Skip to main content
NCBI home page
Cellular and Molecular Neurobiology logoLink to Cellular and Molecular Neurobiology
. 2016 Sep 1;37(5):791–802. doi: 10.1007/s10571-016-0418-4

Piracetam Facilitates the Anti-Amnesic but not Anti-Diabetic Activity of Metformin in Experimentally Induced Type-2 Diabetic Encephalopathic Rats

Shruti Pandey 1, Debapriya Garabadu 1,✉
PMCID: PMC11482147  PMID: 27585927

Abstract

Piracetam exhibits anti-amnesic activity in several animal models of dementia. However, its anti-amnesic potential has yet to be evaluated in type-2 diabetes mellitus (T2DM)-induced encephalopathy. Therefore, in the present study, piracetam (25, 50 and 100 mg/kg) was screened for anti-amnesic and anti-diabetic activity in T2DM-induced encephalopathic male rats. Subsequently, anti-amnesic and anti-diabetic activities were evaluated for piracetam, metformin and their combination in T2DM-induced encephalopathic animals. Rats received streptozotocin (45 mg/kg) and nicotinamide (110 mg/kg) injections on day-1 (D-1) of the experimental schedule and were kept undisturbed for 35 days to exhibit T2DM-induced encephalopathy. All drug treatments were continued from D-7 to D-35 in both experiments. Piracetam (100 mg/kg) attenuated loss in learning and memory in terms of increase in escape latency on D-4 (D-34) and decrease in time spent in the target quadrant on D-5 (D-35) of Morris water maze test protocol, and spatial memory in terms of reduced spontaneous alternation behavior in Y-maze test of encephalopathic rats. Additionally, piracetam attenuated altered levels of fasting plasma glucose and insulin, HOMA-IR and HOMA-B in encephalopathic animals, comparatively lesser than metformin. In the next experiment, combination of piracetam and metformin exhibited better anti-amnesic but not anti-diabetic activity than respective monotherapies in encephalopathic rats. Further, the combination attenuated reduced acetylcholine level and increased acetylcholinesterase activity, increased glycogen synthase kinase-3β level and decreased brain-derived neurotropic factor level in hippocampus and pre-frontal cortex of encephalopathic animals. Thus, piracetam could be used as an adjuvant to metformin in the management of dementia in T2DM-induced encephalopathy.

Keywords: Piracetam, Metformin, Type-2 diabetes mellitus, Memory, Hippocampus, Pre-frontal cortex

Introduction

Diabetes mellitus is considered as one of the most prevailing epidemics of the twenty-first century. The prevalence of type-2 diabetes mellitus (T2DM) reaches approximately 90 % in the diabetic population. It has been suggested that T2DM leads to several complications (Cohen et al. 2007; Rolo and Palmeira 2006) including central nervous system disorder (Ott et al. 1999; Stewart and Liolitsa 1999; Alvarez et al. 2009; Janson et al. 2004), a condition that is referred as “diabetic encephalopathy” (Brands et al. 2003). Clinical and animal studies demonstrate that T2DM can cause multidimensional cognitive deficits (Mehla et al. 2014; Zhang et al. 2014, 2016; Erus et al. 2015). Several strategies have been adopted to mitigate the T2DM-induced cognitive deficits; however, a successful pharmacological agent has not been established to date. Recently, it has been documented that metformin (MET) can ameliorate cognitive deficits in diabetic animals (Oliveira et al. 2016). Thus, it can be assumed that there would be a long-term therapy of MET in the management of cognitive deficits in T2DM-induced encephalopathy. Conversely, the long-term use of MET is limited because of its severe adverse effects including lactic acidosis (Chang et al. 2016). Hence, it is imperative to discover novel strategies in the management of cognitive deficits in T2DM-induced encephalopathy.

Piracetam is a group of racetams that exhibits nootropic activity in experimental animals (Raut et al. 2015; Tkachev 2007). Piracetam also exhibits anti-amnesic activity against experimentally induced amnesia in several animal models (Bhadania et al. 2012; Alikatte et al. 2012; Kosta et al. 2013; Pandareesh et al. 2016). Moreover, it has been reported that piracetam can protect neuronal damage in the brain of alloxan-induced diabetic animals (Zhiliuk et al. 2013, 2015). However, there is no report on the anti-diabetic activity of piracetam in either experimental or clinical studies to date. Furthermore, the anti-amnesic potential of piracetam has not yet been evaluated in T2DM-induced encephalopathy.

Several epidemiological studies have postulated that insulin resistance (IR) is one of the predisposing factors in the pathogenesis of cognitive deficits. However, the underlying molecular mechanism still remains obscure. The pathophysiology of cognitive deficits in T2DM includes the degeneration of cholinergic neurons, which culminates into a decrease in the level of acetylcholine (ACh) in several memory-sensitive brain regions including hippocampus (HIP) and pre-frontal cortex (PFC; Patel et al. 2015; Jiang et al. 2016). Further, it has been reported that an increase in the activity of acetylcholinesterase (AChE) enzyme is another cause of loss in ACh level in these brain regions of animals in such condition (Bhutada et al. 2011). In addition, reduced neurogenesis in terms of deteriorated activity of brain-derived neurotropic factor (Bdnf) is considered as another contributing factor in the degeneration of cholinergic neurons in amnesia (Del Arco et al. 2011; E Dief et al. 2015). Literature review also suggests that there is an upregulation of glycogen synthase kinase-3β (GSK-3β) in memory-sensitive brain areas during amnesic condition (Safar et al. 2016). GSK-3β downregulates TrκB receptors of Bdnf and thus impairs the molecular signaling of Bdnf in such memory sensitive brain regions during amnesic condition (Hu et al. 2013; Devi and Ohno 2015). MET exhibits therapeutic effect on high-fat diet-induced aberration in GSK-3β signaling in liver (Yang et al. 2014). It has also been suggested that MET protects neurodegeneration in MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) challenged animals through increasing the level of Bdnf in specific brain regions (Patil et al. 2014). Piracetam attenuates propofol-induced cognitive deficits, probably through Bdnf-mediated mechanism in the HIP of the animals (Wang et al. 2016). However, there is no report on the Bdnf-mediated mechanism of piracetam in T2DM-induced encephalopathy.

