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Sharma et al. Zinc supplementation prevents against LPS induced neurotoxicity in rats

provided standard animal feed and water ad libitum Active avoidance test
throughout the treatment period. All procedures were Memory impairments can be observed in Active
done in accordance with ethical guidelines laid down Avoidance Test. Apparatus used for this test
[40]
by the Ethics Committee on the Use of Experimental consisted of 2 chambers, which are separated by a
Animals of the Panjab University and in general partition. One of these chambers is enlightened, where
according to the NIH guidelines (Rule No. 23-85, as the animal has to be kept. After 10 s, the buzzer is
revised in 1985). kept on and after 10 s the shock is given. If the animal
jumps to the other side of the compartment as soon as
Chemicals the buzzer is set on, it means the animal has avoided
All the chemicals were purchased from Sigma-Aldrich the test. But in case, if the animal jumps to the other
(St. Louis, USA) and Sisco Research laboratories Pvt., compartment aftershock or does not jump is termed as
Ltd. (Mumbai, India). Zinc sulfate was purchased from escapism. The number of escaped trials/2 min/animal
HiMedia (Mumbai, India). were noted and analyzed statistically.
Experimental design Actophotometer (total locomotor activity)
Rats were preferred for this experiment due to: (1) Total locomotor activity (ambulation’s and rearing) was
shorter gestation length (20-22 days), shorter estrous measured by using a computerized Actophotometer
cycle (4-5 days), litter size of about 7-9, weaning age of (IMCORP, Ambala, India). An array of 16 infrared
about 21 days and relatively short period age of sexual emitter/detector pairs measured animal activity
maturity (7-8 weeks). Rat pregnancies are more size along a single axis of motion, the digital data being
consistent, rats can bred quickly after parturition and displayed on the front panel meters as ambulatory
rat brains show early sexual dimorphism. At the movements. Rats were allowed to acclimatize to the
[38]
commencement of the study 12 female rats of 12-14 observation chamber for a period of 2 min. The activity
weeks of age were randomly distributed into four cages was monitored continuously for a period of 5 min.
with 1 male of 14-16 weeks of age into each cage. Locomotion was expressed in terms of total photo
[38]
Pregnant female rats were then divided into four cages [41]
and were grouped as follows: beam counts per 5 min period per animal.
Rotarod
Group A (control): pregnant females were injected The effect of LPS on the muscle performance was
intraperitoneal with single dose of normal saline
(200 µL) at day 14 of pregnancy; Group B (LPS evaluated using a Rota-rod. Animals were given 2
treated): pregnant females were injected with single initial training trails of 300 s each, approximately 10 min
dose of LPS (200 μg/kg) intraperitoneal at day 14 apart, to maintain posture on a Rota-rod which is 3 cm
of pregnancy; Group C (LPS + zinc supplemented): in diameter and rotating at a constant 25 revolution per
pregnant females were injected single dose of LPS minute. After the initial training trials, a baseline trial of
(200 μg/kg) intraperitoneal at day 14 of pregnancy. 120 s was conducted. The time each animal remained
Animals were also administered with ZnSO (75 mg/L) on the rotarod was recorded; animals not falling off the
4
dissolved in drinking water throughout their gestation rotarod were given a maximum score of 120 s. [42]
period; Group D (zinc supplemented): pregnant
females were supplemented with ZnSO (75 mg/L) Estimation of neurotransmitter levels
4
throughout their gestation period. (epinephrine, nor-epinephrine, dopamine and
serotonin)
Neurobehavioral analysis Biogenic amines dopamine, norepinephrine and
Elevated plus maze test serotonin were measured by high performance
Elevated plus maze (EPM) test is a learning task which liquid chromatography (HPLC) with electrochemical
measures spatial long-term memory and evaluates detector. [43] Water standard system consisting of a
the cognitive behavior. Apparatus used for the EPM high pressure isocratic pump. A 20 µL sample injector
[39]
test consists of 2 open arms (40 cm × 6 cm) and 2 valve, C18 reverse phase column and electrochemical
closed arms of the same size connected with a central device were used. Data was recorded and analyzed
platform (6 cm × 6 cm). The maze was placed 50 cm with the help of Empower software. Mobile phase
above the ground. Rats were allowed to move freely in consisting of 10 mmol/L sodium citrate, 32 mmol/L
this apparatus for 90 min of training. After a few hours, NaH PO , 0.025 mmol/L EDTA, and 0.77 mmol/L
4
2
trained pups were again allowed to move freely for the 1-heptane sulphonic acid was used. pH of mobile phase
same time and time spent in the open arms and closed was adjusted to 4.5 with the help of orthophosphoric
arms were analyzed. The apparatus was cleaned with acid. Electrochemical conditions for the experiment
25% ethanol after testing the pups. were +0.800 V, sensitivity ranges from 5-50 nA.
Neuroimmunology and Neuroinflammation ¦ Volume 4 ¦ March 21, 2017 35