Atorvastatin in improvement of cognitive impairments caused by amyloid β in mice: involvement of inflammatory reaction

The animals were handled according to the guidelines of the Care and Use of Laboratory Animals (NIH USA), and the Animals Scientific Procedures Act (UK).

The study was submitted to and approved by the Ethics Committee of Nanjing Medical University, China (NMU-103-2014).

Male C57BL/6 mice (Oirental Bio Service Inc., Nanjing), weighing 20–25 grams, were housed in a controlled room under a12-h light/dark cycle, and had free access to a standard pellet chow and tap water throughout the study. A total of ninety-six animals were randomly divided into the groups: Aβ (n = 8), Aβ + ATV (n = 8), Aβ + ATV + Farnesol (FOH) (n = 8), Aβ + ATV + LY294002 (n = 8), Aβ + Corticosterone (CORT) (n = 8), Aβ + CORT + ATV (n = 8) and their relevant controls (8 in each). The study was completed 15 days after the administration of Aβ_25–35 (Fig. 1).

The AD model was prepared according to the previously reported method [6]. Briefly, Aβ_25–35 (Sigma Chemical Co., St. Louis, MO, USA) was dissolved in a sterile bi-distilled water at a concentration of 2 mg/ml, and incubated at 37 °C for 4 days. 40 mg/kg of chloral hydrate was intraperitoneally administrated, and the animals were placed in a sterotactic device (Kopf Instruments, Tujunga, CA, USA). A volume of 3 μl of Aβ was injected into the right lateral ventricle, at the following coordinates at 3 mm posterior, 1 mm lateral and 2.5 mm ventral to bregma, using a stepper-motorized micro-syringe at a rate of 0.5 μl/min. The mice serving as the control group were administered with scrambled Aβ25–35 peptide (NeoMPS, Strasbourg, France) dissolved in a sterile bi-distilled water at the same volume.

The animals started with Y-maze task on day 13 after the administration of Aβ_25–35. The Y-maze was performed as described previously [17]. Briefly, both the start arm (27.5 cm long) and two arms forming the Y (both 27.5 cm long and diverged at a 60° angle from the stem arm) were 5 cm in diameter. The home cage was connected to the start arm of the Y-maze. Each mouse was placed at the end of one arm, and allowed to move freely through the maze during an 8 min session. The series of arm entries was recorded visually, and arm entry was considered to be completed when the hind paws of the mouse were completely placed in the arm. Alternation was defined as successive entries into the three arms on overlapping triplet sets. The percentage alternation was calculated as the ratio of actual to possible alternations.

The animals started with Morris water maze on day 5 after the injection of Aβ_25–35. A circular pool with diameter at 120 cm was prepared with the water temperature at 24 ± 1 °C. Ink powder was used to render the water opaque. Swim paths were analyzed using a computer system with a video camera (Neuroscience, Inc., Tokyo, Japan). The platform (7 cm in diameter) was submerged 1 cm below the water surface. Mice were given 90 s in the pool to search the hidden platform. If no platform was found within 90 s, the mouse was guided to the platform, and the trial was terminated. Each mouse started in one of four quadrants in a random manner, with the head facing the wall. Four trials were conducted each day, for six consecutive days. The probe trial was then recorded by removing the platform. The mouse was released from opposite quadrant in which the platform was located, and allowed to swim for 90 s to determine its search patterns. Times spent in platform quadrant, opposite quadrant, adjacent right and left quadrants were measured. The percentage of time spent in each quadrant was determined.

Hippocampal slice preparation was performed as previously reported [18, 19] with modifications. Briefly, the animals were decapitated under deep anesthesia with ethyl ether (400 mg/kg, intraperitoneal injection), and the brains were rapidly removed out. Coronal slices (400 μM) of dorsal hippocampi were cut on a vibrating microtome (Dousaka EM Co, Kyoto, Japan) in an ice-cold cutting solution, and then incubated in artificial cerebrospinal fluid at 30 ± 1 °C for 60 min. After a slice was submerged in a recording chamber, hydraulic micromanipulators (Narishige, Tokyo, Japan) mounted on the microscopy were used to place a stimulating electrode in radiatum layer. Constant current pulses (0.1 ms, 0.06 Hz) were supplied by a stimulator (Nihon Kohden, Japan). The excitatory post-synaptic potential (EPSP) slope was recorded from radiatum layer with a 5 MΩ resistance glass microelectrode, and connected to a neutralized high input-impedance preamplifier. Stability of baseline recordings was established by delivering single pulses (four/min, 0.1 ms pulse width) for 15 min prior to collection of input/output functions. Baseline synaptic transmission was assessed by averaging the response to five pulses (from 0.1 to 1.0 mA) delivered at a rate of 0.06 Hz. Paired-pulse facilitation (PPF) was measured by using the intensity of the test stimulus with an inter-pulse interval of 25–100 ms. Pre-train responses were recorded for 20 min (baseline), high-frequency stimuli (100 Hz, 100 pulse) were used to induce LTP.

