NEUROPROTECTIVE MECHANISM OF ORAL SUPPLEMENTATION OF ALPHA- OR GAMMA-TOCOTRIENOL IN A RAT MODEL OF PARKINSON’S DISEASE

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 to loss of dopaminergic neurons. There are also changes that occur at the genetic level, due to gene mutations, which alter the normal functions of the affected genes. Levodopa (L-dopa), the current available therapeutic drug, is known to cause adverse effects (headache, dizziness, nausea and somnolence) upon long term usage. The toxicity of L-dopa is also linked to oxidative stress (OS); indicating the need to find an agent that can alleviate the build-up of reactive oxygen species (ROS) and eventually be able to inhibit progression of this disease. In the present study, intracisternal injection of neurotoxin 6-hydroxydopamine (6-OHDA) was used, to induce a model of parkinsonism in rat. This study consists of two phases. Phase 1 involved dose- and time-dependent exposure to 6-OHDA to study disease progression and to establish the optimal 6-OHDA dose to be used as well as the exposure time to 6-OHDA. In Phase 2 of this study, two isoforms of Vitamin E- tocotrienols (T3), namely alpha-T3 (T3) and gamma-T3 (T3) were used, as a treatment approach to retard progression of this disease in the same experimental model. The animals injected with 6-OHDA were fed with T3 and the effects of T3 supplementation on disease progression was studied over a period of 28 days. From Phase 1, the lowest dose of 6-OHDA that produced the best results i.e. induced Parkinsonism-like symptoms within four weeks was identified to be a single dose of 250 μg 6-OHDA delivered intracisternally. The animals injected with 250 g 6-OHDA showed a significant increase in latency in both forelimb retraction time (FRT) and hind-limb retraction time (HRT), which was indicative of gradual impaired motor functions. These results were also supported by immunohistochemistry and immunosorbent assay findings, which showed about 63% dopaminergic neuronal cell loss in SN and marked loss of striatal fibres as well as significant decrease in brain dopamine levels. Next, in Phase 1 of the study, the animals were injected intracisternally with the optimised concentration (250 g) of 6-OHDA. The animals were sacrificed at different time points to establish the earliest time that Parkinsonism-like symptoms can be detected in the experimental animals. This is to give an indication of when would be the best time to start oral supplementation of T3; which is an early intervention to minimise disease progression. The data revealed that there was progressive neuroinflammation 6 to 24 hours’ post-injection of 6-OHDA. After 48 hours, the inflammation level was significantly high as indicated by severe astrogliosis, which was evaluated by glial fibrillary acidic protein (GFAP) immunostaining. The level of dopamine neurons in the SN and striatal fibres also reduced significantly by 40% as seen in tyrosine hydroxylase (TH) immunostaining. These findings strongly supported that T3 supplementation to be started 48 hours post injection with 6-OHDA. In Phase 2, the experimental animals received a single injection of 250 μg 6-OHDA intracisternally and 48 hours post injection, oral supplementation with T3 or T3 commenced which was continued for 28 days. The neuroprotective effects of T3 (αT3 or T3) were evaluated using behavioural studies, immunohistochemical and gene expression approaches. Motor deficits were diminished in the T3 (T3 or T3) supplemented animals, which was supported by preventing further reduction of dopamine neuron levels in SN and possibly reversing some neuronal loss. Gene expression using a commercial quantifying polymerase chain reaction (qPCR) array containing 84 genes related to PD strongly suggests that some of the Parkinsonism-like symptoms observed in the rats injected with 6-OHDA could be due to neurotoxin induced mutations in the genes of the PARK family and mitochondrial dysfunction. The expression of PARK1 and PARK2 genes were up-regulated whilst the expression of PARK5, PARK6, PARK7 and PARK13 genes were down-regulated in the brains of rats injected with 250 g of 6-OHDA. Commencement of oral supplementation with T3, 48 hours post 6-OHDA injection markedly reversed the expression of these genes with the exception of PARK1, PARK7 and PARK13 genes. In case of PARK 6 gene, the reversal was observed only with the T3 supplementation. Another set of differentially expressed genes (TH, DDC, SLC18A2, SLC6A3, NURR1) belongs to the dopaminergic pathway category. The 6-OHDA neurotoxicity caused mutations in these neuroprotective genes and mRNA level of these genes were significantly altered. Supplementation with T3 (T3 or T3) was able to reverse this effect except in SLC18A2 gene; and proved to be highly neuroprotective. This study showed that the intracisternal injection of 6-OHDA caused a significant neurotoxicity with altered mRNA levels of neuroprotective genes, leading to Parkinson’s like symptoms in rats. The T3 supplementation reduced 6-OHDA induced oxidative stress by acting as a free radical scavenger and promoter of cellular repair in substantia nigra. There was a significant inverse relationship between T3 supplementation and the 6-OHDA induced neuronal damage in the substantia nigra. In conclusion, the study demonstrates that, T3, in particular αT3 has the potential to be developed as future neuroprotective agents that could further inhibit progression of Parkinson’s disease.