Name: in vitro functional characterization of mouse colorectal afferent endings
Uploaded: 2015-01-21T13:00:00.000+00:00
Duration: 14 min 9 s
Description: Watch this Scientific Journal Video about In vitro Functional Characterization of Mouse Colorectal Afferent Endings at JoVE.com

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>

<ol>
	<li>Naliboff, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+two+distinct+perceptual+alterations+in+irritable+bowel+syndrome.">Evidence for two distinct perceptual alterations in irritable bowel syndrome.</a> Gut. 41, 505-512 (1997).</li><li>Verne, G. N., Robinson, M. E., Vase, L., Price, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversal+of+visceral+and+cutaneous+hyperalgesia+by+local+rectal+anesthesia+in+irritable+bowel+syndrome+(IBS)+patients.">Reversal of visceral and cutaneous hyperalgesia by local rectal anesthesia in irritable bowel syndrome (IBS) patients.</a> Pain. 105, 223-230 (2003).</li><li>Verne, G. N., Sen, A., Price, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrarectal+lidocaine+is+an+effective+treatment+for+abdominal+pain+associated+with+diarrhea-predominant+irritable+bowel+syndrome.">Intrarectal lidocaine is an effective treatment for abdominal pain associated with diarrhea-predominant irritable bowel syndrome.</a> Journal of Pain. 6, 493-496 (2005).</li><li>Chey, W. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linaclotide+for+irritable+bowel+syndrome+with+constipation:+a+26-week,+randomized,+double-blind,+placebo-controlled+trial+to+evaluate+efficacy+and+safety.">Linaclotide for irritable bowel syndrome with constipation: a 26-week, randomized, double-blind, placebo-controlled trial to evaluate efficacy and safety.</a> Am J Gastroenterol. 107, 1702-1712 (2012).</li><li>Rao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+12-week,+randomized,+controlled+trial+with+a+4-week+randomized+withdrawal+period+to+evaluate+the+efficacy+and+safety+of+linaclotide+in+irritable+bowel+syndrome+with+constipation.">A 12-week, randomized, controlled trial with a 4-week randomized withdrawal period to evaluate the efficacy and safety of linaclotide in irritable bowel syndrome with constipation.</a> Am J Gastroenterol. 107, 1714-1724 (2012).</li><li>Busby, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacologic+properties,+metabolism,+and+disposition+of+linaclotide,+a+novel+therapeutic+peptide+approved+for+the+treatment+of+irritable+bowel+syndrome+with+constipation+and+chronic+idiopathic+constipation.">Pharmacologic properties, metabolism, and disposition of linaclotide, a novel therapeutic peptide approved for the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation.</a> J Pharmacol Exp Ther. 344, 196-206 (2013).</li><li>Mei, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+chemosensitivity.">Intestinal chemosensitivity.</a> Physiol Rev. 65, 211-237 (1985).</li><li>Mei, N., Lucchini, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+data+and+ideas+on+digestive+sensitivity.">Current data and ideas on digestive sensitivity.</a> J Auton Nerv Syst. 41, 15-18 (1992).</li><li>Su, X., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosensitive+pelvic+nerve+afferent+fibers+innervating+the+colon+of+the+rat+are+polymodal+in+character.">Mechanosensitive pelvic nerve afferent fibers innervating the colon of the rat are polymodal in character.</a> J Neurophysiol. 80, 2632-2644 (1998).</li><li>McMahon, S. B., Morrison, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+neurones+with+long+projections+activated+from+the+abdominal+viscera+of+the+cat.">Spinal neurones with long projections activated from the abdominal viscera of the cat.</a> J Physiol. 322, 1-20 (1982).</li><li>Cervero, F., Sann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanically+evoked+responses+of+afferent+fibres+innervating+the+guinea-pig's+ureter:+an+in+vitro+study.">Mechanically evoked responses of afferent fibres innervating the guinea-pig's ureter: an in vitro study.