Neuroprotective effect of gallic acid in mice with rotenone-induced neurodegeneration

Abstract

We investigated the effect of gallic acid (Gal) against neurodegenerative pathophysiology relevant to Parkinsion’s disease (PD) in mice with rotenone-induced toxicity. Forty male institute of cancer research (ICR) mice were randomly divided into four groups: sham-veh, PD-veh (received subcutaneous injection with 2.5 mg/kg/48 h of rotenone); PD-Gal50; and PD-Gal100 (the latter two groups received subcutaneous injection with 2.5 mg/kg/48 h of rotenone and oral gavage with gallic acid 50 and 100 mg/kg/48 h, respectively). All treatments continued for 5 weeks with motor ability assessments once per week using hanging and rotarod tests. Brain tissue evaluation of oxidative status, together with striatal and substantia nigra par compacta (SNc) histological and immunohistological assessments were performed. The results indicate that rotenone significantly induced muscle weakness and motor coordination deficit from the first week of rotenone injection, and a significant increase in neuronal degeneration was presented in both the striatum and SNc. Decreased tyrosine hydroxylase and increment of glia fibrillary acidic protein expression in SNc were depicted. The deteriorating effects of rotenone were ameliorated by gallic acid treatment, especially 100 mg/kg dose. Rotenone did not induce a significant change of lipid peroxidation indicated, but gallic acid exhibited a significant inhibitory effect on the lipid peroxidation increment. Rotenone showed a significant reduction of superoxide dismutase activity, and neither 50 nor 100 mg/kg of gallic acid could alleviate this enzyme activity. In conclusion, gallic acid ameliorated motor deficits and preserving SNc neurons which led to maintaining of the dopaminergic source, including a nurturing effect on supporting astrocytes in mice with rotenone-induced neurodegeneration.

Introduction

Parkinson’s disease (PD) is a debilitating movement disorder with pathophysiology effects related to dopaminergic circuitry deprivation in two distinct brain areas, the substantia nigra par compacta (SNc) and the striatum, which are well-known circuits involved in initiating and balancing motor commands [1]. As a neurodegenerative disease, PD is an incurable disease with progressive deterioration and disability, and it is one of the major degenerative diseases worldwide [2, 3]. The risk factors for PD include familial and sporadic factors, while the etiology of PD remain largely unknown. At present, the risk factors for PD are likely to be multifactorial with genetic and environmental factors contributing to disease genesis [4]. Exposure to numerous chemicals and environmental toxins has also been implicated in the onset of PD, for instance exposure to pesticides and heavy metals in agriculture [5, 6].

Rotenone, a potent mitochondrial complex I inhibitor, is used as a pesticide and piscicide against crop destruction and invasive fish species [7]. Its association with PD risk increment has also been indicated [8]. Research on PD pathological and therapeutic strategies in patients is time consuming, so the value of preclinical studies is dependent on how precisely the chosen model replicates features of the disease under investigation. The PD animal model induced by rotenone clearly exhibits the key features of PD, such as progressive degeneration of dopaminergic neurons in the SNc, formation of Lewy-body-like cytoplasmic inclusions in surviving dopaminergic neurons, and behavioral motor symptoms that respond to dopamine therapy [9]. A variety of rotenone doses used with different modes of PD induction result in differences related to sensitivity and induction period, and there is a high mortality rate of experimental animals. Therefore, a specific application for each species with a different physiology is necessary. There are a variety of reproducible PD-like pathologies in mice using rotenone induction. However, similar issues, including high mortality rate, are still a topic [10]. Recently, a mode of PD-like pathology induced by rotenone with a low mortality rate has been established. Rotenone 2.5 mg/kg/48 h has been proven to replicate PD-like pathology features with a low mortality rate in mice. The dose represents PD features, including motor deficit associated with dopaminergic destruction, in both the SNc and the striatum and involves brain oxidative stress and inflammation [11,12,13]. This model therefore has a reproducible ability and may be useful in the present study.

