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 uveitis patients). Randomize female C57BL/6J mice between 6-8 weeks old by weight and age. House the mice&#160;in individually ventilated cages (IVC) in groups of 5-6 mice per cage.</li>
</ol>
2. Immunization of C57BL/6 mice
<ol>
	<li>IRBP1-20 - CFA Emulsion Preparation 
	NOTE: Emulsion preparation is essential to the reproducibility and incidence of disease; as such, every effort must be made to maintain consistency throughout the preparation process and across experiments. When preparing the emulsion, loss should be accounted for in the calculations of all reagents beforehand. This loss can be approximately 1.5x (or 50% extra of the volume prepared), based on the number of mice planned for immunization. Please see the following example below. To immunize 10 mice, prepare 15 mice and use 400 &#181;g (peptide per 20 g mouse) x 15 mice = 6 mg. Each mouse should receive 200 &#181;L for immunization (3 mL total). The final volume comprises a 1:1 ratio of peptide solution and CFA, hence 1.5 mL of peptide solution and 1.5 mL of CFA.
	<ol>
		<li>Prepare all sterile solutions in a laminar flow cabinet using aseptic techniques.</li>
		<li>Weigh out desired amount (400 &#181;g per 20 g mouse) of human IRBP1-20 (LAQGAYRTAVDLESLASQLT) lyophilized peptide. Dissolve peptide in 100% DMSO. Store stock in lyophilized form at -20 &#176;C. 
		NOTE: To ensure the powder is fully dissolved each flake must make contact with the DMSO first and show no sign of residual solid. Add PBS in small portions to reach the final volume. Do not mix with a vortex, instead use gentle agitation with a pipette. The final concentration of DMSO should not exceed 1% of the total peptide preparation volume. Preparing the emulsion in a 20 mL plastic tube with a tapered bottom should allow better accessibility of DMSO to the lyophilized powder.</li>
		<li>Add DMSO-PBS peptide solution at 1:1 v/v to CFA which has already been supplemented with 1.5 mg/mL killed Mycobacterium tuberculosis, to give a final concentration of 2.5 mg/mL. Add dropwise, pipetting gently and frequently to form a viscous and evenly distributed emulsion.</li>
		<li>Aerate the peptide solution and CFA using a 1000 &#181;L pipette (set to 700 &#181;L to prevent further loss) and pipette to generate a creamy thick consistency. This technique involves using the pipette to repeatedly aspirate up and down until reaching the desired thickness.&#160;For optimal results, ensure that the antigen solution and adjuvant are mixed thoroughly before injecting.</li>
	</ol>
	</li>
	<li>Intraperitoneal injection of pertussis toxin
	<ol>
		<li>Suspend 1.5 &#181;g Bordetella pertussis toxin in 100 &#181;L of RPMI 1640 media supplemented with 1% mouse serum11.</li>
		<li>Perform the i.p. injection with a sterile syringe and 23G needle. 
		NOTE: In order to avoid disturbances to the injection site, the pertussis toxin must be administered before injecting the antigen.</li>
		<li>Temporarily transfer each mouse to a separate cage to receive a single 100 &#181;L i.p. injection of Bordetella pertussis toxin.</li>
	</ol>
	</li>
	<li>Subcutaneous injection of IRBP emulsion
	<ol>
		<li>Next, inject the IRBP emulsion subcutaneously. This process requires two animal handlers appropriately attired with protection according to health and safety regulations.</li>
		<li>Have one trained person lightly restrain the mouse on top of the cage in a scruff-like position, with their stomach facing downwards whilst the other trained person pinches the skin to form a tent-like structure on the back of the neck where the needle can be inserted to slot between the finger and thumb. 
		CAUTION: There is a danger of needle stick injury.</li>
		<li>Once the needle is positioned, inject 200 &#181;L of the IRBP emulsion. When removing the needle, rotate the needle head to close the skin before pulling out and apply pressure afterward to the injection site to prevent reflux of the emulsion. 
		CAUTION: The emulsion must not make contact with the mouse skin or fur as this may cause irritation and in more severe cases, a lesion to develop. If this occurs, the area must be wiped immediately and thoroughly using 70% ethanol then dried. 
		&#8203;NOTE: If the draining lymph nodes are needed for examination at the end of the study, the injection site will be different. In this instance, inject 100 &#181;L to both sides of the flank subcutaneously. This will generate a stronger response at the draining inguinal lymph nodes, which can be excised at the time of harvesting. However, if the intended outcome is solely to develop EAU, a single injection of 200 &#181;L at the back of the neck is preferable to avoid discomfort from multiple injection sites.</li>
	</ol>
	</li>
</ol>
3. Clinical Evaluation - Mouse Fundus Examination
NOTE: Clinical disease is to be scored using fundus examination, via bright-field live imaging using a fundoscope and Discover software used for visualization.
