PMID: 29249940 PMCID: PMC5715384 DOI: 10.3389/fncel.2017.00373
Materials and Methods
Animals
Male and female C57/BL6 mice were provided by the Third Military Medical University and housed under a 12 h light/dark cycle in a temperature-controlled room with free access to food and water. All the experimental procedures were performed in accordance with the guidelines for laboratory animal care and use and were approved by Third Military Medical University. Each litter was kept together with its mother throughout the experiment, except for the brief intervals of separation required for the daily injections. At least five mice in each group were analyzed for immunofluorescence staining and three mice for western blot.

Drug Treatment
The day of birth was designated postnatal day 0 (P0). On P7, pups received a vehicle or propofol injection intraperitoneally (i.p.) at a subanesthetic dose of 30 or 60 mg/kg (Cattano et al., 2008; Yang B. et al., 2014), according to our previous study (Huang et al., 2016). The same volume of intralipid was administered i.p. as a vehicle control for propofol. All the neonatal mice were grouped randomly with a random number table base for similar body weight.

To explore the morphological changes in the Purkinje cells, Bergmann glia and granule neuron, pups were sacrificed 24 h (P8) after drug treatment. To evaluate whether propofol affected the radial migration of the granule neurons, a single-dose BrdU injection (50 mg/kg i.p., dissolved in saline) was administered to the pups at P8, which was 1 day after injection with propofol or vehicle. Pups were sacrificed 2 days after the BrdU injection (P10).

To maintain a mouse body temperature of 37°C, the pups were primitively anesthetized in their home cage and then transferred to a Thermocare® ICS therapy warmer unit (Thermocare, Incline Village, NV, USA) after being sedated to keep warm in all the experiments. Meanwhile, mouse normal skin color and respiration were observed.

Immunofluorescence
The dissected cerebella were soaked in 4% paraformaldehyde for 24 h. For cryosections, the tissues (P8) were embedded and sectioned in the sagittal plane at 30 μm. The remaining tissues (P8) and tissues collected on P10 were embedded in paraffin and sagittal sections (5 μm thickness) were collected. Cryosections were used for all the immunofluorescence staining for P8 and paraffin sections were used for Hematoxylin-eosin (HE) staining and BrdU immunofluorescence staining. The sections were pretreated with 3% bovine serum albumin (BSA) (37°C, 1 h) to block non-specific binding and 0.3% Triton X-100 (37°C, 30 min) to increase permeability. Then, the sections were incubated with the following primary antibodies in 1% BSA (4°C, 18 h): (1) mouse anti-calbindin D-28K (CB) (1:1000, Swant, Bellinzona, Switzerland); (2) rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) (1:200, Merck Millipore, Darmstadt, Germany); (3) rabbit polyclonal anti-brain lipid binding protein (BLBP) (1:400, Merck Millipore, Darmstadt, Germany); and (4) mouse anti-neuronal nuclei (NeuN) (1:200, Merck Millipore, Darmstadt, Germany). One percent BSA served as the negative control. After three washing steps with phosphate-buffered saline (PBS, pH 7.4), the sections were incubated with the following secondary antibodies in PBS (room temperature (RT), 3 h): (1) Alexa Fluor 488-conjugated anti-mouse IgG (1:400, Jackson ImmunoResearch, West Grove, PA, USA) for CB and NeuN staining; and (2) cy3-conjugated anti-rabbit IgG (1:400, Jackson ImmunoResearch, West Grove, PA, USA) for BLBP and GFAP staining. All the sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO, USA) and then mounted in Vectashield (Vector Laboratories, Burlingame, CA, USA). For BrdU staining, the paraffin sections were deparaffinized in xylene, rehydrated in graded alcohol and processed for antigen retrieval by boiling in citrate buffer (pH 6.0) for 5 min. After incubation in 2 M HCl (37°C, 30 min) and 0.3% Triton X-100 (37°C, 30 min), the sections were exposed to mouse anti-BrdU IgG (37°C for 2 h and then RT for 22 h) (1:600, BD Pharmingen™, Palo Alto, CA, USA) in 1% BSA, followed by the cy3-conjugated anti-mouse IgG secondary antibody (RT, 3 h) (1:400, Jackson ImmunoResearch, West Grove, PA, USA) and DAPI counterstaining. Fluorescence micrographs of the whole parasagittal cerebellar slices were acquired under a Zeiss (Oberkochen, Germany) Axiovert microscope equipped with a Zeiss AxioCam digital color camera connected to the Zeiss Axiovision 3.0 system. The pictures of the Purkinje dendrite and Bergmann fiber contact points were taken with a TCS-SP8 (Leica, Germany) laser scanning confocal microscope connected to a LAS AF Lite system. A z-stack of images, consisting of 6 image planes taken at 1 μm interval was obtained (for a total stack depth of 5 μm). The 5 μm z-stack was taken from the middle of the section to minimize the potential artificial bias.

