Alzet osmotic pumps model 1002

Il-1 receptor deficient (Il-1r^−/−) mice and Nlrp3 null (Nlrp3^−/−) mice were purchased from Jackson Laboratory. All mice were on C57BL6 background, and mouse genotyping was performed by PCR. To induce estrogen deficiency, bilateral ovariectomy (OVX) or sham surgery (in which ovaries were exteriorized, but replaced intact) were performed under general anesthesia in 4-month old WT or Nlrp3^−/− mice, and bones were analyzed 8 weeks after surgery. Estrogen deficiency was confirmed by uterine atrophy. For PTH experiments, 80 μg/kg/day human PTH1-34 or vehicle were delivered to 4-month old WT or Nlrp3^−/− male mice for 2 weeks via subcutaneously implanted ALZET osmotic pumps (model-1002, DURECT Corporation) with a delivery rate of 0.25 ml/h as previously described^{46 (link)}. GST-RANKL or vehicle was administrated intra-peritonally to 3-month old WT or Nlrp3^−/− male mice at a dose of 1 µg/kg, daily for 2 days. For pharmacologic inhibition of bone resorption, 40 mg/kg zoledronate (Novartis #484188) were injected subcutaneously to 3-month old WT mice, once a week, for 4 weeks prior to GST-RANKL injection. All procedures were approved by the Institutional Animal Care and Use Committee of Washington University School of Medicine in St. Louis. All experiments were performed in accordance with the relevant guidelines and regulations described in the IACUC-approved protocol #20160245.

We chemically synthesized human HCNP (PVDLSKWSGPL). Under anesthesia with 1% isoflurane, a midsagittal incision was made on the scalp, and a subcutaneous tunnel was opened between the shoulder blades, where the Alzet osmotic pumps (model 1002; pumping rate 0.25 μL/h, DURECT Corporation, Cupertino, CA, USA) were implanted. Subsequently, a craniotomy at 0.6 mm posterior and 1.2 mm lateral from the bregma was performed stereotactically. The tip of the pump was carefully inserted into the craniotomy site and was angled at 8°. The cannula was fixed in place using dental cement. The incision was closed with silk sutures and dabbed with Vetbond (3M, St Paul, MN, USA). Intraventricular infusion of the vehicle (bicarbonate buffer) or 0.75 pg/h of HCNP was then performed for 14 d.