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

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 stain (AgNOR) 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.

Axial levodopa-induced dyskinesias and neuronal activity in the dorsal striatum

“…Animals were injected with amphetamine (5 mg/kg; i.p.) and ipsilateral rotations were recorded 30 min post-injection (Healy-Stoffel et al, 2012; Chotibut et al, 2013). Animals that did not show >5 ipsilateral rotations per minute were not included in this study (Chang et al, 1999; Paquette et al, 2009); animals used in the study performed 11±2 ipsilateral rotations per minute.…”

Differential effects of intrastriatal 6-hydroxydopamine on cell number and morphology in midbrain dopaminergic subregions of the rat

“…As previously described in detail (Healy-Stoffel et al, 2012), rats were given single-site unilateral intrastriatal microinjections of 6-OHDA (12.5 μg in 5 μl). Lesions were validated on the basis of d-amphetamine-stimulated rotation (2.5 mg/kg, sc) assessed 7 days after administration of 6-OHDA.…”

“…The partial unilateral lesion results in a moderate loss of midbrain dopamine neurons compared to more extensive lesions (Kirik et al, 1998), and is thus relevant to early-stage PD. Using this model, we have demonstrated decreased SNpc neuronal number and neuronal volume (Healy-Stoffel et al, 2012), as well as decreased volume of the nucleolus in SNpc dopaminergic cells (Healy-Stoffel et al, 2013). The nucleolus, the site of rRNA synthesis, is of particular interest as it is involved in directing the cellular response to stress (Boulon et al, 2010), and is thus a likely participant in the neurodegenerative processes in PD.…”

Altered nucleolar morphology in substantia nigra dopamine neurons following 6-hydroxydopamine lesion in rats

“…Animal husbandry protocol and experimental surgical and lesion validation procedures are previously described in detail [12]. Briefly, male Long-Evans rats (90–100 days old) (n=11) were given single-site unilateral intrastriatal microinjections with 6-OHDA (12.5 μg in 5 μl).…”

“…Design-based stereology is a well-established method for obtaining robust data, and has been extensively used to determine neuronal number and morphology of the SNpc in Parkinson’s disease and in animal models [1, 4, 7, 8, 15, 25, 26]. Accordingly, this study employed the tyrosine hydroxylase-silver nucleolar (TH-AgNOR stain), which combines TH staining for catecholaminergic neurons with silver-binding nucleolar AgNOR staining [12], and stereological analysis techniques to determine the effects of a partial unilateral 6-OHDA model on the SNpc TH-positive neuronal and nucleolar morphology in adult male Long-Evans rats.…”