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Mod Rheumatol, 2015; Early Online: 1–6© 2015 Japan College of Rheumatology
DOI: 10.3109/14397595.2015.1023977

Original Article

A novel rat model of hip pain by intra-articular injection of nerve growth
factor-characteristics of sensory innervation and inflammatory arthritis
Takanori Omae*, Junichi Nakamura*, Seiji Ohtori, Sumihisa Orita, Kazuyo Yamauchi, Shuichi Miyamoto,
Shigeo Hagiwara, Shunji Kishida, and Kazuhisa Takahashi

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Department of Orthopaedic Surgery, Graduate School of Medicine, Chiba University, Chiba City, Chiba, Japan
Abstract

Keywords

Objectives. To determine the direct effects of intra-articular injection of nerve growth factor (NGF)
into normal rat hips and the time course of pain-related mediator appearance.
Methods. Using 36 numbers of 8-week-old male Sprague–Dawley rats, 30 ml of 1% Fluoro-Gold
solution (FG) (Sham-operated group; n  12), 30 ml of 1% FG with 50 mg/ml NGF (NGF50 group;n  12), and 30 ml of 1% FG with 100 mg/ml NGF (NGF100 group; n  12) were injected into theleft hip joints. Neurons in the dorsal root ganglion (DRG) labeled with FG, and FG and calcitonin
gene-related peptide-immunoreactivity (CGRP-IR) were counted. The synovia in the left hip joint
was examined histologically.
Results. The NGF50 and NGF100 groups showed evidence of synovitis without cartilage degeneration compared with the Sham-operated group. At 7 days, the proportions of CGRP-IR FG-labeled to total FG-labeled neurons were 12%, 18%, and 36% in the Sham-operated, NGF50, andNGF100 groups, respectively. At 14 days, the proportions were 13%, 22%, and 35% in the Shamoperated, NGF50, and NGF100 groups, respectively. At 7 and 14 days, the NGF50 and NGF100
groups showed a significantly higher proportion of CGRP-IR FG-labeled neurons than the Shamoperated group.
Conclusions. Intra-articular administration of NGF into the hip joint produces a novel rat modelfor hip pain.

Experimental study, hip joint, intra-articular
injection, nerve growth factor

Introduction
Pain originating from the hip has been classically described aspresenting in the groin. However, referred pain may occur inosteoarthritis (OA) [1–2], osteonecrosis [3–4], acetabular labrallesion [5], and other hip disorders. Pain has been reported to affectthe thigh or lower leg as well as the hip itself. A recent study of OA
of the hip found that referred pain was localized to the groin (89%),
buttock (38%), greater trochanter (27%), anterior thigh (33%),
knee (29%), and lower back (17%), corresponding to the L1 to L5
dermatomes [6]. Thus, it is often difficult to distinguish between
pain referred as from the hip and radicular pain originating from alumbar lesion [7].
It is not clear why the symptoms and pain of inflammatory
or degenerative processes are so varied in the area of the hip.
Birnbaum et al. [8] described the hip as innervated by the obturator, femoral, sciatic, and superior gluteal nerves. Previously, we
reported that dorsal root ganglion (DRG) neurons innervating
the hip were distributed on multiple levels (L1–L4) [9] and that
there is a difference in the level of innervation between the hip andinguinal skin [10]. Moreover, DRG neurons that innervate the hipand inguinal skin may overlap [11]. Animal models of hip pain are

History
Received 2 December 2014
Accepted 23 February 2015
Published online 31 March 2015

essential to clarify the pathogenesis of hip pain. However, such hip
pain model has not been established so far.
Pain is transmitted to the dorsal horn of the spinal cord by small
dark cells in the DRG, and these small DRG neurons are divided
into nerve growth factor (NGF)-sensitive neurons and glial cellline-derived neurotrophic factor (GDNF)-sensitive neurons. NGF
and GDNF regulate the expression of various pain-related molecules, including substance P (SP) and calcitonin gene-related
peptide (CGRP) [12]. NGF-sensitive neurons express two types of
the receptors, i.e., the high-affinity NGF receptor tyrosine kinase A
and the low-affinity NGF receptor p75 [13]. Pain is mainly transmitted by CGRP-immunoreactivity (IR) neurons [9,14]. Previous
studies have suggested that anti-NGF antibodies have analgesic
effects on pathological pain [13]. However, these studies targetedneuropathic pain or pain from cutaneous tissue, and inflammatory
hip pain has been unknown.
The purpose of this study was to determine the direct effects of
intra-articular injection of NGF into normal rat hips and the timecourse of pain-related mediator appearance, focusing particularly
on both inflammatory and neuropathic pain-related states using
local tissues and sensory innervation of the peripheral nervous
system including the DRG.

