Name: analyzing beneficial effects of nutritional supplements on intestinal epithelial barrier functions during experimental colitis
Uploaded: 2017-01-05T15:00:00
Description: Watch this Scientific Journal Video about Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis at JoVE.com

Dette arbejde blev støttet af tilskud fra den mexicanske Råd for Videnskab og Teknologi (CONACYT, 207.268 og 233.395 til Michael Schnoor). KFCO er en modtager af en CONACYT stipendium (396.260) for at opnå en MSc grad.

Alle eksperimenter dyr er blevet godkendt af Institutional Animal Care og brug Udvalg Cinvestav. 1. Udarbejdelse af DSS drikkevand og Induktion af Colitis <ol><li> Der fremstilles en 3,5% w / v dextransulfat-natrium (DSS) opløsning i autoklaveret drikkevand. DSS opløser nemt, og det er ikke nødvendigt at filtrere den endelige løsning. BEMÆRK: 7 dage DSS behandling inducerer svær colitis. Hvis inducere milde colitis, 3 - er 4 dages behandling anbefales. Så længe DSS drikkevand forbliver klar, er ...

<ol>
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For at kunne bruge kosttilskud i kliniske studier, skal fordele og sikkerheden omkring disse tilskud evalueres nøje in vivo i dyreforsøg. I tilfælde af colitis, har flere egnede dyremodeller, der ligner de kliniske tegn på IBD blevet etableret, herunder kemiske modeller med DSS, TNBS eller eddikesyre; knock-out (KO) modeller som IL10-KO; og immun-cellemedieret colitis under anvendelse adoptiv T-celle overdragelse 19-21. The DSS model af colitis er en hurtig og pålidelig metode til at inducere co...

Colitis er en inflammatorisk tilstand i tyktarmen som kan forårsage diarré og mavesmerter. Colitis kan være akut som reaktion på infektion eller stress, eller det kan udvikle sig som en kronisk sygdom, såsom i ulcerativ colitis (UC), der tilhører gruppen af ​​inflammatoriske tarmsygdomme (IBD). Skønt de kliniske tegn på UC er blevet godt beskrevet, er patogenesen stadig dårligt forstået 1. Det accepteres blandt eksperter, at UC er en multifaktoriel sygdom, med genetiske mutationer og afvigende immunreaktioner spiller en stor rolle 2. Men miljømæssige faktorer, såsom livsstil og ernæring, også bidrager til sygdommens udvikling og progression 3. Desværre, IBD er ikke helbredes, men der er mange behandlingsmuligheder, der har til formål at lindre kliniske symptomer. Nuværende behandlinger omfatter anti-inflammatoriske lægemidler, såsom sulfasalazin og kortikosteroider; immunosuppressiva, såsom azathioPrine; monoklonale antistoffer som fanger proinflammatoriske cytokiner, såsom tumornekrosefaktor-α (TNF-α), eller blokere adhæsionsmolekyler, såsom integriner, begrænse overdrevent leukocytrekruttering; og inhibitorer, der er målrettet kinaser, der udløser proinflammatoriske veje, såsom Janus kinase (JAK) 4. Ikke alle patienter reagerer på alle behandlinger, så terapeutiske strategier skal individualiseres 5. Endvidere er de fleste af disse terapeutiske lægemidler interfererer med metabolismen og immunresponser, ofte forårsager alvorlige bivirkninger. Af denne grund har alternative behandlingsmuligheder blevet undersøgt. Alternative behandlinger omfatter probiotika og kosttilskud, der er anvendt i dyremodeller og kliniske forsøg med varierende grad af succes 6,7. Vi har for nylig fundet, at anvendelsen af ​​forskellige enkelt kosttilskud, såsom anti-oxidative vitaminer eller ω3-poly-umættede fedtsyrer (PUFAs), er ringere end anvendelsen af en kombination af sådanne kosttilskud til lindring colitis og hjerte-kar-sygdom symptomer 8,9. Disse undersøgelser blev udført på mus, så kliniske undersøgelser skal udføres på mennesker for at afgøre, om resultaterne også vil være gældende for mennesker. Før kliniske studier igangsættes, skal effektiviteten og sikkerheden af ​​nye behandlingsmuligheder vurderes i dyremodeller. For IBD, har dextransulfat-natrium (DSS) model er ofte blevet brugt til at undersøge mekanismerne for sygdomsudvikling og de gavnlige virkninger af lægemidler og kosttilskud 6,10. I de fleste undersøgelser, kun en akut sygdom i løbet af en periode på syv dage er induceret; ikke desto mindre, de kliniske tegn observeret i disse dyr ligner dem observeret i IBD-patienter (dvs. blodig diarré, vægttab, epithelial dysfunktion og immun celleinfiltration) 10. DSS inducerer erosioner i slimhinden,resulterer i barriere dysfunktion og øget intestinal epithelial permeabilitet 11. Den nøjagtige mekanisme stadig ukendt. En undersøgelse tyder på, at DSS interagerer med medium kædelængde fedtsyrer til dannelse nano-lipocomplexes der er i stand til at indtaste epitelceller og fremkalde inflammatoriske signalveje 12. I denne artikel beskriver vi i detaljer, hvordan colitis induceres og analyseret i mus, hvor kosttilskud der anvendes ved tvangsfodring at sikre en konstant dosering i hvert dyr, og hvordan virkningerne af sådanne kosttilskud på forskellige colitis symptomer er undersøgt.