Therefore, in the first set of experiments, anti-amnesic and anti-diabetic activity of piracetam (25.0, 50.0 and 100.0 mg/kg) was investigated in T2DM-induced encephalopathic rats. In the next set of experiment, anti-amnesic and anti-diabetic activity of the combination of piracetam (effective dose) and MET was compared with respective monotherapies in T2DM-induced encephalopathic animals. Further, the extent of cholinergic dysfunction in terms of the level of ACh and activity of AChE, and levels of expression of GSK-3β and Bdnf were estimated in rat HIP and PFC to derive a plausible molecular mechanism for the combination in such deteriorated cognitive function.

Materials and Methods

Animals

Male Charles-Foster (CF) albino rats (180 ± 20 g) were obtained from the central animal house, Institute of Pharmaceutical Research, GLA University, Mathura and were used in this study. The animals were grouped and housed in poly-acrylic cages lined with husk under standard condition (24 ± 2 °C temperature, 45–55 % relative humidity and 12 h light: 12 h dark cycle). Animals were allowed to freely feed on their standard pellet diet (Lipton India, Ltd., Mumbai) and water ad libitum. Fasted animals were deprived of food for 16–18 h before experimentations, but were allowed to water ad libitum. All the experimental procedures utilized were performed in accordance with the approval of the Institutional Animal Ethics Committee (1260/PO/ERe/S/09/CPCSEA/IAEC/2016/P.Col/R13) under strict compliance of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines for experimental studies. Further, the experiments were conducted in accordance with the principles of laboratory animal care (National Research Council US Committee for the update of the guide for the care and use of laboratory animals 2011).

Chemicals

Streptozotocine, metformin and piracetam were procured from Sigma (St. Louis, MO, USA). Antibodies such as GSK-3β (ab75745), Bdnf (ab24491) and beta-actin (ab8227) were purchased from Abcam Plc., Cambridge, USA. All other chemicals and reagents were available commercially from local suppliers and were of analytical grade.

Animal Model of T2DM-Induced Encephalopathy

The T2DM was induced in overnight fasted rats by a single injection of streptozotocine (45 mg/kg, i.p.) 15 min after nicotinamide (110 mg/kg, i.p.) administration. Streptozotocine was dissolved in 0.1 M citrate buffer (pH 4.5) and nicotinamide was dissolved in physiological saline (Masiello et al. 1998; Garabadu and Krishnamurthy 2014). Rats with fasting serum glucose level ≥13.89 mmol/l and homoeostasis model assessment (HOMA) of IR (HOMA-IR) ≥5.0 on the seventh day of streptozotocine injection were considered T2DM animals and were included in the experimental protocol. Thereafter, these animals were continued in the experimental protocol of 35 days after the day of injection of streptozotocine. Further, the T2DM animals with cognitive deficits were considered as encephalopathic on the last day of the experimental protocol (Liapi et al. 2010).

Experimental Design

The whole study protocol consisted of two independent sets of experiments. The animals were acclimatized for 7 days and were randomly divided into six groups of six animals each namely Control, T2DM, T2DM+P-25, T2DM+P-50, T2DM+P-100 and T2DM+MET in first set of the experimental protocol. The day that animals of all groups (except control) received the streptozotocine and nicotinamide injection was considered as day-1 (D-1). On Day 7 (D-7), after 1 h to blood collection, either piracetam (25.0, 50.0 and 100.0 mg/kg, i.p.; Wang et al. 2016) or metformin (25 mg/kg, per oral (p.o.); Yanardag et al. 2005) was administrated to the T2DM+P-25, T2DM+P-50, T2DM+P-100 and T2DM+MET group animals, respectively. This treatment schedule was continued for 28 consecutive days, i.e., from D-7 to D-35 of the experimental design.

In the second set of experiments, the animals were also acclimatized for 7 days and were randomly divided into five groups of six animals each; namely, Control, T2DM, T2DM+P-100, T2DM+MET and T2DM+P-100+MET. The day that animals of all groups (except control) received the streptozotocine and nicotinamide injection was considered as day 1 (D-1). On D-7, after 1 h to blood collection, either piracetam (100.0 mg/kg, i.p.) or metformin (25 mg/kg, p.o.) or their combination was administrated to the T2DM+P-100, T2DM+MET and T2DM+P-100+MET group animals, respectively. There was a time lag of 30 min between the administration of piracetam (100.0 mg/kg) and MET in the T2DM+P-100+MET group animals. This treatment schedule was continued for 28 consecutive days, i.e., from D-7 to D-35 of the experimental design.

The entire experimental protocol was followed for 35 days in both sets of experiments. In both, the animals were subjected to Morris water maze (MWM) test for five consecutive days, i.e., from D-31 to D-35 of the experimental protocol. Further, the animals were subjected to Y-maze test on D-35 of the experimental protocol after 30 min to MWM test. It is important to note that all the behavioral assessments were started 1 h after drug administration on these days. All the behavioral observations were recorded and quantified with ANY-maze™ (Version-4.96, USA) video-tracking system. Thereafter, all the animals were killed by decapitation. The brains were removed and microdissected (Palkovits and Brownstein 1988) into HIP and PFC and stored immediately at −80 °C until further study.

Assessment of Cognitive Deficits in Different Behavioral Models

Spontaneous Alternation Behavior in Y-Maze Test

The working memory in terms of spontaneous alternation behavior (SAB) in Y-maze was assessed on D-35 of the experimental protocol (Mouri et al. 2007). The Y-maze task was a horizontal maze (40 cm long and 3 cm wide, with walls 12 cm high) made of black painted wood with three arms (labeled A, B, and C) disposed at 120° to each other. Each rat was placed at the center of the apparatus and allowed to move freely through the maze for 8 min. The number of alternations (i.e., consecutive entry sequences of ABC, CAB, or BCA, but not BAB) and the number of arm entries were recorded. Maze arms were thoroughly cleaned between tests with water spray to remove residual odors. The percentage alternation was calculated according to the following equation: percentage alternation = [(number of alternations)/(total arm entries−2)] × 100.

Learning and Memory in Morris Water Maze Test

The MWM test is one of the most commonly used animal models to access learning and memory (Morris 1984; Sharma and Singh 2010). The MWM test procedure was based on the principle of an animal being placed in a large pool of water divided into four equal quadrants. As the animal dislikes swimming, its tendency to escape was accomplished by finding a hidden escape platform. Each animal was subjected to four consecutive training trials (with an inter gap of 5 min) each day for four consecutive days in search for a hidden platform. The D-4 escape latency time to locate the hidden platform in the water maze was taken as the index of acquisition or learning. On the fifth day, the hidden platform was removed. Each animal was allowed to explore the pool for 120 s. The mean time spent in all the quadrants in search of the hidden platform was noted. The mean time spent by the animal in the target quadrant was taken as the index of retrieval or memory.

