Virginia Research Day 2021

Contribution of Mitochondrial Dysfunction in Astrocytic Reactivity following Primary Blast Traumatic Brain Injury

1 Graduate Program in Translational Biology, Medicine, and Health, Virginia Tech, Blacksburg, VA, USA. 2 Biomedical Engineering and Mechanics, Virginia Tech, Blacksburg, VA 24061 3 Veterans Affairs Medical Center, Salem, VA 24153 Fernanda Guilhaume Correa 1 and Pamela VandeVord 2,3

Introduction

Pro-Oxidative Stress Environment Analysis of NOX4, Gene Expression and Protein Quantification, in Primary Rat Astrocytes 24 hours Post-Overpressure Exposure

Shock Wave Generator (SWG)

JC-1 Analysis in 2D monolayer Primary Rat Astrocytes 24 hours and 15 minutes Post-Overpressure Exposure

Blast-induced traumatic brain injury (bTBI) is a modality of injury that has come into prominence at the current time considering the large number of military personnel and civilians, which have experienced a blast wave caused by explosives. Primary blast injury results from the propagation of a high-pressure wave and its subsequent energy transmission within brain tissue. Exposure to this overpressure energy cause a diffuse brain injury that leads to cell damage. Understanding the physiological and pathological mechanism underlying TBI is challenging. Many types of cells within the central nervous system (CNS) contribute to the progressive dysfunction of the brain’s normal architecture and function. There is a constant crosstalk between cells, as well as, paracrine signaling (cell-to-cell communication) following the TBI that remains to be investigated. Very little is known on the mechanism that initiates and/or prolongs the diffuse cellular dysfunction and phenotypic shifts after primary blast injury 1-8 . The proposed experiment aim to identify a unique cellular and molecular mechanism in astrocytes after exposure to the mechanical injury . Precisely, aiming to understand astrocytic mitochondrial dynamics, mainly fission activities, post bTBI in a 2D monolayer of primary astrocytes cell culture at an in vitro primary blast models to then be able to determine the importance of mitochondrial dysfunction to astrocyte reactivity.

A

24 hours

A

A

* P = 0.0498

Pressure Profile

Recorded Data

C

B

B

* P = 0.0079

15 minutes

B

D

Figure 6. Normalized gene expression fold changes at 24 h post-overpressure exposure. q-PCR and Western Blotting were conducted to look at pro-oxidative (NOX4) gene expression and relative protein quantification to identify possible pro-oxidative stress environment. Statistical comparison were conducted between groups using GraphPad Prism 8.4.1 software. Unpaired T-test with Welch’s test correction (assuming different standard deviation) was used to analyze significant difference amongst groups. A) q-PCR reveled a significant downregulation of NOX4 gene expression fold change 24h post-overpressure as compered to sham, n=9/group (data are mean ± SEM; *p-value represents ≤ 0.005). B) Western blot analysis revealed an increase in the pro-oxidative NOX4 protein levels at 24 h post-overpressure as compared to sham, n=9/group (data are mean ± SEM; *p-value represents ≤ 0.005).

Blast Injury Classifications

I. Primary injury is the injury caused by tissue interactions with the blast wave energy. II. Secondary injury is caused by fragments of debris propelled by the blast wave. III. Tertiary injury is due to acceleration of the body or part of the body by the blast wind. IV. Quaternary injury is due to burns or post detonation environmental contaminates.

Conclusion

High-rate overpressure elicits early pro-oxidant signaling mechanisms in astrocytes. The study demonstrated alterations in glial’s mitochondrial structure and function in early response to primary blast injury.

JC-1 Analysis in 2D monolayer Primary Rat Astrocytes 24 hours and 15 minutes Post-8mM Glutamate Treatment Figure 4. Measuring of mitochondrial integrity by using membrane-permeant JC-1 dye. Mitochondrial membrane integrity was assessed by live-cell imagining using JC-1, a radiometric dye. Statistical comparison were conducted between groups using GraphPad Prism 8.4.1 software. Unpaired T-test with Welch’s test correction (assuming different standard deviation) was used to analyze significant difference amongst groups. A) There is no significant difference 24 h post-overpressure as compered to sham, n=8-9/group (data are mean ± SEM ; *p-value represents ≤ 0.005). B) Mitochondria were hyperpolarized at 15 min post-overpressure as compared to sham, n=8-9/group (data are mean ± SEM; *p-value represents ≤ 0.005).

Figure 2. Injury model and pressure profile. A) High-rate overpressure device. 1) Driver for the energy required (high electrical current). 2) Bridge wire mechanism. 3) Cell culture location. Upon wire vaporization, the wave front travels down the test section over cultures denoted by “Cell Culture.” B) Pressure Profile. The underwater pressure wave simulates intracranial high-rate overpressure . C) Representative pressure profile of the shock wave generated by the SWG. D) Primary astrocytes 24 hours post-injury presented ≈ 99% cell viability.

Future Directions

➢ Determine changes in mitochondrial dynamics specifically fission (DRP1 and FIS1) and mitophagy (LC3B) by fluorescence microscopy, gene expression and protein quantification.

Results

Mitochondrial Integrity

24 hours

A

Methods

A

Cell Culture Model

2-D monolayer cell culture and injury model • Brain tissue was obtained from P2 Sprague-Dawley rat pups.

