Published: December 21st, 2018
This paper describes a novel model of primary blast traumatic brain injury. A compressed-air driven shock tube is used to expose in vitro mouse hippocampal slice cultures to a single shock wave. This is a simple and rapid protocol generating a reproducible brain tissue injury with a high throughput.
Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the detonation of explosive devices, however, the mechanisms that underlie the brain damage resulting from blast overpressure exposure are not entirely understood and are believed to be unique to this type of brain injury. Preclinical models are crucial tools that contribute to better understand blast-induced brain injury. A novel in vitro blast TBI model was developed using an open-ended shock tube to simulate real-life open-field blast waves modelled by the Friedlander waveform. C57BL/6N mouse organotypic hippocampal slice cultures were exposed to single shock waves and the development of injury was characterized up to 72 h using propidium iodide, a well-established fluorescent marker of cell damage that only penetrates cells with compromised cellular membranes. Propidium iodide fluorescence was significantly higher in the slices exposed to a blast wave when compared with sham slices throughout the duration of the protocol. The brain tissue injury is very reproducible and proportional to the peak overpressure of the shock wave applied.
Blast traumatic brain injury (TBI) is a complex type of brain injury that results from the detonation of explosive devices1,2. Blast TBI has emerged as a major health issue in the last 15 years with the recent military conflicts in Iraq and Afghanistan2,3. Overall, it is estimated that between 4.4% and 22.8% of soldiers returning from Iraq and Afghanistan have suffered mild TBI, a large proportion of these being blast-related, with a higher reported rate of blast TBI in the US forces compared with the UK forces4,5.
The use of improvised explosive devices has been responsible for most of the blast-associated trauma, including blast TBI, endured by the military forces6. The detonation of an explosive charge results in a very rapid — but transient — increase in pressure, occurring in milliseconds. The resulting overpressure wave from a real-life free-field explosion is modelled by the Friedlander function, with a sudden rise to the peak overpressure followed by an exponential decay7,8. The range of extreme forces and their rapid time course seen in a blast event are usually not experienced in non-blast traumas1,9. The peak overpressure, which is the maximum pressure of the waveform, and the duration of the positive wave are believed to be important contributors to blast brain injury and these depend on the explosive charge and the distance from the detonation10,11.
The trauma that results from an explosive blast is classified as four discrete components, designated as primary, secondary, tertiary and quaternary blast injury10,12,13,14. Each of these components is associated with specific mechanisms of injury. Primary blast injury results from the direct action of the overpressure wave on organs and tissues2,13. Secondary blast injury results from the impact of projectile fragments, causing penetrating and non-penetrating wounds2,15. Tertiary blast injury occurs when the victim's body is displaced against the ground or surrounding objects and is associated with acceleration/deceleration forces1,10,13. Quaternary blast injury describes a heterogeneous group of injuries directly related to the explosion not covered by the first three injury mechanisms described12,13. It includes (but is not limited to) thermal injury, smoke inhalation, radiation, electromagnetic waves, and adverse psychological effects13,15. Most blast-associated TBI results directly from the first three mechanisms of injury, while the quaternary mechanisms of blast injury are usually associated with systemic injury13. The effects of acceleration/deceleration forces (e.g., whiplash), blunt and penetrating traumatic brain injury have been extensively studied in relation to other types of TBI (e.g., motor vehicle crashes, falls, ballistic injury). However, the primary blast overpressure wave is unique to blast injury and its effects on brain tissue are much less well understood16. The primary blast injury mechanisms, associated with an overpressure wave, are the first of the mechanical forces to interact with the brain.