The use of nutraceuticals against degenerative diseases is a focus of research nowadays. A variety of active ingredients with pharmaceutical properties from natural plants and organisms are prodigious on preclinical screening. Oxidative stress and inflammation play a major role in stages of degeneration, so substances with anti-oxidative and anti-inflammatory properties are favored. Gallic acid, one of the most abundant phenolic acids in the kingdom plantae, has a variety of pharmaceutical properties [14]. Preclinical screening of gallic acid’s effects have indicated health benefits, such as antimicrobial, anticancer, anti-inflammation, and antioxidative effects [15]. In preclinical models, the neuroprotective actions of gallic acid have clearly indicated benefits in Alzheimer’s disease, PD, stroke, and psychiatric disorders [14]. Focusing on the PD model, the benefits of gallic acid such as antiapoptotic and modulatory effects on oxidative defense mechanisms have been presented [16]. This information, acquired from a variety of in vitro and in vivo PD models, also has some controversy. Therefore, a qualitive in vivo model that simultaneously investigates the link between brain pathology and behavioral disorder will be more beneficial. The use of an in vivo PD model to evaluate the effect of gallic acid is rare. Research performed on PD models (e.g., in rats) using reserpine, 6-hydroxydopamine (6-OHDA), or tacrine have indicated an ameliorative effect of gallic acid on involuntary oral movements (vacuous chewing movements) and passive avoidance memory as well as modulatory effects on the lipid peroxidation process indicated by malondialdehyde (MDA) level, glutathione peroxidase (GPx), and total thiol [17, 18]. No studies have assessed the effect of gallic acid on movement disorder (i.e., motor strength and coordination) simultaneous with dopaminergic change in specific brain areas such as the SNc and the striatum. Therefore, the present study aimed to investigate the effect of gallic acid against PD-like pathology in vivo using rotenone induction in mice to highlight the motor deficits concurrent with related brain pathology.

Materials and Methods

Animals

Forty (8-week-old) male institute of cancer research (ICR) mice (Mus musculus) weighing 40 ± 2 g were delivered from the National Laboratory Center, Mahidol University (Nakhon Pathom, Thailand). Animals were housed individually in their own cage and fed a standard diet (mouse diet food No. 082G) and reverse osmosis (RO) water ad libitum. The housing room was maintained at a constant room temperature of 25°C with a 12-h light/dark cycle. Animal care and use was approved by the Animal Ethics Committee, Faculty of Science, Kasetsart University (ID#ACKU65-SCI-009).

Chemicals

The chemicals used include rotenone, gallic acid, dimethyl sulfoxide (DMSO), sunflower oil (SO), normal saline solution (NSS), hydrogen peroxide (H₂O₂), phosphate-buffered saline (PBS), cresyl violet, normal goat serum, anti-glia fibrillary acidic protein (GFAP) and anti-tyrosine hydroxylase (TH) antibodies, one-step polymer-horse radish peroxidase (HRP) anti-mouse and rabbit, 3,3′-Diaminobenzidine (DAB), Na₂CO₃, NaOH, CuSO₄-5H₂O, C₄H₄KNaO₆-4H₂O, tricarboxylic, sodium dodecyl sulfate, acetic acid, 5,5′-dithios (2-nitrobenzoic acid), EDTA, epinephrine, MDA, reduced glutathione (GSH), superoxide dismutase (SOD), and thiobarbituric acid. All chemicals and reagents were purchased from Chemical Express Co., Ltd., Merck, Millipore, Darmstadt, Germany and Agilent, CA, USA.

Experimental groups

Animals were divided into four groups (10 in each group): sham-veh (subcutaneous injection of vehicle [veh] with 10% DMSO-SO/48 h alternated with oral administration of NSS 0.04 ml/48 h); PD-veh (subcutaneous rotenone 2.5 mg/kg/48 h dissolved in 10% DMSO-SO alternated with oral administration of NSS 0.04 ml/48 h); PD-Gal50 (subcutaneous injection with rotenone 2.5 mg/kg/48 h dissolved in 10% DMSO-SO alternated with oral administration of gallic acid (Gal) 50 mg/kg/48 h); and PD-Gal100 (subcutaneous injection with rotenone 2.5 mg/kg/48 h dissolved in 10% DMSO-SO alternated with oral administration of gallic acid 100 mg/kg/48 h). All treatments continued for 5 weeks with a motor ability assessment once per week (Fig. 1a).

Fig. 1.

