Argon: neuroprotection in in vitro models of cerebral ischemia and traumatic brain injury

Abstract

Introduction: Recently, it has been shown in several experimental settings that the noble gases xenon and helium have neuroprotective properties. In this study we tested the hypothesis that the noble gas argon has a neuroprotective potential as well. Since traumatic brain injury and stroke are widespread and generate an enormous economic and social burden, we investigated the possible neuroprotective effect in in vitro models of traumatic brain injury and cerebral ischemia.

Methods: Organotypic hippocampal slice cultures from mice pups were subjected to either oxygen-glucose deprivation or to a focal mechanical trauma and subsequently treated with three different concentrations (25, 50 and 74%) of argon immediately after trauma or with a two-or-three-hour delay. After 72 hours of incubation tissue injury assessment was performed using propidium iodide, a staining agent that becomes fluorescent when it diffuses into damaged cells via disintegrated cell membranes.

Results: We could show argon's neuroprotective effects at different concentrations when applied directly after oxygen-glucose deprivation or trauma. Even three hours after application, argon was still neuroprotective.

Conclusions: Argon showed a neuroprotective effect in both in vitro models of oxygen-glucose deprivation and traumatic brain injury. Our promising results justify further in vivo animal research.

Figure 1

Control data. After preparation, 14 days of cultivation and baseline fluorescence imaging, slices were either impaired with oxygen-glucose deprivation (OGD) or traumatic brain injury (TBI) (see panel A or B respectively). For OGD, the slices were incubated in glucose free medium and transferred into an airtight anoxic chamber where they were incubated in an atmosphere of 95% N₂ and 5% CO₂ for 30 minutes. TBI was induced by the impact of a stylus onto the CA1 region of the hippocampus. After trauma, the slices were transferred to an airtight chamber and incubated in an atmosphere of 21% O₂, 5% CO₂ and 74% N₂. The negative control groups' slices were subjected to the same treatment, except for the trauma. After 72 hours the damage was assessed by fluorescence imaging and pixel-based image analysis. In both panels, both curves labelled as a show the histogram of non-traumatized slices (OGD: n = 58 prepared from six mice; TBI: n = 35 prepared from six mice) after 72 hours. The middle line is the mean value; the upper and lower lines represent the upper and lower bounds of the SEM. Curves b present the histogram of traumatized slices (OGD: n = 71 prepared from eight mice; TBI: n = 39 prepared from six mice). The vertical dashed line is the applied threshold at a gray scale value of 100. The sum over all pixel values greater than this threshold were calculated for each group and defined as the trauma intensity. Inserts in panel A and B respectively present the controls normalized to the trauma groups.

Figure 2

Neuroprotective effects of argon. Following trauma (OGD or TBI), slices were incubated for 72 hours in an atmosphere containing either x = 25, 50 or 74% argon in addition to 21% O₂, 5% CO₂ and 74-x% N₂. After fluorescence imaging and image analysis all groups were normalized to their respective trauma control group at t = 72 hours. Panel A shows the results for OGD. For each group an average of 55 slices with a minimum of 42 slices was used (prepared from four to six mice). The trauma intensity in each argon group was significantly lower compared to the trauma control group (*P ≤ 0.001), while there was no significant difference amongst the different argon groups. Panel B shows the results for TBI. An average of 43 slices and a minimum of 35 slices was used for each group (prepared from four to eight mice). The detected trauma for each argon concentration was significantly lower compared to the control group (*P ≤ 0.001). Furthermore there was a significant difference between the three argon gas mixtures (P ≤ 0.004 between 25% and 74% Argon and P ≤ 0.001 between 50% and 74% argon).

Figure 3

Example images. Panels A and B show example images for both OGD (panel A) and TBI (panel B). From left to right: No Trauma, 50% Argon, Trauma control.

Figure 4

Delayed argon application. In this setting, groups were incubated for 72 hours in an atmosphere of 50% Argon, 21% O₂, 5% CO₂ and 24% N₂, either directly after trauma was induced (t = 0) or with a two or three hours delay. All groups were normalized to their respective trauma control group at t = 72 hours. Panel A shows the results for OGD and panel B the results for TBI. In the OGD group an average of 43 slices with a minimum of 22 slices was used (prepared from three mice per group). We found a significant difference between the control group and each tested time point (*P ≤ 0.001). Moreover the trauma intensity between t = 0 hours and t = 3 hours differed significantly (P ≤ 0.05). In the TBI group the detected trauma after zero, two and three hours delay time was significantly lower compared to the trauma control group (*P ≤ 0.001). Furthermore, trauma intensity after three-hour delay time was significantly increased as compared to zero and two-hour delay (P ≤ 0.001). An average of 31 slices and a minimum of 15 slices was used for each group (prepared from two to three mice).