Numerous preclinical TBI models have been developed over the last decades that have been invaluable to understand blast TBI mechanisms of injury and pathophysiology and investigate potential new treatments, which would otherwise be impossible to do exclusively in the clinical setting17,18,19. Although no single preclinical model can reproduce the complexity of clinical blast brain trauma, typically different preclinical TBI models replicate distinct aspects of human TBI. The damaging action of the forces associated with a blast explosion can be studied in isolation or in combination in both in vitro and in vivo blast TBI models. In vitro models have the advantage of allowing a tight control of the experimental environment (tissue physiologic conditions and injury biomechanics), which reduces biological variability and improves reproducibility, permitting the study of specific molecular cascades without the confounders present in animal models20. Our goal was to develop an in vitro model to investigate the effects of primary blast on brain tissue. We aimed to develop a model with a supersonic shockwave with a Friedlander waveform representative of a free-field explosion such as that produced by an improvised explosive device (IED).
The experiments described in this manuscript were done in compliance with the United Kingdom Animals (Scientific procedures) Act of 1986 and have been approved by the Animal Welfare & Ethical Review Body of Imperial College London. Animal care was in compliance with the institutional guidelines of Imperial College London.
1. Hippocampal Organotypic Slice Preparation and Culture
NOTE: This protocol allows the production of organotypic hippocampal slices according to the interface method described by Stoppini and colleagues with minor modifications21,22,23. Ideally, no more than three animals should be euthanized and dissected in one session to ensure each step is done swiftly and to avoid compromising the quality of the slices. Use aseptic technique throughout.
2. Preparation of the Hippocampal Organotypic Slices for the Experimental Blast TBI Protocol
NOTE: All the steps of this section, except imaging, take place in a laminar flow tissue culture hood.
3. Submersion and Transport of the Tissue Culture Inserts with the Hippocampal Organotypic Slices
4. Preparation of the Shock Tube and Hippocampal Organotypic Slice Shock Wave Exposure
5. Hippocampal Organotypic Slice Injury Quantification
The shock tube used in this method allows the generation of overpressure transients that simulate real-life open field explosions modelled by the Friedlander function7,8. Supersonic shockwaves with a velocity of 440 m/s (Mach 1.3) were obtained (Figure 2A). The waveform data reported is from sensor 2, positioned radially at the end of the driven section of the shock tube.
Using the protocol described above, organotypic hippocampal slice cultures exposed to a single shock wave (Figure 2A) develop significant injury quantified using propidium iodide, a highly polar fluorescent dye that only penetrates the cells with compromised cellular membranes24,25 (Figure 2B, C).
Even under optimal conditions, and consistent to other OHSC published models21,22, there is a low level of background propidium iodide fluorescence due, in part, to minor damage resulting from the inherent tissue manipulations (such as media changes during the culture period or removal from the incubator for imaging). This blast TBI protocol involves substantial manipulation that includes the submersion of the slices in medium inside sterile bags and a considerable degree of handling during the shock wave exposure protocol (e.g., clamping the sterile bags to the holder frame). However, if all steps are performed carefully, this additional manipulation does not have an impact on the underlying health of the OHSC as no significant differences were seen between a control group of slices kept in the 6-well plates at all times (i.e., the inserts were not submerged or handled) and the sham group, which included slices that were submerged inside sterile bags clamped to the shock tube (Figure 2B).
The two shock waves chosen, at 50 kPa and 55 kPa peak overpressure, produced significant (p <0.05 and p <0.0001, respectively) and reproducible injury when compared to the uninjured sham slices at all time-points after the blast exposure protocol (Figure 2B) without causing any damage to the tissue culture inserts or the sterile bags. In order to determine the sensitivity of the model to small differences in peak-overpressure, we decided to select values that were different by ~10 %. These results also show that, as expected, the injury resulting from 55 kPa is higher than that after a 50 kPa shock wave.
The data are expressed as mean ± standard error of the mean. Significance was assessed using a 2-way repeated measures analysis of variance using Holm-Sidak post hoc test. Factor 1 was group (control, sham, blast) and factor 2 was time after the injury (-1 h, 24 h, 48 h and 72 h), where factor 1 was the repeated factor. The adjustment of p value for multiple comparisons was used. P values of less than 0.05 were taken to indicate a significant difference between groups. Statistical tests were implemented using a graphing and statistics software package.
