Accuracy of ultrasound-guided intraarticular injections in Guinea pig knees

Abstract

Dunkin Hartley guinea pigs, a commonly used animal model of osteoarthritis, were used to determine if high frequency ultrasound can ensure intra-articular injections are accurately positioned in the knee joint.
A high-resolution small animal ultrasound system with a 40 MHz transducer was used for image-guided injections. A total of 36 guinea pigs were anaesthetised with isoflurane and placed on a heated stage. Sterile needles were inserted directly into the knee joint medially, while the transducer was placed on the lateral surface, allowing the femur, tibia and fat pad to be visualised in the images. B-mode cine loops were acquired during 100 µl. We assessed our ability to visualise 1) important anatomical landmarks, 2) the needle and 3) anatomical changes due to the injection.
From the ultrasound images, we were able to visualise clearly the movement of anatomical landmarks in 75% of the injections. The majority of these showed separation of the fat pad (67.1%), suggesting the injections were correctly delivered in the joint space. We also observed dorsal joint expansion (23%) and patellar tendon movement (10%) in a smaller subset of injections.
The results demonstrate that this image-guided technique can be used to visualise the location of an intra-articular injection in the joints of guinea pigs. Future studies using an ultrasound-guided approach could help improve the injection accuracy in a variety of anatomical locations and animal models, in the hope of developing anti-arthritic therapies. Cite this article: Bone Joint Res 2015;4:1-5.
©2015 The British Editorial Society of Bone & Joint Surgery.

Full text

VOL. 4, No. 1, JANUARY 2015 1
KNEE
Accuracy of ultrasound-guided intra-
articular injections in guinea pig knees
N. Vázquez-
Portalatín,
G. J. Breur,
A. Panitch,
C. J. Goergen
From Purdue
University, West
Lafayette, Indiana,
United States
N. Vázquez-Portalatín, BS,
Graduate Student, Weldon
School of Biomedical Engineering
G. J. Breur, DVM, MS, PhD,
Professor of Small Animal
Surgery, Department of
Veterinary Clinical Sciences
A. Panitch, PhD, Leslie A.
Geddes Professor of Biomedical
Engineering, Weldon School of
Biomedical Engineering
C. J. Goergen, PhD, Assistant
Professor of Biomedical
Engineering, Weldon School of
Biomedical Engineering
Purdue University, West
Lafayette, Indiana 47907, USA.
Correspondence should be sent
to Professor C. J. Goergen; e-mail:
cgoergen@purdue.edu
doi:10.1302/2046-3758.41.
2000370 $2.00
Bone Joint Res 2015;4:1–5.
Received 23 September 2014;
Accepted 10 December 2014
Objective
Dunkin Hartley guinea pigs, a commonly used animal model of osteoarthritis, were used to
determine if high frequency ultrasound can ensure intra-articular injections are accurately
positioned in the knee joint.
Methods
A high-resolution small animal ultrasound system with a 40 MHz transducer was used for
image-guided injections. A total of 36 guinea pigs were anaesthetised with isoflurane and
placed on a heated stage. Sterile needles were inserted directly into the knee joint medially,
while the transducer was placed on the lateral surface, allowing the femur, tibia and fat pad
to be visualised in the images. B-mode cine loops were acquired during 100 μl. We assessed
our ability to visualise 1) important anatomical landmarks, 2) the needle and 3) anatomical
changes due to the injection.
Results
From the ultrasound images, we were able to visualise clearly the movement of anatomical
landmarks in 75% of the injections. The majority of these showed separation of the fat pad
(67.1%), suggesting the injections were correctly delivered in the joint space. We also
observed dorsal joint expansion (23%) and patellar tendon movement (10%) in a smaller
subset of injections.
Conclusion
The results demonstrate that this image-guided technique can be used to visualise the
location of an intra-articular injection in the joints of guinea pigs. Future studies using an
ultrasound-guided approach could help improve the injection accuracy in a variety of
anatomical locations and animal models, in the hope of developing anti-arthritic therapies.
Cite this article: Bone Joint Res 2015;4:1–5.
Article focus
Dunkin Hartley guinea pigs develop
osteoarthritis spontaneously and have
small knee joints.
Injections into the joint space of these ani-
mals are often performed without confir-
mation that the injection is delivered to
the correct location.
Can high frequency ultrasound be used
to improve intra-articular knee injections
in small animals?
Key messages
High frequency ultrasound can be used to
visualise intra-articular needle placement
and injections in the knee joints of guinea
pigs.
Separation of the fat pad showed fluid
accumulation near the cartilage surface,
confirming injections were in the correct
location.
This imaging method could be used with
other animal models to ensure the correct
positioning of injections when develop-
ing anti-osteoarthritic therapies.
Strengths and limitations
Strength: Real-time visualisation of nee-
dle placement and intra-articular injec-
tions with ultrasound can be used to
Freely available online
Keywords : osteoarthritis; car tilage; guinea pig; ultrasound; intra-articular; injection

