Liver growth factor modulated the expression of phospho-Akt and Bcl2 in the striatum and mesencephalon of 6-OHDA-lesioned rats.

<p>As shown in A, the decrease in phospho-Akt/Akt ratio observed in vehicle-treated rats (A, white bar) was prevented 24 hours after the administration of a single injection of LGF (A, lined bar). In the lesioned mesencephalon, the phospho-Akt/Akt ratio was significantly increased 72 hours after the beginning of LGF treatment (B, lined bars), as compared with control (B, dotted bar), and the lesioned mesencephalon of vehicle-treated rats (B, white bar). Panels C and D show Bcl2/Bax ratios in the lesioned striatum (C) and mesencephalon (D) of 6-OHDA-lesioned rats. Note how a single injection of LGF promoted a transitory, but significant, increase in Bcl2/Bax ratio in both structures. Lane 1: control; lane 2: vehicle-treated rats; lane 3∶24-hour LGF-treated rats; lane 3″: lesioned mesencephalon of 48-hour LGF-treated rats; lane 4∶72-hour LGF-treated rats. Results are expressed as percentage of control (naïve striatum and mesencephalon of IP-vehicle treated rats at 13 weeks post-lesion), and represent the mean ± SEM of n individual rats. One way ANOVA were performed in A (p = 0.0064; F_{3, 26} = 5.135, n = 6–11), B (p = 0.014; F_{3, 22} = 4.426, n = 6–7), C (p = 0.0009; F_{3, 22} = 8.003, n = 6–7) and D (p = 0.0047; F_{3, 23} = 5.651, n = 6–7) followed by Newman-Keuls multiple comparison test (*p≤0.05 and **p≤0.01 vs control. +p≤0.05 and ++p≤0.01 vs vehicle. °°p≤0.01 vs 24 hours post IP-LGF. ··p≤0.01 vs 72 hours post-LGF).</p

Liver growth factor modulated TH and DAT immunoreactivity in the striatum of 6-OHDA-lesioned rats.

<p>Panels A-D show TH immunostaining in the naïve striatum of IP-vehicle or LGF-treated rats (A, C red, respectively), and in the lesioned striatum of vehicle-treated rats (B, red), and rats receiving LGF (D, red). E, F, G and H show TH immunostaining in the naïve substantia nigra (SN) of vehicle (E, red) or LGF-treated rats (G, red), and in the lesioned SN of IP-vehicle (F, red) and IP-LGF-treated rats (H, red). Panels I, J and L show double immunostaining for DAT (green) and TH (red). K shows DAT immunostaining (green) and nuclei (blue). DAT immunoreactivity was reduced in the lesioned striatum of vehicle-treated rats (J, green), as compared with the naïve striatum of vehicle-treated rats (I, green) and with the lesioned striatum of the LGF group (K, green). Note how, in the naïve striatum of vehicle-treated, DAT immunostaining (I, green) is confined to small spots in the striatal parenchyma that probably represent neuronal cell bodies (white arrows in I). However, in the lesioned striatum of IP-LGF-treated rats, DAT immunoreactivity is distributed in neuronal cell bodies (asterisks in K) and in the striatal terminals (arrowheads in K). In addition, DAT is mainly expressed in the sprouting TH-positive terminals in the lesioned striatum of rats receiving LGF (L, yellow, clear arrows), while almost no TH/DAT co-labelling is observed in the lesioned striatum of IP-vehicle treated rats (J). Scale bar: A-D, 100 µm, and E-L, 50 µm.</p

Liver growth factor activated glial cells in the striatum of 6-OHDA-lesioned rats.

<p>Panels A, B and D show the effect of IP-LGF treatment on proteins expressed by glial cells. Forty-eight hours after the administration of a single injection of LGF (A, B and D, lined bars), GLUT5, OX6, and GFAP protein levels were significantly higher than those observed in control (A, B and D, dotted bars) or in the lesioned striatum of vehicle-treated rats (A, B and D, white bars). Note how the chronic administration of LGF (A, B and D, black bars) significantly reduced the abovementioned parameters. Panel C shows the effect of IP-LGF administration on PCNA protein expression. Results are expressed as percentage of control (naïve striatum of IP-vehicle treated rats at 13 weeks post-lesion), and represent the mean ± SEM of n individual rats. One way ANOVA were performed in A (p = 0.0149; F_{3, 47} = 3.868, n = 7–18), B (p = 0.006; F_{3, 41} = 7.435, n = 7–12), C (p<0.0001; F_{3, 48} = 21.66, n = 7–18) and D (p<0.0001; F_{3, 48} = 12.75, n = 7–18) followed by followed by Newman-Keuls multiple comparison test (*p≤0.01, and ***p≤0.001 vs control. +p≤0.05, and ++p≤0.01 vs vehicle. &p≤0.05, and &&p≤0.01 vs 48 hours post-LGF). Panels E and F show double immunostaining for OX6 (red) and PCNA (green) in the lesioned striatum of rats receiving a single injection of vehicle or LGF. G and H show the immunoreactivity for GFAP (red) and PCNA (green) in the striatum of IP-vehicle and IP-LGF-treated rats. Note how, 48 hours after the administration of LGF, many OX6-positive cells were PCNA-positive too, and how LGF promoted cell body hypertrophy of GFAP-positive cells (H, white arrow). Scale bar: E-F, 60 µm, and G-H, 30 µm. Panel I shows representative blots for GLUT5, OX6, PCNA, and GFAP. Lane 1: control striatum; lane 2: lesioned striatum of vehicle rats; lane 3: lesioned striatum of 48-hour LGF-treated rats; lane 4: lesioned striatum of chronic LGF-treated rats.</p

Liver growth factor stimulated TH and DAT protein expression in striatum and protected DA neurons in the substantia nigra of 6-OHDA-lesioned rats.

