Investigating the Effects of Magnetic Fields Generated During Transcranial Magnetic Stimulation Using Computer and Biological Models

Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations

2020

Investigating the effects of magnetic fields generated during Transcranial Magnetic Stimulation using computer and biological models

Joseph Boldrey Iowa State University

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Investigating the effects of magnetic fields generated during Transcranial Magnetic Stimulation using computer and biological models

Joseph Corrie Boldrey

A thesis submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

Major: Electrical Engineering (Electromagnetics, Microwave, and Nondestructive Evaluation)

Program of Study Committee: David C. Jiles, Major Professor Long Que Ian Schneider Mani Mina

The student author, whose presentation of the scholarship herein was approved by the program of study committee, is solely responsible for the content of this thesis. The Graduate College will ensure this thesis is globally accessible and will not permit alterations after a degree is conferred.

Iowa State University

Ames, Iowa

2020

TABLE OF CONTENTS Page

ABSTRACT ...... vi

CHAPTER 1. GENERAL INTRODUCTION ...... 1

CHAPTER 2. INFLUENCE OF BRAIN-SCALP DISTANCE ON FOCALITY OF THE QUADRUPLE BUTTERFLY COIL FOR TRANSCRANIAL MAGNETIC STIMULATION ....3 Abstract ...... 3 Introduction ...... 3 Computer Simulations ...... 5 Analysis ...... 9 Conclusion ...... 12 Acknowledgements ...... 13 References ...... 13

CHAPTER 3. ON-CHIP STUDIES OF MAGNETIC STIMULATION EFFECT ON SINGLE NEURAL CELL VIABILITY AND PROLIFERATION ON GLASS AND NANOPOROUS SURFACES ...... 15 Abstract ...... 15 Introduction ...... 16 Materials and Methods ...... 18 Materials ...... 18 Substrates for the Studies of Viability and Behaviors of Neuron Cells N27 ...... 18 Arrayed SU8 Microholder Chip for Studying the Viability and Growth of Single Neural Cell N27...... 20 Substrate Cleaning, Cell Synchronization, Cell Seeding, and SEM Imaging ...... 20 Sterilization ...... 20 Cell Synchronization ...... 20 Cell Seeding ...... 21 Fixing and Staining Cells ...... 21 Imaging Cells ...... 22 Focal Adhesion Analysis ...... 22 Cell Labeling with Fluorophores and Fluorescence Imaging for Live/Dead Assay ..... 23 SEM Imaging ...... 23 Experimental Setup for Applying MS to N27 Neural Cells ...... 23 Statistical Analysis ...... 24 Results and Discussion ...... 26 Spreading of N27 Cells on Different Substrates ...... 26 Viability of N27 Cells on Different Substrates under MS ...... 29 MS Effects on N27 Neural Cell Growth ...... 31 MS effects on morphology (area) of N27 cells on glass and AAO substrates ...... 31 MS effects on the cytoskeleton and paxillin localization...... 32 MS effects on single N27 neural cell on microchips ...... 34 Conclusions ...... 38 Acknowledgements ...... 38 iii

References ...... 39 Appendix. Supporting Information ...... 43 (1) Fabrication Process for SU8 Microholder ...... 43 (2) Experimental Setup for Applying MS ...... 43 (3) Morphometric Analysis ...... 45 (4) Cell Division Process ...... 46 References ...... 46

CHAPTER 4. STUDIES OF SINGLE NEURON CELL GROWTH ON NANOPOROUS SURFACE AND UNDER TRANSCRANIAL MAGNETIC STIMULATION ...... 48 Abstract ...... 48 Introduction ...... 48 Materials and Methods ...... 49 Neuron Cell N27 ...... 49 Preparation of AAO Nanoporous Substrate and Glass Substrate...... 49 Microholder Chip Fabrication ...... 51 Experimental Setup for Applying TMS on Neurons ...... 51 Results and Discussion ...... 53 Neuron Cells Growth on Nanoporous Surface vs. Glass Surface ...... 53 Effect of TMS on the Behavior of Neuron Cells ...... 54 Single Neuron Cell Growth in Microholders ...... 56 Cell synchronization ...... 56 Cells seeding ...... 56 Summary ...... 59 Acknowledgement ...... 59 References ...... 59

CHAPTER 5. INVESTIGATING THE ROLE OF PULSED MAGNETIC FIELD INTENSITY ON THE GROWTH AND PROLIFERATION OF N27 DOPAMINERGIC NEURONAL CELLS ...... 61 Abstract ...... 61 Introduction ...... 62 Experiment Procedure ...... 63 Experimental Timeline ...... 63 2D Extracellular Matrices (ECMs)...... 63 Cell Culture ...... 63 Magnetic Stimulation ...... 63 Measured Magnetic Fields ...... 63 Stimulation Schematic ...... 64 Results ...... 64 Figure-8 Coil Statistical Analysis...... 66 Discussion ...... 66 Acknowledgment ...... 66 References ...... 66

CHAPTER 6. STUDIES OF NEURITE EXTENSIONS ON PC-12 CELLS UNDER TRANSCRANIAL MAGNETIC STIMULATION ...... 68 Abstract ...... 68 Introduction ...... 69 Experiment Procedure ...... 70 Experiment Timeline ...... 70 Cell Preparation ...... 70 Magnetic Stimulation ...... 70 Field Strength of TMS Coil ...... 71 Stimulation Schematic ...... 71 Methods of Measurement ...... 71 Results ...... 72 Statistical Analysis ...... 73 Discussion ...... 73 Acknowledgment ...... 74 References ...... 74

CHAPTER 7. THE INFLUENCE OF PULSED MAGNETIC FIELDS ON THE PROLIFERATION OF ADHERENT AND NON-ADHERENT NEURONAL CELL TYPES ..75 Abstract ...... 75 Introduction ...... 76 Experiment Procedure ...... 77 Experimental Timeline ...... 77 Cell Culture ...... 77 Magnetic Stimulation ...... 77 Measured Magnetic Fields ...... 78 Stimulation Schematic ...... 78 Cell Counting ...... 78 Results ...... 79 Statistical Analysis ...... 80 Cell Viability ...... 80 Conclusion ...... 81 Acknowledgment ...... 81 References ...... 81

CHAPTER 8. THE INFLUENCE OF MAGNETIC FIELD ORIENTATION ON THE GROWTH OF NEURITE EXTENSIONS OF NGF DIFFERENTIATED PC-12 NEURONAL CELLS ...... 82 Abstract ...... 82 Introduction ...... 83 Experiment Procedure ...... 83 Experiment Timeline ...... 83 Cell Preparation ...... 84 Magnetic Stimulation ...... 84 Measured Magnetic Fields ...... 85 Stimulation Schematic ...... 85 Method of Analysis ...... 85 v

Results ...... 86 Statistical Analysis ...... 87 Discussion ...... 87 Acknowledgment ...... 87 References ...... 88

CHAPTER 9. EFFECTS OF MAGNETIC STIMULATION ON THE PROLIFERATION OF DOPAMINERGIC NEURONAL CELLS GROWN IN EXTRACELLULAR MATRIX ...... 89 Abstract ...... 89 Introduction ...... 90 Experiment Procedure ...... 91 Experimental Timeline ...... 91 2D Extracellular Matrix...... 91 Cell Culture ...... 91 Magnetic Stimulation ...... 91 Measured Magnetic Fields ...... 92 Stimulation Schematic ...... 92 Results ...... 92 Statistical Analysis ...... 94 Conclusion ...... 94 Acknowledgment ...... 94 References ...... 95

CHAPTER 10. GENERAL CONCLUSION ...... 96 vi

ABSTRACT

Transcranial Magnetic Stimulation (TMS) is a non-invasive neuromodulation technique that is growing in acceptance by the medical community and the general public. TMS was approved by the U.S. Food and Drug Administration (FDA) for treating major depressive disorder (MDD) in 2008, certain types of migraine headaches in 2013, and obsessive compulsive disorder (OCD) in 2018. Researchers are also investigating how TMS can be used for enhancing the recovery of motor functions after a stroke as well as other debilitating illnesses. Investigating the biological mechanisms involved in the brain’s response to TMS is a growing area of study.

Of the many challenges for researchers to overcome, are the ethical and safety standards that prevent the testing of new TMS devices and protocols on human subjects. To overcome these, researchers have two types of models that are used to recreate the brain and its components for

TMS research. Computer models are used to simulate the neurological system’s responses to the magnetic fields generated during TMS. They are used to simulate the effects of new coil designs and stimulation protocols on different types of head models (i.e., a healthy brain versus a brain damaged by head trauma). Biological models use cell lines developed from animals that recreate the aspects of cells in the human brain that are affected by TMS.

The research presented in this thesis shows the experimental results using both types of models to investigate the underlying mechanisms of TMS. A computer model was used to investigate the effects of brain-scalp distance of healthy subjects on the response to a novel TMS coil design. Two types of biological models were used to investigate, at the cellular level, the effects of magnetic fields generated by a commercial TMS system on neuronal cells in vitro. 1

CHAPTER 1. GENERAL INTRODUCTION

The major principle underlying Transcranial Magnetic Stimulation (TMS) is Faraday's

Law of electromagnetic induction. Whereby the time-varying magnetic field (B) generated by a

TMS coil induces an electric field (E) in the brain. The induced E field, at sufficient strength, causes neurons to fire in a process known as depolarization. The effect of TMS fields on the brain as a system, and at the cellular level, have received considerable attention by the research community, yet there is still much to be learned about these processes.

One of the challenges in the development of TMS therapy is the ability to deliver a focused magnetic field to a specific region in the brain. In the process of delivering a sufficiently strong magnetic field (H) to the target region, surrounding areas of the brain can be stimulated unnecessarily. Stimulating non-target areas can result in side effects ranging from mild discomfort such as tingling sensations in the scalp, headaches, neck pain, and tooth pain to more adverse side effects such as fainting, and seizures. The Quadruple Butterfly Coil (QBC) is a novel design TMS coil that significantly increases the focality of the magnetic field compared to the figure-of-eight (FoE) coil most commonly used in TMS therapy and research today. Focality is measured using the V-Half metric (the volume of the brain exposed to at least one-half of the maximum induced electric field intensity (EMAX)). The QBC generates a sufficiently high E field intensity in order to trigger neuronal depolarization, while stimulating a significantly lower total brain volume than the FoE coil. Multiple studies have shown a direct correlation between the brain-scalp distance (BSD) and the brain’s response to the magnetic pulse generated during

TMS. If the stimulator output is increased to adjust for the increased BSD, there will be an increase in the H field stimulating non-target areas. This might result in a larger total volume of tissue receiving stimulation, which increases the possibility of side effects. The need for 2 increased H field strength along with the reduced focality as the BSD increases can significantly reduce the efficacy of TMS therapy as well as increasing patient discomfort. In order to investigate how the BSD may affect the brain’s response to the QBC in comparison to that of the

FoE coil, computer models using finite element analysis (FEA) were used to run simulations on head models created from MRI scans of healthy individuals.

Much of the biomedical research into the causes of, and treatments for, neurodegenerative diseases (ND) such as Alzheimer’s Disease (AD) and Parkinson’s Disease

(PD) is performed using cell lines from animals that model the behavior of cells in the human brain. Two well-established animal neuronal cell lines used for ND research are the PC-12 and

N27 cell lines. The goal of the biological modeling part of the research was to investigate the effects of pulsed magnetic fields of the same type and intensity that cells in the brain experience during in vivo TMS sessions on the PC-12 and N27 biological models. Multiple experiments were designed and conducted to investigate the effects of the magnetic fields generated during

TMS on these two models. The trials were designed to measure different characteristics of the cells after receiving a predetermined number of pulses from a commercially available monophasic TMS stimulator. Experiments were conducted to measure proliferation rates, and morphology of both cell models after receiving TMS. An investigation was made into the role that the structural growth environment called the extracellular matrix (ECM) may play in the

N27 cells’ response to TMS. Trials were conducted to measure the effects of different H field intensities on the N27 cells’ proliferation rate and morphology. Additionally, experiments designed to measure the effects of TMS on neurite growth on PC-12 cells were conducted.

Aspects of the neurite growth such as the direction of growth, number of neurites per cell, neurite length, and number of branches per neurite were investigated. 3

CHAPTER 2. INFLUENCE OF BRAIN-SCALP DISTANCE ON FOCALITY OF THE QUADRUPLE BUTTERFLY COIL FOR TRANSCRANIAL MAGNETIC STIMULATION

Oluwaponmile F. Afuwape1,2, Joseph Boldrey1, Priyam Rastogi1, Sarah A. Bentil2, and David C. Jiles1, Life Fellow, IEEE

1Department of Electrical and Computer Engineering, Iowa State University 2Department of Mechanical Engineering, Iowa State University

Modified from a manuscript published in IEEE Transactions on Magnetics

Abstract

Transcranial Magnetic Stimulation (TMS) is a neuromodulation technique that non- invasively depolarizes neurons in the brain. During TMS, a pulse (or multiple pulses) of a time- varying magnetic field (H) is delivered to the brain using specialized coils. The Quadruple

Butterfly Coil (QBC) is a novel coil design that shows increased focality of the induced electric field over that of the standard figure-of-eight (FoE) coil. Using 50 different head models created from MRI scans of healthy individuals, our research investigated the role that brain-scalp distance (BSD) plays in the brain’s response to the magnetic fields generated by the QBC and

FoE. The variability of BSD is an inherent characteristic in the human population. As the BSD increases, so does the distance between the brain and TMS coil. Therefore, the anatomical variation of BSD is an independent variable that may play a significant role in the intensity of the induced electric field produced in the brain. Our results show no significant difference of the

QBC’s focality to that of the FoE with respect to BSD.

Introduction

Transcranial Magnetic Stimulation (TMS) is a neuromodulation technique which is capable of non-invasively activating neurons in the brain. During TMS, a pulse (or multiple pulses) of a time-varying magnetic field (H) is delivered to the brain. The magnetic field induces an electric field (E) in the brain which, at sufficient levels, causes neurons to depolarize [1]. The 4

U.S. Food and Drug Administration (FDA) approved TMS for treating major depressive disorder

(MDD) in 2008, certain types of migraine headaches in 2013, and obsessive compulsive disorder

(OCD) in 2018 [2]. Among other indications, TMS is being considered as viable therapy for post-traumatic stress disorder (PTSD) [3] as well as Parkinson’s Disease (PD) [4]. In the future,

TMS may become an alternative to deep brain stimulation (DBS). An inherent risk in DBS arises from neurosurgical implantation of stimulation electrodes into the brain. The risk involved with neurosurgery has brought TMS into consideration as a viable alternative to DBS for treatment of essential tremor, dystonia, PD, and a wider range of clinical depression [5].

