Standard Operating Procedure (SOP) for Combined ICA and Systemic Administration of MPTP: The Overlesioned (Bilateral Asymmetric) Non-human Primate Model
Robert S Turner
Abstract
This standard operating procedure (SOP) describes safe methods for the use of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in non-human primates (adult macaque monkeys).
In both humans and nonhuman primates, the administration of MPTP produces a stable Parkinsonian syndrome. Upon entering the brain, MPTP is converted into the toxic metabolite MPP+ by the enzyme monoamine oxidase B (MAO-B). MPP+ accumulates selectively in neurons possessing a dopamine (DA) uptake system and is further concentrated in mitochondria. The basis of the toxic effects of MPP+ is only partially understood; retrograde axonal transport, melanin binding, and damage to the mitochondrial respiratory system have been implicated in the pathogenesis of injury to DA neurons. MPTP administration reproduces in monkeys a syndrome that closely approximates naturally occurring Parkinson’s disease (PD) with respect to the pattern of DA cell loss, the resultant abnormalities in brain physiology, and ultimately, clinical symptoms.
This SOP describes the procedures for the combined internal carotid artery (ICA) and systemic (intravenous or intramuscular) administration of MPTP in non-human primates. This model produces an overlesioned or bilateral asymmetric model, induced by unilateral ICA plus systemic administration.
This protocol has been adapted from Bankiewicz et al. (1999).
Before start
Confirm IACUC approval and training for all personnel involved in the procedures. * Confirm availability for post-MPTP isolation housing and care: The attending veterinarian and the responsible animal care supervisor must be notified at least 3 days prior to each instance of MPTP use (i.e., prior to each ICA surgery and prior to any course of IV or IM administration). Before proceeding, ensure that appropriate isolation housing is available for animals post-MPTP administration.
- Confirm the availability of required PPE, materials, and selegiline for administration in case of accidental personnel exposure.
Steps
Receipt and storage of MPTP
Place the package with MPTP into a chemical fume hood.
Don appropriate personal protective equipment (Tyvek suit, half-mask respirator, bonnet, face shield, 2 pairs of nitrile gloves) and inspect the package to ensure no damage.
If all packaging and bottles are intact, remove the MPTP bottle from the packaging and safely store it in a locked cabinet.
If an MPTP bottle is damaged, add 1% bleach solution to the vial to inactivate the MPTP. Discard the MPTP in a dedicated biohazard container and contact the Environmental Health and Safety Office to dispose of the biohazard container.
MPTP dosing for ICA administration
MPTP is toxic during only the first pass through the brain. This is because MPTP also undergoes conversion to MPP+ in the periphery, after which it cannot cross the blood-brain barrier. Thus,
for administration, the dose of MPTP is calculated according to brain size e, which tends to be constant over a wide range of body weight, instead of per body weight.
Small animals (3 - 5 kg) : to 2.5mg to 2.5mg MPTP .
Medium-sized (6 - 10 kg) animals: 3mg to MPTP. 3.5mg MPTP.
Large animals (>10 kg): MPTP 4mg MPTP.
Preparation of MPTP solution for ICA administration
Prepare the MPTP solution by first infusing1mL sterile saline into a septum-sealed sterile vial containing 10mg MPTP-HCl powder. After the powder is fully dissolved, aspirate the 1mL solution into a syringe containing 9 ml of sterile saline ( final concentration: 1mg/mL MPTP ).
Transfer the entire 10mL MPTP solution to a septum-sealed sterile vial (10 ml or 30 ml).
For medium or large animals, fill a sterile 60-ml syringe with 3mL to 4mL of the MPTP solution (3mg to 4mg MPTP) and then 56mL to 57mL of sterile saline. The final concentration in the syringe will be 3 to 4 mg/60 ml or 0.05mg/mL to 0.067mg/mL MPTP.
Attach the filled 60mL syringe to an i.v. extension set.
Preparation of the animal for ICA administration of MPTP
Anesthetize a macaque monkey with ketamine ( 10 mg/kg ) and xylazine ( 1 mg/kg ) in the home cage. Transfer the animal to a procedure room.
Shave the calves and neck region of the animal using an electric shaver. Palpate the calf muscle and identify the saphenous vein.
Clean the skin with an alcohol pad. Using a 22-G i.v. catheter, cannulate the vessel and flush with saline.
Intubate the animal with a 3.5 mm to 5 mm (inside diameter) tracheal tube, according to the size of the animal.
Transfer the animal to a surgery room, place it on a surgical table with absorbent blue bench pads, and a heated water pad, or Bair Hugger (3M) at 37°C. Maintain on isoflurane anesthesia (e.g. 1.5% with an oxygen flow of ∼0.3 liters/min).
Hyperextend the head on the surgical table by placing stretch gauze through the canines and applying gentle retraction. Secure the head in position by tying the gauze to the surgery table.
Cover the animal completely with a series of sterile drapes. Cut out an opening in the drape for the neck incision and secure drapes with towel clamps. Scrub the surgery site with Betadine followed by 70% ethanol.
Make a midline incision through the skin of the neck with a sterile scalpel.
