20230808-AI coding-1st round/520 – Burks 2020.txt

PMID: 32413489 DOI: 10.1016/j.ntt.2020.106890
2. Methods
2.1. Animals
Pregnant Sprague-Dawley rats (Charles Rivers, USA) arrived on gestational day 5. Litters were culled to four males and four females on PND 4. A within-litter treatment design was used to evaluate the effect of Sevo. Four animals, 2 males and 2 females, were selected from each litter. One animal of each sex was randomly assigned to the Sevo group with the other being assigned to the vehicle condition. Three animals per sex were assigned to each treatment / timepoint combination (N = 72). Multiple stains were used on tissue from the same animal. Two animals were removed from the 72 h Sevo group. One animal was removed for failing to meet inclusion criteria for oxygenation (no hypoxic animals were included in the study), and a second animal died between exposure and sacrifice. Rats were housed in a light (12 h/12 h light/dark cycle) and temperature (22 ± 2 °C) controlled vivarium and given free access to food and water (NIH41 laboratory animal diet, Envigo, Madison, WI). All animal procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals. Animal use and procedures were approved by the NCTR Institutional Animal Care and Use Committee (IACUC), which has full NIH-OLAW accreditation. Animals were housed in the NCTR facility in isolator top boxes with wooden chip bedding and ad libitum food and water.

2.2. Study design
On PND 7, rats were exposed to vehicle gas alone (75% oxygen/25% nitrogen) or 2.5% Sevo (in vehicle gas) for 6 h. During anesthesia exposure, each pup was placed in an individual airtight acrylic chamber and the selected gas mixture was delivered at a flow rate of 0.75–1 L/min (Walters et al., 2020). The concentration of Sevo was set using a commercial gas analyzer (Riken, USA). Surface body temperature was collected prior to and every 2 h following the start of Sevo exposure using an infrared thermometer (Micro-Epsilon, Ortenburg, Germany). Heating plates located beneath each chamber were used to maintain body temperatures at baseline levels. In addition, arterial oxygen saturation (SpO2), breath rate, heart rate, and pulse distention were monitored in each pup continuously using a pulse oximeter (Starr Life Sciences Corp, USA). The average SPO2 value was calculated every 30 min (Supplemental Table 1); if an individual rat's SPO2 fell below 85% during any of the 30 min intervals, it was excluded from the study. Upon recovery, the pups were removed from the chambers, rubbed with bedding material from their home cage, and returned to their dams. Control animals were treated the same as the experimental group except they were not exposed to anesthesia and there SPO2 was not monitored.

Pups were sacrificed 2 h (PND 7), 24 h (PND 8), and 72 h (PND 10) after the cessation of Sevo or vehicle gas exposure. Briefly, the rats were deeply anesthetized with pentobarbital and transcardially perfused with 0.9% heparinized saline followed by 10% neutral buffered formalin. Brains were removed and post-fixed in 10% neutral buffered formalin for 24 h, cryoprotected in 20% sucrose until they sank, and subsequently frozen on dry ice and stored at −80 °C. Tissue was cut into 30 μm thick coronal sections using a cryostat, stored in 0.08% sodium azide in PBS for up to two weeks and then transferred to freezing solution (0.02 M phosphate buffer (pH 7.4) containing 25% (v/v) glycerol and 30% (v/v) ethylene glycol) until processed for immunohistochemistry or histology.

2.3. FJC immunolabeling
For FJC labeling, a modified method (Bowyer et al., 2018b; Schmued et al., 2005) was used. Briefly, sections of interest were removed from freezing solution and rinsed three times in 0.1 M phosphate buffer (PB, pH 7.4) for 1 min. Sections were then mounted on gelatin coated slides in 0.005 M PB (pH 7.4) and dried at 50 °C for 2 h. Subsequently, slides were immersed for: 3 min in basic alcohol, 2 min in 70% ETOH, 2 min in Millipore water, 11 min in 0.06% potassium permanganate, 2 min in Millipore water, 10 min in FJC (0.00001% in 0.1% glacial acetic acid), and three 2 min washes in Millipore water. Slides were then dried at 50 °C for 5–10 min, cleared with xylene for 1 min, and cover-slipped with DPX mounting media.

