Name: a simple mechanical procedure to create limbal stem cell deficiency in mouse
Uploaded: 2016-11-17T13:00:00
Description: Watch this Scientific Journal Video about A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse at JoVE.com

Forfatterne takker Ruth Zelkha, MS, for hendes generøse bistand i billeddannelse. Denne forskning blev støttet af Clinical Scientist Development Program Award K12EY021475 til ME, give R01 EY024349-01A1 til ARD, core tilskud EY01792 fra National Eye Institute, NIH, og en ubegrænset bevilling fra Research for at forebygge blindhed. ARD er modtageren af ​​en Career Development Award fra Research for at forebygge blindhed.

Alle procedurer udføres på dyrene opfylder Foreningen for Forskning i Vision og Oftalmologi erklæringer til dyr brug i synet forskning. Fjorten fire til seks måneder gamle mandlige / kvindelige C57BL / 6J vildtypemus blev anvendt: ti mus for skaden model og fire som kontroldyr (læderet hornhinder). 1. Dyrepræparation <ol><li> Bedøver en mus med en intraperitoneal injektion af en 100 mg / kg ketamin og 5 mg / kg xylazin-blanding. Efter bedøvelsen har fået virkning, klemmes tå for ...

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Denne artikel beskriver en reproducerbar og forholdsvis simpel teknik til at skabe en musemodel for LSCD. Der er flere vigtige aspekter af denne model, der er værd at bemærke. Først, i modsætning modeller, der bruger kemikalier 11, 12, skaden involverer primært overfladen epitel (og basalmembran), med minimal skade på det underliggende corneale stroma eller andre intraokulære strukturer. Således er der kun begrænset betændelse og ardannelse, som begge kan komplicere proceduren og gøre det vanskelig...

Hornhindens epitel er nødvendig for at opretholde klarhed og integritet af hornhinden. Det er hele tiden fornyes gennem hele livet af de epiteliale stamceller bosiddende i limbus-en smal zone ved krydset af hornhinden og bindehinden. Disse selv-fornyende limbale stamceller spiller en afgørende rolle i at regenerere hornhindeepitelet, både normalt og efter skaden. Delvis eller fuldstændig udtømning af disse stamceller vil resultere i tilbagevendende hornhinde erosioner, smerter, hornhinde ardannelse og neovaskularisering, udseende slimceller, og hvis venstre ubehandlet, hornhinde blindhed. Denne betingelse er kendt som limbal stamcelle-mangel (LSCD) og kan være idiopatisk; arvelig; eller erhvervet som følge af kemiske eller termiske skader, langsigtede kontaktlinser slid, og kronisk inflammation 1-6. Forskning i LSCD kræver en passende dyremodel, at ikke kun efterligner sygdom hos mennesker, men er reproducerbar og bæredygtig, med mindst enmount af skade på andre hornhinde og okulære strukturer. Denne model er nødvendigt at vurdere behandlinger, og at klarlægge de sygdomsmekanismer på molekylære og cellulære niveauer. Som beskrevet før 1, med anvendelse af en roterende Burr, kan man let udvikle en musemodel for LSCD der indeholder de førnævnte fordele og vedvarer i mindst tre måneder. Målet med denne undersøgelse er at præsentere en enkel, reproducerbar, og bæredygtig musemodel af LSCD. Den roterende Burr er et praktisk værktøj, der jævnt fjerner epitel uden at skade den underliggende stroma en. Det er blevet anvendt til at inducere centrale hornhinde erosion 7, 8 ved sårheling undersøgelser. Den hermed præsenteret teknik til at skabe LSCD i en mus er ikke blevet rapporteret før. Tidligere indførte fremgangsmåder til skrabning epithelet med en stump spatel resultat i en mindre ensartet skade-især ved limbus-og en mere variabel fænotype 1, 9, med mere restaurering of normal hornhindeepitel 1. I modsætning til den stumpe skraber, den roterende grat fjerner epitelbasalmembranen samt 7, 8, 10. Andre rapporterede fremgangsmåder til at udsondre stamceller indebærer brug af kemikalier, såsom natriumhydroxid, n-heptanol, og benzalkoniumchlorid, som ikke kun kan inducere uønsket skade på underliggende øjet strukturer, men også kan føre til betydelig inflammation og efterfølgende corneal uigennemsigtighed eller epithelial squamous metaplasi 11-15. Den roterende burr er ikke forbundet med disse alvorlige komplikationer. Kirurgisk fjernelse af limbale epitel, alene eller med anvendelsen af kemikalier 10, 11, 16, er mere vanskelig at udføre og er ikke den bedste løsning i dyr med små øjne og en tynd limbal epitel (som mus). Desuden overraskende limbectomy kan stadig lade nogle af de limbale epitel bag 10. Den nedenfor beskrevne teknik vil resultere i LSCD med neovaskularisering end conjunctivalization der efterligner præsentationen af komplette LSCD hos patienter og varer i mindst tre måneder 1. Den er velegnet til dem, der har til formål at studere LSCD eller sårheling patofysiologi, immunologi, og potentielle behandlinger i mus. Muligvis, med nogle ændringer, denne procedure kan udføres på rotter eller kaniner 10. Som det roterende burr effektivt fjerner epithelet, kan det anvendes i en hvilken som helst forskning vedrørende hornhindeepithelets abrasion / sårdannelse og i enhver tilknyttet behandling eller molekylærbiologiske undersøgelser.

