Name: Video: Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice
Uploaded: 2016-02-03T15:00:00.000+00:00
Duration: 7 min 41 s
Description: 13.9K Views. The University of Melbourne. This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

JCB ו ELH-Y נתמכו תוכנית משרד ההגנה האמריקאי CDMRP אוטיזם מחקר (AR11034). NHMRC (1047674) כדי ELH-Y.The במאי סטיוארט הגזברות-אוניברסיטת אמון מלבורן במימון מלגה MS. אנו מודים עלי טאהר, פטימה Ramalhosa ו גרציה סיגר לתרומות טכנית.

<ol>
	<li>Powell, A. K., O'Brien, S. D., Fida, R., Bywater, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+integrity+is+essential+for+the+propagation+of+colonic+migrating+motor+complexes+in+the+mouse.">Neural integrity is essential for the propagation of colonic migrating motor complexes in the mouse.</a> Neurogastroenterol Motil. 14, 495-504 (2002).</li><li>Furness, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enteric+nervous+system+and+neurogastroenterology.">The enteric nervous system and neurogastroenterology.</a> Nat Rev Gastroenterol Hepatol. 9, 286-294 (2012).</li><li>Gwynne, R. M., Bornstein, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+underlying+nutrient-induced+segmentation+in+isolated+guinea+pig+small+intestine.">Mechanisms underlying nutrient-induced segmentation in isolated guinea pig small intestine.</a> Am J Physiol Gastrointest Liver Physiol. 292, G1162-G1172 (2007).</li><li>Bush, T. G., Spencer, N. J., Watters, N., Sanders, K. M., 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=Spontaneous+migrating+motor+complexes+occur+in+both+the+terminal+ileum+and+colon+of+the+C57BL/6+mouse+in+vitro.">Spontaneous migrating motor complexes occur in both the terminal ileum and colon of the C57BL/6 mouse in vitro.</a> Auton Neurosci. 84, 162-168 (2000).</li><li>Fida, R., Lyster, D. J., Bywater, R. A., Taylor, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonic+migrating+motor+complexes+(CMMCs)+in+the+isolated+mouse+colon.">Colonic migrating motor complexes (CMMCs) in the isolated mouse colon.</a> Neurogastroenterol Motil. 9, 99-107 (1997).</li><li>Neal, K. B., Parry, L. J., Bornstein, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-specific+genetics,+anatomy+and+function+of+enteric+neural+serotonergic+pathways+in+inbred+mice.">Strain-specific genetics, anatomy and function of enteric neural serotonergic pathways in inbred mice.</a> J Physiol. 587, 567-586 (2009).</li><li>Roberts, R. R., Murphy, J. F., Young, H. M., Bornstein, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+colonic+motility+in+the+neonatal+mouse-studies+using+spatiotemporal+maps.">Development of colonic motility in the neonatal mouse-studies using spatiotemporal maps.</a> Am J Physiol Gastrointest Liver Physiol. 292, G930-G938 (2007).</li><li>Spencer, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+migrating+motor+activity+in+the+colon.">Control of migrating motor activity in the colon.</a> Curr Opin Pharmacol. 1, 604-610 (2001).</li><li>Spencer, N. J., Bywater, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+nerve+stimulation+evokes+a+premature+colonic+migrating+motor+complex+in+mouse.">Enteric nerve stimulation evokes a premature colonic migrating motor complex in mouse.</a> Neurogastroenterol Motil. 14, 657-665 (2002).</li><li>Roberts, R. R., Bornstein, J. C., Bergner, A. J., Young, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disturbances+of+colonic+motility+in+mouse+models+of+Hirschsprung's+disease.">Disturbances of colonic motility in mouse models of Hirschsprung's disease.</a> Am J Physiol Gastrointest Liver Physiol. 294, G996-G1008 (2008).</li><li>Tough, I. R., 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=Endogenous+peptide+YY+and+neuropeptide+Y+inhibit+colonic+ion+transport,+contractility+and+transit+differentially+via+Y(1)+and+Y(2)+receptors.">Endogenous peptide YY and neuropeptide Y inhibit colonic ion transport, contractility and transit differentially via Y(1) and Y(2) receptors.</a> Br J Pharmacol. 164, 471-484 (2011).</li><li>Hennig, G. W., Costa, M., Chen, B. N., Brookes, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+peristalsis+in+the+guinea-pig+small+intestine+using+spatio-temporal+maps.">Quantitative analysis of peristalsis in the guinea-pig small intestine using spatio-temporal maps.