(a) The experimental protocol, (b) oxidative parameters, (c) weight, (d) rotarod, (e) and hanging tests. Veh, vehicle; DMSO-SO, dimethyl sulfoxide-sunflower oil; PD, Parkinson’s disease; Gal, gallic acid; MDA, malondialdehyde; GSH, reduced glutathione; CAT, catalase; SOD, superoxide dismutase; TH, tyrosine hydroxylase; GFAP, glia fibrillary acidic protein; SNc, substantia nigra par compacta; w, week. *Statistically significant compared to sham-veh; #statistically significant compared to PD-veh.

Motor coordination test

All behavioral tests were performed in environmental controlled condition, same time test during 6.00 p.m.–12.00 p.m., with constant illumination (200 lx) and quiet background noise (<40 dB). Motor coordination of the mice was assessed with a rotarod (rod diameter 5 cm, length 15 cm, raised 15 cm above the bottom of the rotarod enclosure). The animals were trained for 5 days before the actual test. The rotation speed started from 10 rpm on the first day and reached 15 rpm at the end of the training, with each mouse required to remain on the rotarod for 300 s (baseline). The rotarod test was performed once per week, consisting of three trials with 30-min rest intervals. For the actual test, the animals were placed on a rotating rod at a fixed speed of 20 rpm for 300 s [19]. The latency time that the animals remained on the rotarod was recorded.

Muscle strength test

Muscle strength was assessed using the hanging wire test. A standard wire cage lid was used. In the test, an animal was placed on the top of the lid. After the animal grabbed the lid with all paws and then turned upside down, the mean latency to fall off the wire lid was recorded. Hanging tests were conducted once per week, consisting of three trials with the time extended to 180 s. The hanging test time was adapted to increase the intensity based on our previous studies that used the ICR mice model of rotenone-induced neurodegeneration [11,12,13].

Biochemical analysis

Brains were washed with cold normal saline, homogenated in a 10% w/v PBS (50 mM, pH 7.4). Brain tissue homogenate was kept for MDA and GSH assays. Brain tissue samples weighing 10,000 g were centrifuged at 4°C for 10 min for supernatant analysis of total protein, SOD, and catalase (CAT) activities.

Total brain protein was determined using Lowry’s assay. We used 0.2 ml of the supernatant mixed with 2 ml of solution D (ratio 48:1:1; 2% w/v Na₂CO₃ in 0.1 N NaOH: 0.5% w/v CuSO₄-5 H₂O in distilled water: 1% w/v C₄H₄KNaO₆-4H₂O) and incubated it for 10 min before adding 0.2 ml of 1 N Folin-Ciocalteu reagent (1:1). After 30 min of incubation, the mixture was read at 600 nm. Protein concentration was calculated using the standard curve of bovine serum albumin [20].

MDA level in the brain tissue was determined to represent lipid peroxidation. We mixed 0.2 ml of brain homogenate with 4% sodium dodecyl sulfate, 1.5 ml of 20% acetic acid, and 1.5 ml of 0.5% thiobarbituric acid. The mixture was heated at 95°C for 1 h and was then centrifuged for 10 min at 3,500 rpm before reading the supernatant at 532 nm. MDA concentration was calculated using the standard curve of MDA and presented as µM/mg of protein [21].

GSH was measured by mixing 0.1 ml of homogenate with 10% tricarboxylic acid and centrifuging it for 10 min. We used 0.5 ml of supernatant and mixed it with 5,5′-dithios (2-nitrobenzoic acid) and increased the final volume of the mixture to 3 ml using PBS before reading it at 412 nm. GSH concentration was calculated using the standard curve of GSH and presented as µM/mg of protein [13].

CAT enzyme activity was measured using 50 µl of supernatant and the volume was increased to 3 ml with 0.05 M PBS (pH 7.4) containing 0.01 M H₂O₂. Absorbance was read every 30 s at 240 nm for 3 min. CAT activity was calculated with reference to the extinction coefficient of H₂O₂ and presented as U/mg of protein [12].

SOD enzyme activity was measured by mixing 0.1 ml of supernatant with 0.1 ml of EDTA (0.0001 M), 0.5 ml of carbonate buffer (pH 7.9), and 1 ml of epinephrine (0.0003 M). Absorbance was read every 30 s at 480 nm for 3 min and enzyme activity presented as U/mg of protein, using the standard curve of standard SOD activity 6,150 U/mg [11].

