Growth Mechanism of Rat Dorsal Root Ganglion Neurons

on Slope Substrate

Xiao Li, Yuanyuan Wang, Qi Xu, Fang Chen and Jiping He

Neural Interface and Rehabilitation Technology Research Center,

Huazhong University of Science and Technology, Wuhan, 430074, China

Keywords: Dorsal Root Ganglia (DRG), Axon, Slope Substrate.

Abstract: Neural response to topography depends on the dimensions and shapes of physical features. Most researchers

focused on fabricating different grooves and ridges to study cell adhesion, spreading, alignment, and

morphological changes. Very few papers report about how sloped substrate influences the behavior of

neural cells. In this paper, we made a preliminary experiment to test the reaction of neuronal growth

processes to different slopes. We found that all DRG cells’ axons couldn’t grow across 90 degree slope with

198 μm height. A few axons grew across 90 degree slope with 50 μm height. In addition, we also found that

DRG cells showed preference to grow uphill rather than downhill. In future, we will make more detailed

experiments to study the mechanism of slope modulation. This study will be helpful for the construction of

nerve regenerating scaffolds and neural interface.

1 INTRODUCTION

Every year, thousands of people are disabled by

neurological disease and injury. Successful control

of the cell behavior will open the ways for neural

regeneration and functional rehabilitation. Several

therapies offer significant promise for the restoration

of neuronal function, including the use of growth

factors to prevent cell death following injury

(Vincent and Feldman, 2002), stem cells to rebuild

parts of the nervous system (Horner and Gage,

2000), and the use of functional electrical

stimulation to promote axon regeneration (Al-

Majed, Neumann et al., 2000). During these

treatments, researchers have found that the

interaction of cells with substrate plays a key role in

the cell behavior, such as cell adhesion, spreading,

morphology, proliferation, and even differentiation.

Therefore, the research about how patterned

substrates influence the behavior of neurons appears

especially important right away.

The first experiment which mentioned the

relationship of cells and topography was

accomplished in 1911. Harrison (Harrison, 1911)

grew cells on a spider web and found the cells

followed the fibers of the web. Since then, with the

development of micro- and nano-fabrication

techniques, a large number of studies have shown

that many cell types react strongly to topography. In

general, micropattern substrates were fabricated to

contain repeating rectangular groove-plateau

patterns with varied groove width, varied plateau

width and varied groove depth. Neurons follow the

discontinuities of grooves

and ridges, and attain an

elongated shape due to surface-induced

rearrangements of the cytoskeleton (Curtis, 2004).

Actin and microtubules align along walls and edges,

the microtubules being the first element to be

aligned, followed by actin (Oakley and Brunette,

1993). Grooved surfaces also induce changes in

transcription and the up and down regulation of

several genes, but the explicit mechanism for cell

guidance has yet to be clarified (Dalby, Riehle et al.,

2003).

Most studies focused on the effects of

topography size on neurons. Rajnicek et al

(Rajnicek, Britland et al., 1997) reported that central

nervous system neurites could be guided by shallow

grooves with 14 nm deep and 1 mm wide. Stepien

and coworkers (Stepien, Stanisz et al., 1999)

reported contact guidance for chicken dorsal root

ganglion (DRG) neurons on single scratches with

0.1-0.2 mm wide. If the grooves are greater than 20

mm, no cell type (except red blood cells) has been

found to respond to the guide (Wilkinson, Riehle et

Li X., Wang Y., Xu Q., Chen F. and He J..

Growth Mechanism of Rat Dorsal Root Ganglion Neurons on Slope Substrate.

DOI: 10.5220/0005132600150020

In Proceedings of the 2nd International Congress on Neurotechnology, Electronics and Informatics (NEUROTECHNIX-2014), pages 15-20

ISBN: 978-989-758-056-7

 2014 SCITEPRESS (Science and Technology Publications, Lda.)

al., 2002). Orientation often increases with

increasing depth, but decreases with increasing

groove width (Clark, Connolly et al., 1990). Goldner

et al (Goldner, Bruder et al., 2006) describe an

unusual capability of a subpopulation of DRG

neurons to extend neurites that spanned across the

grooves, with no underlying solid support. The

highest numbers of bridges observed under the

groove width of 30 mm, even few neurites bridge

have been observed spanning a groove of 200 mm.

