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

Traumatic brain injury (TBI) is a common consequence of traffic-related accidents and incidents at work and at home. The annual incidence of TBI in the UK is estimated to be approximately 400 per 100,000 patients per year [1]. The treatment of patients with traumatic injury to the brain accounts for a considerable proportion of the budget spent annually on health care and the subsequent costs for rehabilitation, post-hospital long-term care and disability are a significant burden for the economy and society. It should be noted that all currently available therapy approaches for TBI are symptomatic in nature. To date, no clinically established therapy exists that specifically counteracts the actual pathological mechanisms leading to traumatic brain tissue injury.

A very low level of tissue injury was maintained in all slices prior to inclusion in the study groups. This initial injury could be observed in all slices and is attributable to minimal cell death originating from the preparation procedure and to influential effects regarding the handling and maintenance of the slice cultures over the 14-day cultivation time period. Yet the total trauma signal in the baseline fluorescence measurement was very low when compared with the maximum total trauma signal observed after 72 hours in the trauma control group at 37°C (0.004 ± 0.0004 vs. 1.00 ± 0.14; P = 0.00). The level of injury was persistent over all slices with very little variation. This is demonstrated by the data in Figure 1.

The next step was to identify the characteristics of traumatised and non-traumatised slices with respect to their histograms in order to establish a quantitative measurement tool to express the extent of the tissue trauma. The comparison between traumatised and non-traumatised control group slices yielded very different trauma signals in the fluorescence microscope image histograms (Figure 2). The traumatised slices showed a reduced peak in background signal between gray scale values of 20 and 50 and a shift towards greater gray scale values with a discrete peak at values between 160 and 180. Through these control experiments a threshold was established at a gray scale value of 75. Therefore, gray scale values greater than 75 can be attributed to the traumatised tissue, which became fluorescent due to PI uptake. The portion of the histogram curves with a gray scale value greater than 75 were integrated and the result used as a direct, quantitative figure for the measurement of the extent of traumatic injury.

The trauma intensity increased steadily over time after trauma. This is demonstrated by the data in Figure 3b in slices that were evaluated for trauma development using fluorescence microscopy at 0 hours (0.006 ± 0.002), 24 hours (0.45 ± 0.05), 48 hours (0.69 ± 0.08) and 72 hours (1.00 ± 0.11) post-traumatisation. This slow development of the injury stands in contrast to the quickly achieved equilibrium state of PI binding to the DNA in damaged cells, which has been proven in previous publications [3], so the observed increase in fluorescence over time can be attributed to the ongoing cell death rather than to delayed PI uptake and fluorescence development. The values in panel 3b were normalised against the trauma intensity at t = 72 hours. The results of these control experiments justify the sole measurement of the trauma after 72 hours and therefore we evaluated the tissue trauma only at this time point.

Dimethyl sulfoxide (DMSO), the solving agent needed to solve the lipophilic propofol in the aqueous medium, had no detectable effect on the total tissue trauma when administered. This is demonstrated by the level of total trauma intensities in the control group and the group treated with 0.1% DMSO (1.00 ± 0.14 vs. 1.00 ± 0.09; Figure 3a). The comparison of the trauma intensities between the trauma control group and the non-traumatised group at 37°C yielded a significant difference (1.00 ± 0.14 vs. 0.13 ± 0.04, P = 0.00), as expected.

Propofol at normothermia (37°C) was added to experimental medium at concentrations of 10, 30, 50, 75, 100, 200 and 400 μM. Figure 4a displays the concentration-response curves for both the total (filled circles, upper curve) and secondary injury (open circles, lower curves). The nonlinear regression curves were fitted into the graph to visualise the trend. Figure 4b shows exemplary fluorescence images for total and secondary injury (with applied mask for exclusion of the stylus' direct impact site) in the trauma control group and the group treated with 200 μM propofol. A clearly visible reduction of fluorescent, dead cells can be observed when comparing the images of slices from each group, for total and secondary injury. The total trauma intensities under normothermic conditions were 0.86 ± 0.13 (10 μM), 0.73 ± 0.06 (30 μM), 0.67 ± 0.08 (50 μM), 0.42 ± 0.04 (75 μM), 0.34 ± 0.05 (100 μM), 0.07 ± 0.01 (200 μM) and 0.08 ± 0.02 (400 μM). The secondary injury intensity in the control group under normothermic conditions was 0.43 ± 0.08. The intensities of the observed secondary trauma with propofol treatment after trauma under normothermic conditions were 0.29 ± 0.07 (10 μM), 0.21 ± 0.05 (30 μM), 0.25 ± 0.06 (50 μM), 0.19 ± 0.02 (75 μM), 0.08 ± 0.02 (100 μM), 0.001 ± 0.0001 (200 μM) and 0.02 ± 0.003 (400 μM).

