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. EAE induction
<ol>
	<li>Anesthetize C57BL/6 mice with isofluorane using a vaporizer system. To induce anesthesia in mice, expose the mouse to 4.5% isofluorane in oxygen in a small incubation chamber before transferring the mouse to a facemask with 2.1% isofluorane.&#160;Use an anesthesia unit equipped with a heating pad to prevent hypothermia of the mouse.</li>
	<li>Fix the anesthetized mouse in one hand and first inject 30 &#181;L of myelin oligodendrocyte glycoprotein (MOG)-peptide aa35-55 (MOG35-55)/ complete Freund&#39;s adjuvant (CFA)-emulsion subcutaneously into hind leg flanks (Figure 1: 1 + 2; in total 60 &#181;L; left flank 30 &#181;L and right flank 30 &#181;L) in close proximity to the inguinal lymph nodes.</li>
	<li>Place the mouse on its belly and inject 20 &#181;L of MOGaa35-55/CFA-emulsion into the left and right soft fatty tissue at the tail root (Figure 1: 3 + 4; in total 40 &#181;L; left side tail root 20 &#181;L and right side tail root 20 &#181;L) and a little droplet of MOGaa35-55/CFA-emulsion into the neck of the mouse (Figure 1: 5).</li>
	<li>Inject 100 &#181;L of Pertussis toxin (PTx) solution intraperitoneally into the mouse. Hold the mouse&#39;s head below the body center to avoid injection into the intestine.&#160;</li>
	<li>Replace maintenance diet (extrudate major nutrients: crude protein 18.5%; crude fat 4.5%; crude fiber 4.5%; crude ash 6.5%; starch 35%; metabolic energy: 13.1 MJ/kg) to breeding diet (extrudate major nutrients: crude protein 23.5%; crude fat 5.5%; crude fiber 3%; crude ash 5.7%; starch 36%; metabolic energy: 14.3 MJ/kg) in order to provide the mice with food of higher energy content before and during the expected clinical disease.&#160;</li>
	<li>Remove the face mask and transfer the mouse to its home cage with a warming pad.&#160;After 10 minutes, make sure the mouse is fully awake and motile.&#160;</li>
	<li>Repeat the PTx injection (step 1.4) 48 hours after the first treatment.</li>
</ol>
2. Scoring of EAE mice
<ol>
	<li>Check the health status of EAE mice every morning by taking a look inside the cages.</li>
	<li>Score EAE mice every afternoon.</li>
	<li>Take every individual mouse included in the EAE experiment out of the cage and check whether the tail has tonus by moving it upward with a finger. A healthy mouse will keep its tail up (the tail has a tonus). If clinical EAE has started, the tail tonus will be lower, visible by a gradual drop of the tail. Eventually, the mouse will not be able to lift its tail at all.&#160;</li>
	<li>Place every individual mouse included in the EAE experiment on the clean bench and observe and document the walking behavior. See&#160;Table 1&#160;for scoring criteria for the assessment of disease severity (the EAE score).</li>
	<li>Assess and document the weight of every mouse included in the experiment.</li>
	<li>To ensure adequate food and water uptake by mice displaying a clinical EAE score of 1 supply moistened food in a plastic dish on the bottom of the cage and refresh daily.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Breeding food</td>
			<td>e.g. PROVIMI KLIBA SA</td>
			<td>3336</td>
			<td></td>
		</tr>
		<tr>
			<td>Individually ventilated cages, Blue line Type II or III</td>
			<td>e.g. Tecniplast</td>
			<td>1145T, 1285L</td>
			<td></td>
		</tr>
		<tr>
			<td>Female C57BL/6J mice (8-12 weeks)</td>
			<td>e.g. Janvier Labs</td>
			<td></td>
			<td>Females, 8-12 weeks</td>
		</tr>
		<tr>
			<td>18G x 1&#189;&#39;&#39; (1.2mm x 40mm) injection needle</td>
			<td>e.g. BD, BD Microlance 3</td>
			<td>304622</td>
			<td></td>
		</tr>
		<tr>
			<td>27G x &#190;&#39;&#39; &#8211; Nr. 20 (0.4mm x 19mm) injection needle</td>
			<td>e.g. BD, BD Microlance 3</td>
			<td>302200</td>
			<td></td>
		</tr>
		<tr>
			<td>30G x &#189;&#39;&#39; (0.3 mm x 13 mm) injection needle</td>
			<td>e.g. BD, BD Microlance 3</td>
			<td>304000</td>
			<td></td>
		</tr>
		<tr>
			<td>Incomplete Freund&#39;s adjuvant (IFA)</td>
			<td>e.g. Santa Cruz Biotechnology</td>
			<td>sc-24648</td>
			<td>Store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Maintenance food</td>
			<td>e.g. PROVIMI KLIBA SA</td>
			<td>3436</td>
			<td></td>
		</tr>
		<tr>
			<td>MOGaa35-55 peptide</td>
			<td>e.g. GenScript</td>
			<td></td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr>
			<td>Mycobacterium tuberculosis H37RA</td>
			<td>e.g. BD</td>
			<td>231141</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>NaCl 0.9 %</td>
			<td>B. Braun</td>
			<td>3535789</td>
			<td></td>
		</tr>
		<tr>
			<td>Omnican 50 30G x &#189;&#39;&#39;</td>
			<td>B. Braun</td>
			<td>9151125S</td>
			<td></td>
		</tr>
		<tr>
			<td>Pertussis toxin</td>
			<td>e.g. List biological laboratories, Inc.</td>
			<td>180</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Stitch scissor</td>
			<td>F.S.T</td>
			<td>15012-12</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe 1 ml</td>
			<td>e.g. PRIMO</td>
			<td>62.1002</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe 10 ml</td>
			<td>e.g. CODAN Medical ApS</td>
			<td>2022-05</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of experimental autoimmune encephalomyelitis in a mouse model