Although these researches are important for

constructing high-resolution neural circuit scaffolds

or neural interface, the response of neurons and their

axons to the sloped substrata has not been studied in

detail. Fricke et al (Fricke, Zentis et al., 2011)

constructed a variety of gradient patterns with slight

changes in slope to control neuronal cell position,

the path of neurite growth, and axon directionality.

They found that reduction in the slope of structure

from 0.04 (0.3 mm/7.5 mm) to 0.01 (0.1 mm/7.5

mm) strongly decreased the effects on neurite

growth. However, because the slope substrate

consists of the multiple discontinuous grooves, we

can’t make sure that slope plays a key role in the

difference.

In this paper, we performed a preliminary

experiment to test the reaction of neuronal growth

processes at different slopes. The purpose of the

study was to investigate the preference of axons in

growing uphill or downhill on different angles of the

slope. Present results of this study can be utilized for

nerve regenerating scaffolds or the construction of

neural interface.

2 METHODS

2.1 Construction of 90 Degree Slope

Figure 1: The schematic diagram of 90 degree slope

construction with different height. The powder blue disc is

Petri dish. The light cyan rectangular is 198 μm thick

Aclar film. The green rectangular is 50 μm thick Aclar

film. The blue dot is Dorsal root ganglia explants (DRGs).

The scheme of our experiment is illustrated in

Figure 1. Two Aclar (Aclar® 33C, Electron

Microscopy Sciences Inc., Hatfield, PA) films of

different thickness were plated in the Petri dish. The

dorsal root ganglia explants (DRGs) were plated on

the films or the bottom of Petri dish with different

locations. The 90 degree slopes with 50 or 198 μm

height were produced in this way.

2.2 Construction of Slight Degree Slope

We constructed an axon stretch-growth bioreactor

which contained two independent axon expansion

chambers. The axon expansion chamber consisted of

a stretching frame that formed a lane, an adjustable

towing block that could manipulate cells across the

lane, and a projected towing rod for external

manipulation (Figure 2).

Figure 2: Prototype of the axon expansion chamber.

Dorsal root ganglia (DRG) explants can be cultured in the

chamber.

The 198 μm thick Aclar film was affixed to the

bottom of the stretching frame and spanned the lane.

The 50 μm thick Aclar film was held rigidly by the

towing block. The 50 μm thick Aclar film was

lightly sanded on either side using 1200-grit

waterproof sandpaper (MATADOR, Germany) to

create gradual slope to the border of the exposed

underlying film (Figure 3).

Figure 3: The schematic diagram of slight degree slope

construction by axon stretch-growth system. Two different

Aclar films (green color) were used: one in the horizontal

plane and the other with a slight degree of slope. The blue

dot signifies Dorsal root ganglia explants (DRGs).

NEUROTECHNIX2014-InternationalCongressonNeurotechnology,ElectronicsandInformatics

In order to get more convincing results, we could

move the towing rod (Figure 2 and 3) to divide the

culture by a computer-controlled micro-stepper

motor as described by Smith (Smith, Wolf et al.,

2001). This technique results in the stretch-induced

growth of fascicular tracts of axons spanning the two

membranes. Then we could measure the length and

diameter of the regular axons, which provided a

measure of the relevant ability of axons to climb the

slope substrate.

2.3 Cell Culture

Dorsal root ganglia (DRG) explants were isolated

from 1 day infant Sprague-Dawley rats (purchased

from the Wuhan University Center for Animal

Experiments) as described by Micevych et al.

(Chaban, Mayer et al., 2003). The experimental

protocol was approved by the Ethics Committee for

Animal Research, Huazhong University of Science

and Technology, China. Then dorsal root ganglia

(DRG) explants were plated in the containers as

shown in the Figure 1 and 3. The Aclar films were

washed with laboratory detergent, rinsed with

deionized water, sterilized in 70% ethanol, and

coated with 10 ng/mL high-molecular-weight poly-

D-lysine (Sigma, St.Louis, Mo) before the culture.

The culture medium was Dulbecco’s modified

Eagle’s medium (DMEM, HyClone, Logan, UT)

supplemented with 10% FBS (HyClone, Logan,

UT), 50 ng/ml nerve growth factor (rat β-NGF,

R&D, USA), and 1% penicillin/streptomycin.