*Both the authors equally contributed to this study.

Materials and methods

Correspondence to: Junichi Nakamura, Department of Orthopaedic
Surgery, Graduate School of Medicine, Chiba University. 1-8-1 Inohana,Chuo-ku, Chiba City, Chiba, 260-8677, Japan. Tel:  81-43-226-2117.
Fax:  81-43-226-2116. E-mail: njonedr@chiba-u.jp

We used 36 numbers of 8-week-old male Sprague–Dawley rats
weighing 250–300 g. The protocols for the animal proceduresin these experiments followed the 1996 revision of the National

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T. Omae et al.

Institutes of Health guidelines for the Care and Use of Laboratory Animals and received approval from the ethics committee of
our institution. The rats were anesthetized by an intraperitoneal
injection of sodium pentobarbital (40 mg/kg) and were treatedaseptically throughout the experiments. Using a 26-gauge needle,
the following solutions were injected into the left hip of each rat:
30 ml of 1% Fluoro-Gold solution (FG; Fluorochrome, Denver,
Colorado) in the Sham-operated group rats (n  12), 30 ml of 1%
FG with 50 mg/ml of NGF (recombinant human b-NGF, 256-GF100/CF; R&D Systems, Minneapolis, MN) in the NGF50 grouprats (n  12), and 30 ml of 1% FG with 100 mg/ml of NGF in the
NGF100 group rats (n  12). The injected fluid was restricted tothe joint cavity.
Seven days later, six out of twelve rats in each group were anesthetized with sodium pentobarbital (40 mg/kg, intraperitoneally)and perfused transcardially with 0.9% saline, followed by 500 ml
of 4% paraformaldehyde in phosphate buffer (0.1 M, pH 7.4). The
left hips were exposed and the soft tissues around the joint andcartilage including the synovium and capsule were resected (n  6,each). Then DRGs between T13 and L6 on the left side were also
resected. The resected limbs were prepared for histopathology andthe DRG specimens were prepared for immunohistology. At 14
days, the remaining six rats in each group were processed in thesame manner.

Histopathology of the hip joint
The resected limbs were cut at the pelvis and at the midfemur andimmersed in buffered paraformaldehyde fixative at 4°C for 1 week.
The specimens were demineralized in 10% EDTA for 2 weeks following by standard histological techniques using paraffin blocks
for subsequent coronal sectioning. The samples were serially sectioned in steps of 8 mm, stained using hematoxylin and eosin (HE)used for principal method of pathological change, Safranin O andtoluidine-blue staining used for the detection of cartilage. They
were assessed by light microscopy using the synovitis score of
Krenn et al. [15], which consisted of enlargement of the synovial
lining cell layer (0–3 points), density of the resident cells (0–3
point), and inflammatory infiltrate (0–3 points). In total, 0–1 point
was classified as no synovitis, 2–4 points were low-grade synovitis, and 5–9 points were high-grade synovitis. Cartilage change
was graded as by Mankin score [16], consisted of structure (0–6
points), cells (0–3 points), Safranin O staining (0–4 points), andtidemark integrity (0–1 point). In total, 1–3 points are classified
as slight degeneration, 4–7 points are moderate, and 8–14 points
are severe.

