Page 134 - AN-3-4

Advanced Neurology Drosophila Sirtuin 1 and Alzheimer’s disease

2.3. Nail polish imprint of adult eyes 2.6. Body weight analysis

The external eye morphology of adult Drosophila Body weight analysis was conducted as described in a
was observed following the methodology described previous study. For the present study, flies aged 10, 20,
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previously. For this procedure, flies from the selected and 30 days of the desired genotype were used. The body
33
groups were anesthetized, placed on a clean glass slide, and weights of 20 adult flies were collectively measured using a
decapitated. The heads were coated with small drops of Sartorius weighing balance (Germany) in milligrams (mg).
clear nail polish to create an impression of the eye surface, The experiment was repeated 5 times, using 100 flies from
which was then allowed to dry at 24°C. After drying, the each genotype.
nail polish layer was carefully removed from the eyes using
fine dissecting needles, forming an exact replica of the eye’s 2.7. Survival (lifespan) assay
surface, mimicking the goblet-shaped appearance of the The survival assay was performed as described previously.
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adult eye. The nail polish layer was placed on a glass slide Recently eclosed adult flies were collected, with a total of
with the imprinted side facing upward. To flatten the layer, 100 flies (20 flies per vial) used for each genotype. Every
a coverslip was gently placed over it, and mild pressure was alternate day flies from each vial were transferred to fresh
applied using a needle. The preparations were observed food media, and the number of dead flies was recorded
using differential interference contrast microscopy for daily for up to 50 days. Throughout the assay, flies were
imaging. kept in a temperature-controlled BOD incubator. Median
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2.4. Climbing assay survival was calculated using the Kaplan–Meier method,
and a survival curve was plotted. Statistical analysis was
This assay was conducted as described earlier. For conducted using GraphPad Prism 5.0 software. Significant
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this assay, flies aged 10, 20, and 30 days were placed in a differences between genotypes and major variations in
vertical glass tube (30 cm long × 1.5 cm wide) and allowed median survival were evaluated using the Mantel–Cox log-
to acclimatize for 2 min. Flies were then gently tapped to rank test. 37
the bottom of the tube, and the number of flies crossing
the 8 cm mark within 10 s was counted. Twenty flies of 2.8. Acridine orange (AO) staining
−1
each genotype were placed in the tube, and the experiment AO staining was performed as described previously,
35
was conducted 5 times. In total, 100 flies per genotype were with minor modifications. Larval eye imaginal discs and
used for the climbing assay. The results were expressed as larval brains were dissected in 1× phosphate-buffered
the percentage (%) of flies climbing 8 cm in 10 s under saline (PBS) and then incubated for 2 min in a 1 μg/mL
−1
standard lighting conditions. AO solution (Cat# 877529, Invitrogen, USA) prepared
2.5. Phototaxis assay in 1× PBS. The tissues were washed, mounted in 1×
PBS, and immediately examined using a laser scanning
This assay was also conducted as described previously. confocal microscope (TCS SP5II, Leica Microsystems,
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For this test, 10-day-old flies were placed in a Y-maze glass Wetzlar, Germany). More than 20 larval eye imaginal
tube (with one light arm and one dark arm) and allowed discs and larval brains were collected from each genotype.
to acclimatize for 2 min. Flies were then gently tapped Quantification of AO-positive cells was conducted using
and allowed to move through the Y-maze for 20 s. The ImageJ 5.0 software (NIH, USA).
number of flies moving toward the light and dark paths
was recorded. Twenty flies of the desired genotype were 2.9. Quantitative RT-PCR (qRT-PCR)
placed in the Y-maze, and the experiment was repeated qRT-PCR was conducted as described previously, with
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5 times. For the phototaxis assay, 100 flies per genotype slight modifications. Briefly, 30-day-old fly heads were
were used. The assay was performed under standard isolated for mRNA extraction using TRIzol reagent
lighting conditions (~500 Lux), and results were presented (Cat# 15596026, Invitrogen, USA). cDNA synthesis was
as a light preference index. performed using the Verso cDNA Synthesis Kit (Cat#AB-
Number of Number of 1452/B, Thermo Fisher Scientific, USA) according to the
flies traveling flies traveling manufacturer’s protocol. Amplification of cDNA was
toward the − toward the carried out using gene-specific primers. A 20 μl reaction
SYBR
mixture was prepared, containing PowerUP
TM
TM
Light light path dark path Green Master Mix (Cat. #A25742, Applied Biosystems,

preference index = Total number of flies Thermo Fisher Scientific, USA), primers and cDNA.

Volume 3 Issue 4 (2024) 3 doi: 10.36922/an.4291