Extract
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Nerve fibers in the peripheral nervous system (PNS),
unlike those in the central nervous system (CNS), regrow
toward their original target after injury, and are capable of
functional regeneration[1]. This difference in regeneration
capability between PNS and CNS is attributed to both
intrinsic neuronal determinants and extrinsic environmental
factors. For instance, axonal growth-associated protein
GAP-43 is highly induced by peripheral nerve injury, but not by
axon tract injuries in the spinal
cord[2]. Overexpression of GAP-43 and cytoskeleton-associated protein CAP-23 in
transgenic mice was able to induce the regeneration of
injured spinal cord axons, implying that the supply of
peripheral regeneration factors can similarly induce axonal
regeneration after spinal cord
injury[3]. In addition to the intrinsic
factors, differences in the nature of the molecules in the
surrounding environments of CNS and PNS are critical for
determining the responsiveness of injured axons. Increased
propagation of Schwann cells and activated macrophages
in an area of injured peripheral nerve is important for
promoting axonal regrowth, whereas glial scar tissues containing
chondroitin sulfate proteoglycan, EphB3 and semaphorin 3A
peptidoglycan sulfate, and induced myelin components in
the oligodendrocytes such as Nogo and MAG, inhibit
spinal cord regeneration[4-6].
Although injured peripheral axons have a clear
advantage with respect to regenerative capacity compared with
CNS axons, peripheral nerve repair leading to functional
recovery is seldom perfect. After axotomy, regrowing axons
need to be correctly guided into the growth environment of
the distal nerve stump, which is primarily provided by
activated Schwann cells. Furthermore, targeting is usually
far from perfect, especially if the injury requires regeneration
over long distances. Al-Majed et al have reported that the
correct targeting of sensory and motor neurons after femoral
nerve transection was much improved by electrical
stimulation[7]. Thus, the whole process, including the removal of
nerve debris following Wallerian degeneration, formation of
whole basal lamina/Schwann cell tubes (called the band of
Büngner), and growth cone elongation are equally
important for successful functional recovery.
Most studies on axonal regeneration have been
primarily focused on axonal regrowth and elucidating molecular
factors, and a majority of molecular factors so far reported
are endogenous molecules. Despite the many possibilities
regarding the roles of exogenous molecular factors in axonal
regeneration, little research has been carried out in this field.
Oriental medicinal drugs have a broad spectrum of clinical
uses, including for the cure of cardiovascular and nervous
system diseases, and some of them have been found to have
specific effects on brain diseases such as stroke and
diabetic neuropathy[8,9]. A water extract of Hominis Placenta
(HP) has been used in oriental medicine for the treatment of
diseases of the brain, kidney, and other organs, and to
supplement "vital
essences"[10]. Here, we report that HP had a
growth-promoting activity for injured sciatic axons
in vivo and in cultured dorsal root ganglia (DRG) sensory
neurons.
Materials and methods
Drug preparation Dried human placenta ( HP) was
obtained from Daejeon University Oriental Medicine Hospital
(Daejeon, Korea). HP is approved for in
vivo injection by the Korean Food and Drug Administration (KFDA), and HP
extract was prepared as described
elsewhere[21]. Briefly, dried HP was suspended in 2 L of water, heat-extracted for 3 h, and
filtered 3 times. The filtered fluid was distilled using a rotary
vacuum evaporator. Concentrated solutions were frozen at
-70 oC for 4 h, and freeze-dried for 24 h. The product was kept
at 4 oC, and dissolved in water. The stock solution was
stored at -20 oC and diluted with a physiological saline
solution before use.
