Original Research 413
© 2008 Schattauer GmbH
A quantitative analysis of the nerve fibres
of the acetabular periosteum of dogs
A. Schmaedecke1; J. P. E. Saut2; C. R. A. Ferrigno1
1
Department of Surgery, College of Veterinary Medicine, University of São Paulo, São Paulo, Brazil;
2
Department of Clinics, College of Veterinary Medicine, University of São Paulo, São Paulo, Brazil
Summary
There are many techniques for the treatment of hip
dysplasia, and novel research is currently being under-
taken in the hope of obtaining more efficient and less
traumatic techniques. The denervation of the hip joint
capsule is a simple and effective technique that allows
recovery of the functional activity of the affected limbs
in significantly less time than other techniques. This
surgical procedure consists of removing the acetabular
periosteum, thus eliminating the nerve fibres with con-
sequent analgesia. The aim of this investigation was to
quantify the number of nerve fibres present in different
regions of the acetabular periosteum. The knowledge of
regional differences is potentially valuable for the refin-
ing of the denervation technique of the hip joint cap-
sule. Thirty canine acetabular fragments were used to
compare the nerve fibre density of the periosteum. The
results showed a significant difference between the
mean density of nerve fibres at the cranial and dorsal-
lateral portion (approximately 75 fibres/mm2) and cau-
dal lateral portion (approximately 60 fibres/mm2) of
the acetabulum. Those fibres at the periosteum are al-
most positioned in a sagittal plane, pointing towards
the joint capsule, suggesting the same density in the
latter region. These results indicate a new approach to
the articular denervation technique, thus obtaining
even better results for the treatment of hip dysplasia in
dogs.
Keywords
Acetabulum, nerve fibres, analgesia, hip dysplasia,
dogs
Vet Comp Orthop Traumatol 2008; 21: 413–417
Prepublished online April 11, 2008
doi:10.3415/VCOT-07-10-0098
Received October 19, 2007
Accepted February 5, 2008
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Introduction
The study of the hip joint in dogs has be-
come important after the development of
new surgical techniques that enable the sen-
sory neurotomy of the acetabular region
with the aim of reducing pain. In 2002,
Staszyk and Gasse (1) proposed a multiple
innervation model based on segmented in-
nervations, where the nerve fibres penetrate
distinct areas of the articular capsule, as
well as the ‘indirect’ nerve fibres that con-
tinually penetrate the muscle through the
periosteum and the capsule. In contrast to
the shoulder joint, where the innervations
are not symmetrical, the hip joint has inner-
vations that are bilaterally symmetrical and
individually variable.
The close anatomical association be-
tween the nerve component and the joint is
well established, since the gluteal (2, 3) and
ischiatic (1, 4) nerve endings are frequently
implicated in medical situations, such as
fractures, dysplasia, and surgery.
The periosteum is a highly vascular (5)
structure which contains innumerous nerve
endings. There are various nerve fibres
within the periosteum which are more fre-
quently associated with blood vessels, either
ramifying around or ending at blood vessel
walls. Occasionally, isolated nerve fibres or
small nerve terminations can also be found
between cells, independent of blood vessels
(6).
Articular denervation is a simple and ef-
fective method that is used to treat articular
diseases of the hand and feet in human or-
thopaedics (7, 8). This technique is based on
the selective neurotomy of the sensitive
nerve fibres within the peri-capsular region,
thus resulting in permanent analgesia and
reactivation of the dynamic component of
the joint. Initial studies in veterinary medi-
cine have demonstrated that articular dener-
vation could be useful for the treatment of
diseases of the hip joint, such as hip dyspla-
sia and arthrosis (9).
This technique consists of the surgical
removal of the periosteum, in a semicircular
fashion, beginning at the cranial-dorsal
margin progressing to the ventral margin of
the acetabulum. Additionally, a circular area
is also removed at the insertion of the peri-
articular muscles, thereby eliminating sen-
sory nerve fibres in this area. Preliminary
experiments performed in dogs obtained ex-
cellent results (9).
