Background

The plant Amburana cearensis (Allemao) A.C. Sm. is used traditionally as tea, decoct and syrup for the treatment of various ailments, including headache, migraine and inflammation [1,2,3]. Previous work by our group has demonstrated neuroprotective effects against glutamate-mediated excitotoxicity in PC12 cells and primary cultures of cerebellar cells of the dichloromethane extract of A. cearensis seeds (EDAC) containing coumarin, ethyl ester, methyl esters and γ -sitosterol [4, 5]. Additionally, Amburoside A, a glucoside from A. cearensis trunk bark, has been shown to exhibit antioxidative and neuroprotective effects against 6-OHDA toxicity in rat mesencephalic cell cultures [6]. Together, these studies support the cytoprotective potential of compounds from A. cearensis in the CNS, but their effects on glial cells in brain tissues were unresolved.

Astrocytes and oligodendrocytes are the main glial cell types in the CNS. Oligodendrocytes are specialised to myelinate axons and are essential for the rapid conduction of neural impulses. Notably, oligodendrocytes are one of the most affected brain cells in ischemia, which results in devastating effects on CNS function [7,8,9,10]. For example, oligodendrocytes in the hippocampus are damaged following transient focal cerebral ischemia [11], and have been shown to be highly susceptible to oxygen–glucose deprivation (OGD), a model of ischemia-hypoxia that involves glutamate excitotoxicity [12,13,14]. Glutamate homeostasis is a primary function of astrocytes and they have been shown to protect oligodendrocytes following hypoxia and reperfusion-induced damage by uptake of excess glutamate [15, 16]. Astrocytes respond to glutamate excitotoxicity in a complex spatio-temporal manner that is highly context specific. Astrocyte pathological changes may be marked by a loss of processes and possibly cell death in acute phases and close to the lesion site [17]. At the other extreme, astrocytes may become hypertrophic, with increased glial fibrillary acidic protein (GFAP) and process branching [18, 14]. Since EDAC is cytoprotective for astrocytes and stimulates glutamate homeostasis, this led us to examine whether EDAC is also cytoprotective for oligodendrocytes in OGD in hippocampal slices from SOX10-EGFP mice, which enables us to visualise oligodendrocytes and their processes (Fig. 7A) [8]. Hippocampal slices were maintained in OGN or OGD conditions and treated with DMSO vehicle in controls or 10 µg/ mL EDAC, which we show above is the most effective concentration for cytoprotection and stimulating glutamate homeostasis. After 60 min OGN or OGD, slices were analysed for the total number of SOX10-EGFP + oligodendrocytes and counts of oligodendrocytes that were process-bearing compared to those that had lost their processes, which is an early indication of oligodendrocyte damage (Fig. 7A, insets). There was no significant difference in the total number of SOX10-EGFP + cells between the different treatment groups, indicating that oligodendrocytes survive acute OGD and that EDAC is non-toxic for these cells (Fig. 7A, B). In contrast, OGD resulted in a marked loss of processes in oligodendrocytes, with a significant decrease in process-bearing cells in all three strata of the CA1 region, compared to OGN, with 50% less in the stratum oriens, and 70% less in the strata pyramidale and radiatum (Fig. 7A, C; p values indicated on the graph). Treatment with EDAC protected oligodendrocytes against this damage in response to OGD (Fig. 7A), and the percentage of process-bearing SOX10-EGFP+ cells was not significantly different than OGN controls throughout the CA1 strata (Fig. 7C); EDAC appeared to most effective in stratum radiatum where the proportion of process-bearing oligodendrocytes in EDAC (54.9 ± 22%) was significantly greater (p < 0.05) than in OGD (26 ± 3.4%) (Fig. 7C). The results indicate that EDAC protect oligodendrocytes from OGD-induced acute cellular damage.