Estimation of Plasma Glucose and Insulin

On D-35 of the experimental protocol, 1 ml of blood was collected through retro-orbital puncture from fasted animals and centrifuged at 3000×g for 5 min at 4 °C to obtain plasma for measuring the glucose and insulin levels. The insulin was estimated by ELISA kit following manufacturer’s instructions. The plasma glucose was determined spectrophotometrically (BioTek Instruments Inc., Epoch®, USA) in triplicate using commercial available kits.

Insulin Resistance and β-Cell Function

HOMA-IR and HOMA of β-cell function (HOMA-B) were calculated by the HOMA method using the following equations (Matthews et al. 1985): IR (HOMA-IR) = (fasting glucose (mmol/l) × fasting insulin (mIU/ml))/22·5, and β-cell function (HOMA-B) = (20 × fasting insulin (mIU/ml))/(fasting glucose (mmol/l)– 3·5).

Evaluation of Cholinergic System

Preparation of the Samples

The HIP and PFC tissues were homogenized in 1 ml of 0.1 M perchloric acid with a homogenizer. The homogenate was kept in the polypropylene tubes for 15 min, after which 50 µl of 4 M potassium acetate was added to adjust the pH to 4.0, followed by centrifugation for 15 min at 4000×g (Muthuraju et al. 2009).

Spectrofluorometric Assay of ACh

The amount of ACh in brain tissues was measured using Amplex red assay kit (Molecular Probes, Inc., USA), as described by Zoukhri and Kublin (2001). Briefly, 0.1 ml of control (10 µM H2O2) and tissue homogenate were placed in two separate polypropylene tubes and then 0.1 ml of assay buffer (50 mm Tris–HCl, pH 7.5) containing 0.2 M Amplex red reagent, 2 U/ml horseradish peroxidase, 0.2 U/ml choline oxidase and 10 U/ml AChE were added to each tube. After incubation for 45 min, the fluorescence was recorded with the help of a spectrofluorometer at 530 nm excitation and 590 nm emission wavelengths. The protein content was determined using standard protocol (Lowry et al. 1951).

Estimation of AChE Activity

AChE is considered as a marker for loss of cholinergic neurons in discrete brain regions. The AChE activity was estimated using the Amplex red AChE assay kit (Molecular Probes, Inc., USA). Briefly, 0.1 ml of standard AChE (0.2 U/ml), control (10 µM H2O2) and tissue homogenate were placed in separate polypropylene tubes following addition of 0.1 ml of assay buffer (50 mM Tris–HCl, pH 7.5) containing 400 µM Amplex Red reagent, 2 U/ml horseradish peroxidase, 0.2 U/ml choline oxidase, and 100 µm acetylcholine into each tube and incubated for 30 min. After incubation, the fluorescence was determined with the help of a spectrofluorometer at 530 nm excitation wavelength and 590 nm emission wavelength. The protein content was determined by Lowry method (Lowry et al. 1951).

Immunoblotting

For Western blot analysis, the HIP and PFC were lysed in buffer containing complete protease inhibitor cocktail. Protein concentrations were determined according to standard method (Bradford 1976). A standard plot was generated using bovine serum albumin. Aliquots of each sample were electrophoresed in 10 % SDS-PAGE gels for GSK-3β and Bdnf proteins, transferred to polyvinylidene fluoride membranes and probed with specific antibodies. The membrane was incubated overnight with rabbit anti-GSK-3β (Abcam Plc., Cambridge, USA) and sheep anti-Bdnf (Abcam Plc., Cambridge, USA) polyclonal primary antibody at a dilution of 1:1000. After detection with the secondary antibodies of GSK-3β and Bdnf, the membrane was stripped with stripping buffer (25 mM Glycine pH 2.0, 2 % SDS for 30 min at room temperature) and re-probed overnight with rabbit anti-β-actin polyclonal primary antibody at a dilution of 1:500 to confirm equal loading of protein. Thereafter, membrane was probed with secondary antibodies of β-actin. Immunoreactive band of proteins were detected by chemiluminescence using enhanced chemiluminescence (ECL) reagents (Amersham Bioscience, USA). Quantification of the results was performed by densitometric scan of films. The immunoreactive area was determined by densitometric analysis using Biovis gel documentation software.

Data Analysis

All the data were mean ± standard error of the mean (SEM). All statistical analyses of data were done using one-way analysis of variance (ANOVA) with Newman–Keuls Post-hoc analysis to monitor significance among groups. p < 0.05 was considered as significant.

Results

Piracetam (100 mg/kg) Exhibited Anti-Diabetic Activity in T2DM Rats

Table 1 depicts the effect of piracetam (25.0, 50.0 and 100.0 mg/kg) on T2DM-induced changes in the levels of fasting plasma glucose and insulin, HOMA-IR and HOMA-B in rats. Statistical analysis revealed that there were significant differences in the levels of fasting plasma glucose [F(5,30) = 942.3; p < 0.05] and insulin [F(5,30) = 39.1; p < 0.05], HOMA-IR [F(5,30) = 314.1; p < 0.05] and HOMA-B [F(5,30) = 429.3; p < 0.05] among groups. Post-hoc test revealed that piracetam (100.0 mg/kg) treatment significantly attenuated T2DM-induced increase in the levels of fasting plasma glucose and insulin, and HOMA-IR, and decrease in HOMA-B in the rodents. However, the anti-diabetic activity of piracetam (100.0 mg/kg) was significantly lower compared to standard drug MET in T2DM rats.

Table 1.

