Abstract
Background:
Parkinson’s disease (PD) is a progressive neurodegenerative disorder that is related to neuroinflammation. Vanillin, which possesses both antioxidant, and anti-inflammatory properties, can be a candidate for neuroprotection in PD.
Objective:
This study was aimed to investigate the effects of vanillin on the 6-hydroxydopamine (6-OHDA) rodent model of PD.
Methods:
Male Wistar rats were administrated intraperitoneal (i.p) or oral vanillin at a dose of 20 mg/kg/day for 7 days that was started at three days before or seven days after intracerebral injection of 6-OHDA. The 6-OHDA-induced lesions were assessed behaviorally using the apomorphine rotation test, neurochemically via measuring striatal dopamine concentrations, and through immunohistochemistry.
Results:
Both oral and IP vanillin at three days before or seven days after 6-OHDA lesioning exhbited significantly lower tight contralateral rotations upon apomorphine challenge, and higher striatal dopamine concentrations.
Conclusions:
Vanillin seems to offer protective properties against 6-OHDA lesion via preserving striatal dopamine levels.
Introduction
Parkinson’s disease (PD) is primarily characterized by progressive loss of structure in dopaminergic neurons in the substantia nigra pars compacta (SNpc) (Dias et al., 2014; Frisardi et al., 2016) leading to reduction in dopamine concentrations (Kalia & Lang, 2015). Currently, no cure exists for PD, and finding a new treatment for PD is considered a challenging and critical issue as available therapy is only symptomatic (Redensek, Trost, & Dolzan, 2017).
Since the 1990s, phenolic compounds extracted from plants have been used as a flavoring agent in foods, beverages and pharmaceuticals because of their beneficial effect for human health (Tai, Sawano, Yazama, & Ito, 2011). One such compound vanillin, 4-hydroxy-3-methoxybenzaldehyde, which is extracted from the seedpods of Vanilla planifolia. It is the world’s most popular flavor and fragrance compound and it might be a potential agent for the treatment of neurodegenerative disorder (Chinnasamy Dhanalakshmi, Manivasagam, Nataraj, Justin Thenmozhi, & Essa, 2015; Niazi, Sachdeva, Bansal, Gupta, & Kaur, 2014). Vanillin has good permeability and can cross the BBB (Dhanalakshmi et al., 2016; Kim, Choi, & Jung, 2011). Vanillin possesses various biological activities such as antioxidant, anti-inflammatory and antimicrobial effects (Chinnasamy Dhanalakshmi et al., 2015; Kumar & Khanum, 2012). Xu et al. showed that there is an elevated level of both serotonin and dopamine neurotransmitters in brain tissue after a daily oral dose of vanillin (Xu, Xu, Liu, He, & Li, 2015). These findings suggest that vanillin could have use in the treatment of major depressive disorder (Xu et al., 2015). Additionally, vanillin has proved its benefit in vitro using SH-SY5Y cell line demonstrated by Dhanalakshmi et al., where it was found that the pre-treatment of vanillin inhibited mitochondrial dysfunction, oxidative stress, and apoptosis and suggested that vanillin as a potent therapeutic agent in the treatment of neurodegenerative diseases such as PD (Chinnasamy Dhanalakshmi et al., 2015; Gupta & Sharma, 2014). In the current study, the possible protective effect for vanillin on 6-hydroxydopamine rat model of PD was investigated.
Methods
Animals and chemicals
Male Wistar rats (n = 36) weighing between 200– 250 gm were housed in the animal care facility under hygienic conditions. Animals were grouped as three rats per cage. All experiments followed the ethical principles established in the Guide for the Care and Use of Laboratory Animals, USA, 1986. Vanillin (99%) white powder was purchased from loba chemie (Mumbai, India). Desipramine hydrochloride, pargyline, 6-OHDA, apomorphine, and white crystalline l-ascorbic acid were all obtained from Sigma-Aldrich (Pool, UK), isoflurane was purchased from Hikma pharmaceuticals company (Amman, Jordan). Vanillin was dissolved in normal saline and treated animals were subjected to 20 mg/kg/day either by i.p injection or oral gavage for seven consecutive days, whilst 6-OHDA (8μg/4μl) and apomorphine were dissolved in normal saline containing 2% ascorbic acid.
