This work was partially supported by the German Research Foundation (DFG) 2886 PANDORA Project-No.B3; SCHA 2040/1-1; MU 4240/2-1; CRC1181(C03); TRR241(B04), H2020-FETOPEN-2018-2020 Project 861878, and by the Volkswagen-Stiftung (Grant 97744) to MH.

All procedures involving animals were conducted according to the institutional guidelines on animal welfare and approved by the animal welfare commission of the Friedrich-Alexander-University Erlangen-Nuremberg (FAU) (permit number: 55.2.2-2532-2-1217). Female C57Bl/6 mice, aged 7-9 weeks were used for the present study. The mice were obtained from commercial sources (see Table of Materials) and kept in specific pathogen-free conditions with 12 h day/night cycles.
1. Induction of murine ocular surface inflammation
<ol>
	<li>Perform immunogen preparation for immunization.
	<ol>
		<li>Prepare the immunogen freshly on the day of immunization, mixing ovalbumin (OVA, 50 mg/mL) and pertussis toxin (100 &#181;g/mL) in saline and the adjuvant aluminum hydroxide (40 mg/mL) (see Table of Materials) in a 1:1 proportion. 
		CAUTION: Perform the opening, reconstitution, and immunogen preparation of pertussis toxin in a laminar safety cabinet. Wear protective clothing and avoid any contact with the skin.</li>
		<li>Incubate the immunogen and adjuvant mixture at room temperature for 30 min and load 100 &#181;L into a 1 mL syringe.</li>
		<li>Perform intraperitoneal injection of the immunogen solution in a non-anesthetized mouse.
		<ol>
			<li>Hold the mouse softly by the tail while grasping the cage grid. Hold firmly the skin of 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 of the hand.</li>
			<li>Keep the fixed mouse with its head downward.</li>
			<li>Inject 100 &#181;L of the prepared immunogen solution in the right or left quadrant of the lower abdominal cavity.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Perform ocular surface challenge 2 weeks after immunization.
	<ol>
		<li>Anesthetize the mouse with isoflurane (2.5%).</li>
		<li>Apply 5 &#181;L of OVA (50 mg/mL) or saline (0.9 % NaCl) per eye on both eyes and wait until the drop gets absorbed by the eye. This takes ~5 min.</li>
		<li>Repeat the procedure 1x daily for 7 days. 
		&#8203;NOTE: The topical administration of medications can be done in the same way.</li>
	</ol>
	</li>
</ol>
2. Collection of ocular exudates
<ol>
	<li>Recover the ocular exudates formed during the challenge phase by applying 50 &#181;L of sterile saline to the eye immediately after the challenge.</li>
	<li>To obtain a single-cell suspension, treat the collected ocular discharge with recombinant MNase (2 x 106 gel U/mL, see Table of Materials) containing the cofactor calcium (5 mM) at 37 &#176;C for 20 min.</li>
	<li>Centrifuge at 400 x g for 7 min at room temperature.</li>
	<li>Isolate the supernatant and measure the cytokines and chemokines using multiplexed ELISA according to the manufacturer&#39;s instructions (see Table of Materials). 
	&#8203;NOTE: The obtained supernatant can be used for protein analysis and the pellet for functional assays such as immunophenotyping, phagocytosis, degranulation, and gene expression.</li>
</ol>
3. Excision of ocular surface tissues
<ol>
	<li>Dissect the eyelids and eye globe following the steps below.
	<ol>
		<li>Euthanize the mouse by CO2 asphyxiation and cervical dislocation.</li>
		<li>Place the mouse on an even surface.</li>
		<li>Disinfect the orbital area around the eye with a swab impregnated with 70% ethanol.</li>
		<li>Make an incision between the ear and the retro-orbital sinus and along the surface above the squamous bone vertically, extending the incision horizontally below the lower eyelid along the maxillary bone and above the upper eyelid along the frontal bone. This forms an incision around the eye (Figure 1).</li>
		<li>Carefully hold the dissected tissue around the eye using curved forceps and pull the tissue and eyeball out.</li>
		<li>Place the excised organs in sterile PBS.</li>
		<li>Trim excess facial muscle tissue around the excised upper eyelids using a scalpel and under the stereomicroscope.</li>
	</ol>
	</li>
	<li>Collect the conjunctiva following the steps below.
	<ol>
		<li>Place the upper eyelid on a dry Petri dish under the stereomicroscope.</li>
		<li>Using fine tweezers and a scalpel, peel the whitish-mucous layer very gently out of the inner surface of the eyelid.</li>
	</ol>
	</li>
	<li>Next, dissect the cornea following the steps below.
	<ol>
		<li>Place the eye globe on a new dry Petri dish on top of dry ice for 3 min.</li>
		<li>Take the Petri dish onto the bench surface in a stable position.</li>
		<li>Make a small incision at the border of the cornea next to the limbus using fine sharp scissors.</li>
		<li>Prolong the incision with the scalpel around the eye globe, separating the sclera from the cornea.</li>
		<li>Remove remnants of the iris and the lens from the backside of the cornea by generously flushing with saline solution.</li>
	</ol>
	</li>
</ol>
4. Documentation of Meibomian gland (MG) obstruction
<ol>
	<li>To assess the MG and its orifices, place excised eyelids in an upright position under the stereomicroscope (Figure 2).</li>
	<li>Capture images with white light epi-illumination according to the camera&#39;s exposure time and ISO settings. 
	NOTE: Morphometric analysis of these images provides reliable quantification of the plug size at the outlet of the glands. The quantification can be performed by outlining with the wand tool the eye plugs and executing the &#34;Analyze Particles&#34; command of the Image J software (see Table of Materials).</li>
</ol>
5. Transillumination of eyelids (Meibomian gland morphology)
<ol>
	<li>To assess the MG area, place the excised eyelids in a horizontal position and turn on the backlight of the stereomicroscope equipped with an infrared camera (Figure 3).</li>
	<li>Capture images by adjusting the exposure time according to the camera&#39;s (see Table of Materials) ISO. 
	NOTE: Infrared imaging of these transilluminated eyelids under the stereomicroscope can help measure the shape and size of each acini. Morphometric analysis of these images provides reliable quantification of the size and number of MG. The quantification can be performed by outlining the MG with the wand tool and executing the &#34;Analyze Particles&#34; command of the Image J software.</li>
</ol>

