En modificeret kirurgisk model af hind limb iskæmi i ApoE-/- Mus ved hjælp af en Miniature snit
Summary
Denne artikel demonstrerer en effektiv kirurgisk tilgang til at etablere akut iskæmi hos mus med et lille snit. Denne tilgang kan anvendes af de fleste forskningsgrupper uden laboratorieopgraderinger.
Abstract
Formålet med denne undersøgelse er at indføre og evaluere en modificeret kirurgisk tilgang til at fremkalde akut iskæmi hos mus, der kan implementeres i de fleste dyrelaboratorier. I modsætning til den konventionelle tilgang til dobbelt ligation af lårpulsåren (DLFA), blev der foretaget et mindre snit i den rigtige lyske region for at udsætte den proksimale lårpulsåre (FA) til at udføre DLFA. Derefter, ved hjælp af en 7-0 sutur, snittet blev slæbt til knæet regionen for at afsløre den distale FA. Magnetisk resonans imaging (MRI) på bilaterale bagben blev brugt til at opdage FA okklusion efter operationen. Ved 0, 1, 3, 5 og 7 dage efter operationen blev funktionel genopretning af bagbenene visuelt vurderet og klassificeret ved hjælp af Tarlov-skalaen. Histologisk evaluering blev udført efter aflivning af dyrene 7 dage efter DLFA. Procedurerne blev udført med succes på højre ben i ti ApoE-/- mus, og ingen mus døde under efterfølgende observation. Snitstørrelserne i alle 10 mus var mindre end 5 mm (4,2 ± 0,63 mm). MR-scanningsresultater viste, at FA’s blodgennemstrømning i den iskæmiske side var tydeligt blokeret. Tarlov-skalaresultaterne viste, at bagbensfunktionen faldt betydeligt efter proceduren og langsomt kom sig i løbet af de følgende 7 dage. Histologisk evaluering viste en betydelig inflammatorisk reaktion på den iskæmiske side og reduceret mikrovaskulær tæthed i det iskæmiske bagben. Afslutningsvis introducerer denne undersøgelse en modificeret teknik ved hjælp af et miniature snit til at udføre bagbenskæmi (HLI) ved hjælp af DLFA.
Introduction
Der er et uopfyldt behov for prækliniske dyremodeller til forskning i vaskulære sygdomme som perifer arteriesygdom (PAD). På trods af den avancerede udvikling inden for diagnose og behandling var der mere end 200 millioner patienter med PAD i 20181, og deres antal er konstant stigende. Selv om flere nye terapeutiske tilgange2,3,4,5,6,7 er blevet beskrevet, vellykket oversættelse af disse terapeutiske modaliteter til klinisk anvendelse er stadig en skræmmende opgave. Derfor er der behov for pålidelige og relevante in vivo-eksperimentelle modeller, der simulerer tilstanden af mennesker, for at undersøge den potentielle mekanisme og effektivitet af disse nye terapeutiske tilgange til behandling af PAD6,7.
Hyperlipidæmi og åreforkalkning (AS) er de vigtigste risikofaktorer for udviklingen af PAD. ApoE-/- mus (på en fedtrig kost) udviser unormal fedtstofskifte og hyperlipidæmi og udvikler efterfølgende aterosklerotiske plaques, der gengiver ApoE– / – mus som det bedste valg til at simulere den klinisk relevante PAD. Prækliniske HLI dyremodeller genereres gennem dobbelt ligation af lårpulsåren (DLFA), som er den mest udbredte tilgang i laboratorier over hele verden8,9,10,11,12,13,14,15 for at simulere akut-on-kronisk iskæmi. Denne tilgang kræver dog normalt et relativt stort og invasivt snit. Desuden fører det uundgåeligt til, at dyrene (især mus) lider af øget smerteskade og betændelse, hvilket også påvirker de efterfølgende eksperimentelle resultater5,6,16,17. Dette papir beskriver en akut-on-kronisk HLI model i APOE-/- mus ved hjælp af et meget lille snit.
Protocol
Representative Results
Discussion
Denne undersøgelse rapporterer en modificeret, forenklet og kirurgisk effektiv tilgang til at etablere en HLI-model i ApoE-/- mus ved hjælp af dobbelt ligation i de proksimale og distale områder i FA gennem et 3-4 mm snit uden nødvendige laboratorieopgraderinger. Hovedkarakteristika for denne metode er indsnitmets mindre størrelse sammenlignet med tidligere rapporterede undersøgelser , der beskriver mus HLI-modellerne8,9,<sup class="xr…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Forfatterne takker Viktoria Skude, Alexander Schlund og Felix Hörner for den fremragende tekniske support.