Histological analysis

After the final behavioral test was performed, the mice were euthanized with 50-µl intraperitoneal injection of Zoletil 100 + X-Lazine (4:1) followed by transcardial perfusion with PBS and then by 4% paraformaldehyde. Brains were collected and stored in 4% paraformaldehyde, then further washed in tap water, dehydrated by gradient ethanol, cleared by xylene, embedded in paraffin, and cut into 5-µm thick sections with a rotary microtome. Five slides were selected with a 125-µm interval for each mouse [22]. The sections were rehydrated and stained with 0.1% cresyl violet followed by mounting with cover glass. Photomicrographs were captured with a light microscope (BX51, Olympus, Tokyo, Japan) at 400× magnification in brain areas, specifically the striatum (bregma 1.42 to 0.02 mm) and the SNc (bregma −2.8 to −3.8 mm) referenced with stereotaxic mouse’s brain [23]. Viable and degenerated cells were counted for further representation of the percentage of degeneration in each brain area. The percentage of neuronal degeneration was calculated using the formula: percentage of degeneration = [degenerated cells / (viable cells + degenerated cells)] × 100.

Viable cells were counted according to the number of light purple cresyl-violet-stained cells that presented nucleus and nucleolus. Degenerated cells were counted according to the number of dark purple cresyl-violet-stained cells, pyknotic, and shrinkage with vacuole presenting around cell [24]. We also evaluated neuron density by Nissl positive per area [25]. Cell counts and percentage of Nissl positive areas were analyzed using NIH Image J.

Immunohistochemistry

We used anti-GFAP and anti-TH immunohistochemistry to indicate reactive astrocytes and dopaminergic neurons, respectively. Brain sections were rehydrated and retrieved in a citrate buffer using the microwave method for 20 min. The sections were then rinsed in PBS and incubated with peroxidase blocking solution (0.3% H₂O₂) for 10 min and rinsed with PBS before incubation with 3% normal goat serum blocking solution for 30 min. Sections were incubated overnight with anti-GFAP (1:100) and anti-TH (1:100). Brain sections were rinsed in PBS and incubated with one-step polymer-HRP anti-mouse and rabbit for 30 min, and then they were rinsed in PBS again followed by DAB peroxidase substrate solution for 5–10 min of incubation. They were then rinsed in running tab water, dehydrated, and covered [26]. The photomicrographs of the striatum and SNc areas were captured at 400× magnification for further analysis using national institute of health (NIH) ImageJ. Both immunohistochemistry and histology assessments were performed in a blind fashion with two investigators.

Statistical analysis

All statistical analyses were carried out using the StatView 5.0. The normal distribution and variance homogeneity for each group of data were checked using the Shapiro-Wilk normality test. One-way analysis of variance was conducted and Fisher’s LSD post hoc test for group comparison was used. The data was expressed as mean ± SD. A value of P<0.05 was considered statistically significant.

Results and Discussion

The present study evaluated the neuroprotective effect of gallic acid against rotenone-induced PD-like neurodegeneration in mice. We applied chronic exposure to low-dose rotenone-induced neuronal pathology relevant to PD according to low mortality [27]. Weight of mouse was evaluated and found gradually increase form 1–6 weeks without any statistical significance (P>0.05, Fig. 1c). Our publications using rotenone 2.5 mg/kg/48 h in ICR mice confirmed rotenone could induce motor deficits within 1–6 weeks with low mortality, and motor deficits were found to be concurrent with neuronal destruction in specific brain areas such as the striatum and SNc [11,12,13]. In the present study, motor deficits were observed from the first week after rotenone injection (Figs. 1d and e). The hanging time significantly decreased for PD-veh compared to sham-veh (w1: P=0.0082; w2: P<0.0001; w3: P=0.0008; w4: P<0.0001; w5: P<0.0001, respectively). This result indicates that muscle weakness was significantly induced by the rotenone injection protocol. Additionally, hanging time test protocol in the present study was extended to 180 s, so muscle weakness was rapidly indicated in the present study when compared to the researchers’ previous studies. The rotarod result also showed a motor coordination deficit when comparing PD-veh to sham-veh (w1: P=0.0401; w3: P=0.0031; w5: P=0.0025, respectively). It also showed transient recovery of motor coordination in w2 (P=0.1357) and w4 (P=0.0725) when comparing PD-veh to sham-veh. Transient recovery of motor deficit was previously found in a mice model of rotenone-induced PD-like neuropathology [10, 12]. Transient recovery was driven by an early compensatory mechanism after toxic induction, and then the ability gradually reduced under chronic toxic exposure. Our results indicate a difference in the degree of deterioration due to rotenone’s effect on muscle strength and motor coordination in ICR mice. A dose of 2.5 mg/kg/48 h was able to induce irreversible muscle weakness from the first week of injection, whereas transient recovery of motor coordination was still presented in the early period of toxic exposure.

