Neomycin sulphate

Zebrafish embryos were maintained in our facility according to standard procedures. The apc mutant transgenic line was obtained from Professor Xu Wang. The ages of the zebrafish larvae are described as days post fertilization (dpf). SP600125 (Sigma-Aldrich, St Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) at a stock concentration of 50 mM and further diluted to the desired concentrations in fresh egg water. Dose-response data were obtained by treating larvae with SP600125 (5 μM, 10 μM, and 15 μM) after hair cell damage. For hair cell damage, neomycin sulphate (Sigma-Aldrich) was added to 5 dpf larvae at a final concentration of 400 μM and incubated for 1 h. This was followed by three rinses in fresh egg water, and the zebrafish larvae were allowed to recover for 24 h or 48 h at 28.5°C.

For the generation of bone marrow (BM) chimeras, 12 month-old PSAPP transgenic recipient mice (CD45.2; 12 females and 7 males) were lethally irradiated with two doses of 600 rads each (second irradiation after 12 h). 3 month-old Rag2 ko (Model 461, Taconic, Ry, Denmark) and age-matched WT control donor mice (Model 4001, Taconic, Ry, Denmark) expressing the CD45.1 allele were sacrificed by CO₂ inhalation, and bones (including femur, tibia and pelvis) were flushed with sterile PBS to obtain BM stem cells. A total of 5 x 10⁶ cells in 0.2 ml sterile Hank’s Buffered Salt Solution (HBSS, Life Technologies, Zug, Switzerland) were injected i.v. per animal in a randomized and gender-balanced fashion (10 PSAPP mice (including 6 females and 4 males) received Rag2 ko BM; 9 PSAPP mice (including 6 females and 3 males) received WT BM). BM reconstitution of recipient mice was carried out with BM from gender-matched donors. Autoclaved drinking water supplemented with antibiotics was provided for two weeks (2 g/l Neomycin sulphate; Sigma Aldrich). Reconstitution efficiency with CD45.1 positive cells was assessed on the day of sacrifice (6 months later) by flow cytometry of EDTA whole blood samples.

To prepare the host for the subsequent FMT,^{23 (link)} antibiotics (ATBs) were used to induce a disruption of the gut microbiota.^{24 (link)} Mice were randomly assigned into either control (vehicle/vehicle), ATB-only (ATB/vehicle) or ATB/FMT groups (Fig. 1A). The ATB group had ad libitum access to a non-absorbable ATB cocktail of ertapenem sodium, vancomycin hydrochloride (both from Glentham Life Sciences) and neomycin sulphate (Sigma-Aldrich) in sterile water at a ratio of 1 mg/ml, for 7 days (at Week 8 of age), following a previously published protocol.^{24 (link)} The vehicle of ATB intervention was drinking water. Non-absorbable ATBs have a relatively small degree of systemic absorption, decreasing possible off-target effects. The FMT intervention consisted of oral administration of 150 µl of the prepared 5 g/ml of donor caeca content solution, for 3 days with 2 days of spacing (at 9 weeks of age). The vehicle control for the FMT intervention was oral administration of 7.5% glycerol–PBS. Plastic needles were used to minimize the effect of the gavage on the mice. The control group received neither ATB nor FMT, only the respective vehicle solution (drinking water and 7.5% glycerol–PBS).

Donor mice were killed for collecting bone marrow. Bone marrow cells collected were counted and resuspended in sterile PBS. After irradiation, the recipient mice were inoculated intravenously via the tail vein with 5 million donor cells in a volume of 200 μl. Recipient mice were given mash to eat with antibiotic water containing 2 mg ml⁻¹ of neomycin sulphate (Sigma-Aldrich) and used for experiments after 6 weeks of bone marrow reconstitution.

Mice were given ampicillin (1 g/L; Sigma,St. Louis, MO), vancomycin (500 mg/L; Sigma), neomycin sulphate (1 g/L; Sigma), and metronidazole (1 g/L; Sigma) in drinking water for 4 weeks as described [19 (link)]. Mice were switched to water without antibiotics for 3 days before experimental manipulation. Bacterial load in the feces was quantified after the depletion by isolating DNA and performing quantitative PCR for total bacterial 16S rRNA genes.