<table border="0" cellpadding="0" cellspacing="0" width="1091">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;Rotating burr: AlgerBrush II rust ring remover&#160;</td>
			<td style="width:277px;">Rumex International Co, Clearwater, FL</td>
			<td style="width:144px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 16-141</td>
			<td style="width:351px;">0.5 mm burr. Also available from other companies.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;Surgical microscope</td>
			<td style="width:277px;">Wild Heerbrugg, Switzerland</td>
			<td style="width:144px;">&#160;&#160;&#160;&#160;&#160;&#160; Wild M691</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;Digital camera</td>
			<td style="width:277px;">Nikon, Thailand</td>
			<td style="width:144px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;Nikon FS-2 slit lamp</td>
			<td style="width:277px;">Nikon, Japan</td>
			<td style="width:144px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;Polyclonal anti-CK12 antibody</td>
			<td style="width:277px;">Santa Cruz Blotechnology, CA,</td>
			<td style="width:144px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160; SC-17101</td>
			<td>1:100 concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;Monoclonal anti-CK8 antibody</td>
			<td style="width:277px;">TROMA-I-s, Iowa City, IA</td>
			<td style="width:144px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160; AB_531826</td>
			<td>1:50 concentration</td>
		</tr>
	</tbody>
</table>

a simple mechanical procedure to create limbal stem cell deficiency in mouse

Limbal stamcelle-mangel (LSCD) er en tilstand af fejlfunktion eller tab af limbale epiteliale stamceller, hvorefter hornhindens epitel er erstattet med bindehinde. Patienterne lider af tilbagevendende hornhindedefekter, smerte, inflammation og tab af synet. Tidligere blev en murin model for LSCD beskrevet og sammenlignet med to andre modeller. Målet var at producere en konsistent musemodel for LSCD at begge efterligner fænotype hos mennesker og varer længe nok til at gøre det muligt at undersøge sygdommens patofysiologi og evaluere nye behandlinger. Her er teknikken beskrevet mere detaljeret. En motoriseret værktøj med en roterende grat er designet til at fjerne rust ringe fra hornhindens overflade eller til at udjævne pterygium seng i patienter. Det er en egnet anordning til at skabe den ønskede LSCD model. Det er en let tilgængelig, let at bruge værktøj med en fin spids, der gør det egnet til at arbejde på små øjne, somi mus. Dens anvendelse forhindrer unødige trauma for øjet og det resulterer ikke i uønskede skader, som det ofte er tilfældet med kemisk skade modeller. I modsætning til en stump skraber, det fjerner epithelet med basalmembranen. I denne protokol, blev limbale område slibes to gange, og så er hele hornhindens epitel blev barberet fra limbus til limbus. For at undgå stroma skade, blev der draget omsorg for ikke at børste hornhindeoverfladen når epithelet allerede var fjernet.