2 N. VÁZQUEZ-PORTALATÍN, G. J. BREUR, A. PANITCH, C. J. GOERGEN
BONE & JOINT RESEARCH
improve accuracy and confirm delivery location of
anti-arthritic therapies.
Limitation: We were not able to visualise 25% of the
injections due to the small size of the knee joint, diffi-
culties with probe positioning, variation in the location
of the needle tip and unanticipated animal limb move-
ment during the injection. More practice and longer
recordings could improve this rate of visualisation.
Introduction
Osteoarthritis is a chronic joint condition characterised by
joint pain, crepitus, reduction of range of movement and
variable degrees of inflammation, cartilage erosion and
subchondral changes in bone. It is estimated that some-
where between 15% and 20% of the entire US population
suffers from some form of arthritis.1 While osteoarthritis
occurs at multiple locations, cartilage breakdown in the
knee leads to significant comorbidities since these are
weight-bearing joints.2 The development of locally
injected anti-arthritic drugs to limit or even prevent carti-
lage breakdown is a current area of research,3-6 but it is
difficult to ensure that injected material is deposited adja-
cent to cartilage in the synovial space. These false posi-
tives can lead to the question of whether a therapy is not
working correctly or has simply not been delivered to the
right location. Previous clinical studies in humans have
demonstrated that ultrasound guidance will improve the
accuracy of an intra-articular injection,7-10 but similar
studies that focus on animal disease models have not
been conducted. Indeed, the small size of many labora-
tory animals requires high frequency ultrasound capable
of producing images with good image resolution at shal-
low depths.11- 13
Dunkin Hartley guinea pigs develop spontaneous, age-
related osteoarthritis, characterised by cartilage degener-
ation, osteophyte formation, subchondral bone changes
and synovitis, making these animals a popular disease
model for the human condition.14 These albino guinea
pigs h ave hi stolo gical evid ence of osteo arth rit is as ea rly as
three months old and disease severity continues to
increase with age. Previous work has shown that proteo-
glycan content increases, collagen concentration in carti-
lage decreases, and radiological changes (including
osteophyte formation, sclerosis of subchondral bone,
femoral condyle cyst formation and calcification of collat-
eral ligaments) present more in older animals.14 At the
level of the tibial plateau, their knee joints are typically
2 cm to 3 cm in the cranio-caudal direction and 1 cm to
2 cm in the medio-lateral direction. The joint is < 1 cm
below the skin’s surface in the parapatellar region. These
features thus make visualisation with high frequency
ultrasound possible.
The purpose of this study was to determine if high fre-
quency ultrasound could be used to ensure intra-articular
injections were correctly deposited in the knee joint. While
we focused on the knees of Dunkin Hartley guinea pigs,
the methods of ultrasound-guided injection described
here could be used to improve accuracy of injection in a
variety of small animal models. Indeed, joint visualisation
should be possible as long as the anatomy allows for both
needle insertion and placement of the ultrasound trans-
ducer head. We did not focus on direct visualisation of the
needle with ultrasound, but rather the movement and
changes within the intra-articular space in order to quan-
tify the location of the injection. Future small animal stud-
ies using this ultrasound-guided approach can help
confirm intra-articular deposition of injected fluid and aid
in the development of anti-arthritic therapies.
Materials and Methods
A total of 216 injections in 36 Dunkin Hartley guinea pigs
were imaged for this study: 12 animals received one injec-
tion bilaterally, 12 animals received three injections bilat-
erally, and 12 animals received five injections bilaterally.
The right stifle joints were injected with 100 μl of phos-
phate-buffered saline and the left joints with 100 μl of a
proteoglycan (aggrecan) mimetic. Animals were anaes-
thetised with 2% to 5% isoflurane in 2 L/min O2 using a
chamber and a mask. The hair covering both knee joints
was removed with clippers and the skin was scrubbed
with chlorhexidine gluconate (2% dilution) and sterile
saline. The rate of respiration was monitored visually and
each animal was placed on a heated stage to maintain
body temperature. Depth of anaesthesia was assessed
with periodic toe pinches. Alcohol swabs were used to
clean the transducer and stage between animals.
A high-resolution small animal ultrasound system was
used for the image-guided injections (Vevo2100,
FUJIFILM VisualSonics Inc., Toronto, Canada). A 256-
element, 40 MHz linear transducer with a 7.0 mm geo-
metric focus (MS550D) was clamped to an adjustable
bench-mounted rail system designed for the positioning
of a small animal. The axial, lateral and elevation image
resolution were 40 μm, 90 μm and 193 μm, respectively.
Guinea pigs were positioned in dorsal recumbency on
the heated stage such that the knee joint could be easily
manipulated (Fig. 1).
A sterile 28-gauge needle was then inserted approxi-
mately halfway between the patella and tibial tuberosity,
just medial to the patellar tendon. The needle was
inserted into the knee joint until it was in direct contact
with the medial condyle of the femur, without the benefit
of ultrasound visualisation (Fig. 1). We then applied ster-
ile ultrasound gel over the lateral joint area before lower-
ing the ultrasound probe to the animal. Moderate
extension of the joint and firmly pushing the back of the
leg against the transducer surface is crucial to visualise
the tibial plateau, medial femoral condyle and fat pad at
the same time. We further refined the joint position until
lucent lines separating the fat pad and tibia and fat pad
and femur, representing joint space and articular carti-
lage, were clearly distinguishable (Fig. 2). The needle was

