Yonago Acta medica 2007;50:57–63

Effects of Sciatic Neurectomy on Arthritis and Bone Lossin Rats with Collagen-Induced Arthritis
Masaru Kuwamoto, Toru Okano, Satoru Fukata, Makoto Enokida and Hiroshi
HaginoDivision of Orthopaedic Surgery, Department of Medicine of Sensory and Motor Organs, Tottori
University Faculty of Medicine, Yonago 683-8504 Japan

We investigated the effects of sciatic neurectomy on arthritis and bone mineral density(BMD) in 7-month-old female Sprague-Dawley rats with collagen-induced arthritis (CIA).
After the animals obtained a uniform mean body weight, we divided them at random into
4 groups, and treated them with the following surgical manipulations: (i) sham group (n= 8), sham surgery; (ii) CIA group (n = 9), collagen sensitization and sham surgery; (iii)
NTx group (n = 9), sciatic neurectomy and (iv) CIA + NTx group (n = 9), collagen sensitization and a sciatic neurectomy. Every 2 weeks up to 8 weeks after sensitization, the arthritis score for hind paw swelling was evaluated, and BMD of the cancellous and corticalbones in the proximal tibia was measured by peripheral quantitative computed tomography (pQCT). Both the hind paw swelling and arthritis score in the CIA + NTx groupwere signiﬁcantly lower than those in the CIA group at 6 and 8 weeks after sensitization.
There was no signiﬁcant difference in the cancellous BMD between the CIA and CIA+ NTx groups. The cortical BMD of the tibial metaphysis was signiﬁcantly lower in theNTx and CIA + NTx groups than in the sham group at 4 and 8 weeks, and in the CIA +NTx group than in the CIA group at 4 weeks after sensitization. There was no signiﬁcantdifference between the CIA and sham groups as well as the CIA and CIA + NTx group at
8 weeks after sensitization. It was concluded that sciatic neurectomy suppressed the severity of arthritis, but did not affect the cancellous bone loss in adult CIA rats.
Key words: bone mineral density; collagen-induced arthritis; peripheral quantitative computed
tomography; rheumatoid arthritis; sciatic neurectomy

healthy individuals (Hooyman et al., 1984).
Therefore, control of osteoporosis in RA patientsis an important issue.
RA is a disease to which middle-aged toelderly persons are susceptible, similar to lifestyle related diseases. Paralytic diseases such asstrokes, which are lifestyle related diseases, areknown to cause disuse bone loss and to worsen
osteoporosis (Takamoto et al., 1995). The risk for
fracture may increase when RA patients have ac-One of the common complications of rheumatoid
arthritis (RA) is osteoporosis, which is categorized into generalized osteoporosis and localizedosteoporosis. Periarticular osteoporosis, whichis included in the latter category, is a lesion thatdevelops early in RA and it is an important ﬁnding in the diagnosis of the disease (Alenfeld et al.,2000). In addition, RA patients have a 1.51-foldincrease of risk for hip fracture and a 2.56-foldincreased risk for pelvic fracture compared toAbbreviations: BMC, bone mineral content; BMD, bone mineral density; CIA, collagen-induced arthritis; NTx group,group of sciatic neurectomy; pQCT, peripheral quantitative computed tomography; RA, rheumatoid arthritis

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University Faculty of Medicine. The animals weregiven regular chow (CE-2, Clea Japan, Tokyo,Japan; 1.18 g/100 g Ca, 250 IU/100 g vitamin D3)
and tap water ad libitum. The animals were maintained at a temperature of 24˚C with 12 h lighting(7:00 AM to 7:00 PM). After acclimatization for
2 weeks, the animals obtained a uniform mean
body weight. We divided the animals at random
into 4 groups, and treated them with the following surgical manipulations: (i) sham group (n = 8),sham surgery; (ii) CIA group (n = 9), collagen sensitization and sham surgery; (iii) NTx group (n = 9),
sciatic neurectomy and (iv) CIA + NTx group (n =9), collagen sensitization and sciatic neurectomy.