<ol>
	<li>At disease onset (day 12-14), sedate mice under general anesthesia using a combination of both Ketamine (50 mg/mL) and Domitor (Medetomidine; 1 mg/mL). Dilute 1-part Domitor; 1.5 parts Ketamine and 2.5 parts sterile injectable water, then inject 100 &#181;L per 30 g intraperitoneally. Use 1 mL sterile syringes and 23G needles for the above combination of anesthesia.</li>
	<li>Following this, monitor the mouse to ensure that all reflexes are lost and that it is unresponsive to stimuli.</li>
	<li>Immediately after receiving the i.p. injection and whilst the mouse is still held in a scruff, apply 1% tropicamide and 2.5% phenylephrine topically to each eye for pupil dilation. Aim to completely cover the cornea with both dilating solutions. It may take a few minutes before the pupil is fully dilated.</li>
	<li>Afterward, generously apply to the eye viscotears ointment and maintain throughout the imaging process in order to keep the eye fully lubricated and hydrated.</li>
	<li>In the meantime, open the software (e.g., Discover), and set the fundoscope (e.g., Micron) to capture images under brightfield. Allocate each individual mouse a folder and label images with R or L according to each eye photographed.</li>
	<li>Mount the mouse on a purpose-built stage for live visualization and position the microscope for full access to the retina.</li>
	<li>To get an accurate representation of the disease, take images of the entire retinal area, covering all corners of the periphery in addition to the optic disc. To achieve this, adjust the eyepiece throughout. It is crucial for the eye to remain fully lubricated at all times throughout the imaging process; ensure this by topping up the eye ointment at a constant rate.</li>
	<li>Refer to section 4 (below) at this stage to perform fluorescein angiography.</li>
	<li>Once all imaging is complete, dilute anesthetic reversal anti-sedation (5 mg/mL Antisedan) in injectable water and administer at 0.1 mg/kg i.p. Return the mouse to a cage and place on a pre-heated mat with access to a wet-soaked diet until recovery. Complete recovery is characterized by whole body movement and walking around the cage with steady gait, typically taking a few hours.</li>
	<li>At the designated experimental endpoint (e.g., day 21-23), repeat steps 4.1-4.5 and take photographs of the entire retinal area again, covering the optic disc and all corners of the periphery to capture an accurate representation of disease.</li>
</ol>
4. Fluorescein angiography
<ol>
	<li>To measure vessel leakage in these animals, whilst under anesthesia, give each mouse an injection of 2% fluorescein subcutaneously at the back of the neck and position such that the retina is centralized in the middle of the live image.</li>
	<li>Set the fundoscope to a blue light excitation filter at 465-490 nm. The light captured from excited fluorescein is between 520-530 nm.</li>
	<li>After 1.5 min post fluorescein injection, take a photograph of each retina and repeat again at 7 minutes. 
	&#8203;NOTE: Timing is critical for these events, if unable to capture both then just image one eye.</li>
</ol>
5. Clinical Disease Scoring
<ol>
	<li>Base the clinical assessment on the severity of the following criteria: optic disc inflammation, retinal vessel cuffing, retinal tissue infiltrate and structural damage.</li>
	<li>Award each of these parameters a score on a scale from 0 to 5 and the collective total is representative of clinical disease for the whole eye, with a maximum score of 20 obtainable per eye. Table 1 can be used as a guide for scoring criteria.</li>
</ol>
6. Histology and Histological Scoring
<ol>
	<li>After euthanizing&#160;the mice by cervical dislocation, enucleate the eyes by prizing the eyelids apart for easy access to the entire eye.</li>
	<li>Next, place curved forceps behind the globe with the intention of grasping the orbital connective tissue and optic nerve. Take care to avoid squeezing the globe.</li>
	<li>For fixation, place the eye in 4% glutaraldehyde for a minimum of 15 minutes to minimize retinal detachment, and then transfer to 10% formaldehyde for at least 24 h. 1-2 mL of fixative would give enough volume to cover two eyes.</li>
	<li>Perform embedding in paraffin, sectioning on a microtome, and staining according to standard protocols. 3-4 &#181;m section thickness is recommended for any type of staining.</li>
	<li>Perform histological examination of eyes using standard protocols for Hematoxylin and Eosin (H&#38;E) staining.</li>
	<li>Assign scores on a scale of 0-4, according to the criteria for EAU scoring, based on the extent of the immune cell infiltration within the retina and choroid, the disruption of the retinal layers, the degree of granuloma formation and the extent of retinal detachment, indicating retinal damage, as previously described (Agarwal 2013) and summarized in Table 211.</li>
</ol>