2.4. Mki67 immunolabeling
A buffer of 0.1 M PB (pH 7.4) containing 0.4% Triton X-100 was used in all the steps involving free floating sections agitated on an orbital shaker. Sections containing regions of interest were initially washed in buffer three times (15 min each) to remove excess freezing solution. After a 30 min pre-incubation in 4% normal goat serum, the sections were incubated in 4% serum and chicken polyclonal antibody to Mki67 (1:2000, EnCor Biotechnology, USA) for 1 to 2 h at room temperature followed by 18 to 24 h at 5 °C. Sections were then washed three times for 15 min and incubated in a biotinylated goat anti-chicken antibody (1:350, Invitrogen, USA) for 1 h at room temperature. The sections were then washed three times (15 min per wash) and incubated in Streptavidin TRITC (1:200, Jackson ImmunoResearch, USA) for 1 h. The sections were then washed three times (15 min per wash) and mounted on Superfrost Plus slides (Thermo Fisher Scientific, USA) and dried at room temperature for ≥12 h in the dark. Finally, the slides were cleared in xylene and cover-slipped with DPX mounting medium.

2.5. NeuN immunolabeling
Sections containing the four regions (IG, anterior CPu (CPua), anterior thalamus, and CA1) with the highest per mm2 levels of FJC labeled cells were immunolabeled with an antibody to NeuN in conjunction with DAB visualization. Sections were washed in 0.1 M PB (pH 7.4) for 15 min and then incubated in 0.1 M PB containing 0.05% H2O2 for 10 min to suppress the endogenous peroxidases. From this point on, except for the last step of 3,3′-diaminobenzidine (DAB) processing, incubation and washing solutions consisted of 0.1 M PB containing 0.25% Triton X-100. Sections were then washed three times for 5 min. Following a 20 min pre-incubation in 5% normal goat serum, the sections were incubated in rabbit anti-NeuN (1:1000, Abcam, USA) antibody for 18 to 24 h at room temperature. Sections were then washed three times for 5 min and incubated in a biotinylated goat anti-rabbit antibody (1:300, Thermo Fisher Scientific, USA) for 2 h. The signal was then amplified using the avidin and biotinylated horseradish peroxidase macromolecular complex (Vector Laboratories, USA) and visualized with 0.5 mg/mL of DAB in Tris-HCl buffer. Sections were washed twice for 5 min in Tris-HCl, mounted, and dried on a slide warmer for ≥12 h. Finally, the slides were cleared in xylene and cover-slipped with DPX mounting medium.

2.6. Thionine staining
Thionine staining was performed to verify brain regions. Sections from regions where the highest levels of FJC staining were observed from PND 7, 8 and 10 were mounted from 0.1 M PB (pH 7.4) on Superfrost Plus slides (Thermo Fisher Scientific, USA) and dried at 55 °C for 15 min. They were then immersed in double distilled water for 4 min. Subsequently, the sections were immersed in a solution of 0.1% thionine acetate (Sigma-Aldrich, USA) in double distilled water for 8 min. The sections were then transferred through two washes of water (2 min each) followed by 70% ethanol in water (2 min), 95% ethanol (2 min) and 100% ethanol (2 min). The sections were then transferred to xylene for ≥2 min and cover-slipped as described above.

2.7. Image capturing and analysis
Imaging of brain tissue was conducted using a Nikon Eclipse Ni microscope equipped with digital cameras (Photometrics, USA; Nikon, USA). FJC, Mki67, NeuN, and thionine labeling were quantified in the somatosensory cortex, motor cortex, CPu, thalamus, CA1 region of the hippocampus, septum and amygdala at 10× magnification using NIS Elements AR automated software (Nikon, USA). Brain regions were defined in accordance with brain atlases for adult and neonatal rats (Paxinos and Watson, 2014; Ramachandra and Subramanian, 2011).

2.8. Stereological analysis
The brain regions in which the highest levels of FJC positive neurons were identified with the aid of adult and neonatal atlases as guides. Given the absence of a complete neonatal atlas, these locations were verified using thionine stained sections from the same regions that the FJC sections were taken to determine neurodegeneration from the pups sacrificed at PND 7, 8 and 10 [see Fig. 1]. Subsequent to identifying these regions, unbiased stereological estimates of positively immunolabeled cells/structures were performed from images that were captured with Photometrics (fluorescent) or Nikon (brightfield) digital cameras using NIS elements AR software for analysis.