Effects of MCl-186 on intracellular calcium ion concentration and membrane fluidity of hippocampal neurons in a rat model of Alzheimer’s disease＊＊☆

Ming Yu, Lei Qin, Zhao Wang, Xiaohong Lu, Ying Zhu, Wenhui Leng, Xuan Wang

1Department of Neurology, Affiliated Hospital of Jiangsu University, Zhenjiang 212001, Jiangsu Province, China

2Department of Preventive Medicine, Nanjing University of Chinese Medicine, Nanjing 210046, Jiangsu Province, China

3Department of Biochemistry, Basic Medical College of Jiamusi University, Jiamusi 154007, Heilongjiang Province, China

4Department of Neurology, Affiliated First Hospital of Jiamusi University, Jiamusi 154002, Heilongjiang Province, China

lNTRODUCTlON

Alzheimer's disease (AD) is characterized by extensive synaptic and neuronal loss that leads to impaired memory and cognitive decline[1].The precise mechanisms underlying AD have not been completely explained and no effective therapy has yet been developed.Oxidative stress has attracted increasing attention because many free radical scavengers can slow the development of AD and improve cognitive function in patients with AD[2].

Overactivation of the N-methyl-D-aspartate acid(NMDA)-subtype of glutamate receptor has been known to trigger excessive cellular calcium ion (Ca2+) influx, contributing to neurodegenerative conditions including AD.Such dysregulation of intracellular calcium signaling results in generation of excessive reactive oxygen species; this changes the membrane properties of biological membranes and membrane fluidity is decreased.Oxidative stress affects the homeostasis of intracellular calcium ion levels,and antioxidants play an important role in the prevention and treatment of AD associated with abnormal Ca2+mobilization induced by reactive oxygen species[3].Furthermore, dysregulation of intracellular Ca2+homeostasis was suggested to underlie the occurrence and development of AD.Based on these theories, the Ca2+hypothesis of AD has been proposed[4].

MCI-186 (edaravone, 3-methyl-1-phenyl-2-pyrazolin-5-one), a new type of free radical scavenger, shows unique effects on the treatment of acute cerebrovascular diseases and motor neuron diseases[5-6].In our previous studies, MCI-186 had neuroprotective effects in a cellular model of AD through scavenging of hydroxyl radicals (·OH) and inhibition of apoptosis[7-9].However, it has not yet been elucidated whether calcium ion levels and membrane fluidity play a role in the process of scavenging ·OH by MCI-186.This study investigated the alterations of membrane fluidity and intracellular calcium ion concentration ([Ca2+]i) in hippocampal neurons, and determined the relationships among intracellular Ca2+, membrane fluidity and MCI-186 in AD model rats to further verify the neuroprotective effect of MCI-186 in an animal model of AD and provide a more theoretical basis for treatment with MCI-186 targeting Ca2+homeostasis in AD.

RESULTS

Quantitative analysis of experimental animals

One-hundred Wistar rats were randomly divided into five groups: a normal group in which no treatment was given;a sham operation group in which the skull was drilled, but the hippocampal fimbria-fornix was not transected, and 1 mL of normal saline was injected into the lateral ventricles; a dementia group that received no treatment after successful establishment of an AD rat model; and highand low- concentration MCI-186 treatment groups in which MCI-186, which was diluted with saline to 0.5 mg/mL and 0.2 mg/mL, respectively, was administered at a dose of 3 mg/kg per dayviathe tail vein for 14 days from the 2ndday after the AD rat model was successfully established (doses were determined according to a conversion of the body surface area of man to that of rats).A total of four rats, including two rats in the dementia group and two rats in the high-concentration MCI-186 treatment group, died during dementia model establishment.Six rats were randomly chosen from the survivors in each group on the 0th, 7thand 17thdays following model preparation and included in the final analysis.

Effect of MCl-186 on learning and memory abilities in AD model rats

The escape latency and the spanning platform times during 2 minutes were determined in the Morris water maze on the 0th, 7thand 17thdays following AD model establishment to determine learning and memory abilities in the rats.

