Pomegranate juice exacerbates oxidative stress and nigrostriatal degeneration in Parkinson's disease

Abstract

Numerous factors contribute to the death of substantia nigra (SN) dopamine (DA) neurons in Parkinson's disease (PD). Compelling evidence implicates mitochondrial deficiency, oxidative stress, and inflammation as important pathogenic factors in PD. Chronic exposure of rats to rotenone causes a PD-like syndrome, in part by causing oxidative damage and inflammation in substantia nigra. Pomegranate juice (PJ) has the greatest composite antioxidant potency index among beverages, and it has been demonstrated to have protective effects in a transgenic model of Alzheimer's disease. The present study was designed to examine the potential neuroprotective effects of PJ in the rotenone model of PD. Oral administration of PJ did not mitigate or prevent experimental PD but instead increased nigrostriatal terminal depletion, DA neuron loss, the inflammatory response, and caspase activation, thereby heightening neurodegeneration. The mechanisms underlying this effect are uncertain, but the finding that PJ per se enhanced nitrotyrosine, inducible nitric oxide synthase, and activated caspase-3 expression in nigral DA neurons is consistent with its potential pro-oxidant activity.

Keywords: Inflammation; Mitochondria; Neuroprotection; Neurotoxicity; Oxidative stress; Parkinson's disease; Polyphenols; Pomegranate juice; Rotenone.

Figure 1

Dietary treatment with PJ does not attenuate weight loss or prevent development of a parkinsonian phenotype in the rotenone model. (A) The percent change in body mass, relative to mass at the onset of treatment (day 0) with rotenone (3.0 mg/kg/day), PJ (~7 mL/day) or vehicle. Note that the percent mass change is relatively similar for both groups (VEH+ROT and PJ+ROT) at all-time points. (B) Survival curves following rotenone administration. Rats were euthanized when they developed a debilitating PD phenotype, as characterized by severe bradykinesia, rigidity, and postural instability. No difference in overall survival was observed between the VEH+ROT group (empty squares) and the PJ+ROT group (filled circles).

Figure 2

Neurobehavioral assessment. (A) After 5 days of rotenone treatment, rearing activity was observed over a 5 min period and scored for each group. *** p < 0.001 compared to VEH+VEH, Newman-Keuls post-hoc test; ### p < 0.001 vs PJ+VEH; + p < 0.05 vs VEH+ROT. (B) Postural instability at baseline, prior to administration of rotenone, and 5 days after rotenone administration (two way-ANOVA, ** p < 0.01 vs VEH+VEH; ## p < 0.01 vs PJ+VEH). For all tests, n = 6 for control and n = 11 for rotenone-treated.

Figure 3

Striatal catecholamine levels and turnover. DA (A), DOPAC (B), HVA (C), and DA turnover (DOPAC+HVA)/DA) (D). Rotenone induced a decrease in DA levels and turnover. Supplementation with PJ had no effect either alone or after neurotoxin exposure. Data are means ± S.E.M. ** p < 0.01, * p < 0.05 compared to VEH+VEH, Newman-Keuls post-hoc test; # p < 0.05 compared to PJ+VEH.

Figure 4

Staining for tyrosine hydroxylase fiber loss in the striatum. Representative images of striatal TH immunohistochemistry from two animals in each group are shown. Rotenone led to degeneration of the DA terminals in the striatum, as evidenced by a focal loss of TH immunopositive fibers in anterior and posterior striatal sections (I-L) compared to control animals (A-D). Oral supplementation with PJ alone did not have any effect (E-H), but when administered together with rotenone, induced an overall decrease in striatal TH immunoreactivity (M-P). Green circles denote the area of the lesion. Note that the lesions are strikingly similar in magnitude and localization across animals. Scale bar = 500 µm. Representative immunofluorescence-stained sections were depicted (Q) and quantified (R) with values representing average striatal intensity from three to five sections per animal. ** p < 0.01 vs VEH+VEH, Newman-Keuls post-hoc test; * p < 0.05 vs VEH+ROT.

Figure 5

Immunostaining of tyrosine hydroxylase-positive neurons in the substantia nigra. Examination at low (scale bar = 500 µm) and high magnification (scale bar = 50 µm) of the dorsolateral nigra revealed a dense TH-immunopositive network of cell bodies and fibers in the SN in controls alone (A-D) or with PJ (E-H). After the rotenone injections, there was a reduction in TH immunoreactivity at the level of the SN (I-L). Oral administration of PJ with rotenone caused an increase in DA cell loss and pruning of processes compared to treatment with rotenone alone (M-P). The loss of SN neurons was counted by unbiased stereology, and rats treated with PJ showed potentiation of rotenone-induced neurotoxicity (Q). Bars represent means ± S.E.M. Statistical analyses were carried out using the Newman Keuls post-hoc test. *** p < 0.001 vs VEH+VEH; * p < 0.05 vs VEH+ROT.