</a> J Physiol. 412, 245-266 (1989).</li><li>Sengupta, J. N., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosensitive+properties+of+pelvic+nerve+afferent+fibers+innervating+the+urinary+bladder+of+the+rat.">Mechanosensitive properties of pelvic nerve afferent fibers innervating the urinary bladder of the rat.</a> J Neurophysiol. 72, 2420-2430 (1994).</li><li>Sengupta, J. N., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+mechanosensitive+pelvic+nerve+afferent+fibers+innervating+the+colon+of+the+rat.">Characterization of mechanosensitive pelvic nerve afferent fibers innervating the colon of the rat.</a> J Neurophysiol. 71, 2046-2060 (1994).</li><li>Habler, H. J., Janig, W., Koltzenburg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+unmyelinated+afferent+fibres+by+mechanical+stimuli+and+inflammation+of+the+urinary+bladder+in+the+cat.">Activation of unmyelinated afferent fibres by mechanical stimuli and inflammation of the urinary bladder in the cat.</a> J Physiol. 425, 545-562 (1990).</li><li>Habler, H. J., Janig, W., Koltzenburg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+type+of+unmyelinated+chemosensitive+nociceptor+in+the+acutely+inflamed+urinary+bladder.">A novel type of unmyelinated chemosensitive nociceptor in the acutely inflamed urinary bladder.</a> Agents Actions. 25, 219-221 (1988).</li><li>Brierley, S. M., Jones, R. C., Gebhart, G. F., Blackshaw, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splanchnic+and+pelvic+mechanosensory+afferents+signal+different+qualities+of+colonic+stimuli+in+mice.">Splanchnic and pelvic mechanosensory afferents signal different qualities of colonic stimuli in mice.</a> Gastroenterology. 127, 166-178 (2004).</li><li>Meyer, R. A., Davis, K. D., Cohen, R. H., Treede, R. D., Campbell, 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=Mechanically+insensitive+afferents+(MIAs)+in+cutaneous+nerves+of+monkey.">Mechanically insensitive afferents (MIAs) in cutaneous nerves of monkey.</a> Brain Res. 561, 252-261 (1991).</li><li>Handwerker, H. O., Kilo, S., Reeh, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unresponsive+afferent+nerve+fibres+in+the+sural+nerve+of+the+rat.">Unresponsive afferent nerve fibres in the sural nerve of the rat.</a> J Physiol. 435, 229-242 (1991).</li><li>Feng, B., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+silent+afferents+in+the+pelvic+and+splanchnic+innervations+of+the+mouse+colorectum.">Characterization of silent afferents in the pelvic and splanchnic innervations of the mouse colorectum.</a> Am J Physiol Gastrointest Liver Physiol. 300, G170-G180 (2011).</li><li>Feng, B., La, J. H., Schwartz, E. S., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Irritable+bowel+syndrome:+methods,+mechanisms,+and+pathophysiology.+Neural+and+neuro-immune+mechanisms+of+visceral+hypersensitivity+in+irritable+bowel+syndrome.">Irritable bowel syndrome: methods, mechanisms, and pathophysiology. Neural and neuro-immune mechanisms of visceral hypersensitivity in irritable bowel syndrome.</a> Am J Physiol Gastrointest Liver Physiol. 302, G1085-G1098 (2012).</li><li>Feng, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+sensitization+of+mechanosensitive+and+-insensitive+afferents+in+mice+with+persistent+colorectal+hypersensitivity.">Long-term sensitization of mechanosensitive and -insensitive afferents in mice with persistent colorectal hypersensitivity.</a> Am J Physiol Gastrointest Liver Physiol. 302, G676-G683 (2012).</li><li>Feng, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+colorectal+afferent+function+associated+with+TNBS-induced+visceral+hypersensitivity+in+mice.">Altered colorectal afferent function associated with TNBS-induced visceral hypersensitivity in mice.</a> Am J Physiol Gastrointest Liver Physiol. 303, G817-G824 (2012).