Effect of piracetam (25.0, 50.0 and 100.0 mg/kg) on T2DM-induced changes in the levels of blood glucose and insulin, HOMA-IR and HOMA-B in rats

Groups Fasting blood glucose (mmol/l) Fasting blood insulin (pmol/l) HOMA-IR HOMA-B
Control 5.6 ± 0.5 72.0 ± 6.5 2.6 ± 0.2 96.3 ± 9.3
T2DM 48.0 ± 4.8a 132.9 ± 11.5a 40.8 ± 3.9a 8.5 ± 0.8a
T2DM+P-25 47.7 ± 4.7a 125.9 ± 12.5a 38.4 ± 2.8a 8.1 ± 0.7a
T2DM+P-50 46.8 ± 3.9a 129.8 ± 12.1a 38.9 ± 2.9a 8.6 ± 0.8a
T2DM+P-100 21.6 ± 1.8a,b,c,d 100.4 ± 9.1a,b,c,d 18.4 ± 1.0a,b,c,d 15.9 ± 1.2a,b,c,d
T2DM+MET 15.0 ± 1.2a,b,c,d,e 85.6 ± 7.2a,b,c,d,e 8.2 ± 0.8a,b,c,d,e 21.4 ± 2.1a,b,c,d,e
Open in a new tab

All values are mean ± SEM (n = 6)

a p < 0.05 compared to control

b p < 0.05 compared to T2DM

c p < 0.05 compared to T2DM+P-100

d p < 0.05 compared to T2DM+MET (One-way ANOVA followed by Student–Newman–Keuls test)

Piracetam (100 mg/kg) Exhibited Anti-Amnesic Activity in T2DM Rats

Effect of piracetam (25.0, 50.0 and 100.0 mg/kg) on T2DM-induced changes in the spatial memory in terms of SAB of the animals in Y-maze test paradigm is illustrated in Fig. 1. Statistical analysis revealed that there were significant differences in the SAB [F(5,30) = 46.9; p < 0.05] among groups. Post-hoc test showed that piracetam at the highest dose level significantly increased the T2DM-induced decrease in the SAB of the animals during Y-maze test. MET also significantly increased the T2DM-induced decrease in the SAB of rats during Y-maze test paradigm. However, the anti-amnesic activity of piracetam (100.0 mg/kg) was significantly higher compared to MET in T2DM animals during this test.

Fig. 1.

Fig. 1

Open in a new tab

Effect of piracetam (25.0, 50.0 and 100.0 mg/kg) on T2DM-induced changes in the spatial memory in terms of spontaneous alternation behavior (SAB) of rats in Y-maze test. All values are mean ± SEM (n = 6). a p < 0.05 compared to control, b p < 0.05 compared to T2DM, c p < 0.05 compared to T2DM+P-25, d p < 0.05 compared to T2DM+P-50, and e p < 0.05 compared to T2DM+P-100 (one-way ANOVA followed by Student–Newman–Keuls Post-hoc test)

Figure 2 illustrates the effect of piracetam (25.0, 50.0 and 100.0 mg/kg) on T2DM-induced changes in the learning and memory in terms of escape latency in D-1 (A) and D-4 (B), and time spent in the target quadrant in D-5 (C) in MWM test in rats. Statistical analysis revealed that there were significant differences in the escape latency in D-4 [F(5,30) = 40.1; p < 0.05] and time spent in the target quadrant in D-5 [F(5,30) = 26.6; p < 0.05] among groups. However, there were no significant differences in the escape latency in D-1 [F(5,30) = 0.4; p > 0.05] among groups. Post-hoc test revealed that piracetam (100.0 mg/kg) treatment significantly attenuated T2DM-induced increase in the escape latency in D-4 and decrease in the time spent in the target quadrant in D-5 of the MWM test protocol in the animals. MET also significantly attenuated the T2DM-induced increase in the escape latency in D-4 and decrease in the time spent in the target quadrant in D-5 of the MWM test protocol. However, the anti-amnesic activity of piracetam (100.0 mg/kg) was significantly higher compared to MET in T2DM animals during this test.

Fig. 2.

Fig. 2

Open in a new tab

Effect of piracetam (25.0, 50.0 and 100.0 mg/kg) on T2DM-induced changes in the learning and memory in terms of escape latency in D-1 (a) and D-4 (b), and time spent in the target quadrant in D-5 (c) of rats in Morris water maze (MWM) test. All values are mean ± SEM (n = 6). a p < 0.05 compared to control, b p < 0.05 compared to T2DM, c p < 0.05 compared to T2DM + P-25, d p < 0.05 compared to T2DM+P-50, and e p < 0.05 compared to T2DM+P-100 (one-way ANOVA followed by Student–Newman–Keuls Post-hoc test)

Piracetam and MET Combination Exhibited Better Anti-Diabetic Activity than Piracetam Monotherapy in T2DM Rats

Table 2 depicts the effect of piracetam (100.0 mg/kg), MET and their combination on T2DM-induced changes in the levels of fasting plasma glucose and insulin, HOMA-IR and HOMA-B in rats. Statistical analysis revealed that there were significant differences in the levels of fasting plasma glucose [F(4,25) = 376.8; p < 0.05] and insulin [F(4,25) = 29.4; p < 0.05], HOMA-IR [F(5,30) = 429.9; p < 0.05] and HOMA-B [F(5,30) = 470.1; p < 0.05] among groups. Post-hoc test revealed that piracetam (100.0 mg/kg) treatment significantly attenuated T2DM-induced increase in the levels of fasting plasma glucose and insulin, and HOMA-IR, and decrease in HOMA-B in the rodents. However, the anti-diabetic activity of piracetam (100.0 mg/kg) was significantly lower compared to standard drug MET in T2DM rats. Moreover, the anti-diabetic activity of their combination was significantly higher compared to piracetam (100.0 mg/kg) treatment in the T2DM rats.

Table 2.

Effect of piracetam (100.0 mg/kg), MET and their combination on T2DM-induced changes in the levels of blood glucose and insulin, HOMA-IR and HOMA-B in rats

Groups Fasting blood glucose (mmol/l) Fasting blood insulin (pmol/l) HOMA-IR HOMA-B
Control 6.4 ± 0.6 76.5 ± 7.4 3.1 ± 0.3 74.7 ± 6.5
T2DM 53.0 ± 4.8a 135.2 ± 12.8a 45.3 ± 3.5a 7.6 ± 0.8a
T2DM+P-100 30.7 ± 2.8a,b 110.6 ± 11.0a,b 21.8 ± 2.1a,b 11.7 ± 1.0a,b
T2DM+MET 19.8 ± 1.9a,b,c 90. 8 ± 8.9a,b,c 11.0 ± 1.1a,b,c 15.9 ± 1.5a,b,c
T2DM+P-100+MET 18.7 ± 1.8a,b,c 95. 9 ± 9.5a,b,c 10.3 ± 1.0a,b,c 18.4 ± 1.8a,b,c
Open in a new tab

All values are mean ± SEM (n = 6)

a p < 0.05 compared to control

b p < 0.05 compared to T2DM

c p < 0.05 compared to T2DM+P-100

d p < 0.05 compared to T2DM+MET (one-way ANOVA followed by Student–Newman–Keuls test)

Piracetam and MET Combination Exhibited Better Anti-Amnesic Activity than their Monotherapies in T2DM Rats

Effect of piracetam (100.0 mg/kg), MET and their combination on T2DM-induced changes in the spatial memory in terms of SAB of the animals in Y-maze test paradigm is illustrated in Fig. 3. Statistical analysis revealed that there were significant differences in the SAB [F(4,25) = 32.1; p < 0.05] among groups. Post-hoc test showed that piracetam significantly increased the T2DM-induced decrease in the SAB of the animals during Y-maze test. MET also significantly increased the T2DM-induced decrease in the SAB of the animals during Y-maze test paradigm. However, the anti-amnesic activity of piracetam was significantly higher compared to MET in T2DM animals during this test. In addition, the combination of piracetam and MET exhibited better anti-amnesic activity compared to both piracetam and MET monotherapies in T2DM animals.

