Histological, behavioural and neurochemical evaluation of medial forebrain bundle and striatal 6-OHDA lesions as rat models of Parkinson's disease

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Abstract

We compared the effect of an injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle (MFB) and into the striatum on different parameters for evaluation of motor dysfunction and dopamine denervation in rats, as a function of time. A combination of behavioural, neurochemical and histological techniques was employed.

Amphetamine-induced rotation is shown to provide a first rough estimation of motor impairment. Indeed, the number of rotations observed after amphetamine administration can distinguish between a partial and a near complete (>90%) denervation in the substantia nigra. However, lesion sizes of 50–80% resulted in similar rotational behaviour. Similarly, the elevated body swing test (EBST) can determine severe lesions, but is not sensitive enough in the partial model. In both models, determination of the dopamine tissue content with HPLC is a more precise measure of striatal dopamine innervation than striatal TH-immunostaining. The number of cells estimated by TH- and Nissl-staining correlated well in the striatal model, but there was a discrepancy between both measures in the MFB-lesioned animals. Therefore, additional Nissl-staining is necessary for better estimation of the size of the lesion at the level of the substantia nigra or ventral tegmental area in the severely lesioned animals.

The MFB lesion model mimics end-stage Parkinson's disease. The striatal injection of 6-OHDA described here cannot be considered a progressive model, since there was no change in the number of TH-immunoreactive cells in the substantia nigra up to 8 weeks post-lesioning. However, the partial denervation renders its quite suitable for mimicking early stage Parkinson's disease, and is thus suitable for testing possible neuroprotective and neurotrophic drugs.

Introduction

Parkinson's disease is an age-related neurodegenerative disease, affecting about 1–3% of the population over 50 years of age and characterized by relatively selective nigrostriatal dopaminergic degeneration. The pathogenesis of Parkinson's disease is not completely understood. Until now, there is no therapy available that can stop or at least slow down the neurodegeneration in patients suffering from this disease. To understand the pathophysiology of Parkinson's disease and to develop novel therapies for improved symptomatic management, it is important to have a relevant animal model, in which new pharmacological agents and treatment strategies can be assessed before clinical trials are initiated (Betarbet et al., 2002).

The optimal Parkinson's disease model shows easily detectable parkinsonian motor deficits, selective and gradual loss of dopamine (DA) neurons with aging and production of Lewy bodies-like cytoplasmic inclusions. Parkinson's disease is usually replicated in mice by the systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or in rats by unilateral intracerebral injection of 6-hydroxydopamine (6-OHDA). However, the main difficulty using MPTP toxicity as a model of Parkinson's disease is that it is an acute or sub-acute process, whereas Parkinson's disease is a slow progressive illness. A major advantage of the 6-OHDA model is a quantifiable motor deficit (rotation) and its usefulness in the pharmacological screening of agents that have effects on DA and its receptors (Deumens et al., 2002, Ungerstedt, 1971). The most common model involves a unilateral injection of 6-OHDA into the substantia nigra (SN) or medial forebrain bundle (MFB), leading to rapid cell death, similar to that of the acute MPTP model. The achieved lesion is considered appropriate to study late-stage Parkinson's disease. Another model involves injection of the 6-OHDA directly into the striatum (Berger et al., 1991, Ichitani et al., 1994, Lee et al., 1996, Przedborski et al., 1995, Sauer and Oertel, 1994), causing retrograde degeneration of SN neurons. This causes a slow partial lesion of these neurons and has been used to mimic the slow progress of Parkinson's disease (Shimohama et al., 2003).

In each model, appropriate but simple testing routines should be chosen to allow the assessment of lesion-induced disability and/or treatment-induced recovery (Cenci et al., 2002). Furthermore, behavioural tests should reflect histological and/or functional deficits or preservation of the injured tissue.

Therefore, the aim of the present study was to assess the degenerative changes in the dopaminergic neurons of the SN and the associated behavioural and neurochemical consequences of intrastriatal injections of 6-OHDA in rats and compare them with those after an MFB lesion of the nigrostriatal pathway, as a function of time. Structural deficits were quantified by determination of cell counts in the SN after tyrosine hydroxylase (TH) and Nissl staining as well as determination of striatal DA and DOPAC content (HPLC) and DA innervation (striatal TH-immunostaining). Functional impairment was estimated with the elevated body swing test (EBST) and amphetamine-induced rotation. The data provide insight into the most appropriate parameters for simple testing of potential neuroprotective drugs or other treatment strategies in these two models.

Section snippets

Chemicals

d-Amphetamine sulphate, dopamine HCl, decane sulfonic acid, 6-hydroxydopamine.HBr, phosphate buffered saline (PBS), dibutylamine and sodium octanesulphonate were purchased from Sigma (St. Louis, MO, USA). Sodium disulphite, hydrochloric acid, sodium acetate trihydrate, citric acid monohydrate, acetonitrile, disodium EDTA, formaline were from Merck (Darmstadt, Germany). Ascorbic acid was bought from Roche (Brussels, Belgium) and 3,4-dihydroxybenzylamine was from Janssen (Beerse Belgium). For the

The elevated body swing test (EBST) (Fig. 1)

MFB lesion (severe lesion model): The percentage right-biased swings 3 weeks after the MFB-lesion was significantly higher than that observed in normal rats [76.4 ± 2.5% (n = 10) versus 49.7 ± 0.8% (n = 10)]. Five weeks after the lesion, the percentage right- biased swings was 85.3 ± 1.3% (n = 10). This is also significantly higher compared to the percentage in normal rats, and is significantly different from the 3 weeks lesioned group. Five weeks after lesioning, MFB/VTA lesioned rats exhibited

Discussion

In this study, we investigated the degeneration of DA neurons in the striatum, the SNpc and the VTA after injection of 6-OHDA into the MFB and the striatum, respectively, as a function of time. The aim was to select the most appropriate parameters for future testing of potential antiparkinsonian and/or neuroprotective drugs in these two models.

No biased swinging behaviour was observed in the intact animals whereas right-biased swinging behaviour of more than 70% was shown in the severely

Acknowledgements

The authors wish to acknowledge the animal care by Mr. E. Van Malderen, Mrs. M. Cresens and Mr. F. Bonnaerens. We would like to thank Dr. Peter In’t Veld and Mrs. Vanessa Ghislain, for their assistance in the digital photography. We also thank the excellent technical assistance of Mr. G. De Smet, Mrs. C. De Rijck, Mrs. A Goossen, Mrs. N. Buelens, Mrs. E. De Blay and Mr. P. Verhavert. We also thank Dr. A. Michotte, Dr. M. Marichal and Dr. L. Bouwens for the use of their laboratory facilities.

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