Motor deficits are the primary manifestation of PD [28]. SNc neurodegeneration leads to dopaminergic projection insufficiently supporting the selective programming and motor initiation of the striatum, resulting in an imbalance of striatal motor circuits and misleading motor commands [29]. Histological assessment in related brain areas indicated rotenone significantly increased neuronal degeneration in the striatum (Fig. 2) and the SNc (Fig. 3). In the striatum, the percentage of neuronal degeneration significantly increased in PD-veh compared to sham-veh (P<0.0001). This result presented the same trend when interpreted with the percentage of Nissl positive per area. We found that the percentage of Nissl positive of PD-veh significantly decreased in the striatal area when compared to sham-veh (P=0.0003). In the SNc, rotenone also increased the percentage of neuronal degeneration (P<0.0001) and decreased the percentage of Nissl positive (P<0.0001) when PD-veh was compared with sham-veh. Our results indicated that 2.5 mg/kg/48 h of rotenone exposure for 5 weeks induced significant neuronal degeneration in the striatum and the SNc.

Fig. 2.

Photomicrographs of the striatal area of (a) sham-veh, (b) PD-veh, (c) PD-Gal50, and (d) PD-Gal100 captured at 400× magnification, scale bar=50 µm. Red arrows indicate degenerating cells; transparent arrows indicate viable cells. Processed binary images after adjusting threshold of (e) sham-veh, (f) PD-veh, (g) PD-Gal50, and (h) PD-Gal100 used to analyze the percentage of Nissl area. Veh, vehicle; PD, Parkinson’s disease; Gal, gallic acid. *Statistically significant compared to sham-veh; #statistically significant compared to PD-veh.

Fig. 3.

Photomicrographs of the substantia nigra par compacta area of (a) sham-veh, (b) PD-veh, (c) PD-Gal50, and (d) PD-Gal100 captured at 400× magnification, scale bar=50 µm. Red arrows indicate degenerating cells; transparent arrows indicate viable cells. Processed binary images after adjusting threshold of (e) sham-veh, (f) PD-veh, (g) PD-Gal50, and (h) PD-Gal100 used to analyze the percentage of Nissl area. Veh, vehicle; PD, Parkinson’s disease; Gal, gallic acid; SNc, substantia nigra par compacta. *Statistically significant compared to Sham-veh; #statistically significant compared to PD-veh.

Dopaminergic neurons settled in the SNc have a key role in motor control, such as movement frequency, and are related to bradykinesia [29]. In the present study, immunohistochemistry of TH and GFAP in the striatum and SNc were evaluated (Figs. 4 and 5). We found that rotenone significantly decreased TH expression in the striatum (P=0.0325) and SNc (P=0.0012) when comparing PD-veh with sham-veh. A similar PD mice model used in previous studies indicated significant SNc neuronal degeneration in combination with a reduction of TH immunoreactivity which appeared as early as the fourth week, while a significant reduction of TH had not yet appeared in the striatum at this point [12]. The results from the present study exhibited similar effects in the SNc and a novel presentation of TH being significantly reduced in the striatum in the fifth week of rotenone exposure. Another study extended rotenone exposure to 6 weeks and found that TH significantly reduced in both the SNc and striatum concurrent with significant damage to neurons in these areas [13]. Therefore, corroboration of these studies could validate the reproducibility of this PD mice model.

Fig. 4.

Tyrosine hydroxylase (TH) immunohistochemistry of striatal area of (a) sham-veh, (b) PD-veh, (c) PD-Gal50, and (d) PD-Gal100 captured at 4× magnification, scale bar=1,000 µm. Photomicrographs of the striatal area of (e) sham-veh, (f) PD-veh, (g) PD-Gal50, and (h) PD-Gal100, representing TH positive cells, and captured at 400× magnification, scale bar=20 µm. Photomicrograph of the striatal area of (i) sham-veh, (j) PD-veh, (k) PD-Gal50, and (l) PD-Gal100, representing glia fibrillary acidic protein (GFAP) positive cells, and captured at 400× magnification, scale bar=20 µm. Histogram showing the percentage of TH and GFAP positive cells (% area). Veh, vehicle; PD, Parkinson’s disease; Gal, gallic acid. *Statistically significant compared to sham-veh.

