Establishment of a Novel Method for Spinal Discectomy Surgery in Elderly Rats in an In Vivo Spinal Fusion Model
Abstract
:1. Introduction
2. Experimental Design
2.1. Materials
- Fentanyl 0.05 mg/mL (Sintetica S.A., Mendrisio, Switzerland).
- Midazolam 5 mg/mL (Sintetica S.A., Mendrisio, Switzerland).
- Medetomidine 1 mg/mL (Orion Corporation, Espoo, Finland).
- Meloxicam 5 mg/mL, injection solution (Boehringer Ingelheim GmbH, Basel, Switzerland).
- Meloxicam 0.5 mg/mL, oral suspension (Boehringer Ingelheim GmbH, Basel, Switzerland).
- Buprenorphin 0.3 mg/mL (Streuli Pharma SA, Uznach, Switzerland).
- Flumazenil 0.1 mg/mL (CPS Cito Pharma Services GmbH, Uster, Switzerland).
- Naloxon 0.4 mg/mL (OrPha Swiss GmbH, Küsnacht, Switzerland).
- Atipamezol 5 mg/mL (Orion Corporation, Espoo, Finland).
- Ropivacain 7.5 mg/mL (Fresenius Kabi AG, Kriens, Switzerland).
- Sodiumchloride 0.9% (Bichsel AG, Interlaken, Switzerland).
- Glucose 5% (B. Braun Medical AG, Sempach, Switzerland).
- Pentobarbital 300 mg/mL (Streuli Pharma SA, Uznach, Switzerland).
- Isofluran 99.9% (Provet AG, Lyssach, Switzerland).
- O2 100%
- Sterile ophthalmic lubricant (Bausch & Lomb Swiss AG, Zug, Switzerland).
- Phosphate Buffered Solution (PBS).
- Bovine Serum Albumin (BSA) (Sigma–Aldrich Chemie GmbH, Buchs, Switzerland).
- OpSite transparent spray dressing (Smith & Nephew, Solothurn, Switzerland).
- Tissue culture-treated plate, 60 mm dish (TPP Techno Plastic Products AG, Schaffhausen, Switzerland).
- TOPIC spray (Vetoquinol AG, Bern, Switzerland).
- β-TCP carrier, ∅ 0.5 mm, h = 1.5 mm, 75% porosity (produced by Prof Marc Bohner, Robert Mathys Foundation, Bettlach, Switzerland) (Figure 2a).
- Growth Factors, BMP-2 5 mg/mL and L51P 5 mg/mL. Growth factors were delivered lyophilized and sterile, and were kindly provided (produced by Prof Walter Sebald, Department of Physiological Chemistry, University of Würzburg, Würzburg, Germany).
- Customized external ring fixator (Urs Rohrer, SITEM, Inselspital, University Hospital Bern, Bern, Switzerland) (Figure 2b).
- Customized hex wrench (Urs Rohrer, ARTORG Center, Inselspital, University Hospital Bern, Bern, Switzerland) (Figure 2b).
- Surgical instruments: scalpel holder, needle holder, anatomical forceps, surgical forceps, suture scissors, curette, sharp pliersat, Kerrison rongeur (B. Braun Medical AG, Sempach, Switzerland).
- Kirschner wires 0.8 mm (Synthes GmbH, Oberdorf, Switzerland).
- Carbon steel surgical blades 15 mm (Swann–Morton, Sheffield, United Kingdom).
- Ethanol 70%, 80%, 96%, 100%.
- Sterilium disinfection (Paul Hartmann AG, Heidenheim, Germany).
- Octenisept spray disinfection (Schülke & Mayr GmbH, Norderstedt, Switzerland).
- Sterile surgical gloves (Mölnycke Health Care AB, Göteborg, Sweden).
- Leukosilk silk plasters 2.5 cm × 5 m (BSN medical GmbH, Hamburg, Germany).
- Ethilon Sutures 4.0 (Johnson & Johnson Medical Ltd., Livingston, United Kingdom).
- Vicryl Sutures 5.0 (Johnson & Johnson Medical Ltd., Livingston, United Kingdom).
- Sterile compresses 5 × 5 cm, 10 × 20 cm (IVF Hartmann AG, Neuhausen, Switzerland).
- Sterile covers 20 × 20 cm (IVF Hartmann AG, Neuhausen, Switzerland).
- Cling Film
- Syringes 1 mL, 2.5 mL, 5 mL, 10 mL (Terumo Corporation, Tokyo, Japan).
