Investigation of the therapeutic potential of N-acetyl cysteine and the tools used to define nigrostriatal degeneration in vivo

Abstract

The glutathione precursor N-acetyl-L-cysteine (NAC) is currently being tested on Parkinson's patients for its neuroprotective properties. Our studies have shown that NAC can elicit protection in glutathione-independent manners in vitro. Thus, the goal of the present study was to establish an animal model of NAC-mediated protection in which to dissect the underlying mechanism. Mice were infused intrastriatally with the oxidative neurotoxicant 6-hydroxydopamine (6-OHDA; 4 μg) and administered NAC intraperitoneally (100mg/kg). NAC-treated animals exhibited higher levels of the dopaminergic terminal marker tyrosine hydroxylase (TH) in the striatum 10d after 6-OHDA. As TH expression is subject to stress-induced modulation, we infused the tracer FluoroGold into the striatum to retrogradely label nigrostriatal projection neurons. As expected, nigral FluoroGold staining and cell counts of FluoroGold(+) profiles were both more sensitive measures of nigrostriatal degeneration than measurements relying on TH alone. However, NAC failed to protect dopaminergic neurons 3 weeks following 6-OHDA, an effect verified by four measures: striatal TH levels, nigral TH levels, nigral TH(+) cell counts, and nigral FluoroGold levels. Some degree of mild toxicity of FluoroGold and NAC was evident, suggesting that caution must be exercised when relying on FluoroGold as a neuron-counting tool and when designing experiments with long-term delivery of NAC--such as clinical trials on patients with chronic disorders. Finally, the strengths and limitations of the tools used to define nigrostriatal degeneration are discussed.

Keywords: Dopamine; FluoroGold; N-acetylcysteine; Neurodegeneration; Parkinson's disease; Retrograde; The reference trap.

Fig. 1

NAC raises TH levels in the striatum 10 days after 6-OHDA infusions. Mice were stereotaxically infused with 4 μg 6-OHDA or an equivalent volume of vehicle (0.02% ascorbic acid in saline) into the right striatum. For the next 10 days, mice received daily intraperitoneal injections of 100 mg/kg NAC or an equivalent volume of phosphate-buffered saline (PBS). Forebrain sections were immunostained for the dopaminergic terminal marker tyrosine hydroxylase (TH) and scanned on a high-sensitivity Odyssey Imager. (A) 6-OHDA significantly reduced ipsilateral/contralateral striatal TH levels. NAC did not affect this ratio. (B) Overall TH levels in the contralateral and ipsilateral striata. 6-OHDA reduced TH levels on the side ipsilateral to the infusion. NAC raised TH levels in the contralateral striatum of 6-OHDA-infused animals. There was a trend towards a NAC-mediated increase in TH levels in the ipsilateral hemisphere in 6-OHDA-treated animals (p = 0.058). (C) Striatal area was not affected by 6-OHDA or NAC. (D) TH levels were expressed as a function of striatal area, a measurement that reflects average dopaminergic terminal density (TH per unit area). NAC significantly raised TH density in both hemispheres after 6-OHDA infusions. (E) Representative coronal sections through the forebrain, immunolabeled for TH⁺ dopaminergic terminals and scanned on the Odyssey imager. Shown are the mean and SEM of 7–8 mice per group. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 versus 0 μg 6-OHDA; ⁺p ≤ 0.05, ⁺⁺p ≤ 0.01, ⁺⁺⁺p ≤ 0.001 ipsilateral versus contralateral; ^~p ≤ 0.05, ^~~p ≤ 0.01, ^~~~p ≤ 0.001 versus 0 mg/kg NAC; two or three-way ANOVA followed by Bonferroni post hoc correction.