</a> J Physiol. 517 (Pt 2), 575-590 (1999).</li><li>Hoffman, J. M., Brooks, E. M., 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=Gastrointestinal+Motility+Monitor+(GIMM).">Gastrointestinal Motility Monitor (GIMM).</a> J Vis Exp. , (2010).</li><li>Smith, T. K., Gershon, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rebuttal+from+Terence+K.+Smith+and+Michael+D.+Gershon.">Rebuttal from Terence K. Smith and Michael D. Gershon.</a> J Physiol. 593, 3233 (2015).</li><li>Spencer, N. J., Sia, T. C., Brookes, S. J., Costa, M., Keating, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CrossTalk+opposing+view:+5-HT+is+not+necessary+for+peristalsis.">CrossTalk opposing view: 5-HT is not necessary for peristalsis.</a> J Physiol. 593, 3229-3231 (2015).</li><li>Tabuchi, K., 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+neuroligin-3+mutation+implicated+in+autism+increases+inhibitory+synaptic+transmission+in+mice.">A neuroligin-3 mutation implicated in autism increases inhibitory synaptic transmission in mice.</a> Science. 318, 71-76 (2007).</li><li>Jamain, 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=Mutations+of+the+X-linked+genes+encoding+neuroligins+NLGN3+and+NLGN4+are+associated+with+autism.">Mutations of the X-linked genes encoding neuroligins NLGN3 and NLGN4 are associated with autism.</a> Nat Genet. 34, 27-29 (2003).</li><li>Chaidez, V., Hansen, R. L., Hertz-Picciotto, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+problems+in+children+with+autism,+developmental+delays+or+typical+development.">Gastrointestinal problems in children with autism, developmental delays or typical development.</a> J Autism Dev Disord. 44, 1117-1127 (2014).</li><li>Ibrahim, S. H., Voigt, R. G., Katusic, S. K., Weaver, A. L., Barbaresi, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+gastrointestinal+symptoms+in+children+with+autism:+a+population-based+study.">Incidence of gastrointestinal symptoms in children with autism: a population-based study.</a> Pediatrics. 124, 680-686 (2009).</li><li>Kohane, I. 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=The+co-morbidity+burden+of+children+and+young+adults+with+autism+spectrum+disorders.">The co-morbidity burden of children and young adults with autism spectrum disorders.</a> PloS One. 7, e33224 (2012).</li><li>McElhanon, B. O., McCracken, C., Karpen, S., Sharp, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+symptoms+in+autism+spectrum+disorder:+a+meta-analysis.">Gastrointestinal symptoms in autism spectrum disorder: a meta-analysis.</a> Pediatrics. 133, 872-883 (2014).</li><li>Peters, 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=Rigid-compulsive+behaviors+are+associated+with+mixed+bowel+symptoms+in+autism+spectrum+disorder.">Rigid-compulsive behaviors are associated with mixed bowel symptoms in autism spectrum disorder.</a> J Autism Dev Disord. 44, 1425-1432 (2014).</li><li>Ellis, M., Chambers, J. D., Gwynne, R. M., Bornstein, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+and+cholecystokinin+mediate+nutrient-induced+segmentation+in+guinea+pig+small+intestine.">Serotonin and cholecystokinin mediate nutrient-induced segmentation in guinea pig small intestine.</a> Am J Physiol Gastrointest Liver Physiol. 304, G749-G761 (2013).</li><li>Parracho, H. M., Bingham, M. O., Gibson, G. R., McCartney, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+between+the+gut+microflora+of+children+with+autistic+spectrum+disorders+and+that+of+healthy+children.">Differences between the gut microflora of children with autistic spectrum disorders and that of healthy children.</a> J Med Microbiol. 54, 987-991 (2005).</li><li>Buie, T., 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=Evaluation,+diagnosis,+and+treatment+of+gastrointestinal+disorders+in+individuals+with+ASDs:+a+consensus+report.">Evaluation, diagnosis, and treatment of gastrointestinal disorders in individuals with ASDs: a consensus report.</a> Pediatrics. 125, S1-S18 (2010).