This work was supported by the Interdisciplinary Center for Clinical Research (IZKF) M&#252;nster (SEED 03/12, SB).

1. General Comments for Mouse Experiments
<ol>
	<li>All experiments using mice should be performed in accordance with the guidelines of the respective institutional animal care and use committee.</li>
	<li>Keep the mice under pathogen-free conditions and enable them to have access to food and water ad libitum. Note: It is important to use age- and sex-matched mice in experimental groups because susceptibility to disease can vary with age and gender.</li>
	<li>Depending on the chosen experimental conditions, a sham-immunized control group can be considered where MOG35-55 peptide is replaced by either PBS without antigen or a nonencephalitogenic peptide.</li>
	<li>Please consider methodological aspects prior to starting experiments (see also below). We recommend to involve one or two blinded observers for EAE scoring.</li>
</ol>
2. Preparation of MOG35-55 Emulsion
<ol>
	<li>200 &#181;l of a 1:1 ratio of MOG35-55 peptide solution and CFA should be injected to each mouse. There is some loss of the viscous emulsion while preparing and injecting. Therefore, prepare 1.5-2x&#160;of the needed amount. Calculate the total volume of the emulsion and divide by 2 for the needed amounts of both MOG35-55 peptide solution and CFA.</li>
	<li>Dilute lyophilized MOG35-55 in ddH2O to a final concentration of 2 mg/ml. We usually use 200 &#181;g MOG35-55 peptide per mouse. This amount is contained in 100 &#181;l of the stock solution. The peptide solution should be stored at -20 &#176;C.</li>
	<li>Place the content of 1 vial of desiccated Mycobacterium tuberculosis H37RA (100 mg) into a mortar.
	<ol>
		<li>Ground with mortar and pestle to obtain a thin powder.</li>
		<li>Add 10 ml of incomplete Freund&#39;s adjuvants to obtain a 10 mg/ml CFA stock solution which can be stored at 4 &#176;C.</li>
		<li>Prior to immunization, dilute CFA stock solution with IFA to a final concentration of 2 mg/ml. Mix thoroughly before each use to resuspend particulate material and consider some volume loss of the viscous solution during the experimental preparations.</li>
		<li>Mix 1:1 with MOG35-55 peptide solution until the final concentration of 1 mg/ml is reached. 
		CAUTION: Heat-killed Mycobacterium tuberculosis stimulates the innate immune response. Avoid inhalation, ingestion and contact with skin and eyes.</li>
	</ol>
	</li>
	<li>Precool solutions on ice.
	<ol>
		<li>Draw up 1 ml of 2 mg/ml CFA and 1 ml of 2 mg/ml MOG35-55 solution into two 2 ml syringes. Calculate the number of syringes necessary according to the number of immunized animals. 
		CAUTION: Strictly avoid stitching during preparation of adjuvant as it may cause granulomas or induce autoimmune reactions.</li>
		<li>Use a 27 G cannula for the MOG35-55 solution and a 20 G cannula for CFA. Avoid air bubbles and connect both syringes with a three-way-valve.</li>
		<li>Send the emulsion from one syringe to the other and mix thoroughly for at least 10 min as a good emulsification is a critical step. Near-close the three-way-valve to support emulsification. The solution should be white, stiff and viscous with no separation of phases.</li>
		<li>Emulsion can be stored for several days prior to immunization. Wait at least 30 min after preparing the emulsions to observe whether they are stable. Prior to immunization, draw the solution into one of two syringes and connect a 27 G cannula.</li>
	</ol>
	</li>
</ol>
3. Preparation of Pertussis Toxin
<ol>
	<li>Different amounts of pertussis toxin can be found in the literature which also depends on the route of administration (e.g. intravenously or intraperitoneally). Our laboratory uses 400 ng of pertussis toxin in 200 &#181;l of PBS intraperitoneally at the day of immunization and two days later. CAUTION: Pertussis toxin has many biological effects. Avoid inhalation, ingestion, and contact with skin and eyes.</li>