On the 0thday (before treatment with MCI-186), compared with the normal and sham operation groups, the escape latency was significantly prolonged and the spanning platform times were decreased in the dementia group and in the high- and low-concentration MCI-186 treatment groups (P＜0.01 orP＜0.05).There were no differences in escape latency or spanning platform times among these three groups (P＞0.05).

On the 7thand 17thdays (after treatment with MCI-186), compared with the dementia group, the escape latency was shortened and the spanning platform times were notably increased in the high- and low-concentration MCI-186 treatment groups (P＜0.01 orP＜0.05).This suggests that MCI-186 improved the learning and memory abilities in AD rats.The escape latency was increased and the spanning platform times were decreased in the high- and lowconcentration MCI-186 treatment groups compared with those in the normal group (P＜0.01 orP＜0.05).These results suggest that the learning and memory abilities in AD rats did not recover to the normal level after treatment with MCI-186.The escape latency was shortened and the spanning platform times were increased in the low-concentration MCI-186 treatment group compared with those in the high-concentration MCI-186 treatment group (P＜0.01 orP＜0.05).This suggests that the effect of low-concentration MCI-186 treatment was better than that of the high-concentration MCI-186 treatment(Tables 1, 2).

Table 1 Comparison of escape latency (second)among five groups

Table 2 Comparison of spanning platform times (times in 2 minutes) among five groups

Effect of MCl-186 on intracellular calcium concentration in rats with AD

The intracellular calcium concentration in the hippocampal neurons was assessed by Fura-2/AM fluorimetry.There was a significant difference in intracellular calcium concentration between the three AD model groups and the normal and sham operation groups on the 0thday.This suggests that rats with AD were successfully prepared (P＜0.01 orP＜0.05).No significant difference was observed among the high- and low-concentration MCI-186 treatment groups and the dementia group (P＞ 0.05).The intracellular calcium concentrations in the high- and low-concentration MCI-186 treatment groups were lower than that in the dementia group on the 0thand 7thdays (P＜0.01 orP＜0.05); the difference was greater on the 17thday (P＜0.01).The intracellular calcium concentrations in the high- and low-concentration MCI-186 treatment groups were remarkably lower on the 17thday than before, and the difference in the low-concentration group was extremely significant (P＜0.05; Table 3).

Table 3 Comparison of intracellular calcium concentration(nmol/L) among five groups

Effect of MCl-186 on the mean microviscosity of hippocampal neurons in AD rats

Membrane fluidity in hippocampal neurons isolated from Wistar rats was measured using a fluorescence spectrophotometer.Neurons were enzymatically isolated and loaded with the fluorescent dye, 1, 6-diphenyl-1, 3,5-hexatriene (DPH).Mean microviscosity was used as an index of membrane fluidity.There was a significant difference in the mean microviscosity of hippocampal neurons between the three model groups and the normal and sham operation groups on the 0thday, which suggests that the AD model rats were successfully prepared(P＜0.01).However, there was no statistically significant difference in the mean microviscosity of hippocampal neurons among the high- and low- concentration MCI-186 treatment groups and the dementia group on the 7thday (P＞ 0.05).There was also no statistically significant difference in mean microviscosity in hippocampal neurons among the high- and low-concentration MCI-186 treatment groups and the dementia group on the 7thday (P＞ 0.05).There was a significant difference on the 17thday (P＜0.01).The mean microviscosity of hippocampal neurons was decreased in the high- and low-concentration MCI-186 treatment groups only on the 17thday, and the difference in the low-concentration group was extremely significant(P＜0.01; Table 4).

