Mini osmotic pump

Bumetanide (Sigma-Aldrich, B3023) (dosage of 40 mg/kg body weight/day) and bromodeoxyuridine (Sigma-Aldrich, B5002) (dosage 40 mg/kg body weight/day) were dissolved in H₂O: PEG300 (1:5) with incubation on a thermo shaker on 36 °C, 1000 rpm, overnight. Mini-osmotic pumps from Alzet® (model 2001, DURECT Corporation, Cupertino, CA, USA) with a filling volume of 200 μl and a pump delivery rate of 1 μl/h were used. One day prior to pump implantation, the Mini-osmotic pumps were loaded with appropriate volume (200 μl) of the solution as directed by manufacturer using the provided needle. The loaded Mini-osmotic pumps were primed in sterile NaCl 0.9% at 37 °C overnight according to the supplier’s instruction.

A model of local microglial ablation using CD11b-HSVTK transgenic mice expressing the herpes simplex thymidine kinase (HSVTK) protein in microglia and macrophages [9 (link), 36 (link)] was employed. When these animals are exposed to ganciclovir (GCV), the cells that express HSVTK are eliminated. CD11b-HSVTK (+/-) male mice (gift of Dr. Tsirka, Stonybrook University) were bred with C57BL/6 males. Offspring were genotyped by PCR using primers 5′ -GACTTCCGTGGCTTCTTGCTGC-3′ and 5′ -GTGCTGGCATTACAGGCGTGAG-3′. GL261 glioma cells were implanted into the right cerebral hemisphere of 12–16 week old CD11b-HSVTK (+/-) mice. C57BL/6 mice were used as controls. Tumors were allowed to grow for ten days and then mini-osmotic pumps (Alzet, DURECT, model 2004) were installed for local administration of GCV (Calbiochem, Billerica, MA, USA) to the tumor. Animals were anesthetized and a 3mm brain infusion cannula connected to pump was set up at the previous tumor implantation site using brain infusion kit (Alzet, DURECT). The pumps were placed subcutaneously on the mouse back. The drug was infused at 0.25 μL/h, 1 mg/mL over 7 days. Pumps with normal saline solution were used as a control.

Mini-osmotic pumps were purchased from DURECT Corporation (Model 2006, Cupertino, CA). Angiotensin II (AngII), Honokiol (HKL), FITC-inulin, Bovine Serum Albumin (BSA) were purchased from Sigma Aldrich (Sigma Aldrich, USA). Mouse urine creatinine assay kit was purchased from R&D Systems (R&D, USA). Mouse urine albumin, BUN and Scr assay kit were purchased from AssayPro Corporation (AssayPro, USA). Antibodies against SIRT3 (28kDa), acetylated lysine, Tubulin and GAPDH were purchased from Cell Signaling Technology (CST, USA). Antibodies against KLF15, fibronectin, collagen type IV, synaptopodin, WT-1 were purchased from abcam (Abcam, USA).