eoe sub articles

EoE Sub Articles

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.
<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).&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: See the respective&#160;Discussion&#160;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).&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	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 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.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	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.&#160;&#160;&#160;&#160;&#160; 
	NOTE: Follow all precautionary measures during surgery to 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&#160;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.&#160; 
	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.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: The anchor screws are necessary to stabilize the set-up by holding the dental cement and, thereby, the catheter in place. While 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 while 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.&#160;&#160;&#160;&#160;&#160;&#160; 
	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 (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 postoperative 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; 5 mg/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 while checking for leakage. If no leakage occurs, discard the PBS and remove piston 1 again.</li>
	<li>Pipette 1 mL of incomplete Freund&#39;s adjuvant (IFA) and 50 &#181;g of recombinant myelin oligodendrocyte glycoprotein (rMOG1-125)&#160;together and adjust the mixture to a final volume of 2 mL with sterile PBS (pH 7.4) in a suitable tube.&#160;&#160;&#160;&#160; 
	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&#160;mixture in the open syringe. Insert the piston gently while 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 isoflurane 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. Intracerebral Cytokine Injection
<ol>
	<li>Adjust the length of the connector cannula (2 mm). (See&#160;Figure 4&#160;for the preparation steps.)</li>
	<li>Fill a 1 mL syringe with the cytokine mixture (500 ng/&#181;L of tumor necrosis factor-alpha (TNF-alpha), 300 U/&#181;L of recombinant rat interferon-gamma (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.&#160; 
	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&#160;and&#160;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&#160;and&#160;5E).&#160;&#160;&#160; 
	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>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adult male Dark Agouti rats (300 &#177;25 g)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fentanyl</td>
			<td>Hameln pharma plus, Germany</td>
			<td></td>
			<td>As Fentanyl-Citrate, 50 &#181;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</td>
			<td>Orion Pharma, Finland</td>
			<td></td>
			<td>As Medetomidin hydrochloride, 1mg/ml</td>
		</tr>
		<tr>
			<td>Flumazenil</td>
			<td>Roche, Switzerland</td>
			<td></td>
			<td>0.1 mg/ml</td>
		</tr>
		<tr>
			<td>Atipamezol</td>
			<td>Orion Pharma, Finland</td>
			<td></td>
			<td>As Atipamezol hydrochloride, 5 mg/ml</td>
		</tr>
		<tr>
			<td>10% povidone-iodine complex</td>
			<td>Mundipharma, Austria</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dental cement</td>
			<td>Heraeus Kulzer, Germany</td>
			<td>6603 7633</td>
			<td></td>
		</tr>
		<tr>
			<td>Physiological saline solution</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</td>
			<td>AbbVie, Austria</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lubricating eye drops</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 ddH2O for preparation of 70% v/v</td>
		</tr>
		<tr>
			<td>2.5% Enrofloxacin</td>
			<td>Bayer, Germany</td>
			<td></td>
			<td>Prophylactic antibiotics</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Pfizer, USA</td>
			<td></td>
			<td>Painkillers, 50 mg/ml</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital</td>
			<td>Richter Pharma, Austria</td>
			<td></td>
			<td>Pentobarbital sodium, 400 mg/ml</td>
		</tr>
		<tr>
			<td>Interferon gamma</td>
			<td>PeproTech, USA</td>
			<td>&#160;400-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Tumor necrosis factor alfa</td>
			<td>R&#38;D Systems, USA</td>
			<td>510-RT-050/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>rMOG1-125</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>Incomplete Freund&#8217;s adjuvant</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>&#160;F5506</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</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</td>
			<td>David Kopf Instruments, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Catheters, MRI suitable</td>
			<td>PlasticsOne, USA</td>
			<td>8IC315GPKXXC</td>
			<td></td>
		</tr>
		<tr>
			<td>Dummy cannulas</td>
			<td>PlasticsOne, USA</td>
			<td>8IC315DCNSPC</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic screws, MRI suitable</td>
			<td>PlasticsOne, USA</td>
			<td>8L080X093N01</td>
			<td></td>
		</tr>
		<tr>
			<td>Connector cannula</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</td>
			<td>Proxxon, Germany</td>
			<td>NO 28 472, NO 28 706, NO 28 620,</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill bit, round, 0.5 mm</td>
			<td>Hager &#38; Meisinger, Germany</td>
			<td>REF310 104 001 001 009</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill bit, twisted, 1.3 mm</td>
			<td>Hager &#38; Meisinger, Germany</td>
			<td>REF350 104 417 364 013</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Braun, Germany</td>
			<td>BB510</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>Fine Science Tools, Germany</td>
			<td>91003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton tip applicator</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</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</td>
			<td>Fine Science Tools, Germany</td>
			<td>18050-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Homoeothermic blanket</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</td>
			<td>Guilin Woodpecker Medical, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbable suture</td>
			<td>Johnson &#38; Johnson, Belgium</td>
			<td>V792E</td>
			<td></td>
		</tr>
		<tr>
			<td>Programmable syringe pump</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</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</td>
			<td>Rothacher Medical, Switzerland</td>
			<td>PS-0347</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia mask for rats</td>
			<td>Rothacher Medical, Switzerland</td>
			<td>PS-0307-A</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>Codan, Denmark</td>
			<td>REF 62.1612</td>
			<td></td>
		</tr>
		<tr>
			<td>26 Gauge needle for injection</td>
			<td>Braun, Germany</td>
			<td>4657683</td>
			<td></td>
		</tr>
		<tr>
			<td>20 Gauge needle for cytokine injection and immunization</td>
			<td>Braun, Germany</td>
			<td>4657519</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer lock tip glass syringes</td>
			<td>Poulten &#38; Graf, Germany</td>
			<td>7.140-37</td>
			<td></td>
		</tr>
		<tr>
			<td>3 way stopcock</td>
			<td>Becton Dickinson, Sweden</td>
			<td>394600</td>
			<td></td>
		</tr>
		<tr>
			<td>37&#176;C incubator</td>
			<td>Kendro, Germany</td>
			<td>50042301</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes</td>
			<td>Gilson, USA</td>
			<td>F167350</td>
			<td></td>
		</tr>
	</tbody>
</table>

