Name: Author Spotlight: Creating a Versatile Experimental Autoimmune Encephalomyelitis Model Relevant for Both Male and Female Mice
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This work has been supported by Ministero dell&#39;Istruzione, dell&#39;Universit&#224; e della Ricerca - MIUR project Dipartimenti di Eccellenza 2018-2022 and 2023-2027 to Department of Neuroscience Rita Levi Montalcini; Cavalieri-Ottolenghi Foundation, Orbassano, Italy. BB was fellow of INFRA-P, Piedmont Region (n.378-35) (2022-2023) and PRIN 2020 - 20203AMKTW. We thank Fondazione per la Ricerca Biomedica Onlus (FORB) for the support. The publication fees have been supported by the kind donation of Distretto Rotaract 2031, and particularly, Rotaract Club Torino Nord-Est. We thank Elaine Miller for the proofreading of our manuscript.

The animal care and handling in the present work was performed according to the European Union Council Directive of 22nd September 2010 (2010/63/UE); all the procedures reported in the present study were approved by the Italian Ministry of Health (407/2018-PR) and by the Ethical Committee of the University of Torino (Project n&#176; 360384). We suggest conforming to the experimental design to the ARRIVE guidelines originally published by Kilkenny et al. in 201020. Before starting, ensur...

Induction and Evaluation of Experimental Autoimmune Encephalomyelitis in Mouse

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The MOG35-55-induced EAE protocol that we described led to the development of a chronic form of MS in C57BL/6J mice7,8,13. In these representative results, we reported that the animals of both sexes that underwent the immunization procedure developed a chronic form of the disease (i.e., they do not fully recover after the disease onset, they accumulate deficits, and maintain a CS at least of 1.5 in the chronic phase)....

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65778">Modeling Multiple Sclerosis in the Two Sexes: MOG35-55-Induced Experimental Autoimmune Encephalomyelitis</a>. The Authors section was updated. An additional affiliation (School of Pharmacy, Pharmacology Unit, University of Camerino) was added for author Antonino Casile.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65778">Modeling Multiple Sclerosis in the Two Sexes: MOG35-55-Induced Experimental Autoimmune Encephalomyelitis</a>. The Authors section was updated.

