Dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEAS) protect hippocampal neurons against excitatory amino acid-induced neurotoxicity

Abstract

DHEA, together with DHEAS, is the most abundant steroid in the blood of young adult humans. Levels in humans decline with age and during certain types of illness or stress. We have found that DHEA(S) can prevent or reduce the neurotoxic actions in the hippocampus of the glutamate agonists N-methyl-D-aspartic acid (NMDA) both in vitro and in vivo or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainic acid in vitro. Pre-treatment with DHEA (10-100 nM for 6-8 h) protected primary hippocampal cultures from embryonic day 18 (E18) embryos against NMDA-induced toxicity (0.1, 1, 10, and 50 mM). DHEA added either with NMDA (1 mM) or 1 h later had lesser, but still significant, protective actions. DHEAS also reduced NMDA-induced toxicity (1 mM), although the lowest effective dose of DHEAS (100 nM) was higher than that of DHEA (10 nM). DHEA (100 nM) protected cultured neurons against the neurotoxic actions of either AMPA (25 microM) or kainic acid (1 mM) as well. In vivo, s.c. pellets of DHEA, which resulted in plasma levels that resembled those in young adult humans, protected hippocampal CA1/2 neurons against unilateral infusions of 5 or 10 nmol of NMDA. Because the release of glutamate has been implicated in the neural damage after cerebral ischemia and other neural insults, these results suggest that decreased DHEA levels may contribute significantly to the increased vulnerability of the aging or stressed human brain to such damage.

Figure 1

The effect of DHEA on the survival of cells in hippocampal primary cultures exposed to NMDA. (A) Photomicrographs showing the protective effects of DHEA on NMDA-induced toxicity. (Bar = 10 μm.) NMDA clearly reduced the number of phase-bright, Hoechst, and βT-III-stained cells, although a number of astrocytes remains. In the presence of DHEA, a large number of βT-III-positive cells survived exposure to NMDA. (B) Graph showing that, in the absence of DHEA, there was a decline in the number of βT-III-positive cells (log-transformed data ANOVA: main effect NMDA F(4,5) = 4.70, P < 0.007); this was prevented by the application of 100 nM DHEA (main effect DHEA F(1,5) = 30.34, P < 0.0001). There was a significant interaction between these factors (F = 2.95, P < 0.04). Values are mean ± SEM. Each variable is the mean of 4–6 wells. (C) Graph showing that there were no significant effects of either NMDA (F = 2.17, P > 0.05) or DHEA (F = 3.69, P > 0.05) on the survival of GFAP-stained cells in culture.

Figure 2

The effects of incremental doses of either DHEA (•) or DHEAS (○) on the numbers of neurons in cultures exposed to 1 mM NMDA. The means (± SEM) of 3–4 experiments are shown. ∗, DHEA; †, DHEAS; P < 0.05 compared with baseline (no steroid).

Figure 3

Graph showing the effect of changing the time of application of either DHEA or DHEAS relative to that of NMDA (1 mM) on neuronal survival. DHEA (░⃞) or DHEAS (▪) was added either 6 h before NMDA (Pre), co-administered with NMDA (Co), or applied 60 minutes afterward (Post). ∗, P < 0.05 (Scheffe test) compared with controls (no steroid added); †, univariate comparisons between pretreatment and other steroid treatments.

Figure 4

The effects of DHEA on the lesions induced by NMDA infused into the hippocampus of rats. (A) Photomicrographs of sections through the hippocampus of a sham-operated (CSF infusion) animal, a rat infused with 10 nmol of NMDA and no DHEA, and one implanted with a 100% DHEA pellet and then infused with NMDA [Upper, low power (Bar = 1000 μm); Lower, high power (Bar = 100 μm)]. Sections pass through the center of the lesions at the site of the infusion cannula. Arrowheads show the cannula track, and arrows show healthy pyramidal neurons. NMDA in the absence of DHEA caused a clear, reproducible lesion that was prevented by DHEA pretreatment. (B) Graphs showing the effects of DHEA on two doses of NMDA-induced toxicity in rats after infusions in the hippocampus under halothane anesthesia. CSF infusion results in small, barely detectable cell loss; NMDA without DHEA pretreatment results in a dose-dependent lesion that was prevented by s.c. DHEA implants. ANOVAS were carried out on log-transformed data. Main effects: NMDA F(2,40) = 33.31, P < 0.001; DHEA F(2,40) = 47.63, P < 0.01. Values shown are mean ± SEM. (nd, not done). There were no significant differences between the CSF controls and groups receiving NMDA (either 5 or 10 nmol) and DHEA (F = 1.35, P value not significant) (6–11 rats per group). (C) Graphs showing the effects of s.c. DHEA (either 50 or 100% pellets) on the mean lesion size after NMDA (5 nmol) infusions through preimplanted guide cannula. NMDA in the absence of DHEA induced degeneration of the hippocampal pyramidal neurons, as expected (NMDA main effect F(1,24) = 26.97, P < 0.001). This degeneration was greatly reduced in rats receiving either 50% or 100% DHEA s.c. (main effect DHEA F(2,24) = 11.11, P < 0.001) vs. no DHEA. There was no significant difference between the effects of the two doses of DHEA (post hoc t test; df = 11, t = 0.38, P > 0.05), and lesion size in these two groups was not significantly larger than lesions induced by infusions of CSF into control animals (Scheffe test, P > 0.05) (5–7 rats per group).