Regional susceptibility to domoic acid in primary astrocyte cells cultured from the brain stem and hippocampus

Abstract

Domoic acid is a marine biotoxin associated with harmful algal blooms and is the causative agent of amnesic shellfish poisoning in marine animals and humans. It is also an excitatory amino acid analog to glutamate and kainic acid which acts through glutamate receptors eliciting a very rapid and potent neurotoxic response. The hippocampus, among other brain regions, has been identified as a specific target site having high sensitivity to DOM toxicity. Histopathology evidence indicates that in addition to neurons, the astrocytes were also injured. Electron microscopy data reported in this study further supports the light microscopy findings. Furthermore, the effect of DOM was confirmed by culturing primary astrocytes from the hippocampus and the brain stem and subsequently exposing them to domoic acid. The RNA was extracted and used for biomarker analysis. The biomarker analysis was done for the early response genes including c-fos, c-jun, c-myc, Hsp-72; specific marker for the astrocytes- GFAP and the glutamate receptors including GluR 2, NMDAR 1, NMDAR 2A and B. Although, the astrocyte-GFAP and c-fos were not affected, c-jun and GluR 2 were down-regulated. The microarray analysis revealed that the chemokines / cytokines, tyrosine kinases (Trk), and apoptotic genes were altered. The chemokines that were up-regulated included - IL1-alpha, IL-Beta, IL-6, the small inducible cytokine, interferon protein 10P-10, CXC chemokine LIX, and IGF binding proteins. The Bax, Bcl-2, Trk A and Trk B were all down-regulated. Interestingly, only the hippocampal astrocytes were affected. Our findings suggest that astrocytes may present a possible target for pharmacological interventions for the prevention and treatment of amnesic shellfish poisoning and for other brain pathologies involving excitotoxicity.

Keywords: Domoic acid; astrocytes; electron microscopy; semi-quantitative analysis; susceptibility.

Figure 1

Micrographs from the CA 3 region of the hippocampus of untreated controls and domoic acid treated rats (5.0 mg/kg/day). A). Control animal with well preserved astrocyte-magnification (Bar =1.1μm); B). Astrocyte from treated animal showing minimal vacuolation (V) of the cytoplasm (Barr=1.1 μm); C). Control animal with well preserved pyramidal neurons of the CA 3 region, (Bar=2.5μm); D). Treated animal showing astrocyte cell necrosis (arrow) with disruption of the cytoplasmic membrane and degenerating processes (Bar = 3.5μm).

Figure 2

Graph showing the MTT assays for the astrocytes from the A-Hippocampus (Hp) and B- Brain Stem (Bs). Five different concentrations of domoic acid (DOM) were used. 10 μM of DOM was used for neurotoxicology experiments. A). The MTT assays for the astrocytes from the hippocampus (Hp) and B. MTT assays for the astrocytes cultured from the brain stem (Bs). O.D.- optical density.

Figure 3

(A) The semi-quantitative analysis of the PCR amplification of different apoptotic markers: Bax-2, Bcl-2, Caspase-3, Bad and GluR 2 derived from RT-PCR performed in triplicate. M: molecular biomarkers; A: control samples from the hippocampus; B: astrocytes treated with 10 μM of DOM from the hippocampus; C: control samples from the brain stem; and D: treated samples from the brain stem. The gels were analysed using the Chemimager and the densities were measured and statistically analysed using the t-test (GraphPad, Prism 4, USA). (B) Shows a graph of the statistical analysis of the marker -Bax-2. C-control, T-astrocytes treated with 10 μM of DOM. Hp- astrocytes isolated from the hippocampus region and Bs- astrocytes isolated from the brain stem. The gels were analysed using the Chemimager and the densities were measured and statistically analysed using the t-test (GraphPad, Prism 4, USA).

Figure 4

(A) The semi-quantitative analysis of the PCR amplification of different cytokine markers: TNF-α-(tumour necrotic factor), IP-10, IL-B, IL-α, and the house keeping gene 18S (3:7) derived from RT-PCR performed in triplicates. M: molecular biomarker; A: control samples from the hippocampus; B: astrocytes treated with 10 μM of DOM from hippocampus; C: control samples from the brain stem; and D: treated samples from the brain stem. All the reactions were done in triplicate. The gels were analysed using the Chemimager and the densities were measured and statistically analysed using the t-test (GraphPad, Prism 4, USA). (B) Shows a graph of the statistical analysis of the marker -IL-B. C- control, T- astrocytes treated with 10 μM of DOM. Hp- astrocytes isolated from the hippocampus region and Bs- astrocytes isolated from the brain stem. The gels were analysed using the Chemimager and the densities were measured and statistically analysed using the t-test (GraphPad, Prism 4, USA).