</li><li>Feng, B., Brumovsky, P. R., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+roles+of+stretch-sensitive+pelvic+nerve+afferents+innervating+mouse+distal+colon+and+rectum.">Differential roles of stretch-sensitive pelvic nerve afferents innervating mouse distal colon and rectum.</a> Am J Physiol Gastrointest Liver Physiol. 298, G402-G409 (2010).</li><li>Feng, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+guanylate+cyclase-C+attenuates+stretch+responses+and+sensitization+of+mouse+colorectal+afferents.">Activation of guanylate cyclase-C attenuates stretch responses and sensitization of mouse colorectal afferents.</a> J Neurosci. 33, 9831-9839 (2013).</li><li>Jolliffe, I. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principal component analysis. , (2002).</li><li>La, J. H., Feng, B., Schwartz, E. S., Brumovsky, P. R., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luminal+hypertonicity+and+acidity+modulate+colorectal+afferents+and+induce+persistent+visceral+hypersensitivity.">Luminal hypertonicity and acidity modulate colorectal afferents and induce persistent visceral hypersensitivity.</a> Am J Physiol Gastrointest Liver Physiol. 303, G802-G809 (2012).</li><li>Christianson, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=plasticity+and+modulation+of+visceral+afferents.">plasticity and modulation of visceral afferents.</a> Brain Research Reviews. 60, 171-186 (2009).</li><li>Kiyatkin, M. E., Feng, B., Schwartz, E. S., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+genetic+and+pharmacological+inhibition+of+TRPV1+and+P2X3+attenuates+colorectal+hypersensitivity+and+afferent+sensitization.">Combined genetic and pharmacological inhibition of TRPV1 and P2X3 attenuates colorectal hypersensitivity and afferent sensitization.</a> Am J Physiol Gastrointest Liver Physiol. 305, G638-G648 (2013).</li><li>Brumovsky, P. R., Feng, B., Xu, L., McCarthy, C. J., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystitis+increases+colorectal+afferent+sensitivity+in+the+mouse.">Cystitis increases colorectal afferent sensitivity in the mouse.</a> Am J Physiol Gastrointest Liver Physiol. 297, G1250-G1258 (2009).</li><li>Shinoda, M., Feng, B., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+and+central+P2X+receptor+contributions+to+colon+mechanosensitivity+and+hypersensitivity+in+the+mouse.">Peripheral and central P2X receptor contributions to colon mechanosensitivity and hypersensitivity in the mouse.</a> Gastroenterology. 137, 2096-2104 (2009).</li><li>Tanaka, T., Shinoda, M., Feng, B., Albers, K. M., Gebhart, G. 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><tbody><tr><td>Leica MZ16 stereo microscope</td><td>Leica Microsystems Inc.</td><td></td><td></td></tr><tr><td>Leica IC D camera</td><td>Leica Microsystems Inc.</td><td></td><td></td></tr><tr><td>Amplifier</td><td>World Precision instruments, Inc.</td><td>SYS-DAM80</td><td>Low-noise differential amplifier</td></tr><tr><td>Two-compartment tissue chamber</td><td>Custom made</td><td></td><td></td></tr><tr><td>Power1401</td><td>Cambridge Electronic Design Limited</td><td>Power1401</td><td>Data acquisition, analog signal input/out</td></tr><tr><td>Spike2 v5.02</td><td>Cambridge Electronic Design Limited</td><td></td><td>Software package that works with the Power1401</td></tr><tr><td>Audio monitor</td><td>Natus</td><td>Am 8</td><td></td></tr><tr><td>Square pulse stimulator</td><td>Natus</td><td>S48</td><td>To deliver electrical stimuli</td></tr><tr><td>Photoelectric isolation unit</td><td>Natus</td><td>PSIU6</td><td>Stimulus isolation to reduce noise</td></tr><tr><td>Concentric bipolar microelectrode</td><td>FHC Inc.</td><td>CBFFG75</td><td>To deliver electrical stimuli</td></tr><tr><td>Dual-mode lever system</td><td>Aurora Scientific Inc.</td><td>Series 300C</td><td>To deliver mechanical stimuli</td></tr><tr><td>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 ...

