The authors would like to thank all personnel of the Institute of Biomedical Research at the Medical University Graz for their help and cooperation, as well as Christopher John Wrighton for proofreading the manuscript.

All methods described here have been approved by local authorities (Bundesministerium f&#252;r 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.
<ol>
	<li>Prepare an anesthetic mixture: 0.02 mg/mL of fentanyl, 0.4 mg/mL of midazolam, and 0.2 mg/mL of medetomidine (final concentrations in the mixture). 
	NOTE: See the respective Discussion section for alternative anesthetics.</li>
	<li>Prepare an antidote mixture: 0.07 mg/mL of flumazenil and 0.42 mg/mL of atipamezole (final concentrations in the mixture).</li>
	<li>Assemble the catheter and the catheter cap with the inlet and the screw. Cut the catheter to a length of 2 mm with a scalpel (Figure 1). 
	NOTE: Do not use scissors for this, since they squeeze and distort the circular cross-sectional shape of the catheter tip.</li>
</ol>
2. Surgical Preparation
<ol>
	<li>Anesthetize the rat by an intraperitoneal (i.p.) administration of the anesthetic mixture (1.5 mL/kg of body weight).</li>
	<li>Shave the head of the rat between the ears using the electric shaver. Place a homeothermic blanket on the stereotactic frame before positioning the animal, to avoid hypothermia throughout the surgery.</li>
	<li>Immobilize the rat&#39;s head in the stereotactic frame using the ear bars and bite plate, ensuring that the head is horizontal and stable. Check the stability by applying pressure to the skull with finger or forceps. 
	NOTE: A loose fixation and non-horizontal positioning within the stereotactic frame may cause a deviation from the intended coordinates.</li>
	<li>Apply lubricating eye drops to prevent cornea dryness during the surgery. Cover the eyes with an opaque material to prevent any surgical light exposure.</li>
	<li>Clean the shaven area by alternating the application of 70% ethanol and 10% povidone-iodine complex. 
	NOTE: Follow all precautionary measures during surgery to the avoid infection. The surgery is conducted under aseptic conditions. If asepsis is broken, then the contaminated material has to be replaced.</li>
</ol>
3. Catheter Implantation
<ol>
	<li>Make a longitudinal incision of about 2 cm of length in the middle of the head skin. Use bulldog clamps to hold the skin off to the sides. For an overview of these steps, see Figure 2.</li>
	<li>Remove the blood using a cotton-tipped applicator.</li>
	<li>Remove the skull periosteum. Clean the tissue with the cotton-tip applicator and expose the skull bone. Allow the skull to dry for about 1 min.</li>
	<li>Identify the anatomical landmarks, Lambda, Bregma, and medial suture. With the drill installed on the stereotactic frame, position the drill tip at the Bregma as the starting point. Move 2 mm posterior from the Bregma and move ~2.4 mm laterally to the medial suture.</li>
	<li>Drill a 0.5 mm diameter hole for the catheter at this position. Gently puff away any bone dust. 
	NOTE: It is important that the dura mater remains intact during the drilling. To ensure this, 1) use a drill that can be installed on the stereotactic frame, 2) inspect the hole frequently during the drilling, and 3) drill down in small steps-if too much pressure is applied to the skull, the drill tip will continue into, and damage the brain when the skull is fully penetrated.</li>
	<li>Drill 3 further holes (~1.3 mm in diameter) for the anchor screws a few millimeters away from the first hole. Gently blow bone dust away. 
	NOTE: Select anchor screw locations that provide enough space for the catheter top (~2 mm in diameter) and the anchor screw tops (~1 mm).</li>
	<li>Remove the bone dust by irrigation with around 1 - 2 mL of sterile phosphate-buffered saline (PBS) or physiological saline using a syringe. Clean the skull. Tighten the anchor screws by 2 - 3 full turns. 
	NOTE: The anchor screws are necessary to stabilize the set-up by holding the dental cement, and, thereby, the catheter, in place. Whilst tightening an anchor screw, ensure that it is not easily removable by gently lifting it upwards with forceps. Since the implantation of the catheter itself causes tissue trauma, additional dura injury whilst drilling for, or tightening the anchor screws will lead to multiple traumatic injuries, and possibly hamper the comparability within a group. Tighten the anchor screws first and insert the catheter last.</li>
	<li>Insert the 2-mm length catheter through the first hole, perpendicular to the skull surface. While still holding the catheter in, apply a little dental cement and let it polymerize with a brief (~5 s) exposure to the dental curing light to stabilize the catheter, allowing the use of both hands in the next step. 
	CAUTION: While working with a dental curing light, avoid looking directly at the tip, or at the light reflected from the application area, as the high intensity of this light can cause retinal damage. Use appropriate protective goggles.</li>
	<li>Apply more dental cement around the catheter, anchor the screws, and solidify the dental cement with the dental curing light (~15 - 30 s). Confirm the hardening of the cement with the tip of a forceps.</li>
</ol>
4. Closing of the Wound and Antagonization of Anesthesia
<ol>
	<li>Close the head skin with resorbable sutures, anterior and posterior to the catheter. 
	NOTE: Since there will be a bumpy set-up over the skull at the end of the implantation, do the wound closure accordingly. Lifting up the skin too much will result in discomfort for the animal.</li>
