Rosa26 lsl tdtomato mice

C57BL/6J male and female mice were group-housed, respectively, until 14 weeks of age in a temperature-controlled room under a 12:12 h light-dark cycle with ad lib access to regular chow diet (5V5R-Advanced Protocol PicoLab Select Rodent 50 IF/6F, PicoLab) and water. Forty nine mice were divided into 16 groups by four factors (1) sex: male or female groups; (2) surgeries: receiving sham or castration (CAST)/ovariectomy (OVX) surgeries at 14 weeks of age (see below); (3) dietary interventions: chronic feeding with chow or a high-fat diet (HFD, D12492i Rodent Diet With 60 kcal% Fat, Research Diets, Inc.), (4) nutritional status: fed or fasted at the time of perfuse (see below).
Penk-IRES2-Cre mice were purchased from Jackson Laboratory (#025112) which express Cre recombinase selectively in preproenkephalin (PENK) neurons (Harris et al., 2019 (link); Heinsbroek et al., 2020 (link)). These mice were bred with Rosa26-LSL-tdTOMATO mice (Madisen et al., 2010 (link)) (Jackson Laboratory, #007905) to generate Penk-IRES2-Cre/Rosa26-LSL-tdTOMATO reporter mice, in which PENK neurons are labeled by tdTOMATO. These mice were used to examine whether PNNs enmeshed PENK-expressing neurons, as described below.
Care of all animals and procedures were approved by the Baylor College of Medicine Institutional Animal Care and Use Committee.

Rbpjk^CNULL lungs were generated by the airway-specific ablation of Rbp-jk using methods that have been described previously (6, 7). Upk3a-creERT2 and Rosa26-LSL-Td-Tomato mice were obtained commercially (Jackson Laboratory). For labeling Upk3a-expressing cells, Upk3a^cre/+; Rosa^Td-tomato/+ heterozygotes (6–8 weeks of age) were injected with Tamoxifen (Sigma, 0.25 mg/gm body weight) on three consecutive days to activate the Cre recombinase. Animals were sacrificed 1 week after the last injection s. Naphthalene injury experiments were performed using established protocols [17] (link). Briefly, FVB/n mice aged 8–12 weeks (Charles River) were injected intraperitoneally with corn oil (vehicle) or Naphthalene dissolved in corn oil (300 mg/Kg) between 10 am-12 pm and sacrificed at 50–52 hr. Mice that express enhanced Green Fluorescent Protein (EGFP) under the control of the promoter of the B1 subunit of the Vacuolar ATPase (B1-EGFP, B6CBAF1/J background) have been described previously [16] (link).

Tg(Alk1-cre)-L1 (L1-Cre) mice on an FVB background (19 (link)) and Tg(Cdh5-cre/ERT2) (VE-cadherin-CreER^T2) mice on a C57BL/6 background (42 (link)) have been previously described. Rosa26-lsl-tdTomato mice on a C57BL/6 background (43 (link)) and Ppargc1α^fl/fl mice on a 129 background (44 (link)) were purchased from Jackson Laboratory. C57BL/6J mice were purchased from CLEA Japan, Inc. Tg(Alk1-cre)-L1 (L1-Cre) and Ppargc1α^fl/fl mice were backcrossed onto C57BL/6J background for 10 generations before producing experimental animals. Male mice were used for the experiments. All personnel involved in data collection and analysis were masked to the treatment allocation.
All mice were housed on a 12-hour light/12-hour dark cycle at 24°C ± 1°C and were provided with standard mouse food and water ad libitum. The mice either were housed under standard normoxic conditions or were continuously housed in a hypoxic chamber (8.5% O₂) (12 (link)) at sea-level atmospheric pressure at 8 weeks of age for up to 1 week or 3 weeks, except for a 5-minute interval once a week when the chamber was cleaned or when a drug was percutaneously administered, and once a day when a drug was administered i.p. The hypoxic gas mixture was continuously delivered from a nitrogen gas generator MNT-0.4SI (KOFLOC) to the chamber at a flow rate of approximately 1 L/min.