Sciatic nerve surgery Sprague-Dawley rats (8 weeks
old, male) were housed individually in cages in a
temperature-controlled room with a 12-h light and dark cycle. A total
of 56 rats were used in the present experiment. Animals were
anesthetized by intraperitoneally (ip) injecting a mixture of
ketamine (80 mg/kg) and xylazine (5 mg/kg), and the sciatic
nerve was exposed and crushed with a pair of forceps held
tightly for 30 s twice at 1 min
interval[11]. For drug admini-stration, the Hamilton microsyringe (600 series, gauge 22s;
Hamilton, USA) attached to a microinjection apparatus
(Stoelting, USA) was placed onto the injury site immediately
after the sciatic nerve was crushed, and 5 mL of HP solution
or an equivalent volume of saline was injected slowly for 5
min. The dose-dependent effects of HP on axonal
regeneration were examined by injecting 5 mL of HP solutions of
different concentrations (2, 10, and 40 mg/mL), corresponding
to dosages of 50, 250, and 1000 mg/kg for rats with a body
weight of 200 g, respectively. Animals recovered from
anesthesia and were killed 7 d later. Before being killed, animals
were deeply anesthetized with a mixture of ketamine and
xylazine, and the sciatic nerves were dissected, immediately
frozen, and kept at -75 oC until use. For the purposes of
immunofluorescence staining, the sciatic nerve was prepared
by dividing it into the proximal stump (a 5-mm segment
proximal to the injury site) and the distal stump (5 mm segment
distal to the injury site).
Histology and immunofluorescence stainingNerve segments were embedded into embedding media (Histo Prep,
Fisher Scientific, USA) and frozen at -20
oC. Longitudinal or transverse sections (20
mm thickness) were cut on a cryostat and mounted on glass slides that electrostatically
attract frozen and formalin-fixed tissue sections (Superfrost
Plus, Fisher Scientific). For double immunofluorescence
staining, sections were fixed with 4% paraformaldehyde and
4% sucrose in phosphate-buffered saline (PBS) at room
temperature for 40 min, permeabilized with 0.5 % nonidet P-40 in
PBS, and blocked with 2.5% horse serum and 2.5% bovine
serum albumin for 4 h at room temperature. Sections were
incubated with anti-neurofilament-200 antibody (NF-200,
clone no. N52, mouse monoclonal, diluted 1:200; Sigma,
USA), anti-GAP-43 antibody (H-100, rabbit polyclonal,
diluted 1:200; Santa Cruz Biotech, USA), anti-bIII-tubulin
antibody (TUJ1, rabbit polyclonal, diluted 1:200; Covance,
USA), anti-S100b antibody (rabbit polyclonal, diluted 1:200; Dako,
Denmark), anti-Cdc2 antibody (p34, mouse monoclonal,
diluted 1:200; Santa Cruz Biotech), then incubated with
fluorescein-goat anti-mouse (diluted 1:400; Molecular Probes,
USA) or rhodamine-goat anti-rabbit secondary antibodies
(diluted 1:200, Molecular Probes) in 2.5% horse serum and
2.5% bovine serum albumin for 1 h at room temperature and
coverslipped with gelatin mount medium. We always
included the control sections treated with secondary antibody
alone, which usually did not have any visible images. In
cases when the nonspecific signals were high, no data from
those experiments were analyzed further. Sections were
viewed with a Nikon fluorescence microscope and the
images were captured using a Nikon digital camera (Nikon). To
minimize experimental variation, sections from different
animals in an experiment were always treated with the same
solutions throughout the whole immunostaining process,
and the images for a given experiment were captured after
the same exposure time using Nikon ACT-1 software. Adobe
Photoshop (version 5.5) was used to process the images.
For all the sections from the individual experiments, the
merged images were produced by the layer blending mode
options of the Photoshop program.
Western blot analysisNerve segments were washed
with ice-cold PBS, and sonicated under 50-200 mL of triton
lysis buffer [20 mmol/L Tris, pH 7.4, 137 mmol/L NaCl, 25
mmol/L b-glycerophosphate, pH 7.14, 2 mmol/L sodium
pyrophosphate, 2 mmol/L ethylenediamine tetraacetic acid
(EDTA), 1 mmol/L Na3VO4, 1% Triton X-100, 10% glycerol, 5
mg/mL leupeptin, 5 mg/mL aprotinin, 3 mmol/L benzamidine,
0.5 mmol/L DTT, 1 mmol/L PMSF]. Ten micrograms of
proteins were used for Western blotting analysis using anti-
GAP-43 antibody (H-100, rabbit polyclonal, Santa Cruz
Biotech) or anti-Cdc2 antibody (p34, mouse monoclonal,
Santa Cruz Biotech). Electrophoresis and western blotting
were performed as described
previously[11]. Primary and secondary antibodies were diluted to 1:100 and 1:10 000,
respectively, and used as recommended by the manufacturers.