	<li>Inject the antidote mixture subcutaneously (1.5 mL/kg of body weight) using a 1 mL syringe with a 26 G needle.</li>
	<li>Administer enrofloxacin &#160; (2.5%) by subcutaneous injection (7.5 mg/kg body weight) for prophylactic antibiotic treatment. Administer carprofen (1 mg/mL; 5 mg/kg body weight) and buprenorphine (1.2 mg/kg) for pain relief by subcutaneous injection.</li>
</ol>
5. Post-operative Care and Medication
<ol>
	<li>Return the animal to the modified cage and keep it under observation for 1 - 3 h, with an application of infrared light to avoid hypothermia.&#160;Constantly observe and reposition the animal every 5 to 10 minutes until post-operative recovery. Take special care to avoid constant light exposure to the eyes till recovery.</li>
	<li>Repeat enrofloxacin (7.5 mg/kg body weight) and carprofen (1 mg/mL; 5mg/kg body weight) administrations by subcutaneous injections the day after surgery. Buprenorphine is not necessary to be refreshed as the previous treatment is effective for 72 hours.</li>
</ol>
6. Preparation of Immunization Mixture (at the Earliest 14 Days after Catheter Implantation)
Note: Place the syringes on ice during the preparation procedure.
<ol>
	<li>Connect two 10 mL Luer lock tip glass syringes to the short arms of a 3-way stopcock and close the third outlet with the long arm.</li>
	<li>Ensure the connections are secure and leak-free: add approximately 4 mL of sterile PBS to the open syringe while holding piston 2. Insert piston 1 and push both pistons back and forth whilst checking for leakage. If no leakage occurs, discard the PBS and remove piston 1 again.</li>
	<li>Pipette 1 mL of IFA and 50 &#181;g of rMOG1-125 together and adjust the mixture to a final volume of 2 mL with sterile PBS (pH 7.4) in a suitable tube. 
	NOTE: Due to losses of emulsion on the tips or walls of syringes during the preparation, prepare a larger volume than intended for the administration. It is similarly more practical to prepare for more than 1 animal at once.</li>
	<li>Place the diluted IFA and rMOG1-125 mixture in the open syringe. Insert the piston gently whilst maintaining a loose pressure on the opposite piston (Figure 3A).</li>
	<li>Emulsify the inoculum by driving it from one syringe to the other by pushing the pistons back and forth, until it is white and viscous (Figure 3B).</li>
	<li>Fix a 1 mL Luer lock syringe to the open short arm of the 3-way stopcock and fill it with inoculum (Figure 3C). Distribute all inoculum to 1 mL syringes. Keep it on ice until the injection. Administer the mixture on the day of the preparation.</li>
</ol>
7. Immunization
<ol>
	<li>Anaesthetize the rat with isofluorane in a chamber (~2 min, mixed with oxygen 2 L/min) and then sustain anesthesia through&#160;a mask (mixed with oxygen 1.5 L/min).</li>
	<li>Inject 200 &#181;L of inoculum subcutaneously at the tail base using a 21 G needle. 
	NOTE: Administer the injection slowly as the solution is viscous.</li>
</ol>
8. Determination of Antibody Titers
<ol>
	<li>Withdraw ~200 &#181;L of blood 4 weeks after the immunization in order to determine the anti-MOG antibody titers.</li>
	<li>Coat the wells of a 96-well plate with MOG (5 &#181;g/mL in PBS) and incubate them for 1 h at 37 &#176;C.</li>
	<li>Block the plate with 1% bovine serum albumin (BSA) in PBS for 1 h at room temperature.</li>
	<li>Incubate the plate with rat serum (1:50) and standard it for 2 h at 37 &#176;C. Wash the plate 3x with 200 &#181;L of PBS/Polysorbate 20.</li>
	<li>Incubate the plate with horseradish peroxidase-conjugated anti-rat IgG secondary antibody (1:10,000). Wash the plate 3x with 200 &#181;L of PBS/Polysorbate 20.</li>
	<li>Add 100 &#181;L of peroxidase substrate solution per well and incubate it for 20 - 30 min in the dark at room temperature.</li>
	<li>Measure the optical density at a 405 nm wavelength and calculate the antibody titer from the optical density using a standard curve.</li>
</ol>
9. Intracerebral Cytokine Injection
<ol>
	<li>Adjust the length of the connector cannula (2 mm). (See Figure 4 for the preparation steps.)</li>
	<li>Fill a 1 mL syringe with the cytokine mixture (500 ng/&#181;L of TNF-alpha, 300 U/&#181;L of recombinant rat IFN-gamma in sterile PBS). Connect the syringe to a connector cannula. Fill the cannula with the cytokine mixture. Avoid any bubbles.</li>
	<li>Mount the syringe onto the programmable syringe pump and program it to inject 0.2 &#181;L/min (Figure 5A). Start the pump and keep it working in order to avoid an air bubble formation at the tip of the cannula. 
	NOTE: The injection speed must take into account the inner diameter of the specific syringe used; thereby, the syringe diameter has to be registered during the pump set up.</li>
	<li>Anesthetize the rat with isoflurane in a chamber (~2 min, mixed with 2 L/min of oxygen) and then sustain the anesthesia through a mask (mixed with 1.5 L/min of oxygen) (Figures 5B and 5C).&#160;Apply lubricating eye drops as the animal will be anesthetized for at least 30 min.</li>
	<li>Remove the catheter cap with the inlet. Insert the connector cannula into the catheter and screw and tighten it (Figures 5D and 5E). 
	NOTE: Do not overtighten it, as this will destroy the upper tip of the catheter.</li>
	<li>Allow the injection to proceed for 10 min (the total volume of injection being 2 &#181;L). Stop the pump. Leave the cannula inside the catheter for 20 min to allow the injected volume to fully diffuse.</li>
	<li>Unscrew the connector cannula and remove it slowly to avoid a vacuum effect.</li>
	<li>Reattach the catheter cap with the inlet and screw it. Allow the animal to recover from the anesthesia in a cage.</li>
</ol>

