〔Chiba Medical J.

92E：31 ～ 36，2016〕〔 Original Paper 〕

Gait and inflammatory response in a rat model of hip pain induced byintra-articular injection of nerve growth factor
Takanori Omae, Junichi Nakamura, Seiji Ohtori, Sumihisa Orita
Takane Suzuki, Miyako Suzuki, Shuichi Miyamoto, Shigeo Hagiwara
Takayuki Nakajima, Makoto Takazawa, Tomonori Shigemura, Yasushi Wako
Michiaki Miura, Yuya Kawarai, Masahiko Sugano
Kento Nawata and Kazuhisa Takahashi
Department of Orthopedic Surgery, Graduate School of Medicine, Chiba University, Chiba 260-8670.
（Received March 14, 2016, Accepted March 23, 2016）

SUMMARY
Objectives To determine gait and inflammatory response in a rat hip pain model produced byintra-articular injection of nerve growth factor（NGF）.
Methods Using 36 8-week-old male Sprague Dawley rats, 30 µl of saline（sham-operated group;n＝12）, 30 µL of 50 µg/mL of NGF（NGF50 group; n＝12）, and 30 µL of 100 µg/mL of NGF（NGF100
group; n＝12）were injected into the left hip. The gait of 6 rats in each group was analyzed using a
CatWalk system. The levels of inflammatory cytokines were determined with the synovium and femoral
head.
Results The maximum contact area was significantly decreased in the NGF100 group comparedwith in the sham-operated group on day 14. Swing speed significantly decreased in the NGF50 and
NGF100 groups compared with in the sham-operated group on day 14. Duty cycle significantly
decreased in the NGF50 and NGF100 groups compared with the sham-operated group on day 14.
TNF-α, IL-1β, and IL-6 were significantly elevated in NGF50 and NGF100 groups compared with the
sham-operated group.
Conclusions NGF into the rat hip joint induces an increase of inflammatory cytokines in the
synovium and elicits pain escape behavior.
Key words: Gait, cytokine, hip joint, nerve growth factor

Address correspondence to Dr. Junichi Nakamura.
Department of Orthopedic Surgery, Graduate School of
Medicine, Chiba University, 1-8-1, Inohana, Chuou-ku, Chiba,
260-8670 Japan.
Phone: +81-43-226-2117. Fax: +81-43-226-2116.
E-mail: njonedr@chiba-u.jp

Abbreviations: dorsal root ganglion（DRG）, nerve growth factor（NGF）, osteoarthritis（OA）, enzyme-linked immunosorbent
assay（ELISA）, tumor necrosis factor（TNF）, interleukin（IL）

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Takanori Omae et al.

Ⅰ．Introduction

The purpose of this study was to evaluate painrelated behavior using the CatWalk gait analysis system

The pathogenesis of hip pain is unclear and theand the synovial levels of inflammatory cytokines inroles of sensory innervation and the nature of paina rat model of hip pain produced by intra-articulartransmitting substances in the hip joint have not been

injection of NGF into the hip joint.

fully elucidated. Previously, we reported that dorsal
root ganglion（DRG）neurons innervating the hip are

Ⅱ．Materials and Methods

distributed on multiple levels（L1-L4）［1］, and that
there is a difference in the level of innervation between

We used 36 8 -week-old male Sprague Dawley

the hip and inguinal skin［2］. Moreover, DRG neurons

rats weighing from 250 to 300 g. The protocols forthat innervate the hip and inguinal skin may overlap

the animal procedures in our experiments followed［3］. Animal models of hip pain are essential to clarify

the 1996 revision of the National Institutes of Healththe pathogenesis of hip pain. However, such models

guidelines for the Care and Use of Laboratory Animalsof hip pain have not been widely reported. We haveand received prior approval from the ethics committee

established two novel rat models of hip pain producedof our institution. The rats were anesthetized by an

by intra-articular administration into the hip joint withintraperitoneal injection of sodium pentobarbital（40mono-iodoacetate［4］or nerve growth factor（NGF）［5］.

mg/kg）and were treated aseptically throughout the

NGF is a neurotrophin that is well known to regulate

experiments. Using a 26-gauge needle, the following

neuronal development, survival, and maintenance［6］.

solutions were injected into the left hip of rats assigned

NGF also seems to influence chronic pain states, notably

to various groups according to their treatment: 30 µL

those associated with inflammation, and may be a targetof saline in rats in a sham-operated group（n＝12）, 30

for analgesia in patients with osteoarthritis（OA）［7］.