in-vitro-functional-characterization-mouse-colorectal-afferent

Universidad de Cartagena UDEC

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

Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).

Over the past few years, new generation endoscopes have emerged as important diagnostic research aids for evaluating murine colitis and colorectal tumors. We present herein a detailed protocol for endoscopic assessment of inflammation and colorectal tumors in mice, as well as a novel scoring system that uses decimal identifiers to document the endoscopic severity of colitis and colorectal tumors.

Bir Geçerliliği Endoskopik Puanlama Sisteminin Kullanılması Kolit ve Kolorektal Tümörlerin Şiddetini Değerlendirmek için Farelerde esnek Kolonoskopi

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental ...

Tıp

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

Bir<em&gt; In-vitro</emYetişkin Mouse myenterik Pleksus izole edilen enterik Nöronlar ve Glia bir&gt; hazırlanması

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

Nörobilim

Combined Recording of Mechanically Stimulated Afferent Output and Nerve Terminal Labelling in Mouse Hair Follicle Lanceolate Endings

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The technique is very cost-effective and easily undertaken with materials commonly found in most electrophysiology laboratories, or easily purchased. The dissection is simple and fast, with the mechanical displacement provided by a generic electroceramic wafer controlled by proprietary software. The same software also records and analyses the electroneurogram output. The recording of the evoked nerve activity is through a commercial differential amplifier connected to fire-polished standard glass microelectrodes. Helpful tips are given for improving the quality of the preparation, the stimulation and the recording conditions to optimize recording quality. The system is suitable for assaying the electrophysiological and optical properties of lanceolate terminals of palisade endings of hair follicles, as well as the outcomes from their pharmacological and/or genetic manipulation. An example of combining electrical recording with mechanical stimulation and labeling with a styryl pyridinium vital dye is given.

A simple and novel technique for recording afferent discharge due to mechanical stimulation of lanceolate terminals of palisade endings innervating mouse ear skin hair follicles is presented.

Fare Saç Folikül hançer Endings de Mekanik Uyarılmış Afferent Çıkışı ve Sinir Terminali Etiketleme Kombine Kaydı

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The ...

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the different organ systems from the periphery towards the central nervous system. As such, the increased activity or &#39;sensitization&#39; of mesenteric afferent nerves has been allocated an important role in the pathophysiology of visceral hypersensitivity and abdominal pain syndromes.
Mesenteric afferent nerve activity can be measured in vitro in an isolated intestinal segment that is mounted in a purpose-built organ bath and from which the splanchnic nerve is isolated, allowing researchers to directly assess nerve activity adjacent to the gastrointestinal segment. Activity can be recorded at baseline in standardized conditions, during distension of the segment or following the addition of pharmacological compounds delivered intraluminally or serosally. This technique allows the researcher to easily study the effect of drugs targeting the peripheral nervous system in control specimens; besides, it provides crucial information on how neuronal activity is altered during disease. It should be noted however that measuring afferent neuronal firing activity only constitutes one relay station in the complex neuronal signaling cascade, and researchers should bear in mind not to overlook neuronal activity at other levels (e.g., dorsal root ganglia, spinal cord or central nervous system) in order to fully elucidate the complex neuronal physiology in health and disease.
Commonly used applications include the study of neuronal activity in response to the administration of lipopolysaccharide, and the study of afferent nerve activity in animal models of irritable bowel syndrome. In a more translational approach, the isolated mouse intestinal segment can be exposed to colonic supernatants from IBS patients. Furthermore, a modification of this technique has been recently shown to be applicable in human colonic specimens.