The authors have nothing to disclose.

Our method uses DA rats. The adaptation to mice will likely require the use of a smaller catheter and screws. It should also be borne in mind that the disease course, the inflammatory response, and the extent of demyelination might differ from what is presented here if a different species/strain is used. Such differences have been observed with classical EAE models using different strains of mice. MOG92-106 of rat origin, for instance, resulted in primary progressive or secondary progressive EAE in A.SW mice, whilst it induced relapsing-remitting EAE in SJL/J mice46. Animals of the same strain should, therefore, be used. Gender differences in EAE manifestation have also been reported in various previous studies24. The occurrence of such gender effects might well be expected for the protocol described here yet remains to be validated in further experiments.
The intraperitoneal (IP) administration of an anesthetics mixture comprising 0.02 mg/mL of fentanyl, 0.4 mg/mL of midazolam, and 0.2 mg/mL of medetomidine is used for the surgical intervention. Adult male DA rats weighing 270 - 300 g require around 0.4 - 0.6 mL of this mixture (i.e., ~1.5 mL/kg) to induce an anesthesia lasting 60 - 90 min. Following the surgery, the anesthesia is antagonized by a subcutaneous injection of an antidote comprising 0.07 mg/mL of flumazenil and 0.42 mg/mL of atipamezole in physiological saline (0.9% NaCl). A dose of 1 - 1.5 mL/kg antagonizes the anesthesia within 5 min. Alternatively, the animals can be allowed to wake up spontaneously upon a physiological washing out of the anesthetics, but in that case, the animals would need to be kept under observation until they are fully conscious.
Other anesthesia options frequently used for animal surgeries, such as an IP injection of ketamine and xylazine47 or sodium pentobarbital48, or an inhalation of volatile anesthetics like isofluorane49 and halothane50, can also be considered for the surgery presented here. It is critical, however, to choose an anesthetic agent that does not interfere with the intended downstream intervention(s).
During the immunization and intracerebral cytokine injection, 5% isoflurane is used for the anesthesia. The model described here was established using rats, and the experimental details listed are, thus, specifically applicable to the rat. The catheter implantation coordinates were selected to enable the simultaneous analysis of possible white matter changes (the catheter tip in the corpus callosum). Whilst the catheter insertion site can be varied with respect to the anteroposterior and lateral position, the selection of the central sulcus requires the avoidance of damage to the superior sagittal sinus.
A further feature of the described method is the equivalent demyelination of both ipsi- and contralateral hemispheres, possibly resulting from the carriage of the injected cytokine mixture to the subarachnoid space by the physiological flow of the interstitial fluid from cortical regions51. The injection mode, and not the location of the catheter, therefore, causes demyelination throughout the cerebral cortex, and the choice of right or left parietal cortex should, thus, be immaterial in this regard.
The protocol uses a 26 G catheter, which is small enough to avoid extensive traumatic injury and large enough to avoid an increased rate of clogging of the catheter tip over the long course of the experiment. Certainly, the implantation and the presence of the catheter itself cause an astrocytic and microglial activation, also in the control animals receiving only the catheter implantation; however, this is minor when compared to the cytokine-injected animals.43 To avoid any interference with subsequent analyses, we used MRI-compatible catheters made of poly-ether-ether-ketone (PEEK).
A similar depth of demyelination is, in fact, created in both ipsi- and contralateral regions with the presented method. This implies that the catheter depth/length might not play a major role in the pattern and extent of demyelination in the cortex. Therefore, a modification of the catheter length might be considered in order to reduce the catheter-induced lesion size. Nevertheless, a significantly shorter catheter length might cause a slightly less pronounced cortical demyelination, whilst a conclusive answer would only be obtained by experiments specifically testing for the catheter length.
One advantage of the model is that the implanted catheter allows for the testing of potential therapeutics administered to the cortex via the catheter to allow remyelination at or after the peak of histologically detectable cortical demyelination (day 15 or later), whilst in a pretreatment setting this would be after the immunization but before the cytokine injection. The decision on the time frame when therapeutics would be administered, therefore, will depend on the particular research question and the drug of interest.
Following catheter implantation, it is important to house animals single in the modified (preferably high top) cages in order to avoid catheter removal till the end of the study (Figure 8). Animals might also unscrew the catheter cap with the inlet, although this rarely happens. The animals should be observed daily and removed caps should be replaced with fresh ones, to avoid catheter tip blockage in the absence of an inlet, and to ensure an accurate delivery into the parenchyma following the intracerebral injection. The animals are immunized at the earliest 2 weeks after the catheter implantation to allow the healing and closure of the blood-brain barrier.
Serum anti-MOG antibody titers should be measured after the immunization. A dose-response experiment showed that 5 &#181;g of MOG1-125 (in IFA) provided sufficient immunization within 4 weeks in adult male DA rats. A titer of 5,000 &#181;g/mL and higher would be sufficient, but will certainly depend on several factors, including the MOG preparation and the animal strain and, thus, will have to be determined individually. It is important to avoid excessively high antigen doses potentially resulting in a classical EAE phenotype with paralyzed hind limbs even before the cytokine injection.
Each animal is immunized with 5 &#181;g of recombinant myelin oligodendrocyte glycoprotein (rMOG1-125) emulsified in 200 &#181;L of incomplete Freund&#39;s Adjuvant (IFA). Since some of the emulsion is lost within the syringe during the preparation, it is advisable to prepare more than this amount for each animal. We used recombinant MOG (1-125 from the N-terminus of rat MOG), which was expressed in Escherichia coli and was then purified to homogeneity by chelate chromatography, dissolved in 6 M urea, and dialyzed against PBS to obtain a physiological preparation52,53. Commercially available MOG may, however, also be used.
Other antigen preparations, such as MOG1-116, MOG35-55 or PLP139-151 are used in various EAE models, and antigen and animal strain differences are known to induce distinct disease phenotypes in these models20. These antigen preparations were not tested in DA rats and, if used in preference to rMOG1-125, might induce a disease phenotype or histology results differing from what is presented here.
A connector cannula the same length as the catheter is prepared prior to the intracerebral injection. This can be done by assembling it with a template catheter and cutting it to the same size (2 mm in length) (Figure 4). It is important that the connector cannula be air bubble-free during the cytokine injection-because the injection volume is only 2 &#181;L, even a tiny air bubble at the cannula tip will significantly reduce the volume of the liquid successfully delivered into the brain. This is achieved by keeping the pump running and inserting the cannula only when a growing drop of injection liquid is present at the tip. Following the cannula insertion, the connector is screwed to the catheter while avoiding overtightening so as not to damage the upper tip of the catheter, which will make it difficult to recap after the injection. An injection speed of 0.2 &#181;L/min is used to avoid injection-induced trauma. Moreover, a slow injection, combined with a 20 min waiting period after the injection, ensures the diffusion of the injected liquid into the interstitial fluid and an effective draining into the CSF. The cannula is then removed slowly to avoid a vacuum effect.
The reported method includes surgical intervention and, therefore, requires staff able to perform stereotactic survival surgery. Personnel in direct contact with the animals should have taken the appropriate animal experimentation courses. The remainder of the protocol can be carried out by competent lab members.
The method is intended to produce inflammation-triggered demyelination of the cerebral cortex and does not reproduce all features of human MS (e.g., the occurrence of focal inflammatory white matter lesions, which is a hallmark of human MS).

