Cardiac sympathetic neurodegeneration and dysautonomia affect patients with sporadic and familial Parkinson's disease (PD) and are currently proposed as prodromal signs of PD. We have recently developed a nonhuman primate model of cardiac dysautonomia by iv 6-hydroxydopamine (6-OHDA). Our in vivo findings included decreased cardiac uptake of a sympathetic radioligand and circulating catecholamines; here we report the postmortem characterization of the model. Ten adult rhesus monkeys (5–17 yrs old) were used in this study. Five animals received 6-OHDA (50 mg/kg iv) and five were age-matched controls. Three months post-neurotoxin the animals were euthanized; hearts and adrenal glands were processed for immunohistochemistry. Quantification of immunoreactivity (ir) of stainings was performed by an investigator blind to the treatment group using NIH ImageJ software (for cardiac bundles and adrenals, area above threshold and optical density) and MBF StereoInvestigator (for cardiac fibers, area fraction fractionator probe). Sympathetic cardiac nerve bundle analysis and fiber area density showed a significant reduction in global cardiac tyrosine hydroxylase-ir (TH; catecholaminergic marker) in 6-OHDA animals compared to controls. Quantification of protein gene protein 9.5 (pan-neuronal marker) positive cardiac fibers showed a significant deficit in 6-OHDA monkeys compared to controls and correlated with TH-ir fiber area. Semi-quantitative evaluation of human leukocyte antigen-ir (inflammatory marker) and nitrotyrosine-ir (oxidative stress marker) did not show significant changes 3 months post-neurotoxin. Cardiac nerve bundle α-synuclein-ir (presynaptic protein) was reduced (trend) in 6-OHDA treated monkeys; insoluble proteinase-K resistant α-synuclein (typical of PD pathology) was not observed. In the adrenal medulla, 6-OHDA monkeys had significantly reduced TH-ir and aminoacid decarboxylase-ir. Our results confirm that systemic 6-OHDA dosing to nonhuman primates induces cardiac sympathetic neurodegeneration and loss of catecholaminergic enzymes in the adrenal medulla, and suggests that this model can be used as a platform to evaluate disease-modifying strategies aiming to induce peripheral neuroprotection.

BackgroundWe recently developed a nonhuman primate model of cardiac dysautonomia by systemic dosing of the catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA). The aim of this study was to assess whether systemic 6-OHDA affects the central nervous system of nonhuman primates, in particular the dopaminergic nigrostriatal system.MethodsBrain sections from adult rhesus monkeys that received systemic 6-OHDA (50 mg/kg intravenously; n=5) and were necropsied 3 months later, as well as normal controls (n=5) were used in this study. Tissue was cut frozen at 40 μm on a sliding microtome, processed for immunohistochemistry, and blindly evaluated.ResultsNeither the optical density of tyrosine hydroxylase immunoreactivity (TH-ir; a dopaminergic neuronal marker) in the caudate and putamen nucleus nor the TH-ir cell number and volume in the substantia nigra showed significant differences between groups. Yet within groups, statistical analysis revealed significant individual differences in the 6-OHDA-treated group, with two animals showing a lower cell count and volume. Optical density quantification of α-synuclein-ir in the substantia nigra did not show differences between groups. As α-synuclein intracellular distribution was noted to vary between animals, it was further evaluated with a semiquantitative scale. A greater intensity and presence of α-synuclein-positive nigral cell bodies was associated with larger TH-positive nigral cell volumes. Increased human leukocyte antigen (HLA-DR; a microglial marker) expression was observed in 6-OHDA-treated animals compared with controls. HLA-DR-ir was primarily localized in endothelial cells and perivascular spaces throughout cortical and subcortical structures. Semiquantitative evaluation using a rating scale revealed higher HLA-DR-ir in blood vessels of 6-OHDA-treated animals than controls, specifically in animals with the lowest number of dopaminergic nigral neurons.ConclusionOur results demonstrate that systemic 6-OHDA administration to rhesus monkeys can affect the dopaminergic nigrostriatal system and upregulate inflammatory markers in the cerebrovasculature that persist 3 months post neurotoxin challenge. The variability of the subject response suggests differences in individual sensitivity to 6-OHDA.

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