a technique to induce chronic experimental autoimmune dry eye in rats

A Technique to Induce Chronic Experimental Autoimmune Dry Eye in Rats

On day zero, distribute 200 microliters of the emulsion which contains 1 milligram of lacrimal gland extract and 200 micrograms of ovalbumin, and complete Freund&#39;s adjuvant into 1-milliliter Luer-lock syringes, and equip the syringes with 27-gauge needles.
Without using anesthesia, subcutaneously inject the emulsion at the base of the rat tails. Then, on day 14 in a similar manner, inject 200 microliters of the same antigen and incomplete Freund&#39;s Adjuvant. On the same day, intraperitoneally inject 300 nanograms of pertussis toxin in 100 microliters of PBS.
On day 48, weigh the required amount of ovalbumin and dissolve it in PBS, then, combine ovalbumin with the defrosted lacrimal gland extract prepared earlier in this video, making antigen solution containing 1 milligram per milliliter of ovalbumin and 1 milligram per milliliter of lacrimal gland extract.
After anesthetizing the rats, inject 5 microliters of antigen into the fornicial subconjuctiva of the eye, then, inject 20 microliters of antigen into the lacrimal gland.

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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 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 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 Mycobacterium tuberculosis H37Ra into 10 mL of complete Freund&#39;s adjuvant containing 1 mg/mL Mycobacterium tuberculosis H37Ra and mix well. 
		NOTE: The final complete Freund&#39;s adjuvant contains 5 mg/mL Mycobacterium tuberculosis 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. 
		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 Mycobacterium tuberculosis 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., 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>

<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</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</td>
			<td>Sigma-Aldrich</td>
			<td>F5506-6X10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>pertussis toxin</td>
			<td>Sigma-Aldrich</td>
			<td>P7208-50UG</td>
			<td></td>
		</tr>
		<tr>
			<td>fluorescein sodium solution</td>
			<td>Bausch &#38; Lomb U.K Limited</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Sonics</td>
			<td>Vibra-Cell</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro IV microscope</td>
			<td>Phoenix Research Labs</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> 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.

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

Take a rat previously immunized with an antigen-adjuvant emulsion containing lacrimal gland-derived autoantigens and foreign antigens.
Re-inject the emulsion and intraperitoneally inject pertussis toxin, ensuring maximal T cell activation.
Activated cells differentiate into circulating effector memory T cells, TEMs and tissue-resident memory T cells, TRMs. A percentage of TRMs resides in ocular surface tissues.
After a defined interval, inject the antigens into the forniceal sub-conjunctiva and lacrimal glands, inducing localized inflammation.
Subconjunctival antigen-presenting cells, APCs, present antigens to the TRMs, triggering cytokine release.
Cytokines induce conjunctival epithelial cells to release chemokines, which recruit TEMs.
APCs reactivate the infiltrated TEMs, triggering proinflammatory cytokine release.
Cytokines cause the death of mucin-producing conjunctival goblet cells, decreasing mucin levels, and causing tear film instability.
Cytokines induce lacrimal gland cell death &#8212; reducing tear production &#8212; and stimulate corneal epithelial cells to produce matrix metalloproteinases, damaging the cornea.
Assess the rat for the clinical features indicating successful dry eye induction.

36 次观看. 诱导大鼠慢性实验性自身免疫性干眼症的技术

视频: 诱导大鼠慢性实验性自身免疫性干眼症的技术 - 实验

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.

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).

诱导眼表炎症和受累组织收集

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

科研

JoVE Journal

免疫与感染

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.

由康卡纳林注射到乳原区引起的水性干眼病的兔子模型:药物功效研究的应用

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

医学

Comparing Objective Conjunctival Hyperemia Grading and the Ocular Surface Disease Index Score in Dry Eye Syndrome During COVID-19

The incidence of dry eye syndrome (DES) has increased due to wearing masks, utilizing digital devices, and working remotely during the pandemic. A survey was conducted during the COVID-19 pandemic to determine the prevalence of dry eye syndrome. A cross-sectional study investigated how prevalent DES is during COVID-19 in healthy patients aged 20-45 in the United States. An Ocular Surface Disease Index (OSDI) questionnaire was given to 40 individuals remotely from October 31, 2021, to December 1, 2021. The AOS and the OSDI survey were used to evaluate DES. The subjects were 29 years old on average (SD 14.14), with 23 males (57.5%) and 17 females (42.5%). According to the OSDI survey, low DES, moderate DES, and severe DES had prevalence rates of 15%, 77.5%, and 7.5%, respectively. White (W) people represent 50% of the population, while African Americans (AA) represent 35%, Asians represent 7.5%, and Hispanics represent 7.5%. Mild DES affected 77.5% of subjects, with 64.50% males and 35.50% females. According to the AOS objective grading system, mild (M) DES, moderate (MO) DES, and severe (S) DES had prevalence rates of 40%, 12.5%, and 15%, respectively. Linear regression was used to compare the two grading systems, and it demonstrated a strong relationship between the two grading systems.

The present protocol describes the cross-sectional research performed on 40 healthy subjects between the ages of 20 and 45 to assess the prevalence of dry eye syndrome (DES) during COVID-19. The OSDI survey evaluated DES, and the advanced ophthalmic systems (AOS) software was used to assess limbal redness.

COVID-19期间干眼症患者结膜充血分级与眼表疾病指数评分的比较

The incidence of dry eye syndrome (DES) has increased due to wearing masks, utilizing digital devices, and working remotely during the pandemic. A survey ...

A Technique to Induce Chronic Experimental Autoimmune Dry Eye in Rats

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A Technique to Induce Chronic Experimental Autoimmune Dry Eye in Rats