Astrocyte Viability and Functionality in Spatially Confined

Subscriber access provided by University of Winnipeg Library

Biological and Medical Applications of Materials and Interfaces

Astrocyte Viability and Functionality in Spatially Confined Microcavitation Zone Bo Chen, Jessica Tjahja, Sameep Malla, Caleb Liebman, and Michael Cho ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b21410 • Publication Date (Web): 14 Jan 2019 Downloaded from http://pubs.acs.org on January 15, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Astrocyte Viability and Functionality in Spatially Confined Microcavitation Zone

Bo Chen, Jessica Tjahja, Sameep Malla, Caleb Liebman and Michael Cho*

Department of Bioengineering, University of Texas at Arlington, Arlington, TX

*Corresponding Author University of Texas at Arlington Department of Bioengineering 500 UTA Blvd., Suite 226 Arlington, TX 76019

E-mail: [email protected]

1 ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 34

ABSTRACT Blast-induced traumatic brain injury (bTBI) can result in cell/tissue damages and lead to clinical and neuropsychiatric symptoms. Shockwaves from a blast propagate through the brain and initiate cascades of mechanical and physiological events that can adversely affect the brain function. While studies using animal models and brain slices have shown macroscale changes in the brain tissue in response to blast, systematic elucidation of coupling mechanisms is currently lacking. One mechanism that has been postulated and demonstrated repeatedly is the blast-induced generation and subsequent collapse of micron-size bubbles (i.e., microcavitation). Using a customdesigned exposure system we have previously reported that, upon collapsing of microbubbles, astrocytes exhibited changes in the cell viability, cellular biomechanics, production of reactive oxygen species, and activation of apoptotic signaling pathways. In this paper, we have applied microfabrication techniques and seeded astrocytes in a spatially controlled manner in order to determine the extent of cell damage from the site of the collapse of microbubbles. Such a novel experimental design is proven to facilitate our effort to examine the altered cell viability and functionality by monitoring the transient calcium spiking activity in real-time. We now report that the effect of microcavitation depends on the distance from which cells are seeded, and the cell functionality assessed by calcium dynamics is significantly diminished in the cells located within ~ 800 m of the collapsing microbubbles. Both calcium influx across the cell membrane via Ntype calcium channels and intracellular calcium store are altered in response to microcavitation. Finally, the FDA-approved poloxamer 188 (P188) was used to reconstitute the compromised cell membrane and restore the cell’s reparative capability. This finding may lead to a feasible treatment for partially mitigating the tissue damage associated with bTBI.


Page 3 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


KEYWORDS: blast-induced traumatic brain injury (bTBI), microcavitation, astrocytes, microfabrication, calcium dynamics, poloxamer P188



Page 4 of 34

1. Introduction Blast-induced traumatic brain injury (bTBI) represents a major public health dilemma with over 150,000 military people exposed to blast in the battlefield and diagnosed with a form of bTBI and the consequences can be manifested in a range of various clinical symptoms such fatigue, headache, tinnitus, and irritability.1-2 Blast-induced barotrauma with more than 8000 cases of bTBI were recently evaluated by the Defense and Veterans Brain Injury Center3. In most cases, only the symptoms are diagnosed and treated, and at present, there are no effective therapeutic treatments available. The development of therapeutic treatments would first require establishing a clear understanding of the mechanisms that mediate such injuries. Identification of potential mechanisms of brain tissue damage following blast exposure is compounded by the complexity of brain tissue and a lack of systematic approaches to examine subcellular effects. Multiple experimental studies designed to understand the tissue damage from bTBI have examined blast wave propagation via thoracic mechanism, ischemic brain damage, head acceleration, intracranial pressure increase, and direct skull deformation.4-7 However, an alternate feasible mechanism has emerged that suggests the shock wave induces sudden changes in the intracranial pressure and creates a transient vacuum, leading to the formation of energetic micronsize bubbles (i.e., microcavitation). Such mechanical assault can result in disruption of axonal pathways and to capillaries.8-9 Recently, our laboratory and others have found that blast-induced cavitation was indeed observed in a simulation model of bTBI.10-12 We have studied and reported altered biophysical and subcellular properties in astrocytes in response to exposure to a combination of shock waves and microcavitation. While the cells exposed the shockwave of ~ 10 MPa for several µs do not show any evidence of altered cell viability or functionality,10 subsequent collapses of microbubbles (< 100 µm diameter) induced cell detachment and death, and activation of apoptosis. Astrocyte response to injury is indeed complex. It involves many different pathways such as increased expression of glial fibrillary acidic protein(GFAP) and matrix metalloppeptidase 9 (MMP-9)13, upregulation of chondroitin sulfate proteoglycans (CSPG) 14 and assessment of A1 vs A2 phenotype markers15. Astrocytes are the most abundant cell type in the central nervous system and are crucial for blood-brain barrier homeostasis, the formation of intracellular communication networks, regulation of axonal outgrowth, and response to injury and repair.16 These responses are 4 ACS Paragon Plus Environment

Page 5 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


typically regulated by carefully orchestrated calcium dynamics. Intracellular calcium ions (Ca2+), one of the most versatile biological regulators for secretory functions, enzyme activity, intracellular

transport,

contractile

processes,

membrane

potential,

and

intracellular

communication,17-19 spontaneously fluctuate or exhibit spiking activity. While transiently altered calcium dynamics serves as a critical regulator, any prolonged disruption could be detrimental to the cell. It should not be surprising that unregulated changes in the Ca2+ homeostasis do contribute to the cell injury and death,19 and neurodegulation20 in bTBI. Systematic studies of the altered calcium dynamics in astrocytes in response to bTBI may illuminate potential mechanisms that adversely impact the cell viability and functionality. A clear understanding of the molecular and cellular cascade of events associated with bTBI is essential to the development of efficacious treatment strategies. In this paper, we applied microfabrication techniques to seed astrocytes in spatially defined patterns. By monitoring the calcium dynamics in real-time, the functionality of astrocytes seeded at different locations was determined in response to the collapse of microbubbles. Pharmacological agents are used to block specific calcium influx/efflux pathways and delineate the involvement of calcium transport across the cell that modulates the intracellular calcium dynamics. Furthermore, the FDA-approved P188 was used to reconstitute the compromised cell membrane and restore the cell’s reparative capability.