Cultures were treated with the mitotic inhibitors

formulated with 10 mM 5-fluoro-2`-deoxyuridine

(FdU) (Sigma), and 10 mM uridine (Sigma) to

encourage non-neuronal cell elimination. The

incubation was conducted at 37 ℃ in a humid

atmosphere containing 5% CO

. The growth location

of DRG explants were observed on the Olympus

CKX41 inverted microscope (Olympus Inc. , Tokyo,

Japan), and recorded with a Canon 600D SLR

(Canon Inc., Tokyo, Japan).

3 RESULTS

3.1 Cell Culture on 90 Degree Slope

After one day of incubation, a few glia cells could be

observed by microscope. After three days’

incubation, a large number of axons grew out from

the DRG explants following the path of glial cells

growth (Figure 5-8). Most of the DRG explants were

not in the defined position because the Aclar films

were shook when we changed the medium. Some

growth cones from the DRG explants advanced

more than one millimeter after 6 days (Figure 4).

However, they couldn’t grow down from the Aclar

film. Almost all axons and glial cells grew along the

edge of 198 μm thick Aclar film (Figure 5).

Figure 4: DRG explants cultured in the Petri dish with 90

degree slope. DRG neurite images were taken by a

10×objective, 6 days after the DRG explants were planted

on the Aclar strips. (Scale bar = 200 μm).

Figure 5: DRG explants cultured on 198 μm thick Aclar

film coated with PDL. DRG neurite images were taken by

a 10×objective, 3 days after the DRG explants were

planted on the Aclar strips. (Scale bar = 200 μm).

The morphology of axons grown on the 198 μm

thick Aclar film was obviously different from 50 μm

thick Aclar film (Figure 4 and 6). Therefore, most

axons didn’t grow down from 50 μm thick Aclar

GrowthMechanismofRatDorsalRootGanglionNeuronsonSlopeSubstrate

film to 198 μm thick Aclar film. A few axons grew

across the junction (Figure 7), but we could not

make sure that if these axons grew up from the 198

or 50 μm thick Aclar films.

Figure 6: DRG explants cultured on 50 or 198 μm thick

Aclar film coated with PDL. DRG neurite images were

taken by a 10×objective, 3 days after the DRG explants

were planted on the Aclar strips. (Scale bar = 200 μm).

Figure 7: DRG explants cultured on 50 or 198 μm thick

Aclar film coated with PDL. DRG neurite images were

taken by a 10×objective, 3 days after the DRG explants

were planted on the Aclar strips. (Scale bar = 200 μm).

On the bottom of Petri dish, a lot of axons and

glial cells migrated from the DRG explant (Figure 8).

The paths of axons growth were very clear with few

glial cells around the DRG explant. However, all

DRG explants were too far from the junction with

the 198 μm thick Aclar film to fail to arrive at its

edge. In order to make sure whether axons can grow

up 198 μm from 90 degree slope, we designed

another experiment by two pieces of 198 μm thick

Aclar film. Finally, we found that it is difficult for

axons to grow up on a 90 degree slope (Figure 9).

Figure 8: DRG explants cultured on the smooth bottom of

the Petri dish. DRG neurite images were taken by a

10×objective, 3 days after the DRG explants were planted

in the Petri dish. (Scale bar = 200 μm).

Figure 9: DRG explants cultured on 198μm thick Aclar

film coated with PDL. DRG neurite images were taken by

a 10×objective, 3 days after the DRG explants were

planted on the Aclar strips. (Scale bar = 200μm).

3.2 Cell Culture on Slight Degree Slope

In the axon stretch-growth bioreactor, a lot of axons

grew out from the DRG explants on the 50 μm thick

Aclar film (Figure 10). There were more axons on

the upper side than lower side. Previous studies have

shown that fibroblast cells prefer to grow uphill

NEUROTECHNIX2014-InternationalCongressonNeurotechnology,ElectronicsandInformatics

rather than downhill (Alaerts, De Cupere et al.

2001). In addition, Johansson et al found the axons

of the DRG cells on the original negative (grooved)

pattern were always found on the ridge edges, but

not in the grooves (Johansson, Carlberg et al. 2006).

This may explain why few axons grew down from

the 50 μm thick Aclar film (Figure 11).

