Argon attenuates the emergence of secondary injury after traumatic brain injury within a 2-hour incubation period compared to desflurane: an in vitro study

Abstract

Despite years of research, treatment of traumatic brain injury (TBI) remains challenging. Considerable data exists that some volatile anesthetics might be neuroprotective. However, several studies have also revealed a rather neurotoxic profile of anesthetics. In this study, we investigated the effects of argon 50%, desflurane 6% and their combination in an in vitro TBI model with incubation times similar to narcotic time slots in a daily clinical routine. Organotypic hippocampal brain slices of 5- to 7-day-old mice were cultivated for 14 days before TBI was performed. Slices were eventually incubated for 2 hours in an atmosphere containing no anesthetic gas, argon 50% or desflurane 6% or both. Trauma intensity was evaluated via fluorescent imagery. Our results show that neither argon 50% nor desflurane 6% nor their combination could significantly reduce the trauma intensity in comparison to the standard atmosphere. However, in comparison to desflurane 6%, argon 50% displayed a rather neuroprotective profile within the first 2 hours after a focal mechanical trauma (P = 0.015). A 2-hour incubation in an atmosphere containing both gases, argon 50% and desflurane 6%, did not result in significant effects in comparison to the argon 50% group or the desflurane 6% group. Our findings demonstrate that within a 2-hour incubation time neither argon nor desflurane could affect propidium iodide-detectable cell death in an in vitro TBI model in comparison to the standard atmosphere, although cell death was less with argon 50% than with desflurane 6%. The results show that within this short time period processes concerning the development of secondary injury are already taking place and may be manipulated by argon.

Keywords: argon; desflurane; in vitro model; neuroprotection; organotypic hippocampal brain slices; propidium iodide; secondary injury; traumatic brain injury.

Figure 1

Histogram of traumatized versus non-traumatized organotypic hippocampal brain slices. Note: This figure shows the difference in histograms of traumatized in comparison to non-traumatized slices in the no-intervention groups. Curve a) presents the histogram of non-traumatized slices and curve b) presents the histogram of traumatized slices. Standard error of the mean in length means standard error of the mean. The black line equals the mean value and the grey lines show the scanning electron microscope. The values of pixels above the established threshold (grey scale value of 100) were summed up to evaluate the extent of cell injury. The extent of cell injury amounts to the integrated area under the histogram curve exceeding the threshold. This figure is also part of another publication of ours (unpublished).

Figure 2

Trauma intensity in traumatized vs. non-traumatized organotypic hippocampal brain slices. Note: Slices were subjected to a focal trauma and incubated in the respective atmosphere for 2 hours. In the non-trauma groups no traumatic brain injury was performed. After 2 hours, fluorescence images were taken and the trauma intensity was quantified as described under microscopy and assessment of cell death. The trauma induced resulted in a significant increase of trauma intensity in comparison to the slices not subjected to a trauma (*P < 0.001, no-intervention trauma group vs. no-intervention non-trauma group).

Figure 3

Trauma intensity of traumatized organotypic hippocampal brain slices incubated for 2 hours with standard atmosphere or argon (Arg) 50% or desflurane (Des) 6% or both. Note: 93 slices in the trauma standard atmosphere group, 136 slices in the trauma Arg 50% group, 60 slices in the trauma Arg 50%/Des 6% group and 52 slices in the trauma Des 6% group were considered. One-way analysis of variance did not show a significant difference within the trauma groups (P = 0.070). The difference between the trauma Arg 50% group and trauma Des 6% group was significant (*P = 0.015). The difference between the trauma standard atmosphere group and the trauma Des 6% group was not significant (P = 0.058). The combination of Arg 50% and Des 6% did not result in significant differences in comparison to the other groups.

Figure 4

Trauma intensity of non-traumatized organotypic hippocampal brain slices incubated for 2 hours with standard atmosphere or argon (Arg) 50% or desflurane (Des) 6% or both. Note: In the non-trauma groups, 171 slices in the standard atmosphere group, 166 slices in the Arg 50% group, 92 slices in the Arg 50%/Des 6% group and 118 in the Des 6% group were analyzed. One-way analysis of variance did not show a significant difference within the non-trauma groups (P = 0.056). The difference between the non-trauma Arg 50% group and the non-trauma Des 6% was slightly significant (*P = 0.047). The difference between the non-trauma standard atmosphere group and the non-trauma Des 6% group was not significant (P = 0.075). The combination of Arg 50% and Des 6% did not result in significant differences in comparison to the other groups.