One of the challenges in the development of TMS therapy is the ability to deliver a focused magnetic field to a specific targeted region in the brain. In the process of delivering a sufficiently strong H field to the targeted region, surrounding areas of the brain can be stimulated unnecessarily. Stimulating non-target areas can result in side effects ranging from mild discomfort such as tingling sensations in the scalp, headaches, neck pain, and tooth pain to more adverse side effects such as fainting, and seizures [6]. The figure-of-eight (FoE) TMS coil was developed in 1988 by Ueno et al. [7] and has become the most commonly used coil for TMS therapy and research [6]. The Quadruple Butterfly Coil (QBC) is a novel design TMS coil developed by Rastogi [8] that significantly increases the focality of the magnetic field compared to the FoE coil. The QBC generates a sufficiently high E field intensity in order to trigger neuronal depolarization, while stimulating a significantly lower total brain volume than the FoE coil. The reduction of brain volume (defined as the total volume of grey matter and white matter) exposed to high E field intensities is by 11% when comparing the QBC with the FoE coil.

Focality is measured using the V-Half metric (the volume of the brain exposed to at least one- half of the maximum induced electric field intensity (EMAX) in the brain). 5

Multiple studies have shown a direct correlation between the brain-scalp distance (BSD) and the brain’s response to the magnetic pulse generated during TMS [9], [10]. One study showed that a 3% increase in TMS output is necessary to elicit the same response for every millimeter increase in distance between the coil and the target within the brain [11]. If the stimulator output is increased to adjust for the increased BSD, there will be an increase in the H field passing through the tissue to reach the target site. This might result in a larger total volume of tissue unnecessarily receiving stimulation. This, in turn, increases the possibility of stimulating non-target areas which will increase the occurrences of side effects, and reduce focality [11]. The need for increased H field strength along with the reduced focality as the BSD increases can significantly reduce the efficacy of TMS therapy as well as increasing patient discomfort.

The goal of our research is to investigate the effects of the BSD on the performance of the QBC in comparison to that of the FoE.

Computer Simulations

The QBC was modeled, and finite element (FE) simulations were run, using Sim4Life software [12] on head models derived from Magnetic Resonance Imaging (MRI) scans of 50 healthy subjects. The Sim4Life software is a low frequency magneto-quasi-static solver that uses the FE method to compute the magnetic field and induced electric field within the head models.

The QBC coil configuration, shown in Fig. 1, consists of two coil groups: a large set of coils and a small set of coils. The large coils have the same dimension as the FoE coil (a diameter of 95 mm) and the small coils have dimension of 40% the size of the large coils. These two sets of coils have the same number of turns (9 on both sides of each set which makes for 36 turns in total) and make a right angle with one another. 6

Fig. 1. Three different views of the Quadruple Butterfly Coil.

The QBC was placed at a distance of 5 mm from the vertex of each head model. This distance accounted for the insulation of the QBC, as seen in Fig. 2. The coil was pulsed at a frequency of 2.5 kHz and a current with an amplitude of 5 kA. A total of 50 simulations were run with the coil.

The BSD is obtained in the Sim4Life software using the integrated measuring tool. This distance is taken as the vertical distance from the vertex (origin 0,0,0) to the highest point on the grey matter. However, due to the uniqueness of each head model and the gyri on the right and left hemisphere, the grey matter may not always be on the same level. Fig. 2 shows the FE simulation setup in the Sim4Life software, with the grey matter in place. The vertical distance from the vertex is then measured perpendicularly to the connecting line of the brain lobes and taken as the BSD. The connecting line was established by selecting the highest point (peak) of each lobe and connecting them with a straight line. 7

Fig. 2. Representative images of the FE simulation model in the Sim4Life software. Frontal view (a) and right hemisphere view (b) of the head model, Quadruple Butterfly Coil, and grey matter.

The green dots (enlarged for clarity), in Fig. 2, are the peaks of each hemisphere of the grey matter. The straight lines (enlarged for clarity) in the same figure represent the vertical distance drawn perpendicularly downward from the vertex, and the line connecting each lobe.

The 50 MRI scans of healthy subjects with ages ranging from 25-35 years were sourced from the

Human Connectome Project Model Library [13] and developed using the SimNIBS pipeline by

Lee et al. [14]. Each head model had seven different anatomical structures which included the skin (sn), skull (sk), cerebrospinal fluid (csf), grey matter (gm), white matter (wm), cerebellum

(cb), and ventricles (vc), as shown in Fig 3. 8

Fig. 3. The seven different anatomic variations (with the edges of their voxel shown) are (a) skin (sn), (b) skull (sk), (c) cerebrospinal fluid (csf), (d) grey matter (gm), (e) white matter (wm), (f) cerebellum (cb), and (g) ventricles (vc).

The electrical properties of the anatomical structures, used in the FE simulations, were sourced from the Information Technologies in Society (IT’IS) foundation database [15] and are defined in Table I. The computed EMAX (i.e., maximum induced electric field) values were extracted and exported to MATLAB [16] for post processing and interpretation.

Table 1. Electrical Properties of Anatomical Structures at a Frequency of 2.5 kHz

Structure Name Relative Permittivity Electrical Conductivity (S/m)

Skin (sn) 1140 0.0002 Skull (sk) 1440 0.0203 Cerebrospinal fluid (csf) 109 2 Grey matter (gm) 78100 0.104 White matter (wm) 34300 0.0645 Cerebellum (cb) 78400 0.124 Ventricles (vc) 102 2

Analysis

The EMAX was extracted from the FE simulations for each of the 50 head models. Fig. 4 shows the color map of the induced electric field on the skin and the grey matter, respectively, of one of the head models. A maximum induced value of 210 V/m was observed on the skin while a maximum value of 101 V/m was observed on the grey matter.

Fig. 4. Induced electric field in skin (top) and grey matter (bottom) from one of the head models. 10

A box plot showing the characteristic distribution of the EMAX values, across the 50 head models on the basis of the minimum, maximum, median, first quartile, and third quartile, is presented in Fig. 5.

Fig. 5. Box plot shows the distribution of the maximum induced electric field, for the 50 head models.

The maximum and minimum values of EMAX, among the 50 head models, was calculated as 343 V/m and 79 V/m respectively. The average EMAX and standard deviation value, across the

50 head models, was calculated as 144 V/m and 41 V/m respectively.

A graph showing the variation of EMAX with the BSD for both the QBC and FoE coil is presented in Fig. 6. Our results show that the EMAX generated by both the QBC and FoE coil decreases as the BSD increases, with an R-squared value of 0.4293 for the QBC and an R- squared value of 0.4141 for the FoE coil. These results agree with the conclusions of Lee et al.

[16] that used a FoE coil on the same set of 50 head models and also used the same FE simulation parameters as our study. 11

Fig. 6. Comparison between the maximum induced electric field (EMAX) versus brain-scalp distance for 50 head models, using the Quadruple Butterfly Coil and Figure-of-Eight Coil.

A graph showing the variation of V-Half versus the BSD for both the QBC and FoE coil is presented in Fig. 7. Our results show that the V-Half stimulated by the QBC and FoE coil shows no significant statistical correlation with the increasing BSD. The R-squared values are

0.0899 for the QBC and 0.0351 for the FoE coil. These results are also in good agreement with the conclusions of Lee et al. [16] that used a FoE coil on the same set of 50 head models with the same FE simulation parameters as our study. 12

Fig. 7. Stimulated Volume versus brain-scalp distance for 50 head models using the Quadruple Butterfly Coil.

Conclusion

The variability of BSD plays a role in the brain’s response to TMS. With our study, we conclude that the EMAX in the brain induced by the QBC decreases with BSD. This confirms that, without regards to the coil configuration, the BSD has a significant effect on EMAX. However, there is no significant correlation between BSD and the V-Half (the metric for focality).

We used MRI scans of healthy subjects ranging in age from 25-35 years in our study. To further investigate the role of BSD in TMS, we intend to conduct further simulations using MRI scans from unhealthy adults and older generational cohorts, since BSD has been found to increase with age [16]. 13

Acknowledgements

We thank Erik Lee and Dr. Ravi Hadimani for their support and contributions to the research. This work was supported in part by the Galloway Foundation, and the Stanley Chair in

Interdisciplinary Engineering at Iowa State University.

References

[1] C. Miniussi, W. Paulus, P. Rossini, (2013). “Transcranial Brain Stimulation”. Boca Raton: CRC Press, https://doi.org/10.1201/b14174.

[2] “FDA permits marketing of transcranial magnetic stimulation for treatment of obsessive compulsive disorder | FDA.” [Online]. Available: https://www.fda.gov/newsevents/press-announcements/fda-permits-marketing-transcranial-magnetic-stimulation- treatment-obsessive-compulsive-disorder. [Accessed: 04-May-2020].

[3] R. J. Koek, J. Roach, N. Athanasiou, M. van ‘t Wout-Frank, and N. S. Philip, “Neuromodulatory treatments for post-traumatic stress disorder (PTSD),” Prog. Neuro- Psychopharmacology Biol. Psychiatry, vol. 92, no. December 2018, pp. 148–160, 2019, doi: 10.1016/j.pnpbp.2019.01.004.

[4] D. H. Benninger and M. Hallett, “Non-invasive brain stimulation for Parkinson’s disease: Current concepts and outlook 2015,” NeuroRehabilitation, vol. 37, no. 1, pp. 11–24, Aug. 2015, doi: 10.3233/NRE-151237.

[5] M. D. Fox et al., “Resting-state networks link invasive and noninvasive brain stimulation across diverse psychiatric and neurological diseases,” Proc. Natl. Acad. Sci., vol. 111, no. 41, pp. E4367–E4375, Oct. 2014, doi: 10.1073/pnas.1405003111.

[6] S. Rossi et al., “Safety, ethical considerations, and application guidelines for the use of transcranial magnetic stimulation in clinical practice and research,” Clin. Neurophysiol., vol. 120, no. 12, pp. 2008–2039, 2009.

[7] S. Ueno, T. Tashiro, and K. Harada, “Localized stimulation of neural tissues in the brain by means of a paired configuration of time-varying magnetic fields,” J. Appl. Phys., vol. 64, no. 10, p. 5862, 1988.

[8] P. Rastogi, “Novel coil designs for different neurological disorders in transcranial magnetic stimulation,” PhD Thesis, Iowa State University, Ames, IA, USA, 2019. Available: https://lib.dr.iastate.edu/etd/17547. 14

[9] M. G. Stokes et al., “Biophysical determinants of transcranial magnetic stimulation: Effects of excitability and depth of targeted area,” J. Neurophysiol., vol. 109, no. 2, pp. 437–444, 2013.

[10] K. A. McConnell et al., “The transcranial magnetic stimulation motor threshold depends on the distance from coil to underlying cortex: A replication in healthy adults comparing two methods of assessing the distance to cortex,” Biol. Psychiatry, vol. 49, no. 5, pp. 454– 459, 2001.

[11] M. G. Stokes et al., “Simple metric for scaling motor threshold based on scalp-cortex distance: Application to studies using transcranial magnetic stimulation,” J. Neurophysiol., vol. 94, no. 6, pp. 4520–4527, Dec. 2005, doi: 10.1152/jn.00067.2005.

[12] E. Neufeld, M. C. Gosselin, D. Sczcerba, M. Zefferer, and N. Kuster, “Sim4Life: A Medical Image Data Based Multiphysics.”

[13] D. C. V. Essen et al., “The Human Connectome Project: A data acquisition perspective,” NeuroImage, vol. 62, no. 4, pp. 2222–2231, 2012, doi: https://doi.org/10.1016/j.neuroimage.2012.02.018.

[14] E. G. Lee et al., “Investigational Effect of Brain-Scalp Distance on the Efficacy of Transcranial Magnetic Stimulation Treatment in Depression,” IEEE Trans. Magn., vol. 52, no. 7, pp. 1–4, 2016.

[15] P. Hasgall et al., “‘IT’IS Database for thermal and electromagnetic parameters of biological tissues,’ Version 4.0, May 15, 2018, DOI: 10.13099/VIP21000-04-0. itis. swiss/database.”

[16] “MATLAB and Statistics Toolbox Release 2019a, The MathWorks, Inc., Natick, Massachusetts, United States.” 15

CHAPTER 3. ON-CHIP STUDIES OF MAGNETIC STIMULATION EFFECT ON SINGLE NEURAL CELL VIABILITY AND PROLIFERATION ON GLASS AND NANOPOROUS SURFACES

Xiangchen Che1, Joseph Boldrey1, Xiaojing Zhong1, Shalini Unnikandam-Veettil2, Ian Schneider2, David Jiles1, and Long Que1

1Department of Electrical and Computer Engineering, Iowa State University 2Department of Chemical and Biological Engineering, Iowa State University

Modified from a manuscript published in ACS Applied Materials & Interfaces

Abstract

Transcranial magnetic stimulation (TMS) is a noninvasive neuromodulation technique, an

FDA-approved treatment method for various neurological disorders such as depressive disorder,

Parkinson’s disease, post-traumatic stress disorder, and migraine. However, information concerning the molecular/cellular-level mechanisms of neurons under magnetic simulation (MS), particularly at the single neural cell level, is still lacking, resulting in very little knowledge of the effects of MS on neural cells. In this paper, the effects of MS on the behaviors of neural cell N27 at the single-cell level on coverslip glass substrate and anodic aluminum oxide (AAO) nanoporous substrate are reported for the first time. First, it has been found that the MS has a negligible cytotoxic effect on N27 cells. Second, MS decreases nuclear localization of paxillin, a focal adhesion protein that is known to enter the nucleus and modulate transcription. Third, the effect of MS on N27 cells can be clearly observed over 24 h, the duration of one cell cycle, after

MS is applied to the cells. The size of cells under MS was found to be statistically smaller than that of cells without MS after one cell cycle. Furthermore, directly monitoring cell division process in the microholders on a chip revealed that the cells under MS generated statistically more daughter cells in one average cell cycle time than those without MS. All these results 16 indicate that MS can affect the behavior of N27 cells, promoting their proliferation and regeneration.

Introduction

Transcranial magnetic stimulation (TMS) is a noninvasive neuromodulation technique that uses time-varying short pulses of magnetic fields to induce an electric field in the conductive tissues of the brain, thus modulating the synaptic transmission of neurons. This neuromodulation technique can be used to excite or inhibit the firing rate of neurons as a treatment for various neurological disorders such as major depressive disorder, Parkinson’s disease, post-traumatic stress disorder, and migraine.1,2 While the effect of magnetic stimulation (MS) on other types of cells and biomolecules in vitro has been studied in the past,3−7 information concerning the molecular/cellular mechanisms of neurons under MS, particularly at the single neural cell level, is still lacking. The effects of MS on single neural cells need to be thoroughly understood in order for experts to make the greatest use of MS as a neuromodulation tool for treating neurological disorders, especially those originating from the subcortical regions of the brain. To study the behaviors of single neural cells in vitro, a solid substrate or scaffold is usually needed for the cell’s proper adhesion, spreading, and growth.8 It is particularly desirable to create a substrate or scaffold mimicking the native extracellular environment that can interface with individual neural cells within living tissues and in cell culture. As previous studies have demonstrated, substrates or scaffolds with nanoscale features (i.e., nanostructured biointerfaces) have greatly improved specificity and accuracy for many neural-engineering applications,9−11 including neural probes for Parkinson’s patients and guidance scaffolds for axonal regeneration in patients with traumatic nerve injuries,12,13 just to name a few. Hence, nanostructured biointerfaces have become a rapidly emerging area of study. For instance, over the past decades, 17 which have witnessed the development of nanotechnology and nanomaterial that is safe for biological applications, interfaces between a variety of nanomaterials or nanostructures with biomolecules have been studied.14 Examples of nanobiointerfaces include the interface of the nanoparticle−lipid bilayer,15 the interface of carbon nanotubes (CNTs)−biomolecules,16 and the interface of graphene−biomolecules.17 The studies of nanobiointerfaces primarily aim to understand the dynamic physicochemical interactions, kinetics, and thermodynamics between nanomaterial surfaces and the surfaces of biological components, which include, for example, proteins, membranes, phospholipids, endocytic vesicles, organelles, DNA, and biological fluids.

