All authors had access to the study data and reviewed and approved the final manuscript. All in vivo studies represent 20 animals per group. All in vitro studies are from at least three replicate experiments. All animal experiments were approved and carried out in accordance with the Institutional Animal Care and Use Committee at the Chi Mei Medical Center, Tainan, Taiwan (approved no. 108120110).
The laboratory animals were male Wistar rats with a weight of 250-350 g (BioLASCO Taiwan Co., Ltd.). The rats were housed in an air-conditioned animal facility at 26 ± 0.5°C under a 12-hour light-dark cycle and given unlimited access to water and food. All experiments were conducted during the daytime 10:00-18:00.
The rats were randomly assigned into eight groups, including (1) sham operation with the sedentary and vehicle-treated group (Sham+Sed+Veh); (2) bilateral intracerebroventricular (i.c.v.) injection of Aβ1-42 with the sedentary and vehicle-treated group (Ab+Sed+Veh); (3) i.c.v. injection of Ab1-42 with ER, and vehicle-treated group (Ab+ER+Veh); (4) i.c.v. injection of Ab1-42 with ER and ANA12 administration (Ab+ER+ANA-12); (5) i.c.v. injection of Ab1-42 with astragaloside (AST) and vehicle administration (Ab+AST+Veh); (6) i.c.v. injection of Ab1-42 with AST and ANA12 administration (Ab+AST+ANA12); (7) i.c.v. injection of Ab1-42 with ER and AST combined therapy and vehicle administration (Ab+ER+AST); and (8) i.c.v. injection of Ab1-42 with ER and AST and ANA12 administration (Ab+ER+AST+ANA12). The ALZET osmotic pump full of AST was implanted subcutaneously. The ANA12 was injected intraperitoneally every day from day 8 to day 35. The treadmill ER was performed on day eight post-surgery. The cognitive functions were measured before surgery and every seven days of post-surgery. After the last cognitive evaluation at week-5 of post-surgery, the rats were sacrificed.
Aβ1-42 was obtained from Anaspec (Cat.# AS-20276, Fremont, CA, USA). The lyophilized Aβ peptides were prepared by reconstituting the powder in 1% ammonium hydroxide. Subsequently, this stock was dissolved in 1X PBS to obtain a 30 uM working solution and then subjected to oligomerization. For oligomerization, Aβ peptides were incubated at 4℃overnight and then aliquoted to store at –80 °C until use.
ICV injection of Aβ1-42
Rats were anesthetized with a combination of Zoletil (40 mg/kg; Virbac, Nice, France), xylazine hydrochloride (2 mg/kg; Balanzine, Health-Tech Pharmaceutical Co., Taipei, Taiwan), and atropine sulfate (1 mg/kg; Tai Yu Chemical & Pharmaceutical Co. Ltd., Hsinchu, Taiwan) intraperitoneally (i.p.) and then placed in a standard stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA). A middle sagittal incision was made in the scalp and was sterilized using standard procedures. Bilateral holes were drilled in the skull using a dental drill over the lateral ventricles. Rats were bilaterally and intracerebroventricularly (i.c.v., AP: -0.8mm, ML: ±1.4mm, DV: -4.0mm) injected with 20 μg/μl Aβ1-42 (Cat.# AS-20276, AnaSpec, Inc., Fremont, CA, USA) at a rate of 60 μl/hr by using Hamilton microsyringe and a minipump (Hamilton, Reno, NV, USA). The syringe was removed 5 min after injection. The sham group received sterile 1x PBS. After surgery, the scalp was sutured, and sulfamethoxazole was sprinkled on the wound to prevent infection. In addition, penicillin (40,000U) was injected intramuscularly into the gluteus once a day for 3 days [10, 11].
Treadmill exercise protocol
On day-8 of post-surgery, we trained the rats on a treadmill (model Exer-3/6, Columbus Instruments, Columbus, OH, USA) 5 days a week for four weeks. They were acclimatized to run for 15 min at 20 m/min at 0° for 3 days initially, and then they were running for 30 min at 20 m/min at 10o, 30 min at 30 m/min at 10o, 60 min at 30 m/min at 10o, and 60 min at 30 m/min at 10o after 1, 2, 3, and 4 weeks of training, respectively. Rats in the Sham+ Veh and Aβ+ Veh groups were placed daily on a stationary treadmill .
