Experimental animals
A total of 60 BALB/c male mice were used in the experiment, procured from the National Laboratory Animal Centre, Taipei, Taiwan. All mice were housed in a controlled environment with a 12 hour light / dark cycle photoperiod and raised until they reached 5 weeks of age, weighing between 20 and 25 g. The mice were provided Purina Laboratory food and water ad libitum. They were housed in a specific pathogen-free room at the Animal Centre, Chung-Shan Medical University (Taichung, Taiwan), for more than a week prior to the start of the experimental infection (Chiu and Lai, 2013). Throughout the experiment, mice were monitored daily for access to food, water, bedding and overall health conditions. Following infection with A. cantonensis, mice were closely observed for signs of disease, such as ruffled fur, decreased activity or tachypnea, as well as any weight loss. In particular, no mortality was recorded among mice during the infection and subsequent drug treatment phases. To ensure humane euthanasia, mice were subjected to CO2 exposure until respiratory arrest occurred, followed by cervical dislocation as a confirmatory euthanasia method before necropsy. This study was carried out with the approval of the Institutional Animal Care and Use Committee of Chung-Shan Medical University, and all procedures were carried out in accordance with institutional guidelines for animal experiments.
Antibodies
Polyclonal goat anti-mouse COX-2 (mPGES-1, PGE-2) antibodies were purchased from Santa Cruz Biotechnology (CA, USA). The goat anti-mouse NLRP3 polyclonal antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse anti-mouse β-actin monoclonal antibody was acquired from Sigma (St. Louis, MO, USA).
Larval preparation
Infectious larvae (third stage, L3) of A. cantonensis were obtained from Achatina fulica snails, which had been bred for several months and infected with larvae of A. cantonensis through rat hosts at the Wufeng Experimental Farm (Taichung, Taiwan). L3 larvae were retrieved from tissues using a method previously outlined but with several adjustments (Chen et al., 2022). The nail shells were crushed and tissues were homogenized in a pepsin-HCl solution (pH 1 to 2, 500 IU of pepsin/g of tissue) and digested by agitation at 37 ° C for 2 hours. The L3 larvae in the sediment were collected by sequential washes with double distilled water and enumerated under a microscope.
Animal infection
A total of 18 BALB/c male mice were randomly distributed into 5 experimental groups, labelled D5, D10, D15, D20, and D25, each consisting of 3 mice, together with a control group of 3 mice. Before infection, mice were deprived of food and water for 12 hours. Mice in groups D5, D10, D15, D20 and D25 were orally inoculated with 30 larvae of A. cantonensis and subsequently sacrificed on days 5, 10, 15, 20, or 25 after inoculation (PI), respectively. The control group mice received water only and were sacrificed on day 25 PI. Following sacrifice, the brains were immediately extracted and frozen in liquid nitrogen for further analysis.
Treatment of animals
A total of 42 mice were randomly allocated into six groups, each group consisting of 7 mice. Before infection, food and water were held for 12 hours. The uninfected control mice received oral administration of distilled water only on Day 10 post-inoculation (PI). All other groups, including untreated infected control mice, were infected with 30 A. cantonensis larvae and intraperitoneal injection with distilled water only on day 10 PI. The treatment groups were intraperitoneal injection with DMSO alone (10 mg/kg/day), MCC950 alone (10 mg/kg/day, Sigma-Aldrich, St. Louis, MO, USA), NS398 (10 mg/kg/day, Sigma Aldrich, Saint Louis, MO, USA) and MF63 (30 mg/kg/day, Cayman Chemical, Ann Arbor, MI, USA) for 7 consecutive days, starting on day 10 PI. Subsequently, all mice were sacrificed 22 days after inoculation for further analysis.
Preparation of excretory and secretory products (ESPs)
Third-stage infectious larvae of A. cantonensis were acquired from Achatina fulica snails following the method described by Chen et al. (2022). These larvae were used to infect mice and young adult A. cantonensis was harvested from mouse brains 20 days after infection. Subsequently, the harvested worms were washed thoroughly to eliminate host cells. A total of 200 intracranial larvae were suspended in 400 ml of sterile water. The larvae were then mechanically disrupted using a clear glass pestle and subjected to sonication on ice (75 W for 10 seconds, repeated 10 times, with 2-minute intervals) three times to facilitate the release of soluble antigens. After sonication, the samples were centrifuged at 11,500g for 5 minutes at 4 ° C and the resulting supernatant was collected. This supernatant was further filtered through a 0.22-µm filter. The total protein concentration in the filtrate was determined using the bicinchoninic acid method and the ESPs were stored at -80°C until further use.