Fig. 3.

Fig. 3

Open in a new tab

Effect of piracetam (100.0 mg/kg), MET and their combination on T2DM-induced changes in the spatial memory in terms of SAB of animals in Y-maze test. All values are mean ± SEM (n = 6). a p < 0.05 compared to control, b p < 0.05 compared to T2DM, c p < 0.05 compared to T2DM+P-100, and d p < 0.05 compared to T2DM+MET (one-way ANOVA followed by Student–Newman–Keuls Post-hoc test)

Figure 4 illustrates the effect of piracetam, MET and their combination on T2DM-induced changes in the learning and memory in terms of escape latency in D-1 (A) and D-4 (B), and time spent in the target quadrant in D-5 (C) of MWM test protocol in rats. Statistical analysis revealed that there were significant differences in the escape latency in D-4 [F(5,30) = 28.9; p < 0.05] and time spent in the target quadrant in D-5 [F(5,30) = 16.4; p < 0.05] among groups. Furthermore, there were no significant differences in the escape latency in D-1 [F(4,25) = 2.7; p > 0.05] among groups. Post-hoc test revealed that piracetam treatment significantly attenuated T2DM-induced increase in the escape latency in D-4 and decrease in the time spent in the target quadrant in D-5 of the MWM test protocol in the rodents. MET also significantly attenuated the T2DM-induced increase in the escape latency in D-4 and decrease in the time spent in the target quadrant in D-5 of the MWM test protocol in the rats. However, the anti-amnesic activity of piracetam was significantly higher compared to MET in T2DM animals during this test. Further, the anti-amnesic activity of the combination of piracetam and MET was significantly higher compared to either of their monotherapies in T2DM rats.

Fig. 4.

Fig. 4

Open in a new tab

Effect of piracetam, MET and their combination on T2DM-induced changes in the learning and memory in terms of escape latency in D-1 (a) and D-4 (b), and time spent in the target quadrant in D-5 (c) of animals in MWM test. All values are mean ± SEM (n = 6). a p < 0.05 compared to control, b p < 0.05 compared to T2DM, c p < 0.05 compared to T2DM+P-100, and d p < 0.05 compared to T2DM+MET (One-way ANOVA followed by Student–Newman–Keuls Post-hoc test)

Piracetam and MET Combination Exhibited Better Improvement in Cholinergic Function in HIP and PFC than their Monotherapies in T2DM Rats

Effect of piracetam, MET and their combination on T2DM-induced changes in the cholinergic function in terms of the level of ACh (A) and the activity of AChE (B) in rat HIP and PFC is illustrated in Fig. 5. Statistical analysis revealed that there were significant differences in the level of ACh and activity of AChE in HIP ([F(4,25) = 73.1; p < 0.05] and [F(4,25) = 29.7; p < 0.05] respectively) and PFC ([F(4, 25) = 55.3; p < 0.05] and [F(4,25) = 27.1; p < 0.05], respectively) among groups. Post-hoc test showed that piracetam significantly attenuated the T2DM-induced decrease in the ACh level and increase in the AChE activity in both brain regions of the animals. MET also significantly attenuated the T2DM-induced decrease in the ACh level and increase in the AChE activity in both brain regions of the animals. However, the piracetam treatment executed significantly higher improvement in cholinergic function in both the brain regions compared to MET administration in T2DM animals. Moreover, the combination of piracetam and MET exhibited higher improvement in cholinergic function in all the brain regions compared to either piracetam or MET monotherapy in T2DM animals.

Fig. 5.

Fig. 5

Open in a new tab

Effect of piracetam, MET and their combination on T2DM-induced changes in the cholinergic function in terms of the level of ACh (a) and activity of AChE (b) in rat HIP and PFC. All values are mean ± SEM (n = 6). a p < 0.05 compared to control, b p < 0.05 compared to T2DM, c p < 0.05 compared to T2DM+P-100, and d p < 0.05 compared to T2DM+MET (one-way ANOVA followed by Student–Newman–Keuls Post-hoc test)

Piracetam and MET Combination Exhibited Better Improvement in the Levels of Expression of GSK-3β and Bdnf in HIP and PFC than their Monotherapies in T2DM Rats

The effects of piracetam, MET and their combination on T2DM-induced changes in the levels of expression of GSK-3β and Bdnf in both brain regions of the animals are illustrated in Figs. 6 and 7, respectively. Statistical analysis revealed that there were significant differences in the levels of expression of GSK-3β and Bdnf in HIP ([F(4,10) = 50.1; p < 0.05] and [F(4, 10) = 52.5; p < 0.05] respectively) and PFC ([F(4, 10) = 44.5; p < 0.05] and [F(4,10) = 53.0; p < 0.05] respectively), among groups. Post-hoc test showed that piracetam significantly attenuated the T2DM-induced increase in the level of GSK-3β and decrease in the level of Bdnf in both the rat brain regions. MET also significantly attenuated the T2DM-induced increase in the level of GSK-3β and decrease in the level of Bdnf in HIP and PFC of the rats. However, the improvement in T2DM-induced changes in the levels of expression of these two proteins by piracetam was significantly higher compared to MET in T2DM animals. Moreover, the combination of piracetam and MET exhibited higher improvement in the level of expression of these two proteins in all the brain regions compared to both piracetam and MET monotherapy in T2DM animals.

Fig. 6.

Fig. 6

Open in a new tab

The effect of piracetam, MET and their combination on T2DM-induced changes in the level of expression of GSK-3β in rat HIP and PFC tissues. Blots a are representative of GSK-3β of rat HIP and PFC. The results in the histogram b are expressed as the ratio of relative intensity of level of expression of GSK-3β to β-actin. All values are mean ± SEM (n = 3). a p < 0.05 compared to control, b p < 0.05 compared to T2DM, c p < 0.05 compared to T2DM+P-100, and d p < 0.05 compared to T2DM+MET (one-way ANOVA followed by Student–Newman–Keuls Post-hoc test)

Fig. 7.

