Department of Biomedical Sciences, Section of Neuropsychopharmacology, University of Cagliari, Cagliari, Italy
National Institute of Neuroscience (INN), University of Cagliari, Cagliari, Italy
National Research Council (CNR), Neuroscience Institute, Cagliari, Italy
Address correspondence and reprint requests to Prof. Micaela Morelli, Department of Biomedical Sciences, Section of Neuropsychopharmacology, University of Cagliari, Via Ospedale 72, 09124 Cagliari, Italy. E-mail: email@example.com
Epidemiological studies have indicated an inverse association between high uricemia and incidence of Parkinson's disease (PD). To investigate the link between endogenous urate and neurotoxic changes involving the dopaminergic nigrostriatal system, this study evaluated the modifications in the striatal urate levels in two models of PD. To this end, a partial dopaminergic degeneration was induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice, while a severe dopaminergic degeneration was elicited by unilateral medial forebrain bundle infusion of 6-hydroxydopamine (6-OHDA) in rats. Urate levels were measured by in vivo microdialysis at 7 or 14 days from toxin exposure. The results obtained demonstrated higher urate levels in the dopamine-denervated striatum of 6-OHDA-lesioned rats compared with the intact striatum. Moreover, an inverse correlation between urate and dopamine levels was observed in the same area. In contrast, only a trend to significant increase in striatal urate was observed in MPTP-treated mice. These results demonstrate that a damage to the dopaminergic nigrostriatal system elevates the striatal levels of urate, and suggest that this could be an endogenous compensatory mechanism to attenuate dopaminergic neurodegeneration. This finding may be important in light of the epidemiological and preclinical evidences that indicate a link between urate and development of PD.
This study evaluated the in vivo modifications in the striatal urate levels in the mouse MPTP and the rat 6-OHDA models of Parkinson's disease-like dopaminergic nigrostriatal toxicity. The results obtained demonstrated that dopaminergic neurotoxicity is associated with higher urate levels in the striatum, which would strengthen the idea of a link between endogenous urate and Parkinson's disease.
The increase in oxidative stress has long been envisioned as one of the factors that may promote the degeneration of dopaminergic mesencephalic neurons underlying Parkinson's disease (PD) (Schapira and Jenner 2011). In recent years, intensive study has focused on both the discovery of novel antioxidant-based therapies for the management of PD, and the elucidation of the role played by endogenous oxidant and antioxidant species on PD onset and progression. In this regard, evidence has accumulated to suggest that urate might influence the neuropathology of PD (Chen et al. 2009). Urate (the dissociated form of uric acid) is the final product of purine catabolism, and has marked antioxidant properties (Ames et al. 1981), being responsible for most of the antioxidant capacity in human plasma (Yeum et al. 2004).
Both in vitro and in vivo studies have demonstrated that urate has a protective effect in experimental models of PD. PC12 cell survival is extended by urate application when cells are exposed to substances such as dopamine (DA), rotenone, 1-methyl-4-phenylpyridinium (MPP+), or Fe2+ (Jones et al. 2000; Duan et al. 2002), all of which are used in experimental PD models. Uric acid has likewise been shown to elicit long-term protection in a cell culture model of spontaneous midbrain dopaminergic neuronal death (Guerreiro et al. 2009). Furthermore, chronic administration of uric acid affords neuroprotection in the 6-hydroxydopamine (6-OHDA)-lesioned rat model of PD, by attenuating both the decline of dopaminergic neurons in the substantia nigra pars compacta (SNc) and the decrease in DA levels in the striatum, as well as ameliorating the behavioral impairment caused by dopaminergic denervation (Gong et al. 2012). Finally, transgenic mice that do not express the catabolic enzyme urate oxidase, and display brain levels of urate higher than normal, are less susceptible to the neurotoxic effects of 6-OHDA compared with wild-type mice (Chen et al. 2013).