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Mesenteric afferent nerves convey information from the gastrointestinal tract towards the brain regarding normal homeostasis as well as pathophysiology. Gastrointestinal afferent nerve activity can be assessed by mounting isolated intestinal segments with attached afferent nerves into an organ bath, isolating the nerve, and assessing basal as well as stimulated activity.

Fare jejunal ve Kolon Segmentlerde Mezenterik Afferent Sinir Faaliyet Vitro kaydedilmesi

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the ...

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory bowel diseases and diabetes. Here, we describe a protocol for the in vitro characterization of the electrophysiological properties of colonic afferents in rats. The colorectum, with the intact pelvic ganglion (PG) attached, is removed from the rat; superfused with carbogenated Krebs solution in the recording chamber; and cannulated at the oral and anal ends to allow for distension. A fine nerve bundle emanating from the PG is identified, and the multiunit afferent nerve activity is recorded using a suction electrode. Distension of the colonic segment elicits gradual increases in multiunit discharge. A principal component analysis is conducted to differentiate the low-threshold, the high-threshold, and the wide-dynamic range afferent fibers. Chemical sensitivity of colonic afferents can be studied through the bath or intraluminal administration of test compounds. This protocol can be modified for application to other species, such as mice and guinea pigs, and to study the differences in the electrophysiological properties of thoracolumbar/hypogastric and lumbosacral/pelvic afferents of the descending colon in normal and pathological conditions.

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Abnormal sensory function underlies visceral pain and other symptoms of functional and inflammatory bowel diseases. A protocol for the electrophysiological recording of the colonic afferent nerves in an ex vivo rat colorectum preparation is presented here.

Vitro Sıçanlarda kolon Afferent liflerinin Elektrofizyolojik özellikleri karakterizasyonu

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory ...

Advanced Imaging of Dorsal Root Ganglion Neurons for Sensory Modality Research

In Vivo Calcium Imaging of Dorsal Root Ganglia Neurons' Response to Somatic and Visceral Stimuli

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the protocol for in vivo calcium imaging of the exposed DRG in response to various visceral and somatic stimuli. Pirt-GCaMP6s mice or C57BL6 mice intrathecally injected with AAV viruses packaged with GCaMP6s were utilized to capture Ca2+ transients. The amplitude of these transients indicates sensitivity to specific sensory modalities. Afferent fibers originate from internal organs, with primary neuronal cell bodies in spinal or vagal ganglia. Studies on visceral nociception and acupuncture analgesia can potentially be conducted on primary sensory neurons using advanced imaging technologies like in vivo calcium imaging, allowing for the recording of neuronal activity ensembles in the intact animal during stimulation or intervention. The responses of DRG neuron ensembles to somatic and visceral stimuli applied to their corresponding receptive fields were recorded. This technique illustrates how neuronal populations react to various types of somatic and visceral stimuli. It is possible to comprehensively compare neuronal ensemble responses to different stimuli, which is a particularly valuable approach in research on visceral pain and segmental mechanisms of somatic stimulation, such as acupuncture.

The present protocol outlines in vivo calcium imaging for measuring the responses of ensembles of lumbar-6 DRG neurons to somatic and visceral stimuli. Thorough comparisons can be made among neurons responding to different stimuli. This protocol is valuable for investigating mechanisms of visceral pain and somatic stimulation, such as acupuncture.

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the ...