	<li>Reconstitute 50 &#181;g of pertussis toxin in 500 &#181;l ddH2O for a 100 &#181;g/ml stock solution. Store at 4 &#176;C.</li>
	<li>Dilute 1:50 with PBS. 200 &#181;l now contain 400 ng of PBS. Prepare the necessary amount of syringes and consider an additional excess volume of ~100 &#181;l for each needle hub.</li>
</ol>
4. Animal Immunization
<ol>
	<li>Please refer to the institutional animal care and use committee for specific criteria for required short-term euthanasia. Our laboratory uses brief narcotization with isoflurane while other protocols such as ketamine/xylazine injection or halothan may also be possible. Wait for anesthesia and use a front foot toe pinch to assess the level of anesthesia.</li>
	<li>Make sure that immunization is performed by an experienced person to minimize stress for animals and to ensure optimal immunization.</li>
	<li>Inject 100 &#181;l of antigen/CFA emulsion subcutaneously into two different sites on each hind flank. Ensure that a bulbous mass forms under the skin which should persist throughout the experiment.</li>
	<li>Inject 200 &#181;l of pertussis toxin intraperitonelly.</li>
	<li>Ensure that individual mice can easily be identified for daily evaluation, e.g. by color markings on the tail base.</li>
	<li>Administer a second dose of pertussis toxin on day two after immunization.</li>
</ol>
5. EAE Monitoring
<ol>
	<li>Weight and clinical score should be evaluated daily. The onset of EAE typically correlates with weight loss, which can be used as an indicator of disease activity. Please refer to the institutional animal care and use committee to predefine criteria when individual mice have to be taken out of the experiments. This should be considered when weight loss exceeds 20% of the initial body weight or when severe clinical signs (EAE score 7 or worse) occur. Please refer to the respective guidelines of the respective institutional animal care and use committee for allowed maximum scores.</li>
	<li>When mice have clinical symptoms of EAE, it is important to ensure that the water bottle can still be reached and that food is placed on the cage floor.</li>
	<li>Different scoring symptoms can be used. In our laboratory a 0-10 scale is established.</li>
</ol>
<table border="1">
	<tbody>
		<tr>
			<td>Grade</td>
			<td>Clinical sign</td>
			<td>Comment</td>
		</tr>
		<tr>
			<td>0</td>
			<td>No clinical signs</td>
			<td>Normal gait, tail moves and can be raised, tail wraps around a round object if mouse is held at the base of the tail</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Partially limp tail</td>
			<td>Normal gait, tip of the tail droops</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Paralyzed tail</td>
			<td>Normal gait, tail droops</td>
		</tr>
		<tr>
			<td>3</td>
			<td>Hind limb paresis, uncoordinated movement</td>
			<td>Uncoordinated gait, tail limps, hind limbs respond to pinching</td>
		</tr>
		<tr>
			<td>4</td>
			<td>One hind limb paralyzed</td>
			<td>Uncoordinated gait with one hind limb dragging, tail limps, one hind limb does not respond to pinch</td>
		</tr>
		<tr>
			<td>5</td>
			<td>Both hind limbs paralyzed</td>
			<td>Uncoordinated gait with both hind limbs dragging, tail limps, both hind limbs do not respond to pinch</td>
		</tr>
		<tr>
			<td>6</td>
			<td>Hind limbs paralyzed, weakness in forelimbs</td>
			<td>Uncoordinated gait with forelimbs struggle to pull body, forelimbs reflex after pinching, tail limps</td>
		</tr>
		<tr>
			<td>7</td>
			<td>Hind limbs paralyzed, one forelimb paralyzed</td>
			<td>Mouse cannot move, one forelimb responds to toe pinch, tail limps</td>
		</tr>
		<tr>
			<td>8</td>
			<td>Hind limbs paralyzed, both forelimbs paralyzed</td>
			<td>Mouse cannot move, both forelimbs do not respond to toe pinch, tail limps</td>
		</tr>
		<tr>
			<td>9</td>
			<td>Moribund</td>
			<td>No movement, altered breathing</td>
		</tr>
		<tr>
			<td>10</td>
			<td>Death</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1. Clinical scoring system.