One widely used nanostructured material is anodic aluminum oxide (AAO). The unique properties of nanoporous AAO thin film have greatly contributed to the development of a variety of novel biomedical applications, ranging from biofiltration membranes, lipid bilayer support structures, biosensing devices, and implant coatings to drug delivery systems with AAO capsules and scaffolds for tissue engineering.18−24 Additionally, nanoporous AAO membranes have generated increasing interest and shown great promise as cell-interface substrates for manifold cell types.24 Parkinson’s disease (PD), the second most common neurodegenerative disorder after

Alzheimer’s disease (AD),25 is caused by the death of dopaminergic neurons in the substantia nigra. N27 neural cells have been widely used for their dopaminergic properties as an in vitro model of PD.26 In addition, they have been extensively used for studies of neurotoxicity, oxidative stress, neurodegeneration, and other molecular pathways.27,28 The effects of MS on their functions are currently unknown. Previously, we have found the MS stimulates N27 cells proliferation in a population-based assay.29,30 However, we did not analyze single cells behavior, nor did we assess other cell functions that modulate proliferation such as cytoskeletal structure or adhesion. In this paper, an AAO nanoporous surface is used as one of the substrates to study the 18 behavior of N27 neural cells under MS. The effects of MS on the single-cell level spreading, cytoskeletal structure, adhesion, and proliferation on a glass surface and an AAO nanoporous surface are reported for the first time.

Materials and Methods

Materials

(1) The experiments use neural cell N27 (Millipore Sigma), which stands for immortalized rat mesencephalic cells (1RB3AN27). The following chemicals are used for culturing the N27 neural cell: RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Sigma-Aldrich); 1% L-glutamine (Sigma-Aldrich); penicillin with a concentration of 100 U/mL (Sigma-Aldrich); streptomycin in 100 U/mL (Sigma-Aldrich). (2)

Fluorophores, calcein AM (Sigma-Aldrich), and propidium iodide (Sigma-Aldrich) are used for monitoring cell viability using a fluorescence microscope (Olympus, Inc.). (3) Scanning electron microscopy (SEM) is used to image the neuron cell growth on different substrates. Standard PBS

(Sigma-Aldrich), 4% paraformaldehyde in a 0.1 M PO4 buffer (Sigma-Aldrich), ethanol >99%

(Sigma-Aldrich), and hexamethyldisilazane (HMDS) >99% (Sigma-Aldrich) are used for sample pretreatment before SEM imaging. (4) Aluminum pellets with a purity of 99.999% (Iamaterials,

Inc.), titanium pellets with a purity of 99.995% (Iamaterials, Inc.), and SU-8 3025 (Microchem,

Inc.) are used to fabricate microholder chips.

Substrates for the Studies of Viability and Behaviors of Neuron Cells N27

Different surfaces/substrates were prepared for studying neuron cell growth: standard coverslip glass (Thermo Fisher Scientific, Inc.), AAO substrates fabricated by a one-step anodization process, and AAO substrates fabricated by a two-step anodization process. The procedure for preparing the AAO substrates is described in detail in our previous work.31 Briefly, after a layer of Al is deposited on a glass substrate, a one-step anodization process is carried out 19 to convert the Al into a nanoporous AAO thin film. An SEM image of the AAO substrates fabricated by the one-step anodization process is shown in Figure 1a. The two-step anodization process begins with 10 min of anodizing the Al layer in 0.3 M oxalic acid. Then the samples are etched with a mixture of phosphoric acid (0.4 M) and chromic acid (0.2 M) at 65 °C for 30 min, followed by a 40 min two-step anodization in 0.3 M oxalic acid with the same experimental condition as the one-step anodization. An SEM image of the AAO substrates fabricated by the two-step anodization process is shown in Figure 1b. The average nanopore diameter of the one- step AAO substrates (AAO-substrate1) is ∼20 nm, and its average porosity is 6.6%. The two- step AAO substrates (AAO-substrate2) have an average diameter of ∼80 nm and an average porosity of 84.11%. As shown, the nanopore size and porosity of the AAO-substrate2 are larger and more uniform than those of AAO-substrate1. The two AAO substrates therefore have differently nanostructured topological surfaces.

Figure 1. SEM images of (a) AAO-substrate1: nanopore diameter = 20 nm, porosity = 6.6%. (b) AAO- substrate2: nanopore diameter = 100 nm, porosity = 84.11%. (c) A photo of microholders on-chip and the close-up optical micrographs of arrayed SU8 microholders and the captured single neural cell N27. 20

Arrayed SU8 Microholder Chip for Studying the Viability and Growth of Single Neural Cell N27

Two types of SU8 microholder chips have been designed and fabricated. One type consists of arrayed microholders embedded with glass surfaces; the other type consists of arrayed microholders embedded with nanoporous AAO surfaces. The fabrication processes flow for the

SU8 microholder chip with an AAO nanoporous surface (AAO microchip) and for the SU8 microholder chip with a glass surface (glass microchip) are described in Figure S1 (Appendix.

Supporting Information). Figure 1c shows a photo of an SU8 microholder chip and the close-up optical micrographs of the microholders.

Substrate Cleaning, Cell Synchronization, Cell Seeding, and SEM Imaging

Sterilization

Both AAO nanoporous substrates and glass substrates are cleaned for 5 min with DI water using the ultrasonicator (Branson 1510-Fisher Scientific), and sterilization of these substrates is achieved by cleaning them with acetone and methanol with the ultrasonicator for 5 min in sequence. The substrates are then cleaned with DI water by ultrasonicating for 5 min and dried by a nitrogen gun, followed by air plasma cleaning (Harrick plasma-PCD 32G) for 3 min.

Finally, they are baked for 5 min on a hot plate at 150 °C before use.

Cell Synchronization

In general, cells in a culture will exist in different stages within the cell cycle at any one moment. The biospecific differences of each cell make it difficult to remain synchronized over long times.32 To obtain statistically accurate results of the effect of MS on the cell division process for a large number of cells, cell synchronization, a process that brings cells at different stages of the cell cycle to the same phase, must be implemented. To this end, a simple serum deprivation method is used by changing the percentage of fetal bovine serum from 10% to 1% in 21 the cell culture medium.32,33 Specifically, the medium of 10% fetal bovine serum is replaced with

1% fetal bovine serum 9 h before the cells are seeded into the microholders. A 9 h serum deprivation time was chosen for this study for a number of reasons. First, cell mortality was considered. Because the serum contains essential components for cell growth, cell death increases with the serum deprivation time. On the basis of our experiments, a 9 h serum deprivation does not cause substantial cell death. This 9 h synchronization period ensures that most of the cells have been synchronized at the G1 phase, the first of four phases of the cell cycle that takes place in eukaryotic cell division.33

Cell Seeding

Trypsin is used to detach the cells from the bottom of the flask before the cells are seeded on different substrates. After 2 or 3 min, we pipet a normal medium with 10% fetal bovine serum to the flask. Then the cells are ready to be seeded to the microholder devices.

Fixing and Staining Cells

Cells are plated on either AAO-substrate2 or glass microscope slides in 10% fetal bovine serum media as described above and fixed and stained for F-actin and paxillin with the following protocol. Cells are fixed in 4% paraformaldehyde (Fisher Scientific) w/v dissolved in cytoskeleton buffer at 37 °C for 15 min. Cytoskeleton buffer is a solution containing 10 mM

MES (Sigma-Aldrich), 3 mM MgCl2 (Fisher Scientific), 138 mM KCl (Fisher Scientific), and 2 mM ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) (Sigma-

Aldrich) at pH = 6.1. Cells are permeabilized with a 0.5% triton X solution for 5 min. The samples are then treated with 0.1 M glycine (Fisher Scientific) for 10 min. Both glycine and triton X solutions are prepared in the cytoskeleton buffer. After glycine treatment, cells are washed with Tris-buffered saline (TBS) containing 150 mM NaCl (Fisher Scientific) and 20 mM

Tris-Cl (Fisher Scientific) at pH = 7.4 multiple times. Blocking and F-actin staining is conducted 22 simultaneously. The blocking buffer is made with TBS, 0.1% v/v tween-20 (Fisher Scientific), and 2% w/v bovine serum albumin (BSA) (Sigma-Aldrich). A 1:200 dilution of alexa 488- phalloidin (Molecular Probes) was added to the blocking buffer. The samples are incubated for 1 h at room temperature and are protected from light. After incubation, the cells are washed in TBS multiple times. Cells are then incubated with purified mouse antipaxillin antibody (BD

Transduction Laboratories, clone 349) in blocking buffer at 1:200 ratio for an hour at room temperature and protected from light, followed by washing in TBS multiple times. Then the cells are incubated with donkey anti-mouse Cy3 conjugated antibody (Jackson Immuno Research

Laboratories) in blocking buffer at 1:200 ratio for an hour at room temperature and light protected conditions. Further, the samples are washed in TBS multiple times. Mounting media solution consists of 75% v/v glycerol and 20 mM Tris solution at pH = 8. The samples are inverted onto mounting media on number 1.5 coverslips and sealed with nail polish.

Imaging Cells

Fluorescence imaging is conducted on a fluorescence microscope (Olympus, Inc.) which includes the following filter sets: FITC (excitation filter: 460−495 nm; barrier filter: 500−540 nm); TRITC (excitation filter: 545−565 nm; barrier filter: 580−620 nm). Images are captured through a 40× objective. Exposure times are kept constant for a particular fluorophore (alexa 488 or Cy3). Twenty images from each condition are captured.

Focal Adhesion Analysis

Analysis is conducted using ImageJ (National Institutes of Health). The F-actin images are thresholded and used to specify the outline of the cell for both F-actin and paxillin

images. The paxillin image is thresholded differently to specify the outline of the nucleus.

Whole cell F-actin and paxillin and nuclear average intensity is background subtracted. Cytosolic paxillin intensity is calculated from the integrated whole cell paxillin intensity less the integrated 23 nuclear intensity divided by the whole cell area less the nuclear area. Peripheral and central focal adhesions are manually counted. Binucleated cells are identified as nuclei that could not be segmented using a threshold to find the nuclear outline.

Cell Labeling with Fluorophores and Fluorescence Imaging for Live/Dead Assay

To carry out the live/dead assay and monitor cell viability, we label the cells with fluorophores calcein AM and propidium iodide, using a concentration of 2 μM. Fluorescence images are captured with a fluorescence microscope (Olympus, Inc.).

SEM Imaging

The following steps are taken to obtain SEM images of the cells’ growth on the glass and

AAO surfaces. First, after 3 days of growth, the samples are rinsed with PBS. Then the neuron cells are fixed in 4% paraformaldehyde resolved in a 0.1 M phosphate (PO4) buffer with a pH of

7.4 for 20 min at room temperature. Second, the samples are rinsed five times with PBS at room temperature, and then with a DI water rinse for 10 min. Third, dehydration is achieved by immersing the samples into solutions of 25%, 50%, 70%, 80%, 85%, 90%, 95%, and 100% ethanol for 10 min each, followed by an additional two rinses with 100% ethanol. Last, the samples are rinsed with three mixtures of ethanol and HMDS, with volume ratios of 3:1, 1:1, and

1:3, for 10 min each. Then the samples are rinsed twice with pure HMDS for 10 min and left in the HMDS until completely dry. The samples are then ready for SEM imaging.

Experimental Setup for Applying MS to N27 Neural Cells

The Magstim 2002 monophasic stimulator with an ‘8’-shaped coil (Magstim D702 double

70 mm) is used to apply MS to the cells, as shown in Figure S2a (Appendix. Supporting

Information). The magnetic fields are generated in opposite directions through the two windings and are perpendicular to the surface of the ‘8’-shaped coil. As shown in Figure S2b, an upward magnetic field (MF) is generated from the left side of coil, and a downward MF is introduced 24 from the right. The power level is set to 100%, and the MF strength for the coil is shown in

Figure S2c. The upward peak MF strength is ∼1.5 MA/m, and the downward peak MF is ∼1.25

MA/m.

Statistical Analysis

The study focuses on cell growth under two different experimental conditions. First, the cell morphology (i.e., area) of each single N27 cell on different substrates after a 48 h incubation period has been measured. Second, hundreds of single N27 cells have been captured inside each

SU8 microholder, and the division process of each cell has been monitored by counting cell numbers in each SU8 microholder after a 48 h incubation period. For each case, several trials have been conducted to collect sufficient data for statistical analysis. The experimental conditions and parameters are summarized in Table 1.

Table 1. Summary: Experimental Conditions and Parameters

Substrate/ When MS NT (no. NC (no. Experiments Microchip is applied of trials) of cells)

Cell morphology AAO substrate 1 24 h 3 370 (cell size/area measurement) AAO substrate 2 24 h 3 429

Glass substrate 24 h 3 196

Cell division (cell count) Glass microchip 24 h 5 880

AAO microchip 24 h 5 2073

Specifically, for the morphometric analysis of N27 cells, experiments with N27 cells on three substrates (AAO-substrate1, AAO-substrate2, and glass substrate) have been carried out.

For all experiments, MS is applied when the cells have been seeded and grow on the substrates

24 h later. In other words, the cells settle on the substrate’s surface and are allowed 1 day of 25 growth before the application of MS. Three independent trials (NT = number of trials) have been performed for each substrate. For each trial, at least 65 cells (NC = number of cells) are selected for cell area measurement. More details can be found in Table S1 (Appendix. Supporting

Information). To analyze the N27 cell division process, experiments on two types of microchip

(AAO and glass) are performed. On both microchips, only microholders with single N27 cell captured in each are monitored in order to track cell division accurately. For both AAO and glass microchips, MS is only applied after one day (24 h) growth of the captured cells. Five independent trials are performed for each substrate. For each trial, at least 150 single N27 cells are captured in 150 microholders. More details can be found in Table S2 (Appendix. Supporting

Information). SPSS statistical analysis is used to analyze the experimental data. Tests of normality, outliers, and homogeneity variance are performed for each group of data before a one- way analysis of variance (ANOVA).34 The statistical significance is reported with p < 0.05 and p

< 0.005 for each group of data. The one-way ANOVA test results are shown in the following section, and more detailed information about the statistical analysis can be found in Appendix

(Supporting Information).

Figure 2. N27 neural cell growth on nanoporous AAO-substrate2 (a-e) vs glass coverslip substrate (f-j): (a and f) after one day growth; (b and g) after three days growth (cells are indicated with red arrows); (c and h) AFM images of AAO surface and glass surface, respectively; (d, e and i, j) SEM images for a single N27 neural cell growing on AAO surface and glass surface, respectively. 26

Figure 3. F-actin and paxillin staining of N27 neural cells on nanoporous AAO-substrate2 and glass. (a) Cells stained for F-actin and paxillin after 24 h and 48 h on AAO-substrate2 and glass. The scale bar is 40 μm. Whole cell F-actin as well as nuclear paxillin and cytosolic paxillin staining after (b) 24 h and (c) 48 h. (d) Number of center to peripheral focal adhesions as identified by paxillin staining. (e) Number of binucleated cells. Error bars are 95% confidence intervals and nonoverlapping confidence intervals were considered statistically significant and marked with bars.