Hypothermia at 32°C alone decreased the total tissue trauma in this model by more than 50% (0.48 ± 0.10 vs. 1.00 ± 0.14, P = 0.015), similar to previous reports [2‐4]. A significant reduction of total traumatic injury in hypothermia groups when compared with the corresponding groups treated with the same concentration of propofol under normothermic conditions could be observed at propofol concentrations of 30 μM (0.37 ± 0.03 vs. 0.73 ± 0.06; P = 0.00) 50 μM (0.34 ± 0.02 vs. 0.67 ± 0.08; P = 0.00) 75 μM (0.14 ± 0.03 vs. 0.42 ± 0.04; P = 0.00) and 100 μM (0.13 ± 0.02 vs. 0.34 ± 0.05; P = 0.00), as shown in Figure 5. Thus, the use of mild hypothermia (32°C) in combination with propofol at concentrations between 30 and 100 μM showed a remarkable effect in reducing total tissue trauma. The trauma reduction between hypothermia groups and the corresponding normothermia groups ranged from 48.7% (30 μM) to 66.4% (75 μM) with a mean reduction of 55.7%. The analysis using a two-way ANOVA revealed a statistical significance (P < 0.0001) of both factors (propofol concentrations and temperature) when applied independently. The interaction, beyond the additive effect, of the two factors was not statistically significant (P = 0.397).

In summary, post-traumatic administration of propofol led to a dose-dependent decrease in total as well as secondary tissue trauma, and the combination of propofol and hypothermia were additive in regard to neuroprotection.

We have investigated the potential beneficial neuroprotective effects of propofol in an in vitro setting of TBI utilising the well-established [2, 3, 5, 6, 8‐17] model of organotypic hippocampal slice cultures. We could show that propofol greatly diminishes both total and secondary injury when administered in our in vitro model of TBI. A dose-dependent neuroprotective effect can be observed in the concentration range between 10 and 400 μM propofol (Figure 4).

This method yields easy and open access to the nervous tissue in vitro for manipulation and assessment. Yet, in contrast to dissociated cell cultures, it retains most of the features of tissue organisation as a heterogeneous population of cerebral cells and functional characteristics, e.g. the preservation of synaptic and anatomical organisation, with great similarities to the in vivo state [2, 8, 11, 13, 14, 16, 18‐21]. Hence, organotypic hippocampal slice cultures are an appropriate compromise between models using dissociated cell cultures and experimental in vivo models with whole living animals [4, 13, 22, 23]. The method of selectively traumatising the hippocampal slices has been widely described and used before [3‐6, 22]. To a certain extent, it shares in vitro the characteristics of cerebral traumatic injury in vivo. We selectively traumatised the vulnerable CA1 region of the hippocampus. The subsequent occurrence of focal injury at the primary site of impact and the development of secondary injury distant to that site are also comparable with the in vivo situation. Thus, our model can be used as a testing environment for experimental treatments with a sufficiently high level of confidence.

The development of post-traumatic and post-ischaemic secondary injury has been analysed using this model in previous studies [3, 4]. A number of possible molecular and cellular causes including the activation of pro-apoptotic mediator pathways [24‐26], up-regulation of cell death genes [12], free radical generation, excitotoxicity [8] and cell-to-cell electrical communication possibly involving gap-junctions [21] have been identified. In fact, observations have been made that secondary injury following TBI shares similarities with the post-ischaemic neuronal damage observed in the penumbra surrounding an ischaemic core following stroke [27]. This may give rise to the assumption that similar neuroprotective strategies may be successful in both aetiologies of brain injury.

