JG was awarded a UCL Impact Studentship and Rosetrees Trust funding to support CB. VC was in receipt of a research collaborative grant from Akari Therapeutics Inc. We would like to thank UCL Institute of Ophthalmology, Biological Service Unit especially Ms Alison O&#39;Hara and her team for their technical support.

All experiments were performed in accordance with the UK Animals (Scientific Procedures) Act of 1986, and institutional Animal Welfare and Ethical Review Body (AWERB) guidelines.
1. Housing C57BL/6J mice
<ol>
	<li>House mice in a specific pathogen free environment, on a 12 hour light-dark cycle and food and water available ad libitum.</li>
	<li>Perform all experiments on adult female C57BL/6J (females are preferentially chosen as there is an incidence of women to men 1.4 to 1 in uve...

<ol>
	<li>Lyu, C., 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=TMP778,+a+selective+inhibitor+of+RORgammat,+suppresses+experimental+autoimmune+uveitis+development,+but+affects+both+Th17+and+Th1+cell+populations.">TMP778, a selective inhibitor of RORgammat, suppresses experimental autoimmune uveitis development, but affects both Th17 and Th1 cell populations.</a> The European Journal of Immunology. 48, 1810-1816 (2018).</li><li>Klaska, I. P., Forrester, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+autoimmune+uveitis.">Mouse models of autoimmune uveitis.</a> Current Pharmaceutical Design. 21, 2453-2467 (2015).</li><li>Eskandarpour, M., Alexander, R., Adamson, P., Calder, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+Inhibition+of+Bromodomain+Proteins+Suppresses+Retinal+Inflammatory+Disease+and+Downregulates+Retinal+Th17+Cells.">Pharmacological Inhibition of Bromodomain Proteins Suppresses Retinal Inflammatory Disease and Downregulates Retinal Th17 Cells.</a> The Journal of Immunology. 198, 1093-1103 (2017).</li><li>Mochizuki, M., 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=Preclinical+and+clinical+study+of+FK506+in+uveitis.">Preclinical and clinical study of FK506 in uveitis.</a> Current Eye Research. 11, 87-95 (1992).</li><li>Nguyen, Q. D., 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=Intravitreal+Sirolimus+for+the+Treatment+of+Noninfectious+Uveitis:+Evolution+through+Preclinical+and+Clinical+Studies.">Intravitreal Sirolimus for the Treatment of Noninfectious Uveitis: Evolution through Preclinical and Clinical Studies.</a> Ophthalmology. 125, 1984-1993 (2018).</li><li>Leal, I., 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=Anti-TNF+Drugs+for+Chronic+Uveitis+in+Adults-A+Systematic+Review+and+Meta-Analysis+of+Randomized+Controlled+Trials.">Anti-TNF Drugs for Chronic Uveitis in Adults-A Systematic Review and Meta-Analysis of Randomized Controlled Trials.</a> Frontiers in Medicine (Lausanne). 6, 104 (2019).</li><li>Chen, Y. H., 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=Functionally+distinct+IFN-?++IL-17A++Th+cells+in+experimental+autoimmune+uveitis:+T-cell+heterogeneity,+migration,+and+steroid+response.">Functionally distinct IFN-?+ IL-17A+ Th cells in experimental autoimmune uveitis: T-cell heterogeneity, migration, and steroid response.</a> European Journal of Immunology. 50 (12), 1941-1951 (2020).</li><li>Xu, H., 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=A+clinical+grading+system+for+retinal+inflammation+in+the+chronic+model+of+experimental+autoimmune+uveoretinitis+using+digital+fundus+images.">A clinical grading system for retinal inflammation in the chronic model of experimental autoimmune uveoretinitis using digital fundus images.