Electro-acupuncture induced NGF, BDNF and NT-3 expression

in spared L6 dorsal root ganglion in cats subjected

to removal of adjacent ganglia

Juan Chen a,b, Jian-Guo Qi a,c, Wei Zhang a,c, Xue Zhou a,c, Qing-Shu Meng b,Wei-Min Zhang b, Xu-Yang Wang b, Ting-Hua Wang a,b,c,*

a Institute of Neurological Disease, West China Hospital, Sichuan University, Chengdu 610041, Chinab Institute of Neuroscience, Kunming Medical College, Kunming 650031, China

c Department of Histology, Embryology and Neurobiology, College of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China

Received 25 September 2006; accepted 9 August 2007

Available online 15 August 2007

www.elsevier.com/locate/neures

Neuroscience Research 59 (2007) 399–405

Abstract

This study evaluated the effect of electro-acupuncture (EA) on the NGF, BDNF and NT-3 expression in spared L6 dorsal root ganglion (DRG) in

cats subjected to bilateral removal of L1-L5 and L7-S2 DRG, using immunostaining, in situ hybridization and RT-PCR. The positive products of

NGF, NT-3 protein and mRNA in the small and large neurons of spared L6 DRG in EA side increased greatly more than that of control side, while

the increased BDNF was only noted in small and medium-sized neurons. RT-PCR demonstrated that the mRNA level for three factors was not

influenced by EA in intact DRG, when a significant increase was seen in the spared L6 DRG of EA side. As it has been well known that DRG

neurons project to the spinal cord wherein morphological plasticity has been present after DRG removal, the present results might have some

bearing to the observed phenomenon.

# 2007 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Keywords: NGF; BDNF; NT-3; Dorsal root ganglion; EA; Spinal cord plasticity

1. Introduction

Neuroplasticity in the central nervous system (CNS) was

firstly demonstrated by Liu and Chambers (1958) who

proposed that after surgically removing a series of dorsal root

ganglia (DRG), axonal sprouting from the central processes of

the spared ganglion occurred in Lamina II. This pioneering

work prompted numerous researchers to look into the

mechanism on the axonal regeneration and synaptic reorga-

nization of neurons following traumatic lesions in the

mammalian spinal cord (Leong and Lund, 1973; Guth, 1974;

Steward, 1989; He, 1994; Mendell et al., 2001; Siddall and

Loeser, 2001; Wolpaw and Tennissen, 2001).

* Corresponding author at: Institute of Neurological Disease, West China

Hospital, Sichuan University, Chengdu 610041, China. Tel.: +86 871 5329245;

fax: +86 871 5342766.

E-mail address: tinghua_neuron@263.net (T.-H. Wang).

0168-0102/$ – see front matter # 2007 Elsevier Ireland Ltd and the Japan Neuro

doi:10.1016/j.neures.2007.08.006

Arising from some recent related work is the significant

finding that failure of neurons to spontaneously regenerate after

injury in the adult CNS might be attributed to the nonpermissive

nature of the CNS environment (Aubert et al., 1995), like the

lack of growth-promoting molecules (Varon and Conner, 1994)

and the presence of inhibitory molecules (Fitch and Silver,

1997). Moreover, providing a growth supportive environment

by the administration of neurotrophic factors (NTFs) (Lindsay

et al., 1994) has been partially successful in inducing axonal

regeneration within the adult mammalian CNS.

Though a good number of studies have investigated the roles

of neurotrophic factors in preventing neuronal death or

promoting anatomical reorganization after spinal cord injury

(Huang and Reichardt, 2001; Kim et al., 2001; Liu et al., 2002),

the involvement of endogenous neurotrophic factors in the

dynamic modulation of local circuitry remains to be elucidated.

Acupuncture, an ancient craft originating in China more

than 3000 years ago, has been shown to promote functional

recovery in spinal cord injury (Li et al., 1985; He, 1994).

science Society. All rights reserved.

Fig. 1. Drawing showing xuewei is where EA was applied.