Calcitonin gene-related peptide immunohistochemistryof DRGThe specimens were immersed in the phosphate-buffered paraformaldehyde fixative overnight at 4°C. After storage in 0.01 M
phosphate-buffered saline (PBS) containing 20% sucrose for 20
hours at 4°C, each DRG was sectioned at 12 mm on a cryostat
and mounted on poly-l-lysine-coated slides. Endogenous tissue
peroxidase activity was quenched by soaking the sections for 30
minutes in 0.3% hydrogen peroxide solution in 0.01 M PBS. The
specimens were then treated in a blocking solution, 0.01 M PBS
containing 0.3% Triton X-100 and 3% skimmed milk, overnightat 4°C. They were processed for CGRP immunohistochemistry
using rabbit antibody to CGRP (1:1000; Chemicon), diluted witha blocking solution, and incubated overnight at 4°C. After incubation with labeled antibody, the sections were incubated with goatanti-rabbit Alexa 488 for CGRP-IR (1:1000; Molecular Probes,
Eugene, Oregon). After each step, the sections were rinsed in 0.01
M PBS three times and studied using fluorescence microscopy.

Mod Rheumatol, 2015; Early Online: 1–6­
Figure 1. Fluorescence photomicrographs of the left L4 (dorsal root
ganglia) after injection of Fluoro-Gold solution into the hip. A) FG-labeledneurons in dorsal root ganglia. B) CGRP-IR neurons in dorsal root ganglia.
The CGRP-IR neurons are labeled green. Arrows show two FG-labeledneurons that are also CGRP-IR neurons innervating the hip. (A and B).
(Scale bars are 100 mm).

The numbers of FG-labeled and CGRP-IR FG-labeled neurons
were counted (Figure 1).

Statistical analyses
Differences between the groups in the number of FG-labeled DRG
neurons in the DRGs between T13 and L6 were evaluated usinga Tukey–Kramer test. The proportions of CGRP-IR FG-labeledneurons in the DRGs were compared using a Mann–Whitney U
test followed by a Bonferroni correction. P  0.05 was considered
to be statistically significant.

Results
Histopathology of the hip joint
All the hips in both NGF 50 and NGF 100 groups showed inflammatory change with low-grade synovitis and angiogenesis in day
7. Synovitis score of each group was shown in Table 1. The histological features of synovitis were a single layer of lining cells,slightly increased cellularity, and few, mostly perivascular situatedlymphocytes or plasma cells. However, the hips did not show
osteoarthritis-like appearances such as cartilage loss and degeneration, subchondral bone collapse, or subchondral cystic structure with Safranin O, and toluidine blue staining. The NGF 50 and
NGF 100 groups showed similar appearances (Figures 2 and 3).
Sham-operated hips showed an expected normal appearance(Figure 4). Inflammatory change persisted in both the NGF 50 and
NGF 100 groups until at least day 14. By contrast, osteoarthritic
Table 1. Comparison of synovitis score among sham-operated, NGF50,
and NGF100 groups at day 7.
Sham-operated NGF50 NGF100
group
group
group
Enlargement of the synovial lining
cell layer
Density of the resident cells
Inflammatory infiltrate
Total

0

0.3

0.3

0.1
0.2
0.3*†

1.0
0.9
2.2*

1.2
0.8
2.3†

­Mann–Whitney’s U test followed by Bonferroni correction, *p  0.0074,†p  0.0049.

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DOI 10.3109/14397595.2015.1023977

A novel rat model of hip pain by intra-articular injection of nerve growth factor 3­
Figure 2. Histopathology of the hip joint in a NGF50 rat at day 7. A) Hematoxylin and eosin staining, B) Safranin O staining, and C) Toluidine-blue
staining of 40 magnification (Scale bars are 500 mm). Each shows mild synovitis and angiogenesis (white arrow head) without cartilage degeneration in
the femoral head (white arrow) and the acetabulum (black arrow head). D) Hematoxylin and eosin staining of 400 magnification (Scale bars are 50 mm)
shows synovitis and angiogenesi s (black arrow).

changes were not seen in any groups. Mean Mankin score was 0.3
in Sham-operated group, 0.7 in NGF50 group, and 0.7 in NGF100
group without significant differences (Sham-operated group ver-sus NGF50, P  0.522; Sham-operated group versus NGF100,
P  0.522; NGF50 versus NGF100, P  1.000; respectively,
Mann–Whitney U test followed by a Bonferroni correction).