Fig. 5.

Tyrosine hydroxylase (TH) immunohistochemistry of substantia nigra par compacta (SNc) of (a) sham-veh, (b) PD-veh, (c) PD-Gal50, and (d) PD-Gal100 captured at 4× magnification, scale bar=1,000 µm. Photomicrographs of SNc of (e) sham-veh, (f) PD-veh, (g) PD-Gal50, and (h) PD-Gal100, representing tyrosine hydroxylase (TH) positive cells and captured at 400× magnification, scale bar=20 µm. Photomicrographs of the SNc of (i) sham-veh, (j) PD-veh, (k) PD-Gal50, and (l) PD-Gal100, representing glia fibrillary acidic protein (GFAP) positive cells, and captured at 400× magnification, scale bar=20 µm. Histogram showing the percentage of TH and GFAP positive cells (% area). Veh, vehicle; PD, Parkinson’s disease; Gal, gallic acid; SNc, substantia nigra par compacta. *Statistically significant compared to sham-veh, #statistically significant compared to PD-veh.

Glia activation in the SNc of the PD mice model induced by rotenone has been indicated [30]. In the present study, significant activated GFAP induced by rotenone exposure was only found in the SNc (P=0.0198). It was indicated that reactive astrocytes have both beneficial and harmful effects due to their role in the process of inflammation and neurotoxicity, so they also have a series of adverse effects. The benefits of astrocytes in ionic homeostasis, neuronal energy support, and balancing of oxidative status were clearly depicted [31]. When damage occurs to the brain, reactive astrocytes also gather at the edge of the injury site and can further combine with glycoprotein to form a glial scar. The glial scar aggravates inflammation, inhibits axon growth, and hinders the recovery of motor function [32]. Astrocyte function and disease progression has a strong relationship, so exploration and clarification of the timing and mechanism of astrocytes participating in specific disease stages is required. The PD mice model using rotenone 2.5 mg/kg/day indicated different spatial and temporal expressions of astrocyte activation. A study indicated that the SNc was the most vulnerable brain area specific to rotenone-induced toxicity, in which the astrocyte activation peak followed by neuronal degeneration was presented early as 2 weeks after rotenone exposure, and then astrocyte activation followed by neuronal degeneration appeared in the striatum several weeks later [33]. The present study used 2.5 mg/kg/48 h of rotenone exposure, therefore no evidence including glia activation was found for this model. In this study, the researchers have shown that 5-week rotenone exposure is adequate for neuronal degeneration induction in the SNc and striatum, as well as for significant activation of reactive astrocytes in the SNc but not in the striatum at this time point. However, additional studies will be required to fill the research gap relevant to the spatial and temporal expression of glia activation and the molecular level of pathophysiology progression in the recent PD mice model.

Involvement of oxidative damage due to rotenone-induced neuronal toxicity and support for the evaluation of antioxidant therapies for PD have been reported [30]. We observed the brain’s oxidative status (Fig. 1b) which indicated that only SOD activity was significantly reduced by rotenone exposure when comparing PD-veh with sham-veh (P=0.0219). The effect of rotenone on SOD activity was similar to the previous studies that indicated the inhibition effect of rotenone on SOD activity appeared from the fourth week [12] and continued in the sixth week of rotenone exposure [13]. The present study observed these effects in the fifth week of rotenone exposure and the same result was observed. Therefore, corroboration of these results indicated the prominent inhibitory effect of rotenone on SOD activity in the PD mice model.

