Animals
Study protocols involving animal subjects were approved by the Animal Ethics Committee of Nanjing Medical University (approval #: IACUC-2004043). All experiments were conducted in strict accordance with institutional guidelines. Adult male C57BL/6J mice (6–8 weeks of age, 20–25 g) and neonatal pups (3–4 days of age, used in experiments involving primary culture) were obtained from Qinglongshan Animal Breeding Farm (Nanjing, China). CXCR5−/− mice [B6.129S2 (Cg)-CXCR5tm1Lipp/J, stock number 006659] with a B6 background were kindly provided by Professor Yongjing Gao (Institute of Pain Medicine, Nantong University, China) [23]. Mice were housed in a pathogen-free facility in the Experimental Animal Center at Nanjing First Hospital, and maintained under a 12-hour light-dark cycle with ad libitum access to standard food and water.
Study design
This study consisted of both in vivo and in vitro experiments. The in vivo experiments were conducted in mice (C57BL/6J or CXCR5−/−) with cecal ligation and puncture (CLP; Fig. 1A-B). A group of C57BL/6J mice undergoing sham surgery was included as a control. Tests for cognitive functions included a standard 5-day Morris water maze (14 days after CLP) and then a fear conditioning test (19 days after CLP). Separate groups of mice were sacrificed 3, 7, and 14 days after CLP for Western blotting, transmission electron microscopy and immunohistochemistry analyses in the hippocampus. The in vitro experiments were conducted in primary microglial cells (Fig. 1C). Briefly, cells were seeded at a density of 3.5 × 105 cells/mL in serum-free DMEM (Thermo Fisher Scientific, Waltham, MA, USA) into 6-well plates containing coverslips, incubated at 37 ℃ overnight, and then exposed to lipopolysaccharide (LPS; Thermo Fisher Scientific) at a concentration of 200 ng/mL for 24 h [13]. In some experiments, cultures were pretreated with short interfering RNA (siRNA) targeting CXCR5 (5’-CUGGACAGAUUGGACAACU-3’; GenePharma, Shanghai, China) or with scrambled sequence (5’-UUCUCCGAACGUGUCACGU-3’; GenePharma) at 24 h before LPS challenge [22]. The siRNAs (20 pmol) were dissolved in 100 µL serum-free OptiMEM, mixed with 1 µL Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), and added to the cultures. In some experiments involving CXCR5 siRNA, cells were pretreated with the p38MAPK inhibitor SB203580 (10 mmol/L; Sigma-Aldrich, St Louis, MO, USA), the p38MAPK agonist P79350 (50 mmol/L; Calbiochem, La Jolla, CA, USA) or vehicle for 1 h prior to adding CXCR5 siRNA administration. Cells were collected at 24 h after LPS treatment for Western blotting and immunofluorescence analysis.
CLP
CLP was conducted as previously described [25]. Briefly, mice were anesthetized with intraperitoneal injection of sodium pentobarbital (40 mg/kg). After disinfection, an incision 2–3 cm long was made at 1.5 cm below the xiphoid to expose the abdominal cavity. The cecum was isolated and ligated at half the distance between the distal pole and the base of the cecum, punctured with a 22-gauge needle, and gently squeezed to force the fecal contents into the peritoneal cavity. The cecum was returned to the peritoneal cavity, and the abdomen was closed using 3 − 0 silk sutures. In the case of sham-operated mice, the abdominal cavity was opened to expose the cecum without ligation or puncture.
Core body temperature was maintained at 37 ± 0.5 ℃ using a heating blanket during surgery. At the end of surgery, mice received 40 ml/kg sterile saline via subcutaneous injection and returned to home cage with a warm cotton pad and free access to food and water. No antibiotics were given.
Primary microglial culture
Primary microglia cultures were prepared from neonatal mouse pups as described previously [26, 27]. Pups were disinfected with 70% alcohol, brains were harvested, and the cerebral cortex was carefully dissected and washed in ice-cold D-Hanks Balanced Salt Solution (Thermo Fisher Scientific) supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin (Sigma-Aldrich). Tissue was dissociated by gentle trituration in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich) containing 10% heat-inactivated fetal bovine serum (Sigma-Aldrich) and antibiotics. The cell suspension was filtered through a 100-µm cell sieve (Becton Dickinson AB, Stockholm, Sweden) and centrifuged at 864 g for 5 min at 4 ℃. The pellet was suspended in DMEM, then incubated in a 75-mL culture flask (Thermo Fisher Scientific) for 14 days in a humidified incubator at 37 ℃ in an atmosphere containing 5% CO2. Next, the flask was shaken at 100 rpm at 37 ℃ for 6 h on a rotary shaker to harvesting microglia with centrifugation (288 g at 4 ℃ for 5 min).
