The aim of the present study was to evaluate the effects of levosimendan on brain metabolism, perfusion and inflammatory response resulting from a defined hypoxia/ischaemia injury and to characterise the time-course during subsequent re-oxygenation. We found that levosimendan did not reduce the initial ischaemic/hypoxic neuronal injury if it was administered after the insult and during resuscitation.
Ischaemia/hypoxia leads to a primary energy failure that is accompanied by the dysfunction of ATP-dependent ion channels (Na+
) and an increased intracellular Ca2+
]. Subsequent glutamate release activates postsynaptic AMPA and NMDA receptors, which induce intracellular sodium and calcium overflow that may be detected prior to anoxic depolarisation [27
]. As a consequence of all this cellular oedema and the activation of different proteases, lipases, endonucleases and the generation of free radicals is induced. Importantly, the restoration of blood flow and oxygenation will restore oxidative metabolism within 30 to 60 min, which suggests a therapeutic window during which the neurotoxic cascade can be inhibited. Most neurons will die as a consequence of secondary energy failure, which occurs 6 to 15 h after injury [4
]. The magnitude of primary cell death is dependent on the severity and duration of ischaemia/hypoxia and could not be observed in the neocortex within the first 13 min [28
]. In the present study, we found that an insignificant increase of s100ß could be observed after 90 min of reperfusion. Thus, the neuroprotective effects of levosimendan observed in the neocortex within the first few hours could only be evaluated based on the monitoring of triggers of cell death (i.e., glutamate release, energy metabolism and inflammation).
Previous studies have demonstrated that it is possible to reduce glutamate release during ischaemia/hypoxia by treatment with tiagabine [29
], dantrolene [30
], nimodipine [31
] or magnesium [32
]. In principle, the activation of mKATP
channels should result in smaller increases in intracellular Ca2+
levels and glutamate release during ischaemia. Thus, levosimendan may be as effective as diazoxide [33
]. Studies have suggested that mKATP
agonists may be beneficial for the treatment of brain disorders that are associated with low ATP levels [35
], and levosimendan has been shown to be neuroprotective in the spinal cord when applied prior to [13
] or during ischaemia [14
]. Preservation of the energy status displayed an important mechanism of protection, but was independent of vasodilatation [36
The failure of levosimendan to affect neuroprotection the parameters determined during the present study may be related to the low intracerebral concentrations achieved in the current protocol. Although efficient serum concentrations for cardiac effects (32 μg/L) were achieved, the tissue concentration in the brain reached only 12% of the concentration in the heart. Although this concentration (0.17 ng g-1
) was six times higher compared with animals with an intact blood–brain barrier [37
], it was not sufficient in the in vitro
model to reduce the primary or secondary injury after trauma. In the traumatic brain model injury model, a 100-fold higher concentration was required to achieve significant affects [15
]. The low cerebral concentrations could be a consequence of the rapid redistribution of the levosimendan bolus [37
] and the delayed disruption of the blood–brain barrier related to the insult [22
], which appears to be a prerequisite to achieve higher levosimendan levels within the brain.
Other reasons for the ineffectiveness of levosimendan might be related to the unique differences of individual animal models, such as the observation period after the insult, the methods used to describe the neuronal injury and the region of interest. For example, the basal ganglia and the hippocampus are more susceptible to ischaemia [28
]. The proposed protective effects of levosimendan might become visible after a longer observation period and may not be associated with reduced glutamate release [39
]. The magnitude of neuronal injury after 15 min of ischaemia/hypoxia should be questioned because no lasting effect on metabolism, glutamate release or s100ß increase was observed. Indeed, only the increase in inflammation and cerebral oedema and the disruption of auto regulation and the blood–brain barrier account for cerebral injury. However, if the ischaemia/hypoxia is prolonged in this model, animals may die from cardiovascular complications or brain death. Thus, modifications to the current protocol for levosimendan administration and a longer observation period are necessary for further validation.
The neuroprotective effects of different drugs might be related to effects on the vasculature. For example, the protective effects of nimodipine and magnesium are associated with decreased cerebral blood flow during reperfusion. Studies have previously shown that controlled reperfusion alone can reduce neuronal injury [40
]; thus, the protective actions of drugs may be mediated by a similar mechanism. Indeed, levosimendan affects cerebral perfusion pressure and flow [41
]. Although levosimendan delayed reperfusion, we observed an upward shift in the relationship between CBF and MAP, which might counterbalance levosimendan’s direct protective actions. In this context the activation of mKATP
channels and increase of NO release in the vessels by levosimendan was effective and led to vasodilation in the brain as described earlier in an animal model of subarachnoid haemorrhage [42
]. In addition, the delayed reperfusion during levosimendan treatment may explain the slower normalisation of the lactate/pyruvate ratio. The relevance of this difference is questionable, however, because no differences in the extracellular glucose concentrations were observed.
Other properties associated with cerebral injury were unaffected by levosimendan treatment. The lack of a disturbance of cerebral auto regulation and disruption of the blood–brain barrier resulted in similar cerebral oedema responses between the groups. Similarly, diazoxide also demonstrated protective effects in this context [43
], where activation of KATP
channels reduced the permeability of the BBB and down regulation of occludin after hypoxia [44
]. These differences might be explained by a diverse affinity to receptor subtypes of the substances, which have not been investigated in detail.
Although levosimendan demonstrated anti-inflammatory actions in sepsis, myocardial reperfusion injury and ARDS, no effects on the expression of inflammatory genes in the neocortex were observed. Because inflammation aggravates neuronal injury [45
], the protective effects of levosimendan under inflammatory conditions are more unlikely.