<ol>
	<li>McFarland, H. F., Martin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+sclerosis:+a+complicated+picture+of+autoimmunity.">Multiple sclerosis: a complicated picture of autoimmunity.</a> Nat. Immunol. 8, 913-919 (2007).</li><li>Wiendl, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroinflammation:+the+world+is+not+enough.">Neuroinflammation: the world is not enough.</a> Curr. Opin. Neurol. 25, 302-305 (2012).</li><li>vander Star, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+models+of+multiple+sclerosis.">In vitro and in vivo models of multiple sclerosis.</a> CNS Neurol. Disord. Drug Targets. 11, 570-588 (2012).</li><li>Pachner, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+of+multiple+sclerosis.">Experimental models of multiple sclerosis.</a> Curr. Opin. Neurol. 24, 291-299 (2011).</li><li>Bittner, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TASK1+channel+inhibitor+A293+shows+efficacy+in+a+mouse+model+of+multiple+sclerosis.">The TASK1 channel inhibitor A293 shows efficacy in a mouse model of multiple sclerosis.</a> Exp. Neurol. 238, 149-155 (2012).</li><li>Handel, A. E., Lincoln, M. R., Ramagopalan, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+mice+and+men:+experimental+autoimmune+encephalitis+and+multiple+sclerosis.">Of mice and men: experimental autoimmune encephalitis and multiple sclerosis.</a> Eur. J. Clin. Invest. 41, 1254-1258 (2011).</li><li>Krishnamoorthy, G., Wekerle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EAE:+an+immunologist's+magic+eye.">EAE: an immunologist's magic eye.</a> Eur. J. Immunol. 39, 2031-2035 (2009).</li><li>Stromnes, I. M., Goverman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+induction+of+experimental+allergic+encephalomyelitis.">Active induction of experimental allergic encephalomyelitis.</a> Nat. Protoc. 1, 1810-1819 (2006).</li><li>Fletcher, J. M., Lalor, S. J., Sweeney, C. M., Tubridy, N., Mills, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cells+in+multiple+sclerosis+and+experimental+autoimmune+encephalomyelitis.">T cells in multiple sclerosis and experimental autoimmune encephalomyelitis.</a> Clin. Exp. Immunol. 162, 1-11 (2010).</li><li>Mendel, I., Kerlero de Rosbo, N., Ben-Nun, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+myelin+oligodendrocyte+glycoprotein+peptide+induces+typical+chronic+experimental+autoimmune+encephalomyelitis+in+H-2b+mice:+fine+specificity+and+T+cell+receptor+V+beta+expression+of+encephalitogenic+T+cells.">A myelin oligodendrocyte glycoprotein peptide induces typical chronic experimental autoimmune encephalomyelitis in H-2b mice: fine specificity and T cell receptor V beta expression of encephalitogenic T cells.</a> Eur. J. Immunol. 25, 1951-1959 (1995).</li><li>Hofstetter, H. H., Shive, C. L., Forsthuber, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pertussis+toxin+modulates+the+immune+response+to+neuroantigens+injected+in+incomplete+Freund's+adjuvant:+induction+of+Th1+cells+and+experimental+autoimmune+encephalomyelitis+in+the+presence+of+high+frequencies+of+Th2+cells.">Pertussis toxin modulates the immune response to neuroantigens injected in incomplete Freund's adjuvant: induction of Th1 cells and experimental autoimmune encephalomyelitis in the presence of high frequencies of Th2 cells.</a> J. Immunol. 169, 117-125 (2002).</li></ol>

