**1. Introduction**

Despite years of research pharmacological neuroprotection remains challenging. Neuronal cell death represents the smallest common denominator occurring within the pathophysiological cascade of acute cerebrovascular or traumatic brain diseases. For example, subarachnoid haemorrhage occurs with an incidence of 9/100,000 per year [1], while traumatic brain injury is one of the leading causes for death and disability in the young with reported incidences of 262/100,000 per year [2]. Considering the dreads of brain damage for patients and their dependants, therapeutic means of effective neuroprotection is possibly one of the most aimed-for means in modern medicine.

Although the pathomechanisms of neuronal cell death are yet to be fully understood, one of the first and most frequently investigated events is the elevation of intracellular calcium concentration. Elevated intracellular calcium represents a central part within the early phenomenon of excitotoxicity as well as all the pathways resulting in or from these raised ion concentrations [3–7]. Various drugs blocking the excitotoxicity-induced calcium influx have failed to show effective neuroprotection in the first place or failed in overcoming the translational roadblock from in vivo or animal in vitro experiments to actual clinical application (e.g., trials of glutamate-receptor antagonists) [8,9]. Beside calcium influx via glutamate receptor overactivation, calcium influx may also occur via voltage-gated calcium channels (VGCC) located within neuronal membranes. Additionally, VGCCs are also expressed by vascular smooth muscle cells responsible for intracellular calcium rise and vasoconstriction when activated.

The L-type calcium channel inhibitor nimodipine is well established as an oral agent for the management of delayed cerebral ischaemia (DCI) for patients after subarachnoid haemorrhage (SAH) [10–15]. Although nimodipine induces vascular smooth muscle relaxation [16] and hence is widely believed to reduce vasospasms, research has shown that the beneficial effects in SAH patients with DCI may not be primarily caused by this effect on larger cerebral vessels [12,17], but possibly by a direct neuroprotective effect. Experimental studies have been rather controversial with some arguing towards direct neuroprotection [18–21] and some against [22–25]. In brief, the inhibition of voltage-gated L-type calcium channels is believed to provide protection against raised intracellular calcium concentration once the cell depolarises in ischaemic conditions. However, it remains unclear whether this effect also counts for the observed beneficial effect of VGCC inhibitors in SAH.

Other possible drug targets regarding cerebral ischaemia are sodium- and (to a lesser extent) calcium-permeable acid-sensing ion channels (ASICs), with the ASIC1a subtype channel as the most prominent amongst them [26]. ASIC1a channels can be responsible for neuronal damage in acidosis and provide a neither voltage-gated nor glutamate-dependent mechanism for calcium influx. Amiloride can block this non-specifically [27–29]. Many experiments have already shown that neuroprotection can be achieved by blocking ASIC1a and thus targeting these channels might be promising [9,30,31]. Some studies point out connections between the affinity of ASIC for H<sup>+</sup> depending and the extracellular concentration of calcium [32,33], as it occurs in brain ischemia [34].

In our experiment, we investigated the possible neuroprotective effect of two clinically used drugs, which both block calcium channels: The L-type calcium channel inhibitor nimodipine and the acid-sensing ion channel (ASIC1a) blocker amiloride.

We hypothesised a possible synergistic neuroprotective effect of the two compounds being used in combination since ASIC channels are known to be influenced by external and internal calcium levels [32,33]. To assess our postulation, we used a well-established in vitro model of oxygen–glucose deprivation (OGD) in organotypic hippocampus slices of mice with propidium iodide (PI) staining for cell death assessment. Nimodipine was investigated for neuroprotective effects alone and in combination with amiloride to observe a possible synergistic interaction.

### **2. Results**

#### *2.1. OGD Damage*

Comparing our control group (*n* = 102) with our OGD group (*n* = 96) a significant difference was seen (*p* < 0.0001, Mann–Whitney test), thus demonstrating significant and robust cell damage by our OGD regimen (Figure 1).

**Figure 1.** Damage intensity of the control group (*n* = 101) vs. oxygen–glucose deprivation (OGD) group (*n* = 96). Mann–Whitney U test was used to determine whether our OGD model caused adequate damage. Slice images shown are example slices for control and OGD groups, both at 72 h with typical means for the grey-scale value. Slice images are red-channel-filtered and contrast-enhanced.

#### *2.2. E*ff*ect of DMSO as Vehicle on OGD-Induced Damage*

To rule out possible effects of our vehicle, slices were incubated receiving dimethyl-sulfoxide (DMSO) without any drug in addition either as controls (Figure 2, left) or undergoing OGD (Figure 2, right) at levels of 0.1, 1.0, or 2.0 Vol.% DMSO. For pure vehicle control slices without OGD, there was a tendency towards a concentration dependent effect; however, with significant cell impairment observed only with the highest concentration of 2.0 Vol.% DMSO (*p* = 0.0001, Kruskal–Wallis with post hoc Dunn's test). Following OGD, there was a slight albeit far not significant tendendy towards a protective effect of DMSO (Figure 2, right: DMSO concentration of 1.0 Vol.%, *p* = 0.2237) compared with the OGD slices without vehicle or with other DMSO concentrations.

**Figure 2.** (**Left**): Slices receiving 2.0 Vol.% dimethyl-sulfoxide (DMSO) alone (*n* = 20) without undergoing OGD showed significantly (*p* = 0.0001) more cell damage. This effect can be observed for 0.1 (*n* = 25) and 1.0 Vol.% (*n* = 12) as well but at a much lower level without statistical significance. (**Right**): No significant effects of DMSO in slices undergoing OGD were observed for 0.1 (*n* = 41), 1.0 (*n* = 19) or 2.0 (*n* = 45) Vol.% DMSO compared to the OGD group without DMSO (*n* = 96). The figure depicts a tendency towards less cell damage at 1.0 Vol.% DMSO.
