GAP43 immunofluorescence increased in 6-OHDA monkeys and had minimal colabeling with TH.

<p>Cardiac sections of control (<b>A–D</b>) and 6-OHDA-treated (<b>F–I</b>) animals were labeled for GAP43 (red) and TH (green) immunofluorescence and counterstained with DAPI (blue nuclei). Arrows indicate GAP43 expression in nerve bundles. Arrowheads indicate colocalization of GAP43-ir and TH-ir. Inset (<b>d</b>) shows a positive control for GAP43 labeling in caudorostral diencephalon of rhesus embryonic day 38, run in parallel to these stainings. (<b>E</b>) Averaged scores demonstrate an increase of GAP43-ir in 6-OHDA monkeys. (t(8) = 9.073, <i>P</i><0.001). (<b>J</b>) Global GAP43 scores negatively correlate with AAT of TH-ir in cardiac nerve bundles. ***<i>P</i><0.001. AAT, area above threshold; GAP43, growth associated protein 43; TH, tyrosine hydroxylase.</p

6-OHDA-treated monkeys have decreased PGP9.5-ir in cardiac nerve bundles and fibers.

<p>(<b>A–D</b>) Microphotographs of PGP9.5-positive nerve bundles (<b>A,B</b>) and fibers (<b>C,D</b>) in control and 6-OHDA-treated animals. (<b>E</b>) Global PGP9.5-ir cardiac nerve bundle was reduced for both AAT (t(8) = 3.7, <i>P</i> = 0.006) and OD quantification (t(8) = 4.21, <i>P</i> = 0.003) in 6-OHDA compared to control animals. (<b>F</b>) PGP9.5-ir nerve bundle AAT was also decreased between groups in the lateral wall of the left ventricle. Within the 6-OHDA group, a significant effect of wall (ANOVA: F(3,16) = 4.632, <i>P</i> = 0.016) with the greatest reduction in the lateral compared to the septal wall (^a<i>P</i> = 0.022) was found. (<b>G</b>) Quantification of PGP9.5-ir fiber area density was significantly reduced in 6-OHDA-treated monkeys compared to controls (t(8) = 3.026, <i>P</i> = 0.016). (<b>H</b>) TH to PGP9.5 positive fibers ratio was significantly decreased in the anterior (t(8) = 4.335, <i>P</i> = 0.002) and inferior walls (t(8) = 4.43, <i>P</i> = 0.002) in 6-OHDA animals compared to controls. Within the 6-OHDA group there was a significant reduction in ratio between walls (ANOVA; F(3,16) = 3.299, <i>P</i> = 0.047) with significant loss in the inferior compared to septal wall (^b<i>P</i> = 0.044). Scale bar  = 25 µm. <i>*P</i><0.05, **<i>P</i><0.01, ^a<i>P</i> = 0.02, ^b<i>P</i> = 0.044. AAT, area above threshold; OD, optical density; PGP9.5, protein gene product 9.5; TH, tyrosine hydroxylase.</p

Measures of cardiac TH and PGP9.5 expression correlated with <i>in vivo</i> MHED uptake.

<p>Nerve bundle AAT quantification of TH-ir correlated with TH-ir fibers (<b>A</b>) and AAT of PGP9.5-ir nerve bundles (<b>B</b>). Area density quantification of TH-ir fibers highly correlated with PGP9.5-ir fibers (<b>C</b>). <i>In vivo</i> MHED uptake correlated with TH-ir (<b>D</b>) and PGP9.5-ir fiber area density (<b>E</b>). Despite the significant interaction between MHED and TH-ir fibers, MHED uptake did not correlate with AAT quantification of TH-ir nerve bundles (r² = 0.446, <i>P</i> = 0.070) (not shown). AAT, area above threshold; MHED, meta-hydroxyephedrine; PGP9.5, protein gene product 9.5; TH, tyrosine hydroxylase.</p

Cardiac α-synuclein was moderately affected by 6-OHDA.

<p>(<b>A–D</b>) Microphotographs of nerve bundles immunostained for soluble α-synuclein (<b>A,B</b>), phosphorylated α-synuclein (<b>C,D</b>) and proteinase K resistant α-synuclein (<b>E,F</b>) in control (<b>A,C,E</b>) and 6-OHDA-treated (<b>B,D,F</b>) monkeys. Insets (<b>a–c</b>) display immunostained nigral tissue from a Thy1-α-synuclein mouse processed in parallel for each specific antibody and used as a positive control to validate staining. (<b>G</b>) Regional reduction of soluble α-synuclein expression was found in the septal wall (t(8) = 2.787, <i>P</i> = 0.024) of 6-OHDA compared to control monkeys. Within each group, soluble α-synuclein scores did not show significant differences by wall in control animals (ANOVA; F(3,16) = 3.115, <i>P</i> = 0.056) and in 6-OHDA-treated animals (ANOVA: F(3,16) = 0.772, <i>P</i> = 0.526). (<b>H</b>) Average scores of soluble α-synuclein-ir in nerve bundles correlated with AAT TH-positive nerve bundles (r² = 0.570, <i>P</i> = 0.012). Scale bar  = 25 µm, inset scale bar  = 100 µm. <i>*P</i><0.05. AAT, area above threshold; TH, tyrosine hydroxylase.</p

6-OHDA-treated monkeys have reduced TH-ir in cardiac nerve bundles and fibers.

