150 - The Impact of Acute Exposure to High Dose Cadmium on the Postnatal Expression of Vascular Endothelial Barrier Antigen in the Rat Central Nervous System: A Quantitative Immunofluorescent Investigation

Poster Board Number: 150
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Abstract Body : Clinical and experimental investigations have documented the neurotoxic and behavioral impacts of cadmium (Cd). However, the exact pathophysiological mechanisms of Cd neurotoxicity on the human central nervous system (CNS) is not completely understood. A rat-specific putative BBB indicator protein, the endothelial barrier antigen (EBA), has been previously identified and classified by Sternberger et al. In the rat brain and peripheral nerves, the monoclonal antibody reacted selectively with a 23-30 kDa protein triplet, localized to the luminal surfaces of microvascular endothelial cells associated with barrier function. Microvascular endothelial cells of peripheral organs such as liver and kidney, and CNS endothelial cells of the circumventricular or neurosecretory organs such as the area postrema and organum vasculosum of the lamina terminalis (ovalt), known to lack a permeability barrier function, are not labelled by the anti-EBA monoclonal antibody. Further, experimental studies have documented an inverse correlation between BBB dysfunction and EBA immunoreactivity in rat models of CNS pathologies. 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) served as the control group. All rats were sacrificed 3 days post-injection. Brains were immersion-fixed in 10% neutral-buffered formalin; blocks were paraffin-embedded and 8 µm sections were processed for two-step indirect immunofluorescent microscopy.
Sections treated: dewaxed and rehydrated to PBS; blocked with 5% normal donkey serum, 1 hour; mouse anti-EBA (1:1000) overnight at 4 °C; PBS wash; donkey anti-mouse Alexa Fluor 488-conjugated secondary antibody (1:400), 1 hour; PBS wash. Mounted in Vectashield antifade mounting medium with DAPI. Sections were examined and photographed with an Olympus BX63 microscope outfitted with cellSense software package. EBA-positive microvessels were quantitated by measuring the surface areas per field of view at 40x magnification (0.1 mm2). Grand mean surface areas of all fields of view from control and cadmium-treated brain sections were analyzed using the Welch’s paired t-test.
Results: We found a significant 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-exposed rats compared to normal controls. In the control group, the cerebellar granule cell layer showed greater numbers of EBA-positive microvessels than the molecular layer. The cerebellar white matter is less vascularized by EBA-positive microvessels than the molecular and granule cell layers, Also, statistical analysis by Welch’s two-sample t-test to compare the Grand Mean Surface Area (µm2) for all microscopic fields evaluated for the cerebellum and forebrain yielded a p-value of less than 0.05 (p = 0.02), with higher density of BBB-competent microvessels concentrated in the cerebellum compared to the forebrain (t = 2.28; df = 102.96) 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.