Generation of seipin knockout (seipin-KO) mice
All animal handling procedures followed the guidelines for Laboratory Animal Research of Nanjing Medical University. The use of animals was approved by the Institutional Animal Care and Use Committee of Nanjing Medical University. The mice were maintained in constant environmental conditions (temperature 23 ± 2 °C, humidity 55 ± 5 %, and a 12:12-h light/dark cycle). The neuronal
seipin-KO mice were generated as described elsewhere [
11]. The genotypes of the
seipin-KO mice were identified by PCR using genomic DNA from their tails. The DNA was amplified with the following primers: 5′-CTTGTCTCAAAGGGGTCT-3′ (forward primer for loxP) and 5′-TCAACAGAACAGACGCT-3′ (reverse primer for loxP); and 5′-GCGGTCTGGCAGTAAAAACTATC-3′ (forward primer for nestin-Cre) and 5′-GTGAAACAGCATTGCTGTCACTT-3′ (reverse primer for nestin-Cre). All animals received a standard laboratory diet before and after all procedures. In this study, 16-week-old male
seipin-KO mice (
n = 88) and wild-type (WT) mice (
n = 56) were used at the beginning of all experiments. All mice were randomly assigned to one of the following six experimental groups: WT mice (
n = 24), Aβ
25–35-treated WT mice (Aβ
25–35-mice,
n = 16), Aβ
1–42-treated WT mice (Aβ
1–42-mice,
n = 16),
seipin-KO mice (
n = 32), Aβ
25–35-treated
seipin-KO mice (Aβ
25–35-KO mice,
n = 40), and Aβ
1–42-treated
seipin-KO mice (Aβ
1–42-KO mice,
n = 16). Each experiment was performed by two experimenters who were blinded to the experimental groups. The behavioral tests (
n = 16 mice for each group) and histological observations (
n = 8 mice for each group) or Western blot analyses (
n = 8 mice for each group) were sequentially performed in the same cohorts.
Injection (i.c.v.) of Aβ25–35 and Aβ1–42
The Aβ
25–35 (Sigma, St. Louis, MO, USA) was aggregated by incubation in distilled water (1 mg/ml) at 37 °C for 4 days and diluted to the final concentration with saline immediately before the experiment [
30]. After this treatment, two types of birefringent fibril-like structures and globular aggregates of Aβ
25–35 can be observed by light microscopic observation [
30]. The Aβ
1–42 (Sigma, St. Louis, MO, USA) was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Sigma-Aldrich) and then flash-freezed in liquid nitrogen and lyophilized to completely remove the solvent [
31]. Lyophilized Aβ
1–42 was dissolved in 100 mM NaOH at a concentration of 6 mg/ml, aliquoted in 50 μl volumes [
32]. The mice were anesthetized with ketamine (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) [
33] and placed in a stereotactic apparatus (Motorized Stereotaxic StereoDrive; Neurostar). The “aggregated” Aβ
25–35 (1.2 nmol/mouse at 2 μl) or the Aβ
1–42 (0.1 nmol/2 μl in 0.1 M phosphate-buffered saline) was injected into the ventricles (0.3 mm posterior, 1.0 mm lateral, and 2.5 mm ventral to the bregma) using a stepper-motorized micro-syringe at a rate of 0.3 μl/min [
34]. The injection site was confirmed by the injection of Indian ink in the preliminary experiments. The mice infused with the same volume of vehicle served as the control group.
Behavioral examination
The behavioral performances on days 5–14 after Aβ
25–35 injection were captured via video recording (Winfast PVR; Leadtek Research, Fremont, CA) and analyzed using TopScan Lite 2.0 (Clever Sys., Reston, VA).