Fig. 7

Open in a new tab

The effect of piracetam, MET and their combination on T2DM-induced changes in the level of expression of Bdnf in rat HIP and PFC tissues. Blots a are representative of Bdnf of rat HIP and PFC. The results in the histogram b are expressed as the ratio of relative intensity of level of expression of Bdnf to β-actin. All values are mean ± SEM (n = 3). a p < 0.05 compared to control, b p < 0.05 compared to T2DM, c p < 0.05 compared to T2DM+P-100, and d p < 0.05 compared to T2DM+MET (one-way ANOVA followed by Student–Newman–Keuls Post-hoc test)

Discussion

The present study demonstrated for the first time the fact that piracetam (100.0 mg/kg) exhibited anti-amnesic and anti-diabetic activity in T2DM-induced encephalopathic rats. Further, the combination of piracetam (100.0 mg/kg) and MET exhibited better therapeutic effect in terms of anti-amnesic activity than their monotherapy in animals subjected to T2DM-induced encephalopathy. In addition, the combination exerted better improvement in T2DM-induced alteration in cholinergic, GSK-3β and Bdnf activity in memory-sensitive brain regions. Hence, piracetam can perhaps be considered as an adjuvant to MET in the management of cognitive deficits during T2DM-induced encephalopathy.

Cognitive deficit is considered as one of the symptomatic characteristics of encephalopathy during T2DM condition (Mehla et al. 2014; Zhang et al. 2014, 2016; Erus et al. 2015). In the present study, the highest dose of piracetam ameliorated the T2DM-induced cognitive deficits in Y-maze and MWM test paradigms. In support to our study, Zhiliuk et al. (2013) has reported that piracetam can exhibit anti-amnesic activity in alloxan-induced cognitive deficits in animals. MET also exhibited anti-amnesic activity in the present T2DM-induced encephalopathic rats, which is similar to that of earlier reports (Oliveira et al. 2016; Mostafa et al. 2016; Ghadernezhad et al. 2016). Moreover, the combination of piracetam and MET exhibited better anti-amnesic activity than their respective monotherapies in such T2DM-induced encephalopathy condition, indicating the fact that piracetam may enhance the anti-amnesic potential of MET in T2DM-induced encephalopathy.

In the present study, piracetam at highest dose level exhibited anti-diabetic activity that was comparatively lower than MET in T2DM animals. Literature review suggests that piracetam can attenuate hyperglycemia-induced complications such as platelet (Zhiliuk et al. 2012) and neuronal mitochondrial dysfunction (Zhiliuk et al. 2015). However, this is the first report of piracetam that ameliorates T2DM-induced hyperglycemia, though inferior to the standard drug MET. Moreover, the combination of piracetam and MET imparted better anti-diabetic activity than piracetam monotherapy in these animals, suggesting the fact that piracetam may not facilitate the anti-diabetic activity of MET in this condition.

The degenerated cholinergic activity in several brain regions is well established in the pathophysiology of loss in memory during T2DM-induced encephalopathy (Patel et al. 2015; Jiang et al. 2016). In the present study, piracetam and MET both mitigated T2DM-induced cholinergic dysfunction in terms of decreased ACh level and increased AChE activity in selected brain regions. The therapeutic effect of both of these candidates is reported in other amnesic conditions (Bhutada et al. 2011; Ahmad et al. 2014; Pandareesh et al. 2016), but we for the first time report the anti-amnesic activity of these two drugs in T2DM-induced encephalopathy. Further, the selected combination exhibited better therapeutic effect on cholinergic activity in rat HIP and PFC during this encephalopathy than their respective monotherapies. This observation emphasizes the fact that there could be one or more common targets in the due course of action of the combination.

In the present study, piracetam exhibited better amelioration than MET in T2DM-induced increase and decrease in the level of expression of GSK-3β and Bdnf in all the brain regions. In concurrence with our report, Yang et al. (2014) documented that MET can mitigate the high-fat diet-induced deleterious effect of GSK-3β signaling in liver tissues. Additionally, MET can also improve the Bdnf signaling in MPTP challenged animals (Patil et al. 2014). In support of our findings, it has also been suggested that piracetam attenuates propofol-induced cognitive deficits, probably through Bdnf-mediated mechanism in the brain tissue of the animals (Wang et al. 2016). It is well evidenced that the hyperactivity of GSK-3β signaling and reduced Bdnf activity are common in the pathogenesis of cholinergic dysfunction in amnesia (Bhutada et al. 2011; Del Arco et al. 2011; Hu et al. 2013; E Dief et al. 2015; Devi and Ohno 2015; Safar et al. 2016). Our present finding further reports that the selected combination exerted better attenuation than either of their monotherapies in both of the molecular signals in T2DM-induced encephalopathy. These observations indicate the fact that perhaps these two molecular signals may be common targets for the combination in attenuation of cholinergic dysfunction in such amnesic condition.

In conclusion, piracetam at highest dose level exhibited anti-amnesic and anti-diabetic activity in T2DM-induced encephalopathic rats. Further, the combination of piracetam and MET showed comparatively higher therapeutic effect against amnesia than either monotherapy in animals challenged to T2DM-induced encephalopathy. Moreover, the combination exerted better improvement in cholinergic, GSK-3β and Bdnf activity in memory-sensitive brain regions of T2DM challenged animals. Hence, piracetam could be used as an adjuvant to MET in the management of cognitive deficits during T2DM-induced encephalopathy.

Acknowledgments

SP is thankful to GLA University, Mathura, Uttar Pradesh, India for the financial assistantship.

Authors Contribution

SP and DG designed the study and were responsible for different aspects of data generation. SP performed the experimentation and analyzed the data. SP and DG wrote the manuscript.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interests.