To confirm the immunoreaction specificity of the antibodies
to GAP-43 and Cdc2, a control experiment was performed by
supplying an excess of purified GAP-43 and Cdc2 proteins
(Calbiochem, USA) as antigens, using the protocol provided
by the manufacturer (Acris Antibodies, Germany). Briefly,
2 mg of antibody was mixed with 20 mg of antigen protein in
100 mL of PBS at 37 oC for 2 h, and centrifuged for 15 min at
4 oC at 10 000 r/min. Then, 50 mL of the bottom phase
containing immune complexes in the tube was taken and mixed
with 0.1% triton X-100 in PBS for western blot analysis.
Quantitative analysis of protein levels in the autoradiographic
images was determined by using the i-Solution software
package (Image and Microscope Technology, USA).
Retrograde tracing of motor neurons in the spinal
cordThe sciatic nerves of rats anesthetized with ketamine and
xylazine were exposed, and DiI (5 mL of a 3% solution in
dimethylsulfoxide; Molecular Probes) was applied to the
portion of the sciatic nerve 10 mm distal to the injury site by
using a microsyringe. The incision was sutured, and the
animals were returned to their cages after recovering from
the anesthesia. Forty eight hours later, animals were killed
and all the sections collected (20 mm thickness) were used to
count diI-labeled motor neurons observed at the T11-12
levels. The mean number of total labeled cells in individual
animals was compared among groups by using Student¡¯s
t-test. Cell counting analysis was conducted with the
examiner blinded to the experimental treatment conditions.
Primary DRG sensory neuron culture Glass coverslips
were precoated with a mixture of poly-L-ornithine (0.1
mg/mL; Sigma) and laminin (0.02 mg/mL; Collaborative Research,
USA) in a 37 oC, 5% CO2 incubator. L4 and L5 DRG were
removed from adult male rats, and placed in ice-cold
Dulbecco¡¯s modified Eagle¡¯s medium (DMEM; Gibco, USA).
The ganglia were treated with DMEM containing type XI
collagenase (2500 U/mL; Sigma) for 90 min at 37
oC. Tissues were then washed with DMEM medium and centrifuged at
800 r/min for 1 min to remove the supernatant. After one
more wash, cells were suspended in DMEM, dissociated
gently with 16-20 passages through a flamed Pasteur pipette,
and centrifuged at 800 r/min for 1 min to remove the
supernatant. Cells were then treated with DMEM
containing type SII trypsin (0.5 mg/mL) for 10 min followed by
DMEM containing trypsin inhibitor (100 mg/mL), EDTA (1
mmol/L) and DNase I (80 mg/mL) for 5 min. After cells were
washed with culture medium [DMEM containing 5%
heat-inactivated fetal bovine serum (FBS; Gibco), 5% horse serum,
2 mmol/L glutamine and 1% penicillin-streptomycin],
800-1200 neurons were plated onto 12 mm round coverslips and
cultured for 12 h in a 37 oC, 5%
CO2 incubator with fresh culture medium. DRG neurons were treated with HP (50 mg/mL or 200 mg/mL) or saline vehicle and cultured for 24-48
h. Cells grown on the coverslips were fixed with 4%
paraformaldehyde/4% sucrose solution for 45 min at room
temperature and used for immunofluorescence staining. Images of
immunostained cells were captured on a digital camera, and
neurite arborization and length were quantitatively assessed
by using the i-Solution software package (Image and
Microscope Technology).
Results
Effect of HP treatment on GAP-43 protein levels in
regenerating sciatic nerves Crush injury induced moderate
levels of GAP-43 signaling in both the proximal and distal
stumps compared with intact animals (Figure 1A).