Morris water maze task: The water maze task was consecutively performed to examine the spatial memory [
29]. A pool (diameter = 120 cm) made of black-colored plastic was prepared. The water temperature was maintained at 20 ± 1 °C with a bath heater that was used before the sessions. On days 1–2 of training, a cylindrical black-colored platform (diameter = 7 cm) was placed 0.5 cm above the surface of the water. A mouse was randomly released into one of the four different quadrants and allowed to swim for 90 s. The latency to reach the visible platform was measured. On days 3–7 of the training, the platform was moved to the opposite quadrant of the previously visible platform and submerged 1 cm below the surface of the water. Four trials were conducted each day with an intertrial interval of 30 min. The average swimming speeds (m/s) and latencies (s) to reach the platform were scored for all trials. If the mouse was unable to reach the platform within 90 s, the experimenters gently assisted it onto the platform and allowed it to remain there for 15 s. Each mouse began in one of the four quadrants, which was selected in a random manner. On day 8 of the training, a probe trial was performed by removing the platform. The mouse was released from the opposite quadrant relative to the previous location of the platform and allowed to swim for 90 s. The percentages of swimming time spent in the target quadrant, opposite quadrant, and right and left adjacent quadrants were determined.
Y-maze task: Spatial working memory performance was assessed by recording spontaneous alternation behavior in a Y-maze [
30]. A Y-maze task was performed 48 h after the probe trial task. The Y-maze was constructed of black-painted wood. Each arm was 40-cm long, 13-cm high, 3-cm wide at the bottom, and 10-cm wide at the top, and the arms converged at equal angles. 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 were recorded visually, and arm entries were considered to be completed when the hind paws of the mouse were completely placed in the arm. Alternations were defined as successive entries into the three arms on overlapping triplet sets. The percentage of alternations was calculated as the ratio of actual to possible alternations (defined as the total number of arm entries minus two). The scorers were blinded to the treatment groups.
Histological examination
The mice were anesthetized with chloral hydrate (400 mg/kg, i.p.) and perfused with 4 % paraformaldehyde. The brains were removed and immersed in 4 % paraformaldehyde at 4 °C overnight.
Toluidine blue staining: The brains were processed for paraffin embedding, and coronal sections (5 μm) were cut. The pyramidal cells in the hippocampal CA1 region were identified using a conventional light microscope (Olympus DP70, Tokyo, Japan) with a 60× objective. The healthy pyramidal neurons exhibited round cell bodies with plainly stained nuclei.
Stereological counting CA1 pyramidal cells: The brains were transferred into 30 % sucrose. After gradient dehydration, the coronal sections of the hippocampus (40 μm) were cut with a freezing microtome (Leica, Nussloch, Germany) and stained with toluidine blue. Every fourth section was obtained for consecutive cell quantification analyses. Stereological cell counting was performed by a stereological system, consisting of a light microscope with a CCD camera (Olympus DP70), a motorized specimen stage for automatic sampling (MicroBrightField, Williston, USA), and a computer running Microbrightfield Stereo Investigator software (Microbrightfield, Williston, VT, USA) [
36]. All counts were performed by the same investigator, who was blind to the treatment. The total numbers of healthy pyramidal cells throughout the hippocampal CA1 region were counted using the optical fractionators’ method. The section thickness was measured using a dissector height of 10 μm. The layer of pyramidal cells was defined according to the terminology of Blackstad [
37]. The boundary between the CA3 and CA1 subregions was identified by the differential thickness of the cell layers and by the smaller cell somas of the CA1 subregion. The boundary between CA1 and the subiculum was defined as the line separating the contiguous cells of CA1 from the more widely spaced cells of the subiculum.
Hoechst staining: Coronal paraffin sections (5 μm) were incubated in Hoechst 33342 (1 μg/ml, Cell Signaling Technology, Inc., Boston, MA, USA) for 2 min. Quantitative analyses of Hoechst-positive cells: Hoechst-positive (Hoechst+) cells in hippocampal CA1 region were observed by a conventional light microscope (Olympus DP70, Japan) with 60× objective and counted. The density of Hoechst+ cells was expressed as the number of cells per millimeter length along the pyramidal cells layer.