MS is an immune-mediated, inflammatory disease of the CNS, which primarily damages myelin sheets, but eventually leads to the axonal loss and permanent neuronal damage. MS is the most common immune-mediated disease of the CNS with an estimated prevalence of about 2.3 million people worldwide, according to the National MS Society1, and represents a major personal and socioeconomic burden. The average age of the disease onset is 30 years and leads to the loss of productive years by causing severe disability. MS is currently incurable, and the present treatment modalities aim to manage the symptoms during an acute relapse in relapsing-remitting MS and to modify the course of the disease to decrease the relapse frequency by immunomodulatory therapy2,3. No treatment option has yet proven efficacious for the progressive types4, with the exception of a recent clinical trial of B-cell depleting therapy, which was shown to be efficacious in a subgroup of primary progressive MS (PPMS) patients with active inflammation5. Whilst several potential genetic6 and environmental risk factors7 have been identified, the etiology of MS, however, remains unknown.
MS is characterized by large inflammatory demyelinating plaques in, and diffuse injuries to, the white matter8,9. Focal lesions have been associated with an extensive T-cell mediated attack, oligodendrocyte destruction, reactive astrogliosis, and axonal degeneration, leading to a motor neuron decline. Grey matter demyelination and atrophy have gained recognition as additional histopathological features of the disease9,10,11. The latter has been suggested to contribute to the neurological dysfunction and cognitive decline in patients12,13. Three patterns of cortical demyelination have been distinguished, namely i) leukocortical, contiguous with the white matter lesions (34%), ii) small, perivascular (16%), and iii) subpial (50%). Unlike focal white matter lesions, these grey matter lesions have been reported to lack a T-cell mediated attack and are instead characterized by an enhanced microglial activation, apoptosis, and neuronal loss12.
To date, it has not been possible to recapitulate human MS in a single animal model, largely due to the complexity of the disease. A variety of MS animal models, each simulating different aspects of disease pathogenesis and progression have, instead, been developed14,15. Current animal models mimic three different disease processes: i) focal inflammatory lesions, ii) diffuse white matter injury, and iii) diffuse grey matter pathology.
Animal studies of MS white matter plaques have mostly been conducted in rodent encephalomyelitis (EAE) models. Test animals are actively immunized with an emulsion containing a myelin antigen [usually myelin oligodendrocyte glycoprotein (MOG)16,17, myelin basic protein (MBP)18, or proteolipid protein (PLP)19], together with a complete Freund&#39;s adjuvant (CFA)20. The disease can also be passively induced by an adoptive transfer of myelin-specific T-cells21. The disease course depends on the antigen/mouse strain combination that is used. MOG35-55 in C57BL/6, for instance, results in a monophasic chronic disease22, whilst PLP139-151 in Swiss Jim Lambert (SJL) mice leads to a relapsing-remitting disease course23 in a gender-specific manner24. Rat MOG35-55, further,induces an encephalitogenic T-cell response, whilst human MOG35-55 induces B-cell-dependent inflammation in C57BL/6 mice25. Various EAE models provide an excellent tool to study cell-mediated inflammation primarily in the spinal cord and cerebellum, but forebrain structures like the cortex, corpus callosum, and subcortical structures remain largely spared26. Neither diffuse white matter injury nor grey matter demyelination is, furthermore, adequately replicated in EAE models26,27. Cortical demyelination associated with active EAE induction has been reported in marmosets28,29 and in certain Lewis rat sub strains, in the latter case attributed to the prevailing combinations of MHC class I and class II isotypes and alleles30.
The cuprizone model31 is a useful tool for studying diffuse white matter demyelination and grey matter pathology with an extensive demyelination of the cortical, sub-cortical32, and hippocampal33 regions, as well as the corpus callosum34 and the caudate putamen35. Cuprizone intoxication, which, in principle, results in metabolic stress-induced apoptosis of the oligodendrocytes, mimics some characteristics of the cortical demyelinating lesions of MS brains such as a microglia activation, astrogliosis, and a relative lack of infiltrating peripheral immune cells. The lack of neuronal apoptosis and thalamic atrophy, as well as the complete resolution of demyelination with a robust remyelination observed upon the cessation of dietary cuprizone supplementation32, however, limit the cuprizone intoxication&#39;s usage as a preclinical MS model. Toxic demyelination can also be induced by a focal injection of lysolecithin or ethidium bromide into the white matter tracts36,37, but these methods are rarely used. Toxic demyelination models are especially suitable for the analysis of the complex mechanisms of remyelination, such as the requirement for oligodendrocyte progenitor cells and astrocytes38,39. Detailed information on EAE and intoxication models are provided in two recent reviews15,40.
The cytokine-induced demyelination was initially developed to study spinal white matter lesions in EAE41. Later, it was modified to study cortical grey matter pathologies in MS. Dark Agouti (DA) or Lewis rats are first primed by a sub-clinical immunization with MOG1-12542,43&#160;or MOG1-11644 in an incomplete Freund&#39;s adjuvant (IFA). Unlike classical EAE models, these primed animals do not exhibit clinical symptoms of focal inflammatory lesions in the spinal cord. Instead, an inflammatory response and demyelination in the brain are subsequently achieved by an intracerebral administration of a pro-inflammatory cytokine mixture [tumor necrosis factor alpha (TNF&#945;) and interferon gamma (IFN&#947;)] once the animals have attained a stable titer of anti-MOG antibodies in the blood.
Studies by Merkler et al.42 and Gardner et al.44 have proven the efficacy of a subpial cortical demyelination induction by a sub-clinical MOG immunization and an intracerebral or subarachnoidal cytokine injection. The reported duration of the demyelination was, however, too short-a complete remyelination occurred in 14 days or less, thereby limiting the window for any pharmacological intervention testing. Both models, moreover, utilize a traumatic injection modus, which could per se cause an injection trauma and a blood-brain barrier (BBB) breakdown and thus lead to an uncontrolled recruitment of inflammatory cells into the parenchyma. Both studies, furthermore, demonstrated demyelination that is restricted to a limited area, in the ipsilateral cortex, or in the close vicinity of the site of the cytokine injection.
To overcome these limitations, we implanted a catheter in the right parietal cortex of DA rats, with the catheter tip located just above the corpus callosum. To allow a full recovery of the BBB integrity, the animals were allowed a resting period of 2 weeks after the catheter implantation. Subsequently, the rats were sub-clinically immunized with 5 &#181;g of recombinant MOG1-125 in IFA. Following the attainment of a stable anti-MOG antibody titer after around 4 weeks, 2 &#181;L of cytokine mixture was injected via the catheter within 10 min using a programmable syringe pump. This procedure elicited a widespread cortical demyelination of both the ipsi- and the contralateral cerebral hemispheres in 15 days, with a partial remyelination around 30 days post-cytokine injection43. Multiple demyelination phases could, moreover, be induced by the repeated administration of pro-inflammatory cytokines through the catheter, and global brain atrophy, a common feature of progressive MS subtypes45, could be induced as early as after the second demyelination phase43. Importantly, the implanted catheter could also be used to test pharmacological interventions.
The protocol described below provides a detailed explanation of the experimental steps for a reproducible generation of widespread cortical demyelination in both cerebral hemispheres of DA rats using an intracerebral catheter.

