Un modello chirurgico modificato di ischemia dell'arto posteriore in ApoE-/- Topi utilizzando un'incisione in miniatura
Summary
Questo articolo dimostra un approccio chirurgico efficiente per stabilire l’ischemia acuta nei topi con una piccola incisione. Questo approccio può essere applicato dalla maggior parte dei gruppi di ricerca senza aggiornamenti di laboratorio.
Abstract
Lo scopo di questo studio è quello di introdurre e valutare un approccio chirurgico modificato per indurre ischemia acuta nei topi che può essere implementato nella maggior parte dei laboratori animali. Contrariamente all’approccio convenzionale per la doppia legatura dell’arteria femorale (DLFA), è stata praticata un’incisione più piccola sulla regione inguinale destra per esporre l’arteria femorale prossimale (FA) per eseguire DLFA. Quindi, utilizzando una sutura 7-0, l’incisione è stata trascinata nella regione del ginocchio per esporre la FA distale. La risonanza magnetica (MRI) sugli arti posteriori bilaterali è stata utilizzata per rilevare l’occlusione FA dopo l’intervento chirurgico. A 0, 1, 3, 5 e 7 giorni dopo l’intervento chirurgico, il recupero funzionale degli arti posteriori è stato valutato visivamente e classificato utilizzando la scala di Tarlov. La valutazione istologica è stata eseguita dopo l’eutanasia degli animali 7 giorni dopo il DLFA. Le procedure sono state eseguite con successo sulla gamba destra in dieci topi ApoE-/- e nessun topo è morto durante la successiva osservazione. Le dimensioni dell’incisione in tutti e 10 i topi erano inferiori a 5 mm (4,2 ± 0,63 mm). I risultati della risonanza magnetica hanno mostrato che il flusso sanguigno FA nel lato ischemico era chiaramente bloccato. I risultati della scala di Tarlov hanno dimostrato che la funzione dell’arto posteriore è diminuita significativamente dopo la procedura e lentamente recuperata nei successivi 7 giorni. La valutazione istologica ha mostrato una significativa risposta infiammatoria sul lato ischemico e una ridotta densità microvascolare nell’arto posteriore ischemico. In conclusione, questo studio introduce una tecnica modificata che utilizza un’incisione in miniatura per eseguire l’ischemia degli arti posteriori (HLI) utilizzando DLFA.
Introduction
C’è un bisogno insoddisfatto di modelli animali preclinici per la ricerca nelle malattie vascolari come la malattia delle arterie periferiche (PAD). Nonostante gli sviluppi avanzati nella diagnosi e nel trattamento, ci sono stati più di 200 milioni di pazienti con PAD nel 20181e il loro numero è in costante aumento. Sebbene siano stati descritti diversi nuovi approcci terapeutici2,3,4,5,6,7, la traduzione di successo di queste modalità terapeutiche in applicazione clinica rimane un compito scoraggiante. Pertanto, sono necessari modelli sperimentali in vivo affidabili e pertinenti che simulino la condizione della malattia umana per studiare il potenziale meccanismo e l’efficienza di questi nuovi approcci terapeutici per il trattamento della PAD6,7.
L’iperlipidemia e l’aterosclerosi (AS) sono i principali fattori di rischio per lo sviluppo della PAD. I topi ApoE-/- (con una dieta ricca di grassi) mostrano un metabolismo anormale dei grassi e iperlipidemia e successivamente sviluppano placche aterosclerotiche che rendono i topi ApoE-/- la scelta migliore per simulare la PAD clinicamente rilevante. I modelli animali HLI preclinici sono generati attraverso la doppia legatura dell’arteria femorale (DLFA), che è l’approccio più utilizzato nei laboratori di tutto il mondo8,9,10,11,12,13,14,15 per simulare l’ischemia acuta su cronica. Tuttavia, questo approccio di solito richiede un’incisione relativamente grande e invasiva. Inoltre, porta inevitabilmente agli animali (soprattutto topi) che soffrono di aumento delle lesioni da dolore e dell’infiammazione, che influenza anche i successivi risultati sperimentali5,6,16,17. Questo articolo descrive un modello HLI acuto su cronico nei topi APOE-/- utilizzando un’incisione molto piccola.
Protocol
Representative Results
Discussion
Questo studio riporta un approccio modificato, semplificato e chirurgicamente efficiente per stabilire un modello HLI nei topi ApoE-/- utilizzando la doppia legatura nelle regioni prossimale e distale della FA attraverso un’incisione di 3-4 mm senza alcun aggiornamento di laboratorio richiesto. La caratteristica principale di questo metodo è la dimensione minore dell’incisione rispetto agli studi precedentemente riportati che descrivono i modelli HLI murini8,<sup …
Disclosures
The authors have nothing to disclose.
Acknowledgements
Gli autori ringraziano Viktoria Skude, Alexander Schlund e Felix Hörner per l’eccellente supporto tecnico.
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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