Abstract
Background
Numerous studies suggest that dysfunction of the blood-brain barrier (BBB) is an important step in the neurotoxicity of cadmium. A rat-specific BBB marker protein, the endothelial barrier antigen (EBA), has been previously identified and classified by Sternberger and others.
Hypothesis
We hypothesize that acute exposure to cadmium does not significantly affect postnatal EBA expression in young rats.
Methods
Twenty healthy 40-day-old male Sprague-Dawley rats were randomly divided into two groups: intraperitoneal injection with cadmium chloride (4 mg/kg body weight; n=10) in isotonic saline; intraperitoneal injection with isotonic saline (n=10) to serve as the control group. All rats were sacrificed 3 days post-injection. The brains were fixed in 10% formalin, embedded in paraffin and 8 µm thin sections were processed for two-step indirect immunofluorescent microscopy.
Sections treated as follows: dewaxed and rehydrated to water; treated with 5% normal donkey serum x 1 hour; mouse anti-EBA (1:500) overnight at 4 °C; PBS wash; donkey anti-mouse Alexa Fluor 488 secondary antibody (1:400) x 1 hour; PBS wash. Sections were mounted in Vectashield antifade containing DAPI as a nuclear counterstain. Sections were examined and photographed with an Olympus BX63 outfitted with microscope/cellSense software. EBA-immunoreactive microvessels were quantitated by measuring the surface areas per field of view at 40x magnification (0.1 mm2). Welch’s paired t-test was used to compare grand mean areas of all fields from the control and cadmium-treated brain sections.
Results
We detected a reduction in EBA-positive microvessel surface areas in the forebrain (t = 5.86, df = 1789, p-value < 0.001) and cerebellum (t=73.40, df=1337, p < 0.001) of cadmium-treated rats compared to the normal controls.
Conclusion
EBA is involved in the pathogenesis of cadmium neurotoxicity and could serve as a sensitive and measurable indicator for studying the impact of cadmium on nascent BBB microvessels in the developing rat central nervous system.