</li><li>Etherton, M., 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=Autism-linked+neuroligin-3+R451C+mutation+differentially+alters+hippocampal+and+cortical+synaptic+function.">Autism-linked neuroligin-3 R451C mutation differentially alters hippocampal and cortical synaptic function.</a> Proc Natl Acad Sci U S A. 108, 13764-13769 (2011).</li><li>Etherton, M. R., Tabuchi, K., Sharma, M., Ko, J., Sudhof, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+autism-associated+point+mutation+in+the+neuroligin+cytoplasmic+tail+selectively+impairs+AMPA+receptor-mediated+synaptic+transmission+in+hippocampus.">An autism-associated point mutation in the neuroligin cytoplasmic tail selectively impairs AMPA receptor-mediated synaptic transmission in hippocampus.</a> EMBO J. 30, 2908-2919 (2011).</li><li>Zhang, Q., 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=Expression+of+neurexin+and+neuroligin+in+the+enteric+nervous+system+and+their+down-regulated+expression+levels+in+Hirschsprung+disease.">Expression of neurexin and neuroligin in the enteric nervous system and their down-regulated expression levels in Hirschsprung disease.</a> Mol Biol Rep. 40, 2969-2975 (2013).</li><li>Wang, J., 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=Expression+and+significance+of+neuroligins+in+myenteric+cells+of+Cajal+in+Hirschsprung's+disease.">Expression and significance of neuroligins in myenteric cells of Cajal in Hirschsprung's disease.</a> PloS One. 8, e67205 (2013).</li><li>Yang, H., 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=The+down-regulation+of+neuroligin-2+and+the+correlative+clinical+significance+of+serum+GABA+over-expression+in+Hirschsprung's+disease.">The down-regulation of neuroligin-2 and the correlative clinical significance of serum GABA over-expression in Hirschsprung's disease.</a> Neurochem Res. 39, 1451-1457 (2014).</li><li>Roberts, R. R., 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=The+first+intestinal+motility+patterns+in+fetal+mice+are+not+mediated+by+neurons+or+interstitial+cells+of+Cajal.">The first intestinal motility patterns in fetal mice are not mediated by neurons or interstitial cells of Cajal.</a> J Physiol. 588, 1153-1169 (2010).</li><li>Barnes, K. J., Spencer, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+colonic+migrating+motor+complexes+occur+in+mice+lacking+the+endothelin-3+gene?.">Can colonic migrating motor complexes occur in mice lacking the endothelin-3 gene?.</a> Clin Exp Pharmacol Physiol. 42, 485-495 (2015).</li><li>Chambers, J. D., Bornstein, J. C., Thomas, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+neural+oscillators+and+muscle+feedback+are+required+for+the+intestinal+fed+state+motor+program.">Multiple neural oscillators and muscle feedback are required for the intestinal fed state motor program.</a> PloS One. 6, e19597 (2011).</li><li>Heredia, D. J., 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=Important+role+of+mucosal+serotonin+in+colonic+propulsion+and+peristaltic+reflexes:+in+vitro+analyses+in+mice+lacking+tryptophan+hydroxylase+1.">Important role of mucosal serotonin in colonic propulsion and peristaltic reflexes: in vitro analyses in mice lacking tryptophan hydroxylase 1.</a> J Physiol. 591, 5939-5957 (2013).</li><li>Chambers, J. D., Bornstein, J. C., Thomas, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+mechanisms+of+intestinal+segmentation+in+guinea+pigs:+a+combined+computational+modeling+and+in+vitro+study.">Insights into mechanisms of intestinal segmentation in guinea pigs: a combined computational modeling and in vitro study.</a> Am J Physiol Gastrointest Liver Physiol. 295, G534-G541 (2008).</li><li>Huizinga, J. 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=The+origin+of+segmentation+motor+activity+in+the+intestine.">The origin of segmentation motor activity in the intestine.</a> Nat Commun. 5, 3326 (2014).</li><li>Neild, T. O., Shen, K. Z., Surprenant, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vasodilatation+of+arterioles+by+acetylcholine+released+from+single+neurones+in+the+guinea-pig+submucosal+plexus.">Vasodilatation of arterioles by acetylcholine released from single neurones in the guinea-pig submucosal plexus.</a> J Physiol. 420, 247-265 (1990).</li></ol>

