Axon Outgrowth of Rat Embryonic Hippocampal Neurons in the

Aug 16, 2016 - Thus, to determine an optimal EF for axon outgrowth of mammalian CNS neurons, we applied a wide range of EF to rat hippocampal neurons...
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Axon Outgrowth of Rat Embryonic Hippocampal Neurons in the Presence of an Electric Field Kwang-Min Kim,†,‡,∥ Sung Yeol Kim,†,‡,⊥ and G. Tayhas R. Palmore*,†,‡,§ †

School of Engineering, ‡Center for Biomedical Engineering, and §Department of Chemistry, Brown University, Providence, Rhode Island 02912, United States ABSTRACT: Application of an electric field (EF) has long been used to induce axon outgrowth following nerve injuries. The response of mammalian neurons (e.g., axon length, axon guidance) from the central nervous system (CNS) to an EF, however, remains unclear, whereas those from amphibian or avian neuron models have been well characterized. Thus, to determine an optimal EF for axon outgrowth of mammalian CNS neurons, we applied a wide range of EF to rat hippocampal neurons. Our results showed that EF with either a high magnitude (100 mV/mm or higher) or long exposure time (10 h or longer) with low magnitude (10−30 mV/mm) caused a neurite collapse and cell death. We also investigated whether neuronal response to an EF is altered depending on the growth stage of neuron cultures by applying 30 mV/mm to cells from 1 to 11 days in vitro (DIV). Neurons showed the turnover of axon outgrowth pattern when electrically stimulated between 4−5 DIV at which point neurons have both axonal and dendritic formation. The findings of this study suggest that the developmental stage of neurons is an important factor to consider when using EF as a potential method for axon regeneration in mammalian CNS neurons. KEYWORDS: Nerve regeneration, electrical stimulation, ionic currents, axon outgrowth, mammalian CNS

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EF as functions of neurite alignment8 and neurite extension.9 These results suggest the development of mammalian PNS neurons are more affected by non-neuronal cells (i.e., astrocytes and Schwann cells) in conjunction with the exogenous EF rather than by EF alone. With respect to the effect of EF on the neurons from the mammalian central nervous system (CNS), it has been reported that rat embryonic hippocampal neurons as a CNS model align perpendicularly to an applied EF between 28 and 219 mV/mm for 24 h10 and their dendritic growth cones responded only to focally applied EF (higher than 580 mV/ mm).11 It also has been reported that cells from postnatal rat hippocampal tissues migrated toward the cathode when an EF (120 mV/mm) was applied for 1 h.12 However, no additional studies on the effect of EF on mammalian CNS neurons in vitro have been reported elsewhere. Thus, we still lack evidence showing a significant response of mammalian CNS neurons to an exogenous EF despite its potential for clinical use.13 Accordingly, the exogenous EF application as an approach to the treatment of mammalian nerve injury needs further study. Toward this end, we examined the effect of EF on axon outgrowth using rat hippocampal neurons as a CNS mammalian neuron model using a wide range of magnitudes (10−300 mV/mm) and durations (10 min to 24 h). We also varied the time point for applying EF to neuron cultures from 1

he cells in the vertebrate tissues generate endogenous electric field (EF) for the growth and migration during embryonic development and adult wound healing.1,2 Inspired by this phenomenon, exogenous EF as an electrical stimulation (E-stim) has been applied to nerve tissues for the treatment of brain damage and neurodegenerative diseases. Previous studies demonstrated that the exogenous EF within physiological limits (500 mV/mm)3 generated ionic currents, thereby eliciting axon outgrowth and guidance.4−6 These results varied depending on the species of the vertebrate animal models. For example, neurons from neural tubes of embryonic Xenopus laevis as an amphibian neuron model showed faster and longer extension of neurites toward the cathode under 100−150 mV/mm for 5 h.5,7 As an avian neuron model, both chick dorsal root ganglion neurons5 and sympathetic neurons6 showed the migration of somata toward the anode under 70−140 mV/mm for several hours and 300−400 mV/mm for 3 h, respectively. In contrast, neurons from neural tubes of embryonic zebrafish did not show any changes in either neurite extension or alignment in response to an applied EF with 100 mV/mm.2 The effect of exogenous EF on mammalian neuron models remains controversial. One study demonstrated that rat dorsal root ganglion neurons (DRGs) as a mammalian peripheral nervous system (PNS) model did not respond to either 10 or 500 mV/ mm for 24 h8 while another study demonstrated that DRG neurons showed longer neurite extension under 50 mV/mm for 8 h.9 In studies that show contradicting results, it should be noted that DRG neurons, when cocultured with astrocytes and Schwann cells, showed an enhanced response to the exogenous © 2016 American Chemical Society