Results and Discussion

Spreading of N27 Cells on Different Substrates

The N27 cells are seeded on an AAO nanoporous surface (Figure 2a,b) and on a coverslip glass surface (Figure 2f,g). Cells are then cultured in an RPMI medium supplemented with 10% fetal bovine serum, 1% L-glutamine, penicillin (100 U/mL), and streptomycin (100 U/mL). Petri dishes are used as the container, and all the samples are kept in an incubator (NUAIRE-5700) at

37 °C in a humidified atmosphere of 5% CO2 during the cell culture process. It should be noted that both the coverslip glass surface and the AAO nanopore surface shown in Figure 2 are bare surfaces used directly after the cleaning process without any further surface treatment. As shown in Figure 2a,b, cells spread out clearly on the AAO nanoporous surface over the course of 24 h of 27 growth and 3 days of growth alike. As shown in Figure S3, cells can also spread out clearly on the common cell culture flask, similar to those on the AAO nanoporous surface. By contrast,

“ball-shaped” cells are observed after 1 day of growth on coverslip glass (Figure 2f). After 3 days of growth, over 50% of these cells still grow in clusters (Figure 2g), indicating that they have a relatively slower migration than those on the AAO nanoporous substrate.35−37 AFM images of the AAO substrate and glass substrate are shown in Figure 2c and Figure 2h, respectively. As indicated, the surface roughness of the AAO substrate is significantly greater than that of the coverslip glass. Clearly, the nanoporous surface of the AAO substrate is much preferred for cell adhesion and spreading.38−40 Thus, the morphology has obviously been improved compared to that of the cells on coverslip glass.41 The corresponding SEM images of a single N27 neural cell on the AAO surface and on the glass surface are shown in Figure 2d,e and

Figure 2i,j, respectively. The nanopore structures on the AAO surface can facilitate the attachment, adhesion, and spread for N27 cells compared to the smooth surface of coverslip glass.38−40 It has also been reported that the cell adhesion does play an important role in cell proliferation of cancer cells based on biochemical analysis.42 To characterize the difference in adhesion shown on AAO and glass substrates, we fixed and stained for both F-actin and paxillin in N27 cells (Figure 3a). As suggested in Figure 2, cells on AAO substrates were frequently separated from each other as compared to glass. On glass, cells clustered together. This is frequently seen when cell migration is slow compared to cell proliferation.43 In addition, cells on

AAO surface appeared to be better spread (Figure 3a). The formation of F-actin as well as focal adhesions occurs during adhesion, so we measured the levels of F-actin and paxillin, a protein that is localized to focal adhesions44,45 (Figure 3a). At 24 h after plating, there was a small enhancement in F-actin content in N27 cells plated on glass substrate vs AAO substrate (Figure 28

3b). This difference was eliminated at 48 h (Figure 3c). Interestingly, there appeared to be a significant paxillin signal coming from the nucleus in addition to that seen in focal adhesions

(Figure 3a). Paxillin is known to shuttle into the nucleus to affect gene transcription that leads to proliferation.46,47 Enhanced shuttling to the nucleus appears to be due to turnover of paxillin in focal adhesions.48 The nuclear paxillin signal was the same at 24 h after plating on AAO or glass substrates (Figure 3b). However, at 48 h, the paxillin in the nucleus was significantly higher in cells plated on AAO substrates than cells plated on glass (Figure 3c). The opposite trend was seen for cytosolic paxillin, which was higher at both 24 h and 48 h on glass as it was on AAO substrates. This suggests more mature adhesions related to less motile cells. In addition to the enhanced paxillin localization in the nucleus, cells plated on AAO substrates had a lower number of central focal adhesions as compared to peripheral focal adhesions (Figure 3d). Furthermore, central focal adhesion numbers increased with respect to peripheral focal adhesion numbers as the plating time increased (Figure 3d). Central focal adhesions are a marker for less motile cells and tend to turnover slower.39,40 The absence of these central focal adhesions potentially explains the higher paxillin localization in the nucleus of the cell on AAO substrates. Indeed, others have shown similar focal adhesion characteristics on AAO substrates compared to

glass.39,40 Finally, when cells are highly adherent and lack motility, they tend to produce cytokinesis defects on 2D substrates, resulting in binucleated cells (Figure 3e). Taken together, an AAO substrate appears to better promote migration than a glass substrate, resulting in non- clustered cells. The enhanced migration on AAO substrates is caused by the ability of focal adhesions to turnover quicker, leading to more paxillin in the nucleus. On glass substrates, migration is blocked, and central and stable focal adhesions are formed, which act to inhibit the 29 accumulation of paxillin in the nucleus as well as block proper cytokinesis of cells, resulting in more binucleated cells.

Viability of N27 Cells on Different Substrates under MS

To assess the viability of N27 cells under MS, cellular viability tests by calcein AM and propidium iodide (PI)-based live/dead assay have been performed.49 In these experiments, four substrates have been used, including coverslip glass, AAO substrate, glass microchip, and AAO microchip. All the surfaces of these substrates are coated with poly-D-lysine. MS is applied after the cells grow on these substrates for 24 h (i.e., one cell cycle). As expected,50 the cell morphology on the poly-D-lysine coated coverslip glass in Figure 4a,c is superior to that on bare coverslip glass in Figure 2f,g and closer to that on bare AAO substrate in Figure 2a,b and poly-

D-lysine-coated AAO substrate in Figure 4b,d. As evident from the fluorescence images (Figure

4a-d) and viability quantification of the cells (Figure 4e) on the four substrates, MS has a negligible cytotoxic effect on cells. Furthermore, as shown in Figure 4c,d, cell division can also be observed clearly. For example, in the image from the glass microchip in Figure 4c, a single

N27 cell is captured and divided into two daughter cells 24 h (one cycle) later. At this point, MS is applied, and the two daughter cells grow and are divided into five live cells 24 h later (another cell cycle). Similarly, on the AAO microchip seen in Figure 4d, a single N27 cell is divided into two daughter cells 24 h later without MS. At this point, MS is applied, and the two daughter cells grow and are divided into six daughter cells after another 24 h. 30

Figure 4. Effect of MS on N27 cell viability. Live/dead assay on N27 cells cultured on (a) coverslip glass, inset is the close up image of N27 cells, (b) AAO-substrate2, inset is the close up image of N27 cells, (c) glass microchip (cells are indicated with red arrows), (d) AAO-substrate2 microchip (cells are indicated with red arrows). Green (calcein AM) and red (propidium iodide: PI) denote live cell and dead cell, respectively. MS is applied at 24 h: (e) % viability of N27 cells cultured on four substrates for 24 h (blue) and 48 h (orange), respectively. NS denotes no statistically significant difference. 31

MS Effects on N27 Neural Cell Growth

MS effects on morphology (area) of N27 cells on glass and AAO substrates

In these experiments, both AAO substrates were statistically different from the glass substrate. However, there is no significant statistical difference in the N27 cell morphology on

AAO-substrate1 in comparison to that on the AAO-substrate2, suggesting that differences in nanopore size and porosity among AAO substrates have no clear effect on cell growth. The cell areas on the three substrates (AAOsubstrate1, AAO-substrate2, and glass) with and without MS are shown in Figure 5b (2−4).

Figure 5. (a) Representative images showing N27 cells grown on a nanoporous AAO-substrate1; their sizes (areas) are measured by an imaging analysis software. (b) Quantification of cell size of N27 cells cultured on three different substrates without or with MS at 24 h. Asterisks * and ** denote statistically significant differences with p < 0.05 and p < 0.005, respectively. NS denotes no statistically significant difference. 32

The area of the cell without MS on AAO-substrate1 (∼538 μm2) is larger than that (∼520

μm2) with MS. Similarly, on AAO-substrate2, the cell area without MS (∼551 μm2) is larger than that with MS (∼537 μm2). In terms of the glass substrates, the cell area without MS (∼582

μm2) is also larger than that with MS (∼519 μm2). Detailed statistical analysis can be found in

Appendix (Supporting Information). All the results indicate that MS indeed affects cell spreading. Specifically, for all three substrates, the area of the cells that received MS is statistically smaller than that of the ones that did not receive MS. It is important to note that MS is applied to the cells after 24 h of growth, or one cell cycle, and that the measurements are taken

24 h later.

MS effects on the cytoskeleton and paxillin localization

In addition to spreading area, we analyzed the F-actin content as well as paxillin localization in cells with and without MS. While the substrate type (glass vs AAO substrates) did affect cell morphology, there were no gross changes in the cell morphology after MS (Figure 3a and Figure 6a). In addition, there were no large differences in the cytosolic paxillin fluorescence compared to that with and without MS (Figure 6b). Interestingly, the paxillin level in the nucleus, which was high on AAO substrates, decreases when the cells were treated with MS and could be associated with changes in transcription brought on by MS (Figure 6b). This was not seen on glass and may only be observable in well-spread cells. MS did not seem to change the central to peripheral focal adhesion ratio on AAO substrates, but it did decrease it on glass

(Figure 6c). Finally, there is a small increase in the number of binucleated cells after MS on

AAO substrates (Figure 6d). Taken together, it appears that MS decreases paxillin localization to the nucleus. This likely leads to or is caused by enhanced adhesion, resulting in cytokinesis defects leading to binucleated cells. 33

Figure 6. F-actin and paxillin staining of N27 neural cells on nanoporous AAO-substrate2 after magnetic stimulation (+MS). (a) Cells stained for F-actin and paxillin after 48 h on AAO substrate2 and glass and with or without MS at 24 h. The scale bar is 40 μm. (b) Whole cell F-actin as well as nuclear paxillin and cytosolic paxillin staining after 48 h on AAO or glass substrates with and without MS. (c) Number of center to peripheral focal adhesions as identified by paxillin staining. (d) Number of binucleated cells. Error bars are 95% confidence intervals and nonoverlapping confidence intervals were considered statistically significant and marked with bars. 34

MS effects on single N27 neural cell on microchips

To assess the effect of MS on cells more efficiently, arrayed microholder chips have been used to monitor the division process of single cells subject to MS. First, cell synchronization is needed to ensure that the cells are at the same stage of the cell cycle, which can be achieved using the serum deprivation method described in Materials and Methods.32,33 Second, to monitor the cell division process accurately, one single N27 cell is captured in each microholder, enabling its division process to be tracked over the following days. To this end, several steps are required to maximize the capture of the single cell into each microholder: (1) After cells are detached from the bottom of the cell culture flask, they are pipetted into a sterile culture tube

(Corning) that is put on a vortex mixer (VX200-Labnet) with a rotation speed of 500 rpm. This step ensures that the cells are uniformly distributed in the tube and that there are no aggregated cells. (2) The concentration of cells is controlled close to 0.1 million/mL. On the basis of our experiments, 0.1 million/mL is the optimum cell concentration to maximize the number of microholders on-chip to capture a single cell when the cell solution is delivered to the chip by pipet. (3) The chip is given several gentle rinses with the culture medium (10% fetal bovine serum) to achieve optimum single cell capture. Accordingly, single cell division in a microholder is monitored by taking optical images at 0 h, 24 h, and 48 h, as shown in Figure 7. Figure 7a shows that, when MS is not administered, a cell typically divides into two daughter cells after one cell cycle (∼24 h) and into four cells after another cell cycle (∼48 h in total). However, when MS is applied at the 24 h mark, the two cells divide into more than six cells after another cell cycle (∼48 h in total), as shown in Figure 7b. As a result of MS, more than two new daughter cells manifest within the same period of time compared to the chip with cells that do not receive MS (Figure 7a). Clearly, the microholder chip provides a straightforward technical 35 platform to conduct an accurate analysis of the effect of MS on cell division. Several trials have been performed to study the MS effect. For each trial, three microchips with captured cells are immersed in cell media in three Petri dishes and are stored in an incubator (NUAIRE-5700) at all times save for when the MS is applied. Upward MS and downward MS are applied on two microchips, respectively, the third microchip serving as a control without any MS. The experimental details and analysis are summarized in Table S2 (Appendix. Supporting

Information). First, statistical comparisons of cell division are conducted on the glass microchip and the AAO microchip without applying MS. Because no MS is applied to these cells, any differences in outcome are due solely to the surface properties of the glass and AAO surfaces. As shown in Figure 7c (1), the average number of cells (∼4.08) on the glass microchip is smaller than that (∼4.28) on the AAO microchip, a statistically significant difference. Detailed statistical analysis can be found in Appendix (Supporting Information). In short, the nanoporous surface of the AAO microchip affects N27 cell growth, promoting the cell division process in comparison with the flat surface of the glass microchip.

Second, when upward-MS is applied to cells on a glass microchip after 24 h of growth, as shown in Figure 7c(2), the average cell number after a further 24 h (∼4.87) is greater than the cell number of the control group that received no MS (∼3.87). Similarly, when downward-MS is administered to cells on a glass microchip, the average cell number at the 48 h mark (∼4.64) is larger than that of the control. Detailed statistical analysis can be found in Appendix (Supporting

Information). 36

Figure 7. Representative images showing (a) the division of a single neuron cell in a microholder without TMS, and (b) the division of a single cell with MS. The cells are indicated with red arrows. (c) Quantification of cell number at 48 h in a glass microchip and AAO-substrate2 microchip without or with TMS at 24 h. Asterisks * and ** denote statistically significant differences, with p < 0.05 and p < 0.005, respectively. NS denotes no statistically significant difference. 37

Clearly, the average cell number without applying MS is statistically smaller than that with MS. This result indicates that MS can promote cell division. However, no statistical difference has been found between upward MS and downward MS, suggesting that the direction of MS has no effect on the cell division process in these contexts. Third, when MS is applied to cells on an AAO microchip after a 24 h period of growth, as shown in Figure 7c(3), the average cell number at the 48 h mark is ∼4.73 with upward MS and ∼4.85 with downward MS. By contrast, the average cell number of the control group receiving no MS is ∼4.18. These constitute similar results to those observed on the glass microchip. In both cases, the average number of cells is increased when MS is administered after 24 h (i.e., one cell cycle) of growth, indicating that the proliferation of N27 cells can be expedited by MS. The MS direction has no effect on cell division. Several important elements in the experiments should be noted. First, if the MS is applied immediately after the cells are seeded on the substrates or microchips, the experimental results are not consistent, and the viability of the cells is unpredictable. Allowing the cells to attach to the substrate and allowing them to grow for 1 day before applying MS are crucial steps to ensure consistent experimental results. Hence, we only focus on the experiments in which MS is applied to cells after cells have been given one full cycle of growth (24 h) on the substrate. Second, the effect of MS on cells can be clearly and immediately observed in one cell cycle (i.e., 24 h); hence we mainly focus on monitoring cells’ behaviors during a 24 h period after administration of MS. Notably, experiments that have monitored cell growth 72 h and longer after MS have consistently shown increased cell numbers in comparison with control groups. 38

Conclusions

This study constitutes the first investigation into the effect of MS on the viability, spreading, and proliferation of N27 cells at the single-cell level on different substrates. N27 cells have been found to have clear preference for growing on the bare nanostructured AAO surface as opposed to the bare coverslip glass surface. The nanostructures on the AAO surface allow the

N27 cells to attach, spread, and migrate easily. The AAO surface appears to facilitate paxillin movement into the nucleus as well as limit central adhesions that inhibit or are the result of poor migration. A poly-D-lysine coating on either of these surfaces leads to no discernible difference in the spreading of the cells. A live/dead assay has shown negligible cytotoxic effects of MS on cell viability. Additionally, the study has evaluated the effect of MS on the adhesion and proliferation of single N27 cells by measuring cell sizes and monitoring the cell division process in the microholders on a chip. MS has been found to have a statistically significant effect on both cell morphology and cell division/proliferation, and it results in lower paxillin levels in the nucleus. Specifically, MS can expedite the division of N27 cells regardless of the direction in which it is applied. As a result, more N27 cells can be generated with MS than without. These results indicate that MS may help the proliferation and regeneration of neural cells; more largely, the results enhance current understanding of the potential for MS to be used in the treatment of neurological disorders.