Stereotaxic surgery
Animals were divided into six groups. The control group, which was injected with vehicle. The 6-OHDA group, which received 6-OHDA via intracerebral route. The 6-OHDA + Vanillin IP -3D, which received intraperitoneal (i.p) vanillin (20 mg/kg/day for seven days starting from day -3) and 3 days later (day 0) was administered intracerebral 6-OHDA. The 6-OHDA + Vanillin IP 7D group, which was administrated intracerebral 6-OHDA (day 0), and 7 days later received i.p vanillin (20 mg/kg/day for seven days, day 7 through day 14). Finally, the 6-OHDA + Vanillin oral -3D and 6-OHDA + Vanillin oral 7D groups, which received vanillin (20 mg/kg/day for seven days, using oral gavage and using the same regimens mentioned above), and other than that they had the same manipulation as the 6-OHDA + Vanillin IP -3D and 6-OHDA + Vanillin IP 7D groups, respectively.
The 6-OHDA was administrated using intracerebral unilateral injection via stereotaxic surgery. In that respect, thirty minutes prior to 6-OHDA intracerebral injection, animals were given pargyline (50 mg/kg) i.p and desmethylimipramine (25 mg/kg) i.p. Animals were then anesthetized with isoflurane (4% for induction, 1.5– 2% for maintenance). After that, animals were fixed onto a vernier stereotaxic frame and using a Hamilton microsyringe (10μl) injected with 6-OHDA (8μg/4μl) into the MFB (from bregma A – 4.3 mm, L 1.4 mm, V 8.2 mm) (Paxinos & Watson, 2007). These injections were performed at a rate of 1μl per minute. Once the 6-OHDA injection has been accomplished, the needle was left in place for further 5 minutes to ensure distribution of the 6-OHDA at the injection area. The needle was, then, slowly withdrawn above the injection site to prevent flow back to the needle (Harkavyi et al., 2008).
Apomorphine challenge
Fourteen days after initial surgery, all animals were given an apomorphine (0.5 mg/kg) subcutaneous (s.c) injection; to establish a behavioral effect and assess lesion severity between the right and left-brain hemispheres and determine the integrity of nigrostriatal function. Twenty minutes post injections, animals were randomly selected, and placed into a circular test domain and rotations were monitored, by a blinded observer, for 120 seconds using a stop watch, only complete or full rotation were recorded (Harkavyi et al., 2008; Yan et al., 2017).
Tissue dopamine assay
After completion of treatments and behavioral assessment, the animals were killed and brains were removed from the skull and placed on dry ice, and then, placed in – 80 freezer for further analysis using High-Performance Liquid Chromatography- electrochemical detector (HPLC-ECD). During tissue sectioning, brains were cut 3 mm thick coronal sections, and striatum dissected and homogenized in 1 ml ice-cold phosphate buffer (pH 7.4), Thereafter, 40μl of the supernatant from Striatum homogenate was prepared with 10μl (0.2 M perchloric acid), then centrifuged at 8000×g for 10 min at 4°C using cold centrifuge (Wiltshire, UK). The supernatants were collected into vials with insert and whole tissue dopamine levels were evaluated using HPLC-ECD. The remaining brain parts after dissection were stored, frozen at – 80°C and retained for immunohistochemistry.
Statistical analysis
Data were analyzed using one way ANOVA followed by Bonferonni’s post hoc using statistical package Prizm 5 software package. All data are presented as mean±SEM for six rats in each group and P < 0.05 was considered significant.
Result
Apomorphine challenge
After 14 days, the 6-OHDA-treated rats showed significant apomorphine-induced contralateral rotation as compared to the control group. Vanillin, whether administered before or after 6OHDA, either using IP or oral administration, decreased rotation as compared to the 6OHDA group (Fig. 1, P < 0.05.).
Effects of I.P and oral vanillin on apomorphine-induced rotational behavior in 6-OHDA lesioned rats. Vanillin was administered once daily for seven days where it was started either at 3 days before (-3D) or 7 days after (D7) 6-OHDA treatment. Fourteen days after 6-OHDA injection, number of rotations was measured for 120 seconds, 20 min after apomorphine injection. * indicates significant difference from control group, and ∧ indicates significant differences of the 6-OHDA group (p < 0.05, n = 6 per group).
Figure (2) represents dopamine contents in striatal tissue with either i.p or oral administration of vanillin. The 6-OHDA group had a significant decline of dopamine concentrations compared with all other groups (Fig. 2, P < 0.05). On the other hand, oral and IP vanillin at three days before and seven days after 6-OHDA resulted in a significant increase in striatal dopamine concentrations that, however, did not reach the level of normal animals (Fig. 1, P < 0.05).
