*3.2. Amiloride and Combined L-Type Calcium Channel and ASICs Inhibition*

ASICs have been identified to be accountable for increased levels of intracellular calcium in ischaemic conditions. Under acidic conditions, as it occurs during ischaemia, ASIC1a channels become permeable for calcium and provide a mechanism for calcium influx that is not dependent on depolarisation or excitotoxicity [29]. In our study, the ASIC channel inhibitor amiloride did not show any significant neuroprotective effect. This contrasts with other studies showing a protective effect of ASIC inhibition on brain injury [27]. Positive effects of ASIC inhibition were most prominent in a combined acidosis and OGD model. That may explain the insufficient protection of amiloride in our pure OGD model in which the OGD induced acidosis may be less pronounced. Besides, a combination of L-type calcium channel and ASIC inhibition by nimodipine plus amiloride did also not show a protective effect in our model. These findings point against a significant damaging role of calcium entry in parenchymal cells via VGCCs and ASICS during OGD in models not depending on an intact blood supply. In our study, the amount of damage was investigated 72 h after the insult. It can therefore not be ruled out that an early, only transient, and thus not sustained effect of calcium entry blockade via VGCCs and ASICs may have occurred.

Interestingly, in slices treated with regimes using the possibly harmful concentration of 2.0 Vol.% DMSO, we observed a significant further increase in cell damage only for slices where nimodipine and amiloride were applied in combination. DMSO at this high concentration already caused damage in our control slices without OGD. It is also known from the literature that DMSO higher than 5% may have harmful effects on biological tissue and cells [44,45]. Safe concentrations are described up to 3% in hippocampal neurons [44]. In combination with OGD, 2 Vol.% DMSO did not induce an increase in damage beyond the OGD induced damage. In addition, slices receiving DMSO at this high concentration with additional 100μM amiloride did not significantly differ from the OGD control group. We did not investigate nimodipine alone in a concentration of 2.0 Vol.% DMSO. Thus, we cannot draw conclusions whether this effect is caused by the combination of amiloride and nimodipine in 2.0 Vol.% DMSO, or whether nimodipine combined with high concentration of DMSO alone would have the same effect. The high DMSO concentration of 2 Vol.% may have affected the system in addition to OGD, inducing a setting of higher damage-susceptibility, where calcium channel inhibition (equal whether via nimodipine alone or in combination with amiloride) is even harmful instead of neuroprotective. If we assume a combined mechanism, impairment of regulatory systems responsible for ASIC function by the simultaneous L-type calcium channel inhibition with nimodipine might be the answer. This could either be caused by a reduction in intracellular calcium or by the missing reduction in extracellular calcium. Paukert et al., for example, showed that extracellular calcium influences ASIC function and can be competitively inhibited by calcium ions. Furthermore, they suggest that higher extracellular calcium shifts ASIC sensitivity to more acidic pH levels [33]. Admittedly, we would rather expect less ASIC activity and thus less damage if we follow this argumentation. A further explanation may be based on amiloride's unspecific effects. Amiloride does also interfere with other channels, e.g., Na+/H+- or Na+/Ca2<sup>+</sup>-exchangers and even T-type calcium channels [46]. As aforementioned, calcium extrusion mechanisms appear to be crucial for neuronal integrity. It could be imagined that unspecific blockage mechanisms in combination with L-type inhibition prevent calcium homeostasis mechanisms in the damaging environment of possible toxic effects of DMSO and OGD. Lastly, the concentration of 2.0 Vol.% DMSO is a comparably high choice for the solvent. We recommend avoiding this concentration if feasible.

#### *3.3. Nimodipine—Most Important E*ff*ect on the Vasculature*

Despite years of research, nimodipine is the only drug available that shows an improved functional outcome and positive effects on mortality after SAH. However, the exact underlying mechanism is still unclear [40]. As pointed out in this publication, a direct neuroprotective effect appears to be unlikely. After the CONSCIOUS-1 study [47], the scientific community changed their perspective on vasospasms of large vessels and DCI. The latter is now considered a complex condition with possible pathways in micro-thrombi [48,49], micro-vasculature spasms (mostly described in experiments and possibly a cause for the secondary development of micro-thrombi) [50–52], neuroinflammation [53,54], and cortical spreading depolarization [55]. Indeed, there is evidence from the literature that nimodipine interacts with several of the abovementioned mechanisms rather than directly acting on neurons, finally resulting in neuroprotection and better functional outcome. That explains a lack of neuroprotection in our model using organotypic slice cultures.

#### *3.4. DMSO as Solvent*

DMSO is a commonly used solvent in slice experiments similar to the one presented here. In healthy slices, DMSO applied for 72 h displayed significant damage to the slices at the highest concentration of 2 Vol.% tested. The lower concentrations only induced a concentration-dependent tendency towards slightly enhanced but not relevant damage. In contrast to this effect on slices without OGD, while applying DMSO following OGD, 1.0 Vol.% DMSO alone appears to induce a slight, although not statistically significant, reduction in cell damage. The highest concentration of 2 Vol.% DMSO did not show a protective effect but also did not significantly add to the OGD induced damage. This suggests identical damage pathways for DMSO at this high concentration and OGD. DMSO seems to have a U-shaped concentration-dependent effect on slices after OGD. Neuroprotective effects through DMSO have been described in different experimental designs and models [44,56,57], and thus, DMSO was even suggested as a treatment option for ischaemic brain conditions [58]. Lu and Mattson, e.g., report that DMSO inhibits the glutamate-induced (excitotoxic) calcium influx in hippocampal rat neurons at DMSO levels from 0.5 to 2.0% [44]. Suppression of excitotoxicity may thus also be a possible explanation for our observation of slightly protective effects at 1.0 Vol.%, since neuronal connections stay intact in organotypic hippocampal slices [59]. Another suggested explanation is DMSO's property as a scavenger for free oxygen radicals [58]. However, in our control slices, we observed a dose-depending increase in cell damage with significantly more damage for DMSO at 2.0 Vol.% compared to slices without DMSO or with 0.1 Vol.%. Contrary to the inhibiting effect on excitotoxicity, Galvao et al. have reported apoptosis-inducing effects in retinal cells using DMSO concentrations as low as 1% [60]. Zhang et al. confirmed these observations in experiments with neurons and astrocytes, reporting neuronal alterations at 0.5% DMSO [61]. In addition, even the smallest DMSO concentrations are described to have effects on cell metabolism, and the solvent possibly accumulates in brain slices [62]. From these diverse literature findings, a clear concentration-dependent effect—regardless of protective or harmful—cannot be identified. We consider it an important technical note to this paper that for brain slice experiments addressing mechanisms of neuroprotection, DMSO should be avoided if feasible. If DMSO is utilised, it should only be done so with the utmost caution and proper utilisation of vehicle controls.

#### *3.5. Quality of Slices—Need for Defined Exclusion Criteria*

In our group, we recognised that in many publications using organotypic hippocampus slices the inclusion criteria for slices are unclear or not exactly defined. Organotypic slice cultures are highly sensitive to external influences and can therefore be inhomogeneous with regard to cell damage [63]. We claim that a standardised inclusion pipeline would benefit further research conducted using this method. With the here-suggested pipeline (see Materials and Methods), we are confident that we enhanced the quality of data used for statistical analysis in our experiments.
