Propofol: neuroprotection in an in vitro model of traumatic brain injury

Abstract

Introduction: The anaesthetic agent propofol (2,6-diisopropylphenol) has been shown to be an effective neuroprotective agent in different in vitro models of brain injury induced by oxygen and glucose deprivation. We examined its neuroprotective properties in an in vitro model of traumatic brain injury.

Methods: In this controlled laboratory study organotypic hippocampal brain-slice cultures were gained from six- to eight-day-old mice pups. After 14 days in culture, hippocampal brain slices were subjected to a focal mechanical trauma and subsequently treated with different molar concentrations of propofol under both normo- and hypothermic conditions. After 72 hours of incubation, tissue injury assessment was performed using propidium iodide (PI), a staining agent that becomes fluorescent only when it enters damaged cells via perforated cell membranes. Inside the cell, PI forms a fluorescent complex with nuclear DNA.

Results: A dose-dependent reduction of both total and secondary tissue injury could be observed in the presence of propofol under both normo- and hypothermic conditions. This effect was further amplified when the slices were incubated at 32 degrees C after trauma.

Conclusions: When used in combination, the dose-dependent neuroprotective effect of propofol is additive to the neuroprotective effect of hypothermia in an in vitro model of traumatic brain injury.

Figure 1

After 30 minutes of incubation with propidium iodide, baseline fluorescence imaging was performed on all slices to qualify the level of cell injury prior to trauma. Histograms were computed from images by counting the sum of all pixels sharing the same eight-bit gray scale value (from 0 to 255). (a) The mean with the standard error of the mean (SEM) of a total of 206 slices included in this setting is shown. (b) An enlarged portion of the graph around the applied threshold of 75 (dashed line) is shown. The continuous line is the mean value, and the dotted lines are the upper and lower bounds of the SEM. The data demonstrates the continuous low level of injury throughout the slices prior to traumatisation.

Figure 2

After preparation, cultivation for 14 days and baseline measurement, slices were either traumatised or not by the impact of a stylus onto the CA1 region of the hippocampus. The extent of the trauma was evaluated by fluorescence imaging 72 hours after the induced trauma and pixel-based image analysis. Curve a shows the histogram of non-traumatised slices (n = 17) at t = 72 hours. The straight line is the mean value, the dashed lines are the upper and lower bounds of the standard error of the mean. Curve b shows the histogram of traumatised slices (n = 17). For a better view of the important section the y-axis was split at 12,000 and two different scales were used in both parts. The vertical dashed line is the applied threshold at a gray scale value of 75. The integral of all pixel values greater than the threshold was calculated for each group and defined as the trauma intensity. Two example images for (a) non-traumatised and (b) traumatised slices at t = 72 hours are shown in the upper right corner.

Figure 3

All groups were normalised against the trauma control group at t = 72 hours. (a) No significant difference between the untreated traumatised control group and the group treated only with 0.1% dimethyl sulfoxide (DMSO) could be observed. The addition of 0.1% DMSO was necessary to solve the lipophilic propofol in an aqueous medium. The detected trauma in the non-traumatised group and in the group with hypothermia was significantly lower compared with the trauma control group. * P ≤ 0.05. (b) The trauma intensity increased steadily over time, which has been shown before. This is demonstrated by slices (n = 14). Values were normalised against the trauma intensity at t = 72 hours.

Figure 4

The extent of tissue trauma in the slices was quantified using pixel-based analysis of the acquired fluorescence images at t = 72 hours after trauma. Total injury was defined as the total level of cell death in the slices. A minimum of 12 slices were evaluated per group. Secondary injury was calculated by covering the pin's direct impact site in the images with a defined mask excluding this area from trauma analysis. (a) The concentration-response curves of propofol from 10 to 400 μM for both total (filled circles, upper curve) and secondary injury (open circles, lower curve). (b) Exemplary images for total and secondary injury (showing the impact site exclusion mask) in the control group and at a propofol concentration of 200 μM. Non-linear regression curves were fitted into the graph to visualise the trends.

Figure 5

The use of hypothermia at a temperature of 32°C decreased the trauma intensity. All groups were normalised against the trauma control group at 37°C. The black bars resemble the trauma intensity at normothermia (37°C) and propofol concentrations from 0 to 100 μM after t = 72 hours. The grey bars are the corresponding trauma intensities for slices treated with the same concentrations of propofol but kept at hypothermia (32°C) for 72 hours.