MPTP treatment induces a similar lesion in striatum-substantia nigra of HO-1^−/− mice and their wild type littermates.

<p>Animals received one daily i.p. injection of saline or MPTP (30 mg/kg) for five consecutive days. Pictures show representative coronal sections, 40-µm thick, from VMB and STR stained with anti-TH antibody and counterstained with Nissl. <i>A</i>, Dopaminergic denervation in STR. <i>B</i>, Loss of dopaminergic neurons in VMB. Rectangles indicate SN. <i>C</i>, Stereological quantification of TH-immunoreactive neurons and Nissl positive neurons in SN. <i>D</i>, immunoblot of STR protein lysates. <i>Upper panel</i>, anti-TH antibody. <i>Lower panel</i>, anti-β-actin antibody showing similar protein load per lane. <i>E</i>, densitometric quantifications (arbitrary densitometry units) from representative immunoblots of D after normalization by β-actin densitometry units obtained from the same immunoblot.Results are representative of 5–10 animals per group. Values represent mean ± SD. Two-way ANOVA followed by Bonferroni's test was applied to determine the significance of biochemical differences among groups.</p

MPTP induces a similar deposition of ferric iron in Nrf2^−/− and HO-1^−/− mice.

<p>Iron precipitates were detected by DAB-enhanced Perls reaction followed by Nissl counterstaining. <i>A,</i> simplified scheme of a coronal section of midbrain showing the location of mouse dorsal and ventral substantia nigra on the left (SNc and SNr, respectively). The rectangle on the right side indicates the region shown in panels <i>D, E, F and G</i>). <i>B</i> and <i>C</i>, high magnification pictures of the boundary between SNc and SNr from wild type mice submitted to saline or MPTP treatments, respectively. Yellow arrowheads point some large Perls-negative cells with pale nuclei and dark nucleoli typical of dopaminergic neurons. Green arrowheads point small Perls-positive cells with picnotic nuclei typical of microglia. The red arrowhead points a Perls-positive microglial cell that has been drawn in the inset to show microgrial extensions. The large panels show representative fields of both SNc and SNr (location indicated in the rectangle of <i>A</i> panel), from saline-treated wild type mice (<i>D</i>), MPTP-treated wild type mice (<i>E</i>), MPTP-treated Nrf2^−/− mice (<i>F</i>), and MPTP-treated HO-1^−/− mice (<i>G</i>).</p

HO-1^−/− mice exhibit similar glial activation compared to wild type littermates in response to sub-acute MPTP treatment.

<p>Immunohistochemistry was done in 40-µm thick coronal section of STR with anti-GFAP (astroglia) and anti-Iba1 (microglia) antibodies. <i>A,</i> immunohistochemical staining of STR with anti-GFAP antibody showing no differences in astroglia in HO-1^−/− mice compared to wild type littermates in response to MPTP. <i>B,</i> immunohistochemical staining of STR with anti-Iba1 antibody showing no differences microglial activation in MPTP-treated HO-1^−/− mice compared to wild type littermates. <i>C,</i> immunoblots from STR. <i>Upper panels</i>, anti-GFAP antibody; <i>middle panels</i>, anti-Iba-1 antibody; <i>lower panels</i>, anti-β-actin antibody. <i>D and E</i>, densitometric quantifications (arbitrary densitometry units) from representative immunoblots of C after normalization by β-actin densitometry units obtained from the same immunoblots.Results are representative of 5–10 animals per group. Values represent mean ± SD. Two-way ANOVA followed by Bonferroni's test was applied to determine the significance of biochemical differences among groups.</p

DA and DOPAC levels in STR of MPTP-treated Nrf2^−/− and HO-1^−/− and wild type littermate mice.

<p>Animals received one daily i.p. injection of saline or MPTP (30 mg/kg) for five consecutive days and striatal DA and DOPAC levels were determined by HPLC. <i>A and B,</i> the reduction in DA and DOPAC is more pronounced in MPTP-treated Nrf2^−/− mice than in MPTP-treated wild type littermates. <i>C and D,</i> the reduction in DA and DOPAC is similar between MPTP-treated HO-1^−/− mice and their wild type littermates. Values represent the mean ± SD from five samples. Two-way ANOVA followed by Bonferroni's test was applied to determine the significance of biochemical differences among groups. Asterisks denote significant differences between treatments with <i>P</i><0.05.</p

Sub-acute MPTP treatment induces a more profound lesion in STR and SN of Nrf2^−/− mice in comparison to their wild type littermates.

<p>Animals received one daily i.p. injection of saline or MPTP (30 mg/kg) for five consecutive days. Pictures show representative coronal sections, 40-µm thick, from VMB and STR stained with anti-TH antibody and counterstained with Nissl. <i>A</i>, Dopaminergic denervation in STR. <i>B</i>, Loss of dopaminergic neurons in VMB. Rectangles indicate SN. <i>C</i>, Stereological quantification of TH-immunoreactive neurons and Nissl positive neurons in SN. <i>D</i>, immunoblot of STR protein lysates. <i>Upper panel</i>, anti-TH antibody. <i>Lower panel</i>, anti-β-actin antibody showing similar protein load per lane. <i>E</i>, densitometric quantifications (arbitrary densitometry units) from representative immunoblots of D after normalization by β-actin densitometry units obtained from the same immunoblot. Results are representative of 5–10 animals per group. Values represent mean ± SD. Two-way ANOVA followed by Bonferroni's test was applied to determine the significance of biochemical differences among groups. Asterisks denote significant differences between treatments with <i>P</i><0.05.</p

Impairment of the phase II response in Nrf2^−/− but not in HO-1^−/− mice.

<p><i>A</i>, immunoblots showing STR protein levels of the phase II proteins NQO1, GCL-M and GCL-C and the constitutively expressed enzyme HO-2, in Nrf2^−/− mice, HO-1^−/− mice and their wild type controls under saline or MPTP treatment. <i>B</i>, <i>C</i>, <i>D</i> and <i>E</i>, densitometric quantifications (arbitrary densitometry units) from representative immunoblots of <i>A</i> after normalization by β-actin densitometry units obtained from the same immunoblots.Results are representative of 5–10 animals per group. Values represent mean ± SD. Two-way ANOVA followed by Bonferroni's test was applied to determine the significance of biochemical differences among groups. Asterisks denote significant differences between treatments with <i>P</i><0.05.</p

Nrf2^−/− mice show increased microglial activation compared to wild type littermates in response to sub-acute MPTP treatment.

<p>Immunohistochemistry was done in 40-µm thick coronal section of STR with anti-GFAP (astroglia) and anti-Iba1 (microglia) antibodies. <i>A,</i> immunohistochemical staining of STR with anti-GFAP antibody showing increased basal and MPTP-induced astroglia in Nrf2^−/− mice compared to wild type littermates. <i>B,</i> immunohistochemical staining of STR with anti-Iba1 antibody showing increased microglial activation in MPTP-treated Nrf2^−/− mice compared to wild type littermates. <i>C,</i> immunoblots from STR. <i>Upper panels</i>, anti-GFAP antibody; <i>middle panels</i>, anti-Iba-1 antibody; <i>lower panels</i>, anti-β-actin antibody. <i>D and E</i>, densitometric quantifications (arbitrary densitometry units) from representative immunoblots of C after normalization by β-actin densitometry units obtained from the same immunoblot.Results are representative of 5–10 animals per group. Values represent mean ± SD. Two-way ANOVA followed by Bonferroni's test was applied to determine the significance of biochemical differences among groups. Asterisks denote significant differences between treatments with <i>P</i><0.05.</p