PMID: 28840469 PMCID: PMC5808855 DOI: 10.1007/s12035-017-0730-0
Materials and Methods
Animals
We used 7-day-old (PND7) CD-1 mice (Harlan Laboratories, Indianapolis, IN) for all experiments. We chose this age because 1) it is when rodents are most vulnerable to GA-induced developmental neurotoxicity [16] and, 2) it falls before developmental pruning of the IPB begins [15]. Our ketamine anesthesia protocol was as follows: experimental mouse pups were exposed to 6h of ketamine anesthesia and controls were exposed to 6h of mock anesthesia (vehicle) injected I.M. During anesthesia, pups were carefully monitored. After the administration of anesthesia, mice were reunited with their mothers until sacrifice (from P8 until P65). The weaning was done at P21 using the standard protocol. At the desired age mice were divided randomly into two groups: one group for assessing expression of pro- and the mature form of BDNF using the Western blotting technique and the second group for morphometric studies of IPB development. Our randomization process was designed to provide each group with roughly equal representation of pups from each dam.

The experiments were approved by the Animal Use and Care Committees at the University of Colorado the Office of Animal Resources (OLAR), Aurora, Colorado and the Animal Use and Care Committees of the University of Virginia, Charlottesville, Virginia. The experiments were done in accordance with the Public Health Service's Policy on Humane Care and Use of Laboratory Animals. Efforts were made to minimize the number of animals used while being able to conduct meaningful statistical analyses.

Anesthesia administration
To achieve general anesthesia state, we used a ketamine protocol known to cause significant developmental neurotoxicity in PND7 mice whereby mouse pups received a total of four doses of ketamine, at 75 mg/kg, IM every 90 minutes so that the loss of righting reflex and lack of response to tail pinch could be maintained for 6 hours [14]. For control animals, saline was administered using the same volume and administration schedule. During entire anesthesia procedure, animals were kept away from their mother and were housed in standard, tightly closed mice cages, with free air flow through air filters. Animals were kept in close proximity to each other in the cages, so they could preserve, even under anesthesia, important olfactory cues and stimuli, necessary to bust and sustain their metabolic output. During the experiment, we carefully monitored animals and measured environmental temperature in their breeding cages. We established that the ambient temperature in the breeding cage was around 37.0±1°C. Considering that animals at this age are very sensitive to change in body temperature, they were kept under constant ambient temperature maintained with heating pads conveniently set up around the cages. The ambient temperature was assessed at frequent time intervals using the thermometer.

Western blot studies
For BDNF protein quantification, we dissected the hippocampus immediately after the brains were removed from the individual pups using a dissecting scope (10× magnification). Tissue was collected on ice and was snap-frozen in liquid nitrogen immediately. The protein concentration of the lysates was determined with the Total Protein kit using the Bradford method (Cayman Chemical, Ann Arbor, MI). Approximately 10-25 μg of total protein was heat- denatured, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) through 4-20% Tris-glycine polyacrylamide gradient gels (BioRad, Hercules, CA). Separated proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA), blocked at room temperature for 1h in 3% bovine serum albumin (BSA) followed by incubation at 4°C overnight with primary antibody, anti-BDNF (1:500, Alomone Labs, Jerusalem, Israel), and anti-β-actin antibody (1:10000 Sigma Aldrich, USA) as a loading control.

Membranes were incubated for 1h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies - goat anti-rabbit or goat anti-mouse IgG (1:10000, Santa Cruz, Dallas, TX). Three washes with 0.3% Tween-20 in Tris-buffered saline were performed between all steps. Immunoreactivity was detected using enhanced chemiluminescence substrate (Super Signal west Femto; Thermo Scientific, UT). Images were captured using GBOX (Chemi XR 5, Syngene, MD) and gels were analyzed densitometrically with the computerized image analysis program ImageQuant 5.0 (GE Healthcare, Life Sciences, Piscataway, NJ).

Histological Preparation
Mice were deeply anesthetized with 2% isoflurane and immediately perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (at pH 7.4). Brains were extracted and immersed in fresh 4% paraformaldehyde and incubated at 4°C for additional 2-3 days before being embedded in agar. Briefly, brain coronal sections (50μm thickness) were cut using vibratome. Tissue sections were blocked with 1× TBS contains normal goat serum 5%, 1% BSA and 0.1% triton X-100 for 1h at room temperature before incubated with primary antibodies against calbindin (anti-calbindin D-28K antibody, 1:1000; Gene Tex, CA, USA) overnight at 4°C. Free floating sections were then washed three times with TBS, and then incubated with corresponding HRP-conjugated secondary antibodies (1:200) at room temperature for 2h. Tissue sections were mounted on glass slides and air dried. For detection, we used DAB Peroxidase substrate kit (Vector Laboratories) following manufacturer's instructions.

Histological Morphometric Assessment
The morphometric analyses of IBP developmental shortening (from PND10 until PND65 in both control and ketamine-treated mice) were performed using coronal hippocampal slices (50μm) cut from bregma -1.34mm to -2.30mm (as determined using a mouse brain atlas). The images were scanned at 20× magnification using an Aperio Scanscope XT digital slide scanner (Aperio Technologies Inc., Vista, CA) at University of Virginia, Charlottesville, VA and at University of Colorado, Aurora, CO. The hippocampal area in digital sections (.svs file) was extracted at 600μm scale to convert to a .tiff file and was spatially calibrated using 1000 μm2 grid prior to quantifying using Image-Pro Plus 7.0 software (Media Cybernetics, MD). The morphometric approach used to evaluate so called ‘normalized length of IPB’, which takes into consideration individual variability and developmental growth of hippocampus. The IPB length was approximated from the tip of the inferior blade of the dentate granule cell layer (“a”). The length of CA3 was approximated from the tip of the inferior blade to the apex of the curvature of the CA3 pyramidal cell layer (“b”). Normalized IPB length was taken as a ratio between “a” and “b”. The values from serial sections (n=3-6 serial sections per animal from 6-7 animals per age group) were averaged to provide a single data point and is presented as normalized IPB length. The results from different age groups were statistically analyzed by t test and between both groups by Two-way ANOVA using Graph Pad Prism 5.01 software (Graph Pad, CA). The experimenters were blinded to the experimental condition.

Statistical analysis
Comparisons among groups were made using one-way and two-way ANOVAs followed by Tukey's post hoc test. Using the standard version of GraphPad Prism 5.01 software (Media Cybernetics, Inc, Bethesda, MD), we considered p<0.05 to be statistically significant. All data are presented as mean ±SD or mean ±SEM. The sample sizes reported throughout the Results and in the Figure Legends were based on previous experience.