Page 6 of 34

2. Methods 2.1. Cell Cultures. Mouse astrocytes C8-D1A (CRL-2541) were purchased from American Type Culture Collection (ATCC, Manassas, VA) and cultured with 4500 mg/L high glucose Dulbecco Modified Eagle’s medium (DMEM; Sigma-Aldrich, St. Louis, MO), 5% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO), and 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO) in a 37°C and 5% CO2 incubator. When cells grew confluent between 70 to 90%, they were subcultured and seeded on 22  22 mm glass coverslips at the density of 10,000 cell/ml and cultured for about 6-7 days prior to experimentation. 2.2. Exposure Chamber. The custom-built exposure chamber contains two needle electrodes symmetrically placed in the center and separated 700 μm apart (Figure 1). Full details of the chamber design and characterization have been reported elsewhere.10-11, 21 Briefly, the chamber has a total volume of 2.5 mL and was designed to minimize temperature rise to < 1oC. Short electrical pulses of 4 kV and 800 s were applied across the electrodes to generate shockwaves and subsequent microbubbles. Transducers were placed at the top and bottom of the chamber to measure the strength of the peak pressure of 10 MPa.21 Shockwaves originating from the electrodes propagate to the top and bottom uniformly (i.e., principle of symmetry). Assuming the sound waves travel at 1500 m/s in water, it would require 3 s to traverse the distance from the electrodes to the top or bottom of the exposure chamber. However, the microbubbles rise only to the top in tens of seconds and interact with astrocytes seeded at the top of the exposure chamber. Capturing images of microbubbles collapsing at the focal plane where the cells were seeded allowed detailed analysis of size-distribution and density of microbubbles.10 Our previous results indicate that when microbubbles collapse, they create several areas on the substrate in which cells are detached.10 We therefore coined a term, 2D crater, to describe the areas of cell detachment. Because the formation of craters appears stochastic, One design challenge was to confine the microbubbles in one location. One would be then able to control the predictable location of the crater and study the cells that are seeded outside the crater, not detached but exhibit the molecular signatures of cellular damage and apoptosis.11 By placing a PDMS sheet with an aperture immediately above the electrodes, the microbubbles were observed to rise in a column and reached the coverslip before they can diffusively migrate. With this simple but rather important modification, only one central 6 ACS Paragon Plus Environment

Page 7 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


location of collapsing microbubbles was engineered. We coined the term crater to refer to the area where cells were detached from the substrate in response to microcavitation, and this term is used throughout the manuscript. 2.3. Cell Viability Assay and FAM-FLICA™ Caspase 3 Assay Kit. The Live/Dead Assay (Life Technologies, USA) combines fluorescence reagents (Calcein-AM and Ethidium homodimer-1) to yield two-color discrimination of the population of live (green) or dead cells (red). Following an exposure to shockwave and microbubbles, astrocytes were washed several times with PBS and then incubated with 2 μM Calcein-AM and 4 μM EthD-1 for 30 min at room temperature, washed again and imaged using Nikon microscopes. The FAM-FLICA™ Caspase 3 Assay Kit (Immunochemistry Technologies LLC, Minneapolis, MN) was used to quantitate apoptosis by measuring the caspase activity in astrocytes. 2.4. Live Cell Calcium Signaling. To study the intracellular calcium signaling, the astrocytes were loaded with 5 μM Fluo-4 AM (Thermo Fisher Scientific, Waltham, MA) for 30 min at room temperature, rinsed three times with Hank's balanced salt solution (HBSS, Sigma-Aldrich, St. Louis, MO). Coverslip with Fluo-4 AM loaded astrocytes was mounted on the top of exposure chamber and immersed in an artificial cerebrospinal fluid (ACSF) (125 mM NaCl, 3 mM KCl, 10 mM glucose, 26 mM NaHCO3, 1.1 mM NaH2PO4, 2 mM CaCl2, 1 mM MgSO4; pH adjusted to 7.4), and imaged. Fluorescence signals that represent calcium dynamics were monitored in real time at 5 s intervals for a period of 10 min before and after an exposure to microcavitation (20 min total). Average fluorescence intensities of each cell were determined using ImageJ (https://imagej.nih.gov/ij/) and its built-in segmentation tool. 2.5. Immunofluorescence staining. Cells were fixed with 4 % paraformaldehyde for 15 minutes, washed with PBS, blocked in 5% normal goat serum with 0.1% Triton X-100 and then blocked with 3% bovine serum albumin (BSA), then labeled with a glia marker GFAP Monoclonal Antibody (GA5), eFluor 570(Thermo Fisher Scientific, Waltham, MA). GFAP is known to be a prototypical marker for immunohistochemical identification of astrocytes.22



Page 8 of 34

2.6. Microcontact Printing of Linear Pattern for Controlled Cell Seeding. Micropatterns consisting of parallel linear strips of 100 μm wide were fabricated on silicon wafers using standard photolithographic techniques.23-24 Each adjacent linear strip was separated by 100 μm. Complementary PDMS (polydimethylsiloxane) replicas were generated and used as stamps in subsequent microcontact printing steps to form strip patterns of copolymer poly(ethylene glycol)block-polylactide methyl ether PEG average MW 5000, PLA average MW ~500 (PEG-L-PLA) directly on culture dishes. Briefly, a solution of the polymer (20 μL of a 1.0 wt% solution in dichloroethane for a stamp approximately 1.5 cm2) was spread uniformly over the stamp. After the solvent had not completely dried, the stamp was pressed gently against the surface of the culture dish for a few seconds and peeled away gently. The printed patterns were cured for overnight at 60 °C. 2.7. Pharmacological Agents. To determine potential calcium influx/efflux pathways, various blockers were used. Cells were treated with 1,2-Bis(2-amino-phenoxy)ethane-N,N,N′,N′tetraacetic acid (BAPTA-AM, 20 μM) to chelate intracellular calcium or incubated with calciumfree HBSS (supplemented by 2 mM Mg2+). The role of voltage-sensitive Ca2+ channels (VSCCs) and mechanosensitive cation-selective channels (MSCCs) were determined by treating the cells 30 min with T- and R-type VSCC blockers (nickel, 100 μM25), L-type VSCC blockers (verapamil, 100 μM), N-type VSCC blockers (ɷ-conotoxin GVIA, 100 μM), and non-specific MSCC blocker (GdCl3, 100 μM), respectively. In addition, 2-aminoethoxydiphenyl borate 2-APB (100 μM) was used to block the IP3-dependent signaling.26 All reagents were purchased from Sigma Chemical (Sigma, St. Louis, MO) unless otherwise indicated. 2.8. Statistical analysis. Data represent mean ± standard deviation (SD). Pair-wise comparisons between the means of two different groups were performed using Student t-test. The differences between two groups of data were considered statistically significant or statistically highly significant if p < 0.05(*) or < 0.01(**), respectively. Multiple comparisons were performed using analysis of variance (ANOVA), followed by Tukey's post-test to determine differences.


