Tager det næste skridt: en neurale coaptation Orthotopic Hind Limb Transplant Model at maksimere funktionel helbredelse i Rat
Summary
Denne protokol præsenterer en robust, reproducerbar model af vaskulære komposit allotransplant (VCA) rettet mod samtidig undersøgelse af immunologi og funktionel genopretning. Den tid, der investeres i omhyggelig teknik i en højre midt-låret bagekstremiteten ortopisk transplantation med hånd syet vaskulære anastomoses og neurale coaptation giver mulighed for at studere funktionel helbredelse.
Abstract
Limb transplantation i særdeleshed og vaskulariseret komposit allotransplant (VCA) generelt har bred terapeutisk løfte, der er blevet hæmmet af de nuværende begrænsninger i immunsuppression og funktionelle neuromotoriske opsving. Mange dyremodeller er udviklet til at studere unikke funktioner i VCA, men her præsenterer vi en robust reproducerbar model af ortopisk hind lemmer transplantation hos rotter designet til samtidig at undersøge begge aspekter af den nuværende VCA begrænsning: immunsuppression strategier og funktionelle neuromotoriske opsving. Kernen i modellen hviler en forpligtelse til omhyggelige, tidstestede mikrokirurgiske teknikker såsom håndsyet vaskulære anastomoses og håndsyet neural coaptation af lårbensnerven og iskiasnerven. Denne fremgangsmåde giver holdbare lemmer rekonstruktioner, der giver mulighed for længere levede dyr i stand til rehabilitering, genoptagelse af daglige aktiviteter, og funktionelle test. Med kortvarig behandling af konventionelle immunsuppressive midler overlevede allotransplanterede dyr op til 70 dage efter transplantationen, og isotransplanterede dyr giver langvarige kontroller ud over 200 dage efter operationen. Tegn på neurologisk funktionel helbredelse er til stede ved 30 dage efter operativt. Denne model giver ikke kun en nyttig platform for afhøring af immunologiske spørgsmål, der er unikke for VCA og nerveregenerering, men giver også mulighed for in vivo-test af nye terapeutiske strategier, der er skræddersyet til VCA.
Introduction
Limb transplantation under den bredere kategori af vaskulære-komposit allotransplant (VCA) eller komposit væv allotransplant (CTA) har endnu ikke opfyldt sit terapeutiske løfte. Siden de første vellykkede menneskelige hånd transplantationer i Lyon, Frankrig og Louisville, Kentucky i 1998 og 1999, over 100 øvre ende transplantationer er blevet udført på verdensplan i nøje udvalgte patienter1. Bredere anvendelighed er blevet hæmmet af betydelig immunsuppression og begrænset funktionel neuromotorisk opsving. Nuværende immunsuppressionsstrategier resulterer i 85% forekomst af akut afstødning i lyset af 77% forekomst af opportunistisk infektion2. På den anden side, funktionel opsving efter hånd transplantation opstår; gennemsnitlige scorer for armskuld og hånd (DASH) forbedres fra 71 til 43, men dette funktionsniveau kan stadig betragtes som et handicap2. I betragtning af den ikke-besparende karakter af lemmer transplantation, skal de nuværende teknikker raffineres i dyremodeller til at tage det næste skridt i VCA.
Siden den første rottemodel af lemtransplantation i 19783er der udviklet mange innovative dyremodeller til at fremme feltet af VCA4, der omfatter vaskulære manchet anastomosesforat minimere den udløsendetid 5,6,heterotopisk osteomyokutane transplantationer for at minimere fysiologisk fornærmelse mod recipientdyret7,8,,9,10,11og nye immunologiske tilgange7,12,13,14. Den rotte model af ortotopisk højre bagbensbenseks.
Protocol
Representative Results
Discussion
Limb transplantation, under den bredere kategori af vaskulære komponent allotransplantation (VCA), har bredt anvendelig terapeutisk løfte endnu uopfyldt. De vigtigste vejspærringer ligger i uløste immunologiske spørgsmål unikke for VCA og neuromotoriske opsving teknikker, der anvendes i øjeblikket. Udvikling af nye teknikker vil afhænge af dyremodellering, der er fleksibel, robust og reproducerbar.
