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Neuroprotective effect of nerolidol against neuroinflammation and oxidative stress induced by rotenone

Neuroprotective effect of nerolidol against neuroinflammation and oxidative stress induced by... Background: Parkinson disease (PD) is a movement disorder affecting 1 % of people over the age of 60. The etiology of the disease is unknown; however, accumulating evidence suggests that mitochondrial defects, oxidative stress, and neuroinflammation play important roles in developing the disease. Current medications for PD can only improve its symptoms, but are unable to halt its progressive nature. Although many therapeutic approaches are available, new drugs are urgently needed for the treatment of PD. Thus, the present study was undertaken to investigate the neuroprotective potential of nerolidol, a sesquiterpene alcohol, on a rotenone-induced experimental model of PD, where male Wistar rats intraperitoneally received rotenone (ROT ) at a dose of 2.5 mg/kg of body weight once daily for 4 weeks. Results: Nerolidol, which has antioxidant and anti-inflammatory properties, was injected intraperitoneally at 50 mg/ kg of body weight, once daily for 4 weeks, and at 30 min prior to ROT administration. ROT administration significantly reduced the activities of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT ), and the level of the antioxidant tripeptide glutathione (GSH). Moreover, ROT increased the levels of the lipid peroxidation product malon- dialdehyde (MDA), proinflammatory cytokines (IL-1β, IL-6, and TNF-α), and inflammatory mediators (COX-2 and iNOS) in rat brain tissues. Immunostaining of brain tissue sections revealed a significant increase in the number of activated astrocytes (GFAP) and microglia (Iba-1), along with the concomitant loss of dopamine (DA) neurons in the substantia nigra pars compacta and dopaminergic nerve fibers in the striatum of ROT-treated rats. As expected, nerolidol sup - plementation to ROT-injected rats significantly increased the level of SOD, CAT, and GSH, and decreased the level of MDA. Nerolidol also inhibited the release of proinflammatory cytokines and inflammatory mediators. Finally, nerolidol treatment prevented ROT-induced glial cell activation and the loss of dopaminergic neurons and nerve fibers, and ultimately attenuated ROT-induced dopaminergic neurodegeneration. Conclusion: Our findings are the first to show that the neuroprotective effect of nerolidol is mediated through its anti-oxidant and anti-inflammatory activities, which strongly supports its therapeutic potential for the treatment of PD. Keywords: Parkinson disease, Rotenone, Neurodegeneration, Oxidative stress, Nerolidol *Correspondence: shreeshojha@uaeu.ac.ae; ehaque@uaeu.ac.ae Department of Biochemistry, College of Medicine and Health Sciences, United Arab Emirates University, PO Box 17666, Al Ain, UAE Department of Pharmacology and Therapeutics, College of Medicine and Health Sciences, United Arab Emirates University, PO Box 17666, Al Ain, UAE © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Javed et al. BMC Neurosci (2016) 17:58 Page 2 of 12 obtained from medicinal plants exhibit a variety of bio- Background logical properties, such as anticonvulsant [11], analgesic Parkinson disease is a neurodegenerative disorder that [12], anxiolytic [13], antidepressant [14], antioxidant [15], is characterized by a progressive loss of dopamine (DA) and anti-inflammatory [16] properties. Some of these neurons in the substantia nigra pars compacta (SNc) effects are frequently attributed to terpenes, which are and dopaminergic nerve terminal fibers of the striatum chemical components that are present in these essential that are enriched in the DA transporter [1]. As a result oils [11, 17]. Nerolidol (NRD) [3,7,11-trimethyl-1,6,10- of dopaminergic neurodegeneration, the level of the DA dodecatrien-3-ol] is a sesquiterpene alcohol (Fig.  1) that neurotransmitter (which mediates the control of move- is found in essential oils from plants such as Baccharis ment) is significantly reduced in the striatum. Therefore, dracunculifolia [18], Amaranthus retroflexus [ 19], and dopaminergic neurodegeneration leads to impairments Canarium schweinfurthii [20]. Nerolidol is an aliphatic in movement, a resting tremor, rigidity, and a disturbance sesquiterpene that is commonly used in cosmetics, per- in gait. Although the etiology for dopaminergic neuronal fumes, shampoos, and soaps [21], as well as non-cosmetic degeneration in PD is not completely understood, cur- products (cleansers and detergents). Essential oils from rent evidence suggests that oxidative stress and neuroin- Ferula fukanensis containing nerolidol have been shown flammation play key roles in the pathogenesis of PD [2, to prevent nitric oxide (NO) production and NO-induced 3]. gene expression, suggesting its plausible use as an antioxi- Rotenone (ROT), an inhibitor of mitochondrial com- dant agent. NRD is hydrophobic in nature with an XlogP3 plex I, has been widely used as a herbicide/pesticide in value of 4.6 (>2.5 is needed for efficient transport across agriculture. A ROT challenge, due to its complex I inhibi- blood brain barrier), and thus NRD can cross easily the tory property, induces pathological features in animals blood brain barrier. NRD significantly reduces the level that are similar to those seen in human PD patients. of lipid peroxidation and nitrite content in the hippocam- u Th s, ROT-treated animals represent a promising ani - pus of mice, which protects against oxidative stress [15], mal model with construct validity [4–6]. The ROT model suggesting an important antioxidant property for NRD. recapitulates most of the pathological features observed A recent study reported that NRD reduces interleukin-1β in human PD pathogenesis, including the loss of DA neu- (IL-1β) production in lipopolysaccharide-induced perito- rons in SNc, and enhanced oxidative stress and neuroin- neal macrophages [16]. Additionally, NRD shows potent flammation in the nigrostriatal dopaminergic pathway [2, anti-inflammatory activity through the suppression of 6–8]. Moreover, a ROT challenge to rodents induced the IL-1β and tumor necrosis factor-alpha (TNF-α) levels in formation of α-synuclein cytoplasmic inclusions in DA an experimental mouse model of pain [16]. Therefore, the neurons, Lewy pathology, DJ-1 acidification and trans - main objective of the present study was to investigate the location, proteasomal dysfunction, and nigral iron accu- anti-oxidative and anti-inflammatory effects of NRD on mulation [9]. Therefore, the ROT-treated rat is a suitable ROT-induced neurodegeneration in rats. model for investigating novel therapeutic agents target- ing oxidative stress and neuroinflammation in PD. Methods Currently available drugs alleviate the symptoms of Drugs and chemicals PD, but are inadequate in terms of halting the progres- Polyclonal rabbit anti-cyclooxygenase-2 (COX-2), anti- sion of the disease. Additionally, motor complications inducible nitric oxide synthase (iNOS), and anti-glial during advanced stages of the disease, the adverse effects fibrillary acidic protein (GFAP) antibodies were pur - of the available drugs, and non-motor symptoms remain chased from Abcam, Cambridge, MA, USA. Anti-ionized huge challenges for long-term therapy. Therefore, newer calcium-binding adaptor molecule-1 (Iba-1) polyclonal therapeutic agents and approaches are urgently needed rabbit antibody was procured from Wako Chemicals, to stop or delay the progressive nature of the disease [10]. Richmond, VA, USA. Polyclonal rabbit anti-tyrosine PD is intimately connected to excessive oxidative stress hydroxylase antibody was obtained from Novus Biologi- that damages brain areas and accelerates the process of cals, Littleton, CO, USA. Alexa fluor 488 conjugated sec - neurodegeneration, especially in the nigrostriatal area. ondary goat anti-rabbit antibodies were purchased from This process is an important breakpoint for controlling Life Technologies, Grand Island, NY, USA. Biotinylated the disease. Consequently, in addition to other therapeu- secondary anti-rabbit antibody was purchased from tic approaches, treatment with antioxidants is gradually Jackson Immunoresearch, West Grove, PA, USA. Nero- gaining importance in the pharmacotherapy of PD. lidol was purchased from Santa Cruz Biotechnology Inc., Plants are a potential source of novel drugs to benefit CA, USA. ROT and the assay kit for reduced glutathione mankind. Much research effort has focused on discover - (GSH) and other reagents of analytical grade were pur- ing new antioxidant compounds from plants. An increas- chased from Sigma-Aldrich, St. Louis, MO, USA. ing number of studies have shown that essential oils Javed et al. BMC Neurosci (2016) 17:58 Page 3 of 12 Tissue collection At the end of the experiment, the animals from all groups were anaesthetized with pentobarbital (40 mg/kg of body weight) and a cardiac perfusion was performed using 0.01  M of phosphate-buffered saline (PBS) pH 7.4 to completely clear the blood. The skull of the rat was open Fig. 1 Chemical structure of nerolidol to quickly isolate the brain. The brain was placed on an ice-plate and cut along the midline to separate the 2 cer- ebral hemispheres. For biochemical assay, the midbrain Experimental animals and striatum regions of the brain were isolated from 1 We used male Wistar rats at 6–7  months old (weighing hemisphere and immediately fresh frozen in liquid nitro- 280–300 g) from the Animal Research Facility of the Col- gen for further use. The other hemisphere of the brain lege of Medicine and Health Sciences, United Arab Emir- was incubated in 4 % paraformaldehyde solution for 48 h ates University, UAE. Prior to the start of the experiment, and subsequently exchanged with 10  % sucrose solution a maximum of 4 rats were housed per cage, and rats were containing 0.1  M of phosphate buffer (PB) 3 times daily acclimatized for 1 week to the laboratory conditions. The for 3 consecutive days at 4 °C prior to cryostat sectioning. cages of the animals were changed twice a week. The animals were housed and kept under standard labora- Sample preparation for biochemical assay tory light and dark cycle conditions. The animals were For biochemical assay, the midbrain tissue of each ani- fed with commercially available rodent food and water mal was homogenized in