Page 9 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3. RESULTS 3.1. Characterization of Crater Size and Location. The distribution of the sizes of microbubbles, biological responses in astrocytes, and cell detachment from the substrate were all observed to depend on repetitive exposures to microcavitation. While we were able to control the density of microbubbles as demonstrated in our previous work10, the microbubbles vertically moved to the top of the chamber in an uncontrollable manner. Indeed, the locations where cells detached (i.e., 2D craters) due to the collapsing microbubbles were many, unpredictable and varied in size (Figure 1A). In order to overcome this technical limitation, a PDMS sheet with a single aperture at the center was fabricated and inserted 1 mm above the two electrodes (Figure 1B). This simple but important modification confined the microbubbles to rise in a column and restricted them to one central crater whose size and location can be predicted and regulated. As a control experiment, we fabricated the same aperture but placed it away from the electrodes (Figure 1C). The rationale was to confirm a very few, if any, of the microbubbles was able to escape through the aperture and thus no microbubbles reached the top of the chamber where astrocytes were seeded. Moreover, there was no cell detachment and the astrocytes still maintained normal calcium dynamics. These observations further confirmed the cell detachment was due to the collapse of microbubbles. The configuration described in Figure 1B was used for all of the experiments in this study. Several different sizes of aperture (3.2 to 9.6 mm in diameter) were fabricated and histograms were constructed to determine the distribution of the number and size of microbubbles under repetitive blast conditions (Supporting Information S1). Most of the microbubbles measured were ~ 30 µm radius without an aperture but the craters were stochastically formed. However, the radii became larger using a 3.2 or 4.8 mm aperture, and essentially no changes in the microbubble size were detectable using even a larger 9.6 mm PDMS aperture (Figure 1D). Average radii also depended on the number of electrical pulses (i.e., repetitive blasts) applied to generate microbubbles. This characterization is rather important because the severity of bTBI is often correlated with repetitive exposures to blast. Using either the 3.2 or 4.8 mm aperture, average microbubble sizes in response to 5 repetitive pulses were observed indistinguishable (Figure 1E). These characterization studies provided us with a set of optimized parameters; a 4.8 mm aperture



Page 10 of 34

to produce a column of microbubbles rising to the top of the chamber with an average radius of ~ 55 m. 3.2. Determination of Pressure of Collapsing Microbubbles. The collapsing microbubbles were observed to immediately detach astrocytes from the substrate and spatially defined regions (i.e., crater) without any cells remaining attached. We reported that a typical crater was rapidly expanded and reached the size that is comparable to the aperture dimension (e.g., ~ 5 mm) in less than 20 s.10 This observation suggested that the pressure associated with collapsing microbubbles could be substantial. However, the magnitude of pressure generated by the collapse of microbubbles remains unknown and needs be estimated. Using the pressure-sensitive film sensors provided to us by Sensor Products, Inc (Madison, NJ; www.sensorprod.com), the film was cut into 22 × 22 mm squares, placed on a coverslip and mounted onto the top of exposure chamber in HBSS buffer. Microbubbles were generated by electrical pulses and allowed to collapse on the sensors. The film strip was retrieved and shipped to the manufacturer for image analysis. As shown in Figure 2A, the pressure associated with collapsing microbubbles produced the change of colors that have been calibrated. Average pressure measured using the pressure-sensitive film sensors indicated that the collapsing microbubbles were capable of generating 55.8±8.0 kPa (Figure 2B). While this secondary shear pressure (sometimes referred to as microjet in literature) is approximately more than two orders of magnitude smaller than the peak shockwave pressure measured using transponders (~ 10 MPa)21 but nonetheless large enough to cause cell detachment. 3.3. Cell Detachment and Viability. One challenge is to identify whether the cell detachment is due to the collapse of microbubbles instead of the shockwave of ~ 10 MPa peak pressure. We exploited the principle of symmetry and seeded astrocytes directly above and below the electrodes (4.8 mm in either direction). Shockwaves produced by the electrodes should propagate to the top and bottom of the chamber, while the microbubbles can rise only to the top through the inserted aperture (see Figure 1). We have previously reported that cell detachment is observed only at the top of the chamber, and the cells seeded at the bottom of the chamber did not show any indication of cell detachment or apoptosis.10 In contrast, astrocytes seeded at the top of the chamber showed visible evidence of cell detachment. A Live/Dead cell viability assay was first used to determine the extent of cell death and detachment. In response to one pulse blast, cell viability remained unchanged (Figure 3A and 3B). Exposure of astrocytes to 5 repetitive pulses led to the formation of only one crater as well as cell detachment within the crater (Figure 3C and 3D). It is interesting 10 ACS Paragon Plus Environment

Page 11 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


to note that the 5 pulse treatment caused a single crater whose dimension was comparable to that of the aperture (4.8 mm). About 95 % of the cells inside of the aperture area were detached, and the cells outside of the aperture area remained attached to the substrate. Insertion of an aperture consistently produced a predictable crater that allowed us to design experiments and probe the impact of the collapsing microbubbles as a function of distance from the crater’s edge. We also note there appears to be a fraction of cells at the crater’s periphery (Figure 3D) that may be nonviable but still remained attached to the substrate. 3.4. Calcium Dynamics. Having characterized and modified the exposure system using apertures, we next monitored the calcium dynamics by loading astrocytes with a calcium-sensitive fluorophore (Fluo-4 AM), and changes in the intracellular calcium concentration were recorded fluorescently at 5 s intervals. Representative images of control astrocytes loaded with Fluo-4 AM (Figure 4A) and the astrocytes following microcavitation (Figure 4B) are shown. Of these cells, we randomly selected 4 cells and monitored their calcium spiking activity as a function of time. A calcium spike is defined as the peak fluorescence intensity that exceeds at least 15% of the baseline intensity. In the control cell (labeled #1; trace 1 in Figure 4C), 7 calcium spikes were identified with the 20 min observation time; 10 min prior to a sham microcavitation and 10 min afterward. The calcium spiking can also be monitored in real-time throughout the collapse of microcavitation. For example, the trace 2 (Figure 4C) shows the altered calcium spiking pattern in a cell located near the crater’s edge (~ 80 m). Following an exposure to microcavitation (indicated by an arrow), there appears to be a large increase in the intracellular calcium concentration and then a steady decrease without any spiking activity was observed. The cell maintained an elevated intracellular calcium concentration level, suggesting an influx of extracellular calcium ions was likely. Examining other cells following the collapse of microbubbles, the calcium spiking was either diminished over time (trace 3; Figure 4D) or no such activity could be observed at all (trace 4; Figure 4D). These findings suggest that the astrocytes that were plated near the site of the collapse of microbubbles remained attached to the substrate but lost the cell functionality. These findings cannot be attributed to the primary shock waves but rather to the secondary pressure (55.8±8.0 kPa) generated by the collapse of microbubbles that could have produced a short-lived shear stress or a microjet.27-28 The four traces of intracellular calcium responses shown in Figure 4 are meant to illustrate different calcium spiking profiles. Quantitative data analyses are provided below. 11 ACS Paragon Plus Environment


