Intrahippocampal Administration of Ibotenic Acid Induced Cholinergic Dysfunction via NR2A/NR2B Expression: Implications of Resveratrol against Alzheimer Disease Pathophysiology

Abstract

Although several drugs revealed moderate amelioration of symptoms, none of them have sufficient potency to prevent or reverse the progression toward Alzheimer's disease (AD) pathology. Resveratrol (RSV), a polyphenolic compound has shown an outstanding therapeutic effect on a broad spectrum of diseases like age-associated neurodegeneration, inflammation etc. The present study was thus conducted to assess the therapeutic efficacy of RSV in ameliorating the deleterious effects of Ibotenic acid (IBO) in male Wistar rats. Stereotactic intrahippocampal administration of IBO (5 μg/μl) lesioned rats impairs cholinergic transmission, learning and memory performance that is rather related to AD and thus chosen as a suitable model to understand the drug efficacy in preventing AD pathophysiology. Since IBO is an agonist of glutamate, it is expected to exhibit an excitotoxic effect by altering glutamatergic receptors like NMDA receptor. The current study displayed significant alterations in the mRNA expression of NR2A and NR2B subunits of NMDA receptors, and further it is surprising to note that cholinergic receptors decreased in expression particularly α7-nAChR with increased m1AChR. RSV administration (20 mg/kg body weight, i.p.) significantly reduced these changes in IBO induced rats. Glutamatergic and cholinergic receptor alterations were associated with significant changes in the behavioral parameters of rats induced by IBO. While RSV improved spatial learning performance, attenuated immobility, and improvised open field activity in IBO induced rats. NR2B activation in the present study might mediate cell death through oxidative stress that form the basis of abnormal behavioral pattern in IBO induced rats. Interestingly, RSV that could efficiently encounter oxidative stress have significantly decreased stress markers viz., nitrite, PCO, and MDA levels by enhancing antioxidant status. Histopathological analysis displayed significant reduction in the hippocampal pyramidal layer thickness and live neurons in IBO induced rats, with slight pathological changes in the entorhinal cortex (EC) of rat brain, which was prevented on RSV administration. Our study thus concludes that RSV administration significantly ameliorated the deleterious effects in the IBO lesioned rat model for AD by alleviating cholinergic pathways, reducing oxidative stress and thereby improving spatial memory.

Keywords: Alzheimer's disease; NMDAR; acetylcholine receptors; hippocampus; ibotenic acid; memory; resveratrol.

Figure 1

Schematic representation of time-line for RSV treatment in IBO induced experimental animals.

Figure 2

mRNA expression of NMDA receptors (NR2A and NR2B) in control and experimental animals. NMDARs expression levels were evaluated through semi-quantitative RT (reverse transcriptase)-PCR. (A,B), Shows NR2A and NR2B mRNA expression in hippocampus of control and experimental animals with corresponding bar diagram that denotes relative mRNA expression levels which is normalized with β-actin. Values are represented as mean ± SEM (n = 3). Statistical significance was performed by one-way ANOVA followed by Tukey's multiple comparison test. Values are statistically significant at ^*P < 0.05; ^**P < 0.01; ns.

Figure 3

Histopathological analysis of control and experimental group of rat brain regions: Photomicrographs showing the effect of RSV on IBO induced in vivo toxicity in hippocampus of rats (A). Sections were obtained from rat hippocampus 15 days after injection of IBO (n = 3). The morphology of the hippocampus and sub-regions are examined using H & E staining. In the IBO-model group, neuron arrangement is disrupted, severe lesions such as neuronal cell loss (black arrow), pycnotic cells (green arrow), and dead cells (red arrow) are observed in the nucleus and cytoplasm. Further prevention of degeneration through neuronal loss was attempted through RSV administration (A, magnification 40X). CA1, cornus ammonis 1; CA2, cornus ammonis 2; CA3, cornus ammonis 3; CA4, cornus ammonis 4; DG, Dentate gyrus. H and E staining of hippocampus associated region like entorhinal cortex (B) is examined for the presence of apoptotic cells, degeneration pattern etc., identifying IBO mediated pathology in associated regions from site of induction (magnification 40X).