<p>(<b>A,B</b>) Microphotographs of H&E stained nerve bundles identified as a collection of nerve fibers (white asterisk) and intact epineurium indicated by white arrows. (<b>C</b>) Global TH-ir in cardiac nerve bundle was reduced for both AAT (t(8) = 3.508, <i>P</i> = 0.008) and OD quantification (t(8) = 3.403, <i>P</i> = 0.009) in 6-OHDA compared to control animals. (<b>D,E</b>) Microphotographs of TH-ir in matching nerve bundles. (<b>F</b>) Regional wall quantification showed reduced AAT of TH-ir in anterior (t(8) = 5.913, <i>P</i><0.0001), inferior (t(8) = 3.096, <i>P</i> = 0.015), and septal (t(8) = 3.009, <i>P</i> = 0.017) walls in 6-OHDA-treated monkeys compared to controls. In the control group there were also significant differences in OD quantification of TH (F(3,16) = 5.213, <i>P</i> = 0.011) with more TH-ir in the septal wall compared to the lateral (<i>P</i> = 0.043) and inferior walls (<i>P</i> = 0.12) (<i>post hoc</i> Bonferroni multiple comparisons analysis; not shown). (<b>G,H,J,K</b>) Microphotographs of cardiac TH positive fibers in either transverse (<b>G,H</b>) or longitudinal orientation (<b>J,K</b>) in control and 6-OHDA monkeys; black arrows indicate TH-ir fibers. (<b>I,L</b>) TH-ir fiber area density was significantly reduced in 6-OHDA compared to control animals for global (<b>I</b>) analysis (t(8) = 4.934, <i>P</i> = 0.001) and between all walls of the left ventricle (<b>L</b>) including the anterior (t(8) = 4.324, <i>P</i> = 0.003), inferior (t(8) = 5.37, <i>P</i> = 0.001), lateral (t(8) = 5.081,<i>P</i> = 0.001) and septal walls (t(8) = 3.866, <i>P</i> = 0.005). Scale bar  = 25 µm. <i>*P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001. AAT, area above threshold; OD, optical density; TH, tyrosine hydroxylase.</p

α-synuclein expression in the adrenal medulla was not affected by 6-OHDA treatment.

<p>Microphotographs of adrenal medulla immunostained for soluble α-synuclein (<b>A,B,C</b>), phosphorylated α-synuclein (<b>D,E,F</b>) and proteinase K resistant α-synuclein (<b>G,H,K</b>) in 6-OHDA-treated (<b>C,F,I</b>) and low or high α-synuclein expressing control monkeys. Insets display nigral tissue from a Thy1-α-synuclein mouse immunolabeled for each antibody in parallel and used as a positive control to validate processing. Scale bar  = 25 µm, inset scale bar  = 50 µm.</p

6-OHDA-treated monkeys have reduced TH-ir and AADC-ir in the adrenal medulla.

<p>(<b>A-F</b>) Microphotographs of adrenal medulla sections stained for H&E (<b>A,B</b>), TH (<b>C,D</b>) and AADC (<b>E,F</b>) from control and 6-OHDA monkeys. Squares in A-F corresponds to respective insets a-f showing a higher magnification image of the selected area. (<b>G</b>) OD (TH: t(8) = 3.455, <i>P</i> = 0.009; AADC: t(8) = 2.192, <i>P</i> = 0.06) and (<b>H</b>) AAT (TH: t(5.791) = 4.437, <i>P</i> = 0.005; AADC: t(8) = 3.180, <i>P</i> = 0.013) quantifications of immunostainings showed significant reductions in 6-OHDA monkeys. Scale bar  = 50 µm; inset scale bar  = 50 µm. <i>*P</i><0.05, **<i>P</i><0.01. AADC, aromatic l-amino acid; AAT, area above threshold; OD, optical density; TH, tyrosine hydroxylase.</p

PGP9.5 and TH immunofluorescense demonstrated colabeling and decreased expression in cardiac nerve bundles of 6-OHDA monkeys.

<p>Microphotographs of immunofluorescence staining of PGP9.5 (red; <b>A,E</b>), TH (green; <b>B,F</b>) and counterstained with DAPI (blue nuclei; <b>C,G</b>) in controls (A–D) and 6-OHDA-treated animals (E–H). Colocalization of PGP9.5 and TH expression is identified as yellow. PGP9.5, protein gene product 9.5; TH, tyrosine hydroxylase.</p

Similar nitrotyrosine-ir and HLA-DR-ir were observed across groups.

<p>(<b>A–D</b>) Microphotographs of a region of the left ventricle containing a nerve bundle, blood vessel and cardiomyocytes immunostained for nitrotyrosine (<b>A,B</b>) and human leukocyte antigen (HLA-DR) (<b>C,D</b>) in control (<b>A,C</b>) and 6-OHDA-treated monkeys (<b>B,D</b>). (<b>E</b>) The sum of nitrotyrosine scores in 6-OHDA-treated animals was significantly lower in the subepicardial compared to the subendocardial layer (t(8) = 2.921, <i>P</i> = 0.019), while no difference were found between layers in the control group. (<b>F</b>) Summed nitrotyrosine scores in nerve bundles by wall were not different between groups, but within 6-OHDA-treated animals a significant effect was found between walls (ANOVA: F(3,16) = 3.803, <i>P</i> = 0.031), with greater nitrotyrosine-ir scores in the septal than the anterior wall (^a<i>P</i> = 0.031). Scale bar  = 25 µm. <i>*P</i> = 0.019, ^a<i>P</i> = 0.031.</p