TITLE: 6-OHDA mouse model of Parkinson&#39;s disease
AUTHORS: Beatriz E Nielsen

[DESCRIPTION]
This protocol describes the steps for generating a 6-hydroxy-dopamine (6-OHDA) mouse model of Parkinson's disease.
Low or high doses of 6-OHDA are injected into the medial forebrain bundle to induce a partial or a more complete dopaminergic lesion respectively.


[STEPS]
SECTION: Drugs preparation
3. Preparation of low (1 µg/µL) and high dose (4 µg/µL) 6-OHDA solutions (freshly immediately before surgery):


SECTION: Drugs preparation
3.1. 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
 

SECTION: Pre-operative care
1. 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 ).
SECTION: Drugs preparation
3.3. Wrap in aluminum foil, vortex until drug is dissolved and place in an ice bucket.
SECTION: Drugs preparation
3.2. Add 1 mL of sterile saline or sterile saline containing ascorbic acid 0.2 % volume.

 

SECTION: Stereotaxic surgery
4. Gather supplies needed for surgery plus desipramine/pargyline and 6-OHDA solutions.

 

SECTION: Stereotaxic surgery
5. 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).
SECTION: Post-operative care
8. 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:
SECTION: Stereotaxic surgery
7. To prevent dehydration, inject 1 mL of sterile saline (or 5 % volume sterile glucose) subcutaneously (s.c.).

SECTION: Post-operative care
8.2. Keep house cages halfway onto the heating pad.
SECTION: Post-operative care
8.3. Keep providing supplementary (e.g. food pellets soaked in water, sunflower seeds, nutritionally fortified water gel ).
SECTION: Post-operative care
8.1. Check mice daily and monitor general health, weight (every 3-4 days) and signs of pain/distress, dehydration, hypothermia, aphagia and adipsia.
SECTION: Post-operative care
8.4. 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).
SECTION: Post-operative care
8.5. 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.
SECTION: Stereotaxic surgery
6. 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).
 

SECTION: Drugs preparation
2. Preparation of desipramine (2.5 mg/mL) and pargyline (0.5 mg/mL) solution 

SECTION: Drugs preparation
2.1. 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
SECTION: Drugs preparation
2.2. Add 7-8 mL of sterile saline and sonicate with heat 37-45 °C until the mixture is completely dissolved.
SECTION: Drugs preparation
2.3. As the pH of this solution will be acid (~3), add 1 Molarity (m) NaOH until pH=7.4.

SECTION: Drugs preparation
2.4. Bring volume up to 10 mL with sterile saline.
SECTION: Drugs preparation
2.5. Aliquot in 1.5 mL tubes and store them at -80 °C.