Source: Üçal, M. et al., Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines. J. Vis. Exp. (2021)
The video demonstrates a method for generating a rat model with widespread cerebral cortical demyelination. This involves disrupting the blood-brain barrier by injecting pro-inflammatory cytokines into mice primed with recombinant myelin sheath protein. This process facilitates the infiltration of immune cells, resulting in widespread cerebral cortical demyelination.
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Material Preparation
NOTE: The surgery is conducted under aseptic conditions. Prior to starting, make sure that all surgical instruments, including the drill bits, are cleaned with an appropriate disinfectant.
2. Surgical Preparation
3. Catheter Implantation
4. Closing of the Wound and Antagonization of Anesthesia
5. Post-operative Care and Medication
6. Preparation of Immunization Mixture (at the Earliest 14 Days after Catheter Implantation)
NOTE: Place the syringes on ice during the preparation procedure.
7. Immunization
8. Intracerebral Cytokine Injection
Figure 1: Preparation of the catheter. (A and B) The guide cannula and the dummy cannula (catheter cap with inlet) are assembled and screwed. (C) Then the catheter is cut to 2 mm in size with the help of a scalpel. The microscopic observation showed that the usage of scissors for that purpose distorts the circular shape of the cannula tip, and, thereby, must be avoided.
Figure 2: The implantation of the catheter. (A) The surgery starts with a longitudinal incision and removal of the periosteum. (B, C) This panel shows the marking of the place for the catheter at 2 mm posterior from Bregma and 2.4 mm lateral to the right from the sagittal suture; as well as the places for the holes intended for the three anchor screws with an appropriate distance from the catheter and Lambda. (D) After drilling the catheter hole (a 0.5 mm diameter, with a round drill tip) and the holes for the anchor screws (1.3 mm-diameter with a twisted drill tip), the anchor screws are tightened. (E, F) Then the catheter is inserted and the whole setup is stabilized with polymerizing dental cement. (G) The wound is stitched with two or three knots anterior and posterior to the catheter. B = Bregma; L = Lambda; C = Catheter; S1, S2 and S3 = places for the holes for the three anchor screws. The scale bars = 1 cm.
Figure 3: Preparation of rMOG/IFA emulsion. (A) The mixture of rMOG, PBS, and IFA is emulsified by pressing the inoculum from one syringe to another by pushing the pistons back and forth, (B) until it is white and viscous. (C) Subsequently, the inoculum is distributed to 1 mL syringes for the injection. 5 µg of rMOG is used in 200 µL of PBS/IFA mixture to sub clinically immunize one rat; however, due to the losses at the tips and walls of the syringes during the preparation, a larger volume should be prepared.
Figure 4: Preparation of the connector cannula. (A -cD) These panels show how a connector and an internal are assembled with a 2-mm size template guide cannula. (E) The internal is cut to the same size as the guide cannula with the help of a scalpel (F) and the template guide is then unscrewed. (G) The other end of the connector cannula is fixed to a 1 mL syringe, which contains the injection mix, with a 20 G needle. The scale bars = 3 cm.
Figure 5: Intracerebral injection. (A) A programmable syringe pump is adjusted for a 2 µL/min injection speed, and the 1 mL syringe filled with cytokine mix (or sterile PBS for the controls) is mounted to the pump. (B) The animal is first anesthetized in the chamber using 5% isoflurane with a 2 L/min oxygen flow and then (C) the anesthesia is sustained through the mask using 2.5% isoflurane with a 1.5 L/min oxygen flow. (D) The catheter cap with the inlet (the dummy cannula) is screwed off and the injection cannula is inserted through the implanted catheter. Since the volume of the injection is very small, the investigator should be cautious to avoid air bubbles at the tip of the cannula. For that reason, it is important to start the insertion while the pump is in operation and only when there is a growing liquid bubble at the tip. The extra volume will not go into the brain anyway, as it breaks down on top of the catheter before the insertion. (E) Then the connector cannula is tightened, and the pump is let to operate for 10 min. After 10 min of injection, the pump is stopped, and the cannula is left inside for 15 – 20 min to allow for the diffusion of the injected volume to the interstitial fluid. The scale bars = 5 cm unless indicated otherwise.
The authors have nothing to disclose.
