The glial antioxidant network and neuronal ascorbate: protective yet permissive for H(2)O(2) signaling

Abstract

Increasing evidence implicates reactive oxygen species, particularly hydrogen peroxide (H(2)O(2)), as intracellular and intercellular messengers in the brain. This raises the question of how the antioxidant network in the brain can be sufficiently permissive to allow messages to be conveyed yet, at the same time, provide adequate protection against oxidative damage. Here we present evidence that this is accomplished in part by differential antioxidant compartmentalization between glia and neurons. Based on the rationale that the glia-to-neuron ratio is higher in guinea-pig brain than in rat brain, we examined the neuroprotective role of the glial antioxidant network by comparing the consequences of elevated H(2)O(2) in guinea-pig and rat brain slices. The effects of exogenously applied H(2)O(2) on evoked population spikes in hippocampal slices and on edema formation in forebrain slices were assessed. In contrast to the epileptiform activity observed in rat hippocampal slices after H(2)O(2) exposure, no pathophysiology was seen in guinea-pig hippocampal slices. Similarly, elevated H(2)O(2) caused edema in rat brain slices, whereas this did not occur in guinea-pig brain tissue. The resistance of guinea-pig brain tissue to H(2)O(2) challenge was lost, however, when glutathione (GSH) synthesis was inhibited (by buthionine sulfoximine), GSH peroxidase activity was inhibited (by mercaptosuccinate), or catalase was inhibited (by 3-amino-1,2,4,-triazole). Strikingly, exogenously applied ascorbate, a predominantly neuronal antioxidant, was able to compensate for loss of any other single component of the antioxidant network. Together, these data imply significant roles for glial antioxidants and neuronal ascorbate in the prevention of pathophysiological consequences of the endogenous neuromodulator, H(2)O(2).

Fig. 1. Greater tolerance of guinea-pig than rat brain tissue to H₂O₂ elevation

A) Electrophysiological recordings of the extracellular PS evoked by stimulation of the Schaffer collaterals in CA1 before H₂O₂ exposure (Control), after 15 min superfusion with H₂O₂ (1.5 mM), and after 30 min washout of H₂O₂ (Wash) in rat and guinea hippocampal slices. Recovery of the primary PS was accompanied by mild epileptiform activity, indicated by an additional PS after washout (arrow; n = 9) in rat, but not guinea pig (n = 8). Shown are the averaged PS responses for each species under each condition. B) Tissue water content in rat and guinea-pig forebrain slices incubated for 3 h at 35 °C in ACSF alone (Control) and with H₂O₂ (1.5 mM). The water content of rat brain slices was significantly higher after H₂O₂ challenge than in control slices incubated in ACSF alone (***p < 0.001; n = 22–44). Although guinea-pig brain slices also gained water during incubation (n = 12), final water content was significantly lower than that seen in rat brain slices incubated under identical conditions (p < 0.001 guinea pig vs. rat incubation in ACSF). Moreover, no further water gain occurred in guinea-pig slices incubated with H₂O₂ (n = 19) (p > 0.05), so the final water content remained below that in rat control slices (indicated by dashed line; p < 0.01 vs. rat control).

Figure 2. Ascorbate (Asc) and GSH contents in intact rat and guinea-pig cortex and hippocampus

A) The GSH:Asc ratio in rat cortex is strain independent (Long-Evans, L-E, from Kume Kick et al., 1996; Sprague-Dawley, S-D, from Rice et al., 1994); Asc content is also gender dependent (+++p < 0.001 female vs. male L-E; Kume Kick et al., 1996) (n = 42–43 for each). GSH levels are higher and ascorbate levels lower in guinea-pig cortex (n = 15) than in the cortex of any rat group examined (***p < 0.001 vs. any rat group; data from male and female guinea pigs are pooled (from Rice and Russo-Menna, 1998). B) GSH and ascorbate contents in rat and guinea-pig hippocampus. In rat hippocampus, GSH:Asc ratio is strain independent (Kume Kick et al., 1996; Rice et al., 1994), with lower Asc content in female than male L-E rats (+++p < 0.001 female vs. male; Kume Kick et al., 1996) (rat data n = 35–41). In guinea-pig hippocampus (n = 18), GSH content was higher and ascorbate content lower than in the hippocampus of the rats examined (***p < 0.001; data from male and female guinea pigs are pooled).