Treatment with HP at concentrations of 50 mg/kg, 250
mg/kg and 1 mg/kg enhanced GAP-43 protein signaling in both the
proximal and distal segments of the injured sciatic nerves in a
dose-dependent manner (Figure 1A, 1B); that is, GAP-43
signals in the injured sciatic nerve with a dosage of 250
mg/kg of HP were stronger than those in nerves treated with 50
mg/kg HP, and reached similar levels as those achieved in nerves
treated with 1 mg/kg. Thus, we used an HP dose of 250
mg/kg for the remaining in vivo experiment in the present study. HP
administration (250 mg/kg) into the intact nerve did not
induce any GAP-43 protein (Figure 1A). Immunostaining of
the same nerve sections with the neurofilament protein
NF-200, a neuronal marker, revealed that signals for the GAP-43
protein mostly overlapped with those for NF-200 (shown in
yellow in the merged image; Figure 1C).
To determine the GAP-43 expression levels in crushed
nerves after HP treatment, proteins in the sciatic nerves were
analyzed by Western blotting. GAP-43 protein was induced
in the injured sciatic nerves, particularly in the proximal stump
(Figure 2A, 2B). HP treatment further increased the amount
of GAP-43 protein in the distal as well as the proximal stump
of the sciatic nerves. Western blotting analysis of nerve
tissues after preincubation of the anti-GAP-43 antibody with
excess GAP-43 protein completely eliminated GAP-43 signals,
indicating the reaction specificity between the anti-GAP-43
antibody and the protein samples (Figure 2C).
Upregulation of Cdc2 protein levels in the injured sciatic nerve caused by HP treatment We recently found that
cell division cycle 2 (Cdc2) kinase, a key regulatory protein
for the progression from G2 to M phase in the cell
cycle[12], is strongly induced in injured
nerves[11,13], and is required for axonal regeneration (unpublished data). In the present study,
we examined whether HP regulates Cdc2 protein levels in the
regenerating sciatic nerve. Cdc2 protein was not detected in
the uninjured sciatic nerve, but there was a strong induction
in the distal, but not in the proximal, stump of the nerves 7 d
after injury (saline control in Figure 3A, 3B). HP treatment
further increased the levels of Cdc2 protein in the distal stump
of the injured sciatic nerve. Western blotting analysis of
nerve tissues after preincubation of anti-Cdc2 antibody with
excess Cdc2 protein completely inhibited Cdc2 signals,
indicating the reaction specificity between the anti-Cdc2
antibody and protein samples (Figure 3C).
Immunofluorescence staining also indicated an increased
Cdc2 protein signal in the injured nerves with HP treatment
(Figure 4A). To localize induced Cdc2 protein signals in the
nerve tissues, double immunofluorescence staining was
performed using transverse sections of the sciatic nerve 3 mm
distal to the injury site. S100b is the myelin protein that is
selectively expressed in Schwann cells. Induced Cdc2 signal
in the injured sciatic nerve with HP treatment mostly
overlapped with the S100b signal, but not the axon-specific
bIII-tubulin signals (Figure 4B). Next, we examined the cell
numbers in the areas of the nerves undergoing axonal
regenera-tion. The non-neuronal cell population in the distal stump
was visualized by nuclear staining of longitudinal nerve
sections with the Hoechst 33258 dye. Nuclear counts were
increased in the injured nerves relative to the uninjured
control group, and further increases were observed with HP
treatment in the corresponding area of the sciatic nerve (Figure
4C). These data, along with increased Cdc2 protein
expression, suggest that HP treatment causes increased
Schwann cell proliferation in injured sciatic nerves.
Improved axonal regeneration caused by HP treatment
In an animal group with no injury, injection of diI into the
nerve labeled motor neuron cell bodies (Figure 5A). In a
group with sciatic nerve injury that was injected with saline,
the number of diI-labeled motor neurons was much less
compared with the control group, suggesting that the majority of
injured nerve fibers did not regenerate to a position 10 mm
distal to the injury site by d 7 post crush. In the injured
nerves treated with HP, the number of diI-labeled neurons
was enhanced, reaching similar levels as in the control. To
confirm the microscopic observations, all diI-labeled cells
from individual sections were summed, and a numerical
comparison was made. As shown in Figure 5B, the number of
diI-labeled cells in the HP-treated injured neurons was
significantly greater relative to the number in the saline-treated
injury group. These data suggest that HP is effective in
promoting sciatic nerve fiber regeneration after crush injury.