ACCURACY OF ULTRASOUND-GUIDED INTRA-ARTICULAR INJECTIONS IN GUINEA PIG KNEES 3
VOL. 4, No. 1, JANUARY 2015
inserted at an angle of 45° to the long axis of the ultra-
sound transducer in order to image the entire joint,
including the femur, tibia and fat pad. Once an accept-
able orientation was obtained, a 500-frame B-mode cine
loop of approximately five seconds was acquired during
the 100 μl injection of aggrecan mimic or PBS (control).
For each animal, one knee received aggrecan mimic injec-
tions while the contralateral joint received PBS injections.
We then assessed our ability to visualise 1) important ana-
tomical landmarks, 2) the needle and 3) anatomical
changes due to the injection.
Results
Anatomical landmarks of the knee joint could be distin-
guished from the ultrasound images in all animals (Fig. 2)
and clearly visualised in 75% of the injections (Table I). We
defined ‘visualised’ injections as those where separation,
movement, or expansion due to the build-up of intra-
articular fluid was observed of the fat pad, of the joint
space dorsal to the patella and trochlea, or of the patellar
tendon. Separation of the fat pad was identified when the
fat pad was physically pushed away from the femur or
tibia (Fig. 3). Dorsal joint expansion was identified when
fluid or bubbles moved into the joint space proximal to
the patella or trochlea. Finally, movement of the patellar
tendon was identified as injections that led to a pressure
increase that caused the tendon to bend or arch. No differ-
ences between injections of aggrecan mimic or PBS were
observed. Table I summarises our results by identifying
and characterising several anatomical features and
observed movement due to the injections.
The vast majority of the ultrasound images where the
injection was visualised also showed separation of the fat
pad (145 of 162; 89.5%), providing confidence that the
injected fluid was delivered to the correct location.
Furthermore, proximal/superior joint expansion was
observed both with and without separation of the fat
pad. Only 12 of the 50 injections where dorsal joint space
expansion was observed occurred without separation of
the fat pad. We also observed small bubbles in 28 of the
images (Fig. 4a), an occurrence that increased echo-
genicity of the injected fluid and helped with visualisa-
tion. Finally, five injections led to subcutaneous
expansion below the skin surface but above the patellar
tendon (Fig. 4b).
Discussion
The results of this study suggest that high frequency
ultrasound can be used to measure the accuracy of intra-
articular injections in the knee joints of Dunkin Hartley
guinea pigs. Because of the size of the stifle joint and the
need to maintain aseptic technique, insertion of the nee-
dle under ultrasound guidance was not possible. How-
ever, we were able to demonstrate that 75% of the
injections were deposited intra-articularly. The small knee
joint size, difficulties with probe positioning, variation in
the location of the needle tip and unanticipated animal
limb movement during the injection are likely reasons
why we were not able to visualise 25% of the injections.
Furthermore, even though we made a strong effort to
keep the ultrasound probe position consistent, the innate
variation between animals and the fast injection times
meant that visualisation was not always possible. In
future studies, longer cine loops of more than 500 frames
can be obtained to help ensure the exact time of injection
is acquired with the ultrasound system.
We realised during this study that several small
changes could be made to improve visualisation of the
injection. First, we began adding a small amount of air
into the syringes in order to create bubbles during the
injection. These bubbles helped us distinguish exactly
where the injected fluid appeared and spread. Similarly,
contrast-enhanced ultrasound using gas-filled micro-
bubbles has become common in clinical imaging.15
Fig. 1
Photograph of intra-articular injections in the right knee of a guinea
pig. Image shows the ultrasound transducer, needle and knee during
the injection. The right hand of the operator held the syringe that was
inserted in the joint, while the left hand controlled the positioning of
the limb of the guinea pig. The ultrasound probe, clamped to an
adjustable rail system (not shown), was oriented in a craniolateral-cau-
domedial oblique direction.
Fig. 2
Example ultrasound image of a knee joint with labels over the femur
(‘F’), tibia (‘T’), superior joint space and fat pad. These anatomical
landmarks were observed for each injection.

