assessment of cornea and tear smoothness in an autoimmune dry eye rat model

Assessment of Cornea and Tear Smoothness in an Autoimmune Dry Eye Rat Model

To measure the cornea tear smoothness, place the rat under a stereomicroscope, equipped with a ring illuminator and a camera. Then, apply 5 microliters of saline to the rat cornea. With gloved fingers, passively blink by moving the upper and lower eyelids approximately five times to spread the saline. Under 1.6 times magnification, focus the ring illuminator on the middle of the cornea surface, then, after 10 seconds, acquire photographic images.

assessment-cornea-tear-smoothness-an-autoimmune-dry-eye-rat

Research

JoVE Encyclopedia of Experiments

Immunology

Immunopathology

Assay and Analysis

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. Preparation of Lacrimal Gland Extract
NOTE: Rats were anesthetized with the intraperitoneal injection of ketamine (75 mg/kg) and xylazine (10 mg/kg). Proper anesthetization was confirmed by toe pinching and tail pinching. Ophthalmic gel was applied to the rat&#39;s eyes to prevent dryness after each procedure. The anesthetized rats were placed under far infrared lights to keep the animals warm until they fully recovered. During the procedures and recovery time, animals were closely monitored by researchers. All the materials and surgery tools were sterile before use. At the end of the experiment, rats were euthanized by the intraperitoneal injection of pentobarbital (80 mg/kg). Complete euthanasia was verified by lack of cardiac pulse and no&#160;blink reflex by touching the eyeball. Rats were housed in standard conditions: room temperature, 21-23 &#176;C; relative humidity, 30-70%; light-dark cycle, alternating 12 h (7 AM to 7 PM).
<ol>
	<li>Place the euthanized female Sprague-Dawley rats (age range from 8 weeks to 16 weeks) flat, with one ear against the table and the other facing up. Make a 10-mm incision superior-inferiorly under the exposed ear with a pair of spring scissors. Remove the lacrimal gland by dissecting it from the surrounding connective tissue and from the drainage duct.
	<ol>
		<li>Store the glands at -80 &#176;C until required. Thaw the glands on ice. Mince as finely as possible on ice with scissors. Add 150 &#181;L of phosphate-buffered saline or PBS with 1x protease inhibitor per lacrimal gland.</li>
	</ol>
	</li>
	<li>Sonicate the samples on ice for 5 min at 20 kHz with the sonicator set at 10 s on, and 10 s off at 30% amplitude. Centrifuge the sonicated samples for 20 min at 13,000 x g and 4 &#176;C.</li>
	<li>Pipette the supernatant and transfer it to a new tube. Aliquot the supernatant to 1.5 mL tubes and store them at -80 &#176;C. Measure the protein concentration with a bicinchoninic acid assay, as per the manufacturer&#39;s instructions.</li>
</ol>
2. Preparation of Emulsion and Pertussis Toxin
<ol>
	<li>Emulsion
	<ol>
		<li>Add 40 mg of heat-killed&#160;Mycobacterium tuberculosis&#160;H37Ra into 10 mL of complete Freund&#39;s adjuvant containing 1 mg/mL&#160;Mycobacterium tuberculosis&#160;H37Ra and mix well.&#160;&#160;&#160;&#160;&#160; 
		NOTE: The final complete Freund&#39;s adjuvant contains 5 mg/mL&#160;Mycobacterium tuberculosis&#160;H37Ra.</li>
		<li>Weigh the ovalbumin, dissolve it in PBS, and prepare an antigen mixture containing 2 mg/mL ovalbumin and 10 mg/mL lacrimal gland extract. Aiming for an injection volume of 200 &#181;L for each animal, prepare a master mixture based on the animal numbers.</li>
		<li>Transfer incomplete Freund&#39;s adjuvant or complete Freund&#39;s adjuvant to a 50 mL tube, ensuring that the volume of adjuvant to the antigen mixture is in a 1:1 ratio. Add the antigen mixture drop-by-drop to the adjuvant while vortexing with the highest speed so that does not result in spillage. Continue vortexing for 5 min after all the antigen has been added.</li>
		<li>Transfer the emulsion to a 5 mL syringe and link it with another 5 mL syringe through a syringe connector. Push the emulsion from one syringe to the other to mix it.&#160; 
		NOTE: The emulsion is ready if a single droplet remains as a sphere when placed in water. Only freshly prepared emulsion or emulsion stored overnight at 4 &#176;C should be used.</li>
	</ol>
	</li>
	<li>Pertussis toxin
	<ol>
		<li>Reconstitute 50 &#181;g of pertussis toxin in 500 &#181;L of water to make a final concentration of 100 ng/&#181;L. Votex the container for 30 seconds to make sure the toxin dissolves completely. 
		NOTE: Do not sterilize by filtration, as this will result in a loss of material. Do not freeze. This solution remains active for at least 6 months at 4 &#176;C.</li>
		<li>Dilute 100 ng/&#181;L of pertussis toxin to 3 ng/&#181;L using PBS. Transfer 100 &#181;L of the diluted pertussis toxin to a 1 mL Luer-lock injection syringe with a 27 G needle.</li>
	</ol>
	</li>
</ol>
3. Immunization of the Lewis Rats
<ol>
	<li>On day 0, mix the emulsion a few times. Distribute 200 &#181;L of the emulsion, which contains 1 mg of lacrimal gland extract and 200 &#181;g of ovalbumin in complete Freund&#39;s adjuvant, into 1-mL Luer-lock syringes with 27G needles. Inject the emulsion subcutaneously at the base of the rat tails without anesthesia. 
	NOTE: The tail does not need to be pre-warmed before the injection. Each rat is immunized with 1 mg of lacrimal gland extract and 200 &#181;g of ovalbumin in complete Freund&#39;s adjuvant with 500 &#181;g of&#160;Mycobacterium tuberculosis&#160;H37Ra.</li>
	<li>On day 14, inject 200 &#181;L of the emulsion, which contains 1 mg of lacrimal gland extract and 200 &#181;g of ovalbumin in incomplete Freund&#39;s adjuvant, in the same manner as on day 0. Inject 300 ng of pertussis toxin in 100 &#181;L of PBS intraperitoneally per rat on the same day.</li>
</ol>
4. Injection of the Antigen Mixture into the Forniceal Subconjunctiva and Lacrimal Gland to Recruit Antigen-specific Immune Cells and Cause Local Inflammation
<ol>
	<li>Calculate the amount of ovalbumin and lacrimal gland extract based on the number of rats in the experiment. Weigh the required amount of ovalbumin and combine it with the defrosted lacrimal gland extract prepared in step 1, making antigen solution containing 1 mg/mL ovalbumin and 1 mg/mL lacrimal gland extract.</li>
	<li>Anesthetize the rats with ketamine (75 mg/kg) and xylazine (10 mg/kg) by intraperitoneal injection. Pinch the toes of rats to ensure that proper anesthesia has been achieved (i.e.,&#160;no responsive movement is observed after the pinch). Inject 5 &#181;L of antigen into the forniceal subconjunctiva of the eye. Inject 20 &#181;L of antigen into the lacrimal gland.</li>
</ol>
5. Assessment of Dry Eye Features
NOTE: The anesthetized rats should be held gently by a gloved hand in an upright position on a flat surface to avoid movement.
<ol>
	<li>Measure the tear volume with phenol red thread.
	<ol>
		<li>Use one pair of forceps to hold the thread and another to pull the lower eyelid of the rat. Place the thread at the proximal corner of the lower fornix for 1 minute and then remove the thread.&#160;&#160;&#160;&#160;&#160; 
		NOTE: Tears make the wetted part of the thread red in color.</li>
		<li>Take an image of the length of the wetted part of the thread next to a ruler with millimeter markings. Measure the wetted length to the tenth of a millimeter using an image software (e.g.,&#160;ImageJ).</li>
	</ol>
	</li>
	<li>Measure cornea/tear smoothness.
	<ol>
		<li>Place the rat under a stereomicroscope equipped with a ring illuminator and a camera. Apply 5 &#181;L of saline to the rat cornea. Passively blink by moving the upper and lower eyelids with gloved fingers ~5 times to spread the saline.</li>
		<li>Focus the ring illuminator on the middle of the cornea surface under 1.6x magnification. Acquire photographic images after 10 s.&#160;&#160;&#160;&#160;&#160;&#160; 
		NOTE: The ring illuminator projects two circular rows of dot images on the corneas of the animal. The regular spacing of the undistorted dots suggests a smooth cornea/tear layer.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>P2714-1BTL</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermal Scientific</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td>Mycobacterium tuberculosis H37Ra&#160;</td>
			<td>Becton, Dickinson and company</td>
			<td>231141</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete Freund&#39;s adjuvant</td>
			<td>Becton, Dickinson and company</td>
			<td>231131</td>
			<td></td>
		</tr>
		<tr>
			<td>Ovalbumin</td>
			<td>Sigma-Aldrich</td>
			<td>A5503-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Incomplete Freund&#39;s adjuvant&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F5506-6X10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Pertussis toxin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P7208-50UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Sonics&#160;</td>
			<td>Vibra-Cell</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope with ring light illuminator and camera</td>
			<td>Carl Zeiss</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

