role of neurotrophin 3 in spinal neuroplasticity in rats subjected to cord transection

Role of Neurotrophin 3 in spinal neuroplasticity in rats subjected to cordtransection

HUI-JUAN YANG1,2,†, XIAO-YAN YANG2,†, YING-CHUN BA2, JIANG-XIA PANG2,

BU-LIANG MENG2, NA LIN1,2, LI-YAN LI2, XIN-YI DONG3, YU ZHAO3,

CHANG-FU TIAN3, & TING-HUA WANG1,2

1Institute of Neurological Disease, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu,

610041 China, 2Institute of Neuroscience, KunmingMedical College, Kunming 650031, P.R. China, and 3Experimental Center

for Medical Science, Kunming Medical College, Kunming 650031, P.R. China

(Received 9 October 2008; revised 9 December 2008; accepted 6 May 2009)

AbstractThat neuroplasticity occurs in mammalian spinal cord is well known, though the underlying mechanism still awaitselucidation. This study evaluated the role of endogenous Neurotrophin-3 (NT-3) in the spinal neuroplasticity. Following cordtransection at the junction between T9 and T10, the hindlimb locomotor functions of rats showed gradual but significantimprovement from 7 to 28 days post-operation. Corresponding to this was a significant increase in the level of NT-3 in the cordsegments caudal to injury site. Significantly, after NT-3-antibody administration, the spinal transected rats displayed poorhindlimb locomotor functions and a decrease in the number of neurons in spinal laminae VIII–IX. Whether NT-3-antibodywas administered, corticospinal tract regeneration and somatosensory evoked potentials could not be detected. Our findingssuggested that endogenous NT-3 could play an important role in spinal plasticity in adult spinal cords subjected to transection,possibly through a regulation of neuronal activity in the local circuitry.

Keywords: NT-3, role, neuroplasticity, spinal cord, transection, rat

Introdution

Spinal cord injuries (SCI) in the form of crush injury,

contusion, hemisection, and transection are com-

monly encountered in clinical practice. There are

about 15–40 SCI cases per million persons annually

worldwide (Lim and Tow 2007). SCI usually results in

severe neurologic dysfunction and disability. It is

expected that spinal cord transection would have a

high mortality. The severe consequences of SCI have

prompted numerous researchers to look into the

mechanism involved in axonal regeneration and

synaptic reorganization of neurons following trau-

matic lesions in mammalian spinal cords (Leong and

Lund 1973; Guth 1974; Steward 1989; He and

McCarthy 1994; Siddall and Loeser 2001; Wolpaw

and Tennissen 2001). Over the past several years, it

has been shown that the administration of exogenous

neurotrophic factors (NTFs) results in partial func-

tional recovery following SCI (Widenfalk et al. 2001),

indicating the potential value of NTFs in the

treatment of SCI.

Neurotrophin 3 (NT-3) is one of four related

polypeptide growth factors and shares structural and

functional homology with nerve growth factor (NGF)

(Tessarollo et al. 1994). It plays an important role in

maintaining neuronal survival and promoting neurite

growth in both physiological and pathological con-

ditions (Diener and Bregman 1994; Escandon et al.

1994; Kahane and Kalcheim 1994; Schnell et al. 1994;

Tessarollo et al. 1994; Tojo et al. 1995; Ye and Houle

ISSN 0897-7194 print/ISSN 1029-2292 online q 2009 Informa UK Ltd.

DOI: 10.1080/08977190903024298

Correspondence: T.-H. Wang, Institute of Neurological Disease, State Key Laboratory of Biotherapy, West China Hospital, SichuanUniversity, Chengdu, 610041 China. Tel: 86 28 85501518. Fax: 86 28 85501518. E-mail: [email protected]

†H.-J. Yang and X.-Y. Yang contributed equally to this work.

Growth Factors, August 2009; 27(4): 237–246

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1997; Ruitenberg et al. 2003; Hagg et al. 2005;

Kłopotowska and Strzadała 2005). NT-3 also alleviates

secondary response through preventing the secondary

cell loss that commonly occurs in injured spinal cords

(Bradbury et al. 1998). In addition, the transplantation

of NT-3-producing cells has a beneficial role in

enhancing the survival of spinal cord neurons

(Zhang et al. 1998; Uchida et al. 2003) and regeneration

of thecorticospinal tract followingSCI(Ruitenbergetal.