PI was used in this method as a staining agent to assess the degree of tissue injury. PI labelling has been proven to correlate well with the extent of neuronal injury [7, 17, 28] and used as a cell viability marker in previous studies [3‐5, 9, 14, 17, 29]. It is generally accepted that there is a linear correlation between the cumulative fluorescence emission in PI-treated tissue and the number of damaged cells when compared with cell viability assessment relying on morphological criteria [4, 14, 17].

The exact pharmacodynamic mechanism of action of propofol is not fully known as yet. However, evidence indicates that it primarily acts by potentiating the function of the gamma-aminobutyric acid (GABA)_A and possibly glycine-receptors [30‐32]. Additionally, recent results suggest that propofol may interact with the endocannabinoid system [33, 34], which could contribute to its anaesthetic properties. Propofol has previously been under investigation in both in vivo and in vitro models of ischaemia-reperfusion injury and oxygen-glucose deprivation. The in vitro studies have yielded both positive [9, 35‐38] and negative [10, 15, 39] results for the neuroprotective benefits of propofol. In various in vivo studies a neuroprotective effect in terms of post-ischaemic cerebral damage reduction could be demonstrated in models of transient [40‐44] but not permanent [45] focal ischaemia. The effects of propofol in mechanical TBI have rarely been investigated to date, although there is evidence that propofol can protect neurons from acute mechanically induced cell death following dendrotomy by potentiation of GABA_A-receptor functions [46].

Two in vivo studies in rodent models yielded negative results concerning the neuroprotective effect of propofol [47, 48]. The results of these studies are in contrast to the findings presented in this study. This may be due to different circumstances found in experimental models using whole living animals with all present systemic variables, which are absent in our model of TBI. In addition, the six-hour period of post-traumatic propofol application until the final assessment of brain injury was significantly shorter than the three-day period used in our model. There is also a difference in the propofol concentrations used and the point of application, which is beyond the blood-brain barrier in our study. Recent studies have focused on the concentrations of propofol in the blood serum, cerebrospinal fluid and brain parenchyma and found that when propofol is administered directly via the nutrient medium in a model of organotypic brain slices a final equilibrium concentration of propofol in the brain parenchyma is not reached until 360 minutes after the start of propofol administration [49].

Hypothermia at 32°C alone had a strong effect in reducing the trauma intensity by more than 50% (Figure 3). These results are not entirely surprising because the neuroprotective benefit of hypothermia has been demonstrated before [2‐4, 50]. When hypothermia was combined with propofol at concentrations between 30 and 100 μM, a further reduction in total traumatic injury could be achieved (Figure 5).

There are several issues in this study that must be clarified. First, the maximum clinically feasible propofol concentration in the cerebral tissue remains unclear. Some authors consider the concentrations used in this study (10 to 400 μM) to be reasonable [9, 10, 51‐54], whereas they are considered to be too high by others [49, 55, 56]. Second, the hippocampal slice culture model, besides its many favourable advantages, bears certain disadvantages that need to be mentioned. Due to the nature of the model it excludes mechanisms of injury that may influence brain damage in the in vivo situation such as the absence of any injury pathways related or due to brain swelling inside an enclosed skull, reperfusion injury, global or local ischaemia and/or hypoxia and other systemic variables. Third, propofol was administered directly following the traumatisation procedure excluding the effects of a delay possibly encountered in clinical routine management of patients with TBI.

In this study we could show that propofol is an effective neuroprotective agent when administered after TBI in the hippocampal slice culture model. Propofol reduced both the total tissue injury as well as the secondary injury distant to the primary site of brain injury. This effect was dose-dependent and increased up to 400 μM, the greatest concentration of propofol that was tested. Hypothermia at 32°C alone reduced the tissue injury by about factor two. When hypothermia and propofol were combined a cumulative effect could be observed and the extent of brain injury was further reduced throughout all concentrations that underwent investigation.