</a> Experimental Eye Research. 87, 319-326 (2008).</li><li>Copland, D. A., 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+clinical+time-course+of+experimental+autoimmune+uveoretinitis+using+topical+endoscopic+fundal+imaging+with+histologic+and+cellular+infiltrate+correlation.">The clinical time-course of experimental autoimmune uveoretinitis using topical endoscopic fundal imaging with histologic and cellular infiltrate correlation.</a> Investigative Ophthalmology &amp; Visual Science. 49, 5458-5465 (2008).</li><li>Dick, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doyne+lecture+2016:+intraocular+health+and+the+many+faces+of+inflammation.">Doyne lecture 2016: intraocular health and the many faces of inflammation.</a> Eye (Lond). 31, 87-96 (2017).</li><li>Agarwal, R. K., Silver, P. B., Caspi, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+experimental+autoimmune+uveitis.">Rodent models of experimental autoimmune uveitis.</a> Methods in Molecular Biology. 900, 443-469 (2012).</li><li>Caspi, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+look+at+autoimmunity+and+inflammation+in+the+eye.">A look at autoimmunity and inflammation in the eye.</a> Journal of Clininical Investigation. 120, 3073-3083 (2010).</li><li>Caspi, R. R., 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=Mouse+models+of+experimental+autoimmune+uveitis.">Mouse models of experimental autoimmune uveitis.</a> Ophthalmic Research. 40, 169-174 (2008).</li><li>Shao, H., 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=Severe+chronic+experimental+autoimmune+uveitis+(EAU)+of+the+C57BL/6+mouse+induced+by+adoptive+transfer+of+IRBP1-20-specific+T+cells.">Severe chronic experimental autoimmune uveitis (EAU) of the C57BL/6 mouse induced by adoptive transfer of IRBP1-20-specific T cells.</a> Experimental Eye Research. 82, 323-331 (2006).</li><li>Horai, R., 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=Microbiota-Dependent+Activation+of+an+Autoreactive+T+Cell+Receptor+Provokes+Autoimmunity+in+an+Immunologically+Privileged+Site.">Microbiota-Dependent Activation of an Autoreactive T Cell Receptor Provokes Autoimmunity in an Immunologically Privileged Site.</a> Immunity. 43, 343-353 (2015).</li><li>Chen, 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=Comparative+analysis+of+induced+vs.+spontaneous+models+of+autoimmune+uveitis+targeting+the+interphotoreceptor+retinoid+binding+protein.">Comparative analysis of induced vs. spontaneous models of autoimmune uveitis targeting the interphotoreceptor retinoid binding protein.</a> PLoS One. 8, 72161 (2013).</li><li>Chen, J., Qian, H., Horai, R., Chan, C. C., Caspi, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+optical+coherence+tomography+and+electroretinography+to+evaluate+retinal+pathology+in+a+mouse+model+of+autoimmune+uveitis.">Use of optical coherence tomography and electroretinography to evaluate retinal pathology in a mouse model of autoimmune uveitis.</a> PLoS One. 8, 63904 (2013).</li><li>Harry, R., 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=Suppression+of+autoimmune+retinal+disease+by+lovastatin+does+not+require+Th2+cytokine+induction.">Suppression of autoimmune retinal disease by lovastatin does not require Th2 cytokine induction.</a> Journal of Immunology. 174, 2327-2335 (2005).</li></ol>