Received: June 29, 2016 Accepted: August 16, 2016 Published: August 16, 2016 1325

DOI: 10.1021/acschemneuro.6b00191 ACS Chem. Neurosci. 2016, 7, 1325−1330

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Figure 1. Procedures for electrical stimulation of rat hippocampal neurons. (a) Neurons dissociated from hippocampi are seeded on both chambers of the culture platform. (b) Culture platforms are placed in an incubator until cells are stimulated and imaged. (c) During the application of EF, an aluminum cover with transparent foil is fixed to the top of the platform to prevent the evaporation and contamination of culture media. Salt bridges (2% agar) connect cell-containing chambers to side wells that have immersed platinum wires. EF is applied through the platinum wires. (d) Neurons are stimulated by an EF (10, 30, 100, 200, or 300 mV/mm) for 10 min, 1, 2, 5, 10, or 24 h. Panels (c) and (d) were adapted from our previous study.14 (e) Phase contrast images of live neurons before and after the application of EF. Δaxon length is obtained by the axon length difference starting immediately before the onset of EF and ending 2 h after removal of EF. When an axon has a branch node (length B and C), the longer length (B) is chosen for analysis. Scale bar = 20 μm.

day in vitro (DIV) through 11 DIV in order to determine whether neuronal response to an applied EF changes depending on the different developmental stage of neurons. Experimental design and procedures are briefly illustrated in Figure 1.

studies that used an EF with a range of 10−30 mV/mm. Wood and Willits applied 25 mV/mm to chick DRG neurons for only 10 min and demonstrated enhanced axon lengths.15 Koppes et al. also demonstrated that the application of both 10 and 50 mV/mm for 8 h resulted in longer axons of rat DRG neurons when compared to those without EF.9 The discrepancy between these reports and our study suggest that cell type (e.g., cells from CNS or PNS) or species (e.g., Xenopus, chick, rat) determines the response of neurons to an applied EF. When stimulated by 100 mV/mm and higher EF for 1, 2, 5, 10, and 24 h, all hippocampal neurons died between 30 min and 1 h after the onset of E-stim (Figure 2b). The application of 100 mV/mm for 10 min also resulted in neurite collapse and neurons eventually died several hours after the EF of 10 mV/ mm was removed. These results indicate that rat hippocampal neurons in vitro cannot grow or survive under a strong EF between 100 and 300 mV/mm, which is within the range of endogenous physiological EF.3,16 Our results are contradictory to many previous studies that claimed a strong EF could elicit neurite outgrowth and alignment. In previous studies, neurons from the neural tubes of Xenopus laevis showed longer neurites directed toward the cathode under 100−150 mV/mm for 5 h.5,7 Neurites of chick sympathetic neurons aligned perpendicular to the direction of EF under 300−400 mV/mm for 3 h.6 It was also shown that mammalian astrocytes, non-neuronal cells in the brain, aligned perpendicular to the direction of EF under 500 mV/mm for 24 h.8,17 The contradicting results between our studies and previous studies suggest that mammalian CNS neurons have much lower resistance to an applied EF than other cell types including not only non-neuronal cells (e.g., astrocytes, Schwann cells) and neurons from different species (e.g., amphibian, avian) but also neurons from the same species (i.e., mammalian PNS). Applying Electrical Stimulation at Different Growth Stages of Hippocampal Neurons in Vitro. We could not find an optimal EF condition for axon outgrowth of rat hippocampal neuron cultures between 1 and 2 DIV. Neurons were electrically stimulated from 1 to 11 DIV to determine whether a neuronal response to EF depends on their

