Biaxial basal tone og passiv testning af murine reproduktive system ved hjælp af en Tryk Myograph
Summary
Denne protokol udnyttede et kommercielt tilgængeligt tryk myograph-system til at udføre tryk myograph test på murine vagina og livmoderhalsen. Ved hjælp af medier med og uden calcium blev bidragene fra de glatte muskelceller (SMC) basal tone og passiv ekstracellulær matrix (ECM) isoleret for organerne underestimerede fysiologiske forhold.
Abstract
De kvindelige reproduktive organer, især vagina og livmoderhalsen, er sammensat af forskellige cellulære komponenter og en unik ekstracellulær matrix (ECM). Glatte muskelceller udviser en kontraktile funktion inden for vaginal og cervikal vægge. Afhængigt af det biokemiske miljø og den mekaniske udspiling af orgel væggene, de glatte muskelceller ændre kontraktile betingelser. Bidraget fra de glatte muskelceller under baseline fysiologiske betingelser er klassificeret som en basal tone. Mere specifikt, en basal tone er den baseline delvis konstriktion af glatte muskelceller i fravær af hormonelle og neurale stimulation. Desuden, ECM giver strukturel støtte til orgel vægge og funktioner som et reservoir for biokemiske signaler. Disse biokemiske signaler er afgørende for forskellige organfunktioner, såsom tilskyndelse til vækst og opretholdelse af homøostase. ECM af hvert organ er sammensat primært af kollagen fibre (for det meste kollagen typer I, III, og V), elastiske fibre, og glycosaminoglycans/proteoglycans. Sammensætningen og organiseringen af ECM diktere de mekaniske egenskaber af hvert organ. En ændring i ECM sammensætning kan føre til udvikling af reproduktive patologier, såsom bækkenorganprolaps eller tidlig cervikal remodeling. Endvidere, ændringer i ECM mikrostruktur og stivhed kan ændre glatte muskel celle aktivitet og fænotype, hvilket resulterer i tab af kontraktile kraft.
I dette arbejde, de rapporterede protokoller anvendes til at vurdere basal tone og passive mekaniske egenskaber af den ikke-gravide murine vagina og livmoderhalsen på 4-6 måneder i estrus. Organerne blev monteret i et kommercielt tilgængeligt tryk myograph, og både tryk-diameter og Force-længde tests blev udført. Prøve data og dataanalyse teknikker til mekanisk karakterisering af de reproduktive organer er inkluderet. Sådanne oplysninger kan være nyttige til at konstruere matematiske modeller og rationelt designe terapeutiske interventioner for kvinders sundhedsmæssige patologier.
Introduction
Den vaginale væg består af fire lag, Epitelet, lamina propria, muscularis og adventitia. Epitel består primært af epiteliale celler. Den lamina propria har en stor mængde af elastiske og fibrillar kollagen fibre. Muscularis er også sammensat af elastin og kollagen fibre, men har en øget mængde af glatte muskelceller. Adventitien består af elastin, kollagen og fibroblaster, omend i reducerede koncentrationer sammenlignet med de tidligere lag. De glatte muskelceller er af interesse for biomekorisk motiverede forskergrupper, da de spiller en rolle i den kontraktile karakter af organerne. Som sådan, kvantificere den glatte muskel celleområde fraktion og organisation er nøglen til at forstå den mekaniske funktion. Tidligere undersøgelser tyder på, at den glatte muskel indhold i den vaginale væg primært er organiseret i den omgående og længdeakse. Histologisk analyse tyder på, at den glatte muskel område fraktion er ca 35% for både proksimale og distale sektioner af væggen1.