Page 12 of 34

Immunostaining for glial fibrillary acidic protein (GFAP) has been used to assess reactive astrocytes and cell damage.22 Astrocytes prior to an exposure to microcavitation exhibit a baseline expression of GFAP (Figure 5A). As expected, in response to microcavitation, there was no cell found in the crater, e.g., astrocytes detachment. In addition, the astrocytes that survived the impact of the secondary shear pressure and remained attached to the substrate showed a noticeably higher level of GFAP expression (Figure 5B). The higher GFAP expression was particularly noticeable in astrocytes that were at the edge of the crater. Taken together with the altered calcium dynamics, these findings indicate the astrocytes may have sustained an injury. Micro-patterned technique becomes an effective tool to study the cell-surface interactions. 24, 2930

One important issue remains unresolved as to whether we can measure the critical shielding

distance beyond which the brain cells are not adversely impacted by the collapse of microbubbles. Moreover, it is difficult to separate the cells because they are typically connected to form a network. To overcome these limitations, a convenient microcontact printing technique was used to spatially control cell seeding. For example, linear patterns were successfully fabricated and a fluorophore staining method was used to validate the pattern quality (Figure 6A and 6B). Astrocytes were seeded on the linearly patterned strips of 100 m width each and with 100 µm separation between two adjacent strips. A brightfield image of astrocytes also illustrated the boundary of a crater and blurred image of electrodes located 4.8 mm below the focal plane, as indicated by the two arrows (Figure 6C). Within the crater, ~ 95% of cells detached after microbubble exposure (Figure 6D). Because the location of cell detachment can be predicted and regulated, the distance-dependent calcium spiking in these astrocytes was monitored and measured as a function of distance away from the crater’s edge (Figure 7A). Although the 100 m strips, fabricated using PEG-PLA, were found to autofluorescence, the signals from the cells loaded with Fluo-4 AM provide sufficient fluorescence intensities to monitor the calcium dynamics. Cells seeded within the first 200 m from the crater’s edge showed a significantly diminished spiking activity in response to microcavitation (Figure 7B). As the distance from the crater’s edge increased, the calcium spiking appears to be gradually restored. For example, astrocytes seeded at a distance between > 800 m resembled the typical spiking profile observed in control cells. Statistical analyses indeed confirmed the observation (Figure 7C).


Page 13 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


We next examined the apoptosis following microcavitation. Using the FAM-FLICA™ Caspase 3 assay kit, the fluorescent signals from the active Caspase 3 enzyme activity was measured (Figure 8). Upregulation of such signals indicates activation of the Caspase signaling pathways that lead to apoptosis. Consistent with the altered calcium dynamics shown in Figure 7, activation of the Caspase signaling was significantly elevated in the cells seeded between 200 to 600 m from the crater’s edge. However, other cells seeded beyond the shielding distance (e.g., 800 m) showed at least a 6-fold decrease in the Caspase enzyme activity. 3.5. Calcium Influx/Efflux Pathways. The calcium signaling mechanisms in astrocytes are complex.31-33 In order to delineate potential pathways by which the intracellular calcium concentration may be regulated, we applied several pharmacological agents without exposing astrocytes to microcavitation. To determine the role of extracellular calcium and/or internal calcium store in control cells, either a calcium-free buffer was used, or a selective calcium chelator (BAPTA-AM; 20 μM) was applied, respectively. As shown in Figure 9, both extracellular calcium influx and release of calcium from the internal store are required to sustain proper calcium spiking activity. Furthermore, to determine specific calcium influx pathways, astrocytes were first treated with Gd3+ (mechanically activated cation channel blocker; 100 μM) or verapamil (L-type calcium channel blockers; 100 μM). Neither blockers had any impact in control astrocytes, and the calcium spiking was not modulated (Figure 9A). However, when the N-type calcium channels were inhibited by ɷ-conotoxin GVIA (100 μM), the calcium spiking was noticeably diminished (Figure 9B), suggesting the calcium influx may be mediated primarily through the N-type channels. Interestingly, after the cells were exposed to microcavitation, the same N-type blocker (i.e., conotoxin) was capable of sustaining the calcium spiking activity in the cells presumed to be damaged and undergoing apoptosis. Incubation of the cells with both ɷ-conotoxin and verapamil together showed no additional beneficial effect. Finally, we tested the hypothesis that astrocytes that were partially damaged by the secondary shear pressure may be rescued by sealing the cell membrane using the FDA-approved P188 (0.5 mM). If the plasma membrane of the cells near the crater is compromised, the potential rescue of the affected cells by P188 should restore the calcium dynamics. Therefore, we exposed astrocytes to microcavitation and treated the cells with varying duration of the P188 incubation time. Of astrocytes seeded within 600 m from the crater’s edge (i.e., cells are still affected by 13 ACS Paragon Plus Environment


Page 14 of 34

microcavitation), a partial restoration of the calcium spiking by the P188 treatment was evident and determined to be time-dependent (Figure 10). A 6 hr incubation with P188 following an exposure to microcavitation significantly restored the calcium spiking whereas non-treated cells incubated in the media alone without P188 exhibited essentially no calcium activity, as expected. A longer incubation time with P188 did not further increase the capability to sustain calcium spiking in these rescued astrocytes. These results suggest that the cell membrane integrity is critical to maintaining the cell functionality and that the membrane sealing by P188 might lead to the development of a therapeutic treatment.


Page 15 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4. DISCUSSION The details of the engineering approaches and techniques to produce pressurized microbubbles and monitoring their collapsing have been reported elsewhere.10-11,

21

It is important to note that,

applying the principle of symmetry, the impact of shock waves (peak pressure measured ~ 10 MPa with 3 s interaction time with astrocytes) did not alter cell viability or cell functionality,10 indicating the observed biological effects reported in this paper and elsewhere are likely due to the collapse of microbubbles. Advancing further our engineering strategies, at least two additional novel ideas were incorporated. First, an aperture was selectively used to control the movement of microbubbles in a column that allowed the formation of only one crater (i.e., area of cell detachment). It provided us with a capability to reproducibly create the craters, and accurately predict their location. Second, microfabrication techniques were applied to seed astrocytes in a distance-dependent manner. These simple but creative ideas made possible to examine the impact of microcavitation on the cells plated some distance away from the crater site, and thereby achieving one of the goals of the current studies of quantitative determination of the “shielding distance”. Beyond this critical distance, cells are likely shielded from the secondary shear pressure or waterjet, presumably produced by the collapse of microcavitation. Moreover, we are not aware of any published work that directly measured the pressure of collapsing microbubbles. As shown in Figure 2, we utilized a pressure-sensitive film to record such pressures estimated ~ 55 kPa. The secondary shear pressure was demonstrated to adversely impact the cells seeded within ~ 800 m from the crater’s periphery. Finally, use of the FDA-approved P188 was highlighted to mitigate and minimize the detrimental effects of microcavitation by sealing the cell membrane and restoring the cell functionality. The spontaneous calcium spiking is typically found in normal astrocytes. These cells integrate synaptic transmission by modulating the calcium activation and therefore loss of such a capacity to regulate the calcium dynamics could imply dysfunctional astrocytes or a pathway to cell death. The astrocyte network was also shown to propagate the calcium activity of the blast-induced shockwaves.13 One simple but quantitative measure of the astrocyte’s calcium dynamics is to determine the cell’s calcium spiking rate. For example, astrocytes were found to exhibit calcium spiking at the rate of 0.5/min.34 It is interesting to compare that we observed a similar rate (0.57 spikes/min; see Figure 9A control). While transient changes in the calcium spiking activity are thought to be necessary for the cells to respond to stimuli, the prolonged and sustained elevation 15 ACS Paragon Plus Environment


