Chotosan ameliorates cognitive and emotional deficits in an animal model of type 2 diabetes: possible involvement of cholinergic and VEGF/PDGF mechanisms in the brain

Abstract

Background: Diabetes is one of the risk factors for cognitive deficits such as Alzheimer's disease. To obtain a better understanding of the anti-dementia effect of chotosan (CTS), a Kampo formula, we investigated its effects on cognitive and emotional deficits of type 2 diabetic db/db mice and putative mechanism(s) underlying the effects.

Methods: Seven-week-old db/db mice received daily administration of CTS (375 - 750 mg/kg, p.o.) and the reference drug tacrine (THA: 2.5 mg/kg, i.p.) during an experimental period of 7 weeks. From the age of 9-week-old, the animals underwent the novel object recognition test, the modified Y-maze test, and the water maze test to elucidate cognitive performance and the elevated plus maze test to elucidate anxiety-related behavior. After completing behavioral studies, Western blotting and immunohistochemical studies were conducted.

Results: Compared with age-matched non-diabetic control strain (m/m) mice, db/db mice exhibited impaired cognitive performance and an increased level of anxiety. CTS ameliorated cognitive and emotional deficits of db/db mice, whereas THA improved only cognitive performance. The phosphorylated levels of Akt and PKCα in the hippocampus were significantly lower and higher, respectively, in db/db mice than in m/m mice. Expression levels of the hippocampal cholinergic marker proteins and the number of the septal cholinergic neurons were also reduced in db/db mice compared with those in m/m mice. Moreover, the db/db mice had significantly reduced levels of vasculogenesis/angiogenesis factors, vascular endothelial growth factor (VEGF), VEGF receptor type 2, platelet-derived growth factor-B, and PDGF receptor β, in the hippocampus. CTS and THA treatment reversed these neurochemical and histological alterations caused by diabetes.

Conclusion: These results suggest that CTS ameliorates diabetes-induced cognitive deficits by protecting central cholinergic and VEGF/PDGF systems via Akt signaling pathway and that CTS exhibits the anxiolytic effect via neuronal mechanism(s) independent of cholinergic or VEGF/PDGF systems in db/db mice.

Figure 1

Schematic drawing of experimental schedule. After one week of acclimatization, the db/db mice were randomly divided into 5 groups. Age-matched m/m and db/db mice were used as controls in the behavioral and neurochemical experiments.

Figure 2

Effects of CTS and THA on object discrimination performance of db/db mice in the ORT. The ORT was conducted on days 19–21 after starting drug administration. Each datum represents the mean ± S.D. (6 – 9 mice per group). (A) The data from the sample trials of the ORT. The animal was placed into the arena where two identical sample objects made of glass (objects A1 and A2) were placed in two adjacent corners of the arena and was allowed to explore for five minutes. (B) The data from the test phase trials conducted ten minutes after the sample phase trials. In the test phase trials, the time animals spent exploring a familiar object or a new object was measured during a 5-minute observation period. ***P < 0.001 and **P < 0.01 vs. the time spent exploring a familiar object (paired t-test). (C) Discrimination index (DI) in the ORT. DI was calculated as described in the text. **P < =0.01 vs. vehicle-treated m/m group (t-test). *P < 0.05 vs. vehicle-treated db/db group Tukey test.

Figure 3

Diabetes-induced spatial working memory deficit in the MYM test and its reversal by test drugs.A) Schematic drawings of the Y-maze and the experimental procedure. The maze was surrounded by different spatial cues. A) Experimental protocol of the MYM test. The sample trial and test trials were conducted for 5 min at a 30-min interval. (B) Each test was conducted 30 min (THA) or 60 min after daily oral (CTS) or i.p. (THA) administration of test drugs. % Time spent in a novel arm (B) and total locomotion distance during a 5-min observation period were analyzed. Each data column represents the mean ± S.D. (n = 6–9). #P < 0.05 and ###P < 0.001 compared with vehicle-treated m/m group. *P < 0.05 compared with vehicle-treated db/db group (Tukey test).

Figure 4

Effects of CTS and THA on water maze performance of db/db mice. Learning performance of the animals was analyzed in the training test by latency (A) and swimming speed (B). Each data point indicates the mean ± S.D. for 6 – 9 animals in each group. ^###P < 0.001 vs. vehicle-treated m/m group, and ^**P < 0.01 vs. vehicle-treated db/db group (one-way repeated measures ANOVA). Memory retrieval performance (D) elucidated in the probe test. Each datum represents the mean of time spent in the target quadrant ± S.D. ^###P < 0.001 vs. vehicle-treated m/m group (t-test). ^*P < 0.05 vs. respective vehicle-treated db/db group (one-way repeated measure ANOVA followed by Tukey test).

