The effect of vanillin on KBrO3-induced motor performance was investigated using a rotarod task on day 15, as shown in Figure 1A. We found that muscle grip strength was significantly reduced (P < 0.01) in the KBrO3 as compared to the control group. Mice that were co-administered vanillin showed significantly increased muscle grip strength as compared to the KBrO3 group (P < 0.01). There was no significant difference in motor performance between vanillin and control groups (Figure 1A).
Figure 1. Effect of vanillin and KBrO3 on mice muscle grip strength (rotarod test, A) and mobility (open field test, B). Values from the rotarod test are expressed as time in seconds and compared to the performance of the control group (mean ± SD). In the open field test, values are expressed as number of squares crossed, head dips, and rearing frequency. Means not sharing the same letters (a, b) are significantly different (P < 0.05).
The open field test showed that significantly fewer squares were crossed by mice in the KBrO3 group compared to those in the control group (P < 0.01) (Figure 1B). In mice treated with vanillin, we saw an improvement in mobility, measured by an increase in the number of squares crossed compared to the KBrO3 group (P < 0.01). Similarly, we found that rearing activity decreased in KBrO3-treated animals, whereas vanillin treatment increased the rearing activity and mobility on day 15, as compared to the KBrO3 group (P < 0.01) (Figure 1B). We did not see any significant difference in rearing between the KBrO3+vanillin and the KBrO3 group. In the vanillin group, there was an increase in rearing, however, this difference was not significant when compared to the control group.
Lipid Peroxidation The lipid peroxidation product was assessed by measuring MDA levels. As shown in Table 1, mice exposed to KBrO3 had a significant increase in lipid peroxidation in the cerebellum (F = 1.47; P = 0.094) compared to controls. Co-administration of vanillin significantly decreased (P < 0.01) the level of lipid peroxidation compared to the KBrO3 group. Further, there was no significant difference in lipid peroxidation between the vanillin and control groups.
Parameters & Treatments Control KBrO3 KBrO3+Vanillin Vanillin F P MDA (nmol MDA/g tissue) 92.920 ± 12.830a 133.540 ± 11.300b 102.330 ± 10.950a 94.510 ± 17.670a 1.470 0.094 LOOH (nmol/mg protein) 1.220 ± 0.130a 3.150 ± 0.180b 2.070 ± 0.080a, b 1.300 ± 0.100a 3.470 0.030 H2O2 (μmol/mg of protein) 0.020 ± 0.007c 0.080 ± 0.009a 0.060 ± 0.008b 0.030 ± 0.007c 37.450 < 0.001 AOPP (µmol/mg of protein) 0.400 ± 0.090a 0.810 ± 0.040b 0.540 ± 0.070a, b 0.380 ± 0.050a 5.670 0.120 Mg2+ ATPase (μmol Pi/h/mg protein) 0.110 ± 0.008 a 0.010 ± 0.003a 0.050 ± 0.004b 0.082 ±0.090a 130.541 < 0.001 Na+-K+ ATPase (μmol Pi/h/mg protein) 0.073 ± 0.004a 0.079 ± 0.008a 0.029 ± 0.009c 0.046 ± 0.005b 35.371 < 0.001 Note. Values are means ± SD for twelve mice in each group. Means not sharing the same letters (a-c) within a column are significantly different (P < 0.05). MDA, Malondialdehyde; LOOH, Lipid Hydroperoxide; H2O2, Hydrogen Peroxide; AOPP, Advanced Oxidation Protein Product.
Table 1. Effect of Potassium Bromate (KBrO3), Vanillin and their Combination (KBrO3+vanillin) on Oxidative Stress Markers
H2O2 Generation As shown in Table 1, we detected higher levels of H2O2 in the cerebellum (F = 3.47; F = 37.45) of KBrO3-treated mice than in controls. Co-administration of vanillin significantly decreased the levels of H2O2 (Table 1).