The authors have nothing to disclose

באמצעות טכניקה הדמיה בסרטון הזה, תדירות CMMC נמדדה כאינדיקציה תנועתיות במעי הגס בקרב סוג בר עכברים R451C NL3, במודל של עכברים של הפרעת ספקטרום האוטיזם 17. התוצאות שלנו מצביעים על ירידה במספר CMMCs בעכברים מוטציה NL3 R451C בהשוואה לעכברים סוג בר בנוכחות של אנטגונ?...

מערכת העצבים enteric (ENS) היא הרשת העצבית הפנימי של מערכת העיכול ומווסת פונקציות שונות כגון עיכול של תוכן המעי, ספיגת המזון בגוף ואת הפרשת ספיגה חוזרת של נוזלים. נוירונים של ENS ממוקמים plexuses myenteric ו submucosal. מקלעת myenteric משחק תפקיד מרכזי בוויסות תנועתיות מערכת העיכול 1 ואילו מקלעת Submucosal מטפל בעיקר בבקרה של הפרשת 2,3. מקלעת myenteric ממוקמת בין שכבות שריר אורך וחוזר של קיר העיכול. הפעילות התכווצות של שכבות שריר חלק של לדופן המעי הופך לפשוט יותר את הפונקציות העיקריות של מערכת העיכול על ידי ערבוב הדוחף תוכן המעי לאורך המעי 3. למרות ההיצע העצב החיצוני אל מערכת העיכול מן CNS תורם לתפקוד עיכול in vivo, את ENS מסוגל ויסות תפקוד מערכת העיכול באופן עצמאי. מאפיין ייחודי זה מאפשר חקירה תפקודית של מעגלים עצביים מעיים ותרומתם תנועתיות מערכת העיכול vivo לשעבר. גס נודדים מתחמי מנוע (CMMCs) הם ספונטניים, אירועים נוירוגנית כי הם דפוס מנוע השולט שנצפה מעי גס עכבר מבודד בהעדר כדורי הצואה 4-9. CMMCs מוגדרים התכווצויות קצובות כי להפיץ לאורך המרחק האופקי הוא לפחות מחצית האורך הכולל של המעי הגס (כלומר, מן cecum אל פי הטבעת) 10. הקשר בין CMMCs ואת דפוסי ההתכווצות שמניעי כדורי צואה הוא עדיין לא נקבע בבירור, עם זאת כמה הבדלים תרופתיים דווחו 11. עם זאת, היכולת של ENS לתפקד באופן עצמאי של מערכת עצבים מרכזיים ואת קיומם של דפוסי מנוע בתיווך עצבי כלומרהמעי הגס olated מספק מערכת assay אידיאלי לבחון הפרעות תנועתיות כתוצאה מחוסר תפקוד ENS הבסיסית. הספונטניות של דפוסי מנוע עיכול מאפשרת שינויים תפקודיים בתגובה לגירויים תרופתית להיבדק. השימוש הדמיה וידאו ומיפוי spatiotemporal פותחה לראשונה לבחון הפריסטלטיקה של המעי הדק כמותית שרקנים 12. הנה, טכניקת vivo לשעבר מתוארת המאפשרת חקר דפוסי תנועתיות עכבר גסים באמצעות הדמיה וידאו וניתוח של הקלטות אלה לבנות ברזולוציה גבוהה (~ 100 מיקרומטר, 33 msec) מפות של בקוטר גס כפונקציה של מיקום לאורך המעי הגס ושל זמן (מפות spatiotemporal). שימוש ללא צורך במיקור חוץ בתוכנת איתור קצה (Analyse2; על פי דרישה), נתונים מתחומי הגס באורך מלא קבלנות בזמן אמת מעובדים ליצור מפות spatiotemporal עבור כל ניסוי. בשלב זה, וידאו (AVI) הקבצים הם ייתרוrized ועל המרה מפות spatiotemporal באמצעות Analyse2. מפות Spatiotemporal (איור 2) מתארים התכווצות לאורך זמן ולאפשר מדידת פרמטרים מרובים כולל מהירות התפשטות, גודל, אורך ומשך. קוטר גוט נרשם גם לאורך כל תקופת הניסוי כאמצעי של ההתכווצות הכוללת של מגזר הרקמות. שיטה זו יכולה להיות מיושמת לזהות הבדלי נקודת חניכה של מתחמי התכווצות מה שיכול להעיד קישוריות עצבית מעיים שונה. פרוטוקול הדמיה וידאו דומה שנועד להעריך הנעה גלולה ב שרקנים דווחה 13 אולם כאן נתאר את יישום הגישה הדמיה וידאו כימות של תנועתיות המעי הגס ספונטנית (כלומר, בהעדר כדורי). כמו כן אנו מספקים מידע מפורט כדי לסייע לנתיחה והכנת רקמות gastrointestinal עבור הגישה הדמיה וידאו. זֶהפרוטוקול מספק לחוקרים עם כלי נגיש לשכפל בקלות לניתוח שליטה עצבית מעיים של פונקצית עיכול במודלים של בעלי חיים של מחלה כולל מודלי עכבר גנטי. טכניקת דימות וידאו מאפשרת ניתוח של תנועתיות המעי הגס בתגובה סוכני תרופתי שונים. סמים יכולים להיות מנוהלים באמצעות לומן המעי או באמבטית האיברים החיצוני להכנה הגסה. באזורים שונים של מערכת העיכול העכבר מפגינים דפוסי תנועתיות ספציפיות כגון פילוח של המעי הדק CMMCs במעי הגס. טכניקה זו נעשתה שימוש כדי לזהות הבדלי זן בתפקוד המעי דק; רגישות ההפרש ל 5-HT 3 ו 5-HT 4 אנטגוניסטים נצפו מְעִי צָם של Balb / c ועכברים C57 / BL6 בשל אופי צורות של הגן tph2 מתבטא 6 שני זנים. השפעת עיכוב 5-HT על תנועתיות נשאר conשנוי במחלוקת, כפי דווחו נתונים סותרים על החשיבות של 5-HT אנדוגני על הפריסטלטיקה במעי גס, CMMCs 