Figure 3. Histopathology of the hip joint in a NGF100 rat at day 7. A) Hematoxylin and eosin staining, B) Safranin O staining, and C) Toluidine-blue
staining of 40 magnification (Scale bars are 500 mm). Each shows mild synovitis and angiogenesis (white arrow head) without cartilage degeneration in
the femoral head (white arrow) and the acetabulum (black arrow head). D) Hematoxylin and eosin staining of 400 magnification (Scale bars are 50 mm)
shows synovitis and angiogenesis (black arrow).

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T. Omae et al.

Mod Rheumatol, 2015; Early Online: 1–6­
Figure 4. Histopathology of the hip joint in a Sham-operated rat at day 7. A) Hematoxylin and eosin staining, B) Safranin O staining, and C) Toluidineblue staining of 40 magnification (Scale bars are 500 mm). Each shows normal appearance of synovia (white arrow head), the femoral head (white arrow)and the acetabulum (black arrow head). D) Hematoxylin and eosin staining of 400 magnification (Scale bars are 50 mm).

Fluoro-Gold-labeled neurons in dorsal-root ganglia
Neurons with FG-labeling, in which FG was transported fromthe hip, were distributed in the left DRGs between T13 and L6
at day 7 (Figure 5). The labeled neurons were mainly found in
the L3 and L4 DRG, and the proportion of FG-labeled neurons
in the L1, L2, L3, and L4 DRGs was significantly higher thanthose in the L6 DRG (Tukey–Kramer test, P  0.05). Very similar distributions were observed in each group (data not shown).
Furthermore, the proportions of FG-labeled neurons were notsignificantly different for each level of DRG among these groups.

The distributions and the proportions of FG-labeled neurons in
DRG were not significantly different on day 7 and day 14 among
the three groups.
Fluoro-Gold-labeled calcitonin gene-related
peptide-immunoreactive neurons
FG-labeled CGRP-IR neurons were mainly present in the left
DRGs between L1 and L4. Of the FG-labeled neurons on day 7,
the mean proportion of CGRP-IR FG-labeled neurons in DRGs
was 11.5% in the Sham-operated group, 18.1% in the NGF50
group, and 36.2% in the NGF100 group. The proportion increased
dose dependently (Sham versus NGF50, P  0.0084; NGF50
versus NGF100, P  0.0007; NGF100 versus Sham, P  0.0001;
respectively, Mann–Whitney U test followed by a Bonferroni correction, Figure 6). On day 14, the proportion was 13.1% in the
Sham-operated group, 22.1% in the NGF50 group, and 34.7% in the
NGF100 group. The proportion increased dose dependently (Shamversus NGF50, P  0.0001; NGF50 versus NGF100, P  0.0005;
NGF100 versus Sham, P  0.0001; respectively, Mann–Whitney
U test followed by a Bonferroni correction, Figure 6). By contrast,
the proportions in each group were not significantly different
between days 7 and 14.

Discussion

Figure 5. Distribution of the mean number of Fluoro-Gold-labeled dorsal
root ganglia neurons innervating the hip at day 7. Bar chart showingthat the proportions of FG-labeled dorsal root ganglia neurons are notsignificantly different between Sham, NGF50, and NGF100 group rats(n  6 each), respectively, at all the levels. The proportions of FG-labeled
dorsal root ganglia neurons at L1, L2, L3, and L4 are significantly higher
than those at L6 in Sham, NGF50, and NGF100 groups, respectively. Error
bars represent the standard deviation. *P  0.05 (Tukey–Kramer test).

We demonstrated that intra-articular injection of NGF into the rat
hip produced microinflammation without osteoarthritic change.
To our knowledge, this is the first report describing an NGFinduced rat model of hip pain. Ashrafs et al. [17] reported intraarticular injection of NGF into the rat OA knee. OA was induced
in rat knees by meniscal transection or intra-articular monosodium
iodoacetate injection, and then NGF added to evoke pain-related
behavior. However, there are no reports to our knowledge of intraarticular injection of NGF into rat normal hips. We found invasionof inflammatory cells in synovial tissue and angiogenesis in rat
hips. Walsh et al. [18] also described angiogenesis and NGF at theMod Rheumatol Downloaded from informahealthcare.com by Chiba University on 05/10/15
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DOI 10.3109/14397595.2015.1023977