<table><tbody><tr><td>Adult male Dark Agouti rats (300 &amp;plusmn;25 g)&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Fentanyl</td><td>Hameln pharma plus, Germany</td><td></td><td>&amp;nbsp;as Fentanyl-Citrate, 50 &amp;micro;g/ml</td></tr><tr><td>Midazolam</td><td>ERWO Pharma, Austria</td><td>50039017</td><td>5 mg/ml</td></tr><tr><td>Medetomidin&amp;nbsp;</td><td>Orion Pharma, Finland</td><td></td><td>as Medetomidin hydrochloride, 1mg/ml</td></tr><tr><td>Flumazenil&amp;nbsp;</td><td>Roche, Switzerland</td><td></td><td>0.1 mg/ml</td></tr><tr><td>Atipamezol&amp;nbsp;</td><td>Orion Pharma, Finland</td><td></td><td>as Atipamezol hydrochloride, 5 mg/ml</td></tr><tr><td>10% povidone-iodine complex&amp;nbsp;</td><td>Mundipharma, Austria</td><td></td><td></td></tr><tr><td>Dental cement&amp;nbsp;</td><td>Heraeus Kulzer, Germany</td><td>6603 7633</td><td></td></tr><tr><td>Physiological saline solution&amp;nbsp;</td><td>Fresenius Kabi, Austria</td><td></td><td>0.9% NaCl</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich, Germany</td><td>P3813</td><td></td></tr><tr><td>Isofluorane&amp;nbsp;</td><td>AbbVie, Austria</td><td></td><td></td></tr><tr><td>Lubricating eye drops&amp;nbsp;</td><td>Thea Pharma, Austria</td><td></td><td></td></tr><tr><td>70% EtOH</td><td>Merck, Germany</td><td>1070172511</td><td>Absolute ethanol was diluted in ddH&lt;sub&gt;2&lt;/sub&gt;O for preparation of 70% v/v</td></tr><tr><td>2.5% enrofloxacin</td><td>Bayer, Germany</td><td></td><td>Prophylactic antibiotics&amp;nbsp;</td></tr><tr><td>carprofen</td><td>Pfizer, USA</td><td></td><td>Painkillers, 50 mg/ml&amp;nbsp;</td></tr><tr><td>Tween-20&amp;nbsp;</td><td>Sigma-Aldrich, Germany</td><td>P9416</td><td></td></tr><tr><td>Pentobarbital&amp;nbsp;</td><td>Richter Pharma, Austria</td><td></td><td>pentobarbital sodium, 400 mg/ml</td></tr><tr><td>Interferon gamma&amp;nbsp;</td><td>PeproTech, USA</td><td>&amp;nbsp;400-20</td><td></td></tr><tr><td>Tumor necrosis factor alfa</td><td>R&amp;amp;D Systems, USA</td><td>510-RT-050/CF</td><td></td></tr><tr><td>rMOG&lt;sub&gt;1-125&lt;/sub&gt;</td><td>own product at the Centre of Molecular Medicine, Karolinska Institute, Sweden</td><td></td><td>Recombinant rat myelin oligodendrocyte glycoprotein, amino acids 1-125 from the N-terminus, also commercially available: AnaSpec, AS-55152-500, USA</td></tr><tr><td>Anti-MOG antibody&amp;nbsp;</td><td>Ana Spec/Kaneka Corporation, Japan</td><td>AS-555157</td><td>Standard from ELISA-Kit; Ana Spec/Kaneka&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Incomplete Freund&amp;rsquo;s adjuvant&amp;nbsp;</td><td>Sigma-Aldrich, Germany</td><td>&amp;nbsp;F5506</td><td></td></tr><tr><td>Horse radish peroxidase conjugated anti-rat IgG secondary antibody&amp;nbsp;</td><td>Ana Spec/Kaneka Corporation, Japan</td><td>AS-555157</td><td>Secondary Antibody from ELISA-Kit; Ana Spec/Kaneka Corporation</td></tr><tr><td>Bovine serum albumin&amp;nbsp;</td><td>Sigma-Aldrich, Germany</td><td>A9576</td><td></td></tr><tr><td>Peroxidase substrate solution</td><td>Vector Laboratories, USA</td><td>SK-45000</td><td></td></tr><tr><td>Stereotactic frame&amp;nbsp;</td><td>David Kopf Instruments, USA</td><td></td><td></td></tr><tr><td>Catheters, MRI suitable&amp;nbsp;</td><td>PlasticsOne, USA</td><td>8IC315GPKXXC</td><td></td></tr><tr><td>Dummy cannulas&amp;nbsp;</td><td>PlasticsOne, USA</td><td>8IC315DCNSPC</td><td></td></tr><tr><td>Plastic screws, MRI