6-OHDA mouse model of Parkinson's disease

1 to 3 days prior to surgery, provide mice (7-8 week-old, preferrably in groups of at least 3 mice/cage) with:

• Cage enrichment with hiding places (e.g. igloos) and nesting material (avoid cage changes the day before surgery).
• Supplementary food to prevent food neophobia post-surgery (e.g. food pellets soaked in water, sunflower seeds, nutritionally fortified water gel).

Preparation of desipramine (2.5mg/mL) and pargyline (0.5mg/mL) solution

For a final volume of 10 mL, weigh the appropriate amount of each drug, accounting for the weight of the salt component such that the following concentrations are achieved:

• Desipramine: 25 mg
• Pargyline: 5 mg

Add 7-8 mL of sterile saline and sonicate with heat 37-45°C until the mixture is completely dissolved.

As the pH of this solution will be acid (~3), add 1Molarity (M) NaOH until pH=7.4.

Bring volume up to 10 mL with sterile saline.

Aliquot in 1.5 mL tubes and store them at -80°C.

Preparation of low (1µg/µL) and high dose (4µg/µL) 6-OHDA solutions (freshly immediately before surgery):

For a final volume of 1 mL, weigh the appropriate amount of drug accounting for the weight of the salt component such that the following concentrations are achieved:

• 6-OHDA high dose: 5 mg 6-OHDA
• 6-OHDA low dose: 1 mg 6-OHDA
  Note

  As 6-OHDA is light and heat sensitive, avoid exposure to light, and place on ice prior to weighing.When injecting high and low doses of 6-OHDA the same day, the low dose solution can be prepared by dilution from high dose solution. For 1 mL of low dose 6-OHDA, take 250 µL of the high dose solution and add 750 µL of vehicle.

Add 1 mL of sterile saline or sterile saline containing ascorbic acid 0.2% volume.

Wrap in aluminum foil, vortex until drug is dissolved and place in an ice bucket.

Gather supplies needed for surgery plus desipramine/pargyline and 6-OHDA solutions.

30 minutes prior to surgery, weigh each mouse and administer desipramine/pargyline solution at 10 mL/kg by intraperitoneal injection (i.p). (Final doses per mouse: desipramine 25 mg/kg and pargyline 5 mg/kg).

Start stereotaxic surgery. The full detailed protocol for performing stereotaxic surgery is available below, and specific considerations for 6-OHDA lesion model of Parkinson's disease are described here:

• After loading injector with 6-OHDA solutions, carefully wrap the micropipette with aluminum foil, but leaving the tip exposed to enter the brain.
• Inject 1 µL of high or low dose 6-OHDA for experimental mice and 1 µL of vehicle (saline or saline + ascorbic acid) for control mice, into the medial forebrain bundle (MFB) (coordinates: AP: -1.2 ML: +/- 1.3 DV: -4.75). Stereotaxic Surgery

To prevent dehydration, inject 1 mL of sterile saline (or 5% volume sterile glucose) subcutaneously (s.c.).

As 6-OHDA lesion is associated to significant mortality rates, post-operative care is conducted for at least 1 week post-surgery to increase survival rates:

Check mice daily and monitor general health, weight (every 3-4 days) and signs of pain/distress, dehydration, hypothermia, aphagia and adipsia.

Keep house cages halfway onto the heating pad.

Keep providing supplementary (e.g. food pellets soaked in water, sunflower seeds, nutritionally fortified water gel).

Dehydration is a common complication (slow retraction of the skin following skin pinch). Inject daily 1 mL of sterile saline i.p. or s.c. (or 5% volume sterile glucose).

In male mice, also monitor genitals, since penis prolapse (paraphimosis) and uretral plugs (obstructive uropathy) are usual complications. If there is an urethral plug, apply a lubricant (e.g. oftalmic ointment, mineral oil) and local analgesic cream if needed and try to remove it. Then use a lubricated stainless-steel probe to reinsert the penis into the prepuce if possible, or leave the penis lubricated and check for spontaneous resolution.