Purine receptors are required for DHA-mediated neuroprotection against oxygen and glucose deprivation in hippocampal slices

Abstract

Docosahexaenoic acid (DHA) is important for central nervous system function during pathological states such as ischemia. DHA reduces neuronal injury in experimental brain ischemia; however, the underlying mechanisms are not well understood. In the present study, we investigated the effects of DHA on acute hippocampal slices subjected to experimental ischemia by transient oxygen and glucose deprivation (OGD) and re-oxygenation and the possible involvement of purinergic receptors as the mechanism underlying DHA-mediated neuroprotection. We observed that cellular viability reduction induced by experimental ischemia as well as cell damage and thiobarbituric acid reactive substances (TBARS) production induced by glutamate (10 mM) were prevented by hippocampal slices pretreated with DHA (5 μM). However, glutamate uptake reduction induced by OGD and re-oxygenation was not prevented by DHA. The beneficial effect of DHA against cellular viability reduction induced by OGD and re-oxygenation was blocked with PPADS (3 μM), a nonselective P2X1-5 receptor antagonist as well as with a combination of TNP-APT (100 nM) plus brilliant blue (100 nM), which blocked P2X1, P2X3, P2X2/3, and P2X7 receptors, respectively. Moreover, adenosine receptors blockade with A1 receptor antagonist DPCPX (100 nM) or with A2B receptor antagonist alloxazine (100 nM) inhibited DHA-mediated neuroprotection. The addition of an A2A receptor antagonist ZM241385 (50 nM), or A3 receptor antagonist VUF5574 (1 μM) was ineffective. Taken together, our results indicated that neuroprotective actions of DHA may depend on P2X, A1, and A2B purinergic receptors activation. Our results reinforce the notion that dietary DHA may act as a local purinergic modulator in order to prevent neurodegenerative diseases.

Fig. 1

Neuroprotective properties of DHA against oxygen/glucose deprivation and re-oxygenation (OGD)-induced toxicity in brain hippocampal slices. a Effect of increasing concentrations of DHA on cellular viability of hippocampal slices incubated in control conditions (1–50 μM). b Effect of DHA (1–50 μM) when added to hippocampal slices 15 min before OGD and maintained through OGD. c Effect of DHA (1–50 μM) incubation during the re-oxygenation period. Control group (dashed lines) was considered as 100 %. These results represent means ± SEM of six experiments carried out in triplicates. *P < 0.05 when compared to control group (100 % of cellular viability); ^# P < 0.05 when compared to OGD treated group (one-way ANOVA followed by Tukey’s test)

Fig. 2

Effect of DHA on glutamate-induced excitotoxicity and on glutamate uptake into hippocampal slices subjected to oxygen/glucose deprivation and re-oxygenation (OGD). Effect of DHA (5 μM) on a cellular viability and b TBARS levels in hippocampal slices incubated with 10 mM glutamate (Glu) for 2 h. Control group (dashed lines) was considered as 100 %. These results represent means ± SEM of six experiments carried out in triplicates. Two asterisk indicate means significantly different from control group (P < 0.01) and number sign indicates mean significantly different from glutamate (Glu) group (P < 0.05) (one-way ANOVA followed by Tukey’s test). c Effect of DHA (5 μM) on glutamate uptake impairment induced by oxygen/glucose deprivation and re-oxygenation (OGD). The values are expressed as nmol of L-[³H]glutamate taken up/mg protein/min and represent means ± SEM of five experiments carried out in triplicates. Asterisk indicates means significantly different from control group (P < 0.05) (one-way ANOVA followed by Tukey’s test)

Fig. 3

Blockade of purinergic P2 receptors prevents DHA-mediated protective effect against oxygen/glucose deprivation and re-oxygenation (OGD). The P2 receptors selective antagonists (PPADS, TNP-ATP, or BBG) were incubated for 15 min prior to DHA exposure and maintained throughout OGD in the presence or absence of DHA. Control group (dashed lines) was considered as 100 %. The values are expressed as percentage of control group (considered as 100 %) and represent means ± SEM of 5–7 experiments carried out in triplicates. a Blockade of P2 receptor with the nonselective antagonist PPADS. In this figure, three asterisks (P < 0.001) indicate means significantly different from control group; number sign represents mean different from OGD and PPADS + DHA + OGD group (P < 0.05). b Blockade of P2X₁, P2X₃, and P2X_2/3 receptors with the selective antagonist TNP-ATP. In this figure, two asterisks (P < 0.01) and asterisk (P < 0.05) represent means significantly different from control group; two number signs (P < 0.01) and number sign (P < 0.05) represent means different from OGD. c Blockade of P2X₇ receptors with the selective antagonist BBG. In this figure, three asterisks (P < 0.001) indicate means significantly different from control group and number sign (P < 0.05) represents mean different from OGD group. d Blockade of P2X₁, P2X₃, P2X_2/3, and P2X₇ receptors with the combination of selective antagonists TNP-ATP and BBG. In this figure, three asterisks (P < 0.001) represent means significantly different from control group and two number signs (P < 0.01) represent means significantly different from OGD and TNP + BBG + DHA + OGD groups. Statistical analysis was performed by ANOVA followed by Tukey’s test

Fig. 4

Blockade of adenosine receptors prevents DHA-mediated protective effect against oxygen/glucose deprivation and re-oxygenation (OGD). Adenosine receptors selective antagonists (DPCPX, ZM241385, alloxazine, and VUF5574) were incubated for 15 min prior to DHA exposure and maintained throughout OGD. Control group (dashed lines) was considered as 100 %. The values are expressed as percentage of control and represent means ± SEM of 5–7 experiments carried out in triplicates. a Blockade of A₁ receptor with the selective antagonist DPCPX. In this figure, asterisk (P < 0.05) represents means significantly different from control group and number sign (P < 0.05) represents mean different from OGD and DPCPX + DHA + OGD group. b Blockade of A_2A receptor with the selective antagonist ZM241385. In this figure, asterisk (P < 0.05) represents mean significantly different from control group and number sign (P < 0.05) represents means different from OGD group. c Blockade of A_2B receptors with the selective antagonist alloxazine. In this figure, two asterisks and asterisk represent means significantly different from control group (P < 0.01 and 0.05, respectively); number sign (P < 0.05) represents mean different from OGD and Alox + DHA + OGD group. d Blockade of A3 receptors with VUF5574. In this figure, three asterisks represent means significantly different from control (P < 0.001); two asterisks and asterisks indicate means different from OGD group (P < 0.01 and 0.05, respectively). Statistical analysis was performed by ANOVA followed by Tukey’s test