14,15. שינויים טרום תנועתיות לאחר הלידה במהלך הפיתוח 7, ואת ההשפעות של מוטציות גנטיות על תנועתיות הקיבה והמעיים במודלים של בעלי חיים של מחלת 10 יכול גם להיבדק על ידי ניצול הדמיה וידאו. כאן אנו מדגימים שימוש בשיטת לצורכי מחקר על תנועתיות המעי הגס במודל של אוטיזם העכבר R451C NL3, המבטאת מוטציה missense בגן Nlgn3 המקודד את החלבון הידבקות הסינפטי Neuroligin-3 16. המוטציה זו זוהתה לראשונה בחולים שאובחנו עם הפרעת ספקטרום האוטיזם (ASD) 17, אשר מזוהה מאוד עם תפקוד לקוי GI 18-22. חקרנו אם סינפטיים מוטצית R451C NL3 משפיעה פלט עצבי ENS באמצעות טכניקת דימות וידאו. אנו מציגים נתונים מאפיינים CMMCs בתחילת מחקר בתגובת 5H serotonergicT 3/4 קולטן אנטגוניסט tropisetron במודל של אוטיזם העכבר R451C NL3.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NaCl (MW: 58.44)</td><td>Sigma-Aldrich</td><td>S7653-250G</td><td></td></tr><tr><td>KCl (MW: 74.55)</td><td>Sigma-Aldrich</td><td>P9333-500G</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;.2H&lt;sub&gt;2&lt;/sub&gt;O (MW: 156.01)</td><td>Chem Supply</td><td>471-500G</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;0 (MW: 246.48)</td><td>Chem Supply</td><td>MA048</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;.2H&lt;sub&gt;2&lt;/sub&gt;O (MW: 147.02)</td><td>Chem Supply</td><td>CA033</td><td></td></tr><tr><td>D-Glucose anhydrous (MW: 180.16)</td><td>Chem Supply</td><td>GA018-500G</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3 &lt;/sub&gt;(MW: 84.01)</td><td>Chem Supply</td><td>GA018-500G</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Two chambered organ bath&lt;br/&gt; Dimentions: 14 cm x 8 cm x 3 cm</td><td>Custom Made</td><td></td><td>Contact Laboratory Directly&amp;nbsp;</td></tr><tr><td>&amp;nbsp;732 MULTI -PURPOSE SEALANT CLEAR</td><td>Dow Corning Australia Pty Ltd</td><td>1890573</td><td></td></tr><tr><td>SYLGARD 184 SILICONE ELASTOMER KIT&amp;nbsp;</td><td>Dow Corning Australia Pty Ltd</td><td>1064291</td><td></td></tr><tr><td>STOPCOCK 3 WAY FEM-ML L/LOCK S</td><td>Terumo Medical Corporation</td><td>0912-2006</td><td></td></tr><tr><td>SYRINGES with Luer Lock Tips 50 ml, 20 ml, 10 ml</td><td>Terumo Medical Corporation</td><td>N/A</td><td></td></tr><tr><td>1.57 mm (ID) x 3.16 mm (OD) - Silastic Tubing</td><td>Masterflex</td><td>508-008</td><td></td></tr><tr><td>1.02 mm (ID) x 2.16 mm (OD) - Silastic Tubing</td><td>Masterflex</td><td>508-005</td><td></td></tr><tr><td>1.50 mm (ID) x 2.50 mm (OD) - Silastic Tubing</td><td>Masterflex</td><td>508-007</td><td></td></tr><tr><td>1.60 mm (ID) - Platinum cured silicone tubing&amp;nbsp;</td><td>Masterflex</td><td>96410 - 14</td><td></td></tr><tr><td>4.40 mm (ID) - Platinum cured silicone tubing&amp;nbsp;</td><td>Masterflex</td><td>96410 - 15&amp;nbsp;</td><td></td></tr><tr><td>3.10 mm (ID) - Platinum cured silicone tubing&amp;nbsp;</td><td>Masterflex</td><td>96410 -16</td><td></td></tr><tr><td>Graduated Laboratory Glass Bottles - 500 ml&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Thermofisher Scientific&amp;nbsp;</td><td>100-400</td><td></td></tr><tr><td>CHEMICAL RUBBER STOPPER 57 x 65 mm&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>CHEMICAL RUBBER STOPPER 29 mm x 32 mm</td><td></td><td></td><td></td></tr><tr><td>Water heater&amp;nbsp; (thermo regulator)&amp;nbsp;</td><td>Ratek&amp;nbsp;</td><td>TH7000&amp;nbsp;</td><td></td></tr><tr><td>Logitech Webcam</td><td>Logitech</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Virtual Dub - 1.9 11</td><td>virtualdub.org</td><td></td><td></td></tr><tr><td>MATLAB R2012a&amp;nbsp;</td><td>Graph Pad</td><td></td><td></td></tr><tr><td>Logitech Webcam Software</td><td>Logitech</td><td></td><td></td></tr></tbody></table>