KCl buffer (10-mM Tris–HCl, ad  libitum. All experiments were conducted between 140-mM-NaCl, 300-mM KCl, 1-mM EDTA, and 0.5  % 0900 and 1500  hours. The Animal Ethics Committee of Triton X-100) at pH 8.0 supplemented with protease the United Arab Emirates University, UAE, approved the and phosphatase inhibitor, keeping the samples on ice. experimental protocol for animal experimentation. The tissue homogenates of each sample were centrifuged at 14,000×g for 20  min at 4  °C to obtain the post-mito- Experimental design chondrial supernatant (PMS) fraction. This PMS fraction To induce the PD in rats, ROT (2.5 mg/kg body weight) was was used to estimate the levels of the antioxidant enzyme administered intraperitoneally (i.p.) once daily for 4 weeks. GSH, lipid peroxidation product, and proinflammatory The regimen used in the current study for the induction cytokines using spectrophotometric measurements and of Parkinsonism in rats was adopted as reported earlier an enzyme-linked immunosorbent assay (ELISA) follow- [22]. Briefly, ROT was first dissolved in dimethyl sulfox - ing a standard protocol, as reported earlier [22]. ide (DMSO) to prepare a 50X stock solution and stored at −80 °C for further use. Before injection, the stock solu- Assay for lipid peroxidation tion of ROT was thawed and further diluted in sunflower A malondialdehyde (MDA) assay kit procured from oil to obtain a final concentration of 2.5 mg/mL. To evalu - Northwest Life Science (Vancouver, WA, USA) was ate its neuroprotective efficacy, NRD was initially diluted in used to determine the amount of lipid peroxidation, as olive oil to obtain a final concentration of 50 mg/2 mL, and reported earlier [22]. Briefly, the samples or standards injected i.p. 30 min prior to ROT administration at a dose (250  µL) were incubated in the presence of acid reagent of 50  mg/kg of body weight once daily for 4  weeks. The (250  µL) and thiobarbituric acid (250  µL) and vortexed dose of NRD was selected based on dose–response stud- vigorously. Samples were further incubated for 60 min at ies (data not shown) as well as earlier reports [15, 23]. Rats 60 °C followed by centrifugation at 10,000×g for 2–3 min. that were used as controls received an equivalent volume of The reaction mixture was then aseptically transferred vehicle only. The animals were sacrificed 48 h after the last without disturbing the pellet to a cuvette and the absorb- injection of NRD or ROT or both in combination to com- ance was recorded at 532 nm. The concentration of MDA pletely eliminate these drugs from the body. The rats were was calculated using a standard curve and expressed as divided into 4 experimental groups where each group con- µM MDA/mg protein. sisted of 8 animals; the groups were named as follows: Estimation of GSH Group I, the vehicle-injected control group (CONT). A GSH kit was used to estimate the GSH level, as reported Group II, the rotenone-injected and vehicle-treated earlier [22]. Briefly, the samples were first deproteinized group (ROT). with 5  % 5-sulfosalicylic acid solution and centrifuged Group III, the rotenone-injected and NRD-treated to remove the precipitated protein. The supernatant was group (ROT + NRD). used to measure the GSH level. Samples and standards Group IV, the NRD-only injected group (NRD). Javed et al. BMC Neurosci (2016) 17:58 Page 4 of 12 of different concentrations (10  µL) were added in each of activated GFAP-positive astrocytes and Iba-1-positive well of a 96-well plate and incubated for 5  min with the microglia. The brain sections were first washed twice working mixture (150  µL; assay buffer  +  5,5′-dithiobis with PBS and incubated with blocking reagent (10 % nor- (2-nitrobenzoic acid)  +  glutathione reductase). Diluted mal goat serum in PBS containing 0.3 % Triton-X 100) for NADPH solution (50  µL) was added to each well and 1 h. The sections were then incubated overnight with the mixed properly. The absorbance of the samples was primary polyclonal rabbit antibodies ant-GFAP (1:1000) measured at 412  nm with kinetics capability for 5  min and anti-Iba-1 (1:1000) at 4 °C. The sections were washed using a microplate reader. The results were expressed as and incubated with fluorescent secondary anti-rabbit µM GSH/mg protein. Alexa fluor 488 antibody for 1  h at room temperature. Sections were then washed, mounted on slides, and Assay for antioxidant enzymes activity coverslipped using the mounting medium Fluoroshield The activity of antioxidant enzymes such as superoxide (Sigma Aldrich, St. Louis, MO, USA). The images were dismutase (SOD) and catalase (CAT) were determined captured using a fluorescence microscope EVOS FL following the manufacturer’s instructions of a kit (Cay- (Thermo Fisher Scientific, Waltham, MA, USA). man Chemicals Company, Ann Arbor, MI, USA), as reported earlier [22]. The CAT activity was expressed Assessment of TH‑immunopositive (TH+) dopamine (DA) as nmol/min/mg protein and the SOD activity was neurons in the SNc and TH‑immunoreactive (TH‑ir) DA expressed as U/mg protein. nerve fibers in the striatum To evaluate the ROT-induced neurodegeneration and Assay for pro‑inflammatory cytokines neuroprotective effect of NRD, the total number of Commercially available ELISA kits for IL-1β, IL-6, and TH+ DA neurons at 3 different anatomical levels of the TNF-α were purchased from R&D systems, Minneapo- SNc (−4.8, −5.04, and −5.28  mm of the bregma) were lis, MN, USA. The level of IL-1β, IL-6, and TNF-α were counted. The average number of TH+ neurons were estimated as described earlier [22]. The results were calculated and converted as a percentage with reference expressed as pg/mg protein. to the control. The loss of striatal fibers was evaluated by measuring the intensity of TH-ir dopaminergic fibers Immunohistochemistry for tyrosine hydroxylase (TH) in the striatum using Image J software (NIH, Bethesda, expression MD, USA). The intensity of the TH-immunoreactive The one hemisphere of the brains collected from each nerve fibers in 3 different fields of brain sections (3 sec - animal were serially cut after fixation and processed for tions per animal) within the striatal region (adjacent to immunohistochemical analysis. Briefly, 14 μm-thick cor - 0.3  mm of the bregma) was measured to examine the onal brain sections were cut at the level of the striatum DA nerve fibers loss. An average of the 3 sections was and SNc using a cryostat (Leica, Wetzlar, Germany) for calculated and presented as a percentage with refer- the immunohistochemical analysis of TH. Sections were ence to the values of the control group. The intensity of washed twice with 0.01 M of PBS, pH 7.4, and then incu- the overlying cortex area was used as the background bated with blocking reagent (10 % normal goat serum in measurement and subtracted from the value generated PBS containing 0.3 % Triton-X 100) for 1 h. Next, the sec- from the striatum. An investigator that was ‘blind’ to the tions were incubated overnight with a primary polyclonal experimental groups counted the TH+ DA neurons and rabbit antibody against TH (1:500) at 4 °C. Sections were determined the immunoreaction intensity of the TH washed and incubated with biotinylated secondary anti- fibers. rabbit (1:1000) antibody for 1  h at room temperature. The brain sections were incubated with avidin–biotin Assessment of activated astrocytes and microglia in the complex (Vector Laboratories Ltd. Burlingame, CA, striatum USA) and 3,3′ diaminobenzidine (DAB) to visualize and Three coronal sections from a comparable anatomical analyze the TH immunoreactivity. Finally, the sections level of striatum from each animal were used to analyze were coverslipped using DPX mounting medium. The and count the number of activated astrocytes and micro- slides were then viewed under a light microscope (Olym- glia. Activated astrocytes and microglia were considered pus, Hamburg, Germany) and images were acquired for for counting based on the intense immunoreactivity of analysis. GFAP- and Iba-1-labeled cells, whose characteristic mor- phological features include hypertrophied and extended Immunofluorescence staining of GFAP and Iba‑1 glial processes. The total number of activated astrocytes Immunofluorescence staining was performed using and microglia were counted from 3 randomly chosen 14  µm-thick striatum sections to quantify the number fields of an equal area in each section using the Image Javed et al. BMC Neurosci (2016) 17:58 Page 5 of 12 J software (NIH, Bethesda, MD, USA), and the results that chronic ROT treatment caused a significant loss were presented as a percentage. (p  <  0.05) of DA neurons in the SNc area as expected when compared to vehicle-injected control rats (Fig.  2a, Western blot analysis of COX‑2 and iNOS expression c). In contrast, NRD supplementation prior to ROT Western blot analysis was carried out to measure the injection in rats provided significant (p < 0.05) protection level of COX-2 and iNOS expression in different groups to DA neurons when compared to only ROT injection of animals following the protocol, as reported earlier of rats. The DA neurons of the SNc project their nerve [22]. Briefly, striatal tissues isolated from each animal terminals to the striatum where they are highly enriched were homogenized in radioimmuno-precipitation buffer with the dopamine transporter (DAT). Degeneration with protease and phosphatase inhibitors. The cell lysates of DA neurons in the SNc decreases DAT expression in were then centrifuged at 15,000  rpm for 20  min. The the striatum, which further confirms the neuronal loss. supernatant containing cytoplasmic fractions was iso- Therefore, we examined whether the loss of DA neu - lated, and protein concentration was measured as men- rons in the SNc area is correlated with DA-terminal loss tioned below. The cytoplasmic fraction containing equal by measuring the intensity of TH-ir dopaminergic nerve amounts of protein (35  μg) were loaded and separated terminal fibers of the striatum. A significant decrease using 10 % SDS–polyacrylamide gel electrophoresis. The (p < 0.05) was observed in the intensity of TH-ir fibers in proteins were then transferred onto a PVDF membrane ROT-injected animals when compared to vehicle-treated and incubated overnight at 4  °C with specific primary controls. However, NRD supplementation prior to ROT rabbit polyclonal antibodies against COX-2 (1:1000) and administration