The Inuence of Brain Injury or Peripheral Nerve Injury on Calcitonin

Gene-Related Peptide Concentration Variation and

Fractures Healing Process
Dianying Zhang, Peixun Zhang, Yanhua Wang, Na Han, Chi Tang and
Baoguo Jiang
Department of Trauma Orthopedics, Peoples Hospital, Peking University, Beijing, China
Abstract: To investigate the changes of calcitonin gene-related peptide (CGRP) in rats blood plasma, spinal anterior motorneuron,
and dorsal root ganglion (DRG) after fractures combined with central or peripheral nerve injuries and its influence on fracture healing,
72 healthy adult SD rats (male or female) were divided into 4 groups (18 rats in each group): group A, simple(left) tibial fracture; group
B, left tibial fracture combined with left sciatic nerve injury; group C, left tibial fracture combined with T9-11 spinal cord transection
injury; group D, left tibial fracture combined with right cerebral cortex injury. Group A was the control group. The concentration of
serum CGRP was measured immediately, 1w, 2w, and 4w after injury using radio immunoassay. X-ray photograph was taken at 1w,
2w, and 4w after injury to assess fracture healing. The concentration of serum CGRP in spinal anterior motorneuron and dorsal root
ganglion was measured 1w, 2w, and 4w after injury. Bony callus at 2w after injury using H.E.staining was observed. 1w and 2w after
injury, the fracture line was still clear on the X-ray of all groups, but 4w after injury the fracture line disappeared with complete healing
except the peripheral nerve injury group. By H.E. staining, we found lesser bony callus contents in the peripheral nerve injury group
than the simple fracture group at 2w after injury; irregular bone trabecula and healing defect were found in the former group. While the
spinal injury group and cerebral cortex injury group represented more bony callus than the simple fracture group, increased bone
trabecula and regularity, medullary cavity occluded and finally solid bony connections were found. CGRP concentration in blood
plasma and spinal anterior motorneuron represented no apparent differences among all groups during each observation period. For the
dorsal root ganglion group, 1w after fracture, there was no apparent difference of CGRP concentration in the peripheral nerve injury
group and cerebral cortex group compared with the control group (P0.05), but the spinal injury group showed more CGRP than the
control group (PB0.01). 2w after injury, the peripheral nerve injury group and cerebral cortex group also showed no difference
compared with the control group, but the cerebral cortex group had more CGRP contents than the peripheral nerve injury group
(PB0.05), and the spinal injury group showed more CGRP than the control group (PB0.01). 4w after injury, the peripheral nerve
group, spinal injury group, and cerebral cortex injury group all showed higher concentration of CGRP than the control group. Among
the 3 groups, the spinal injury group is the highest (PB0.01). When fracture combined with peripheral nerve injury, the healing process
can be slowed down. In contrast, fracture combined with spinal injury and cerebral cortex injury will accelerate the healing process. The
CGRP in dorsal root ganglion in spinal injury group and cerebral cortex injury group increased, which may have positive effects on
fracture healing.
Keywords: Injury of nerve, fracture, calcitonin gene-related peptide
In our clinical work, we found that when fracture
combined with peripheral nerve injury, the healing
process can be slowed down. In contrast, fracture
combined with spinal injury and cerebral cortex injury
will accelerate the healing process. Our research was
aimed to investigate the changes of calcitonin gene-
related peptide (CGRP) in rats blood serum, spinal
anterior motorneuron, and dorsal root ganglion (DRG)
after fractures combined with central or peripheral nerve
injuries, and its influence on fracture healing.
This research project was funded by Chinese National Natural Science Fund for Outstanding Youth (30625036), Chinese 973
Project Planning (2005CB522604), Chinese National Natural Science Youth Fund (30801169), Beijing City Science & Technology
New Star Classification A-2008-10 and Chinese ministry of education for Doctor Position New Teacher (20070001780).
Address correspondence to Prof. Baoguo Jiang, Department of Trauma and Orthopedics, Peoples Hospital, Peking University,
Beijing 100044, China. E-mail: jianbaoguo@vip.sina.com
Artificial Cells, Blood Substitutes, and Biotechnology, 37: 8591
Copyright # 2009 Informa UK Ltd.
