Tocotrienols Ameliorate Neurodegeneration and Motor Deficits in the 6-OHDA-Induced Rat Model of Parkinsonism: Behavioural and Immunohistochemistry Analysis

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Tocotrienols Ameliorate Neurodegeneration and Motor Deficits in the 6-OHDA-Induced Rat Model of Parkinsonism: Behavioural and Immunohistochemistry Analysis

Mangala Kumari 1,*, 

Premdass Ramdas 2, 

Ammu Kutty Radhakrishnan 3, 

Methil Kannan Kutty 4 

and Nagaraja Haleagrahara 5






Citation: Kumari, M.; Ramdas, P.;

Radhakrishnan, A.K.; Kutty, M.K.;

Haleagrahara, N.

1 Department of Anatomy, Division of Human Biology, School of Medicine, International Medical University,

Kuala Lumpur 57000, Malaysia

2 Division of Applied Biomedical Sciences and Biotechnology, School of Health Sciences, International Medical

University, Kuala Lumpur 57000, Malaysia; premdass_ramdas@imu.edu.my

3

Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Bandar Sunway,Selangor 47500, Malaysia; Ammu.Radhakrishnan@monash.edu4 Department of Medicine, Lincoln University College, Kelana Jaya, Selangor 47301, Malaysia;methilkannan@gmail.com 5 College of Medicine and Dentistry, James Cook University, Townsville, QLD 4811, Australia; haleagrahara.nagaraja@jcu.edu.au * Correspondence: mangala_kumari@imu.edu.my 

Abstract: 

Parkinson’s disease (PD) is a debilitating neurodegenerative disease, which progresses

over time, causing pathological depigmentation of the substantia nigra (SN) in the midbrain due toloss of dopaminergic neurons. Emerging studies revealed the promising effects of some nutrient compounds in reducing the risk of PD. One such nutrient compound that possess neuroprotective effects and prevents neurodegeneration is tocotrienol (T3), a vitamin E family member. In the present study, a single dose intracisternal injection of 250 µg 6-hydroxydopamine (6-OHDA) was used to induce parkinsonism in male Sprague Dawley (SD) rats. Forty-eight hours post injection, the SD rats were orally supplemented with alpha (α)- and gamma (γ)-T3 for 28 days. The neuroprotective effects of α- and γ-T3 were evaluated using behavioural studies and immunohistochemistry (IHC).The findings from this study revealed that supplementation of α- and γ-T3 was able to ameliorate the motor deficits induced by 6-OHDA and improve the neuronal functions by reducing inflammation, reversing the neuronal degradation, and preventing further reduction of dopaminergic neurons in the SN and striatum (STR) fibre density. Keywords: vitamin E; tocotrienols; tyrosine hydroxylase; Parkinson’s disease; neurofunctions; 

1. Introduction 

Parkinson’s disease (PD) is the second most common progressive neurodegenerative disease in humans after Alzheimer’s disease ¹. The number of people with PD is estimated to be between 4 to 6 million in the most populous nations. Longitudinal meta-analysis data showed that the prevalence of PD steadily increased with age. Besides, the geographical prevalence is 2.5 times higher in people from Australia, Europe, and America compared to the individuals from Asia ². The selective loss of dopaminergic neurons in the pars compacta region of the substantia nigra (SN) of the midbrain and reduced striatal dopamine (DA) levels in the brain are the main pathological features that lead to the development of PD. These pathologies cause disturbances in the nigrostriatal dopaminergic pathway, which result in significant clinical abnormalities that manifest as motor and non-motor symptoms [3]. The motor symptoms of PD include tremor at rest, muscle rigidity, bradykinesia, postural instability, and autonomic disturbances. Some of the non-motor symptoms of PD include sleep disturbance, cognitive-related deficits, anosmia, depression, and gastrointestinal disorders [4].Neuroinflammation induced by activated neuroglia is also linked to the pathogenesisof PD. Activated neuroglia secretes pro-inflammatory cytokines, which further aggravate the disease [5]. Researchers postulated that drugs that target activated neuroglia and reducethe levels of pro-inflammatory cytokines in the brain are suitable candidate drug(s) for PD, as these types of drugs can reduce neuroinflammation and also alleviate neuronal oxidative stress [6]. Interestingly, some nutrients have been known to target activated neuroglia and have been reported to have profound effects in regulating the proinflammatory cytokines. Studies have revealed the role of some nutrients in reducing the risk of PD by preventingneurodegeneration or halting the disease progression ⁷.One such natural compound is tocotrienols (T3), a vitamin E family member, which is a naturally occurring fat-soluble vitamin ⁸. Tocotrienols are found naturally in annatto seeds, rice bran, and particularly palm oil and rice bran oil in which higher amounts of tocotrienols are present ⁹. Several studies have shown that T3 possesses many healthenhancing effects such as anti-cancer ^10,11, anti-proliferative ^12,13, anti-thrombotic ¹⁴, anti-inflammatory ¹⁵, antioxidant ¹⁶, and neuroprotective ^17,18 effects. Palm oilderived tocotrienol-rich fraction (TRF) has been reported to provide immense protection against cytotoxicity caused by oxidative stress (OS) in rat striatal cells ¹⁹. A recent systematic review on the safety and neuroprotective efficacy of palm-oil and TRF concluded that T3 could enhance the healthy animals’ cognitive function and attenuate OS ²⁰. According to Khanna et al., α-T3 exhibits cellular protection of the nerves through two main pathways: 12-lipoxygenase (LOX) and c-Src pathways ²¹. However, the reports on the neuroprotective effects of γ-T3 are minimal, in particular, the effects of γ-T3 supplementation on the 6-OHDA-induced rat model of parkinsonism are not available in the literature. A previous clinical trial showed the protective effects of mixed T3 in decreasing the progression of white matter lesions (WML) in 121 human volunteers ²². Moreover, palm vitamin E has achieved the “generally regarded as safe (GRAS)” status by the Food and Drug Administration (FDA), USA ²³. It is considered safe for consumption and has been attributed with many health-enhancing effects including brain health ²⁴. Animal models using various types of neurotoxins are popular tools that have been used for the study of PD over the past couple of decades and have imparted significant knowledge on the aetiology and pathogenesis of PD ²⁵. The specific target of neurotoxins in the animal models is the catecholaminergic neurons, which induce PD-related symptoms and pathology when impaired by neurotoxins. The neurotoxin 6-OHDA cannot cross the blood–brain barrier; hence, systemic administration will not cause parkinsonism ²⁶. The degree of the lesion with 6-OHDA depends on the route of administration (intracerebral or intracisternal), concentration, and the species used ^27–29. Hence, this study aimed to evaluate the neuroprotective efficacy of oral supplementation of isomers of T3 (α- and γ-T3) on the intracisternal 6-OHDA-injected rat model. With this study, we sought to determine whether isomers of T3 (α- and γ-T3) could reverse parkinsonian motor symptoms on 6-OHDA-injected SD rats and prevent the DA neuronal loss in agreement with the behavioural observation of the corresponding animals.