B

• Cells were cultured for up to 14 days before initial passage, with a mechanical purification step (24-48 hours shaking) at seven days post-isolation. • Primary rat astrocytes were exposed to a single overpressure wave by a high-rate overpressure device. • Astrocytes were exposure to a peak overpressure of approximately 20psi, previously identified as a mild injury in animals model 4 . • Following overpressure exposure, samples were collected at 15 minutes and 24 hours for analyzes purpose.

I would like to thanks Dr. VandeVord and all the laboratory members for the support, guidance and learning. ➢ Determine reactive oxygen species (ROS) by analyzing free radicals superoxide and hydrogen peroxide with DCFDA and MitoSox technique. ➢ Data will be translated into an in vivo blast model by using an advanced blast simulator which re-creates free field blast exposure. Astrocytes cells from adult rat brain will be isolated through Magnetic-Activated Cell Sorting technique (MACs). Acknowledgements 1. Sajja, V. S. et al. Blast-induced neurotrauma leads to neurochemical changes and neuronal degeneration in the rat Hippocampus. NMR Biomed. 25, 1331–1339 (2012). 2. Cho, H. J., Sajja, V. S., Vandevord, P. J. & Lee, Y. W. Blast induces oxidative stress, inflammation, neuronal loss and subsequent short-term memory impairment in rats. Neuroscience. 3, 9–20 (2013). 3. Hlavac, N., & VandeVord, P. J. (2019). Astrocyte mechano-activation by high-rate overpressure involves alterations in structural and junctional proteins. Frontiers in neurology, 10, 99. 4. Hubbard, W. B., Hall, C., Sajja, V. S. S. S., Lavik, E., & Vandevord, P. (2014). Examining lethality risk for rodent studies of primary blast lung injury. Biomedical sciences instrumentation, 50, 92. 5. Fischer, T. D., Hylin, M. J., Zhao, J., Moore, A. N., Waxham, M. N., & Dash, P. K. (2016). Altered mitochondrial dynamics and TBI pathophysiology. Frontiers in systems neuroscience, 10, 29. DOI: 10.3389/fnsys.2016.00029. 6. Motori, E., Puyal, J., Toni, N., Ghanem, A., Angeloni, C., Malaguti, M., ... & Winklhofer, K. F. (2013). Inflammation-induced alteration of astrocyte mitochondrial dynamics requires autophagy for mitochondrial network maintenance. Cell metabolism, 18(6), 844-859. 7. Fiebig, C., Keiner, S., Ebert, B., Schäffner, I., Jagasia, R., Lie, D. C., & Beckervordersandforth, R. (2019). Mitochondrial dysfunction in astrocytes impairs the generation of reactive astrocytes and enhances neuronal cell death in the cortex upon photothrombotic lesion. Frontiers in molecular neuroscience, 12, 40. 8. Arun, P., Abu-Taleb, R., Oguntayo, S., Wang, Y., Valiyaveettil, M., Long, J. B., & Nambiar, M. P. (2013). Acute mitochondrial dysfunction after blast exposure: potential role of mitochondrial glutamate oxaloacetate transaminase. Journal of neurotrauma, 30(19), 1645-1651. Figure 7. Co-stain Mitotracer red + LC3B at a 60X fluorescence microscopy. Samples of 2D monolayer primary astrocytes at 24h post-overpressure exposure. References

15 minutes

B

C

Figure 3. Measuring of mitochondrial health by using membrane-permeant JC-1 dye. JC-1 is a cationic dye (green) exhibits potential-dependent accumulation in mitochondria where it starts forming J aggregates (red). Mitochondrial depolarization is indicated by a fluorescence emission shift from green to red. Average ratio of intensity for green and red fluorescence emission were obtained using Mat Lab software and the data were normalized to average sham. A) Schematic illustration of JC-1 mechanism. Upon depolarization, JC-1 remains as monomer showing green fluorescence. B) Image of green (~529 nm) and red (~590 nm) fluorescence. All images were taken under same exposure time and used the same microscopy magnification (20X). C) Image collected from MATLAB software from proper data analysis. Grayscale image were obtained from ImageJ by calculating appropriate threshold from each fluorescence emission (green/Red) prior to use the software. A custom MATLAB code was created (by Nora Hlavac and Carly Norris) to help to assess pixel-to-pixel ratios between the red (mitochondrial) and green (cytoplasmic) fluorescent stains.

Figure 5. Measuring of mitochondrial integrity by using membrane-permeant JC-1 dye. Mitochondrial membrane integrity was assessed by live-cell imagining using JC-1, a radiometric dye. Statistical comparison were conducted between groups using GraphPad Prism 8.4.1 software. Unpaired T-test with Welch’s test correction (assuming different standard deviation) was used to analyze significant difference amongst groups. This experiment aim to mimic an excitotoxicity environment which is a hallmark of TBI injury post mechanical (primary) etiology. A) There is no statistical significant difference 24 hours post-8mM glutamate treatment without blasting injury as compared to sham, n=3/group (data are mean ± SEM; *p-value represents ≤ 0.005). B) There is no statistical significant difference 15 min post-8mM glutamate treatment without blasting injury as compared to sham, n=3/group (data are mean ± SEM; *p-value represents ≤ 0.005). In summary, no significance difference was identified which could indicate that during this acute time point the changes in mitochondrial integrity are mainly due to the mechanical injury itself.

Figure 1. Immunofluorescence: Glial fibrillary acidic protein (GFAP – green) as astrocyte marker and the nuclear counter stain DAPI (Blue). Images were taken at 20x.

123 2 0 2 1 R e s e a r c h R e c o g n i t i o n D a y