Ethilon 5 0

Once tumor masses were detected in the brain, mice were randomized in two groups based on flux values, prior to treatment. Anti-NRR1 was purified with PD Columns from GE Heathcare, according to manufactures instructions. To achieve continuous intraventricular CNS administration of anti-NRR1, pumps (Alzet Co., Model 1004, flow rate 0.11 μL hr⁻¹) were loaded with 1 μg μL⁻¹ of antibody or PBS (CTL). Pumps were coupled to brain infusion kits (Alzet Co., Model 8851) and primed for 48 h at 37 °C, 5% CO₂. Osmotic pumps were planted subcutaneously on the dorsum, slightly caudal to the scapulae. Using a stereotaxic apparatus, brain cannulae were inserted intraventricular per predefined coordinates (on the coronal suture, 2 mm right lateral to midline, 4 mm into the lateral ventricle) following removal of periosteal connective tissue and secured with dental cement (Stoelting Co.) and continuous sutures in the skin (Ethilon 5-0, Johnson & Johnson). Pumps containing anti-NRR1 stayed implanted until mice were sacrificed (between 10 and 50 days).

Mice were anesthetized with isoflurane 3% and placed on a heating pad in lateral decubitus. The fur was clipped on the left lateral side of the abdomen, from the thoraco-lumbar junction to the iliac crest. Skin was disinfected with chlorhexidine 5 mg/ml, (Fresenius Kabi, Halden, Norway) and 70% ethanol (Kemetyl, Vestby, Norway). A 5 mm incision was made in the skin and abdominal wall, parallel and ventral to the spine, midway and between the last rib and the iliac crest. The ovarian fat pads were exteriorized and the ovaries were held in position facing the surgeon with the oviduct ventral, using a serrefine clamp. The cell suspensions (10 µL) containing 1×10⁴ SKOV-3^luc+ cells, were inoculated inserting the needle (30 gauge) at the junction between the bursa and the fat pad. Before closing muscles and skin with continuous 5-0 monofilament non-absorbable sutures (Ethilon 5-0, Johnson & Johnson, New Brunswick, NJ, USA) the ovaries were put back to the original position. After the surgery the animals received 0.1 mg/kg buprenorphine hydrochloride (Temgesic, Reckitt Benckiser, Berkshire, UK) and were placed in a warm environment until full recovery.

Four-week-old female C57BL/6 mice were purchased from Nippon Bio-Supp (Japan). All animal experiments were approved by the Animal Research Committee of the University of Tokyo. The chambers made of silicon were synthesized by a 3D printer^{20 (link)}. Briefly, a mold of a cap-shaped silicon chamber with a 5 mm wide flange was made using a 3D printer, and silicon was poured into the chamber by mixing two liquids. The synthesized silicon chamber was sterilized by autoclave.
The method of attachment was based on the method that we have previously reported for a hair reconstruction assay of skin ulceration^{20 (link)}. The inter-scapular area was chosen for the chamber attachment site because of the high stability of the chamber fixation and the effectiveness in maintaining the ulcer surface over time. Under general anesthesia with isoflurane, the chamber attachment site was shaved thoroughly. Circular areas (1 cm in diameter) of the skin and subcutaneous tissue were surgically removed under the panniculus carnosus. The chamber was inserted, and the brim and overlying skin was sutured at 4 positions using 5-0 Ethilon^® (Johnson and Johnson, USA).

Surgical chambers were developed previously (Jeong et al., 2015 (link)) to follow tumor metastasis and custom milled for our purposes. They consist of a top and bottom plate of titanium containing a circular cut out for placement of an 11.7 mm circumference coverslip (Figure 1). These two plates are superimposed in a mirror configuration with a section of skin brought to the lateral border of the plates which is then sutured in place. Surgical supplies were those used for basic microsurgical technique, fine microsurgical instruments (Fine Science Tools, Foster City, CA), 5–0 ethilon and 5–0 stainless steel sutures (Ethicon, Somerville, NJ).

RabPXL01 was prepared in HA as previously described (2 (link)), using a concentration of 20 mg/mL rabPXL01 in 1.5% HA (molecular weight 1.5–8.1 × 10⁶ Da) (3 (link)). After surgery, the paws of the rabbits were randomized either to receive rabPXL01 in HA treatment or to be left untreated (paired control to account for any possible genetic influences on healing). RabPXL01 in HA was administered to the paw randomized to receive treatment (treatment paws were randomized for each animal and blinded to the surgeon up to the moment of product administration, and to all personnel involved in the evaluation) using a BD Neoflon 24GA catheter (Becton, Dickinson Infusion Therapy AB, Helsingborg, Sweden) connected to a 1 mL syringe containing the rabPXL01 in HA formula. The Neoflon catheter was inserted into the opening of the sheath and was still within the tendon sheath as the tendon sheath was closed with a running 6-0 PDS suture (Ethicon). When performing the final sutures, 0.3 mL of the rabPXL01 in HA formula was injected and the tendon sheath fully closed. The same procedure was performed in the untreated paw, but without injecting rabPXL01 in HA formula. The skin was closed with a running suture (5-0 Ethilon, Ethicon).