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RESULTS AND DISCUSSION Application of Various EF Conditions for Axonal Outgrowth. To investigate the effect of the exogenous EF on the behavior of mammalian neurons (e.g., neurite elongation, neurite orientation), rat hippocampal neuron cultures in the platforms between 1 and 2 DIV (i.e., 36 h) were electrically stimulated by EF of 10, 30, 100, 200, and 300 mV/mm with durations of 10 min, 1, 2, 5, 10, and 24 h. When hippocampal neurons were exposed to 10 mV/mm, short (10 min) and intermediate (1, 2, and 5 h) durations of EF did not result in either an increase or decrease of axon length compared to those of the control group (i.e., neurons without EF), which are 11.57, 21.8, 34.9, and 49.8 μm at 10 min, 1, 2, and 5 h, respectively (Figure 2a). However, a long duration (10 and 24 h) of 10 mV/mm resulted in collapsed neurites and eventually led to cell death (i.e., neurons with ruptured somata or detached from the substrate) between 15−20 h after the onset of E-stim (Figure 2b). These results indicate that a weak EF 1) is not effective for inducing enhanced axon outgrowth of mammalian CNS neurons, and 2) has negative effects on the neuronal health when applied for a long time. When 30 mV/mm was applied to hippocampal neurons for 2 h or longer, all neurites began collapsing or ruptured between 2−4 h after the onset of E-stim (Figure 2b). A short (10 min) and intermediate (1 h) duration of 30 mV/mm did not cause a significant change in axon length when compared to those of the control group (Figure 2a). These results were similar to those of the experiment using 10 mV/mm in that (1) neurons did not respond to a weak EF (10−30 mV/mm) for up to 1 h, and (2) a longer exposure of neurons to the exogenous EF showed a negative effect on survival and outgrowth of neurons. Interestingly, these results are distinct from those from previous 1326

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Figure 2. Electrical stimulation of E18 hippocampal neurons using various EF conditions. (a) Graphs showing Δaxon length of neurons stimulated by 10, 30, 100, 200, and 300 mV/mm for 10 min, 1, 2, 5, 10, and 24 h. The average value of Δaxon length from neurons with 10 mV/mm and 30 mV/mm did not show significant change up to 10 and 2 h, respectively, when compared to that without EF (i.e., no EF). The average value of Δaxon length from neurons with 100, 200, and 300 mV/mm showed negative values (i.e., neurite collapse) from 10 min stimulation. After 24 h EF, all the EF conditions resulted in negative values of Δaxon length between −47.1 and −55.9 μm, which indicates a complete loss of neurite since the first measurement of neurites at immediately prior to applying an EF (i.e., neurite collapse). (b) Timeline of neurite collapse and cell death under 10, 30, and 100 mV/mm. (c) Phase contrast images of live neurons showing neurite collapse after a long time exposure of neurons to an EF. Scale bar = 20 μm.

and 3DIV, respectively). However, neurons stimulated from 5 DIV showed similar Δaxon length values to those from the control (p > 0.05). At 9 DIV, electrically stimulated neurons showed 3.1 fold higher value of Δaxon length than that of the control even though there was no statistical difference between two groups (p = 0.11). These results suggest that rat hippocampal neurons become more resistant to an applied EF after 4 DIV and Δaxon length shows an increasing trend as the culture day increases. It should be noted that the value of Δaxon length turned from negative to positive between 3 and 5 DIV (Figure 3b). The alteration in the response of neurons to an EF between 3 and 5 DIV may be related to the developmental stage of hippocampal neurons in vitro. According to the developmental stage of hippocampal neurons,19,20 neurons between 0 and 1 DIV begin to sprout neurites. One of these neurites develops into an axon between 1 and 2 DIV. The remaining neurites become dendrites between 3 and 4 DIV. Further maturation such as longer extension and more branching of neurites occurs at between 5 and 7 DIV or thereafter. If we correlate our results with the developmental stages of hippocampal neurons, the time point when the value of Δaxon length turns positive is exactly aligned