video imaging and spatiotemporal maps to analyze gastrointestinal motility in mice

מערכת העצבים enteric (ENS) ממלא תפקיד חשוב בוויסות של מערכת העיכול (GI) תנועתיות יכול לתפקד באופן עצמאי של מערכת העצבים המרכזית. שינויים בתפקוד ENS הם הגורם העיקרי של תסמינים במערכת העיכול ומחלות ועשויים לתרום סימפטומים במערכת העיכול דיווחה בהפרעות נוירופסיכיאטריות כולל אוטיזם. היא מבוססת היטב כי חלקי מעי גס מבודד ליצור התכווצויות ספונטניות, קצבית המכונה גס נודד Motor מכלולים (CMMCs). הליך לנתח את התקנה העצבית המעיים של CMMCs ב לשעבר הכנות vivo של מעי גס עכבר מתוארות. המעי הגס הוא גזור מן החיה והדיח להסיר תוכן צואתי בטרם cannulated באמבט איברים. הנתונים נרכשו באמצעות מצלמת וידאו מוצבת מעל באמבטית האיברים ועל מרת מפות spatiotemporal ברזולוציה גבוהה באמצעות חבילת תוכנה ללא צורך במיקור חוץ. באמצעות טכניקה זו, דפוסי התכווצות בסיס ואפקטים תרופתיים על תפקוד ENS במעי גס שלegments ניתן להשוות מעל 3-4 שעות. בנוסף, אורך התפשטות ומהירות CMMCs ניתן להקליט כמו גם משינויים בשיעורי קוטר הבטן ותדירות התכווצות. טכניקה זו שימושית לאפיון דפוסי תנועתיות מערכת העיכול במודלים עכבר מהונדס (וגם מינים אחרים כולל חולדות שרקן). בדרך זו, הנגרמת פרמקולוגית שינויים CMMCs נרשמים בעכברי סוג ברים במודל עכבר R451C Neuroligin-3 של אוטיזם. יתר על כן, טכניקה זו יכולה להיות מיושם על אזורים אחרים של מערכת העיכול כולל התריסריון, מְעִי צָם ואת מעי ובגילאים התפתחותיים שונים בעכברים.

