Late-onset Parkinsonism in NFκB/c-Rel-deficient mice methods
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L-DOPA treatment
l -DOPA ( l -3,4-dihydroxyphenylalanine methyl ester hydrochloride) and benserazide hydrochloride were purchased from Sigma Aldrich and freshly dissolved in a saline solution at a final injection volume of 5 ml/kg. Both l -DOPA (20 mg/kg) and benserazide hydrochloride (12.5 mg/kg) or vehicle (saline) were injected intraperitoneally into mice 1 h before the beginning of the behavioural tests.
Materials and methods
C57BL/6 mice carrying the c-Rel gene null mutation (c-rel -/- ) were originally generated by inserting the neomycin cassette into the fifth exon of the c-Rel gene ( ). The c-rel -/- mice were backcrossed to C57BL/6 J mice for nine generations before being used in this study. The genotypes were verified by PCR analysis (wild-type c-rel forward primer, 5′-AAGTGGGGTTACAGGTGCTCA-3′; wild-type c-rel reverse primer, 5′-TTGCCAATAGGCTTAGTCAAATA-3′; c-rel neomycin reverse primer, 5′-CTCTCGTGGGATCATTGTTTTTC-3′). PCR conditions were 94°C for 30 s, 57°C for 30 s, 72° for 30 s (30 cycles). The c-rel -/- and c-rel +/+ (wild-type) lines were continued by homozygous breeding. Animals were housed in standard cages and maintained under a 12h/12h light:dark cycle with food and water available ad libitum. Humidity was...
Immunohistochemistry
Mice were anaesthetized with chloral hydrate (400 mg/kg intraperitoneally) and transcardially perfused with PBS (SIGMA), 4% (w/v) ice-cold paraformaldehyde and 14% (w/v) ice-cold picric acid (SIGMA). Coronal slices (30 or 10 µm thickness) were cut to obtain serial sections of the following cerebral areas using bregma-based coordinates ( ): substantia nigra pars compacta (anterior-posterior -2.54 to -3.40 mm), ventral tegmental area (anterior-posterior -2.92 to -3.80 mm), medial septal area (anterior-posterior 1.18 to 0.38 mm), nucleus basalis magnocellularis (anterior-posterior -0.34 to -1.34 mm) and striatum (anterior-posterior 1.70 to -2.30 mm).
Immunohistochemistry
For ferric iron, a Prussian blue stain with diaminobenzidine enhancement was used ( ). Sections were incubated with 7% potassium ferrocyanide (Perls reagent) in 3% HCl for 1 h at 37°C followed by distilled water and 0.015% H 2 O 2 -activated 0.75 mg/ml diaminobenzidine in 0.1 M PBS for 10 min.
Immunohistochemistry
Double immunofluorescence staining was performed in sections (10 µm) incubated with α-synuclein antibody overnight at 4°C followed by a goat anti-mouse secondary antibody conjugated with Cy3 (1:400 Jackson ImmunoResearch) for 30 min at room temperature. Slices were then incubated with a primary rabbit anti-tyrosine hydroxylase (1:200 Chemicon) antibody overnight at 4°C followed by incubation in Alexa Fluor® 405-conjugated secondary antibodies (1:400 Jackson ImmunoResearch). For thioflavin S/α-synuclein double staining, sections were incubated with 0.3% KMnO 4 for 3-5 min, washed with water and incubated with a solution of 0.1% NaBH 4 for 5 min and then placed in a high-concentration PO 4 buffer (411 mM NaCl, 8.1 mM KCl, 30 mM NaHPO 4, 5.2 mM KH 2 PO 4 ) pH 7.2. After washing, thioflavin S (Sigma-Aldrich) and α-synuclein immunostaining was perf...
Cell quantification
The number of cholinergic neurons in the basal forebrain was determined by counting ChAT-positive cells in the medial septal area and in the nucleus basalis magnocellularis. For the striatum, NeuN-positive cells with identifiable nuclei were counted in three regions selected between lateral ±1.00 to ±2.00 mm and ventral ±2.00 to ±3.00 mm, (520 × 380 µm; left and right). Values are expressed as the mean ± SEM.