developmental stage. For this study, we used 30 mV/mm as a potential E-stim to elicit axon outgrowth because neurons showed high vulnerability to stronger EF (100, 200, 300 mV/ mm) regardless of applied time (Figure 2). When applying an EF, we alternated between applied EF (20 min) and paused (20 min) four times (80 min EF in total) (Figure 3a) because even a weak (10 mV/mm) and intermediate EF (30 mV/mm) can induce neurite damage and cell death if applied continuously for a long time as shown in Figure 2. Inserting a pause between applied EF has been used to promote neurite outgrowth of chick DRG neurons.18 When multiple EFs were applied to rat hippocamapal neurons from 1 to 11 DIV, neurons at different culture day responded differently to the exogenous EF (Figure 3b). As culture days increased, the values of Δaxon length were gradually increased until 9 DIV. The control group (neurons without EF) did not show significant changes in values of Δaxon length from 1 to 11 DIV, which were between 11−35 μm (Figure 3b). In particular, neurons electrically stimulated between 1 and 3 DIV showed significantly lower values of Δaxon length when compared to those of the control experiment (p = 0.001, 0.002, and 0.02 for 1 DIV, 2 DIV, 1327

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Figure 3. Applying multiple EFs to hippocampal neurons at different culture day. (a) Multiple EFs of 30 mV/mm (alternating between 20 min EF and 20 min pause) were applied to hippocampal neuron cultures in 11 different platforms from 1 to 11 DIV to investigate the response of neurons to an EF at different growth stages of neurons. (b) Δaxon length of electrically stimulated neurons gradually increased from 2 to 9 DIV whereas Δaxon length of the control group did not show significant change from 1 through 11 DIV. The turnover of Δaxon length from negative to positive value is highlighted in the red dashed box. (c) Illustrations showing the developmental stages of rat embryonic hippocampal neurons in vitro (modified reprint from previous papers19,20). The turnover of Δaxon length is aligned to the stage of 3−4 DIV where neurons show dendritic outgrowth. (d) Phase contrast images of live neurons at 2, 5, and 9 DIV. Red arrows indicate the tips of axons that were used in measuring Δaxon length. At 2 DIV, neurons with short dendrites (white arrows) (i.e., minor processes of dendrites indicated as blue arrows in panel c) showed contraction of an axon and loss of dendrites following EF. Neurons at 5 and 9 DIV with longer extensions of dendrites (i.e., dendritic outgrowth) showed increased value of Δaxon length, which is similar to or longer than that of neurons without EF. Scale bar = 20 μm.

exogenous EF has a negative impact on axon outgrowth of rat hippocampal neurons until 4−5 DIV is reached, which corresponds to the growth state when these types of neurons are polarized (i.e., showing both axonal and dendritic outgrowth). As a neuron develops, the axon outgrowth in response to an EF gradually increases, showing a higher value than that of the control group (neurons without EF) at 8 and 9 DIV despite a lack of statistical difference (p = 0.11). This result suggests that the response of rat hippocampal neurons to the exogenous EF is dependent on the developmental stage of neurons, which should be taken into consideration when applying electrical stimulation to mammalian nerve tissue. We speculate that the molecular pathways involved in neuronal polarization (e.g., neurotrophin signaling and cytoskeletal organization signaling)25 are susceptible to an applied EF before neurons are polarized. Comparing gene expression profiles before and after neuronal polarization may be required to probe other possible mechanisms underlying the response of mammalian CNS neurons to an exogenous EF. These possibilities are the subject of ongoing studies and will be reported elsewhere. The findings and discussion in this study provide a useful guide in evaluating electrical stimulation as an approach to repair and regenerate damaged mammalian CNS.

