All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Assessment of Dry Eye Features
Measure corneal damage with fluorescein staining.
<ol>
	<li>Add 2 &#181;L of 0.2% fluorescein to the rat cornea. Passively open and close the rat eyelids 3 times with a gloved finger to spread the fluorescein dye on the surface of the eye.</li>
	<li>After 1 min, draw 1 mL of saline with a 3 mL syringe (without any needle attachment), position the syringe about 2-3 mm anterior to the cornea, and gently propel saline onto the eye.</li>
	<li>Acquire images under the cobalt blue filter (i.e., ~400 nm) of an ocular-imaging microscope with the background lights switched off. 
	NOTE: Images were subsequently analyzed by a grading system modified from previous publications. Images were then analyzed by subjectively grading the number, area, and intensity of the green spots from 0 to 2, where 0 indicates their absence, 1 indicates punctate staining of less than 50 spots, and 2 indicates punctate staining of more than 50 spots.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td></td>
			<td></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>Stereo microscope with ring light illuminator and camera</td>
			<td>Carl Zeiss</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

fluorescein staining in an autoimmune dry eye rat model to examine corneal damage

Source: Hou Ahiua, 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 the staining of the damaged cornea using fluorescein dye to evaluate the extent of autoimmune dry eye disease in a rat model. The green fluorescence spots of the stained collagen fibers indicate corneal epithelial damaged cells.

Fluorescein Staining in an Autoimmune Dry Eye Rat Model to Examine Corneal Damage

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

Start with an anesthetized rat presenting symptoms of autoimmune dry eye disease.
Apply an appropriate amount of fluorescein dye over the rat's cornea.
Close and open the rat's eyelid to ensure the fluorescein spreads evenly over the cornea.
Allow the dye to settle and adhere to the cornea.
In autoimmune dry eye disease, the immune cells mistakenly target and attack the lacrimal glands, decreasing tear production.
This leads to the desiccation of the corneal epithelium and damage to the underlying Bowman's membrane &#8212; an acellular zone anterior to the stroma.
The fluorescein molecules permeate through the damaged corneal barriers to reach the underlying collagen-rich stromal layer.
Here, the fluorescein molecules bind to collagen, resulting in the staining of the collagen fibers.
Using an appropriate imaging technique, acquire images of the stained cornea.
Analyze the number, area, and intensity of the green fluorescent spots to measure the extent of corneal damage.

Use fluorescein scanning to measure corneal damage by adding 2 microliters of 0.2% fluorescein to the rat cornea. With a gloved finger, passively open and close the rat eyelids three times to spread the fluorescein dye on the surface of the eye.
After 1 minute, use a 3-milliliter syringe to draw 1 milliliter of saline. Then, position the syringe about 2 to 3 millimeters anterior to the cornea, and gently apply saline onto the eye. With the background lights switched off, acquire images under the cobalt blue filter of an ocular imaging microscope.

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1.3K Views. Start with an anesthetized rat presenting symptoms of autoimmune dry eye disease. Apply an appropriate amount of fluorescein dye over the rat's cornea. Close and open the rat's eyelid to ensure the fluorescein spreads evenly over the cornea. Allow the dye to settle and adhere to the cornea. In autoimmune dry eye disease, the immune cells mistakenly target and attack the lacrimal glands, decreasing tear production. This leads to the desiccation of the corneal...

Name	Company	Catalog Number
Reagent
Fluorescein sodium solution	Bausch & Lomb U.K Limited	NA
Equipment
Stereo microscope with ring light illuminator and camera	Carl Zeiss	NA

Fluorescein Staining in an Autoimmune Dry Eye Rat Model to Examine Corneal Damage

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