<html> עד 90% מהחולים עם ASD לחוות מערך של הפרעות במערכת העיכול, כולל שלשול ועצירות 18,24,25. עם זאת, הגורמים הבסיסיים של בעיות במערכת עיכול אלה אינם ידועים. מוטציות רבות זיהו בחולים עם ASD משויכים תורמים חלבונים סינפטיים לשינויים והפרעות בשידור סינפטי או...

Watch this Scientific Journal Video about Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice at JoVE.com

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

הדמיה וידאו ומפות Spatiotemporal לנתח תנועתיות מערכת העיכול של עכברים

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central ...

video-imaging-spatiotemporal-maps-to-analyze-gastrointestinal

Research

JoVE Journal

Neuroscience

13.9K Views.  The University of Melbourne. This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

Video: Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

Fabrication and Implantation of Miniature Dual-element Strain Gages for Measuring In Vivo Gastrointestinal Contractions in Rodents.

Gastrointestinal dysfunction remains a major cause of morbidity and mortality. Indeed, gastrointestinal (GI) motility in health and disease remains an area of productive research with over 1,400 published animal studies in just the last 5 years. Numerous techniques have been developed for quantifying smooth muscle activity of the stomach, small intestine, and colon. In vitro and ex vivo techniques offer powerful tools for mechanistic studies of GI function, but outside the context of the integrated systems inherent to an intact organism. Typically, measuring in vivo smooth muscle contractions of the stomach has involved an anesthetized preparation coupled with the introduction of a surgically placed pressure sensor, a static pressure load such as a mildly inflated balloon or by distending the stomach with fluid under barostatically-controlled feedback. Yet many of these approaches present unique disadvantages regarding both the interpretation of results as well as applicability for in vivo use in conscious experimental animal models. The use of dual element strain gages that have been affixed to the serosal surface of the GI tract has offered numerous experimental advantages, which may continue to outweigh the disadvantages. Since these gages are not commercially available, this video presentation provides a detailed, step-by-step guide to the fabrication of the current design of these gages. The strain gage described in this protocol is a design for recording gastric motility in rats. This design has been modified for recording smooth muscle activity along the entire GI tract and requires only subtle variation in the overall fabrication. Representative data from the entire GI tract are included as well as discussion of analysis methods, data interpretation and presentation.

Fabrication and Implantation of Miniature Dual-element Strain Gages for Measuring In Vivo Gastrointestinal Contractions in Rodents.

The in vivo measurement of smooth muscle contractions along the gastrointestinal tract of laboratory animals remains a powerful, though underutilized, technique. Flexible, dual element strain gages are not commercially available and require fabrication. This protocol describes the construction of reliable, inexpensive strain gages for acute or chronic implantation in rodents.

ייצור והשרשה של מדי עיבור Dual-אלמנט הזעיר למדידה<em&gt; בVivo</em&gt; במערכת העיכול התכווצויות במכרסמים.

Gastrointestinal dysfunction remains a major cause of morbidity and mortality. Indeed, gastrointestinal (GI) motility in health and disease remains an ...

Bioengineering

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

הדמיה של Dynamics התאים מיאלואידית בזמן אמת<em&gt; Apc<sup&gt; מינימום / +</sup</em&gt; מעיים גידולים על ידי דיסק מסתובב Confocal מיקרוסקופית

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Immunology and Infection

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

העכברי אנדוסקופיה ל<em&gt; בVivo</em&gt; ריפוי Multimodal הדמיה של היווצרות הסרטן וההערכה של מעיים פצע ודלקת

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Medicine

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

הדמיה נויטרופילים ומונוציטים בmesenteric ורידים על ידי מיקרוסקופית Intravital על עכברים מורדמים בזמן אמת

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

הערכה לא פולשנית של היעילות של ניו Therapeutics למעי פתולוגיות שימוש הדמיה אנדוסקופי סידורי של עכברים חיים

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

Gastrointestinal Motility Monitor (GIMM)

