Materials and Reagents

3 ml disposable transfer pipettes (VWR, catalog number: 414004-037)
0.2 µm sterile syringe filter (VWR, catalog number: 28145-477)
Latex exam gloves (VWR, catalog number: 414004-429)
Lab coat (VWR, catalog number: 37000-922)
3 month female adult SVJ 129 mouse
3 month adult female Sprague Dawley rat
PNU-282987 (N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride) (Sigma-Aldrich, catalog number: P6499) (desiccate, store at 4 °C)
BrdU (5-Bromo-2’-deoxyuridine) (Sigma-Aldrich, catalog number: 19-160) (store for up to 2 years at -20 °C)
PBS (Phosphate buffered saline 10x concentrate) (Sigma-Aldrich, catalog number: P5493) (store at room temperature)
DMSO (Sigma-Aldrich, catalog number: D8418) (store at room temperature)
Cheerios (Local grocery store, store at room temperature)
PNU-282987/BrdU solution (see Recipes)

Equipment

30 ml clear glass bottles (VWR, catalog number: 10862-356)
4 °C refrigerator
Fume hood

Procedure

Animals are given one eye drop once a day from a backfilled 3 ml plastic disposable transfer pipette for various amounts of time depending on the experimental design of the study. One drop of solution from the transfer pipette contains 30 µl of diluted PNU-282987/BrdU. Eye drops are delivered directly to the center of the bulbar conjunctiva of the eye; a clear membrane that covers the anterior face of the sclera and the transparent surface epithelium of the cornea. A new disposable 3 ml transfer pipette is used each time eye drops are applied. Both sexes of adult rats and mice produce BrdU positive cells after 3 days of eye drop treatments. In vehicle control studies, eye drops containing PBS or eye drops containing PBS with 1% DMSO are applied as negative controls.

Adding eye drops to adult mice
Notes:
1. The safe restraint and gentle handling of these rodents is a key part of the experimental procedures. All rodent handlings wear a lab coat and latex gloves in the animal facility. All handlers have Institutional Animal Care and Use Committee training before proceeding.
2. Animals should be approached with confidence and the animals should be handled gently, but firmly to avoid bites or scratches to the handler or injuries to the animal.
3. One handed mouse restraint is usually performed with the non-dominant hand, leaving the dominant hand free for applying eye drops.
1. Lift adult mice (ranging from 3-6 months) by the base of the tail from their cage and place them on the cage lid. Tuck the base of the tail between the 3^rd and 4^thfinger (Figure 2A).
2. Then immobilize the adult mice (ranging from 3-6 months) by scruffing the skin over their neck. Using the same hand that is holding the tail, grasp the skin near the base of the head with the index finger and thumb (Figure 2B).
  Note: Be sure to apply just enough pressure, or firmness, to the skin around the neck to ensure the eyes are opened and to prevent the mouse from turning or twisting out of the hand restraint.
3. Once the mouse is firmly scruffed, place one drop of diluted PNU-282987/BrdU solution on the bulbar conjunctiva of the right eye. The right eye should be positioned up by twisting the wrist of the restrainer’s hand. The dilution should be dropped just over the eye. Do not bring the transfer pipette in direct contact with the cornea. The drop will cover the entire eye and should sit on top of the eye as a small ball for 1-2 s (Figure 2C).
  Note: If treatment is needed in the animal’s contralateral eye, the restrainer’s wrist containing the same scruffed animal should be turned so that the left eye is situated up. At this point, another single drop can be added to the left eye and allowed to sit in a small ball over the eye for 1-2 s.
4. Once eyes have been treated, release the animal back into its cage. It is not unusual for the animal to shake its head once it is released to remove any excess fluid that remains over its eye (see Notes).
  
  Figure 2. Eye drop application to adult mice. A. A 3-month-old female adult SVJ 129 mouse is removed from cage and positioned for handling. B. The mouse is held for eye drop delivery. C. Eye drop applied to scruffed animal. Scale bar represents 1.25 cm.
Adding eye drops to adult rats
1. Deliver eye drops to rats (aged 3-6 months) that are not physically restrained. Remove the top of a rat cage and add an eye drop containing a dilution of PNU-282987 and BrdU to the center of the rat eye using a disposable 3 ml backfilled transfer pipette (Figure 3A). Place the eye dropper within an inch of the eye before releasing one drop of fluid containing 30 µl of diluted PNU-282987/BrdU onto the bulbar conjunctiva. The rat will blink and shake its head when an eye drop has been successfully applied. Do not allow the transfer pipette to come in direct contact with the cornea.
2. When one drop has been added, a drop can also then be added to the contralateral eye depending on the experimental design of the study.
3. After all drops have been administered, give each rat 2-3 Cheerios before the top of the cage is replaced (example in Figure 3B) and then return the cage containing the treated animal to the colony. All animals are housed singly.
  