<ol><li>Lyu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TMP778%2C+a+selective+inhibitor+of+RORgammat%2C+suppresses+experimental+autoimmune+uveitis+development%2C+but+affects+both+Th17+and+Th1+cell+populations%5BTitle%5D)+AND+%22The+European+Journal+of+Immunology%22%5BJournal%5D)">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/pubmed?term=((Mouse+models+of+autoimmune+uveitis%5BTitle%5D)+AND+%22Current+Pharmaceutical+Design%22%5BJournal%5D)">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/pubmed?term=((Pharmacological+Inhibition+of+Bromodomain+Proteins+Suppresses+Retinal+Inflammatory+Disease+and+Downregulates+Retinal+Th17+Cells%5BTitle%5D)+AND+%22The+Journal+of+Immunology%22%5BJournal%5D)">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/pubmed?term=((Preclinical+and+clinical+study+of+FK506+in+uveitis%5BTitle%5D)+AND+%22Current+Eye+Research%22%5BJournal%5D)">Preclinical and clinical study of FK506 in uveitis.</a> Current Eye Research. 11, Suppl 87-95 (1992).</li>
<li>Nguyen, Q. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravitreal+Sirolimus+for+the+Treatment+of+Noninfectious+Uveitis%3A+Evolution+through+Preclinical+and+Clinical+Studies%5BTitle%5D)+AND+%22Ophthalmology%22%5BJournal%5D)">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/pubmed?term=((Anti-TNF+Drugs+for+Chronic+Uveitis+in+Adults-A+Systematic+Review+and+Meta-Analysis+of+Randomized+Controlled+Trials%5BTitle%5D)+AND+%22Frontiers+in+Medicine+%28Lausanne%29%22%5BJournal%5D)">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/pubmed?term=((Functionally+distinct+IFN-%3F%2B+IL-17A%2B+Th+cells+in+experimental+autoimmune+uveitis%3A+T-cell+heterogeneity%2C+migration%2C+and+steroid+response%5BTitle%5D)+AND+%22European+Journal+of+Immunology%22%5BJournal%5D)">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/pubmed?term=((A+clinical+grading+system+for+retinal+inflammation+in+the+chronic+model+of+experimental+autoimmune+uveoretinitis+using+digital+fundus+images%5BTitle%5D)+AND+%22Experimental+Eye+Research%22%5BJournal%5D)">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/pubmed?term=((The+clinical+time-course+of+experimental+autoimmune+uveoretinitis+using+topical+endoscopic+fundal+imaging+with+histologic+and+cellular+infiltrate+correlation%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">The clinical time-course of experimental autoimmune uveoretinitis using topical endoscopic fundal imaging with histologic and cellular infiltrate correlation.</a> Investigative Ophthalmology & Visual Science. 49, 5458-5465 (2008).</li>
<li>Dick, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Doyne+lecture+2016%3A+intraocular+health+and+the+many+faces+of+inflammation%5BTitle%5D)+AND+%22Eye+%28Lond%29%22%5BJournal%5D)">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/pubmed?term=((Rodent+models+of+experimental+autoimmune+uveitis%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">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/pubmed?term=((A+look+at+autoimmunity+and+inflammation+in+the+eye%5BTitle%5D)+AND+%22Journal+of+Clininical+Investigation%22%5BJournal%5D)">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/pubmed?term=((Mouse+models+of+experimental+autoimmune+uveitis%5BTitle%5D)+AND+%22Ophthalmic+Research%22%5BJournal%5D)">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/pubmed?term=((Severe+chronic+experimental+autoimmune+uveitis+%28EAU%29+of+the+C57BL%2F6+mouse+induced+by+adoptive+transfer+of+IRBP1-20-specific+T+cells%5BTitle%5D)+AND+%22Experimental+Eye+Research%22%5BJournal%5D)">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/pubmed?term=((Microbiota-Dependent+Activation+of+an+Autoreactive+T+Cell+Receptor+Provokes+Autoimmunity+in+an+Immunologically+Privileged+Site%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">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/pubmed?term=((Comparative+analysis+of+induced+vs.+spontaneous+models+of+autoimmune+uveitis+targeting+the+interphotoreceptor+retinoid+binding+protein%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Use+of+optical+coherence+tomography+and+electroretinography+to+evaluate+retinal+pathology+in+a+mouse+model+of+autoimmune+uveitis%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Suppression+of+autoimmune+retinal+disease+by+lovastatin+does+not+require+Th2+cytokine+induction%5BTitle%5D)+AND+%22Journal+of+Immunology%22%5BJournal%5D)">Suppression of autoimmune retinal disease by lovastatin does not require Th2 cytokine induction.</a> Journal of Immunology. 174, 2327-2335 (2005).</li>
</ol>