Livmoderhalsen er en meget kollagenous struktur, der indtil for nylig, blev anset for at have minimal glat muskel celleindhold2,3. Nylige undersøgelser, dog, har antydet, at glatte muskelceller kan have en større overflod og rolle i livmoderhalsen4,5. Livmoderhalsen udviser en gradient af glatte muskelceller. Det interne operativsystem indeholder 50-60% glatte muskelceller, hvor det eksterne operativsystem kun indeholder 10%. Mus undersøgelser, dog, rapportere livmoderhalsen, der skal bestå af 10-15% glatte muskelceller og 85-90% fibrøst bindevæv uden omtale af regionale forskelle6,7,8. I betragtning af, at musemodel adskiller sig fra den hyppigt rapporterede menneskelige model, yderligere undersøgelser vedrørende mus livmoderhalsen er nødvendige.
Formålet med denne protokol var at belyse de mekaniske egenskaber af murine vagina og livmoderhalsen. Dette blev opnået ved hjælp af en Tryk myograph enhed, der muliggør vurdering af mekaniske egenskaber i de circumferentielle og aksiale retninger samtidig samtidig bevare indfødte celle-matrix interaktioner og organ geometri. Organerne blev monteret på to brugerdefinerede kanyle og sikret med silke 6-0 suturer. Prøvning af tryk diameter blev udført omkring den anslåede fysiologiske aksiale strækning for at bestemme overensstemmelsen og tangent moduli9. Der blev udført Force-length-tests for at bekræfte den anslåede aksiale strækning og for at sikre, at de mekaniske egenskaber blev kvantificeret i det fysiologiske område. Forsøgsprotokollen blev udført på den ikke-gravide murine vagina og livmoderhalsen ved 4-6 måneders alderen i estrus.
Protokollen er opdelt i to vigtigste mekaniske test sektioner: basal tone og passiv test. En basal tone er defineret som baseline partielle konstriktion af glatte muskelceller, selv i fravær af eksterne lokale, hormonelle og neurale stimulation10. Denne baseline kontraktile natur af vagina og livmoderhalsen giver karakteristisk mekanisk adfærd, som derefter måles ved tryk myograph system. De passive egenskaber vurderes ved at fjerne det intercellulære calcium, der bevarer den grundlæggende tilstand af sammentrækning, hvilket resulterer i afslapning af de glatte muskelceller. I den passive tilstand, kollagen og elastin fibre giver den dominerende bidrag til de mekaniske egenskaber af organerne.
Murine-modellen anvendes i udstrakt grad til at studere patologier i kvinders reproduktive sundhed. Musen giver flere fordele ved at kvantificere de udviklende relationer mellem ECM og mekaniske egenskaber i det reproduktive system11,12,13,14. Disse fordele omfatter korte og velkarakteriserede estrous cyklusser, relativt lave omkostninger, nem håndtering, og en relativt kort gestationstid15. Desuden er genomet af laboratorie mus godt kortlagt og genetisk modificerede mus er værdifulde værktøjer til at teste mekanistiske hypoteser16,17,18.
Kommercielt tilgængelige tryk myograph systemer anvendes i udstrakt grad til at kvantificere de mekaniske reaktioner af forskellige væv og organer. Nogle bemærkelsesværdige strukturer analyseret på trykket myograph system omfatter elastiske arterier19,20,21,22, vener og væv manipuleret vaskulære grafts23,24, spiserøret25, og de store tarme26. Tryk myograph-teknologien muliggør samtidig vurdering af egenskaber i de aksiale og circumferentielle retninger, samtidig med at de indfødte celle-ECM-interaktioner og in vivo-geometri bevares. På trods af den omfattende brug af myograph systemer i blødt væv og orgel mekanik, en protokol, der udnytter trykket myograph teknologi ikke tidligere var blevet udviklet til vagina og livmoderhalsen. Forudgående undersøgelser af de mekaniske egenskaber i vagina og livmoderhalsen blev vurderet uniaksialt27,28. Disse organer, dog oplever multiaksial lastning i kroppen29,30, således kvantificere deres biksimale mekaniske respons er vigtigt.
Desuden tyder nylige arbejde på, at glatte muskelceller kan spille en potentiel rolle i bløddels patologier5,28,31,32. Dette giver en anden attraktion ved at udnytte trykket myograph teknologi, da det bevarer den indfødte celle-matrix interaktioner, hvilket tillader afgrænsning af det bidrag, glatte muskelceller spiller i fysiologiske og patofysiologiske Betingelser. Heri, foreslår vi en protokol til at kvantificere de multiaksiale mekaniske egenskaber i vagina og livmoderhalsen under både basal tone og passive betingelser.
