Fig. 5

Extracellular NM and neuronophagia in AAV-hTyr-injected rats. a Left, extracellular NM aggregates in ipsilateral SNpc from AAV-hTyr-injected rats. *p < 0.05, compared to 0.5&1 m; #p < 0.05, compared to 2 m (one-way ANOVA; Student–Newman–Keuls post-hoc test). Middle, hematoxylin-eosin (H&E)-stained ipsilateral SNpc sections from AAV-hTyr-injected rats (4 m). Scale bar, 25 μm. Right, Pearson correlation analysis between total SNpc DA neurons and extracellular NM. b Left, neuronophagia in ipsilateral SNpc from AAV-hTyr-injected rats. *p < 0.05, compared to 0.5 m (ANOVA on ranks; Dunn’s post-hoc test). Middle, H&E-stained ipsilateral SNpc sections from AAV-hTyr-injected rats (4 and 12 m). Scale bar, 62.5 μm. Right, Pearson correlation analysis between extracellular NM and neuronophagia. c H&E-stained midbrain sections from PD post-mortem brains and AAV-hTyr-injected rats. Scale bars, 12.5 μm (top & bottom) and 25 μm (middle). d Ipsilateral SNpc sections from AAV-hTyr-injected rats (2–12 m) immunostained for the microglial marker Iba1 (blue, top) or the macrophage marker CD68 (blue, bottom). Scale bars, 375 μm (Iba1, low magnification), 12.5 μm (Iba1, high magnification), 150 μm (CD68, low magnification), and 25 μm (CD68, high magnification). e Ipsilateral SNpc sections from AAV-hTyr-injected rats (4 m) immunostained for TH (red) and Iba1 (green) and co-stained with the nuclear marker Hoechst (blue). Arrowheads, intact TH-positive NM-laden neurons; single arrow, neuronophagia directed at extracellular NM debris; double arrows, neuronophagia directed at a TH-immunonegative NM-laden neuron. Scale bar, 25 μm. f Top, Ipsilateral SNpc section from an AAV-hTyr-injected rat (4 m) immunostained with Iba1 (green) and astrocytic marker GFAP (purple), co-stained with blood vessel marker tomato lectin (red) and Hoechst (blue). Arrowheads, microglial cells carrying NM towards blood vessels. Arrow, perivascular NM. Scale bar, 25 μm. Bottom, Pearson correlation analysis between neuronophagia and perivascular NM. In a, b histograms, values are mean ± SEM. In a, n = 8 (0.5 m), n = 7 (1 m), n = 5 (2 m), n = 8 (4 m), n = 7 (12 m), n = 6 (24 m). In b, n = 6 (0.5 m), n = 5 (1 m), n = 5 (2 m), n = 7 (4 m), n = 6 (12 m), n = 6 (24 m). For Pearson correlation analyses each point represents the average value for the corresponding parameter at any given time post-AAV-hTyr injection (0.5, 1, 2, 4, 12, and 24 m). BF bright-field. In all panels, unstained NM appears as brown. Photomicrographs correspond to 5-μm-thick sections