The authors have no conflicts of interest to declare with this work.

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><tbody><tr><td>antisedan</td><td>ZOETIS, USA</td><td></td><td>for waking up</td></tr><tr><td>Complete Freund&amp;rsquo;s Adjuvant; CFA</td><td>Sigma, UK</td><td>F5881</td><td>for immunisation&amp;nbsp;</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>IRBP&lt;sub&gt;1-20&lt;/sub&gt;</td><td>Chamberidge peptide, UK</td><td></td><td>peptide;antigen&amp;nbsp;</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&amp;nbsp;</td></tr><tr><td>Mycobacterium terberculosis</td><td>Sigma, UK</td><td>344289</td><td>for immunisation&amp;nbsp;</td></tr><tr><td>Pertussis Toxin</td><td>Sigma, UK</td><td>P2980</td><td>for immunisation&amp;nbsp;</td></tr><tr><td>phenylephrine hydrochloride 2.5%&amp;nbsp;</td><td>Bausch &amp;amp; Lomb UK&amp;nbsp;</td><td>PHEN25</td><td>for dilation&amp;nbsp;</td></tr><tr><td>Tropicamide 1%</td><td>SANDOZ</td><td></td><td>for dilation&amp;nbsp;</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 ...

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Immunology and Infection

4.7K Views.  University College London. 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. 

Video: Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable dry eye animal model to test novel therapeutics to treat autoimmune dry eye conditions. This protocol describes a chronic autoimmune dry eye rat model. Lewis rats were immunized with an emulsion containing lacrimal gland extract, ovalbumin, and complete Freund&#39;s adjuvant. A second immunization with the same antigens in incomplete Freund&#39;s adjuvant was administered two weeks later. These immunizations were administered subcutaneously at the base of the tail. To boost the immune response at the ocular surface and lacrimal glands, lacrimal gland extract and ovalbumin were injected into the forniceal subconjunctiva and lacrimal glands 6 weeks after the first immunization. The rats developed dry eye features, including reduced tear production, decreased tear stability, and increased corneal damage. Immune profiling by flow cytometry showed a preponderance of CD3+ effector memory T cells in the eyeball.

This report describes a method to induce chronic experimental autoimmune dry eye in Lewis rats through immunization with an emulsion of rat lacrimal gland extract, ovalbumin, and complete Freund's adjuvant, followed by the injection of lacrimal gland extract and ovalbumin into the forniceal subconjunctiva and lacrimal glands six weeks later.

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable ...

Intravitreal Injection and Quantitation of Infection Parameters in a Mouse Model of Bacterial Endophthalmitis

Intraocular bacterial infections are a danger to the vision. Researchers use animal models to investigate the host and bacterial factors and immune response pathways associated with infection to identify viable therapeutic targets and to test drugs to prevent blindness. The intravitreal injection technique is used to inject organisms, drugs, or other substances directly into the vitreous cavity in the posterior segment of the eye. Here, we demonstrated this injection technique to initiate infection in the mouse eye and the technique of quantifying intraocular bacteria. Bacillus cereus was grown in brain heart infusion liquid media for 18 hours and resuspended to a concentration 100 colony forming units (CFU)/0.5 &#181;L. A C57BL/6J mouse was anesthetized using a combination of ketamine and xylazine. Using a picoliter microinjector and glass capillary needles, 0.5 &#181;L of the Bacillus suspension was injected into the mid vitreous of the mouse eye. The contralateral control eye was either injected with sterile media (surgical control) or was not injected (absolute control). At 10 hours post infection, mice were euthanized, and eyes were harvested using sterile surgical tweezers and placed into a tube containing 400 &#181;L sterile PBS and 1 mm sterile glass beads. For ELISAs or myeloperoxidase assays, proteinase inhibitor was added to the tubes. For RNA extraction, the appropriate lysis buffer was added. Eyes were homogenized in a tissue homogenizer for 1-2 minutes. Homogenates were serially diluted 10-fold in PBS and track diluted onto agar plates. The remainder of the homogenates were stored at -80 &#176;C for additional assays. Plates were incubated for 24 hours and CFU per eye was quantified. These techniques result in reproducible infections in mouse eyes and facilitate quantitation of viable bacteria, the host immune response, and omics of host and bacterial gene expression.