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			<td height="21" style="height:21px;width:439px;">0.3% of gelatin&#160;</td>
			<td style="width:184px;">Bioxon</td>
			<td style="width:139px;">158</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">10% formaldehyde</td>
			<td style="width:184px;">J.T. Baker</td>
			<td style="width:139px;">2106-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">96-well plate with flat bottom</td>
			<td style="width:184px;">Corning</td>
			<td style="width:139px;">3368</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Absolute ethanol&#160;</td>
			<td style="width:184px;">J.T. Baker</td>
			<td style="width:139px;">9000-03</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Absolute xylene</td>
			<td style="width:184px;">J.T. Baker</td>
			<td style="width:139px;">9490-03</td>
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			<td height="21" style="height:21px;width:439px;">ABTS</td>
			<td style="width:184px;">Sigma Aldrich</td>
			<td style="width:139px;">H5882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Bovine Serum Albumin</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:139px;">9048-46-8</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">ColoScreen</td>
			<td style="width:184px;">Helena Laboratories</td>
			<td style="width:139px;">5072</td>
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			<td height="21" style="height:21px;width:439px;">Corabion (Kindly provided by Merck, Naucalpan, Mexico)</td>
			<td style="width:184px;">Merck</td>
			<td style="width:139px;"></td>
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			<td height="21" style="height:21px;width:439px;">Dextran Sulfate Sodium Salt</td>
			<td style="width:184px;">Affymetrix</td>
			<td style="width:139px;">9011-18-1</td>
			<td style="width:191px;">(M.W. 40,000-50,000)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Dihydroethidium</td>
			<td style="width:184px;">Life Technology</td>
			<td style="width:139px;">D11347</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Eosin-Y&#160;</td>
			<td style="width:184px;">J.T. Baker</td>
			<td style="width:139px;">L087-03</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Evans blue</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:139px;">314-13-6</td>
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		</tr>
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			<td height="21" style="height:21px;width:439px;">Feeding&#160; needle</td>
			<td style="width:184px;">Cadence Science Inc.</td>
			<td style="width:139px;">9921</td>
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			<td height="21" style="height:21px;width:439px;">Glass slide rack with handle</td>
			<td style="width:184px;">Electron Microscopy</td>
			<td style="width:139px;">70312-16</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Glass Slides</td>
			<td style="width:184px;">Corning</td>
			<td style="width:139px;">2947</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Harris hematoxylin&#160;</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:139px;">H3136</td>
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			<td height="21" style="height:21px;width:439px;">Hexadecyltrimethyammonium (HTAB)</td>
			<td style="width:184px;">Sigma Aldrich</td>
			<td style="width:139px;">H6269</td>
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			<td height="21" style="height:21px;width:439px;">Histosette (Embedding cassette)</td>
			<td style="width:184px;">Simport</td>
			<td style="width:139px;">M498.2</td>
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			<td height="21" style="height:21px;width:439px;">Hydrochloric acid 1 M</td>
			<td style="width:184px;">J.T. Baker</td>
			<td style="width:139px;">9535-62</td>
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			<td height="21" style="height:21px;width:439px;">Hydrogen peroxide</td>
			<td style="width:184px;">Sigma Aldrich</td>
			<td style="width:139px;">H3410</td>
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			<td height="21" style="height:21px;width:439px;">Ketamine</td>
			<td style="width:184px;">PiSA Agropecuaria</td>
			<td style="width:139px;">Q-7833-028</td>
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			<td height="21" style="height:21px;width:439px;">Liquid paraffin&#160;</td>
			<td style="width:184px;">Paraplast</td>
			<td style="width:139px;">39501-006</td>
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			<td height="21" style="height:21px;width:439px;">Lithium carbonate&#160;</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:139px;">2362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">N,N-dimethylformamide</td>
			<td style="width:184px;">J.T. Baker</td>
			<td style="width:139px;">68-12-2</td>
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			<td height="21" style="height:21px;width:439px;">N-acetylcysteine</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:139px;">A9165</td>
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			<td height="21" style="height:21px;width:439px;">Plastic cubes</td>
			<td style="width:184px;">Electron Microscopy</td>
			<td style="width:139px;">70181</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Poly-L-Lysine Solution</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:139px;">25988-63-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Prism 5 statistical software</td>
			<td style="width:184px;">GraphPad Software</td>
			<td style="width:139px;">Prism 5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Saline Solution 0.9% NaCl</td>
			<td style="width:184px;">CS PiSA</td>
			<td style="width:139px;">Q-7833-009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Sodium citrate</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:139px;">W302600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Synthetic resin&#160;</td>
			<td style="width:184px;">Poly Mont</td>
			<td style="width:139px;">7987</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Taq DNA polymerase</td>
			<td style="width:184px;">Invitrogen</td>
			<td style="width:139px;">11615-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Tissue-tek. O.C.T Compound</td>
			<td style="width:184px;">Sakura Finetek</td>
			<td style="width:139px;">4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Tuberculine Syringe</td>
			<td style="width:184px;">BD Plastipak</td>
			<td style="width:139px;">305945</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Tween 20</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:139px;">9005-64-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">VECTASHIELD Antifade Mounting Medium with DAPI</td>
			<td style="width:184px;">Vector Laboratories</td>
			<td style="width:139px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Xylazine</td>
			<td style="width:184px;">PiSA Agropecuaria</td>
			<td style="width:139px;">Q-7833-099</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing beneficial effects of nutritional supplements on intestinal epithelial barrier functions during experimental colitis