Astragaloside (AST, molecular formula: C28H32O17; molecular weight = 640.55, was purchased from Fusol Material Co., Ltd, Tainan, Taiwan) was dissolved in 2 ml of saline with 5% EtOH and administered through an ALZET osmotic minipump (Alzet 2ML4; Alza, Palo Alto, CA, USA) at a dose of 40 mg/kg/day after Ab1-42 injection [3, 13, 14].
The rats received another i.p. injection of vehicle (1% dimethylsulfoxide in physiological saline, 1 ml/kg; Sigma-Aldrich, St. Louis, MO, USA) or N-[2-[[(Hexahydro-2-oxo-1H-azepin-3-yl) amino]carbonyl] phenyl] benzo [b]thiophene-2-carboxamide (ANA12, a TrkB receptor antagonist, 1 mg/kg/day, i.p.; Tocris Bioscience, Bristol, UK), respectively .
Radial arm maze task
The maze comprised of 8 arms, extending radially from a central area. Before the training, rats were placed to explore the maze for 5 min and consume food freely. They were trained for five days to run to the end of the arms and consumed the baited food. The training trial continued until 5 min has passed. After adaption, each animal was checked for working and reference memory, in which the same four arms (no. 2, 4, 6, and 8) were baited. All the rats were trained with one trial: (1) before Aβ1-42 injection per day for five days; and (2) after Aβ1-42 injection 7, 14, 21, 28, and 35 days. Finally, the number of long-term memory (reference memory) errors (entrance into an arm that never baited) and short-term memory (working memory) errors (repeat entrance into an arm) were counted . Latency was measured as the time elapsed from the beginning to the end of each trial. All measures were averaged across each trial.
Y maze task
Short-term spatial recognition memory was assessed by analysis of spontaneous alternation in Y-maze, as reported before [15, 16]. In brief, each rat was placed at the end of one arm and allowed to move through the arms for 8 min, and a sequence of arm entries was recorded. Alternation was obtained as successive entries into the three arms on overlapping triplet sets. The maximum number of possible spontaneous alternations was obtained as the total number of arms entered minus 2, and the alternation was calculated as the ratio of actual to possible alternations × 100.
Rotarod motor coordination test
Rotarod accelerating test was also performed on each animal to evaluate motor coordination impairment . It was to examine the possible defects in neuromuscular coordination that might occur on the Aβ1-42 injection rats. Before the stereotaxic surgery, each rat was placed in a rotarod apparatus and subjected to an accelerating test. The rat was placed on the rotating rod (at the slowest speed, 4 rpm) for 1 minute and acceleration to its maximum speed of 30 rpm at 3 min. Each rat that could not hold at the acceleration rod for more than 1 minute was excluded from the further experiments. For the qualified rats starting from the 2nd day before surgery, the rats were trained per day as described above for 2 days. On days 7, 14, 21, 28, and 35, after surgery, the rotational speed of the rod was then accelerated to its maximum speed of 30 rpm. The length of the time that rats could grasp at the rod was measured. The test score is the average number of seconds that rats could hold onto the rod per trial.
Formalin-fixed brains were embedded in paraffin blocks. Serial sections through the hippocampus and cortex were stained with hematoxylin and eosin for microscopic examination. An examiner who was blinded to the experimental conditions evaluated the extent of neuronal damage for each section. The damage scores were determined by two grading systems. In the first system, Honório et al.  determined that damage scores from 0 to 4 in which “0”, “1”, “2”, “3”, and “4” denote no pathological changes, lesions involving 25% of the field, lesions involving 25% to 50%, lesions involving 50% to 75%, and lesions involving 75% to 100%, respectively. In another system, Liu et al.  used 0, 1, 2, and 3 denote normal morphology, minor damage (edema, few pyknotic cells), moderate damage (structural disorganization, edema, moderate pyknotic cells, vacuolization, inflammatory cell infiltration), intense damage (structural disorganization, edema, intense pyknotic cells, vacuolization, inflammatory cell infiltration), respectively. In our present study, we multiplied these two grading scores to present damage scores.
