Alzet brain infusion kit 3

For anti-miR release into the CSF, the ICV and ITh routes were used in BALB/c-nu mice. ICV injection was carried out via stereotaxic surgery, and anti-miRs were administered into the right cerebroventricular space (A/P: +0.2 mm; lateral: +1.0 mm; depth: −2.5 mm from the bregma; Figure 1B). ITh injection was performed in the mouse after anesthesia at the L3, L4 intervertebral space. A volume of 5 μL was administered using a 32 G mouse intrathecal catheter (Alzet^® brain infusion kit3, Durect Corporation, Cupertino, CA, USA) connected to a 50 μL Hamilton syringe. The animal was lightly restrained to maintain the dorsal recumbent position and for puncture of the dura through spinous processes by the catheter. Anti-miRs were released into the mouse epidural space, and animals were sacrificed after the final treatment to analyze their distribution in the brain. The detailed course of events is described in Figure 1C.

Osmotic minipump
(Alzet 1004, Durect; flow rate of 0.11 μL/h) implantation was
performed 4 weeks after virus vector injections in a stereotaxic operation
as described in the study by Svarcbahs et al.^{47 (link)} Minipumps were filled with 16 mM KYP-2047 solution (0.2% dimethyl
sulfoxide (DMSO) in PBS) or 16 mM HUP-55 in 5% Tween
in saline (Braun) and primed according to producer’s instructions.
5% Tween in saline was used as a vehicle. A cannula (Alzet Brain Infusion
Kit 3, Durect) was implanted in the left hemisphere at 0.7 mm anterior
and 1.4 mm lateral to bregma as described in the study by Hof et al.,⁶¹ and was lowered 2.5 mm deep to lateral ventricle
(stereotaxic coordinates according to Franklin and Paxinos⁵⁹ ) and the attached osmotic minipump was implanted
subcutaneously in the intracapsular region. Topical lidocaine (10
mg/mL), buprenorphine, (0.1 mg/kg) and carprofen (5 mg/kg) s.c. injections
were provided as pre- and postoperative pain management. Osmotic minipumps
were kept in mice for 28 days.

The administration of the IFNγ (50 ng/ml) at a low flow rate of 0.5 μl/h during 7 days in the third ventricle was carried out by continuous infusion with an Alzet® osmotic minipump (model 1007D) and Alzet® Brain Infusion Kit 3 (DURECT Corporation, Cupertino, CA, USA) as previously described (Pérez-Asensio et al., 2013 (link)). The delivery of saline as the vehicle or IFNγ to the third ventricle was achieved by inserting the cannula 1.7 mm depth from the brain surface at −0.1 mm posterior, and 0.6 lateral coordinates (Franklin and Paxinos, 2008 ), after exposing Bregma. The contralateral hemisphere (left) was always considered for histological analysis and some ipsilateral hemispheres were used for biochemical studies.

The chemotherapeutic agent temozolomide (TMZ), Temodal® 2.5 mg/ml (Merck Sharp & Dohme, Sweden) was used for the in vivo experiments. The powder was dissolved in sterile PBS (GIBCO-Life technologies) according to the manufacturer’s protocol and adjusted to the desired concentration. 3-day mini-osmotic pumps Alzet® model 1003D, fill volume 100 μl, pumping rate 1 μl/h (DURECT Corporation) were used for CED-TMZ. TMZ solution concentration was 2.5 mg/ml which corresponds to the dose of 2.4 mg/Kg/day in a mouse weighing 25 g. The total administered dose is 180 μg in 3 days. The mini-osmotic pumps were filled with 100 μl of solutions containing TMZ and coupled to the Alzet® brain infusion kit 3 (DURECT Corporation) with a 2 cm catheter tube according to the manufacturer’s protocol. The pumps assemblies were incubated at 37 °C overnight in sterile PBS (GIBCO-Life technologies) before use.

For intraventricular administration, GPPGPAG was dissolved in artificial cerebrospinal fluid (ACSF; 130 mM NaCl, 24 mM NaHCO₃, 3.5 mM KCl, 1.3 mM Na₂HPO₄, 2 mM CaCl₂, 2 mM MgCl₂ 6H₂O, and 10 mM glucose at pH 7.4) for use. GPPGPAG or ACSF was continuously injected for 28 days into the left ventricle (bregma: −0.2 mm, lateral: 1.0 mm, depth: −3.0 mm) using Alzet Osmotic Pump (1004, DURECT Corporation, CA, United States) and ALZET Brain Infusion Kit 3 (DURECT Corporation). Based on the amount of CSF produced which was 18 μL/h and the pump flow rate of 0.11 μL/h, the peptide in the pump was estimated to be diluted 164 times in the CSF to reach a final concentration 10 nM which was the effective dose in the cultured neuron experiments. Therefore 1.64 µM GPPGPAG was loaded into the pump. Stability of GPPGPAG in saline, mouse plasma and mouse cerebral cortex was confirmed by LC-MS quantification. GPPGPAG was mixed with saline or plasma at 5 μg/ml concentration. Otherwise, GPPGPAG was mixed with cortical lysate at 6.5 μg/g of cortex. After incubation at 37°C for 0, 10, 60, and 180 min, samples were dried up and served to LC-MS. Areas of MS peaks of GPPGPAG were quantified.
For oral administration; GPPGPAG (1, 10 mg/kg/day) or a solvent (saline) was orally administered once a day, totally for 26 days.