The present study found that 5-week rotenone exposure did not induce a significant change of lipid peroxidation indicated by MDA level (P=0.1935), GSH level (P=0.5291), and CAT activity (P=0.8949) when comparing PD-veh with sham-veh. Our previous study [12] and the present study showed a nonsignificant change of MDA level during 4–5 weeks of rotenone exposure. However, the significant activation of MDA level was clearly presented in the sixth week of rotenone exposure in this PD mice model [11, 13]. A similar effect was found for CAT activity in which a nonsignificant change was indicated at 4–5 weeks, but a significant reduction of CAT activity presented in the sixth week of rotenone exposure [13]. MDA product referred to lipid peroxidation status, indicating that this PD mice model (2.5 mg/kg/48 h) induced a gradual increase of lipid peroxidation during 4–5 weeks and increased to a significant level in the sixth week of rotenone exposure. The results show rotenone’s effect on oxidative stress induction ranged from a mild to a severe degree in response to a short or long period of rotenone exposure, indicating that oxidative stress associated with rotenone exposure resulted in neuronal degeneration being retained in the present study.

We evaluated the effect of gallic acid on rotenone-induced PD-like pathology in mice and found that mice treated with gallic acid showed no motor deficit in either hanging and rotarod tests indicated by a nonsignificant difference of latency time from sham-veh (P>0.05). Mice treated with 50 mg/kg of gallic acid exhibited greater muscle strength indicated by hanging time compared to PD-veh (w1: P=0.0168; w2: P<0.0001; w3: P=0.0025; w4: P<0.0001, and w5: P=0.0004, respectively). A similar effect was found in mice with 100 mg/kg of gallic acid administration (w2: P<0.0001; w3: P=0.0011; w4: P<0.0001, and w5: P=0.0002, respectively; Fig. 1c). Motor coordination in the rotarod test also exhibited significant difference when comparing PD-Gal50 (w3: P=0.0023; w5: P=0.0038) to PD-veh and PD-Gal100 (w3: P=0020; w5: P=0.0038) to PD-veh (Fig. 1d). These results indicated an ameliorative effect of gallic acid against rotenone-induced motor deficits in mice. Previous research reported the benefit of gallic acid on vacuous chewing movements and catalepsy in rats (reserpine) and mice (tacrine) that were used as in vivo PD models [14]. In the present study, the alleviated effects of gallic acid on motor deficit indicated an association with preservation of neuronal and TH intensity in related brain areas. Mice treated with 50 mg/kg and 100 mg/kg gallic acid doses showed they were protected against rotenone-induced neuronal degeneration in both the striatum (Fig. 2) and SNc (Fig. 3). A significant preventative effect against neuronal degeneration in the striatum (P<0.0001 and<0.0001) and the SNc (P<0.0001 and<0.0001) was clearly exhibited when comparing PD-Gal50 and PD-Gal100 to PD-veh. When interpreted using the percentage of Nissl positive, gallic acid was found to prevent the decrease of the percentage of Nissl positive induced by rotenone. The significant difference of the percentage of Nissl positive was clearly presented when comparing PD-Gal50 and PD-Gal100 to PD-veh in both the striatum (P=0.0193 and 0.0334) and the SNc (P=0.0417 and 0.0459). In addition, mice treated with gallic acid exhibited a preventative effect against TH reduction in both the striatum and the SNc, which was indicated by a nonsignificant difference when comparing PD-Gal50 (P>0.05) and PD-Gal100 (P>0.05) to sham-veh. Only a dose of 100 mg/kg of gallic acid showed a significant difference in the TH level in the SNc area when compared to PD-veh (P=0.0341). Dose-dependent effect was presented in SNc area that gallic acid 100 mg/kg showed significantly preserved the dopaminergic neurons but not 50 mg/kg when compared to PD-veh group. Our results indicated both doses of gallica acid can prevent neuronal degeneration in SNc (indicated by Nissl staining), however, when focus on specific dopaminergic neurons (indicated by TH immunostaining), the effective dose was at 100 mg/kg that clearly exhibited nurturing effect on both neuronal structure and function. In addition, in the SNc, both 50 and 100 mg/kg doses of gallic acid maintained an astrocytic stage comparable to sham-veh (P>0.05) and GFAP significantly lower than PD-veh (P=0.0201 and P<0.0001, respectively). GFAP immunoreactivity also indicated that treatment with gallic acid significantly reduced reactive astrocytes in the SNc when comparing PD-Gal50 (P<0.0001) and PD-Gal100 (P<0.0001) to PD-veh (Fig. 5). Recently, a study indicated gallic acid’s effect on attenuated reactive gliosis resulting in an inflammatory modulatory effect in an animal model of Alzheimer’s disease [34]. The present study first indicated the role of gallic acid in attenuating reactive astrocytes in a PD mice model. Therefore, our results depict the protective effect of gallic acid against neuronal degeneration and maintenance of dopaminergic source, including a nurturing effect on supporting astrocytes.