Aβ Plaque stain
Amyloid plaques were stained with Thioflavin-S. The deparaffinized and hydrated sections were incubated in 0.25% potassium permanganate solution for 20 min, rinsed in distilled water, and incubated in bleaching solution (2% oxalic acid and 1% potassium metabisulfite) for 2 min. After rinsed in distilled water, the sections were transferred to a blocking solution (1% sodium hydroxide and 0.9% hydrogen peroxide) for 20 min, incubated for 5 s in 0.25% acidic acid, washed in distilled water, and stained for 5 min with 0.0125% Thioflavin-S in 50% ethanol. The sections were washed with 50% ethanol, placed in distilled water, and then covered with a glass cover using a mounting solution .
Triple immunofluorescence staining
Dissected brains were fixed in 4% phosphate-buffered formalin at 4 °C, and subjected to immunohistochemical staining. The following antibodies were used: Iba1 (1:200; #GTX10042, GeneTex Inc., Irvine, CA, USA), CD86 (1:200, #ab53004, Abcam Inc., Boston, MA, USA), and Neu-N (1:200, MAB377, Merck Millipore, Billerica, MA, USA). The staining was visualized using Alexa Fluor 568- and Alexa Fluor 488-conjugated secondary antibodies (1:500, Invitrogen, Carlsbad, CA, USA) and 4’, 6-diamidino-2 phenylindole (DAPI, 1:5000, #D1306, Invitrogen).
For degenerative neuron detection, after secondary antibody incubation, the brain tissue slides were immersed in a solution of 5% sodium hydroxide and 100% ethanol for 5 min, followed by immersion in 70% ethanol for 2 min, distilled water for 2 min, and 0.06% potassium permanganate solution for 10 min. The sections were then rinsed in distilled water for 2 min/2 times and placed in a 0.0004% Fluoro-Jade B solution made by adding 4 ml of a 0.01% stock solution of Fluoro-Jade B to 96 ml of 0.1% acetic acid. After 20 min in the Fluoro-Jade B staining solution, the stained slides were thoroughly washed in distilled water, dehydrated, and coverslipped [21, 22].
Before the primary antibody incubation, the brain slides were treated with proteinase K (20 µg/ml) for 15 min at room temperature for neuronal apoptosis detection. Subsequently, equilibration buffer was applied for 10 sec, and the brain slides were immersed and incubated for 1 h in working strength terminal deoxynucleotidyl transferase (TdT) enzyme solution at 37˚C. Following incubation in stop/wash buffer for 10 min to terminate the reaction, brain slides were incubated for 30 min in working strength anti-digoxigenin conjugate at room temperature in the dark to visualize the DNA fragments. Proteinase K, equilibration buffer, and stop/wash buffer were all included in the terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL) assay kit (#630108, Takara Bio USA, Inc., CA, USA) . TUNEL-positive neurons with condensed nuclei were identified as dead neurons.
After a final wash with PBS, slides were mounted in glycerol gelatine mounting medium (#GG1-15 ML, Sigma-Aldrich, St. Louis, MO, USA) and viewed using an upright fluorescence microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) at excitation/emission wavelengths of 578/603 nm (rhodamine, red) and 490/525 nm (FITC, green). A digital camera linked to a computer running Axioscope version 4 (Carl Zeiss) was used to capture images. A pathologist counted the percentage of Fluoro-Jade+NeuN/DAPI and TUNEL+NeuN/DAPI double-labeled cells in 6 fields per section in cortex and hippocampus (x 400 magnification).