Source: Tietz, S. M. et al. <a href="https://app.jove.com/v/59249">Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo.</a> J. Vis. Exp.&#160; (2019)
This video demonstrates the method of inducing experimental autoimmune encephalomyelitis (EAE) by injecting myelin-derived peptides with an adjuvant to activate autoreactive T cells, which migrate to the brain. Pertussis toxin is administered to increase blood-brain barrier permeability, allowing these T cells and other immune cells to enter the brain. These immune cells cause demyelination, leading to impaired nerve transmission and development of EAE.

<img alt="Figure 1" src="/files/ftp_upload/22986/22986_Figure1.jpg" /> 
Figure 1: Graphical representation of injection sites for EAE induction.&#160;1) Injection site for 30 &#181;L MOG-emulsion into the right flank, 2) injection site for 30 &#181;L MOG-emulsion into the left flank, 3) injection site for 20 &#181;L MOG-emulsion into the left tail root, 4) injection site for 30 &#181;L MOG-emulsion into the right tail root, 5) injection site for a little droplet of M...

Generation of Experimental Autoimmune Encephalomyelitis in a Mouse Model

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

Inject the myelin-derived peptides with an adjuvant near the peripheral lymph nodes of an anesthetized mouse.
The peptide-adjuvant conjugates activate the antigen-presenting cells, or APCs, which recognize and process the peptides.
The APCs then migrate to the lymph nodes and activate the autoreactive T cells.
Periodically inject the pertussis toxin intraperitoneally, which disrupts the tight junctions between the endothelial cells and increases the blood-brain barrier permeability.
The autoreactive T cells circulate in the blood and cross the blood-brain barrier to enter the brain tissue.
These cells recognize the peptides on the myelin sheath and release the cytokines.
These cytokines recruit macrophages and B cells to the site.
Macrophages release reactive oxygen species that damage the myelin-producing oligodendrocytes, while the B cells interact with T cells, become activated, and produce autoantibodies targeting the myelin sheath.
This leads to demyelination, impairing nerve transmission.
Lift the mouse tail; a gradual drop indicates nerve damage, signaling the onset of autoimmune encephalomyelitis, or EAE.

