Elsevier

Journal of Neuroscience Methods

Volume 210, Issue 2, 30 September 2012, Pages 187-194
Journal of Neuroscience Methods

Basic Neuroscience
A novel use of combined tyrosine hydroxylase and silver nucleolar staining to determine the effects of a unilateral intrastriatal 6-hydroxydopamine lesion in the substantia nigra: A stereological study

https://doi.org/10.1016/j.jneumeth.2012.07.013Get rights and content

Abstract

Neurotoxic lesions of the nigrostriatal pathway model the deficits found in Parkinson's disease. This study used stereology and a novel staining method to examine the effects of a partial unilateral striatal 6-hydroxydopamine (6-OHDA) lesion on substantia nigra pars compacta (SNpc) dopamine neuron number and morphology in rats. Adult male Long-Evans rats were subjected to unilateral lesion of the SNpc by intrastriatal microinjection of 6-OHDA (12.5 μg). Lesions were verified by d-amphetamine-stimulated rotation (2.5 mg/kg, sc) by force-plate rotometry 7 days post-surgery. Seven days after rotation testing, rats were euthanized, and brains were prepared for either histology (n = 12) or determination of striatal dopamine content by HPLC-EC (n = 20). Brains prepared for histology were stained for tyrosine hydroxylase (TH) combined with a silver nucleolar (AgNOR) stain using a modified protocol developed for stereological assessment. The AgNOR counterstain allowed for precise definition of the nucleolus of the cells, facilitating both counting and qualitative morphometry of TH-positive neurons. Stereological quantitation determined a 54% decrease in TH-positive neuron number (P < 0.01), and a 14% decrease in neuron volume (P < 0.05) on the lesioned side. Striatal dopamine concentration was decreased by 92% (P < 0.01), suggesting that striatal dopamine analysis may overestimate the numbers of SNpc neurons lost. These findings demonstrate that combined use of TH and AgNOR staining provides improved characterization of 6-OHDA-induced pathology. Furthermore, the data suggest that decreased neuronal volume as well as number contributes to the functional deficits observed after unilateral intrastriatal 6-OHDA lesion.

Highlights

► Tyrosine hydroxylase-silver nucleolar (TH-AgNOR) stain was used in 6-OHDA lesioned rats. ► AgNOR staining improves visibility of the nucleolus for stereological analysis. ► Neuron number and volume was decreased in the SNpc of unilaterally lesioned rats. ► The magnitude of SNpc cell loss was smaller than the decrease in striatal dopamine content.

Introduction

Parkinson's disease is a chronic neurodegenerative disorder marked by loss of nigrostriatal dopamine neurons, resulting in clinical signs when about 80% of striatal dopamine is depleted (Calne and Langston, 1983). Current treatments are largely palliative, emphasizing the urgent need for preventative and disease-modifying treatments. However, the development of such early-stage treatments is fraught with challenges. The relationships between the causative factors of Parkinson's disease and the effects of neuroprotective treatments are still unclear (Ybot-Gorrin et al., 2011), and a better understanding of neuronal morphology is needed to elucidate the mechanisms of neurodegenerative disease (Przedborski et al., 2003). Thus the importance of early- to moderate-stage Parkinson's disease models is vital to advancing the treatment of the disease.

The partial unilateral intrastriatal 6-hydroxydopamine (6-OHDA) model is a clinically relevant model of early-stage Parkinson's disease (Deumens et al., 2002, Kirik et al., 1998). This technique has been widely used as an investigational tool for both screening potential therapeutics and for understanding the underlying mechanisms of early Parkinson's disease. Although rotational behavior and striatal dopamine concentration are routinely quantified after neurotoxic lesions to verify a functional dopaminergic deficit, advanced histological analysis is needed to assess the effects of the lesion and potential treatments on morphologic changes to the dopaminergic neurons. Histological analysis of partial 6-OHDA lesions in the substantia nigra pars compacta (SNpc) is challenging, however, because moderate degenerative changes to neuron morphology can be difficult to quantify. Stereological analysis using tyrosine hydroxylase (TH) staining of dopamine neurons has been used to study the morphological changes in several animal models of Parkinson's disease, but the morphometric outcomes vary according to study. The variation in the results of Parkinson's disease model morphology studies has been attributed to the varying lesion, staining and quantitation methods used (Stark and Pakkenberg, 2004); and while a certain amount of inter-study variation is unavoidable, a clear understanding of the morphology of dopaminergic neurons has undoubtedly been clouded by the inconsistent findings in the literature.

