compartment tissue chamber</td>
			<td style="width:291px;">Custom made</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Power1401</td>
			<td>Cambridge Electronic Design Limited</td>
			<td style="width:137px;">Power1401</td>
			<td>Data acquisition, analog signal input/out</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Spike2 v5.02</td>
			<td>Cambridge Electronic Design Limited</td>
			<td style="width:137px;"></td>
			<td>Software package that works with the Power1401</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Audio monitor</td>
			<td>Natus</td>
			<td style="width:137px;">Am 8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Square pulse stimulator</td>
			<td>Natus</td>
			<td style="width:137px;">S48</td>
			<td>To deliver electrical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Photoelectric isolation unit</td>
			<td>Natus</td>
			<td style="width:137px;">PSIU6</td>
			<td>Stimulus isolation to reduce noise</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Concentric bipolar microelectrode</td>
			<td style="width:291px;">FHC Inc.</td>
			<td style="width:137px;">CBFFG75</td>
			<td>To deliver electrical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dual-mode lever system</td>
			<td>Aurora Scientific Inc.</td>
			<td style="width:137px;">Series 300C&#160;</td>
			<td>To deliver mechanical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td>forceps with fine tips</td>
		</tr>
	</tbody>
</table>

in vitro functional characterization of mouse colorectal afferent endings

Bu video ayrıntılı olarak, bir fare kolorektum sinir preparatı kullanılarak bir in vitro tek lif elektrofizyolojik kayıt protokolünü gösterir. yaklaşımı tarafsız kimlik ve bireysel kolorektal afferentlerin fonksiyonel karakterizasyonu sağlar. Bir veya birkaç afferent kaynaklanan yayılan aksiyon potansiyeli (AP) dışı kayıtları (yani, tek-lif) kolorektumda alıcı alanlar (RFS) alay sinir lifi fasiküller yapılır. kolorektum pelvis (PN) veya bel splanknik (LSN) sinir bağlı ve boylamasına açılmıştır ya çıkarılır. Doku düz tutturulmuş ve oksijene edilmiş Krebs çözeltisi ile perfüze, bir kayıt bölümü içinde yer almaktadır. Odak elektriksel stimülasyon işlevsel beş mechanosensi içine afferentler kategorize (sondalama mukozal okşayarak ve çevresel streç künt) ayrıca üç farklı mekanik uyaranlarla test edilmektedir kolorektal afferent sonlar, bulmak için kullanılırtive sınıflar. Bu mekanik uyaranlara hiçbiri yanıt sonlar mekanik duyarsız afferentleri (MIAS) olarak kategorize edilir. Kimyasallara RFS lokalize maruz mechanosensitive ve MIAS sensitizasyon (yani, gelişmiş yanıt, azaltılmış eşik, ve / veya mechanosensitivity edinimi) için değerlendirilebilir Hem (örneğin, iltihabi çorba (IS), kapsaisin, adenozin trifosfat (ATP)). Biz ekipman ve kolorektum-sinir kayıt hazırlık, ekli PN veya LSN, sinir liflerinin gelen kolorektum, tek-lif kayıt RFS belirlenmesi ile kolorektumun hasat ve RF kimyasalların lokalize uygulamasını açıklar. Buna ek olarak, hazırlık ve standart mekanik uyarım uygulama zorlukları da tartışılmıştır.

Kurulum, Şekil 1 &#39;de gösterilmiştir. Bu, bir silikon-kaplı banyo bölmesi ve bitişik bir mineral yağ ile doldurulmuş bölmesine bağlı sinir kolorektal barındıran bir ölçüye doku odasını kapsamaktadır. İki bölmeli odası CNC makinesi ile akrilik plastik bir katı blok işlenmiş edildi; Her iki bölmelerin alt sonradan kolorektal doku aşağı kolay pimini izin firma silikon ile kaplı. Alay sinir liflerinin hücre dışı AP yüksek ortak mod reddetme oranı (CMRR ~ 60 dB) ile dü...

