Hydroxychloroquine attenuated motor impairment and oxidative stress in a rat 6-hydroxydopamine model of Parkinson’s disease

Abstract Purpose Parkinson’s disease (PD) is associated with the destruction of dopaminergic neurons in the substantia nigra (SN). Hydroxychloroquine (HCQ) has the capability to cross the blood–brain barrier and promote a neuroprotective potential. This study evaluated the effects of HCQ on the 6-hydroxydopamine (6-OHDA)-induced PD model in rats. Methods Wistar rats were randomly divided into sham, PD, PD + levodopa and PD + HCQ groups. The PD model was induced by a stereotactic administration of 6-OHDA into the left SN pars compacta (SNpc) and confirmed by rotation and the Murprogo’s tests. HCQ (100 mg/kg, p.o.) and levodopa (12 mg/kg, p.o.) were administered once a day for 21 days. Three weeks after surgery, the behavioral tests were performed. Brain lipid peroxidation index (MDA), glutathione peroxidase activity (GPx), total antioxidant capacity (TAC) levels and α-synuclein protein expression in the SN were also measured. Results The behavioral tests demonstrated that induction of PD increased the muscle rigidity and the number of rotations, which were reversed by HCQ treatment. Also, induction of PD was associated with an increase in α-synuclein protein levels and MDA and decreased TAC levels and GPx activity. However, HCQ decreased α-synuclein and MDA levels while increased TAC levels and GPx activity. In addition, histopathological data showed that HCQ protects dopaminergic neurons against 6-OHDA-induced toxicity. Conclusion According to the results, HCQ has a beneficial effect in improving PD-related pathophysiology, in part, by mitigating oxidative stress and protecting the dopaminergic neurons in the SN.


Introduction
Neurological diseases are the main cause of disability and premature death among aged people worldwide, owing to the gradual destruction of neurons and age-associated motor and cognitive impairments [1].Parkinson's disease (PD) is a neurodegenerative disease, which is characterized by accumulation of α-synuclein (called lewy Bodies) and progressive loss of dopaminergic neurons in the midbrain [2]. it is estimated that 60% of dopaminergic neurons are destroyed at the beginning of the disease preceding diagnosis [2].
6-hydroxydopamine (6-OhDa), a hydroxylated analog of dopamine, is easily absorbed by dopaminergic neurons and oxidized by reactive oxygen species (ROs).it induces neurotoxicity by inhibiting the mitochondrial electron transport chain and provoking oxidative stress [3].6-OhDa lesion is widely used to test new treatment strategies in PD [4].6-OhDa administration into the substantia nigra pars compacta (sNpc) or the striatum results in specific biochemical and motor dysfunctions, which mimic those of human PD [5].
current available PD treatments include dopamine analogs, dopamine-degrading enzyme inhibitors and deep brain stimulation, all aimed at improving secondary motor symptoms, but no solution has been reported to stop progressive neuronal death [6].Management of moderate to severe stages of PD is challenging because in the later stages of the disease, response to levodopa is progressively declined, and some levodopa-resistant symptoms, such as falling and freezing, dysarthria, dysphagia, and dementia are elicited [2].hydroxychloroquine (hcQ), a chloroquine hydroxyl-derivative, crosses the blood-brain barrier [7] and exer ts anti-malarial, anti-rheumatic, anti-inflammatory and neuroprotective effects [8].Kim et al. [9] reported that some anti-malarial drugs, including chloroquine, improved motor disturbances in 6-OhDa-lesioned rats.evidence also shows that hcQ alleviates neurological symptoms in patients with sarcoidosis [10].Recently, hedya et al. [8] demonstrated that hcQ ameliorated motor deficits induced in an animal model of PD induced by rotenone through suppression of striatal inflammatory cytokines, restoring tyrosine hydroxylase (th) levels, GsK-3β inhibition and upregulation of the orphan nuclear receptor mRNa expression.
hence, this study investigated the effects of hcQ administration on motor function, oxidative stress markers and histopathological changes in a rat 6-OhDa model of PD.