significantly increased (p  <  0.05) TH-ir iNOS (1:500). The membrane was washed and then incu - intensity in nerve terminal fibers, suggesting that NRD bated with horseradish peroxidase-conjugated secondary has a potent neuroprotective effect against ROT-induced anti-rabbit antibody. The protein recognized by the anti - dopaminergic neurodegeneration (Fig. 2b, d). body was visualized using an enhanced chemilumines- cence Pico kit (Thermo Fisher Scientific, Rockford, IL, NRD treatment suppresses excessive lipid peroxidation USA). The blots were stripped and re-probed for β-actin and ameliorates GSH levels (1:5000; monoclonal mouse, Millipore, MA, USA) as a Next, we tested whether NRD mediated the neuropro- loading control. The intensity of the bands was measured tective effect that we observed in our model is due to using densitometry and quantified using Image J software its antioxidant activity because NRD is an antioxidant. (NIH, Bethesda, USA). To clarify this issue, the role of NRD in ROT-induced neurodegeneration was investigated by examining the Protein estimation level of MDA, a marker for lipid peroxidation in the The concentration of protein in each sample was esti - midbrain region. As shown in Fig.  3a, ROT treatment mated using the Pierce BCA protein assay kit (Thermo resulted in a massive amount of MDA production in the Fisher Scientific, Rockford, IL, USA) following the manu - ROT group of animals when compared to control rats. facturer’s instructions. Treatment with NRD significantly abolished (p  <  0.05) the elevated MDA production. Increased MDA produc- Statistical analyses tion due to oxidative stress correlated with a decrease in The data were expressed as the mean value  ± SEM. The the bioavailability of the tripeptide antioxidant GSH in data were analyzed with Graph Pad (InStat software, midbrain cells. The ROT-challenged rats also showed a La Jolla, CA, USA) using a one-way analysis of variance significantly decreased (p < 0.05) level of GSH compared (ANOVA) followed by a Tukey’s test to determine the to vehicle-injected control rats (Fig.  3b). Supplementa- statistical significance between various groups. For all of tion with NRD prior to ROT administration significantly the tests, the criterion for any statistically significant dif - attenuated (p  <  0.05) the level of GSH compared to the ference was set at p < 0.05. ROT-injected rats. Taken together, these results suggest that the neuroprotective effect of NRD on ROT-induced Results neurodegeneration might be mediated through its anti- NRD administration prevents the loss of DA neurons in the oxidative effect. SNc and DA nerve fibers in the striatum To investigate the beneficial effect of NRD on DA neu - NRD reverses the ROT‑induced decrease in the activity rodegeneration in ROT-injected rats, TH immunohisto- of antioxidant enzymes chemical analysis was performed to assess the expression We also measured the activity of antioxidant enzymes of healthy TH+ DA neurons in the SNc and the TH-ir SOD and CAT. ROT administration significantly DA nerve fibers density in the striatum. The results show decreased (p  <  0.01) SOD and CAT activity when Javed et al. BMC Neurosci (2016) 17:58 Page 6 of 12 Fig. 2 Expression of tyrosine hydroxylase ( TH)-immunopositive ( TH+) dopamine (DA) neurons in the substantia nigra pars compacta (SNc) and TH-immunoreactive ( TH-ir) dopaminergic nerve fibers in the striatum. The scale bar is 100 µm. a The number of TH+ neurons was decreased in the SNc region of the rotenone (ROT )-injected rats compared to the control (CONT ) group. In contrast, nerolidol (NRD) administration rescued the TH+ neurons in the ROT + NRD injected rats compared to the ROT rats. b The expression of TH-ir fibers in the striatum of the CONT, ROT, ROT + NRD, and NRD-only group of rats. c The number of TH+ positive DA neurons in the SNc was counted from each group. A significant decrease (*p < 0.05) in the number of DA neurons was observed in the SNc of the ROT group compared to the CONT group. NRD treatment significantly ( p < 0.05) pro- tected the DA neurons from the ROT-induced neuronal death. No significant difference was observed in the DA neurons of the CONT and NRD-only group. Values are expressed as percent mean ± SEM (n = 3). d A significant decrease (*p < 0.05) in the TH-ir fibers was observed in the ROT group compared to the CONT group. NRD treatment significantly inhibited ( p < 0.05) the loss of TH-ir fibers in the ROT + NRD group compared to the ROT group. The CONT rats and NRD-only-injected rats did not show a remarkable loss of TH-ir fibers compared with the vehicle-injected control group. How- Immunofluorescence staining showed a significant ever, NRD supplementation significantly increased increase (p < 0.01) in the number of activated astrocytes (p < 0.05) the activities of SOD (Fig. 3c) and CAT (Fig. 3d) and microglia in ROT-injected animals compared to when compared with the ROT-injected animals. Control the vehicle-injected control animals (Fig.  4a–d). Inter- animals did not show any significant changes in the activ - estingly, NRD treatment to the ROT-injected rats sig- ities of SOD and CAT when compared to the NRD-only- nificantly decreased (p  <  0.05) the number of activated injected rats. astrocytes and microglia compared to the ROT-injected rats. Animals injected with NRD alone did not show any Neuroprotective effects of NRD involves the inhibition remarkable activation of astrocytes and microglia when of glial cell activation compared to control animals. Glial cell (astrocyte and microglia) activation follow- ing ROT administration has been observed, and is con- NRD attenuates the ROT‑induced release sidered to be an index of the inflammatory response. of proinflammatory cytokines We observed an increase in the expression of GFAP The persistent activation of astrocytes and microglia and Iba-1, which are markers of activated astrocytes accompanied by the sustained secretion of inflamma - and microglia, respectively, upon ROT administration. tory mediators is thought to be involved in the neuronal Javed et al. BMC Neurosci (2016) 17:58 Page 7 of 12 Fig. 3 Quantification of malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT ) in midbrain tissue. Rotenone (ROT ) exposure induced a significant increase (**p < 0.01) in the MDA level (a) and a decrease in the GSH level (b) in the midbrain of the ROT- challenged rats compared to the vehicle injected control (CONT ) rats. NRD supplementation to the ROT-administered rats significantly decreased # ## ( p < 0.05) the level of MDA, and increased ( p < 0.01) the level of GSH. ROT injection also significantly decreased (**p < 0.01) the activity of SOD (c) and CAT (d) compared to the CONT rats. NRD supplementation significantly inhibited ( p < 0.05) the ROT-induced decrease in SOD and CAT activity compared to the ROT-injected rats. Values are expressed as the mean ± SEM (n = 6–8) injury in PD. Therefore, the release of proinflammatory the ROT-administered rats, a remarkable reduction in cytokines such as IL-1β, IL-6, and TNF-α in response the level of COX-2 (148.16 vs. 217.42  %) was observed to ROT exposure was examined and quantified using when compared to the ROT-treated rats. ROT adminis- ELISA. A significant increase (p  <  0.01) in the IL-1β, tered animals also showed a significant increase (p < 0.05, IL-6, and TNF-α level was observed in the ROT-injected 206.35 vs. 100 %) in iNOS expression (Fig. 6d) when com- animals when compared to the vehicle-injected control pared to the control animals. Interestingly, NRD supple- animals (Fig.  5a–c). However, NRD supplementation to mentation to the ROT-injected rats modestly decreased the ROT-administered animals significantly decreased (156.16 vs. 206.35  %) iNOS induction when compared (p  <  0.05) the level of these proinflammatory cytokines to the ROT-treated rats. Treatment with only NRD when compared to the ROT-injected animals (Fig. 5a–c). decreased COX-2 expression by 10.1  % (89.9 vs. 100  %), Animals injected with NRD alone did not show any sig- whereas a slight increase of 2.76  % (102.76 vs. 100  %) in nificant changes in the level of analyzed proinflammatory iNOS expression was observed when compared to the cytokines when compared to control animals. control group. Eec ff t of NRD on the expression of COX‑2 and iNOS Discussion The expressions of COX-2 and iNOS were also exam - PD is one of the most common progressive neurodegen- ined using Western blot in tissue lysates that were iso- erative disorders with a complex pathogenesis. The cur - lated from the striatum region (Fig.  6a, b). A significant rent hypothesis suggests that mitochondrial dysfunction, increase (p  <  0.05) in COX-2 expression (Fig.  6c) was oxidative stress, and neuroinflammation play a crucial observed in ROT-treated animals (217.42 vs. 100  %) role in the development of PD. Systemic administration when compared to the vehicle-injected control group of ROT to rats has been shown to induce behavioral and of animals. However, following treatment with NRD in pathological symptoms of PD, such as motor dysfunction, Javed et al. BMC Neurosci (2016) 17:58 Page 8 of 12 Fig. 4 Striatal glial fibrillary acidic protein (GFAP)-positive astrocytes and ionized calcium binding adaptor molecule-1 (Iba-1)-positive microglia. Profound expression of GFAP-positive astrocytes (green, a) and Iba-1-positive microglia (green, b) was found in the ROT-administered rats compared to the vehicle-injected CONT rats. In contrast, NRD supplementation to the ROT injected rats showed moderate staining of GFAP and Iba-1 com- pared to the ROT-injected rats (scale bar = 200 µm). Quantitative analysis of activated astrocytes (c) and microglia (d) revealed a significant increase (**p < 0.01) in the number of activated astrocytes and microglia in the ROT group rats compared to the CONT group. However, NRD supplemen- tation significantly reduced ( p < 0.05) the number of activated astrocytes and microglia in the ROT + NRD group compared to the ROT group. CONT rats and NRD-only-injected rats did not show any marked difference in the activation of astrocytes and microglia. Values are expressed as the percent mean ± SEM (n = 3) dopaminergic neurodegeneration, oxidative stress, 70  % of the dopaminergic neurons are dead resulting in inflammation, and α-synucleinopathy [6, 22, 24, 25]. In the retraction of dopaminergic nerve terminals