Acknowledgements

The authors express gratitude for the technical support from Dr. Wai Leung at

Microelectronics Research Center (MRC) at Iowa State University, from Dr. Kanthasamy’s lab at the College of Veterinary Medicine (CVM) at Iowa State University. The authors also gratefully acknowledge the financial support of the National Science Foundation (Award EECS-

1610967). 39

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Appendix. Supporting Information

(1) Fabrication Process for SU8 Microholder

The fabrication process flow for SU8 microholder chip with AAO nanoporous surface

(AAO microchip) is described in Fig. S1. For SU8 microholder chip with glass surface (glass microchip), the fabrication process flow is similar to that in Fig. S1(a) but requiring only from step 4 to step 5 to fabricate SU8 microholders on glass substrate.

Fig. S1: Arrayed SU8 microholder chip for studying the single N27 cell behaviors. (a) Fabrication process flow for AAO microholder chip. (b) A photo of microholders on chip and the close-up optical micrographs of arrayed SU8 microholders and the captured single neural cell N27.

(2) Experimental Setup for Applying MS

The Magstim 2002 monophasic stimulator with ‘8’ shape coil (Magstim D702 double 70 mm) is used to apply MS on cells as shown in Fig. S2a. The directions of generated magnetic fields are opposite in the two windings and perpendicular to the surface of the ‘8’ shape coil. As shown is Fig. S2b, upward magnetic field (MF) is generated from left side of coil and downward

MF is introduced from right side coil. The power level is set to 100% which is the maximum 44 power level of the stimulator, and measured MF strength for the ‘8’ shape coil is shown in Fig.

S2c. The upward peak MF strength is ~1.5 MA/m and the downward peak MF is ~1.25 MA/m for the ‘8’ shape coil, respectively.

Fig. S2: (a) Experimental setup for applying MS on cells inside microholder chips, the chips are kept in petri dish holders filled with cell media; the magnetic field direction is perpendicular to the chip surface, one is upward, the other is downward. No magnetic field is applied on the chip for the control experiment; (b) Sketch of the cross-section of the experimental setup; (c) The measured magnetic field strength and distribution generated by the MS generator (‘8’ shape coil). 45

Fig. S3: N27 cells cultured on three substrates after one-day growth and three-day growth (a-b) on bare AAO-substrate2; (c-d) on cell culture flask; (e-f) on bare coverslip glass.

(3) Morphometric Analysis

The morphometric analysis of N27 cells and experiments of N27 cells on three substrates

(AAO substrate1, AAO substrate2, and glass) are summarized in Table S1.

Table S1. Statistical results of MS effect on cell morphology on different substrates.

Cell Mean cell size SD SD err Substrate & MS treatment P-value numbers (area: µm2) (area: µm2) (area: µm2)

AAO-substrate1 (no MS) 199 538.67 56.25 3.99 0.002 AAO-substrate1 (with MS) 171 520.52 55.48 4.24

AAO-substrate2 (no MS) 252 551.36 68.94 4.34 0.032 AAO-substrate2 (with MS) 177 537.04 68.41 4.99

Coverslip glass (no MS) 100 582.12 90.92 9.09 0.0 Coverslip glass (with MS) 96 519.15 97.26 9.93 46

(4) Cell Division Process

Cell division process analysis of N27 cells and experiments on two microchips (glass microchip and AAO microchip) are summarized in Table S2.

Table S2. Statistical results of MS effect on cell division on microchip

Mean cell number in each SD SD err No. of Microchip and MS treatment microholder after 48 h (one (no. of (no. of P-value cells cell per microholder at 0 h) cells) cells)

a) Glass microchip w/o MS 348 3.87 1.79 0.10

b) Glass microchip w/ (a, b) = 0.002 285 4.87 2.17 0.13 upward-MS at 24 h (a, c) = 0.000 (b, c) = 0.432 c) Glass microchip w/ 247 4.64 2.18 0.14 downward-MS at 24 h

e) AAO microchip w/o MS 625 4.18 2.03 0.08

f) AAO microchip w/ (e, f) = 0.000 728 4.73 1.99 0.07 upward-MS at 24 h (e, g) = 0.000 (f, g) = 0.514 g) AAO microchip w/ 720 4.85 2.26 0.08 downward-MS at 24 h

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CHAPTER 4. STUDIES OF SINGLE NEURON CELL GROWTH ON NANOPOROUS SURFACE AND UNDER TRANSCRANIAL MAGNETIC STIMULATION

Xiangchen Che1, Joseph Boldrey1, Xiaojing Zhong1, Ian Schneider2, David Jiles1, Life Fellow, IEEE, and Long Que1, Member, IEEE

1Department of Electrical and Computer Engineering, Iowa State University 2Department of Chemical and Biological Engineering, Iowa State University

Modified from a manuscript published in the Proceedings of the 2017 IEEE International Conference on Nanotechnology, Pittsburgh, USA

Abstract

This paper reports the behaviors of neuron cell N27 growth on nanostructured surfaces, and under transcranial magnetic stimulation (TMS) at single cell level for the first time. The growth of neuron cell N27 on anodic aluminum oxide (AAO) nanoporous surfaces has been studied. It has been found that cells show preference to grow on the nanostructured surface compared with the flat coverslip glass surface. The sizes of cells grown on AAO nanoporous surfaces with and without TMS were studied. It was found that the sizes of cells with TMS treatment are statistically smaller than those without TMS treatment in the same period of time, indicating the TMS might speed up the cell division. To verify this observation, the growth of single N27 cells inside SU8 microholders with and without TMS has been investigated. It has been found that up to 17% more daughter cells can be produced when the cells are subjected to

TMS compared to those without TMS. All these results suggest the TMS can contribute to the growth of N27 cells.

Introduction

Nanostructured bio-interfaces are one of the fastest emerging fields in biomedical engineering. For instance, over the past years, the unique properties of nanoporous anodic alumina oxide (AAO) thin film have greatly contributed to the development of a variety of novel 49 biomedical and medicine applications, ranging from biofiltration membranes, lipid bilayer support structures, biosensing devices, and implant coatings to drug delivery systems with AAO capsules and scaffolds for tissue engineering [1-6]. Additionally, nanoporous AAO membranes have attracted increasing interest as cell-interface substrates for manifold cell types [7]. On the other hand, transcranial magnetic stimulation (TMS) is a non-invasive neuromodulation technique that uses time varying short pulses of magnetic field to induce an electric field in the tissues of the brain, thus modulating the synaptic transmission of neurons. This neuromodulation technique can be used to excite or inhibit the firing rate of neurons as a treatment for various neurological disorders such as major depressive disorder, Parkinson's disease, post-traumatic stress disorder and, migraine [8-9]. The molecular/cellular mechanisms of neurons under TMS are complex. Hence, the effects of TMS on individual neurons needs to be thoroughly understood in order to fully utilize the benefits of TMS as a neuromodulation tool for treating neurological disorders.

Herein, we report on the studies of neuron cell growth on the AAO nanoporous surface and also the effect of TMS on single neuron cells using a microchip.

Materials and Methods

Neuron Cell N27

Immortalized rat mesencephalic cells (1RB3AN27, N27 for short) are used in the experiments, these cells are grown in RPMI medium supplemented with 10% fetal bovine serum,

1% L-glutamine, penicillin (100 U/ml), and streptomycin (100 U/ml), maintained at 37 °C in a humidified atmosphere of 5% CO2.

Preparation of AAO Nanoporous Substrate and Glass Substrate

Preparation of AAO nanoporous substrate is briefly summarized as follows. After a layer of Al is deposited on a glass substrate, a one-step anodization process is carried out to convert Al 50 into nanoporous AAO thin film [10]. A SEM image of the AAO thin film is shown in Fig. 1.

Both AAO nanoporous substrates and glass substrates, were cleaned with DI water by ultrasonicator (BARNSON 1510 - Fisher Scientific) for 5 mins, then disinfection of these substrates is achieved by cleaning with Acetone and Methanol by ultra sonicator for 5 mins in sequence. Then they were cleaned with DI water by ultra sonicator for 5 mins and dried by a nitrogen gun, followed by air plasma cleaning (Harrick plasma-Pcd 32G) for 3 mins. Finally, they were baked on a hot plate at 150°C for 5 mins before they are ready for use. In some experiments, poly-D-lysine is coated on the substrates before the cells are seeded for experiments.

Figure 1: SEM image of the AAO nanoporous surface fabricated using one-step anodization process. 51

Microholder Chip Fabrication

Microholders on a glass substrate are fabricated from polymer SU8 using a standard optical lithography process. The size of the microholders was designed as 180 μm × 180 μm so that the N27 cells can fully grow without any spatial limitations. A photo of the fabricated SU8 microholder chip and a close-up optical micrograph of four microholders are shown in Fig. 2.

Figure 2: Photo of the arrayed SU8 microholders and their close-up for studying the behaviors of single neuron cell.

Experimental Setup for Applying TMS on Neurons

A figure-of-eight coil was used to apply TMS to cells as shown in Fig. 3(a-b). On the left side of the coil, the direction of the magnetic field (MF) is upward and perpendicular to the chip holder, while the direction of the MF on the right side of the coil is downward. Three chips in three holders were used for the experiments. The magnetic field (H) was measured using a

Gaussmeter and an axial Hall probe. The measured MF strength of the figure-of-eight coil is shown in Fig. 3(b). The upward peak MF strength is ~1.5 MA/m and the downward peak MF is

~1.25 MA/m for the figure 8 coil, respectively. 52

Figure 3: (a) Experimental setup for applying TMS to cells inside microholder chips, the chips are kept in petri dish holders filled with cell media; the magnetic field direction is perpendicular to the chip surface, one is upward, the other is downward. No magnetic field is applied on the chip for the control experiment; (b) sketch of the cross-section of the experimental setup; (c) the measured magnetic field strength and distribution generated by the TMS generator using the figure-of-eight coil. 53

Results and Discussion

Neuron Cells Growth on Nanoporous Surface vs. Glass Surface

The following phenomena have been observed: for the AAO and glass substrates, (1) if both substrates are not coated with poly-D-lysine, after the cells are seeded on them, the growth

(adhesion and spreading) of N27 cells on the AAO surface are much better than that on the glass substrate; (2) if both substrates are coated with poly-D-lysine, the growth of N27 cells on poly-

D-lysine coated glass substrate becomes better, but not as good as that on poly-D-lysine coated

AAO substrate. Representative optical images of the N27 cells on AAO, and glass substrates after one day growth are shown in Fig. 4(a) and Fig. 4(d), respectively, and after three days are shown in Fig. 4(b) and Fig. 4(e), respectively. The AFM images of the AAO substrate and glass substrate are shown in Fig. 4(c) and Fig. 4(f), respectively. As shown, the roughness of the surface of AAO substrate is much larger than that of the coverslip. Clearly, the nanoporous surface of the AAO substrate is much preferred, in comparison with the surface of the glass coverslip, for cells’ adhesion and spreading [11].

Another observation is that, if the glass substrate is subjected to an air plasma treatment, then coated with ploy-D-lysine several times, the growth of N27 cells becomes essentially similar to that on AAO nanoporous substrate. It should be noted that even without any plasma treatment and poly-D-lysine coating, the growth of N27 on AAO nanoporous substrates is always very good, again indicating the N27 cells’ preference to grow on the nanostructured surface. 54

Figure 4: Optical images showing the N27 cell growth on nanoporous substrate (a-b) vs. glass coverslip substrate (d-e): (a) and (d) after one day growth; (b) and (e) after three day growth (cells are pointed by red arrows); AFM images of (c) AAO nanoporous surfaces and (f) glass surface.

Effect of TMS on the Behavior of Neuron Cells

Several trials have been conducted to study the TMS effect on the behavior of N27 cells by monitoring the size (area) of the cells. In these experiments, cells seeded on poly-D-lysine coated AAO surface have been investigated. The direction of the applied TMS on cells is illustrated in Fig. 3.

Representative images showing the sizes (areas) of N27 cells, which are measured by an imaging analysis software, are given in Fig. 5(a). 55

Figure 5: (a) Representative images showing N27 cells grown on a nanoporous AAO substrate; their sizes (areas) are measured by an imaging analysis software; N27 cells on poly-D-Lysine coated AAO nanoporous substrate (b) with a thickness of 1.3 μm and (c) with a thickness of 2.5 μm with TMS vs without TMS. 56

In Fig. 5(b-c), the measured sizes (areas) of the cells with and without TMS are given. It is clearly shown that the average size of the cells with TMS is about 4.5% smaller than that of cells without TMS during the same period of time. The measured sizes (areas) of cells with TMS are statistically smaller than those without TMS, suggesting that the division of cells with TMS might occur earlier than that of cells without TMS, namely the TMS might speed up the division of the cells.

Single Neuron Cell Growth in Microholders

In order to examine if the TMS can speed up the division of N27 cells, single N27 cell growth behavior in a microholder without and with TMS has been studied.

Cell synchronization

In order to monitor the cell division accurately, cell synchronization, a process by which cells at different stages of the cell cycle in a culture are brought to the same phase, has been implemented in our experiments. The method we used is serum deprivation. Serum deprivation is a simple and reliable method by changing the percentage of fetal bovine serum from 10% to

1% in the medium. 9 hours before seeding the cells to the microholders, we replaced the medium with 10% fetal bovine serum with that with 1% fetal bovine serum to the cells. Because the bovine serum is essential for the growth of N27 cells, the risk of cell death increases with the serum deprivation time. We chose 9 hours to make sure the cells will not die because of the serum deprivation.

Cells seeding

Before we seeded cells from the flask to microholder devices, trypsin was used to detach the cells from the bottom of flask. After 2 or 3 minutes, we pipetted normal medium with 10% fetal bovine serum to the flask. Then the cells were ready to be seeded into microholder devices. 57

Representative optical images showing the single cell growth in microholders without

TMS and with TMS are shown in Fig. 6(a) and Fig. 6(b), respectively. As shown in Fig. 6(a), without TMS, as expected, in most cases, one cell divides into two cells after one cell cycle (~24 hours), then the two cells divide into four cells after another cell cycle (~48 hours). In contrast, in

Fig. 6(b), one cell divides into two cells after one cell cycle (~24 hours), immediately at this point, the TMS is applied. After another cell cycle (~48 hours), the two cells divide into more than six cells. As a result, due to TMS, more daughter cells have been produced.