Enhanced neurite outgrowth of DRG sensory neurons
caused by HP treatment Neurite outgrowth in the group
with sciatic nerve injury (designated the "preconditioned"
group) involved elongation and arborization to a much greater
extent compared with the non-injury control, indicating that
the lesion signals in response to nerve crush induce neurite
outgrowth of DRG sensory neurons in culture (data not
shown). In order to examine the effect of HP on axonal
regeneration of DRG sensory neurons in terms of neurite
outgrowth, cells were exposed to HP for 24 h. Neurons treated
with 50 mg/mL HP had significantly longer neurite outgrowth
processes and branches compared with the saline-treated
group (Figure 6A-C). When HP concentration was increased
to 200 mg/mL, there were further increase in neurite
out-growth.
Discussion
The present study provides compelling evidence that
HP is a biologically active drug that promotes the recovery
of impaired nerves. Local administration of HP upregulated
GAP-43 and Cdc2 protein levels in regenerating nerves.
Observations of regenerating axons in vivo using retrograde
tracers and of in vitro regeneration potential using
preconditioned DRG sensory neurons indicated that there was
enhanced axonal re-growth in HP-treated nerves compared with
non-treated controls.
Axonal growth-associated protein GAP-43 is selectively
expressed in the neural system and is highly localized in the
axons. Previous studies have shown that increased
synthesis of GAP-43 protein is closely linked with axonal
regeneration processes as an intrinsic determinant for axonal
elongation in both peripheral and spinal cord
axons[3,14]. In the current study, we screened several different drugs used in
oriental medicine, all of which are used clinically for the
treatment of nervous system diseases. Of all drugs examined, the
induction levels of GAP-43 protein were highest in the
sciatic nerves treated with HP at the injury site (data not shown).
GAP-43 protein induced by HP treatment of the sciatic nerve
mostly colocalized with axon-specific protein NF-200 signals,
and was strongly induced in the distal stump where active
axonal re-growth occurs following Wallerian
degeneration[1,4].
Our data also indicate strong induction of the Cdc2
protein in the distal stump of the regenerating sciatic nerves
with HP treatment. Cdc2 protein, a prototypical cyclin-dependent kinase, regulates the mitotic phase of the cell
cycle[15]. Recent studies have also demonstrated the
involvement of Cdc2 in cell migration
processes[16] and in the process of neuronal apoptosis via the activation of proapoptotic
protein Bad[17]. We have recently found strong induction of
the Cdc2 protein in regenerating nerves such as facial or
sciatic nerves[11,13]. The induced Cdc2 protein signals mostly
overlapped with the S100b protein signals, indicating
predominant Cdc2 expression in peripheral non-neuronal cells
such as Schwann cells, although the possible induction and
role of Cdc2 protein in the regenerating axon itself cannot be
completely ruled out. Interestingly, HP-treated injured nerves
had increased numbers of Hoechst-stained nuclei in the
injured sciatic nerves. Because Schwann cells are important
for guiding growing axons toward their target muscles or
sensory organs, it is possible that HP might act as a positive
regulator for the growth-promoting actions of Schwann cells
via increased Cdc2 activity. Recent characterization of Cdc2
substrates in vivo has suggested diverse molecular
interactions for cell-cycle regulation and other cell
functions[18]. Experiments using a Cdc2 kinase inhibitor to regulate Schwann
cell proliferation would be informative regarding the role of
the Cdc2 protein in the axonal regeneration process.