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The authors have no conflicts of interest to disclose.

The oily secretion of the Meibomian glands is of great importance for a healthy eye22. However, the obstruction of these sebaceous glands by aggregated neutrophil extracellular traps (aggNETs) that line up as parallel strands located on the tarsal plates of both eyelids can disrupt the tear film23. This disruption results in Meibomian gland dysfunction (MGD)1 and accelerated tear evaporation and conditions the damage of the ocular surface<sup class="...

Every blink of an eye replenishes the smooth tear film dispersed over the cornea. The ocular surface epithelia facilitate the distribution and correct orientation of the tear film on the ocular surface. Mucins are provided by the cornea and conjunctiva epithelial cells to help position the aqueous part of the tear film coming from the lacrimal glands on the eyes&#39; surface. Finally, MG secretes lipids that create a covering layer that prevents the evaporation of the aqueous part of the tear film1,2,3. In this fashion, the coordinated functions of all the ocular organs protect the ocular surface from invading pathogens or injury and support crystal clear vision without any pain or discomfort.
In a healthy ocular surface, the ocular flowing discharge or eye rheum sweeps away dust, dead epithelial cells, bacteria, mucus, and immune cells. Aggregated neutrophil extracellular traps (aggNETs) formulate a mesh-like matrix composed of extracellular chromatin and incorporate these components in the eye rheum. AggNETs resolve inflammation by the proteolytic degradation of pro-inflammatory cytokines and chemokines4. However, when they become dysfunctional, these aberrant aggNETs drive the pathogenesis of diseases such as vascular occlusions in COVID-195, gallstones6, and sialolithiasis7. Similarly, aggNETs on the ocular surface play a protective role and contribute to resolving inflammation of the highly exposed surface8. Either an exaggerated formation or lack of aggNETs in the ocular surface can impair the tear film stability and/or cause corneal wounds, cicatrizing conjunctivitis, and dry eye disease. For example, the obstruction of MG is a leading cause of dry eye disease9. AggNETs are also known to plug the flow of lipid secretion from the ducts of MG and cause Meibomian gland dysfunction (MGD). The congestion of MG orifices by aggNETs causes a lack of fatty fluid enveloping the ocular surface and retrograde bottled-up fluid, resulting in dysfunction of the gland function and acinar damage. This dysfunction can result in tear film evaporation, fibrosis of the margins on the eyelids, eye inflammation, and detrimental damage to the MG10,11.
Several animal models have been developed over the years to imitate the pathological process of MGD in humans. For example, C57BL/6 mice aged 1 year have helped study age-related effects on dry eye disease (DED) and MGD, reflecting the ocular disease pathology in patients aged 50 years and older12,13,14. Furthermore, rabbits are appropriate models for investigating the effects of pharmacological interventions. Therefore, inducing MGD in rabbits has been reported by either the topical administration of epinephrine or the systemic introduction of 13-cis-retinoic acid (isotretinoin)15,16,17,18,19.
Although these animal models were adequate for determining the different factors contributing to the pathophysiology of MGD, they were restricted in their utilization. For instance, the murine model of age-related MGD was ideal for deciphering elements in older adults only, and hence, rabbits appeared to be the most suitable animal model to study ocular surface diseases, as they enable the investigation of multiple pathophysiological mechanisms. However, due to the lack of comprehensive analytical tools to detect proteins at the ocular surface and because many parts of the rabbit genome are unannotated, they are limited for investigations20,21.
In addition, these animal models used to investigate the pathogenesis of dry eye disease did not provide adequate details to analyze the immunological arm of the disorder that instigates the inflammation of the ocular surface. Accordingly, the murine model of MGD developed by Reyes et al. showed an association between allergic eye disease in mice and MGD in humans and highlighted the immune etiology responsible for obstructive MGD21. This model associates allergic eye disease with a TH17 response that recruits neutrophils to the conjunctiva and eyelid, causing MGD and chronic ocular inflammation21. The induction of MGD and ocular inflammation in this murine model is a valuable tool for investigating upstream events during the development of local inflammation driven by an ongoing immune response21. The current protocol describes the ocular surface inflammation accompanied by obstructive MGD. In this method, mice are immunized and, after 2 weeks, challenged on the ocular surface with the immunogen for 7 days. Furthermore, the steps to isolate ocular exudate and the associated ocular organs during acute inflammation and the dissection of the cornea, conjunctiva, and eyelids are described.