Fig. 7
figure 7

EDAC is cytoprotective for oligodendrocytes in OGD in hippocampal slices. Hippocampal slices from P10-12 SOX10-EGFP mice were incubated in aCSF for 1 h in OGN (normal oxygen + glucose) or OGD (no oxygen or glucose), with EDAC (10 mg/mL), or DMSO vehicle in controls (as indicated). A Representative confocal images of the CA1 region of the hippocampus illustrating the effects of OGD and EDAC on SOX10-EGFP + oligodendrocytes (green) counterstained with Hoechst for cell nuclei (blue). Insets are high magnification z-stacks illustrating oligodendrocyte morphology, illustrating that in OGN oligodendrocytes appear normal with bright somata and numerous fine processes, which support myelin sheaths, whereas in OGD there is a marked loss of processes and somata appear dim, and EDAC protected oligodendrocytes against these effects of OGD. Scale bar 50 µm in main panels and 30 μm in insets. B, C Counts were performed of total SOX10-EGFP + cells (B) and process-bearing SOX10-EGFP + cells (C, expressed as % of total SOX10-EGFP + cells) in constant FOV in the CA1 strata (as indicated), and plotted as mean ± SEM (n = 4 for each treatment group). Data were tested for significance using Kruskal–Wallis test between the control and several samples with post-hoc Dunns Multiple Comparison or standardized with the Mann–Whitney test of variance between two groups; **p < 0.01 indicates comparisons with OGN control, and #p < 0.05 indicates comparison with OGD control

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Discussion

A number of constituents in extracts obtained from seeds of Amburana cearensis have been shown to be protective against glutamate cytotoxicity in PC12 cells [5] and primary cultures of cerebellar cells [4]. Here, we examined the effect of dichloromethane extract of A. cearensis seeds (EDAC) in hippocampal slices. The key finding of this study is that EDAC is cytoprotective against glutamate excitotoxicity and ischemia ex vivo in hippocampus slice cultures by stimulating astrocyte glutamate homeostasis, as summarised in Fig. 8. Glutamate dyshomeostasis is a major factor in stroke and other pathologies, including AD and MS [13], and our results suggest the natural product EDAC could provide a potential adjunct therapy in these diseases.

Fig. 8
figure 8

Proposed model of the cytoprotective effects of EDAC. Extract obtained from Amburana cearensis seeds (EDAC) is protective against astrocytes and oligodendrocytes in oxygen–glucose deprivation (OGD) and glutamate excitotoxicity in CA1 region of hippocampal slices. The protective effect of EDAC is associated with stimulation of astrocyte increased glutamate homeostasis

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Our NMR analysis verified that EDAC is rich in coumarin, which encompasses a class of benzopyrones (1,2-benzopyrones or 2H-1-benzopyran-2-ones) phytochemicals and their derivatives. There are over 1,300 subtypes of coumarin and its derivatives and they are known to have antioxidant, anti-inflammatory, anti-bacterial and anti-viral activity [31, 32]. The most recognized pharmacological activity of derivatives from synthetic coumarins is anticoagulant activity, via its VitK inhibitory actions, hence coumarin derivatives are already in use as treatment for stroke and other vascular diseases [33]. Previously, metabolite profiling by Pereira and colleagues [5] using gas chromatography and mass spectrometry found that EDAC is composed of coumarin, methyl esters, γ-sitosterol and ethyl hexadecanoate. Our NMR analysis shows that coumarin is the main constituent of EDAC, comprising approximately one-third of the extract, and the recognised anti-oxidant properties of coumarin are likely to play a key role in the cytoprotective effects of EDAC demonstrated in the present study. Nonetheless, other components of EDAC may also play an important role, and two of them, methyl hexadecanoate and γ-sitosterol, have relevant pharmacological potential in the CNS. For example, methyl hexadecanoate has been shown to improve neuronal survival in the CA1 region of the hippocampus, as well as recovery of learning and memory, in a rat model of global cerebral ischemia [34], and γ-sitosterol was identified in neuroprotective terpenoid-rich extracts from orange juice [35]. Further studies are required to unravel the relative contributions of EDAC constituents to cytoprotection in the CNS, and although it is likely that coumarin is of major importance in the effects observed in the present study, an exciting possibility is that the multiple compounds in EDAC have combinatorial or even synergistic effects.

