Timed-pregnant Long Evans hooded rats (Charles River Laboratories; Portage, MI) were individually housed in polyethylene cages in rooms maintained on a 12:12 hour photoperiod, L:D (0700: 1900), temperature (70E± 2E F), and humidity (50% ± 5%). Twenty-four hours following birth, postnatal day 1 (PND 1), all pups were pooled and new litters consisting of eight pups, a minimum of 7 males, were randomly selected and placed with each dam. Dams and litters were randomly assigned to experimental exposure conditions. Pb-exposure was initiated on PND1 with the addition of Pb acetate (0.2%, Sigma Chemical Co., St. Louis, MO) to the deionized boiled drinking water of the dam and continued until PND 20. Control animals received deionized boiled drinking water. This exposure equated to an average daily dose to the dam of 23 mg/kg body weight. This exposure level was chosen based upon previous studies demonstrating alterations in neuronal arborization, astrocyte structures, and associated developmentally regulated genes in the cortex, hippocampus, and cerebellum (14, 15). This exposure did not produce signs of maternal toxicity; growth and weight gain of pups were within normal range and no differences were seen in regional brain weights. Food and water were available ad libitum throughout the study.
Male pups within each litter were randomly assigned for examination at PND 9, 12, 15, 20, or 25. At the removal of a pup from each litter, a filler pup was added to maintain a litter size of eight throughout the study. At each time point, animals were lightly anesthetized with CO2, decapitated, brain excised, and the cerebellum, hippocampus, and neocortical tissue were dissected and stored at -80EC. All experiments were conducted in compliance with animal protocols approved by the NIEHS/NIH Animal Care and Use Committee.
On PND 15 and 20, one previously randomly selected male pup per litter was used to determine lead levels in the cortex, hippocampus, and cerebellum. At each time, animals were deeply anesthetized with pentobarbitol and perfused with sterile saline via cardiac puncture; the brain excised, brain regions dissected using EDTA rinsed teflon coated forceps. Each standard and sample was analyzed with an atomic absorption spectroscopy graphite furnace (Perkin-Elmer 3030: wavelength 283.3 nm; argon flow, atomization at 2300°C). Each sample was sequentially wet ashed by microwave in nitric acid followed by 30% hydrogen peroxide, filtered through Whatman 541 filters, brought to a final volume of 15 ml with deionized water and a 20ul aliquot was used to determine lead concentration. The concentration of lead in each sample was calculated by the analysis of the calibration standards. The mean value of three absorbance readings for the standards and the mean value of two absorbance readings for each sample were used to calculate the concentration of lead. Based on the addition of external lead standards to the samples at various stages of the assay, overall lead recoveries from the samples averaged 104% (83 - 125%). Method spike recoveries ranged from 90.6 to 105%. (Radian Corp., Research Triangle Park, NC). Trunk blood was collected following decapitation for determination of blood lead levels at PND 20. The lower limit of detection was 2 ug Pb/dl blood.
Ribonuclease Protection Assay (RPA)
Total RNA was isolated from cortical, hippocampal, or cerebellar tissue from individual animals by the Trizol method (Gibco BRL, Frederick, MD). Ten μg aliquots from each sample were subjected to ribonuclease protection assays using commercially available probe sets for brain specific growth factors and apoptosis factors (Pharmingen; San Diego, CA). The first set contained probes for Fas Ligand Receptor, bcl-x, Fas ligand, caspase 1, caspase 3, caspase 2, bax, and bcl-2 (rAPO-1). The second set contained probes for IL-1α, bcl-x, BDNF, caspase 3, caspase 2, bax, and TNFα. Each set contained probes for two housekeeping gene products, ribosomal fraction (L32) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). A 1 μl aliquot of an equimolar pool of plasmid templates was used for the synthesis of 32[P]-labeled cRNA probe set using a T7 RNA polymerase. Briefly, a 10 μg aliquot of total RNA was hybridized overnight at 56°C with 8 μl of hybridization buffer and 4 × 105 cpm of 32[P] labeled probe set. Single-stranded RNA was digested with Ribonuclease (1:100; Promega, Madison, WI) in 10 mM Tris, 300 mM MgCl, and 5 mM EDTA, pH 7.5. The protected fragments were extracted and precipitated and separated by gel electrophoresis on a 5% acrylamide/8M urea sequencing gel. Radioactivity in each fragment was visualized by autoradiography and quantitation of radioactivity in each fragment was conducted by phosphorimaging with the relation volume of each determined using Imagequant (Molecular Dynamics, Sunnyvale, CA) with the addition of a background subtraction. No significant differences as a function of age or exposure were demonstrated for either L32 or GAPDH. Given the potential for biological variance due to the developmental process, each individual RPA gel represented 3 full replicates of control and lead dosed samples over the developmental period. Thus, each brain region was analyzed on a minimum of 2 RPA gels.
The response of each mRNA was calculated relative to corresponding L32 mRNA level for 5-6 animals per group. Litter was the unit of measure and the minimum sample size for each age and treatment was 5. The experimental design for the mRNA levels was a randomized block design, with dams as blocks, and age (5 levels) and treatment (2 levels) as main factors in the design. For each brain region, data for mRNA transcripts were analyzed by an analysis of variance (ANOVA). Given the high degree of variability that can be inherent to developmental ontogenic data, RPA data for each specific mRNA transcript relative to L32 were logarithmically transformed and analyzed by ANOVA to assess main effect of age and treatment and interactions between these factors. Following determinations of significant main effects for each region (p<0.05), multiple comparisons by a Fisher’s LSD procedure were performed to determine significant effects for each transcript in a given region. All data are reported as mean ± SEM. Brain and blood lead levels were analyzed by an ANOVA followed by a Fisher’s LSD. In all cases the level of statistical significance was set at p<0.05.