The authors declare no competing financial interests.

A multitude of different EAE models with active immunization protocols has been described over the last decades. While rat models have been widely used until recently, mice are now the most popular model organism for EAE research. This development is among others due to the broad and ever increasing repertoire of available transgenic mice. Immunization of C57BL/6 mice with MOG35-55 peptide is one of the most widely distributed EAE models and can be considered as a reliable, replicable and well-to-use animal model. In many neuroimmunological laboratories, MOG35-55 induced EAE is established as the model of choice while other EAE models are used for more specific experimental questions.
A critical point to consider is the planning of the experimental settings to ensure that EAE experiments are performed methodologically correct. For internal validity, blinded scoring of disease symptoms is highly recommended. Experimental groups should be age-, weight-, and sex-matched and mice should be randomly allocated to treatment groups. Experiments should always be performed in compliance with animal welfare regulations. EAE studies are often underpowered and do not take into account statistical type II errors. Therefore, prior to experiments, sample size calculations should be performed. Necessary group sizes depend on the expected effect size. Consultation of an expert for statistical analysis might be considered before starting EAE experiments.
Some limitations of the protocol need to be kept in mind. Most importantly, interpretation of aEAE data is compromised by the mode of immunization with the use of adjuvant and pertussis toxin which have both additional influence on the immunological reaction. It should also be considered that the MOG35-55 EAE model shows mainly a CD4+ T cell driven immunological response. CD8+ T cells and B cells play a less prominent role and alternative protocols should be considered when addressing these cell types. The expected disease course is acute, monophasic and self-limited. Alternatively, a relapsing-remitting disease course can also be achieved in alternative EAE models. An additional important limitation of the protocol is a certain bias towards the immunological component of the MS pathophysiology. During the last years, it has become increasingly clear that MS has a strong neurodegenerative component. The death of oligodendrocytes and neurons results in a progressive accumulation of neurological deficits. It must be taken into account that the EAE model may not be fully suited to address experiment questions concerning neurodegenerative mechanisms of autoimmune inflammation. Alternative animal models with a focus on CNS pathology might be considered &#8211; e.g. the cuprizone model which compromises toxic demyelination without involvement of the peripheral immune system.
The described protocol be considered as a basic neuroimmunological experimental model and may be modified for other applications. The experimental procedure described above can be easily applied to other EAE protocols by varying mice strains or the type and amount of protein (e.g. use PLP139-151 peptide and SJL mice for a relapsing-remitting EAE disease course which is especially suited for assessing therapeutic effects on relapses). The described protocol can also be used for adoptive-transfer experiments (passive EAE). In this model, C57BL/6 mice are immunized with MOG35-55 peptide and CFA as described above. In contrast, pertussis toxin is not required. After 7-15 days, spleens or lymph nodes are isolated and immune cells are restimulated in vitro with MOG35-55 peptide and various cytokines prior to transfer into a new group of C57BL/6se mice. These recipient mice develop EAE a few days earlier than upon classical immunization. In vitro conditions can be varied for specific immunological questions (e.g. polarization into TH1 or TH17 cells).
Sometimes, low disease incidence or weak symptoms might be an experimental challenge. Some recommendations for troubleshooting are:
- Disease severity can be varied with different amounts of peptide / mouse.
- Optimal CFA concentration may vary from 1-5 mg/ml. Consider a titration of CFA when establishing the experiments. Please refer to the respective guidelines of the respective institutional animal care and use committee for allowed CFA concentrations as many regulations forbid CFA concentrations exceeding 2 mg/ml.
- Different methods are described for preparing the emulsion. Alternative methods such as vortexing for 1 hr or sonication might be considered if poor emulsification is considered as possible error source.
- Age, gender, season of the year and environmental conditions within the animal facility are important factors that influence EAE susceptibility. It should be ensured that conditions are comparable between independent experiments.
As described above, the mentioned protocol can be used as starting point for adoptive EAE experiments. This model is especially suited for separating peripheral and CNS effects of a genetic phenotype (e.g. by transferring encephalitogenic knockout cells into wildtype recipient mice) and for specific immunological questions as the phenotype of the transferred cells can be characterized thoroughly. The latest development in EAE research during the last years are T cell receptor transgenic mice. These mice develop EAE symptoms spontaneously without external influence circumventing the problem of adjuvant inoculation. However, this model requires large amounts of animals for breeding to ensure sufficient group sizes. Evaluation of knockout mice requires crossbreeding prior to EAE experiments in contrast to aEAE. As each mouse develops disease symptoms on a different day, evaluation of novel substances can be rather complicated. Therefore, the value of classical aEAE for neuroimmunological remains unchallenged.