We describe here a method of intravitreal injection and subsequent bacterial quantitation in mouse model of bacterial endophthalmitis. This protocol can be extended for measuring host immune responses and bacterial and host gene expression.

Intraocular bacterial infections are a danger to the vision. Researchers use animal models to investigate the host and bacterial factors and immune ...

Primed Mycobacterial Uveitis (PMU) as a Model for Post-Infectious Uveitis

The term 'uveitis' describes a heterogeneous set of conditions that all feature intraocular inflammation. Broadly, uveitis is defined by etiology: infection or autoimmunity. Infectious uveitis requires treatment with the appropriate antimicrobial agents, while autoimmune uveitis requires treatment with corticosteroids or other immunosuppressive agents. Post-infectious uveitis is a form of chronic uveitis that requires corticosteroids to control immune sequela following the initial infection. Uveitis associated with Mycobacterium tuberculosis (Mtb) infection is a well-recognized form of post-infectious uveitis, but the mechanisms of disease are not fully understood. To understand the role mycobacterial antigens and innate ligands play in stimulating chronic ocular inflammation following mTB infection, the model Primed Mycobacterial Uveitis (PMU) was developed for use in mice. This manuscript outlines the methods for generating PMU and monitoring the clinical course of inflammation using color fundus and optical coherence tomography (OCT) imaging. PMU is induced by immunization with heat-killed mycobacterial extract followed by intravitreal injection of the same extract into one eye seven days later. Ocular inflammation is monitored longitudinally using in vivo imaging and followed by sample collection for a wide range of assays, including histology, flow cytometry, cytokine analysis, qPCR, or mRNA sequencing. The mouse model of PMU is a useful new tool for studying the ocular responses to mTB, the mechanism of chronic uveitis, and for preclinical effectiveness tests of new anti-inflammatory therapies.

Primed Mycobacterial Uveitis PMU as a Model for Post-Infectious Uveitis

This protocol outlines the steps for inducing&#160;Primed Mycobacterial Uveitis (PMU) in mice. This method outlines the steps to help produce reliable and robust ocular inflammation in the mouse model system. Using this protocol, we generated uveitic eyes and uninflamed fellow eyes from single animals for further evaluation with immunologic, transcriptomic, and proteomic assays.

The term 'uveitis' describes a heterogeneous set of conditions that all feature intraocular inflammation. Broadly, uveitis is defined by etiology:  ...

Subcutaneous Injection of Bacterial Antigen to Induce Systemic Inflammation in a Murine Model

Source: John, S. et al., <a href="https://app.jove.com/v/62925">Primed Mycobacterial Uveitis (PMU) as a Model for Post-Infectious Uveitis.</a>&#160;J. Vis. Exp. (2021)
This video demonstrates the induction of systemic inflammation in an anesthetized mouse. The injection of mycobacterial antigen-adjuvant depots triggers cytokine release and localized inflammation. Lymphatic vessels open, enabling immune cell entry into lymph nodes and activation of T cells, which release pro-inflammatory cytokines, leading to widespread inflammation.

Source: John, S. et al., Primed Mycobacterial Uveitis (PMU) as a Model for Post-Infectious Uveitis.&#160;J. Vis. Exp. (2021)
This video demonstrates the ...

JoVE Encyclopedia of Experiments

Immunology

Immunopathology

Host-Pathogen Interaction Models

A Murine Model of Primed Mycobacterial Uveitis to Study Post-Infectious Uveitis

Source: John, S., et al. <a href="https://app.jove.com/v/62925">Primed Mycobacterial Uveitis (PMU) as a Model for Post-Infectious Uveitis.</a> J. Vis. Exp. (2021).
This video demonstrates a method to generate a mouse model of primed mycobacterial uveitis (PMU) for studying post-infectious uveitis. Upon induction of PMU by intravitreal injection of heat-killed Mycobacterium tuberculosis in a mouse previously immunized with the same immunogen, the progression of chronic inflammation in the eye is analyzed using imaging and histopathological examination

Source: John, S., et al. Primed Mycobacterial Uveitis (PMU) as a Model for Post-Infectious Uveitis. J. Vis. Exp. (2021).
This video demonstrates a method ...

