The standard strain of serotype III S. pneumoniae from American Type Culture Collection (ATCC, Manassas, VA, USA) was cultured on blood agar plates for 18 hours and then transferred into Vital Aer Broth (R&D Systems, Minneapolis, MN, USA) for another 18 hours at 37 °C in air with 5% CO2 to achieve the logarithmic growth phase. Then, the bacteria were centrifuged for 20 min at 5000g, washed twice, and resuspended in sterile saline to approximate 1 × 104 colony forming units (CFU)/ml using a nephelometer (bio-Merieux, Marcy-l’Étoile, France).
Rat Model of Streptococcus pneumonia Meningitis
Three-week-old Sprague-Dawley rats were obtained from the Shanghai Laboratory Animal Management Center (Shanghai, China). A well-established rat model of pneumococcal meningitis was used as described previously [21, 39]. In brief, rats were anesthetized with pentobarbital sodium (50 mg/kg), 30 μL cerebrospinal fluid (CSF) was slowly removed via intracisternal puncture, then infected intracisternal with 30 μL volume containing either 1 × 104 CFU/mL S.pneumoniae or pyrogen-free saline. At 24 h post-infection, rats were weighed and a clinical score evaluated the severity of the disease (1= coma, 2 = does not turn upright, 3 = turns upright within 30 s; 4 = turns upright within <5 s, and 5 = normal) in a blinded manner. Animals were sacrificed and perfused with pyrogen-free saline. The brain tissues were removed and separated immediately, half of the hemispheres were frozen in liquid nitrogen and half of the hemispheres were fixed in 4% paraformaldehyde. Samples of cerebellar and spleen homogenates were plated in serial dilutions on the sheep blood agar plates under 37◦C and 5% CO2 overnight to determine the bacterial titers.
Experimental Design and Drug injection
A total of 172 rats were included in this study, which were randomly divided into the following experimental, as shown in Fig.1.
In the first experiment, 36 animals were assigned to two groups: Sham group and PM group. All rats from day 7, 14 and 28 groups received antibiotic treatment (100 mg/kg ceftriaxone; Beyotime Biotech, Shanghai, China) continuously through subcutaneous injection for 5 days, starting from 24hours post-infection. Rats were scarified after 24 hours (n=6 each), days 7 (n=4 each), 14 (n=4 each), or 28 (n=4 each) to evaluate the expression of proBDNF/p75NTR.
In the second experiment, 60 animals were divided into 4 groups: Sham+Vehicle (water, 60μL/day) group (n=10), Sham+LM11A-31 (15μg/60μL/day) group (n=10), PM+Vehicle group (n=20) and PM+LM11A-31group (n=20). LM11A-31(2-amino-3-methyl-N-[2-(4-morpholinyl)ethyl]-pentanamide) was obtained from MedChemExpress (Monmouth Junction, NJ, USA). For intranasal compound delivery, LM11A-31 was dissolved in water at a concentration of 0.25 mg/ml, instilled in alternating nares for 2 minutes, repeated five times per nares, for 60ul (15ug of the compound). Animals were pretreated once daily for consecutive three days of LM11A-31 in water or water alone, and the PM model as mentioned above was constructed 30min after the last pretreatment. At 24 h post-infection, rats were scarified to investigate brain injury and neuroinflammation during acute PM.
In the third experiment, 20 animals were divided into 2 groups: shSCR group (n=10) and shp75NTR group (n=10). p75NTR knockdown was performed by RNA interference using adenovirus transfection. The shRNA sequence targeting p75NTR (shp75NTR, 2×1010pfu/mL) and a scrambled sequence (shSCR, 2.4×1010pfu/mL) used as a nontargeting shRNA control were supplied by Genechem Co. Ltd. (Shanghai, China). The sequences used were as follows: 5′- GAGGTGCCAAGGAGACATGTT -3′ for p75NTR, and 5′- TTCTCCGAACGTGTCACGT -3′for scrambled sequence. Control adenovirus was diluted to 2×1010 pfu/mL in enhanced transfection solution (GeneChem) before intracerebroventricular injection. Rats were stereotactically injected with 3 μL adenovirus solution (2×1010pfu/mL) with shp75NTR or shSCR into the right lateral ventricle at a rate of 0.2μL/min with 10 μL-Hamilton syringe 7 days before PM. The stereotaxic coordinates were 3.8mm rostral to the lambdoid suture of the skull, 2 mm lateral to the right side from the midline of the skull, and 2.5 mm from the skull surface. At 24 h post-infection, rats were studied to assess the effect of p75NTR knockdown on neuroinflammation.