Multiple sclerosis (MS) is a chronic demyelinating inflammatory disease of the central nervous system wherein the destruction of oligodendrocytes and neurons results in heterogenic and accumulating clinical symptoms. MS is considered as a prototypic autoimmune disorder of the central nervous system (CNS) and animal models have been developed to shed light on its complex pathogenesis. Furthermore, current therapies are only partially effective and target mainly the inflammatory phase of the disease while the neurodegenerative component is probably the major challenge for future therapeutic approaches1,2.
While the exact etiology of the disease is yet unclear, an autoimmune reaction against epitopes on the myelin sheath of the axons in the CNS is assumed to provoke the onset of the disease. Dysregulation of the immune system, genetic vulnerability and environmental factors (e.g. infections, Vitamin D) are believed to influence central aspects of the pathophysiological mechanisms of MS.
Three different types of animal models are currently established for the exploration of pathologic patterns of MS: Viral models like Theiler&#39;s murine encephalomyelitis virus (TMEV), models induced by toxic agents like cuprizone, and finally different variants of experimental autoimmune encephalomyelitis (EAE)3,4. Although they all mimic features of MS, they differ tremendously in underlying pathological features like the involvement of the adaptive immune system. EAE is the most common animal model as it is especially useful to investigate neuroinflammatory pathways and often serves as a "proof-of-principle" model for the efficacy of novel treatment strategies5,6. EAE can be induced in many different animals (e.g. mice, rats, miniswine, guinea pigs, chickens, or primates). However, mice have become the most widely use species which is at least partly due to expanding repertoire of sophisticated transgenic or knockout mice7.
The pathophysiology of EAE is based on the reaction of the immune system against brain-specific antigens. This reaction induces inflammation and destruction of the antigen carrying structures, resulting in neurological and pathological features comparable which those observed in MS patients. Three different approaches can be distinguished: Actively-induced EAE (aEAE; active immunization), passively transferred EAE (pEAE; transfer of encephalitogenic cells from an immunized animal), and more recently spontaneous EAE mouse models (sEAE) which allow the study of autoimmune mechanisms without exogenous manipulation. The easiest inducible model is aEAE in mice yielding in fast and robust results. This model is considered as the "gold standard" of neuroimmunological animal models by many researchers in the field8.
For aEAE induction, the animal is immunized with a subcutaneous injection of an emulsion consisting of the chosen antigen and complete Freund&#39;s adjuvans (CFA) accompanied by an intraperitoneal injection of pertussis toxin on the day of immunization and two days later. Consequently, myelin-specific T lymphocytes are activated in the periphery and migrate into the CNS across the blood-brain-barrier. Upon entry into the CNS, T cells are reactivated by local and infiltrating antigen-presenting cells resulting in subsequent inflammatory cascades, involvement of other cells like monocytes or macrophages and eventually in demyelination and axonal cell death9. Depending upon the immunization protocol and combination of mouse strain (e.g. C57BL/6, SJL/J, Biozzi) and antigen (e.g. myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), myelin proteolipid protein (PLP)), the disease course can take an acute, chronic progressive or relapsing remitting disease course.
C57BL/6 mice have become the most commonly used strain for transgenic mice construction and a growing multitude of knockout or transgenic mice is available. We here describe a protocol for immunization of C57BL/6 mice with MOG35-55 peptide10 which results in a monophasic EAE with first symptoms after 9-14 days, disease maximum about 3-5 days after disease onset and slow and partial symptom recovery over the next 10-20 days. As the immunogenic potential of MOG35-55 peptide alone is not sufficient to induce disease, adjuvants such as CFA are necessary. It is assumed, that the components of CFA activate mononuclear phagocytes inducing the phagocytosis of these molecules and the secretion of cytokines. This results in the prolongation of the presence of antigens and a more efficient transport of these to the lymphatic system. EAE induction is facilitated by application of pertussis toxin (PT) which has among others been suggested to modulate the blood-brain barrier and the immunological responsiveness itself11. After disease induction, special care must be taken for daily evaluation of mice for disease symptoms.

<table border="0" cellpadding="0" cellspacing="0" width="707">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Female C57BL/6 mice, age 10-12 weeks, ~20 g weight</td>
			<td style="width:205px;">e.g. Jackson Laboratory, Charles River</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MOG35-55 (MEVGWYRSPFSRVVHLYRNGK)</td>
			<td style="width:205px;">e.g. Pepceuticals Ltd</td>
			<td style="width:119px;"></td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Incomplete Freund&#8217;s adjuvant (IFA)</td>
			<td style="width:205px;">e.g. Sigma-Aldrich Co.</td>
			<td style="width:119px;">F5506</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Syringes, 1 ml with 26G3/8 needle&#160;</td>
			<td style="width:205px;">e.g. Becton Dickinson &#38; Co.</td>
			<td style="width:119px;">309625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringes, 2 ml</td>
			<td style="width:205px;">e.g. B. Braun</td>
			<td style="width:119px;">7389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Needles, 25G5/8 and 30G1/2&#160;</td>
			<td style="width:205px;">e.g. Becton Dickinson &#38; Co.,</td>
			<td style="width:119px;">305122, 305106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Three way halve</td>
			<td style="width:205px;">e.g. B. Braun</td>
			<td style="width:119px;">16494 C</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pertussis toxin, lyophilized in buffer</td>
			<td style="width:205px;">Enzo Life Sciences Inc.</td>
			<td style="width:119px;">BML-G100</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">M. tuberculosis H37 RA</td>
			<td style="width:205px;">BD Difco&#160;</td>
			<td style="width:119px;">231141</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
	</tbody>
</table>

myelin oligodendrocyte glycoprotein (mog35-55) induced experimental autoimmune encephalomyelitis (eae) in c57bl/6 mice