Cell culture and stimulation assay
The mouse microglial cell line N9, obtained from the American Type Culture Collection, was cultured in RPMI 1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum (Gibco, Carlsbad, CA, USA) and maintained at 37 ° C in a humidified atmosphere containing 5% CO2. Cells were seeded in 12-well culture plates (Corning, NY, USA) at a density of 1×105 cells per well. Once a monolayer was formed, the cells were stimulated with 50 µg/mL of ESPs as a positive control. For the negative control group, an equal volume of liquid was added, obtained following the ESPs preparation procedure without culturing A. cantonensis.
Small interfering RNA (siRNA) transfection
siRNA transfections were performed using Lipofectamine 2000 (11668027, Life Technologies, CA) following the manufacturer’s protocol. Briefly, cells were transfected with targeted siRNA or scrambled siRNA (20 pM) mixed with 6 µl of Lipofectamine 2000 diluted in 150 µl of reduced serum medium (# 31985062, Life Technologies, CA). The resulting siRNA-lipid complexes were added to cells, incubated for 6 hours, and then the medium was replaced and maintained for an additional 24 hours. Subsequently, cells were treated with cocaine (10 µM) or left untreated, and harvested after an additional 24 hours as previously described (Chivero et al., 2021). The knockdown efficiencies were evaluated by Western blotting.
Collection of cerebrospinal fluid (CSF)
A single CSF sample was collected from the magna cisterna for ELISA and zymography assays. Mice were anaesthetised by intraperitoneal injection of urethane (1.25 g/kg). Each mouse was placed in a stationary apparatus at a 135 ° angle with respect to the head and body. The skin over the neck area was shaved and cleaned three times with 70% ethanol. Subsequently, the subcutaneous tissue and muscles were carefully separated. A capillary tube was then inserted through the dura mater into the cisterna magna and CSF was aspirated into the capillary tube following established procedures (Chiu and Lai, 2014). The collected CSF was transferred to a 0.5 ml Eppendorf tube and centrifuged at 3000 × g at 4 ° C for 5 minutes. The resulting supernatant was carefully collected in another 0.5 ml Eppendorf tube and stored in a freezer at -80°C until further analysis.
Western blot analysis
Electrophoresis, coupled with subsequent Western blot analysis, stands as an essential methodology for probing protein alterations within cerebrum tissues. Initially, cerebrum tissue homogenates were centrifuged at 10,000 × g at 4°C for 10 minutes to remove debris. Subsequently, protein concentrations (30 µg) in the supernatant were determined using protein assay kits (Bio-Rad, Hercules, CA, USA), using bovine serum albumin (Sigma-Aldrich Corporation, St. Louis, MO, USA) as standard. These proteins were then diluted 1:1 in a loading buffer composed of 10% sodium dodecyl sulphate (SDS), 5% bromophenol blue, 2% glycerol, 2-mercaptoethanol, and 0.5 M Tris-HCl (pH 6.8). Subsequently, the sample mixtures were boiled for 5 minutes before 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed at room temperature and 110 V for 90 minutes, followed by electrotransfer to polyvinylidene fluoride membranes (PVDF) (Pall Corporation, Coral Gables, FL, USA) at a constant current of 30 V and at 4 ° C overnight. Following electrotransfer, PVDF membranes were subjected to two 10-minute washes in phosphate buffered saline (PBS) containing 0.1% Tween 20 (PBS-T) at room temperature, repeating this process three times. The membrane surface was then blocked with 5% fat-free dry milk in PBS at 37 ° C for 1 hour, followed by three saturations with PBS-T for 10 minutes at room temperature. Subsequently, the membranes were incubated with primary antibodies diluted at 1:1000 at 37 ° C for 1 hour. After incubation, PVDF membranes were washed three times with PBS-T before being incubated with horseradish peroxidase-conjugated secondary antibodies diluted at 1:10,000 at 37 ° C for 1 hour, facilitating the detection of bound primary antibodies. Finally, the labelled proteins were visualised utilizing an enhanced chemiluminescence detection system (Amersham Biosciences, Amersham, UK), and the densities of specific immunoreactive bands were quantified using a computer-assisted imaging densitometer system.
