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Neuroprotective properties of levosimendan in an in vitro model of traumatic brain injury
- Anna B Roehl†1Email author,
- Marc Hein†1,
- Philipp D Loetscher1,
- Jan Rossaint1,
- Joachim Weis2,
- Rolf Rossaint1 and
- Mark Coburn1
© Roehl et al; licensee BioMed Central Ltd. 2010
Received: 25 March 2010
Accepted: 21 October 2010
Published: 21 October 2010
We investigated the neuroprotective properties of levosimendan, a novel inodilator, in an in vitro model of traumatic brain injury.
Organotypic hippocampal brain slices from mouse pups were subjected to a focal mechanical trauma. Slices were treated after the injury with three different concentrations of levosimendan (0.001, 0.01 and 0.1 μM) and compared to vehicle-treated slices. After 72 hrs, the trauma was quantified using propidium iodide to mark the injured cells.
A significant dose-dependent reduction of both total and secondary tissue injury was observed in cells treated with either 0.01 or 0.1 μM levosimendan compared to vehicle-treated slices.
Levosimendan represents a promising new pharmacological tool for neuroprotection after brain injury and warrants further investigation in an in vivo model.
Traumatic brain injury (TBI) is common, carries high rates of morbidity and mortality and lacks specific treatment. In our study of TBI, the initial lesion results from direct mechanical damage at the impact site. Subsequently, several cellular and molecular processes expand the local injury. This so-called secondary injury is due to several factors: excitotoxicity; mitochondrial dysfunction resulting in the up-regulation of cell-death genes and the formation of free radicals; and proapoptotic mediator pathway activation . At present, medical intervention cannot rescue directly traumatised, dying cells. Therefore, current neuroprotective drugs target the surviving cells near the impact site . Hypotension, hypoxia, hyper- and hypocapnia, and hyper- and hypoglycemia remain potentially avoidable insults, all of which aggravate the outcomes of TBI . Levosimendan is a novel inodilator that enhances myocardial performance without leading to substantial changes in oxygen consumption. Levosimendan's positive inotropic and vasodilator effects are tied to its abilities to increase calcium sensitivity and open ATP-sensitive K+ channels (mitoKATPchannels) . In a swine model of cardiac arrest, levosimendan significantly improved the initial resuscitation success, increased coronary perfusion pressure and elevated regional brain oxygen saturation . Levosimendan favourably affects mitochondrial adenosine triphosphate synthesis, conferring cardioprotection and possible neuronal protection during ischemic insults. In a model of spinal cord injury, levosimendan has been reported to attenuate neurologic motor dysfunction . This finding is supported by the fact that the selective mitoKATPchannel opener, diazoxide, is an effective neuroprotectant, as has been demonstrated in an ischemia reperfusion study in rats . In fact, it has been observed that the secondary injury following a traumatic brain injury is similar to the post-ischemic neuronal damage observed in the penumbra surrounding the ischemic core after a stroke . In the present study, we tested the hypothesis that levosimendan would provide neuroprotection for these selectively vulnerable neurons in an in vitro, organotypic, hippocampal slice model of cerebral trauma.
To calculate the extent of the secondary injury, ImageJ was used to create a mask with the same diameter as the stylus. The mask was positioned directly over the stylus' impact site in the images, and that area was then excluded from the pixel analysis and subsequent calculations of trauma. The same mask was applied to every image when calculating the secondary injury. All values were normalised in reference to the control injury, which was defined as 100%. Both the mean value and the standard deviation (SD) were calculated for the trauma intensities of the slices in each group. One-way analysis of variance (ANOVA) with Bonferoni post-hoc analysis was used to test for statistical significance. A P-value of less than or equal to 0.05 was set as the threshold for statistical significance (SPSS 17.0, SPSS Inc., Chicago, IL, USA).
Results and Discussion
The precise mechanisms by which mitoKATPchannel activation protects the brain are unclear. One possible explanation is that the acute activation of mitoKATP channels results in K+ influx, organelle depolarisation, and the expansion of mitochondrial matrix volume. Mitochondrial Ca2+ overload has been closely correlated with mitochondrial damage, which can result in both necrotic and apoptotic forms of cell death . Thus, despite the profound differences in cellular physiology and sensitivity to anoxic injury that exist between myocardial cells and neurons, the same pharmacological approach has been shown to protect the heart  and, possibly, the brain, thereby saving both brain and heart tissues. The occurrence of focal injury at the primary site of impact and the subsequent development of secondary injury distant from the site are also comparable to the in vivo situation. Thus, this model can be confidently used as the testing environment for experimental treatments. In fact, we found that the secondary injury following traumatic brain injury displayed many similarities to the post-ischemic neuronal damage that can be observed in the penumbra surrounding the ischemic core following a stroke . This likeness suggests that similar neuroprotective strategies may be successful in both etiologies of brain injury. The nature of the model excludes mechanisms of injury that are specific to brain damage in the in vivo situation, such as injury pathways related to brain swelling inside an enclosed skull, reperfusion injury, global or local ischemia, global or local hypoxia and other systemic variables . Another limitation of this study is that levosimendan was administered directly following traumatisation, thus failing to account for the effects of delayed treatment that may be encountered in the routine clinical management of patients with traumatic brain injury, global ischemia or local ischemia.
The present in vitro study demonstrated that levosimendan worked as an effective neuroprotectant in an in vitro model of traumatic brain injury. Levosimendan reduced both the total tissue injury and the secondary injury distant from the primary site of brain injury. The effects were observed at two different concentrations (0.01 and 0.1 μM) of levosimendan.
Special thanks to Rosemarie Blaumeiser-Debarry for her help with data acquisition, and gratitude to the teams at the Department of Neuropathology, Pathology and Animal Research at the University Hospital, Aachen.
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