Erratum: 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 shows the presence of perivascular infiltration of inflammatory cells, demyelination, axonal loss, and gliosis1. Its etiology remains unknown, and its clinical aspects, radiographic, and pathological features suggest remarkable heterogeneity in the disease2.
Due to its unknown etiology and complexity, at present, no animal model recapitulates all the clinical and radiological features displayed in human MS3,4. However, various animal models are employed to study different aspects of MS3,4. In these models, disease initiation is typically extremely artificial, and the timeframe of the onset of clinical signs is different between humans and mice. For example, in humans, the pathophysiological processes underlying the disease are undetected for years before the onset of clinical manifestations. Conversely, the experimenters can detect symptoms in animal models within weeks or even days after MS induction4.
Three basic animal models produce the features of demyelination that are characteristic of MS: those that are virus-induced (e.g., Theiler&#39;s murine encephalomyelitis virus), those that are induced by toxic agents (e.g., cuprizone, lysolecithin), and the different variants of experimental autoimmune encephalomyelitis (EAE)5. Each model helps study some specific facets of the disease but none replicates all the features of MS6. Thus, it is critical to choose the correct model considering the specific experimental needs and the scientific questions to be addressed.
Thanks to immunization procedures against myelin-derived antigens, EAE is induced by triggering an autoimmune response to CNS components in susceptible mice. The interplay between a wide range of immunopathological and neuropathological mechanisms causes the development of principal pathological traits of MS (i.e., inflammation, demyelination, axonal loss, and gliosis) in the immunized mice7,8. Mice begin to show clinical symptoms around the second week after immunization and generally show ascending paralysis from the tail to the limb and forelimb. The clinical score (i.e., quantification of the accumulation of disease-related deficits) is generally assessed using a 5-point scale7.
Active immunization with protein or peptide or passive transfer of encephalitogenic T cells can be used to induce EAE in mice with different genetic backgrounds (e.g., SJL/J, C57BL/6, and non-obese-diabetic (NOD) mice). Myelin proteolipid protein (PLP), myelin basic protein (MBP), and myelin oligodendrocyte glycoprotein (MOG) are examples of self-CNS proteins from which immunogens are usually produced. Particularly, SJL/J mice immunized with the immunodominant epitope of PLP (PLP139&#8722;151) develop a relapsing-remitting (RR) disease course, while C57BL/6J mice immunized with the immunodominant MOG35-55 peptide show EAE of a chronic nature1. Despite some limitations, such as providing very little information about MS progression, the role of B cells in the disease, the inside-out mechanisms, or difficulties in studying remyelination, the EAE models have hugely contributed to the understanding of autoimmune and neuroinflammatory processes, increasing the knowledge in the MS field and thus allowing the development of novel therapeutic approaches for this disease4,6.
In the present work, we focused on a particular form of active EAE, the myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55)-induced form9,10,11,12. The MOG35-55-induced EAE models a chronic form of MS. After immunization, the mice undergo an asymptomatic phase within the first week after immunization, then the disease typically arises during the second week after immunization, while between the third and fourth weeks after immunization, the disease becomes chronic, with no possibility of full recovery from the accumulated deficits7,8,13. Interestingly, no differences between males and females in incidence, disease onset, course, or progression are observed in most of the studies present in the literature14, even if fewer studies compare the disease in males and females.
In contrast, in humans, these parameters are known to be strongly sexually dimorphic2. MS affects more women than men; however, men generally develop a more aggressive form of the disease2. This evidence has suggested an essential, as well as complex, role of the gonadal hormones15; nevertheless, the role and the mechanism of action of sex hormones in the pathology remain unclear. Moreover, data from animal models support the idea that both estrogens and androgens exert positive effects on different tracts of the pathology in a sex-specific manner16,17.
Some studies also suggest neuroprotective, promyelinating, and anti-inflammatory effects of progesterone18 and, although evidence in MS patients is scarce18, neuroactive steroids (i.e., de novo synthetized steroids by the nervous system, such as pregnenolone, tetrahydroprogesterone, and dihydroprogesterone) might also affect the pathological course19. Collectively, these data support the idea that sex hormones produced both peripherally and inside the CNS have an important and sex-specific role in disease onset and progression. Therefore, in the present work, we urge the collection of separate data from both male and female animals.
From the histopathological point of view, the white matter of the spinal cord serves as the principal site of CNS injury in this model, which is characterized by multifocal, confluent regions of mononuclear inflammatory infiltration and demyelination8. Thus, in describing this protocol for the induction of MOG35-55-induced EAE in C57BL/6J mice, we will take into account the disease outcome in the two sexes and provide some histopathological insights regarding the spinal cords.