Fig. 2

NAC fails to protect dopaminergic neurons in the substantia nigra from 6-OHDA toxicity. Mice were stereotaxically infused with 4 μg 6-OHDA or an equivalent volume of vehicle (0.02% ascorbic acid in saline) into the right striatum. For the next 10 days, mice received daily intraperitoneal injections of 100 mg/kg NAC or an equivalent volume of phosphate-buffered saline (PBS). Midbrain sections were stained for the dopaminergic terminal marker tyrosine hydroxylase (TH). (A) Overall TH levels in the contralateral and ipsilateral nigrae. The ipsilateral nigra had lower TH levels than the nigra contralateral to the 6-OHDA infusion site. (B) Nigral area was significantly reduced by 6-OHDA in both hemispheres in the absence or presence of NAC. (C) TH levels were expressed as a function of nigral area, a measurement that reflects average TH density (TH per unit area). 6-OHDA raised TH density in the contralateral nigra and NAC did not significantly affect this measure. (D) 6-OHDA significantly reduced ipsilateral/contralateral nigral TH levels. Although 6-OHDA did not cause a significant loss of this measure in NAC-treated animals, NAC did not significantly increase this ratio. (E) Representative coronal sections through the mesencephalon, immunolabeled for TH⁺ cell bodies and scanned on the Odyssey Imager. (F) TH cell counts in the nigra reveal that NAC has no impact on the loss of dopaminergic cells in this structure. Shown are the mean and SEM of 7–8 mice per group. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 versus 0 μg 6-OHDA; ⁺p ≤ 0.05, ⁺⁺p ≤ 0.01, ⁺⁺⁺p ≤ 0.001 ipsilateral versus contralateral; ^~p ≤ 0.05, ^~~p ≤ 0.01, ^~~~p ≤ 0.001 versus 0 mg/kg NAC; two or three-way ANOVA followed by Bonferroni post hoc correction.

Fig. 3

Impact of the retrograde tracer FluoroGold on 6-OHDA toxicity in the striatum. (A) Mice were stereotaxically infused with 6 μg FluoroGold (FG). Seven days later, mice were sacrificed and brain sections viewed under UV illumination on an epifluorescent microscope. The ipsilateral nigrostriatal pathway was retrogradely labeled. cc, Corpus callosum; CPu, caudatoputamen; SNpc, substantia nigra, pars compacta; SNpr, substantia nigra, pars reticulata. (B) In a separate series of experiments, mice were infused with 6 μg FluoroGold or an equivalent volume of phosphate-buffered saline (PBS) into the right striatum. Seven days after FluoroGold infusions, mice were infused with 4 μg 6-OHDA or an equivalent volume of vehicle (0.02% ascorbic acid in saline) in the same striatal location. One week following 6-OHDA infusions, forebrain sections were immunostained for the dopaminergic terminal marker tyrosine hydroxylase (TH) and FluoroGold and scanned on the Odyssey Imager. (b) The impact of 6-OHDA toxicity on overall striatal TH levels was not affected by FluoroGold. (C) FluoroGold slightly reduced striatal area in the hemisphere contralateral to the infusion. 6-OHDA prevented this effect. (D) FluoroGold did not change TH levels per unit area in the striatum of vehicle or 6-OHDA-infused animals. (E) Representative coronal sections through the forebrain, immunolabeled for TH⁺ dopaminergic terminals (green) and FluoroGold (red). Shown are the mean and SEM of 5–6 mice per group. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 versus 0 μg 6-OHDA; ⁺p ≤ 0.05, ⁺⁺p ≤ 0.01, ⁺⁺⁺p ≤ 0.001 ipsilateral versus contralateral; ^~p ≤ 0.05, ^~~p ≤ 0.01, ^~~~p ≤ 0.001 versus 0 μg FG; three-way ANOVA followed by Bonferroni post hoc correction. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Impact of the retrograde tracer FluoroGold on 6-OHDA toxicity in the substantia nigra. Mice were stereotaxically infused with 6 μg FluoroGold (FG) or an equivalent volume of phosphate-buffered saline (PBS) into the right striatum. Seven days later, mice were infused in the same location with 4 μg 6-OHDA or an equivalent volume of vehicle (0.02% ascorbic acid in saline). One week later, brain sections were stained for the dopaminergic terminal marker tyrosine hydroxylase (TH) and FluoroGold. (A) The impact of 6-OHDA on overall TH levels in the nigra was not affected by FluoroGold. (B) FluoroGold slightly reduced nigral area in both hemispheres of 6-OHDA-infused animals. (C) FluoroGold did not change TH levels per unit area in the nigra of vehicle or 6-OHDA-infused animals. (D) Overall FluoroGold levels in the nigra were significantly reduced by 6-OHDA. (E) FluoroGold did not significantly change TH⁺ cell counts in the nigra in vehicle or 6-OHDA-infused animals. (F) The number of FluoroGold⁺ cells of the nigrostriatal pathway was significantly reduced by 6-OHDA. (G) Representative coronal sections through the midbrain, immunolabeled for TH⁺ dopaminergic neurons (green) and FluoroGold⁺ cells (red) and scanned on the Odyssey Imager. (H–I) Microscopic images of FluoroGold⁺ (h) and TH⁺ (i) cells in the ventral midbrain in 6-OHDA/saline and FluoroGold/PBS-infused animals. The arrow points to the lateral nigra, the area most vulnerable to loss in Parkinson's disease. (J) Confocal analysis of FluoroGold and TH dual labeling. Arrows point to some of the cells that contain both FluoroGold and TH. Asterisks overlie FluoroGold-labeled nigrostriatal cells that do not express TH. FluoroGold⁺ cells were viewed under UV illumination and pseudocolored red for presentation. Shown are the mean and SEM of 5–6 mice per group. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 versus 0 μg 6-OHDA; ⁺p ≤ 0.05, ⁺⁺p ≤ 0.01, ⁺⁺⁺p ≤ 0.001 ipsilateral versus contralateral; ^~p ≤ 0.05, ^~~p ≤ 0.01, ^~~~p ≤ 0.001 versus 0 μg FG; three-way ANOVA followed by Bonferroni post hoc correction or two-tailed Student's t-test for panels d and f. IPN, interpeduncular nucleus; ml, medial lemniscus; SNpc, substantia nigra, pars compacta; SNpr, substantia nigra, pars reticulata; VTA, ventral tegmental area. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

