Hamilton syringe

G. mellonella larvae obtained from a commercial supplier (BioBichos, Ltd., Chillan, Chile) were used. All experiments were performed according to the protocols described by various authors [15 (link),41 (link),42 (link)]. Briefly, study groups consisted of 10 final stages of G. mellonella larvae, with an approximate weight of 250 mg each. They were randomly selected. A Hamilton^® syringe (Merck^® Ltd., Kenilworth, NJ, USA) was used to inject the larvae—inoculation volume used in each test was 10 uL. MRSA inoculum (CFU/mL) that decreases the survival of G. mellonella larvae was studied. Inoculum that managed to reduce survival to less than 20% in 72 h was selected. Once the inoculum of bacteria was chosen and the safety of the compounds to be tested was determined, the larvae were inoculated with the chosen suspension. After 1 h, they were injected with the compound to be studied at different concentrations. After each treatment, the larvae were incubated for 5 days at 37 °C, 80% humidity, and their survival was determined daily. One group of larvae was injected with phosphate buffer solution, and another group was not subjected to treatment. Both groups were used as controls in each of the experiments. The results of any experiment with more than two dead larvae in any control group were discarded.

After the rats were anesthetized with sodium pentobarbital (45 mg/kg, i.p.), they were mounted on a stereotaxic frame (Stoelting Co.). A midline incision was made to expose the skull and one burr hole was drilled. Bilateral stereotaxic injection was performed. The injection of 5 μl of anti-NMDAR1 (50 ng/μl, dissolved in CSF; Merck Millipore, Billerica, MA, USA) into dentate gyri [coordinates: 5.2 mm posterior, ±4.3 mm lateral, 4.8 mm deep (relative to bregma)] was performed at each side with a Hamilton syringe connected to a syringe pump. In control rats, 5 μl of CSF was injected in the similar way. The injection was performed at a rate of 0.25 μl/min (over 20 min) via a perfusion pump. One to seven days following the injection learning behavior experiments and electrophysiological experiments were performed accordingly.
In a subset of animals, histological examinations were performed to examine the localization of the stereotactic injection into the dentate gyrus. In this procedure, 0.5 μl of 2% Evans blue was given through the dentate gyrus. Then, the animals were anesthetized with sodium pentobarbital and intra-cardiacally perfused with physiological saline followed by 4% of paraformaldehyde solution. The hippocampus was sectioned and the location of injection sites was verified by identification of blue dye according to the atlas of Swanson (21 ).

The moth Galleria mellonella larvae were used to assess the potential toxicity of fungal and bacterial EVs according to the previously published protocols [29 (link)]. Larvae in their final instar stage (10 per group) were randomly chosen for the experiment and then inoculated in the last left proleg with 10 µl of the solution of fungal or bacterial EVs in sterile DPBS buffer, pH 7.50 ± 0.30 (Biowest) using a 10 μl Hamilton syringe (Merck). DPBS buffer served as a control for injection. The caterpillars were further incubated at 37 °C, and the number of dead larvae was scored daily; they were considered dead when they did not show movement in response to touch. The experiment was carried out in three biological replicates, and the averaged results are presented. The survival curves of EV-treated and control animals were compared using the Mantel-Cox log-rank test with GraphPad Prism 7.0. A value of p < 0.05 was considered significant.

To assess the impacts of blocking striatum miR-200b-3p expression in SHR, rat miR-200b-3p antagomir (AT) and the antagomir-negative control (ATNC) were purchased (BioLion Technology Co., Ltd., Taipei, Taiwan). Five nmol miR-200b-3p AT and ATNC in 1 μL PBS were mixed with 1 μL HiPerFect transfection reagent (Cat. #: 301705, Qiagen, Germantown, MA, USA) prior to injection into the striatum of SHR. The striatal stereo-taxic injection was performed as described elsewhere [35 (link)]. Briefly, SHR were intraperitoneally injected with urethane (1.25 g/kg) to anesthetize them, and they were placed on an animal heating pad. Next, the rats were fixed in a stereotactic apparatus, and a hole was drilled in the skull. The mixed solution was then injected (1 μL/min) into the left striatum of rats using a 10 μL Hamilton syringe (Sigma-Aldrich, St. Louis, MO, USA) connected to a microinfusion pump (Stoelting Co., Wood Dale, IL, USA). The skin was sutured after injection.

Delivery of TNF-α, IL-1β ± HB-EGF by i.c.v. injection was performed as described previously^{18 (link),20 (link),53 (link)}. In brief, mice were anesthetized using 1% isoflurane mixed with oxygen. Heads were shaved and cleaned using 70% ethanol and lidocain gel followed by a medial incision of the skin to expose the skull. The ventricles were targeted bilaterally using the coordinates: ±1.0 (lateral), −0.44 (posterior), −2.2 (ventral) relative to Bregma. Mice were injected with 10 μl of vehicle or cytokine solution containing 100 ng of TNF-α (Peprotech, no. AF-315-01A), IL-1β (Peprotech, no. 211-11B) ± HB-EGF (Novus Biologicals, no. 35069) using a 10-μl Hamilton syringe (Sigma Aldrich, no. 20787) on a Stereotaxic Alignment System (Kopf, no. 1900), sutured and permitted to recover in a separate clean cage. Mice received a subcutaneous injection of 1 mg kg⁻¹ meloxicam post i.c.v. injection and were analyzed after 24 h.

G. mellonella was used as an in vivo virulence model, as previously described [26 (link)] to compare the in vivo virulence of Sal199, Sal147, and Sal280, in comparison with to S. Typhimurium LT2. Briefly, Salmonella grown in Mueller-Hinton broth for 2, 16 and 24h at 37°C, was harvested to give a final concentration of 10⁸ bacteria in 1 mL 0.1 M PBS (pH 7.2). Ten larvae were injected with a 10 μL 10⁴ CFU mL^-1 in the right fore pro-leg, using a Hamilton syringe (Sigma, UK). All larvae were incubated at 37°C for 24 h before determining rates of survival and macroscopic appearance. Non-injected and 0.1 M PBS-injected animals were included in each experiment as controls. The exact dose was determined after plating the inoculum on LB agar. Representative results were obtained from three independent experiments. Data are expressed as percentage of survival, and analysed using the statistical package GraphPad Prism 5 (GraphPad Software, Inc., California, USA). A p-value of < 0.05 was taken to indicate statistical significance.