Figure 10: DRG explants cultured on 50 μm thick Aclar

film coated with PDL. DRG neurite images were taken by

a 10×objective, 13 days after the DRG explants were

planted on the Aclar strips. (Scale bar = 200 μm).

Figure 11: DRG explants cultured on 50 or 198 μm thick

Aclar film coated with PDL. DRG neurite images were

taken by a 10×objective, 6 days after the DRG explants

were planted on the Aclar strips. (Scale bar = 200 μm).

After seven days’ culture in the axon stretch-

growth bioreactor, only a few of axons extended on

the bottom substrate. Stretch was applied by towing

the neuronal soma away from the growth cones by

taking a series of short 1

μm

steps. After two and a

half days’ stretch, the axons grew more than 1.75

mm and showed a regular alignment on the bottom

Aclar film (Figure 12). However, the axons didn’t

cross the 50 μm thick Aclar film, so we could not

assess the ability of stretched axons to climb on the

slope substrate.

Figure 12: DRG explants cultured on 50 or 198 μm thick

Aclar film coated with PDL. The blue line is more than

1.75 mm long. DRG neurite images were taken by a

4×objective, 10 days after the DRG explants were planted

on the Aclar strips. (Scale bar = 500 μm).

4 CONCLUSIONS

After nerve injury, neuronal connections are not

easily re-established. In the natural environment,

neurons are not growing on flat surface but in

complex three-dimensional microenvironment

formed by other cells or extra-cellular matrix. Along

with the chemical signals, neural behavior is also

determined by mechanical signals. As scarred tissue

is regenerates around the injury site, it is difficult for

the regenerating neurites to cross the injury gap. A

detailed analysis for the interaction of cells with

sloped substrate will not only support the

“regenerating axon” to cross the lesion in vivo but

also be helpful for the three-dimensional neural cell

cultures in vitro.

In this study, we performed a preliminary

experiment to test the reaction of neuronal growth

processes on the substrate slopes of different angles.

We found that all DRG cells’ axons couldn’t grow

across the 90 degree slope with 198 μm height. A

few axons grew across 90 degree slope with 50 μm

height. In addition, we also found that DRG cells

may also prefer to grow uphill rather than downhill.

This study didn’t provide the sufficient details

about the mechanisms which actually guide the

growth of neuronal cells on the different angles of

GrowthMechanismofRatDorsalRootGanglionNeuronsonSlopeSubstrate

substrate slopes, but it was reasonable to assume that

the guidance relied on extra-cellular cues, which

triggered some reorganization mechanisms in the

cytoskeleton. The actin cytoskeleton in cells

(fibroblasts, endothelia, and macrophages) reacting

to topography is organized in a way which we

believe to be appropriate for movement. Some

proteins, like semaphorines and ephrins, can inhibit

axons to grow the wrong way while other proteins

can attract axons to grow on the right way (Cook,

Tannahill et al. 1998). Compared with flat surface,

growth cones of the growing neurites on the slope

would get larger mechanical stress which can affect

the strength of the integrin–cytoskeleton links and

the integrin receptor distribution and conformation,

thus activating intracellular pathways active in cell

development and behaviour.

In future, we aim to make more experiments to

study the mechanism of slope modulation. For

example, we will put DRG explants on different

slope substrates to measure the growth rate of

neurites and observe the cytoskeleton morphology.

Even we will use quantitative real-time polymerase

chain reaction (qRT-PCR) to measure the magnitude

of changes in the expression of gene compliment

which regulates the neuron cell growth on different

topologies of substrates. Moreover, neurons are

usually not very likely to be the first cells to

encounter an implant as any topography may be

covered and obscured by glia cells (Franze 2013).

Therefore, we also need to study how slope affect

the glial cells.

ACKNOWLEDGEMENTS

This study was supported by the Natural Science

Foundation of China (grant number 61233015), the

National Basic Research Program of China (973

Program) (grant number SQ2012CB037202) and in

part by independent innovation fund of Huazhong

University of Science and Technology, Wuhan, P. R.

China (grant number 2013YGYL004). The authors

would like to thank Dr. Jun Ma and Dr. Jianfeng Xu

for their instruction in the experiments.

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NEUROTECHNIX2014-InternationalCongressonNeurotechnology,ElectronicsandInformatics