Inflammatoriske tarmsygdomme (IBD), herunder Crohns sygdom og ulcerativ colitis, er kronisk tilbagevendende sygdomme i tarmene. De forårsager alvorlige problemer, såsom mavekramper, blodig diarré og vægttab, i berørte individer. Desværre er der ingen kur endnu, og behandlinger kun til formål at lindre symptomerne. Aktuelle behandlinger omfatter anti-inflammatoriske og immunosuppressive lægemidler, der kan forårsage alvorlige bivirkninger. Det forudsætter en søgen efter alternative behandlingsmuligheder, såsom kosttilskud, som ikke forårsager bivirkninger. Før deres anvendelse i kliniske studier, skal sådanne forbindelser være grundigt testet for effektivitet og sikkerhed i dyremodeller. En pålidelig eksperimentel model er den dextransulfat-natrium (DSS) colitis model i mus, som gengiver mange af de kliniske tegn på colitis ulcerosa i mennesker. Vi har for nylig anvendt denne model til at teste de gavnlige virkninger af et kosttilskud indeholdendevitamin C og E, L-arginin, og w3-polyumættede fedtsyrer (PUFA). Vi analyserede forskellige sygdomstilstande parametre og fandt, at dette supplement kunne lindre ødemdannelse, vævsbeskadigelse, leukocytinfiltration, oxidativ stress, og produktionen af ​​pro-inflammatoriske cytokiner, der fører til en generel forbedring af sygdomsaktiviteten indeks. I denne artikel, vi forklare i detaljer den korrekte anvendelse af kosttilskud ved hjælp af DSS colitis modellen i C57BL / 6 mus, samt hvordan sygdom parametre såsom histologi, oxidativ stress, og inflammation vurderes. Analysere de gavnlige virkninger af forskellige kosttilskud kan så i sidste ende åbne nye muligheder for udvikling af alternative behandlingsstrategier, der lindrer IBD symptomer og / eller at forlænge faser remission uden at forårsage alvorlige bivirkninger.