Figure 1: Schematic of the shock tube device with the sterile bag holder frame. (A). The shock tube is a 3.8 m long stainless-steel tube, made of three 1.22 m long sections, connected by gaskets and flanges, with an internal diameter of 59 mm. (B) Inset shows the double breech assembly. One or two Mylar diaphragms can be clamped in the assembly with seal provided by rubber o-rings. (C) Sterile bag holder frame. The body of the frame consists of two metal plates with a centered circular hole (59 mm diameter) that aligns with the shock tube outlet. Two thin (4 mm) sheets of silicone elastomer are fitted between the two metal plates. The purpose of these sheets is to provide an even and non-slippery surface to clamp the sterile bags. The distance between the bag and the outlet of the shock tube is 7 cm. Please click here to view a larger version of this figure.
Figure 2: Typical shockwave and resulting injury in organotypic hippocampal slice cultures. (A) Representative example of shock wave obtained using 23 µm-thick polyester film, 2.16 bar burst pressure, 55 kPa peak overpressure, 0.4 ms positive wave duration, 10.1 kPa·ms impulse. Waveform data were obtained from sensor 2 radially mounted on the distal flange of the shock tube driven section. The shock wave velocity was 440 m/s (Mach 1.3). (B) The development of injury is proportional to the intensity of the shock wave. Both 50 kPa and 55 kPa peak overpressure shock waves caused significant injury that developed throughout the 72 h protocol when compared with the sham group. The injury resulting from a 55 kPa peak overpressure wave exposure was significantly higher than after 50 kPa at 48 h and 72 h. Sham slices were treated identically to blast slices but shock tube was not fired. Control slices were maintained in 6 well plates in an incubator without any manipulation. Bars represent mean values and error bars are standard errors (n = 7, controls; n = 48, sham; n = 30, blast 50 kPa; n = 51, blast 55 kPa; n = number of slices, from 6 separate experiments). *p <0.05, ****p <0.0001 compared with sham. #p <0.05, ##p <0.01 compared with blast 55 kPa. (C) Representative propidium iodide fluorescence images of organotypic slices from (i) sham, (ii) blast 50 kPa and (iii) blast 55 kPa groups at 72 h after injury. The sham slice shows low levels of fluorescence, i.e., injury, and the blast exposed slices show high levels of diffuse injury, more pronounced on the 55 kPa peak overpressure exposed slice (Scale bar = 500 µm). Please click here to view a larger version of this figure.
Among all the mechanisms of injury associated with blast TBI (primary, secondary and tertiary blast injury mechanisms), primary blast injury is unique to blast trauma and it is the least understood of the blast-associated mechanisms1,2. The novel protocol described here was developed to study primary blast TBI using an open-ended shock tube to expose in vitro mouse hippocampal slice cultures to a single shock wave using a simple and rapid protocol that allows the creation of a reproducible primary blast TBI with a high throughput.
The first in vitro primary blast TBI models applied hydrostatic pressure waves to cells26,27. However, the pressure output did not model the Friedlander function as the duration of a hydrostatic pressure pulse was much longer than airborne blast overpressure waves13. The characteristic Friedlander function can be easily modelled in the laboratory using a shock tube1,8. The shock tube can produce shock waves that simulate real-life open field explosions in a conventional laboratory environment, while allowing the precise control of wave parameters, such as peak overpressure, positive wave duration and impulse, by varying the diaphragm material and thickness, and the driver volume8,28,29.
