The effects of mango leaf extract during adolescence and adulthood in a rat model of schizophrenia

Abstract

There is evidence that in schizophrenia, imbalances in inflammatory and oxidative processes occur during pregnancy and in the early postnatal period, generating interest in the potential therapeutic efficacy of anti-inflammatory and antioxidant compounds. Mangiferin is a polyphenolic compound abundant in the leaves of Mangifera indica L. that has robust antioxidant and anti-inflammatory properties, making it a potential candidate for preventive or co-adjuvant therapy in schizophrenia. Hence, this study set-out to evaluate the effect of mango leaf extract (MLE) in a model of schizophrenia based on maternal immune activation, in which Poly I:C (4 mg/kg) is administered intravenously to pregnant rats. Young adult (postnatal day 60-70) or adolescent (postnatal day 35-49) male offspring received MLE (50 mg/kg of mangiferin) daily, and the effects of MLE in adolescence were compared to those of risperidone, assessing behavior, brain magnetic resonance imaging (MRI), and oxidative/inflammatory and antioxidant mediators in the adult offspring. MLE treatment in adulthood reversed the deficit in prepulse inhibition (PPI) but it failed to attenuate the sensitivity to amphetamine and the deficit in novel object recognition (NOR) induced. By contrast, adolescent MLE treatment prevented the sensorimotor gating deficit in the PPI test, producing an effect similar to that of risperidone. This MLE treatment also produced a reduction in grooming behavior, but it had no effect on anxiety or novel object recognition memory. MRI studies revealed that adolescent MLE administration partially counteracted the cortical shrinkage, and cerebellum and ventricle enlargement. In addition, MLE administration in adolescence reduced iNOS mediated inflammatory activation and it promoted the expression of biomarkers of compensatory antioxidant activity in the prefrontal cortex and hippocampus, as witnessed through the reduction of Keap1 and the accumulation of NRF2 and HO1. Together, these findings suggest that MLE might be an alternative therapeutic or preventive add-on strategy to improve the clinical expression of schizophrenia in adulthood, while also modifying the time course of this disease at earlier stages in populations at high-risk.

Keywords: Poly I:C; magnetic resonace imaging (MRI); mangiferin; neuroinflammation; oxidative/nitrosative stress; schizophrenia.

FIGURE 1

The effect of MLE treatment in adulthood on the behavior of the Poly I:C offspring. (A) Experimental timeline showing the design to study the effect of MLE treatment in adults. Maternal immune activation was induced in pregnant dams by administering Poly I:C (4 mg/kg i.v., intravenously) or the vehicle alone (controls) on gestational day (GD) 15. Male offspring were treated orally (p.o.) with MLE at a daily dose of 50 mg/kg of mangiferin in young adults since postnatal day (PND) 60. Then, behavioral evaluation was performed (PND 70-80). (B) In the prepulse inhibition (PPI) test, the effect of MLE treatment in adulthood is represented as the percentage PPI for 74, 80 and 86 dB prepulse intensity. (C,D) The effect of MLE treatment in adulthood on the object discrimination index for the short-term (STM) and long-term memory (LTM) phases of the novel object recognition test (NOR). (E) The effect of MLE treatment in adulthood on amphetamine sensitivity in the Poly I:C offspring. The total distance travelled in the open field is represented in 5 min (min) blocks, before and after amphetamine injection (shaded area, 2.5 mg/kg, i.p.). In addition, the area under the curve (AUC) values of amphetamine-induced activity were represented from 5 min after amphetamine injection. (F) The effect of MLE treatment in adulthood on anxiety-like behavior. The time spent (%) in the center is represented during the baseline period of free exploration in the open field. (G) The effect of MLE treatment in adulthood on grooming behavior. The total time spent grooming and number of grooming events are represented during the last 10 min of the baseline period in the open field. The data are represented as the mean ± SEM of 8–13 animals per group in the PPI test, n = 5-11 animals per group in STM and LTM phases of NOR test, and 9-11 animals per group in amphetamine-induced activity, anxiety-like and grooming behaviors. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 vs. VH-H₂O; ^## p < 0.01 vs. Poly I:C-H₂O, as assessed by two-way or two-way RM ANOVA followed by the LSD post-hoc test.

