Nf33bv 2

To evaluate the effect of FGF10 on cell proliferation during chronic inflammation, we injected FGF10 (five mice per group; 10 LGs) or BSA (five mice per group; 10 LGs) into the LGs of NOD.B10.H2^b mice, performed cell proliferation assays (see above), and determined focus scores. Four-month-old NOD.B10.H2^b and TSP-1^–/– mice were anesthetized, and the hair on both sides of each mouse was removed using hair clippers. Skin was disinfected with 10% povidone–iodine solution.⁴² After skin preparation, an approximately 0.5-cm full-thickness skin incision was made in the center of the prepared area using a sterile surgical instrument, and the extra-orbital LG was slowly injected with 2 µL of human recombinant FGF10 (100 ng/µL in PBS) using a 33-gauge needle (#NF33BV-2; World Precision Instruments, Sarasota, FL, USA) and a nanofil syringe (World Precision Instruments). Control LGs were injected with vehicle (PBS). Three injections in each gland into different lobules were performed to ensure larger areas of gland treatment. Analyses of LGs were carried out 3 days after injection.

Injection of AAV was conducted as previously described [48 (link)] with minor modifications. Briefly, mice were anesthetized with isoflurane and placed in a stereotaxic frame (51733U, Stoelting, Wood Dale, IL, USA). The skull was exposed, and a small hole was drilled through the skull at the coordinates for central amygdala (AP: −1.22 mm, ML: ±2.9 mm, DV: −4.6 mm). The designer receptors exclusively activated by designer drug (DREADD) virus particles AAV-hSyn-DIO-hM3D (Gq) or AAV-hSyn-DIO-hM4D (Gi)- mCherry (Neurophotonics, Quebec, QC, Canada) or AAV9.hsyn. eGFP/AAV9.hsyn. eGFP-Cre (University of Pennsylvania vector core) were injected by using microliter syringe (NanoFil, World Precision Instruments, Sarasota, FL, USA) with a 33-gauge beveled needle (NF33BV-2, World Precision Instruments). The injection needle was lowered, and virus particles were delivered at a rate of 1nl/sec using a UMP3 micro-syringe pump (World Precision Instruments). The volume of injections was kept at 150 nL to obtain the precise local infection and to avoid the leak into other brain regions. The needle was left in place for additional 10 min at the injection site and was slowly withdrawn over the period of 5 min. The incision was sealed with surgical tissue adhesive (1469SB, 3M, Maplewood, MN, USA). Only mice that had virus injection restricted to the CeA were included in the study.