Povidone Iodine

Postnatal day 49–63 (P49–P63) male and female C57BL/6 mice were used in all experiments in accordance with institutional guidelines. All surgical procedures were conducted under general anesthesia using continuous isoflurane (induction at 5%, maintenance at 1–2.5% vol/vol). Depth of anesthesia was monitored continuously and adjusted when necessary. After induction of anesthesia, the mice were fitted into a stereotaxic frame, with their heads secured by blunt ear bars and their noses placed into an anesthesia and ventilation system (David Kopf Instruments). Mice were administered 0.05 ml buprenorphine (0.1 mg/ml; Buprenex) subcutaneously before surgery. The surgical incision site was then cleaned three times with 10% povidone iodine and 70% ethanol. Skin incisions were made, followed by craniotomies of 2–3 mm in diameter above the left parietal cortex using a small steel burr (Fine Science Tools) powered by a high speed drill (K.1070; Foredom). Saline (0.9%) was applied onto the skull to reduce heating caused by drilling. Unilateral viral injections were performed by using stereotaxic apparatus (David Kopf Instruments) to guide the placement of beveled glass pipettes (World Precision Instruments) into the left hippocampus (2 mm posterior to bregma, 1.5 mm lateral to midline, and 1.6 mm from the pial surface). Either 2 µl AAV2/5 gfaABC₁D Lck-GCaMP3 (1.2 × 10¹³ gc/ml), 1.5 µl AAV2/5 gfaABC₁D GCaMP3 (1.5 × 10¹³ gc/ml), 1.5 µl AAV2/5 gfaABC₁D Lck-GFP (2.41 × 10¹³ gc/ml), or 1.0 µl AAV2/5 gfaABC₁D tdTomato (2.5 × 10¹³ gc/ml) was injected using a syringe pump (Pump11 PicoPlus Elite; Harvard Apparatus). Glass pipettes were left in place for at least 10 min. Surgical wounds were closed with single external 5–0 nylon sutures. After surgery, animals were allowed to recover overnight in cages placed partially on a low voltage heating pad. Buprenorphine was administered two times per day for up to 2 d after surgery. In addition, trimethoprim sulfamethoxazole (40 and 200 mg, respectively, per 500 ml water) was dispensed in the drinking water for 1 wk. Mice were killed 12–20 d after surgery for imaging (typically 13–15 d). We chose this period because generally it takes ∼2 wk to achieve GECI expression in cells by AAV infection and because it has been suggested that long-term expression (>3 wk after AAV injection) can cause toxicity in neurons (Akerboom et al., 2012 (link)).

Porcine eyes were obtained from the local abattoir and processed within approximately 2 hours of death. The eyelids and adnexal structures were excised, eyes dipped into 5% povidone-iodine ophthalmic solution (betadine 5%; Alcon, Fort Worth, TX, USA) for 30 seconds and then transferred into sterile PBS (Dulbecco's PBS; MP Biomedicals, LLC, Santa Ana, CA, USA). In a tissue culture hood, the eyes were hemisected along the equator followed by removal of the vitreous, lens, ciliary body, iris, retina, and choroid. Pigment shedding was avoided by carefully dissecting the choroid off the sclera directly along the equatorial incision in one piece while leaving the vitreous and lens in place as a barrier toward the anterior chamber. The eyes were irrigated with 10 mL PBS to remove pigment and cellular remnants. The anterior segments were then immediately mounted in perfusion chambers connected to a microinfusion pump (PHD 22/2000; Harvard Apparatus, Holliston, MA, USA) and perfused with serum-free media without phenol red (DMEM with penicillin G sodium/streptomycin sulfate [100 units/mL and 100 μg/mL, respectively]) at a constant flow rate of 3 μL/min. Anterior segments were maintained at 37°C in 5% CO₂. The intraocular pressure was continuously monitored with pressure transducers (physiological pressure transducer, SP844; MEMSCAP, Skoppum, Norway) and recorded using a software system (LabChart; ADInstruments, Colorado Springs, CO, USA). The perfusion system was calibrated using a pressure transducer tester (Veri-Cal; Utah Medical Products, Midvale, UT, USA). Eyes that experienced a contamination, showed erroneous IOP recordings in the negative pressure range, or readings above 30 mm Hg during the first 24 hours were considered a failure. Such negative IOP recordings were observed for instance when debris blocked the transducer lumen while early high IOP was interpreted as relative TM failure or blockage.
Gravity perfused anterior segments (CO_gr) were similarly processed and mounted in perfusion chambers connected to a gravity flow system and perfused with serum free clear DMEM supplemented with penicillin G sodium and streptomycin sulfate (100 units/mL and 100 μg/mL, respectively). The gravity flow system utilized a fluid column at a constant height of 20.4 cm above perfusion chambers to maintain pressures at 15 mm Hg. Anterior segments were maintained at 37°C in 5% CO₂.

The animals were draped in a sterile fashion, and the skin was prepped with povidone-iodine and alcohol. Following exposure of the femoral and cephalic veins, a 7-French (Fr) sheath (Sungwon Medical Co., Ltd.) was inserted, and 200 IU/kg of heparin (JW Pharmaceutical) was injected into the sheath.
A 7-cm-long ablation catheter (VS or CF) for segmental ablation was inserted through the sheath and advanced to the treatment site. Proper positioning of the catheter was confirmed through ultrasonography (US; HD15, Philips) using a specialized transducer (L15-7io, Philips).
The veins of 1 dog to be sacrificed on the day of the procedure were ablated without tumescent injection of normal saline. For the remaining dogs, a sufficient amount of normal saline was injected around the target veins to be subjected to ablation.
When using the VS, an RF generator (VVR Generator, STARmed) connected to the ablation catheter was operated, and ablation was performed at 30 W for 25 seconds. The RF power was fixed at 30 W for all experiments in this study. When using the CF, segmental energy at 120 ℃ was delivered in 20-second cycles.
In cases of cephalic vein, ablation was performed twice at the proximal segment, the catheter was then withdrawn to the next segment (middle) through the graduation mark on the catheter, and then ablation was performed once. Subsequently, the catheter was withdrawn to the distal side of the target vein, and ablation was performed once again. Four ablations were performed in the cephalic vein. For femoral vein, ablations at the proximal and middle segments were performed thrice. However, the procedure was performed by adjusting the number of ablations considering the length of the target vein for each subject.
At the end of the procedure, the 7-Fr sheath was removed and the vein was repaired. The subcutaneous tissue and skin were then sutured. After the procedure, cephradine 100 mg/mL (Panzedin, Hankook Korus Pharm Co., Ltd.) was injected intramuscularly for 6 days.