Neuronal regeneration after acute spinal cord injury in adult rats

Neuronal regeneration after acute spinal cord injury in adult rats

Accepted Manuscript Title: Neuronal regeneration after acute spinal cord injury in adult rats Author: He B, Nan G PII: DOI: Reference: S1529-9430(16)...

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Accepted Manuscript Title: Neuronal regeneration after acute spinal cord injury in adult rats Author: He B, Nan G PII: DOI: Reference:

S1529-9430(16)30278-9 http://dx.doi.org/doi: 10.1016/j.spinee.2016.06.020 SPINEE 57067

To appear in:

The Spine Journal

Received date: Revised date: Accepted date:

31-12-2015 1-6-2016 22-6-2016

Please cite this article as: He B, Nan G, Neuronal regeneration after acute spinal cord injury in adult rats, The Spine Journal (2016), http://dx.doi.org/doi: 10.1016/j.spinee.2016.06.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Neuronal regeneration after acute spinal cord

1

injury in adult rats

2

He B1#, Nan G1#*

3 4 5

1

6

Laboratory of Pediatrics in Chongqing, Chongqing International Science and

7

Technology Cooperation Center for Child Development and Disorders

Ministry of Education Key Laboratory of Child Development and Disorders,Key

8 9 10 11 12 13 14 15 16

#

Current Address: Department of Orthopaedics Children’s Hospital of Chongqing

Medical University, Chongqing, China *Corresponding author: [email protected]

Nan

G,

PhD,

fax:

+86

(023)63632084,

email:

First author: He B, Dr, email: [email protected]

17

Abstract

18

Background context

19

The most common cause of the spinal cord injury (SCI) is traumatic traffic accidents,

20

falls, and violence. SCI greatly affects a patient’s mental and physical conditions and

21

causes substantial economic impact to society. There are many methods such as high

22

doses of corticosteroids, surgical stabilization, decompression and stem cell

23

transplantation for functional recovery after SCI, but the effect is still not satisfied.

24

Purpose

25

This study investigated the role of neuronal regeneration and the location of the

26

neuronal regeneration after spinal cord injury (SCI) in rats.

27

Study Design 1

Page 1 of 24

1

An experimental animal study of ASCI investigating the neuronal regeneration after

2

SCI. Double immunofluorescence staining of NF-200 and BrdU was performed to

3

detect the location of the neuronal regeneration.

4

Methods

5

Forty-five adult Wistar rats were tested. Allen’s hit model (10 g) induced acute SCI

6

sites

7

immunohistochemistry. Double immunofluorescence staining of neurofilament 200

8

(NF-200) and 5-bromo-2'-deoxyuridine (BrdU) was performed 10 mm away from the

9

spinal cord center. Neural functional recovery was determined using the Basso,

10

Beattie, and Bresnahan (BBB) score and electro-physiological examination. The

11

study was funded by Natural Science Foundation of China(NSFC, 81272172). The

12

funder of this study had no capacity to influence the scholarly conduct of the research,

13

interpretation of results or the dissemination of study outcomes.

14

Results

15

BrdU- and NF-200-positive cells were rarely detected and absent at 3w and 4w,

16

respectively. We also detected the BrdU and NF-200 co-expressed cells are at 3-5 mm

17

away from the injured site, and no co-expressed cells were detected at the injured site

18

in this SCI model. The The BBB score and electro-physiological examination of the

19

nervous system were significantly different at 4w.

20

Conclusions

21

To our knowledge, this is the first study to demonstrate that neurons are regenerated

22

3-5 mm away from the injured site, and no neurons are regenerated at the injured site

targeted

at

T10

segments.

Nestin

expression

was

detected

via

2

Page 2 of 24

1

in this SCI model, which suggests a novel train of thought regarding SCI treatment.

2 3

Keywords: Spinal cord injury; neurons regeneration; stem cells; rats

4 5

Introduction

6

The most common cause of the spinal cord injury (SCI) is traumatic traffic accidents,

7

falls, and violence [1]. In SCI, the force of the injury destroys or damages neural

8

tissues, which causes a sudden loss of neurological function. SCI greatly affects a

9

patient’s mental and physical conditions and causes substantial economic impact to

10

society [2]. Treatment methods during the acute phase are often limited to high doses

11

of corticosteroids, as well as surgical stabilization and decompression, to potentially

12

attenuate further damage [3-4]. Stem cell transplantation is a new method for

13

treatment of spinal cord injury. However, the roles of these methods in functional

14

recovery after SCI remain controversial.

