20230808-AI coding-1st round/407 – Istaphanous 2013.txt

PMID: 23460572 DOI: 10.1213/ANE.0b013e318281e988
METHODS
All procedures were approved by the Institutional Animal Care and Use Committee and conformed to the guidelines for ethical treatment of animals. Efforts were made to minimize the number of animals used. Breeding pairs of male CD1 and female C57BL/6 mice were housed in a 12/12-hour light-dark cycle at 22°C with free access to food and water. This hybrid was selected because they exhibit robust anesthesia-induced apoptosis with acceptable survival.3

Isoflurane Treatment
For caspase 3 immunohistochemistry, 7-day-old CD1 and C57BL/6 hybrid littermates (n = 14) were randomly assigned to a 6-hour exposure to 1.5% isoflurane (approximately 0.6 minimum alveolar concentration in these mice) in 30% oxygen (anesthesia, n = 8) or to 6 hours in room air (control, n = 6). Immediately after treatment, animals were euthanized with an overdose of ketamine, acepromazine, and xylazine. Brains were immersion-fixed in 4% paraformaldehyde in phosphate-buffered saline (pH 7.4), postfixed overnight at 4°C, and cryopreserved in 25% sucrose. Brains were snap frozen and 40-μm coronal sections were cut on a cryostat (Thermo Electronics, Kalamazoo, MI). Sections were mounted to charged slides and stored at −80°C until use.

For protein analyses, a separate set of animals (n = 22) was treated and euthanized as described above. The left hemispheres were cut into 4 coronal sections, frozen in liquid nitrogen, and stored at −80°C until use. At a later date, sections of neocortex around bregma −3 mm were separated with a razor blade on dry ice and then homogenized twice in cell lysis buffer solution for approximately 10 seconds each time at 4°C. The homogenate was then centrifuged at 13,000 rpm using a refrigerated microcentrifuge (Fresco centrifuge, Sorvall, Buckinghamshire, UK). The supernatant was removed and used for testing for the specific proteins.

Immunohistochemistry
Slide-mounted brain sections were blocked for 1 hour in normal goat serum, followed by incubation in rabbit antiactivated caspase 3 polyclonal antibodies (1:100, 9661L; Cell Signaling, Danvers, MA) for 18 hours at −4°C, combined with one of the following antibodies: (1) mouse anti–Neuronal Nuclei (NeuN) monoclonal antibodies (NeuN, 1:500, Chemicon, MAB377; Millipore, Billerica, MA), (2) mouse antiglutamate decarboxylase isoform 67 (antiglutamic acid decarboxylase [GAD]67, 1:2000, MAB5406; Chemicon), (3) mouse anti-S100β (1:500, CB1040; Millipore), or (4) chicken antiglial fibrillary acidic protein (GFAP) (1:500, AB5541; Chemicon). Sections were then rinsed in blocker and incubated in Alexa Fluor 488 goat antirabbit secondary antibodies (1:200, A11034, Molecular Probes Inc.; Invitrogen, Carlsbad, CA) for 4 hours at 20°C, combined with either Alexa Fluor 594 goat antimouse (1:250, A11032, Molecular Probes) or Alexa Fluor 594 goat antichicken (1:250, A11042, Molecular Probes) secondary antibodies, as appropriate for the primary antibody species. After immunostaining, sections were dehydrated in an ascending ethanol series, cleared in xylenes, and mounted with Krystalon (EMD, Gibbstown, NJ).

Identification of Cellular Phenotype
To determine the phenotype of degenerating cells, brain sections from anesthesia-treated and control animals, corresponding to Bregma −2.46 to −2.70 (figures 51–53 in the mouse brain atlas by Paxinos and Franklin11) and double-immunostained for caspase 3 and NeuN or triple stained for caspase 3, S100β, and GFAP, were examined by an observer unaware of group assignment. NeuN and S100β stains cannot be combined in the same section, because both secondary antibodies are raised in the same species.

Caspase 3 immunostaining was excited using the 488-nm laser line, and emission wavelengths between 510 and 540 nm were collected to identify caspase-positive cells in layers II/III from retrosplenial cortex to piriform cortex using an SP5 confocal microscope set up on a DMI6000 stand (Leica Microsystems, Wetzlar, Germany) equipped with a 63× objective (NA 1.4). This region was selected because it has repeatedly demonstrated increased numbers of apoptotic cells in immature rodents.2,3 Immunostaining for NeuN, S100β, or GFAP was excited using the 543-nm laser line, and emission wavelengths between 600 and 650 nm were collected. Confocal optical sections were collected through the midpoint of the caspase 3–positive cell (pinhole = 1 Airy unit). Data are expressed as the percentage of caspase 3–immunoreactive cells that were also NeuN- or GFAP-positive, respectively.