GFAP and Iba1 staining: The hippocampal sections (40 μm) were cut using a cryostat. Every fourth section was obtained with a randomly chosen starting section. The free-floating sections were blocked with 3 % normal goat serum for 60 min. Glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba1) immuno-staining were performed using the following primary antibodies and incubation at 4 °C overnight: mouse anti-GFAP antibody (1:500; Millipore, Billerican, MA, USA) and goat polyclonal anti-Iba1 antibody (1:500; Abcam, Cambridge, UK). Then, the sections were incubated in biotin-labeled goat anti-mouse IgG antibody (1:400; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or biotin-labeled rabbit anti-goat IgG antibody (1:500; Bioworld Technology, Inc., St. Louis Park, MN, USA) for 2 h at room temperature. Immuno-reactivity was visualized based on the avidin-biotin-horseradish peroxidase complex (ABC Elite; Vector Laboratories, Inc., Burlingame, CA, USA).
Counting GFAP- and Iba1-positive cells in the hippocampal CA1: The GFAP-positive (GFAP
+) cells and Iba1-positive (Iba1
+) cells in the hippocampal CA1 radiatum layer were counted using a conventional light microscope (Olympus DP70). The radiatum layer was outlined at ×5 magnification. Systematic random sampling was achieved with a uniform sampling grid superimposed randomly over the region. A counting frame was placed in each square of the grid, and the cells were counted within that frame at ×40 magnification. A cell was counted if the nucleus was stained and was (a) in focus, (b) within the counting frame or crossed the green counting frame inclusion line, and (c) within the optical disector. Total estimated cell number per section = number of counted cells × (1/ssf) × (1/asf) × (1/hsf), where ssf is the section sampling fraction, asf is the area sampling fraction, and hsf is the height sampling fraction [
38]. The densities of GFAP
+ cells and Iba1
+ cells are expressed as the mean numbers per mm
3, which were normalized to the control value obtained from WT mice [
39].
Seipin/GFAP or Iba1 double immuno-staining: The coronal sections (5 μm in thickness) were placed in gelatine-coated slides, blocked with 3 % normal goat serum, and then incubated in rabbit anti-seipin antibody (1:200; Abcam) at 4 °C overnight, which was revealed using CY3-labeled anti-rabbit IgG antibody (1:200; Millipore) or FITC-labeled anti-rabbit antibody (1:200; Millipore) and mouse monoclonal anti-GFAP antibody (1:200; Millipore) or goat polyclonal anti-Iba1 antibody (1:500; Abcam), which was revealed using FITC-labeled anti-mouse antibody (1:50; Millipore) or CY3-labeled anti-goat IgG antibody (1:200; Millipore). The immune-positive cells/fibers were observed by a confocal laser-scanning microscope (Leica, Heidelberg, Germany).
Western blot analyses
The mice were decapitated under deep anesthesia with chloral hydrate. The hippocampus was quickly removed and homogenized in a lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 1 mM sodium orthovanadate, 1 % Triton X-100, 0.5 % sodium deoxycholate, 1 mM phenyl-methylsulfonyl fluoride, and a protease inhibitor cocktail (Complete; Roche, Mannheim, Germany). The protein concentration was determined with a BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA). The total proteins (20 μg) were separated by SDS-poly-acrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane. The membranes were incubated with 5 % nonfat dried milk in Tris-buffered saline containing 0.1 % Tween 20 for 60 min at room temperature and were then incubated with a mouse monoclonal anti-phospho-GSK3β (Tyr216) antibody (1:1000; Cell Signaling Technology), a rabbit monoclonal anti-phospho-GSK3β (Ser9) antibody, and an anti-phospho-STAT3 antibody (1:1000; Cell Signaling Technology); a rabbit polyclonal anti-IL-6 antibody (1:1000; Abcam), a mouse monoclonal anti-TNF-α antibody and a PPARγ antibody (1:200; Santa Cruz Biotechnology); or a mouse monoclonal anti-β-actin antibody (1:1000; Cell Signaling Technology) and a rabbit monoclonal anti-GAPDH antibody (1:1000; Cell Signaling Technology) at 4 °C overnight. The membranes were then incubated with HRP-labeled secondary antibodies (1:10,000; Millipore). The blots were stripped by incubation in a stripping buffer (Restore, Pierce) for 5 min, incubated with rabbit polyclonal antibodies for GSK3β or STAT3 (1:1000; Cell Signaling Technology) and developed using an ECL Detection Kit (Millipore). The Western blot bands were scanned and analyzed with an image analysis software package (ImageJ; NIH Image, Bethesda, MD, USA).