<table><tbody><tr><td>1x PBS</td><td>Gibco</td><td></td><td></td></tr><tr><td>Aluminium Hydroxide</td><td>Imject alum Adjuvant</td><td>77161</td><td>40 mg/ mL&lt;br/&gt; &lt;strong&gt;Final Concentration&lt;/strong&gt;: &lt;em&gt;in vivo&lt;/em&gt;: 1 mg/ 100 &amp;micro;L</td></tr><tr><td>C57Bl/6 mice, aged 7&amp;ndash;9 weeks</td><td>Charles River Laboratories&amp;nbsp;</td><td></td><td></td></tr><tr><td>Calcium</td><td>Carl roth</td><td>CN93.1</td><td>1 M&lt;br/&gt; &lt;strong&gt;Final Concentration&lt;/strong&gt;: 5 mM</td></tr><tr><td>Curved forceps</td><td>FST by Dumont SWITZERLAND</td><td>5/45 11251-35</td><td></td></tr><tr><td>Fine sharp scissor</td><td>FST Stainless steel, Germany</td><td>15001-08</td><td></td></tr><tr><td>Laminar safety cabinet</td><td>Herasafe</td><td></td><td></td></tr><tr><td>Macrophotography Camera</td><td>Canon</td><td>EOS6D</td><td></td></tr><tr><td>Macrophotography Camera (without IR filter)</td><td>Nikon</td><td>D5300</td><td></td></tr><tr><td>Mnase</td><td>New England biolabs</td><td>M0247S</td><td>2 x 10&lt;sup&gt;6&lt;/sup&gt; gel U/mL</td></tr><tr><td>Multi-analyte flow assay kit (Custom mouse 13-plex panel)</td><td>Biolegend</td><td>CLPX-200421AM-UERLAN</td><td></td></tr><tr><td>NaCl 0,9% (Saline)</td><td>B.Braun</td><td></td><td></td></tr><tr><td>Ovalbumin (OVA)</td><td>Endofit, Invivogen</td><td>9006-59-1</td><td>10 mg/200 &amp;micro;L in saline</td></tr><tr><td>Pertussis toxin&amp;nbsp;</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>PHZ1174</td><td>50 &amp;micro;g/ 500 &amp;micro;L in saline&lt;br/&gt; &lt;strong&gt;Final Concentration&lt;/strong&gt;: &lt;em&gt;in vivo&lt;/em&gt;: 100 &amp;micro;g/ 100 &amp;micro;L</td></tr><tr><td>Petridish</td><td>Greiner bio-one</td><td>628160</td><td></td></tr><tr><td>Scalpel</td><td>Feather disposable scalpel</td><td>No. 21&amp;nbsp;</td><td>&lt;strong&gt;Final Concentration&lt;/strong&gt;: &lt;em&gt;in vivo&lt;/em&gt;:&amp;nbsp; 300 ng/ 100 &amp;micro;L</td></tr><tr><td>Stereomicroscope</td><td>Zeiss</td><td>Stemi508</td><td></td></tr><tr><td>Syringe (corneal/iris washing)</td><td>BD Microlane</td><td>27 G x 3/4 - Nr.20 0,4 x 19 mm</td><td></td></tr><tr><td>Syringe (i.p immunization)</td><td>BD Microlane</td><td>24 G1&quot;-Nr 17, 055* 25 mm</td><td></td></tr></tbody></table>

