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

Separation was carried out at a flow of 1 mL/min. Lipid peroxidation (MDA) assay
Samples (20 µL) were injected manually. On the Lipid peroxide formation was assayed by the method
day of experiment frozen samples of midbrain were of Wills. An aliquot containing 0.5 mL of tissue
[45]
homogenized in the homogenizing solution containing homogenate (10% w/v), diluted to 1.0 mL using 0.1 mL
0.1 mol/L perchloric acid. After that samples were Tris-HCl buffer (pH = 7.4) was shaken. Samples were
centrifuged at 12,000 g for 5 min. The supernatant incubated at 37 °C for 2 h with constant shaking.
was further filtered through 0.25 micron nylon filters After incubation, 1 mL of ice cold 10% TCA was
before injecting in the HPLC injection pump. Data added and mixed it thoroughly; the reaction mixture
was recorded and analyzed with the help of Empower was centrifuged at 800 g for 10 min. One mL of
software. [43] 0.67% thiobarbituric acid (TBA) was added to 1 mL
of supernatant and color developed at 100 °C for
Biochemical estimations 10 min. Samples were cooled and diluted with 1 mL
All the tissues (10% w/v) were homogenized in 10 mmol/L double distilled water. The absorbance was read at
PBS, pH = 7.4. Homogenate was made using 532 nm. The amount of Malondialdehyde formed was
mechanically driven Teflon Potter-Elvejhem type calculated on the basis of molar extinction coefficient
homogenizer for total disruption of cells. Homogenate of MDA-TBA chromophore (1.56 × 10 M cm ) and
-1
-1
5
was centrifuged first at 10,000 g for 30 min at 40 °C. results were expressed as nmoles of MDA/mg protein.
Pellet was discarded and supernatant was used for
various biochemical estimations. Catalase
The enzymatic activity of catalase was estimated by UV
NO spectrophotometer method described by Luck. H O
[46]
2
2
NO was estimated by the method of Raddassi et al. The was used as substrate. The UV absorption of H O
[44]
2
2
level of NO was estimated as nitrite, a NO metabolite solution is measured at 240 nm on decomposition of
and remains stored in tissues as Nitrates (NO ) or H O with catalase. The amount of H O decomposed
3-
2
2
2
2
Nitrite (NO ). Thus, NO concentration can be estimated was calculated on the basis of molar extinction
2-
by measuring concentrations of NO and NO in coefficient of H O (39.4 M cm ) and results were
3-
2-
-1
-1
2
2
combination. The simplest technique is the monitoring expressed as μ moles of H O decomposed/min/mg
2
2
of reduction of NO to NO by nitrate reductase or protein.
2-
3-
metallic catalyst, followed by the calorimetric Griess
Reaction to measure NO levels (nitrite levels). A Superoxide dismutase
2-
standard curve was made with serial dilutions of sodium Superoxide dismutase (SOD) estimation was assayed
nitrite by making its volume to 100 μL; add 100 μL of by the method of Kono. The principle of SOD activity
[47]
Griess reagent in wells of ELISA reader plate. Instead assay was based on the inhibition of nitrobluetetrazolium
of sodium nitrite 100 μL of homogenate was added (NBT) reduction using the following reagents. Solution
along with 100 μL of Griess reagent in subsequent A: EDTA (0.1 mmol/L) containing 50 mmol/L sodium
wells of ELISA plate. The colorimetric reaction was carbonate, pH 10.0; solution B: NBT (90 mmol/L) in
allowed to proceed for 10 min at room temperature solution A; solution C: Triton-X (0.6%) in solution A;
in dark, and optical density was measured at 550 nm solution D: hydroxylamine hydrochloride (20 mmol/L)
using ELISA reader. pH 6.0. The reaction mixture contained 1.3 mL of
solution A, 0.5 mL of solution B and 0.1 mL of solution
Glutathione reduced C. The reaction is initiated by the addition of 0.1 mL
Estimation of glutathione reduced (GSH) was of solution D to the reaction mixture and the rate of
performed in the tissue homogenate by the method reduction of NBT in the absence of enzyme source
of Moron et al. The assay was performed by mixing was recorded at 560 nm for 3 min, which is considered
[34]
100 μL of 2.5% homogenate and trichloroacetic acid as reference. Following this an appropriate amount of
(TCA). The precipitated proteins were separated by the enzyme source (PMS 20-50 µL) was added and
centrifugation at 2,000 g for 15 min; 0.1 mL supernatant the rate of reduction was noted for 3 min at 560 nm.
was diluted to 1 mL with 0.2 mol/L phosphate buffer (pH Percentage inhibition in the rate of NBT reduction
= 8.0). Further, 2 mL of freshly prepared 0.6 mmol/L is calculated and one unit of the enzyme that is the
5,5’-dithiobis (2-nitrobenzoic acid) (DTNB) in buffer inverse of the amount of the protein (mg) required to
was added. In this method, DTNB is reduced by -SH inhibiting the reduction rate by 50%. The results are
groups to form 1 mole of 2-nitro-5-mercaptabenzoic expressed as IU/mg of protein.
acid per mole of SH. The nitro mercaptobenzoic acid
anion released has intense yellow color and can be Histopathology
used to measure -SH groups at 412 nm. Histology of brain tissues was done by the method
36 Neuroimmunology and Neuroinflammation ¦ Volume 4 ¦ March 21, 2017