Regarding oxidative stress, treatment with gallic acid 50 mg/kg (P=0.0210 and 0.9791) or 100 mg/kg (P=0.0250 and 0.9326) did not show an ameliorative effect on SOD activity when compared to sham-veh and PD-veh, respectively. Mori et al. (2020) indicated the decreasing effect of gallic acid on SOD1 gene expression against disease induced an increased SOD1 gene expression and implied the stabilization of the brain oxidative mechanism [34]. However, the present study evaluated only SOD enzyme activity and not SOD1 gene expression and may not be conclusive at this time. Treatment with gallic acid 50 mg/kg had an enhancing effect on CAT activity when compared to sham-veh (P=0.0062) and PD-veh (P=0.0051), while the high dose at 100 mg/kg did not and indicated the difference response for CAT activity. Nowadays, the reference for gallic acid dose’s effect on CAT and SOD activities in PD mice model is rare. Varying doses of gallic acid were used in variety animal models of neurodegenerative disease and also varying outcomes [16]. Therefore, researches are needed to elucidate the effect of dose response of gallic acid on difference parameters in each disease’s model. In addition, the present study found that mice treated with 50 mg/kg (P=0.0045 and 0.0007) and 100 mg/kg (P=0.0012 and 0.0002) showed significantly reduced MDA levels when compared to sham-veh and PD-veh, respectively. These results indicated the beneficial effect of gallic acid, especially against lipid peroxidation and previous report also indicated the ameliorative effect of gallic acid on MDA in mouse brain as well [35]. The potential anti-PD mechanism of gallic acid acquired from previous in vivo research also indicated the antioxidant features of gallic acid. Mansouri et al. (2013) reported that treatment with gallic acid at doses of 50, 100, and 200 mg/kg prevented memory deficit and cerebral oxidative stress mediated by 6-OHDA injected in the medial forebrain bundle in rats of the PD model. They indicated gallic acid enhanced the total thiol and GPx, as well as diminished MDA and TBARS levels [17]. In addition, an in vitro PD model using a human dopaminergic cell line determined the antiapoptotic effect of gallic acid (i.e., reverted the up-regulation of Keap-1 and caspase-3 and down-regulation of Nrf2, BDNF, and p-CREB, as well as diminished the ratio of Bax and Bcl-2 proteins) [36]. Unlike previous studies, this research is the first to investigate the effect of gallic acid on PD pathology in mice with rotenone exposure, and we only claim that gallic acid exhibits an alleviating effect on motor deficit concurrent with protection against neuronal degeneration, leading to TH maintenance and nurturing astrocytic function. However, the aspect of oxidative status is unclear, and further study is required to elucidate these unclear results, especially the effect of gallic acid on SOD activity.

Conclusion

The present study indicated beneficial effects of gallic acid against rotenone-induced neurotoxicity relevant to PD-like pathology in mice. Using chronic low-dose of rotenone (2.5 mg/kg/48 h) induction for 5 weeks in ICR mice significantly induced motor deficits associated with dopaminergic neuronal degeneration both in SNc and striatum, as well as significant activated reactive astrocyte in SNc. Gallic acid is found to exhibit an alleviating effect on motor deficit concurrent with protection against neuronal degeneration, leading to dopaminergic maintenance and nurturing astrocytic function in mice with rotenone-induced PD-like pathology.

Author Contribution

Wachiryah Thong-asa conceived and designed research, behavioral, biochemical, immunohistological and histopathological analyses, data analysis and integration, artworks and wrote the manuscript. Chatrung Wassana, Kunyarat Sukkasem, Pichcha Innoi, Montira Dechakul, Pattraporn Timda performed the experiment i.e., animal care and treatments, behavioral assessments, brain tissue collection, biochemical and histological evaluation.

Declarations

Ethics approval “All applicable international and national guidelines for the care and use of animals were followed” The experimental procedure was approved by the Animal Ethics committee, Faculty of Science, Kasetsart University (ID#ACKU65-SCI-009).

Consent to Participate

All authors agree to participate.

Consent for Publication

All authors agree to publish.

Conflict of Interest

The authors declare that they have no conflict of interest.

Data Availability

Available upon request.