Animals and experimental design
Forty-eight adult male Wistar rats, weighing 250 ± 20 g, were used in this study.animals were obtained from Pasteur institute (tehran, iran) and housed 4 rats per cage under a standard laboratory condition (12-h light/ dark cycles starting at 8:00 h, room temperature 23 ± 2 °c). the diet and water were freely provided.all the experimental procedures were carried out in accordance with the guidelines of ethical committee of tabriz university of Medical sciences for care and use of laboratory animals (iR.tBZMeD.VcR.Rec.1400.024),and all efforts were made to decrease the number of animals used and minimize the suffering of rats during the study.
Following a week of adaptation to the new conditions, the animals were randomly divided into four groups (n = 12) as follows: sham, PD, PD + levodopa and PD + hcQ groups.animals in the sham group underwent stereotaxic surgery, and the vehicle of 6-OhDa was injected into the sNpc, and then treated with normal saline (2 ml/kg, p.o.) for 21 days.animals in the PD, PD + levodopa and PD + hcQ received unilateral intra sNpc injection of 6-OhDa.three weeks following neurotoxin administration, the model was confirmed by behavioral tests.animals with rotation more than 7 times/min were considered as an established PD model [11].then, the PD animals were treated with normal saline (2 ml/kg, p.o.), levodopa (12 mg/kg, p.o., twice daily) [12] and hcQ (100 mg/kg, p.o.) [8], for 21 consecutive days, respectively.

PD model
animals were anesthetized by injection of ketamine (80 mg/kg, i.p.) and xylazine (8 mg/kg, i.p.), and fixed in the stereotaxic frame symmetrically.6-OhDa toxin (12.5 µg in 5 µl of a solution containing % 0.2 ascorbic acid) was injected into the left sNpc according to the corresponding area in Paxinos atlas coordination (bregma: anteroposterior (aP) = -5.5 mm, Dorsoventral (DV) = -7.3mm, Mediolateral (Ml) = -2.6 mm) [13]. in the sham group, the same volume of normal saline containing ascorbic acid was injected.Five minutes after the solution infusion (1 µl/min), the needle was removed.Vehicle and toxin were administered slowly at a rate of 1 µl/min using a hamilton syringe.

Behavioral tests
the behavioral tests, including the Murprogo's test and apomorphine-induced rotational behavior, were carried out 21 days after 6-OhDa injection, for confirmation of the model, and at the end of the treatments, by a blinded experimenter.

Rotational behavior
apomorphine-induced rotation is a common test for assessment of the extent of motor impairment induced by 6-OhDa. in this study, all animals were tested for rotational behavior 21 days after 6-OhDa injection (before treatment) and 21 days after lesion (after treatment).animals were individually positioned in a circular cage, and after at least 10 min of habituation, they received a single injection of apomorphine hydrochloride (0.5 mg/kg, s.c.).Following one min of injection, full numbers of rotations were counted for 30 min at 10-min intervals.the number of contralateral turns to the lesion side was considered as positive scores for apomorphine, and the mean rotational numbers were calculated by the positive scores minus the negative scores [14].

Murprogo's test
to study muscle stiffness, the animals were individually placed on a flat surface.the animal received a score of zero if the animal's standing and walking were normal.if the animal remained immobile due to muscle stiffness or had difficulty in moving the arms and legs, it received a score of 0.5.then the right hand of the animals was placed on a wooden platform with 3 cm height; if the animal held its hand on the platform for at least 10 s, it received a score of 0.5.this step was repeated for the opposite hand, and if the hand was held steady, they received another 0.5 points.then, the animal's right hand was placed on a 9 cm platform, and they were given a score of 1 if they held their hand for 10 s. this step was repeated for the opposite hand, and if the hand was held steady after 10 s, they received another 1 point.the PD animal received a score of 3.5, indicating full rigidity, and the healthy animal (the sham group) received a score of zero [15].

Tissue collection
at the end of the behavioral tests, all rats were anesthetized by a mixture of ketamine (100 mg/kg, i.p) and xylazine (10 mg/kg, i.p) and sacrificed by decapitation.the whole brain was extracted from the skull carefully.For biochemical assessments and Western blotting, brain samples were sliced using a tissue chopper, and the sN was dissected on a cold plate and stored in a -80 °c freezer.For histological examination, the brain was immersed in 4% buffered paraformaldehyde fixation solution for 48 h.

Assessment of oxidative stress status
Brain samples were homogenized in ice-cold 1.15% Kcl solution and then centrifuged at 12,000 rpm for 15 min at 4 °c to obtain the supernatant.the Bradford method was used to estimate protein concentration in the supernatant.