Mice were anesthetized by intraperitoneal injection of chloral hydrate (40 mg/kg), and transcardially perfused with ice-cold phosphate-buffered saline followed by 4 % para-formaldehyde. The brains were quickly taken out, immersed in 4 % para-formaldehyde for fixation at 4 °C overnight, and processed for paraffin embedding. Coronal sections (5 μm) of hippocampus were prepared for the histological examination. The sections were treated with 3 % H₂O₂ for 10 min, and then incubated in 5 % goat serum for 30 min. For Iba-1 staining, the sections were incubated with a goat polyclonal anti-Iba-1 antibody (Abcam, Cambridge, UK), and then incubated in biotin-labeled anti-goat IgG antibody (Bioworld Technology, Inc., St. Louis Park, MN, USA) for 2 h at room temperature. The immunoreactivity was visualized by the standard avidin-biotin complex reaction with 3, 3′-diaminobenzidine (Vector Laboratories, Burlingame, CA, USA). Iba-1 positive cells were counted using a light microscope (Olympus, Japan). The density of Iba-1 positive cells was expressed as the mean number of Iba-1 positive cells per mm².

Hippocampal slice cultures were prepared according to the previously reported method [20]. Briefly, mice were decapitated 15 days after Aβ25-35 administration. The brain was removed, and 400 μm of hippocampal slices were prepared, and separated in ice-cold HBSS solution with a pH value of 7.2. On average, six slices from the middle of hippocampus were obtained and placed in a 12-wells plate. The cells served as control group were from the animals without administration of Aβ25-35. The cell cultures were maintained in an incubator for 1 h before performing cell viability.

The animals without Aβ25-35 administration were decapitated. Hippocampal slice cultures were prepared as reported previously [20]. To examine the dose-dependent protective effects of ATV, different doses at 0.5, 1 and 2.5 μmol/L in the culture medium were used. The cells cultures were maintained in an incubator with a 5 % CO₂ mixed with 95 % O₂ at 37 °C for 14 days, and the medium were replaced at every exchange of culture medium. The cells served as control group were treated with DMSO only in culture medium. The doses of ATV was chosen as described previously [21], which was confirmed to generate the dose-dependently inhibited effects.

Cell death was assessed by using fluorescent exclusion dye propidium iodide (PI) uptake. PI is a polar compound that penetrates damaged cells only, and binds to nuclear DNA to generate a bright red fluorescence. The appearance of PI uptake is cellular membrane injury [22]. After 14 days of ATV exposure, 7 μm/ml of PI was added to the culture medium, and incubated for 40 min at 37 °C. Cultures were observed with an inverted microscope (Leica Microsystems Inc., Wetzlar, Germany) using a standard rhodamine filter set. The images were taken with an Olympus camera, and analyzed using Scion Image Software (http://​scion-image.​updatestar.​com).

Mice were decapitated under a deep anesthesia with chloral hydrate. Hippocampus was quickly taken out, and homogenized in a lysis buffer (Roche, Mannheim, Germany). Protein concentration was determined according to the instruction of the Protein Assay Kit (Pierce Biotechnology, Inc., Rockford, IL, USA). The membranes were incubated and developed by following the instruction of the ECL detection Kit (Millipore, Massachusetts, USA). Western blot bands were scanned and analyzed with the image analysis software package (NIH Image, Bethesda, MD, USA).

Hippocampus was micro-dissected, and stored at −80 °C until assayed. RNA was isolated using Trizol reagent (Invitrogen, Camarillo, CA, USA), and reverse-transcribed into cDNA according to the instruction of Prime Script RT reagent kit (Takara Bio. Mountain View, CA., USA) for quantitative PCR in the presence of a fluorescent dye (Takara Bio. Mountain View, CA., USA). Relative expression of genes was determined using the reported method [23]. The levels of IL-1β and TNF-α mRNA were normalized by controls. The primers of IL-1β are 5′-CCATGGCACATTCTGTTCAAA-3′ and 5′-GCCCATCAGAGGCAAGGA-3′; the primers of TNF-α are 5′-ACGGCATGGATCTCAAAGAC-3′ and 5′-CGGACTCCGCAAAGTCTAAG-3′; the primers of GAPDH are 5′-ACCACAGTCCATGCCATCAC-3′ and 5′-TCCACCACCACCCTGTTGCTGTA-3′.

A cell culture experiment would help to clarify if ATV treatment reduces the secretion of pro-inflammatory cytokines. However, the results from our study showed that the paradigms of cellular death caused by Aβ25–35 and ATV protection observed in vitro culture were similar with those in animal model. In addition, the inflammatory cytokines associated with the ATV neuro-protection following Aβ administration has been evidenced in the recent study [29].