15

Endogenous neural stem cells (NSCs) are tissue-specific somatic stem cells that are

16

present in NSCs. They are characterized by their multi-lineage potential and

17

self-renewal activity [6-7]. In an adult mammalian central nervous system (CNS), a

18

major population of NSCs is located in the periventricular area throughout the

19

neuraxis from the forebrain to the spinal cord [8]. Previous studies have demonstrated

20

that pluripotent NSCs have been isolated from the rat spinal cord [9-10]. Pluripotent

21

NSCs proliferate following stimulation, such as damage and demyelination, and

22

widely migrate to the spinal cord white matter, which plays an important role in 3

Page 3 of 24

1

neural functional recovery after SCI [11]. Although previous studies have

2

demonstrated that NSCs proliferate after SCI, nearly all NSCs differentiate into

3

astrocytes rather than neurons and oligodendrocytes [12-14]. Recent studies have

4

identified newborn neurons in the adult mammalian brain ependymal area and

5

hippocampal dentate gyrus after brain injury [15-16]. Thus, the question arises as to

6

whether neuronal regeneration occurs after acute SCI.

7

This study aimed to use endogenous NSCs to repair SCI via factors that stimulate the

8

proliferation and differentiation potential of stem cells to replace damaged nerve cells,

9

which prompts the self-repair of damaged spinal cord tissues. Compared with NSC

10

transplantation in the treatment of CNS damage, endogenous NSCs are used to repair

11

SCI because the use of autologous resources can avoid cause a cell hybrid

12

phenomenon in the process of cell transplantation, tumorigenicity after transplantation,

13

immune rejection reactions, and many other issues [16]. Therefore, the use of

14

endogenous NSCs to repair a damaged CNS is the current trend in SCI treatment.

15

BrdU is broadly used to label new born cells, and is an indicator of DNA synthesis

16

marker [17-18]. NF-200 is a neurofilament protein that comprises a biological marker

17

for mature neuronal cells in the CNS. In the present study, NF-200 and BrdU staining

18

were used to evaluate NSC proliferation. We suggest that BrdU and NF-200

19

co-expressed cells should represent newborn neuronsa. Reproducible rat model of

20

traumatic SCI was used to investigate neuronal regeneration after SCI.

21 22

Materials and Methods 4

Page 4 of 24

1 2

Sample Preparation and Assay Kits

3

BrdU, paraformaldehyde, BrdU antibody, and neurofilament 200 (NF-200) antibodies

4

were purchased from Sigma Biotechnology Co., Ltd. (St. Louis, MO, USA). Nestin

5

antibody was purchased from DHSB Biotechnology Co., Ltd. A Power Vision kit was

6

purchased

7

Tetramethylrhodamine isothiocyanate (TRITC)-conjugated Goat anti-Mouse IgG and

8

fluorescein isothiocyanate (FITC)-conjugated Goat anti-Mouse IgG were purchased

9

from Zhongshan Biotechnology Co., Ltd (Beijing, China). A laser scanning confocal

10

microscope was purchased from Leica Co., Ltd. (Wetzlar, Germany), and a

11

multi-channel physiology recorder was purchased from PowerLab Co., Ltd. (Shanghai,

12

China).

from

Changdao

Biotechnology

Co.,

Ltd.

(Shanghai,

China).

13 14

Animals

15

This study was conducted in strict accordance with the National Institutes of Health

16

Guide for the Care and Use of Laboratory Animals. It was approved by the

17

Institutional Animal Care and Use Committee accredited by the Association for the

18

Assessment and Accreditation of Laboratory Animal Care International in China and

19

the Experimental Animal Committee of Chongqing Medical University (Permit

20

numbers: SCXK (Yu) 2012 - 0001 and SYXK (Yu) 2012 - 0001). All surgeries were

21

performed under sodium pentobarbital anesthesia, and all efforts were made to

22

minimize suffering. 5

Page 5 of 24

1

Forty-five adult Wistar rats (22 males and 23 females; weight, 240-250 g) were

2

provided by the Animal Center of Chongqing Medical University. The animals were

3

bred at room temperature (between 20-25°C) with 40-60% relative humidity and a

4

day/night cycle of 12/12 hours. Food and water were provided ad libitum. The rats

5

were randomly divided into two groups: sham group (SH, n = 10; five males and five

6

females) and injury group (IN, n = 35; 17 males and 18 females). The IN group was

7

divided into seven subgroups according to different time points: 1 day (1d), 3d, 1

8

week (1w), 2w, 3w, 4w, and 8w (n = 5 per group).