<p>Panel A shows TH-positive innervation in the striatum of lesioned rats treated with vehicle (A, white bars) or with LGF (A, black bars). Data in B represent the number of TH-positive neurons in the naïve (B, dotted bar) and lesioned substantia nigra (SN) of rats treated with vehicle (B, white bar) or LGF (B, black bar). Panels C and D show TH and DAT protein expression in the naïve (control, dotted bars) and lesioned striatum of rats receiving vehicle (white bars) or LGF (black bars). Results are represented as the mean ± SEM of n individual animals. T-tests were performed in A (n = 4–9. +p≤0.05, ++p≤0.01, and +++p≤0.001 vs vehicle). One way ANOVA were performed in B (p<0.0001; F_{2, 24} = 59.26, n = 8–11), C (p<0.0001; F_{2, 41} = 126.9, n = 11–17) and D (p = 0.0216; F_{2, 25} = 4.490, n = 5–12) followed by Newman-Keuls multiple comparison test (*p≤0.05 and ***p≤0.001 vs control. +p≤0.05 vs vehicle). C, D, lane 1: control striatum; C, D, lane 2: lesioned striatum of vehicle-treated rats; C, D, lane 3: lesioned striatum of LGF rats.</p

Liver growth factor activated the MAPK/ERK1/2 signalling pathway and elicited the phosphorylation of CREB in the striatum of 6-OHDA-lesioned rats.

<p>A single injection of LGF promoted a transient increase in the phosphorylation of ERK1/2, which was observed 24 hours after the administration of the factor (A, B, lined bars). Moreover, 72 hours after LGF treatment, phospho-CREB levels were significantly raised (C, lined bars), as compared with control (C, dotted bar) and vehicle-treated rats (C, white bars). Lane 1: control; lane 2: lesioned striatum of vehicle rats; lane 3: lesioned striatum of 24-hour LGF-treated rats; lane 4: lesioned striatum of 48-hour LGF-treated rats; lane 5: lesioned striatum of 72-hour LGF-treated rats. Results are expressed as percentage of control (naïve striatum of IP-vehicle treated rats at 13 weeks post-lesion), and represent the mean ± SEM of n individual rats. One way ANOVA were performed in A (p = 0.0006; F_{4, 28} = 6.791, n = 6–7), B (p = 0.0028; F_{4, 29} = 5.178, n = 6–7) and C (p<0.0001; F_{4, 29} = 11.58, n = 6–8), followed by Newman-Keuls multiple comparison test (*p≤0.05, **p≤0.01, and ***p≤0.001 vs control. +p≤0.05, ++p≤0.01, and +++p≤0.001 vs vehicle. °p≤0.05, °°p≤0.01 vs 24 hours post-LGF. ···p≤0.001 vs 72 hours post-LGF). Panels D-G show the immunohistochemistry for phospho-ERK1/2 (D and F) and phospho-CREB (E and G) in the lesioned striatum of vehicle (D and E), 24-hours LGF-treated rats (F), and 72-hours LGF-treated rats (G). Note how IP-LGF increased phospho-ERK1/2 (F) and phospho-CREB (G) immunostaining, and how phospho-ERK1/2-positive cells exhibit the morphology of ramified glia (F, inset). Scale bar: 100 µm.</p

Representative scheme showing the methodological procedure followed for the repeated administration of vehicle and LGF to 6-OHDA-lesioned rats.

<p>Representative scheme showing the methodological procedure followed for the repeated administration of vehicle and LGF to 6-OHDA-lesioned rats.</p

Change in cognitive measures at week 48.

<p>Change in cognitive measures at week 48.</p

Changes in NPZ-7 and in information processing speed and executive functioning domains.

<p>(a) Mean change in NPZ-7. (b) Mean change in information processing speed. (c) Mean change in executive functioning. Values are expressed as z-score means, bars as standard errors. P values and d sizes are provided for rivastigmine vs control group comparisons. Abbreviations: CG, control group; EF, executive functioning; IPS, information processing speed; LG, lithium group; RG, rivastigmine group.</p

LGF ameliorated motor deficits in 6-OHDA-lesioned rats.

<p>Between 10 and 13 weeks post-lesion, the number of apomorphine-induced contralateral rotations was significantly reduced in 6-OHDA-lesioned rats receiving LGF (A, black bars), as compared with rats treated with vehicle (A, white bars). Results represent the mean ± SEM of 23 to 24 individual rats. Motor performance, as assessed by the rotarod test, was reduced in lesioned rats receiving vehicle (B, white bars). Note how, between 11 and 13 weeks post-lesion, LGF significantly improved this parameter (B, black bars). Results represent the mean ± SEM of 4 individual animals. Two-way ANOVA was performed in A (p<0.0001; F_{1, 351} = 26.08) and B (p = 0.0166; F_{1, 48} = 6.16) followed by Bonferroni multiple comparison test (+p≤0.05, ++p≤0.01 vs vehicle). Start of treatments (↓).</p

Baseline characteristics of study participants.

<p>Baseline characteristics of study participants.</p