PMID: 17959201 PMCID: PMC2170454 DOI: 10.1016/j.neuropharm.2007.09.005
2. Materials and methods
2.1. Animals
Institute of Animal Care and Use Committee (IACUC) at the University of Pennsylvania approved all experimental procedures and protocols used in this study. All efforts were made to minimize the number of animals used and their suffering. Sprague–Dawley pregnant rats (Charles River Laboratories, Inc Wilmington, MA) were housed in polypropylene cages and the room temperature was maintained at 22 °C, with a 12 h light–dark cycle. Pregnant rats at gestation day 21 (E21) were used for all experiments because it approximately corresponds to mid-gestation in human beings according to the theory of brain growth spurt (Dobbing and Sands, 1979, Jevtovic-Todorovic et al., 2003), and is a common time for most fetal surgeries (18–25 weeks) (Myers et al., 2002).

We have designed the following three related studies: (1) pilot study; (2) neurodegeneration study; (3) finally a behavioral study. A pilot study was first conducted to find the highest concentration of isoflurane not accompanied by significant arterial blood gas (ABG) and mean arterial blood pressure (MABP) changes in the mothers. A neurodegeneration study was used to determine the appearance of apoptosis by detection of caspase-3 and TUNEL (terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling) positive cells in the fetal brain (2 and 18 h post-exposure) or neonatal brain at postnatal day 5 (P5). We have chosen the above time points to detect apoptosis in fetal or newborn brains, based on previously published work (Jevtovic-Todorovic et al., 2003). The behavioral study was performed to investigate the effects of fetal exposure to isoflurane on postnatal memory and learning. The pregnant rats used in each study were not reused in the other two studies. Within each study described above, animals were randomly divided into either isoflurane treatment or sham control groups. Pregnant rats in the isoflurane treatment groups inhaled isoflurane for 6 h, while those in the sham control group only inhaled a carrier gas (30% oxygen, balanced with nitrogen) for 6 h under the same experimental conditions. The distribution of pregnant rats and pups in all three groups is illustrated in Fig. 1.
2.2. Anesthetic exposure
Isoflurane is used clinically at a wide range of concentrations (about 0.2–3%), depending on the presence of other kinds of anesthetics or narcotics and the type and duration of surgery. As isoflurane neurotoxicity is concentration-dependent (Jevtovic-Todorovic et al., 2003, Wei et al., 2005), a primary goal of this study was to investigate if the highest isoflurane concentration used clinically is harmful to the fetal brain. Due to our concern that the physiological side effects of these drugs would contaminate the interpretation, we conducted a pilot study to determine the highest anesthetic concentration we could use without invasive support (tracheal intubation and ventilation) that would not significantly affect arterial blood gas (ABG) and mean arterial blood pressure (MABP) in the mothers, and then used this concentration in the subsequent formal study. We wanted to avoid tracheal intubation, as it could possibly affect the hemodynamics of pregnant rats and the apoptosis in the fetal brains. In addition, this makes it more difficult to set up the sham control groups without anesthesia. In the pilot study, five pregnant rats were initially anesthetized with 2% isoflurane in 30% oxygen via a snout cone for approximately 1 h and the right femoral artery was catheterized for blood sample collection and measurement of MABP by a pressure transducer/amplifier (AD Instruments Inc., Colorado Springs, CO, USA). The rats were recovered for 2 h and then exposed to isoflurane, starting at 1.5% in a humidified carrier gas of 30% oxygen, balance nitrogen for 6 h in a monitored chamber in hood. The pregnant rats breathed spontaneously without intubation or other support while being warmed using a deltaphase isothermal pad (Braintree Scientific Inc, Braintree, MA, USA). The rectal temperature was maintained (Fisher Scientific, Pittsburgh, PA, USA) at 37 ± 0.5 °C. We monitored isoflurane concentration in the chamber using IR absorbance (Ohmeda 5330, Detex-Ohmeda, Louisville, CO, USA). Arterial blood (0.1 ml) from previously placed femoral arterial catheter was collected and ABG determined every 2 h for up to 6 h by an ABG analyzer (Nova Biomedical, Waltham, MA, USA). Blood glucose was simultaneously measured with a glucometer (ACCU-CHECK Advantage, Roche Diagnostics Corporations, Indianapolis, IN, USA). Control rats were exposed only to humidified 30% O2 balanced by N2 (carrier gas for isoflurane in the treatment group) for 6 h in the same chamber under the same experimental conditions as in the treatment group. Because one pregnant rat treated with 1.5% isoflurane showed obvious acidemia (which reversed after termination of anesthesia), we decreased the isoflurane concentration to 1.3%, and subsequently found no significant changes in the ABG or MABP between the treatment group and the sham control group (Table 1). Therefore, 1.3% isoflurane was used in the ensuing neurodegeneration and behavioral studies.
In the behavioral study, pregnant rats were treated with 1.3% isoflurane (n = 8) or carrier gas (sham controls, n = 7) for 6 h. The monitoring was the same as that in the pilot study except that femoral artery catheters were not placed. After the exposures, the animals were returned to their cages and the rat pups were delivered naturally. Four rat pups from each pregnant mother were raised to P28 (Juvenile) and P118 (adult), and then used to determine memory and learning ability with a Morris Water Maze (MWM). Two rat pups from the control group and one from the isoflurane group died unexpectedly, leaving a total of 26 and 31 rat pups in the control and isoflurane treatment groups respectively (Fig. 1).