A novel rat model of hip pain by intra-articular injection of nerve growth factor 5

Figure 6. Mean proportions of CGRP-IR Fluoro-Gold solution-labeled
dorsal root ganglia neurons at day 7. The proportion increases dose
dependently between Sham, NGF50, and NGF100 group rats (n  6 each).
Data are shown as mean  standard deviation. *P  0.05 (Mann–Whitney
U test followed by Bonferroni correction).

osteochondral junction in patients with rheumatoid arthritis andosteoarthritis. By contrast, we did not observe any joint space narrowing or cartilage degeneration. Furthermore, invasion of inflammatory cells in synovial tissue and angiogenesis were sustained forat least until 14 days after NGF application. NGF is a neurotrophinthat regulates neuronal development, survival, and maintenance[19]. NGF plays a key role in many persistent pain states, notably
those associated with inflammation [20–23]. We hypothesize that
direct application of NGF into the hip evokes an inflammatory
condition without degeneration of the articular cartilage and that
NGF can be used to model inflammatory hip pain in rats.
In the current study, CGRP was upregulated in DRG neurons byintra-articular injection of NGF. CGRP is a marker of sensory neurons typically involved in nociception [24–25]. Pain from the hip is
most likely transmitted by CGRP-IR DRG neurons [9]. Orita et al.
reported that the concentration of NGF was significantly elevatedafter 7 days in synovium in a rat model of knee MIA [26]. NGF
is retrogradely transported to DRG and mediates CGRP production. Thus, intra-articular injection of NGF influenced nociceptive
transmission at the DRG via production of CGRP. However, it isinteresting that the NGF50 group showed a significantly lower
proportion of CGRP-IR neurons than the NGF100 group despite
a similar extent of inflammation. Further study is needed to determine the minimum amount of NGF that causes inflammatorychange and increase in CGRP.
The mechanism of pain transmission in hip disorders is notcompletely understood. Nakajima et al. [9] reported that CGRP-IR
neurons play an important role in the perception of pain in the
hip joint in rats. They also documented that FG-labeled neurons
were distributed throughout the ipsilateral DRGs from T13 toL5, primarily at L1, L2, L3, and L4 in the hip joint. The current
study showed almost similar distributions and the distribution was
not changed despite NGF administration. These results explain
the clinical observation that pain is referred to originate in thethigh and lower leg in patients with disorders of the hip [6]. In
patients with OA of the hip, sensory innervation and inflammatory
cytokines in hypertrophic synovia are associated with nociception[14,27]. Shirai et al. [28] stated that hip pain occurs following theinvasion of the labrum with blood vessels and nerve fibers frominflamed synovial tissue following labral degeneration in the hip
joint affected by OA. Shigemura et al. [2] reported that 6% of
patients with OA of the hip showed neuropathic pain. These find-ings may provide the development of novel analgesic therapies for
hip joint pain including the treatments targeting NGF. Recently, arandomized clinical trial of an NGF inhibitor in OA of the hip andknee was reported to reduce joint pain [29–31].
There are several limitations to this study. First, the distributionof cytokines was not examined in histopathology of the hip. The
hip joint is a complex structure consisting of synovium, cartilage,
and bone. The level of inflammatory cytokines and their receptors,
the macrophages and endothelial cell markers in each structure
should be investigated quantitatively using protein determinationor gene expression. Second, hip pain was suggested by immunohistochemical determination of CGRP expression. Further study
is needed to determine pain-related behavior using other methods
such as evaluation of weight bearing in walking animals [32–33].
Third, dose dependency could not be found, since only NGF50 and
NGF100 groups were documented. Higher dose and lower dose
NGF models should be studied in the future.
In conclusion, a novel rat model of hip pain was produced byintra-articular administration of NGF into the hip joint. NGF elicited mild synovitis and expression of CGRP-IR in sensory nerves.
We suggest that NGF is crucially involved in the expression andtransmission of pain from the hip joint.­­

Acknowledgements
The source of support in the form of grants or industrial support: The corresponding author, JN, received Grants-in-Aid for
Scientific Research (Research Project Number: 25870125) for thiswork. The other authors do not receive any funding or financial
support that may be perceived to have biased the study.

Conflict of interest
None.

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