Using blunt dissection technique, locate and open the carotid sheath, exposing the common carotid artery, internal jugular vein, and vagus nerve. Isolate the common carotid artery below the carotid bifurcation.
Locate the superior thyroid artery and the external carotid artery and temporarily clamp the vessels using mosquito hemostats.
Attach a 27-G needle to the i.v. extension tubing and drain all the air from the needle by activating an infusion pump for several seconds. Drain all excess MPTP onto a sponge soaked with 1% bleach solution.
Intracarotid artery (ICA) infusion
Insert the 27-G needle (with attached extension set and 60-ml MPTP syringe) into the internal carotid artery in a direction retrograde to the direction of blood flow.
Infuse the entire volume from the syringe at 4 ml/min (3mg to 4mg MPTP).
Remove the vascular clamps (or mosquito hemostat) from the superior thyroid and external carotid arteries. Withdraw the needle from the common carotid artery and apply pressure for 0h 5m 0s (until bleeding has stopped).
Clean the wound with peroxide, close the incision site at the neck with 3-0 Vicryl, and clean the sutured area.
Allow the animal to recover from anesthesia (~15 minutes) before placing in a cage in the isolation room for 72h 0m 0s post-MPTP injection.
Intravenous (IV) and Intramuscular (IM) Administration of MPTP
Systemic administration (IV or IM) will be used when it is necessary to supplement the effects of ICA MPTP administration so as to produce a chronic moderate Parkinsonian syndrome.
Following ICA MPTP infusion, house animals in their home cage for at least 1 week and assess motor function before further IV or IM administration of MPTP.
Intravenous (IV) Administration of MPTP
Weigh the animal to determine the total dose of MPTP required. For IV administration the recommended dose is 0.3 mg/kg body weight.
Anesthetize the animal with ketamine ( 10 mg/kg ) and xylazine ( 1 mg/kg ) in its home cage. Transfer the animal to a procedure room.
Shave the calves using an electric shaver. Palpate the calf muscle and identify the saphenous vein. Clean the area with an alcohol pad.
Using a 22-G angiocatheter, cannulate the vessel and flush with sterile saline.
Fill a sterile 6mLsyringe with the appropriate volume of 1mg/mL MPTP solution ( 0.3 ml/kg or 0.3 mg/kg ) and infuse the MPTP into the vein over 0h 3m 0s .
Flush the line with 3mL sterile saline.
Remove the needle and apply pressure to the saphenous vein until bleeding stops.
The animal will be allowed to recover from anesthesia (~15 minutes) and then be placed in a cage in an isolation room for 3 days (72h 0m 0s) post-MPTP injection
Intramuscular (IM) Administration of MPTP
Move the animal to a cage in an isolation room prior to IM MPTP administration
Lightly anesthetize the animal with ketamine ( 4 - 10 mg/kg ) and transfer to a procedure table in the isolation room or to a procedure room.
Weigh the animal to determine the total dose of MPTP required ( 0.3 - 1 mg/kg )
Inject the MPTP into the thigh muscle at one or multiple sites (if the volume is larger than 1 ml) using sterile syringes.
Allow the animal to recover from light sedation (5 - 10 minutes) and place in a cage in an isolation room for 3 days (72h 0m 0s) post-MPTP injection
Animal Care Post-MPTP
For at least 72h 0m 0s following the administration of MPTP, house the animal in an isolation room that is physically separated from other animals in the colony that are not receiving MPTP).
Given safety concerns (i.e. onset of motor signs), animals should not be housed in double-tier cages during MPTP administration and isolation period.
Post signs warning: ‘ DANGER: MPTP – Neurotoxin Use Area – Entry Restricted’ on the doors to the quarantine space.
During this quarantine period, only trained laboratory personnel (and not the general animal care staff) can provide care to the animal. Lab personnel supply the animal with standard food and water rations plus enhanced enrichment throughout the period of isolation.
Laboratory personnel who enter the enclosure to feed or otherwise interact with the animal need to wear appropriate PPE. The PPE consists of a one-piece garment (with an attached hood), elasticized wrists, and attached boots made of chemically- and biologically inert, non-absorbent material (Tyvek fabric). Personnel also need to wear a full- or half-mask respirator with a removable HEPA filter (NIOSH/MSHA-approved) which has been fit-tested to the individual. Personnel using respirators undergo annual health assessments and a fit test for respirator use. Personnel also wear splash-proof goggles or a face shield and puncture-resistant gloves during all procedures.
Line the base of the cage, the drop pan, and the floor surrounding the cage with plastic-backed absorbent pads.
Every day for three days, laboratory personnel wearing full PPE dampen the contaminated bedding material and absorbent pads with 1% bleach solution, allow the solution to soak for 10 minutes, and then place the materials in a plastic bag. After sealing this bag, it is wiped with 1% bleach and then placed in a second biohazard bag before disposal as biohazardous waste and incinerated.
At the end of the confinement period, returned to its home cage. Spray the cages liberally with 1% bleach solution and allow to soak for 10 minutes. Once the animal(s) have been transferred to clean cages, and the room has been decontaminated, the use of special MPTP PPE is not required, and the room may be cleaned according to standard husbandry procedures.