induction of ocular surface inflammation and collection of involved tissues

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular surface gland network. Meibomian glands (MG) secrete lipids that create a covering layer that prevents the evaporation of the aqueous part of the tear film. Neutrophils and extracellular DNA traps populate MG and the ocular surface in a mouse model of allergic eye disease. Aggregated neutrophil extracellular traps (aggNETs) formulate a mesh-like matrix composed of extracellular chromatin that occludes MG outlets and conditions MG dysfunction. Here, a method for inducing ocular surface inflammation and MG dysfunction is presented. The procedures for collecting organs related to the ocular surface, such as the cornea, conjunctiva, and eyelids, are described in detail. Using established techniques for processing each organ, the major morphological and histopathological features of MG dysfunction are also shown. Ocular exudates offer the opportunity to assess the inflammatory state of the ocular surface. These procedures enable the investigation of topical and systemic anti-inflammatory interventions at the preclinical level.

The present protocol describes the sequential steps for establishing a murine model of ocular surface inflammation. The protocols aim to show how to apply therapeutics locally, obtain ocular exudates, and excise associated accessory organs such as healthy and inflamed eyelids (Figure 2), the cornea, and the conjunctiva. Attention must be paid when the upper eyelids are dissected for the isolation of the conjunctiva, and it must be stored in 1x PBS during the dissection of the cornea. This wi...

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Induction of Ocular Surface Inflammation and Collection of Involved Tissues

Ocular surface inflammation harms the ocular surface tissues and compromises vital functions of the eye. The present protocol describes a method to induce ocular inflammation and collect compromised tissues in a mouse model of Meibomian gland dysfunction (MGD).

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular ...

induction-ocular-surface-inflammation-collection-involved

Research

JoVE Journal

Immunology and Infection

2.2K Views.  Friedrich-Alexander-University Erlangen-Nürnberg and Universitätsklinikum Erlangen. Dit protocol beschrijft achtereenvolgens de stappen die betrokken zijn bij de inductie van oogletselontsteking en de verzameling van geassocieerde aangetaste organen die betrokken zijn bij meibomklierdisfunctie. Dit nieuwe model van MGD maakt het onderzoek mogelijk naar het aangeboren immuunaspect van de ziekte, met name de vorming van neutrofiele extracellulaire vallen die de Meibom-klier afsluiten. De techniek die in het protocol wordt getoond, kan worden uitgebreid om actuele therapeutisch...

Video: Induction of Ocular Surface Inflammation and Collection of Involved Tissues

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...

Impression Cytology of the Lid Wiper Area

Few reports on the cellular anatomy of the lid wiper (LW) area of the inner eyelid exist and only one report makes use of cytological methods. The optimization of a method of collecting, staining and imaging cells from the LW region using impression cytology (IC) is described in this study. Cells are collected from the inner surface of the upper eyelid of human subjects using hydrophilic polytetrafluoroethylene (PTFE) membranes, and stained with cytological dyes to reveal the presence of goblet cells, mucins, cell nuclei and various degrees of pre- and para-keratinization. Immunocytochemical dyes show cell esterase activity and compromised cell membranes by the use of a confocal scanning laser microscope. Up to 100 microscopic digital images are captured for each sample and stitched into a high-resolution, large scale image of the entire IC span. We demonstrate a higher sensitivity of IC than reported before, appropriate for identifying cellular morphologies and metabolic activity in the LW area. To our knowledge, this is the first time this selection of fluorescent dyes was used to image LW IC membranes. This protocol will be effective in future studies to reveal undocumented details of the LW area, such as assessing cellular particularities of contact lens wearers or patients with dry eye or lid wiper epitheliopathy.