Our data further demonstrate that HP is involved in
axonal regeneration after injury. Measurement of regenerating
motor neuron axons in vivo by using a retrograde tracer
showed increased numbers of regenerating motor neurons
in the spinal cord in animals treated with HP. We also
examined the effects of HP treatment on the neurite outgrowth of
cultured DRG sensory neurons. DRG sensory neurons are
useful for studying regeneration in vitro because of their
anatomical structure, which extends to both the peripheral
and central nervous systems[19]. DRG sensory neurons have
increased neurite outgrowth potential when sciatic nerve
injury is inflicted a week before DRG
culture[20]. The addition of HP at concentrations of 50-200
mg/mL induced increased neurite outgrowth of DRG sensory neurons,
suggesting that HP has a growth-promoting activity in
regenerating DRG sensory axons.
Although HP has been used for a long period of time in
oriental medicine for the cure of physiological abnormalities
in human organs, the explanations of its effects have tended
to be descriptive rather than quantitative. HP treatment has
been reported to have an alleviating effect on arthritic
symptoms in a rat model when treatment was accompanied by
acupuncture therapy[21]. HP treatment has also been reported
to induce hematopoiesis[22]. HP preparation in clinical
oriental medicine involves several processes, including drying,
boiling, and freeze-drying human placenta, and thus the major
biologically active proteins or other macromolecules in the
tissues would be degraded or inactivated, and small
molecules such as estradiol, progesterone, and several
monosaccharides and amino acids would remain as the active
components[10]. According to the present data, HP exerted an
axonal growth promotion effect at multiple levels, including
induction of Cdc2 and GAP-43 proteins, sciatic nerve
elongation, and neurite outgrowth of DRG sensory neurons,
thus it is likely that more than one chemical component of
HP might act on the axonal regeneration processes.
Despite the growing body of evidence regarding the role
of diverse molecular factors in axonal regeneration in both
the PNS and CNS, there are only a few reports on the effects
of herbal drugs on axonal regeneration. Injection of
a buyang huanwu decoction (Radix hedysari) into injured sciatic
nerves has been reported to promote axonal
regeneration[23,24]. Ginsenoside Rb1 has also been shown to be
effective for peripheral nerve regeneration and for the survival of injured
spinal cord neurons[25,26]. Administration of herbal mixtures
is reported to enhance the survival and regeneration of
axoto-mized retinal ganglion cells[27]. Although these studies
suggest that herbal drugs may be potentially useful for axonal
regeneration, the molecular mechanisms underlying their
actions remain to be investigated. An initial step toward this
goal would be to identify the major chemical components in
the herbal drugs and investigate their effects at the
molecular level. In this respect, our findings regarding the
induction of GAP-43 and Cdc2 protein levels by HP treatment in
regenerating nerves are significant because the study
provides important parameters for assessing regeneration in a
quantitative way. Future studies to characterize and
examine the chemical components of HP would be useful to
elucidate molecular mechanisms by which it exerts its effects, and
to develop drug therapies for functional regeneration.
References
1 Fawcett JW, Keynes RJ. Peripheral nerve regeneration. Annu
Rev Neurosci 1990; 13: 43-60.
2 Schreyer DJ, Skene JH. Injury-associated induction of GAP-43
expression displays axon branch specificity in rat dorsal root
ganglion neurons. J Neurobiol 1993; 24: 959-70.
3 Bomze HM, Bulsara KR, Iskandar BJ, Caroni P, Skene JH. Spinal
axon regeneration evoked by replacing two growth cone
proteins in adult neurons. Nat Neurosci 2001; 4: 38-43.
4 Ide C. Peripheral nerve regeneration. Neurosci Res 1996; 25:
101-21.
5 Behar O, Mizuno K, Neumann S, Woolf CJ. Putting the spinal
cord together again. Neuron 2000; 26: 291-3.
6 Davies SJ, Fitch MT, Memberg SP, Hall AK, Raisman G. Silver J.
Regeneration of adult axons in white matter tracts of the central
nervous system. Nature 1997; 390: 680-3.
7 Al-Majed AA, Neumann CM, Brushart TM, Gordon T. Brief
electrical stimulation promotes the speed and accuracy of motor
axonal regeneration. J Neurosci 2000; 20: 2602-8.
8 Gong X, Sucher NJ. Stroke therapy in traditional Chinese
medicine (TCM): prospects for drug discovery and development. Trends
Pharmacol Sci 1999; 20: 191-6.