NAC fails to protect dopaminergic terminals in the striatum three weeks following 6-OHDA infusions. Mice were stereotaxically infused with 6 μg FluoroGold (FG) into the right striatum. Seven days later, mice were infused in the same location with 4 μg 6-OHDA or an equivalent volume of vehicle (0.02% ascorbic acid in saline). For the next three weeks, mice received daily intraperitoneal injections of 100 mg/kg NAC or an equivalent volume of PBS. Forebrain sections were stained for the dopaminergic terminal marker tyrosine hydroxylase (TH) and FluoroGold (FG). (A) NAC reduced overall TH levels in the striatum contralateral to vehicle infusion and did not protect against 6-OHDA-induced loss of TH. (B) Striatal area was not significantly affected by NAC or 6-OHDA. (C) TH levels were expressed as a function of striatal area, reflecting TH density (TH per unit area). NAC reduced this measure in the contralateral hemisphere of vehicle-infused animals. (D) 6-OHDA significantly reduced ipsilateral/contralateral striatal TH levels and NAC did not impact this measure. (E) Representative coronal sections through the forebrain, immunolabeled for TH⁺ terminals (green) and FluoroGold (red). Shown are the mean and SEM of 5–8 mice per group. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 versus 0 μg 6-OHDA; ⁺p ≤ 0.05, ⁺⁺p ≤ 0.01, ⁺⁺⁺p ≤ 0.001 ipsilateral versus contralateral; ^~p ≤ 0.05, ^~~p ≤ 0.01, ^~~~p ≤ 0.001 versus 0 mg/kg NAC; two or three-way ANOVA followed by Bonferroni post hoc correction. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

NAC fails to protect dopaminergic neurons in the substantia nigra three weeks following 6-OHDA infusions. Mice were stereotaxically infused with 6 μg FluoroGold (FG) into the right striatum. Seven days later, mice were infused in the same location with 4 μg 6-OHDA or an equivalent volume of vehicle (0.02% ascorbic acid in saline). For the next three weeks, mice received daily intraperitoneal injections of 100 mg/kg NAC or an equivalent volume of PBS. Midbrain sections were stained for the dopaminergic terminal marker tyrosine hydroxylase (TH) and FluoroGold (FG). (A) NAC reduced overall TH levels in the nigra in all groups except for the vehicle-infused ipsilateral striatum. (B) Nigral area was increased by infusion of vehicle into the ipsilateral hemisphere and 6-OHDA prevented this effect. NAC had no impact on this measure. (C) TH levels were expressed as a function of nigral area, reflecting TH density (TH per unit area). NAC did not raise TH density in the ipsilateral nigra. (D) 6-OHDA significantly reduced ipsilateral/contralateral nigral TH levels and NAC did not impact this ratio. (E) Overall FluoroGold levels were reduced by 6-OHDA in the ipsilateral nigra. NAC decreased FluoroGold levels in vehicle-infused animals. (F) Representative coronal sections through the mesencephalon, immunolabeled for TH⁺ (green) and FluoroGold⁺ (red) cells. Shown are the mean and SEM of 5–8 mice per group. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 versus 0 μg 6-OHDA; ⁺p ≤ 0.05, ⁺⁺p ≤ 0.01, ⁺⁺⁺p ≤ 0.001 ipsilateral versus contralateral; ^~p ≤ 0.05, ^~~p ≤ 0.01, ^~~~p ≤ 0.001 versus 0 mg/kg NAC; two or three-way ANOVA followed by Bonferroni post hoc correction. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)