Alzet model 2002

Eight-week-old male C57BL/6 mice were purchased from Japan SLC (Shizuoka, Japan). These mice were randomly assigned into three groups as follows: normal saline (NS), Ang II infusion (AT) or Ang II infusion with hyperthermia treatment (AT + heat). Under isoflurane anesthesia (2%), a mini-osmotic pump (ALZET, model 2002; DURECT Corporation, CA, USA) filled with either Ang II (1.5 μg/kg/min; Sigma-Aldrich, MO, USA) or normal saline was inserted underneath the skin^{29 (link)}. Hyperthermia treatments were performed using a heating plate (NEO HOTPLATE HI-1000; ASONE, Japan) at 42 °C for 60 min per every day and core temperatures were monitored by measurement of the rectal temperature. Body weight (BW), systolic blood pressure (SBP), and heart rate (HR) were measured before, 1 week, and 2 weeks after Ang II infusion. SBP was measured with a non-invasive computerized tail-cuff system (BP-98AL; Softron, Japan). After 2 weeks of Ang II infusion, all mice were euthanized with isoflurane and the heart was rapidly excised. Half of the left ventricle (LV) was fixed in 10% formaldehyde solution for histological staining and total RNA was collected from another half of the LV.

Mini-osmotic pumps developed for in vivo experiments (Alzet model 2002; Durect Corporation, Cupertino, CA, USA; infusion rate 0.5 µL/h, 200 µL nominal reservoir, two weeks factor release) were used as drug source for long-term delivery. A 6 well tissue culture plate (TPP, Trasadingen, Switzerland) was modified to enable pump immersing next to the NOC for a parallel performance of N = 4 experiments (Figure 3). Small holes (Ø1.8 mm) were drilled into the multi-well plate at a height of 1 cm to allow a connection between the separated NOCs and osmotic pumps without compression of the catheter. The catheters (Rat Femoral Catheter; Durect Corporation, Cupertino, CA, USA; outer diameter of 1.02 mm) were cut into 3 cm long pieces, linked to the pumps, threaded through the drilled holes, and inserted in the inlet of the NOC. Two of the 6 wells served as a depot for two mini-osmotic pumps each, whereas the other four wells hosted the NOCs. Wells were filled with saline (NaCl 0.9%; B. Braun, Melsungen, Germany) until the pumps were completely covered to ensure a continuous drug release. Due to the drilled holes, saline diffused to all wells of the plate and prevented an evaporation of the medium in the NOCs. A placement on 3D printed pedestals (white PLA, height: 11 mm; thickness: 7 mm; diameter: 30 mm) elevated the NOCs above the saline level.

Wildtype (WT) C57Bl/6 male mice, obtained from The Jackson Laboratory were studied at 3 months of age. Ang II (490 ng/kg/min) or vehicle (sham) was infused for 6 days via osmotic minipumps (Alzet, Model 2002; DURECT Corporation, Cupertino, CA, USA) as previously described.⁵ (link)^,6 (link) All animal procedures were approved by Vanderbilt University’s Institutional Animal Care and Use Committee (IACUC) where the mice were housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals, US Department of Health and Human Services.

C57BL/6 mice (male, 6-8 weeks old and 20±2 g) were maintained under a specific pathogen-free condition with free access to tap water and regular mice chow pellet. A cardiac hypertrophic mouse model was established using an Ang II infusion as described previously [14 (link)]. Briefly, 50 mg/kg sodium pentobarbital was used to anesthetize the mice, followed with implantation of 1.46 mg/kg/d of Ang II with osmotic minipump (Alzet model 2002; DURECT, Cupertino, CA) for 14 days in the scapular area. Saline infused mice served as sham group. Blood pressure (systolic and diastolic), were taken using tail-cuff method that is noninvasive (CODA System, Kent Scientific, Torrington, CT) at baseline and 14 days after the Ang II infusion. After 2 weeks, cardiac dimensions and function were analyzed by echocardiography as described below. Then, the mice were sacrificed by cervical dislocation. The hearts were immediately excised, washed in phosphate-buffered saline, gently dried, and weighed. Afterwards, ventricles were separated and stored for histological analysis and RNA/protein isolation.

As described previously [19] (link), [20] (link), rats were anesthetized with halothane (5% to induce, 2–3% to maintain), and their right external jugular vein was catheterized with a vincristine-filled miniosmotic pump (0.5 µl/h, 14 days; Alzet Model 2002, Durect Corporation, Cupertino, CA, USA) that had been primed overnight to deliver 30 mg/kg/day vincristine sulfate (Sigma-Aldrich, St Louis, MO, USA) (Vincristine group). Control rats were implanted with primed mini-osmotic pump containing 0.9% saline (Sham group).

The device (Cochlear Ltd., Sydney, Australia) consisted of six platinum contacts, 0.3 mm each, with 0.4 mm spacing (Figure 2). Beginning on the electrode tip, only the first and fourth contacts were linked to the connector via parylene-insulated platinum–iridium wires. The wires were embedded in a silicone matrix of about 450 µm outer diameter. The silicone matrix included a drug delivery channel (diameter: ~200 µm) with a single opening at the electrode tip. Wires and the drug channel separated in a distance of 22 mm from the tip (Figure 2A). During surgery, the silicone tube for drug delivery was connected to the flow moderator of a mini-osmotic pump with an infusion rate of 0.5 µL/h, suitable for 14 days delivery (Alzet model 2002; Durect Corp., Cupertino, CA, USA). The day before surgery, the pumps were either filled with AP with addition of 0.1% guinea pig serum albumin (Sigma-Aldrich, Steinheim, Germany) [19 (link)] or 50 ng BDNF (R&D Systems, Wiesbaden, Germany) diluted in 1 mL of serum albumin containing AP and primed in saline at 37 °C overnight.

Six-week-old male db/db mice were purchased from CLEA Japan Inc (Tokyo, Japan). Mice were maintained in a pathogen-free facility under controlled environmental conditions and exposed to a 12:12 h light:dark cycle. After 2 weeks of acclimation, mice were fed HF diets (HFD-32; CLEA Japan Inc., Tokyo, Japan) with or without TA-1887 treatment (0.01% w/w in chow). To assess effects of chronic insulin treatment, animals attached to either insulin or normal saline pumps (Alzet, model 2002; DURECT, Cupertino, CA) were similarly fed and received insulin (3 μg/g/day) or control saline, respectively. Blood glucose levels of insulin-treated mice were adjusted to ~200 mg/dl by additional administration of long-acting insulin (Insulin Glargine, Sanofi, Gentilly, France). Animal experiments were approved by the institutional review board at Kumamoto University, and all animals received humane care.