Morris water maze
Learning and memory were examined using a standard 5-day Morris water maze test, as described previously [28]. A circular water pool (125-cm diameter and 40-cm height) was filled with opaque water to a depth of 30 cm and maintained at 23 ± 1 ℃. The escape platform (10-cm diameter) was placed 1 cm below the water surface in the target quadrant. Each mouse was given 4 daily trials with a 20-min intertrial interval for 4 consecutive days to find the platform, and individually placed in the pool at one of 4 quadrant locations. Mice that failed to locate the escape platform within 60 s were manually guided to the platform, and allowed to stay for 20 s. The escape latency from four sessions on the same day was averaged. On the fifth day, the platform was removed to allow for probe testing. During the 60-s session, the number of crossings over the target quadrant and the total time spent in target quadrant were recorded. Training and probe tests were analyzed using motion detection software (Actimetrics Software, Evanston, IL, USA).
Fear conditioning test
The fear conditioning test was conducted on the next day after the Morris water maze experiments were completed, using a standard fear conditioning chamber with metal grid floor [28]. Mice were allowed to the chamber for 2 min prior to 30-sec mono-frequency sound (2 kHZ, 80 dB); during the last 2 sec of the sound, foot shock (1 mA) was delivered. After 3 min, this process was repeated once. On the next day, mice were placed in the same chamber again, but without any stimulation. Freezing was defined as a completely immobile posture except for respiration [29].
Transmission electron microscopy
Transmission electron microscopy was performed as described previously [30]. Hippocampal tissue (1 mm3) was fixed with 2.5% glutaraldehyde at 4 ℃ for 12 h, washed in 0.1 M cacodylate buffer three times, post-fixed in 1% osmium tetroxide for 2 h, and then dehydrated through a graded series of acetone solutions (30%, 50%, 70%, 80%, 90% and 100%). Tissues were cut into 50-nm sections using an ultrathin microtome (Leica, Wetzlar, Germany), contrasted with 2% uranyl acetate for 10 min and lead citrate for 5 min, and observed under an FEI Tecnai G2 Spirit Bio TWIN transmission electron microscope (Thermo Fisher Scientific).
Western blotting
Tissue or cells were lysed in a lysis buffer (Thermo Fisher Scientific), separated with 10% SDS-PAGE, and then transferred to polyvinylidene fluoride membranes (Thermo Fisher Scientific). Membranes were blocked 2% bovine serum albumin (BSA; Thermo Fisher Scientific) for 2 h, then incubated overnight at 4 ℃ with a primary antibody against CXCR5 (1:2000), beclin-1 (1:1000), Atg-5 (1:2000), p62 (1:2000), Iba-1 (1:1000), IL-1β (1:500), IL-6 or GAPDH (1:1000) (all from Abcam, Cambridge, MA, UK); or against LC3 (1:1000), p38MAPK (1:1000) or p-p38MAPK (1:1000) (all from Cell Signaling Technology, Boston, MA, USA). After thorough washing, blots were incubated with an appropriate peroxidase-labeled secondary antibody (1:500; Abcam) for 1 h at room temperature. Protein bands were detected using enhanced chemiluminescence (Bio-Rad Laboratories, Hercules, CA, USA) and quantitated using Image J software (NIH, Bethesda, MD, USA). Band intensities were normalized to GAPDH.
Immunofluorescence
Brains were post-fixed in 4% paraformaldehyde overnight at room temperature, embedded in an optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA), and cut into 5-µm coronal sections. The sections were blocked with 1% BSA (Thermo Fisher Scientific). For LC3 or Iba-1 staining, sections were incubated overnight at 4 ℃ with a primary antibody (Abcam) against LC3 (1:200) or Iba-1 (1:100), and then with a goat anti-rabbit IgG H&L (Cy3®) (1:100; Abcam). For co-labeling of CXCR5 and Iba-1, sections were incubated overnight at 4°C with primary antibodies (Invitrogen) against CXCR5 (1:300) and Iba-1 (1:300), then incubated with an anti-mouse or anti-rabbit IgG (1:1000; Cell Signaling Technology) conjugated with Alexa Fluor® 488 and Alexa Fluor® 594. Sections were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature, then observed and imaged under an Axio Observer A1/D1/Z1 fluorescence microscope (Carl Zeiss, Oberkochen, Baden-Wurttemberg, Germany).
Cells on coverslips were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.5% Triton X-100 for 15 min and blocked with 1% BSA for 2 h at 37 ℃. Cells were treated overnight at 4 ℃ with primary antibodies (Abcam) against LC3 (1:200) and Iba-1 (1:500), followed by goat anti-rabbit IgG H&L (Cy3®) (1:100; Abcam) for 2 h. Nuclei were stained with DAPI. Images were taken using a TCS SP8 fluorescence microscope (Leica, Weztlar, Germany).
Statistical analysis
Statistical analysis was performed using GraphPad Prism 9.0.0 (Graph Pad Software, San Diego, CA, USA). All continuous variables followed normal distribution (Shapiro-Wilk test; data not shown), and were reported as mean ± standard deviation (SD). Data from Morris water maze training were analyzed using two-way ANOVA for repeated-measures, followed by the Bonferroni post hoc test for multiple comparisons. All other variables were analyzed using Student's t test (for comparisons between two groups), or one-way ANOVA followed by Tukey’s multiple test (for comparisons among at least three groups). P < 0.05 was considered statistically significant.