Ophthalmic Drug Delivery for Noninvasive Neuromodulation in a Mouse Model

Samenvatting

Source: Zhan, J., et al. Non-invasive Strategies for Chronic Manipulation of DREADD-controlled Neuronal Activity. J. Vis. Exp. (2019)

This video demonstrates a non-invasive ophthalmic drug delivery method for targeted neuromodulation. The drug is delivered via eye drops, absorbed into the circulation, crosses the blood-brain barrier, and activates specific neuronal receptors.

Protocol

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

1. Repetitive clozapine-N-oxide (CNO) delivery using eye-drops

1. Acclimate the animals to handling by scruffing each mouse 3 minutes daily for 3-4 days prior to the administration of eye drops.
2. Dissolve clozapine-N-oxide (CNO, 5 mg) in 1 mL of sterile 0.9% saline solution (stock solution: 5 mg CNO/ mL). Keep the solution refrigerated at 4°C.
3. Weigh each mouse before starting the experiment to determine the amount of CNO to be delivered. Use 1-3 μL drop (per eye) to achieve 1.0 mg CNO/ kg body weight.
   NOTE: As an example, a 20 g mouse should receive bilateral (2 μL each) eye drops.
4. Deliver the eye drops during the inactive (light) phase of mice, 2 h before the lights turn off (zeitgeber time (ZT) 10). In cases where CNO needs to be delivered during the active (dark) phase of mice, ensure the presence of dim red light for proper animal handling.
   NOTE: Precautions should be taken to avoid disrupting the circadian (and light/dark) cycle of experimental animals.
   1. Using a P10 micropipette, load the required amount (1-3 μL) of CNO solution to achieve 1.0 mg CNO/ kg.
      NOTE: Use a new and sterile pipette tip for each eye drop. In this set of experiments, bilateral eye drops of CNO were performed; however, if a lower CNO concentration is required, unilateral eye drops could also be applied.
   2. Immobilize the mouse via the scruff.
   3. Slowly expel the solution until a stable droplet forms on the pipette tip.
   4. Carefully bring the droplet close to the cornea of the mouse’s eye until the solution is delivered. The pipette tip should never contact the mouse’s eye.
   5. Release the mouse, placing it back in its home cage.
5. Repeat this procedure every day for 5 days.
   NOTE: This duration can be adjusted as per the experimental requirements.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
BSA	Sigma life science	#A2153-100G	Lyophilized powder ≥96% (agarose gel electrophoresis)
C57BL/6J mice	The Jackson laboratory	#000664	male mice, 3 months old
Capillaries	Drummond Scientific Company	#3-000-203-G/X	Outer diameter: 1.14 in.
Clozapine-N-oxide	Sigma	#C0832	5mg
Forane	Baxter	#NDC 10019-360-60	Isoflurane, USP
Microinjector III	Drummond Scientific Company	#3-000-207	Nanoject III - Programmable Nanoliter Injector
Mounting media	Invitrogen	#P36930	Prolong Gold antifade reagent