ISSN: 1073-1199 print / 1532-4184 online
DOI: 10.1080/10731190902743149
85
MATERIALS AND METHODS
Animal Models
Seventy-two healthy adult SD rats (male or female,
weight 220-250g) were divided into 4 groups according
to the operation methods, with 18 rats each group. All the
operations were allowed by the animal operation experi-
ment committee of Peking University.
Group A, simple(left) tibial fracture;
Group B, left tibial fracture combined with left
sciatic nerve injury;
Group C, left tibial fracture combined with T9-11
spinal cord transection injury;
Group D, left tibial fracture combined with right
cerebral cortex injury.
Group A was designed as control group.All the rats
were provided by the Science Animal Center of Peking
University Health Science Center.
SD rats were anaesthesized with 5% ketamine
hydrochloride (0.2.03 ml/100 g) by intraperitoneal
injection. We built the superior segment of the rat left
tibial fracture model, after anatomy reestablishment, and
fixed the bone with a Kirschner wire. Group B: left tibial
fracture combined with left sciatic nerve injury models
were constructed by cutting the left sciatic nerve at 10mm
above sciatic nerve crotch, turning over the distal end and
suturing it to the subcutaneous. Group C: we made a
median incision at the back of the rat, exposed the
thoracic vertebra, cut off and resected about 2mm spinal
cord at the 911 lever simultaneously, built the left tibial
fracture combined with T9-11 spinal cord transection
injury model. Group D: the right cerebral cortex injury
model was constructed by the Allen method; we built the
left tibial fracture combined with right cerebral cortex
injury model. The rats were raised in different cages
according to the grouping; we supplied the rats with
enough food and water, and gave a massage to the
bladder of the spinal cord injury rats every 6h after
operation.
Dection Items
X-ray photographs were taken at 1w, 2w, and 4w after
injury to assess the fracture healing. Bony callus at 2w
after injury using H.E staining was observed.
The concentration of serum CGRP was measured
immediately 1w, 2w, and 4w after injury using radio
immunoassay.
2 ml blood were drawn from the angular vein
immediately 1w, 2w, and 4w after injury. We put it into
a centrifuge tube within 30 ml EDTA (100 g/L)and 40 ml
aprotinin, 3000r/min centrifuge for 10 min, drew the clear
supernatant liquid 1ml, put it into a sample tube and
preserved it in 708C profound hypothermia environ-
ment.
We executed 6 rats in each group immediately and at
1w, 2w, and 4w after injury, cut for 1.5 cm T
12
S
1
ventro-spinal cord and 3 pairs of spinal cord dorsal root
ganglion, ground for a short while and put it into a sample
tube within 1ml HAC (1%), heated it to boil for 15 min,
bene tritum to get homogenate, then 3000r/min centrifuge
for 15 min. to draw the clear supernatant liquid. We put it
into a sample tube and preserved it in 708C profound
hypothermia environment, then detected the CGRP con-
centration using radioimmunity method (CGRP kit was
bought from Ferrer Biotechnology Companies in Beij-
ing).
Statistical Analysis
The data were analyzed by SPSS 11.0 and compared by
t-test method. A probability where pB0.05 was consid-
ered significant for all statistical comparison. All values
were presented as the mean9SD.
RESULTS
Observation According to the X-ray
The overview morphous of the fracture is shown in
Figure 1: 1w and 2w after injury, the fracture line was still
clear on the X-ray of all groups, but 4w after injury, the
fracture line disappeared with complete healing except
peripheral nerve injury group.
H.E. Staining of the Bony Callus
As Figure 2 shows, by H.E. staining, we found lesser
bony callus contents in the peripheral nerve injury group
than the simple fracture group at 2w after injury; irregular
bone trabecula and healing defect were found in the
former group. While the spinal injury group and cerebral
cortex injury group represented more bony callus than the
simple fracture group, increased bone trabecula and
regularity, medullary cavity occluded and finally, solid
bony connections were found.
The Variation of CGRP Concentration in Blood
Serum
As can be seen in Table 1, the CGRP concentration in
blood serum represented no apparent differences among
all groups during each observation period.
86 D. Zhang et al.
Figure 1. The X-ray to display the fracture healing conditions in 4 different groups in different time.
The Influence of Brain Injury or Peripheral Nerve Injury on CGRP Concentration Variation 87
The Variation of CGRP Concentration in Spinal
Anterior Motor Neuron
As shown in Table 2, CGRP concentration in spinal
anterior motorneuron represented no apparent differences
among all groups during each observation period.