Page 16 of 34

of intracellular calcium concentration is probably detrimental.35 Thus cells possess various mechanisms to self-regulate and achieve calcium homeostasis. This would likely involve changes in the intracellular calcium concentration ([Ca2+]i) mediated by influx/efflux across the cell membrane and activation of internal calcium store (e.g, endoplasmic reticulum31). As shown in Figure 9A, either depletion of extracellular Ca2+ or inhibiting the release of the internal store can abolish the calcium spiking activity in control astrocytes. This finding is not surprising and provides additional evidence that the calcium homeostasis in astrocytes is regulated by complex intricate interplays between the extracellular and intracellular Ca2+.36 Interestingly, the Ca2+ influx does not appear to be mediated by the stretch receptors or the L-type calcium channels because incubation of cells with either Gd3+ or verapamil, respectively, did not alter the calcium dynamics. However, a treatment of cells with an N-type channel blocker (-conotoxin) significantly decreased the calcium spiking activity in control astrocytes not exposed to microcavitation. More interestingly, a treatment of cells with the N-type channel blocker after exposing them to microcavitation was observed to restore the calcium dynamics (Figure 9B). The implication could be that microcavitation causes a calcium influx via the N-type channels, and such an influx interferes with the complex machinery in the cell and abolishes the calcium spiking activity. Therefore, blocking extracellular Ca2+ entry via the N-type channels can help restore the cell viability and functionality. Moreover, this finding is consistent with the previously reported results that demonstrated several N-type channel blockers provided neuroprotection37-38 and in some cases cardioprotection.39-40 We can now demonstrate that the N-type channel blockers protect astrocytes that were mechanically impacted by the collapse of microbubbles. Since bTBI was caused by a mechanical means, it is difficult to contemplate that microcavitation specifically targets the N-type channels. Rather, it is more likely the N-type channels in astrocytes perhaps better mediate the Ca2+ currents than other types of calcium channels. As an example, astrocytes were shown to regulate the synapse development through the N-type channels.41 P188 is a nonionic, amphiphilic, linear copolymer, including a central hydrophobic group that is linked on both sides by two hydrophilic chains of polyoxyethylene.42 This copolymer was approved by U.S. FDA nearly 6 decades ago as a therapeutic reagent to reduce viscosity in the blood before transfusions.43 It has been demonstrated to be safe when given for up to 72 hours in humans with a half-life of 18 hours.44 One of the remarkable characteristics of P188 is its ability to incorporate into the phospholipid bilayer and lowering the surface tension.26 These 16 ACS Paragon Plus Environment

Page 17 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


characteristics are prerequisite for repairing the damaged cell membrane.45-47 As the membrane repair is achieved with tighter lipid packing, the P188 should be squeezed out from the membrane. 47-48

The similar reparative mechanism appears to be applicable when the partially comprised

astrocytes were treated with P188 following an exposure to microcavitation (Figure 10). A 6-hr treatment with P188 showed a considerable and statistically significant restoration of the calcium dynamics. P188 may be considered as a therapeutic agent for the development of an effective therapy for bTBI patients. Our results are consistent with the recently published papers that examined the effect of P188 using both in vitro and in vivo TBI models. For example, an in vitro shearing device was applied to mechanically injure primary neurons and the injured cells were treated with P188, which demonstrated neuroprotection.49 Serbest et al.50 also used a mechanical injury model to show that multiple events are involved in the long-term response of neurons, and the P188 appeared to provide a significant protection from necrosis and apoptosis. Studies of in vivo models indicate P188 attenuates TBI-induced brain enema by resealing the blood brain barrier51 and contributes to neuroprotection and anti-inflammation in rats in response to TBI induced by cortical impact.52 It is indeed exciting to observe both the N-type channel blocker (-conotoxin) and P188 can reverse and restore the functionality of astrocytes. Since these two compounds are thought to affect different molecular mechanisms, one interesting question was raised. Would the combined treatment of both -conotoxin and P188 show additive or synergistic effects? The preliminary experimental results suggest that astrocytes pre-treated with -conotoxin, exposed to microcavitation and post-treated with P188 for 3 hrs exhibited similar calcium spikes when compared to the post-treatment with P188 alone for 3 hrs (Figure 10). The extent of restored cell functionality is not significantly altered. Because P188 is not expected to specifically block the Nchannels, an interesting postulate can be formulated as follows. The combined application of an N-channel blocker and P188 may not provide a synergistic therapeutic treatment for those who may have suffered traumatic brain injuries. However, systematic treatment with N-channel blockers may require delivery strategies that are specific to the injured cells. In contrast, P188 is expected to incorporate into the plasma membrane of the compromised cells in response to microcavitation, suggesting P188 may be more efficacious in response to injury associated with bTBI.



Page 18 of 34

5. CONCLUSIONS This study proposed a direct microfabrication technique to seed astrocytes in a spatially controlled manner in order to determine the extent of cell damage from the site of the collapse of microbubbles. This novel experimental design is proven to facilitate our effort to examine the altered cell viability and functionality by monitoring the transient calcium spiking activity in realtime. We find that the effect of microcavitation depends on the distance from which cells are seeded, and the cell functionality assessed by calcium dynamics is significantly diminished in the cells located within ~ 800 m. Both calcium influx across the cell membrane via N-type calcium channels and intracellular calcium store are altered in response to microcavitation. We are currently developing experimental designs to utilize two-color imaging techniques to monitor simultaneously the calcium spiking activity and up-regulation of the Caspase 3 expression and establish potential correlation between the modulated calcium dynamics and cell viability (e.g., Caspase 3 activity). Finally, P188 was used to reconstitute the compromised cell membrane and restore the cell’s reparative capability. This finding may lead to a feasible treatment for partially mitigating the tissue damage associated with bTBI. Although there are potential concerns with toxicity with long-term application and bioavailability in some cases, there still appears to be promise for the application of poloxamers in the treatment of various diseases in the future.