Materials
| 10x Phosphate buffer saline | Roth | 9143.1 | Used for haematoxylin and eosin stain and immunohistochemistry stain |
| 30% H2O2 | Roth | 9681.2 | Used for immunohistochemistry stain |
| 6-0 absorbable sutures | PROLENE | 8776H | Used for stitching the skin |
| 6-0 absroable suture | PROLENE | EP8706 | Used in Surgery |
| 7-0 absorbable sutures | PROLENE | EH8021E | Used for ligating the artery |
| 7-0 absroable suture | PROLENE | EP8755 | Used in Surgery |
| Acetic acid | Roth | 6755.1 | Used for haematoxylin and eosin stain |
| Albumin Fraktion V | Roth | 8076.2 | Used for immunohistochemistry stain |
| Autoclave | Systec GmbH | Systec VX-150 | Used for the sterilisation of the surgical instruments |
| Axio vert A1 microscope | Carl Zeiss | ZEISS Axio Vert.A1 | Used for viewing and taking the pictures from haematoxylin and eosin stain and immunohistochemistry stain |
| Bruker BioSpec 94/20 AVIII | Bruker Biospin MRI GmbH | N/A | Scan the femoral artery blockage |
| Buprenovet Sine 0,3mg/ml | Bayer AG | 2542 (WDT) | Used in post operative pain-management. Dose – 0.1 mg/kg body weight every 8 hours for 48 h after operation |
| CD31 antibody | Abcam | ab28364 | Used for immunohistochemistry stain |
| Eosin Y solution 0.5 % in water | Roth | X883.1 | Used for haematoxylin and eosin stain |
| Epitope Retrieval Solution pH 6 | Leica Biosystems | 6046945 | Used for immunohistochemistry stain |
| Ethanol ≥ 99,5 % | Roth | 5054.1 | Used for haematoxylin and eosin stain and immunohistochemistry stain |
| Fentanyl | Cayman Chemical | 437-38-7 | Used for anesthesia |
| Fine point forceps | Medixplus | 93-4505S | Used for separating the artery from nerve and vein |
| Glass bead sterilisator | Simon Keller | Type 250 | Used for sterilisation of the surgical instruments |
| Graefe iris forceps curved | VUBU | VUBU-02-72207 | Used for blunt separation of skin and subcutaneous tissue |
| Hair Remover cream, Veet (with aloe vera) | Reckitt Benckiser | 108972 | Remove hair from mice hind limbs |
| Heating plate | STÖRK-TRONIC | 7042092 | Keep the satble temperature of mice |
| Hematoxylin | Roth | T865.2 | Used for haematoxylin and eosin stain and immunohistochemistry stain |
| Leica surgical microscope | Leica | M651 | Enlarge the field of view to facilitate the operation |
| Liquid DAB+Substrate Chromogen System | Dako | K3468 | Used for immunohistochemistry stain |
| Male ApoE-/- mice | Charles River Laboratories | N/A | Used for establish the Peripheral artery disease mice model |
| Medetomidine | Cayman Chemical | 128366-50-7 | Used for anesthesia |
| Micro Needle Holder | Black & Black Surgical | B3B-18-8 | Holding the needle |
| Micro suture tying forceps | Life Saver Surgical Industries | PS-MSF-145 | Used to assist in knotting during surgery |
| Microtome | Biobase | Bk-Mt268m | Used for tissue sectioning |
| Midazolam | Ratiopharm | 44856.01.00 | Used for anesthesia |
| MR-compatible Small Animal Monitoring and Gating System Model 1025 | SA Instruments | N/a | monitoring vital signs of animal during MRI scan |
| Octeniderm farblos | Schülke & Mayr GmbH | 180212 | used for disinfection of the skin |
| Ointment for the eyes and nose | Bayer AG | 1578675 | Keep the eyes wet under the anesthesia |
| Paraformaldehyde | Roth | 0335.1 | Used for fixation of the tissue |
| Pentobarbital | Nembutal | 76-74-4 | Used for anesthesia |
| Saline | DeltaSelect | 1299.99.99 | Used for anesthesia |
| Spring handle scissors with fine, sharp tips | Black & Black Surgical | B66167 | Used for cutting the artery |
| SuperCut Scissors | Black & Black Surgical | B55992 | Used for cutting the skin |
| Triton X-100 | Roth | 9002-93-1 | Used for immunohistochemistry stain |
| Western diet, 1.25% Cholesterol | ssniff Spezialdiäten GmbH | E15723-34 | Diet for the mice |
| Xylene | Roth | 4436.3 | Used for haematoxylin and eosin stain and immunohistochemistry stain |
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