INTRODUCTION:

The neurodegenerative diseases are one of the major areas of concern in research. Neurodegeneration is the chronic process of degeneration of neurons which hampers the physiology and ultimately leads to pathological complications. The brain has the highest metabolic rate and almost negligible regeneration ability in comparison to other organs in the human body, making it more vulnerable to structural and functional damages^1,2. Many factors are involved in neurodegeneration which include protein deposition or disintegration, inadequate protein degradation, activation of autophagy pathway, formation and release of reactive oxygen species, mitochondrial dysfunction, neuroinflammation.

As a virtue of these factors the neuronal cell death occurs either by apoptosis, autophagy, necrosis and necroptosis^3-5. Thus, the need to explore the novel therapeutic modalities is mandatory to reverse or stop the neurodegeneration. The invitro method which can be used to screen the different agents for neuroprotection is way useful to get the lead drug in the research.

Rotenone, a pesticide, is an important mitochondrial toxin which is highly lipophilic in nature. The rotenone has various mechanism which causes neuronal cell death. The main mechanism involves inhibition of Complex I in mitochondria. The complex I is responsible to oxidizes the NADH and transferring the electron to the ubiquinone. The complex I is also involved in maintaining the calcium dependent mitochondrial permeability. Thus, inhibition of complex I will lead to breakdown of mitochondrial integrity. Further rotenone inhibits NADH oxidase which embarks further reactive oxygen species generation^6-8. Rotenone hence is useful as a powerful tool to induce mitochondrial disruption which mimics the neurodegeneration⁹. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is widely applicable to determine the cell viability, mainly because of the ability of viable cells to convert MTT in to it’s formazan which can be quantified by scanning the absorbance^10-12.

Thus, we have attempted to determine the rotenone concentration to be used in MTT assay which can be useful to screen the potential targets as neuroprotective agents

MATERIALS AND METHODS:

Materials:

The Rotenone and MTT were purchased from Sigma-Aldrich while all other reagents were procured from S.D. Fine- chem Ltd. All the reagents were freshly prepared using ice cold deionized water and used for the study.

Animals:

Sixteen male Wistar albino rats weighing approximately 250gm were used for the study. The animals were acclimatized for a week before initializing the study. The study was carried out as per guidelines set by Committee for purpose of control and supervision of experiments on animals. The animals were caged in polypropylene cages and maintained in 12hour light dark cycle at temperature 23±2˚C and relative humidity as 60±5%. The standard food pellets and water was provided ad libitum throughout the acclimatization period.

Rat brain synaptosomes preparation:

The method of authors P. Kamat et al was slightly modified and used. Briefly, the rats were sacrificed and immediately the intracardiac perfusion of ice-cold saline was carried out to ensure complete removal of blood from the brain tissue. The brain was then isolated and stored in 0.9% ice cold saline. The 10% W/V brain homogenate was prepared in ice cold 0.3 M sucrose HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer with approximate 10 up and down stroke with the homogenizer at 800 rpm. The obtained homogenate was cold centrifuged at 4 to 8˚C for 10m at 3000xg. The supernatant thus obtained was mixed in equal proportion with ice cold 1.3M sucrose HEPES buffer. The obtained mixture for further centrifuged at 4˚C for 15m at 12000xg. The residual pellets obtained were resuspended in ice cold 0.8M sucrose HEPES buffer pH7.4 to get aliquots of synaptosomes fraction^13-16. The obtained aliquots of synaptosomes fraction (ASF) were stored in deep freezer at -20˚C; until the MTT reduction assay was performed.

MTT assay:

The 400µL of ASF was taken in different test tubes to which 25, 50, 100 and 200µL of 1mM rotenone was added. The test tubes were then incubated for 37˚C for 2 h. After the incubation period 10µL MTT (5mg/ml) was added. The final volume was made up to 2500µL. The obtained mixture was further incubated for 24h at 37˚C. The reduction of MTT leads to formation of violet colour product which was quantified at a wavelength of 570nm using UV spectrophotometer. The intensity of purple colour is directly proportional to the MTT reduction in cell indicating cell viability and directly proportional to the absorbance ^10-13. The experiment was performed in sextuplet for statistical analysis.

RESULT AND DISCUSSION:

Table 1: Cell viability in presence of rotenone

Test tube	Rotenone Concentration (µl)	Percentage Cell Viability
Control	0	64.43 ± 4.20
Rotenone 1	25	62.50 ± 3.26
Rotenone 2	50	53.97 ± 4.31*
Rotenone 3	100	45.37 ± 2.49*
Rotenone 4	200	29.00 ± 2.93*

The percent cell viability values are indicated as average ± standard deviation, where experiment was performed in sextuplet. * Indicates significant (P < 0.05) difference in comparison to control when calculated with ANNOVA as statistical analysis method

The MTT assay is widely applicable to check the cell viability and hence study cell cytotoxicity studies ¹². The MTT readily penetrates the viable cell and the NADH in the mitochondria of the cell or the mitochondrial enzymes are responsible to cause the tetrazolium reduction. Thus, MTT is converted into violet coloured formazan which can be detected by recording the absorbance at 570nm. The formazan colour intensity is directly proportional to the tetrazolium reduction and hence the number of viable cells^10,11,17. Different mitochondrial toxins like Methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP), 3-nitropropionic (3- NPA) have been used to induce mitochondrial dysfunction specifically in the brain synaptosomes⁶. The rotenone is a pesticide which has high lipophilicity and is able to inhibit the complex I of mitochondrial chain reaction. This potent inhibitor of Complex I thus leads to cell death^7,9. The synaptosomal cell are enriched with synaptic protein that reflects the normal neuronal physiology¹³. The use of rotenone invitro will lead to microtubule disruption and hence mitochondrial cell death. Hence the MTT is used to evaluate the cell viability when the synaptosomes are exposed to rotenone which leads to cell death. The tetrazolium reduction is decreased in presence of rotenone and will indicate the mitochondrial disruption. Thus, the MTT assay can be used to screen different potential neuroprotective agents. In current study the different concentrations of rotenone were used to understand the suitable concentration of rotenone which causes significant and 50 percent of mitochondrial disruption. It was found that 50 and 100µl of rotenone (1mM) concentration were more effective in causing significant mitochondrial dysfunction.

CONCLUSION:

The 50 and 100µl of rotenone (1mM) concentration can be successfully used to induce mitochondrial dysfunction and hence synaptosomal cell death. The MTT is the tetrazolium which when reduced to formazan will show the colour change indicating the synaptosomal cell viability. Thus, MTT assay along with rotenone a potent mitochondrial inhibitor can be utilised to screen and evaluate the potential neuroprotective activity of test drug by in vitro method.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 08.06.2022 Modified on 19.10.2023

Research J. Pharm. and Tech. 2024; 17(8):3543-3545.

DOI: 10.52711/0974-360X.2024.00553