Page 19 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Supporting Information: Histograms of distribution of microbubbles genereated using 4 different aperture sizes; (a) 0, (b) 3.2 mm, (c) 4.8 mm, and (d) 9.6 mm diameter aperture.



Page 20 of 34

ACKNOWLEDGEMENTS

This work was supported by a grant (N00014-16-1-2140) from the Office of Naval Research.


Page 21 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


REFERENCES (1) Hoge, C. W.; McGurk, D.; Thomas, J. L.; Cox, A. L.; Engel, C. C.; Castro, C. A. Mild Traumatic Brain Injury in U.S. Soldiers Returning from Iraq. N. Engl. J. Med. 2008, 358 (5), 453463. (2) Ling, G.; Bandak, F.; Armonda, R.; Grant, G.; Ecklund, J. Explosive Blast Neurotrauma. J. Neurotrauma. 2009, 26 (6), 815-825. (3) Benzinger, T. L.; Brody, D.; Cardin, S.; Curley, K. C.; Mintun, M. A.; Mun, S. K.; Wong, K. H.; Wrathall, J. R. Blast-Related Brain Injury: Imaging for Clinical and Research Applications: Report of the 2008 St. Louis Workshop. J Neurotrauma. 2009, 26 (12), 2127-2144. (4) Long, J. B.; Bentley, T. L.; Wessner, K. A.; Cerone, C.; Sweeney, S.; Bauman, R. A. Blast Overpressure in Rats: Recreating a Battlefield Injury in the Laboratory. J. Neurotrauma. 2009, 26 (6), 827-840. (5) Leonardi, A. D. C.; Bir, C. A.; Ritzel, D. V.; VandeVord, P. J. Intracranial Pressure Increases During Exposure to a Shock Wave. J. Neurotrauma. 2011, 28 (1), 85-94. (6) Moss, W. C.; King, M. J.; Blackman, E. G. Skull Flexure from Blast Waves: A Mechanism for Brain Injury with Implications for Helmet Design. Phys. Rev. Lett. 2009, 103 (10), 108702. (7) Courtney, M. W.; Courtney, A. C. Working toward Exposure Thresholds for Blast-Induced Traumatic Brain Injury: Thoracic and Acceleration Mechanisms. Neuroimage 2011, 54, S55-S61. (8) Nakagawa, A.; Manley, G. T.; Gean, A. D.; Ohtani, K.; Armonda, R.; Tsukamoto, A.; Yamamoto, H.; Takayama, K.; Tominaga, T. Mechanisms of Primary Blast-Induced Traumatic Brain Injury: Insights from Shock-Wave Research. J. Neurotrauma. 2011, 28 (6), 1101-1119. (9) Wolf, S. J.; Bebarta, V. S.; Bonnett, C. J.; Pons, P. T.; Cantrill, S. V. Blast Injuries. The Lancet 2009, 374 (9687), 405-415. (10) Sun, S.; Kanagaraj, J.; Cho, L.; Kang, D.; Xiao, S.; Cho, M. Characterization of Subcellular Responses Induced by Exposure of Microbubbles to Astrocytes. J. Neurotrauma. 2015, 32 (19), 1441-1448. (11) Kanagaraj, J.; Chen, B.; Xiao, S.; Cho, M. Reparative Effects of Poloxamer P188 in Astrocytes Exposed to Controlled Microcavitation. Ann. Biomed. Eng. 2018, 46 (2), 354-364. (12) Goeller, J.; Wardlaw, A.; Treichler, D.; O'Bruba, J.; Weiss, G. Investigation of Cavitation as a Possible Damage Mechanism in Blast-Induced Traumatic Brain Injury. J. Neurotrauma. 2012, 29 (10), 1970-1981. (13) Ravin, R.; Blank, P. S.; Busse, B.; Ravin, N.; Vira, S.; Bezrukov, L.; Waters, H.; GuerreroCazares, H.; Quinones-Hinojosa, A.; Lee, P. R. Blast Shockwaves Propagate Ca2+ Activity Via Purinergic Astrocyte Networks in Human Central Nervous System Cells. Sci. Rep. 2016, 6, DOI: 10.1038/srep25713. (14) Asher, R. A.; Morgenstern, D. A.; Fidler, P. S.; Adcock, K. H.; Oohira, A.; Braistead, J. E.; Levine, J. M.; Margolis, R. U.; Rogers, J. H.; Fawcett, J. W. Neurocan Is Upregulated in Injured Brain and in Cytokine-Treated Astrocytes. J. Neurosci. 2000, 20 (7), 2427-2438. (15) Liddelow, S. A.; Barres, B. A. Reactive Astrocytes: Production, Function, and Therapeutic Potential. Immunity 2017, 46 (6), 957-967. (16) Burda, J. E.; Bernstein, A. M.; Sofroniew, M. V. Astrocyte Roles in Traumatic Brain Injury. Exp. Neurol. 2016, 275, 305-315. (17) Petersen, O. H.; Michalak, M.; Verkhratsky, A. Calcium Signalling: Past, Present and Future. Cell Calcium 2005, 38 (3), 161-169.