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Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Cerebrovascular Diseases

72 Views. Źródło: Tietz, S. M. et al. Wizualizacja upośledzenia barier śródbłonkowych i glejowych jednostki nerwowo-naczyniowej podczas eksperymentalnego autoimmunologicznego zapalenia mózgu i rdzenia kręgowego In Vivo. J. Vis. Exp.(2019 rok) Ten film demonstruje metodę wywoływania eksperymentalnego autoimmunologicznego zapalenia mózgu i rdzenia (EAE) poprzez wstrzykiwanie peptydów pochodzących z mieliny z adiuwantem w celu aktywacji autoreaktywnych limfocytów T, które migrują do mózgu....

Video: Generowanie eksperymentalnego autoimmunologicznego zapalenia mózgu i rdzenia kręgowego w modelu mysim - Concept

Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice

Experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a model for relapsing-remitting multiple sclerosis (RRMS). Clinical EAE scores describing motor function deficits are basic readouts of the immune-mediated inflammation of the spinal cord. However, scores and body weight do not allow for an in vivo assessment of brain inflammation and optic neuritis. The latter is an early and frequent manifestation in about 2/3 of MS patients. Here, we show methods for bioluminescence and near-infrared live imaging to assess EAE evoked optic neuritis, brain inflammation, and blood-brain barrier (BBB) disruption in living mice using an in vivo imaging system. A bioluminescent substrate activated by oxidases primarily showed optic neuritis. The signal was specific and allowed the visualization of medication effects and disease time courses, which paralleled the clinical scores. Pegylated fluorescent nanoparticles that remained within the vasculature for extended periods of time were used to assess the BBB integrity. Near-infrared imaging revealed a BBB leak at the peak of the disease. The signal was the strongest around the eyes. A near-infrared substrate for matrix metalloproteinases was used to assess EAE-evoked inflammation. Auto-fluorescence interfered with the signal, requiring spectral unmixing for quantification. Overall, bioluminescence imaging was a reliable method to assess EAE-associated optic neuritis and medication effects and was superior to the near-infrared techniques in terms of signal specificity, robustness, ease of quantification, and cost.

We show a technique for in vivo live bioluminescence and near-infrared imaging of optic neuritis and encephalitis in the experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis in SJL/J mice.

Experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a model for relapsing-remitting multiple sclerosis (RRMS). Clinical EAE scores describing ...

JoVE Journal

Medicine

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological deficits and disability as the disease progresses. B lymphocytes play a complex and critical role in MS pathology and are the target of several therapeutics in clinical trials. Currently, there is no way to accurately select patients for specific anti-B cell therapies or to non-invasively quantify the effects of these treatments on B cell load in the CNS and peripheral organs. Positron emission tomography (PET) imaging has enormous potential to provide highly specific, quantitative information regarding the in vivo spatiotemporal distribution and burden of B cells in living subjects.
This paper reports methods to synthesize and employ a PET tracer specific for human CD19+ B cells in a well-established B cell-driven mouse model of MS, experimental autoimmune encephalomyelitis (EAE), which is induced with human recombinant myelin oligodendrocyte glycoprotein 1-125. Described here are optimized techniques to detect and quantify CD19+ B cells in the brain and spinal cord using in vivo PET imaging. Additionally, this paper reports streamlined methods for ex vivo gamma counting of disease-relevant organs, including bone marrow, spinal cord, and spleen, together with high-resolution autoradiography of CD19 tracer binding in CNS tissues.

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

This paper details methodology for radiolabeling a human-specific anti-CD19 monoclonal antibody and how to use it to quantify B cells in the central nervous system and peripheral tissues of a mouse model of multiple sclerosis using in vivo PET imaging, ex vivo gamma counting, and autoradiography approaches.

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological ...

Generation of Experimental Autoimmune Encephalomyelitis in a Mouse Model

Transkrypcja

Generation of Experimental Autoimmune Encephalomyelitis in a Mouse Model