9

Following SCI, the rats were assessed every four hours on the first day after the

10

operation and every 12 hours on subsequent days to determine whether they attacked

11

each other, exhibited self-destructive behavior, or were breathing. The rats were

12

monitored during the acute post-injury phase via examination of the bladder size,

13

suture, and body weight twice per day. The bladder was gently squeezed twice per

14

day for seven days to avoid urinary tract infections (the urine could be cloudy, bloody,

15

or contain precipitates). The integrity of the rats was also assessed to determine

16

whether they were injured or exhibited behaviors in typical food consumption. If the

17

rats bit each other and/or exhibited self-destructive behavior, they were individually

18

housed and fed alone. If these rats continued to exhibit self-destructive behavior

19

following individual housing, they were euthanized using carbon dioxide. Intragastric

20

artificial feeding was utilized for the rats that were unable to feed themselves.

21 22

Allen’s method for spinal cord lesions 6

Page 6 of 24

1

Prior to skin preparation and routine sterilization, the rats were anesthetized via an

2

intraperitoneal injection of 10 g/L sodium pentobarbital (40 mg/kg) and were fixed in

3

a stereotaxic frame on an operating table. A 2.0-cm incision around the T10 lamina

4

was exposed, and the spinal process was separated. The erector spine was exposed,

5

and the T10 lamina was removed, with the exception of the dura mater. In the IN

6

group, a modified Allen’s method was used to induce contusion injury [19]. Briefly, a

7

thin copper slice was used to cover the exposed spinal cord, which was vertical to a

8

graduated glass tube. A 10 g metal block was dropped onto the copper slice from a

9

height of 2.5 cm, and the copper slice was immediately removed. If a rat’s tail curved

10

or swung and the lower limbs exhibited paralysis, the injury was considered

11

successful. In the SH group, the rats underwent the same operation without spinal

12

cord contusion. Following surgery, forced urination was performed three times per

13

day.

14 15

Drug dose injection and approach

16

The rats were anesthetized via an intraperitoneal injection of 10 g/L sodium

17

pentobarbital at 40 mg/kg of body weight prior to the operation. All rats received 150

18

mg/kg of body weight of BrdU solution via intraperitoneal injection every 12 hours

19

for three days prior to specimen collection. Following surgery, intraperitoneal

20

injections of penicillin (20,000 U/kg) were administered twice per day for three

21

consecutive days.

22 7

Page 7 of 24

1

Basso, Beattie, and Bresnahan (BBB) locomotor rating scale

2

Motor function recovery after SCI was assessed using an open field walking test.

3

Double blind evaluations were performed by investigators. The locomotor assessment

4

was initiated one week after injury and performed every two weeks for eight weeks.

5

Each hind limb was individually graded during free locomotion on the floor according

6

to the BBB locomotor score [20].

7 8

Electro-physiological examination of the nervous system

9

A multi-channel physiology recorder was used to examine the nervous system. After

10

the rats were anesthetized, the left sciatic nerve and contralateral sensorimotor cortex

11

area were electrically stimulated. The somatosensory-evoked potential (SEP) and

12

motor-evoked potential (MEP) waveform curves were recorded, and the latency was

13

calculated.

14

The SEP and MEP stimulus parameters were as follows: stimulation frequency = 4 Hz,

15

pulse width = 0.2 ms, and intensity = 10-40 mV. The signal was magnified

16

one hundred thousand times by the pre-amplifier, and the average latency was

17

calculated after it was superimposed 64 times by a computer.

18 19

Specimen collection

20

All surgical procedures were standardized and performed by the same surgeon,

21

including the separation of the spinal cord from the spine. The specimens were

22

contained approximately 10 mm from rostral and caudal to the injury site. The spinal 8

Page 8 of 24

1

cord samples were fixed in 4% paraformaldehyde for paraffin sectioning.

2 3

Histopathology

4

Spinal cord tissues were fixed in 4% paraformaldehyde, dehydrated, embedded in

5

paraffin wax, and serially sectioned (5-m thick). The thin sections were dewaxed

6

with xylene and gradient dehydrated with alcohol for hematoxylin and eosin (H&E)

7

staining.