  Figure 3. Eye drop application to adult rats. A. A 3-month-old adult female Sprague Dawley rat is positioned to receive one drop of PNU-282987/BrdU solution. B. The Cheerio reward is delivered after a successful eye drop delivery. Scale bar represents 2.5 cm.

Notes

The first time an eye drop is delivered to a rat eye, the animal will be unfamiliar with the procedure and may not be still long enough for an eye drop to be deposited to the center of the cornea (1-2 s is needed). To minimize this behavior, the transfer pipette containing the eye drop should be positioned and held steady above the cage until the animal settles down. The time for the animals to settle down the first time eye drops are delivered varies depending on each animal’s personality. However, the first time, it can take an average of 2 or 3 min before the animal gets use to the gloved hand holding the transfer pipette and stays still long enough to deposit an eye drop. After drops have been added to the rats two or three times however, the rats stop moving as soon as the top of the cage is removed, as they become familiar with the procedure and know they will get the Cheerio reward after its completion. Future studies may benefit from a training period for the rats to improve tolerance for getting the eye drops.
Care using BrdU:
BrdU replaces thymidine during DNA replication and can cause DNA mutations. Therefore, appropriate precautions should be taken to avoid exposure. Pregnant and lactating women should avoid exposure to BrdU and animals that have been administered BrdU eye drops. As BrdU exposure can impair the immune system; immunocompromised individuals should also use extreme caution when handling BrdU.
When eye drops are added to the animals, small amounts of solution may fall into the animal’s bedding when the animal shakes its head. This bedding should be regularly removed and autoclaved by animal facility staff that wear gloves and lab coats during the animal’s normal care. Any accidental spills of PNU-282987/BrdU should be treated with a hazardous spill kit.
Crossover:
In some instances, only one rat eye was treated with the PNU/BrdU solution, while the contralateral eye acted as an untreated control. Under these conditions, there was no evidence of crossover effects. No BrdU staining appeared in control untreated eyes (Webster et al., 2017). However, this was not the case in adult mice. When one mouse eye was treated with the PNU/BrdU solution and the contralateral eye was left untreated, crossover effects occurred. For instance, in adult mice, both eyes typically demonstrate BrdU positive cells, even if only one eye was treated with the PNU/BrdU solution. This crossover effect in mice is likely due to the relative closeness of the two eyes. As a result, contralateral eyes can be used as internal controls in adult rats, but should not be used as untreated internal controls in adult mice.

Recipes

PNU-282987/BrdU solution
Notes:
1. When preparing PNU-282987 and BrdU stock solutions, latex gloves and lab coats are worn.
2. Unused PNU-282987/BrdU is disposed of in a hazardous materials container.
1. Preparing 10 mM PNU-282987 stock solution
  A stock solution of 10 mM PNU-282987 is made using dimethyl sulfoxide (DMSO). The stock solution is kept at 4 °C for up to 3 months
2. Preparing BrdU stock solution
  Please specify how to prepare BrdU stock solution.
3. When needed, the PNU-282987 stock is diluted using sterile phosphate buffered saline (PBS) to various concentrations between 1 and 1,000 nM for eye drop studies
4. BrdU is then added directly to the diluted PNU-282987 solution to make a final BrdU concentration of 1 mg/ml
5. The final PNU-282987/BrdU solution is sterile filtered with a 0.2 µm syringe filter before use under a fume hood
6. Dilutions of PNU-282987/BrdU are kept in a refrigerator at 4 °C in labeled 30 ml glass bottles for up to one week.

Acknowledgments

The funding for this procedure is provided by NIH NEI # EY022795 and EY027970 to Dr. C. Linn.
Previous studies including Webster et al. (2017) have utilized this eye drop protocol.

Competing interests

No conflicts of interest.

Ethics

Rats and mice were kept at Western Michigan University’s animal facility until needed and were cared for in accordance with the approved guidelines of the Institutional Animal Care and Use Committee (IACUC).

References

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