Adult male Dark Agouti rats (300 ±25 g) | |||
Fentanyl | Hameln pharma plus, Germany | As Fentanyl-Citrate, 50 µg/ml | |
Midazolam | ERWO Pharma, Austria | 50039017 | 5 mg/ml |
Medetomidin | Orion Pharma, Finland | As Medetomidin hydrochloride, 1mg/ml | |
Flumazenil | Roche, Switzerland | 0.1 mg/ml | |
Atipamezol | Orion Pharma, Finland | As Atipamezol hydrochloride, 5 mg/ml | |
10% povidone-iodine complex | Mundipharma, Austria | ||
Dental cement | Heraeus Kulzer, Germany | 6603 7633 | |
Physiological saline solution | Fresenius Kabi, Austria | 0.9% NaCl | |
Phosphate buffered saline (PBS) | Sigma-Aldrich, Germany | P3813 | |
Isofluorane | AbbVie, Austria | ||
Lubricating eye drops | Thea Pharma, Austria | ||
70% EtOH | Merck, Germany | 1070172511 | Absolute ethanol was diluted in ddH2O for preparation of 70% v/v |
2.5% Enrofloxacin | Bayer, Germany | Prophylactic antibiotics | |
Carprofen | Pfizer, USA | Painkillers, 50 mg/ml | |
Tween-20 | Sigma-Aldrich, Germany | P9416 | |
Pentobarbital | Richter Pharma, Austria | Pentobarbital sodium, 400 mg/ml | |
Interferon gamma | PeproTech, USA | 400-20 | |
Tumor necrosis factor alfa | R&D Systems, USA | 510-RT-050/CF | |
rMOG1-125 | own product at the Centre of Molecular Medicine, Karolinska Institute, Sweden | Recombinant rat myelin oligodendrocyte glycoprotein, amino acids 1-125 from the N-terminus, also commercially available: AnaSpec, AS-55152-500, USA | |
Incomplete Freund’s adjuvant | Sigma-Aldrich, Germany | F5506 | |
Bovine serum albumin | Sigma-Aldrich, Germany | A9576 | |
Peroxidase substrate solution | Vector Laboratories, USA | SK-45000 | |
Stereotactic frame | David Kopf Instruments, USA | ||
Catheters, MRI suitable | PlasticsOne, USA | 8IC315GPKXXC | |
Dummy cannulas | PlasticsOne, USA | 8IC315DCNSPC | |
Plastic screws, MRI suitable | PlasticsOne, USA | 8L080X093N01 | |
Connector cannula | PlasticsOne, USA | 8IC313CXSPCC | |
Screw driver with 2mm tip-size | |||
Drill with flexible shaft extension | Proxxon, Germany | NO 28 472, NO 28 706, NO 28 620, | |
Drill bit, round, 0.5 mm | Hager & Meisinger, Germany | REF310 104 001 001 009 | |
Drill bit, twisted, 1.3 mm | Hager & Meisinger, Germany | REF350 104 417 364 013 | |
Scalpel | Braun, Germany | BB510 | |
Scalpel handle | Fine Science Tools, Germany | 91003-12 | |
Cotton tip applicator | Henry Schein Medical, Austria | 900-3155 | |
Surgical scissors | Fine Science Tools, Germany | 14101-14, 14088-10 | |
Surgical forceps | Fine Science Tools, Germany | 11002-12, 11251-35 | |
Bulldog clamps | Fine Science Tools, Germany | 18050-35 | |
Homoeothermic blanket | TSE systems, Germany | ||
Infrared Lamp | Beurer, Germany | 616.51 | |
Dental curing light | Guilin Woodpecker Medical, China | ||
Absorbable suture | Johnson & Johnson, Belgium | V792E | |
Programmable syringe pump | World Precision Instruments, USA | AL-1000 | |
Exam gloves | |||
Surgical gown | |||
Electric Shaver | Aesculap, Germany | GT420 | |
Volatile anesthetic vaporizer | Rothacher Medical, Switzerland | CV 30-301-D | |
Oxygen source for volatile anesthetic vaporizer | Air Liquide, Austria | 19,113 | |
Volatile anesthesia chamber | Rothacher Medical, Switzerland | PS-0347 | |
Anesthesia mask for rats | Rothacher Medical, Switzerland | PS-0307-A | |
1 ml syringe | Codan, Denmark | REF 62.1612 | |
26 Gauge needle for injection | Braun, Germany | 4657683 | |
20 Gauge needle for cytokine injection and immunization | Braun, Germany | 4657519 | |
Luer lock tip glass syringes | Poulten & Graf, Germany | 7.140-37 | |
3 way stopcock | Becton Dickinson, Sweden | 394600 | |
37°C incubator | Kendro, Germany | 50042301 | |
Micropipettes | Gilson, USA | F167350 |
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