Quantification of CD11b- and glial fibrillary acidic protein-positive cells
Quantification of CD11b and GFAP immunostaining was carried out in brain slices sections processed on the same day in order to avoid any difference in staining intensity among animals. All pictures were captured within the same session in order to avoid any difference in any differences in lighting conditions. In each section, the entire left and right substantia nigra pars compacta were analysed, whereas for the striatum evaluation, one portion from the dorsolateral striatum (lateral from ±1.65 to ±2.15; ventral from -2.00 to -2.40) and one from the ventromedial striatum (lateral from ±1.25 to ±1.75; ventral from -1.75 to -2.15, 520 × 380 µm; left and right) were analysed. For each animal, three sections from the substantia nigra pars compacta (anterior-posterior -2.92 mm, -3.28 mm and -3.64 mm from bregma,...
α-Synuclein extraction
Twenty milligrams of brain tissue were homogenized in five volumes of Tris-buffered saline plus (TBS + ) buffer (Tris-HCl 50 mM pH 7.4, NaCl 175 mM, EDTA 5 mM, PMSF 0.1 mM and N -ethylmaleimide 1 mM; Sigma) using a glass homogenizer on ice. The homogenate was centrifuged at 120 000 g for 30 min at 4°C. The supernatant (S1) was saved, and the pellet was dissolved in TBS + buffer with 1% Triton™ X-100 and centrifuged at 120 000 g for 30 min at 4°C to obtain the second supernatant (S2). The procedure was repeated, the supernatant (S3) was retained and the pellet was dissolved in urea 8 M (SIGMA) with SDS 5% to obtain the supernatant S4.
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Evidence quotes (8)
l -DOPA ( l -3,4-dihydroxyphenylalanine methyl ester hydrochloride) and benserazide hydrochloride were purchased from Sigma Aldrich and freshly dissolved in a saline solution at a final injection volume of 5 ml/kg. Both l -DOPA (20 mg/kg) and benserazide hydrochloride (12.5 mg/kg) or vehicle (saline) were injected intraperitoneally into mice 1 h before the beginning of the behavioural tests.
C57BL/6 mice carrying the c-Rel gene null mutation (c-rel -/- ) were originally generated by inserting the neomycin cassette into the fifth exon of the c-Rel gene ( ). The c-rel -/- mice were backcrossed to C57BL/6 J mice for nine generations before being used in this study. The genotypes were verified by PCR analysis (wild-type c-rel forward primer, 5′-AAGTGGGGTTACAGGTGCTCA-3′; wild-type c-rel reverse primer, 5′-TTGCCAATAGGCTTAGTCAAATA-3′; c-rel neomycin reverse primer, 5′-CTCTCGTGGGATCATTGTTTTTC-3′). PCR conditions were 94°C for 30 s, 57°C for 30 s, 72° for 30 s (30 cycles). The c-rel -/- and c-rel +/+ (wild-type) lines were continued by homozygous breeding. Animals were housed in standard cages and maintained under a 12h/12h light:dark cycle with food and water available ad libitum. Humidity was kept at a constant level, and the room temperature was maintained at 22-23°C. All animal experiments were authorized by the Italian Ministry of Health and by the University of Brescia Animal Care Committee in compliance with the Italian guidelines for animal care (DL 116/92) and the Euro...
Mice were anaesthetized with chloral hydrate (400 mg/kg intraperitoneally) and transcardially perfused with PBS (SIGMA), 4% (w/v) ice-cold paraformaldehyde and 14% (w/v) ice-cold picric acid (SIGMA). Coronal slices (30 or 10 µm thickness) were cut to obtain serial sections of the following cerebral areas using bregma-based coordinates ( ): substantia nigra pars compacta (anterior-posterior -2.54 to -3.40 mm), ventral tegmental area (anterior-posterior -2.92 to -3.80 mm), medial septal area (anterior-posterior 1.18 to 0.38 mm), nucleus basalis magnocellularis (anterior-posterior -0.34 to -1.34 mm) and striatum (anterior-posterior 1.70 to -2.30 mm).
