Thirty-six 2-month-old male guinea pigs weighing 300-350 g were used in the present study. All animals were housed in acrylic box cages and freely accessible to rodent chow and water. Animals were maintained under conditions of constant temperature (25 °C), humidity (50 ± 10%) and lighting cycle (12:12 h). Rat retinal sections from another study involving experimental glaucoma were used for assay control. All experimental procedures used in the present study were approved by the Animal Ethics Committee of Xiangya School of Medicine, Central South University, in accordance with the experimental animal use and welfare requirements set by the Ministry of Health of China as well as the NIH guidelines for use and care of laboratory animals.
Chronic lead exposure and BrdU injection
Eighteen animals were used as Pb exposure groups, and they received 0.2% lead acetate in drinking water for 2, 4 and 6 months, respectively (n = 6/time point). The remaining 18 age-matched control animals received the same type of drinking water without the addition of Pb. In each group, half of the animals were used for morphological study, with the rest used for blood Pb level measurements. BrdU pulse-chasing study was applied to the 4 and 6 months surviving group. Thus, two BrdU injections (50 mg/kg, i.p., 8 h apart, B5002, Sigma-Aldrich, MO, USA) were given after 2 months dosing of Pb or vehicle.
Measurement of blood Pb concentration
After surviving for 2, 4 and 6 months, animals were anesthetized with chloral hydrate (4 mg/kg, i.p.), and 100 μL of blood were collected from the left ventricle of the heart immediately following thoraxitomy. For each analysis, blood Pb concentration was measured by BH2100 graphite furnace atomic absorption spectrophotometry (Bohui, Beijing, China) spectrophotometer in the clinical laboratory of the Third Xiangya Hospital of Central South University . The final concentration of blood Pb was expressed as μg/dL.
Animals were anesthetized with chloral hydrate (4 mg/kg, i.p.) and then perfused with saline followed by 4% paraformaldehyde in 0.01 M phosphate-buffered saline (PBS) at 4 °C. Brains were dissected and post-fixed by immersion in the same fixative at 4 °C for 24 h, and were passed in gradual concentrations of sucrose (10%, 20% and 30%) until they sunk. The brains were cut coronally in a cryostat at 30 μm and 10 μm, with sections collected alternatively in two 12-well culture plates. For immunohistochemistry using the avidin-biotin complex method, a set of sections were treated with 1% H2O2 in 0.01 M PBS for 30 min, and pre-incubated in 5% normal horse serum (Sigma-Aldrich, MO, USA) in PBS with 0.3% Triton X-100 for 1 h at room temperature, followed by incubation with the goat anti-DCX antibody (polyclone, sc-8066, Santa-Cruz, CA, USA, 1:1000) overnight at 4 °C . Sections were further reacted with a biotinylated pan-specific secondary antibody (horse anti-mouse, rabbit and goat IgG, Vector, CA, USA, 1:400) for 2 h., and subsequently with avidin-biotin complex reagents (Vector, CA, USA, 1:400) for 1 h. Immunoreaction product was visualized using 0.003% hydrogen peroxide and 0.05% diaminobenzidine. Three 10-min washes with PBS were used between incubations. Sections were mounted on microslides, allowed to air-dry, and coverslippered. The specificity of this DCX antibody was evaluated previously by multiple groups of investigators .
For double immunofluorescence, sections (10 μm, thaw-mounted onto slides) were blocked with 5% donkey serum (Sigma-Aldrich, MO, USA) for 30 min, and then reacted with goat anti-DCX and one of the following antibodies: rat anti-BrdU (monoclonal, MCA2060, Serotec, CA, USA, 1:2000), mouse anti-NeuN (MAB377, Millipore, MA, USA, 1:1000) and rabbit anti caspase-3 (ab3235, Abcam, MA, USA, 1:1000). Sections subjected to BrdU immunolabeling were treated in 1 X SSC and 50% formamide for 1 h at 65 °C, then in 2 N HCl for 30 min at 37 °C prior to primary antibody incubation. Immunoreaction products were visualized using Alexa Fluor 488 and 594 conjugated secondary antibodies generated in donkey (Jackson ImmunoResearch, PA, USA, 1:200). Fluoro-Jade C stain was carried out according to manufacturer’s instruction (AG325, Millipore, MA, USA). In brief, the sections were air dried at room temperature overnight after DCX reaction of immunofluorescence consummated, rehydrated for 2 min in distilled water and then transferred to the 0.06% potassium permanganate solution for 10 min. The slides were then rinsed for 2 min in distilled water, transferred to the Fluoro-Jade C working solution for 10 min and then rinsed, air dehydrated, xylene cleared and coverslipped with DPX. All sections were counterstained with DAPI (C1002, Beyotime, Beijing, China, 1:10000), washed and counterstained with DAPI before microscopic examination.
Imaging and densitometry
Immunostained sections were examined and imaged on microscope (BX40, Olympus, Tokyo, Japan) using 4×, 10× and/or 40× objective lens. Four cortical areas were arranged for systematic imaging and densitometry, including the frontal cortex, parietal cortex, temporal cortex and occipital cortex. The methodology for cell counting with a Motic pathology picture HD analysis (Motic, Xiamen, China) was described previously . In brief, DCX + cells around layer II were counted in montaged 10× images taken along the cortical surface, and cell density was calculated based on the number of labeled cells underneath unit length of the pial surface (i.e., # of cells/1 mm × 30 μm). During cell counting, DCX + cells with a longer somal diameter <10 μm (ranged from 5-10 μm) were marked with a cross sign “×”, whereas those >10 μm (ranged from 10-20 μm) marked with a small ruler bar (-) of 10 μm in length. The number of the cells was then recorded and relative density calculated after obtaining the corresponding pial length of the measured cortical area.
Data were analyzed using SPSS 10.0 (SPSS, IL, USA). Means of blood Pb levels and DCX + cell densities were calculated for individual and groups of animals. Statistical comparisons were conducted using one-way ANOVA or Student’s t-test, with P ≤ 0.05 being considered statistically significance.