Coherent with preclinical findings, epidemiological studies have indicated the existence of an inverse association between high plasma levels of urate and the risk of developing PD (Davis et al. 1996; De Lau et al. 2005; Weisskopf et al. 2007; Ascherio et al. 2009; Chen et al. 2009). Moreover, a study by Weisskopf et al. (2007) has shown that the inverse association between uricemia and PD described by epidemiological studies was not influenced by a series of habits and lifestyles (e.g. caffeine consumption, smoking) which had been previously suggested to independently modify either PD incidence or uricemia. This finding further corroborates the hypothesis that urate and PD progression may be specifically linked. However, while the results of epidemiological studies are intriguing, they do not clarify whether the inverse association between urate and PD progression is merely due to a pre-existing high uricemia, or the levels of endogenous urate can dynamically change during dopaminergic nigrostriatal degeneration.
Starting from these considerations, this study has evaluated the changes in the extracellular striatal levels of urate by in vivo microdialysis in two different experimental models of PD: the partial dopaminergic degeneration induced by systemic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the mouse, and the severe dopaminergic degeneration elicited by unilateral medial forebrain bundle infusion of 6-OHDA in the rat. This approach was taken to ascertain the possible relationship between dopaminergic degeneration and changes in brain urate levels.
Materials and methods
Male C57BL/6J mice (28–30 g) and Sprague–Dawley rats (275–300 g) were used. Animals were purchased from Charles River (Calco, Italy), and maintained at constant temperature (21 ± 1°C) and humidity (60%) under a 12 h light–dark cycle, with free access to food and water, except during the microdialysis experiments. Experimental procedures were approved by the Ethical Committee of the University of Cagliari, in compliance with the Italian guidelines for the care and use of experimental animals (DL 116/92) and the European Communities Council Directive (2010/63/EEC). Efforts were made to minimize the number of animals used and maximize humane treatment.
Drugs were obtained from the following sources: Santa Cruz Biotechnology, Santa Cruz, CA, USA (MPTP hydrochloride); Sigma-Aldrich, Milan, Italy (desipramine and 6-OHDA hydrochloride); Carlo Erba, Milan, Italy (chloral hydrate and sodium pentobarbital). MPTP, sodium pentobarbital, chloral hydrate, and desipramine were dissolved in distilled water, while 6-OHDA was dissolved in saline containing 0.05% ascorbic acid. MPTP and sodium pentobarbital were administered to mice i.p. in a volume of 10 mL/kg. Desipramine and chloral hydrate were administered to rats i.p. in a volume of 3 mL/kg. Distilled water was administered to vehicle-treated mice.
Dopaminergic neurodegeneration was induced in mice by the administration of MPTP (20 mg/kg) on four consecutive days as described by Costa et al. (2013), and in rats by the acute intracranial infusion of 6-OHDA (4 μg/μL, for a total of 4 μL) in the medial forebrain bundle as described by Frau et al. (2013). 6-OHDA infusion was preceded by the administration of desipramine (10 mg/kg, 30 min before), to prevent damage to the noradrenergic neurons. Extracellular DA and urate were then sampled by in vivo microdialysis at 7 days from MPTP administration in mice, and at 14 days from 6-OHDA infusion in rats. The protocols of dopaminergic degeneration used in this study have been previously shown to cause a decline in dopaminergic mesencephalic neurons of approximately 30% for MPTP and 95% for 6-OHDA (Deumens et al. 2002; Costa et al. 2013).
