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J Neurosci Methods. 2012 Sep 30;210(2):187-94. doi: 10.1016/j.jneumeth.2012.07.013. Epub 2012 Jul 28.

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.

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Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160, USA.


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.

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