Early (6 weeks post treatment) toxic effects of hα
-syn-carrying AAV vectors. (a-d) Rats received a single injection of vehicle (control animals, n = 3), hα-syn-AAV prep 1 (n = 7) or hα-syn-AAV prep 2 (n = 7) into the vagus nerve. (a) Stereological counts of Nissl-stained DMnX neurons were performed on tissue from the left (ipsilateral to AAV injections, solid bars) and right (contralateral to AAV injections, dotted bars) MO. Values are means ± SEM. (b,c) Representative sections of the MO (Bregma: −13.68 mm) were obtained from rats treated with (b) hα-syn-AAV prep 1 or (c) prep 2. Tissues were immunostained with an anti-hα-syn antibody. Images generated by z-stacking of two representative DMnX neurons are compared, showing an apparent decrease in cell size after transduction with hα-syn-AAV prep 1 (b). Scale bar in (c) = 10 μm. (d) MO tissue sections were stained with either an antibody against ChAT (control tissue) or an anti-hα-syn antibody (rats injected with hα-syn-AAV prep 1 or prep 2). The volume (μm3) of ChAT-positive or hα-syn-immunoreactive neurons was estimated using a six-ray isotropic nucleator probe . Measurements were made on 695 cells from control animals, 671 cells from prep 1-treated rats and 878 cells from prep 2-injected animals. For each group, single cell values were averaged. Values are means ± SEM. *P <0.0001 vs. the control or prep 2 groups (one-way ANOVA followed by Tukey’s post hoc test, F2,2244 = 51.15). (e) MO tissue sections from rats injected with hα-syn-AAV prep 1 or prep 2 were double-stained with antibodies against hα-syn and cleaved PARP (cPARP). Rare neurons (one of these neurons is shown in the upper panels) immunoreactive for both cPARP and hα-syn could be detected in hα-syn-AAV prep 1-treated animals. Scale bar = 20 μm. (f-h) DMnX-containing sections of the MO were immunostained with an antibody that preferentially reacts with aggregated forms of α-syn . (f,g) Representative images of DMnX neurons from rats treated with (f) hα-syn-AAV prep 1 or (g) prep 2. Scale bar in (g) = 10 μm. (h) Fluorescence intensity of immunostained DMnX neurons was measured by optical densitometry in animals treated with hα-syn-AAV prep 1 (n = 4) or prep 2 (n = 4). Values (pixels) are means ± SEM.