Mange dyremodeller er blevet etableret i VCA, hver med særlige fordele<sup class="xref"…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev finansieret af Frankel Foundation og Northwestern Memorial Hospital McCormick Grant (Operation RESTORE). Forskning rapporteret i denne publikation blev støttet af National Institute of General Medicial Sciences af National Institutes of Health under Award Nummer T32GM008152. Dette arbejde blev støttet af Northwestern University Microsurgery Core og Behavioral Fænotypebestemmelse Core.
Materials
| Anesthesia machine | Vet Equip | 911103 | |
| 0.5cc syringe | Exel | 26018 | |
| 18-gauge needle | BD | 305196 | |
| 1cc syringe | BD | 309659 | |
| 22-gauge needle | BD | 305156 | |
| 24-gauge angiocatheter | Sur-Vet | SROX2419V | |
| 25-gauge needle | Exel | 26403 | |
| 3 cc syringe | BD | 309657 | |
| 5cc syringe | Exel | 26230 | |
| Alcohol | Fisher Scientific | HC-600-1GAL | |
| Anesthesia induction chamber | Vet Equip | 941443 | |
| Anesthetic gas scavenger system | Vet Equip | 931401 | |
| Bipolar electrocautery | Aura | 26-500 | |
| Bitter Spray Mist | Henry Schein | 5553 | |
| Bone wax | CP Medical | CPB31A | |
| Breathing circuit | Vet Equip | 921413 | |
| Buprenophine | Reckitt Benckiser | 12496075705 | |
| Castro-Viejos needle drivers | Roboz | RS-6416 | |
| Cordless rotary saw | Dremel | 8050-N/18 | |
| Cotton swab stick | Fisher Scientific | 23-400-101 | For hemostasis |
| DigiGait Appparatus and Software | Mouse Specifics | MSI-DIG, DIG-SOFT | |
| Dumont forceps (#4) | Roboz | RS-4972 | |
| Dumont forceps (#5) | Roboz | RS-5035 | |
| Enrofloxacin | Norbrook | ANADA 200-495 | |
| FK-506 | Astellas | 301601 | |
| Gauze | Kendall | 1903 | |
| Gauze | Covidien | 8044 | |
| Gloves | Microflex | DGP-350-M | |
| Hair clippers | Oster | 078005-010-003 | |
| Handheld monopolar electrocautery | Bovie | AA00 | |
| Hargreaves Apparatus | Ugo Basile S.R.L. Gemonio, Italy | 37370 | |
| Heating pad | Walgreens | 126987 | |
| Heparin | Fresenius Kabi | 42592K | |
| Hot plate | Corning | PC-351 | For warming resusscitation fluid |
| Isoflurane | Henry Schein | 29405 | |
| Lactated ringers | Baxter | 2B2074 | |
| Large petri dish | Fisher Scientific | FB0875713 | For donor graft while in chilled saline |
| Meloxicam | Henry Schein | 49755 | |
| micro Collin Hartmann retractor | |||
| Micro dissecting scissors | Roboz | RS-5841 | |
| Microfibrillar collagen powder | BD | 1010590 | For hemostasis |
| Microvascular clips | Roboz | RS-5420 | |
| Normal saline | Baxter | 2F7124 | |
| Opthalmic lube | Dechra | IS4398 | |
| Rapmycin | MedChem Express | HY-10219 | |
| Small petri dish | Fisher Scientific | FB0875713A | For warmed resusscitation fluid |
| Sterile drapes | ProAdvantage | N207100 | |
| Surgical gown | Cardinal Health | 9511 | |
| Surgical mask | 3M | 1805 | |
| Surgical microscope, optic model OPMIMD | Zeiss | 169756 | |
| Surgical microscope, Universal S3 | Zeiss | 243188 | |
| Suture 10-0 nylon | Covidien | N2512 | |
| Suture 5-0 vicryl | Ethicon | J213H | |
| Suture 7-0 silk tie | Teleflex | 103-S | |
| Tape | 3M | 1530-1 | |
| Ultrasonic instrument cleaner | Roboz | RS-9911 | |
| Vessel dilation forceps | Roboz | RS-5047 |
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