In the fetal brain apoptosis study, pregnant rats were treated with either 1.3% isoflurane or carrier gas for 6 h. At 2 and 18 h after exposure, the rat pups were delivered by C-section under sodium pentobarbital (100 mg/kg, i.p.) anesthesia. The fetal brains were removed and snap frozen for immunohistochemical analysis. Two fetal brains from each pregnant rat were studied. In addition, newborn brains from the rat pups born to the pregnant rats in the behavioral study group (one pup from each pregnant rat, treatment n = 8, control n = 7) were also obtained at postnatal day 5 and prepared for the apoptosis study at P5 (Fig. 1).

2.3. Measurement of isoflurane concentration in the brain tissues
To confirm that the isoflurane concentration in the fetal brain correlated with the inhaled concentration and brain concentration in the pregnant mothers, we measured the brain isoflurane concentrations in the fetus and the mother simultaneously in one rat. Briefly, after the pregnant rat was exposed to 1.3% isoflurane for 6 h, the brains of both mother and fetuses were removed and the brain tissue was immediately placed into 4 ml of 0.02 M phosphate buffer (with 1 mM halothane as internal standard) and homogenized in a glass homogenizer. The homogenate was centrifuged (30,000 × g at 4 °C for 30 min), the supernatant collected and then loaded onto C18 cartridge that had been conditioned with 2 ml methanol and washed with water, for solid phase extraction. The final sample was eluted with 0.5 ml solution of methanol and 2-propanol (vol:vol 2:1) with 0.1% trifluoroacetic acid. All procedures were performed in the cool room (4 °C). A 250 μl aliquot of each final elute was injected into a high performance liquid chromatography (HPLC) system, equipped with a refractive index monitor, for quantitation.

2.4. Tissue preparation
After treatment with isoflurane or carrier gas alone (control), pregnant rats were anesthetized with sodium pentobarbital intraperitoneally (i.p. 100 mg/kg) at either 2 or 18 h after the end of isoflurane exposure, and the fetuses removed by cesarean section. Likewise, postnatal pups at day 5 (P5) were given the same dose of sodium pentobarbital. All fetuses and pups were then perfused transcardially with ice-cold normal saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were then removed and post-fixed overnight in the same fixative at 4 °C, and cryoprotected in 30% (wt/vol) sucrose in 0.1 M phosphate buffer (pH 7.4) at 4 °C for 24 h. Thereafter, the brains were frozen in isopentane at −20 °C and stored at −80 °C until use. Serial coronal sections (10 μm) were cut in a cryostat (Dolbey–Jamison Optical Company, Inc., Pottstown, PA, USA), mounted on gelatin-coated slides and stored at −80 °C. Coronal brain sections from the same brain corresponding to figure 96 of the rat fetal brain atlas (Paxinos et al., 1990) were chosen for detection of apoptosis by caspase-3 immunohistochemistry and TUNEL staining. In the initial examination of brains sections from the neurodegeneration study, we noticed that apoptosis was most apparent in the hippocampus CA1 region and the retrosplenial cortex, and thus we chose these two brain regions to quantify apoptosis.