Experimental animal models are necessary tools for studying disease pathogenesis and preclinical testing of novel therapeutic paradigms. In the current protocol, we have discussed a methodology for inducing, monitoring, and scoring EAU, an experimental model of intraocular inflammatory uveitis. This EAU model has more than 95% disease incidence when all procedures are performed according to the protocol outlined herein, and results in the development of chronic, monophasic EAU. To achieve this incidence level, we stress ...

This protocol will demonstrate how to induce Experimental Autoimmune Uveitis (EAU) in the C57BL/6J mouse by a single subcutaneous injection of a retinal antigen in an emulsified adjuvant. Methods for monitoring and assessing disease progression will be detailed through fundoscopic imaging and histological examination, with measurement parameters outlined within. In addition, fluorescein angiography, a technique for examining retinal blood vessel structure and permeability will be discussed.
This EAU model recapitulates central features of non-infectious posterior uveitis in humans with regards to clinicopathologic characteristics and the basic cellular and molecular mechanisms that drive disease. EAU is mediated by Th1 and/or Th17 subsets of self-reactive CD4+T lymphocytes, as shown in adoptive transfer experiments and with IFN&#947;-depleted mice1. Much of our understanding of the potential roles for these cells in uveitis comes from studying EAU2 where both Th1 and Th17 cells are detected within the retinal tissues3. Often, EAU is used as a preclinical model to assess the utility of novel therapies in attenuating disease. Therapeutic approaches that have successfully modulated EAU disease have shown some efficacy in the clinic and reached FDA approved status. Examples of these are groups of immunoregulatory drugs such as the T cell-targeting therapies: cyclosporine, FK-506, and rapamycin4,5,6. Recently, interventions targeting novel pathways have also been explored in this model to investigate both mechanism and effect on disease outcome. These include targeting transcriptional regulation through chromatin reader Bromodomain Extra-Terminal (BET) proteins and P-TEFb inhibitors3. Moreover, more conventional approaches such as a VLA-4 inhibitor have recently demonstrated suppression in EAU via modulation of effector CD4+ T cells7. In addition, targeting Th17 cells with TMP778, a ROR&#947;t inverse agonist, has also been found to significantly suppress EAU8. Furthermore, this model offers an opportunity to study chronic autoimmune inflammation in the retina and the accompanying underlying mechanisms such as lymphocyte priming.
The primary readouts for EAU preclinical studies are clinical assessment by performing retinal fundoscopy imaging and less frequently, by assessing retinal integrity by Optical Coherence Tomography (OCT). Retinal histopathological evaluation and immunophenotyping of retinal cells by flow cytometry are then undertaken at termination. Fundoscopy is an easy-to-use live imaging system that allows for rapid and reproducible clinical assessment of the whole retina. For immunohistochemical assessments, the techniques are based on the preparation of retinal sections that allow us to study tissue architecture for the degree of inflammation and structural damage9. The assessment criteria and conventional scoring systems, for all techniques used, will be outlined within this protocol. The extent of damage recorded using fundoscopic imaging often closely correlates with histological changes. This dual approach to monitoring and assessing disease severity affords greater sensitivity and more reliable measurement outcomes.
EAU is a well-established, commonly used model for preclinical testing and investigation of immune-mediated eye disease. This model is reliable and reproducible with &#62;95% disease incidence and generates comprehensive data that can be used to validate or repudiate new therapies for the treatment of intraocular inflammatory disease that represents a major cause of working-age blindness worldwide10.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>antisedan</td>
			<td>ZOETIS, USA</td>
			<td></td>
			<td>for waking up</td>
		</tr>
		<tr>
			<td>Complete Freund&#8217;s Adjuvant; CFA</td>
			<td>Sigma, UK</td>
			<td>F5881</td>
			<td>for immunisation&#160;</td>
		</tr>
		<tr>
			<td>Domitor</td>
			<td>Orion Pharma, Finland</td>
			<td></td>
			<td>for anesthesia</td>
		</tr>
		<tr>
			<td>Flourescein</td>
			<td>Sigma, UK</td>
			<td>F2456</td>
			<td>for Angiography</td>
		</tr>
		<tr>
			<td>IRBP1-20</td>
			<td>Chamberidge peptide, UK</td>
			<td></td>
			<td>peptide;antigen&#160;</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Orion Pharma, Finland</td>
			<td></td>
			<td>for anesthesia</td>
		</tr>
		<tr>
			<td>Micron III</td>
			<td>Phoenix Research, USA</td>
			<td></td>
			<td>for fundoscopy</td>
		</tr>
		<tr>
			<td>Mouse Serum</td>
			<td>Sigma, UK</td>
			<td>M5905</td>
			<td>for immunisation&#160;</td>
		</tr>
		<tr>
			<td>Mycobacterium terberculosis</td>
			<td>Sigma, UK</td>
			<td>344289</td>
			<td>for immunisation&#160;</td>
		</tr>
		<tr>
			<td>Pertussis Toxin</td>
			<td>Sigma, UK</td>
			<td>P2980</td>
			<td>for immunisation&#160;</td>
		</tr>
		<tr>
			<td>phenylephrine hydrochloride 2.5%&#160;</td>
			<td>Bausch &#38; Lomb UK&#160;</td>
			<td>PHEN25</td>
			<td>for dilation&#160;</td>
		</tr>
		<tr>
			<td>Tropicamide 1%</td>
			<td>SANDOZ</td>
			<td></td>
			<td>for dilation&#160;</td>
		</tr>
		<tr>
			<td>Viscotears</td>
			<td>WELDRICKS Pharmacy, UK</td>
			<td>2082642</td>
			<td>for eye lubrication</td>
		</tr>
	</tbody>
</table>