In 2002, Kinzel et al. (9) reported on 17
dogs that had been treated using this tech-
nique. 90.6% of these animals clinically re-
covered, while 56% showed remarkably de-
creased lameness scores three days after the
surgery. One dog remained free of signs
after 10 years of treatment.
With the aid of a radiofrequency percut-
aneous equipment, Kawaguchi et al. (10)
used transitory block process of the sensory
nerve response of the obturator and femoral
nerves to treat articular pain in human hip
joints and obtained satisfactory results in
86% of the patients during a period of one to
11 months.
However, the greatest advantage of de-
nervation is the simplicity of the technique.
There is no need for sophisticated equip-
ment and the operation is restricted to the
periosteum, thus eliminating the nerve
fibres within this area, without disturbing
the articular capsule or the joint. Such sim-
plicity renders this surgical procedure feas-
ible for young and old patients. Other ad-
vantages of this technique include the re-
duced surgical time and the shorter recovery
period in comparison to other pre-existing
procedures. Furthermore, in the authors’ex-
perience, the low cost of the operation is yet
another factor that easily persuades owners
Vet Comp Orthop Traumatol 5/2008
414
Schmaedecke et al.
to proceed with the treatment. Even owners
of older dogs seem to prefer this technique
in comparison to other forms of treatment
which are commonly associated with pro-
longed recuperation periods or onerous con-
servative treatments.
The objective of this study was to quan-
tify the nerve fibres of the acetabular peri-
osteum of dogs aiming to refine a low-cost,
precise and efficient denervation technique
for canine hip joints.
Material and methods
Thirty hip joints from eight male and seven
female mongrel dogs, weighing between 22
to 62 kg, from two to eight years of age,
without any evidence of bone disease, that
had been sacrificed for reasons unrelated to
the study (in accordance with the bioethics
committee of the University of São Paulo)
were studied. All of the specimens were col-
lected and placed in fixative within three
hours after euthanasia. The acetabular re-
gion was dissected, and released from all
muscular and neurovascular insertions. The
joint capsule and ligaments were then re-
moved, followed by luxation of the femoral
head at the point of incision and rupture of
the femoral head ligament. A fragment of
this region extending from 1 cm cranially
and 1 cm caudally to the acetabular margin
was removed.
This fragment was measured, divided in
frontal plane sections into three equal parts
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Fig. 1
Image showing the divi-
sion and subdivion of the
acetabulum.
and labeled division A (cranial region), B
(dorsal-lateral region), and C (caudal re-
gion); these were further subdivided and la-
beled 1, 2, and 3 (Fig. 1).
From each subdivision, central 1 cm2
fragments were obtained. Each fragment
was obtained after removing the cancellous
sub-cortical bone; only the periosteum and
the cortical bone were utilized. These frag-
ments were then identified based on their
place of origin, denominated A1, A2, and
A3, or B1, B2, and B3, or C1, C2, and C3.
The segments were individually fixed in
10% buffered neutral formalin solution for
five days.
After fixation, all of the fragments were
decalcified in solution made up of 5% hy-
drochloric acid, 3% acetic acid, and 10%
Fig. 2 Photomicrography from cranial lateral aspect of
the acetabulum. Nerve fibres identified by silver impreg-
nation (magnification 400X).
chloroform, 10% distilled water, and 73%
alcohol at 95% (V/V). The time for dec-
alcification varied according to each frag-
ment, and was checked daily by penetration
of the bone with a 6 metric (22 gauge)
needle. Decalcification was assumed to be
achieved when the needle passed freely
through each section.The time for decalcifi-
cation varied between six and 15 days. The
fragments were then washed with water to
remove the excess of decalcification sol-
ution and subsequently processed for histo-
pathology.
Silver impregnation
Nerve fibres were identified by silver im-
pregnation (Fig. 2) using the method pro-
posed by Linder (11), with some adap-
tations. This method involved the use of a
1% silver nitrate solution with pH con-
trolled by addition of a buffer solution of
0.5% borax/ boric acid until a pH of 7.8 was
obtained (6).
Randomization
This study was randomized, considering
equally each region evaluated, independent
of the animal studied. Of all of the animals
only one of the areas (1, 2 or 3) from each of
the regions (A, B or C) was selected and
then analyzed. Consequently, in this study
30 acetabular samples were utilized (15
from the left and 15 from the right antime-
rus).