We collected, stained and imaged cells from the conjunctiva of the inner eyelid margin of human subjects. By characterizing cell morphology and metabolic activity, this method may further our understanding of dry eye and the role that friction between the ocular surfaces may play in perceiving ocular discomfort.

Few reports on the cellular anatomy of the lid wiper (LW) area of the inner eyelid exist and only one report makes use of cytological methods. The ...

Medicine

A Rabbit Model of Aqueous-Deficient Dry Eye Disease Induced by Concanavalin A Injection into the Lacrimal Glands: Application to Drug Efficacy Studies

Dry eye disease (DED), a multifactorial inflammatory disease of the ocular surface, affects 1 in 6 humans worldwide with staggering implications for quality of life and health care costs. The lack of informative animal models that recapitulate its key features impedes the search for new therapeutic agents for DED. Available DED animal models have limited reproducibility and efficacy. A model is presented here in which DED is induced by injecting the mitogen concanavalin A (Con A) into the orbital lacrimal glands of rabbits. Innovative aspects of this model are the use of ultrasound (US) guidance to ensure optimal and reproducible injection of Con A into the inferior lacrimal gland; injection of Con A into all orbital lacrimal glands that limits compensatory production of tears; and use of periodic repeat injections of Con A that prolong the state of DED at will. DED and its response to test agents are monitored with a panel of parameters that assess tear production, the stability of the tear film, and the status of the corneal and conjunctival mucosa. They include tear osmolarity, tear break-up time, Schirmer&#39;s tear test, rose bengal staining, and tear lactoferrin levels. The induction of DED and the monitoring of its parameters are described in detail. This model is simple, robust, reproducible, and informative. This animal model is suitable for the study of tear physiology and of the pathophysiology of DED as well as for the assessment of the efficacy and safety of candidate agents for the treatment of DED.

This article describes the development of a method to induce acute or chronic dry eye disease in rabbits by injecting concanavalin A to all portions of the orbital lacrimal gland system. This method, superior to those already reported, generates a reproducible, stable model of dry eye suitable for the study of pharmacological agents.

Dry eye disease (DED), a multifactorial inflammatory disease of the ocular surface, affects 1 in 6 humans worldwide with staggering implications for ...

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360&#176; thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic ...

Neuroscience

Isolation of Intact Eyeball to Obtain Integral Ocular Surface Tissue for Histological Examination and Immunohistochemistry

Ocular surface (OS) consists of an epithelial sheet with three connected parts: palpebral conjunctiva, bulbar conjunctiva and corneal epithelium. Disruption of OS would lead to keratitis, conjunctivitis or both (keratoconjunctivitis). In experimental animal models with certain genetic modifications or artificial operations, it is useful to examine all parts of the OS epithelial sheet to evaluate relative pathogenetic changes of each part in parallel. However, dissection of OS tissue as a whole without distortion or damage has been challenging, primarily due to the softness and thinness of the OS affixed to physically separate yet movable eyelids and eyeball. Additionally, the deep eye socket formed by the hard skull/orbital bones is fully occupied by the eyeball leaving limited space for operating dissections. As a result, direct dissection of the eyeball with associated OS tissues from the facial side would often lead to tissue damages, especially the palpebral and bulbar conjunctiva. In this protocol, we described a method to remove the skull and orbital bones sequentially from a bisected mouse head, leaving eyelids, ocular surface, lens and retina altogether in one piece. The integrity of the OS sheet was well preserved and could be examined by histology or immunostaining in a single section.

This protocol describes a method for the isolation of the mouse eyeball with eyelid, ocular surface, anterior and posterior segments in relatively intact position.

Ocular surface (OS) consists of an epithelial sheet with three connected parts: palpebral conjunctiva, bulbar conjunctiva and corneal epithelium. ...

Biology

Establishing a Severe Corneal Inflammation Model in Rats Based on Corneal Epithelium Curettage Combined with Corneal Sutures