to the stage between 3 and 5 DIV, which is when hippocampal neurons are polarized (i.e., showing both axonal and dendritic outgrowth) (compare Figure 3b,c). If neurons were stimulated at the stage prior to showing minor processes of dendrites (i.e., 2 DIV), they failed to promote axon outgrowth as shown in Figure 3d. This result suggests that the optimal time point to apply electrical stimulation to mammalian CNS neurons is after neurons are polarized. In conclusion, we investigated the effect of exogenous EF on neuronal outgrowth using a wide range of EF conditions. Our results reveal that mammalian CNS neurons are significantly more sensitive to the application of EF than other neuron types (e.g., amphibian, avian, and mammalian PNS neurons). In addition, application of an exogenous EF with either high strength or long duration to rat hippocampal neurons leads to neurite collapse and cell death, indicating these modes of EF are not a good approach for nerve regeneration. As such, these modes of EF may be the reason so few examples demonstrate exogenous EF as an effective cue for regulating the outgrowth and guidance of mammalian CNS neurons10,11,21 in contrast to biochemical cues (e.g., extracellular matrix proteins, lipoproteins), which elicit effective outgrowth and guidance of mammalian CNS neurons.22−24 Our results also show that 1328

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kept constant by the incubation controller. The glass surface of the chamber (18 mm × 3 mm) was scanned to search for healthy neurons showing neurites. Only the neurons that had a neurite longer than the size of a soma were chosen for analysis. Phase-contrast images of cells in both the control and experimental chambers were captured every 30 min from before the initiation of E-stim to 2 h after the end of E-stim. Ten fields of view showing healthy neurons in the control and experimental chambers were used for image analysis. Image Analysis and Statistics. Captured images were analyzed using imaging software (AxioVision 4.0, Zeiss) to measure the axon length. The length of axon was measured by manually tracing a neurite from the border of a soma to the tip of an axon. The longest neurite was chosen as an axon when a neuron has multiple neurites.19 The axons connected to adjacent neurons were not measured. The effect of an applied EF on axon outgrowth was determined by the differences of axon lengths before and after application of EF (Δaxon length) (Figure 1e): Δaxon length = (axon length 2 h after the end of EF) − (axon length immediately before the onset of EF). All the data were obtained from three to four independent experiments for 10, 30, and 100 mV/mm, and 2 independent experiments for 200 and 300 mV/mm. The bar graphs are presented as means ± SEM. Differences were analyzed using two-tailed unpaired Student’s t test unless otherwise indicated, and were considered significant when p < 0.05.