Figure 4

Effects of RSV on relative mRNA expression of α7-nicotinic acetylcholine receptors and Muscarinic acetylcholine receptors 1 in IBO-induced hippocampus of rats. (A,B) Shows α7-nAChR and m1AChR mRNA expression in the hippocampus of control and experimental animals with corresponding bar diagram that denotes relative mRNA expression levels which are normalized with β-actin. Values are represented as mean ± SEM (n = 3). Statistical significance was performed by one-way ANOVA followed by Tukey's multiple comparison test. Values are statistically significant at ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ns.

Figure 5

RSV mitigates AChE activity on IBO-induced cholinergic impairments in rats. Acetylcholinesterase activity expressed as μmols of substrate hydrolyzed/min/mg protein in the hippocampus of control and experimental animals. Values are represented as mean ± SEM (n = 4). Statistical significance was performed by one-way ANOVA followed by Tukey's multiple comparison test. Values are statistically significant at ^*P < 0.05; ^**P < 0.01; ns.

Figure 6

Effect of RSV on spatial learning and memory of control and experimental rats using radial arm maze (RAM) task. The overall memory score was calculated on the basis of both training and the test phase (n = 7). (A) Shows the comparison of Retention time (latency period) of animals on 8-arm radial arm maze (RAM) task. The bar diagram represents the retention time of acquired task, where the retention time was assessed for 4 days following the procurement of the task. (B) Represents % correct response as depicted by the performance of animals in 8-arm RAM baited task. The bar diagram depicts the mean percentage of correct choices made by the rats as a function of time. Each animal received five trials/day for 4 days. Values are represented as mean ± SEM. Statistical significance was performed by one-way ANOVA followed by Tukey's multiple comparison test. Values are statistically significant at ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ns.

Figure 7

Effect of RSV on ambulation and locomotor activity of rats assessed in the open field test for the 5 min period for two successive days. Open field test activity was measured as the number grid crossing in 5 min. (A) Depicts the period of immobility in seconds by animals in the open field maze; (B) Represents ambulatory activity of the animals in the open field task. Values are represented as mean ± SEM (n = 7). Statistical significance was performed by one-way ANOVA followed by Tukey's multiple comparison test. Values are statistically significant at ^**P < 0.01; ^***P < 0.001; ns.

Figure 8

Effect of RSV on nitrite levels and its relative mRNA expression of nNOS gene in the hippocampus of IBO lesioned rats. Panel (A) denotes nNOS expression levels of control and experimental animals in representative gel image with corresponding bar diagram that indicates relative mRNA expression of nNOS normalized with β-actin (n = 3). Panel (B) Nitrite levels expressed as nmol/mg protein in the hippocampus of control and experimental animals (n = 4). Values are represented as mean ± SEM. Statistical significance was performed by one-way ANOVA followed by Tukey's multiple comparison test. Values are statistically significant at ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ns.

Figure 9

RSV exhibits antioxidative potential by mitigating IBO-induced oxidative stress. Antioxidant efficiency of RSV on IBO induced rats was assessed by determining enzymatic antioxidants like (A) glutathione peroxidase (μg of glutathione consumed/min/mg protein), (B) reduced glutathione levels (μg of glutathione/mg protein) and its efficacy in reducing oxidative stress was determined by evaluating (C) protein carbonyl content (nmol/mg protein), and (D) malondialdehyde levels (mmol/mg protein) in the hippocampus of control and experimental rats. Rats were exposed to IBO lesions (5 μg/μl) for 15 days and effect on these parameters assessed immediately after evaluating the behavioral performance. Values are represented as mean ± SEM (n = 4). Statistical significance was performed by one-way ANOVA followed by Tukey's multiple comparison test. Values are statistically significant at ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ns.