Mus nyretransplantation: modeller af allotransplantatafstødning
Summary
Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.
Abstract
Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.
Introduction
Vellykket nyretransplantation til behandling af nyresvigt blev første gang beskrevet i 1955 mellem enæggede tvillinger 1, siden da er det blevet en revolutionerende behandling til patienter med slutstadiet nyresvigt i hele verden, der tilbyder både forbedring i længden og livskvalitet 2. Men på lang sigt transplantat overlevelse er blevet hæmmet af en lang række patologiske processer resulterer i kronisk allograft skade 3.
Afvisning af den transplanterede nyre hos mennesker er fortsat en væsentlig årsag til sygelighed, til trods for væsentlige forbedringer i immunosupporessive regimer. Henblik på at udvikle en musemodel for nyretransplantation er at nøje replikere processen og patologi fundet i human nyretransplantation 4. Skoskiewicz et al. Først beskrev musemodel for nyretransplantation i 1973 5. Selv avancerede mikrokirurgiske færdigheder der kræves, er det en værdifuld tool af flere grunde: musegenomet er blevet godt karakteriseret, og der er et stort udvalg af eksperimentelle metoder og teknikker til rådighed til undersøgelser mus.
Mange grupper under anvendelse af musemodel for nyretransplantation har brugt den transplanterede nyre som en livsbevarende organ, men i andre undersøgelser og i vores beskrevne metode en af modtagerens mus native nyrer efterlades in situ for eksperimentets varighed 4. Fordelen er, at musen undergår en enkelt anæstesi og drift og derved reducere morbiditet til musen og risikoen for at dø af en anden procedure. Derudover musen ikke lider under de negative virkninger af en gradvis nyresvigt.
Selvom modeller af allogene afvisning findes i andre organer som hjerte og hud, er disse ikke altid er direkte relevante for nyretransplantation. Der er beviser for, at disse modeller fremkalde forskellige tilstande og dynamik for afvisning, for eksempel tidsforløbet for afvisning i hjertets allograft og nyre transplanteret adskiller sig væsentligt i visse stamme kombinationer 6. Vi har beskrevet akutte nyreallotransplantatafstødning mønstre i BALB / c donorer til ikke-transgene FVB / NJ mus, denne model viste cellemedieret skade med ophobning af T-celler og makrofager 7. Alternativt har vi også beskrevet en model af kronisk allograft skader, der udviser interstitiel fibrose og tubulær atrofi, dette skyldes at transplantere en nyre fra C57BL / 6 BM12 donorer i C57BL / 6 modtagere, da disse mus er kendetegnet ved en enkelt MHC klasse II-loci mis -match 8.
Flere aspekter af transplantation er blevet undersøgt ved anvendelse af musemodel for nyretransplantation herunder akut afstødning, cellulære og humorale afstødning, iskæmisk reperfusionsbeskadigelse og trialing hidtil ukendte terapeutiske midler. Vi har ændret den kirurgiske technique at reducere driftstiden og forbedre den lethed af operationen. Især har vi beskrevet samtidig donor og modtager forberedelse og en forenklet vaskulær anastomose teknik ved at anvende en kontinuerlig aorta patch anastomose. Denne video og manuskript vil give centrale punkter for at hjælpe i etableringen af denne teknik.
Protocol
Representative Results
Discussion
Den mest beskrevne måde at udføre arteriel anastomose er at bruge den distale aorta donor, med den renale arterie i forlængelse i en ende-til-side-måde til modtageren aorta. Vi beskriver brugen af en aorta patch, svarende til 'Carrell patch' spejling, der udføres i den menneskelige nyretransplantation, som vi mener at være mere praktisk. Selvom rapporter i litteraturen af donor og modtager operationstid er sparsomme mener vi, at udnytte en aorta patch til modtager aorta i stedet for ende-til-si…
Declarações
The authors have nothing to disclose.
Acknowledgements
Finansiering fra Nyre Research UK, The Royal College of Surgeons i Edinburgh og European Society of organtransplantation støttede denne undersøgelse.