For ferric iron, a Prussian blue stain with diaminobenzidine enhancement was used ( ). Sections were incubated with 7% potassium ferrocyanide (Perls reagent) in 3% HCl for 1 h at 37°C followed by distilled water and 0.015% H 2 O 2 -activated 0.75 mg/ml diaminobenzidine in 0.1 M PBS for 10 min.
Double immunofluorescence staining was performed in sections (10 µm) incubated with α-synuclein antibody overnight at 4°C followed by a goat anti-mouse secondary antibody conjugated with Cy3 (1:400 Jackson ImmunoResearch) for 30 min at room temperature. Slices were then incubated with a primary rabbit anti-tyrosine hydroxylase (1:200 Chemicon) antibody overnight at 4°C followed by incubation in Alexa Fluor® 405-conjugated secondary antibodies (1:400 Jackson ImmunoResearch). For thioflavin S/α-synuclein double staining, sections were incubated with 0.3% KMnO 4 for 3-5 min, washed with water and incubated with a solution of 0.1% NaBH 4 for 5 min and then placed in a high-concentration PO 4 buffer (411 mM NaCl, 8.1 mM KCl, 30 mM NaHPO 4, 5.2 mM KH 2 PO 4 ) pH 7.2. After washing, thioflavin S (Sigma-Aldrich) and α-synuclein immunostaining was performed and the sections examined with a Zeiss, LSM 510 META confocal microscope (Carl Zeiss), with the laser set at λ = 405-488-543 nm and the height of the section scanning = 1 mm. Images (512 × 512 pixels) were then re-constructed using LSM Image Examiner (Carl Zeiss) and Adob...
The number of cholinergic neurons in the basal forebrain was determined by counting ChAT-positive cells in the medial septal area and in the nucleus basalis magnocellularis. For the striatum, NeuN-positive cells with identifiable nuclei were counted in three regions selected between lateral ±1.00 to ±2.00 mm and ventral ±2.00 to ±3.00 mm, (520 × 380 µm; left and right). Values are expressed as the mean ± SEM.
Quantification of CD11b and GFAP immunostaining was carried out in brain slices sections processed on the same day in order to avoid any difference in staining intensity among animals. All pictures were captured within the same session in order to avoid any difference in any differences in lighting conditions. In each section, the entire left and right substantia nigra pars compacta were analysed, whereas for the striatum evaluation, one portion from the dorsolateral striatum (lateral from ±1.65 to ±2.15; ventral from -2.00 to -2.40) and one from the ventromedial striatum (lateral from ±1.25 to ±1.75; ventral from -1.75 to -2.15, 520 × 380 µm; left and right) were analysed. For each animal, three sections from the substantia nigra pars compacta (anterior-posterior -2.92 mm, -3.28 mm and -3.64 mm from bregma, according to the mouse brain atlas by ) and three sections from the striatum (anterior-posterior -1.10 mm, 0.74 mm and 0.38 mm from bregma) were analysed. CD11b immunostaining was evaluated with the Scion Image analysis program (Scion Corp.). Inside each frame, the area occupied by grey...
Twenty milligrams of brain tissue were homogenized in five volumes of Tris-buffered saline plus (TBS + ) buffer (Tris-HCl 50 mM pH 7.4, NaCl 175 mM, EDTA 5 mM, PMSF 0.1 mM and N -ethylmaleimide 1 mM; Sigma) using a glass homogenizer on ice. The homogenate was centrifuged at 120 000 g for 30 min at 4°C. The supernatant (S1) was saved, and the pellet was dissolved in TBS + buffer with 1% Triton™ X-100 and centrifuged at 120 000 g for 30 min at 4°C to obtain the second supernatant (S2). The procedure was repeated, the supernatant (S3) was retained and the pellet was dissolved in urea 8 M (SIGMA) with SDS 5% to obtain the supernatant S4.
Machine-readable layer
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