Page 22 of 34

(18) Racay, P.; Lehotsky, J. Intracellular and Molecular Aspects of Ca2+-Mediated Signal Transduction in Neuronal Cells. Gen. Physiol. Biophys. 1996, 15, 273-289. (19) Weber, J. T. Calcium Homeostasis Following Traumatic Neuronal Injury. Curr. Neurovasc. Res. 2004, 1 (2), 151-171. (20) Small, D. H. Dysregulation of Calcium Homeostasis in Alzheimer’s Disease. Neurochem. Res. 2009, 34 (10), 1824-1829. (21) Kang, D.; Nah, J. B.; Cho, M.; Xiao, S. Shock Wave Generation in Water for Biological Studies. IEEE Trans. Plasma Sci. 2014, 42 (10), 3231-3238. (22) Sofroniew, M. V.; Vinters, H. V. Astrocytes: Biology and Pathology. Acta Neuropathol. 2010, 119 (1), 7-35. (23) Qin, D.; Xia, Y.; Whitesides, G. M. Soft Lithography for Micro-and Nanoscale Patterning. Nat. Protoc. 2010, 5 (3), 491-502. (24) Singh, A. V.; Raymond, M.; Pace, F.; Certo, A.; Zuidema, J. M.; McKay, C. A.; Gilbert, R. J.; Lu, X. L.; Wan, L. Q. Astrocytes Increase Atp Exocytosis Mediated Calcium Signaling in Response to Microgroove Structures. Sci. Rep. 2015, 5, DOI: 10.1038/srep07847. (25) Treinys, R.; Kaselis, A.; Jover, E.; Bagnard, D.; Šatkauskas, S. R-Type Calcium Channels Are Crucial for Semaphorin 3a-Induced Drg Axon Growth Cone Collapse. PLoS One 2014, 9 (7), DOI: e102357. (26) Young, S. Z.; Platel, J. C.; Nielsen, J. V.; Jensen, N.; Bordey, A. Gabaa Increases Calcium in Subventricular Zone Astrocyte-Like Cells through L-and T-Type Voltage-Gated Calcium Channels. Front. Cell Neurosci. 2010, 4 (8), DOI: 10.3389/fncel.2010.00008. (27) Lohse, D.; Zijm, W. H. M. Bubble Puzzles. Phys. Today 2003, 56 (2), 36-41. (28) Yang, E.; Li, J.; Cho, M.; Xiao, S. Cell Fragmentation and Permeabilization by a 1 Ns Pulse Driven Triple-Point Electrode. Biomed Res. Int. 2018, 2018, 1-10. (29) Singh, A. V.; Patil, R.; Thombre, D. K.; Gade, W. Micro‐Nanopatterning as Tool to Study the Role of Physicochemical Properties on Cell–Surface Interactions. J. Biomed. Mater. Res. A 2013, 101 (10), 3019-3032. (30) Singh, A. V.; Gharat, T.; Batuwangala, M.; Park, B. W.; Endlein, T.; Sitti, M. ThreeDimensional Patterning in Biomedicine: Importance and Applications in Neuropharmacology. J. Biomed. Mater. Res. B Appl. Biomater. 2018, 106 (3), 1369-1382. (31) Rusakov, D. A. Disentangling Calcium-Driven Astrocyte Physiology. Nat. Rev. Neurosci. 2015, 16 (4), 226-233. (32) Bazargani, N.; Attwell, D. Astrocyte Calcium Signaling: The Third Wave. Nat. Neurosci. 2016, 19 (2), 182-189. (33) Volterra, A.; Liaudet, N.; Savtchouk, I. Astrocyte Ca2+ Signalling: An Unexpected Complexity. Nat. Rev. Neurosci. 2014, 15 (5), 327-335. (34) Sakuragi, S.; Niwa, F.; Oda, Y.; Mikoshiba, K.; Bannai, H. Astroglial Ca2+ Signaling Is Generated by the Coordination of Ip3r and Store-Operated Ca2+ Channels. Biochem. Biophys. Res. Commun. 2017, 486 (4), 879-885. (35) Weber, J. T. Altered Calcium Signaling Following Traumatic Brain Injury. Front. Pharmacol. 2012, 3 (60), 1-16. (36) Petravicz, J.; Boyt, K. M.; McCarthy, K. D. Astrocyte Ip3r2-Dependent Ca2+ Signaling Is Not a Major Modulator of Neuronal Pathways Governing Behavior. Front. Behav. Neurosci. 2014, 8, 384. (37) Valentino, K.; Newcomb, R.; Gadbois, T.; Singh, T.; Bowersox, S.; Bitner, S.; Justice, A.; Yamashiro, D.; Hoffman, B.; Ciaranello, R. A Selective N-Type Calcium Channel Antagonist 22 ACS Paragon Plus Environment

Page 23 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Protects against Neuronal Loss after Global Cerebral Ischemia. Proc. Natl. Acad. Sci. U.S.A. 1993, 90 (16), 7894-7897. (38) Pringle, A.; Benham, C.; Sim, L.; Kennedy, J.; Iannotti, F.; Sundstrom, L. Selective N-Type Calcium Channel Antagonist Omega Conotoxin Mviia Is Neuroprotective against Hypoxic Neurodegeneration in Organotypic Hippocampal-Slice Cultures. Stroke 1996, 27 (11), 2124-2130. (39) Nattel, S. N-Type Calcium Channel Blockade: A New Approach to Preventing Sudden Cardiac Death? Cardiovas. Res. 2014, 104, 1-2. (40) Kosaka, T.; Nakagawa, M.; Ishida, M.; Iino, K.; Watanabe, H.; Hasegawa, H.; Ito, H. Cardioprotective Effect of an L/N-Type Calcium Channel Blocker in Patients with Hypertensive Heart Disease. J. Cardiol. 2009, 54 (2), 262-272. (41) Mazzanti, M.; Haydon, P. G. Astrocytes Selectively Enhance N‐Type Calcium Current in Hippocampal Neurons. Glia 2003, 41 (2), 128-136. (42) Marks, J. D.; Pan, C. Y.; Bushell, T.; Cromie, W.; Lee, R. C. Amphiphilic, Tri-Block Copolymers Provide Potent Membrane-Targeted Neuroprotection. FASEB J. 2001, 15 (6), 11071109. (43) Moloughney, J. G.; Weisleder, N. Poloxamer 188 (P188) as a Membrane Resealing Reagent in Biomedical Applications. Recent Pat. Biotechnol. 2012, 6 (3), 200-211. (44) Adams-Graves, P.; Kedar, A.; Koshy, M.; Steinberg, M.; Veith, R.; Ward, D.; Crawford, R.; Edwards, S.; Bustrack, J.; Emanuele, M. Rheothrx (Poloxamer 188) Injection for the Acute Painful Episode of Sickle Cell Disease: A Pilot Study. Blood 1997, 90 (5), 2041-2046. (45) Lee, R. C.; River, L. P.; Pan, F. S.; Ji, L.; Wollmann, R. L. Surfactant-Induced Sealing of Electropermeabilized Skeletal Muscle Membranes in Vivo. Proc. Natl. Acad. Sci. U.S.A. 1992, 89 (10), 4524-4528. (46) Maskarinec, S. A.; Hannig, J.; Lee, R. C.; Lee, K. Y. C. Direct Observation of Poloxamer 188 Insertion into Lipid Monolayers. Biophys. J. 2002, 82 (3), 1453-1459. (47) Collins, J. M.; Despa, F.; Lee, R. C. Structural and Functional Recovery of Electropermeabilized Skeletal Muscle in-Vivo after Treatment with Surfactant Poloxamer 188. Biochim. Biophys. Acta, Biomembr. 2007, 1768 (5), 1238-1246. (48) Wu, G.; Majewski, J.; Ege, C.; Kjaer, K.; Weygand, M. J.; Lee, K. Y. C. Lipid Corralling and Poloxamer Squeeze-out in Membranes. Phys. Rev. Lett. 2004, 93 (2), DOI: 10.1103/1193.028101. (49) Luo, C. L.; Chen, X. P.; Li, L. L.; Li, Q. Q.; Li, B. X.; Xue, A. M.; Xu, H. F.; Dai, D. K.; Shen, Y. W.; Tao, L. Y. Poloxamer 188 Attenuates in Vitro Traumatic Brain Injury-Induced Mitochondrial and Lysosomal Membrane Permeabilization Damage in Cultured Primary Neurons. J. Neurotrauma. 2013, 30 (7), 597-607. (50) Serbest, G.; Horwitz, J.; Jost, M.; Barbee, K. Mechanisms of Cell Death and Neuroprotection by Poloxamer 188 after Mechanical Trauma. FASEB J. 2006, 20 (2), 308-310. (51) Bao, H. J.; Wang, T.; Zhang, M. Y.; Liu, R.; Dai, D. K.; Wang, Y. Q.; Wang, L.; Zhang, L.; Gao, Y. Z.; Qin, Z. H. Poloxamer-188 Attenuates Tbi-Induced Blood–Brain Barrier Damage Leading to Decreased Brain Edema and Reduced Cellular Death. Neurochem. Res. 2012, 37 (12), 2856-2867. (52) Zhang, Y.; Chopp, M.; Emanuele, M.; Zhang, L.; Zhang, Z. G.; Lu, M.; Zhang, T.; Mahmood, A.; Xiong, Y. Treatment of Traumatic Brain Injury with Vepoloxamer (Purified Poloxamer 188). J. Neurotrauma. 2018, 35 (4), 661-670.



