All methods described here have been approved by local authorities (Bundesministerium für Wissenschaft und Forschung (Austrian Ministry of Science and Research); License Number: 66.010/0132-WF/V/3b/2014). The adult male DA rats (10 – 12 weeks of age) were housed in a 12/12 h light/dark cycle with free access to food and water.
1. Material Preparation
NOTE: The surgery is conducted under aseptic conditions. Prior to start, 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. Determination of Antibody Titers
9. Intracerebral Cytokine Injection
Cortical demyelination could be assessed at different time points after a cytokine injection by immunohistochemistry for proteolipid protein (PLP) (Figure 6). Figure 6A shows intact PLP immunoreactivity at day 15 in a MOG-immunized control animal that received only sterile PBS through the implanted catheter. On day 1 after the cytokine injection, demyelination could already be detected in MOG-primed animals, albeit only in the close vicinity of the catheterized area (Figure 6B). The PLP immunoreactivity stays intact in the contralateral cortex 1-day post-cytokine injection. On day 3, a gradual increase in the loss of the PLP immunoreactivity, which spreads in the ipsilateral cortex (Figure 6C), could be observed. Contralateral cortical demyelination could also be detected at day 3 (Figure 6D), but it is rather restricted to the area beneath the anchor screws, possibly due to a low-flow area of interstitial fluid caused by the anchor screws43. The absence of a similar observation in the PBS-injected control animals excludes the possibility of trauma-induced demyelination stemming from the anchor screw.
Between days 9 – 15, demyelination affects large parts of the cortex of both hemispheres (Figures 6E, 6F, and 6G). This coincides with an observation of slow behavior, though without a statistically significant decrease in motor skills in a rotarod test43. The cortical demyelination is sustained for up to 30 days post-cytokine injection in both hemispheres (Figures 6H and 6I) with only a partial remyelination. Figure 6J shows a quantification of PLP loss in the cortical grey matter after the intracerebral cytokine injection. It should be noted that PLP immunoreactivity has not yet been assessed after periods longer than 30 days; thereby, the instantaneous resolution of remyelination, if there is any, remains to be assessed by further experimentation. A second administration of cytokine mixture through the implanted catheter 30 days after the first injection results in marked brain atrophy at day 15 (Figure 7).
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Figure 4: Preparation of the connector cannula. (A – D) 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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Figure 6: PLP immunoreactivity in coronal brain sections. (A) This panel shows a control brain (MOG-primed) with a PBS injection (day 15), which does not result in a cortical demyelination. (B) As early as day 1 post-cytokine injection, demyelination is apparent in the catheter area. (C) A broader loss of PLP immunoreactivity is observed in the ipsilateral cortex at day 3, (D) as well as at the contralateral side. Widespread loss of PLP immunoreactivity is observed in both hemispheres at day 15, as (E) this panel shows the ipsilateral cortex and (F) this panel shows the contralateral cortex. (G) An overview of both hemispheres is given in showing the widespread loss of PLP at day 15. At day 30, as (H) this panel shows the ipsilateral cortex and (I) this panel shows for contralateral cortex, there is still remarkable demyelination, but also some remyelinated areas could be observed. (J) This panel shows the quantification of the demyelination (PLP loss in mm2/hemisphere). 1.5 – 2 µm coronal brain sections were used for the PLP detection with MS anti-PLP with a dilution factor of 1:500. The sections were counterstained with hematoxylin for cell nuclei. For detailed information on the immunohistochemistry, see Ucal et al.43. Panel G and J were modified from Ucal et al.43. Please click here to view a larger version of this figure.
Figure 7: Brain atrophy after second cytokine injection. (A) This panel shows a control brain (MOG-primed) with a PBS injection (day 15). (B) At day 15 after the first cytokine injection, a second injection leads to brain atrophy within 15 days. 1.5 – 2 µm coronal brain sections were used for the PLP detection with MS anti-PLP with a dilution factor of 1:500. The sections were counterstained with hematoxylin for cell nuclei. For detailed information on immunohistochemistry, see Blakemore37. The scale bars = 500 µm. Please click here to view a larger version of this figure.