Western Blot
Cerebella were harvested on P8 and then isolated and homogenized in ice-cold RIPA Lysis buffer (Beyotime, Shanghai, China). After centrifuging the lysates (15,000× g, 5 min at 4°C), the protein concentration was calculated using the Bicinchoninic Acid Kit (Beyotime, Shanghai, China). Then, 50 μg of protein from each sample was separated by 10% SDS-polyacrylamide electrophoresis (120 min 80 V) and then transferred to a nitrocellulose (NC) membrane (90 min at 210 mA). The membranes were incubated in 5% fat-free milk in Tris-buffered saline containing 0.1% Tween 20 (3 h at RT). Membranes were then incubated with the following primary antibodies (4°C, overnight): (1) hamster monoclonal anti-Notch1 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA); (2) rabbit polyclonal anti-Jagged1 (1:500, Santa Cruz Biotechnology, USA); (3) mouse anti-β-actin (1:1000, Cell Cwbio, Beijing, China); and (4) rabbit anti-GAPDH (1:1000, Cell Cwbio, China), followed by the following peroxidase-conjugated secondary antibodies (RT, 2 h): (1) goat anti-mouse IgG (1:1000, Santa Cruz Biotechnology, USA); (2) goat anti-rabbit IgG (1:1000, Santa Cruz Biotechnology, USA); and (3) goat anti-Syrian hamster IgG (1:1000, Santa Cruz Biotechnology, USA). All the bands were exposed to X-ray films (Kodak, Rochester, NY, USA), detected using an enhanced chemiluminescence detection kit (Pierce, Rockford, IL, USA), and analyzed with the Gel-Pro analyzer (Quantity One 4.0; Bio-Rad Laboratories, Hercules, CA, USA). Quantification of Jagged1 and Notch1 were normalized to the internal reference protein β-actin or GAPDH, and then normalized to the control values.

Quantification
The quantification was obtained from regional analysis of lobe IX. All the sections were taken from similar medial-lateral position within the cerebellum, and each count area was chosen from the same field by the middle of the lobe IX. Calbindin-positive cells were analyzed along the long axis for 500 μm in the middle, and the dendrite length of Purkinje cells was evaluated by measuring the primary dendrite from the soma up to the surface of the ML (three Purkinje dendrites were measured per picture). Number of the NeuN-positive granule neurons were analyzed in the center region of the IGL in lobe IX along the long axis (unit area 2000 μm2). The number of BLBP- and GFAP-positive Bergmann fibers was counted from a 100-μm length in the middle area of lobe IX according to our previous methods (Yamada et al., 2000; Eiraku et al., 2005; Yang Y. et al., 2014). To analyze the astrocytes in the deep white matter, we compared the intensity of the GFAP-positive cells and fibers. Both the background integrated optical density (IOD) and surveyed area (same center area of the white matter from each group) were acquired, and the relative optical density (ROD) was calculated by subtracting the background from the IOD of the positive staining (Bao et al., 2017). Contact points between the calbindin-positive Purkinje cells and GFAP-positive Bergmann fibers were defined as where the tips of growing Purkinje cell dendrites were aligned parallel and attached directly to the rod-like domain of Bergmann fibers, entering the base of the overlying EGL, as previously reported (Yamada et al., 2000; Yamada and Watanabe, 2002; Lordkipanidze and Dunaevsky, 2005). Points were counted per image (212.5-μm length) at the interface between the EGL and ML. Only the yellow dots at the end of the dendrites in the direction of the Bergmann fiber were included, while the crossed ones were excluded in case of false positive. For quantifying granule neuron migration, BrdU-labeled cells were counted in a rectangular box (200 μm width and about 100 cells were counted) extending from the pial surface to the end of the IGL; this value was expressed as a percentage of the total number of BrdU-labeled cells. At least five sections were analyzed in each mouse and five mice from each group. All the quantitative statistics were performed blind to the experimental treatment.

Statistical Analysis
All the data were presented as the mean ± standard deviation and analyzed using one-way analysis of variance (ANOVA) followed by Fisher’s protected least- significant difference post hoc test or a least-significant difference multiple-comparison. The differences were statistically significant when the P value was less than 0.05. Statistical analysis was performed using the SPSS 19.0 software (SPSS Inc., Chicago, IL, USA).