Table 4 Comparison of the mean microviscosity (ηp)of hippocampal neurons among five groups

DlSSCUSlON

The relationship between intracellular Ca2+and aging-associated neurodegeneration in the brain is receiving increasing attention[10-12].Ca2+participates in presynaptic neurotransmitter release or affects NMDA receptors in the neuronal postsynaptic membrane[13].Ca2+interferes with the formation of long-term potentiation (LTP) and acts as a key factor controlling neural plasticity[14-15].On the contrary, overload of intracellular Ca2+not only accompanies many pathophysiological processes including tau hyperphosphorylation, mutation of presenilins, beta amyloid (Aβ) production and apoptotic induction, but also increases the threshold of synaptic transmission and the inhibition of synaptic facilitation and influences synaptic transmission,neurotransmitter release and signal transduction[16-17].Therefore, overload of intracellular calcium reduces learning and memory abilities in patients with AD[18-19].Intracellular calcium, a second messenger in the cytoplasm, is an apoptotic signal transduction molecule and effector molecule that regulates almost all physiological activities of the body and participates in the process of apoptosis[20].

The results from this study showed that a rat model of AD can be successfully established by hippocampal fimbria-fornix disconnection combined with intraventricular injection of Aβ25-35.Compared with the normal and sham-operation groups, the concentration of intracellular calcium and the mean microviscosity of hippocampal neurons were significantly increased, the escape latency prolonged and the spanning platform times were decreased in the AD model rats.This study further confirmed that changes in intracellular calcium and membrane fluidity affect learning and memory abilities in AD model rats.Many studies have indicated that Aβ protein oligomerization, a process thought to be central to AD, causes the degeneration of synapses and neurons, finally inducing the pathogenesis of AD.Numerous findings have showed that reactive oxygen species (ROS), especially the ·OH, which is the most active and destructive species, can be generated outside neurons when Aβ aggregates into insoluble fibers from monomers[21].These oxygen-derived free radicals and hydrogen peroxide have been suggested to contribute to cell damage.Extracellularly generated free radicals including·OH may initiate toxic reactions with the unsaturated fatty acids present in membranes at the plasma membrane.Phosopholipids, resulting from peroxidation of the unsaturated fatty acids of the cell membrane, cause alterations of membrane structural and functional characteristics[22].Lipid peroxidation also affects the physical properties of membranes, including membrane fluidity.The decrease in membrane fluidity caused by hydrogen peroxide may be partially related to an increase in intracellular calcium concentration and free radicals may directly change membrane fluidity by lipid peroxidation.They may also indirectly change membrane fluidity as a result of increasing the intracellular calcium concentration.Aβ-induced changes in membrane fluidity can be explained by physico-chemical interactions of the peptide with membrane components such as cholesterol, phospholipids and gangliosides.Intracellular calcium overload also resulted from calcium ion influx through ion channels formed by Aβ itself[23].Thus, Aβ disturbs the membrane properties of biological membranes leading to calcium ion influx.Then, several downstream calcium-conducting signaling pathways,involving the Ca2+-dependent protein phosphatase calcineurin, Ca2+/calmodulin-dependent protein kinase II,protein phosphatase 1, and cAMP response element-binding protein, are dysregulated[24].Dysregulation of calcium signaling, in turn, causes excessive production of reactive oxygen species.Finally, cell organelles,cell membranes and the cytoskeleton are damaged,leading to cell apoptosis or even cell death[25].Because Aβ disrupts the balance of calcium ions inside and outside neurons and changes signal transduction through calcium-conducting channels, Aβ-induced Ca2+dysregulation may contribute to many neuropathologic features of AD[26-27].

Compared with the normal and sham-operation groups,the concentration of intracellular calcium and the mean microviscosity of hippocampus neurons were significantly decreased, the escape latency shortened and the spanning platform times increased after treatment with MCI-186.The results also showed that low-concentration MCI-186 was superior to high-concentration MCI-186 in decreasing intracellular calcium concentration and mean microviscosity in hippocampal neurons, shortening the escape latency and increasing the spanning platform times, which suggested that low-concentration MCI-186 has more advantages than high- concentration MCI-186 in improving learning and memory abilities in AD model rats.These results suggest that MCI-186 could improve learning and memory abilities in AD model rats through scavenging hydroxyl radicals generated from Aβ, decreasing intracelluar calcium ion concentration and improving membrane fluidity.However, the precise mechanism by which MCI-186 improves the imbalance of intracellular calcium is still unclear.