The Variation of CGRP Concentration in Dorsal Root
Ganglion
For the dorsal root ganglion group, 1w after fracture, there
was no apparent difference of CGRP concentration in the
peripheral nerve injury group and cerebral cortex group
Figure 2. The HE staining to display the bone callus condition in different groups at 2 weeks.
Table 1. The variation of CGRP concentration in blood plasma (pg/ml)
Time of after injury (d)
Groups 0d 2d 7d 14d 28d
Group A 8.8096.10 5.7994.69 3.0390.87 13.32916.39 4.4591.21
Group B 7.5197.09 2.9191.47 2.6491.76 4.1992.71 78.899118.90
Group C 7.4693.72 3.1791.46 2.4990.77 6.0494.73 5.1391.19
Group D 8.7394.24 5.3992.95 2.8791.11 6.0091.63 9.2092.33
The variation column diagram of CGRP concentration in blood serum at different times after fracture of animals.
88 D. Zhang et al.
compared with the control group (P0.05), but the spinal
injury group showed more CGRP than the control group
(PB0.01). 2w after injury, the peripheral nerve injury
group and cerebral cortex group also showed no differ-
ence compared with the control group, but the cerebral
cortex group had more CGRP contents than the peripheral
nerve injure group (PB0.05), and the spinal injury group
showed more CGRP than the control group (PB0.01).
4w after injury, the peripheral nerve group, spinal injury
group, and cerebral cortex injury group all showed higher
concentration of CGRP than the control group. Among
the 3 groups, the spinal injury group is the highest
(PB0.01).
DISCUSSION
Calcitonin gene-related peptide (CGRP) is one of several
biologically active neuropeptides founded by Rosenfeld
[1] through DNA recombination and molecular biology
techniques in 1983. CGRP is coded by calcitionin gene;
the calcitionin gene is composed of 2800 bp, which
Table 2. The variation of CGRP concentration in spinal anterior motorneuron (pg/ml)
Time of after injury (d)
Groups 7d 14d 28d
Group A 2396.199819.78 2176.6991006.92 1786.389644.88
Group B 2188.429398.05 1991.0491642.31 1968.879603.50
Group C 2148.459304.09 1786.089820.96 1824.1691628.37
Group D 2305.349587.43 2210.589529.81 2333.7191027.86
The variation column diagram of CGRP concentration in spinal anterior motor neuron at different times after fracture of animals.
Table 3. The variation of CGRP concentration in dorsal root ganglion (pg/ml)
Time of after injury (d)
Groups 7d 14d 28d
Group A 37200.73920315.64 12143.6793129.19 8908.3093999.77
Group B 46486.54915052.26 5739.0992679.03 29236.4096185.04
Group C 11207.7091604.74 27621.74913120.23 80162.31915711.83
Group D 22378.2299122.83 14667.3798658.43 28356.07912610.88
The variation column diagram of CGRP concentration in in dorsal root ganglion at different times after fracture of animals.
The Influence of Brain Injury or Peripheral Nerve Injury on CGRP Concentration Variation 89
contains 5 piece intervening sequence and 6 piece exons.
They undertake recombination in different tissues to
present different productions. For example, the calcitonin
gene transcribes to calcitonin in the thyroid gland, but in
the nervous tissue it transcribes to CGRP. The relative
molecular mass of CGRP is 3786.91, and it contains 37
amino-acid residues [1]. At the present realization, CGRP
is the most fortis vasodilator material, and its regulation
effect to bone metabolism is clearer as the investigation
developed [2].
Hukkanen [3] et al. verified that there was rapid
proliferation of calcitonin gene-related peptide-immunor-
eactive nerves during healing of rat tibial fracture,
suggesting that neural involvement is present in the
bone growth and remodeling process. In addition, Onuoha
[4] designed a study to examine the circulatory levels of
wound modulatory peptidescalcitonin gene-related pep-
tide (CGRP) in patients with muscle injuries with bone
fractures and within 24 h of the injury. The peripheral
plasma levels of the sensory nerve peptide were measured
on hospital admission (OA) and 24 h post-injury (PI)
using ELISA technique. The result of this study shows
that mean (s.d) ng/liter of CGRP was higher in patients
OA than the controls; sensory nerve peptides are
increased in bone-fracture-related injuries up to 24h after
injury. It hints that CGRP may participate in the tissue
repairment, and an intact nociceptor system of primary
afferent sensory nerves is important for the initiation of
successful tissue repair as dysfunction of this system
could be a contributing factor for a delayed wound
healing.