2003). The biological effects of NT-3 are mediated

through a specific high affinity tyrosine kinase receptor

type 3 (trkC) (Mocchetti and Wrathall 1995). Lastly,

NT-3 mRNA and protein have been demonstrated in

both the peripheral and central nervous systems

(Zhou and Rush 1994; Chen et al. 2007).

Although many experiments have extensively

investigated the role of exogenous NTFs in promoting

neuroplasticity following trauma, there were not many

studies to investigate the involvement of endogenous

NTFs in the dynamic modulation of local circuitry

in injured spinal cord. Our previous study suggested

that endogenous NT-3 could be involved in spinal

plasticity following SCI (Qin et al. 2006); the present

study sought to look at the exact role of endogenous

NT-3 in spinal neuroplasticity observed in the spinal

cord of rats subjected to transection in the lower part

of the spinal cord. As it was the hindlimbs that were

most affected after cord transection, this study mainly

focuses on the caudal part of injured spinal cords.

Materials and methods

Characterization of antibodies

The specificities of the two antibodies against NT-3

(gifts from Professor Xin-Fu Zhou, the Department of

Human Physiology and Centre for Neuroscience,

Flinders University of South Australia) (Deng et al.

2000; Zhou et al. 2000) and trkC (Neuromics, Edina,

Minnesota, USA) were confirmed by Western Blot

analysis, using spinal cord homogenates of rats in our

lab. The polyclonal antisera for NT-3 and monoclonal

antisera for trkC were specific for the appropriate

antigens and no cross-reaction with other antigens was

detected. Control of immunostaining specificity was

performed by omitting the primary antibodies and

antibodies pre-adsorbed with the NT-3 or trkC. These

controls did not exhibit any specific immunostaining.

Preparation of probe for in situ hybridization

For detection of NT-3 cellular expression in situ, we

used Digoxigenin-labeled oligonucleotide probes

(designed by Primer premier 5.0 package) which were

complementary to the rat’s NT-3 gene sequence

(25mer, 50-ATTACCAGAGCACCCTGCCCA-

AAGC-30) and trkC gene sequence (31mer, 50-CCT-

TGAGATGCCGTGATGTTGATACTGGCGT-30).

Each antisense DNA single-stranded oligonucleotide

probe was synthesized by Takara Biotechnology

Company (Shiga, Japan).

Animal grouping for various experimental procedures

Sprague-Dawley rats of either sex (weighing 200–

220 g) were obtained from the Animal Experimental

Centre, Sichuan University of Medical Sciences. They

were individually housed in a 12/12 h light/dark, quiet

and non-strong-light vivarium with free access to

water and food. Spinal cord transection was

performed at the junction between T9 and T10. The

operated rats were allowed to survive 1, 3, 7, 14 and

28 days post-operation (dpo), respectively. For sham

control, the operation was just short of transecting the

spinal cord. Laboratory procedures include immuno-

histochemistry, in situ hybridization, RT-PCR, Wes-

tern blot analysis, BDA tracing of the corticospinal

tract; and the cord transected rats were injected with

NT-3 antibodies or control serum Ig. In addition,

culturing motonurons in vitro from five normal rats

were treated with NT-3 antibodies or control serum Ig

to investigate the changes in the number of neurons

in vitro. To determine the possibility of a corticospinal

influence on hindlmb locomotor functions, BDA

tracing and SEP recording were also done. The animal

grouping and the number of animals used in the

various experimental procedures are shown in Table I.

Every effort was taken to reduce the number of

animals used and their suffering during the exper-

iments. All the experiments using animals were carried

out according to the guidelines of the NIH Guide

for Care and Use of Laboratory Animal. For cord

transection, the rats were anesthetized with 3.6%

chloral hydrate (1 ml/100 g). A midline incision was

made in the back, and the T7–T8 spinous processes

and the vertebral laminae were removed to expose the

spinal cord at T9–T10 level. The cord was transected

between T9 and T10 segments with a pair of

microscissors. After surgery, the superficial back

muscles and the skin were sutured along the midline.

Assessment of hindlimb locomotor functions

We used the BBB Locomotor Rating Scale (Basso et al.

1995) for the post-surgical analysis of the animal’s

hindlimb locomotor functions, including frequency

and quality of hindlimb movement as well as

forelimb/hindlimb coordination. The BBB Loco-

motor Rating Scale for evaluation of hindlimb

locomotor functions ranges from 0, which corre-

sponds to flaccid paralysis, to 21, which is normal gait.