Western blot assay
Western blotting was performed by using the standard method. The total proteins, obtained from cells cultures or brain tissues, were extracted by the modified RIPA buffer (50mM Tris-HCl, pH7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1mM EDTA) containing protease and phosphatase inhibitors (Sigma, St Louis, Mo, USA). Protein concentrations were quantified by the Bradford method (Bio-Rad, Hercules, CA, USA). For blot analysis, protein extracts were boiled for 10 min in loading buffer, separated on SDS-PAGE, and transferred to polyvinylidene difluoride membrane (Pall Corporation, East Hills, NY, USA) using a wet transfer system (Bio-Rad, Hercules, CA, USA). The membranes were blocked in 5% non-fat milk in PBS containing 0.05% Tween-20 (TBS-T) for 1hr at room temperature. The membranes were hybridized with Cathepsin D (#SC-46656, Santa Cruz Biotechnology, Santa Cruz, CA, USA), BDNF (#ab108319, Cell Signaling Technology, Inc., Beverly, MA, USA), TrkB (#4603, Cell Signaling), Phospho-TrkB (#ABN1381, Millipore), Phospho-Akt (phosphoresidue serine 473, #4060, Cell Signaling), total Akt (#9272, Cell Signaling), Phospho-GSK-3β (phosphoresidue serine 9, #9336, Cell Signaling), total GSK-3β (#9315, Cell Signaling), b-catenin (#9562, Cell Signaling), and β-actin (#SC-47778, Santa Cruz) antibodies for overnight at 4˚C. After washing with TBS-T, the membranes were continuously incubated with appropriate secondary antibodies coupled to horseradish peroxidase (Cell Signaling Technology, Inc.) for 1hr at room temperature. The blots were developed in the ECL Western detection reagents (PerkinElmer, Waltham, MA, USA) and exposed to Hyperfilm ECL (GE Healthcare, Piscataway, NJ, USA). Protein bands were scanned and quantified using ImageMaster TotalLab image analysis software (GE healthcare).
Ab-induced primary cortical neuron injury model
Neuronal cultures were prepared from the cortex of embryonic day 18 rat embryos. In brief, embryos were removed and their brain cortices dissected. The neurons were obtained by digestion of papain (2 mg/ml for 30 min at 30 ℃, Millipore, Billerica, MA, USA) followed by trituration with a 1 ml pipette. The cells were plated onto poly-L-lysine (Trevigen, Gaithersburg, MD, USA)-coated 6 well plates or dishes and maintained in neurobasal media supplemented with 2% B27 and 0.5 mM GlutaMax (Invitrogen, Carlsbad, CA, USA) at 37℃ in a humidified 5 % CO2 incubator. Culture media were exchanged every 4 days, and cells were grown for 8 days in vitro (DIV) before Aβ25-35 treatment. Ab25-35 was purchased from Kelowna International Scientific Inc. (Taipei, Taiwan) and dissolved in water with vortex. The solution was allowed to aggregate for 6 days at 37 oC before use. BDNF protein obtained from GenScript (Piscataway, NJ, USA) was dissolved (100 ug/ml) in distilled water. The Chinese herb Astragaloside (AST) was made up as a 100 mg/ml stock in DMSO. ANA12 (TrkB selective antagonist) was purchased from Selleckchem (Houston, TX, USA) and dissolved in DMSO. To induce cell cytotoxicity, the cortical neurons were exposed to 60 uM Ab25-35 for 24h. BDNF, AST, and ANA12 were administered at 400 ng/ml, 400 ug/ml, and 35 uM, respectively.
MTT assay for cell viability
Cell viability was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After treatment, cells were treated with the MTT solution (final concentration, 5 μg/ml) for 2 h. The dark blue formazan crystals formed in intact cells were solubilized with lysis buffer (20% sodium dodecylsulfate in 50% aqueous N, N-dimethylformamide). The absorbance of the sample was read at 540-595 nm with a MultiSkan GO microplate reader (Thermo Fisher Scientific, MA, USA). Results were expressed as the percentage (%) of MTT reduction, assuming that the absorbance of control cells was 100 %.