In the final experiment, 56 animals were divided into 4 groups: Sham+Vehicle (water, 60μL/day) group, Sham+LM11A-31 (15μg/60μL/day) group, PM+Vehicle group and PM+LM11A-31group. Rats from all groups received antibiotic treatment in lines with the first experiment. Meanwhile, rats were treated once daily for consecutive 7 days with LM11A-31 in water, or water alone, starting 24 hours after infection. The dose and mode of administration were same as in the second experiment. On day 7 (n=18), 14 (n=18), or 28 (n=20), animals were sacrificed to evaluate hippocampal neurogenesis.
Ethynyl Deoxyuridine Treatment
Ethynyl Deoxyuridine (EdU) acts as a marker of dividing cells to monitor neurogenesis. To evaluate cell proliferation, EdU (50 mg/kg; Santa Cruz Biotechnology, Dallas, TX, USA) diluted in 5% DMSO was given every four hours, starting 30 hours before sacrifice on day 7 post-infection, a total of four times. Animals were sacrificed 18 hours after the last EdU treatment. To evaluate the differentiation of NSCs, animals were injected with the same dose of EdU twice daily on days 4-7 after infection, and animals were sacrificed on day 14 or 28 post-infection.
Western Blot Analysis
The brain tissues (cortex and hippocampus) lysates were homogenized in cold radioimmunoprecipitation assay (RIPA) buffer containing a protease inhibitor cocktail (#87786, Thermo Scientific, Waltham, MA, USA). Total protein was quantified using the BCA assay reagent (#23227, Thermo Fisher Scientific, Waltham, MA, USA). Aliquots (60 μg total proteins) were loaded into SDS-polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking in 5% fat-free milk (#9999S, Cell Signaling Technology, Danvers, MA, USA), the membranes were incubated overnight at 4°C with the following primary antibodies: mouse anti-β-actin (1:1000, #3700, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-proBDNF (1:1000, #PA5-77533, Thermo Fisher Scientific, Waltham, MA, USA ), rabbit anti-p75NTR (1:1000, #4201, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-NF-kB p65 or phospho-NF-kB p65 (1:1000, #8242, #3033, Cell Signaling Technology, Danvers, MA, USA) and rabbit anti-C/EBPβ (1:1000, #ab32358, Abcam, Cambridge, UK). Primary antibodies were detected with horseradish peroxidase-conjugated IgG secondary antibody, followed by exposure to a chemiluminescence system and visualized using the ChemiDOC XRS+ imaging system (BIO-RAD, Hercules, CA). Protein expression was normalized to the same sample expression of β-actin. The relative band intensity was quantified by ImageJ software (National Institutes of Health, USA).
Tissue pathology, Fluoro-Jade B and terminal deoxynucleotidyl transferase dUTP-nick-end labeling staining
Animals were anesthetized and perfused through the heart with sterile saline 24h after infection. Then, brains were fixed in 4% paraformaldehyde at 4°C. After 24h of fixation, the brain tissues were embedded in paraffin wax on the oriented edge and cut into coronal 5-μm-thick sections for hematoxylin and eosin (H&E) staining. The TdT-mediated dUTP nick end labeling (TUNEL) assay was performed using the In Situ Cell Death Detection kit (Roche, Basel, Switzerland), according to the manufacturer’s instructions. In brief, deparaffinized tissue sections were incubated with proteinase K (20 µg/mL) for 15 minutes at room temperature and immersed in 3% H2O2 in methanol for 10 minutes. Permeabilization with 0.3% Triton-X-100 for 10 minutes was performed on ice. The sections were incubated with the TUNEL reaction mixture at 37°C in a humidified chamber for 2 hours, followed by incubation with fresh prepared 4’,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA) reagent for 10 minutes in a dark room. For Fluoro-Jade B (FJB) staining, deparaffinized tissue sections were stained with 0.25% (50% glacial acetic acid as solvent) FJB overnight at 4°C and nuclear staining with DAPI the next day following product instructions.
