PMID: 33437390 PMCID: PMC7791508
Materials and methods
Materials and reagents
Sevoflurane was purchased from Henry Schein, Inc. (Melville, NY, USA), and insulin (Humulin R U-100) from Eli Lily (Indianapolis, IN, USA). Primary antibodies used in this study are listed in Table 1. Peroxidase-conjugated anti-mouse and anti-rabbit IgG were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). The enhanced chemiluminescence (ECL) kit was from Pierce (Rockford, IL, USA). Other chemicals were from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated.
Animals and animal treatments
The breeding pairs of C57BL/6J mice were initially obtained from Jackson Laboratory (New Harbor, ME, USA). The mice were bred in our air-conditioned animal facility and housed with a 12/12 hr light/dark cycle and with ad libitum access to food and water. The housing, breeding, and animal experiments were approved by the Institutional Animal Care and Use Committee of the New York State Institute for Basic Research in Developmental Disabilities and were in accordance with the PHS Policy on Human Care and Use of Laboratory Animals (revised March 15, 2010).

Induction of anesthesia was carried out by placing neonatal mice at the age of postnatal (P) days 7 in an anesthesia chamber (25 cm × 15 cm × 13 cm) filled with 5% sevoflurane in a mixture of O2 and N2 (50%/50%). The sevoflurane concentration was reduced to 2.5% after the induction period of 3 min and was maintained for 6 hr. The air flow rate was 0.9-1.0 L/min during anesthesia. A small petri dish of water was placed into the anesthesia chamber to maintain moisture. At the end of anesthesia, the sevoflurane was turned off, and the mouse pups were kept in the same chamber with O2 and N2 for one hour to allow their recovery from anesthesia. A warm pad was placed in the anesthesia chamber to maintain the body temperature of the neonatal mice to 35-36°C during the procedure. After they awakened from anesthesia, the mouse pups were returned to their parents’ cages. Neonatal mice of control groups were removed from the parents’ cages and left in the experiment room for the same periods of time as the anesthetized group.

Neonatal mice received a total of 7.0 μl insulin (140 mU/mouse) or saline treatment through intranasal delivery 30 min before the beginning of anesthesia. The manual intranasal administration method was modified from that for adult mice reported previously [23]. Briefly, the P7 mouse pups were held in a supine position in hand, and 1.0 μl insulin or saline was delivered into the left nare by using a 2.5-μl Eppendorf pipette. The pups were given 15-20 sec to allow the fluid to be inhaled before repeating the administration six times.

Neonatal mice (P7, both male and female) from various litters were randomly assigned into four groups: (1) control (Con) group, which received intranasal administration of saline instead of insulin and were not anesthetized; (2) sevoflurane (Sevo) group, which received intranasal saline followed by anesthesia with sevoflurane; (3) sevoflurane plus insulin (Sevo+Ins) group, which received both; and (4) control insulin (Ins) group, which received insulin but not sevoflurane. Mouse pups at the age of P7 with body weight less than 3.0 grams were excluded from the study. A total of 10 and 6 mouse pups were included in each group and time point for Western blot analyses and immunohistochemistry, respectively. To eliminate any potential bias caused by litter variations, a similar number of mouse pups from each litter was assigned to each group, and each group included pups from several litters.

Western blot analysis
The mouse pups were sacrificed by decapitation, and the forebrains were removed and homogenized in pre-chilled buffer containing 50 mM Tris-HCl (pH 7.4), 50 mM GlcNAc, 20 µM UDP, 2.0 mM EGTA, 2.0 mM Na3VO4, 50 mM NaF, 20 mM glycerophosphate, 0.5 mM AEBSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 4 µg/ml pepstatin A. Protein concentrations of the homogenates were determined by using the Pierce 660-nm Protein Assay (Rockford, IL, USA). The homogenate samples were resolved by 10% SDS-PAGE and electro-transferred onto Immobilon-P membrane (Millipore, Bedford, MA, USA). The blots were then probed with primary antibodies and developed with the corresponding horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescent kit.

Immunofluorescence
Mouse brains were immersion-fixed in 4% paraformaldehyde at 4°C for 24 hr, followed by dehydration in 30% sucrose at 4°C for 48 hr. Coronal brain sections (40-µm thick) were cut by using a freezing sliding microtome. The sections were stored in antifreeze solution, consisting of glycerol, ethylene glycol, and phosphate-buffered saline (PBS) at the ratio of 3:3:4, at -20°C till immunofluorescence staining at a later time.

Coronal mouse brain sections at the same plate, as evidenced by the identical hippocampal size and structure in the sections, were chosen for immunofluorescence studies. The brain sections were first washed with PBS three times, 15 min each, followed by incubation in 0.5% Triton X-100 in PBS for 20 min. The sections were then washed with PBS for another 10 min and blocked in PBS containing 5% normal goat serum and 0.1% Triton X-100 for 30 min, followed by incubation overnight at 4°C with antibody against cleaved caspase-3. After washing with PBS again, the sections were incubated with Alexa 488-conjugated goat anti-mouse IgG (1:1000) at room temperature for 2 hr. The sections were washed for a last time, mounted, and cover-slipped by using Prolong ® gold anti-fade mountant (Invitrogen, Carlsbad, CA, USA). The immunostaining was analyzed by using a laser scanning confocal microscope (PCM 200, Nikon). The immuno-positive cells were counted manually from three sections per mouse brain and six brains per group.

Statistical analysis
The quantitative data were analyzed by one-way ANOVA plus post hoc test, if applicable, by using Graphpad. All data are presented as means ± SEM, and P < 0.05 was considered statistically significant.