Corneal inflammation, especially severe corneal inflammation, plays a significant role in the development of corneal limbal stem cell dysfunction. Constructing appropriate animal models can help us focus on the effects of severe inflammation on corneal limbal stem cells. A 2 mm rust remover&#160;was&#160;used to remove the central corneal epithelium of Sprague Dawley (SD) rats to create an injury. Then, the central stroma of the cornea was sutured with nylon sutures to induce persistent inflammation. In this way, a corneal inflammation model with central corneal epithelium abrasion and central stroma suturing was constructed, which induced severe corneal inflammation. The changes in corneal inflammation and the condition of the limbal stem cells at 1, 3, and 7 days post-modeling were observed. On the 3rd day after modeling, the rats&#39; corneal limbus was severely edematous, with obvious neovascularization and local hyperplasia, which are typical signs of limbal stem cell deficiency. By the 7th day, the corneal edema gradually worsened, and the neovascularization continued to increase. Through quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunofluorescence staining, we found that the corneal epithelial inflammatory factors were significantly upregulated, the corneal epithelial differentiation was abnormal, the corneal epithelial stem cells were significantly reduced, and the cell proliferation and stemness had also decreased. Therefore, this model demonstrates that severe inflammation can induce limbal stem cell damage without directly damaging the limbal stem cells. The model is beneficial for observing the effects of severe inflammation on the biological mechanisms of stem cells and provides an ideal platform for studying the mechanisms of corneal epithelial stem cell dysfunction induced by inflammation.

We developed a rat model of severe corneal inflammation through corneal epithelium curettage combined with corneal sutures. The study evaluated corneal inflammation patterns, epithelial proliferation, and changes in limbal stem cells under inflammatory conditions.

Corneal inflammation, especially severe corneal inflammation, plays a significant role in the development of corneal limbal stem cell dysfunction. ...

A Method to Induce Ocular Surface Inflammation in a Murine Model

Source: Singh, J., et al. <a href="https://app.jove.com/v/63890">Induction of Ocular Surface Inflammation and Collection of Involved Tissues.</a> J. Vis. Exp. (2022)
This video demonstrates a method to induce ocular surface inflammation and meibomian gland dysfunction in a murine model. Stimulation of the mouse&#39;s immune system using an immunogen and later restimulation with the same immunogen promotes ocular surface inflammation, which causes plugging of meibomian gland secretion through neutrophil-produced aggNETs and leads to dry eye disease.

Source: Singh, J., et al. Induction of Ocular Surface Inflammation and Collection of Involved Tissues. J. Vis. Exp. (2022)
This video demonstrates a ...

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Immune Modulation

A Technique to Assess the Immune Response against an Antigen by Inducing Skin Inflammation

Source: Yao, Y., et al. <a href="https://app.jove.com/v/54070">Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice.</a> J. Vis. Exp. (2016).
This video demonstrates an in vivo technique to assess a test antigen by inducing skin inflammation in a murine model. The test antigen&#8212;a hapten in an oil/acetone solution&#8212;is topically applied to the skin of the mouse ear, and inflammation of the skin is assessed after an adequate period.

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

A Technique to Induce Chronic Experimental Autoimmune Dry Eye in Rats

Source: Hou, A., et al. <a href="https://app.jove.com/v/55592">A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue.</a> J. Vis. Exp. (2017).
This video demonstrates a technique to induce chronic experimental autoimmune dry eye disease in rats. Upon injecting an antigen-adjuvant emulsion containing autoantigens and foreign antigens into a rat at predefined intervals, chronic eye inflammation is induced in the immunized rat by injecting the same antigens into the ocular surface tissues.

Immunopathology

Host-Pathogen Interaction Models

Inducing Experimental Glaucoma Induction via Iridocorneal Angle Obstruction with Magnetic Beads

After anesthetizing the rats, apply ocular ointment to the unoperated contralateral eye to keep it moist during anesthesia. Apply 0.5% pro procaine hydrochloride local anesthetic to the operative eye. Next, wash this eye with 5% povidone iodine in water. After 5 minutes, wick off the povidone iodine and wash the eye with 0.9% sterile saline solution.
Keep the eye moist during anesthesia with regular application of sterile saline. Now, place a toroidal magnet around the eye. To minimize the risk of iris trauma and minimize bead loss, orient the needle tangential to the corneal surface as parallel to the iris as possible. Proceed to inject 25 microliters of 30 milligrams per millimeter gamma irradiated magnetic microspheres into the anterior chamber.
Leave the needle in place for one minute post-injection, to ensure that beads settle into the iridocorneal angle to impede aqueous drainage from the trabecular meshwork. Slightly angle the needle a few seconds after the beads have initially settled to allow some leakage of aqueous, and to minimize transient increases in intraocular pressure.
To ensure continued use of the needle in separate procedures, flush the needle with PBS, then 70% ethanol, followed by distilled water. At this stage, if necessary, remove the magnet and use it to draw beads into areas of incomplete coverage. Leave the magnet in place around the eye for a further 10 minutes post-injection, to ensure beads settle well into the iridocorneal angle.

Induction of Ocular Surface Inflammation and Collection of Involved Tissues

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