Induction of Experimental Autoimmune Encephalomyelitis in a Mouse Model

Source: Schmitz, K., et al. <a href="https://app.jove.com/v/55321">Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice.</a> J. Vis. Exp (2017).
In this video, a mouse model of multiple sclerosis is established using experimental autoimmune encephalomyelitis (EAE). The mouse is injected with an emulsion containing a neuronal peptide and an adjuvant, triggering an immune response that activates autoreactive T cells. This is followed by an injection of pertussis toxin (PTX) to increase blood-brain barrier permeability, allowing the autoreactive T-cells to infiltrate the brain. This infiltration initiates a signaling cascade that ultimately results in myelin damage, mimicking the demyelinating condition of multiple sclerosis.

1. EAE Induction in SJL/J Mice

	Mice
	
		Use 11-week-old female SJL/J mice and allow them to habituate to the experimental room for about 7 days. Use n = ...

Neuroscience

Neuropathology

Neuromuscular Disorders

Bacterial Endophthalmitis Mouse Model: A Method for Generating Bacterial Endophthalmitis via Intravitreal Injection of Bacterial Suspension into Eye of Mouse

Source: Mursalin, M. H. et al., <a href="https://www.jove.com/v/61749">Intravitreal Injection and Quantitation of Infection Parameters in a Mouse Model of Bacterial Endophthalmitis.</a> J. Vis. Exp. (2021).
This video describes a method for generating a mouse model that mimics bacterial endophthalmitis &#8211; an intraocular infection resulting from a bacterial invasion into the eye&#8217;s vitreous cavity &#8211; via intravitreal injection of a bacterial suspension into the eye. This mouse model helps study the disease mechanism and evaluate therapeutics for it.

Source: Mursalin, M. H. et al., Intravitreal Injection and Quantitation of Infection Parameters in a Mouse Model of Bacterial Endophthalmitis. J. Vis. ...

Biology

Rodent Models

Adult

A Method to Induce Ocular Surface Inflammation in a Murine Model

To perform intraperitoneal injection of the immunogen solution, hold the 7 to 9-weeks-old C57 Black 6 non-anesthetized female mouse softly by the tail while grasping the cage grid. Then, firmly hold the skin at the back and the neck region between the thumb and index finger and fix the tail and lower limbs between the ring and little finger against the palm. Keeping the mouse fixed with its head downward, inject 100 microliters of the prepared immunogen solution in the right or left quadrant of the lower abdominal cavity.
To perform an ocular surface challenge after immunization, apply 5 microliters of ovalbumin, or ova, or saline per eye of an anesthetized mouse, and wait for 5 minutes to get the drop absorbed by the eye. Next, collect the ocular exudates by applying 50 microliters of sterile saline to the eye immediately after the challenge.

Immune Response

Immune Modulation

EoE Sub Articles

Optical Coherence Tomography Imaging for Assessing Uveitis in a Mouse Model

After anesthetizing the mouse, dilate the pupil with one drop of 2.5% phenylephrine, avoiding excess droplets entering the nose or mouth. After two to three minutes, dab off the excess liquid, and cover the eye with 0.3% hypromellose gel. Next, wrap the mouse in a layer of surgical gauze to maintain body warmth, then place the mouse on the animal cassette, and position the head with the bite bar.
To acquire the optical coherence tomography, or OCT images, turn on the OCT imaging system, and open the imaging software. For posterior chamber imaging, bring the OCT close to the eye&#39;s surface, taking care not to bring the surface of the lens in contact with the eye.
Once the eye is correctly positioned, stop the Fast Scan, select the Volume Scan protocol, and activate the scan with the Aim option. For posterior segment images, adjust until the optic nerve is centered in the horizontal B-Scan alignment image and the retina is aligned with the vertical alignment axis. For anterior segment images, adjust the position to center the apex of the cornea in both the horizontal B-Scan alignment image and vertical B-Scan alignment image. Finally, click on Snapshot to capture the volume scan image. Then click on Save.