µL of 50 µg/mL of NGF（recombinant human b-NGF,

Intra-articular injection of NGF into rat hips produced

256-GF-100/CF; R&D Systems, Minneapolis, MN）in

microinflammation without osteoarthritic change［5］.

rats in a NGF50 group（n＝12）, and 30 µL of 100µg/

Intra-articular injection of NGF into rat knees evoked

mL of NGF in rats in a NGF100 group（n＝12）. The

pain-related behavior［8］. NGF is associated withintra-articular injections were limited to the joint cavity.

angiogenesis at the osteochondral junction in patients

The rats were allowed unrestricted cage activity after thewith arthritis［9］. We hypothesized that direct application

injection.

of NGF into the hip joint evokes an inflammatory
condition without degeneration of the articular cartilage

Experiment 1: gait parameters

and that NGF can be used to model inflammatory hip

The CatWalk system（Noldus Information

pain in rats. However, the direct effects of intra-articularTechnology, Wageningen, The Netherlands）was used

injection of NGF into normal rat hips on pain mediators

to evaluate gait characteristics in the present study.

and pain-related behavior are not yet known.

Rats were placed on a glass plate located in a darkened

A CatWalk gait analysis system is able to perform

room and allowed to walk freely. A light beam from adetailed gait analysis noninvasively and repeatedly［10］.

fluorescent lamp was shone through the glass plate. The

This system has been described in detail elsewherelight was normally reflected internally, except in areas［11-14］. The time course of pain-related mediator

where the rat’s paw touched the glass plate, where theappearance can be analyzed using enzyme-linked

light was refracted on the opposite side. This produced aimmunosorbent assay（ELISA）to determine the levels

sharp bright image of the paw print as seen from underof inflammatory cytokines, focusing on inflammatory

the plate where the rat walked, and the entire run wasand neuropathic pain-related states using local tissues

recorded using a video camera. The data were acquired,and peripheral sensory innervation including DRGs.

compressed, and analyzed using CatWalk system

33

NGF hip pain

software. In the present study, the gait patterns of 6

decreased in rats in the NGF100 group compared withrats in each group were recorded 3 times and analyzed

rats in the sham-operated group on day 14（ 81 . 3 ％automatically using the CatWalk system.

versus 95 . 8 ％, P＝0 . 0002 , Figure 1 ）. Swing speed

We compared 18 parameters of hind paw gait

tended to decrease from day 0 to day 7 after NGF

between the 3 groups. The ratios of the affected side to

injection in rats in all 3 groups and then significantly

the unaffected side were taken as adjusted data for eachdecreased in rats in both the NGF 50 and NGF100

parameter. The rat gait patterns were recorded on day

groups compared with rats in the sham-operated group0（before injection, n＝6）and again on days 7 and 14

on day 14（80.3％ versus 82.2％ versus 96.2％, P＝after the injection（before they were humanely killed,

0.0001 and P＝0.0005, respectively, Figure 2）. Dutyn＝6, respectively）.

cycle tended to decrease from day 0 to day 7 after NGF
injection in rats in all 3 groups and then significantly

Experiment 2: inflammatory response
For each group, 6 rats were killed after Experiment
1 on day 7, while the remaining 6 were killed on day
14 . Rats were deeply anesthetized using 40 mg/kg

sodium pentobarbital and their left hips were exposed;
the synovium and femoral head cartilage were resected
for cytokine ELISAs. Tumor necrosis factor（TNF）-α,interleukin（IL）-1β, and IL- 6 production in the
synovium and the femoral head cartilage were quantified
using ELISAs in accordance with the manufacturer’sprotocols（R&D Systems, Minneapolis, MN）. Tissue
protein was assayed using a kit in accordance with the
manufacturer’s protocol（Bio-Rad, Hercules, CA）, andcytokine levels were normalized to tissue protein levels.
Statistical Analysis

Fig. 1

Maximum contact area.