Rats were allowed to walk around freely in a spacious

field for about 4 min while movements of the

hindlimbs were closely observed. As the grading

could be biased by subjectivity, three observers who

had no knowledge of the operative procedure and

survival time were requested to assess the locomotor

H.-J. Yang et al.238

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functions of both hindlimbs in all the operated and

sham operated animals. The scores of the three

observers were equated to get the average. All the

behavior evaluations were performed at 8–9 a.m. at

various periods after operation.

Measurement of somatosensory evoked potentials (SEP)

The right peroneal nerve of the rats was dissected and a

stimulus electrode was placed on it while a hole

(3–4 mm in diameter) was made in the skull (2 mm to

the left of the midline and 2 mm in front of the fonticulus

posterior). An electrode was placed on the dura over the

left-brain cortex to record the SEP. The reference

electrode was inserted at the nose epithelium. A ground

electrode was inserted at the tail. The stimulus intensity

was set high enough (about 1.1 mA) to produce a

marked muscle twitch in the left hindlimb; the

amplitude was 0.2 ms and the frequency 3 Hz. The

SEP Tracings represented the average of 200 responses.

Immunohistochemistry

The Groups I and IV rat spinal cords with the

administration of NT-3-antibody or control serum Ig

were perfused with 0.9% saline, followed by 4%

paraformaldehyde in 0.1 M phosphate buffered saline

(PBS) at pH 7.2. The spinal cord segments caudal to the

site of transection (checked to be at T11) were obtained

and placed in 20% sucrose solution in 0.1 M phosphate

buffer (PB). After the specimens had sunk to the bottom

of the bottle, they were placed on a freezing microtome

(Leica CM1900, Wetzlar, Hesse, Germany) and serial

horizontal sections were cut at 20mm thickness. For

consistent representation of the data, five sections, the

10th, 20th, 30th, 40th, and 50th sections of each animal

were processed for NT-3 immunohistochemistry. The

11th, 21st, 31st, 41st, and 51st sections in the

corresponding segments of the sham operated rats

were processed for trkC immunohistochemistry.

After washing three times (5 min each) in 0.1 M

PBS, the sections were incubated free-floating in 3%

hydrogen peroxide for 30 min to inactivate the possible

existence of endogenous peroxidase, and soaked for

30 min at 378C in 5% normal goat serum containing

0.3% TritonX-100. They were then transferred to a

solution (containing 2% normal goat serum and 0.3%

Triton X-100) of the respective primary antibodies

(rabbit polyclonal antisera for NT-3, diluted 1:1000

and mouse monoclonal antisera for trkC, diluted

1:100) for 24 h at 48C. After washing again with 0.1 M

PBS three times each for 5 min, the sections were

incubated in Reagents I and II from the Reagent Kit

(Chemicon, Anti-Rabbit/Mouse Poly-HRP IHC

Detection Kit, Temecula, CA, USA), each for

30 min at 378C. This was followed by three washings,

each for 5 min in 0.1 M PBS. The immunoreactive

products were then visualized by placing the sections

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Role of NT-3 in spinal neuroplasticity 239

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in a staining solution containing 0.05% 3,30-diamino-

benzidine 0.1% nickel sulfate and 0.01% hydrogen

peroxide for 10 min. Sections were observed in a light

microscope (Leica DMI 6000 B inverted microscope)

coupled with a computer assisted video camera

(Leica).

In situ hybridization

Frozen sections of the caudal spinal segments of some

the perfused sham operated rats were cut horizontally at

25mm thickness. For in situ hybridization, sections were

fixed in 4% paraformaldehyde in 0.1 M PBS, pH 7.2

(all treatments were performed at room temperature

unless otherwise indicated), and further treated with

0.3% TritonX-100 solution for 10 min and proteinase

K (5mg/ml) at 378C for 25 min, re-fixed with 4%

paraformaldehyde for 5 min, immersed again in 0.1 M

PBS, then acetylated with 0.25% acetic anhydride in

0.1 M triethanolamine (pH 8.0) to prevent non-specific

binding of the probes. The sections were washed with

2 £ SSC (pH 7.0), then pre-hybridized in a hybridiz-

ation solution (50% formamide, 10% dextran sulfate,

1 £ Denhardt’s solution, 0.2 mg/ml Herring sperm

DNA, and 10 mM dithiothreitol) without probes at

378C for 2 h before hybridization, then hybridized in

100ml hybridization solution containing 1ml probes at

378C for 12–16 h in a moist chamber. This was

followed by washing in decreasing concentrations of

SSC, from 4 £ SSC (pH 7.0) at 378C for 20 min, 2 £

SSC (pH 7.0) at 428C for 20 min, 1 £ SSC (pH 7.0) at

488C for 20 min and ending with 0.5 £ SSC (pH 7.0)