Page 24 of 34

Figure Legends Figure 1. Schematics of tunable PDMS aperture. (A) Control without a PDMS aperture. Multiple craters of different shape and dimension were formed by collapsing microbubbles. The pink plane represents the focal plane where astrocytes were plated. (B) When an aperture is placed directly above the electrodes, only one central crater was formed. (C) When the same aperture was placed sufficiently away from the electrodes, microbubbles did not reach the focal plane. Not drawn to scale. (D) Histograms were constructed to demonstrate the distribution of the number and size of the microbubbles in response to 5 repetitive pulse application with different PDMS aperture sizes ranging from 0 (control) to 9.6 mm. At least 250 microbubbles for each case were captured at the focal plane where cells were seeded and analyzed using an image processor (Element, Nikon). (E) Using apertures ~ 5 mm in diameter consistently produced microbubbles of similar size. In contrast, using a ~ 10 mm aperture, the microbubble size was much smaller and resembled to that observed when no aperture was used, suggesting that an aperture of > 10 mm diameter mimics the experimental condition without using any aperture.

Figure 2. Pressure measurement of collapsing microbubbles. (A) image of film sensors demonstrating the change of colors that correspond to a different pressure. The film was cut to a 22 × 22 mm area. (B) The histogram was constructed by the manufacturer based on the calibrated standard. Figure 3. LIVE/DEAD viability. A two-color assay was applied to determine the cell viability. The plasma membrane integrity can be probed, and the esterase activity in live cells can be visualized. (A) and (C) are brightfield images of mouse astrocytes exposed to microcavitation from one or five repetitive blasts, respectively. (B) and (D) show live cells in green color indicating the fluorophores were cleaved by esterase enzyme, and red color indicates dead cells (non-permeable fluorophores able to enter the compromised cell membrane). Scale bar = 100 m. Figure 4. Representative image of astrocytes loaded with the calcium-specific Fluo-4 AM shown before (A) and after (B) exposure to microcavitation. An arc was drawn in (B) to illustrate the boundary of the crater. For illustration purposes, the four randomly chosen cells were monitored 24 ACS Paragon Plus Environment

Page 25 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and their calcium spiking activity was recorded in real-time. (C) The calcium spiking in control cells was recorded and shown in Trace 1. The algorithm we used to determine the number of calcium spikes correctly counted 7 such spikes. Trace 2 demonstrated changes in the calcium spiking in a cell located near the crater ( ~ 80 m) during microcavitation. The arrow indicates the time of a blast. (D) Traces 3 and 4 illustrated the noticeably altered calcium spiking patterns after the collapse of microbubbles is completed. Figure 5. Immunostaining of astrocytes with GFAP. Antibodies against GFAP were used to visualize the GFAP expression in control astrocytes (A) and the astrocytes exposed to microcavitation (B).

Figure 6. Microfabricated linear patterns. (A) Brightfield image of linear strips and (B) fluorescent image labeled with rhodamine-conjugated BSA proteins to validate the micron-scale patterns. Cell adhesion was prevented by polyethylene glycol-treated surface. (C) Astrocytes were seeded and imaged. A brighter circle indicates the location and dimension of the aperture. The arrows indicate the position of electrodes. (D) An image capturing microbubbles of various sizes and cell detachment induced by collapsing microbubbles. Figure 7. Distance-dependent calcium spiking. (A) Astrocytes loaded with Fluo-4 AM are seeded in the patterned 100 m strips. (B) Representative calcium spiking profiles observed as a function of distance away from the crater’s edge. (C) Analysis of calcium spiking observed at each specified distance in at least 30 cells from three independent experiments. Data represent Mean ± SD. * indicates p < 0.05.

Figure 8. Activation of the Caspase signaling pathway. The Caspase signaling was quantitatively determined. Data represent mean ± SD of at least 30 cells from three independent experiments. ** indicates p < 0.01. Figure 9. Effect of pharmacological agents inhibiting specific calcium pathway. (A) no exposure to microcavitation, and the calcium spikes were significantly inhibited by either a calcium-free buffer or a treatment with BAPTA-AM. (B) The calcium spiking activity was monitored before or 25 ACS Paragon Plus Environment


Page 26 of 34

after exposure to microcavitation using pharmacological agents. Cells exposed to microvatation were subsequently incubated with these agents. The calcium spiking was noticeably inhibited by ɷ-conotoxin GVIA before microcavitation was introduced. However, this N-type calcium channel blocker was capable of sustaining the calcium spiking activity after the cells were exposed to microcavitation. Data represent Mean ± SE of at least 30 cells from three independent experiments. ** indicates p < 0.01. Figure 10. Changes in the calcium spiking with and without P188 (0.5 mM) treatment. Incubation of astrocytes with P188 did not alter the calcium spiking activity. Following exposure of astrocyte to microcavitation, the cells were incubated with P188 for 3 to 18 hours. If left untreated, the calcium spiking essentially disappeared in about 6 hours. However, when treated with P188, the calcium spikes were observable. The FDA-approved P188 likely sealed the compromised cell membrane and restored the cell’s ability to generate and sustain spontaneous calcium spiking activity. Data represent Mean ± SD of at least 100 cells from three independent experiments. * indicates p < 0.05, and ** p < 0.01.


Page 27 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1

Figure 2



Page 28 of 34

Figure 3


Page 29 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4



Page 30 of 34

Figure 5

Figure 6


Page 31 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7



Page 32 of 34

Figure 8

Figure 9


Page 33 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 10



TOC 338x190mm (96 x 96 DPI)

ACS Paragon Plus Environment

Page 34 of 34

Astrocyte Viability and Functionality in Spatially Confined

Recommend Documents