Although calcium imbalance plays a role in the pathogenesis of AD, other pathogenic factors are also involved.A shortage of calcium ions and energy leads to a decrease in the activity of Ca2+-transporting ATPase and a deposition of Aβ protein and microtubule-associated protein, thus building a link between the theory of calcium ion imbalance and the theory of Aβ and microtubule-associated protein[28-30].Therefore, it is suggested there is a harmful process involving abnormal metabolism of amyloid precursor protein (APP), oxidative damage, inhibition of energy metabolism and calcium imbalance.

In conclusion,the present study showed that MCI-186 improved the learning and memory abilities, decreased the concentration of intracellular calcium and increased membrane fluidity of hippocampal neurons (that is, decreased the mean microviscosity) to protect these neurons in AD model rats.

MATERlALS AND METHODS

Design

A randomized controlledin vivoanimal experiment.

Time and setting

This experiment was performed at the Laboratory of Neurology, Affiliated Hospital of Jiangsu University,China, from August 2008 to October 2009.

Materials

One-hundred healthy male clean Wistar rats, aged 4 months and weighing 200-250 g, were provided by the Experimental Animal Center of Jiangsu University (certification No.SCXK (Su) 2007-0001).All experiments were performed under the supervision of theRegulations for the Administration ofAffairs Concerning Experimental Animalspublished by the Ministry of Science and Technology of the People’s Republic of China[31].

Methods

Establishment of AD rat models

AD rat modelswere established as described previously[32](hippocampal fimbria-fornix transection), with some modifications[33](Aβ25-35intraventricular injection).Rat heads were fixed on a stereotaxic apparatus(Shanghai Alcott Biotech Co., Ltd., Shanghai, China)after intraperitoneal anesthesia.The skull was drilled and the periosteum was broken with a dental drill at 1.2 mm posterior to the Bregma and 1.0 mm lateral to the midline on each side.A double-edged blade was used to make a coronal incision of the brain surface.The initial incision was 4.5 mm deep and 1.0 mm in an outward direction;then, the incision was made 1.0 mm deeper and continued a further 1.5 mm outward.Finally, the blade was lifted up and down 20 times.Before the incision was sutured, the blade was drawn out and a little penicillin powder was sprayed on the wound.Aβ25-35, which had been incubated at 37°C in an incubator (Sheldon Manufacturing, Cornelius, Oregon, USA), was injected into the lateral ventricle for 7 days.Finally, 2 μL of methylene blue solution was injected in order to confirm that the injection site was in the lateral ventricles.

Preparation of hippocampal tissue single cell suspensions

Six rats from each group were killed after inhalation of carbon dioxide.The brains were quickly removed from the cranial cavities and the hippocampi were separated from the cortices.After three washes in D-Hanks solution,the meninges and blood vessels were taken off, and the hippocampi were cut into many pieces.After discarding the D-Hanks solution in the centrifuge tubes, 5 mL of 0.25% trypsin was added before the hippocampus was digested at 37°C for 15 minutes.Digestion was terminated after cold Dulbecco's modified Eagle's medium(DMEM) supplemented with 10% bovine serum albumin(BSA) was added.Then the tube was centrifuged at 1 000 ×gfor 5 minutes before the stop solution was removed from the tube.D-Hanks solution was poured into the tube again and centrifuged at 1 000 ×gfor another time.After filtering with 200-mesh sieve, the filtrate was centrifuged at 1 000 ×g, washed twice in D-Hanks solution and treated with DMEM supplemented with 10% BSA to produce single cell suspension.Single cell suspension from hippocampal tissue could be used when the cell viability was up to 95%, as detected by Trypan blue staining[34].