- 25 G 16 mm, 22 G 30 mm, 20 G 30 mm needles (B. Braun Melsungen AG, Melsungen, Germany).
- Falcon Tubes, 50 mL (BD Falcon, New York, NY, USA).
2.2. Equipment
- Biolaboratory safety class II
- Specific-pathogen-free (SPF) animal facility with rodent operation room
- Anaesthesia station (Vet Tech Solutions, Congleton, Great Britain).
- Rodent Surgical Monitor including heating pad, thermometer, and pulse oximeter, respiratory rate and heart rate (UNO B.V., PC Zevenaar, Netherlands).
- Bairhugger, heated air inflatable blanket (3 M Schweiz GmbH, Rüschlikon, Switzerland).
- Stryker System 8 cordless driver (Stryker, Selzach, Switzerland).
2.3. Animals
3. Procedure
3.1. Coating of β-TCP-Carrier
- CRITICAL STEP Do not vortex; shake by hand for approximately 10 min.
3.2. Anaesthesia
- CRITICAL STEP Anaesthetic mixture should be applied subcutaneously in loose restrained handling into the nuchal fold. Make sure to vent the needle prior to injection and use a different injection needle (22 G 30 mm) than the needle used for drawing up the anaesthetic mixture to avoid a blunt needle tip. Directly after injection, release loose restrained handling to avoid reflux of the fluid (Figure 3a,b).
3.3. Surgical Setup
3.4. Surgical Procedure
- CRITICAL STEP Insert the k-wires in a slightly posterolateral direction to avoid damaging the vessels running laterally to the vertebrae.
- CRITICAL STEP Wound closure should be performed in a two-step process with subcutaneous closure using 4.0 Vycril and cutaneous closure with 5.0 Ethilone to ensure complete wound closure and prevent animal wound biting and wound infection.
3.5. Postoperative Regimen
- Analgesic water mixture (per rat for 72 h):
- 360 mL autoclaved H2O
- + 10 mL Glucose (5%)
- + 6 mL Buprenorphine (0.3 mg/mL)
- CRITICAL STEP Check on the correct positioning of the external ring fixator and closed wounds during the animal checkups.
3.6. Euthanasia
4. Expected Results
4.1. Effectiveness of the Protocol
4.1.1. Anaesthesia
4.1.2. Surgical Procedure
4.1.3. Pain Management
4.1.4. Complications
4.1.5. Euthanasia
4.2. Data Analysis and Interpretation
4.2.1. X-rays and µCT
4.2.2. PMMA Histology and Immunohistology
4.3. Data Application to Human Clinical Trials
4.4. Comparison of Surgical Technique
5. Reagents Setup
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Parameter | |||
---|---|---|---|
Fusion | none | incomplete | complete |
On-growth on proximal endplate in % of endplate surface | none | <50 | >50 |
On-growth on distal endplate in % of endplate surface | none | <50 | >50 |
In-growth through carrier in % of carrier surface | none | <50 | >50 |
Bridging callus formation in % of endplate surface | none | <50 | >50 |
Carrier absorption in % of carrier surface | none | <50 | >50 |
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Oswald, K.A.C.; Bigdon, S.F.; Croft, A.S.; Bermudez-Lekerika, P.; Bergadano, A.; Gantenbein, B.; Albers, C.E. Establishment of a Novel Method for Spinal Discectomy Surgery in Elderly Rats in an In Vivo Spinal Fusion Model. Methods Protoc. 2021, 4, 79. https://doi.org/10.3390/mps4040079
Oswald KAC, Bigdon SF, Croft AS, Bermudez-Lekerika P, Bergadano A, Gantenbein B, Albers CE. Establishment of a Novel Method for Spinal Discectomy Surgery in Elderly Rats in an In Vivo Spinal Fusion Model. Methods and Protocols. 2021; 4(4):79. https://doi.org/10.3390/mps4040079
Chicago/Turabian StyleOswald, Katharina A. C., Sebastian F. Bigdon, Andreas S. Croft, Paola Bermudez-Lekerika, Alessandra Bergadano, Benjamin Gantenbein, and Christoph E. Albers. 2021. "Establishment of a Novel Method for Spinal Discectomy Surgery in Elderly Rats in an In Vivo Spinal Fusion Model" Methods and Protocols 4, no. 4: 79. https://doi.org/10.3390/mps4040079