The cells were fixed with 4% paraformaldehyde in PBS, permeabilized, and blocked with 0.3 % Triton-X 100 plus and 5 % normal bovine serum in PBS. The cultures were then incubated with combinations of the primary antibodies against the following targets: microtubule-associated protein 2 (MAP-2; a neuronal marker; Santa Cruz) and synaptophysin (is an integral membrane protein localized to synaptic vesicles; Santa Cruz) and diluted in 5% BSA overnight at 4 °C. Then, cortical neurons were incubated with the appropriate secondary antibodies (Alexa Fluor 568-conjugated goat anti-mouse IgG or Alexa Fluor 488-conjugated goat anti-mouse) for 1 h at room temperature. The neurons were also stained with DAPI (Sigma-Aldrich) as a nuclear marker. Their digital images were captured with a 20x objective (N.A. 0.75) by a fluorescence microscope system (Carl Zeiss Microscopy GmbH, Jena, Germany) with Zen Software (Carl Zeiss). Synaptic density was measured as a synaptophysin-positive area. At least 10 fields obtained from different experiments were conducted in triplicate from 3 cultures, were captured and measured for each condition. Values obtained for each field (0.15 mm2) were pooled to obtained mean values for each culture.
Mitochondrial membrane potential analysis
After culturing, JC-1 (5,5’,6,6’-Tetrachloro-1,1’,3,3’-tetraethyl-imidacarbocyanine iodide; BD Mitoscreen kit, San Jose, CA, USA) was added to a final concentration of 2.5 mM, and cells were shaken in the dark at 37 oC for 15 min. Following incubation, 400 ul staining buffer was added to each sample and analyzed on the Novocyte flow cytometry (ACEA Biosciences, CA, USA). A total of 10,000 events were measured using FL-1 channel (525 nm) and results were expressed as a percentage.
Sub-G1 phase analysis
At the end of the treatment period, the cells were harvested, washed with PBS, and fixed in cold 70% ethanol at -20 °C overnight. The fixed cells were washed with PBS and treated with propidium iodide (PI) staining solution containing 10 ug/ml PI (ThermoFisher Sci)、100 ug/ml RNase (ThermoFisher Sci) and 0.1% Triton X-100 (Sigma-Alrich) for 30 min at 37℃. Finally, the samples were analyzed in the Novocyte flow cytometer using NovoExpress software to analyze sub-G1 distribution.
Evaluation of DNA fragmentation
DNA was extracted by the use of a commercially available Wizard genomic DNA purification kit (Promega, Madison, WI, USA) following the manufacturer’s instructions. Neuron cell was lysed in Nuclei lysis solution and then digested all RNAs with RNase A solution for 30 min at 37 oC water bath. Protein precipitation solution was added, vortexed vigorously, and kept on ice for 5 min. After short centrifugation, the supernatant was transferred to a new microtube containing 600 ul isopropanol, mixed by inversion, and centrifuged. The supernatant was removed, and the pellet was washed in 600 µl 70% ethanol. Finally, the pellet was air-dried, then rehydrated with hydration solution, and determined the DNA concentration. Two ug of extracted DNA was loaded to 1.8% agarose gel electrophoresis in Tris-borate-EDTA buffer system, stained with ethidium bromide, and visualized through a Quantum CX5 UV-transilluminator (Vilber Lourmat, Torcy, France) and photographed to determine the DNA concentration.
The person charged with functional outcome measurements was the only one that was blinded to treatments among those working on animals (single-blind). She used cage and animal codes to recognize individuals and to report repeated measurements on data collection forms. Statistical analyses were performed using GraphPad Prism 7.01 (GraphPad Software Inc., CA, USA). Parameters such as histological scores and the immunofluorescence staining data with non-normal distribution were analyzed by the Kruskal-Wallis test with Dunn's post-hoc test. One-way analysis of variance (ANOVA) with Tukey's post hoc test was used by an investigator blinded to the treatment groups and the flow cytometry and Western blotting data. We performed two-way ANOVA with Tukey's multiple comparisons tests to analyze behavioral performance data. A Student's t-test was used to compare variables for two groups if there was a significant difference. All data were expressed as mean ± standard deviation (SD). P-values < 0.05 were considered statistically significant.