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

作者：Healy Stoffel Michelle; Ahmad S Omar; Stanford John A; Levant Beth^*

来源：Journal of Neuroscience Methods, 2012, 210(2): 187-194.

DOI：10.1016/j.jneumeth.2012.07.013

• Silver nucleolar stain
• 6-Hydroxydopamine
• Stereology
• Substantia nigra pars compacta
• Neuronal volume
• Neuronal number

Neurotoxic lesions of the nigrostriatal pathway model the deficits found in Parkinson%26apos;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 mu g). 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 %26lt; 0.01), and a 14% decrease in neuron volume (P %26lt; 0.05) on the lesioned side. Striatal dopamine concentration was decreased by 92% (P %26lt; 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.

• 出版日期2012-9-30