at 508C for 20 min. Then sections were incubated at

378C in 1% blocking buffer (Roche, Mannheim,

Baden-Wurttemberg, Germany) for 1 h, subsequently

reacted in 1:1000 sheep anti-digoxygenin-alkaline

phosphatase (AP) antibody in 1% blocking buffer

at 48C overnight. Lastly, AP activity was detected

using nitroblue tetrazolium (NBT)/5-bromo-4-chloro-

3-indolyl phosphate (BCIP) substrate (Roche).

The sections were visualized with blue and purple

sedimentation and observed with a light microscope.

Western blotting

Caudal spinal cord segments from Group III were

obtained. After carefully removing the spinal

meninges, the cord segments were homogenized on

ice in a Lysis Buffer, containing 0.05 M Tris–HCL

(pH 7.4, Amresco, Solon, OH, USA), 0.5 M EDTA

(Amresco), 30% TritonX-100 (Amresco), NaCl

(Amresco), 10% SDS (Sigma, St Louis, MO, USA)

and 1 mM PMSF (Amresco), then centrifuged at

12,000g for 30 min. The supernatant was obtained

and stored at 2808C for later use. Protein concen-

tration was assayed with BCA reagent (Sigma). A 20ml

aliquot of the samples was loaded on to each lane and

electrophoresed on 12% SDS–polyacrylamide gel

(SDS–PAGE) for 2.5 h at a constant voltage of 120 V.

Proteins were transferred from the gel to a nitrocel-

lulose membrane for 435 min at 24 V. The membrane

was blocked with PBS containing 0.05% Tween-20

(PBST) with 10% non-fat dry milk overnight at 48C.

The membrane was rinsed with PBST and incubated

with the primary antibody for NT-3 (1:200) at 48C.

The membrane was incubated with a HRP-conju-

gated goat anti-rabbit IgG (1:5000; Vector Labora-

tories, Burlingame, CA, USA) for 2 h at room

temperature and was developed in ECM kit and

exposed against X-ray film in a darkroom. Densito-

metry analysis for the level of NT-3 protein was

performed by Bio-Gel Imagining system equipped

with Genius synaptic gene tool software. b-actin (the

primary antibody, 1:200, the secondary antibody,

1:5000; Santa Cruz Biotechnology, Inc., Santa Cruz,

CA, USA) was used as an internal control.

Reverse transcription polymerase chain reaction

(RT-PCR)

RT-PCR was used to determine the expression level of

NT-3 in the caudal spinal segments of Group II. Total

RNA was isolated from the spinal cord sample

(weighing 50 mg), using Trizol (Invitrogen, Carlsbad,

CA, USA), according to the manufacturer’s instruc-

tion. The concentration was measured using a

Nanodrop spectrophotometer (ND-1000), and the

total RNA was subjected to reverse transcription

procedure. cDNA was generated using the Revert Aid

First Strand cDNA Synthesis Kit (Fermentas,

Burlington, Ontario, Canada). Total RNA (4mg/reac-

tion) was added to a “master mix” (12ml) containing

final concentrations of total RNA, oligo-dT primer

and DEPC-treated water. Sample mixtures were then

heated to 708C for 5 min. Then 5 £ reaction buffer,

RNase inhibitor and 10 mM dNTP Mix were added.

After heating at 378C for 5 min, M-MLV reverse

transcriptase was added to a final concentration of

20ml. The mixed samples were then incubated at

428C for 60 min and stopped by incubation at 708C for

10 min for reverse transcription. This was followed by

further PCR amplification for each cDNA sample,

using rat specific primers as follows: b-actin (227 bp)

sense: 50-GTAAAGACCTCTATGCCAACA-30 and

antisense: 50-GGACTCATCGTACTCCTGCT-30;

NT-3 (754 bp) sense: 50-GATATTTCTTGCTTAT-

CTCCG-30; antisense: 50-ATGTTCTTCCAATTT-

TTCTCG-30. PCR was performed using 2 £ PCR

Master Mix (Fermentas), and reacted in 0.2 ml thin-

walled reaction tubes, using the “hot-start” method.

