For success of the analysis, definition of several parameters is important: probably, changes in calcium channel function and expression occur at different time points reflecting a dynamic process of induction of cerebral vasospasm after aneurysmal SAH. Therefore, definition of the time range, respectively, the time points of ion channel analysis seems to be critical. The time course varies among different animal models of SAH-induced cerebral vasospasm probably due to species-related differences in the clot clearance and structural differences in the vascular and perivascular elements (14–19). In various animal models, delayed cerebral vasospasm could be distinguished from direct acute effects of oxy-hemoglobin on voltage-gated ion channels. Acute effects were suggested to depend on suppression of voltage-gated K⁺ channel currents, while delayed vasospasm involves also expression of voltage-gated Ca²⁺ channels (20). At best, ion channel assessment should reflect changes in channel expression and function over time with direct effects, like changed electrophysiological properties, a putative different behavior toward interaction partners or a putative enhancement or suppression by certain agents, and chronic effect depending on long-lasting changes or on alteration of channel expression.

For assessment of changes in ion channels expression, knowledge about ion channel expression in healthy vessels is crucial. Only few studies addressed ion channel expression in cerebral arteries: Transcripts of the voltage-gated K⁺ channel subunit K_v1.1–K_v1.6, K_v2.1, and K_v2.2 were detected in healthy rat cerebral vessels but only the K_v1.2, K_v1.5, and K_v2.1 subunits were found to be expressed (21, 22). Furthermore, ATP-sensitive K⁺ channels (among other: Kir6.1/SUR2B KATP channels, reviewed in refs. (23, 24)), large conductance Ca²⁺-activated K⁺ channels (BK_Ca) and inwardly rectifying K⁺ channels (Kir) were proven (25, 26). For voltage-gated Ca²⁺ channels, L-type Ca²⁺ channel were traditionally believed to control calcium influx in myocytes of cerebral arteries, but two recent studies showed expression of various isoforms. In basilar myocytes of healthy dogs, Nikitina et al. (27) identified besides the L-type subunits Ca_v1.2 and Ca_v1.3 also the Ca_v2.2 subunit (N-type calcium channel) and the T-type subunits Ca_v3.1 and Ca_v3.3. In rat basilar arteries, the Ca_v1.2, an alternative splice variant of Ca_v1.2 containing exon 9*, Ca_v1.3, Ca_v3.1, and Ca_v3.2 subunit were found to be strongly expressed besides a weak expression of the Ca_v2.3 subunit (28).

In response of SAH, voltage-gated K⁺ channel (K_v) are functionally impaired and expression of the K_v1.5 and K_v2 channels and K_v-mediated currents are markedly reduced (4, 5, 19, 29, 30). While expression of large conductance Ca²⁺-activated K⁺ channels (BK_ca) were unchanged after SAH, expression of Kir channels (Kir 2.1 subunit) was found to be increased which might be a compensatory mechanism (29, 31). Changes of Ca²⁺ channel expression in response to SAH occur later and have only partially been subjected. The recent studies analyzing expression of Ca²⁺ channels during vasospasm only carried out a molecular analysis for the Ca_v1.2 (L-type calcium channel) and the Ca_v2.3 subunit (E-/R-type calcium channels) in cerebral arteries (32, 33). In a rabbit model, cerebral arteries of healthy animals solely express Ca_v1.2 Ca²⁺ channels. Here, Ca²⁺ influx after pressure-induced vasospasm depends on L-type Ca²⁺ channels and spasm could completely reversed by the L-type Ca²⁺ channel blocker diltiazem (32). In contrast after SAH, oxy-hemoglobin induces an upregulation of the Ca_v2.3 subunit and a downregulation of Ca_v1.2 in small arterial vessels and cytoplasmatic Ca²⁺ influx is mediated by E-/R-type Ca²⁺ channels in up to 20%. These “resistant” Ca²⁺ currents were not assessable for L-type Ca²⁺ channel blockers (33). Furthermore, oxy-hemoglobin leads to a reduced sensitivity of L-type Ca²⁺ channels for diltiazem (33). Here, further studies evaluating expression and associated currents of all voltage-gated Ca²⁺ channels in arterial myocytes are needed.

Several factors besides subarachnoid blood have great influence on ion channel expression and function and should therefore be considered. Here, a fussy definition and declaration of the general condition is crucial. Ion channel regulation is highly dynamically regulated during maturation or in response of other external influences (4, 34–39). Also yet not been studied for myocytes, several examples have documented developmental changes of Ca²⁺ channels, for example, in the calyx of Held, where transmission at early developmental stages is mediated by a cooperative action of spatially intermingled N-, P/Q-, and probably also R-type Ca²⁺ channels and N- and R-type Ca²⁺ channel are replaced by P/Q-type calcium channels during maturation (34, 35). Similar changes occur also in aging brains (40) and developmental changes have also been documented for K⁺ channels (41). Here, a study evaluating possible differences in ion channel expression after SAH at different points of maturation might be interesting. Furthermore, expression and function of ion channels are regulated by several other parameters, like in response of several hormones, pain, etc. (42–45). During planning of studies addressing ion channel expression, function or analysis of corresponding currents, those parameters influencing channel expression and function, like age of animals, should be considered and defined.

Standard analysis of ion channel expression and of their mediated currents focused yet on cerebral vessel myocytes. Assessment of ion channel expression and dysfunction might also include other vascular layers. Oxy-hemoglobin-induced apoptotic changes in cultured vascular endothelial cells have been described; also they could not be prevented by standard Ca²⁺ channel blockers (46). Furthermore, evaluation of alterations of ion channel expression and function in the cerebral cortex in response to SAH might be interesting and has yet not been investigated. One might speculate, if subarachnoid bleed and its break-down products impair neurological transmission. For hippocampal CA1 neurons, such an influence on neuronal transmission has not been found for hemoglobin itself but for its degradation products ferrous chloride and hemin which produce an irreversible depression of field excitatory postsynaptic potential (47).