Microdialysis, sample collection, and histology
Microdialysis probes with a dialyzing portion of 2 mm were prepared with AN69 fibers (Hospal Dasco, Bologna, Italy). Mice were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and implanted unilaterally in the striatum (A = −0.8, L = +1.6 from bregma, V = −3.7 from dura) according to the mouse brain atlas of Paxinos and Franklin (2008). Rats were anesthetized with chloral hydrate (300 mg/kg, i.p.) and implanted bilaterally in the striatum (A = −0.6, L = +1.5 from bregma, V = −4.5 from dura), according to the rat brain atlas of Paxinos and Watson (1998). The day after surgery, the probes were connected to an infusion pump and perfused with Ringer solution (147 mM NaCl, 4 mM KCl, 2.2 mM CaCl2) at a constant rate of 1 μL/min. The first hour of perfusate was discarded and DA samples collected thereafter. Following that, the perfusion rate was increased to 2 μL/min and samples collected for urate levels. At the end of the microdialysis experiments, the animals were transcardially perfused with saline and 4% formaldehyde in phosphate-buffered saline (pH = 7.4). Afterwards, the probes were removed and the brains extracted and cut on a vibratome in serial coronal slices to identify the probe location, based on either the mouse or rat brain atlas. Sections from the SNc were processed for tyrosine hydroxylase (TH) immunohistochemistry and the number of TH-positive mesencephalic neurons was quantified by stereological counting according to the procedure reported by Costa et al. (2013).
Dopamine and urate detection
For DA detection, dialysate samples (20 μL) were taken every 20 min and injected without any purification into a high-performance liquid chromatography system equipped with a reverse-phase column (LC-18 DB; 15 cm, 5 μm particle size, Supelco, Bellefonte, PA, USA) and a coulometric detector (Coulochem II; ESA, Bedford, MA, USA). The first electrode of the detector was set at +130 mV (oxidation) and the second at −175 mV (reduction). The composition of the mobile phase was as follows: 50 mM NaH2PO4, 0.1 mM Na2-EDTA, 0.5 mM N-octyl sodium sulfate, 15% (v/v) methanol, pH 5.5. The sensitivity of the assay for DA was 5 fmol per sample.
For urate detection, dialysate samples (60 μL) were taken every 30 min and immediately kept at −80°C until processed for lyophilization, to avoid urate degradation over time. Lyophilized samples were kept at −20°C until the gas chromatography–mass spectrometry (GC/MS) analysis. Before GC/MS analysis, uric acid was derivatized as described by Jenner et al. (1998), and diluted 1 : 10 with hexane. Five microliters from each sample were injected into a GC/MS system and analyzed using a Trace DSQ Thermo Electron Corporation (Waltham, MA, USA) mass spectrometer coupled to a trace gas chromatograph Ultra (Waltham, MA, USA). A thin-film capillary methylpolysiloxane column VB-5 (VICI Gig Harbor Group Inc., Gig Harbor, WA, USA: length, 30 m; i.d., 0.25 mm; film thickness, 0.25 μm) was used to resolve the chromatography. The oven temperature was raised from 60 to 230°C at 30°C/min, 230 to 250°C at 1°C/min, and 250 to 320°C at 30°C/min. The injector and transfer line temperatures were maintained at 300 and 310°C, respectively. Derivatives were first qualitatively analyzed by full scanning in the mass range of 250–950. For quantification, the mass spectrometer was operated in the selected ion-monitoring mode. The temperatures of mass spectrometer source and quadrupole were set at 200°C and 100°C, respectively. For each animal, four to six consecutive dialysate samples of either DA or urate were analyzed, and the values obtained were averaged thereafter for each animal.
Values are reported as mean ± SEM of the levels of DA (fmol) or urate (μmol), and of the percentage of TH-positive neurons in the SNc. In mice, data obtained in MPTP-treated animals were compared with data collected in vehicle-treated animals. In rats, data obtained from the striatum and SNc corresponding to the side of 6-OHDA infusion were compared with data collected in the contralateral intact striata and SNc of the same animals. The presence of significant differences between groups was assessed by Student's t-test. Moreover, Pearson's test was applied to the data obtained from each individual 6-OHDA-lesioned rat to ascertain whether the levels of urate measured in the DA-denervated striatum correlated with those of DA in the same area.