Due to its maintenance of cell–cell interactions and cytoarchitecture, hippocampal organotypic cultures exposed to glutamate excess have been widely used as an ex vivo model for bioprospection of neuroprotective compounds [21]. Additionally, the CA1 region of the hippocampus receptors is of clinical importance in neuropathology and is highly susceptible to glutamate due to the high density of glutamatergic neurons and NMDA receptors, which are strongly implicated in glutamate-mediated excitotoxicity [36, 37]. Here, we show that EDAC prevents cell death induced by cytotoxic levels of glutamate in hippocampal slices. We also demonstrate that EDAC stimulates astrocyte glutamate homeostatic mechanisms in the face of cytotoxic levels of glutamate. Astrocytes are the main detoxifying cells for excess glutamate, but they are also excited by activation of their NMDA and AMPA receptors [38], which can result in their damage by excess glutamate [39, 40]. In the present study, elevated glutamate induced an overall reduction in GFAP-EGFP in the hippocampus, indicating acute morphological disruption of astrocytes and cell loss, consistent with other studies [17]. Notably, the cytotoxic effect of glutamate on astrocytes was completely overturned by treatment with EDAC, which increased the morphological complexity and overall GFAP-EGFP fluorescence, and an equivalent effect of EDAC was observed in ischemia. Hence, the primary action of EDAC on astrocytes was to protect against cellular atrophy, which is generally associated with downregulation of glutamate transporters and GS and contributes to neuronal and oligodendrocyte demise [13]. GS is pivotal for depleting high levels of glutamate in astrocytes and its upregulation in astrocytes is an important mechanism for neuroprotection against glutamate-mediated excitotoxicity in vitro [20, 21] and in vivo [41]. Here, in addition to a marked increase of GS, we observed increases in glutamate transporters GLT-1 and GLAST in hippocampal organotypic slices treated with EDAC in excitotoxic glutamate. These transporters are expressed by morphologically distinct GFAP astrocytes [42], and their up regulation has been suggested as a neuroprotective mechanism of action for natural compounds against glutamate excitotoxicity [20, 21]. The increased of GFAP, GLT1, GLAST, and GS observed in the slices treated with EDAC + glutamate was associated with an increased number of GFAP-positive cells, which strongly suggests the astrocytes activation as the potential mechanism of EDAC protective effect.

Additionally, the increase in astrocyte processes induced by EDAC reflects greater coverage in the hippocampus and combined with enhanced glutamate homeostasis is a primary mechanism of EDAC cytoprotective activity in high glutamate, applied experimentally or in response to ischemia. Significantly, we show that EDAC also protects oligodendrocytes against ischemia. Oligodendrocyte viability and myelination are disrupted in ischemic stroke [43]. It has previously been shown that OGD induces the loss of oligodendrocyte processes which is mediated by glutamate excitotoxicity and precedes cellular death [14], and similar morphological changes in response to ischemia have been shown in oligodendrocyte precursors [44]. In the present study, we demonstrate OGD induced morphological atrophy in oligodendrocytes in the hippocampus and EDAC significantly protected oligodendroglial cells against these acute responses to ischemia. However, EDAC was not completely cytoprotective for oligodendrocytes, which is consistent with acute ischemic damage to these cells involving multiple mechanisms besides glutamate, including raised extracellular ATP and activation of P2X7 receptors [13, 45].

Conclusion

In conclusion, EDAC protects the hippocampus from glutamate excitotoxicity and ischemia. An important cytoprotective action of EDAC is to preserve astrocyte and oligodendrocyte integrity by stimulating glutamate homeostatic mechanisms. The results demonstrate EDAC is neuroprotective and support the need for further detailed analyses of the mechanisms underlying its biological effects in complex systems.