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While the exact etiology of the disease is yet unclear, autoreactive T lymphocytes are thought to play a central role in its pathophysiology. MS therapy is only partially effective so far and research efforts continue to expand our knowledge on the pathophysiology of the disease and to develop novel treatment strategies. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for MS sharing many clinical and pathophysiological features. There is a broad diversity of EAE models which reflect different clinical, immunological and histological aspects of human MS. Actively-induced EAE in mice is the easiest inducible model with robust and replicable results. It is especially suited for investigating the effects of drugs or of particular genes by using transgenic mice challenged by autoimmune neuroinflammation. Therefore, mice are immunized with CNS homogenates or peptides of myelin proteins. Due to the low immunogenic potential of these peptides, strong adjuvants are used. EAE susceptibility and phenotype depends on the chosen antigen and rodent strain. C57BL/6 mice are the commonly used strain for transgenic mouse construction and respond among others to myelin oligodendrocyte glycoprotein (MOG). The immunogenic epitope MOG35-55 is suspended in complete Freund's adjuvant (CFA) prior to immunization and pertussis toxin is applied on the day of immunization and two days later. Mice develop a "classic" self-limited monophasic EAE with ascending flaccid paralysis within 9-14 days after immunization. Mice are evaluated daily using a clinical scoring system for 25-50 days. Special considerations for care taking of animals with EAE as well as potential applications and limitations of this model are discussed.

After immunization, mice have to be evaluated daily for changes in weight and clinical symptoms. Disease onset is typically correlated with a reduction of weight which might begin 1-2 days before EAE symptoms are visible. Clinical signs of EAE usually begin between day 9 and 14 post-immunization. As lesions are predominantly localized to the spinal cord in MOG-EAE in C57BL/6 mice, they typically develop predominantly motoric symptoms in a caudal to rostral pattern. An exemplary picture of an immunized mouse with EAE symptoms (score 6) is depicted in Figure 1A. Some atypical EAE symptoms might also occur such as rolling in axial-rotary manner, hunched appearance or hypersensitivity which are not reflected within the classical EAE score depicted in Table 1. As motor symptoms are the main characteristic in this model, this score does give us a good indication of disease severity. Different monitoring systems (e.g. score 0-5 or 0-6) and more complex composite scores assessing different deficits have also been published. Please note that mice with severe symptoms have to be taken out of the experiment according to the respective institutional animal care and use committee. Mice show generally partial recovery of symptoms over the next 10-20 days. A representative disease course is depicted in Figure 1B. There are different time points during EAE which are of interest for assessing outcome parameters. Some typical assays which can often be found are described very briefly. At disease maximum, mice can be evaluated for cytokine production or proliferation after restimulation of isolated immune cells with MOG35-55 peptide in vitro (MOG recall assay). Immune cells can also be isolated from the brain and spinal cords from mice with EAE symptoms and further processed, e.g. by flow cytometry. Histological analysis of spinal cord sections can be performed at disease maximum for lesion load and demyelination, while at later time points markers for neurodegeneration and neuronal cell death might be of interest.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51275/51275fig1highres.jpg" src="/files/ftp_upload/51275/51275fig1.jpg" width="600px" /> 
Figure 1. Representative EAE results. A.&#160;Representative picture of an immunized C57BL/6 mouse with EAE symptoms (EAE score 6). B. Representative disease course after immunization over 30 days. Data are shown as mean and standard error of the mean (n = 5). <a href="https://www.jove.com/files/ftp_upload/51275/51275fig1highres.jpg" target="_blank">Click here to view larger image</a>.

Watch this Scientific Journal Video about Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice at JoVE.com

Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

Experimental autoimmune encephalomyelitis (EAE) is an established animal model of multiple sclerosis. C57BL/6 mice are immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (MOG35-55), resulting in an ascending flaccid paralysis caused by autoreactive immune cells in the central nervous system. Protocols for disease induction and monitoring will be discussed.

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

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JoVE Journal

Immunology and Infection

81.4K Views.  University of Münster.  Экспериментальный аутоиммунный энцефаломиелит (ЕАЕ) является признанным животной модели рассеянного склероза. C57BL / 6 мышей иммунизируют миелина олигодендроцитов гликопротеина (MOG) пептида 35-55 (MOG 35-55), в результате чего восходящей периферического парал?...