References

  1. Ahmad A, Ramasamy K, Jaafar SM, Majeed AB, Mani V (2014) Total isoflavones from soybean and tempeh reversed scopolamine-induced amnesia, improved cholinergic activities and reduced neuroinflammation in brain. Food Chem Toxicol 65:120–128 [DOI] [PubMed] [Google Scholar]
  2. Alikatte KL, Akondi BR, Yerragunta VG, Veerareddy PR, Palle S (2012) Antiamnesic activity of Syzygium cumini against scopolamine induced spatial memory impairments in rats. Brain Dev 34:844–851 [DOI] [PubMed] [Google Scholar]
  3. Alvarez EO, Beauquis J, Revsin Y, Banzan AM, Roig P, De Nicola AF, Saravia F (2009) Cognitive dysfunction and hippocampal changes in experimental type 1 diabetes. Behav Brain Res 198:224–230 [DOI] [PubMed] [Google Scholar]
  4. Bhadania M, Joshi H, Patel P, Kulkarni VH (2012) Protective effect of menthol on β-amyloid peptide induced cognitive deficits in mice. Eur J Pharmacol 681:50–54 [DOI] [PubMed] [Google Scholar]
  5. Bhutada P, Mundhada Y, Bansod K, Tawari S, Patil S, Dixit P, Umathe S, Mundhada D (2011) Protection of cholinergic and antioxidant system contributes to the effect of berberine ameliorating memory dysfunction in rat model of streptozotocin-induced diabetes. Behav Brain Res 220:30–41 [DOI] [PubMed] [Google Scholar]
  6. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 [DOI] [PubMed] [Google Scholar]
  7. Brands AM, Henselmans JM, de Haan EH, Biessels GJ (2003) Diabetic encephalopathy: an underexposed complication of diabetes mellitus. Ned Tijdschr Geneeskd 147:11–14 [PubMed] [Google Scholar]
  8. Chang CH, Sakaguchi M, Dolin P (2016) Epidemiology of lactic acidosis in type 2 diabetes patients with metformin in Japan. Pharmacoepidemiol Drug Saf. doi:10.1002/pds.4030 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cohen G, Riahi Y, Alpert E, Gruzman A, Sasson S (2007) The roles of hyperglycaemia and oxidative stress in the rise and collapse of the natural protective mechanism against vascular endothelial cell dysfunction in diabetes. Arch Physiol Biochem 113:259–267 [DOI] [PubMed] [Google Scholar]
  10. Del Arco A, Segovia G, de Blas M, Garrido P, Acuña-Castroviejo D, Pamplona R, Mora F (2011) Prefrontal cortex, caloric restriction and stress during aging: studies on dopamine and acetylcholine release, BDNF and working memory. Behav Brain Res 216:136–145 [DOI] [PubMed] [Google Scholar]
  11. Devi L, Ohno M (2015) TrkB reduction exacerbates Alzheimer’s disease-like signaling aberrations and memory deficits without affecting β-amyloidosis in 5XFAD mice. Transl Psychiatry 5:e562 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dief AE, Samy DM, Dowedar IF (2015) Impact of exercise and vitamin B1 intake on hippocampal brain-derived neurotrophic factor and spatial memory performance in a rat model of stress. J Nutr Sci Vitaminol (Tokyo) 61:1–7 [DOI] [PubMed] [Google Scholar]
  13. Erus G, Battapady H, Zhang T, Lovato J, Miller ME, Williamson JD, Launer LJ, Bryan RN, Davatzikos C (2015) Spatial patterns of structural brain changes in type 2 diabetic patients and their longitudinal progression with intensive control of blood glucose. Diabetes Care 38:97–104 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Garabadu D, Krishnamurthy S (2014) Diazepam potentiates the antidiabetic, antistress and anxiolytic activities of metformin in type-2 diabetes mellitus with co-occurring stress in experimental animals. Biomed Res Int 2014:693074 [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  15. Ghadernezhad N, Khalaj L, Pazoki-Toroudi H, Mirmasoumi M, Ashabi G (2016) Metformin pretreatment enhanced learning and memory in cerebral forebrain ischaemia: the role of the AMPK/BDNF/P70SK signalling pathway. Pharm Biol 4:1–9 [DOI] [PubMed] [Google Scholar]
  16. Hu YS, Long N, Pigino G, Brady ST, Lazarov O (2013) Molecular mechanisms of environmental enrichment: impairments in Akt/GSK3β, neurotrophin-3 and CREB signaling. PLoS One 8:e64460 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Janson J, Laedtke T, Parisi JE, O’Brien P, Petersen RC, Butler PC (2004) Increased risk of type 2 diabetes in Alzheimer disease. Diabetes 53:474–481 [DOI] [PubMed] [Google Scholar]
  18. Jiang B, Song L, Huang C, Zhang W (2016) P7C3 attenuates the scopolamine-induced memory impairments in C57BL/6 J Mice. Neurochem Res 41:1010–1019 [DOI] [PubMed] [Google Scholar]
  19. Kosta P, Mehta AK, Sharma AK, Khanna N, Mediratta PK, Mundhada DR, Suke S (2013) Effect of piracetam and vitamin E on phosphamidon-induced impairment of memory and oxidative stress in rats. Drug Chem Toxicol 36:48–54 [DOI] [PubMed] [Google Scholar]
  20. Liapi C, Kyriakaki A, Zarros A, Galanopoulou P, Al-Humadi H, Dontas I, Voumvourakis K, Tsakiris S (2010) Choline-deprivation alters crucial brain enzyme activities in a rat model of diabetic encephalopathy. Metab Brain Dis 25:269–276 [DOI] [PubMed] [Google Scholar]
  21. Lowry OH, Rosenborough NJ, Farr AL, Randall RJ (1951) Protein measurement with folin phenol reagent. J Biol Chem 193:265–275 [PubMed] [Google Scholar]
  22. Masiello P, Broca C, Gross R, Roye M, Manteghetti M, Hillaire-Buys D, Novelli M, Ribes G (1998) Experimental NIDDM: development of a new model in adult rats administered streptozotocin and nicotinamide. Diabetes 47:224–229 [DOI] [PubMed] [Google Scholar]
  23. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC (1985) Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 28:412–419 [DOI] [PubMed] [Google Scholar]
  24. Mehla J, Chauhan BC, Chauhan NB (2014) Experimental induction of type 2 diabetes in aging-accelerated mice triggered Alzheimer-like pathology and memory deficits. J Alzheimers Dis 39:145–162 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Morris R (1984) Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods 11:47–60 [DOI] [PubMed] [Google Scholar]