Figure 6

Representative 100x confocal images for NT and 4-HNE immunostaining in SN sections. NT and 4-HNE were weakly expressed in controls (B and C). Increased expression of NT was noticed after oral treatment of PJ (F). Rotenone administration strongly enhanced NT immunoreactivity (J), which was further augmented when PJ was co-administered with the neurotoxin (N). A similar rotenone effect was observed for 4-HNE expression; however, PJ+ROT-treated animals did not exhibit significant differences relative to the rotenone-treated group (O vs K). White: TH⁺; red: NT; green: 4-HNE. Quantification of the percentage of fluorescence intensity for NT levels (Q) and 4-HNE (R) was assessed, with data representing average fluorescence immunoreactivity from 5 SN sections per animal (200–300 neurons per animal). Each treatment group was comprised of 4 rats. Scale bar = 20 µm. *** p < 0.001, ** p < 0.01, * p < 0.05 compared to VEH+VEH or VEH+ROT, Newman-Keuls post-hoc test.

Figure 7

Microscopy images of the immunohistochemistry for activated microglia. Immunofluorescence images of the entire SN were acquired. Untreated animals revealed a minimum expression of Iba1 (A and inset B) and oral administration of PJ did not alter Iba1 immunoreactivity (C and inset D). However, under pathological conditions, chronic rotenone treatment induced significant alterations in the morphology of nigral Iba1 positive cells (E and inset F). Iba1 expression remains unaltered after ROT+PJ treatment (G and inset H) relative to the rotenone-treated group. Quantitative analysis of Iba1 was carried out in 5 nigral sections and data is provided for “morphological activation” (I), number of Iba1 cells (J), and total area occupied by the microglial particles (K). Rotenone-induced toxicity caused a significant increase in the average size of Iba1 cells, but no changes were appreciated in the total number of cells. Oral administration of PJ did not induce microglial activation either by itself or combined with rotenone. Green: TH⁺; red: Iba-1. Scale bar: 50 µm. The histogram values represent the mean ± S.E.M. Significant differences between groups were determined by Newman-Keuls post-hoc test. *** p < 0.001, ** p < 0.01 vs VEH+VEH. ### p < 0.001, ## p < 0.01, # p < 0.05 vs PJ+VEH.

Figure 8

Nigral proinflammatory mediators. Confocal images at 100x revealed an absence of iNOS expression in the vehicle group (A2) but oral administration of PJ significantly increased iNOS immunoreactivity (A5). Following rotenone injection, a marked expression of iNOS was detected (A8) and this effect was even greater when PJ was co-administered with rotenone (A11). Quantification of fluorescence levels of iNOS was expressed as the fold increased versus untreated controls (A13) and is representative of the average of 200–300 DA neurons corresponding to 5 SN sections per animal. Results are expressed as the mean ± S.E.M. of 4 rats per group. Scale bar = 20 µm. *** p < 0.001, * p < 0.05 vs VEH+VEH, Newman-Keuls posthoc test; ** p < 0.01 vs VEH+ROT. Representative western blots of different proinflammatory markers in SN brain homogenates (B and C). Densitometric analysis disclosed that the amount of protein for the enzyme COX-2 (~ 74 kDa), the cytokine IL-1β (~ 17 kDa), and TNF-α (~ 17 kDa) remained unchanged (B2, B3, and C3, respectively). Nevertheless, NF-kB p65 (~ 65 kDa) expression levels were significantly enhanced after dietary supplementation with PJ (C2). The histogram values represent the mean ± S.E.M in arbitrary units of the optical density of 4 independent measurements in triplicate (A-C are biological replicates). β-Actin was used as a reference control. Significant differences between groups were determined by one-tailed Student’s t-test. ** p < 0.01 and * p < 0.05 compared to rotenone.

Figure 9

Caspase-3 activation. Ventral midbrain tissue lysate was subjected to western blot with antibodies against procaspase-3 and its active form cleaved caspase-3 (illustrated in A). Semi-quantitative values of activated caspase-3 were determined by optical density measurements on western blot autoradiograms (B). Mean values of band densities and their corresponding S.E.M. are obtained from four different animals per group and normalized to percentages of control (VEH+VEH). Significant differences between groups were determined by Newman-Keuls post-hoc test. *** p < 0.001, ** p < 0.01, and * p < 0.05 compared to VEH+VEH. ^## p < 0.01 compared to PJ+VEH.