Protocol
Representative Results
Discussion
Den protokol, der er fastsat i denne artikel præsenterer en metode til bestemmelse af de mekaniske egenskaber af murine vagina og livmoderhalsen. De mekaniske egenskaber, der analyseres i denne protokol, omfatter både de passive og basale tone forhold i organerne. Passive og basale tone forhold induceres ved at ændre det biokemiske miljø, hvor orglet er nedsænket. For denne protokol indeholder de medier, der er involveret i basal testning, calcium. Afprøvning af basal tone tilstand tillader isolering af den glatte …
Declarações
The authors have nothing to disclose.
Acknowledgements
Arbejdet blev finansieret af NSF CAREER Award Grant #1751050.
Materials
| 2F catheter | Millar | SPR-320 | catheter to measure cervical pressure |
| 6-0 Suture | Fine Science Tools | 18020-60 | larger suture ties |
| CaCl2 (anhydrous) | VWR | 97062-590 | HBSS concentration: 140 mg/ mL |
| CaCl2-2H20 | Fischer chemical | BDH9224-1KG | KRB concentration: 3.68 g/L |
| Dextrose (D-glucose) | VWR | 101172-434 | HBSS concentration: 1000 mg/mL KRB concentration: 19.8 g/L |
| Dumont #5/45 Forceps | Fine Science Tools | 11251-35 | curved forceps |
| Dumont SS Forceps | Fine Science Tools | 11203-25 | straight forceps |
| Eclipse | Nikon | E200 | microscope used for imaging |
| Flow meter | Danish MyoTechnologies | 161FM | flow meter within the testing apparatus |
| Force Transducer – 110P | Danish MyoTechnologies | 100079 | force transducer |
| ImageJ | SciJava | ImageJ1 | used to measure volume |
| Instrument Cases | Fine Science Tools | 20830-00 | casing to hold dissection tools |
| KCl | Fisher Chemical | 97061-566 | HBSS concentration: 400 mg/ mL KRB concentration: 3.5 g/L |
| KH2PO4 | G-Biosciences | 71003-454 | HBSS concentration: 60 mg/ mL |
| MgCl2 | VWR | 97064-150 | KRB concentration: 1.14 g/L |
| MgCl2-6H2O | VWR | BDH9244-500G | HBSS concentration: 100 mg/ mL |
| MgSO4-7H20 | VWR | 97062-134 | HBSS concentration: 48 mg/ mL |
| Mircosoft excel | Microsoft | 6278402 | program used for spreadsheet |
| Na2HPO4 (dibasic anhydrous) | VWR | 97061-588 | HBSS concentration: 48 mg/mL KRB concentration: 1.44 g/L |
| NaCl | VWR | 97061-274 | HBSS concentration: 8000 mg/mL KRB concentration: 70.1 g/L |
| NaHCO3 | VWR | 97062-460 | HBSS concentration: 350 mg/ mL KRB concentration: 21.0 g/L |
| Pressure myograph systems | Danish MyoTechnologies | 110P and 120CP | Pressure myograph system: prorgram, cannulation device, and controller unit |
| Pressure Transducer | Danish MyoTechnologies | 100106 | pressure transducer |
| Student Dumont #5 Forceps | Fine Science Tools | 91150-20 | straight forceps |
| Student Vannas Spring Scissors | Fine Science Tools | 91500-09 | micro-scissors |
| Tissue dye | Bradley Products | 1101-3 | ink to measure in vivo stretch |
| Ultrasound transducer | FujiFilm Visual Sonics | LZ-550 | ultrasound transducer used; 256 elements, 40 MHz center frequency |
| VEVO2100 | FujiFilm Visual Sonics | VS-20035 | ultrasound used for imaging |
| Wagner Scissors | Fine Science Tools | 14069-12 | larger scissors |
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