In order to analyze the effect of TMS more accurately on the cell division, the following experiments and analysis schemes have been carried out. Under ideal growth conditions, one parent cell divides into two daughter cells in the first cell cycle (~24 hours), then into four daughter cells in the second cell cycle (~48 hours) [12]. The difference between the number of cells with/without TMS and the number of cells under ideal growth condition can be calculated using the formulas:

Δdifference = Nw / TMS - Nideal and Δdifference = Nw/o TMS - Nideal where

Nideal = number of cells under ideal culturing conditions

Nw / TMS = number of cells for the group that received TMS

Nw/o TMS = number of cells for group that did not receive TMS

The ratio R = Δdifference / Nideal is used to evaluate the TMS effect on cell division. First, the TMS is applied immediately after the cells are seeded into microholders. As shown in Fig.

6(c), two trials have been carried out. In Trial 1, the ratio R is about +2.44% when an upward

TMS is applied, the ratio is about +2.77% under a downward TMS. In the control experiment, the ratio is about -9.09% without applying TMS. In Trial 2, the ratio R is about +4.67% when an 58 upward TMS is applied, the ratio is about +5.49% under a downward TMS. In the control experiment, the ratio is about -6.22%. For both trials, the net ratio R = R - Rcontrol is 10.89% -

11.53% under an upward TMS, and 11.71% - 11.86% under a downward TMS, respectively.

Figure 6: Representative images showing (a) the division of a single neuron cell in a microholder without TMS, (b) the division of a single cell with TMS (cells are pointed by red arrows); the measured ratio (Δdifference / Nideal) under TMS vs. no TMS: (c) TMS applied immediately after cells seeded into microholders; (d) TMS applied after cells’ growth inside microholders for 24 h.

In Fig. 6(d), the TMS is applied after 24-hour cell growth. In Trial 1, the ratio is about

+18.72% when an upward TMS is applied, the ratio is about +18.62% under a downward TMS.

In the control experiment, the ratio is about 2.67%. In Trial 2, the ratio is about +21.93% when 59 an upward TMS is applied, the ratio is about +19.75% under a downward TMS. In the control experiment, the ratio is about 5.11%. For both trials, the net ratio R=R-Rcontrol is 16.05-16.56% under an upward TMS, and 14.64-15.95% under a downward TMS, respectively.

Note that in each trial, the growth of about 100 single cells has been monitored. Based on these experiments, the TMS effect on cell growth is obvious. When the TMS is applied after cells’24 h growth, the effect becomes even stronger.

Summary

In this paper, a nanoporous AAO surface has been evaluated for the growth of N27 cells.

It has been found that N27 cells have clear preference to grow on the nanostructured surface rather than the flat surface. In addition, the effect of TMS on the growth of single N27 cells has been studied by measuring their size and monitoring their cell division in the microholders. It has been found that TMS can increase cell division, up to ~17% more daughter cells are divided in the same cell cycle compared to those without TMS.

Acknowledgement

The authors thank the technical supports from Dr. Wai Leung at Microelectronics

Research Center (MRC) at Iowa State University.

References

[1] S. Majd, E. Yusko, Y. Billeh, M. Macrae, J. Yang and M. Mayer, "Applications of biological pores in nanomedicine, sensing, and nanoelectronics", Current Opinion in Biotechnology, vol. 21, no. 4, pp. 439-476, 2010.

[2] L. Movileanu, "Interrogating single proteins through nanopores: challenges and opportunities", Trends in Biotechnology, vol. 27, no. 6, pp. 333-341, 2009.

[3] L. Gu and J. Shim, "Single molecule sensing by nanopores and nanopore devices", The Analyst, vol. 135, no. 3, pp. 441-451, 2010.

[4] Y. He, X. Li and L. Que, "A Transparent Nanostructured Optical Biosensor", Journal of Biomedical Nanotechnology, vol. 10, no. 5, pp. 767-774, 2014. 60

[5] A. Md Jani, D. Losic and N. Voelcker, "Nanoporous anodic aluminium oxide: Advances in surface engineering and emerging applications", Progress in Materials Science, vol. 58, no. 5, pp. 636-704, 2013.

[6] X. Li, Y. He and L. Que, "Fluorescence Detection and Imaging of Biomolecules Using the Micropatterned Nanostructured Aluminum Oxide", Langmuir, vol. 29, no. 7, pp. 2439- 2445, 2013.

[7] D. Brüggemann, "Nanoporous Aluminium Oxide Membranes as Cell Interfaces", Journal of Nanomaterials, vol. 2013, pp. 1-18, 2013.

[8] J. O'Reardon, H. Solvason, P. Janicak, S. Sampson, K. Isenberg, Z. Nahas, W. McDonald, D. Avery, P. Fitzgerald, C. Loo, M. Demitrack, M. George, H. Sackeim, "Efficacy and Safety of Transcranial Magnetic Stimulation in the Acute Treatment of Major Depression: A Multisite Randomized Controlled Trial", Biological Psychiatry, vol. 62, no. 11, pp. 1208-1216, 2007.

[9] F. Fregni, "Repetitive transcranial magnetic stimulation is as effective as fluoxetine in the treatment of depression in patients with Parkinson's disease", Journal of Neurology, Neurosurgery & Psychiatry, vol. 75, no. 8, pp. 1171-1174, 2004.

[10] X. Che, Y. He, H. Yin and L. Que, "A molecular beacon biosensor based on the nanostructured aluminum oxide surface", Biosensors and Bioelectronics, vol. 72, pp. 255- 260, 2015.

[11] F. Gentile, R. La Rocca, G. Marinaro, A. Nicastri, A. Toma, F. Paonessa, G. Cojoc, C. Liberale, F. Benfenati, E. di Fabrizio and P. Decuzzi, "Differential Cell Adhesion on Mesoporous Silicon Substrates", ACS Applied Materials & Interfaces, vol. 4, no. 6, pp. 2903-2911, 2012.

[12] "Cell division", En.wikipedia.org, 2017. [Online]. Available: https://en.wikipedia.org/wiki/Cell_division. [Accessed: 28- May-2017]. 61

CHAPTER 5. INVESTIGATING THE ROLE OF PULSED MAGNETIC FIELD INTENSITY ON THE GROWTH AND PROLIFERATION OF N27 DOPAMINERGIC NEURONAL CELLS

Joseph Boldrey1, Long Que1, Ian Schneider2, David Jiles1, and Ravi Hadimani3

1Department of Electrical and Computer Engineering, Iowa State University 2Department of Chemical and Biological Engineering, Iowa State University 3Department of Mechanical and Nuclear Engineering, Virginia Commonwealth University

Modified from a presentation given at the 2019 IEEE Magnetics Society Conference on Magnetism and Magnetic Materials, Las Vegas, USA

Abstract

Much of the biomedical research to find the causes of and treatments for Parkinson's

Disease (PD) and Alzheimer's Disease (AD) is performed using cell lines from animals that model the behavior of cells in the human brain that are linked to PD and AD. The N27 dopaminergic cell line is well-established in neurodegenerative disease research. [1] [2].

Transcranial Magnetic Stimulation (TMS) has been approved by the U.S. Food and Drug

Administration (FDA) as a method for treating major depressive disorder (MDD), obsessive- compulsive disorder (OCD) and certain types of migraine headaches [3]. However, the effect of

TMS type fields at the cellular level are not fully understood. Recent studies have shown that monophasic pulsed magnetic fields generated by a TMS stimulator increases growth and proliferation of N27 cells in vitro [4] [5] [6].

In TMS therapy, each session is tailored to the specific patient by establishing a base threshold level of magnetic field strength (H). This is performed by aiming the stimulation coil at the patient’s motor cortex and finding the minimum intensity level to make the patients thumb visibly twitch. This H field intensity baseline, called the motor threshold (MT), is then multiplied by a specified percentage set by the therapy protocol established by the FDA. Different patients 62 have different MTs. Therefore, the intensity of the H field delivered to the brain cells of different patients should also vary.

Recent studies have shown that monophasic pulsed magnetic fields generated by a TMS stimulator increases growth and proliferation of N27 cells in vitro [4] [5] [6]. The goal of our research is to investigate the effects of different H field intensities on N27 cells. By modulating the H field, an exploration was made into the role of variable H field intensity experienced during TMS therapy due to different MT between patients. The results of our research will increase our understanding of the effects of magnetic fields at the cellular level, increase the knowledge of the underlying mechanisms behind TMS, and contribute the ongoing research into treatments for PD and AD.

Introduction

Transcranial Magnetic Stimulation (TMS) is a neuromodulation technique which is capable of non-invasively activating neurons in the brain. TMS is approved by the U.S. Food and

Drug Administration (FDA) for treating major depressive disorder, obsessive compulsive disorder, and certain types of migraine headaches. In TMS therapy, each session is tailored to the patient by initially establishing a baseline of magnetic field strength (H) called the Motor

Threshold (MT). The MT is then multiplied by a specified percentage established by the FDA for each type of therapy. Different patients have different MTs. Therefore, the intensity of the H field delivered to the brain cells of different patients should also vary. The goal of our research is to investigate the effects of different H field intensities on N27 dopaminergic cells. We chose the

N27 cell line because it is well established in biomedical research for neurodegenerative diseases. By modulating the H field, an exploration was made into the role of variable H field intensity experienced by brain cells during TMS therapy. 63

Experiment Procedure

Experimental Timeline

The chart in Fig. 1 shows the timeline followed during all of the experimental trials.

Fig. 1. The major events of the experimental trials at their respective time points during each trial.

2D Extracellular Matrices (ECMs)

Collagen coated glass cover slips, and poly-D-lysine (PDL) coated glass cover slips were used as the structural growth media for experiments.

Cell Culture

Rat mesencephalic cells (N27) were cultured in RPMI 1640 [+ L-Glutamine] medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 U/ml), maintained at 37 °C in a humidified atmosphere of 5% CO2.

Magnetic Stimulation

A commercially available Magstim 2002 monophasic stimulator, along with two commercially available coils from the Magstim company, were used to apply to magnetic stimulation to the cells. The two coils used were the D702 figure-8 coil and the single 90 mm circular coil. The stimulator was set at 100% power output for the stimulation sessions. The cells received 150 pulses in single 12 min session. The pulse rise time was ~100 µs with a duration of

1 ms. The frequency of the stimulation pulse was 1 kHz.

Measured Magnetic Fields

The magnetic field strength (H) for each coil was measured using a Gaussmeter and an axial Hall probe. The placement of the Hall probe was 20 mm from each coil, respectively. This 64 is the distance between the coils and the ECM coated cover slips during the experiments. The measured peak H field for the D702 figure-8 coil was 0.5593 T (0.4451 MA/m). The measured peak H field for the 90 mm circular coil was 0.4728 T (0.3762 MA/m). The peak H field of figure-8 coil is 18.31% greater than the peak H field of the single 90 mm coil.

Stimulation Schematic

The placement of the well plates that held the glass cover slips during the experiment trials with respect to each of the stimulation coils are shown in Fig. 2.

Fig 2. Location of the glass coverslips used for growing the N27 cells with respect to the TMS coils during stimulation using the (a) D702 figure-8 coil and the (b) 90 mm circular coil.

Results

The numbers of cells counted at each time point during the trials are plotted in Fig. 3 for the 90 mm circular coil, and Fig 4. for the D702 figure-8 coil. 65

Fig. 3. Comparison of cell numbers with respect to time. Magnetic stimulation applied using the 90 mm circular coil at 24 hr time point after seeding cells on collagen and PDL ECMs, respectively.

Fig. 4. Comparison of cell numbers with respect to time. Magnetic stimulation applied using the D702 figure-8 coil at 24 hr time point after seeding cells on collagen and PDL ECMs, respectively. 66

Figure-8 Coil Statistical Analysis

Fig. 5 shows the analysis of the cells (stimulated compared with non-stimulated) using the figure-8 coil for stimulation while being grown in the different ECMs.

Fig. 5. The groups that received TMS, show an increase in proliferation rate compared to the groups that did not receive TMS. This increase occurred on both ECMs, collagen and poly-D-lysine.

Discussion

Monophasic TMS fields, at sufficient intensity, and given in a single session, increase the proliferation rate of N27 neuronal cells. This increase occurs on collagen and on poly-D-lysine

ECMs. By modulating the H field, a threshold of the sufficient intensity is shown to exist between 0.4728 T and 0.5593 T. Further research into the effects of TMS on the internal cellular processes of N27 cells are currently in progress.

Acknowledgment

Funded by the National Science Foundation, the James and Barbara Palmer Foundation, and the Stanley Chair in Interdisciplinary Engineering at Iowa State University.

References

[1] Rocca, Walter A. “Time, Sex, Gender, History, and Dementia.” Alzheimer disease and associated disorders 31.1 (2017): 76–79. PMC. Web. 1 Aug. 2018. 67

[2] “N27 Rat Dopaminergic Neural Cell Line SCC048.” [Online]. Available: http://www.emdmillipore.com/US/en/product/N27-Rat-Dopaminergic-Neural-Cell- Line,MM_NF-SCC048. [Accessed: 10-Apr-2018].

[3] “FDA permits marketing of transcranial magnetic stimulation for treatment of obsessive compulsive disorder,” Case Medical Research, 2018. [Online]. Available: https://www.fda.gov/newsevents/newsroom/pressannouncements/ucm617244.htm. [Accessed: 18-Mar-2019].

[4] "Cellular level studies and coil system design for transcranial magnetic stimulation", Meng, Yiwen, (2015) Graduate Theses and Dissertations, https://lib.dr.iastate.edu/etd/14511

[5] "On-Chip Studies of Magnetic Stimulation Effect on Single Neural Cell Viability and Proliferation on Glass and Nanoporous Surfaces", Xiangchen Che, Shalini Unnikandam- Veettil and Ian Schneider, ACS Applied Materials & Interfaces 2018 10 (34)

[6] "Effects of Magnetic Stimulation on the Proliferation of Dopaminergic Neuronal Cells Grown in Extracellular Matrix", Xiaojing Zhong, Long Que and Ian Schneider, INTERMAG Conference, Singapore, April 23-27, 2018 68

CHAPTER 6. STUDIES OF NEURITE EXTENSIONS ON PC-12 CELLS UNDER TRANSCRANIAL MAGNETIC STIMULATION

Joseph Boldrey1, Ian Schneider2, Long Que1, and David Jiles1

1Department of Electrical and Computer Engineering, Iowa State University 2Department of Chemical and Biological Engineering, Iowa State University

Modified from a presentation given at the 2019 IEEE Magnetics Society Summer School, Richmond, USA

Abstract

Transcranial Magnetic Stimulation (TMS) is an approved treatment for major depressive disorder (MDD) and obsessive compulsive disorder (OCD) by the U.S. Food and Drug

Administration (FDA). The underlying mechanisms that make TMS effective are still being discovered as researchers continue to discover more applications for TMS.