in the the present study, the ROT-induced PD model was used striatum and depletion of DA in the striatum [27]. There - to investigate the neuroprotective potential of the NRD fore, the death of dopaminergic neurons in the SNc is phytochemical, which recently showed potential antiox- believed to decrease the level of striatal dopamine. The idant activity [15]. ROT at a dose of 2.5  mg/kg of body loss of dopaminergic neurons in the SNc and reduced weight once daily was used for 4  weeks to induce dopa- density of striatal nerve terminals are considered to be minergic neurodegeneration. Our results show the novel the pathological hallmark of human PD [28]. potential benefits of NRD in attenuating dopaminergic In the present study, immunohistochemical examina- neurodegeneration, improving antioxidant enzymes, and tion of TH revealed that ROT causes a significant loss of inhibiting inflammatory mediators and lipid peroxidation TH+ dopaminergic neurons in the SNc area and the den- in the brain. sity of dopaminergic nerve terminal fibers in the striatum, Tyrosine hydroxylase (TH) is a rate-limiting enzyme which concurs with previous reports [22, 25]. Interestingly, that catalyzes the synthesis of the DA neurotransmit- NRD supplementation to ROT-treated rats significantly ter, which is synthesized in the dopaminergic neurons protected against the ROT-induced loss of dopaminergic of the SNc area. DA is stored in synaptic vesicles and, in neurons in the SNc and reduced striatal nerve fiber density response to stimuli, released in the striatum to exert its in the striatum. This result indicates that NRD has neuro - physiological function [26]. In PD patients, more than protective effects that prevent dopaminergic neuron loss Javed et al. BMC Neurosci (2016) 17:58 Page 9 of 12 Fig. 5 Proinflammatory cytokines in the midbrain tissue of the CONT, ROT, ROT + NRD, and NRD-only groups. The level of IL-1β (a), IL-6 (b), and TNF-α (c) was significantly increased (**p < 0.01) in the ROT-treated group when compared to the CONT group. However, NRD treatment signifi- cantly decreased ( p < 0.05) the ROT-induced increase of these proinflammatory cytokines in the ROT + NRD group. No significant differences were observed in these cytokines between the CONT and NRD-only group. Values are expressed as the mean ± SEM (n = 6–7) Fig. 6 Western blot analysis of COX-2 and iNOS expression in striatal tissue. COX-2 (a) and iNOS (b) expression levels were determined using Western blotting in the striatum. The ROT-administered group showed significant increase (*p < 0.05, 217.42 vs. 100 %) in the COX-2 level compared to the CONT group. NRD supplementation to the ROT-injected rats remarkably decreased the expression level of COX-2 (148.16 vs. 217.42 %) compared to the ROT group (c). Likewise, iNOS expression was also significantly increased (*p < 0.05, 206.35 vs. 100 %) in the ROT group compared to the CONT group. NRD treatment markedly decreased the iNOS expression (156.16 vs. 206.35 %) compared to the ROT group (d). NRD-only treat- ment decreased (89.9 vs. 100 %) COX-2 expression, while an increase (102.76 vs. 100 %) in iNOS expression was observed when compared to the CONT group (n = 3) Javed et al. BMC Neurosci (2016) 17:58 Page 10 of 12 and striatal nerve terminal retraction in ROT-induced PD stress in ROT-treated rats. Complex-I of the mitochon- in rats. A convincing number of studies have shown that drial respiratory chain is the major source of superoxide dopaminergic neurons exist in a state of constant oxida- radicals through inhibition of the electron transport chain tive stress, as brain cells have a low antioxidant defense [28]. The diminished activity of SOD would be detrimen - capacity and a tendency to generate reactive oxygen spe- tal in the scenario when superoxide radical production is cies due to the presence of highly oxidizable DA [29, increased. Our present data showed decreased activity of 30]. In our study, NRD administration protected against SOD in ROT-challenged rats compared to control rats. ROT-induced dopaminergic neurodegeneration possibly The administration of NRD was beneficial, because this through its strong antioxidant action, as NRD shows effi - treatment efficiently restored the SOD activity in ROT- cient antioxidant activity in the hippocampus [15]. injected rats. This result corroborated a previous report Defects in complex-I of the mitochondrial electron showing an increase in SOD activity after NRD adminis- transport chain leads to a massive release of free radi- tration [15]. CAT is an enzyme that is responsible for cat- cals, and consequently, cellular death [31]. ROT, which is alyzing the decomposition of H O . The maintenance of a 2 2 an inhibitor of complex-I of the mitochondrial electron normal metabolism of reactive oxygen species is impor- transport chain, upon systemic administration to rats, tant for proper cell function in different body parts [35, causes the loss of ATP production and subsequently gen- 36]. In the present study, CAT activity was significantly erates reactive oxygen species [30]. Furthermore, reactive diminished in ROT-injected rats, while the administration oxygen species induce the oxidation of polyunsaturated of NRD significantly reversed the decreased activity of fatty acid in a process called lipid peroxidation, which is CAT in NRD-supplemented rats. NRD supplementation characterized by the formation of MDA, a major prod- reduced oxidative damage, as observed by the decrease in uct of lipid peroxidation that forms adducts with DNA lipid peroxidation, restoration of the GSH level, and activ- bases and proteins, thereby causing cellular damage. The ities of the antioxidant enzymes (SOD and CAT) follow- increased level of oxidative damage to DNA, proteins, ing ROT administration in rats. and lipids, and decreased level of antioxidants have been Furthermore, ROT-treated animals showed an increase reported in the brain of PD patients [4]. Significantly in neuroinflammation that is triggered and sustained increased levels of MDA in the ROT-treated rats were through various mechanisms. Dysfunction of complex-I observed compared to the vehicle-injected control rats. of the mitochondrial respiratory chain due to ROT treat- Interestingly, NRD supplementation to the ROT-injected ment increased the release of reactive oxygen species, rats significantly lowered the levels of MDA compared to which can trigger the activation of glial cells [37]. A large the ROT-injected rats. Notably, NRD ameliorates against number of studies report the involvement of neuroin- oxidative stress due to lipid peroxidation in the mouse flammation in PD, which is largely characterized by an hippocampus [15]. The inhibition of lipid peroxidation accumulation of activated microglia [38]. The activation by NRD in the current study may have been mediated of astrocytes and microglia results in increased expres- through the detoxification of peroxy radicals and reactive sion of GFAP and Iba-1, respectively. Microglial activa- oxygen species, which further supports an antioxidative tion also causes NO· overproduction via iNOS induction role for NRD. [39]. Robust microgliosis and increased expression of GSH is an important antioxidant and has a crucial role iNOS have also been reported in postmortem brain sam- in scavenging hydrogen peroxide. The decreased lev - ples of PD patients [40, 41]. Proinflammatory cytokines els of GSH in the brain might indicate a state of oxida- such as IL-1β, IL-6, and TNF-α, and enzymes such as tive stress. Decreased levels of GSH have been observed iNOS and COX-2 appear to be involved in DA toxicity, in surviving dopaminergic neurons of the SNc of PD and have been observed in the cerebrospinal fluid and patients compared to age-matched controls [32]. In the postmortem brain tissue samples of PD patients [40, present study, the GSH level was significantly lower in 42–45]. ROT-treated rats than in control rats. Decreased levels In addition, inflammatory processes that are associ - of GSH lead to oxidative damage to DNA, protein, and ated with the increased expression of COX-2 and iNOS lipids in PD [32–34]. Administration of NRD significantly are involved in the cascade of deleterious events that increased GSH levels compared to the ROT injected and leads to neurodegeneration in PD [46]. In line with this vehicle-treated rats (Group II). The effect of NRD on report, here we showed an increase in activated astro- GSH may involve the direct antioxidant effects of NRD or cytes and microglia with a concomitantly enhanced level the prevention of ROT-induced GSH oxidation. of the proinflammatory cytokines IL-1β, IL-6, and TNF-α SOD and CAT are the main antioxidant enzymes in ROT-treated rats, which was significantly attenuated involved in detoxifying free radicals. The loss of SOD following NRD supplementation. Additionally, NRD sup- activity further contributes to an increase in oxidative plementation normalized the expression of COX-2 and Javed et al. BMC Neurosci (2016) 17:58 Page 11 of 12 Ethical approval animal experiments iNOS in the ROT-injected rats. Therefore, the present The Animal Ethics Committee of United Arab Emirates University, UAE, findings further suggest that NRD exerts its anti-inflam - approved the experimental protocol for animal experimentation. The protocol matory effects through inhibiting the increased level of number is A23-14. COX-2 and iNOS, as well as proinflammatory cytokines. Funding The NRD-mediated suppression of inflammation, as We duly acknowledge the grants supporting this research from the United observed in the present study, could be facilitated by the Arab Emirates University and the National Research Foundation, United Arab Emirates to MEH (Grant # 31M123) and SO (Grant # 31M278). strong anti-inflammatory activity of NRD that has been reported in other studies [47, 48]. NRD has also been Received: 28 January 2016 Accepted: 11 August 2016 shown to inhibit the lipopolysaccharide-induced release of pro-inflammatory cytokines in bone marrow-derived dendritic cells, and diminish the induction of inflamma - tory mediators in an in  vivo acute model of inflamma - References tion [49, 50]. Oxidative stress and neuroinflammation 1. Moore DJ, West AB, Dawson VL, Dawson TM. 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Neuroprotective effect of nerolidol against neuroinflammation and oxidative stress induced by rotenone