Malondialdehyde levels
thiobarbituric acid reaction (tBaR) colorimetric assay was used to evaluate the concentration of malondialdehyde (MDa), as a lipid peroxidation marker.Briefly, 200 μl of the supernatant was mixed with 1 ml of thiobarbituric acid, trichloroacetic acid and 0.9 ml of tris-hcl (ph 7.4) and incubated for 20 min at 100 °c.Following centrifugation of the samples at 3,000 rpm for 10 min, optical density of the supernatant was read at 540 nm in a plate reader, and the results were presented as nmol/mg protein [16].

Glutathione peroxidase
enzyme activity of glutathione peroxidase (GPx) was assessed spectrophotometrically using the RaNsel kit (Randox laboratories ltd.) based on the method of Paglia and Valentine.the absorbance was read at 340 nm at 37 °c, and the results were presented as nmol/mg protein.

Total antioxidant capacity levels
to determine brain total antioxidant capacity (tac) levels, the 2′-azino-bis (3-ethylbenzothiazoline-6 -sulfonic acid) (aBts) method was applied using a Randox total antioxidant status kit (Randox laboratories ltd., crumlin, united Kingdom).a spectrophotometer was used to measure the absorbance at 600 nm, and the results were expressed as µmol/mg protein.

Western blotting
sN samples were homogenized (1:10, wt:vol) in 20 mM tris-hcl (ph 7.4) buffer containing 1 mM NaF, 150 mM Nacl, 1% triton-100, and protease inhibitor cocktail.the homogenate was centrifuged at 12,000g for 10 min at 4 °c to obtain supernatant.Protein concentrations were determined in the supernatant using the Bradford method.an equal amount of protein (40 μg) was subjected to electrophoresis on 12.5% sodium Dodecyl sulfate (sDs)-polyacrylamide gel and then transferred onto a Polyvinylidene Difluoride (PVDF) (Roche, uK) membrane.Membranes were blocked in 5% dried milk in 15 mM tris-hcl (ph 7.4), 150 mM Nacl, and 0.05% tween 20 (tBst) for 1 h.subsequently, the membranes were incubated with the primary antibodies (santa cruz, ca, usa) against α-synuclein (sc-12767) and β-actin (sc-47778) as an internal loading control overnight at 4 °c.after washing three times with tBst, the membranes were incubated with horseradish peroxidase (hRP)-conjugated goat anti-rabbit igG secondary antibody (1:5,000, sc-2004) for 2 h.Following washing the membranes with PBs, the membranes were placed in enhanced chemiluminescence (ecl) detection solution (amersham, uK) and exposed to X-ray film to visualize the signals.the density of protein bands was evaluated by image J software [17].

Histological examination
the blocks of sN were embedded in paraffin, and slides were prepared by dehydration in ascending alcohol series and then cleared in xylene.subsequently, coronal sections (40 μm) were serially collected (-4.52 mm to -6.04 mm from bregma) from midbrains [18] using a microtome and stained with 0.1% cresyl violet.the representative images of the left hemisphere sNpc were captured, and the numbers of Nissl-stained neurons of sNpc were counted manually under light microscopy (magnification ×200) for three equivalent sections per brain by a blinded person to the study groups.

Statistical analysis
GraphPad Prism 6.01 (GraphPad software inc., la Jolla, ca, usa) was used to analyze the data.Values were presented as mean ± seM.One-way aNOVa followed by tukey post hoc test was performed for comparison of the differences among the groups.the criterion for statistical significance was set at a p value <0.05.

Rotational behaviors with apomorphine
there was no significant difference in the number of rotations before the induction of the PD among groups.the mean number of rotations was significantly increased in the normal saline-received PD group compared to the sham animals (p < 0.001).Besides, treatment with levodopa and hcQ markedly reduced the number of rotations compared to the PD animal treating with normal saline (Figure 1(B), p < 0.001 for both).interestingly, the average number of rotations in the PD + hcQ group was significantly lower than the PD + levodopa group (p < 0.05).

Muscle stiffness
the result of this test indicated that PD induction significantly increased muscle rigidity in the 6-OhDalesioned groups compared to the sham group.however, rigidity was markedly decreased following treatment with levodopa and hcQ compared to the PD animals (Figure 2(B), p < 0.001 for both).there was no significant difference between the PD + levodopa and the PD + hcQ groups regarding the mean scores obtained from the Murprogo's test after treatment.