Reagents were assembled in a final volume of 25ml,

and the final concentrations of reagents were as

follows: 1.5ml first strand cDNA, 0.5ml forward

primer, 0.5ml reverse primer, 12.5ml 2 £ PCR

Master Mix, and 10ml RNase-free water to 25ml.

Samples were initially denatured at 948C for 5 min,


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then 1 min denaturation at 948C, 1 min annealing at

52.58C for b-actin and 548C for NT-3, and 1 min

extension at 728C. The whole reverse transcription

procedure was repeated for 30 cycles with a final

extension step at 728C for 10 min. Aliquots (25ml) of

the PCR reaction were run on 1% agarosegels, and the

size of the reaction products was determined after

ethidium bromide staining. Rat b-actin was amplified

as an internal control, using rat-specific prime.

BDA anterograde tracing

At 14 dpo, the cord transected rats for BDA

anterograde tracing were anesthetized and fixed in

stereotaxic head-holder device (David Kopf Instru-

ments, Tujunga, CA, USA). Burr holes were made in

the dorsal cranium, and 10% solution of BDA

(Molecular Probes, Eugene, Oregon, USA) was

microinjected into eight sites at a depth of 0.3 mm

from the cortical surface (0.5ml/site) to cover the

hindlimb region. Animals were then sacrificed 2 weeks

later to allow sufficient time for axonal transport of

BDA in the corticospinal tract. The spinal cords were

removed and post-fixed at 3 days in cold 4%

paraformaldehyde in 0.1 M PBS (pH 7.2). Transverse

sections (30mm) of the spinal cord at the transection

site and the neighboring rostral and caudal areas were

processed for the presence of BDA-labeled axons by

incubation in avidin-HRP (Molecular Probes). Lastly,

the positive staining fibres, which appeared brown

were visualized by DAB staining.

NT-3 antibody neutralization in vivo

Each cord transected rat in Group IV was intraper-

itoneally injected with 0.5 ml (30 mg/ml) of anti-NT-3

solution once every 2 days until 14 dpo. Control rats

(Group IV) were injected with serum Ig of the same

isotype to rule out non-specific effects. To determine

the effects of NT-3 antibody administration, the

hindlimb locomotor functions were evaluated by BBB

scores at 1, 3, 7, 14 and 28 dpo. Lastly, at 28 dpo, the

numbers of neuron in spinal laminae VIII–IX of the

caudal spinal segments in treated with NT-3 antibody

and control serum Ig rats were counted.

NT-3 antibody neutralization in cultured motoneurons

in vitro

Rats were anaesthetized with intraperitoneal injection

of 3.6% chloral hydrate (1 ml/100 g). The spinal cords

were rapidly taken out following a dorsal laminectomy.

Both the right and left ventral horns of the L2–L6

spinal cord were harvested with the help of an

operation microscope. They were dissected into

D-Hanks’ balanced salt solution (HBSS) and digested

with 0.25% trypsin at 36–36.58C for 20 min. The cell

suspension was then prepared and sieved through

a steel mesh. This was followed by centrifuging at

1000 rpm for 5 min and the cell sedimentation was

harvested and transferred into Dulbecco’s modified

Eagle’s medium (DMEM)/F12 mix containing 1 ml

100 U/ml penicillin and streptomycin and sup-

plemented with 100 ml/l calf serum. Cells were plated

at a density of 1 £ 105 cells/ml of culture medium by

leucocyte counting plate into 24-well trays coated with

0.5 g/l poly-l-lysine and 400ml per well. Then half of

the cells were treated with NT-3 antibody (1:100) to

neutralize the endogenous NT-3, and the remaining

cells were used as control treated with serum Ig.

To inhibit non-neuronal growth 10ml 100mg/ml

cytarabine solution were added into each well and

the cells were incubated at 378C in a 5% CO2

incubator. The medium was replaced after 24 h and

every 3 days thereafter till 12 days. Lastly, the cultured

cells were immunostained for identification of

motoneurons. Briefly, they were fixed for 20 min in

4% paraformaldehyde-PBS on day 12 in culture,

permeabilized with methanol for 10 min, incubated

overnight at 48C with rabbit anti-peripherin (1:1000;

Chemicon International), then incubated with bioti-

nylated swine anti-rabbit antibodies (1:500; Dako,

Glostrup, Denmark) for 1 h. Diaminobenzidine was

used to develop the stain. All the numbers were

calculated manually in 15 random equal campus

visualis under high magnification (400 £ ).

Statistical analysis

All data were expressed as the mean ^ SEM. They

were analyzed using one-way ANOVA and LSD-q test

by SPSS software package. Statistical significance was

defined as p , 0.05.

Results

Localization of NT-3 and trkC in the spinal cord

Immunohistochemical products of NT-3 were seen in

the neurons located in spinal laminae I–VII and VIII–

IX of sham operated rats. Both the cytoplasm and the

nucleus of NT-3 immnoreactive (IR) neurons dis-

played intense staining (Figure 1A–C). TrkC IR

neurons were also seen in spinal laminae I–VII and

IX. Their cellular membrane and cytoplasm were

obviously staining (Figure 1D–F).

In sham operated rats, signals of hybridization for

NT-3 mRNA were intense in the neurons of spinal

lamina I–III, IX and central canal, but less so in the

neurons in other filed (Figure 1G–I). TrkC mRNA-

positive neurons which were obviously staining were

observed in spinal laminae I–V, IX and central canal,

and neurons which were lightly staining presentd in

spinal laminae VI–XIII (Figure 1J–L).


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Changes in NT-3 mRNA and protein expressions in cord

transected and sham operated rats

In the caudal spinal segments of both cord transected

and sham operated rats, a single positive band was

observed at a molecular weight of about 13.6 KD,

which corresponds to the molecular weight of NT-3.

There were no significant changes in NT-3 at 1 dpo

( p . 0.05). Significant increases were observed at 3

dpo ( p , 0.05), peaked at 7 dpo ( p , 0.05), and

remained so up to 14 dpo, then returned to the sham

operated level at 28 dpo (Figure 2).

The expressions of NT-3 mRNA were similar to

those of NT-3 protein, except that they appeared

earlier than the NT-3 protein. Compared with the

sham operated group, the level of NT-3 mRNA in the

caudal spinal segments of the cord transected rats

showed a rapid upregulation at 1 dpo ( p , 0.05), and

remained so at 3 dpo till 7 dpo ( p , 0.05), but

returned to sham operated group level at 14 dpo, up till

28 dpo ( p . 0.05) (Figure 3).

Effects of NT-3 antibody administration on NT-3

expression in vivo

NT-3 immunopositive cells were markedly present in

the motoneurons of the ventral horn of the caudal

spinal segments of both in sham contral and serum

Ig injected rats. The stained IR products occurred

Figure 1. The presence of NT-3-IR (A–C), TrkC-IR (D–F), NT-3 mRNA (G–I), TrkC-mRNA (J–L) in the sham operated rats. NT-3-IR

neurons were localized in thoracal spinal cord (A, 50 £ ), with the NT-3 immunoreactive products present in both cytoplasm and nucleus

(B, arrow, 400 £ ). Control cord section with PBS replacing the NT-3 antibodies is shown in (C, 50 £ ). TrkC-IR neurons were localized in

thoracal spinal cord (D, 50 £ ) with the immunoreactive products present in the cellular membrane and cytoplasm (E, arrow, 400 £ ). Control

cord section with PBS replacing the TrkC antibodies is shown in (F). NT-3 mRNA was markedly present in thoracal spinal cord (G, 50 £ ),

showing blue and purple staining in the cytoplasm (H, arrow, 400 £ ). Control cord section without NT-3 probes is shown in (I, 50 £ ). TrkC

mRNA-positive neurons in thoracal spinal cord (J, 50 £ ) showing blue and purple staining in the cytoplasm (K, arrow, 400 £ ). Control cord

section without TrkC probes is shown in (L, 50 £ ).


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mainly in the cytoplasm of the cell body and its

processes. Following NT-3 antibody administration,

NT-3 immunostaining in the motoneurons was

markedly reduced, compared with that in rats treated

with serum Ig (Figure 4).

Changes in the numbers of neurons in vivo and in vitro

The numbers of motoneurons in spinal laminae

VIII–IX in the cord segments rostral and caudal to

transection site in vivo (Table II), and cultured

motoneurons in vitro (Table III) decreased significantly

(p , 0.05) in NT-3 antibody treated rats, compared

with the control serum Ig treated group (Figure 5).

Behavior tests

The BBB scores for locomotor functions in the sham

operated group declined rapidly at 3 dpo, then

Figure 2. Results from Western Blotting in cord segments caudal

to cord transection site of sham operated and cord transection rats.

b-actin was used as control. Lane 1 in the left side displays the

protein band in the sham-operated group, and lanes 2–6 show the

protein band at 1, 3, 7, 14 and 28 dpo, respectively. The bands in

lanes 4 (7 dpo) and 5 (14 dpo) are the most intense.

Table II. Numbers of neurons in spinal laminae VIII–IX in control

serum Ig and NT-3-antibody treated rats subjected to cord

transection at 28 dpo.

Lam VIII–IX of

cord rostral to the

transection side

Lam VIII–IX of

cord caudal to the

transection side

Control serum Ig

group at 28 dpo

6.40 ^ 1.91 4.60 ^ 2.01

NT-3 antibody treated

group at 28 dpo

4.55 ^ 2.83* 3.25 ^ 2.26#

Numbers refer to mean numbers (M) per equal area

(281.25mm2) ^ SEM. dpo: Days post-operation. *p , 0.05, com-

pared with control ones. #p , 0.05, compared with control ones.

Figure 3. RT-PCR results in cord segments caudal to cord

transection site of sham operated and cord transection rats. b-actin

was used as control. Marker in the left side is shown in lane 1; band

of NT-3 mRNA in the sham operated group in lane 2; NT-3 mRNA

in cord transected group at 1, 3, 7, 14 and 28 dpo in lanes 3–7,

respectively. The bands in lane 3 (1 dpo), 4 (3 dpo), 5 (7 dpo) are

obviously brighter than lane 6 (14 dpo) and lane 7 (28 dpo).

Table III. Numbers of spinal motoneurons incubated 12 days

in vitro (mean ^ SEM).

Control serum Ig group NT-3 antibody neutralized group

12 ^ 3 7 ^ 2*

*p , 0.05, compared with the control serum Ig group in which

without NT-3 neutralization.

Figure 4. NT-3 IR neurons in NT-3 antibody injected rats versus

serum Ig injected rats. Intensely stained NT-3 IR spinal

motoneurons in sham control (A) and in serum Ig (B) injected

rats as compared to the lightly stained NT-3-IR neurons in NT-3

antibody treated rats (C). Absence of staining in negative control

section (D).


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returned to the value of 21 by 7 dpo. They increased

gradually from 7 to 28 dpo in the cord transected and

control serum Ig treated groups, but were not

significantly different between the two groups. NT-3-

antibody treated group showed a gradual improve-

ment in the BBB scores from 7 to 28 dpo, but

compared with either cord transection or control

serum Ig treated group there was a significant decrease

in the BBB scores ( p , 0.05) (Table IV).

Somatosensory evoked potentials

SEP are electrical signals generated by the cerebral

cortex in response to peripheral sensory stimuli. In this

study, they consist of a series of waves that reflect

sequential activation of neural structures along the

somatosensory pathways following electrical stimu-

lation of the peroneal nerve. SEP (P1N1P2 type) was

recorded in the control rats and shown in Figure 6.

Throughout the experimental periods, no SEP could

be recorded in rats subjected to cord transection

(Figure 6).

BDA tracing

BDA-positive axons which appeared as brown short

silky strands were detected in the cord segments

rostral to transection site (Figure 7A), but not in the

scar tissue (Figure 7B), nor in the cord segments

caudal to transection site (Figure 7C).

Discussion

The occurrence of NT-3 and trkC IR products in the

ventral motoneurons in sham operated rats in this

study indicates that NT-3 is involved in the

maintenance of their normal physiological functions.

The observation that NT-3 mRNA and trC mRNA

(as revealed by in situ hybridization) as well as NT-3

and trkC proteins (immunohistochemically demon-

strated) were both present in the motoneurons of

ventral horns indicates that the NT-3 is endogenously

produced. This finding is in agreement with previous

results that NT-3 mRNAs are widely distributed in a

variety of neuronal and non-neuronal cells in embryos

and adults (Friedman et al. 1991; Lauterborn et al.

1994; Ernfors et al. 1995).

The SEP of cord transected rats was absent

throughout the entire experimental period, indicating

a loss of sensory function in the hindlimbs. On the

other hand, though hindlimb locomotor functions

were totally lost at 1–3 dpo, they returned at 7 dpo

and slowly increased thereafter, as demonstrated by

the gradual increase in the BBB scores during this

period. Could the return of hindlimb locomotor

functions be the result of regeneration of the

corticospinal tract fibres across the transection site to

the caudal segments? The absence of any of such fibres

with BDA tracing rules out this possibility. It is

possible that the return of locomotor function is

related to the modulation of the spinal circuitry

dependant on the role of endogenous NTFs, one of

which is NT-3, demonstrated to be present in both

the cord segments caudal to the transection site of

sham operated rats in this study. As stated above,

the endogenous nature of the NT-3 had been

Table IV. Mean values of BBB scores in cord transected rats

(mean ^ SEM).

Group 3 dpo 7 dpo 14 dpo 28 dpo

Sham-operated group 15 ^ 3 21 ^ 0 21 ^ 0 21 ^ 0

Untreated cord

transected group

0 ^ 0 0.9 ^ 0.3 2.5 ^ 0.7 4.5 ^ 1.1

Control serum Ig

treated group

0 ^ 0 0.8 ^ 0.2 2.4 ^ 0.6 4.4 ^ 0.9

NT-3-antibody treated

group

0 ^ 0 0.2 ^ 0.1 0.8 ^ 0.3 2.1 ^ 0.6

BBB score in sham-operated rats presented a rapid decline at 3 dpo,

then returned to the normal level at 7 dpo, and remained so up to 28

dpo. The scoring between the transected group only subjected to the

surgery and the serum Ig treated group had no statistical significance

( p . 0.05), while there was a significant decrease of BBB score in

NT-3-antibody treated group from 7 to 28 dpo, compared with

transected group and control serum Ig treated group ( p , 0.05).

Figure 5. Peripherin-IR cultured neurons. Peripherin-IR neurons

cultured from the serum Ig injected (A), and NT-3 antibody

neutralized (B) rats. Magnification: 400 £ .

Figure 6. Results of SEP. SEP (P1N1P2type) recorded in a sham

operated rat. Latency of SEP: P1 (10.3 ^ 0.26) ms, N1

(8.81 ^ 0.34) ms, P2 (15.5 ^ 0.43) ms, amplitude: P1

(2.34 ^ 0.02), N1 (16.3 ^ 0.14), P2 (25.06 ^ 0.05). And no

SEP could be recorded in rats subjected to cord transection.


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demonstrated in this study. That the endogenous

NT-3 could play an important role is indicated by its

up-regulation following cord transection. This is

further supported by the observation that the BBB

scores in NT-3-antibody treated rats was less than

those not receiving the antibody treatment.

The numbers of neurons in laminae VIII–IX of cord

segments rostral and caudal to the transection site also

decreased in the NT-3 antibody treated group. Our

study also showed that NT-3 could play a part in

motoneuronal survival in vivo and in vitro, for, without

NT-3 the numbers of NT-3 IR neuron in spinal

laminae VIII–IX and of cultured peripherin-positive

spinal motoneurons were significantly reduced. These

suggested that endogeous NT-3 plays a role in

improving locomotor functions recovery.

A good number of studies have shown that the

administration of exogenous NT-3 by transgenic

therapy or direct injection into spinal cord could

improve the recovery of locomotor functions (Chi-

kawa et al. 2001; Widenfalk et al. 2001; Tuszynski

et al. 2003; Girard et al. 2005; Ruitenberg et al. 2005;

Mitsui et al. 2005; Zhang et al. 2007). Our results are

the first to show that endogenous NT-3 also plays an

important role in maintaining the survival of

motoneurons and improving locomotor functions of

the hindlimbs of rats subjected to cord transection.

The demonstration of NT-3 and trkC mRNA-

positive products and proteins in the motoneurons of

the ventral horn suggests that NT-3 could exert its

function via autocrine and paracrine mechanisms.

Of course, one cannot rule out a possible retrograde

transport of NT-3 from a target tissue to neuronal cell

bodies (DiStefano et al. 1992; Curtis et al. 1998;

Haase et al. 1998).

To conclude, we hypothesize that NT-3 most likely

plays an important role in the neuroplasticity of the

spinal cord in adult rats subjected to cord transection,

probably through the regulation of local circuit activity

in the spinal cord segments caudal to the site of

transection.

Acknowledgements

This work was supported by The National Science

Foundation of China (No. 30260125) and the CMB

Grant (CMB-00-72). We thank Mr Kevin J. Foehrkolb

and Dr Leong Seng Kee for their invaluable

comments in the writing of this manuscript.

Declaration of interest: The authors report no

conflicts of interest. The authors alone are responsible

for the content and writing of the paper.

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This paper was first published online on iFirst on 8 June 2009


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role of neurotrophin 3 in spinal neuroplasticity in rats subjected to cord transection

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