Besides analysis of ion channel expression, distribution and their mediated currents, evaluation of their physiological and pathophysiological role is out of outstanding interest. Use of inhibitors enables specific blockade of particular ion channels in the plasma membrane and a subsequent analysis of their function and comparison to controls. Ion channels inhibitors should carefully be selected due to their inhibition pattern to prevent unwanted side effects. Furthermore, dosage and corresponding ED₅₀ values should be considered. Importantly, nearly all ion channel inhibitors show unspecific side reactions in high concentrations, like the specific blocker of E-/R-type Ca²⁺ channels inhibiting L-type Ca²⁺ channels in high concentration over 200 nM.

Effects of ion channels, respectively, their blockage could be monitored in different ways and depend on the analyzed question. Often, functional analysis focuses on quantification of the contribution of ion channels due to cerebral vasospasm. Degree of vasospasm can be analyzed histopathologically, in either in vivo or in vitro models like isometric tension recordings or in vivo models for example by imaging methods like the traditional X-ray angiography or Doppler-Ultrasound measurement. These chapters were considered to discuss vasospasm assessments (Chap. 47), neuroimaging assessments, X-ray and angiographic assessments and MRI assessments of cerebral vasospasms (Chaps 58–60). But functional analysis includes also other methods, like evaluation of mediated currents or visualization of ion channel influx or efflux. Especially for voltage-gated Ca²⁺ channels, calcium influx can be visualized be specific dyes. Blockade of influx from the extracellular space allows also assessment of cytoplasmatic calcium influx from intracellular stores. Also these methods are presented elsewhere (Chap. 56)

The physiological role of ion channels in cerebral vessels and in particular their pathophysiological role during SAH-induced vasospasm has not completely been elucidated and remains partially unclear. Previous studies of the physiological role of potassium channels revealed that they are involved in the maintenance of the resting membrane potential of vascular myocytes, that they are involved in limiting depolarization of the membrane potential by repolarization through K+ efflux and thus regulate diameter of cerebral arteries (6, 21, 22). K_ATP channels have shown to be involved in vasodilatation, reactive hyperemia in cerebral circulation, and cerebral autoregulation while their inhibition leads to vasoconstrictions (reviewed in ref. (6)). The role of Ca²⁺ channels remains unclear: Navarro-Gonzalez et al. (28) suggested that L-type Ca²⁺ channels were responsible for vasomotion, whereas non-L-type Ca²⁺ channel control the vascular tone. In contrast, an alternative splice variant of Ca_v1.2 containing exon 9* was found to be mainly involved in K⁺-induced arterial constriction (48). After SAH, decrease and dysfunction of K_v channels were mentioned to contribute partially to depolarization and to have a smaller contribution toward maintenance of the membrane potential. Therefore, they may be involved in the genesis and maintenance of cerebral vasospasm, also their role remains controversial (5). While BK_Ca channels are unchanged after SAH, Kir channels might compensatorily be upregulated (29, 31). Voltage-gated K⁺ channels were suggested to trigger acute effects, while expression changes of voltage-gated Ca²⁺ channels occur during delayed vasospasm (20). However, the role of expression changes of voltage-gated Ca²⁺ channels is yet not understood.

Besides these methods, usage of an SAH-knockout model is a possibility for an evaluation of the physiological and pathophysiological role of a particular ion channel. Such an analysis has recently been performed for the role of adenosine A receptors in early ischemic vascular injury after SAH (49). Comparison of wild type and gene-manipulated animals might reveal striking differences and subsequently might give insights in ion channel function. Meanwhile, Ca²⁺ channel-deficient mice have been established for nearly all pore-forming subunits, but to our knowledge never used in SAH models. In part, results from experiments of ion channel deficient animals are hard to interpret. One should consider that loss of a particular ion channel usually leads to partial compensation by others. As an example, such a compensatory upregulation R-type and N-type Ca²⁺ channel occurs at the neuromuscular endplate of Ca_v1.2-deficient mice (36, 37).

Ion channels are embedded in the functional or pathophysiologically disturbed network of the cell. Therefore, assessment has not only to focus on particular ion channels but also on their role in this network. The evaluation is important for understanding the mechanisms following SAH-induced cerebral vasospasm. Here, interaction partner plays an important role. Several regulatory proteins underlie molecular changes following SAH. As one example, protein kinase C (PKC) is mainly involved in the pathogenesis of cerebral vasospasms following SAH with an activation of the PKC-dependent contractile system (50, 51). Hemoglobin changes levels of PKC expression and different isoforms of the protein kinase are translocated from the cytosol to the plasma membrane after SAH (PKC-δ on day 4 and PKC-α on day 7) (52). Therefore, it was considered that PKC-δ is involved in initiation of SAH-induced vasospasm, whereas PKC-α plays a role in its maintenance (53, 54). As a classical target of modulation, PKC phosphorylates the Ca_v1.2 subunit of L-type calcium channels and leads to dual modulation with inhibitory and stimulatory effects in vascular smooth muscle cells. Further, PKC is involved in the Ca²⁺-dependent stimulation of Ca_v2.3. Interestingly, calmodulin, another regulatory protein of voltage-gated Ca²⁺ channels, was considered to decrease within 48 h after SAH in a canine model (55). One might speculate, if imbalance of calmodulin-mediated inactivation and PKC-mediated Ca²⁺-dependent stimulation of R-type calcium channels might lead to self-perpetuating Ca²⁺ influx during vasospasm. Therefore, further studies should also address interaction partners of voltage-gated calcium channels and elucidate their role within the cellular network.