Figure 8: Modified cages to prevent catheter removal by the animal. In the standard cages, the food holder space on the grid is located closer to the cage bottom, creating a risky narrow space that increases the chance of the catheter to tangle with the grid and, thereby, its removal. To avoid this, the cage has to be modified. This narrow space was blocked with a transparent plane, allowing the observance of the animal. The food has to be given inside the cage in these modified cages. Please click here to view a larger version of this figure.
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 | |
Anti-MOG antibody | Ana Spec/Kaneka Corporation, Japan | AS-555157 | Standard from ELISA-Kit; Ana Spec/Kaneka |
Incomplete Freund’s adjuvant | Sigma-Aldrich, Germany | F5506 | |
Horse radish peroxidase conjugated anti-rat IgG secondary antibody | Ana Spec/Kaneka Corporation, Japan | AS-555157 | Secondary Antibody from ELISA-Kit; Ana Spec/Kaneka Corporation |
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 | |
96-well plate | Thermo Fisher Scientific, USA | 442404 | |
Plate reader | Cole-Parmer, USA | EW-1396-00 | |
37°C incubator | Kendro, Germany | 50042301 | |
Micropipettes | Gilson, USA | F167350 |
Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and death, caused by white matter lesions in the spinal cord and cerebellum, as well as by demyelination in grey matter. Whilst conventional models of experimental allergic encephalomyelitis are suitable for the investigation of the cell-mediated inflammation in the spinal and cerebellar white matter, they fail to address grey matter pathologies. Here, we present the experimental protocol for a novel rat model of cortical demyelination allowing the investigation of the pathological and molecular mechanisms leading to cortical lesions. The demyelination is induced by an immunization with low-dose myelin oligodendrocyte glycoprotein (MOG) in an incomplete Freund's adjuvant followed by a catheter-mediated intracerebral delivery of pro-inflammatory cytokines. The catheter, moreover, enables multiple rounds of demyelination without causing injection-induced trauma, as well as the intracerebral delivery of potential therapeutic drugs undergoing a preclinical investigation. The method is also ethically favorable as animal pain and distress or disability are controlled and relatively minimal. The expected timeframe for the implementation of the entire protocol is around 8 – 10 weeks.
Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and death, caused by white matter lesions in the spinal cord and cerebellum, as well as by demyelination in grey matter. Whilst conventional models of experimental allergic encephalomyelitis are suitable for the investigation of the cell-mediated inflammation in the spinal and cerebellar white matter, they fail to address grey matter pathologies. Here, we present the experimental protocol for a novel rat model of cortical demyelination allowing the investigation of the pathological and molecular mechanisms leading to cortical lesions. The demyelination is induced by an immunization with low-dose myelin oligodendrocyte glycoprotein (MOG) in an incomplete Freund's adjuvant followed by a catheter-mediated intracerebral delivery of pro-inflammatory cytokines. The catheter, moreover, enables multiple rounds of demyelination without causing injection-induced trauma, as well as the intracerebral delivery of potential therapeutic drugs undergoing a preclinical investigation. The method is also ethically favorable as animal pain and distress or disability are controlled and relatively minimal. The expected timeframe for the implementation of the entire protocol is around 8 – 10 weeks.
Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and death, caused by white matter lesions in the spinal cord and cerebellum, as well as by demyelination in grey matter. Whilst conventional models of experimental allergic encephalomyelitis are suitable for the investigation of the cell-mediated inflammation in the spinal and cerebellar white matter, they fail to address grey matter pathologies. Here, we present the experimental protocol for a novel rat model of cortical demyelination allowing the investigation of the pathological and molecular mechanisms leading to cortical lesions. The demyelination is induced by an immunization with low-dose myelin oligodendrocyte glycoprotein (MOG) in an incomplete Freund's adjuvant followed by a catheter-mediated intracerebral delivery of pro-inflammatory cytokines. The catheter, moreover, enables multiple rounds of demyelination without causing injection-induced trauma, as well as the intracerebral delivery of potential therapeutic drugs undergoing a preclinical investigation. The method is also ethically favorable as animal pain and distress or disability are controlled and relatively minimal. The expected timeframe for the implementation of the entire protocol is around 8 – 10 weeks.