2.5. Immunohistochemistry for caspase-3
Caspase-3 positive cells were detected using immunohistochemical methods described previously (Gown and Willingham, 2002). Briefly, brain sections were first incubated in 3% hydrogen peroxide in methanol for 20 min to quench endogenous peroxidase activity. Sections were then incubated with blocking solution containing 10% normal goat serum in 0.1% phosphate buffered saline with 0.1% Tween 20 (PBST) for 1 h at room temperature after washing with 0.1% PBST. The anti-activated caspase-3 primary antibody (1/200, Cell Signaling Technology, Inc Danvers, MA, USA) was then applied in blocking solution and incubated at 4 °C overnight. Tissue sections were biotinylated with goat anti-rabbit antibody (1/200, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) in 0.1% PBST for 40 min, followed by incubation with the avidin–biotinylated peroxidase complex (Vectostain ABC-Kit, Vector Lab, Burlingame, CA, USA) for 40 min. Tissue sections were colorized with diaminobenzidine (DAB, Vector Laboratories, Burlingame, CA, USA) for 8 min and counterstained with modified hematoxylin. Negative control sections were incubated in blocking solution that did not contain primary antibody. Images were acquired and assessed at 200× using IP lab 7.0 software linked to an Olympus IX70 microscope (Olympus Corporation, Japan) equipped with a Cooke SensiCam camera (Cooke Corporation, Romulus, MI, USA). Three brain tissue sections at 10 μm corresponding to the Atlas of the Developing Rat Brain, Figure 96 (Paxinos et al., 1990) were chosen from each animal and analyzed for caspase-3 positive cells in the two brain regions. Two persons blinded to the treatments counted the total number of caspase-3 positive cells in the hippocampal CA1 region and retrosplenial cortex. The areas of entire hippocampal CA1 region and retrosplenial cortex were defined according to the Atlas of the Developing Rat Brain, Figure 96 (Paxinos et al., 1990) and the area measured using IPLab Suite v3.7 imaging processing and analysis software (Biovision Technologies, Exton, PA, http://www.BioVis.com). The density of caspase-3 positive cells in a particular brain region was calculated by dividing the number of caspase-3 positive cells by the area of that brain region.

2.6. TUNEL for DNA fragmentation
Three brain sections (10 μm) adjacent to the sections used for caspase-3 detection were used for TUNEL staining using the DeadEnd™ Colorimetric TUNEL System Kit (Promega Corporation, Madison, WI, USA) according to the manufacturer’s protocol (Gavrieli et al., 1992). Briefly, sections were permeabilized by proteinase K solution (20 μg/ml) for 8 min, incubated in equilibration buffer for 10 min and the terminal deoxynucleotidyl transferase (TdT) and biotinylated nucleotide were added to the section and incubated in a humidified chamber at 37 °C for 1 h. The reaction was then stopped, followed by incubation with horseradish peroxidase-labeled streptavidin, colorization with DAB/ H2O2 and counterstained with modified hematoxylin. For positive-controls, the tissue sections were first treated with DNase I (1000 U/ml, pH 7.6) for 10 min at room temperature to initiate breakdown of DNA. Incubation of sections in reaction buffer without TdT provided negative controls. Images were acquired, and TUNEL quantitation performed as described above for caspase-3.