We had observed the fracture healing status and the
variation of CGRP concentration in different positions,
seen from results of this experiment. When fracture
combined with peripheral nerve injury, the healing
process can be slowed down. In contrast, fracture
combined with spinal injury and cerebral cortex injury
will accelerate the healing process. By H.E. staining, we
found lesser bony callus contents in the peripheral nerve
injury group than the central nerve injury group; the
different type of injury of the nerve can result in different
speed of fracture healing. It demonstrated that nerve
factors can affect the healing process of the fracture. The
theory that nerve factors can affect the healing process of
the fracture had already been put forward by Aro et al. [5]
and was confirmed by some researchers in the last decade,
but its mechanism still remains unclear. So we presume
that CGRP is one of the nerve factors that affect the
healing process of fracture. J.M. Garcia-Castellano [6]
demonstrated that CGRP has a great effect on angioecta-
sia to capillary, so it can accelerate the proliferation of
osteocyte.
In our research, we observed that the CGRP
concentration in blood plasma represented no apparent
differences among all groups during each observation
period, so we presume that the way of CGRP regulated
healing process of fracture is not by humoral regulation.
Zaidi et al. [7] thought that the CGRP in osseous tissue
plays its role by means of paracrine secretion, and the
CGRP in normal serum has no effect on bone metabolism.
The CGRP concentration in spinal anterior motorneuron
represented no apparent differences among all groups
during each observation period. It demonstrated that
spinal anterior motorneuron did not participate in the
regulation to the healing process of fracture. In other
words, the regulation of CGRP to healing process of
fracture is not carried by means of the spinal anterior
motor neuron.
As we all know, the distribution of CGRP is mainly
concentrated in dorsal root ganglion. Through the analysis
of the variation of CGRP concentration in dorsal root
ganglion, we can see that CGRP concentration is the
highest, and decreased gradually as time passed. There
were two possible reasons for the CGRP concentration
decrease: (1)The reduction of synthesis; (2) The augmen-
tation of consumption. CGRP is a neurotransmitter, which
is synthesized and secreted by neurone, but there is no
obvious variation of CGRP concentration in spinal
anterior motor neuron in the simple fracture group. So
we can presume that the synthesis of CGRP did not
decrease after fracture, but the consumption of CGRP is
increasing for its participation in the regulation of the
fracture healing process. Compared with the control
group, the CGRP concentration in dorsal root ganglion
degraded during the two weeks after injury in the
peripheral nerve injury group, and the extent of degrade
is obvious. The result agrees with what K.C. Kajander [8]
had observed: the CGRP concentration in dorsal root
ganglion degraded obviously when the peripheral nerve
was injured. However, in our research, compared with the
control group, the CGRP concentration of peripheral
nerve injury group decreased obviously at 4 weeks after
injury. We presumed that because o the CGRP cannot be
transported to the target organ through axoplasmic
transportation and accumulated in dorsal root ganglion,
so the CGRP concentration in dorsal root ganglion
increased relatively. The CGRP concentration in dorsal
root ganglion is high, but it cannot be transported to the
target organ and play its regulation role, so the speed of
healing is slower than the normal group, and contents of
local bony callus is low. Compared with the control
group, the CGRP concentration in spinal cord dorsal root
ganglion of spinal cord injury group is lower, and
increased gradually at 2 weeks and 4 weeks after injury.
The extent is higher than the control group. The speed of
fracture healing in the spinal cord injury group is faster
than the control group, and contents of local bony callus
are high. It demonstrated that the reason spinal cord injury
can accelerate the healing speed is the participation of
massive CGRP synthesized and secreted by neuron.
90 D. Zhang et al.
There was no apparent variation of CGRP concentra-
tion of the cerebral cortex injury group, respecting the
serum CGRP concentration of the simple fracture group
degraded gradually after injury, so we can conclude that
after the cerebral cortex injuring the synthesis and
secretion of CGRP increased compared with the simple
fracture group, but the extent of CGRP increasing is lower
than the spinal cord injury group.
When hematencephalon, hemorrhagic shock, endo-
toxic shock, and septic shock happened,the secretion of
CGRP increased obviously [9]; meanwhile, the mRNA
lever of CGRP in spinal cord dorsal root ganglion
increased simultaneously. Why did the content of
CGRP increase? The mechanism is still unclear.