Determination of intracellular calcium concentration

Cell suspension from hippocampal tissue was pre-warmed at 37°C for 5 minutes.Following the addition of Fura-2/AM into the cell suspension, the mixture was loaded into centrifuge tubes at 37°C for 30 minutes and then centrifuged at 1 000 r/min for 5 minutes.After the supernatant was discarded, the suspension was washed twice in the D-Hanks solution and the cell concentration was adjusted to 1 × 106/mL.Cell suspension (1 μL) was placed into a colorimetric plate cuvette and the intracellular calcium ion concentration was detected using a fluorescence spectrophotometer (Perkin-Elmer Corp., Norwalk, NJ, USA).Upon binding Ca2+, the excitation spectrum of Fura-2 shifted to shorter wavelengths of 300-400 nm, while the peak emission remained steady at around 510 nm.There was a peak in fluorescence intensity at 340 nm, which suggests that Fura-2/AM had entered hippocampal cells.The cytoplasmic calcium ion concentration was continuously monitored using 340 nm as the excitation wavelength.The intracellular calcium ion concentration was calculated by measuring the fluorescence (F) of solutions with different Ca2+concentrations using the equation[35][Ca2+]=Kd(F0-Fmin)/(Fmax-F0), where Kd is the dissociation constant of the chemical reaction for Ca2+buffering by the fluorescence dye andF0,FmaxandFminare instantaneous, maximal and minimal dye fluorescence emissions, respectively.The Kd is 224 nmol/L.These values were determined using internal solutions containing 10 mmol/L EGTA (0 Ca2+) and 10 mmol/L CaCl2(max.Ca2+).

Determination of learning and memory abilities of AD rats

The learning and memory abilities of all surviving rats were determined through the use of the Morris water maze.

Navigation test: the rats were placed into water from four different directions twice every morning and afternoon,and the time required to find the platform and the escape latency were recorded.If the rats did not find the platform within 2 minutes, they were directed to the platform by experimenters.The escape latency was set to be 120 seconds and the interval between two training sessions was 60 seconds.

Spatial probe test: the platform was removed after the navigation test had finished and the rats were placed into the water from any direction.Thereafter, the spanning platform times were determined within 120 seconds.The same experiment was performed again twice a day on the 0th, 7thand 17thdays following successful preparation of AD rat models, and each experiment lasted 4 days.The average results were calculated.

Measurement of membrane fluidity

DPH, at 10 μmol/L, was added to one aliquot of suspension for incorporation into the hydrocarbon chains of the cell membrane lipids.The suspension was shaken at 37°C for 30 minutes, then diluted in PBS and centrifuged at 1 500 ×gfor 5 minutes, twice.The pellets were resuspended in PBS for detection.An MPF-4 spectrofluorophotometer was used for fluorescence determination at 22-24°C.After DPH loading, the peak excitation spectrum was shifted from 382 nm for the simple DPH solution to 362 nm for the samples labeled with DPH, and the peak emission spectrum was shifted from 442 nm for the simple DPH solution to 432 nm for the samples loaded with DPH.Mean microviscosity (η) was used as index of membrane fluidity.The value of η was calculated based on a standard formula[36].The value was negatively correlated to the membrane fluidity.

Statistical analysis

All measurement data were statistically analyzed using SPSS 15.0 software (SPSS, Chicago, IL, USA) and are expressed as means±SD.One-way analysis of variance was used for comparisons among the five groups.The least significant difference test was used for pairwise comparisons between two groups.APvalue of less than 0.05 was considered statistically significant.

Author contributions:Ming Yu and Xiaohong Lu participated in study design and provided guidance.Lei Qin provided technical and data support.Zhao Wang, Ying Zhu, Wenhui Leng and Xuan Wang performed all experiments.All authors read and approved the final manuscript.

Conflicts of interest:None declared.

Funding:The study was supported by the Natural Science Foundation of Heilongjiang Province, No.D2008072; Projects of Clinical Medicine in Science and Technology Development of Jiangsu University, No.JLY20080013.

Ethical approval:Removal of brain tissue from rats was approved by the Animal Ethics Committee of Jiangsu University,China.

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