Protein Oxidative Damage The degree of protein oxidation was estimated by measuring AOPP and LOOH levels. We found that there was a significant increase in AOPP and LOOH levels in the cerebellum (F = 5.67; P < 0.001; P = 0.03; P < 0.001 respectively) of KBrO3-treated mice when compared to controls (Table 1). Co-administration of vanillin significantly decreased these levels (Table 1). Further, there was no significant difference in the AOPP and LOOH levels between the vanillin and control groups.
Table 1 shows the activity of the membrane-bound Na+-K+ and Mg2+ ATPase in the cerebellum tissue of mice. KBrO3 administration caused a marked decrease (F = 130.541 and F = 35.371, respectively; P < 0.001) in the Na+-K+ and Mg2+ ATPase activity compared to the control. Vanillin co-administration significantly (P < 0.001) protected against the KBrO3-induced decrease in enzyme activity in the KBrO3+vanillin group. Vanillin administration alone did not produce any significant changes, as there was no significant difference in enzyme activity between the vanillin and control groups.
Intracellular activity and gene expression of antioxidant enzymes, such as SOD and GPx, were measured in the cerebellum following 15 days of KBrO3 and vanillin exposure (Figure 2). We found a significant increase (P < 0.01) in SOD and GPx activity in KBrO3-treated mice as compared to controls. Semi-quantitative RT-PCR analysis confirmed our biochemical results and revealed a marked decrease in SOD and GPx mRNA levels after normalization to the β-actin gene in the KBrO3 group (Figure 2). Co-administration of vanillin improved the antioxidant status and gene expression in cerebellar tissue compared to the KBrO3 group. Conversely, the vanillin group showed no significant difference in enzyme activity and gene expression compared to the control group.
Figure 2. Effect of potassium bromate (KBrO3), vanillin (Van), and their combination (KBrO3+vanillin) on the activities and mRNA expression of superoxide dismutase (SOD) and glutathione peroxidase (GPx) in the cerebellum of adult mice. Values are expressed as mean ± SD (n = 3). C: Controls. Means not sharing the same letters (a, b) are significantly different (P < 0.05).
The effect of vanillin, either co-administered with KBrO3 or administered alone, on proinflammatory gene expression was examined by measuring TNF-α and IL-6 mRNA accumulation using RT-PCR (Figure 3). We found a concomitant significant increase in TNF-α and IL-6 mRNA in the cerebellum of KBrO3-treated mice. Importantly, co-administration of vanillin significantly inhibited this increase in proinflammatory cytokines (TNF-α and IL-6 gene expression).
Figure 3. Effect of potassium bromate (KBrO3), vanillin (Van), and their combination (KBrO3+vanillin) on TNF-α and IL-6 mRNA expression in the cerebellum of adult mice. Values are expressed as mean ± SD (n = 3). C: Controls. Means not sharing the same letters (a, b) are significantly different (P < 0.05).
Administration of KBrO3 caused cerebellar histological changes in mice (Figure 4C). The most conspicuous damage in KBrO3-treated animals was found in the Purkinje cell layer, where there was a marked reduction in the number of cells. Moreover, edema caused a widening of the Purkinje cell layer (Figure 4C). Co-administration of vanillin protected the cerebellum from severe damage induced by KBrO3 (Figure 4D). The histological pattern in cerebellar tissue was normal in mice treated only with vanillin, similar to the control mice (Figure 4B). Semi-quantitative histological analysis by hematoxylin and eosin staining indicated significant apoptosis and reduction in the number of Purkinje cells in the KBrO3 group.
Figure 4. Cerebellum histological sections of controls (A), vanillin (B), KBrO3 (C), and KBrO3+vanillin (D) groups. The score of Purkinje cell edema (E) in adult mice. ML, molecular layer; PCL, Purkinje cell layer; IGL, internal granular layer. Arrows indicate →pyknotic Purkinje cells and
Purkinje cell edema. Sections were stained with hematoxylin-eosin and observed with light microscopy, (×400), C, controls; Van, vanillin. Means not sharing the same letters (a, b) are significantly different (P < 0.05).