Fig. 3. Effect of GSH synthesis inhibition on cortical GSH content in guinea-pig brain slices

Normalized data are given as percent of GSH content after recovery in ACSF alone (% rec; left y-axis), with absolute tissue content indicated (right y-axis). To achieve and maintain GSH synthesis inhibition, BSO (5 mM) was present throughout recovery (2 hours at room temperature) and incubation (3 h at 35 °C). GSH content was unaltered by prolonged incubation whether in ACSF alone or in the presence of H₂O₂ (+H₂O₂) (p > 0.05 versus recovery) (n = 13 per group). However, GSH levels were significantly lower in BSO-treated than control slices for each condition tested (**, P < 0.01; ***, P < 0.001; n = 12–13). Exposure to H₂O₂ caused a significant decrease in GSH content in BSO-treated slices, but not in control slices (+ p < 0.05, BSO incubated vs. BSO + H₂O₂; p > 0.05 control incubated vs. control H₂O₂). Ascorbate content in cortical samples from guinea-pig brain slices after recovery was not altered by BSO; however, ascorbate loss during incubation with and without H₂O₂ was enhanced in BSO-treated slice (see text).

Figure 4. H₂O₂-induced pathophysiology in guinea-pig hippocampal slices after GSH-synthesis inhibition by BSO

Evoked PS in guinea-pig hippocampal slices under control conditions, after 15 min exposure to H₂O₂ (1.5 mM), and after 30 min H₂O₂ washout. GSH synthesis was inhibited by BSO (5 mM), which was present throughout the experiment. Neither the initial evoked PS nor H₂O₂-induced PS suppression were altered by BSO, however, epileptiform activity was seen after H₂O₂ washout (n = 9) (arrow). Inclusion of ascorbate (400 μM; +Asc) during H₂O₂ washout prevented this secondary pathophysiology (lower panel) (n = 8). Shown are the averaged PS responses for each condition; n = 8–9.

Figure 5. H₂O₂-induced pathophysiology in guinea-pig hippocampal slices after GSH-peroxidase inhibition

A) Evoked PS in guinea-pig hippocampal slices under control conditions, after 15 min exposure to H₂O₂ (1.5 mM), and after 30 min H₂O₂ washout in ACSF alone (ACSF), in the presence of MCS (1 mM) to inhibit GSH peroxidase, and in MCS when H₂O₂ was washed out with ACSF plus ascorbate (400 μM; +Asc). Superfusion of MCS for 30 min had no effect on hippocampal PS amplitude. However, in the continued presence of MCS, exposure to H₂O₂ caused a larger suppression of the evoked PS than in slices with an intact antioxidant network exposed to H₂O₂ in ACSF alone (p <0.01 MCS vs. ACSF; n = 7). Recovery of the PS was accompanied by epileptiform activity (arrow); the presence of ascorbate during H₂O₂ washout prevented this secondary pathophysiology (lower panel) (n = 5). Shown are the averaged responses for each condition (n = 5–8). B) Time course of PS amplitude changes during H₂O₂ exposure and washout in guinea-pig hippocampal slices with and without MCS, and with ascorbate present during H₂O₂ washout. PS suppression during H₂O₂ application was more pronounced after GSH peroxidase inhibition by MCS, and recovery was also delayed compared with companion slices exposed to H₂O₂ in ACSF alone (p < 0.01 washout in MCS vs. ACSF; ANOVA). When ascorbate was present during H₂O₂ washout in GSH-peroxidase inhibited slices, the time course of recovery was indistinguishable from that in ACSF alone (n = 5) (p > 0.05 washout in MCS + Asc vs. ACSF; ANOVA).