4 N. VÁZQUEZ-PORTALATÍN, G. J. BREUR, A. PANITCH, C. J. GOERGEN
BONE & JOINT RESEARCH
Second, the knee joints were also placed on the left side
of the image such that both the fat pad and superior joint
space could be visualised at the same time. This joint
placement facilitated observation of injections that
spread from the centre of the joint into the superior joint
space. Finally, small movements of the needle, before the
injection, allowed us to localise the tip location by either
visualisation of the metal artefact caused by the needle, or
from movement of adjacent tissue. While the 90° orienta-
tion difference between the probe and needle reduced
our ability to directly visualise the tip, small amounts of
movement of the needle helped to ensure tip placement
near the cartilage surface. Our experience also suggests
that both the success of the injection and quality of the
image improves with practice.
This high frequency technique of ultrasound injection
could be used to help guide intra-articular needle place-
ment for studies that focus on developing treatments for
osteoarthritis with a variety of animal models. While we
focused on the knee in this study, the hip, shoulder,
elbow, or any other joint where arthritis is common,
could also benefit from the use of ultrasound to improve
the accuracy of injections. This is particularly important
as advancements in protein and tissue engineering have
led to exciting new potential candidates that may one day
slow, prevent, or even reverse cartilage damage.5,6
The choice of animal models and anatomy will influ-
ence the ideal ultrasound frequency and position of the
probe. Larger animals or joints deep within the body
would likely require transducer frequencies of < 40 MHz
due to the inverse relationship between frequency and
depth. In other words, lower frequency ultrasound can
penetrate deeper into the body, but then produces
images with a lower resolution. Furthermore, joint
anatomy and positioning are important to consider as
ultrasound does not easily penetrate calcified bone. The
dense femur and tibia comprising the knee required us to
place the joint in moderate flexion such that the intra-
articular space could be visualised. Thus, ultrasound
probe placement, in addition to needle insertion, may
Tab l e I . Summary of total injections visualised and anatomical landmarks observed. We quantified the number and percentage of injections where the
needle or movement of the fat pad, superior joint space and/or patellar tendon was clearly observed.
Number of
injections Visualised
Needle
observed
Fat pad
observed
Separation
of fat pad
Superior joint
space observed
Superior joint
space expansion
Patellar tendon
observed
Patellar tendon
movement
n (%) 216 162 (75 . 0)21 (9.7) 205 (95.0) 145 (67.1) 108 (50.0)50 (23.1) 92 (42.6) 22 (10 .2 )
Fig. 3a
Ultrasound images showing substantial separation of the fat pad from the tibia a) before and b) after an injection.
Fig. 3b
Fig. 4a
Ultrasound images showing a) that bubbles helped visualise the injections and were clearly observed in the joint space between femur and
fat pad. The needle b) and subsequent metal shadow are clearly seen during an injection that created subcutaneous expansion.
Fig. 4b

ACCURACY OF ULTRASOUND-GUIDED INTRA-ARTICULAR INJECTIONS IN GUINEA PIG KNEES 5
VOL. 4, No. 1, JANUARY 2015
require some adjustments depending on the size, depth
and orientation of the region of interest. If the desired
location is not reached, an injection can always be
repeated to ensure proper administration. Indeed, clinical
studies have shown that the use of ultrasound improves
the accuracy of injection in the knee7,10 and hip.9
In conclusion, these data suggest that high frequency
ultrasound can be used to visualise intra-articular needle
placement and injections in small animals. Separation of
the fat pad from the cartilage surface showed that fluid
accumulates in the correct location within the knee joint.
This method could also be used with different frequency
probes for other animal models to ensure the correct
positioning of injections when developing anti-
osteoarthritic therapies.
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Funding statement:
This article was funded by the Purdue Research Foundation Trask Innovation Fund.
A. Panitch reports that monies have been received by the NIH and Symic Biomedical to
Purdue University which is not related to this ar ticle.
Author contributions:
N. Vázquez-Portalatín: Data collection, Data analysis, Injection assistance, Writing the
paper.
G. J. Breur: Data analysis, Injection assistance, Edit ing the paper.
A. Panitch: Data analysis, Editing the paper, Providing financial support.
C. J. Goergen: Data collection, Data analysis, Injection assistance, Writing and editing
the paper.
ICMJE Conflict of Interest:
None declared
©2015 The British Editorial Society of Bone & Joint Surgery. This is an open-access arti-
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