Figure 5

Effects of CTS administration on EPM performance of db/db mice. The animals received an EPM test at around the age of 13 weeks old. The 7-week-old db/db mice were orally administered water (vehicle), 375 – 750 mg/kg CTS, 2.5 mg/kg THA once daily during the experimental period. The EMT was conducted 1 hr after CTS administration. The age-matched naïve db/db mice received i.p. injection of 1 mg/kg diazepam 30 min before the test. Each datum represents the mean ± S.D. (6 – 9 animals per group). The proportion of time spent in open arms (A) and the total locomotion distance on the maze (B) were calculated. The data are expressed as the mean ± S.D. ###P < 0.001 vs. vehicle-treated m/m group (Student’s t-test). *P < 0.05, **P < 0.01 vs. vehicle-treated db/db group (one-way ANOVA followed by Tukey test).

Figure 6

Effects of CTS and THA treatment on Akt, p-Akt, PKCα, p-PKCα/βII, and β-actin in the hippocampi of db/db mice. After completing the behavioral studies, the animals were decapitated and proteins were extracted from the hippocampi in each animal group. A) Typical photos indicating the expression levels of each factor in the hippocampus of vehicle-treated m/m (lane a), and vehicle (lane b)-, CTS (325 mg/kg per day: lane c; 750 mg/kg per day: lane d)-, and THA (2.5 mg/kg per day; lane e)-treated db/db mice. B) Quantitative comparisons of test drug-induced changes in Akt, p-Akt, PKCα, p-PKCα/βII, and β-actin in the hippocampi of db/db mice. The data are expressed as the percentage of the value obtained from naïve control m/m mice. Each data column represents the mean ± S.D. obtained from 5-6 brain samples. #P < 0.05 or ##P < 0.01 vs. vehicle-treated SAMR1 group (Student’s t-test). ##P < 0.01, *P < 0.05, ***P < 0.001 vs. respective vehicle-treated db/db group (one-way ANOVA followed by Tukey test).

Figure 7

Effects of CTS and THA on VEGF/VEGFR2 and PDGF/PDGFR expression in the hippocampus of db/db mouse.A) Typical photos indicating the expression levels of each factor in the hippocampus of vehicle-treated m/m (lane a), and vehicle (lane b)-, CTS (325 mg/kg per day: lane c; 750 mg/kg per day: lane d)-, and THA (2.5 mg/kg per day; lane e)-treated db/db mice. B) Quantitative comparisons of each factor among different animal groups were conducted as described in the text. The data are expressed as the percentage of the value obtained from naïve control m/m mice. Each data column represents the mean ± S.D. obtained from 5 – 6 brain samples. #P < 0.05, ##P < 0.01 vs. vehicle-treated m/m group (Student’s t-test). *P < 0.05, **P < 0.01, ***P < 0.001 vs. respective vehicle-treated db/db group (one-way ANOVA followed by Tukey test).

Figure 8

Effects of CTS and THA on VEGF expression in the retinal tissue in db/db mice. The retinal tissues were obtained from the animals perfused with paraformaldehyde as described in the text. The arrows a, b, c, and d represent pigment epithelium, outer segments, inner segments, and outer plexiform layers, respectively. VEGF-positive portions were identified by VEGF immunostaining. Photos 1, 2, 3, and 4 were from the retinal tissues of m/m control, vehicle-treated db/db, CTS (750 mg/kg per day)-treated db/db, and THA (2.5 mg/kg per day)-treated db/db group, respectively. Arrows a, b, c, and d represent pigment epithelium, outer segments, inner segments, and outer plexiform layer, respectively.

Figure 9

Effects of CTS and THA on ChAT, M₁, M_3,and M₅receptor protein expression in the hippocampus of db/db mouse. Typical photos indicating the expression levels of each factor in the hippocampus of vehicle-treated m/m (lane a), and vehicle (lane b)-, CTS (325 mg/kg per day: lane c; 750 mg/kg per day: lane d)-, and THA (2.5 mg/kg per day; lane e)-treated db/db mice. B) Quantitative comparisons of each factor among different animal groups were conducted as described in the text. The data are expressed as the percentage of the value obtained from naïve control m/m mice. Each data column represents the mean ± S.D. obtained from 5-6 brain samples. ##P < 0.01, ###P < 0.01 vs. vehicle-treated m/m group (Student’s t-test). **P < 0.01, ***P < 0.001 vs. respective vehicle-treated db/db group (one-way ANOVA followed by Tukey test).

Figure 10

Effects of CTS and THA treatment on diabetes-induced cholinergic neuron degeneration in the medial septum. Cholinergic neurons were identified by ChAT immunostaining in vehicle-treated m/m (1), and vehicle (2)-, CTS (750 mg/kg per day: 3)-, and THA (2.5 mg/kg per day; 4)-treated db/db mice. Cholinergic neurons were stained with anti-ChAT antibody, showing that predominant ChAT expression occurred in the cell body of cholinergic neurons. Scale bar = 100 μm.