Effects of I.P and oral vanillin on striatal dopamine levels in 6-OHDA lesioned rats. Dopmine levels in the striatum were reduced in 6-OHDA groups, and this reduction was partially improved by oral and I.P vanillin administered at -3D and D7. *indicates a significant difference from all other group and **indicate a significant difference from the control group (p < 0.05). -3D: three days before and D7: seven days after 6_HDA treatment.
This study reported a neuroprotective effect for vanillin against 6-OHDA model of PD. Current results indicated that oral and IP vanillin at 3 days before or 7 days after 6-OHDA lesioning afforded improvement in apomorphine chalenge, and straital dopamine levels in cells. In previous studies, orally administered vanillin was shown to be helpful in improving cognitive function and possibility increasing endogenous neuronal proliferation in the brain (Kim & Park, 2017). Similarly, vanillin enhanced the integration of granule cells in the dentate gyrus, which was capable of enhancing neurogenesis and improving various neurological diseases (Kim & Park, 2017).
Unilateral injection of 6-OHDA into the MFB induced degeneration of the nigrostriatal pathway and striatal dopamine depletion, closely mimicking events that occur in PD, which is consistent with previous studies (Hernandez-Baltazar, Zavala-Flores, & Villanueva-Olivo, 2017; Stępkowski, Wasyk, Grzelak, & Kruszewski, 2015). It is known that the 6-OHDA lesion produces a phenotypic behavior, upon subcutaneous administration of apomorphine revealed as an increase of contralateral rotations towards the lesioned side (Lai et al., 2019; Tieu, 2011). Apomorphine-induced rotation test is commonly used as a quantitative predictive marker to evaluate dopaminergic neurons deficits and efficacy of treatments (Harkavyi et al., 2008). Current results have demonstrated that i.p and oral administration of vanillin either before or after 6-OHDA significantly attenuated 6-OHDA-induced rotations during apomorphine challenge.
It was shown previously that pre-treatment microglial cells with vanillin inhibited mitochondrial dysfunction, oxidative stress, and apoptosis, and thus, vanillin was suggested as therapeutic agent in the treatment of neurodegenerative diseases such as PD (Dhanalakshmi et al., 2016; Yan et al., 2017). This is in support of the results of the current study where vanillin administration significantly increased both striatal tissue dopamine content in 6-OHDA lesioned animals. Striatal tissue dopamine content is a key indicators of nigrostriatal neurodegeneration (Xu et al., 2015; Yan et al., 2017). This is possibly due to enhancing neurogenesis of cells with a dopaminergic phenotype by vanillin (Abuirmeileh, Harkavyi, Lever, Biggs, & Whitton, 2007; Chinnasamy Dhanalakshmi et al., 2015; Xu et al., 2015).
The underlying cellular mechanisms responsible for finding of the current study are still unclear. Previous studies suggest that the ability of vanillin to reduce release of free radical following oxidative stress and protect dopaminergic neurons from damage (Dhanalakshmi et al., 2016). In that respect, vanillin showed significant brain protective properties via reducing lipid peroxidation and increasing activities of antioxidant such as glutathione and superoxide dismutase enzyme (Gupta & Sharma, 2014). On the other hand, vanillin demonstrated an anti-inflammatory effect mediated through counteracting microglial cells activation by reducing expression of pro-inflammatory cytokines (Dhanalakshmi et al., 2016; Kim, Na, Park, & Lee, 2019; Wu et al., 2009). Kim et al. found that vanillin significantly reduced the production of nitric oxide and pro-inflammatory cytokines such as IL -1β and IL-6 (Kim et al., 2019). Additionally, cells pretreated with vanillin and incubated for 24 hrs with neurotoxin showed a reduction in the protein levels of inducible nitric oxide synthase, which is responsible for nitric oxide production, thus, inhibiting enzyme complexes I and IV of the mitochondrial electron transport chain, leading to reactive oxygen species generation (Kim et al., 2019). Wu et al. found that vanillin has anti-inflammatory effects by inhibiting microglial cell activation with its possible use as a therapeutic agent for inflammatory bowel disease (Wu et al., 2009). Furthermore, pretreatment of cells with vanillin showed reduced NFκB protein expression, which is important in modulating the expression of pro-inflammatory mediators and elevating their levels by phosphorylation, leading to microglial activation (Yan et al., 2017).
In conclusion, the current study results indicate the beneficial effects of vanillin protective properties in reducing the effects of 6-OHDA lesion via preserving striatal dopamine levels, thus, vanillin seems to be a promising agent for PD.
Footnotes
Acknowledgment
Financial Support was via grant, from the Israa University, Amman, Jordan.