FIGURE 2

The effect of MLE or risperidone (RIS) treatment during adolescence on the behavior of the Poly I:C offspring. (A) Experimental timeline showing the design to study the effect of MLE treatment during adolescence. Maternal immune activation was induced in pregnant dams by administering Poly I:C (4 mg/kg i.v., intravenously) or the vehicle alone (controls) on gestational day (GD) 15. Male offspring were treated orally (p.o.) with MLE at a daily dose of 50 mg/kg of mangiferin during adolescence (postnatal day, PND 39-54) or administered intraperitoneally (i.p.) with risperidone (RIS, 0.3 mg/kg) as an adolescence reference treatment. Behavioral evaluation and magnetic resonance imaging (MRI) studies were performed at adulthood (PND 70-80 and PND 115-120, respectively). Finally, brain samples were collected at the end of the experiments. (B) The effect of adolescent MLE or RIS treatment on prepulse inhibition (PPI) test. The percentage PPI is represented for a 74, 80 and 86 dB prepulse intensity. (C,D) The effect of adolescent MLE or RIS treatment on the short-term (STM) and long-term memory (LTM) discrimination index between objects in the novel object recognition test (NOR). (E) The effect of adolescent MLE (left) or RIS (right) treatment on amphetamine sensitivity. The total distance travelled in the open field is represented in 5 min (min) blocks, before and after amphetamine injection (shaded area, 2.5 mg/kg i.p.). In addition, the area under the curve (AUC) values of amphetamine-induced activity were represented from 5 min after amphetamine injection. (F) The effect of adolescent MLE or RIS treatment on anxiety-like behavior. The time spent (%) in the center is represented during the baseline period of free exploration in the open field. (G) The effect of adolescent MLE or RIS treatment on grooming behavior. The total time spent grooming and the number of grooming events are represented during the last 10 min of the baseline period in the open field. The data are represented as the mean ± SEM of 10–13 animals per group in PPI test, 7-11 animals per group in the STM and LTM phases of the NOR test, and 7-12 animals per group for amphetamine induced activity, anxiety-like and grooming behaviors. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 vs. VH-H₂O; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. Poly I:C-H₂O as assessed by two-way or two-way RM ANOVA followed by the LSD post-hoc test.

FIGURE 3

The effect of MLE or risperidone (RIS) treatment during adolescence on brain volumetric changes measured by MRI. (A) Voxel-based morphometry (VBM) and (B) regions of interest (ROI) analysis in the Poly I:C model (phenotype). VBM results are represented in T-maps overlaid on a T2-MR template showing the volumetric changes in the gray matter (GM), white matter (WM) and cerebrospinal fluid (CSF) in Poly I:C-H₂O animals relative to the controls (VH-H₂O). The color bars represent the T-values corresponding to volumetric enlargement (warm) and shrinkage (cold). Tables show the phenotype-related effects on brain volumetric changes in the GM, WM and CSF of Poly I:C animals from the VBM analysis. The ROI results are represented in column plots of global and regional volumetric changes in whole brain, hippocampus (Hipp), frontal lobe (FL), all ventricles and the fourth ventricle (4 V) of Poly I:C- H₂O animals relative to the controls (VH-H₂O). (C) VBM results after adolescent treatment with MLE or (D) RIS. The VBM results are represented in T-maps overlaid on a T2-MR template showing the volumetric changes in the GM, WM and CSF of Poly I:C offspring treated with MLE or RIS relative to Poly I:C-H₂O animals. The color bars represent the T-values corresponding to volumetric enlargement (warm) and shrinkage (cold). The tables show treatment-related effects on the brain volumetric changes in the GM, WM and CSF of Poly I:C animals in the VBM analysis. (E) The ROI results after MLE or RIS treatment are represented as column plots of global and regional volumetric changes in whole brain, Hipp, FL, all ventricles and the 4 V compared to Poly I:C-H₂O animals. The VBM tables include: side, right (R) and left (L); T, t value; k, cluster size; volume, increase (↑) or decrease (↓); p unc., p value uncorrected; FDR, false discovery rate. The data are represented as the mean ± SEM of 10 animals per group. ^* p < 0.05, ^** p < 0.01 vs. VH-H₂O; ^## p < 0.01 vs. Poly I:C-H₂O as assessed with a Student’s t-test (unpaired, two-tailed), or one-way ANOVA followed by the LSD post-hoc test. Abbreviations: AA, amygdaloid area; ac, anterior commissure; C, cortex; Cb, cerebellum; icp, inferior cerebellar peduncle; LR4V, lateral recess of the fourth ventricle; mfb, medial forebrain bundle; rs, rubrospinal tract; RSA, retrosplenial area; vHipp, ventral hippocampus.