Figure 6. H₂O₂-induced pathophysiology in guinea-pig hippocampal slices after catalase inhibition

A) Evoked PS in guinea-pig hippocampal slices under control conditions, after 15 min exposure to H₂O₂ (1.5 mM), and after 30 min H₂O₂ washout in ACSF alone (ACSF), in the presence of ATZ (10 mM), in the presence of ATZ to inhibit catalase, and in ATZ when H₂O₂ was washed out with ACSF plus ascorbate (400 μM; +Asc). Superfusion of ATZ (10 mM) for 30 min had no effect on hippocampal PS amplitude; however, in the continued presence of ATZ, exposure to H₂O₂ caused a larger suppression of the evoked PS than during H₂O₂ exposure in slices with an intact antioxidant network exposed to H₂O₂ in ACSF alone (p <0.001 ATZ vs. ACSF; n = 7). Recovery of the PS was accompanied by epileptiform activity (arrow); the presence of ascorbate during H₂O₂ washout prevented this secondary pathophysiology (lower panel) (n = 8). Shown are the averaged PS responses for each condition (n = 7–8). B) Time course of PS-amplitude changes during H₂O₂ exposure and washout in guinea-pig hippocampal slices with and without ATZ, and with ascorbate present during H₂O₂ washout. PS suppression during H₂O₂ application was more pronounced after catalase inhibition by ATZ. Moreover, recovery was less complete (p < 0.05 ATZ vs. ACSF) and delayed compared to that seen during H₂O₂ washout in companion slices exposed to H₂O₂ in ACSF alone (p < 0.01 washout in ATZ vs. ACSF; ANOVA). The presence of ascorbate during H₂O₂ washout in catalase-inhibited slices returned the time course of recovery to that seen in control slices (n = 8) (p > 0.05 washout in ATZ + Asc vs. ACSF; ANOVA).

Figure 7. Roles of GSH, GSH peroxidase, and catalase in prevention of H₂O₂-enhanced edema in guinea-pig slices

A) Water content of guinea-pig forebrain slices after incubation for 3 h at 35 °C with or without H₂O₂ (1.5 mM) in ASCF alone (Control) and after inhibition of GSH synthesis (BSO, 5 mM), GSH peroxidase (MCS, 1 mM), or catalase (ATZ, 10 mM). Water content after incubation with BSO, MCS, or ATZ did not differ from those in ACSF alone (p > 0.05). Incubation with H₂O₂ did not increase water content in ACSF alone; however, edema was enhanced significantly by H₂O₂ in slices treated with either BSO, MCS, or ATZ (***p < 0.001 for H₂O₂ + inhibitor vs. inhibitor alone); the final water contents of antioxidant-compromised slices after H₂O₂ challenge were also significantly greater than in H₂O₂-exposed control slices (+++p < 0.001) (n = 12–21). Dotted line indicates the H₂O₂-enhanced water content of rat brain slices (see Fig. 1B). B) Absolute water gain after H₂O₂ incubation in rat brain slices compared to that in guinea-pig slices with and without antioxidant inhibition. The H₂O₂-enhanced water gain in control guinea-pig was significantly less than in rat or in guinea-pig after inhibition of GSH synthesis (+BSO), GSH peroxidase (+MCS), or catalase (+ATZ) (***p < 0.001 vs. guinea-pig H₂O₂). In guinea-pig brain slices, the H₂O₂-enhanced water gain in the presence of BSO or MCS did not differ from the H₂O₂-enhanced water gain in rat slices. However, after catalase inhibition in guinea-pig slices, the H₂O₂-enhanced water gain was greater than that in rat brain slices (++p < 0.01, +++p < 0.001 vs. rat H₂O₂ alone).