An approach for studying the direct effect of shock waves on neuronal cell structure and function.

doi:10.21203/rs.3.rs-4908725/v1

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An approach for studying the direct effect of shock waves on neuronal cell structure and function.

https://doi.org/10.21203/rs.3.rs-4908725/v1

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Recent U.S. military conflicts have underscored the knowledge-gap regarding blast-induced traumatic brain injury (bTBI). In vitro models of TBI, have the advantage of following the neuronal response to biomechanical perturbations in real-time that can be exceedingly difficult in animal models. Here we sought to develop an in vitro approach with controlled blast biomechanics to study the direct effects of the primary shock wave at the neuronal level.

An in-vitro blast injury apparatus that simulates human anatomy was developed. Primary neuronal cells from Sprague-Dawley rat embryos were cultured inside the apparatus. On day 10 in vitro the neuronal cultures were exposed to 70 kPa peak blast overpressure using helium gas in a blast tube. Incident pressure as well as apparatus pressure were measured. 24hrs post injury cell viability was measured.

We were able to successfully blast injured cells without detaching them and caused a significant change in viability from a single blast. The Model also allowed adjustable level of bTBI based on the cover thickness which is an added value not present in other bTBI models. Results also stress the importance of pressure wave frequency as a significant factor for cell viability in bTBI. For the same peak pressure cell can survive low frequency wave even if they have higher amplitude.

Physical sciences/Engineering/Biomedical engineering

Biological sciences/Neuroscience/Cell death in the nervous system

Recent U.S. military conflicts have underscored the knowledge-gap regarding blast-induced traumatic brain injury (bTBI). Since 2001, 60–80% of all U.S. service member casualties in Iraq and Afghanistan have resulted from improvised explosive devices^{[1, 2]}. In the traumatic brain injury (TBI) field, it is well accepted that injury to neurons occurs from the rapid deformation of the brain tissue ^[3–11]. The initiating event is a dynamic mechanical loading (blunt, inertial, blast) to the head that can differ significantly terms of speed, direction, ^[12–14]. Indeed, the initiation of TBI from blast is different than blunt forms of head injury and the mechanical mechanism leading to injury is much less understood. In the case of bTBI, how the mechanical perturbation induces injury is unknown.

Currently there is a significant effort in animal TBI models of blast exposure^{[15, 16]}. In vitro models of TBI, however, have the advantage of following the neuronal response to biomechanical perturbations in real-time that can be exceeding difficult in animal models^[17]. How rapid dynamic mechanical deformation of the neuron leads to injury progression has been best described in reduced in vitro models^[18–22]. Here we sought to develop an in vitro approach with controlled blast biomechanics to study the direct effects of the primary shock wave at the neuronal level.

A limited effort of in vitro blast modeling has been performed using a few different sources to create the shock wave ^{[17, 22–28]}. The results from these models have not revealed a significant change in cell viability at exposure levels comparable to human exposure, however, changes in intracellular sodium ions, calcium ions, cell permeability, and reactive oxygen species have been reported ^{[2, 29, 30]}. In these models, the designs are such that the shock wave is attenuated the through a large water barrier or the applied blast wave had a very short duration of less than 1ms ^{[25, 31]}. One model simulated human anatomy by enclosing cell cultures in a plastic cover to represent the human skull^[28]. To better understand the effects of primary blast, an in vitro model needs to be designed such that the neurons are exposed to similar shock waveforms within the structure of a human head. Our goal is to consider a model that includes the features of the skull, cerebral spinal fluid (CSF) and ménages of the brain compartment to better more closely replicate the effects from the direct action of a shock wave on the cells of the brain. Importantly, these features can have a large effect on the transmission of the shock wave on the brain. In this study we sought to create a model that can be exposed to a Friedlander shock wave using a compressed gas shock tube with adjustable parameters for the skull and CSF ^[32–34].

Inspired by previously published research on surrogate head models, our preliminary experiments started by culturing neurons inside a full-size surrogate head model and then exposing them to a shock wave^{[35, 36]}. However, a full replicate of the human head presented significant challenges and confounding experimental variables for a reduced model including a large physical size and weight, variations in neuronal sample placement, and shock wave exposure. In this study, we designed a simplified enclosure that allows a precise shockwave exposure to a primary neuronal culture. A round cylindrical enclosure symmetrically holds a sample amenable to cell culture methodology, a watertight cover that can be adjusted to varying skull material properties and thicknesses, and a surrounding media simulating cerebrospinal fluid.

In vitro Blast Injury Apparatus

One consideration with existing in vitro models is that they orient cells parallel to the shock wave propagation where cells in the front would receive a higher pressure than cell in the back^{[23, 30]}. To guarantee that all cells receive the same blast intensity, our design ensured cells were on a plane normal the direction of blast wave motion. In addition, our initial experiments found that the use of tissue culture plastic led to 100% cell detachment after blast. We found that a compliant silicone-based cell culture substrate resolved these issues.

To develop a soft cell culture substrate for the blast apparatus, we used polydimethylsiloxane (PDMS) to create custom culture wells. This choice was guided by our previous experience in culturing neurons on PDMS in neuronal stretch injury systems^{[37, 38]}. PDMS has many advantages that were relevant to our application. PDMS is simple to process, biocompatible and transparent for microscopy^[39]. The PDMS cell culture substrate was designed with a plating area (2680 mm²) accommodating about 2 million cells per sample which is adequate for western blotting. The bottom thickness of the PDMS cell culture substrate was limited to 3mm to allow for sufficient microscopic examination. Since the design did not provide mechanical stability for handling and insertion into the blast apparatus, 10 mm thick supporting sides were formed surrounding the plating area, Fig. 1. The PDMS mold was drawn in Autodesk Fusion 360, converted into CNC machining code, and machined from a 101mm square block of aluminum on a HAAS CNC mill. The mold was polished using diamond lapping paste to achieve the best optical clarity in the PDMS. Polishing was done in 6 steps starting with 12-micron diamond paste then 8,4,2,1,0.5 micron respectively.

To cast the PDMS culture well, a PDMS kit (Sylgard 184, Dow Corning, Midland, MI, USA) was prepared by mixing base & curing agent, in a ratio of 10 parts base to 1 part curing agent by weight, agitated to remove air bubbles, poured into the mold, and cured for 45 min at 75°C. The PDMS was removed from the mold, washed with soap and water, dried, plasma cleaned for 3 minutes, and autoclaved prior to plating cells. The final product of PDMS culture wells as well as aluminum mold are shown in Figs. 1 and 2.

The blast injury apparatus consists of a custom-machined cylindrical container made from autoclavable polypropylene and a clear acrylic cover to house the neuronal culture within the shock tube, Fig. 2. The polypropylene cylinder was fabricated to fit the PDMS cell culture substrate and a rubber O-ring to liquid seal the apparatus. Two Luer lock openings on the side allow for injecting media into the sealed container and release air bubbles. We tested three different cover thicknesses (1/8” [3.125 mm], 3/16” [4.8 mm], and 1/4” [6.35 mm]) representing three skull thicknesses within the published range of human skull (2–16 mm) ^[40–43]. In addition, Young’s modulus of acrylic is 2.76–3.00 GPa, which is within the range of the human cranial bone tensile modulus of 0.45–10.0 GPa ^[43–47]. Cell culture media is a good simulant of cerebrospinal fluid, both with viscosity comparable to water ^{[48, 49]}. The cylinder was designed in Autodesk Fusion 360 and machined with a similar procedure as explained before for the mold Fig. 1C.

Primary Neuronal Culture

All procedures followed the guidelines established in the Guide for the Care and Use of Laboratory Animals and were approved by the Rutgers University Institutional Animal Care and Use Committee. Pregnant Sprague Dawley rats were obtained from Charles River Laboratories and then anesthetized with CO₂ and euthanized using CO₂ after embryos extraction. Rat brain cortices were isolated from E17 Sprague-Dawley rat embryos and stored in ice-cold HBSS containing Ca²⁺ and Mg²⁺. After rinsing with Ca²⁺free and Mg²⁺free HBSS, the cortices were mechanically disrupted by pipetting up and down (3–5 times). The media containing the cells was filtered with a nylon mesh filter with 100-µm pores and then a filter with 40-µm pores to remove large pieces of tissue. The remaining cortical cells were cultured on a poly-L-lysine (0.14 mg/mL)-coated custom-made polydimethylsiloxane [PDMS] wells (2,000,000 cells per well in 57-mm) in NeuroBasal media containing 2% B-27, 1% penicillin-streptomycin, and 0.4 mM L-glutamine at 37°C in a CO₂ (5%) incubator. All methods here are reported according to ARRIVE guidelines.

Shockwave Exposure

Before adding cultured cells and media into the blast injury apparatus, the cylindrical container was autoclaved, and the acrylic cover sterilized using 70% ethanol. Inside a biological safety cabinet, PDMS neuronal cultures (day 10 in-vitro) were placed inside the cylindrical container, and the clear acrylic cover was then bolted to the container. Last, warm culture media (37°C) was injected into the apparatus through the Luer lock connections (Fig. 2c).

A compressed gas shock tube was used to replicate a free-field blast wave characterized by the Friedlander function ^{[32, 34, 50]}. The assembled blast apparatus with neuronal culture was quickly mounted inside the tube, facing the blast wave, and blasted at 70 kPa peak blast overpressure using helium gas, Fig. 2. Pressures were measured using high-frequency Tourmaline pressure transducers model 134A24 (1,000 psi maximum pressure, resonant frequency ≥ 1,500 kHz, 0.2 µs rise time, PCB Piezotronics, Depew, NY, USA). A series of pressure sensors were distributed along the length of the shock tube to measure pressure-time profiles including the incident shock wave at the location of the in vitro culture, Fig. 5. The apparatus pressure transducer was mounted though the side wall of the cylindrical housing and in contact with the medium surrounding the PDMS culture well, Fig. 2 (red arrow). This allowed for the measurement of the transmitted pressure wave form the incident shock wave and representative of the pressure profile presented to the normal cells. All data were recorded at 1.0 MHz sampling frequency, and the typical acquisition time ranged from 50 to 200 ms^[32]. After blast, the apparatus was then disassembled under the sterile hood, media was changed and the PDMS culture wells with cells returned to the incubator for further examination.

Pressure Wave Analysis

All data were analyzed using MATLAB; the Fourier transform for the pressure wave signal was calculated using MATLAB’s fft function, the mean pressure using the mean function, and the pressure impulse using the trapz function. The average pressure wave amplitude, which is the arithmetic average of the absolute values of the deviations of the peak pressure from the mean pressure, was calculated using the following equations:

$$\:WA=\frac{1}{N}\sum\:_{i=1}^{N}\left|{x}_{i}-\mu\:\right|\:$$

$$\:\mu\:=\frac{1}{N}\sum\:_{i=1}^{N}{x}_{i}$$

where WA is the average pressure wave amplitude.

The incident blast pressure profiles from the experiments were analyzed. The mean maximum pressure was 15.79 psi ± 3.74, mean duration was 4.16 ms ± 0.64 and mean impulse was 17.82 psi.ms ± 3.57. Based on the measured mean incident blast wave duration of 4.16 ms; we limited our analysis of the apparatus pressure (intercranial pressure) to 4ms following the start of blast overpressure.

Cell Viability

Using a colloidal dye assay, we assessed cell viability by the cells’ ability to exclude trypan blue. The cells were incubated in 0.4% trypan blue solution for 30 minutes and then washed and counted using bright-field optics. Dead cells were distinguishable by their dark blue staining. Cells were injured on day 10 in vitro and viability counts were done 24 hours after injury.

Statistics

All the experiments included a minimum of three technical replicates, and all results are expressed as mean ± standard deviation. One-way ANOVAs was used to determine the differences between the control and experimental conditions, and a probability value of p < 0.05 was considered significant.

Cell viability after blast

We hypothesized that the magnitude of the shockwave would injure neurons in a dose dependent manner. Indeed, the transmission of a shock wave to neuronal cells can be affected by the thickness of the skull ^{[51, 52]}. Here, three blast apparatus cover thickness of 0.125” (3.125 mm), 0.1875 (4.8 mm) and 0. 250” (6.35 mm) were used to test injury to neurons. Cell viability of the neuronal culture was measured from uninjured shams and injured groups (n = 12) including 4 wells from 3 different cell isolations to remove potential bias. The uninjured sham had the same handling as the injured group except that the cells were not subjected to a shockwave.

The cover thickness had a large effect on cell viability following blast exposure. The control cultures had a viability of 0.87 ± 0.11, while the cells blasted with a cover thickness of 1/8” (3.125 mm) had a viability of 0.41 ± 0.32 (F(120) = 106.51, p < 0.05), those blasted with a cover thickness of 3/16” (4.8 mm) had a viability of 0.65 ± 0.24 (F(134) = 40.53, p < 0.05), and those blasted with a cover thickness of 1/4” (6.35 mm) had a viability of 0.79 ± 0.14 (F(138) = 13.92, p < 0.05) (Fig. 3&4). As expected, the thicker cover (analogous to skull thickness) attenuates the immediate shockwave damage to neurons.

Pressure wave analysis.

To understand the link between external blast pressure exposure and cell viability, we examined pressure profiles inside the blast tube (incident pressure) as well as transmitted pressure (intercranial pressure) inside the apparatus. Our blast tube allows pressure measurement at 6 different positions along the length of the tube ^[32]. Figure 5 illustrates pressure measurement positions and distance between them. The cell blast apparatus was placed right under pressure sensor T4. After carefully examining the pressure profiles before the cell blast apparatus (B1, C1 and T4) we noticed a reflected wave demonstrated by a second peak in the pressure profile (see red arrow in Fig. 4B). The reflected wave was likely caused by the large surface area of the blast apparatus compared with the blast tube cross sectional area. The effect of the reflected wave on our model will be discussed later when analyzing the apparatus pressure wave.

The blast tube pressure wave was analyzed, and maximum pressure, duration and impulse were calculated. Maximum pressure was reported as the largest recorded pressure during the blast event. Duration was calculated as the distance between two dotted red lines drawn at the point where the pressure signal crosses the x axis, Fig. 5c. Impulse is the area under the graph between the two dotted lines. Twenty-one blast pressure profiles from the overall experiments were analyzed. The mean maximum pressure was 15.79 psi ± 3.74, mean duration was 4.16 ms ± 0.64 and mean impulse was 17.82 psi.ms ± 3.57.

While the incident shock wave is the defined mechanical perturbation to the in vitro model, it is essential to understand how the blast wave is transmitted through the apparatus cover into the culture medium and neuronal cells. This is analogous to the transmission of a shock wave through the skull and CSF to the brain (intercranial pressure). The internal apparatus pressure wave had 1.3 to 1.9 times higher peak pressure and the pressure impulse was 1.6 to 2.1 higher in the apparatus pressure compared with the incident pressure (Fig. 6B &6C). In addition, careful examination of the pressure profiles inside the apparatus and the blast tube suggests that the reflected wave in the blast tube translated into a pressure peak inside the apparatus. Notice the lagging peak labeled with blue arrow in Fig. 6A that follows the reflected wave labelled in red arrow.

We further examined the transmitted pressure profiles inside the blast apparatus across the three different cover thicknesses (Fig. 7A). Initial evaluation of the pressure profiles found no significant differences for peak pressure, impulse, or average pressure. Peak pressure for 1/8” (3.125 mm) was 27.6 ± 2.8 psi, for 3/16” (4.8 mm) was 26.3 ± 4.8 psi, and for 1/4” (6.35 mm) was 28.6 ± 3.6 psi. Average pressure for 1/8” (3.125 mm) was 27.6 ± 2.8 psi, for 3/16” (4.8 mm) was 22.9 ± 0.46 psi, and for 1/4” (6.35 mm) was 24.3 ± 0.9 psi. Pressure impulse for 1/8” (3.125 mm) was 29.74 ± 1.12 psi.ms, for 3/16” (4.8 mm) was 31.25 ± 3.08 psi.ms, and for 1/4” (6.35 mm) was 32.94 ± 3.01 psi.ms.

Given we see significant difference in viability between different cover thickness but no difference in analysed pressure wave parameters, we looked into the dynamics of pressure waveforms. A Fourier transform analysis of the pressure profile revealed higher frequencies were present for the 1/8” (3.125 mm) cover than for the 3/16” (4.8 mm) and 1/4” (6.35 mm) covers (Fig. 7B). To confirm the higher frequencies for the thinner covers, we calculated the mean time between peaks for each pressure signal, which were 6.9e-5 ± 0.25e-5 sec for 1/8” (3.125 mm), 7.9e-5 ± 0.08e-5 sec for 3/16” (4.8 mm), and 10.3e-5 ± 1.5e-5 sec for 1/4” (6.35 mm). The mean time between peaks was significantly different between the 1/8” (3.125 mm) and 3/16” (4.8 mm) (F(5) = 28.9, p < 0.05) covers and between the 1/8” (3.125 mm) and 1/4” (4.8 mm) covers (F(5) = 10.09, p < 0.05) (Fig. 7C).

Finally, the average pressure wave amplitude was calculated, which is the arithmetic average of the deviations of the absolute values of the peak pressure from the mean pressure (Fig. 7). The average pressure wave amplitude, equation WA in methods, for 1/8” (3.125 mm) was 5.38 ± 0.2 psi, for 3/16” (4.8 mm) was 6.22 ± 0.62 psi, and for 1/4” (6.35 mm) was 6.9 ± 0.5 psi. The average pressure wave amplitude was significantly different between the 1/8” (3.125 mm) and 1/4” (4.8 mm) covers (F(5) = 15.66, p < 0.05) (Fig. 7D).

The overall goal of this study was to replicate the CSF and skull thickness barriers of human anatomy and study their effect on the cell viability of neuronal cultures exposed to a blast wave produced by a laboratory shock tube. This study demonstrated that in vitro neuronal cultures can be successfully adapted to shockwave in air exposure and used to address relevant questions on the direct effects of shock on the neuronal cell. The results of this study revealed that neuronal cell viability correlated with the thicknesses of the acrylic covers (3.125–6.35 mm) which varied within the published range of human skull thicknesses (2–16 mm) ^[40–43]. Interestingly, the thicker cover attenuated the pressure wave frequency inside the blast apparatus but had no effect on the peak pressure or impulse. This finding suggests a potential dependency between pressure wave frequency and cell viability.

This study produced successful methods to recreate a realistic model with anatomic features. From experience with previous blast models, cells cultured on hard plastic substrates tend to detach when subjected to blast injury. This is likely due to the shock interacting with a large change in material properties between the plastic substrate and the surrounding liquid media ^{[53, 54]}. Furthermore, this is an unrealistic material interface that does not exist in the brain. In the current model, we tested a soft PDMS substrate on which cells remained attached after blast exposure. The blast apparatus was designed with an acrylic cover representing the human skull—the Young’s modulus of acrylic is 2.76–3.00 GPa, which is within the range of the human cranial bone tensile modulus of 0.45–10.0 GPa ^[43–47]. The media surrounding the cultured cells was used as a simulant of cerebrospinal fluid, both having viscosity similar to water ^{[48, 49]}.

The transmitted pressure wave measured inside the apparatus was within the range of published intercranial pressures from both cadaveric blast testing and surrogate head models ^{[33, 55–58]}. Our measured intracranial pressure ranged between 23 to 30 psi which is similar to computational^[33] and other surrogate head models^[55]. Interestingly, our model showed that the measured intercranial pressure was higher than incident pressure by a ratio of 1.3 to 1.9 depending on the cover thickness. This increase in peak pressure has also been demonstrated in a cadaveric model reporting an intercranial pressure of 37 psi for an incident pressure of 15 psi which is within the range of our reported data ^[56]. This experimental increase in intercranial pressure has also been investigated using a computational model with a similar experimental setup ^{[53, 55, 56]}. Here a shock wave traveling in a blast tube interacted with a plastic cover then water and finally endothelial cells. The model showed blast waveforms similar to those shown in Fig. 5c. This study proposed the effect was caused by the continuity of pressure at the interface between different material with different compressibilities. In order to keep the pressure at the interface between air and plastic cover continuous, the transmitted wave amplitude has to be the sum of the incident wave and the reflected wave. When the compressibility is very high in the air and very low in plastic and water, the air/plastic interface will behave similarly to a solid wall. In this case, the reflected wave will have an amplitude almost equal to the incident wave, the transmitted wave could have an amplitude almost twice as big as that of the incident wave.

Unexpectedly, this study revealed that cell viability scales with pressure wave frequency and not peak pressure. Frequency and amplitude analysis are important measures in brain injury due to the viscoelastic properties of the brain^{[59, 60]}. Viscoelastic materials such as brain tissue behave with both and an elastic response (spring) and damping response (dashpot) that includes dissipation (damping) and dispersion (delay or phase shift) when undergoing deformation^[61–64]. Specially, the material will behave with increased stiffness with increased frequency or rate. Indeed, it has been shown that strain rate affects neural viability in vitro ^{[59, 60, 64, 65]}. This finding suggests that the rate of change and oscillation of the pressure wave is important to neuronal injury under blast.

Incident wave reflections within the shock tube was evident in this model most likely to the large surface area of the blast apparatus compared with the cross-sectional surface area of the blast tube. It has been reported that when the specimen occupies less than 20% of the area inside the shock tube, the shock wave structure and the measured pressure profiles on the surface of the specimen are relatively unaffected by reflections ^{[66, 67]}. Our current apparatus design occupies 36% of the shock tube cross sectional area. Accordingly, fully understanding the effect of the reflected wave on cell viability can’t be determined without control of the wave reflection. However, our data suggest that the reflected incident wave is transmitted through the injury apparatus and its effects can be seen as a second pressure peak in the transmitted wave Fig. 6A.

In conclusion, this model successfully blasts injured cells without detaching them and caused a significant change in viability from a single blast. This model allows adjustable level of bTBI based on the cover thickness which is an added value not present in other bTBI models. This model highlights the importance of pressure wave frequency as a significant factor for cell viability in bTBI. For the same peak pressure cell can survive low frequency wave even if they have higher amplitude.

Author Contribution

M.H did the experimental design, ran the experiments, analyzed the data and drafted the manuscriptB.P Supervised the experimental design, data collection, data analysis and drafted the manuscript

Data Availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

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No competing interests reported.

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An approach for studying the direct effect of shock waves on neuronal cell structure and function.

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Abstract

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Introduction

Methods

Primary Neuronal Culture

Shockwave Exposure

Statistics

Results

Cell viability after blast

Discussion

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Author Contribution

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References

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