Inden for en måned efter at have udført denne teknik, 100% af hornhinderne udviklede overfladisk neovaskularisering (figur 1A. Billeder blev taget med et kamera forbundet til en spaltelampe under et lyst felt og kobolt blåt filter). I 18/20 (90%) af øjnene, neovaskularisering involverede hele hornhindens overflade (figur 1A). I 2/20 (10%) blev neovaskularisering observeret i tre ud af fire hornhinde kvadranter, men det stadig nået hornhindens centru...

Watch this Scientific Journal Video about A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse at JoVE.com

A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse

Følgende artikel giver en nem, reproducerbar teknik til effektivt at skabe et bæredygtigt musemodel af limbal stamcelle-mangel (LSCD). Denne dyremodel er nyttig til at teste og sammenligne effektiviteten af ​​behandlinger for limbale stamcellelinjer sygdomme.

En simpel mekanisk fremgangsmåde til at oprette limbal Stem Cell Mangel i Mus

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with ...

The overall goal of this procedure is to create a reproducible, sustainable, and persistent mouse model of limbal stem cell deficiency. This simple technique provides a consistent animal model of limbal stem cell deficiency, without inducing unwanted injury to the eye, which is difficult to achieve with the standard chemical injury models. This model is useful for studying the disease process in long-term at cellular and molecular level.And for testing or comparing the efficacy of novel treatments. After anesthetizing the mouse, pinch the toe to ensure adequate depth of anesthesia. Inspect the eye under a slit lamp to verify the absence of any previous corneal injury.Instill a drop of 1%tetracaine hydrochloride onto the eye, and wait for 60 seconds. Next, gently apply 5%povidone iodine on the eye and the hair around it. After one minute, wipe off the drop or spread it onto the hair around the eye to prevent the hair from interfering with the tip of the tool.Prepare a sterile rotating burr and a pair of sterile bent tweezers, and keep them on a sterile sheet during the procedure. Now, stabilize the eye by grabbing the lids from the nasal side using the bent tweezers, and gently proctozing the eye. Be careful not to apply too much pressure.Under a surgical microscope, shave around the limbal area twice using the sterile rotating burr. Remove the whole corneal epithelium from limbus to limbus with the rotating burr in a circular fashion, and work with the lateral side of its tip. To avoid stroma injury, do not re-brush corneal parts from which the epithelium has been removed.Do not apply any pressure on the eye. Clean the brush tip as required. Apply a drop of sterile PBS on the cornea, and wipe away any detached epithelial sheets with a soft cleaning tissue.Then, shave the remaining epithelium, if any. Pay attention to the mouse tail to ensure adequate depth of anesthesia during the surgery. Apply a drop of Flourescein sodium onto the cornea.Clean the cornea after 30 seconds, and examine it under a microscope with a cobalt blue filter to verify the complete removal of the epithelium. After the procedure, place the animal in its cage on a heating blanket and observe it for 30-45 minutes until complete recovery from anesthesia. In the meantime, apply 1%Erythromycin Ophthalmic ointment to the eye and continue the application of the antibiotic daily for a week.In addition, inject a 0.1mg/kg of Buprenorphine subcutaneously immediately, and 12 hours after the procedure, and continue the injection twice a day for two days. This is the cornea phenotype one month after the procedure. You can see extensive neovascularization which is a feature of limbal stem cell deficiency.A normal cornea expresses Cytokeratin 12 but is devoid of Cytokeratin 8 or goblet cells. In limbal stem cell deficiency, Cytokeratin 8 and goblet cells appear on the cornea, while Cytokeratin 12 is lost. Once mastered, this technique will result in corneal neovascularization that will cover almost the entire mouse corneal surface in one month, and is stable up to three months.It is important to avoid re-brushing the corneal parts that the epithelium has already been removed. This can cause undesired stroma inflammation and opacification. Following development of limbal stem cell deficiency, experimental treatment methods can be tested and compared.Alternatively, treatments can be started shortly after the injury. This video displayed how to effectively remove the limbal and corneal epithelium to induce limbal stem cell deficiency in mouse. With some modifications, it can be performed on other animals as well.

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