Darkly pigmented TH antibody immunostaining can mask morphological markers such as the nucleolus and individual neuronal body outlines in regions thickly populated with TH-positive neurons. With the goal of better understanding the effects of 6-OHDA on dopaminergic morphology, this study used stereological methods and a newly available commercial TH stain combined with a silver nucleolar (TH-AgNOR) stain to determine the effects of 6-OHDA partial unilateral intrastriatal lesions on cell number and morphology. AgNOR staining is a silver nitrate preparation that targets chromosomal proteins in the nucleolus known as nucleolar organizing regions (NORs). This stain, which has been extensively used by cancer pathologists to assess cell proliferation (Trerè, 2000), has recently been exploited for use as a co-stain in dopaminergic neurodegeneration studies (Switzer et al., 2011). We will show that the combined use of TH and AgNOR staining provides improved characterization of 6-OHDA-induced pathology by facilitating the identification and quantification of neuronal structures used in stereology. In addition, our findings suggest that decreased neuronal volume as well as decreased neuronal number contributes to the functional deficits observed after unilateral 6-OHDA lesion.

Section snippets

Materials and methods

All experiments were performed in compliance with the NIH Guide for the Care and Use of Animals and were approved by the University of Kansas Medical Center Institutional Care and Use Committee.

Amphetamine-stimulated rotation

The mean number of amphetamine-stimulated rotations completed by lesioned rats during the 60-min observation period was 145 ± 24 (n = 31), indicating a functional dopaminergic deficit in the ipsilateral striatum.

Striatal dopamine concentrations

Dopamine concentration was decreased between the ipsilateral and contralateral striata of all rats subjected to 6-OHDA lesion, with a mean within-subject decrease in striatal dopamine of 92 ± 2.1% (P < 0.01) (Fig. 2). The dopamine levels measured in the unlesioned hemispheres were comparable to

Discussion

The 6-OHDA toxin, a structural analog of dopamine, is transported by the monoamine transporter into catecholaminergic cells, leading to their destruction by oxidative mechanisms (Glinka et al., 1997, Kostrzewa and Jacobowitz, 1974). Although lacking the Lewy bodies and the progressive course of clinical Parkinson's disease, the 6-OHDA lesion nonetheless produces marked degeneration of targeted dopaminergic neurons, providing a functional Parkinson's disease model quantifiable by characteristic

Conclusions

In conclusion, we find that TH-AgNOR staining facilitates stereological analysis of the effects of 6-OHDA lesions on the SNpc of rats when compared to standard TH-thionine staining. Using this staining protocol combined with stereological analysis to determine the effects of a unilateral intrastriatal 6-OHDA lesion revealed a moderate loss of SNpc TH-positive neurons despite severe striatal dopamine depletion. In addition, volumetric measurements confirmed SNpc TH-positive neuronal atrophy 14

Role of funding source

The funding sources had no involvement in the study design, collection, analysis and interpretation of the data, the writing of the report, or the decision to submit the paper for publication.

Conflicts of interest

None.

Acknowledgements

The authors thank Drs. Robert Switzer and Stanley Benkovic of NeuroScience Associates for preparing the AgNOR stained sections and facilitating our use of this staining technique in this study. Supported by NIH Grants NS067422 (BL), RR016475 (BL), HD02528 (BL JAS), the Institute for Advancing Medical Innovation (MH-S), the Mabel A. Woodyard Fellowship in Neurodegenerative Disorders (MH-S), KU Endowment, and the Institute for Neurological Disorders.

References (37)

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