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Springer Journals
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Copyright © 2016 by The Author(s)
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Biomedicine; Neurosciences; Neurobiology; Animal Models
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Abstract

Background: Parkinson disease (PD) is a movement disorder affecting 1 % of people over the age of 60. The etiology of the disease is unknown; however, accumulating evidence suggests that mitochondrial defects, oxidative stress, and neuroinflammation play important roles in developing the disease. Current medications for PD can only improve its symptoms, but are unable to halt its progressive nature. Although many therapeutic approaches are available, new drugs are urgently needed for the treatment of PD. Thus, the present study was undertaken to investigate the neuroprotective potential of nerolidol, a sesquiterpene alcohol, on a rotenone-induced experimental model of PD, where male Wistar rats intraperitoneally received rotenone (ROT ) at a dose of 2.5 mg/kg of body weight once daily for 4 weeks. Results: Nerolidol, which has antioxidant and anti-inflammatory properties, was injected intraperitoneally at 50 mg/ kg of body weight, once daily for 4 weeks, and at 30 min prior to ROT administration. ROT administration significantly reduced the activities of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT ), and the level of the antioxidant tripeptide glutathione (GSH). Moreover, ROT increased the levels of the lipid peroxidation product malon- dialdehyde (MDA), proinflammatory cytokines (IL-1β, IL-6, and TNF-α), and inflammatory mediators (COX-2 and iNOS) in rat brain tissues. Immunostaining of brain tissue sections revealed a significant increase in the number of activated astrocytes (GFAP) and microglia (Iba-1), along with the concomitant loss of dopamine (DA) neurons in the substantia nigra pars compacta and dopaminergic nerve fibers in the striatum of ROT-treated rats. As expected, nerolidol sup - plementation to ROT-injected rats significantly increased the level of SOD, CAT, and GSH, and decreased the level of MDA. Nerolidol also inhibited the release of proinflammatory cytokines and inflammatory mediators. Finally, nerolidol treatment prevented ROT-induced glial cell activation and the loss of dopaminergic neurons and nerve fibers, and ultimately attenuated ROT-induced dopaminergic neurodegeneration. Conclusion: Our findings are the first to show that the neuroprotective effect of nerolidol is mediated through its anti-oxidant and anti-inflammatory activities, which strongly supports its therapeutic potential for the treatment of PD. Keywords: Parkinson disease, Rotenone, Neurodegeneration, Oxidative stress, Nerolidol *Correspondence: shreeshojha@uaeu.ac.ae; ehaque@uaeu.ac.ae Department of Biochemistry, College of Medicine and Health Sciences, United Arab Emirates University, PO Box 17666, Al Ain, UAE Department of Pharmacology and Therapeutics, College of Medicine and Health Sciences, United Arab Emirates University, PO Box 17666, Al Ain, UAE © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Javed et al. BMC Neurosci (2016) 17:58 Page 2 of 12 obtained from medicinal plants exhibit a variety of bio- Background logical properties, such as anticonvulsant [11], analgesic Parkinson disease is a neurodegenerative disorder that [12], anxiolytic [13], antidepressant [14], antioxidant [15], is characterized by a progressive loss of dopamine (DA) and anti-inflammatory [16] properties. Some of these neurons in the substantia nigra pars compacta (SNc) effects are frequently attributed to terpenes, which are and dopaminergic nerve terminal fibers of the striatum chemical components that are present in these essential that are enriched in the DA transporter [1]. As a result oils [11, 17]. Nerolidol (NRD) [3,7,11-trimethyl-1,6,10- of dopaminergic neurodegeneration, the level of the DA dodecatrien-3-ol] is a sesquiterpene alcohol (Fig.  1) that neurotransmitter (which mediates the control of move- is found in essential oils from plants such as Baccharis ment) is significantly reduced in the striatum. Therefore, dracunculifolia [18], Amaranthus retroflexus [ 19], and dopaminergic neurodegeneration leads to impairments Canarium schweinfurthii [20]. Nerolidol is an aliphatic in movement, a resting tremor, rigidity, and a disturbance sesquiterpene that is commonly used in cosmetics, per- in gait. Although the etiology for dopaminergic neuronal fumes, shampoos, and soaps [21], as well as non-cosmetic degeneration in PD is not completely understood, cur- products (cleansers and detergents). Essential oils from rent evidence suggests that oxidative stress and neuroin- Ferula fukanensis containing nerolidol have been shown flammation play key roles in the pathogenesis of PD [2, to prevent nitric oxide (NO) production and NO-induced 3]. gene expression, suggesting its plausible use as an antioxi- Rotenone (ROT), an inhibitor of mitochondrial com- dant agent. NRD is hydrophobic in nature with an XlogP3 plex I, has been widely used as a herbicide/pesticide in value of 4.6 (>2.5 is needed for efficient transport across agriculture. A ROT challenge, due to its complex I inhibi- blood brain barrier), and thus NRD can cross easily the tory property, induces pathological features in animals blood brain barrier. NRD significantly reduces the level that are similar to those seen in human PD patients. of lipid peroxidation and nitrite content in the hippocam- u Th s, ROT-treated animals represent a promising ani - pus of mice, which protects against oxidative stress [15], mal model with construct validity [4–6]. The ROT model suggesting an important antioxidant property for NRD. recapitulates most of the pathological features observed A recent study reported that NRD reduces interleukin-1β in human PD pathogenesis, including the loss of DA neu- (IL-1β) production in lipopolysaccharide-induced perito- rons in SNc, and enhanced oxidative stress and neuroin- neal macrophages [16]. Additionally, NRD shows potent flammation in the nigrostriatal dopaminergic pathway [2, anti-inflammatory activity through the suppression of 6–8]. Moreover, a ROT challenge to rodents induced the IL-1β and tumor necrosis factor-alpha (TNF-α) levels in formation of α-synuclein cytoplasmic inclusions in DA an experimental mouse model of pain [16]. Therefore, the neurons, Lewy pathology, DJ-1 acidification and trans - main objective of the present study was to investigate the location, proteasomal dysfunction, and nigral iron accu- anti-oxidative and anti-inflammatory effects of NRD on mulation [9]. Therefore, the ROT-treated rat is a suitable ROT-induced neurodegeneration in rats. model for investigating novel therapeutic agents target- ing oxidative stress and neuroinflammation in PD. Methods Currently available drugs alleviate the symptoms of Drugs and chemicals PD, but are inadequate in terms of halting the progres- Polyclonal rabbit anti-cyclooxygenase-2 (COX-2), anti- sion of the disease. Additionally, motor complications inducible nitric oxide synthase (iNOS), and anti-glial during advanced stages of the disease, the adverse effects fibrillary acidic protein (GFAP) antibodies were pur - of the available drugs, and non-motor symptoms remain chased from Abcam, Cambridge, MA, USA. Anti-ionized huge challenges for long-term therapy. Therefore, newer calcium-binding adaptor molecule-1 (Iba-1) polyclonal therapeutic agents and approaches are urgently needed rabbit antibody was procured from Wako Chemicals, to stop or delay the progressive nature of the disease [10]. Richmond, VA, USA. Polyclonal rabbit anti-tyrosine PD is intimately connected to excessive oxidative stress hydroxylase antibody was obtained from Novus Biologi- that damages brain areas and accelerates the process of cals, Littleton, CO, USA. Alexa fluor 488 conjugated sec - neurodegeneration, especially in the nigrostriatal area. ondary goat anti-rabbit antibodies were purchased from This process is an important breakpoint for controlling Life Technologies, Grand Island, NY, USA. Biotinylated the disease. Consequently, in addition to other therapeu- secondary anti-rabbit antibody was purchased from tic approaches, treatment with antioxidants is gradually Jackson Immunoresearch, West Grove, PA, USA. Nero- gaining importance in the pharmacotherapy of PD. lidol was purchased from Santa Cruz Biotechnology Inc., Plants are a potential source of novel drugs to benefit CA, USA. ROT and the assay kit for reduced glutathione mankind. Much research effort has focused on discover - (GSH) and other reagents of analytical grade were pur- ing new antioxidant compounds from plants. An increas- chased from Sigma-Aldrich, St. Louis, MO, USA. ing number of studies have shown that essential oils Javed et al. BMC Neurosci (2016) 17:58 Page 3 of 12 Tissue collection At the end of the experiment, the animals from all groups were anaesthetized with pentobarbital (40 mg/kg of body weight) and a cardiac perfusion was performed using 0.01  M of phosphate-buffered saline (PBS) pH 7.4 to completely clear the blood. The skull of the rat was open Fig. 1 Chemical structure of nerolidol to quickly isolate the brain. The brain was placed on an ice-plate and cut along the midline to separate the 2 cer- ebral hemispheres. For biochemical assay, the midbrain Experimental animals and striatum regions of the brain were isolated from 1 We used male Wistar rats at 6–7  months old (weighing hemisphere and immediately fresh frozen in liquid nitro- 280–300 g) from the Animal Research Facility of the Col- gen for further use. The other hemisphere of the brain lege of Medicine and Health Sciences, United Arab Emir- was incubated in 4 % paraformaldehyde solution for 48 h ates University, UAE. Prior to the start of the experiment, and subsequently exchanged with 10  % sucrose solution a maximum of 4 rats were housed per cage, and rats were containing 0.1  M of phosphate buffer (PB) 3 times daily acclimatized for 1 week to the laboratory conditions. The for 3 consecutive days at 4 °C prior to cryostat sectioning. cages of the animals were changed twice a week. The animals were housed and kept under standard labora- Sample preparation for biochemical assay tory light and dark cycle conditions. The animals were For biochemical assay, the midbrain tissue