2.7. Spatial reference memory and learning performance
2.7.1. Morris Water Maze (MWM)
Pregnant rats were allowed to deliver after the isoflurane treatment and 4 pups per litter (2 females and 2 males) were raised. The body weights of the rat pups were recorded at P0, P3, P5, P11, P17 and P28 to determine growth rate. We determined spatial reference memory and learning with the MWM as reported previously with some modification (Jevtovic-Todorovic et al., 2003). A schematic of the experimental paradigm is shown in Fig. 2. A round, fiberglass pool, 150 cm in diameter and 60 cm in height, was filled with water to a height of 1.5 cm above the top of the movable clear 15 cm diameter platform. The pool was located in a room with numerous visual cues (including computers, posters and desks) that remained constant during the studies. Water was kept at 20 °C and opacified with titanium dioxide throughout all training and testing. A video tracking system recorded the swimming motions of animals and the data were analyzed using motion-detection software for the MWM (Actimetrics Software, Evanston, IL, USA). After every trial, each rat was placed in a holding cage, under an infrared heat lamp, before returning to its regular cage.
2.7.2. Cued trials
The cued trials were performed only for postnatal rats at P28 and P29 (28 rats in control group and 31 rats in treatment group) to determine whether any non-cognitive performance impairments (e.g. visual impairments and/or swimming difficulties) were present, which might affect performance on the place or probe trials. A white curtain surrounded the pool to prevent confounding visual cues. All rats received 4 trials per day. On each trial, rats were placed in a fixed position in the swimming pool facing the wall and were allowed to swim to a platform with a rod (cue) 20 cm above water level randomly placed in any of the 4 quadrants of the swimming pool. They were allotted 60 s to find the platform upon which they sat for 30 s before being removed from the pool. If a rat did not find the platform within 60 s, the rat was gently guided to the platform and allowed to remain there for 30 s. The time for each rat to reach the cued platform and the swim speed was recorded and the data at P28/29 were analyzed.

2.7.3. Place trials
After completion of cued trials, we used the same rats to perform the place trials to determine the rat's ability to learn the spatial relationship between distant cues and the escape platform (submerged, no cue rod), which remained in the same location for all place trials. The starting points were random for each rat. The time to reach the platform was recorded for each trial. The less time it took a rat to reach the platform, the better the learning ability. The juvenile rats (P32) received two blocks of trials (two trials per block, 30 s apart, 60 s maximum for each trial and 2 h rest between blocks) each day for 5 days. The adult rats (P115) received only one block of trials each day for 5 days using a new platform location in an effort to increase task difficulty and improve test sensitivity.

2.7.4. Probe trials
Probe trials were conducted after the last place trials for the juveniles (P36) and adults (P119) to evaluate memory retention capabilities. After all rats completed the last place trial on the fifth day, the platform was removed from the water maze and rat was started to swim in the quadrant opposite to one the platform was placed before. The rats were allowed to swim for 60 s during each probe trial and the time the rats spent in each quadrant was recorded. The percentage of the swimming time spent in the target (probe) quadrant where the platform was placed before was calculated. The time spent in the target quadrant compared to other quadrants was an indication of memory retention.

2.7.5. Learning to reach criterion test
After the last probe test for the adult rats, the animals performed the learning to reach criterion test during the next 9 days as described previously (Chen et al., 2000). The experimental procedure was similar to the place trial except that the platform location was changed. For each rat, the platform was moved between nine different locations set up by the computer. Each rat received up to eight trials per day. In order to advance to the next platform location, each rat had to reach the criterion of three successive trials with escape latency of 20 s or less. If a rat reached a criterion in 8 or less trials, a new platform location would be selected the following day. The numbers of learned platforms and the number of trials used to reach the criteria were recorded and compared. The number of platforms learned and the number of trials to reach a criterion indicated the learning ability of the rats.

2.8. Statistical analysis
To reduce variance from different size litters, we averaged the data from all fetal or postnatal rats from the same mother and considered them as a single sample. Results of weight gain of postnatal rat pups, ABG and MABP of pregnant rats and place trials of postnatal rats were analyzed using 2-way ANOVA for repeated measurements. Data for immunohistochemistry, TUNEL and other behavioral studies were analyzed using Student’s t-test for comparison of two groups or by ANOVA followed by Fisher's post hoc multiple comparison tests for those with more than two groups. In all experiments, difference were considered statistically significant at P < 0.05.