companying paralysis. On the other hand, neuralparalysis is known to have a controlling effecton osteoarthritis and rheumatoid arthritis. For
instance, it has been reported that the progressionof osteoarthritis is suppressed in a leg paralyzed
by stroke (Coste et al., 1935) and peripheral nerve
palsy (McEwen, 1940). In addition, many studies
have reported that the occurrence and progressionof rheumatoid arthritis are suppressed in legs paralyzed by stroke (Bland et al., 1968) and poliomyelitis (Glick, 1967). Okumura et al. (1987) havereported that sciatic neurectomy performed on
adult female rats caused osteoporosis in the paralyzed legs and worsened osteoporosis when it wasperformed on rats with oophorectomy. Weinreb
et al. (1989) have reported that sciatic neurectomy
caused cancellous bone loss in the proximal tibia.
Thus, sciatic neurectomy is considered to be an
appropriate method for studying the effects ofneural paralysis on arthritis and osteoporosis.
There have so far been no reports on the effect of neural paralysis on osteoporosis associatedwith RA in either animals and humans. Collageninduced arthritis (CIA) in rats has been used as amodel for studying the pathology and therapy ofRA. Enokida et al. (2001) have reported that adult
CIA rats were more appropriate than young CIA
rats for studying secondary osteoporosis associatedwith RA, because the bone loss in adult CIA rats
occurred mainly in periarticular regions, which resembled secondary osteoporosis in the early stageof RA. The aim of this study was to investigate theeffects of sciatic neurectomy on arthritis and periarticular osteoporosis in adult CIA rats.

Preparation of the CIA model
After intraperitoneal anesthesia with 50 mg/kg
of ketamine hydrochloride and 10 mg/kg bodyweight of xylazine, 1 mL of emulsion containing
500 μg of bovine type II collagen (0.3% acetic
acid solution, k-41; Cosmo-Bio, Tokyo) and 500
μg of incomplete adjuvant (521-00021, Difco Laboratories, Detroit, MI) was intracutaneously injected at 3 sites on the back of each rat. For additional sensitization, 0.5 mL of the same emulsion
was intracutaneously injected into the posterior ofboth hip joints 1 week after the initial sensitization (Trentham, 1977). Physiological saline wasintracutaneously injected by the same procedurein the sham and NTx groups.
Sciatic neurectomy
Under anesthesia with the same method, a dorsolateral incision was made on the bilateral hips at thesame time of the initial sensitization. The sciatic
nerves were exposed, and 0.5 cm sections were excised. The muscle and skin were then sutured.

Materials and Methods
Animals
Seven-month-old female Sprague-Dawley rats
weighing 290 to 350 g (Simizu Laboratory Supply, Kyoto, Japan) were used in the present study.
The study was carried out in accordance with theGuidelines for Animal Experimentation of Tottori

Gastrocunemius muscle weight
All animals were anesthetized with 50 mg/kg ketamine injected intraperitoneally together with 10mg/kg xylazine, and killed by exsanguination at58

Sciatic neurectomy in CIA rats

4.6% with repositioning. The BMD value at thetime of initial sensitization (week 0) was regarded
as 100 (baseline), and the BMD values at 4 and 8weeks after sensitization in each group were presented as the percentage to the value at week 0.

8 weeks after the sensitization. The bilateral gastrocunemius muscles were removed, and immediately weighed, using a Sartorius balance (Northern
Balance and Scale, Blooming, MN).
Evaluation of arthritis

Statistical analysis

Hind paw swelling was quantiﬁed by measuring
the ankle width from the medial malleolus to thelateral malleolus with a constant-tension caliper at
2 week intervals for 8 weeks. Soft X-ray imagesof both hind paws were taken with a soft X-ray radiographic apparatus (SPO-M50; Sofron, Tokyo),operating at 40 kV peak and 2 mA at an exposure
time of 30 s at 2 week intervals for 8 weeks. The
radiologic arthritis score of the right ankle andsubtalar joints was assessed according to Engelhardt’s method (Engelhardt et al., 1995) using
X-ray images. The body weight, hind paw swelling and radiologic arthritis score were measured
every 2 weeks for 8 weeks.

All data values are presented as the mean and SE.
Dunn’s test was used for comparisons among multiple groups, and Dunnet’s test was used for comparisons between the sham and other groups. The
effects of neurectomy and arthritis on BMD wereanalyzed by the two-way analysis of variance. All
statistical analyses were performed using Stat View
J-5.0 (SAS Institute, Cary, NC) on Windows. A
signiﬁcant level of P < 0.05 was used for all comparisons.