of each ani- fed with commercially available rodent food and water mal was homogenized in KCl buffer (10-mM Tris–HCl, ad  libitum. All experiments were conducted between 140-mM-NaCl, 300-mM KCl, 1-mM EDTA, and 0.5  % 0900 and 1500  hours. The Animal Ethics Committee of Triton X-100) at pH 8.0 supplemented with protease the United Arab Emirates University, UAE, approved the and phosphatase inhibitor, keeping the samples on ice. experimental protocol for animal experimentation. The tissue homogenates of each sample were centrifuged at 14,000×g for 20  min at 4  °C to obtain the post-mito- Experimental design chondrial supernatant (PMS) fraction. This PMS fraction To induce the PD in rats, ROT (2.5 mg/kg body weight) was was used to estimate the levels of the antioxidant enzyme administered intraperitoneally (i.p.) once daily for 4 weeks. GSH, lipid peroxidation product, and proinflammatory The regimen used in the current study for the induction cytokines using spectrophotometric measurements and of Parkinsonism in rats was adopted as reported earlier an enzyme-linked immunosorbent assay (ELISA) follow- [22]. Briefly, ROT was first dissolved in dimethyl sulfox - ing a standard protocol, as reported earlier [22]. ide (DMSO) to prepare a 50X stock solution and stored at −80 °C for further use. Before injection, the stock solu- Assay for lipid peroxidation tion of ROT was thawed and further diluted in sunflower A malondialdehyde (MDA) assay kit procured from oil to obtain a final concentration of 2.5 mg/mL. To evalu - Northwest Life Science (Vancouver, WA, USA) was ate its neuroprotective efficacy, NRD was initially diluted in used to determine the amount of lipid peroxidation, as olive oil to obtain a final concentration of 50 mg/2 mL, and reported earlier [22]. Briefly, the samples or standards injected i.p. 30 min prior to ROT administration at a dose (250  µL) were incubated in the presence of acid reagent of 50  mg/kg of body weight once daily for 4  weeks. The (250  µL) and thiobarbituric acid (250  µL) and vortexed dose of NRD was selected based on dose–response stud- vigorously. Samples were further incubated for 60 min at ies (data not shown) as well as earlier reports [15, 23]. Rats 60 °C followed by centrifugation at 10,000×g for 2–3 min. that were used as controls received an equivalent volume of The reaction mixture was then aseptically transferred vehicle only. The animals were sacrificed 48 h after the last without disturbing the pellet to a cuvette and the absorb- injection of NRD or ROT or both in combination to com- ance was recorded at 532 nm. The concentration of MDA pletely eliminate these drugs from the body. The rats were was calculated using a standard curve and expressed as divided into 4 experimental groups where each group con- µM MDA/mg protein. sisted of 8 animals; the groups were named as follows: Estimation of GSH Group I, the vehicle-injected control group (CONT). A GSH kit was used to estimate the GSH level, as reported Group II, the rotenone-injected and vehicle-treated earlier [22]. Briefly, the samples were first deproteinized group (ROT). with 5  % 5-sulfosalicylic acid solution and centrifuged Group III, the rotenone-injected and NRD-treated to remove the precipitated protein. The supernatant was group (ROT + NRD). used to measure the GSH level. Samples and standards Group IV, the NRD-only injected group (NRD). Javed et al. BMC Neurosci (2016) 17:58 Page 4 of 12 of different concentrations (10  µL) were added in each of activated GFAP-positive astrocytes and Iba-1-positive well of a 96-well plate and incubated for 5  min with the microglia. The brain sections were first washed twice working mixture (150  µL; assay buffer  +  5,5′-dithiobis with PBS and incubated with blocking reagent (10 % nor- (2-nitrobenzoic acid)  +  glutathione reductase). Diluted mal goat serum in PBS containing 0.3 % Triton-X 100) for NADPH solution (50  µL) was added to each well and 1 h. The sections were then incubated overnight with the mixed properly. The absorbance of the samples was primary polyclonal rabbit antibodies ant-GFAP (1:1000) measured at 412  nm with kinetics capability for 5  min and anti-Iba-1 (1:1000) at 4 °C. The sections were washed using a microplate reader. The results were expressed as and incubated with fluorescent secondary anti-rabbit µM GSH/mg protein. Alexa fluor 488 antibody for 1  h at room temperature. Sections were then washed, mounted on slides, and Assay for antioxidant enzymes activity coverslipped using the mounting medium Fluoroshield The activity of antioxidant enzymes such as superoxide (Sigma Aldrich, St. Louis, MO, USA). The images were dismutase (SOD) and catalase (CAT) were determined captured using a fluorescence microscope EVOS FL following the manufacturer’s instructions of a kit (Cay- (Thermo Fisher Scientific, Waltham, MA, USA). man Chemicals Company, Ann Arbor, MI, USA), as reported earlier [22]. The CAT activity was expressed Assessment of TH‑immunopositive (TH+) dopamine (DA) as nmol/min/mg protein and the SOD activity was neurons in the SNc and TH‑immunoreactive (TH‑ir) DA expressed as U/mg protein. nerve fibers in the striatum To evaluate the ROT-induced neurodegeneration and Assay for pro‑inflammatory cytokines neuroprotective effect of NRD, the total number of Commercially available ELISA kits for IL-1β, IL-6, and TH+ DA neurons at 3 different anatomical levels of the TNF-α were purchased from R&D systems, Minneapo- SNc (−4.8, −5.04, and −5.28  mm of the bregma) were lis, MN, USA. The level of IL-1β, IL-6, and TNF-α were counted. The average number of TH+ neurons were estimated as described earlier [22]. The results were calculated and converted as a percentage with reference expressed as pg/mg protein. to the control. The loss of striatal fibers was evaluated by measuring the intensity of TH-ir dopaminergic fibers Immunohistochemistry for tyrosine hydroxylase (TH) in the striatum using Image J software (NIH, Bethesda, expression MD, USA). The intensity of the TH-immunoreactive The one hemisphere of the brains collected from each nerve fibers in 3 different fields of brain sections (3 sec - animal were serially cut after fixation and processed for tions per animal) within the striatal region (adjacent to immunohistochemical analysis. Briefly, 14 μm-thick cor - 0.3  mm of the bregma) was measured to examine the onal brain sections were cut at the level of the striatum DA nerve fibers loss. An average of the 3 sections was and SNc using a cryostat (Leica, Wetzlar, Germany) for calculated and presented as a percentage with refer- the immunohistochemical analysis of TH. Sections were ence to the values of the control group. The intensity of washed twice with 0.01 M of PBS, pH 7.4, and then incu- the overlying cortex area was used as the background bated with blocking reagent (10 % normal goat serum in measurement and subtracted from the value generated PBS containing 0.3 % Triton-X 100) for 1 h. Next, the sec- from the striatum. An investigator that was ‘blind’ to the tions were incubated overnight with a primary polyclonal experimental groups counted the TH+ DA neurons and rabbit antibody against TH (1:500) at 4 °C. Sections were determined the immunoreaction intensity of the TH washed and incubated with biotinylated secondary anti- fibers. rabbit (1:1000) antibody for 1  h at room temperature. The brain sections were incubated with avidin–biotin Assessment of activated astrocytes and microglia in the complex (Vector Laboratories Ltd. Burlingame, CA, striatum USA) and 3,3′ diaminobenzidine (DAB) to visualize and Three coronal sections from a comparable anatomical analyze the TH immunoreactivity. Finally, the sections level of striatum from each animal were used to analyze were coverslipped using DPX mounting medium. The and count the number of activated astrocytes and micro- slides were then viewed under a light microscope (Olym- glia. Activated astrocytes and microglia were considered pus, Hamburg, Germany) and images were acquired for for counting based on the intense immunoreactivity of analysis. GFAP- and Iba-1-labeled cells, whose characteristic mor- phological features include hypertrophied and extended Immunofluorescence staining of GFAP and Iba‑1 glial processes. The total number of activated astrocytes Immunofluorescence staining was performed using and microglia were counted from 3 randomly chosen 14  µm-thick striatum sections to quantify the number fields of an equal area in each section using the Image Javed et al. BMC Neurosci (2016) 17:58 Page 5 of 12 J software (NIH, Bethesda, MD, USA), and the results that chronic ROT treatment caused a significant loss were presented as a percentage. (p  <  0.05) of DA neurons in the SNc area as expected when compared to vehicle-injected control rats (Fig.  2a, Western blot analysis of COX‑2 and iNOS expression c). In contrast, NRD supplementation prior to ROT Western blot analysis was carried out to measure the injection in rats provided significant (p < 0.05) protection level of COX-2 and iNOS expression in different groups to DA neurons when compared to only ROT injection of animals following the protocol, as reported earlier of rats. The DA neurons of the SNc project their nerve [22]. Briefly, striatal tissues isolated from each animal terminals to the striatum where they are highly enriched were homogenized in radioimmuno-precipitation buffer with the dopamine transporter (DAT). Degeneration with protease and phosphatase inhibitors. The cell lysates of DA neurons in the SNc decreases DAT expression in were then centrifuged at 15,000  rpm for 20  min. The the striatum, which further confirms the neuronal loss. supernatant containing cytoplasmic fractions was iso- Therefore, we examined whether the loss of DA neu - lated, and protein concentration was measured as men- rons in the SNc area is correlated with DA-terminal loss tioned below. The cytoplasmic fraction containing equal by measuring the intensity of TH-ir dopaminergic nerve amounts of protein (35  μg) were loaded and separated terminal fibers of the striatum. A significant decrease using 10 % SDS–polyacrylamide gel electrophoresis. The (p < 0.05) was observed in the intensity of TH-ir fibers in proteins were then transferred onto a PVDF membrane ROT-injected animals when compared to vehicle-treated and incubated overnight at 4  °C with specific primary controls. However, NRD supplementation