Seen from the results of this experiment, when
fracture combined with peripheral nerve injury, the
healing process can be slowed down. In contrast, fracture
combined with spinal injury and cerebral cortex injury
will accelerate the healing process. The CGRP in dorsal
root ganglion in the spinal injury group and cerebral
cortex injury group increased, which may have positive
effects on fracture healing. CGRP is one of the most
powerful endogenous hemangiectasia peptidehasten. It
was transported to the fracture region by means of
axoplasmic transport and played its hemangiectasia roles,
which could increase the blood supply of the fracture
region and hasten the healing of fracture.
Aoki et al. [11] demonstrated that CGRP is abun-
dantly distributed in bone via sensory nerves, especially
in the epiphyseal trabecular bones. CGRP is shown to be
expressed endogenously by the osteoblasts. Transgenic
mice with osteoblasts overexpressing CGRP are charac-
terized by increased bone formation rate and enhanced
bone volume, suggesting that CGRP indeed acts on bone
metabolism not only via the nervous route but also via
autocrine loop. Calcitonin gene-related peptide has an
osteogenic stimulating effect, either by stimulating stem
cell mitosis or osteoprogenitor cell differentiation (or
both) [12] in vitro. CGRP also inhibited bone resorption
potentially [13].
Our study provided powerful evidence that CGRP
may play a local role in bone metabolism to promote the
healing of fracture, but the mechanism of its action need
further research.
REFERENCES
1. Rosenfeld, M.G., Mermod, J.J., Amara, S.G. et al. (1983).
Production of novel neuropeptide encoded by the calcitonin
gene via issue-specic RNA processing. Nature 304:
129135.
2. Bjurholm, A., Kreicbergs, A., Brodin, E. et al. (1988).
Substance P- and CGRP-immunoreactive nerves in bone.
Peptides 9:165171.
3. Hukkanen, M., Konttinen, Y.T., Santavirta, S. et al. (1993).
Rapid proliferation of calcitonin gene-related peptide-
immunoreactive nerves during healing of rat tibial fracture
suggests neural involvement in bone growth and remodel-
ing. Neuroscience 54:969979.
4. Onuoha, G.N. (2001). Circulating sensory peptide levels
within 24 h of human bone fracture. Peptides 22:
11071110.
5. Aro, H., Eerola, E., Aho, A. J. (1985). Development of
nonunions in the rat bula after removal of periosteral
neural mechanoreceptors. Clin Orthop 199:292299.
6. Garcia-Castellano, J.M., Diaz-Herrera, P., Morcuende, J.A.
(2000). Is bone a target-tissue for the nervous system? New
advances on the understanding of their interactions. Iowa
Orthop J. 20:4958.
7. Zaidi, M., Chambers, T.J., Bevis, P.J.R. et al. (1988).
Effects of peptides from the calcitonin genes in bone and
bone cells. Q J Exp Physiol 173:471485.
8. Kajander, K.C., Xu, J. (1995). Quantitative evaluation of
calcitonin gene-related peptide and substance P levels in rat
spinal cord following peripheral nerve injury. Neurosci Lett
186(23):1848.
9. Wang, X., Han, C.D., Fiscus, R.R. et al. (1991). Hyperten-
sion and endotoxin induced alterations in calcitonin gene-
related peptide: Modulation by dexamethasone. Circ Shock
34:217223.
10. Poyner D, Marshall I, Brain SD. (1999). The CGRP Family.
1st eds. Landes Bioscience.USA:Texas, 173252.
11. Aoki, M., Tamai, K., Saotome, K. (1994). Substance P and
calcitonin gene-related peptide-immunouorescent nerves
in the repair of experimental bone defects. Int Orthop
18:317324.
12. Shih, C., Bernard, G.W. (1997). Calcitonin gene-related
peptide enhances bone colony development in vitro [J].
Clin Orthop 334:335344.
13. Valentijn, K., Gutow, A.P., Troiano, N., Gundberg, C.,
Gilligan, J.P., Vignery, A. (1997). Effects of calcitonin
gene-related peptide on bone turn over in ovariectomized
rats [J]. Bone 21(3):269274.
This paper was first published online on iFirst on 26 February 2009.
The Influence of Brain Injury or Peripheral Nerve Injury on CGRP Concentration Variation 91