Quantification of Apoptotic Cells Using the Optical Dissector Method
Further quantification of the effects of isoflurane exposure on cortical neurons and on GABAergic interneurons was performed as previously described.3,8 Briefly, confocal image stacks of caspase 3/GAD67 double labeling were collected at 1-µm increments through the entire Z-depth of the tissue (40 μm) using 1× optical zoom. Six image stacks were collected from layers II/III of visual cortex, corresponding to figures 51 to 53 in the mouse brain atlas by Paxinos and Franklin,11 from each animal, as follows: for each hemisphere, 3 adjacent confocal image stack frames were collected beginning 750 μm from the midline and moving laterally (Leica SP5, 63× 1.4 NA objective, 1-μm steps). Image stacks, which were 120 × 120 µm in dimension for NeuN and 240 × 240 µm for GAD67, because of the significantly lower cellular density for the latter stain compared with NeuN, were transferred to Neurolucida software (v7.50.4; MBF Bioscience, Williston, VT) for analysis. Using the optical dissector method, an observer unaware of group assignment quantified the respective numbers of NeuN-positive or GAD67-positive cells, the corresponding number of caspase 3–positive cells, and the number of caspase 3/GAD67 or caspase 3/NeuN double-positive cells in each field.12,13 Cells were considered positive if their fluorescence intensity was 2 times or greater than the background intensity. Counts from all 6 respective image stacks were averaged for each animal.

Quantification of GAD67 and GAD65 Expression Using Competitive Enzyme-Linked Immunosorbent Assay
We used a competitive enzyme-linked immunosorbent assay to quantify the expression of the two γ-aminobutyric acid A (GABAA) synthesizing enzymes, GAD67 and GAD65. Rat antiglutamate decarboxylase isoform 67 (Anti GAD67, 1:5000, 671-C; Alpha Diagnostics Inc., San Antonio, TX) and goat antiglutamate decarboxylase isoform 65 (Anti GAD65, 1:32,000, Ab67725; Abcam, Cambridge, MA) antibodies were incubated overnight with the homogenized cortical tissue samples. These bound antibody/antigen complexes were then added to a GAD67 or GAD65 antigen-coated well blocked with 5% bovine serum albumin. Rabbit antirat and rabbit antigoat secondary antibodies were added to GAD67 and GAD65 complexes, respectively. The secondary antibodies were covalently bound to horseradish peroxidase, an enzyme that cleaves the peroxide in the chromophore 3,3′,5,5′-tetramethylbenzidine. This enzyme activation turned on the chromophore and emitted a blue signal, which when treated with 2 M sulfuric acid turned to a yellow color, which was measured at 450 nm using a spectrophotometer (Jenway Genova Life Science Spectrophotometer; Bibby Scientific Limited, Staffordshire, UK). Absorbancy was then compared with a standard curve allowing for the determination of the isoforms’ concentrations.

Statistical Analysis
All sample sizes for group assignment were made a priori. For each animal, the total NeuN-positive cells were counted over the 6 fields. The number of caspase 3/NeuN double-positive cells was defined as an event. The data were normalized to events (caspase 3/NeuN double-positive cells) per 400 NeuN-positive cells counted, the lower end of cells encountered in each animal, to avoid extrapolation. Gross inspection of the raw data revealed that caspase 3 activation in NeuN-positive cells was a rare event with a mean incidence of 2.4% and a maximal incidence of 3.6% in the anesthesia-treated animals. This event rate met the criteria for analysis using the Poisson distribution.

The Poisson mean event rate, λ, and its 95% CI were determined using the MATLAB® function [lambdahat, lambdaci] = poissfit(data, alpha). The vector “data” represented the number of events per 400 counted NeuN cells for each animal in the group of interest and α = (1 − CI). The mean event rates, λ, derived from the MATLAB function, were used to construct probability distribution function curves for the 2 groups (see Appendix).

The raw event counts were used to compute the ratio of events in the anesthesia-treated group to the control group using equations 6 and 7 in Graham et al.14 This method was used as an independent means to assess the mean event ratio and to determine the 95% CI for the event ratio.

All other data are presented as means ± SEM. Group comparisons were made using the Mann-Whitney U test. Statistical calculations were analyzed using Stata/IC 10.1 for Mac OS X (Stata Corp., College Station, TX). Statistical significance was accepted at P < 0.05.