8 9

Nestin immumohistochemical staining

10

Spinal cord tissues were fixed in 4% paraformaldehyde, dehydrated, embedded in

11

paraffin wax, and serially sectioned (5-m thick) using a RM2065-type paraffin

12

section machine (Leica Inc., Wetzlar, Germany). After deparaffinization and

13

rehydration, the paraffin sections were placed in Na+ citrate buffer (0.1 M, pH 6.0),

14

microwave irradiated for 10 min, and cooled by the addition of double-distilled H2O.

15

Hydrogen peroxide (H2O2, 0.3%) was used to block endogenous peroxidase. The

16

tissues were washed in phosphate buffered saline (PBS, 2 x 10 min) and incubated

17

with Nestin antibody (1:500 dilution) for 60 min at 37°C. The tissues were

18

subsequently washed in PBS (3 x 10 min), incubated with a PowerVision reagent for

19

30 min at 37°C, rinsed three times with PBS, DAB color developed, and

20

counterstained with hematoxylin. A densitometric analysis of the positive cells in

21

each group was performed using Image-Pro Plus (Media Cybernetics, Rockville, MD,

22

USA). 9

Page 9 of 24

1 2

BrdU and NF-200 immunofluorescence double staining

3

Spinal cord tissues were fixed in 4% paraformaldehyde, dehydrated, embedded in

4

paraffin wax, and serially sectioned (5-m thick) using a RM2065-type paraffin

5

section machine (Leica Inc., Wetzlar, Germany). After deparaffinization and

6

rehydration, the paraffin sections were incubated with 3% goat serum for 30 min,

7

followed by incubation with BrdU antibody (dilution of 1:2,500; Sigma

8

Biotechnology Co., Ltd., St. Louis, MO, USA) at 4°C overnight. The tissues were

9

washed in PBS (3 x 10 min) and incubated with TRITC-conjugated Goat anti-Mouse

10

IgG (1:100 dilution, Zhongshan Co., Ltd., China) at 37°C for 1 hour. Then, the tissues

11

were washed again in PBS (3 x 10 min), and the paraffin sections were incubated in

12

3% goat serum for 30 min, followed by incubation with NF-200 antibody (1:1,000

13

dilution; Sigma Biotechnology Co., Ltd., St. Louis, MO, USA) at 4°C overnight. The

14

tissues were further washed in PBS (3 x 10 min) and incubated in FITC-conjugated

15

Goat anti-Mouse IgG (1:100 dilution; Zhongshan Co., Ltd., China) at 37°C for 1 hour,

16

followed by an additional wash with PBS (3 x 10 min). The slides were covered with

17

cover slips. Fluorescent images were observed under a confocal laser-scanning

18

microscope using a Leica TCS SP confocal system attached to a Leica DM IRBE

19

microscope. Goat serum (1:50) was used instead of BrdU and NF-200 antibody; the

20

remaining procedures were the same as the previous experiments to determine the

21

specificity of the immunofluorescence.

22 10

Page 10 of 24

1

Statistical analysis

2

All data were analyzed with SPSS 13.0 statistical software (IBM Corp, Armonk, NY,

3

USA). All experiments were repeat performed at least three times, and all data are

4

expressed as the mean ± standard deviation (SD). Differences between the two groups

5

in the same time point were assessed using Student’s t-tests, and differences

6

within-group at the multiple time points was assessed using one-way anova. A value

7

of P<0.05 was considered statistically significant.

8 9

Results

10 11

Survival rate

12

Seven rats died in the two groups, and these rats were replaced with additional rats.

13

One rat in the SH group died during the operation, and one rat in the IN group died

14

prior to anesthesia recovery. These deaths may have resulted from excess anesthesia.

15

Following the completion of the model and anesthesia recovery, no deaths occurred in

16

the SH group, whereas five rats died in the IN group. One rat was euthanized at 2w

17

because of self-destructive behavior (Table 1).

18 19

BBB locomotor rating scale

20

All rats exhibited normal limb function and obtained a BBB score of 21 prior to SCI.

21

There

22

which each obtained a score of 21 throughout the study. Serious hind limb locomotor

was

no

locomotor

dysfunction

in

the

rates

in

the

SH

group,

11

Page 11 of 24

1

dysfunction (complete paralysis) was identified in the rats in the IN group (P < 0.05)

2

at 1w, 2w, 4w, 6w, and 8w. The BBB score was significantly increased at four weeks

3

after SCI (P < 0.05 compared with the SH group; Fig. 1).