Brain MDA levels and antioxidant status
We found that 6-OhDa injection in the PD groups significantly increased brain MDa production compared to the sham group (Figure 3(a), p < 0.001).however, administration of levodopa or hcQ had no significant effect on the brain MDa levels in the PD animals (p > 0.05).
Moreover, PD animals demonstrated lower GPx enzyme ((Figure 3(B)) activity and brain tac ((Figure 3(c)) than the sham group (p < 0.001 for all comparisons).On the other hand, levodopa (p < 0.001) and hcQ (p < 0.05) significantly increased GPx activity in the PD animals.Nevertheless, levodopa or hcQ had no significant effects on the brain tac levels compared to the PD group (p > 0.05).

Levels of α-synuclein in the SNpc
Western blot analysis demonstrated a significant increase in levels of α-synuclein in the sNpc of the PD groups compared to the sham group (Figure 4, p < 0.001 for all comparisons).however, protein levels significantly decreased in the PD + levodopa group compared to the PD group (p < 0.001).also, animals in the PD + hcQ showed lower protein levels of α-synuclein compared to the PD group (p < 0.001).On the other hand, the PD + hcQ group showed a significant reduction in protein levels of α-synuclein compared to the PD + levodopa group (p < 0.001).

Number of neurons in the SNpc
the results of histological examination (table 1 and Figure 5) revealed no significant difference in the number of dopaminergic neurons on the right and left sides of sN in the sham group.however, the PD group had lower numbers of Nissl-stained neurons in the left side of sN than the sham group (p < 0.001).Meanwhile, treatment with levodopa and hcQ markedly increased the number of neurons on the lesion side of the sN compared to the PD group (p < 0.001 for both).

