Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Summary

Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.

Abstract

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key symptoms, such as tremor, rigidity and bradikinesia, develop when about 3/4 of dopaminergic cells are lost in the substantia nigra, and fail to provide for the smooth, coordinated regulation of striatal motor circuits. Depression and hallucinations are common, and dementia eventually occurs in 20% of patients. At this time, there is no treatment to delay or stop the progression of PD. Rather, the medications currently available aim more towards the alleviation of these symptoms. New surgical strategies may reversibly switch on the functionally damaged circuits through the electrical stimulation of deep brain structures, but although deep brain stimulation is a major advance, it is not suitable for all patients. It remains therefore necessary to test new cell therapy approaches in preclinical models.

Selective neurotoxic disruption of dopaminergic pathways can be reproduced by injection of 6-hydroxydopamine (6-OHDA) or MPTP (1-methyl-4-phenyl-1,2,3,6-tertahydropyridine) whereas depleting drugs and oxidative-damaging chemicals may also reproduce specific features of PD in rodents. Unlike MPTP, 6-OHDA lesions cause massive irreversible neuronal loss, and can be uni- or bilateral. The 6-OHDA lesion model is reliable, leads to robust motor deficits, and is the most widely used after 40 years of research in rats1. As interactions between grafted cells and host can now be studied more thoroughly in mice rather than in rats, the model has been transposed to mice^2,3, where it has been recently characterized⁴.

In this video, we demonstrate how to lesion the left nigro-striatal pathway of anesthetized mice by slowly delivering 2.0 μL of 6-OHDA through a stereotaxically inserted micro-syringe needle. The loss of dopaminergic input occurs within days, and the functional impairments can be monitored over post-operative weeks and months by rating animal rotations induced by dopaminergic agents⁵. Here, we show full-body contralateral rotations occurring 10 minutes after a single subcutaneous administration of apomorphine, measured one month after the lesion. Outcomes and drawbacks are discussed below.

Protocol

All chemicals stored in solid form, such as 6-OHDA and apomorphine, were diluted in sterile injection water and filtered inside a laminar flow hood, to avoid contamination. In addition, neurotoxins and neuroactive substances were stored, prepared, handled and disposed according to regulations set by international guidelines⁶ and the local Biosafety Committee.

1 - Anesthesia and surgery

Adult male Swiss mice (25-30g) are premedicated with Atropine (0.2 mg/Kg, s.c.) to reduce vagal tone and then anesthetized with an intraperitoneal injection of Ketamine (90-120 mg/Kg) and Xylazine (10 mg/Kg). The animal....

Discussion

Extensive unilateral lesion of the nigro-striatal pathway can be achieved reliably in mice by a single stereotaxic injection of 6-OHDA into the striatum. The extent of the lesion can be verified post-mortem by immunohistochemistry for Tyrosine Hydroxylase (which catalyses the rate limiting stage of the synthesis of DA, i.e. the conversion of L-Tyrosine to dihydroxyphenylalanine), as illustrated in the video.

In vivo, contralateral rotations induced by apomorphine are a better predictor of max.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ketamine hydrochloride	König		90-120 mg/Kg
Xylazine hydrochloride	König		10 mg/Kg
Atropine Sulfate	Isofarma		Isofarma
6 – Hydroxydopamine Hydrochloride	Sigma-Aldrich		5 μg/μL
L - Ascorbic Acid	USB Corp., Affymetrix		0.2%
Xylocaine (Lidocaine Chloridrate)	AstraZeneca		5% 50 mg
Nebacetin (Neomycin Sulfate + Bacitracin)	Altana Pharma		5 mg/g + 250 Ul/g
R-(-)-Apomorphine hydrochloride	Sigma-Aldrich		0.5 mg/Kg
GenTeal lOphthalmic ointment Hypromellose	Novartis AG		0.33%
Ibuprofen	Abbott Laboratories		4 mg/ 100 ml drinking water
Povidine	Johnson Diversey
R-(-)-Apomorphine hydrochloride	Sigma-Aldrich		0.5 mg/Kg