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility in isolated segments of guinea pig distal colon. The complete system consists of a computer, video camera, illuminated organ bath, peristaltic and heated water bath circulating pumps, and custom GIMM software to record and analyze data. Compared with traditional methods of monitoring colonic peristalsis, the GIMM system allows for continuous, quantitative evaluation of motility. The guinea pig distal colon is bathed in warmed, oxygenated Krebs solution, and fecal pellets inserted in the oral end are propelled along the segment of colon at a rate of about 2 mm/sec. Movies of the fecal pellet proceeding along the segment are captured, and the GIMM software can be used track the progress of the fecal pellet. Rates of propulsive motility can be obtained for the entire segment or for any particular region of interest. In addition to analysis of bolus-induced motility patterns, spatiotemporal maps can be constructed from captured video segments to assess spontaneous motor activity patterns. Applications of this system include pharmacological evaluation of the effects of receptor agonists and antagonists on propulsive motility, as well as assessment of changes that result from pathophysiological conditions, such as inflammation or stress. The guinea pig distal colon propulsive motility assay, using the GIMM system, is straightforward and simple to learn, and it provides a reliable and reproducible method of assessing propulsive motility.

Gastrointestinal Motility Monitor GIMM

Evaluation of colonic motility in the guinea pig distal colon with the Gastrointestinal Motility Monitor (GIMM) is a straightforward and simple to learn approach to quantitatively evaluate propulsive motility in the gastrointestinal tract.

צג תנועתיות מערכת העיכול (GIMM)

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

Biology

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1. Different subtypes of ICC form distinct networks in the muscularis of the GI tract 2,3. Loss or injury to these networks is associated with a number of motility disorders4. ICC cells express the KIT receptor tyrosine kinase on the plasma membrane and KIT immunostaining has been used for the past 15 years to label the ICC network5,6. Importantly, normal KIT activity is required for ICC development5,6. Neoplastic transformation of ICC cells results in gastrointestinal stromal tumor (GIST), that frequently harbor gain-of-function KIT mutations7,8. We recently showed that ETV1 is a lineage-specific survival factor expressed in the ICC/GIST lineage and is a master transcriptional regulator required for both normal ICC network formation and for of GIST tumorigenesis9. We further demonstrate that it cooperates with activating KIT mutations in tumorigenesis. Here, we describe methods for visualization of ICC networks in mice, largely based on previously published protocols10,11. More recently, the chloride channel anoctamin 1 (ANO1) has also been characterized as a specific membrane marker of ICC11,12. Because of their plasma membrane localization, immunofluorescence of both proteins can be used to visualize the ICC networks. Here, we describe visualization of the ICC networks by fixed-frozen cyrosections and whole mount preparations.

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

ויזואליזציה של תאים ביניים של רשת (ICC) קחאל ב עכברים

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

אינטרטל הדמיה של לימפוציטים תוך-אפיתל במעי קטן מוריין

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

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.

מיפוי Spatiotemporal של תנועתיות ב<em&gt; Ex Vivo</em&gt; תכשירים מהמעיים

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

Studying Murine Small Bowel Mechanosensing of Luminal Particulates

Gastrointestinal (GI) motility is critical for normal digestion and absorption. In the small bowel, which absorbs nutrients, motility optimizes digestion and absorption. For this reason, some of the motility patterns in the small bowel include segmentation for mixing of luminal contents and peristalsis for their propulsion. Physical properties of luminal contents modulate the patterns of small bowel motility. The mechanical stimulation of GI mechanosensory circuits by transiting luminal contents and underlying gut motility initiate and modulate complex GI motor patterns. Yet, the mechanosensory mechanisms that drive this process remain poorly understood. This is primarily due to a lack of tools to dissect how the small bowel handles materials of different physical properties. To study how the small bowel handles particulates of varying sizes, we have modified an established in vivo method to determine small bowel transit. We gavage live mice with fluorescent liquid or tiny fluorescent beads. After 30 minutes, we dissect out the bowels to image the distribution of fluorescent contents across the entirety of the GI tract. In addition to high-resolution measurements of the geometric center, we use variable size binning and spectral analysis to determine how different materials affect small bowel transit. We have explored how a recently discovered "gut touch" mechanism affects small bowel motility using this approach.

To study how the small bowel handles particulates of varying sizes, we have modified an established in vivo method to determine small bowel transit.

חקר מורין מעיים קטנים מכנוסנס של חלקיקים לומינליים

Gastrointestinal (GI) motility is critical for normal digestion and absorption. In the small bowel, which absorbs nutrients, motility optimizes digestion ...

הדמיה וידאו ומפות Spatiotemporal לנתח תנועתיות מערכת העיכול של עכברים

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ייצור והשרשה של מדי עיבור Dual-אלמנט הזעיר למדידה

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