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			<td>18 G x 1 &#189;&#8220; 1.2 x 40 mm needle for the glass syringe&#160;</td>
			<td>Terumo</td>
			<td>TER-HYP-18G-112-PIN</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital camera connected to the optical microscope</td>
			<td>NIKON DS-U1 digital camera</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic precision balance</td>
			<td>Merck</td>
			<td>Mod. Kern-440-47N, resolution 0.1 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y</td>
			<td>Sigma-Aldrich</td>
			<td>HT110216</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass syringe pipet &#8220;ultra asept&#8221; 10 ml</td>
			<td>Sacco System&#160;</td>
			<td>L003465</td>
			<td></td>
		</tr>
		<tr>
			<td>Glassware (i.e., becker to prepare the emulsion)</td>
			<td>VWR</td>
			<td>213-1170, 213-1172</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin (Mayer&#8217;s)</td>
			<td>Sigma-Aldrich</td>
			<td>MHS32</td>
			<td>Filter before using it.&#160;</td>
		</tr>
		<tr>
			<td>Image analysis Software</td>
			<td>Fiji</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incomplete Freund&#8217;s adjuvant (IFA)</td>
			<td>Sigma-Aldrich</td>
			<td>F5506</td>
			<td>Store at +4 &#176;C.&#160;</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Wellona Pharma</td>
			<td></td>
			<td>This drug is used as inhalational anaesthetic.</td>
		</tr>
		<tr>
			<td>Male and female C57BL/6J mice</td>
			<td>Jackson Laboratory, Envigo</td>
			<td></td>
			<td>Age 8-10 weeks, optimal body weight of ~20 g.&#160;</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica HistoCore BIOCUT R</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting Medium&#160;</td>
			<td>Merck</td>
			<td>107961</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Rotarod</td>
			<td>Ugo Basile&#160;</td>
			<td>#47600</td>
			<td></td>
		</tr>
		<tr>
			<td>Mycobacterium tuberculosis (MT), strain H37Ra&#160;</td>
			<td>Difco Laboratories Inc.&#160;</td>
			<td>231141</td>
			<td>Store at +4 &#176;C.</td>
		</tr>
		<tr>
			<td>Myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55)</td>
			<td>Espikem</td>
			<td>EPK1</td>
			<td>Store at -80 &#176;C diluted (2 mg/mL) in physiological solution; prepare it on the day of the immunization to avoid, as much as possible, alterations or contaminations.&#160;</td>
		</tr>
		<tr>
			<td>Optical microscope</td>
			<td>NIKON eclipse 90i</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td>Store at +4 &#176;C once diluted (4%) in phosphate buffer.&#160;</td>
		</tr>
		<tr>
			<td>Pertussis toxin (PT)</td>
			<td>Duotech&#160;</td>
			<td>PT.181</td>
			<td>Store at -80&#176;C diluted (concentration 5 &#181;g/mL) in physiological solution&#160;</td>
		</tr>
		<tr>
			<td>Physiological solution (sodium chloride 0.9% solution)</td>
			<td>B. Eurospital</td>
			<td>A 032182038</td>
			<td>Store at +4 &#176;C once opened.</td>
		</tr>
		<tr>
			<td>Saline phosphate buffer (PBS)</td>
			<td>Thermo Scientific</td>
			<td>J61196.AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Software for image acquisition</td>
			<td>&#160;NIS-Element AR 2.10</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes U-100 0.5 mL with 30 G x 5/16&#8221; (0.30 x 8 mm) in fixed needle</td>
			<td>&#160;Nipro</td>
			<td>SYMS-0.5U100-3008B-EC</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes U-100 1 mL with 26G x &#189;&#8221; (0.45 x 12.7 mm) in needle</td>
			<td>PIC</td>
			<td>20,71,26,03,00,354</td>
			<td></td>
		</tr>
		<tr>
			<td>Vet ointment for eyes</td>
			<td>Lacrilube, Lacrigel Europhta</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Rompun</td>
			<td></td>
			<td>This mixture of drug is used as injectable anaesthetic and sedative.&#160;</td>
		</tr>
		<tr>
			<td>Zolazepam and Tiletamine</td>
			<td>Zoletil&#160; 100</td>
			<td></td>
			<td>This drug is used as injectable anaesthetic, sedative, muscle relaxer, and analgesic</td>
		</tr>
	</tbody>
</table>

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 

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

Our research focuses on the experimental autoimmune encephalomyelitis, or EAE model, which can significantly announce the understanding of peripheral immune-mediated mechanism and evaluate neuroinflammatory and partially delineating processes. This model has also been widely used to develop and test a wide range of therapeutics drugs. Due to the unknown etiology and complexity of multiple sclerosis, no animal model currently recapitulates all the clinical and radiological features displayed in humans.Thus, it is quite challenging for the researchers to select the animal model most fitting for a specific experimental question. EAE is a widely used model of multiple sclerosis. However, to do several issue, only some studies performing this model considered both sexes.With our protocol, we want a light possibility of including both sexes in the study conducted in this model. Specifically, our protocol proposed a clinical evaluation based of two methods:the clinical score assessment and the rotator test, leading a more quantitative, less subjective and more precise clinical assessment of the disease course. Even though this model does not entirely reproduce the sexual dimorphism of multiple sclerosis, it has potential advantages.The combination of the induction with other possible risk factor, especially the environmental ones, can end in understanding specific effects of such factors and identifying the specific role in some sexually dimorphic aspect of the disease.

EAE follow-up after immunization 
This was assessed as described below.
Body weight and food intake 
The two-way analysis of variance (ANOVA) (sex and time as independent variables) shows a decrease in the BW of EAE animals of both sexes, especially within the second week post induction (F(1,57) = 4.952, p &#60; 0.001; Figure 2A). However, the sexual dimorphism in BW is always maintained (<st...

Watch this Scientific Journal Video about Creating a Versatile Experimental Autoimmune Encephalomyelitis Model Relevant for Both Male and Female Mice at JoVE.com

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.