Table 1.
Immature rats' weight gain exposed to Mn in vivo.
Figure 1.
Effects of short-term Mn exposure on metal accumulation in the hippocampus, striatum and cerebral cortex of immature rats.
The panels show the accumulation of Mn (A) and Fe (B). Rat pups were treated for five days (PN8-12) with saline (control; NaCl 0.9%) or MnCl2 at doses of 5, 10 or 20 mg/kg. The tissues were analyzed on PN14. Results represent mean ± S.E.M and are expressed in µg metal/g tissue derived from four independent experiments. Statistical analysis was performed by ANOVA followed by Duncan's test. * p<0.05, ** p<0.01, *** p<0.001 compared to control.
Figure 2.
Histological evaluation of the brain of immature rats exposed to Mn in vivo.
The panel shows representative sections from eight independent experiments of (I) striatum, (II) hippocampus and (III) cerebral cortex from rats treated for five days (PN8-12) with saline (control; NaCl 0.9%) or MnCl2 at doses of 5, 10 or 20 mg/kg. The structures analyzed on PN14. The sections were stained with hematoxylin and eosin staining. Magnification ×40.
Figure 3.
Effects of in vivo exposure to Mn for five days on the phosphorylation of MAPKs, AKT, CREB and DARPP-32 in the striatum of immature rats.
The panels show representative immunoblotting and quantification of phosphorylation of ERK1/2 (A), AKT (B), DARPP-32-Thr-34 and -Thr-75 (C) and JNK1/2, p38MAPK, CREB and β-Actin (D) from rats treated for five days (PN8-12) with saline (control; NaCl 0.9%) or MnCl2 at doses of 5, 10 or 20 mg/kg/day. The structures analyzed on PN14. Total and phosphorylated forms of each protein were detected by specific antibodies and the reaction was developed by chemiluminescence. The phosphorylation level of each protein was determined as a ratio of the O.D. of the phosphorylated band over the O.D. of the total band and the data are expressed as percentage of the control (considered as 100%) and the values are presented as mean ± S.E.M derived from twelve independent experiments. Statistical analysis was performed by ANOVA followed by Duncan's test. * p<0.05, ** p<0.01, *** p<0.001 compared to control.
Figure 4.
Mn induces oxidative stress in the striatum.
Oxidative stress was analyzed by DCF fluorescence in the striatum of young rats treated with Mn. The graphic shows the DCF fluorescence from rats treated for five days (PN8-12) with saline (control; NaCl 0.9%) or MnCl2 at doses of 5, 10 or 20 mg/kg/day. The structures were analyzed on PN14. The data are expressed as percentage of the control and the values are mean ± S.E.M derived from eight independent experiments. Statistical analysis was performed by ANOVA followed by Duncan's test. ** p<0.01, *** p<0.001 compared to control.
Figure 5.
Mn induces F2-IsoPs production.
Striatum from immature rats (PN14) exposed in vivo to Mn (PN8-12) were evaluated for F2-IsoPs levels expressed as ng/g tissue. The graphic shows a dose-dependent increase of F2-IsoPs levels by MnCl2 (5, 10 or 20 mg/kg/day). The values were obtained from four independent experiments. Pearson's correlation showed r2 = 0.38 with p = 0.011.
Figure 6.
Effects of Mn on striatal activity of mitochondrial respiratory chain complexes in immature rats.
(A) Activity of mitochondrial complex I. (B) Activity of mitochondrial complex II. (C) Activity of mitochondrial complex IV. Rat pups were treated for five days (PN8-12) with saline (control; NaCl 0.9%) or MnCl2 at doses of 5, 10 or 20 mg/kg. Activities were analyzed on PN14. Results represent mean ± S.E.M and are expressed as nmol min−1/mg protein−1 or mmol min−1/mg protein−1 derived from four independent experiments. Statistical analysis was performed by ANOVA followed by Duncan's test. * p<0.05 compared to control.
Figure 7.
Mn treatment stimulates caspase activity in the striatum of immature rats.
Caspase activities were measured by DEVD cleavage. The panel shows the DEVD cleavage test from rats treated for five days (PN8-12) with saline (control; NaCl 0.9%) or MnCl2 at doses of 5, 10 or 20 mg/kg/day. Activities were analyzed on PN14. Results represent mean ± S.E.M and are expressed as percentage of control (100%) derived from eight independent experiments. Statistical analysis was performed by ANOVA followed by Duncan's test. * p<0.05, ** p<0.01 compared to control.
