eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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&#160;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. 
		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>
</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&#160;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>

<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</td>
			<td></td>
			<td></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>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</td>
			<td>ThermoFisher Scientific</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:&#160;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>

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.

<img alt="Figure 1" src="/files/ftp_upload/21604/21604_Figure1.jpg" /> 
Figure 1: Excision of the facial tissue surrounding the orbital region.&#160;The incision trail is indicated in red. This allows for excising the ocular organs and investigating the influence and effects of various topically administered therapeutics on accessory ocular organs such as the conjunctiva and cornea.

A Method to Induce Ocular Surface Inflammation in a Murine Model

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

The ocular surface system comprises the cornea, conjunctiva, and an associated gland network. A tear film protects the ocular surface.
The evaporation of the tear film&#39;s aqueous layer is inhibited by a lipid layer secreted by the meibomian glands. Ocular surface inflammation obstructs lipid secretion, leading to dry eye disease.
To induce ocular surface inflammation in a murine model, take a mouse and inject an immunogen solution intraperitoneally. The immunogen activates helper T cells, or Th cells, to elicit an immune response. The activated cells spread to the eyes through systemic circulation.
Post-incubation, perform an ocular surface challenge by applying the immunogen on the eye, generating an exaggerated immune response.
Antigen-presenting cells present the immunogen to the activated Th cells. Restimulation of the Th cells causes the release of cytokines and chemoattractants, leading to ocular surface inflammation.
The chemoattractants cause an influx of neutrophils. Binding to the cytokines activates a signaling cascade inside the neutrophils, generating reactive oxygen species or ROS. The generated ROS causes the release of antimicrobial proteins from cytoplasmic granules and chromatin decondensation.
The decondensed chromatin fibers and granular proteins form an extracellular web-like structure termed an aggregated neutrophil extracellular trap or aggNET. The excessive aggNET production clogs meibomian gland openings &#8212; preventing lipid secretion.
The murine model of ocular surface inflammation is ready for downstream analysis.

a-method-to-induce-ocular-surface-inflammation-in-a-murine-model

Research

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Immune Modulation

142 Views. Source: Singh, J., et al. Induction of Ocular Surface Inflammation and Collection of Involved Tissues. 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'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 ...

Video: A Method to Induce Ocular Surface Inflammation in a Murine Model - Concept

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 ...

JoVE Journal

Immunology and Infection

Intense Pulsed Light for the Treatment of Dry Eye Owing to Meibomian Gland Dysfunction

Dry eye disease (DED) is an increasingly common condition and one of the most common complaints of patients. The vast majority of DED is caused by the so-called &#34;evaporative&#34; subtype, that is mainly caused by meibomian gland dysfunction (MGD). Intense pulsed light (IPL) devices employ high intensity pulses of polychromatic lights with a broad range of wavelength (515-1200 nm). IPL treatment has been utilized for years in the field of dermatology, and then its use was applied to ophthalmology for the treatment of MGD. Recently, a new device employing IPL was specifically designed for the periocular application. This procedure determines the thermal selective coagulation and ablation of superficial blood vessels and telangiectasias of the eyelids skin, reducing the release of inflammatory mediators and tear cytokines levels, and improving meibomian glands outflow. IPL treatment is noninvasive and easy to perform, lasts for only a few minutes and can be conducted in an office setting. In the present study, 19 patients underwent 3 sessions of IPL treatment. After treatment, both mean noninvasive break-up time and lipid layer thickness grade significantly increased, as a result of an improvement of tear film stability and quality, respectively. Conversely, no statistically significant changes were found for meibomian gland loss and tear osmolarity. Furthermore, the vast majority of the treated patients (17/19; 89.5% of the total) perceived an improvement of their ocular discomfort symptoms after IPL treatment. Although IPL treatment provides an improvement of both ocular surface parameters and ocular discomfort symptoms after one cycle of three sessions, regular repeated treatments are usually required to maintain the persistence over the time of its beneficial effects.

Dry eye disease is an increasingly common condition, which strongly impair patients&#39; life quality. Recently, a new device employing intense pulsed light, specifically designed for the periocular area, has been shown to improve tear film stability and ocular discomfort symptoms in dry eye disease owing to meibomian gland dysfunction.

Dry eye disease (DED) is an increasingly common condition and one of the most common complaints of patients. The vast majority of DED is caused by the ...

Medicine

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.

A Method to Induce Ocular Surface Inflammation in a Murine Model

Transcript

A Method to Induce Ocular Surface Inflammation in a Murine Model

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

Intense Pulsed Light for the Treatment of Dry Eye Owing to Meibomian Gland Dysfunction

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