Changes in striatal levels of DA and 5-HT after rotenone infusion.

<p>For the dose-response (3-, 6-, 12-µg groups) studies, the animals were sacrificed at the fourth week following the ST infusion of rotenone, and the (A) DA and (C) 5-HT contents were measured. For the time-response studies, the animals were infused with 3 µg of rotenone and sacrificed on the second, fourth, sixth, and eighth week (B and D). The results are expressed in ng/mg (wet tissue), and the data were given as the mean±SD (n = 6). *P<0.05 as compared to contralateral side.</p

Pathological changes in peripheral organs in Parkinsonian rats.

<p>A: The alveolar walls of the ST infusion rats were thin and delicate; B: neutrophilic exudates were detected in the pulmonary alveoli; C: the normal hepatic lobules and central veins; D: the central vein of the hepatic lobule was fractured in SYS rats; E and H: the normal renal glomerulus and renal medulla; F and I: Red blood cells were increased in the renal glomeruli and renal medulla; J: the normal white pulp of the spleen; K: hemorrhage and hemosiderin deposition in the spleen of the SYS rats; L and M: normal heart and stomach of SYS rats (Scale bars = 50 µm).</p

Comparison of our rotenone ST models with other models.

<p>Data on the rotenone ST and SYS administration models are from our study and other studies, while data on the MPTP models are all from reports by others. Animal, animal used for the research; Instrument, special instrument or routes of administration; Nutritional status, the nutritional status of PD animal models and whether supplementation is required; Peripheral toxicity, peripheral organ toxicity caused by the neurotoxin; Application, whether the model is suitable for the study of pathogenesis, pathophysiological research, or drug screening of PD; Slow progression, idiopathic PD-like slow progression; Selective degeneration, selective degeneration of DA neurons; Long-term progression, long-term (more than three months) progression without intervention; Extensive involvement, ability to reproduce extensive pathological involvement observed in PD patients; Cost, costs of neurotoxin drugs, instruments, nutritional supplementation (ST, Stereotaxical Infusion; SYS, Systemic administration; S.C., subcutaneous injection).</p

Double immunohistostaining of alpha-synuclein and TH.

<p>Samples of the double immunohistostained alpha-synuclein and TH of the lesioned SNc were taken from the rats sacrificed four weeks after surgery (3-µg group: A–H; 12-µg group: I–L). Alpha-synuclein was visualized by FITC-conjugated donkey-anti-mouse IgG, TH was labeled by the Cy3-conjugated goat-anti-rabbit IgG, and the nucleus was stained by the Hoechst 33258. The last graph of the three rows was obtained by the overlapping the first three graphs (Scale bars = 50 µm). Compared with the contralateral side of the ST models (A-D), a dose-dependent increase in alpha-synuclein expression in the lesioned brain and a decrease in the number of TH-positive neurons are shown in E–H (3-µg group) and I–L (12-µg group). The quantitative data were shown as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007878#pone-0007878-g004" target="_blank">Figure 4L</a>.</p

Differential effect of APO on the ST infusion rats.

<p>The APO-induced rotations were counted on the 1^st, 2^nd, 3^rd, 4^th, 6^th, 8^th, 10^th, 12^th, 16^th, 20^th, and 24^th weeks following the 3-µg rotenone infusion and on the 2^nd, 3^rd, 4^th, 6^th, and 8^th weeks following the 6-µg and 12-µg rotenone infusions. The control animals did not exhibit rotations. The total number of rotations is expressed as the mean±SD (n = 6).</p

Effect of rotenone on the striatum, VTA, and SNc TH immunoreactivity.

<p>Pictures of the TH immunostaining of coronal sections were taken at the level of the striatum from the DMSO- or rotenone-infused rats (3-µg group) four weeks after infusion. Each treatment group had four rats. The left striatum represents the intact side of the brain, and the right striatum represents the lesioned side of the same animal. The TH immunoreactivity on the ipsilateral side of the striatum was decreased by 66.4% (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007878#pone-0007878-g004" target="_blank">Figure 4I–J</a>; *P<0.05, as compared to the contralateral side). The ST-infused rats (3- and 12-µg groups) and SYS models were sacrificed four weeks after the rotenone infusion, and coronal sections were HE-stained and immunohistochemically stained with TH. HE-staining showed a significant decrease in the number of neurons in the VTA and SNc (A, 3 µg group). The number of TH-positive neurons (E) was decreased by 43.7% (B), 53.6% (C), 59.0%, and 75.8% (D) in the right side of the 3- and 12-µg ST models and in the left and right sides of SYS models, respectively, as compared to the contralateral side of the ST models. The integrated intensity of the VTA and SNc was decreased by 38.1%, 54.5%, 46.4%, and 95.3% in the right side of the 3- and 12-µg ST models and in the left and right sides of the SYS models, respectively (F) (*P<0.05, as compared with the contralateral side; #P<0.05, as compared with the lesioned side of 3-µg group; and P<0.05, as compared with the other side of the SYS group) (Scale bars = 50 µm).</p

Ultrastructural changes of the SNc.

<p>The ultrastructural changes of the SNc were set in order: (1)mitochondrial swelling, (2)mitochondrial crest fracture, (3)mitochondrial vacuolar degeneration, (4)dilated and broken rough endoplasmic reticula, (5)liberation of ribosomes from the rough endoplasmic reticula, (6)lipofuscin deposition, (7)perinuclear space augmentation. A and B: normal mitochondria, rough endoplasmic reticulum. and ribosomes in the contralateral SNc of the 3-µg group animals one week or four weeks after surgery; C: (1), (2), (4) and (5) in lesioned SNc of the 3-µg group animals one week after surgery; D: (1), (2) and (3) in the lesioned SNc of the 3-µg group animals two weeks after surgery; E: (1), (2), (2), (4) and (5) in the lesioned SNc of the 3-µg group animals three weeks after surgery; F: (6) in the lesioned SNc of the 3-µg group animals one week after surgery; G: (1), (2), (3) and (7) in the lesioned SNc of the 3-µg group animals four weeks after surgery; H: the rough endoplasmic reticula and increased lysosomes in glial cells of lesioned SNc of 3-µg group animals four weeks after surgery; I and J: (1), (2) and (3) in both sides of the SNc of the SYS model four weeks after rotenone administration.</p

Effects of ST infusion of rotenone on SNc oxidative stress levels.

<p>(1) Dose response: the rats were sacrificed on the fourth week following the ST infusion of different doses (3-, 6-, or 12-µg) of rotenone, and the GSH activity (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007878#pone-0007878-g003" target="_blank">Figure 3A</a>), SOD activity (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007878#pone-0007878-g003" target="_blank">Figure 3C</a>), and MDA level (E) in the midbrain on the left and right sides were spectrophotometrically measured. (2) Time response: The animals infused with 3-µg rotenone were sacrificed on the second, fourth, sixth, and eighth week for the GSH, SOD, and MDA determination. *P<0.05, as compared to contralateral side. Data were expressed as the means±SD (n = 6).</p