Functional Assessment of Intestinal Tight Junction Barrier and Ion Permeability in Native Tissue by Ussing Chamber Technique

The Ussing chamber technique was first invented by the Danish scientist Hans Ussing in 1951 to study the transcellular transport of sodium across frog skin. Since then, this technique has been applied to many different tissues to study the physiological parameters of transport across membranes. The Ussing chamber method is preferable to other methods because native tissue can be used, making it more applicable to what is happening in vivo. However, because native tissue is used, throughput is low, time is limited, and tissue preparation requires skill and training. These chambers have been used to study specific transporter proteins in various tissues, understand disease pathophysiology such as in Cystic Fibrosis, study drug transport and uptake, and especially contributed to the understanding of nutrient transport in the intestine. Given the whole epithelial transport process of a tissue, not only transepithelial pathways, but also paracellular pathways are important. Tight junctions are a key determinant of tissue specific paracellular permeability across the intestine. In this article, the Ussing chamber technique will be used to assess paracellular permselectivity of ions by measuring transepithelial conductance and dilution potentials.

Intestinal epithelium confers not only nutrient absorption but protection against noxious substances. The apical-most epithelial intercellular junction, i.e., the tight junction, regulates paracellular solute and ion permeability. Here, a protocol for the preparation of mucosal sheets and assessment of the ion selectivity of tight junctions using Ussing chamber technique is described.

Doğal Dokuda İntestinal Sıkı Bileşke Bariyeri ve İyon Geçirgenliğinin Ussing Oda Tekniği ile Fonksiyonel Olarak Değerlendirilmesi

The Ussing chamber technique was first invented by the Danish scientist Hans Ussing in 1951 to study the transcellular transport of sodium across frog ...

Biyoloji

Generation of Mouse Proximal Colon Loop Model: A Surgical Procedure to Generate Proximal Colon Loop and Measure Intestinal Permeability by Detection of Fluorescent Markers in Serum Following Intraluminal Injection

Source: Boerner, K. et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/62093">Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model.</a> J. Vis. Exp. (2021).
In this video, we describe a surgical procedure to generate mouse proximal colon loop or pcLoop segment followed by intraluminal injection of fluorescent markers into the segment to study the intestinal epithelial permeability.

Fare Proksimal Kolon Döngü Modelinin Oluşturulması: İntraluminal Enjeksiyonu Takiben Serumda Floresan İşaretleyicilerin Saptanmasıyla Proksimal Kolon Halkası Oluşturmak ve Bağırsak Geçirgenliğini Ölçmek İçin Cerrahi Bir Prosedür

Source: Boerner, K. et al. Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized ...

JoVE Encyclopedia of Experiments

Kemirgen Modelleri

Yetişkin

Electrophysiological Recording from Colonic Afferent Nerves in an Ex Vivo Rat Colon

Source: Meng, Y. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/56090">In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats.</a> J. Vis. Exp. (2017)
This video demonstrates a method to record the activity of colonic afferent nerves in an ex-vivo rat colon, cannulated at both ends and infused with saline to increase intraluminal pressure. As pressure increases, the nerve signal rises, indicating its stimulation.

Bir Ex Vivo Sıçan Kolonunda Kolon Afferent Sinirlerinden Elektrofizyolojik Kayıt

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Neurophysiology

Electrophysiology Techniques

Recording of Mesenteric Afferent Nerve Activity in an Ex Vivo Mouse Jejunum Segment

In this procedure, using a micromanipulator, lower the tip of the suction electrode into the organ bath. Then, gently aspirate some Krebs solution from the organ bath so that the tip of the electrode is submerged in the Krebs solution. Ensure that the Krebs solution covers the wire electrode inside the suction electrode.
Next, position the tip of the suction electrode immediately next to the transected afferent nerve strand, and draw the transected nerve strand into the capillary over its entire length. Maneuver the tip of the electrode towards the adipose tissue, and aspirate it into the glass capillary with the plunger, thereby mechanically sealing the nerve in the capillary from the contents of the organ bath.
To verify the recording of afferent nerve activity, perform a ramp distension-induced increase in afferent firing by closing the output port to lead to a gradual rise in pressure in the intestinal segment. Only perform the desired experimental protocol when three consecutive ramp distensions yield a reproducible multi-unit discharge.

Fare Kolorektal Afferent Endings in vitro Fonksiyonel Karakterizasyonu içinde

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler