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HIGH-RATE MECHANICAL INSULT CONTRIBUTES TO ALTERATIONS IN BRAIN CELL SIGNALING AND REACTIVITY.

INTRODUCTION

Traumatic brain injury (TBI) is defined broadly as the complex cellular and molecular sequelae caused by external physical force enacting damage on the brain tissue. TBI can present clinically as long-term impaired cognition, mood changes, and increased anxiety [1]. Importantly, no therapeutic strategies have yet been successful in completely mitigating the symptoms of TBI. Many approaches are aimed at general hallmarks of cellular injury, yet there is still a need to understand the scope of mechanobiological and molecular contributors in brain cell injury to properly target them. In vitro TBI models have shown differential cellular outcomes associated with a range of injury mechanics, including injury severity and rate, and evidence shows that brain cells are differentially susceptible to strain rate, in particular, depending on cell type and mechanics [2-4]. In this study, high-rate mechanical insult was the focus for exploring cellular dysfunction in neuronal-glial cultures. High-rate mechanical insult was defined as one millisecond or less exposure to a transient overpressure compression wave. The specific injury model was designed to simulate military-relevant conditions that result from exposure to explosive devices. Blast neurotrauma affects a significant number of Veterans and presents clinically in conjunction with post-traumatic stress disorder [5, 6]. It is debated how the shock wave from an explosive event interacts with the skull and brain to create pressure gradients inside the tissue leading to cognitive dysfunction [7].

Well-characterized cellular hallmarks of blast neurotrauma include oxidative stress, neuroinflammation, glial reactivity, and neuronal damage [8, 9]. However, little is known about the mechanisms which may initiate and/or prolong diffuse cellular dysfunction and phenotypic shifts in cells after blast injury. Glial cells, especially astrocytes, are both biochemically and structurally coupled with neuronal networks, and thus, the feedback mechanisms between these cell types have numerous implications for the prolonging secondary injury mechanisms like neuroinflammation and oxidative stress. Moreover, Sword et al showed that there is a complex relationship between biochemical alterations and structural disruption of neuron-astrocyte coupling after trauma [10]. This may initiate or influence glial contributions to neurodegeneration and cell death after injury [11]. It is therefore critical to identify mechanisms of glial cell reactivity after injury in order to harness their potential to repair rather than instigate further damage to the brain. Previous work has established a mechanical basis for astrocyte reactivity following simulate blast events [12]. The objective of this study was to expand understanding of mechanobiological mechanisms in astrocyte reactivity to include intercellular signaling with neurons following high-rate insult exposure. It is hypothesized that the presence of other cells may significantly influence astrocytic signaling potentiation in the secondary sequelae associated with blast injury. METHODS

Primary cell cultures. Two types of primary cell cultures were prepared for these studies: (1) mixed brain cells and

(2) astrocyte-only. Primary rat mixed brain cell preparations were obtained as a gift from Dr. Beverly Rzigalinski (Edward Via College of Osteopathic Medicine, Blacksburg VA). Culture preparation and maintenance is described by Ahmed et al [13]. Briefly, cortices were extracted from one-two day old rat pups and seeded in equal volume amounts (~1x[10.sup.6] cells) per well. Previous characterization showed that glial cells adhere to the culture plates with neurons adhering on top. Astrocyte-only cultures were also extracted from two day old rat pup cortices and mechanically purified from other cells by gentle shaking for 48 hours. Cells were maintained up to 14 days after extraction before use and were seeded at 1x[10.sup.4]/[cm.sup.2] in standard six-well plates for 6-7 days prior to testing. Samples were routinely stained for glial fibrillary acidic protein (GFAP) to assess cell population composition. For testing, samples were prepared in DMEM and sealed with parafilm (ensuring no air bubbles) before being placed in the overpressure generator, described below. Sham groups were treated equally apart from overpressure exposure.

High-rate insult device. Figure 1 shows the custom water-filled, temperature-controlled chamber used for in vitro exposure to high-rate compression wave which mimics a shock wave profile. This device was fabricated as a means to isolate and study pressure mechanisms associated with high-rate injury. It works by an exploding bridge wire mechanism, in which a thin wire is vaporized at the smaller end of the chamber using high electrical current. The vaporization creates an underwater explosion which propagates down the conical section of the tube exposing cells that were located adjacent to the pressure wave. Pressure measurements were captured using Meggitt 8350C or PCB 113B21 sensor at a location directly above and adjacent to the cell cultures.

Gene expression. At either 24 or 48 hours post exposure, TRIzol reagent (Ambion 15596018) was added to the cell samples for RNA and protein extraction. Manufacturer's protocols were followed for the RNA extraction followed by DNase digestion (Promega M6101) to remove potential contaminants. RNA was quantified using a spectrophotometer, and a 260/280 of 1.8-2 was considered suitable for further analysis. Gene expression analysis was conducted by reverse transcription real-time polymerase chain reaction. Complementary DNA was prepared using random hexamers with one microgram of

RNA. Gene target primers were obtained from PrimerExpress (Thermo-Fisher), and optimized in a reaction with SYBR green (Qiagen 330523) detection. A reference gene, glyceraldehyde triphosphate dehydrogenase (GAPDH) was used for computing normalized gene expression by a delta-Ct method.

Protein expression. Protein lysates were prepared by two methods. First, proteins were precipitated from the phenol phase after RNA was isolated from TRIzol using propanol. Pellets were washed in guanidine-HCl in 95% ethanol. Resuspension buffer for proteins was a 1:1 ratio of 1% sodium dodecyl sulfate to 8M urea in 1M Tris-HCl (pH=8) with protease inhibitor cocktail (Sigma P8340). To facilitate better resuspension, samples were sonicated and heated for 10 minutes at 55[degrees]C three times. Otherwise, protein samples were prepared by incubation/shaking with lysis buffer containing 40 mM Tris-HCl (pH=7.5), 150 mM NaCl, 2.5 mM EDTA, 1% Triton-X, and protease inhibitor. Relative quantification of protein targets was completed using an automatic Simple Western apparatus, Wes (Protein Simple). Manufacturer's protocol was followed for sample preparation. Antibodies used for this analysis were anti-GFAP (Abcam 7260), anti-connexin43 (CX43, Novus NB100-91717), anti-superoxide dismutase 2 (SOD2, Novus NB100-1992), p-actin (Sigma A5441) and anti-GAPDH (Novus NB600-502).

Statistics. Statistical comparisons were made using JMP software (Virginia Tech license). ANOVA analysis was used to determine differences between groups. Assumptions for normality were confirmed by ShapiroWilk test, and Kruskal-Wallis (KW) test was used for nonparametric data sets. Equal variances were assessed using Brown-Forsythe test. If necessary, data was transformed using natural log. Post-hoc student's t-test with a p-value<0.05 was considered statistically significant for comparisons between groups. Statistical blocks were used in gene expression analysis for replicates in each sample set. For graphical presentation, non-transformed data is shown. It should be noted that Figure 4 is presented as box plots with medians, but statistical comparisons were computed between means by methods described here.

RESULTS

High-rate insult mechanics. Table 1 provides the average pressure wave parameters for this study. Samples were exposed to an average of 18.37 psi (127 kPa) peak overpressure, which corresponds to mild severity injury in preclinical rodent models [14, 15]. Previous studies with underwater transient pressure chambers have shown that 17 psi overpressure causes minimal astrocyte death or detachment immediately following exposure [12]. It should be noted that pressure data was not obtained for one mixed cell plate due to sensor malfunction, but it was exposed to the same conditions as the other samples and thus was considered to be well represented by the average parameters listed here.

Phenotypic alterations. Gene expression for structural proteins was measured in mixed cell samples at 24 and 48 hours post overpressure (Fig. 2). The neuronal marker [beta]-tubulin was significantly increased relative to sham (p-value=0.031) at 24 hours but returned to sham levels by 48 hours. Astrocyte-specific GFAP was also increased relative to sham but was delayed to the 48 hour time point (p-value=0.022). This reactive response was preceded by a significant increase in CX43 gene expression (p-value=0.011) at 24 hours and was coupled with decreased CX43 expression at 48 hours (p-value=0.018). Increased GFAP gene expression at 48 hours also corresponded to a decreased protein levels as compared to sham (p-value=0.037, Fig. 3). The increased gene expression may therefore be a compensatory mechanism in response to disruption of the protein by high-rate overpressure and/or cellular sequelae.

Metabolic and signaling aberrations. Mixed brain cells displayed increased protein expression of CX43 at 24 hours as compared to sham and astrocyte-only cultures (p-value<0.0001, Fig. 4A). While astrocyte-only cultures had a similar trend in increased CX43 expression compared to sham, mixed brain cells had a more substantial and variable increase. A similar pattern was found with SOD2, a mitochondrial-linked antioxidant molecule. Figure 4B shows no change in SOD2 protein levels in the astrocyte-only cultures with a significant increase in the mixed cells compared to sham and astrocyte-only groups at 24 hours (p-value=0.0094).

DISCUSSION

Shock wave overpressure is a unique injury paradigm with many aspects that remain unclear. This study found that there is a mechanical basis for structural alterations in both neurons and astrocytes following exposure to high-rate insult. Neurons can experience disruption of tubulin proteins as a result of mechanical deformation of axons [16]. Therefore, increased gene expression may indicate initial repair to compensate for damage to neuronal extensions [17]. Although not evaluated, this response may have been coordinated by support mechanisms from surrounding astrocytes. Because astrocytes are central for repair and signaling processes in the brain, they were largely the focus of this study. Astrocyte reactivity is one of the most common features across a variety of central nervous system insults, including most TBI modes. Prior work conducted with astrocyte-only cultures suggested that astrocytes undergo classical reactivity (as measured by increased GFAP expression) in response to high-rate insult [12, 18]. However, here we find that while increased GFAP gene expression occurs, there was decreased protein levels at 48 hours after exposure. Because this response has only been noted in the presence of other cell types, it suggests that disruption or controlled downregulation of GFAP may be influenced by crosstalk between cell types. It is possible that astrocyte stiffness is modulated to facilitate repair, or astrocyte disruption could precede neuronal death as they compensate for neuronal damage via increased CX43 and other signaling processes [11, 19].

Astrocytes displayed dynamic changes in mRNA and protein levels of specialized gap junctional protein CX43. CX43 is specifically expressed within astrocyte networks for small molecule communication and transport. It has been implicated in astrocyte reactivity to multiple injury mechanisms [20, 21]. Although CX43 regulation is still unclear, differential CX43 expression in astrocytes is influenced by the presence of injured neurons [22]. Spatial distribution of enhanced CX43 is highly varied depending on insult and has roles in both detriment and repair of neurons [11, 21, 23]. Earlier studies showed that CX43-mediated astrocyte communication was imperative to clearance of excess neurotransmitter release and neuroprotection [21, 23], but recent studies have concluded that negative consequences involve increased cell death [11]. Results of this study were consistent with previous findings that neurons influence CX43 regulation, as expression was higher in the mixed cell cultures as compared to astrocytes alone. This may be critical for astrocyte roles in coordinating inflammatory and metabolic processes after injury and suggests that neuronal signaling specifically influences astrocytic network communication [21, 24]. CX43 upregulation also temporally corresponded to increased SOD2 protein in mixed brain cell cultures after overpressure exposure. Astrocytes have a robust antioxidant potential, and thus, may be responsible for coordinating this response to address high susceptibility of neurons to oxidative stresses [25]. No studies have previously implicated CX43 in blast neurotrauma mechanisms, however the results here suggest that future work should focus on CX43 influence in outcomes related to neuronal repair and oxidative stress.

While cell-specific alterations occurred by the 48 hour time point, the insult did not elicit measureable signs of compensatory gene expression of [beta]-actin or vinculin. Taken together, these results indicate that neuronal and astrocytic reactivity to high-rate insult were largely a progressive response to signaling sequelae between cells. There could be a variety of factors that influence these outcomes, from inflammation to cellular adhesion. Moreover, high-rate insult caused increased signaling potential within astrocyte networks both in the presence and absence of neurons. These results may imply that the initiating factors for astrocyte reactivity are mechanically based, while neuronal signaling may influence the extent of astrocyte reactivity. Future work will employ functional manipulations of cell-specific pathways to understand their precise contributions to outcomes observed in this study.

CONCLUSIONS

High-rate insults initiated sub-acute changes in mixed brain cells at 24-48 hours post exposure. Cell-specific proteins were differentially expressed in neurons and astrocytes. Importantly, the presence of neurons influences the potentiation of gap junctional and antioxidant signaling mechanisms in astrocytes. CX43 may be an important mediator of early intercellular signaling after high rate mechanical insult to the brain and is regulated to some extent by non-mechanical cues. This work motivates future studies which will decipher and differentiate the molecular basis for mechanical versus cellular signaling in driving astrocytes ability to assume a reparative phenotype.

ACKNOWLEDGMENTS

The authors would like to acknowledge Samuel Miller for his contributions to sample collection and preparation. We would also like to thank Dr. Beverly Rzigalinski for her generous contribution of mixed cell preparations for use in these studies.

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Nora Hlavac (1), Pamela VandeVord (1,2)

(1) Department of Biomedical Engineering and Mechanics, Virginia Tech, Blacksburg, VA, USA

(2) SalemVeteran Affairs Medical Center, Salem, VA, USA

Caption: Figure 1. The overpressure generator was used for in vitro high-rate overpressure testing. Upon wire vaporization, the wave front travels down the test section over cultures denoted by "Cell Plate." The underwater pressure wave as represented in the pressure trace is meant to mimic a Friedlander waveform.

Caption: Figure 2. High-rate insult induced gene expression alterations in both neuron and astrocyte-specific molecular targets. All values are mean [+ or -] standard error and are normalized to average sham (i.e. sham=1). *p-value<0.05 as compared to respective sham, #p-value=0.055 as compared to respective sham

Caption: Figure 3. GFAP disruption which was found at 48 hours post-overpressure exposure. OP denotes pressure exposed group. Data are mean [+ or -] standard error and are normalized to sham. *p-value<0.05

Caption: Figure 4. Mixed brain cells displayed increased (A) CX43 and (B) SOD2 protein levels compared to astrocyteonly cultures at 24 hours after overpressure exposure. OP denotes exposed group. Data are normalized to average sham and are shown as box plots (centerline=median) to represent distributions across groups. For significance, means were compared across groups by ANOVA/KW and post-hoc student's t-test. Bar indicates p-value<0.05 as
Table 11 Summary of overpressure parameters

                               Avg [+ or -] Std Dev

Peak Overpressure            18.37 [+ or -] 3.66 psi
Positive Peak Duration        1.03 [+ or -] 0.17 ms
Rise Time                     0.48 [+ or -] 0.24 ms
Impulse                   803.02 [+ or -] 107.53 psi*ms
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Author:Hlavac, Nora; VandeVord, Pamela
Publication:Journal of the Mississippi Academy of Sciences
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Date:Apr 1, 2018
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