Much of the biomedical research to find the causes of and treatments for Parkinson's

Disease (PD) and Alzheimer's Disease (AD) is performed using PC-12 neuronal cells (Rattus norvegicus, pheochromocytoma) that model the behavior of cells in the human brain that are linked to PD and AD. Many studies have reported the effects of magnetic stimulation on neurite growth of PC-12 neuronal cells. However, interpreting the effects of electromagnetic fields on neuronal cells and comparing results from multiple studies can be challenging due to the many variables involved in these types of experiments: monophasic vs bi-phasic time-dependent signal, type of magnetic field; AC vs DC, amplitude of the magnetic field, duration/frequency of time-varying magnetic pulses, number of total pulses and length of time under a steady state field. The aim of this study is to investigate the effects of time-dependent magnetic fields on neurite growth of PC-12 cells using the equivalent dose given during TMS treatments.

During TMS treatment, neurites will experience the applied field according to their orientation within the brain. Our research probed the consequence of applying the equivalent 69 dose magnetic field experienced in TMS sessions in different orientations with respect to the growth surface plane of the neurites. Each stimulated cell group had a control group that was subjected to all the same experimental steps except TMS.

Introduction

Transcranial Magnetic Stimulation (TMS) is approved by the U.S. Food and Drug

Administration (FDA) as a method for treating major depressive disorder (MDD), obsessive compulsive disorder (OCD), and certain types of migraine headaches. However, the effect of

TMS type fields on the brain has yet to be fully investigated at the cellular level. Our research investigated the effects of monophasic pulsed magnetic fields generated by a commercial TMS stimulator and coil on the growth of neurite extensions of PC-12 cells stimulated with nerve growth factor beta (NGFβ).

Our previous work showed that a single session of stimulation applied orthogonal to the growth surface increased the average length of neurite extensions on PC-12 cells. Our current work is exploring the effects multiple stimulation sessions on PC-12 neurite growth when the field is applied orthogonal, and when it is applied parallel to the growth surface. The cells were given multiple short stimulation sessions that were repeated daily for several days. The protocol is the same that is used in TMS therapy and clinical research. Each stimulated cell group had a control group that was subjected to all the same experimental steps except magnetic stimulation.

Experiment Procedure

Experiment Timeline

The chart in Fig. 1 shows the timeline followed during the experimental trials.

Fig. 1. The major events of the experimental trials at their respective time points during each trial.

Cell Preparation

The PC-12 cells were cultured in 1640 RPMI [+ L Glutamine] medium supplemented with 10% horse serum, 5% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100

U/ml), maintained at 37 °C in a humidified atmosphere of 5% CO2. The extracellular matrix used was Matrigel (50 μg/ml diluted 1:8 with 1640 RPMI). Prior to seeding the cells, the Matrigel was applied to bottom of the wells, then incubated at room temperature for 60 min. The cells were seeded with 1640 RPMI [+ L Glutamine] medium supplemented with 1% horse serum, penicillin

(100 U/ml), and streptomycin (100 U/ml), and 50 ng/ml NGFβ.

Magnetic Stimulation

The cells received 150 pulses in single 12 min session. The pulse rise time was ~100 µs with a 71 duration of 1 ms. The frequency of the stimulation pulse was 1 kHz. Stimulation sessions were repeated every 24 hours for a total of 4 stimulation sessions per trial.

Field Strength of TMS Coil

The magnetic field strength (H) generated by the coil was measured using a Gaussmeter and an axial Hall probe. The placement of the Hall probe was 12 mm from the. This is the distance between the coil and the cells in the wells during the experiments. The maximum field strengths were measured multiple times and averaged. The averaged measured peak H field for the D702 figure-8 coil was calculated to be 0.509 T (0.405 MA/m).

Stimulation Schematic

The placement of the well plates that held the cells during the experiments with respect to the stimulation coil is shown in Fig. 2.

Fig 2. Location of the cells in the well plates with respect to the TMS coil during stimulation sessions. The white arrows indicate the direction of the applied magnetic field.

Methods of Measurement

Image J software was used to measure the angle of growth of the neurite extensions from the main cell body with respect to a fixed reference point. Two types of measurements were taken of each extension as shown by the examples in Fig. 3. 72

Fig. 3. Examples of how the neurite extensions were measured using Image J software. On the left is a measurement from the base of the extension to the tip of the longest branch. On the right is an example of measuring the angle of the primary branch of the extension as it grows from the main cell body.

Results

Measurement data of the neurite extensions were plotted as histograms in polar coordinates as shown in Fig. 4. This plotting method allows the analysis of the distribution of the neurite’s direction of growth with respect to the main cell body, and the direction of the applied magnetic field.

Fig. 4. The histograms show the directional distribution of neurite growth with respect to the direction of the applied magnetic field as shown in the stimulation schematic in Fig. 2. 73

Statistical Analysis

The method used to statistically analyze the measured angles of the extensions is called the Directionality Index (DI). The DI = -cos (θ), where θ is the angle of the primary branch or the angle of the base to tip measurement with respect to magnetic field direction. Fig. 5 shows box plots that represent the average DI for each experimental group. Statistical analysis shows that the there is no significant preference in the direction of new neurite extensions or overall direction of neurite extension growth with respect to the direction of the applied magnetic field for any group.

Fig. 5. Box plots representing the average Directionality Index (DI) for each experimental group with error bars that represent the 95% confidence interval of the standard deviation for each group.

Discussion

The magnetic stimulation was applied in short sessions and repeated daily over several days. This method is the same that is used in TMS therapy and clinical research. The stimulation protocol did not result in a directional bias of new neurites or a directional bias of overall neurite growth after several days. These results show that PC-12 neurite extensions experienced no adverse effects on directionality of growth as a result of magnetic fields used in TMS therapy and clinical research. 74

Acknowledgment

Funded by the National Science Foundation and partially funded by the Stanley Chair in

Interdisciplinary Engineering at Iowa State University.

References

[1] M. Isserles et al., “Effectiveness of deep transcranial magnetic stimulation combined with a brief exposure procedure in post-traumatic stress disorder-a pilot study,” Brain Stimul. 6(3), 377–383 (2013).

[2] “PC-12 ATCC ® CRL-1721TM Rattus norvegicus adrenal gland pheoc.” [Online]. Available: https://www.atcc.org/products/all/CRL-1721.aspx. [Accessed: 10-Apr-2018].

[3] The Magstim Company Limited, “Magstim 2002 Operating Manual (P/N 3001-23-04),” 28 (March 2005).

[4] Joe Boldrey, Xiaojing Zhong, Long Que, Ian Schneider, David Jiles, "The Influence of Pulsed Magnetic Fields on NGF Differentiated PC-12 Neuronal Cells", Minnesota Neuromodulation Symposium, Minneapolis, USA, April 12-13, 2018.

[5] S. Rossi et al., “Safety, ethical considerations, and application guidelines for the use of transcranial magnetic stimulation in clinical practice and research,” Clin. Neurophysiol., vol. 120, no. 12, pp. 2008–2039, 2009. 75

CHAPTER 7. THE INFLUENCE OF PULSED MAGNETIC FIELDS ON THE PROLIFERATION OF ADHERENT AND NON-ADHERENT NEURONAL CELL TYPES

Joseph Boldrey1, Xiaojing Zhong1, Long Que1, Ian Schneider2, and David Jiles1

1Department of Electrical and Computer Engineering, Iowa State University 2Department of Chemical and Biological Engineering, Iowa State University

Modified from a presentation given at the 2019 IEEE Magnetics Society Joint MMM-Intermag Conference, Washington D.C., USA

Abstract

Much of the biomedical research to find the causes of and treatments for Parkinson's

Disease (PD) and Alzheimer's Disease (AD) is performed using cell lines from animals that model the behavior of cells in the human brain that are linked to PD and AD. Two of the animal cell lines used for PD and AD research are the PC-12 neuronal cells and the N27 dopaminergic neuronal cells. PC-12 cells are non-adherent cells that do not attach to surfaces during their growth cycle. N27 cells are adherent cells that attach and spread out on surfaces during their growth cycle. [1] [2] [3] [4]. We chose these two cell types for our research because of their well-established results in neurodegenerative disease research.

Transcranial Magnetic Stimulation (TMS) has been approved as treatment for major depressive disorders by the U.S. Food and Drug Administration (FDA) since 2008 [5]. The major effect of time dependent magnetic fields on the brain has yet to be fully investigated at the cellular level. Our research used the stimulation protocol used in TMS therapy. A predetermined number of magnetic pulses were applied within a short time period using a Magstim 2002 monophasic stimulator with the Magstim D702 figure eight dual 70 mm high-focality coil [6].

Using this protocol, we observed the effects of magnetic fields that are experienced by cells in

TMS therapy in vivo. Our research showed that pulsed magnetic fields increased the 76 proliferation of N27 cells grown on collagen coated and Poly-D-Lysine coated glass cover slips as shown in Figure 1. Each stimulated cell group had a control group that was subjected to all the same experimental steps except TMS [7].

Our current work is exploring the effects of magnetic stimulation on the growth and proliferation of PC-12 cells free floating in growth media and while in a collagen-hyaluronan 2D extra cellular matrix. The results of our research will increase our understanding of the effects of magnetic fields at the cellular level, increase the knowledge of the underlying mechanisms behind TMS, and contribute the ongoing research into treatments for PD and AD.

Introduction

Much of the biomedical research investigating the causes of, and treatments for,

Parkinson's Disease (PD) and Alzheimer's Disease (AD) is performed using animal cell lines that model the behavior of cells in the human brain. Two of the animal neuronal cell lines used for

PD and AD research are the PC-12 and the N27. PC-12 cells are non-adherent type cells that do not attach to surfaces during their growth cycle. N27 cells are adherent type cells that attach and spread out on surfaces during their growth cycle.

Transcranial Magnetic Stimulation (TMS) is a U.S. Food and Drug Administration

(FDA) approved method for treating major depressive disorder and obsessive-compulsive disorder. The effect of time-dependent magnetic fields used in TMS have yet to be fully investigated at the cellular level. Our previous research showed that pulsed magnetic fields generated by a TMS stimulator increased the proliferation of N27 cells grown on collagen coated and Poly-D-Lysine coated substrates. Our current work explored the effects of the same stimulation protocol on the growth and proliferation of PC-12 cells free floating in growth media. 77

The results of our research will increase our understanding of the effects of magnetic fields used in TMS at the cellular level, increase the knowledge of the underlying mechanisms behind TMS, and contribute the ongoing research into treatments for PD and AD.

Experiment Procedure

Experimental Timeline

The chart in Fig. 1 shows the timeline followed during the experimental trials.

Fig. 1. The major events of the experimental trials at their respective time points during each trial.

Cell Culture

The PC-12 cells were cultured in 1640 RPMI [+ L-Glutamine] medium supplemented with 10% horse serum, 5% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100

U/ml), maintained at 37 °C in a humidified atmosphere of 5% CO2.

Magnetic Stimulation

A commercially available Magstim 2002 monophasic stimulator was used along with a commercially available D702 figure-8 coil from the Magstim company to apply magnetic stimulation to the cells. The stimulator was set at 100% power output for the stimulation sessions. The cells received 150 pulses in single 12 min session. The pulse rise time was ~100 µs with a duration of 1 ms. The frequency of the stimulation pulse was 1 kHz. 78

Measured Magnetic Fields

The magnetic field strength (H) generated by the coil was measured using a Gaussmeter and an axial Hall probe. The placement of the Hall probe was 20 mm from the coil. This is the distance between the coil and the cells in the wells during the experiments. The maximum field strength was measured multiple times and averaged. The average measured peak H field for the

D702 figure-8 coil was calculated to be 0.559 T (0.445 MA/m).

Stimulation Schematic

The placement of the well plates that held the cells during the experiments with respect to the stimulation coil is shown in Fig. 2.

Fig 2. Location of the cells in the well plates with respect to the TMS coil during stimulation sessions.

Cell Counting

At each time point, a single well from each of the well plates, non-stimulated and stimulated were counted. The cells were transferred to a 15 ml test tube and the well was rinsed with buffer solution 3x with the rinse added to the tube containing the cells. Then the cells were isolated from the media via centrifuging. Next, an EDTA-Trypsin (0.25%) solution was used to break up cell clumps into single cells. Finally, an Invitrogen Countess cell counter was used to count two samples from each well and the results were averaged. 79

Results

Representative images in Fig. 3 show the morphology comparison between the stimulated groups and the non-stimulated groups. The plots in Fig. 4 show the cell numbers that were counted at each time point during the trials.

Fig. 3. Images of PC-12 cell cultures during various time-points of the experiment showing no morphological difference between the stimulated and non-stimulated groups.

Fig. 4. Non-stimulated and stimulated PC-12 cell proliferation over 8 day time period. Cells were seeded in wells at 0 h time point. Magnetic stimulation was applied at 24 h time point. 80

Statistical Analysis

Fig. 5 shows the results of the analysis of the stimulated compared with non-stimulated cell groups.

Fig. 5. The analysis shows that the groups are statistically the same. No change in proliferation rate for the stimulated group when compared to the non-stimulated group.

Cell Viability

Trypan Blue staining method was used to check cell viability at the 216 h time point. All replicates were tested. The results in Table 1 show that both groups had very high viability and were statistically the same.

Table 1. Average cell viability for all replicates using Trypan Blue staining.

Cell Group Cell Viability

Non-Stimulated 96 ± 3%

Stimulated 95 ± 3%

Conclusion

The identical protocol of magnetic field stimulation that increases proliferation of N27 adherent neuronal cells was shown not to increase the proliferation of PC-12 non-adherent neuronal cells. The magnetic field stimulation did not affect PC-12 cell viability as shown by

Trypan Blue viability testing. Nor did the stimulation affect PC-12 cell morphology.

Acknowledgment

Funded by the National Science Foundation and partially funded by the Stanley Chair in

Interdisciplinary Engineering at Iowa State University.

References

[1] Rocca, Walter A. “Time, Sex, Gender, History, and Dementia.” Alzheimer disease and associated disorders 31.1 (2017): 76–79. PMC. Web. 1 Aug. 2018.

[2] Westerink, R. H. S., and A. G. Ewing. “The PC-12 Cell as Model for Neurosecretion.” Acta physiologica (Oxford, England) 192.2 (2008): 273–285. PMC. Web. 1 Aug. 2018.

[3] “PC-12 ATCC ® CRL-1721TM Rattus norvegicus adrenal gland pheoc.” [Online]. Available: https://www.atcc.org/products/all/CRL-1721.aspx. [Accessed: 10-Apr-2018].

[4] “N27 Rat Dopaminergic Neural Cell Line | SCC048.” [Online]. Available: http://www.emdmillipore.com/US/en/product/N27-Rat-Dopaminergic-Neural-Cell- Line,MM_NF-SCC048. [Accessed: 10-Apr-2018].

[5] M. Isserles et al., “Effectiveness of deep transcranial magnetic stimulation combined with a brief exposure procedure in post-traumatic stress disorder-a pilot study,” Brain Stimul. 6(3), 377–383 (2013).