Materials
| Surgical Instruments | |||
| Blunt Dissecting Scissors | Fine Science Tools | 14072-10 | For skin cutting |
| Curved Castoviejo scissors | Fine Science Tools | 15017-10 | For tissue cutting |
| Spring Scissors – straight | Fine Science Tools | 15000-08 | For suture cutting |
| Toothed forceps 1×2 teeth | Fine Science Tools | 11021-12 | |
| 2 x Fine Tip forceps (Dumont No.5) | Fine Science Tools | 11251-20 | |
| Angled Fine Tip forceps (Dumont No. 5/45) | Fine Science Tools | 11253-25 | For blunt dissecting |
| Curved Fine Tip forcep (Dumont No.7) | Fine Science Tools | 11273-22 | Useful to pass around vessels |
| Curved Crile Haemostat | Fine Science Tools | 1300-04 | |
| Micro clip applicator with lock | Fine Science Tools | 18056-14 | |
| 2 x Micro serrefines spring width 2mm, jaw length 4mm | Fine Science Tools | 18055-04 | Microvascular clamps |
| 2 x Colibri 3cm wire retractor | Fine Science Tools | 17000-03 | |
| Castroviejo needle holder with lock | Fine Science Tools | 120660-01 | |
| Wound clip applicator | Fine Science Tools | 12031-07 | |
| 7mm wound clips | Fine Science Tools | 12032-07 | Remove 7 to 10 days after surgery |
| Equipment | |||
| OPMI pico microscope | Carl Zeiss | S100 | |
| Thermal cautery unit with fine tip | Geiger | 150A | |
| Heat electronic pad | Cozee Cumfort | n/a | |
| Euroklav 23-S | Melag | n/a | Autoclave |
| Disposable equipment | |||
| 7/O Silk braided suture | Pearsall | 30514 | |
| 10/O Dafilon (polyamide) suture | B-Braun | G1118099 | |
| 6/O Vicryl (plygalectin) | Ethicon | W9537 | |
| Regular bevel needle, 1 inch, 21G | Bection, Dickinson and Company | 305175 | For ureteric anastamosis |
| Regular bevel needle, 5/8 inch, 25G | Bection, Dickinson and Company | 305122 | |
| Regular bevel needle, 1/2 inch, 30G | Bection, Dickinson and Company | 304000 | |
| Insulin needle 1ml, 29G | Bection, Dickinson and Company | 324827 | |
| Insulin needle 0.3ml, 30G | Bection, Dickinson and Company | 324826 | |
| 1 ml syringe slip tip | Bection, Dickinson and Company | 300184 | |
| 5 ml syringe slip tip | Bection, Dickinson and Company | 302187 | |
| Wypall paper swabs | Kimberley-Clark | L40 | sterilised by autoclave |
| Cotton wool buds | Johnson and Johnson | n/a | sterilised by autoclave |
| Plain drapes | Guardian | CB03 | sterilised by autoclave |
| Cell culture dish 60mm x 15mm | Corning Incorporated | 430166 | |
| Dispensing Pin | B-Braun | DP3500L / 413501 | Used with NaCl 0.9% |
| Re-agents and Drugs | |||
| (Lacri-Lube) White soft paraffin 57.3%, mineral oil 42.5% and lanolin alcohols 0.2% | Allergan Ltd | 21956GB10X | |
| (Videne) Povidone-iodine 10% | Ecolab Ltd | PL 04509/0041 | |
| (Vetalar V) Ketamine hydrochloride | Pfizer Animal Health | Vm 42058/4165 | 100mg/ml solution (dose 200mg/kg) |
| (Domitor) Medetomidine hydrochloride | Orion Pharma | Vm 06043/4003 | 1mg/ml (dose 0.5mg/kg) |
| (Vetergesic) Bupernorphine hydrochloride | Alsto Animal Health | Vm 00063/4002 | 0.3mg/ml (dose 0.05mg/kg) |
| (Antisedan) Atipamezole hydrochoride | Orion Pharma | Vm 06043/4004 | 5mg/ml (dose 2mg/kg) |
| University of Wisconsin Solution | Belzer Bridge to Life | n/a | dose approximately 500 microlitres/mouse |
| NaCl 0.9% | Baxter | FKE1323 | |
| Heparin Sulphate | non-proprietary | n/a | 5000units/ml (dose 5units/mouse) |
Referências
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