Isoflurane

Mice were anesthetized using isoflurane (Hospira, Inc., Lake Forest, IL, USA) with a dose of 4-5% for induction and 1-2% for maintenance (Oh et al., 2010 (link)). Onset of anesthesia was judged by diminished righting reflex and decreased pedal withdrawal. The core temperature was maintained at 34°C with a heating lamp. The stainless-steel needle electrodes (Natus Biomedical, Madison, WI, USA) were cleaned with 70% alcohol between animals. Sural sensory NCV was determined by recording at the dorsum of the foot and antidromically stimulating with supramaximal stimulation at the ankle. NCV was calculated by dividing the distance by the take-off latency of the sensory nerve action potential. Sciatic-tibial motor NCV was determined by recording at the dorsum of the foot and orthodromically stimulating with supramaximal stimulation first at the ankle, then at the sciatic notch. Latencies were measured in each case from the initial onset of the compound muscle action potential. The motor NCV was calculated by subtracting the measured ankle distance from the measured notch distance. The resultant distance was then divided by the difference in the ankle and notch latencies for a final NCV.

Isoflurane (Hospira, Lake Forest, IL, USA) was used to anesthetize mice, which were induced with a 4-5% dose and maintained at 1-2%. The animals’ core temperature was held at 34°C with a heating lamp. Anesthesia onset was judged by impaired righting reflex and decreased pedal withdrawal. Using stainless steel needle electrodes (Natus Medical, Pleasanton, CA, USA), sural sensory NCVs were recorded at the foot dorsum following antidromic supramaximal stimulation at the ankle. Sural sensory NCVs were calculated by dividing the distance by the sensory nerve action potential take-off latency. Sciatic-tibial motor NCVs were recorded at the foot dorsum following orthodromic supramaximal stimulation, first at the ankle then at the sciatic notch. Latencies were measured in each case from the initial onset of the compound muscle action potential. Sciatic-tibial motor NCVs were calculated by subtracting the measured ankle distance from the measured notch distance. The resultant distance was then divided by the difference in ankle and notch latencies for a final motor NCV.

Before in vitro brain slice recording, all male and female mice (six months old and older) were maintained at 20–21°C on a 12/12 h L:D cycle (light onset 6 A.M., zeitgeber time 00:00) in an environmental chamber (Percival Scientific), with free access to food and water for a minimum of six weeks. The mice were deeply anesthetized with isoflurane (Hospira, Inc) during the light phase, and brains were removed and submerged in ice-cold Krebs slicing solution consisting of: 82.7 mM NaCl, 2.4 mM KCl, 0.5 mM CaCl₂, 6.8 mM MgCl₂, 1.4 mM NaH₂PO₄, 23.8 mM NaHCO₃, 23.7 mM dextrose, and 60 mM sucrose, saturated with 95% O₂ and 5% CO₂; pH 7.3–7.4, 308–310 mOsm. Coronal (200–250 µm) hypothalamic brain slices containing the SCN were cut with a vibrating-blade microtome (Leica VT 1000 S, Leica Biosystems GmbH). Slices were incubated in the slicing solution 1–1.5 h at 30°C before electrophysiological recordings were initiated.

Lentiviral constructs were previously described (SBE-NLSmCherry-P2A-CreERT2 PGK-rtTA3)^{6 (link)} or cloned in E.F.’s laboratory (SBE-NLSmCherry PGK-rtTA3, Lepr peak reporter-eGFP PGK-rtTA3, TRE-Lepr-IRES-eGFP, PGK-rtTA3, TRE-VegfaEEF1A1-rtTA3, TRE-STOP EEF1A1-rtTA3). The lentiviral production and in utero injection were performed as previously described^{6 (link),23 (link)}. In brief, pregnant female mice with a doxycycline-inducible HRAS^G12V transgene were anaesthetized with isoflurane (Hospira) when their embryos were at E9.5. Lentivirus (500 nl to 1 µl) was injected into the amniotic sacs of the embryos to selectively transduce a small number of individual epidermal progenitors within the surface monolayer that gives rise to the skin epithelium^{49 (link)}. Postnatal induction of tumorigenesis in clonal patches was achieved by doxycycline administration (2 mg per g) through the feed.

The Ethical Committees of Nihon University and Kindai University Faculty of Pharmacy Committee Guidelines for the Care of Laboratory Animals in accordance with the principles of the Association of Research in Vision and Ophthalmology approved the animal experiments in the current study.
One week before the experiment, 5-week-old male C57BL/KsJ-db/db mice (BKS.Cg-Dock7^m +/+ Lepr^db/J; n = 12) and 13-week-old male db/m (congenic nondiabetic littermates, n = 6) control mice were purchased from Charles River Laboratories Japan, Inc. (Yokohama, Japan). Japanese albino rabbits (n = 10) weighing approximately 2.7 kg were provided by Shimizu Laboratory Supplies Co., Ltd. (Kyoto, Japan). The mice were housed in a temperature-controlled room with a 12-h dark and light cycle and free access to food and water.
The animals were anesthetized with inhaled 2% isoflurane (Pfizer, Tokyo, Japan) using a constant flow rate of 1.5 L/min during the experiment. A heated blanket maintained the rectal temperature from 37 °C to 38 °C. Pupils were dilated with 0.5% tropicamide (Santen Pharmaceutical Co., Osaka, Japan). Blood glucose concentrations were measured from the tail vein (glucose assay kit; Abbott Laboratories, Abbott Park, IL, USA).

The Nihon University Ethical Committee approved the animal experiments in the present study, and all experiments were conducted following the tenets of the Association of Research in Vision and Ophthalmology.
One week before the experiment, 5-week-old male C57BL/KsJ-db/db mice (BKS.Cg-Dock7^m+/+Lepr^db/J; n = 12) and 13-week-old male db/m (non-diabetic congenic littermates, n = 5) control mice were purchased from Charles River Laboratories Japan, Inc. (Yokohama, Japan). The mice were housed in a temperature-controlled room with a 12-h dark and light cycle and had free access to food and water. The light level in our animal house was approximately 150–300 lux.
The mice were anesthetized with inhaled 2% isoflurane (Pfizer, Tokyo, Japan) using a constant flow rate of 1.5 L/min for the duration of the experiment. A heated blanket was used to maintain rectal temperature between 37 and 38°C. Blood glucose concentrations were measured from the tail vein (glucose assay kit; Abbott Laboratories, Abbott Park, IL). The pupils were dilated with 0.5% tropicamide (Santen Pharmaceutical Co., Osaka, Japan).