Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

Summary

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing >95% loss of striatal dopamine and 90.63±-4.02 % ipsiversive rotational bias towards the side of the lesion.

Abstract

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients^1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise^3,5.

In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)⁸, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice^9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer¹¹. More recent studies have generated intra-nigral lesions with both a low mortality rate >80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia^11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse¹⁵. Whilst this model has proven useful in the assessment of potential neuroprotective agents¹⁶, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion^{17, 18}.

Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes >95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

Protocol

1. Housing and preparation of mice

1. Maintain a colony of bacterial artificial chromosome (BAC) driven transgenic mice⁸ (Mutant Mouse Regional Resource Center (MMRRC) FVB in a 12:12 h light-dark cycle with free access to food and water. These mice are pure FVB mice, and it is not necessary to cross these mice with any other strain, either for breeding purposes, or to ensure success of the 6-OHDA lesion procedure.
2. In order to generate a parkinsonian model, adult mice aged postnatal day 31-42.......

Discussion

This protocol describes a method for the generation of a stable unilateral 6-OHDA-lesioned mouse model of Parkinson's disease, which is extremely reproducible, with a high lesion success rate, and a low mortality rate. The success of 6-OHDA lesion surgery can be easily estimated by the measurement of spontaneous rotational behaviour with >70% ipsiversive rotations indicative of >95% dopamine depletion in the lesioned striatum²⁷. Quantification of striatal dopamine levels is the most accurate measurement .......

Materials

Name	Company	Catalog Number	Comments
Name of the reagent	Company	Catalogue number	Comments (optional)
desipramine HCl	Sigma-Aldrich, Oakville, ON, Canada	D125	25mg/kg
pargyline HCl	Sigma-Aldrich, Oakville, ON, Canada	P8013	5mg/kg
6-OHDA HBr	Sigma-Aldrich, Oakville, ON, Canada	H116	3mg / mouse
stereotaxic Frame	Kopf Instruments, Tujunga, CA, USA	Model 900
mouse ear cups	Kopf Instruments, Tujunga, CA, USA	Model 921 Zygoma Ear Cups
mouse incisor bar	Kopf Instruments, Tujunga, CA, USA	Model 923B
mouse anaesthesia mask	Kopf Instruments, Tujunga, CA, USA	Model 923B
priming kit (containing 250ml syringe)	Hamilton Company, Reno, NV, USA	PRMKIT 81120
RN compression fitting kit (1 mm)	Hamilton Company, Reno, NV, USA	55750-01
PEEK tubing from RN compression fitting kit< (1/16th inch)	Hamilton Company, Reno, NV, USA	55751-01
dual small hub RN Coupler	Hamilton Company, Reno, NV, USA	55752-01
luer to small hub RN adaptor	Hamilton Company, Reno, NV, USA	55753-01
1ml 25S syringe model 7001KH	Hamilton Company, Reno, NV, USA	80100
*33G removable needle (RN) pack of 6. . Custom 1 inch with 45<° bevel	Hamilton Company, Reno, NV, USA	7803-05
Scissors	Fine Science Tools, Vancouver, BC, Canada.	14084-08
Scalpel	Fine Science Tools, Vancouver, BC, Canada	10003-12
Scalpel blades	Fine Science Tools, Vancouver, BC, Canada	10035-20
Forcep	Fine Science Tools, Vancouver, BC, Canada	11608-15
Hemostats	Fine Science Tools, Vancouver, BC, Canada.	13004-14
Isoflurane	Abbot	02241315	2-3%
Suters (Vicryl 4.0)	Syneture	SS-683
Steriliser	Fine Science Tools, Vancouver, BC, Canada	18000-45
Infusion Pump	Harvard Apparatus	PhD 22/2000
Needles (27G)	Becton Dickinson	305109
Needles (25G)	Becton Dickinson	305127
Syringes (1ml)	BD syringe	309692
Anaesthesia trolley	LEI medical	M2000
Baytril	CDMV, St. hyacinthe, QC	102207
Lidocaine	CDMV, St. hyacinthe, QC	3914
Betadine solution	CDMV, St. hyacinthe, QC	19955