generating the widespread cerebral cortical demyelination in a rat model

Source: &#220;&#231;al, M. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/57879">Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines.</a> 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.

<img alt="Figure 1" src="/files/ftp_upload/22250/22250_Figure1.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, t...

Generating the Widespread Cerebral Cortical Demyelination in a Rat Model

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Begin with an emulsion of recombinant myelin sheath protein with an adjuvant.
Inject this mixture into the subcutaneous layer at the tail of an anesthetized rat, which has a pre-positioned catheter in the brain.
The adjuvant enhances immune responses to antigens in the subcutaneous layer and lymph nodes, generating effector T cells that enter the circulation.
Using the pre-implanted catheter, administer pro-inflammatory cytokines directly into the brain.
These cytokines spread in the cerebral cortex and disrupt the endothelial cell junctions in the blood vessels.
This allows the entry of various immune cells into the cerebral cortex.
Monocytes differentiate into macrophages, releasing reactive oxygen species that cause myelin sheath-producing oligodendrocyte death.
B cells interact with effector T cells, differentiating the B cells that secrete antibodies against myelin sheath protein.
These antibodies interact with myelin sheath protein, damaging the myelin sheath around the neurons.
This results in widespread cerebral cortical demyelination in a rat model.

generating-widespread-cerebral-cortical-demyelination-rat

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Model Development and Disease Study

94 Views. 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, result...

Video: Generating the Widespread Cerebral Cortical Demyelination in a Rat Model - Concept

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) ...

JoVE Journal

Immunology and Infection

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). ...

Neuroscience

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 ...

Generating the Widespread Cerebral Cortical Demyelination in a Rat Model

Transcript

Generating the Widespread Cerebral Cortical Demyelination in a Rat Model

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

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

Induction and Diverse Assessment Indicators of Experimental Autoimmune Encephalomyelitis