The number of nigral dopaminergic neurons is not affected by siRNA-induced α-synuclein suppression.

<p>Squirrel monkeys received a unilateral nigral infusion of siRNA targeting α-synuclein. Both the number of TH-immunoreactive cells and the total number of dopaminergic neurons were counted stereologically in the substantia nigra. Values (mean ± SEM) were not different between the right (untreated) and left (siRNA-infused) hemisphere.</p

Effect of α-synuclein siRNA on α-synuclein protein in the monkey substantia nigra.

<p>α-Synuclein or luciferase siRNA was unilaterally infused into the left substantia nigra. Midbrain sections were immunostained with an antibody against α-synuclein. Representative images from an animal receiving α-synuclein siRNA show more robust α-synuclein immunoreactivity within the neuropil of the right (untreated, <b>A</b>) <i>vs.</i> left (siRNA-infused, <b>B</b>) substantia nigra. Scale bar = 5 µm. (<b>C</b>) Optical density measurements of nigral α-synuclein immunoreactivity. Data are expressed as percent of the control value in the right (untreated) substantia nigra and represent mean ± SEM. A significant decrease is caused by α-synuclein but not luciferase siRNA in the left (siRNA-infused) hemisphere. *p<0.03.</p

Treatment of squirrel monkeys with siRNA.

<p>Animals were unilaterally implanted with a cannula connected to an Alzet minipump delivering siRNA into the left substantia nigra. (<b>A</b>) Sequence of the α-synuclein siRNA. “A,C,G,U” indicate ribonucleotides, “T” designates deoxythymidine, “c” and “u” specify 2′-O-Me-modified pyrimidines and “s” denotes a phosphorothioate linkage. (<b>B</b>) Midbrain sections were immunostained for tyrosine hydroxylase (brown) and counterstained with cresyl violet (purple). A representative section shows placement of the cannula approximately 1 mm dorsal to the substantia nigra (SN). The location of the cannula is indicated by the square box, and the asterisk denotes the exit of the third nerve. Scale bar = 800 µm.</p

Levels of striatal dopamine, DOPAC and HVA are unaffected by α-synuclein siRNA infusion.

<p>α-Synuclein siRNA was unilaterally infused into the left substantia nigra. Values are expressed as ng/mg protein. Data are shown as mean ± SEM. LC, lateral caudate; MC, medial caudate; DP, dorsal putamen; VP, ventral putamen.</p

Lack of microglial activation following siRNA infusion.

<p>α-Synuclein siRNA was unilaterally infused through a cannula positioned approximately 1 mm dorsal to the substantia nigra. Representative midbrain sections were immunostained for microglial cells using an antibody against ionizing calcium-binding adaptor molecule 1 (Iba-1, brown) and counterstained with cresyl violet (purple). Images are from the right (untreated, <b>A</b> and <b>C</b>) and left (siRNA-infused, <b>B</b> and <b>D</b>) substantia nigra. At higher magnification (<b>C</b> and <b>D</b>), Iba-1-positive cells with morphological features of resting microglia are shown close to dopaminergic neurons containing neuromelanin (black granules). The arrows indicate one of these neurons in each panel. Scale bars = 20 µm (A and B) and 10 µm (C and D). (<b>E</b>) The number of Iba-1-immunoreactive cells was counted in the right (R) and left (L) substantia nigra. Data are shown as mean ± SEM. (<b>F</b>) A representative section from the left midbrain shows Iba-1 immunoreactivity close to the tip of the infusion cannula (arrow) but not within the nearby parenchyma. This robust immunoreactivity was observed within cells with morphological characteristics of activated microglia (inset). Scale bars = 250 µm (panel F) and 10 µm (inset).</p

Reduction of α-synuclein mRNA in the substantia nigra infused with α-synuclein siRNA.

<p>Squirrel monkeys received a unilateral nigral infusion of siRNA targeting α-synuclein (<b>A</b>) or luciferase (<b>B</b>). Midbrain sections at the level of the exit of the 3^rd nerve were used for α-synuclein <i>in situ</i> hybridization using digoxigenin-labeled antisense riboprobes. Representative images compare α-synuclein mRNA in the right (untreated) <i>vs.</i> left (siRNA-infused) substantia nigra. Scale bar = 100 µm.</p