Kost Kosttilskud kan beskytte fra DSS Colitis Anti-inflammatoriske og anti-oxidative kosttilskud, såsom vitamin C, vitamin E, nitrogenoxid (NO)-source L-arginin og w3-polyumættede fedtsyrer (w3-PUFA), er blevet anvendt med varierende succes i dyr modeller til at lindre symptomer på inflammatoriske sygdomme 3,6,15. Fejlernæring, oxidativ stress, og produktionen af proinflammatoriske cytokiner kan udløse både akutt...

Watch this Scientific Journal Video about Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis at JoVE.com

Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis

Nuværende behandlinger af inflammatoriske tarmsygdomme (IBD) sigter mod at afhjælpe sygdomssymptomer, men kan forårsage alvorlige bivirkninger. Således er alternative strategier, der undersøges i animalske colitis modeller. Her forklarer vi, hvordan de gavnlige virkninger af kosttilskud på kliniske IBD tegn analyseres i et sådant colitis model.

Analyse Gavnlige virkninger af kosttilskud på Intestinal epitelbarriere funktioner under Eksperimentel Colitis

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause ...

Research

JoVE Journal

Medicine

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated ...

DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

DNBS / TNBS Colitis modeller: giver indsigt i inflammatorisk tarmsygdom og Effekter af kosten fedt

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently ...

Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs

Langsigtet kateterisation af Intestinal lymfeknuder Trunk og Indsamling af lymfeknuder i neonatale Pigs

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

En trin for trin Protokol for subretinal kirurgi i kaniner

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing

Multimodal Kvantitativ Phase Imaging med Digital Holografisk Microscopy Præcist Vurderer tarmbetændelse og Epithelial Sårheling

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The ...

Effects of Allogeneic Platelet-Rich Plasma (PRP) on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

Effects of Allogeneic Platelet-Rich Plasma PRP on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

Virkningerne af allogene trombocyt-Rich Plasma (PRP) på den helbredende proces af sektioneret Achilles sener af rotter: en metodologisk beskrivelse

This article describes the experimental procedures used to observe if PRP can positively affect tendon healing. There are 4 main steps to follow: induce a ...

Ultrasonography in Experimental Reproductive Investigations on Rats

Ultralyd i eksperimentel reproduktive undersøgelser på rotter

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

Forbedret metode til etablering af et In Vitro blod - hjerne barrieren Model baseret på svin hjernen Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

Mikroskopi baseret metoder til vurdering af epitelcelle Migration under In Vitro sårheling

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

En In Vivo metode til evaluering af Gut-blod barriere og lever metabolisme af mikrobiota produkter

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity ...