Standardiseret histomorforisk evaluering af slidgigt i en kirurgisk musemodel
Summary
Den nuværende protokol indfører en streng og reproducerbar metode til kvantificering af morfologiske fælles ændringer, der ledsager slidgigt. Anvendelse af denne protokol kan være værdifuld i overvågning af sygdomsprogression og evaluering terapeutiske indgreb i slidgigt.
Abstract
En af de mest udbredte ledsygdomme i USA, slidgigt (OA) er karakteriseret ved progressiv degeneration af ledbrusk, primært i hofte-og knæled, hvilket resulterer i betydelige konsekvenser for patientens mobilitet og livskvalitet. Til dato er der ingen eksisterende helbredende behandlinger for OA i stand til at bremse eller hæmme brusk degeneration. I øjeblikket er der en omfattende mængde af igangværende forskning for at forstå OA patologi og opdage nye terapeutiske tilgange eller agenter, der effektivt kan bremse, stoppe, eller endda vende OA. Det er således afgørende at have en kvantitativ og reproducerbar tilgang til præcist at evaluere OA-relaterede patologiske ændringer i den fælles brusk, synovium og subchondral knogle. I øjeblikket, OA sværhedsgrad og progression er primært vurderes ved hjælp af Slidgigt Research Society International (OARSI) eller Mankin scoring systemer. På trods af betydningen af disse scoringssystemer er de semikvantitative og kan påvirkes af brugerens subjektivitet. Endnu vigtigere, de undlader at præcist at vurdere subtile, men vigtigt, ændringer i brusk i de tidlige sygdomstilstande eller tidlige behandlingsfaser. Den protokol, vi beskriver her, bruger et edb-og semiautomatiseret histomorfometrisk softwaresystem til at etablere en standardiseret, stringent og reproducerbar kvantitativ metode til evaluering af fælles ændringer i OA. Denne protokol udgør et stærkt supplement til de eksisterende systemer og giver mulighed for mere effektiv påvisning af patologiske ændringer i leddet.
Introduction
En af de mest udbredte ledsygdomme i USA, OA er karakteriseret ved progressiv degeneration af ledbrusk, primært i hofte-og knæled, hvilket resulterer i betydelige konsekvenser for patientens mobilitet og livskvalitet1,2,3. Ledbrusk er den specialiserede bindevæv af diarthrodialsamlinger designet til at minimere friktion, lette bevægelse, og udholde fælles kompression4. Ledbrusk består af to primære komponenter: chondrocytter og ekstracellulær matrix. Chondrocytter er specialiserede, metabolisk aktive celler, der spiller en primær rolle i udviklingen, vedligeholdelsen og reparationen af den ekstracellulære matrix4. Chondrocyt hypertrofi (CH) er en af de vigtigste patologiske tegn på OA udvikling. Det er kendetegnet ved øget cellulær størrelse, nedsat proteoglycan produktion, og øget produktion af brusk matrix-nedbrydende enzymer, der i sidste ende fører til brusk degeneration5,6,7. Yderligere, patologiske ændringer i subchondral knogle og synovium af den fælles spiller en vigtig rolle i OA udvikling og progression8,9,10,11,12. Til dato er der ingen eksisterende helbredende behandlinger, der hæmmer bruskdedegeneration1,2,3,13,14. Således er der omfattende igangværende forskning, der har til formål at forstå OA patologi og opdage nye terapeutiske tilgange, der er i stand til at bremse eller endda stoppe OA. Derfor er der et stigende behov for en kvantitativ og reproducerbar tilgang, der muliggør nøjagtig evaluering af OA-relaterede patologiske ændringer i brusk, synovium og subchondral knogle i leddet.
I øjeblikket vurderes OA-sværhedsgrad og progression primært ved hjælp af OARSI- eller Mankin-scoringssystemerne15. Men disse scoringssystemer er kun semikvantitative og kan påvirkes af brugerens subjektivitet. Endnu vigtigere, de undlader præcist at vurdere subtile ændringer, der opstår i leddet under sygdom eller som reaktion på genetisk manipulation eller en terapeutisk intervention. Der er sporadiske rapporter i litteraturen, der beskriver histomorforometriske analyser af brusk, synovium eller subchondral knogle16,17,18,19,20,21. Der mangler imidlertid stadig en detaljeret protokol for streng og reproducerbar histomorfometrisk analyse af alle disse fælles komponenter, hvilket skaber et udækket behov på området.
For at studere patologiske ændringer i OA ved hjælp af histomorfometrisk analyse, brugte vi en kirurgisk OA mus model til at fremkalde OA via destabilisering af den mediale menisk (DMM). Blandt de etablerede modeller af murine OA, DMM blev udvalgt til vores undersøgelse, fordi det indebærer en mindre traumatisk mekanisme af skade22,23,24,25,26. I forhold til menisk-ligamentous skade (MLI) eller forreste korsbånd skade (ACLI) operationer, DMM fremmer en mere gradvis progression af OA, svarende til OA udvikling hos mennesker22,24,25,26. Mus blev aflivet tolv uger efter DMM kirurgi til at evaluere ændringer i ledbrusk, subchondral knogle, og synovium.
Målet med denne protokol er at etablere en standardiseret, stringent og kvantitativ tilgang til evaluering af fælles ændringer, der ledsager OA.
Protocol
Representative Results
Discussion
Nylige osteoarthritis forskning har forbedret vores forståelse af krydstale mellem de forskellige væv i den fælles og den rolle hvert væv spiller i sygdomsstart eller progression8,9,10,35,36. Det er derfor blevet klart, at vurderingen af OA ikke bør begrænses til analyse af brusen, men også bør omfatte en analyse af subchondralknoglen og synovium. På …
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Vi vil gerne anerkende bistand fra Department of Comparative Medicine personale og molekylær og histopatologi kerne på Penn State Milton S. Hershey Medical Center. Finansieringskilder: NIH NIAMS 1RO1AR071968-01A1 (F.K.), ANRF Arthritis Research Grant (F.K.).
Materials
| 10% Buffered Formalin Phosphate | Fisher Chemical | SF100-20 | For sample fixation following harvest |
| Acetic Acid, Glacial (Certified A.C.S.) | Fisher Chemical | A38S-212 | For Decalcification Buffer preparation and acetic acid solution preparation for staining |
| Cintiq 27QHD Creative Pen Display | Wacom | https://www.wacom.com/en-es/products/pen-displays/cintiq-27-qhd-touch | For histomorphometric analysis and imaging |
| Cintiq Ergo stand | Wacom | https://www.wacom.com/en-es/products/pen-displays/cintiq-27-qhd-touch | For histomorphometric analysis and imaging |
| Ethylenediaminetetraacetic acid, tetrasodium salt dihydrate, 99% | Acros Organics | AC446080010 | For Decalcification Buffer preparation |
| Fast Green stain | SIGMA Life Sciences | F7258 | For sample staining |
| Fisherbrand Superfrost Plus Microscope Slides | Fisher | 12-550-15 | For sample section collection |
| HistoPrep Xylene | Fisherbrand | HC-700-1GAL | For sample deparrafinization and staining |
| Histosette II Tissue Cassettes – Combination Lid and Base | Fisher | 15-182-701A | For sample processing and embedding |
| HP Z440 Workstation | HP | Product number: Y5C77US#ABA | For histomorphometric analysis and imaging |
| Manual Rotary Microtome | Leica | RM 2235 | For sample sectioning |
| Marking pens | Leica | 3801880 | For sample labeling, cassettes and slides |
| OLYMPUS BX53 Microscope | OLYMPUS | https://www.olympus-lifescience.com/en/microscopes/upright/bx53f2/ | For histomorphometric analysis and imaging |
| OLYMPUS DP 73 Microscope Camera | OLYMPUS | https://www.olympus-lifescience.com/en/camera/color/dp73/ | For histomorphometric analysis and imaging (discontinued) |
| ORION STAR A211 pH meter | Thermo Scientific | STARA2110 | For Decalcification Buffer preparation |
| OsteoMeasure Software | OsteoMetrics | https://www.osteometrics.com/index.htm | For histomorphometric measurement and analysis |
| Perfusion Two Automated Pressure Perfusion system | Leica | Model # 39471005 | For mouse knee harvest |
| PRISM 7 Software | GraphPad | Institutional Access Account | Statistical Analysis |
| Safranin-O stain | SIGMA Life Sciences | S8884 | For sample staining |
| ThinkBoneStage – Rotating Microscope Stage | Think Bone Consulting Inc. – OsteoMetrics (supplier) | http://thinkboneconsulting.com/index_files/Slideholder.php | For histomorphometric analysis and imaging |
| Wacom Pro Pen Stylus | Wacom | https://www.wacom.com/en-es/products/pen-displays/cintiq-27-qhd-touch | For histomorphometric analysis and imaging |
| Weigerts Iron Hematoxylin A | Fisher | 5029713 | For hematoxylin staining |
| Weigerts Iron Hematoxylin B | Fisher | 5029714 | For hematoxylin staining |
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