suitable&amp;nbsp;</td><td>PlasticsOne, USA</td><td>8L080X093N01</td><td></td></tr><tr><td>Connector cannula&amp;nbsp;</td><td>PlasticsOne, USA</td><td>8IC313CXSPCC</td><td></td></tr><tr><td>Screw driver with 2mm tip-size</td><td></td><td></td><td></td></tr><tr><td>Drill with flexible shaft extension&amp;nbsp;</td><td>Proxxon, Germany</td><td>NO 28 472, NO 28 706, NO 28 620,&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Drill bit, round, 0.5 mm&amp;nbsp;</td><td>Hager &amp;amp; Meisinger, Germany&amp;nbsp;</td><td>REF310 104 001 001 009</td><td></td></tr><tr><td>Drill bit, twisted, 1.3 mm&amp;nbsp;</td><td>Hager &amp;amp; Meisinger, Germany&amp;nbsp;</td><td>REF350 104 417 364 013</td><td></td></tr><tr><td>Scalpel&amp;nbsp;</td><td>Braun, Germany</td><td>BB510</td><td></td></tr><tr><td>Scalpel handle&amp;nbsp;</td><td>Fine Science Tools, Germany</td><td>91003-12</td><td></td></tr><tr><td>Cotton tip applicator&amp;nbsp;</td><td>Henry Schein Medical, Austria</td><td>900-3155</td><td></td></tr><tr><td>Surgical scissors</td><td>Fine Science Tools, Germany</td><td>14101-14, 14088-10&amp;nbsp;</td><td></td></tr><tr><td>Surgical forceps</td><td>Fine Science Tools, Germany</td><td>11002-12, 11251-35</td><td></td></tr><tr><td>Bulldog clamps&amp;nbsp;</td><td>Fine Science Tools, Germany</td><td>18050-35</td><td></td></tr><tr><td>Homoeothermic blanket&amp;nbsp;</td><td>TSE systems, Germany</td><td></td><td></td></tr><tr><td>Infrared Lamp</td><td>Beurer, Germany</td><td>616.51</td><td></td></tr><tr><td>Dental curing light&amp;nbsp;</td><td>Guilin Woodpecker Medical, China</td><td></td><td></td></tr><tr><td>Absorbable suture&amp;nbsp;</td><td>Johnson &amp;amp; Johnson, Belgium</td><td>V792E</td><td></td></tr><tr><td>Programmable syringe pump&amp;nbsp;</td><td>World Precision Instruments, USA</td><td>AL-1000</td><td></td></tr><tr><td>Exam gloves</td><td></td><td></td><td></td></tr><tr><td>Surgical gown</td><td></td><td></td><td></td></tr><tr><td>Electric Shaver</td><td>Aesculap, Germany</td><td>GT420</td><td></td></tr><tr><td>Volatile anesthetic vaporizer&amp;nbsp;</td><td>Rothacher Medical, Switzerland</td><td>CV 30-301-D</td><td></td></tr><tr><td>Oxygen source for volatile anesthetic vaporizer</td><td>Air Liquide, Austria</td><td>19,113</td><td></td></tr><tr><td>Volatile anesthesia chamber&amp;nbsp;</td><td>Rothacher Medical, Switzerland</td><td>PS-0347</td><td></td></tr><tr><td>Anesthesia mask for rats&amp;nbsp;</td><td>Rothacher Medical, Switzerland</td><td>PS-0307-A</td><td></td></tr><tr><td>1 ml syringe&amp;nbsp;</td><td>Codan, Denmark</td><td>REF 62.1612</td><td></td></tr><tr><td>26 Gauge needle for injection&amp;nbsp;</td><td>Braun, Germany</td><td>4657683</td><td></td></tr><tr><td>20 Gauge needle for cytokine injection and immunization&amp;nbsp;</td><td>Braun, Germany</td><td>4657519</td><td></td></tr><tr><td>Luer lock tip glass syringes&amp;nbsp;</td><td>Poulten &amp;amp; Graf, Germany</td><td>7.140-37</td><td></td></tr><tr><td>3 way stopcock&amp;nbsp;</td><td>Becton Dickinson, Sweden</td><td>394600</td><td></td></tr><tr><td>96-well plate&amp;nbsp;</td><td>Thermo Fisher Scientific, USA</td><td>442404</td><td></td></tr><tr><td>Plate reader&amp;nbsp;</td><td>Cole-Parmer, USA</td><td>EW-1396-00</td><td></td></tr><tr><td>37&amp;deg;C incubator</td><td>Kendro, Germany</td><td>50042301</td><td></td></tr><tr><td>Micropipettes&amp;nbsp;</td><td>Gilson, USA</td><td>F167350</td><td></td></tr></tbody></table>