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>&#160;J. Vis. Exp. (2017)
This video demonstrates a method for assessing corneal smoothness and tear film stability in an autoimmune rat model. It highlights the impact of gland dysfunction on tear film stability, crucial for understanding dry eye disease.

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

The tear film covering the cornea consists of lipid, water, and mucin produced by various secretory glands. Dysfunctioning of lacrimal and meibomian glands affects tear film stability and cornea smoothness, resulting in dry eye disease.
To measure cornea and tear smoothness, position an anesthetized autoimmune rat model under a stereomicroscope equipped with a ring illuminator featuring evenly spaced LED&#160; lights encircling the objective lens.
Moisten the ocular surface with saline and spread it evenly for accurate smoothness assessment.
Focus the ring illuminator on the cornea&#39;s center, generating an illuminator ring image with two circular rows of dots.
In the unaffected control rat, the illuminator ring image on the ocular surface is round with evenly spaced dots, indicating a smooth cornea with a stable tear film.
Conversely, the distorted image of an autoimmune rat model indicates reduced corneal smoothness and unstable tear film, confirming dry eye disease.

39 Aufrufe. Beurteilung der Hornhaut- und Tränenglätte in einem Autoimmunmodell für Ratten mit trockenem Auge

Serie: Beurteilung der Hornhaut- und Tränenglätte in einem Autoimmunmodell für Ratten mit trockenem Auge - Experiment

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.

Intense Pulsed Light für die Behandlung des trockenen Auges aufgrund der Meibom-Drüse Dysfunktion

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 ...

Forschung

JoVE Journal

Medizin

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.

A Rabbit Model of Aqueous-Deficient Dry Eye Disease Induced by Concanavalin A Injection into the Lacrimal Dlands: 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 ...

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer&#39;s tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

Etablierung eines schweren Trockenauges modell mit vollständiger Dakryoadenektomie bei Kaninchen

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...

Assessment of Cornea and Tear Smoothness in an Autoimmune Dry Eye Rat Model

Transkript

Assessment of Cornea and Tear Smoothness in an Autoimmune Dry Eye Rat Model