Immuno-histochemistry
The mouse cerebrum was individual fixation in 10% neutral buffered formalin for a duration of 24 hours. After fixation, the specimens were subjected to dehydration through a series of graded ethanol (50%, 75%, 100%) and xylene, eventually was embedded in paraffin at 55°C for 24 hours. For immunohistochemical purposes, relatively thick (10-µm) serial sections of the paraffin-embedded brains were meticulously cut and mounted onto glass slides. These sections were then dewaxed with xylene and rehydrated using a series of ethanol solutions (100%, 95% and 75%) for 5 minutes each. Subsequently, they were treated with 3% H2O2 in methanol for 10 minutes to deactivate any endogenous peroxidase activity, followed by three 5-minute washes with PBS. Subsequently, the sections were blocked with 3% BSA at room temperature for 1 hour, before being incubated with primary antibodies at a 1:50 dilution in 1% BSA at 37 ° C for 1 hour. After another round of three washes in PBS, the sections were exposed to HRP-conjugated rabbit anti-goat IgG (Jackson ImmunoResearch Laboratories, USA) at a 1:100 dilution in 1% BSA at 37°C for 1 hour, followed by three additional washes in PBS. Finally, sections were incubated for 3 minutes at room temperature with 3,3'-diaminobenzidine (0.3 mg/ml) in 100 mM Tris (pH 7.5) containing 0.3 µl/ml H2O2. After this incubation, the sections were subjected to a final round of three washes in PBS before being mounted in 50% glycerol in PBS for examination under a light microscope.
Gelatin zymography
The procedures were performed based on zymography utilizing gelatin-containing SDS polyacrylamide gels, as previously detailed (Chen et al., 2017). Unboiled protein samples (30 µg) were mixed with an equal volume of standard loading buffer before loading. The samples were loaded onto polyacrylamide gels at a concentration of 7.5% (mass/volume), which included copolymerised substrate gelatin (0.1%) for SDS-PAGE (Sigma-Aldrich Corporation, St. Louis, MO, USA) to evaluate gelatinase activities. SDS-PAGE was carried out in a running buffer (1% SDS, 25 mM Tris, and 250 mM glycine) at room temperature and 110 V for 1 hour. Subsequent to electrophoresis, each gel was washed for two 30-minutes in denaturing buffer (2.5% Triton X-100) at room temperature, followed by two washes with double distilled water at room temperature for 10 minutes each. The gel was then incubated in a reaction buffer (50 mM Tris-HCl, pH 7.5; containing 0.02% Brij-35, 0.01% NaN3 and 10 mM CaCl2) at 37°C for 18 hours. After incubation, the gel was stained with 0.25% Coomassie Brilliant Blue R-250 (Bio-Rad, Hercules, CA, USA) for 1 hour and destained using a solution comprising 15% methanol and 7.5% acetic acid. The resulting gel displayed a uniform background, except in regions where gelatinases had migrated and cleaved their respective substrates. Quantitative analysis of gelatinases was performed using a computer-assisted imaging densitometer system (UN-SCAN-ITTM gel Version 5.1, Silk Scientific, Provo, Utah, USA).
Evaluation of BBB permeability
BBB permeability was evaluated by measuring Evans blue concentrations in the mouse cerebrum, following the methodology outlined by Chen et al. (2019). Initially, mice received an injection of Evans blue dye (5 ml/kg body weight; Sigma, St. Louis, MO, USA) in saline via the tail vein. After a 2-hour circulation period, mice were anaesthetised and subjected to transcardial perfusion with saline to eliminate intravascular dye. The mouse cerebrum was weighed and homogenised in a 50% trichloroacetic acid solution. The resulting homogenates were centrifuged at 12,000 × g for 10 minutes and the supernatants were carefully collected. Each supernatant was then subjected to absorbance measurement at 620 nm using a spectrophotometer (Hitachi U3000, Tokyo, Japan) to quantify Evans Blue concentrations.
Eosinophil counts in the CSF
The CSF was collected and placed in a centrifuge, spinning at 400×g for 10 minutes. The resulting sediments were then gently mixed with 2 ml of acetic acid and 100 mL of Unopette buffer (Vacutainer System, Becton Dickinson, Franklin Lakes, NJ, USA). A hemocytometer cell counting chamber (Paul Marienfeld, Lauda-Koenigshofen, Germany) was used to count eosinophils in the sample.
Statistical analysis
Differences among the various groups of mice were evaluated using Kruskal-Wallis nonparametric analysis followed by Dunn multiple comparison tests. All results were expressed as means ± standard deviation (SD). Statistical significance was considered for P values < 0.05.