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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Immunology and Infection

Induktion af øjenoverfladebetændelse og indsamling af involverede væv

Published: August 4th, 2022

DOI:

10.3791/63890

1Department of Internal Medicine 3-Rheumatology and Immunology, Friedrich-Alexander-University Erlangen-Nürnberg and Universitätsklinikum Erlangen, 2Deutsches Zentrum für Immuntherapie, Friedrich-Alexander-University Erlangen-Nürnberg and Universitätsklinikum Erlangen

Okulær overfladebetændelse skader det okulære overfladevæv og kompromitterer vitale funktioner i øjet. Den nuværende protokol beskriver en metode til at inducere okulær betændelse og indsamle kompromitterede væv i en musemodel af Meibomian kirtel dysfunktion (MGD).

Okulære overfladesygdomme omfatter en række lidelser, der forstyrrer hornhindens, bindehindens funktioner og strukturer og det tilhørende okulære overfladekirtelnetværk. Meibomiakirtler (MG) udskiller lipider, der skaber et dæklag, der forhindrer fordampning af den vandige del af tårefilmen. Neutrofiler og ekstracellulære DNA-fælder befolker MG og den okulære overflade i en musemodel af allergisk øjensygdom. Aggregerede neutrofile ekstracellulære fælder (aggNET'er) formulerer en mesh-lignende matrix sammensat af ekstracellulært kromatin, der okkluderer MG-udløb og betingelser MG-dysfunktion. Her præsenteres en metode til inducering af okulær overfladebetændelse og MG-dysfunktion. Procedurerne for indsamling af organer relateret til den okulære overflade, såsom hornhinden, bindehinden og øjenlågene, er beskrevet detaljeret. Ved hjælp af etablerede teknikker til behandling af hvert organ vises også de vigtigste morfologiske og histopatologiske træk ved MG-dysfunktion. Okulære ekssudater giver mulighed for at vurdere den okulære overflades inflammatoriske tilstand. Disse procedurer muliggør undersøgelse af topiske og systemiske antiinflammatoriske interventioner på præklinisk niveau.

Hvert blink med øjet genopfylder den glatte tårefilm spredt over hornhinden. Den okulære overfladeepitel letter fordelingen og korrekt orientering af tårefilmen på den okulære overflade. Muciner leveres af hornhinden og bindehindeepitelcellerne for at hjælpe med at placere den vandige del af tårefilmen, der kommer fra lacrimalkirtlerne på øjnenes overflade. Endelig udskiller MG lipider, der skaber et dækkende lag, der forhindrer fordampning af den vandige del af tårefilmen 1,2,3. På denne måde beskytter de koordinerede funktioner i alle de okulære organer den okulære overflad....

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Alle procedurer, der involverede dyr, blev udført i henhold til de institutionelle retningslinjer for dyrevelfærd og godkendt af dyrevelfærdskommissionen ved Friedrich-Alexander-University Erlangen-Nürnberg (FAU) (tilladelsesnummer: 55.2.2-2532-2-1217). Hunmus med C57Bl/6 i alderen 7-9 uger blev anvendt til denne undersøgelse. Musene blev hentet fra kommercielle kilder (se Materialetabel) og holdt under specifikke patogenfrie forhold med 12 timers dag/nat-cyklusser.

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Den nuværende protokol beskriver de sekventielle trin til etablering af en murinmodel af okulær overfladebetændelse. Protokollerne sigter mod at vise, hvordan man anvender terapi lokalt, får okulære ekssudater og punktafgiftsrelaterede tilbehørsorganer såsom sunde og betændte øjenlåg (figur 2), hornhinden og bindehinden. Der skal tages hensyn, når de øvre øjenlåg dissekeres til isolering af bindehinden, og den skal opbevares i 1x PBS under dissektion af hornhinden. Dette forhin.......

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Den olieagtige sekretion af de meibomiske kirtler er af stor betydning for et sundt øje22. Imidlertid kan obstruktionen af disse talgkirtler ved aggregerede neutrofile ekstracellulære fælder (aggNET'er), der stiller op som parallelle tråde placeret på tarsalpladerne på begge øjenlåg, forstyrre tårefilmen23. Denne forstyrrelse resulterer i Meibomian kirtel dysfunktion (MGD)1 og accelereret tårefordampning og betingelser skaden på den okulæ.......

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Dette arbejde blev delvist støttet af den tyske forskningsfond (DFG) 2886 PANDORA-projekt-nr. B3; SCHA 2040/1-1; MU 4240/2-1; CRC1181(C03); TRR241(B04), H2020-FETOPEN-2018-2020 Projekt 861878 og af Volkswagen-Stiftung (Grant 97744) til MH.

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NameCompanyCatalog NumberComments
1x PBSGibco
Aluminium HydroxideImject alum Adjuvant7716140 mg/ mL
Final Concentration: in vivo: 1 mg/ 100 µL
C57Bl/6 mice, aged 7–9 weeksCharles River Laboratories 
CalciumCarl rothCN93.11 M
Final Concentration: 5 mM
Curved forcepsFST by Dumont SWITZERLAND5/45 11251-35
Fine sharp scissorFST Stainless steel, Germany15001-08
Laminar safety cabinetHerasafe
Macrophotography CameraCanonEOS6D
Macrophotography Camera (without IR filter)NikonD5300
MnaseNew England biolabsM0247S2 x 106 gel U/mL
Multi-analyte flow assay kit (Custom mouse 13-plex panel)BiolegendCLPX-200421AM-UERLAN
NaCl 0,9% (Saline)B.Braun
Ovalbumin (OVA)Endofit, Invivogen9006-59-110 mg/200 µL in saline
Pertussis toxin ThermoFisher Scientific PHZ117450 µg/ 500 µL in saline
Final Concentration: in vivo: 100 µg/ 100 µL
PetridishGreiner bio-one628160
ScalpelFeather disposable scalpelNo. 21 Final Concentration: in vivo:  300 ng/ 100 µL
StereomicroscopeZaissStemi508
Syringe (corneal/iris washing)BD Microlane27 G x 3/4 - Nr.20 0,4 x 19 mm
Syringe (i.p immunization)BD Microlane24 G1"-Nr 17, 055* 25 mm

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