4

MEP and SEP

5

After SCI, the MEP for the SH group was 7.72 ± 0.86, 7.78 ± 0.67, and 7.76 ± 0.64

6

ms at 2w, 4w, and 8w, respectively. The MEP for the IN group gradually decreased

7

over time after SCI and was 21.27 ± 1.52, 15.12 ± 1.37, and 11.06 ± 1.18 ms at 2w,

8

4w, and 8w, respectively (P < 0.05 compared with the SH group; Figs. 2 and 4). After

9

the sham operation, the SEP in the SH group was 11.89 ± 0.64, 11.92 ± 0.90, and 12.1

10

± 0.65 ms at 2w, 4w, and 8w, respectively. The SEP also gradually decreased over

11

time after the sham operation and was 26.01 ± 2.21, 16.25 ± 1.83, and 15.25 ± 1.04

12

ms at 2w, 4w, and 8w, respectively. Importantly, the SEP was significantly decreased

13

at 4w after SCI. However, there was no significant change in the SEP at 4w-8w after

14

SCI. (P < 0.05 compared with the SH group; Figs. 3 and 4).

15 16

General histopathology

17

H&E staining of tissue paraffin sections indicated there were no significant changes in

18

the morphology of the spinal cord tissues or cells However, a substantial number of

19

neurons were identified in the SH group (Fig. 5a). In the IN group (Fig. 5b),

20

hemorrhage in the gray matter at the central damaged area occurred 24 hours after

21

SCI, and only a small proportion of neurons survived, in which swollen axons and a

22

substantial number of vacuoles were visible in the white matter. 12

Page 12 of 24

1 2

Nestin protein expression

3

In the SH group, Nestin expression was identified in the cytoplasm of a limited

4

number of spinal cord ependymal cells, whereas there was almost no expression in the

5

white matter. In the IN group, Nestin was expressed in a subgroup of ependymal cells

6

at 24 h after SCI. Three days after SCI, Nestin expression was identified in ependymal

7

cells and at the pial surface, which extended into the arborizing processes centripetally

8

from the pial surface towards the central canal. Point sheet and filamentous

9

expressions were present in the gray and white matter. Nestin expression peaked at 1w

10

after SCI, mainly in the ependymal cells and less in the gray matter. The Nestin

11

expression began to decrease 2w after SCI, significantly decreased at 4w, and had low

12

or no expression at 8w. Nestin-positive dendritic protrusions were present and

13

extended to the gray matter and were expressed in the gray matter mainly around the

14

vessel and neurons 4w after SCI. Neuron-like Nestin-positive cells were identified in

15

the gray matter 3w after SCI. The expression of the Nestin positive area of each group

16

was measured at different time points (P < 0.05 compared with the SH group, Table

17

2).

18 19

Expression of BrdU and NF-200

20

In the SH group, minimal BrdU-positive cells and a substantial number of

21

NF-200-positive cells were identified in the white and gray matter. However, BrdU

22

and NF-200 co-expressed cells were absent (Fig. 7a). The NF-200-positive cells were 13

Page 13 of 24

1

significantly decreased 1d after SCI in the IN group compared with the SH group. A

2

small number of BrdU-positive cells were identified; however, no co-expressed cells

3

were present in the spinal cord tissues 1d after SCI. The number of BrdU-positive

4

cells continued to increase, whereas the NF-200-positive cells decreased in the white

5

and gray matter 3d after SCI. However, there was no co-expressed cells in the IN

6

group (Fig. 7b). As time progressed, the number of BrdU-positive cells continued to

7

increase, and BrdU and NF-200 co-expressed cells were occasionally identified 1w

8

after SCI (Figs. 7c-7e). Two weeks after SCI, a substantial number of BrdU and

9

NF-200 co-expressed cells were identified in the area 3-5 mm away from the injured

10

site (Figs. 7f-7h). As time increased after SCI, the number of co-expressed cells

11

significantly decreased; no co-expressed cells were identified at 4w or 8w after SCI

12

(Fig. 7i, Table 6; P < 0.05)

13 14

Discussion

15 16

In this paper, we described a method to obtain a reproducible model of traumatic SCI

17

using an Infinite Horizon Impactor at a force of 10 g (severe). The use of a larger

18

force paradigm (15 g) causes more severe injury, which is unfortunately associated

19

with increased mortality. To avoid this concern, a moderate force paradigm (10 g) was

20

implemented, which is associated with a repeatable lesion, gradual recovery of

21

function, and lower mortality.