experimental autoimmune uveitis: an intraocular inflammatory mouse model

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular inflammatory disease model recapitulate the clinical phenotype of posterior uveitis affecting humans. EAU has been used reliably to study the efficacy of novel inflammatory therapeutics, their mode of action and to further investigate the mechanisms that underpin disease progression of intraocular disorders. Here, we provide a detailed protocol on EAU induction in the C57BL/6J mouse - the most widely used model organism with susceptibility to this disease. Clinical assessment of disease severity and progression will be demonstrated using fundoscopy, histological examination and fluorescein angiography. The induction procedure involves subcutaneous&#160;injection of an emulsion containing a peptide (IRBP1-20) from the ocular protein interphotoreceptor retinoid binding protein (also known as retinol binding protein 3), Complete Freund&#39;s Adjuvant (CFA) and supplemented with killed Mycobacterium tuberculosis. Injection of this viscous emulsion on the back of the neck is followed by a single intraperitoneal injection of Bordetella pertussis toxin. At the onset of symptoms (day 12-14) and under general anesthesia, fundoscopic images are taken to assess disease progression through clinical examination. These data can be directly compared with those at later timepoints and peak disease (day 20-22) with differences analyzed. At the same time, this protocol allows the investigator to assess potential differences in vessel permeability and damage using fluorescein angiography. EAU can be induced in other mouse strains - both wildtype or genetically modified - and combined with novel therapies offering flexibility for studying drug efficacy and/or disease mechanisms.

In this protocol, we describe a step-by-step method for inducing a model of experimental autoimmune uveitis (EAU) by immunizing mice with a uveitogenic retinal peptide derived from IRBP. The assessment of disease employing widely used and readily accessible approaches are covered although these are not exclusive and may be added to, or partially replaced, by other imaging techniques. The first signs of EAU in C57BL/6J mice can be detected two weeks post-immunization and peak disease reached within three weeks as illustra...

Watch this Scientific Journal Video about Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model at JoVE.com

Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model

In this report we present a protocol that allows the investigator to generate a mouse model of intraocular uveitis. More commonly referred to as experimental autoimmune uveitis (EAU), this established model captures many aspects of human disease. Herein, we will describe how to induce and monitor disease progression using several readouts.

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular ...