Video: Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 &mu;g/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 &mu;g/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

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) ...

Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses targeting myelin. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model of CNS autoimmune disease, and is typically induced via immunization with short peptides representing immunodominant CD4+ T cell epitopes of myelin proteins. However, B cells recognize unprocessed protein directly, and immunization with short peptide does not activate B cells that recognize the native protein. As recent clinical trials of B cell-depleting therapies in MS have suggested a role for B cells in driving disease in humans, there is an urgent need for animal models that incorporate B cell-recognition of autoantigen. To this end, we have generated a new fusion protein containing the extracellular domain of the mouse version of myelin oligodendrocyte glycoprotein (MOG) as well as N-terminal fusions of a His-tag for purification purposes and the thioredoxin protein to improve solubility (MOGtag). A tobacco etch virus (TEV) protease cleavage site was incorporated to allow the removal of all tag sequences, leaving only the pure MOG1-125 extracellular domain. Here, we describe a simple protocol using only standard laboratory equipment to produce large quantities of pure MOGtag or MOG1-125. This protocol consistently generates over 200 mg of MOGtag protein. Immunization with either MOGtag or MOG1-125 generates an autoimmune response that includes pathogenic B cells that recognize the native mouse MOG.

We describe a simple protocol using only basic lab equipment to generate and purify large quantities of a fusion protein that contains mouse Myelin Oligodendrocyte Glycoprotein. This protein can be used to induce experimental autoimmune encephalomyelitis driven by both T and B cells.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses ...

Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with embedded pericytes, and the glia limitans composed by the parenchymal basement membrane and astrocytic end-feed embracing the abluminal aspect of central nervous system (CNS) microvessels. In addition to maintaining CNS homeostasis the NVU controls immune cell trafficking into the CNS. During immunosurveillance of the CNS low numbers of activated lymphocytes can cross the endothelial barrier without causing BBB dysfunction or clinical disease. In contrast, during neuroinflammation such as in multiple sclerosis or its animal model experimental autoimmune encephalomyelitis (EAE) a large number of immune cells can cross the BBB and subsequently the glia limitans eventually reaching the CNS parenchyma leading to clinical disease. 
Immune cell migration into the CNS parenchyma is thus a two-step process that involves a sequential migration across the endothelial and glial barrier of the NVU employing distinct molecular mechanisms. If following their passage across the endothelial barrier, T cells encounter their cognate antigen on perivascular antigen-presenting cells their local reactivation will initiate subsequent mechanisms leading to the focal activation of gelatinases, which will enable the T cells to cross the glial barrier and enter the CNS parenchyma. Thus, assessing both, BBB permeability and MMP activity in spatial correlation to immune cell accumulation in the CNS during EAE allows to specify loss of integrity of the endothelial and glial barriers of the NVU. 
We here show how to induce EAE in C57BL/6 mice by active immunization and how to subsequently analyze BBB permeability in vivo using a combination of exogenous fluorescent tracers. We further show, how to visualize and localize gelatinase activity in EAE brains by in situ zymogaphy coupled to immunofluorescent stainings of BBB basement membranes and CD45+ invading immune cells.

Here, we present protocols to investigate impairment of the neurovascular unit during experimental autoimmune encephalomyelitis in vivo. We specifically address how to determine blood-brain barrier permeability and gelatinase activity involved in leukocyte migration across the glia limitans.&#160;

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

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 ...

Immunogenicity Enhancement by &lt;em&gt;Mycobacterium paratuberculosis&lt;/em&gt; in Autoimmune Encephalomyelitis

Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in complete Freund&#39;s adjuvant (CFA) containing inactivated Mycobacterium tuberculosis. The antigenic components of the mycobacterium activate dendritic cells to stimulate T-cells to produce cytokines that promote the Th1 response via toll-like receptors. Therefore, the amount and species of mycobacteria present during the antigenic challenge are directly related to the development of EAE. This methods paper presents an alternative protocol to induce EAE in C57BL/6 mice using a modified incomplete Freund&#39;s adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis strain K-10.
M. paratuberculosis, a member of the Mycobacterium avium complex, is the causative agent of Johne&#39;s disease in ruminants and has been identified as a risk factor for several human T-cell-mediated disorders, including multiple sclerosis. Overall, mice immunized with Mycobacterium paratuberculosis showed earlier onset and greater disease severity than mice immunized with CFA containing the strain of M. tuberculosis H37Ra at the same doses of 4 mg/mL. The antigenic determinants of Mycobacterium avium subspecies paratuberculosis (MAP)&#160;strain K-10 were able to induce a strong Th1 cellular response during the effector phase, characterized by significantly higher numbers of T-lymphocytes (CD4+ CD27+), dendritic cells (CD11c+ I-A/I-E+), and monocytes (CD11b+ CD115+) in the spleen compared to mice immunized with CFA. Furthermore, the proliferative T-cell response to the MOG peptide appeared to be highest in M. paratuberculosis-immunized mice. The use of an encephalitogen (e.g., MOG35-55) emulsified in an adjuvant containing M. paratuberculosis in the formulation may be an alternative and validated method to activate dendritic cells for priming myelin epitope-specific CD4+ T-cells during the induction phase of EAE.