In mice, administration of MPTP (4 × 20 mg/kg, i.p.) caused a significant reduction in both the percentage of TH-positive nigral neurons and striatal DA levels, as measured by in vivo microdialysis, compared with vehicle administration. Student's t-test revealed that these effects were statistically significant (neurons: t =19.7, df=11, p <0.01; DA levels: t =2.45, df=11, p =0.044, Fig. 1a and b). Striatal levels of urate measured by microdialysis in MPTP-treated mice (1.29 ± 0.19 μM) displayed a trend toward an increase compared with vehicle-treated mice (0.89 ± 0.13 μM), but Student's t-test revealed no significant differences in this effect (t =1.48, df =11, p =0.17, Fig. 1c).
In rats, unilateral 6-OHDA infusion in the medial forebrain bundle produced a marked reduction in both the percentage of TH-positive nigral neurons and striatal DA levels, as measured by in vivo microdialysis, in the side of the brain ipsilateral to toxin infusion, compared with the intact non-infused side. Student's t-test showed that these effects were statistically significant (neurons: t =39.2, df=14, p <0.01; DA levels: t =3.74, df=14, p =0.002; Fig. 1d and e). In the same rats, the striatal levels of urate measured by microdialysis were found to be significantly higher in the DA-denervated striatum (2.03 ± 0.19 μM) than in the intact striatum (1.36 ± 0.23 μM), as indicated by Student's t-test (t =2.19, df=14, p =0.04; Fig. 1f). Finally, Pearson's test revealed a significant inverse correlation between the levels of urate and DA in the DA-denervated striatum (r = −0.89; p = 0.02).
The possibility that endogenous urate may protect the integrity of the nigrostriatal dopaminergic system has been suggested by epidemiological studies that have indicated the existence of an inverse association between high uricemia and incidence of PD (Davis et al. 1996; De Lau et al. 2005; Weisskopf et al. 2007; Ascherio et al. 2009; Chen et al. 2009). In line with this, previous preclinical findings have demonstrated a crucial role for purinergic signaling in neurotoxicity and neuroprotection phenomena (Gomes et al. 2011). By demonstrating an elevation in the striatal levels of urate in two experimental models of PD, this study extends previous evidence obtained in post-mortem striatal samples of 6-OHDA-treated mice (Chen et al. 2013) and provides the first in vivo evidence that DA neuron degeneration may be associated with a perturbation in brain levels of urate.
The present study employed two distinct experimental models of PD in order to attain different extents of dopaminergic denervation, and observed a significant increase in the in vivo urate levels in 6-OHDA-lesioned rats, but only a trend toward this effect in MPTP-treated mice. While the regimen of MPTP administration used here is known to cause only a partial reduction in nigral dopaminergic neurons and striatal DA (Costa et al. 2013), 6-OHDA causes a robust drop in both dopaminergic neurons and DA levels, indicative of severe damage to the nigrostriatal system (Deumens et al. 2002). Therefore, it can be hypothesized that severe insults targeting the nigrostriatal dopaminergic system may be associated with higher increases in the levels of striatal urate. Supporting this view is also the finding that the levels of urate measured in the DA-denervated striatum of 6-OHDA-lesioned rats were negatively correlated with the levels of DA measured in the same area. The changes in the striatal levels of urate observed in 6-OHDA-lesioned rats could be explained considering both the mechanism that underlies the effects of this neurotoxin, which are chiefly due to the elevation of oxidative stress (Simola et al. 2007), and the marked antioxidant properties of urate (Ames et al. 1981). However, it has to be acknowledged that the increase in oxidative stress is also one of the mechanisms by which MPTP elicits its neurotoxic effects (Dauer and Przedborski 2003); hence, a significant elevation in urate levels would have been expected in MPTP-treated mice as well. Nevertheless, it is feasible that persistent changes in the striatal levels of urate could occur in mice exposed to MPTP at higher doses and/or for a longer time.
