All experiments were performed in compliance with the local institution's Ethical Review Committee and were approved by an animal protection representative at the Institute of Animal Research at the RWTH Aachen University Hospital, in accordance with German Animal Protection aw §4, Section 3. Organotypic hippocampal slices were prepared as reported previously, using the brains from six- to eight-day-old C57/BL6 mouse pups (Charles River Laboratories, Sulzfeld, Germany) [2, 8, 9]. Unless otherwise stated, all chemicals were obtained from PAA Laboratories GmbH (Pasching, Austria). The slices were maintained in culture for 14 days before experimentation. Traumatic brain injury was produced using a specially designed apparatus, which was also as previously reported [8]. Under stereomicroscopic supervision, a 1.65-mm diameter stylus was positioned 7 mm above the CA1 region of the hippocampal slices with the aid of a three-axis micromanipulator; the stylus was then dropped onto the slice with constant and reproducible impact energy of 5.26 μJ. For the experimental groups, the medium was exchanged immediately after trauma with an experimental medium containing levosimendan (Simdax®, 2.5 mg/ml, Orion Pharma, Espoo, Finland) at concentrations of 0.001 μM (n = 41), 0.01 μM (n = 41) and 0.1 μM (n = 52). Injured, untreated slices were considered the control group (n = 30). The slices were cultivated for 72 hrs in an incubator with an atmosphere of 95% air and 5% carbon dioxide at 37°C, after which cell death was quantified by measuring the intensity of propidium iodide (PI) fluorescence in the CA1 area (Figure 1).
Two types of tissue injury were examined: "total injury" was defined as the complete injury over the slice, whereas "secondary injury" described the injured area on the slice that excludes the primary impact site of the stylus. The relationship between the cumulative fluorescence emissions in PI-treated tissues and the number of damaged cells when compared to cell viability was assumed to be linear. Tissue injury in the slices was measured by pixel-based image analysis using ImageJ software (NIH; USA, http://rsb.info.nih.gov). This method has been proved effective in several previous studies [2, 8–10]. The fluorescence images were digitalised at eight bits, allowing us to classify the images on a spectrum of 256 (from 0 to 255) greyscale levels. Damaged regions with high PI uptake emitted fluorescence at a high greyscale level, while vital regions showed only minor emissions. The red channel of each image was analysed with ImageJ software. For each image, ImageJ generated a histogram that showed the absolute number of pixels sharing the same greyscale value. Histograms from non-traumatised slices showed that the vast majority of all pixels had greyscale values between 10 and 100, representing mostly background fluorescence. In contrast, traumatised slices showed, in addition to their background fluorescence, a well-defined peak of values between 160 and 185 (Figure 2). As in previous publications [2, 8, 9], we established a threshold (in this instance, at a greyscale value of 100) that proved to be valid for distinguishing between traumatised and non-traumatised cells. Integrating the area under the histogram curve for all pixel values exceeding the threshold then allowed us to quantify cell injury.
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).