[6] The Magstim Company Limited, “Magstim 2002 Operating Manual (P/N 3001-23-04),” 28 (March 2005)

[7] Xiaojing Zhong, Joe Boldrey, Long Que, Ian Schneider, David Jiles, "Effects of Magnetic Stimulation on the Proliferation of Dopaminergic Neuronal Cells Grown in Extracellular Matrix", INTERMAG Conference, Singapore, April 23-27, 2018

[8] L. J. Crowther, R. L. Hadimani, and D. C. Jiles, "Effect of Anatomical Brain Development on Induced Electric Fields During Transcranial Magnetic Stimulation," IEEE Transactions on Magnetics, vol. 50, no. 11, p. 5102304, 2014. 82

CHAPTER 8. THE INFLUENCE OF MAGNETIC FIELD ORIENTATION ON THE GROWTH OF NEURITE EXTENSIONS OF NGF DIFFERENTIATED PC-12 NEURONAL CELLS

Joseph Boldrey1, Ian Schneider 2, Long Que1, and David Jiles1

1Department of Electrical and Computer Engineering, Iowa State University 2Department of Chemical and Biological Engineering, Iowa State University

Modified from a presentation given at the 2019 Minnesota Neuromodulation Symposium, Minneapolis, USA

Abstract

Transcranial Magnetic Stimulation (TMS) is a U.S. Food and Drug Administration

(FDA) approved method for treating major depressive disorder and obsessive-compulsive disorder. The effect of time dependent magnetic fields on the brain has yet to be fully investigated at the cellular level. Our research investigated the effects of pulsed magnetic fields generated by TMS stimulators on the growth of neurite extensions of Nerve Growth Factor

(NGF) differentiated PC-12 neuronal cells. A predetermined number of magnetic pulses were applied within a short time period using a Magstim 2002 monophasic stimulator with the

Magstim D702 figure-eight dual 70 mm high-focality coil. The stimulation session was repeated daily for the duration of the experimental trials.

When TMS is applied to a patient, neurites will experience the applied field according to their orientation within the brain. Our research probed the consequence of applying the magnetic field in different orientations with respect to the growth surface plane of neurites. Each stimulated cell group had a control group that was subjected to all the same experimental steps except magnetic stimulation.

Results showed that pulsed magnetic stimulation applied orthogonal to the growth surface increases the average length of neurite extensions on PC-12 cells. Our current work is 83 exploring the effects of magnetic stimulation on neurite growth when the field is applied parallel to the growth surface as well as at different orientation angles with respect the cell. The results of this work will increase our understanding of the effects of magnetic fields at the cellular level as well as the underlying mechanisms behind TMS.

Introduction

Transcranial Magnetic Stimulation (TMS) is approved by the U.S. Food and Drug

Administration (FDA) as a method for treating major depressive disorder (MDD) and obsessive- compulsive disorder (OCD). However, the effect of TMS type fields on the brain has yet to be fully investigated at the cellular level. Our research investigated the effects of monophasic pulsed magnetic fields generated by a TMS stimulator on the growth of neurite extensions of PC-12 cells stimulated with nerve growth factor beta (NGFβ).

When TMS is applied to a patient, neurites will experience the applied field according to their orientation within the brain. Our research probed the consequence of applying the magnetic field in different orientations with respect to the growth surface plane of the neurites. Each stimulated cell group had a control group that was subjected to all the same experimental steps except magnetic stimulation.

Previous work showed that a single session of stimulation applied orthogonal to the growth surface increased the average length of neurite extensions on PC-12 cells. Our current work is exploring the effects of multiple stimulation sessions on neurite growth when the field is applied orthogonal and when applied parallel to the growth surface.

Experiment Procedure

Experiment Timeline

The chart in Fig. 1 shows the timeline followed during the experimental trials. 84

Fig. 1. The major events of the experimental trials at their respective time points during each trial.

Cell Preparation

The PC-12 cells were cultured in 1640 RPMI [+ L Glutamine] medium supplemented with 10% horse serum, 5% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100

(100 U/ml), and streptomycin (100 U/ml), and 50 ng/ml NGFβ.

Magnetic Stimulation

Measured Magnetic Fields

Stimulation Schematic

The placement of the well plates that held the cells during the experiments with respect to the stimulation coil is shown in Fig. 2.

Fig 2. Location of the cells in the well plates with respect to the TMS coil during stimulation sessions.

Method of Analysis

Image J software was used to measure the angle of growth of the neurite extensions from the main cell body with respect to the direction of the applied magnetic field. Three types of measurements were taken of each extension as described in Fig. 3. Only extensions ≥ 11 µm were considered for taking measurements and counting. 86

Fig. 3. Examples of how the neurite extensions were measured using Image J software. Measurements in a straight line from the base of the extension to the tip of the longest branch. Measurements of the entire length of the extension. Measurements of the angle of the primary branch of the extension as it grows from the main cell body.

Results

Plots comparing the lengths of the neurite extensions of the stimulated cells with the non- stimulated cells are shown for the groups stimulated orthogonally to the substrate in Fig. 4, and parallel to the substrate in Fig 5.

Fig. 4. Average neurite lengths of PC-12 cells stimulated orthogonally to the plane of cell growth. 87

Fig. 5. Average neurite lengths of PC-12 cells stimulated parallel to the plane of growth.

Statistical Analysis

The error bars on the chart in Fig. 4 and Fig. 5 are the standard error of the means. A two- tailed t-Test was applied for the 95% confidence interval to determine if there is a significant difference between each pair (non-stimulated and stimulated) at 120 hours.

Discussion

Multiple pulsed stimulation sessions from a production TMS stimulator resulted in a significant reduction in average neurite length for the stimulated group when the field was applied orthogonal to the plane of growth. However, the stimulation protocol did not result in a significant difference between non-stimulated and stimulated groups when the field was applied parallel to the plane of growth. Nor did the pulsed stimulation protocol result in a significant difference of extension branching.

Further analysis of the direction of neurite growth of the primary neurites, and the direction of a straight line base-to-tip, with respect to the applied field is currently under way.

Acknowledgment

Funded by the National Science Foundation and partially funded by the Stanley Chair in

Interdisciplinary Engineering at Iowa State University. 88

References

[2] “PC-12 ATCC ® CRL-1721TM Rattus norvegicus adrenal gland pheoc.” [Online]. Available: https://www.atcc.org/products/all/CRL-1721.aspx. [Accessed: 10-Apr-2018].

[3] The Magstim Company Limited, “Magstim 2002 Operating Manual (P/N 3001-23-04),” 28 (March 2005)

[4] L. J. Crowther, R. L. Hadimani, and D. C. Jiles, “Effect of anatomical brain development on induced electric fields during transcranial magnetic stimulation,” 50(11) (2014).

[5] Joe Boldrey, Xiaojing Zhong, Long Que, Ian Schneider, David Jiles, "The Influence of Pulsed Magnetic Fields on NGF Differentiated PC-12 Neuronal Cells", Minnesota Neuromodulation Symposium, Minneapolis, USA, April 12-13, 2018 89

CHAPTER 9. EFFECTS OF MAGNETIC STIMULATION ON THE PROLIFERATION OF DOPAMINERGIC NEURONAL CELLS GROWN IN EXTRACELLULAR MATRIX

Joseph Boldrey1, Xiaojing Zhong1, Long Que1, Ian Schneider2, and David Jiles1

1Department of Electrical and Computer Engineering, Iowa State University 2Department of Chemical and Biological Engineering, Iowa State University

Modified from a presentation given at the 2018 IEEE Magnetics Society Intermag Conference, Singapore

Abstract

Transcranial Magnetic Stimulation (TMS) is a neuromodulation technique which is capable of non-invasively activating the neurons in the brain. TMS has been an approved method of treatment for major depressive disorders by the U.S. Food and Drug Administration (FDA) since 2008. The major principle in TMS is electromagnetic induction. The time varying magnetic field generated by the TMS stimulator induces an electric field in conductive brain tissues which activates neurons by depolarization. However, limited studies have reported the effects of the magnetic fields used in TMS on individual neurons and in cell cultures, especially on neuronal cells grown in a three-dimensional extracellular matrix (3D ECM). The main function of a 3D

ECM is to provide the biochemical and structural support to the surrounding cells that is found in vivo. The protein collagen is the major component of the ECM that plays an important role in giving cells strength and structural integrity. Hyaluronan is a polysaccharide found in the ECM of all vertebrates. It is known to provide support and protection to the cells within the ECM.

Poly-D-Lysine (PDL) is a positively charged amino acid polymer used to help cells bind to substrates such as glass and plastic.

Based on these theories, we investigated the effects of magnetic fields generated by a monophasic TMS stimulator using a 90 mm circular coil on the N27 rat dopaminergic neuronal 90 cell line grown on two-dimensional (2D) and in three-dimensional (3D) ECMs. In our experiments, collagen-coated functionalized glass cover slips and PDL coated cover slips were used as 2D ECM for cell growth. Images of both, stimulated group, and non-stimulated group, were captured at five time points from the stimulation time (0 hour) to 72 hours. Cells were counted, averaged, and plotted. The results of our experiments showed that the magnetic stimulation decreased the growth rate of the N27 cells in comparison to the non-stimulated control group. However, it was observed that the collagen coating helped the cells to be more resistant to this negative effect caused by magnetic stimulation. We concluded that the magnetic stimulation used in our study decreased the proliferation of N27 dopaminergic neuronal cells and that collagen plays a role in protecting the cells from adverse effects of strong magnetic fields.

Experiments currently underway are studying the effects that magnetic fields have on N27 cells within 3D ECM of collagen and 3D ECM of collagen/hyaluronan.

Introduction

TMS on individual neurons and in cell culture. This study investigated the effects of time- dependent magnetic fields generated by a monophasic TMS stimulator using a 70 mm figure-of- eight dual coil on the N27 rat dopaminergic neuronal cell line grown on two-dimensional (2D) extracellular matrix (ECM). The N27 cell line is widely used in biomedical studies as a model for Parkinson's disease research. 91

Experiment Procedure

Experimental Timeline

The chart in Fig. 1 shows the timeline followed during the experimental trials.

Fig. 1. The major events of the experimental trials at their respective time points during each trial.

2D Extracellular Matrix

Collagen coated functionalized glass cover slips and poly-D-lysine (PDL) coated glass cover slips were used as the 2D ECM for cell growth.

Cell Culture

Rat mesencephalic cells (N27) were grown in RPMI medium supplemented with 10% fetal bovine serum, 1% L-glutamine, penicillin (100 U/ml), and streptomycin (100 U/ml), maintained at 37 °C in a humidified atmosphere of 5% CO2.

Magnetic Stimulation

Measured Magnetic Fields

D702 figure-8 coil was calculated to be 0.559 T (0.445 MA/m).

Stimulation Schematic

The placement of the well plates that held the glass cover slips during the experiment trials with respect to the stimulation coil is shown in Fig. 2.

Fig 2. Location of the glass coverslips used for growing the N27 cells with respect to the TMS coil during stimulation sessions.

Results

Representative images in Fig. 3 show no difference in morphology for the cells grown on poly-D-lysine compared to the cells grown on collagen. The numbers of cells counted at each time point during the trials are plotted in Fig. 4. 93

Fig. 3. Images of N27 cell cultures during various time-points of the experiment showing no morphological difference between cells grown on poly-D-lysine and collagen ECMs, respectively.

Fig. 4. Comparison of cell numbers with respect to time. Magnetic stimulation applied at 24hr time point after seeding cells on collagen and PDL ECMs, respectively. 94

Statistical Analysis

Fig. 5 shows the analysis of the cells (stimulated compared with non-stimulated) using the figure-8 coil for stimulation while being grown in the different ECMs.

Fig. 5. The groups that received magnetic stimulation show an increase in proliferation rate compared to the groups that did not receive magnetic stimulation. This increase occurred on both ECMs, collagen and poly-D-lysine.

Conclusion

Single-phase time-dependent magnetic field stimulation increases growth and proliferation of N27 neuronal cells on collagen and on poly-D-lysine coated 2D extracellular matrices (ECM). Direct observation of the cell cultures show that the N27 cells grow better on the collagen coated surface than the PDL coated surface. The cells attach to and spread more on the collagen. This is an indication of a better growth environment. Future experiments will study the effects in 2.5 dimensions and 3 dimensions ECMs.

Acknowledgment

Funded by the National Science Foundation and partially funded by the Stanley Chair in

Interdisciplinary Engineering at Iowa State University. 95

References

[2] “N27 Rat Dopaminergic Neural Cell Line | SCC048.” [Online]. Available: http://www.emdmillipore.com/US/en/product/N27-Rat-Dopaminergic-Neural-Cell- Line,MM_NF-SCC048. [Accessed: 10-Apr-2018].

[3] L. J. Crowther, R. L. Hadimani, and D. C. Jiles, “Effect of anatomical brain development on induced electric fields during transcranial magnetic stimulation,” 50(11) (2014).

[4] The Magstim Company Limited, “Magstim 2002 Operating Manual (P/N 3001-23-04),” 28 (March 2005). 96

CHAPTER 10. GENERAL CONCLUSION

In this research, two modeling techniques were used to investigate the effects of magnetic fields generated during TMS treatment of neuronal cells.

A computer model was used to investigate the variability of brain-scalp distance (BSD) on the brain’s response to TMS from the QBC and the FoE coils. It was concluded that the EMAX in the brain induced by the QBC decreases with BSD. This result was also found using the FoE coil. The R-value statistical correlation of EMAX to the BSD for the two coils are 0.4293 for the

QBC and 0.4141 for the FoE coil. These results confirm that the BSD has a moderate correlation to the EMAX regardless of the coil used. However, there is no significant correlation between

BSD and the V-Half (the parameter for focality). This result indicates that the advantages of the increased focality of the QBC over the FoE are applicable over a range of subjects with differing

BSD.

Two biological models were used to investigate various aspects of neuronal cells’ response, at the cellular level, to magnetic fields generated during TMS. Results show that monophasic TMS fields, at sufficient intensity, increase the proliferation rate of N27 neuronal cells. This increase occurs on collagen and on poly-D-lysine ECMs. By modulating the H field, a threshold of the sufficient intensity is shown to exist between 0.4728 T and 0.5593 T. The identical protocol of magnetic field stimulation that increases proliferation of N27 adherent neuronal cells was shown not to increase the proliferation of PC-12 non-adherent neuronal cells.

The magnetic field stimulation did not affect the N27 cell viability as shown by the calcein AM and propidium iodide fluorescence microscopy testing. Nor did the stimulation affect PC-12 cell viability, as shown by the Trypan Blue viability testing. Empirical observation showed that neither cell line’s morphology was affected by the magnetic stimulation. It was shown that 97 monophasic TMS fields applied to PC-12 cells in multiple stimulation sessions resulted in a significant reduction in average neurite length for the group that was stimulated with the H field orthogonal to the plane of growth. However, the stimulation protocol did not result in a significant difference in neurite length when the H field was applied parallel to the plane of growth. Regardless of the direction of applied field, the results show no significant difference between the stimulated and non-stimulated groups when measuring directional bias of new neurites, directional bias of overall neurite growth, or extension branching for PC-12 cells.

The modeling techniques shown in this paper demonstrate how computer models and biological models can be used to increase our understanding of the mechanisms behind the brain’s response to the magnetic fields applied during TMS.