  26. Mostafa DK, Ismail CA, Ghareeb DA (2016) Differential metformin dose-dependent effects on cognition in rats: role of Akt. Psychopharmacology (Berl). doi:10.1007/s00213-016-4301-2 [DOI] [PubMed] [Google Scholar]
  27. Mouri A, Noda Y, Hara H, Mizoguchi H, Tabira T, Nabeshima T (2007) Oral vaccination with a viral vector containing Aβ cDNA attenuates age-related Aβ accumulation and memory deficits without causing inflammation in a mouse Alzheimer model. FASEB J 21:2135–2148 [DOI] [PubMed] [Google Scholar]
  28. Muthuraju S, Maiti P, Solanki P, Sharma AK, Amitabh Singh SB, Prasad D, Ilavazhagan G (2009) Acetylcholinesterase inhibitors enhance cognitive functions in rats following hypobaric hypoxia. Behav Brain Res 203:1–14 [DOI] [PubMed] [Google Scholar]
  29. National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals (2011) Guide for the care and use of laboratory animals, 8th edn. National Academies Press, Washington [PubMed] [Google Scholar]
  30. Oliveira WH, Nunes AK, França ME, Santos LA, Lós DB, Rocha SW, Barbosa KP, Rodrigues GB, Peixoto CA (2016) Effects of metformin on inflammation and short-term memory in streptozotocin-induced diabetic mice. Brain Res 1644:149–160 [DOI] [PubMed] [Google Scholar]
  31. Ott A, Stolk RP, van Harskamp F, Pols HA, Hofman A, Breteler MM (1999) Diabetes mellitus and the risk of dementia: the Rotterdam study. Neurology 53:1937–1942 [DOI] [PubMed] [Google Scholar]
  32. Palkovits M, Brownstein MJ (1988) Maps and guide to microdissection of the rat brain. Elsevier, New York [Google Scholar]
  33. Pandareesh MD, Anand T, Khanum F (2016) Cognition enhancing and neuromodulatory propensity of bacopa monniera extract against scopolamine induced cognitive impairments in rat hippocampus. Neurochem Res 41:985–999 [DOI] [PubMed] [Google Scholar]
  34. Patel SS, Parashar A, Udayabanu M (2015) Urtica dioica leaves modulates muscarinic cholinergic system in the hippocampus of streptozotocin-induced diabetic mice. Metab Brain Dis 30:803–811 [DOI] [PubMed] [Google Scholar]
  35. Patil SP, Jain PD, Ghumatkar PJ, Tambe R, Sathaye S (2014) Neuroprotective effect of metformin in MPTP-induced Parkinson’s disease in mice. Neuroscience 277:747–754 [DOI] [PubMed] [Google Scholar]
  36. Raut SB, Parekar RR, Jadhav KS, Marathe PA, Rege NN (2015) Effect of Jyotīṣmatī seed oil on spatial and fear memory using scopolamine induced amnesia in mice. Anc Sci Life 34:130–133 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rolo AP, Palmeira CM (2006) Diabetes and mitochondrial function: role of hyperglycemia and oxidative stress. Toxicol Appl Pharmacol 212:167–178 [DOI] [PubMed] [Google Scholar]
  38. Safar MM, Arab HH, Rizk SM, El-Maraghy SA (2016) Bone marrow-derived endothelial progenitor cells protect against scopolamine-induced alzheimer-like pathological aberrations. Mol Neurobiol 53:1403–1418 [DOI] [PubMed] [Google Scholar]
  39. Sharma B, Singh N (2010) Pitavastatin and 4′-hydroxy-3-methoxyace tophenone HMAP reduce cognitive dysfunction in vascular dementia during experimental diabetes. Curr Neurovasc Res 7:180–191 [DOI] [PubMed] [Google Scholar]
  40. Stewart R, Liolitsa D (1999) Type 2 diabetes mellitus, cognitive impairment and dementia. Diabet Med 16:93–112 [DOI] [PubMed] [Google Scholar]
  41. Tkachev AV (2007) Application of nootropic agents in complex treatment of patients with concussion of the brain. Lik Sprava 5–6:82–85 [PubMed] [Google Scholar]
  42. Wang YL, Li F, Chen X (2016) Piracetam prevents memory deficit induced by postnatal propofol exposure in mice. Eur J Pharmacol 779:59–65 [DOI] [PubMed] [Google Scholar]
  43. Yanardag R, Ozsoy-Sacan O, Bolkent S, Orak H, Karabulut-Bulan O (2005) Protective effects of metformin treatment on the liver injury of streptozotocin-diabetic rats. Hum Exp Toxicol 24:129–135 [DOI] [PubMed] [Google Scholar]
  44. Yang Y, Li W, Liu Y, Li Y, Gao L, Zhao JJ (2014) Alpha-lipoic acid attenuates insulin resistance and improves glucose metabolism in high fat diet-fed mice. Acta Pharmacol Sin 35:1285–1292 [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Zhang Z, Lovato J, Battapady H, Davatzikos C, Gerstein HC, Ismail-Beigi F, Launer LJ, Murray A, Punthakee Z, Tirado AA, Williamson J, Bryan RN, Miller ME (2014) Effect of hypoglycemia on brain structure in people with type 2 diabetes: epidemiological analysis of the ACCORD-MIND MRI trial. Diabetes Care 37:3279–3285 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zhang Y, Lu S, Liu C, Zhang H, Zhou X, Ni C, Qin W, Zhang Q (2016) Altered brain activation and functional connectivity in working memory related networks in patients with type 2 diabetes: an ICA-based analysis. Sci Rep 6:23767 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zhiliuk VI, Levykh AÉ, Mamchur VI (2012) Platelet hyperreactivity and antiaggregatory properties of nootropic drugs under conditions of alloxan-induced diabetes in rats. Eksp Klin Farmakol 75:38–41 [PubMed] [Google Scholar]
  48. Zhiliuk VI, Levykh AE, Mamchur VI (2013) Study of acetylsalicylic acid role in the potentiation of antiamnesic and neuroprotective properties of piracetam in rats with alloxan diabetes. Georgian Med News 220–221:64–71 [PubMed] [Google Scholar]
  49. Zhiliuk VI, Mamchur VI, Pavlov SV (2015) Role of functional state of neuronal mitochondria of cerebral cortex in mechanisms of nootropic activity of neuroprotectors in rats with alloxan hyperglycemia. Eksp Klin Farmakol 78:10–14 [PubMed] [Google Scholar]
  50. Zoukhri D, Kublin CL (2001) Impaired neurotransmitter release from lacrimal and salivary gland nerves of a murine model of sjögren’s syndrome. Invest Ophthalmol Vis Sci 42:925–932 [PMC free article] [PubMed] [Google Scholar]

Articles from Cellular and Molecular Neurobiology are provided here courtesy of Springer

RESOURCES