The values are adjusted by the ratio of the affected
to unaffected side（％）. Maximum contact area wassignificantly decreased in rats in the NGF 100 groupcompared with rats in the sham-operated group on day 14.
Bars represent standard deviation. *P ＜0.05.

All values were calculated and statisticallycompared using unpaired t test followed by Bonferroni’scorrection. A P ＜0.05 was considered to be significant.

Ⅲ．Results
Experiment 1: gait parameters
Of 18 parameters for hind paws, maximum contactarea（the maximum area of paw contact with the glass
plate）, swing speed（the speed of the paw during gait）,
and duty cycle（duration of paw contact divided by time
between consecutive paw contacts: contact/（contact＋
swing）×100％）were determined to be significantly
different between the 3 groups. Maximum contact
area tended to decrease from day 0 to day 7 after NGF
injection in rats in all 3 groups and then significantly

Fig. 2

Swing speed.

The values are adjusted by the ratio of the affected
to unaffected side（％）. Swing speed was significantly
decreased in rats in both the NGF50 and NGF100 groups
compared with that in rats in the sham-operated group on
day 14. Bars represent standard deviation. *P ＜0.05.

34

Takanori Omae et al.

decreased in rats in both the NGF 50 and NGF100

1.55 versus 1.12, P＝0.0004, respectively, Figure 4）.

groups compared with rats in the sham-operated group

The TNF-α levels were also significantly higher in rats

on day 14（82.4％ versus 83.0％ versus 96.7％, P＝in the NGF100 group than in rats in the sham-operated

0.0007 and P＝0.0005, respectively, Figure 3）.

group on day 14（ 1 . 55 versus 1 . 22 , P＝0 . 0025 ）.
Synovial membrane levels of IL-1β（pg/mg protein）

Experiment 2: inflammatory response

were significantly higher in rats in both the NGF100

Synovial membrane levels of TNF-α（pg/mg

and NGF50 groups than in rats in the sham-operated

protein）were significantly elevated on day 14 after thegroup on day 7（11.2 versus 9.8 versus 7.8, P＝0.0001

injection compared with day 7 in rats in both the NGF50

and P＝0.0006, respectively, Figure 5）. However, theand NGF100 groups（1.46 versus 1.08, P＝0.0019 anddifference was no longer significant on day 14. Synovial
membrane levels of IL- 6（pg/mg protein）were
significantly higher in rats in the NGF100 group thanin rats in both the sham-operated and NGF50 groups

Fig. 3

Duty cycle.

The values are adjusted by the ratio of the affected
to unaffected side（％）. Duty cycle was significantly
decreased in rats in both the NGF50 and NGF100 groups
compared with that in rats in the sham-operated group on
day 14. Bars represent standard deviation. *P ＜0.05.

Fig. 4

Fig. 5

Synovial levels of IL-1β.

The synovial levels of IL-1β were significantly higher
in rats in the NGF100 group than in rats in the shamoperated group on day 7. Bars represent standard deviation.
*P ＜0.05.

Synovial levels of TNF-α.

The synovial levels of TNF-α were significantly elevatedon day 14 after the intra-articular injection of NGF comparedwith levels on day 7 in rats in both the NGF50 and NGF100
groups. The synovial levels of TNF-α were significantly
higher in rats in NGF100 group than in rats in the shamoperated group on day 14. Bars represent standard deviation.
*P ＜0.05.

Fig. 6

Synovial levels of IL-6.

The synovial levels of IL-6 were significantly higher inrats in the NGF100 group than in rats in both the shamoperated and NGF50 groups on day 14. Bars represent
standard deviation. *P ＜0.05, **P ＜0.01.

35

NGF hip pain

on day 14（39.4 versus 33.1 versus 30.8, P＝0.0007

contact intensity（the mean intensity of all the pixels

and P＝0.0001, respectively, Figure 6）, although nocontributing to the maximum paw print area）were

significant difference was found on day 7.

significantly less than in the control group.