Five-μm-thick paraffin-embedded sections of the brain were prepared as described above. To determine p75NTR expression patterns in different kinds of cells in brain，double-labeling immunofluorescence with p75NTR&NeuN, p75NTR &Iba-1, p75NTR &GFAP was performed in rats after 24h-infection. Immunofluorescence single-labeling immunofluorescence was used to detect the activation of astrocytes and microglia in the cerebral cortex and hippocampus. In addition, for immunofluorescence evaluation of EdU+ and DCX+, NeuN+ cells in the hippocampal dentate gyrus, on days 7 and 14 post-treated with LM11A-31, EdU&DCX was used to determine neuronal progenitor cells proliferation. On day 28 post-treated with LM11A-31, EdU&NeuN was used to determine neurogenesis. The brain sections were permeabilized for 10 min with 0.3% Triton-X100, and blocked with 5%BSA for 1h at room temperature. The following primary antibodies: rabbit anti-p75NTR (1:200, #ab52987, Abcam, Cambridge, UK), mouse anti-NGFR p75 (1:200, sc-271708, Santa Cruz Biotechnology, Dallas, TX, USA), goat anti-Iba-1 (1:500, #ab5076 Abcam, Cambridge, UK), mouse anti-GFAP (1:500, #GB12096, Servicebio, Wuhan, China), rabbit anti-DCX (1:500, #GB11317, Servicebio, Wuhan, China) and rabbit anti-NeuN (1:500, #ab177487, Abcam, Cambridge, UK) incubated slices overnight at 4°C. After washing 3 times with PBS, slices were incubated with secondary antibodies for 1h at room temperature in the dark, and then mounted with DAPI reagent to stain for nuclei. To detect EdU staining, sections were incubated with an EdU imaging kit (#C10310-1, Ribobio, Guangzhou, China) followed after washing the secondary antibody with PBS. Then, stained sections were rinsed thoroughly with PBS and observed by a fluorescence microscope (Nikon, Japan).
RNA isolation and quantitative real-time PCR
Total RNA was isolated from brain tissues, including the cerebral cortex and hippocampus, with a Total RNA Kit (TaKaRa, Shiga, Japan, catalog #9767), following the manufacturer’s instructions. The RNA concentration was quantified using a NanoDrop 2000 (Thermo Fisher Scientific) and was reverse transcribed to cDNA using the PrimeScript™ RT Master Mix (TaKaRa, Shiga, Japan, catalog #RR036A). Quantitative real-time PCR was carried out with SYBR Premix Dimmer Eraser kit (TaKaRa, Shiga, Japan, catalog #RR420A) in 20µl of final volume using QuantStudio 3 system (Applied Biosystems, Carlsbad, CA, USA). Primers for real-time PCR (RT-PCR) were designed using Premier 5 software, and the sequences are listed in Supplementary Table 1. The fold change of target genes was determined from the observed Ct (cycle threshold) values and calculated using the 2−ΔΔCt method, and β-actin served as the reference gene.
In tissue sections, FJB‐positive cells, Iba-1- positive cells and GFAP- positive cells in cortex and hippocampus were observed from three randomly selected microscopic fields at × 200 magnification. Positive cells count was undertaken on a microscope (Nikon Eclipse C1, Japan) with digitalization software CaseViewer 2.0 (3D HISTECH, Hungary). Randomly selected three fields of view from coronal cortex and DG section to count the cell number, then averaged the results per group. For TUNEL-positive cells, EdU&DCX- and EdU&NeuN- labeled cells count in hippocampus, coronal section of the DG (including adjacent sub-granular zone [SGZ], granule cell layer [GCL], and molecular layer [ML]) within hippocampus was analyzed from each section. We obtained the cell number of the DG from each animal. All counts were performed by the same observer who was blinded to experimental groups.
All the data are presented as mean±SEM. Differences between the two groups were detected by unpaired Student t-test (parametric data). Two-way ANOVA followed by Tukey’s post-hoc test was used to compare differences between multiple groups. The survival curve was performed using the Kaplan-Meier method and analyzed by the log-rank test. All statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). p-value <0.05 was considered statistically significant.