FIGURE 4

The effect of adolescent MLE or risperidone (RIS) treatment on the expression of oxidative/inflammatory mediators in the PFC. (A–C) The expression of the inflammatory mediators iNOS, COX2 and p38 relative to the controls, represented as the percentage (%) change, except for p38 which is represented as the ratio of phosphorylated relative to total p38 protein. (D,E) The concentrations of the indicators of oxidative/nitrosative damage (nitrites and 4-HNE) are expressed as µM/µg of protein, and as the % of the control expression. (F–P) Evaluation of the biomarkers of compensatory antioxidant mechanisms (Keap1, NRF2, SOD, CAT, GSH/GSSG, GPx, NQO1, HO1, TAOC). Keap1, NQO1 and HO1 expression is represented as the % of the controls; NRF2 activity is expressed in arbitrary units (AU); SOD and CAT enzyme activities are expressed as U/mg of protein; the levels of total (GSH_total), oxidized (GSSG) and reduced (GSH_free) glutathione are expressed as μM/µg of protein; Glutathione Peroxidase (GPx) is expressed as nmol/min/ml; and the Total Antioxidant Capacity (TAOC) is expressed in nmol/L. The data are represented as the mean ± SEM of 7-8 PFC samples per group. Representative bands of iNOS, COX2, 4-HNE, Keap1, NQO1 and HO1 (upper bands), and of the β-actin loading control (lower bands), are shown above their corresponding bars in the graph. For p38 protein expression, phosphorylated and total p38 representative bands are shown relative to β-actin (lower bands). ^* p < 0.05, ^** p < 0.01 vs. VH-H₂O; ^$ p < 0.05, ^$$followed by the LSD post-hoc test.

FIGURE 5

The effect of adolescent MLE or risperidone (RIS) treatment on the expression of oxidative/inflammatory mediators in the hippocampus. (A–C) The expression of the inflammatory mediators iNOS, COX2 and p38 represented as the percentage (%) of the control expression, with the exception of p38 which is represented as the ratio of phosphorylated relative to total p38 protein. (D,E) Indicators of oxidative/nitrosative damage (nitrites and 4-HNE) are expressed as their concentration (µM/µg of protein) and the % of the control expression, respectively. (F–P) The evaluation of biomarkers of compensatory antioxidant mechanisms (Keap1, NRF2, SOD, CAT, GSH/GSSG, GPx, NQO1, HO1, TAOC). Keap1, NQO1 and HO1 expression is represented as the % of the control expression; NRF2 activity is expressed in arbitrary units (AU); SOD and CAT enzyme activities are expressed as U/mg of protein; the levels of total (GSH_total), oxidized (GSSG) and reduced (GSH_free) glutathione are expressed as μM/µg of protein; Glutathione Peroxidase (GPx) is expressed in nmol/min/ml; and the Total Antioxidant Capacity (TAOC) is expressed in nmol/L. The data are represented as the mean ± SEM of 7-8 hippocampal samples per group. Representative bands of iNOS, COX2, 4-HNE, Keap1, NQO1 and HO1 (upper bands) and the β-actin loading control (lower bands) are shown above their corresponding bars in the graph. For p38 protein expression, phosphorylated and total p38 representative bands are shown relative to β-actin (lower bands). ^* p < 0.05, ^** p < 0.01, ^*** p < 0.01 vs. VH-H₂O; ^$ p < 0.05, ^$$ p < 0.01, ^$$$ p < 0.001 vs. VH-MLE or VH-RIS; ^# p < 0.05, ^## p < 0.01 vs. Poly I:C-H₂O as assessed by two-way ANOVA followed by the LSD post-hoc test.