There were no significant differences in cytokine

The current report is also the first describing alevels in the femoral head cartilage at any time after the

rat model of hip pain associated with an increase

various treatments. The TNF-α levels in the femoral

in synovial levels of proinflammatory cytokines,

head cartilage were 1.22, 1.21, and 1.23 pg/mg protein

including TNF-α, IL-1β, and IL-6 levels as assessedin rats in the sham-operated, NGF50 , and NGF100

by ELISA. Intra-articular NGF administration into thegroups on day 7, and 1.25, 1.27, and 1.29 pg/mg protein

rat hip was associated with an increase in the synovial

on day 14, respectively. IL-1β levels in the femoral

level of inflammatory cytokines. Sensory innervation

head cartilage were 23.7, 24.6, and 24.9 pg/mg protein

and inflammatory cytokines in hypertrophic synovial

in rats in the sham-operated, NGF50 , and NGF100

membranes are associated with nociception in OA of thegroups on day 7, and 23.8, 26.3, and 27.3 pg/mg protein

hip in humans［15］. IL-6 and TNF-α levels are elevatedon day 14, respectively. IL-6 levels in the femoral headin synovial joint fluid in patients with hip disease,cartilage were 106.1, 112.3, and 116.8 pg/mg protein inreflecting tissue destruction in OA［16］. In the present

rats in the sham-operated, NGF50, and NGF100 groups

study, synovial levels of TNF-α were significantly

on day 7, and 105.4, 105.6, and 97.7 pg/mg protein on

elevated on day 14 after the injection compared withday 14, respectively.

the levels on day 7 in rats in both the NGF 50 and 100
groups. Synovial levels of IL-1β were significantly

Ⅳ．Discussion

higher in rats in both the NGF 50 and 100 groups than inrats in the sham-operated group on day 7, although the

To our knowledge, this is the first report describing

difference was no longer significant on day 14. IL-1β

hip pain-associated changes in gait parametersis therefore likely to play a role in early inflammation.

determined by CatWalk, including maximum contact

Synovial levels of IL-6 were significantly higher in ratsarea, swing speed, and duty cycle. Intra-articularin the NGF 100 group than in rats in the sham-operated

injection of NGF into the hip joint apparently elicits

and NGF 50 groups on day 14, although the levels were

pain escape behavior in rats. Miyagi et al. reported

not significantly different on day 7. Thus, IL-6 is likely

a rat model of myofascial inflammation using pain-

to play a role in delayed inflammation. Our findings

associated changes of gait parameters assessed usingsuggest that NGF is crucially involved in the behavioral

the CatWalk system［11］. In the model of myofascial

expression and neural transmission of pain from the hip

inflammation, duty cycle of front and hind paws was

joint.

significantly greater, stride length（the distance between

There are several limitations to this study. First,

successive placements of the same paw）of each paw

dose dependency was not determined because only 2

was significantly shorter, and minimum contact intensity

doses of NGF were examined. Further doses of NGF

of the complete paw and mean contact intensity ofshould be studied to determine dose dependency.

each paw in the myofascial inflammation group were

Second, gait parameters and inflammatory response

significantly greater compared with the control group.

were evaluated at only 2 time points. Pain-related

Yamazaki et al. reported a rat model of unstabilized

behavior was significantly different between rats inrotator cuff defect using pain-associated changes ofthe various treatment groups on day 14. In a previous

gait parameters assessed using the CatWalk systemstudy, pathology of the hip and immunohistochemistry［13］. In the model of unstabilized rotator cuff defect,of the DRG showed mild synovitis and expression of

stride length, print area（the area of the paw contact

calcitonin gene-related peptide-immunoreactive neurons

with the glass runway surface during locomotion）, andin sensory nerves on day 7 that persisted on day 14［5］.

36Takanori Omae et al.

Determination of longer and shorter-term outcomes of
intra-articular NGF injection is warranted. Third, in the
CatWalk system, it is not possible to assess whether
parameters are related to pain or muscle dysfunction
without pain, and so the possibility that effects are
related to muscle dysfunction cannot be excluded.
In conclusion, an increase in synovial levels of
inflammatory cytokines and pain-related escape behavior
is observed in a novel rat model of hip pain induced byintra-articular administration of NGF into the hip joint.

Acknowledgements
Corresponding author, Junichi Nakamura received
JSPS KAKENHI Grant Number 25870125.
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