9 Heng X, Zhang F. Advances in treatment of diabetic neuropathy
by traditional Chinese medicine. J Tradit Chin Med 1998; 18:
146-52. Chinese.
10 Liu G. Chinese herbal medicine. Beijing: Hua Xia Medical
Publishing; 2001.
11 Namgung U, Choi BH, Park S, Lee JU, Seo HS, Suh BC,
et al. Activation of cyclin-dependent kinase 5 is involved in axonal
regeneration. Mol Cell Neurosci 2004; 25: 422-32.
12 Dorée M, Galas S. The cyclin-dependent protein kinases and the
control of cell division. FASEB J 1994; 8: 1114-21.
13 Seo TB, Han IS, You BG, Jeong IG, Yoon JW, Namgung U.
Induction of Cdc2 protein by sciatic nerve injury and up-regulation
by exercise in rat. Society for Neurosci Abstr 2004; 618.8.
14 Benowitz LI, Routtenberg A. GAP-43: an intrinsic determinant
of neuronal development and plasticity. Trends Neurosci 1997,
20: 84-91.
15 Pines J. Four-dimensional control of the cell cycle. Nat Cell
Biol 1999; 1: E73-9.
16 Manes T, Zheng DQ, Tognin S, Woodard AS, Marchisio PC,
Languino LR. Alpha(v)beta3 integrin expression up-regulates
cdc2, which modulates cell migration. J Cell Biol 2003; 161:
817-26.
17 Konishi Y, Lehtinen M, Donovan N, Bonni A. Cdc2
phosphorylation of BAD links the cell cycle to the cell death machinery.
Mol Cell 2002; 9: 1005-16.
18 Ubersax JA, Woodbury EL, Quang PN, Paraz M, Blethrow JD,
Shah K, et al. Targets of the cyclin-dependent kinase Cdk1.
Nature 2003; 425: 859-64.
19 Richardson PM, Issa VM. Peripheral injury enhances central
regeneration of primary sensory neurones. Nature 1984; 309:
791-3.
20 Smith DS, Skene JH. A transcription-dependent switch controls
competence of adult neurons for distinct modes of axon growth.
J Neurosci 1997; 17: 646-58.
21 Yeom MJ, Lee HC, Kim GH, Shim I, Lee HJ, Hahm DH.
Therapeutic effects of Hominis placenta injection into an acupuncture
point on the inflammatory responses in subchondral bone region
of adjuvant-induced polyarthritic rat. Biol Pharm Bull 2003; 26:
1472-7.
22 Son CG, Han SH, Cho JH, Shin JW, Cho CH, Lee YW,
et al. Induction of hemopoiesis by saenghyuldan, a mixture of Ginseng
radix, Paeoniae radix alba, and Hominis placenta extracts. Acta
Pharmacol Sin 2003; 24: 120-6.
23 Xu H, Jiang B, Zhang D, Fu Z, Zhang H. Compound injection of
radix Hedysari to promote peripheral nerve regeneration in rats.
Chin J Traumatol 2002; 5: 107-11.
24 Cheng YS, Cheng WC, Yao CH, Hsieh CL, Lin JG, Lai TY,
et al. Effects of buyang huanwu decoction on peripheral nerve
regeneration using silicone rubber chambers. Am J Chin Med 2001; 29:
423-32.
25 Chen YS, Wu CH, Yao CH, Chen CT. Ginsenoside Rb1 enhances
peripheral nerve regeneration across wide gaps in silicone rubber
chambers. Int J Artif Organs 2002; 25: 1103-8.
26 Liao B, Newmark H, Zhou R. Neuroprotective effects of ginseng
total saponin and ginsenosides Rb1 and Rg1 on spinal cord
neurons in vitro. Exp Neurol 2002; 173: 224-34.
27 Cheung ZH, So KF, Lu Q, Yip HK, Wu W, Shan JJ,
et al. Enhanced survival and regeneration of axotomized retinal ganglion
cells by a mixture of herbal extracts. J Neurotrauma 2002; 19:
369-78.
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