J. Chen et al. / Neuroscience Research 59 (2007) 399–405400

Electro-acupuncture (EA) is similar to the traditional acu-

puncture but where in EA, electrical stimuli are delivered

instead of manual twist stimuli in the traditional acupuncture. It

has been known that EA can increase the number of axonal

terminal derived from spared DRG, indicating the effect of EA

in meliorating synaptic reconstruction. Previous researches had

demonstrated a correlation between the increase of some

neurotrophic factors like nerve growth factor (NGF), brain

derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3)

and neurotrophin 4/5 (NT-4/5) and the development, survival

and maintenance of neuronal function in both the peripheral

and the central nervous system (Isackson, 1995; McAllister

et al., 1999; Sendtner et al., 2000; Huang and Reichardt, 2001),

in addition endogenous neurotrophic factors may be involved in

neurite-outgrowth enhancement induced by dorsal root gang-

lionectomy (Xue et al., 1994). Moreover, our recent studies also

determined some other neurochemicals expression in DRG

after EA (Wang et al., 2005a,b, 2006). This experiment was

therefore undertaken to observe the possible effects of EA in the

expression of NGF, BDNF and NT-3 and their respective

mRNA in the spared DRG neurons of cats after adjacent DRG

removal. It is hoped that the results derived from immunohis-

tochemistry, in situ hybridization and RT-PCR can provide

some evidences for EA promoting the spinal cord plasticity,

involved in the NGF, BDNF and NT-3 expressions.

2. Materials and methods

2.1. Animals

Twenty adult male cats, weighing 3–3.5 kg for each animal, were used in this

study. The animals were provided by the Laboratory Animal Center of Kunming

Medical College. Every effort was taken to reduce the number of animals and

suffering during the experiments. Group I, consisting of 15 cats were used for

immunohistochemistry, in situ hybridization and RT-PCR for each five, respec-

tively. Animals Group II (n = 5) was also used to detect the mRNA expression

changes in the normal cats after EA by RT-PCR. The animals were maintained

under conditions of controlled light, temperature, food and water.

2.2. Surgical procedures

All experimental procedures were performed under anesthesia achieved

with intraperitoneal injection of 3.5% sodium pentobarbital (1.3 ml/kg body

weight). The DRG of cats in Group I associated with the first through the fifth

lumbar (L1-L5) and the seventh lumbar through the second sacral (L7-S2)

spinal nerves were first bilaterally removed, sparing the L6 DRG, then followed

immediately with EA on the left side at acupuncture points. Group II were used

as normal cats, and performed by EA as group I.

2.3. EA

On the day after surgery, all the cats were subjected immediately to

unilateral electrical stimulation at acupuncture points (Fig. 1) (xuewei)

zusanli-xuanzhong, and futu-sanyinjiao, both of which are known to lie in

L6 dermatome (Xue et al., 1994). Zusanli (ST36) is located 1.5 cm below the

front of the fibula head; xuanzhong (GB39), 1.5 cm above the front of the lateral

malleolus; futu (ST32), 2–3 cm above the lower end of the patella; and

sanyinjiao (SP6), 1.5 cm above the posterior end of the medial malleolus.

The pairs of acupoints were stimulated alternately at the frequency of 98 HZ for

30 min every day. During acupuncture, the electrodes were changed every

15 min. EA is similar to traditional acupuncture except that electrical stimuli are

delivered instead of manual stimuli in the traditional method.

2.4. Immunohistochemistry

The cats were perfused under anesthesia on day 7 after EA. Their L6 DRG on

both sides were removed and immersed in a 4% paraformaldehyde solution for

12 h, then placed in a 20% sucrosed phosphate buffered solution (PBS) over night.

Serial longitudinal sections of the frozen DRG were sectioned into 20 mm slices.

For an unbiased and accurate representation of the data, 5 sections were taken

randomly, representing all the samples in one animal. The selected sections were

processed for immunohistochemical demonstration of NGF, BDNF and NT-3,

using specific NGF (rabbit anti-human 1:100), BDNF (rabbit anti-human 1:500)

and NT-3 (rabbit anti-human 1:1500) antibodies using ABC (Avidin-Biotin

Complex) method. Then the sections were stained by DAB (3,30-diaminobenzi-

dine). All antibodies, bought from Chemicon, have been identified by the

Western-Blot method in our lab and shown to possess specificity for their own

antigens. In addition, control experiments in which PBS was substituted for the

primary antibody were performed to ascertain specificity of antibody staining.

2.5. In situ hybridization

The use of double-stranded cDNA as molecular probes for in situ hybri-

dization to chromosomal preparations has been effective for localizing the

position of particular genes like those of NGF, BDNF and NT-3. Slide-mounted

DRG sections were warmed to room temperature (25 8C), postfixed in 4%

paraformaldehyde (pH 7.4) for 5 min at 4 8C, rinsed in PBS, and treated with

0.25% acetic anhydride (in 0.1 M triethanoLaminae, pH 8.0) for 10 min at room

temperature. After rinsing 2� in standard saline citrate (SSC) for 10 min and

dehydration through a graded series of alcohols, the sections were delipidated in

chloroform for 5 min at room temperature and subsequently re-hydrated to 95%

ethanol in descending concentrations of alcohols and then air-dried. They were

next washed in Tris (trihydroxymethyl aminomethane) at pH 9.4 and

NaCl:MgCl2 (20:1) for 2 min, then color-developed in Tris, pH 9.4 and

NaCl:MgCl2 (20:1) in the presence of nitroblue tetrazolium (NBT) and 5-

bromo-4-chloro-2-indolyl phosphate (BCIP) for 5 h. The sections were finally

rinsed several times and mounted on glass slides with Aqua Poly/Mount

(Polysciences; Warrington, PA).

Controls for in situ hybridization: Preincubation with RNAase was used to

assess the nonspecific binding of the probes to the section. Moreover, to

demonstrate the validity of the probes of three neurotrophic factors, the cRNA

probes were substituted with double distilled water without RNAase or the

unmarked probes. No positive reaction was observed in both control experiments.

2.6. Cell number counting

The distributions of the immunopositive neurons for NGF, BDNF, NT-3 and

their respective mRNA in different sized DRG neurons on the acupunctured and

non-acupunctured side were observed and photographed in an Olympus light

microscope at 40�. In order to calculate the average number of immunoreactive

cells, photographs of DRG were scanned and projected on the screen of a

computer. Then the areas of the DRG and the number of positive neurons were

respectively measured using several manufactured square grids (0.8 cm per

square grid). Therefore the average number in the area designated as 100 grids

was acquired so as to compare the number of positive neurons in equal areas in

different sections. The average numbers of labeled neurons were recorded in

Tables 1–3.

Table 1

Average numbers of NGF and its mRNA positive neurons in the left L6 DRG

Average number NGF-IR positive neurons NGF mRNA positive neurons

Non-EA

side

EA side Non-EA

side

EA side

Large neurons 4.1 � 1.2 6.4 � 1.7* 3.3 � 1.7 5.2 � 2.3*

Medium-sized

neurons

13.2 � 2.7 11.2 � 3.9** 11.2 � 3.3 10.4 � 4.7**

Small neurons 10.4 � 3.9 15.5 � 3.4* 9.1 � 3.6 14.7 � 4.9*

* P < 0.05, compared with non-electro-acupunctured side.** P > 0.05, compared with non-electro-acupunctured side.

J. Chen et al. / Neuroscience Research 59 (2007) 399–405 401

2.7. RT-PCR

RNA preparation: Animals were allowed to survive for 7 days after EA.

Total RNA was isolated from the L6 DRG on the both sides in each cat using

TRIzol Reagent (Invitrogen, USA) according to the manufacturer’s instructions.

The RNA concentration of each sample was determined from the absorbance at

260 nm.

RT-PCR: One microgram of total RNAwas reverse-transcribed to cDNA in a

12 ml reaction volume with anchored oligo dT primer using RevertAidTM First

Stand cDNA Synthesis Kit (Fermentas, Lithuania). Three microliter of the RT

product was then used as template for PCR using 2�PCR Master Mix (Fermentas,

Lithuania). RT and PCR were performed according to the manufacturer’s pro-

tocols on PCR Express thermal cyclers (BioRad, USA). Primers were designed

using Primer Premier 5.0 software and synthesized commercially (TaKaRa,

Dalian, China). The primers used for amplification, together with their specific

optimum cycling conditions, were as follows: NGF: sense 50-GTG GCA GGG

CAG ACC CGC AAC AT-30, antisense 50-AGC ACCACC CGC CTC CAA GTC

CA-30, annealing temperature (TA) 65 8C; 30 cycles, yielding a 144 bp products;

BDNF: sense 50-TGA CAT CCT TGG CTG ACA CTT T-30, antisense 50-ACT

GGG AGT TCC AAT GCC TTT T-30, annealing temperature (TA) 55 8C; 30

cycles, yielding a 456 bp products. NT-3: sense, 50-CTC ATG GAG GAT TAC

GTG GGA-30, antisense, 50-GCC TGG CTT CTT TAC ATC TCG-30, annealing

temperature (TA) 56.5 8C; 30 cycles, yielding a 244 bp products. The primers for

b-actin used for internal contral were 50-GCTACA GTT TCA CCA CCA CCG-30

(sense), 50-ATG CCA CAG GAC TCC ATA CCC-30 (antisense), annealing

temperature (TA) 58 8C, 30 cycles, yielding a 232 bp products.

Table 2

Average numbers of BDNF and its mRNA positive neurons in the left L6 DRG

Average number BDNF-IR positive neurons

Non-EA side EA sid

Large neurons 0.98 � 0.35 1.13 �Medium-sized neurons 12.86 � 0.66 14.40 �Small neurons 34.18 � 0.98 41.30 �

* P < 0.05, compared with non-electro-acupunctured side.** P > 0.05, compared with non-electro-acupunctured side.

*** P < 0.01, compared with non-electro-acupunctured side.

Table 3

Average numbers of NT-3 and its mRNA positive neurons in the left L6 DRG

Average number NT3-IR positive neurons

Non-EA side EA sid

Large neuron 19.68 � 0.31 23.48 �Medium-sized neurons 12.14 � 1.14 12.52 �Small neuron 28.62 � 0.78 32.02 �

* P < 0.05, compared with non-electro-acupunctured side.** P > 0.05, compared with non-electro-acupunctured side.

*** P < 0.01, compared with non-electro-acupunctured side.

The amplification parameters for PCR were an initial 5 min denaturation

step followed by cycles consisting of denaturation at 94 8C for 50 s, annealing at

the specified optimum temperature for 50 s, and extension at 72 8C for 50 s. The

specified optimum total number of cycles was followed by a 10 min extension at

72 8C. 10 ml PCR products were electrophoresed through an ethidium bromide

estained 1% agarose gel, and the band intensity was analyzed using quantitative

scanning densitometry. The ratio of NGF, BDNF and NT-3 to b-actin served as

the levels of mRNA expressions.

2.8. Statistical analysis

Student’s t-tests were also applied bilaterally for comparison. A P value is

considered significant if it is less than 0.05. The 95% confidence interval (CI)

was given when P is less than 0.05. All analyses were performed using the SPSS

software.

3. Results

3.1. Immunoreactive expression of NGF, BDNF and NT-3

and their respective mRNA in L6 DRG neurons

The diameters of the immunopositive neurons in L6 DRG

were measured with a line drawn through the nucleus. The

method of measurement and classification followed the scheme

of a previous report, which showed the L6 DRG neurons of adult

cat consist of small (<42 mm), medium-sized (42–57 mm) and

large (>57 mm) neurons (Wang et al., 2000). In this experiment,

the immunoreactive products of NGF, BDNF, NT-3 appeared as a

brownish deposit in the cytoplasm of the stained DRG neurons,

and those of the genes, as a blue deposit in the cytoplasm.

3.1.1. Immunoreactive expression of protein and mRNA for

NGF in L6 DRG

The numbers of NGF immunopositive small and large

neurons both protein and mRNA in the spared DRG on the

acupunctured side (Fig. 2B and D) increased more than those on

BDNF mRNA positive neurons

e Non-EA side EA side

0.57** 5.01 � 1.06 4.50 � 0.90**

0.55* 14.46 � 0.75 19.23 � 0.75*

1.49*** 17.08 � 1.01 20.36 � 1.34***

NT3 mRNA positive neurons

e Non-EA side EA side

1.85* 12.82 � 1.26 17.38 � 1.54*

1.37** 6.12 � 0.58 6.18 � 0.68**

1.59*** 26.78 � 1.02 31.16 � 1.25***

Fig. 2. Expression of NGF (left column), BDNF (middle column) and NT-3 (right column) (A, B, E, F, I, J, Hematoxylin counterstained in A and B) immunoreactivity

in the spared L6 DRG and their respective mRNA (C, D, G, H, K, L) in spared DRG. Symbol I was denoted non-electro-acupunctured side and symbol II was denoted

electro-acupunctured side. Magnification: 40� (A, B, C, D); 200� (E, F, I, J); 400� (G, H, K, L). Positive neurons were indicated by symbol (!).

J. Chen et al. / Neuroscience Research 59 (2007) 399–405402

the non-acupunctured side (Fig. 2A and C) (P < 0.05), while

there was no significant difference (P > 0.05) in medium-sized

neurons (Table 1).

3.1.2. Immunopositive expression of protein and mRNA for

BDNF in L6 DRG

The numbers of BDNF immunopositive small and medium-

sized neurons both protein and mRNA in the spared DRG on the

acupunctured side (Fig. 2F and H) increased more than those on

the non-acupunctured side (Fig. 2E and G) (P < 0.05), when

there was no significant difference (P > 0.05) in large neurons

(Table 2).

3.1.3. Immunopositive expression of protein and mRNA for

NT-3 in L6 DRG

The numbers of NT-3 immunopositive small and large

neurons both protein and mRNA in the spared DRG on the

acupunctured side (Fig. 2J and L) increased more than those on

the non-acupunctured side (Fig. 2I and K) (P < 0.05).

However, there was no significant difference (P > 0.05) in

the numbers of NT-3 protein and NT-3 mRNA immunopositive

medium-sized neurons in the spared DRG (Table 3).

3.2. Quantitative changes in mRNA level for NGF, BDNF

and NT-3

The reverse transcription-polymerase chain reaction (RT-

PCR) was performed to detect the mRNA expression patterns in

both cells and small quantities of tissue. Under rational PCR

conditions, at least one specifically amplified band in each

sample clearly identified NGF, BDNF and NT-3 (Fig. 3).

In the L6 DRG, NGF, BDNF and NT-3 mRNAwere detected

in two groups, and EA did not influence three growth factor

expressions between acupunctured side and non-acupunctured

side in intact cats. However, following partial deafferentation

combined with acupuncture at two pairs of points that are

located within the innervating area of L6 spinal nerve on the

one side for 7 days, all expressions of mRNA for NGF, BDNF

and NT-3 on the acupunctured-side was significantly up-

regulated (Table 4).

Fig. 3. Quantitative changes in mRNA level for NGF, BDNF and NT-3 in the L6

DRG of non-electro-acupunctured side (line 1 and 3) and electro-acupunctured

side (line 2 and 4) in two Groups rats. RT-PCR products using primers for b-

actin were used as control.

Table 4

Comparison of quantitative changes in mRNA level for NGF, BDNF and NT-3

mRNA expressions Group I Group II

Non-EA side EA side Non-EA side EA side

NGF 0.54 � 0.17 0.63 � 0.16* 0.89 � 0.22** 1.37 � 0.13***

BDNF 0.66 � 0.13 0.69 � 0.11* 0.97 � 0.14** 1.44 � 0.16***

NT-3 0.84 � 0.12 0.91 � 0.17* 0.93 � 0.18* 1.57 � 0.22***

* P > 0.05, compared with non-electro-acupunctured side in Group I.** P < 0.05, compared with non-electro-acupunctured side in Group I.

*** P < 0.05, compared with non-electro-acupunctured side in Group II.

J. Chen et al. / Neuroscience Research 59 (2007) 399–405 403

4. Discussion

Previous study showed that acupuncture treatment could

obviously control the descending of spinal cord blood flow

(SCBF) in rats injured spinal cord. This suggested that

acupuncture could effectly control the descending of SCBF in

the early stage of spinal injured, improved the microcircula-

tion of spinal, abated and delayed the occurrence of

secondary lesion, promoted the recovery of function of

nerve (Wu et al., 1995). EA therapy is usually done by the

insertion of thin metal needles to the acupoints, and this is

followed by electrical stimulated action. It has been reported

that more anti-neurofilament (NF) positive labelings have

been found in the EA treatment group than seen in the control

group on the seventh day after the injury. The results

suggested that EA could be useful to the injured spinal cord

repair (Jin and Tao, 1997). In addition, functional improve-

ment was also found in rats which had been treated with

acupuncture 15 min after injury relative to those that received

no acupuncture treatment. This was accompanied by

minimization of post-traumatic cord shrinkage in acupunc-

ture-treated animals. Results point to a usefulness of

acupuncture as adjunct treatment during early stages after

spinal cord injury (Politis and Korchinski, 1990).

The neuron locating in the dorsal root ganglia consists of the

nerve cell body and the axon, a long protrusion extending into

the periphery. The length of the axon may be 10,000 to 15,000

times the diameter of the cell body. Previous study in our lab

(Wang et al., 2005a,b) had detected the average neurite length

of the normal or intact group was shorter than that of the spared

DRG group, and the spared DRG group’s was shorter than the

EA group’s at the seventh day in vitro (data are not shown). This

indicated that DRG had plasticity and EA probably promoted

the plasticity.

The neurons of DRG, with their axons projecting to the

spinal cord, provide a good approach to study the phenomenon

on the collateral sprouting deprived from sensory neurons in

spinal cord. In this study, the immunoreactive products both

protein and mRNA for NGF increased in small and large

neurons in the spared L6 ganglion after EA, this demonstrates

NGF expression has been upregulated following EA. NGF, as a

phenotype growth factor, shows not only a positive effect on the

survival of neurons, but also promotes the differentiation of

neurons as well as regulates their connection patterns (Jiang and

Smith, 1993; Orike et al., 2001). Zhang observed that NGF

increase in the spinal cord is relevant to spinal cord self-

recovery in the rat (Zhang et al., 1995). As the central processes

of small and large neurons project mainly to spinal Lamina II

and nucleus dorsalis (ND), we postulate that the increased NGF

expression in small and large neurons may be available for the

NGF transportation from DRG to ND or spinal lamina II. It

might also provide suitable conditions for some morphological

changes such as axonal sprouting and neurite-outgrowth of

DRG neurons expressing endogenous NGF.

BDNF protein is widely distributed in the CNS, beginning

early in development and extending throughout the organism’s

life span. Previous study reported that BDNF expressed in a

subpopulation of small and medium-sized neurons in the L6

DRG of normal cats. This study showed that the number of

small and medium-sized neurons for BDNF protein and mRNA

in spared DRG increased than seen in the non-acupunctured

side. The increase could clearly be attributed to the EA

procedure. BDNF can directly induce pluripotential neural

crest cells to differentiate along the sensory neuron lineage in

cultured chick neural crest cells (Sieber-Blum, 1991; Sieber-

Blum et al., 1993). Some studies also reported that endogenous

BDNF is required for peripheral nerve regeneration and

J. Chen et al. / Neuroscience Research 59 (2007) 399–405404

remyelination after injury (Zhang et al., 2000). Other results

clearly showed that BDNF, synthesized and secreted from the

DRG, is involved in the sympathetic sprouting in the DRG

following peripheral nerve injury as well (Deng et al., 2000).

The up-regulation of BDNF protein and mRNA in small and

medium-sized DRG neurons might be also useful to furnish

more BDNF to lamina II, based on the axon projection in spinal

Lamina II derived from small and medium sized neuron. BDNF

can be transported from DRG to lamina II (Zhou and Rush,

1996), indicating a possible role for BDNF in synaptic

plasticity (Wolpaw and Tennissen, 2001). In this study, we

postulate that EA promoting the spinal cord plasticity may be

linked to the up-regulation of BDNF and its transportation.

NT-3 and their mRNA in DRG were present mainly in some

small and large DRG neurons in normal cats. After adjacent

DRG removal, immunoreactive numbers for NT-3 in the spared

DRG significantly increased on the EA side, suggesting that EA

might be responsible for the up-regulation of endogenous NT-3

in small and large DRG neurons. As it is well known that small

DRG neurons project mainly to spinal lamina II whereas large

ones mainly to the nucleus dorsalis (ND), as well as nucleus

gracilis and nucleus cuneatus and NT-3 could be transported in

an anterograde direction, it is expected that more NT-3 would

be transported to these regions when there is more of it in the

DRG neurons. The earliest requirement for NT-3 has been

reported in cultured chick neural crest cells, where NT-3

functions as a mitogenic factor (Kalcheim et al., 1992; Pinco

et al., 1993). The increased amount of NT-3 derived from the

spared DRG might contribute towards sprouting within the

spinal cord as NT-3 injected into the spinal cord increases the

regenerative sprouting of the transected corticospinal tract in

adult rats (Schnell et al., 1994). NT-3-enhanced axonal

regeneration could also exert a beneficial effect on the motor

target organ (Sterne et al., 1997). These reports showed that NT-

3 might play a role in neural plasticity. The present results

indicated that the endogenous NT-3 derived from the spared

DRG might be related to the spinal cord plasticity after EA.

Interestingly, mRNA expressions for NGF, BDNF and NT-3

were not present apparently change on the two sides of L6 DRG

in normal cats, while statistically significant accumulation on

the acupunctured-side in Group I. This suggests that EA

exerting the treatment effect may be only linked to the injury

condition in our observation. In animal experiments, low-

magnitude extraneural compression was noted to decrease

intraneural microvascular flow, impair axonal transport, and

alter nerve structure and function (Rydevik et al., 1981). At

pressures rise to higher grade, all intraneural blood flow ceased.

Moreover, high-magnitude extraneural compression inhibited

retrograde axonal transport (Dahlin and McLean, 1986). That’s

to say, cell nutrition and the intraneurnal communication

system are compromised at elevated extraneural pressures. The

cat has seven lumbar vertebrae as compared with five in

humans. The spinal lumbar enlargement in humans is located at

T11-L1 and in cats at L4-L6. In the present study, the DRG of

cats in Group I associated with L1-L5 and L7-S2 spinal nerves

were first bilaterally removed, then followed immediately with

electrical-acupuncture on the left side at acupuncture points,

which are known to lie in L6 dermatome. More expressions for

NGF, BDNF and NT-3 mRNAwere found in the EA side than in

the control side with a significant difference on the seventh day

after the injury, indicating the expressions of the three

neurotrophic factors inducing by EA have been triggered.

Although it is well-established that EA could promote

morphological and functional plasticity in the spinal cord, the

mechanism is not fully understood. The present study proposed

that EA could induce an up-regulation of NGF, BDNF and NT-3

in the L6 DRG, after removal of the adjacent DRG and this

could be the factors for the axonal sprouting of L6 DRG

neurons.

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