This model can be used to study cellular mechanisms involving the pathogenesis of immune-mediated human posterior uveitis. The implication of this technique extend toward the translational study of human uveitis in monitoring novel drug efficacy and for providing mechanistic insight into chemical disease progression. The main advantage of using this model is the reliability of disease induction and use of non-invasive endoscopic techniques that allow us to monitor disease progression.Here we describe how to induce the disease and assess the pathology using several readouts To prepare IRBP 1-20 complete Freund's Adjuvant or CFA, resuspend a calculated amount of the peptide in a minimum volume of 100%DMSO. When the powder has completely dissolved, add small volumes of PBS to the tube until reaching to the final calculated volume of PBS according to the number of mice to be injected using gentle agitation to mix the solution. After preparing the complete Freund's Adjuvant, gently and frequently pipette the solution to form a viscus and evenly distributed emulsion while adding the DMSO PBS peptide solution dropwise at a one-to-one ratio to the suspension.To aerate the solutions, use a 1000 microliter pipette set to 700 microliters to repeatedly pipette the solution until a thick, creamy emulsion has been obtained. Before injecting the peptide suspension, place each mouse to be injected into a separate cage and use a one milliliter syringe equipped with a 23 gauge needle to intraperitoneal deliver 1.5 micrograms of Bordetella pertussis toxin in 100 microliters of RPMI 1640 medium supplemented with 1%mouse serum to each animal. When pertussis toxin solution has been injected, mouse should be held in a scruff like position and the skin pinched to form a tent like structure on the back of the neck.Thread the needle to the space between the finger and thumb, and inject 200 microliters of the IRBP emulsion to the tented skin, maintaining pressure after the injection and rotating the needle head to close the skin before retraction. To score the clinical disease, on day 21 after injection, confirm a lack of response to petal reflex in the anesthetized peptide injected mouse and restrain the animal in a scruff. Topically apply 1%tropicamide and 2%phenylephrine to the cornea of each eye immediately after injection.When the pupils have fully dilated, place the mouse onto a purpose-built microscope stage. Position the microscope for full access to the retina. Adjust the eye piece throughout the imaging process as necessary to see the optic disc and retina and acquire images of the entire retinal area, adjusting and covering all of the corners of the periphery.To measure vessel leakage, administer 100 microliters of 2%fluorescein subcutaneously at the back of the neck and reposition the mouse such that the retina is center on the middle of the live image. Set the fundoscope to a blue light excitation filter at 465 to 490 nanometers before acquiring an image of each at 1.5 and seven minutes after fluorescent injection. When all of the images have been acquired, intraperitoneally deliver an anti sedation injectable for recovery and place the mouse in a cage on a preheated mat with access to wet soaked diet with monitoring until the mouse regains consciousness.For clinical disease scoring of the images, base the clinical assessment on the severity of the optic disc inflammation, retinal vessel cuffing, retinal tissue infiltrate, and structural damage. Score each of these parameters on a scale from one to five. The collective total is representative of the clinical disease for the whole eye with a maximum score of 20 obtainable per eye.After fundus imaging for the final clinical endpoint of the study, euthanize the mouse. For histological analysis, prize apart the eyelids for access to the entire eye, then place curved forceps behind the globe to grasp the orbital connective tissue and optic nerve and enucleate the eye. Place the enucleated eye in one to five milliliters of 4%glutaraldehyde for a minimum of 15 minutes before transferring the organ to one to five milliliters of 10%formaldehyde for at least 24 hours.After histological tissue staining, according to standard protocols, assigned scores on a scale of zero to three based on the level of immune cell infiltration and the degree of retinal damage. Fundoscopic changes are classified during disease progression as inflammatory changes that include retinal tissue and vascular and optic disc inflammation and retinal structural damage in addition to histological changes based on infiltrating immune cells and structural impairments. These clinical and histopathological changes can be graded and scored to assess disease progression and to evaluate therapeutic intervention efficacy.Vascular leakage is also a pathological feature of the model and of human uveitis. As illustrated in this analysis, fluorescein can be used to visualize vascular leakage as another readout of this model. It is essential for researchers to ensure the emulsion is prepared correctly without signs of separation between the solutions.They should be mixed thoroughly and the final product should be smooth in texture. This procedure can be combined with other methods of drug administration and with for cytometry for phenotyping infiltrating retinal immune cell populations.

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Immunology and Infection

4.2K 조회수.  University College London. 이 모델은 면역-매개 인간 후방 포도막염의 발병기전을 포함하는 세포 메카니즘을 연구하는데 사용될 수 있다. 이 기술의 의미는 새로운 약물 효능을 모니터링하고 화학적 질병 진행에 대한 기계론적 통찰력을 제공하는 인간 포도막염의 중개 연구로 확장됩니다. 이 모델을 사용하는 주요 이점은 질병 유도의 신뢰성과 질병 진행을 모니터링 할 수있는 비 침습적 내시경 기술?...