rat model of widespread cerebral cortical demyelination induced by an intracerebral injection of pro-inflammatory cytokines

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&#39;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.

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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57879/57879fig1.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57879/57879fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57879/57879fig2.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57879/57879fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57879/57879fig3.jpg" /> 
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 &#181;g of rMOG is used in 200 &#181;L 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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57879/57879fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57879/57879fig4.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57879/57879fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57879/57879fig5.jpg" /> 
Figure 5: Intracerebral injection. (A) A programmable syringe pump is adjusted for a 2 &#181;L/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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57879/57879fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57879/57879fig6.jpg" /> 
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 &#181;m 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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57879/57879fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57879/57879fig7.jpg" /> 
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 &#181;m 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 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57879/57879fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/57879/57879fig8.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57879/57879fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines at JoVE.com

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines

The protocol presented here allows the reproduction of a widespread grey matter demyelination of both cortical hemispheres in adult male Dark Agouti rats. The method comprises of intracerebral implantation of a catheter, subclinical immunization against myelin oligodendrocyte glycoprotein, and intracerebral injection of a pro-inflammatory cytokine mixture through the implanted catheter.

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and ...

rat-model-widespread-cerebral-cortical-demyelination-induced-an

Nanyang Technological University

Research

JoVE Journal

Neuroscience

4.6K Views.  Medical University Graz. Het algemene doel van deze procedure is het genereren van een wijdverspreide demyelinisatie van de cortex van beide hersenhelften in een rattenmodel met behulp van subklinische immunisatie tegen myeline oligodendrocyt glycoproteïne, gevolgd door interesse cerebrale injectie van cytokinen via een geïmplanteerde katheter. Deze methode kan helpen bij het beantwoorden van belangrijke vragen bij inflammatoire demyelinerende ziekten van de hersenen, zoals multiple sclerose. Het belangrijkste voor...

Video: Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Immunology and Infection

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in myelin destruction, which not only slows conduction of nerve impulses, but causes axonal injury resulting in motor and cognitive decline. Current treatments for MS focus on attenuating immune cell infiltration into the central nervous system (CNS). These treatments decrease the number of relapses, improving quality of life, but do not completely eliminate relapses so long-term disability is not improved. Therefore, therapeutic agents that protect the CNS are warranted. In both animal models as well as human patients with MS, T cell entry into the CNS is generally considered the initiating inflammatory event. In order to assess if a drug protects the CNS, any potential effects on immune cell infiltration or proliferation in the periphery must be ruled out. This protocol describes how to determine whether CNS protection observed after drug intervention is a consequence of attenuating CNS-infiltrating immune cells or blocking death of CNS cells during inflammatory insults. The ability to examine MS treatments that are protective to the CNS during inflammatory insults is highly critical for the advancement of therapeutic strategies since current treatments reduce, but do not completely eliminate, relapses (i.e., immune cell infiltration), leaving the CNS vulnerable to degeneration.

This protocol describes how to determine whether pharmacological treatments for experimental autoimmune encephalomyelitis show CNS protection as a consequence of suppressing immune cell infiltration or are neuroprotective during the onslaught of immune cell infiltration.

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

Histological Staining of Spinal Cord for Assessing EAE Disease Severity

Scoring Central Nervous System Inflammation, Demyelination, and Axon Injury in Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a common immune-based model of multiple sclerosis (MS). This disease can be induced in rodents by active immunization with protein components of the myelin sheath and Complete Freund&#39;s adjuvant (CFA) or by the transfer of myelin-specific T effector cells from rodents primed with myelin protein/CFA into na&#239;ve rodents. The severity of EAE is typically scored on a 5-point clinical scale that measures the degree of ascending paralysis, but this scale is not optimal for assessing the extent of recovery from EAE. For example, clinical scores remain high in some EAE models (e.g., myelin oligodendrocyte glycoprotein [MOG] peptide-induced model of EAE) despite the resolution of inflammation. Thus, it is important to complement clinical scoring with histological scoring of EAE, which also provides a means to study the underlying mechanisms of cellular injury in the central nervous system (CNS).
Here, a simple protocol is presented to prepare and stain spinal cord and brain sections from mice and to score inflammation, demyelination, and axonal injury in the spinal cord. The method for scoring leukocyte infiltration in the spinal cord can also be applied to score brain inflammation in EAE. A protocol for measuring soluble neurofilament light (sNF-L) in the serum of mice using a Small Molecule Assay (SIMOA) assay is also described, which provides&#160;feedback on the extent of overall CNS injury in live mice.

Author Spotlight: Unveiling the Pathway Linking Obesity to Autoimmune Inflammation in Multiple Sclerosis 

Experimental autoimmune encephalomyelitis (EAE) serves as an animal model of multiple sclerosis. This article describes an approach for scoring spinal cord inflammation, demyelination, and axonal injury in EAE. Additionally, a method to quantify soluble neurofilament light levels in the mice serum is presented, facilitating the assessment of axonal injury in live mice.

Experimental autoimmune encephalomyelitis (EAE) is a common immune-based model of multiple sclerosis (MS). This disease can be induced in rodents by ...

A Stably Established Two-Point Injection of Lysophosphatidylcholine-Induced Focal Demyelination Model in Mice

Receptor-mediated lysophospholipid signaling contributes to the pathophysiology of diverse neurological diseases, especially multiple sclerosis (MS). Lysophosphatidylcholine (LPC) is an endogenous lysophospholipid associated with inflammation, and it could induce rapid damage with toxicity to myelin lipids, leading to focal demyelination. Here, a detailed protocol is presented for stereotactic two-point LPC injection that could directly cause severe demyelination and replicate the experimental demyelination injury quickly and stably in mice by surgical procedure. Thus, this model is highly relevant to demyelination diseases, especially MS, and it can contribute to the related advancing clinically-relevant research. Also, immunofluorescence and Luxol fast blue staining methods were used to depict the time course of demyelination in the corpus callosum of mice injected with LPC. In addition, the behavioral method was used to evaluate the cognitive function of mice after modeling. Overall, the two-point injection of lysophosphatidylcholine via a stereotaxic frame is a stable and reproducible method to generate a demyelination model in mice for further study.

The present protocol describes a two-point injection of lysophosphatidylcholine via a stereotaxic frame to generate a stable and reproducible demyelination model in mice.

Receptor-mediated lysophospholipid signaling contributes to the pathophysiology of diverse neurological diseases, especially multiple sclerosis (MS). ...

Induction and Evaluation of Experimental Autoimmune Encephalomyelitis in Mouse

Modeling Multiple Sclerosis in the Two Sexes: MOG35-55-Induced Experimental Autoimmune Encephalomyelitis

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease affecting the central nervous system (CNS). It is characterized by different prevalence in the sexes, affecting more women than men, and different outcomes, showing more aggressive forms in men than in women. Furthermore, MS is highly heterogeneous in terms of clinical aspects, radiological, and pathological features. Thus, it is necessary to take advantage of experimental animal models that allow the investigation of as many aspects of the pathology as possible. Experimental autoimmune encephalomyelitis (EAE) represents one of the most used models of MS in mice, modeling different disease features, from the activation of the immune system to CNS damage. Here we describe a protocol for the induction of EAE in both male and female C57BL/6J mice using myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) immunization, which leads to the development of a chronic form of the disease. We also report the evaluation of the daily clinical score and motor performance of these mice for 28 days post immunization (28 dpi). Lastly, we illustrate some basic histological analysis at the CNS level, focusing on the spinal cord as the primary site of disease-induced damage.