Discussion
the main findings of this study revealed that treatment of PD animals with hcQ decreased apomorphine-induced rotational behavior and muscle rigidity.these results were accompanied by the reduction in oxidative stress and α-synuclein protein levels and the increased number of Nissl-stained neurons in the sN.according to the dopaminergic theory, reduction in the midbrain and striatal dopamine levels causes motor impairment, including resting tremor, muscle stiffness, bradykinesia and postural instability [19,20].
Pathologically, the underlying mechanisms leading to the degeneration of dopaminergic neurons in PD are not fully understood.though levodopa, a dopamine precursor, is the main treatment of PD, upon long-term administration (after 5 years), its efficacy wears off, and abnormal involuntary movements or dyskinesia are developed that limit its utility [21].hence, identifying new therapeutic interventions that can prevent or reverse the neuronal loss and dampen motor deficits are highly demanded.unilateral 6-OhDa injection into different parts of the nigrostriatal pathway is a widely utilized and reproducible method to develop the hemiparkinsonian model in animals [22].this neurotoxin results in asymmetric motor behaviors and dopaminergic neural loss corresponding to the degree of the lesion [22,23].6-OhDa enters dopaminergic neurons through dopamine transporters and accumulates in the cytosol, resulting in the degradation of dopaminergic neurons by disrupting the mitochondrial respiratory chain (i and iV complexes) and augmentation of oxidative stress [24,25]. in line with previous reports [26][27][28], our results showed that unilateral 6-OhDa microinjection into the left sNpc increased rotational behavior contralateral to the lesion side and muscle stiffness.however, treatment with levodopa reduced the number of rotations induced by apomorphine and decreased rigidity in the Murprogo's test.interestingly, the effect of hcQ on abolishing these motor deficits was superior to the effect of levodopa in the lesioned animals.
hcQ is a chloroquine derivative in which one of the N-ethyl groups is β-hydroxylated [9].evidence shows that hcQ has less toxicity than chloroquine [29].interestingly, amodiaquine and chloroquine significantly improved behavioral deficits in the 6-OhDalesioned rat PD model without any noticeable signs of dyskinesia-like behavior [8], which is consistent with the present study.Moreover, hcQ has been shown to improve motor performance and coordination in the rotenone-induced PD model [10].
accumulating preclinical and clinical evidence supported the causative role of oxidative stress and ).***p < 0.001 vs. sham group; ### p < 0.001 vs. PD animals.@@@ p < 0.001 vs. PD + levodopa group.PD, Parkinson's disease; HcQ, hydroxychloroquine; snpc: substantia nigra pars compacta.mitochondrial dysfunction in mediating the cascade of events that result in PD-associated dopaminergic neuron loss and motor deficits [30][31][32][33].cells usually protect themselves against free radical damage through a variety of mechanisms.superoxide dismutase (sOD), catalase (cat), GPx and glutathione reductase are endogenous antioxidant enzymes that play an essentiall role in cellular protection [34]. in this regard, the effects of antioxidants in preventing or reducing damage caused by free radicals have been investigated in PD [35].
alongside inducing ROs accumulation, 6-OhDa diminishes intracellular antioxidant enzymes, leading to abnormalities in the cell structure and metabolism, and hence neuronal deterioration [36]. in this study, MDa levels, a lipid peroxidation marker, were increased while tac levels and GPx activity were decreased in the brain of 6-lesioned animals, suggesting 6-OhDa neurotoxicity. in line with our study, previous studies demonstrated that injection of 6-OhDa into the sN causes a significant increase in the production of free radicals and deteriorates the antioxidant defensive system in the midbrain and striatum [37][38][39].these results suggest that the neurotoxicity of 6-OhDa occurs through the induction of severe oxidative stress in dopaminergic neurons [40].
although levodopa and hcQ treatments for 21 days failed to increase brain tac levels, these treatments increased brain GPx activity in the unilateral 6-OhDa injected animals.Few studies investigated the effect of hcQ treatment on oxidative stress.Olatunde Farombi et al. [41] reported that hcQ treatment in patients with malaria induces systemic oxidative stress.animal studies also showed that hcQ induces oxidative stress and does not improve the body's antioxidant status [42][43][44].another study reported that acute and chronic chloroquine administration increased NaDPh-induced lipid peroxidation in the rat retina [43].
Pathologically, PD is associated with progressive loss of dopaminergic neurons in the midbrain and aggregation of α-synuclein protein, resulting in motor impairments [45].Previous studies supported the contributing role of excessive production of ROs and decreased capacity of the antioxidant defense system to scavenge toxic-free radicals in the deterioration of dopaminergic neurons and the aggregation of α-synuclein in PD [32,46,47].Zeng et al. [48] demonstrated that unilateral 6-OhDa increased the mRNa expression of α-synuclein in the sN, and levodopa had no effect on its mRNa expression.li et al. [49] also reported that 6-OhDa upregulated α-synuclein protein levels in Pc12 cells.the aggregation of α-synuclein is linked to impaired lipid metabolism and hence mitochondrial dysfunction in dopaminergic neurons [50][51][52].likewise, we found that intra-striatal 6-OhDa injection upregulated α-synuclein in the sN.Nevertheless, levodopa and hcQ administration markedly downregulated α-synuclein protein expression.interestingly, hcQ treatment was more effective than levodopa in decreasing α-synuclein protein levels.autophagy disruption results in the accumulation of abnormal proteins like α-synuclein contributing to the deterioration of PD prognosis [53].Notably, hcQ accumulates within lysosomes, resulting in lysosome neutralization and the inhibition of autophagic flux [54].
Moreover, dopaminergic neurons are more susceptible to oxidative damage due to their high baseline ROs levels and limited antioxidant activity, namely GPx [55]. in agreement with previous reports [56][57][58][59], our histological examination revealed decreased Nissl-stained neurons in the ipsilateral sNpc of the 6-OhDa-lesioned animals compared to the sham group, while we did not observe decreased Nissl-stained neurons in the contralateral sNpc.conversely, levodopa and hcQ significantly increased the number of Nissl-stained neurons, suggesting that these treatments protected dopaminergic neurons from neurotoxicity of 6-OhDa in the sN.hcQ protects striatal new Da neurons in the rotenone-induced PD model by upregulation of the orphan nuclear receptor (Nurr1) gene, a crucial factor involved in the preservation and differentiation of dopaminergic neurons and attenuation of striatal inflammatory mediators [10].

Conclusion
hcQ treatment ameliorated 6-OhDa-induced motor impairment and neuronal loss in the sN possibly by enhancing GPx activity and downregulation of α-synuclein protein levels.More studies are needed to clarify the exact mechanism of the neuroprotective effect of hcQ in PD models.

Table 1 .
comparison of the mean number of dopaminergic neurons on the lesion side in the sn in the experimental groups.