prior to ROT rabbit polyclonal antibodies against COX-2 (1:1000) and administration significantly increased (p  <  0.05) TH-ir iNOS (1:500). The membrane was washed and then incu - intensity in nerve terminal fibers, suggesting that NRD bated with horseradish peroxidase-conjugated secondary has a potent neuroprotective effect against ROT-induced anti-rabbit antibody. The protein recognized by the anti - dopaminergic neurodegeneration (Fig. 2b, d). body was visualized using an enhanced chemilumines- cence Pico kit (Thermo Fisher Scientific, Rockford, IL, NRD treatment suppresses excessive lipid peroxidation USA). The blots were stripped and re-probed for β-actin and ameliorates GSH levels (1:5000; monoclonal mouse, Millipore, MA, USA) as a Next, we tested whether NRD mediated the neuropro- loading control. The intensity of the bands was measured tective effect that we observed in our model is due to using densitometry and quantified using Image J software its antioxidant activity because NRD is an antioxidant. (NIH, Bethesda, USA). To clarify this issue, the role of NRD in ROT-induced neurodegeneration was investigated by examining the Protein estimation level of MDA, a marker for lipid peroxidation in the The concentration of protein in each sample was esti - midbrain region. As shown in Fig.  3a, ROT treatment mated using the Pierce BCA protein assay kit (Thermo resulted in a massive amount of MDA production in the Fisher Scientific, Rockford, IL, USA) following the manu - ROT group of animals when compared to control rats. facturer’s instructions. Treatment with NRD significantly abolished (p  <  0.05) the elevated MDA production. Increased MDA produc- Statistical analyses tion due to oxidative stress correlated with a decrease in The data were expressed as the mean value  ± SEM. The the bioavailability of the tripeptide antioxidant GSH in data were analyzed with Graph Pad (InStat software, midbrain cells. The ROT-challenged rats also showed a La Jolla, CA, USA) using a one-way analysis of variance significantly decreased (p < 0.05) level of GSH compared (ANOVA) followed by a Tukey’s test to determine the to vehicle-injected control rats (Fig.  3b). Supplementa- statistical significance between various groups. For all of tion with NRD prior to ROT administration significantly the tests, the criterion for any statistically significant dif - attenuated (p  <  0.05) the level of GSH compared to the ference was set at p < 0.05. ROT-injected rats. Taken together, these results suggest that the neuroprotective effect of NRD on ROT-induced Results neurodegeneration might be mediated through its anti- NRD administration prevents the loss of DA neurons in the oxidative effect. SNc and DA nerve fibers in the striatum To investigate the beneficial effect of NRD on DA neu - NRD reverses the ROT‑induced decrease in the activity rodegeneration in ROT-injected rats, TH immunohisto- of antioxidant enzymes chemical analysis was performed to assess the expression We also measured the activity of antioxidant enzymes of healthy TH+ DA neurons in the SNc and the TH-ir SOD and CAT. ROT administration significantly DA nerve fibers density in the striatum. The results show decreased (p  <  0.01) SOD and CAT activity when Javed et al. BMC Neurosci (2016) 17:58 Page 6 of 12 Fig. 2 Expression of tyrosine hydroxylase ( TH)-immunopositive ( TH+) dopamine (DA) neurons in the substantia nigra pars compacta (SNc) and TH-immunoreactive ( TH-ir) dopaminergic nerve fibers in the striatum. The scale bar is 100 µm. a The number of TH+ neurons was decreased in the SNc region of the rotenone (ROT )-injected rats compared to the control (CONT ) group. In contrast, nerolidol (NRD) administration rescued the TH+ neurons in the ROT + NRD injected rats compared to the ROT rats. b The expression of TH-ir fibers in the striatum of the CONT, ROT, ROT + NRD, and NRD-only group of rats. c The number of TH+ positive DA neurons in the SNc was counted from each group. A significant decrease (*p < 0.05) in the number of DA neurons was observed in the SNc of the ROT group compared to the CONT group. NRD treatment significantly ( p < 0.05) pro- tected the DA neurons from the ROT-induced neuronal death. No significant difference was observed in the DA neurons of the CONT and NRD-only group. Values are expressed as percent mean ± SEM (n = 3). d A significant decrease (*p < 0.05) in the TH-ir fibers was observed in the ROT group compared to the CONT group. NRD treatment significantly inhibited ( p < 0.05) the loss of TH-ir fibers in the ROT + NRD group compared to the ROT group. The CONT rats and NRD-only-injected rats did not show a remarkable loss of TH-ir fibers compared with the vehicle-injected control group. How- Immunofluorescence staining showed a significant ever, NRD supplementation significantly increased increase (p < 0.01) in the number of activated astrocytes (p < 0.05) the activities of SOD (Fig. 3c) and CAT (Fig. 3d) and microglia in ROT-injected animals compared to when compared with the ROT-injected animals. Control the vehicle-injected control animals (Fig.  4a–d). Inter- animals did not show any significant changes in the activ - estingly, NRD treatment to the ROT-injected rats sig- ities of SOD and CAT when compared to the NRD-only- nificantly decreased (p  <  0.05) the number of activated injected rats. astrocytes and microglia compared to the ROT-injected rats. Animals injected with NRD alone did not show any Neuroprotective effects of NRD involves the inhibition remarkable activation of astrocytes and microglia when of glial cell activation compared to control animals. Glial cell (astrocyte and microglia) activation follow- ing ROT administration has been observed, and is con- NRD attenuates the ROT‑induced release sidered to be an index of the inflammatory response. of proinflammatory cytokines We observed an increase in the expression of GFAP The persistent activation of astrocytes and microglia and Iba-1, which are markers of activated astrocytes accompanied by the sustained secretion of inflamma - and microglia, respectively, upon ROT administration. tory mediators is thought to be involved in the neuronal Javed et al. BMC Neurosci (2016) 17:58 Page 7 of 12 Fig. 3 Quantification of malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT ) in midbrain tissue. Rotenone (ROT ) exposure induced a significant increase (**p < 0.01) in the MDA level (a) and a decrease in the GSH level (b) in the midbrain of the ROT- challenged rats compared to the vehicle injected control (CONT ) rats. NRD supplementation to the ROT-administered rats significantly decreased # ## ( p < 0.05) the level of MDA, and increased ( p < 0.01) the level of GSH. ROT injection also significantly decreased (**p < 0.01) the activity of SOD (c) and CAT (d) compared to the CONT rats. NRD supplementation significantly inhibited ( p < 0.05) the ROT-induced decrease in SOD and CAT activity compared to the ROT-injected rats. Values are expressed as the mean ± SEM (n = 6–8) injury in PD. Therefore, the release of proinflammatory the ROT-administered rats, a remarkable reduction in cytokines such as IL-1β, IL-6, and TNF-α in response the level of COX-2 (148.16 vs. 217.42  %) was observed to ROT exposure was examined and quantified using when compared to the ROT-treated rats. ROT adminis- ELISA. A significant increase (p  <  0.01) in the IL-1β, tered animals also showed a significant increase (p < 0.05, IL-6, and TNF-α level was observed in the ROT-injected 206.35 vs. 100 %) in iNOS expression (Fig. 6d) when com- animals when compared to the vehicle-injected control pared to the control animals. Interestingly, NRD supple- animals (Fig.  5a–c). However, NRD supplementation to mentation to the ROT-injected rats modestly decreased the ROT-administered animals significantly decreased (156.16 vs. 206.35  %) iNOS induction when compared (p  <  0.05) the level of these proinflammatory cytokines to the ROT-treated rats. Treatment with only NRD when compared to the ROT-injected animals (Fig. 5a–c). decreased COX-2 expression by 10.1  % (89.9 vs. 100  %), Animals injected with NRD alone did not show any sig- whereas a slight increase of 2.76  % (102.76 vs. 100  %) in nificant changes in the level of analyzed proinflammatory iNOS expression was observed when compared to the cytokines when compared to control animals. control group. Eec ff t of NRD on the expression of COX‑2 and iNOS Discussion The expressions of COX-2 and iNOS were also exam - PD is one of the most common progressive neurodegen- ined using Western blot in tissue lysates that were iso- erative disorders with a complex pathogenesis. The cur - lated from the striatum region (Fig.  6a, b). A significant rent hypothesis suggests that mitochondrial dysfunction, increase (p  <  0.05) in COX-2 expression (Fig.  6c) was oxidative stress, and neuroinflammation play a crucial observed in ROT-treated animals (217.42 vs. 100  %) role in the development of PD. Systemic administration when compared to the vehicle-injected control group of ROT to rats has been shown to induce behavioral and of animals. However, following treatment with NRD in pathological symptoms of PD, such as motor dysfunction, Javed et al. BMC Neurosci (2016) 17:58 Page 8 of 12 Fig. 4 Striatal glial fibrillary acidic protein (GFAP)-positive astrocytes and ionized calcium binding adaptor molecule-1 (Iba-1)-positive microglia. Profound expression of GFAP-positive astrocytes (green, a) and Iba-1-positive microglia (green, b) was found in the ROT-administered rats compared to the vehicle-injected CONT rats. In contrast, NRD supplementation to the ROT injected rats showed moderate staining of GFAP and Iba-1 com- pared to the ROT-injected rats (scale bar = 200 µm). Quantitative analysis of activated astrocytes (c) and microglia (d) revealed a significant increase (**p < 0.01) in the number of activated astrocytes and microglia in the ROT group rats compared to the CONT group. However, NRD supplemen- tation significantly reduced ( p < 0.05) the number of activated astrocytes and microglia in the ROT + NRD group compared to the ROT group. CONT rats and NRD-only-injected rats did not show any marked difference in the activation of astrocytes and microglia. Values are expressed as the percent mean ± SEM (n = 3) dopaminergic neurodegeneration, oxidative stress, 70  % of the dopaminergic neurons are dead resulting in inflammation, and