Author Spotlight: Creating a Versatile Experimental Autoimmune Encephalomyelitis Model Relevant for Both Male and Female Mice 

Experimental autoimmune encephalomyelitis is one of the most widely used murine models of multiple sclerosis. In the current protocol, C57BL/6J mice of both sexes are immunized with myelin oligodendrocyte glycoprotein peptide, resulting mainly in ascending paresis of the tail and limbs. Here we discuss the protocol of EAE induction and evaluation.

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease affecting the central nervous system (CNS). It is characterized by different ...

Role of CD4 T Cell in Relapsing-Remitting Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease characterized by the infiltration of immune cells and demyelination in the central nervous system (CNS). Experimental autoimmune encephalomyelitis (EAE) serves as a prototypic animal model for studying MS. In this study, we aimed to investigate the role of CD4 T cells in the initiation and relapse of EAE, focusing on the activation phase and immune response. To create the EAE mice model, female mice were immunized with myelin oligodendrocyte glycoprotein (MOG)35-55 emulsified with complete Freund&#39;s adjuvant (CFA). Clinical scores were assessed daily, and results demonstrated that mice in the EAE group exhibited a classic relapsing-remitting pattern. Hematoxylin-eosin (H&#38;E) and luxol fast blue (LFB) staining analysis revealed significant infiltration of inflammatory cells in the CNS and demyelination in EAE mice. Regarding the activation phase, both CD4+CD69+ effector T (Teff) cells and CD4+CD44+CD62L- effector memory T (Tem) cells may contribute to the initiation&#160;of EAE, however, the relapse stage was probably dominated by CD4+CD44+CD62L- Tem cells. Additionally, in terms of immune function, helper T (Th)1 cells are primarily involved in initiating the EAE. However, both Th1 and Th17 cells contribute to the relapse stage, and the immunosuppressive function of regulatory T (Treg) cells was inhibited during the EAE pathological process.

An experimental autoimmune encephalomyelitis (EAE) model in mice was employed to investigate the role of CD4 T cells in the initial and relapse of EAE from the perspective of the activation phase and immune effect function.

Multiple sclerosis (MS) is an autoimmune disease characterized by the infiltration of immune cells and demyelination in the central nervous system (CNS). ...

Stab-Wound Mouse Model for Studying Hemorrhage and Inflammation in Traumatic Brain Injury

Traumatic brain injury (TBI) results from physical damage, often caused by accidents or sports-related incidents. The causes of TBI are diverse, including concussions, brain contusions, hematomas, and skull fractures. To replicate these different causes, various TBI mouse models have been developed using distinct protocols. Physical brain injury leads to both primary and secondary brain injuries, which exacerbate neuronal loss. Primary injury occurs immediately after the damage, often due to hemorrhage, and subsequently triggers secondary injuries, including inflammation around the lesion. Developing a TBI model suitable for assessing hemorrhage extension and inflammatory severity is therefore crucial. This protocol introduces a method for mimicking penetrating brain injury, referred to as the stab-wound TBI mouse model, to study mechanisms of hemorrhage, inflammation, and neuronal loss associated with TBI pathology. This model is created by puncturing the skull and brain with needles and is simple to execute without the need for specialized experimental equipment. Additionally, the minor injury inflicted on the mouse cerebral cortex using a needle does not affect the animal's behavior post-surgery. This feature allows researchers to study the localized effects of brain injury without concerns about broader behavioral consequences. Sample data from stab-wounded mouse cerebral cortices demonstrate the model's effectiveness in assessing blood leakage into the parenchyma, glial activation, and inflammatory cytokine production. Furthermore, this protocol facilitates the evaluation of blood coagulants and anti-inflammatory compounds, aiding in the development of therapeutic agents for TBI.

Simplified traumatic brain injury (TBI) models have facilitated the development of therapeutic approaches. This protocol outlines the creation of a stab-wound mouse cortex using needles, enabling the analysis of hemorrhage and inflammation. The stab-wound TBI mouse model offers the advantage of being performed without requiring specialized equipment.

Traumatic brain injury (TBI) results from physical damage, often caused by accidents or sports-related incidents. The causes of TBI are diverse, including ...

Induction and Diverse Assessment Indicators of Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and axonal damage. Currently, there are no measures to cure MS completely, but multiple disease-modifying therapies (DMT) are available to control and mitigate disease progression. There are significant similarities between the CNS pathological features of experimental autoimmune encephalomyelitis (EAE) and MS patients. EAE has been widely used as a representative model to determine MS drugs' efficacy and explore the development of new therapies for MS disease. Active induction of EAE in mice has a stable and reproducible effect and is particularly suitable for studying the effects of drugs or genes on autoimmune neuroinflammation. The method of immunizing C57BL/6J mice with myelin oligodendrocyte glycoprotein (MOG35-55) and the daily assessment of disease symptoms using a clinical scoring system is mainly shared. Given the complex etiology of MS with diverse clinical manifestations, the existing clinical scoring system can't satisfy the assessment of disease treatment. To avoid the shortcomings of a single intervention, new indicators to assess EAE based on clinical manifestations of anxiety-like moods and osteoporosis in MS patients are created to provide a more comprehensive assessment of MS treatment.

The present protocol describes the induction of experimental autoimmune encephalomyelitis in a mouse model using myelin oligodendrocyte glycoprotein and monitoring the disease process using a clinical scoring system. Experimental autoimmune encephalomyelitis-related symptoms are analyzed using mouse femur micro-computed tomography analysis and open field test to assess the disease process comprehensively.

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and ...

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines

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