Based on the preclinical and epidemiological evidences that suggest a protective role of urate on the dopaminergic system, one possible interpretation of the results of this study could be that urate levels increase to attenuate dopaminergic neurotoxicity. In this regard, it is, however, important to mention that the striatal concentrations of urate detected in our study (about 1.29 μM in mice and 2.03 μM in rats) are lower than those known to elicit neuroprotection in in vitro studies, that are 50 μM or higher (see Gong et al. 2012). Nevertheless, it is also noteworthy that urate was here sampled after the dopaminergic degeneration had already considerably progressed (i.e. 7 days from MPTP in mice and 14 days from 6-OHDA in rats). Furthermore, it has to be considered that the experimental models of PD used here, while reproducing the degeneration of dopaminergic neurons, do not mimic the temporal features of the neuronal demise underlying human PD, which is slow and progressive as it takes place over several years. Thus, the increase in urate levels attained here might be not sufficient to afford neuroprotection in both the MPTP and 6-OHDA models, because of the very rapid dopaminergic neurodegeneration. Therefore, while the hypothesis that endogenous urate may protect the dopaminergic system from degenerating has to be conclusively demonstrated, the present results are nevertheless of great interest. In fact, this study demonstrates that the exposure to insults directed toward the dopaminergic system is associated with changes in the striatal levels of endogenous urate, substantiating the idea of a possible role of this antioxidant in the pathophysiological changes underlying PD. It could be hypothesized that neuroprotection by endogenous urate may be evident only when the brain is exposed to noxious insults of milder intensity protracted over time, similar to the condition thought to underlie neurotoxicity in human PD.
In light of the proposed neuroprotective potential of urate, manipulation of the cerebral levels of this antioxidant molecule may represent a new promising pharmacological strategy and a major step forward in the management of PD. In fact, this approach would tackle the underlying cause of disease, namely neuronal degeneration, and not just provide symptomatic relief, like the pharmacological therapies currently available do. Notably, a very recent clinical trial has demonstrated the feasibility of manipulating urate levels in PD patients, by showing that the administration of the metabolic precursor inosine resulted in the elevation of urate concentrations in plasma and cerebrospinal fluid, and that this practice was associated with negligible adverse effects (Parkinson Study Group SURE-PD Investigators 2014). Moreover, preliminary observations performed during the same trial have suggested the possibility that treatment with inosine may be associated with disease-modifying effects, although at this stage more refined clinical studies appear mandatory to substantiate this hypothesis (Parkinson Study Group SURE-PD Investigators 2014).
In conclusion, by demonstrating for the first time that the in vivo striatal levels of urate are increased in experimental models of PD, this study strengthens the recent idea of a link between urate and PD progression suggested by epidemiological evidence. Hence, the present results would suggest that elevation in urate levels could represent a potential compensatory endogenous mechanism to attenuate neurotoxic damage of the dopaminergic nigrostriatal system. Moreover, this study may represent a starting point for future investigations aimed at elucidating the specific factors, such as type, severity, and duration of the neurotoxic insults, and molecular mechanisms that could promote the modifications of urate levels in conditions of dopaminergic nigrostriatal degeneration.
Acknowledgments and conflict of interest disclosure
This study was supported by funds from the Regione Autonoma della Sardegna (Legge Regionale 7 Agosto 2007, N.7, annualità 2010) and the University of Valencia (UV-INV_PRECOMP13-115500). Dr Nicola Simola gratefully acknowledges the Sardinian Regional Government for financial support (P.O.R. Sardegna F.S.E. Operational Programme of the Autonomous Region of Sardinia, European Social Fund 2007–2013–Axis IV Human Resources, Objective l.3, Line of Activity l.3.1 ‘Avviso di chiamata per il finanziamento di Assegni di Ricerca’). The authors are grateful to Dr Annalisa Pinna and Dr Giulia Costa for their help with TH immunohistochemistry, and to Dr Francesca Marongiu for the lyophilization of microdialysis samples. The authors declare that there are no conflicts of interest, and acknowledge any funding bodies used to support the authors' own work.
All experiments were conducted in compliance with the ARRIVE guidelines.