Simple in vitro models such as cell cultures usually lack the heterogeneity of cell types and synaptic connectivity30. Recently, the effect of blast on in vitro brain cell 'spheroids' incorporating different cell types has been investigated31. Further investigation of these interesting preparations is merited; however, it is not clear how their cellular organization and connectivity mirrors the intact brain. OHSC are a well-established in vitro experimental model23,32, are easy to culture and their three-dimensional tissue cytoarchitecture, cell differentiation and synaptic connectivity are well preserved and very similar to that in vivo33,34,35,36. OHSCs represent an intermediate level of complexity between cell culture and an in vivo model23,32. OHSCs have been demonstrated to reproduce in vitro pathological neurodegenerative cascades seen in in vivo models and have been very useful in the screening of potential neuroprotective drugs and in understanding their mechanisms of action17,21,22,37,38. Finally, the anatomic area studied, the hippocampus, is highly relevant in translational TBI studies, as this region is frequently damaged in TBI patients39,40,41.OHSC have been used to model blast TBI28,42,43,44, however, our model is relatively simple and can be adapted to existing shock-tubes in either horizontal or vertical configurations without complex adaptations.
OHSC can be kept in culture for many days, which facilitates the investigation of biological processes over time34. In this model, the tissue injury that resulted from shock wave exposure was measured daily over three days, following the blast exposure using propidium iodide, a well-established marker of cell damage. Propidium iodide is a nontoxic highly polar dye that penetrates the cells with compromised cellular membranes, where it binds to nucleic acids and exhibits a characteristic bright red fluorescence24,25,45. The fluorescence measured with propidium iodide has been shown to have a good correlation with injured cell count using Nissl staining46,47.
Given that the injury produced in this model was diffuse (Figure 2C), the fluorescence of the whole slice was measured when performing the analysis, similar to previously published work in other brain injury paradigms21,22, instead of using specific regions, as has been done in other in vitro blast TBI models28,43,44,48. The global approach used in the model described in this article also eliminates the potential variability that is introduced when outlining defined regions of interest and provides a more comprehensive picture of the blast-related injury. Both shock wave peak overpressures, 50 kPa and 55 kPa, produced significant (p <0.05 and p <0.0001, respectively) injury when compared to sham slices (Figure 2B). As anticipated, the shock wave with the highest peak overpressure, 55 kPa, produced more injury than the 50 kPa wave. In an in vitro model with isolated brain tissue directly exposed to a shockwave, how to accurately scale to the whole organism or a human being is not straightforward. Nevertheless, the shockwaves we used are within the range of peak overpressures observed in the field, typically 50–1,000 kPa8,49.
In order to maintain the OHSC exposed to physiologic temperature and levels of oxygen and carbon dioxide, while ensuring that they were free from contamination throughout the shock wave exposure protocol, the tissue culture inserts were sealed into sterile polyethylene bags following an aseptic technique, submerged in experimental medium warmed to 37 °C and freshly bubbled with 95% oxygen and 5% carbon dioxide, similarly to previously published work28,43,44,48. Contrary to these models where complex devices were used to hold the sterile bags during shock wave exposure, in this protocol, a simple and rapid method was used to suspend the OHSC tissue culture inserts in front of the shock tube outlet (Figure 1A, C). The model described in this paper allows rapid processing and high throughput, while minimizing the risk of hypothermia. These aspects are particularly relevant for neuroprotection studies given that some therapeutic interventions may have a very limited time window of potential application after TBI. This novel shock wave exposure protocol allows 6 to 9 tissue culture inserts (typically 36 to 54 hippocampal organotypic tissue slices) to be exposed to a shock wave in a short interval of time (approximately 1 h).
The OHSCs require good aseptic technique throughout. It is important to use an aseptic laminar flow hood throughout the culturing and when transferring to the sterile bags for blast. In order to carry out the slice imaging under aseptic conditions with the lids of the 6-well plates in place, we use custom-made metal rings to raise the cell culture inserts to the focal plane of the microscope. An important part of our protocol is that we include uninjured sham slices in every experiment. Sham slices are treated identically to blast slices with the exception that the shock-tube is not fired; another important step is that all slices are imaged 1 h before injury or sham treatment, to ensure that the health of the population of slices used are identical (Figure 2B).