Author Spotlight: Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis  

Here, we present an alternative protocol to actively induce experimental autoimmune encephalomyelitis in C57BL/6 mice, using the immunogenic epitope myelin oligodendrocyte glycoprotein (MOG)35-55 suspended in incomplete Freund's adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis.

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in ...

Preparation and Injection of Myelin Oligodendrocyte Glycoprotein (MOG35-55) Emulsion

Preparation and Injection of Myelin Oligodendrocyte Glycoprotein (MOG35-55) Emulsion  

Begin by grinding 10 milligrams of freeze-dried Mycobacterium paratuberculosis to a fine powder using a mortar and pestle. Add five milliliters of Incomplete Freund's adjuvant to obtain a 10 milligram per milliliter stock solution. Store the stock at minus four degrees Celsius.Before immunization, dilute the stock solution. Then, dilute the MOG 35-55 peptide to two milligrams per milliliter and store it at minus 20 degrees Celsius. Mix equal volumes of the MOG 35-55 with the adjuvant in a five milliliter tube with a homogenizer until a thick emulsion is formed.After every 10 seconds of mixing, place the solution on ice for 20 seconds and spin the tube to recover all the solution. Transfer the emulsion into a one milliliter syringe, ensuring it is free of air bubbles. Then, fit it with a 27-gauge needle.Subcutaneously, inject 200 microliters of the emulsion into the lower back of an anesthetized mouse. Then, intraperitoneally inject a dose of pertussis toxin on day zero and two after immunization. All mice manifested an acute monophasic disease, characterized by a single peak of disability observed at 14 to 17 days, followed by a partial recovery of symptoms over the next 10 days.Mice immunized with M.paratuberculosis showed an earlier onset after immunization and greater severity in the acute phase. Both groups demonstrated similar body weights. Proliferation assay with titrated thymidine incorporation performed on the EAE mice spleen cells showed a strong proliferative response to the peptide MOG 35-55, but not to ovalbumin.Cytofluorometric analysis showed increased T-lymphocytes, dendritic cells, and monocytes in the spleen of M.paratuberculosis-immunized EAE mice compared to CFA-immunized mice. Hematoxylin and and eosin tissue analysis revealed typical paravascular and meningeal mononuclear inflammatory infiltration in the brain and spinal cord.

Investigating Humoral Response in Autoimmune Demyelination

Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological and molecular levels. EAE is induced by immunizing experimental animals via subcutaneous injection of short myelin peptides together with specific adjuvants to boost the immune response. Like the human counterpart, EAE mice develop demyelinating lesions, immune cell infiltration into the central nervous system (CNS), glia activation and neuronal injury. A consistent body of evidence also supports a mechanistic role for B cell dysfunction in the etiology of both MS and EAE. B cells can serve as antigen-presenting cells as well as a primary source of pro-inflammatory cytokines and autoantibodies. In EAE, antibodies are generated against the myelin peptides that were employed to induce the disease. Such autoantibodies have been shown to mediate either myelin loss or pathogenic T cell reactivation into the CNS. This article describes an efficient ELISA-based protocol to quantify autoantibodies in the serum of C57BL/6J mice immunized with the myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) peptide. The proposed method serves as a powerful tool to investigate the specificity and magnitude of the aberrant humoral response in the context of autoimmune demyelination.

Author Spotlight: Novel Assay for Studying B-Cell Responses in Multiple Sclerosis Research

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), which shares with the human disease a robust humoral autoimmune response. Here, we report a simple and flexible ELISA protocol to quantify autoantibodies in the serum of EAE immunized mice.

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological ...