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, China b 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 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). 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). www.elsevier.com/locate/neures Neuroscience Research 59 (2007) 399–405 * 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: [email protected](T.-H. Wang). 0168-0102/$ – see front matter # 2007 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2007.08.006
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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.
* 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