Exclusion criteria
The following criteria for exclusion of dogs
were established:
● Dogs that did not meet the previously
mentioned requirements;
● Dogs whose clinical record revealed any
presumptive or definitive diagnosis of
any infectious disease;
● Dogs that demonstrated clinical signs of
degenerative articular disease (12).
 415
Nerve fibres of periosteal acetabulum

Negative controls
The specificity of the impregnation utilized
in this study was verified by the usage of
histological popliteal hyaline cartilage
samples from the same studied animals,
since this structure normally lacks nerve
fibres (Fig. 3).

Measurements Fig. 3
Photomicrography. Hya-
Digitalized 400X photomicrographs of the line cartilage by silver
area of each of the fragments were obtained staining. Note the absence
of nerve fibres (magnifi-
using a digital camera (Canon E820, cation 400X)
Canon Inc., Tokio, Japan) affixed to a
microscope. Each fibre was identified and
determined by the software Image Pro Plus
v 4.5 (Media Cybernetics, Silver Spring,
Table 1
MD, USA). Each fragment was analyzed in Mean values, standard Parameters Antimere
three different areas and the average number deviation, and amplitude Right Left
of nerves per square mm was calculated. variation of the number of
nerve fibres of the acet- Mean (fibres/mm2) 69.84 68.27
abular periosteum of dogs Standard deviation (fibres/mm2) 8.21 8.32
(distribution based on the Interval of data (fibres/mm2) 54 – 83 51–85
Statistical analysis antimere analyzed)
Note: Mean values do not present significant statistical differences by Tukey test.
Statistical analyses were performed using a
computer statistical package (Minitab, Re-
lease 13, 2000 – Minitab, Inc., State Col-
lege, PA, USA). The data obtained were
tested for normality using the Kolmogorov-
Smirnov Test. Parametric distributions were
obtained and the variance analysis was de-
termined by one-way ANOVA with Tukey
post-hoc test. The differences were con-
sidered as significant when P ≤ 0.05.

Results
Significant statistical differences were not
observed regarding the mean density of
nerve fibres of the acetabular periosteum of
the right antimere (69.84 ± 8.21), when
compared to the left (68.27 ± 8.32), P=0.09.
Furthermore, there was a lack of significant
differences between each division of the
stipulated regions in both antimera. Never-
theless, marked significant differences were Fig. 4 Distribution of nerve fibres in the acetabular periosteum based on the analysis of each region. The regions A, B and
observed between regions A and B (cranial C indicate anatomical cranial, dorsal-lateral and caudal positions, respectively. The non-coincident letters, a, b, and c, indicate
significant statistical difference between regions, independent of the antimerus analyzed – Tukey test (p ≤ 0,05); The non-
and dorsal-lateral regions) of each antimere, coincident letters, A and B, indicate significant statistical difference between within the antimerus analyzed Tukey test (p ≤
when these were compared to region C (cau- 0,05); Horizontal lines represent medians; Vertical lines represent interval of data; The bar represents 75% of the values. Full
dal region) (Table 1 and Fig. 4). circles represent averages; and *depicts discrepant value.

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416
Schmaedecke et al.
Discussion
The relative sensitivity of a bone to a painful
stimulus and its relation to the innervation
density of the periosteum is well known and
has been discussed in depth in the veterinary
literature (6, 13, 14). Although the inner-
vation of the periosteum is mainly respon-
sible for the painful sensation of the bone
tissue, it is certainly not the only factor as-
sociated with bone nociception (15, 16). Pa-
tients with painful bones, due to diseases re-
stricted to cancellous bone or partial lesions
that involve small portions of cortical bone,
did not exhibit signs of periosteal damage
(15, 16).
It has been reported that blood vessels
and nerve fibres occur together in the peri-
osteum (17–19). The silver impregnation
technique that was used in this study was not
specific enough to study the association of
the direction of nerve fibres and blood
vessels. Thurston (6) investigated the den-
sity of the nerve fibre population present at
the periosteal area of long bones of human
beings by using the same impregnation
technique. Again, an association between
the course of blood vessels and nerve fibres
could not be found.