Watch this Scientific Journal Video about In vitro Functional Characterization of Mouse Colorectal Afferent Endings at JoVE.com

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

Fare Kolorektal Afferent Endings in vitro Fonksiyonel Karakterizasyonu içinde

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

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Research

JoVE Journal

Medicine

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

9.5K Görüntüleme.  University of Pittsburgh. This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation. 

Video: In vitro Functional Characterization of Mouse Colorectal Afferent Endings

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

Bilinçli Mesnetlenmemiş Fare hiperinsülinemik öglisemik Kelepçeleri

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

Tıp

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

Parkinson Hastalığı Alanında Mitokondriyal Fenotiplerinin İzleme İlköğretim İnsan Fibroblast Kullanımı

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

Birincil Fare Colon Tümörler in vitro organoid Kültür

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

Taze Cerrahi Örneklerden İzolasyon ve İnsan Ventriküler kardiyomiyositlerin Fonksiyonel Karakterizasyonu

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

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. ...

Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable noninvasive method is essential to assess real-time cardiac morphological and functional changes in animal models of heart disease. Transthoracic echocardiography represents an important tool for noninvasive assessment of cardiac structure and function2. Here we used a high-resolution ultrasound imaging system to monitor myocardial remodeling and heart failure progression over time in a mouse model of TAC. B-mode, M-mode, and Doppler imaging were used to precisely assess cardiac hypertrophy, ventricular dilatation, and functional deterioration in mice following TAC. Color and pulse wave (PW) Doppler imaging was used to noninvasively measure pressure gradient across the aortic constriction created by TAC and to assess transmitral blood flow in mice. Thus transthoracic echocardiographic imaging provides comprehensive noninvasive measurements of cardiac dimensions and function in mouse models of heart disease.

The goal of this protocol is to noninvasively assess cardiac structural and functional changes in a mouse model of heart disease created by transverse aortic constriction, using B- and M-mode echocardiography and color/pulse wave Doppler imaging.

Ekokardiyografik Imaging tarafından Enine Aort Konstrüksiyon Mouse Modeli Kardiyak Morfolojik Değerlendirilmesi ve Fonksiyonel Değişiklikler

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

uzunlamasına<em&gt; İn Vivo</em&gt; Cerebrovasculature Görüntüleme: MSS Hastalıkları uygunluk

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

Hazırlık ve<em&gt; İn Vitro</em&gt; Mıknatıslanmış karakterizasyonu Endotel Hücreleri miR-modifiye

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

Geliştirme ve işlevsel fare Tolerogenic dendritik hücrelerin karakterizasyonu

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

Functional Characterization of Endogenously Expressed Human RYR1 Variants

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia susceptibility, core myopathies and centronuclear myopathy. Because of the diverse phenotypes linked to RYR1 mutations it is fundamental to characterize their functional effects to classify variants carried by patients for future therapeutic interventions and identify non-pathogenic variants. Many laboratories have been interested in developing methods to functionally characterize RYR1 mutations expressed in patients&#39; cells. This approach has numerous advantages, including: mutations are endogenously expressed, RyR1 is not over-expressed, use of heterologous RyR1 expressing cells is avoided. However, since patients may present mutations in different genes aside RYR1, it is important to compare results from biological material from individuals harboring the same mutation, with different genetic backgrounds. The present manuscript describes methods developed to study the functional effects of endogenously expressed RYR1 variants in: (a) Epstein Barr virus immortalized human B-lymphocytes and (b) satellite cells derived from muscle biopsies and differentiated into myotubes. Changes in the intracellular calcium concentration triggered by the addition of a pharmacological RyR1 activators are then monitored. The selected cell type is loaded with a ratiometric fluorescent calcium indicator and intracellular [Ca2+] changes are monitored either at the single cell level by fluorescence microscopy or in cell populations using a spectrofluorometer. The resting [Ca2+], agonist dose response curves are then compared between cells from healthy controls and patients harboring RYR1 variants leading to insight into the functional effect of a given variant.

Here methods used to study the functional effect of RYR1 mutations endogenously expressed in Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy derived satellite cells differentiated into myotubes are described.