METHODS

Fabrication of a Platform for Applying EF and Imaging Neurons. Based on our previous study,14 a special platform for applying EF to neurons was designed with two parallel chambers to allow for the simultaneous study of neurons with and without electrical stimulation (Figure 1). Briefly, the sides of the chambers were molded out of poly(dimethylsiloxane) (PDMS) (Sylgard 184, Dow Corning). A glass microscope slide (25 × 75 mm2) (Fisher Scientific) was subjected to plasma etching prior to attachment to the PDMS to ensure a good seal. Small wells (side wells) distal to the chambers were included in the platform to prevent contamination of the cell culture by possible chemistry at the electrodes. Platinum wire electrodes were placed in the side wells to apply electrical stimulation. Salt bridges between side wells and chambers were made of U-shaped glass tubes (cross-sectional diameter: 5 mm) and filled with a 2% agar gel soaked with Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen). The glass bottom of the chambers was coated with laminin (50 μg/mL in Hank’s balanced salt solution, HBSS) followed by (1) rinse with a mixture of HCl and methanol (1:1 v/v) to increase the number of reactive SiOH groups26 and (2) 30 s plasma etch. Laminin-containing platforms are agitated on a shaker for 1h at room temperature, and then rinsed with PBS prior to cell plating. Primary Hippocampal Neuron Cultures. For the dissociation of neurons, E18 Sprague−Dawley rat hippocampal tissues (BrainBits, Springfield, IL) were incubated in 2 mL of Hibernate E medium without calcium (BrainBits) containing 4 mg papain (Worthington, Lakewood, NJ) at 30 °C for 30 min. For further dissociation, a firepolished Pasteur pipet was used to triturate the hippocampal tissue, and then the supernatant was transferred to the centrifuge tube for removing tissue not dissociated and debris. After the centrifugation at 1100 rpm for 1 min, the cell pellet was resuspended in 2 mL of serumfree culture medium consisting of Neurobasal medium supplemented with 2% B-27 supplement, 0.5 mM L-glutamine (Fisher Scientific), and 1% antibiotic solution, penicillin (100 U/mL)/streptomycin (100 mg/ mL). Glutamic acid was not added to the culture medium because it can induce apoptosis of hippocampal neurons.27 Cells were seeded on both chambers of the platform described in the previous section at a density of 5000 cells/chamber (Figure 1a). Seeded cells were incubated at 37 °C and 5% CO2 under a humidified environment until the platform was placed on microscope stage for electrical stimulation and live-cell imaging (Figure 1b). Electrical Stimulation of Neurons. To prevent evaporation and contamination of the media during E-stim, a transparent culture foil was affixed to the PDMS mold with aluminum caps (Figure 1c). The culture foil is made of fluorethylene-propylene that is permeable only to gas (i.e., O2 and CO2). Hippocampal neurons were electrically stimulated by applying EF (10, 30, 100, 200, and 300 mV/mm) to one of the two chambers in the platform. The other chamber was not stimulated and functioned as the control experiment (Figure 1d). The duration of EF was selected among 10 min, 1, 2, 5, 10, and 24 h for each platform. The combinations of various magnitudes and durations of EF were applied to rat hippocampal cultures at 36 h after plating because hippocampal neurons exhibit polarity and axon elongation between 1 and 2 DIV.20 To ensure application of a constant EF, a potentiostat/galvanostat (EG&G, model 263) maintained a constant current by varying the potential between 1.5 V - 22 V. The applied current was calculated using the formula: E = Iρ/A, where ρ is resistivity of culture media (80 Ω cm) and A is the cross-sectional area of culture media in the chamber (3 mm2; width = 3 mm, height = 1 mm). Then, the EF condition that showed the longest axon extension was applied to hippocampal neuron cultures from 1 DIV through 11 DIV in order to correlate neuronal response to EF with the developmental stage of neurons in vitro. Real-Time Imaging of Cells. An inverted microscope (Axiovert 200M, Zeiss) with a motorized stage was used to image cells in real time. An incubation chamber (Incubator S, Pecon) was attached on the microscope stage to enable electrical stimulation and imaging of cells simultaneously in the platform during the E-stim experiments. Both the temperature (37 °C) and concentration of CO2 (5%) were

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AUTHOR INFORMATION

Corresponding Author

*Mailing address: 182 Hope Street, Box D, Providence, RI 02912, USA. Phone: 401-863-2856. E-mail: tayhas_palmore@ brown.edu. Present Addresses ∥

K.-M.K.: Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA 94305, USA. ⊥ S.Y.K.: School of Mechanical Engineering, Kyungpook National University, Daegu 702-701, South Korea. Author Contributions

K.-M.K. and G.T.R.P. conceived and developed the study. K.M.K. and S.Y.K. designed and fabricated microculture platforms for cell culture and imaging. K.M.K. cultured neurons and analyzed data. K.M.K. and G.T.R.P. wrote the manuscript. Funding

This work was supported by the NIH (1R01EB005722-01A2). Notes

The authors declare no competing financial interest.

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