Figure 8.
Mn exposure on PN8-12 causes later life onset motor deficits in rats.
To evaluate motor coordination, control animals (saline) and rats treated with Mn (PN8-12) were tested on 22, 29 and 36 days of age (3, 4 and 5 week-old) on the rotarod task. The graphic shows the overall latency for falling in rats treated with saline (control; NaCl 0.9%) or MnCl2 at doses of 5, 10 or 20 mg/kg/day. Results represent median ± interquartile range and are expressed as seconds (s) to latency for falling derived from twelve independent experiments. Statistical analysis was performed by Kruskal-Wallis followed by Dunn's post-hoc test. * p<0.05 compared to control.
Figure 9.
Trolox™ blocked the Mn-induced striatal oxidative stress.
DCF fluorescence in the striatum from rats treated on PN8-12 with saline (control; NaCl 0.9%), MnCl2 20 mg/kg (Mn), Trolox™ 1 mg/kg (Tr) or MnCl2 20 mg/kg plus Trolox™ 1 mg/kg (Mn + Tr) was analyzed on PN14. Results represent mean ± S.E.M and are expressed as percent of control (100%) to DCF fluorescence derived from eight independent experiments. Statistical analysis was performed by ANOVA followed by Duncan's test. * p<0.05, compared to control; # p<0.05 compared to MnCl2 20 mg/kg group.
Figure 10.
Effects of Trolox™ on the phosphorylation of ERK1/2 and AKT in the striatum of immature rats exposed to Mn.
The panels (A) and (B) show representative immunoblotting and quantification of ERK1/2 and AKT phosphorylation, respectively, from rats treated for five days (PN8-12) with saline (control; NaCl 0.9%), MnCl2 20 mg/kg (Mn), Trolox™ 1 mg/kg (Tr) or MnCl2 20 mg/kg plus Trolox™ 1 mg/kg (Mn + Tr). The tissues were harvested from the rats on PN14. Total and phosphorylated forms of each protein were detected by specific antibodies and the reaction was developed by chemiluminescence. The phosphorylation of each protein was determined as a ratio of the O.D. of the phosphorylated band over the O.D. of the total band and the data are expressed as percentage of the control (considered as 100%) and the values are presented as mean ± S.E.M derived from twelve independent experiments. Statistical analysis was performed by ANOVA followed by Duncan's test. * p<0.05, ** p<0.01, *** p<0.001 compared to control; # p<0.05 and ### p<0.001 compared to MnCl2 20 mg/kg group.
Figure 11.
Effects of Trolox™ on the motor coordination in immature rats exposed to Mn.
Rats were treated for five days (PN8-12) with saline (control; NaCl 0.9%), MnCl2 20 mg/kg (Mn), Trolox™ 1 mg/kg (Tr) or MnCl2 20 mg/kg plus Trolox™ 1 mg/kg (Mn + Tr), and tested on 22, 29 and 36 days of age (3, 4 and 5 week old) at the rotarod task. Results represent median ± interquartile range and are expressed as seconds (s) to latency for falling derived from twelve independent experiments. Statistical analysis was performed by Kruskal-Wallis followed by Dunn's post-hoc test. * p<0.05 compared to control.
Table 2.
Protective effect of antioxidant Trolox™ on body weight gain in immature rats exposed to Mn in vivo.
Figure 12.
Motor behavioral effects in rats exposed to manganese and Trolox™.
The animals were tested in the circular open field for 10 min to evaluate possible motor changes induced by different treatments used. The panels show the parameters analyzed from rats treated for five days (PN8-12) with saline (control; NaCl 0.9%), MnCl2 20 mg/kg (Mn), Trolox™ 1 mg/kg (Tr) or MnCl2 20 mg/kg plus Trolox™ 1 mg/kg (Mn + Tr). Panel (A) show the distance (m), (B) speed (m/s), (C) grooming frequency and (D) rearing frequency. Results represent mean ± S.E.M derived from twelve independent experiments. Statistical analysis was performed by ANOVA followed by Newman-Keuls test. * p<0.05 compared to control; # p<0.05 compared to MnCl2 20 mg/kg group.