Results

Bone mineral density

Body weight and gastrocnemius muscle
weightThe bone mineral density (BMD) in the proximal
metaphysis (2 mm distant from the epiphyseal line)of the left tibia was measured every 2 weeks for 8weeks after the initial sensitization by peripheral
quantitative computed tomography (pQCT; Model
XCT-960, Norland Stratec, Prorzhem, Germany).
Under the anesthesia just described, the animals
were restrained so that the bone axis of the proximal metaphysis was vertical to the scan beam. Voxel size and slice thickness were set to 0.295 mm and1 mm, respectively. The metaphysis was identiﬁed
and BMD was measured. The analysis parameters
were set to contmode 2, peelmode 2 and cortmode 1,and the measurement parameters were set to bone
area (square millimeters), bone mineral content,and BMD (milligrams per cubic centimeter) for
total bone (total BMD), trabecular bone (trabecularBMD) and cortical bone (intra-cortical BMD). The
threshold value was set to 0.65 for cortical bone.
The coefﬁcient of variation for the measurement ofBMD by pQCT was 1.7% without repositioning andThe mean body weight decreased gradually in theCIA, NTx and CIA + NTx groups. After the initial
sensitization, the body weight loss was signiﬁcantat 6 and 8 weeks in the CIA group, and at 8 weeks
in the NTx and CIA + NTx groups in comparison
with the sham group (Fig. 1A). The weight of gastrocnemius muscle at 8 weeks after the initial sensitization was signiﬁcantly lower in the CIA, NTxand CIA + NTx groups than in the sham group (Fig.
1B), and was also significantly lower in the NTxand CIA+NTx groups than in the CIA group.
Changes in hind paw swelling
The incidence of arthritis was 100% in both theCIA group and CIA + NTx group. Significant
increase of swelling of the hind paws was notedin the CIA groups at 4, 6 and 8 weeks after theinitial sensitization compared with the sham andNTx groups. In the CIA + NTx group, the swell59

M. Kuwamoto et al.

ing was signiﬁcantly higher than in the sham andNTx groups, and was signiﬁcantly lower than in
the CIA group at 6 and 8 weeks (Fig. 2A).

8 weeks after the initial sensitization (Fig. 2B).
Typical X-ray photographs of the ankle and subtalar joints in CIA and CIA + NTx groups (Fig. 3)
showed that arthritis was less severe in the CIA +NTx group (Fig. 3B) than in the CIA group (Fig.
3A).

Changes in the radiologic arthritis score of
the hind paw
The radiologic arthritis score of the right hindpaws was significantly higher in the CIA thanin the sham and NTx groups at 4, 6 and 8 weeks
after the initial sensitization. In the CIA + NTx
group, the score was signiﬁcantly higher than inthe sham and NTx groups at 4, 6 and 8 weeks,
and was lower than in the CIA group at 6 andChanges in the BMD
The total BMD in the CIA, NTx and CIA + NTx
groups was significantly lower in the NTx andCIA + NTx groups at 4 and 8 weeks, and in theCIA group at 8 weeks after the initial sensitization than in the sham group. However, there wasno significant difference among the CIA, NTx
and CIA + NTx groups (Fig. 4A). The cancellous

Fig. 1. Changes in body weight of rats in each group(A) and weight of the gastrocunemius muscle at 8 weeks
after the initial sensitization (B). aP < 0.05 and bP <0.01 compared with sham rats (Dunnett’s test). fP < 0.01
compared with CIA rats (Dunn’s test). Values expressed
as mean ± SE. CIA, collagen sensitization; NTx, sciatic
neurectomy.

Fig. 2. Changes in hind paw swelling (A) and radiological arthritis score (B) in rats of each group. aP < 0.05
and bP < 0.01 compared with sham rats (Dunnett’s test).
cP < 0.05 and dP < 0.01 compared with NTx rats (Dunn’s test). eP < 0.05 and fP < 0.01 compared with CIA rats
(Dunn’s test). Values expressed as mean ± SE. CIA, collagen sensitization; NTx , sciatic neurectomy.

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