Name: induction of ocular surface inflammation and collection of involved tissues
Uploaded: 2022-08-04T13:10:00
Description: Watch this Scientific Journal Video about Induction of Ocular Surface Inflammation and Collection of Involved Tissues at JoVE.com

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. Ind...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meibomian+gland+dysfunction.+II.+The+role+of+keratinization+in+a+rabbit+model+of+MGD.">Meibomian gland dysfunction. II. The role of keratinization in a rabbit model of MGD.</a> Investigative Ophthalmology and Visual Science. 30 (5), 936-945 (1989).</li><li>Jester, J. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+biomicroscopy+and+photography+of+meibomian+glands+in+a+rabbit+model+of+meibomian+gland+dysfunction.">In vivo biomicroscopy and photography of meibomian glands in a rabbit model of meibomian gland dysfunction.</a> Investigative Ophthalmology and Visual Science. 22 (5), 660-667 (1982).</li><li>Lambert, R., Smith, R. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+ELOVL4+in+the+mouse+meibomian+gland+and+sebocyte+cell+biology.">A role for ELOVL4 in the mouse meibomian gland and sebocyte cell biology.</a> Investigative Ophthalmology and Visual Science. 55 (5), 2832-2840 (2014).</li><li>Miyake, H., Oda, T., Katsuta, O., Seno, M., Nakamura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meibomian+gland+dysfunction+model+in+hairless+mice+fed+a+special+diet+with+limited+lipid+content.">Meibomian gland dysfunction model in hairless mice fed a special diet with limited lipid content.</a> Investigative Ophthalmology and Visual Science. 57 (7), 3268-3275 (2016).</li><li>Schaumberg, D. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 
			Final Concentration: in vivo: 1 mg/ 100 &#181;L</td>
		</tr>
		<tr>
			<td>C57Bl/6 mice, aged 7&#8211;9 weeks</td>
			<td>Charles River Laboratories&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium</td>
			<td>Carl roth</td>
			<td>CN93.1</td>
			<td>1 M 
			Final Concentration: 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 106 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 &#181;L in saline</td>
		</tr>
		<tr>
			<td>Pertussis toxin&#160;</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>PHZ1174</td>
			<td>50 &#181;g/ 500 &#181;L in saline 
			Final Concentration: in vivo: 100 &#181;g/ 100 &#181;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&#160;</td>
			<td>Final Concentration: in vivo:&#160; 300 ng/ 100 &#181;L</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Zaiss</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&#34;-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 ...

This protocol sequentially describes the steps involved in the induction of ocular surface inflammation and the collection of associated affected organs involved in Meibomian gland dysfunction. This new model of MGD enables the investigation of innate immune aspect of the disease, particularly the formation of neutrophil extracellular traps occluding the Meibomian gland. The technique exhibited in the protocol can be extended to introduce topical therapeutic interventions in a pre-clinical stage.Moreover, the collection of ocular exudates and organs reflect the efficacy of the treatment. To perform intraperitoneal injection of the immunogen solution, hold the seven to nine weeks old C57 black six non anesthetized female mouse softly by the tail while grasping the cage grid. Then firmly hold 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.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 five microliters of ovalbumin or ova or saline per eye of an anesthetized mouse, and wait for five 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.For the excision of ocular surface tissues, place the mouse on an even surface and disinfect the orbital area around the eye with a 70%ethanol swab. To form an incision around the eye, make an incision between the ear and the retroorbital 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. Carefully hold the dissected tissue around the eye using curved forceps, and pull the tissue and eyeball out.Place the excised organs in sterile PBS. Then using a scalpel under the stereo microscope, trim the excess facial muscle tissue around the excised upper eyelids. To collect the conjunctiva place the upper eyelid on a dry Petri dish.Then using fine tweezers and a scalpel peel the whitish mucus layer gently out of the inner surface of the eyelid. Next to dissect the cornea, place the eye globe on a new dry Petri dish on top of dry ice. After three minutes, place the dish onto the bench surface in a stable position.Use fine, sharp scissors to make a small incision at the border of the cornea next to the limbus. Continue the incision with the scalpel around the eye globe, separating the sclera from the cornea. Then remove the iris and lens remnants from the cornea's backside by generously flushing with saline solution.To assess the Meibomian gland and its orifices, place the excised eyelids in an upright position under the stereo microscope. Then according to the camera exposure time and ISO settings, capture images under white light epi-illumination. To assess the Meibomian gland area, place the excised eyelids in a horizontal position and turn on the backlight of the stereo microscope equipped with an infrared camera.Capture images by adjusting the exposure time according to the camera ISO. Saline administered mice showed healthy ocular surfaces with eyes wide open. In comparison, ova application induces severe ocular surface inflammation, the narrow opening of the eyes, and signs of chemosis.The excised eyelids showed large occlusions plugging the orifices of the Meibomian gland and edema, in contrast to the healthy eyelids showing small plugs of the gland lining the eyelid. Further examination of the gland's infrared transillumination reveals the racemic appearance of the acini of the Meibomian gland. The eyelids of saline challenged mice showed the round acini.In comparison, ova applications induced the destruction and loss of some glands with the allergic eye disease or AED. The histological analysis of the eyelids displayed dilated Meibomian glands compared to mice administered with only saline. Incubating the eyeball on dry ice allowed incision at the cornea next to the limbus and separation of the cornea from the sclera.This study facilitated the collection of the cornea. In addition, ova administration inflicted severe inflammation to the conjunctiva with a hyperemic appearance. Elevated levels of the chemokine CXCL-1 facilitate neutrophil extravasation, phagocytosis, and degranulation, and interleukin-6 mediating the acute phase response and neutrophil infiltration were observed in the supernatants of AED mice.On the other hand, the concentration of interleukin-10 showed no significant changes in both naive and AED mice. This new model enables the development and the evaluation of efficacy of drugs that target the innate immune pathways, and is of great help in data mining the way of administration of anti-inflammatory agents. After appropriate collection of organs, highly multiplex methods like single cell sequencing and imaging mass cytometry are compatible with this protocol.

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