α-synucleinopathy [6, 22, 24, 25]. In the retraction of dopaminergic nerve terminals in the the present study, the ROT-induced PD model was used striatum and depletion of DA in the striatum [27]. There - to investigate the neuroprotective potential of the NRD fore, the death of dopaminergic neurons in the SNc is phytochemical, which recently showed potential antiox- believed to decrease the level of striatal dopamine. The idant activity [15]. ROT at a dose of 2.5  mg/kg of body loss of dopaminergic neurons in the SNc and reduced weight once daily was used for 4  weeks to induce dopa- density of striatal nerve terminals are considered to be minergic neurodegeneration. Our results show the novel the pathological hallmark of human PD [28]. potential benefits of NRD in attenuating dopaminergic In the present study, immunohistochemical examina- neurodegeneration, improving antioxidant enzymes, and tion of TH revealed that ROT causes a significant loss of inhibiting inflammatory mediators and lipid peroxidation TH+ dopaminergic neurons in the SNc area and the den- in the brain. sity of dopaminergic nerve terminal fibers in the striatum, Tyrosine hydroxylase (TH) is a rate-limiting enzyme which concurs with previous reports [22, 25]. Interestingly, that catalyzes the synthesis of the DA neurotransmit- NRD supplementation to ROT-treated rats significantly ter, which is synthesized in the dopaminergic neurons protected against the ROT-induced loss of dopaminergic of the SNc area. DA is stored in synaptic vesicles and, in neurons in the SNc and reduced striatal nerve fiber density response to stimuli, released in the striatum to exert its in the striatum. This result indicates that NRD has neuro - physiological function [26]. In PD patients, more than protective effects that prevent dopaminergic neuron loss Javed et al. BMC Neurosci (2016) 17:58 Page 9 of 12 Fig. 5 Proinflammatory cytokines in the midbrain tissue of the CONT, ROT, ROT + NRD, and NRD-only groups. The level of IL-1β (a), IL-6 (b), and TNF-α (c) was significantly increased (**p < 0.01) in the ROT-treated group when compared to the CONT group. However, NRD treatment signifi- cantly decreased ( p < 0.05) the ROT-induced increase of these proinflammatory cytokines in the ROT + NRD group. No significant differences were observed in these cytokines between the CONT and NRD-only group. Values are expressed as the mean ± SEM (n = 6–7) Fig. 6 Western blot analysis of COX-2 and iNOS expression in striatal tissue. COX-2 (a) and iNOS (b) expression levels were determined using Western blotting in the striatum. The ROT-administered group showed significant increase (*p < 0.05, 217.42 vs. 100 %) in the COX-2 level compared to the CONT group. NRD supplementation to the ROT-injected rats remarkably decreased the expression level of COX-2 (148.16 vs. 217.42 %) compared to the ROT group (c). Likewise, iNOS expression was also significantly increased (*p < 0.05, 206.35 vs. 100 %) in the ROT group compared to the CONT group. NRD treatment markedly decreased the iNOS expression (156.16 vs. 206.35 %) compared to the ROT group (d). NRD-only treat- ment decreased (89.9 vs. 100 %) COX-2 expression, while an increase (102.76 vs. 100 %) in iNOS expression was observed when compared to the CONT group (n = 3) Javed et al. BMC Neurosci (2016) 17:58 Page 10 of 12 and striatal nerve terminal retraction in ROT-induced PD stress in ROT-treated rats. Complex-I of the mitochon- in rats. A convincing number of studies have shown that drial respiratory chain is the major source of superoxide dopaminergic neurons exist in a state of constant oxida- radicals through inhibition of the electron transport chain tive stress, as brain cells have a low antioxidant defense [28]. The diminished activity of SOD would be detrimen - capacity and a tendency to generate reactive oxygen spe- tal in the scenario when superoxide radical production is cies due to the presence of highly oxidizable DA [29, increased. Our present data showed decreased activity of 30]. In our study, NRD administration protected against SOD in ROT-challenged rats compared to control rats. ROT-induced dopaminergic neurodegeneration possibly The administration of NRD was beneficial, because this through its strong antioxidant action, as NRD shows effi - treatment efficiently restored the SOD activity in ROT- cient antioxidant activity in the hippocampus [15]. injected rats. This result corroborated a previous report Defects in complex-I of the mitochondrial electron showing an increase in SOD activity after NRD adminis- transport chain leads to a massive release of free radi- tration [15]. CAT is an enzyme that is responsible for cat- cals, and consequently, cellular death [31]. ROT, which is alyzing the decomposition of H O . The maintenance of a 2 2 an inhibitor of complex-I of the mitochondrial electron normal metabolism of reactive oxygen species is impor- transport chain, upon systemic administration to rats, tant for proper cell function in different body parts [35, causes the loss of ATP production and subsequently gen- 36]. In the present study, CAT activity was significantly erates reactive oxygen species [30]. Furthermore, reactive diminished in ROT-injected rats, while the administration oxygen species induce the oxidation of polyunsaturated of NRD significantly reversed the decreased activity of fatty acid in a process called lipid peroxidation, which is CAT in NRD-supplemented rats. NRD supplementation characterized by the formation of MDA, a major prod- reduced oxidative damage, as observed by the decrease in uct of lipid peroxidation that forms adducts with DNA lipid peroxidation, restoration of the GSH level, and activ- bases and proteins, thereby causing cellular damage. The ities of the antioxidant enzymes (SOD and CAT) follow- increased level of oxidative damage to DNA, proteins, ing ROT administration in rats. and lipids, and decreased level of antioxidants have been Furthermore, ROT-treated animals showed an increase reported in the brain of PD patients [4]. Significantly in neuroinflammation that is triggered and sustained increased levels of MDA in the ROT-treated rats were through various mechanisms. Dysfunction of complex-I observed compared to the vehicle-injected control rats. of the mitochondrial respiratory chain due to ROT treat- Interestingly, NRD supplementation to the ROT-injected ment increased the release of reactive oxygen species, rats significantly lowered the levels of MDA compared to which can trigger the activation of glial cells [37]. A large the ROT-injected rats. Notably, NRD ameliorates against number of studies report the involvement of neuroin- oxidative stress due to lipid peroxidation in the mouse flammation in PD, which is largely characterized by an hippocampus [15]. The inhibition of lipid peroxidation accumulation of activated microglia [38]. The activation by NRD in the current study may have been mediated of astrocytes and microglia results in increased expres- through the detoxification of peroxy radicals and reactive sion of GFAP and Iba-1, respectively. Microglial activa- oxygen species, which further supports an antioxidative tion also causes NO· overproduction via iNOS induction role for NRD. [39]. Robust microgliosis and increased expression of GSH is an important antioxidant and has a crucial role iNOS have also been reported in postmortem brain sam- in scavenging hydrogen peroxide. The decreased lev - ples of PD patients [40, 41]. Proinflammatory cytokines els of GSH in the brain might indicate a state of oxida- such as IL-1β, IL-6, and TNF-α, and enzymes such as tive stress. Decreased levels of GSH have been observed iNOS and COX-2 appear to be involved in DA toxicity, in surviving dopaminergic neurons of the SNc of PD and have been observed in the cerebrospinal fluid and patients compared to age-matched controls [32]. In the postmortem brain tissue samples of PD patients [40, present study, the GSH level was significantly lower in 42–45]. ROT-treated rats than in control rats. Decreased levels In addition, inflammatory processes that are associ - of GSH lead to oxidative damage to DNA, protein, and ated with the increased expression of COX-2 and iNOS lipids in PD [32–34]. Administration of NRD significantly are involved in the cascade of deleterious events that increased GSH levels compared to the ROT injected and leads to neurodegeneration in PD [46]. In line with this vehicle-treated rats (Group II). The effect of NRD on report, here we showed an increase in activated astro- GSH may involve the direct antioxidant effects of NRD or cytes and microglia with a concomitantly enhanced level the prevention of ROT-induced GSH oxidation. of the proinflammatory cytokines IL-1β, IL-6, and TNF-α SOD and CAT are the main antioxidant enzymes in ROT-treated rats, which was significantly attenuated involved in detoxifying free radicals. The loss of SOD following NRD supplementation. Additionally, NRD sup- activity further contributes to an increase in oxidative plementation normalized the expression of COX-2 and Javed et al. BMC Neurosci (2016) 17:58 Page 11 of 12 Ethical approval animal experiments iNOS in the ROT-injected rats. Therefore, the present The Animal Ethics Committee of United Arab Emirates University, UAE, findings further suggest that NRD exerts its anti-inflam - approved the experimental protocol for animal experimentation. The protocol matory effects through inhibiting the increased level of number is A23-14. COX-2 and iNOS, as well as proinflammatory cytokines. Funding The NRD-mediated suppression of inflammation, as We duly acknowledge the grants supporting this research from the United observed in the present study, could be facilitated by the Arab Emirates University and the National Research Foundation, United Arab Emirates to MEH (Grant # 31M123) and SO (Grant # 31M278). strong anti-inflammatory activity of NRD that has been reported in other studies [47, 48]. NRD has also been Received: 28 January 2016 Accepted: 11 August 2016 shown to inhibit the lipopolysaccharide-induced release of pro-inflammatory cytokines in bone marrow-derived dendritic cells, and diminish the induction of inflamma - tory mediators in an in  vivo acute model of inflamma - References tion [49, 50]. Oxidative stress and neuroinflammation 1. Moore DJ, West AB, Dawson VL, Dawson TM. 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