22

SCI destroys neural and glial elements and severs axonal connections between the 14

Page 14 of 24

1

motor and sensory systems, which leads to permanent and often devastating loss of

2

function. Although many promising molecular strategies have emerged to reduce

3

secondary injury and promote axonal regrowth, there is no effective cure to date, and

4

recovery of function remains limited [21]. In recent decades, the therapeutic promise

5

of replacing lost neurons and glia via transplantation has gained significant

6

momentum and has resulted in the initiation of clinical trials [22]. Nevertheless, the

7

potential tumorigenicity of pluripotent stem cells and their progeny must be addressed

8

before these cells can be implemented in clinical practice.

9

NSCs are characterized by their multi-lineage potential and self-renewal activity.

10

Although other researchers previously isolated and cultured pluripotent stem cells

11

from the rat spinal cord [23], the exact site of the spinal cord could not be determined.

12

Lendahl et al. [24] described a gene with an expression that distinguishes stem cells

13

from more differentiated cells in the neural tube. This gene was named Nestin because

14

it is specifically expressed in neuroepithelial stem cells. The predicted amino acid

15

sequence of the Nestin gene product indicates that Nestin comprises a distinct sixth

16

class of intermediate filament proteins. These observations extend a model in which

17

the transitions in intermediate filament gene expression reflect major steps in the

18

neural differentiation pathway [24]. Thus, Nestin protein was used to mark stem cells

19

in the current study. Previous studies that have used glial fibrillary acidic protein

20

(GFAP) and Nestin to mark cells after SCI have demonstrated that some

21

Nestin-positive cells may be astrocytes after SCI. However, Nestin-positive cells in

22

the ependymal do not express GFAP, which suggests that these cells are not 15

Page 15 of 24

1

astrocytes [25]. In our study, some ependymal cells expressed Nestin 24 h after SCI in

2

the IN group. Nestin expression peaked at 1w after SCI, mainly in ependymal cells

3

and less in the gray matter. The Nestin expression subsequently began to decrease 2w

4

after SCI. This decrease was significant at 4w and exhibited minimal or no expression

5

at 8w. Large Nestin-positive cells similar to neurons were identified in the gray matter

6

3w after SCI. We suspect that these cells may reflect astrocytes and neuronal

7

differentiation from NSCs. This finding suggests that there are NSCs that have the

8

potential capability to differentiate and proliferate.

9

BrdU served as a synthetic nucleoside that can be incorporated into newly synthesized

10

DNA during the S phase of the cell cycle. BrdU is widely used to detect proliferating

11

cells in living tissues [26]. NF-200 is a neurofilament protein that comprises a

12

biological marker for mature neuronal cells in the CNS. In the current study, NF-200

13

and BrdU staining were used to evaluate NSC proliferation. We suggest that BrdU and

14

NF-200 co-expressed cells should represent newborn neurons. Our findings indicate

15

that neuronal regeneration significantly increased after SCI, with most new neurons

16

concentrated in the area 3-5 mm away from the injured site. However, new neurons

17

were not identified at the center of the damaged area. We also identified significantly

18

increased Nestin-positive cells at the injury point of the spinal cord, which suggests

19

that the injury point exhibited endogenous NSC proliferation. However, it is difficult

20

to differentiate into neurons, and this may be associated with the different

21

microenvironments of NSCs [27]. Especially during to past decade the experimental

22

studies about the spinal cord injury have been forwarded from the medical therapies 16

Page 16 of 24

1

toward stem cell therapy. However, due to many factors such as poor

2

microenvironment at the injury site, and apoptosis, which precludes the successful

3

effect of the therapy, the clinical use of the stem cell therapy is not widely used. The

4

other important issue of the stem cell therapy is delivery route. In this study, the

5

authors observed neuronal regeneration only 3-5 mm away from the injury site and

6

this result is consisting with the negative effect of the microenvironment on the neural

7

regeneration at the injury site. This result supports the idea of using local treatment of

8

stem cell therapy rather than systemic use.