Silver impregnation of nerve fibres is a
well-established technique (13). When
silver impregnation was compared with im-
munohistochemical staining techniques for
the identification of nerve fibres in the
femur of rats, a marked difference was not
observed (14). Moreover, the extreme spe-
cificity of each reagent used in immunohis-
tochemical methods must be taken into con-
sideration.
The silver impregnation technique used
in this study did not stain any of the struc-
tures in the hyaline cartilage examined be-
cause of its nerve tissue staining specificity.
Consequently, due to these parameters of
specificity and selectivity in association
with the low cost of staining, the silver stain-
ing technique that was applied in this study
was used to identify nerve fibres in the acet-
abular periosteum.
In a microscopic study in mice femora
using imunohistochemstry staining of nerve
fibres, Mach et al. (14) divided the whole
bone into three regions, based on anatomi-
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cal marks, in order to obtain more accurate
results. To quantify the fibres, a computer
programme was used (Image pro-plus 4.5,
Media Cybernetics, Silver Spring, MD,
USA). In our study, in agreement with the
aforementioned model, the acetabulum was
divided in three smaller fragments and, with
the aim of minimizing the influence of po-
tential artifacts provoked by osseous irregu-
larities in order to get more precise results,
these fragments were further divided into
three, thus making nine portions from each
antimere. The final image was processed
using the same software.
The results obtained in this investigation
are similar to those described by Staszyk
and Gasse in 2002 (1) but differ to those
found by Kinzel and coworkers in 1998
(20). The former group demonstrated that
by using acetylcholinesterase, the periosteal
innervation present in the cranial-lateral and
caudal-lateral regions of the dog’s acetabu-
lum comes from nerve fibres which were
originally innervating muscular fibres. Kin-
zel et al. (20) demonstrated the presence of
nerve fibres only at the cranial-lateral re-
gion, showing an absence of nerve fibres at
the caudal-lateral region. In our study, we
were able to prove the presence of nerve
fibres in all regions of the acetabulum, thus
proving that the density of nerve fibres
differs between the cranial-lateral (regions
A and B of our study) and caudal-lateral (re-
gion C of our study).
The density of the nerve fibres obtained
in this study, expressed as an average
(fibres/mm2) has not previously been de-
scribed in the literature in relation to the
anatomical region analyzed.
Judging by the degree of sclerosis ob-
served, in severe hip dysplasia cases, par-
ticularly at the cranial-lateral acetabular
margin, with subsequent cranial-dorsal sub-
luxation of the hip joint, it had been hypo-
thesized that this region is the most affected
by abnormal force behaviours caused by ar-
ticular incongruence (9). Investigations of
human hip joints demonstrated that the
higher concentration of nerve terminations
is localized where compressing forces are
acting. Kinzel et al. (20) reported that the
highest density of nerve terminations
relative to pain is localized in the cranial-lat-
eral region of acetabulum of dogs. At the
time that this group published their results,
there were not any reports about the inner-
vation of the caudo-lateral and ventral por-
tions of hip joint. Because of this, Kinzel et
al. (9) only suggest a cranial-lateral acetabu-
lar periosteum removal as an efficient form
of denervation of hip joint capsule. As a re-
sult, a 90% improvement is reported in pa-
tients on whom this technique was perform-
ed. In our study, we demonstrated the pres-
ence of a significant number of nerve fibres
in the caudal-lateral region of acetabular
periosteum in all analyzed areas.
With these data, Ferrigno et al. (21, 22)
had demonstrated a new surgical approach
to the denervation technique, thus improv-
ing freedom of movement and quality of life
in 95% of the patients.
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Correspondence to:
Alexandre Schmaedecke, DMV, MSc, PhD
University of São Paulo – Surgery
Av Prof Orlando Marqus de Paiva
87 Cidade Universitária São Paulo
São Paulo 05508–900, Brazil
Phone: +55 11 8127 0005, Fax: +55 41 3350 5623
E-mail: alexsvet@usp.br
Vet Comp Orthop Traumatol 5/2008