In addition to quantifying cell injury in the slices over time, the tissue can be fixed at the end of the experiment for conventional immunohistochemistry50. We developed and evaluated the method using mouse hippocampal slices. However, our technique could be easily adapted to use other tissue that can be grown in culture, such as spinal cord, retina, lung or epithelial tissue. In this paper and our previous work with the model, we investigated only the effect of exposure to a single blast. However, the model would be well suited to investigate the effects of repeated low-level blasts on brain or other tissue. OHSCs can be kept in culture for many weeks or even months, allowing chronic effects to be investigated.
In vitro models, being simpler than in vivo models, have a higher throughput, are less expensive and experiments can usually be completed on a shorter time scale17. However, the results obtained using in vitro models need to be validated in animal models as in vitro cultured tissues are kept in an artificial environment and may respond to injury differently from what they would in vivo17. Nonetheless, in vitro models have been extremely valuable in increasing our understanding of brain injury cascades and in screening neuroprotective drugs before the use of more complex in vivo models17,22,51,52. Despite the many advantages offered by this model, it is important to note that in vitro models lack the key features of TBI present in animals and in vivo models, such as the effects on vascular system, increased intracranial pressure, systemic immune response and functional behavioral impairment, which highlights the need to validate the results found in in vitro models in the whole animal. Nevertheless, in vitro models such as the model described in this paper are extremely useful translationally relevant scientific tools.
In conclusion, this work describes a simple and straightforward novel method where mouse organotypic hippocampal tissue cultures are exposed to tightly controlled and reproducible real-life relevant shock waves using a laboratory shock tube. The resulting global injury, which was quantified using propidium iodide, a well-established marker of cell damage, is very reproducible and is proportional to the peak overpressure of the shock waves applied.
The authors have no competing financial interests.
Supported by: Royal Centre for Defence Medicine, Birmingham, United Kingdom, Royal British Legion Centre for Blast Injury Studies, Imperial College London, United Kingdom. Medical Research Council, London, United Kingdom (MC_PC_13064; MR/N027736/1). The Gas Safety Trust, London, United Kingdom. Rita Campos-Pires was the recipient of a doctoral training award from the Fundação para a Ciência e a Tecnologia, Lisbon, Portugal. Katie Harris was the recipient of a PhD studentship from the Westminster Medical School Research Trust, London, United Kingdom.
This model was developed with the support of the Royal British Legion Centre for Blast Injury Studies (RBLCBIS) at Imperial College. We would like to acknowledge the financial support of the Royal British Legion. Researchers interested in collaborations or further detail may contact the authors or RBLCBIS.
We thank Dr Amarjit Samra, Director of Research, Royal Centre for Defence Medicine, Birmingham, United Kingdom, for supporting this work, Scott Armstrong, Department of Surgery & Cancer, Imperial College London, for assistance with preliminary experiments, Theofano Eftaxiopolou, Hari Arora & Luz Ngoc Nguyen, Department of Bioengineering Imperial College London, & William Proud, Department of Physics Imperial College London, for advice on the shock-tube, Raquel Yustos, research technician, Department of Life Sciences, Imperial College London, for technical support, Paul Brown MBE, workshop manager and Steve Nelson, workshop technician, Department of Physics, Imperial College London, for making the metal rings, Neal Powell of the Department of Physics, Imperial College London, for artwork.
|Geys balanced salt solution
|Minimum essential medium Eagle
|Hanks balanced salt solution
|VWR Prolabo, Belgium
|Tissue culture inserts
|Millicell CM 30 mm low height Millipore
|PICM ORG 50
|Sterile polyethylene bags - Twirl'em sterile sample bags
|Portex Avon Kwill Filling Tube 5" (127mm)
|Smiths Medical Supplies
|NIKON Eclipse 80i, UK
|Nikon Plan UW magn. 2x, NA 0.06, WC 7.5 mm
|Nikon G-2B (longpass emission)
|Mylar electrical insulating film, 304 mm x 200 mm x 0.023 mm
|RS Components UK
|Dytran Instruments Inc.
|Mickle Laboratory Engineering Co., Guildford, Surrey, United Kingdom.
|Mcllwain tissue chopper
|Dow Corning, USA
|Graphing & statistics software
|GraphPad Software, USA
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