9

In the current study, a substantial increase in new neuronal regeneration was present

10

in the area 3-5 mm away from the injury at 2w after SCI. As time progressed, the

11

number of new neurons significantly decreased; new neurons were no longer present

12

at 4w and 8w after SCI. Furthermore, the BBB score was not significantly different

13

4w-8w after SCI and the SEP and MEP exhibited the most prominent changes 4w

14

after injury. Previous research has indicated that the combined electro-physiological

15

exploration of MEP and SEP represent a useful tool for monitoring patients with

16

severe SCI [28]. These findings indicate that neuronal regeneration was most

17

significant 2w after SCI and was maintained up to 4w after SCI. This finding is in

18

accordance with the time of SEP, MEP, and BBB score detection of neural functional

19

recovery and suggests that the co-expressed cells represent new neurons.

20

Early after SCI, the gray matter in the central damaged area undergoes hemorrhage,

21

necrosis, ischemia, hypoxia, and the release of a substantial number of inflammatory

22

mediators, such as tumor necrosis factor, interleukin-6, and interleukin-1. This 17

Page 17 of 24

1

makes this area not suitable for endogenous NSCs to differentiate into neurons.

2

Nevertheless, the immediate area approximately 3-5 mm away from the center of the

3

injury

4

microenvironment, which may be more suitable for activating NSCs and promoting

5

their differentiation into neurons. Thus, it is necessary to investigate the

6

microenvironment associated with the region and time period after SCI, as well as to

7

identify a suitable microenvironment for NSCs to differentiate into neurons. The

8

identification of a factor that can induce NSC differentiation into neurons may greatly

9

improve the efficiency of self-healing after SCI.

exhibits

hyperemia,

capillary

regeneration,

and

alterations

in

the

10

There are several limitations that must be addressed in the interpretation of these

11

findings. In the present study, it is not clear why neuronal regeneration only occurred

12

in the early stage after SCI and in an area 3-5 mm away from the injured site. Thus,

13

additional investigations regarding neuronal regeneration microenvironments and

14

function are required. Furthermore, although the small sample size of five animals per

15

group and the use of an animal model revealed valid results, this approach limits the

16

translation to human studies. Nevertheless, these novel findings indicate that neuronal

17

regeneration occurs after SCI and may thus represent an important treatment target in

18

future studies.

19 20

References

21

1. Alabama. Cord Injury Statistical Center: spinal cord injury facts and figures at

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the treatment of acute spinal cord injury: a foundation for best medical practice. J

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4. Furlan, J. C., Noonan, V., Cadotte, D. W, et al. Timing of decompressive surgery of

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5. Okano H. The stem cell biology of the central nervous system.J. Neurosci. Res. 69

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(2002) 698-707.

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6. Okano H. Neural stem cells: progression of basic research and perspective for

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7. Okano H. Adult neural stem cells and central nervous system repair. Ernst Schering

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8. Temple S. and Alvarez-Buylla A. Stem cells in the adult mammalian central nervous

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system. Curr. Opin. Neurobiol. 9 (1999) 135–141.

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9.Okano H, Sakaguchi M, Ohki K, et al. Regeneration of the central nervous system

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using endogenous repair mechanisms. J Neurochem. 102 (2007) 1459–1465.

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10.Weiss S, Dunne C, Hewson J, et al. Multipotent CNS.stem cells are present in the

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adult mammalianspinal cord and ventricular neuroaxis. J Neurosci. 16 (1996)

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7599–7609.

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11. Wallace MC, Tator CH, Lewis AJ: Chronic regenerative changes in the spinal cord 19

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15. Gage FH.Mammalian neural stem cells. Science. 28 (2000) 1433–1438.

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female forebrain mediated by prolactin. Science. 29 (2003) 117-120.

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18. Ming G. L., Song H. Adult neurogenesis in the mammalian brain: significant

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answers and significant questions.Neuron. 70 (2011) 687–702.

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19. Allen AR. Surgery of experimental lesions of spinal cord equivalent to crush injury

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of fracture dislocation Preliminary Report. JAMA. 57 (1911) 878-880.

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20. Basso DM, Beattie MS, Bresnahan JC. A sensitive and reliable locomotor rating

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21.Samuel E. Nutt, Eun-Ah Chang, Steven T. Suhr, et al.Caudalized human

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491–503. 20

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22. Fehlings MG, Vawda R. Cellular treatments for spinal cord injury: the time is right

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for clinical trials. Neurotherapeutics. 8 (2011) 704–720.

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23. Gage FH, Coates PW, Palmer TD, et al. Survival and differentiation of adult

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neuronal progenitor cells transplanted to the adult brain. Proc Natl Acad Sci USA. 92

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(1995) 11879-11883.

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24. Lendahl U, Zimmerman LB, McKay RD. CNS stem cells express a new class of

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intermediate filament protein. Cell. 60 (1990) 585-595.

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25.Shibuya S, Miyamoto O, Auer RN, et a1. Embryonic intermediate filament, Nestin,

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expression following traumatic spinal cord injury in adult rats. Neuroscience. 114

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(2002) 905-916.

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26.Lehner B, Sandner B, Marschallinger J, et al. The dark side of BrdU in neural stem

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cell biology: detri-mental effects on cell cycle, differentiation and survival. Cell Tissue

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Res. 345 (2011) 313-328.

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27.Garbossa D, Boido M,Fontanella M. et al. Recent therapeutic strategies for spinal

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cord injury. treatment: possible role of stem cells. Neurosurg Rev. 35 (2012) 293-311

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28. Cheliout-Heraut F,Loubert G,Masri-Zada T,et al. Evaluation of early motor and

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39-55

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1 2 3 4 5 6 7 8 9 10 11 12 13 14

Figure Legends

15

Figure 1. BBB scores in rats at different time points post-injury.

16

Figure 2. Latency of MEP.

17

Figure 3. Latency of SEP.

18

Figure 4. MEP and SEP of each group.

19

a: MEP at 8w in the sham group. b: MEP at 8w in the injury group. c: SEP at 8w in

20

the sham group. d: SEP at 8w in the injury group.

21

Figure 5. General histopathology of the spinal cord.

22

a: Cross-section of a normal spinal cord (H&E x 200). b: Hemorrhage in gray matter 22

Page 22 of 24

1

24 h after injury (H&E x 100).

2

Figure 6. Nestin expression in each group.

3

a: Nestin-positive cells in the sham group (DAB x 200). b: Nestin-positive cells at 24

4

h after SCI (DAB x 200). c: Nestin-positive cells at 1w after SCI (DAB x 100). d:

5

Nestin-positive cells near ependymal cells 1w after SCI (DAB x 400). e:

6

Nestin-positive cells near vessels and neurons 4w after SCI (DAB x 200). f:

7

Neuron-like Nestin-positive cells in the gray matter after SCI (DAB x 400).

8

Figure 7. BrdU and NF-200 expression.

9

a: Few BrdU-positive cells (red) and a substantial number of NF-200-positive cells

10

(green) were present; however, there were no co-expressed cells in the sham group (x

11

100). b: A substantial number of BrdU-positive cells (red) and NF-200-positive cells

12

(green) were present; however, there were no co-expressed cells in the injury group 3d

13

after SCI (x 100). c: A substantial number of BrdU-positive cells were identified in the

14

injury group 1w after SCI (x 50). d: NF-200-positive cells were present in the injury

15

group 1w after SCI (x 50). e: Co-expressed cells were occasionally identified in the

16

injury group 1w after SCI (x 50). f: NF-200-positive cells were present in the injury

17

group 2w after SCI (x 200). g: BrdU-positive cells were present in the injury group

18

2w after SCI (x 200). h: Co-expressed cells increased in the injury group 2w after SCI

19

(x 200). i: Co-expressed cells were absent in the injury group 4w after SCI (x 50).

20

23

Page 23 of 24

Tables 1 Table 2 1. Mortality per group. Group

Time

Operation

Prior to anesthesia recovery

3d

1w

2w

4w

8w

Sham group

1

0

0

0

0

0

0

Injury group

0

1

3

1

1

0

0

3

Table 4 2. Nestin-positive areas in the spinal cord at different time points ( x Time

1d

3d

1w

98.45 ± 6.21

98.45 ± 6.21

98.45 ± 6.21

Group

Sham group Injury group

2w

 SD

4w

µm2). 8w

98.45 ± 6.21 98.45 ± 6.21 98.45 ± 6.21

410.19 ± 21.34 678.58 ± 48.23 987.34 ± 46.43 768.30 ± 36.26

543.21 ± 41.89

112.08 ± 10.15

5

Table 6 3. Number of NF-200 and BrdU co-expressed cells at different time points. Time

3d

1w

2w

3w

4w

8w

Sham group

0

0

0

0

0

0

Injury group

0

2.4±1.2

6.1±2.3

1.4±0.8

0

0

Group

7 8

24

Page 24 of 24