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Quantitation of the changes in vascularity during arthritis in the knee joint of a mouse with a digital image analysis system.

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THE ANATOMICAL RECORD 262:420 – 428 (2001)
Quantitation of the Changes in
Vascularity During Arthritis in the
Knee Joint of a Mouse With a Digital
Image Analysis System
PIETER BUMA,1* MARTIN GROENENBERG,1 PAUL F.J.W. RIJKEN,2
WIM B. VAN DEN BERG,3 LEO JOOSTEN,3 AND HANS PETERS2
1
Department of Orthopaedics, Orthopaedic Research Laboratory,
University Medical Center, Nijmegen, The Netherlands
2
Department of Radiotherapy, University Medical Center, Nijmegen, The Netherlands
3
Department of Rheumatology, University Medical Center, Nijmegen,
The Netherlands
ABSTRACT
Many joint and bone diseases are caused by, or associated with vascular changes.
Particularly in rheumatoid arthritis, vascular sprouting of synovial vessels plays a major role
in the generation of joint pathology. To assess the effects of pharmaceuticals that are
designed to inhibit neovascularization, we developed a quantitative procedure to measure
vascular changes in cross-sections of the mouse knee joint during arthritic inflammation.
Arthritis was induced in the knee joint of C57Black6 mice by a single subpatellar injection of
methylated BSA after previous immunization. Total vascularity was visualized with a specific monoclonal rat anti-mouse antibody (9F1). Functional vessels were detected with the
fluorescent perfusion marker Hoechst 33342. The localization of Hoechst and the vascular
marker 9F1 were analyzed in separate images with an automated digital image processing
system. By combining the two images, total vascularity and the perfusion status of the
vessels during arthritis could be established. The digital image system measures synovial
area (SA), number of all blood vessels (NBV) and the number of perfused blood vessels
(NpBV). From these parameters the percentage of perfused vessels (perfusion fraction; PF),
the vessel density (VD ⫽ NBV/SA) and the density of perfused vessels (VDp ⫽ NpBV/SA) can
be calculated. The measurements showed that the area of synovial tissue had increased
during arthritis. Moreover, both the number of blood vessels (NBV) and the number of
perfused vessels (NpBV) in the synovial area had increased significantly on Days 4 and 7
after arthritis induction. This procedure enabled quantitation of total vascularity and of
functional blood vessels in cross-sections of synovial tissue. It is expected to be a powerful
tool, not only to analyze the effects of anti-angiogenic therapies in animal models of arthritis,
but could also be applicable to study vascular and perfusion changes in vascular related
diseases of the skeleton. Anat Rec 262:420 – 428, 2001. © 2001 Wiley-Liss, Inc.
Key words: joint pathology; rheumatoid arthritis; neovascularization; digital
image processing; synovial tissue
Many joint diseases are associated with vascular
changes. For instance, changes in vascularity may play a
role in the etiology of osteoarthritis, avascular necrosis
and bone marrow edema. Particularly in rheumatoid arthritis, the synovium shows a chronic inflammatory reaction, with neovascularization, infiltration of lymphocytes,
plasma cells and macrophages. Cells in this tissue that
may even grow out as pannus tissue over the surface of the
cartilage, produce catabolic factors for cartilage (Ben et
al., 1995; Colville-Nash and Scott, 1992; Fava et al., 1994;
©
2001 WILEY-LISS, INC.
FitzGerald et al., 1991). The expansion of this hyperplastic synovial tissue is associated with an extensive prolif-
*Correspondence to: Dr. P. Buma, Dept. Orthopaedics, Orthopaedic Research Laboratory, PO Box 9101, 6500 HB Nijmegen,
The Netherlands. E-mail: P. Buma@orthp.azn.nl
Received 17 July 2000; Accepted 19 October 2000
Published online 28 February 2001
DIGITAL IMAGE ANALYSIS OF MOUSE KNEE JOINT
eration of blood vessels (Blake et al., 1989; Buma et al.,
2000; Colville-Nash and Scott, 1992). The regulation of
neovascularization in the synovium of arthritic joints is
complex and various growth factors and inflammatory
mediators may be involved (Ben et al., 1995; Blake et al.,
1989; Claffey and Robinson 1996; FitzGerald et al., 1991;
Nagashima et al., 1995; Stevens et al., 1991).
Inflammatory cells that may contribute to the catabolic
effect on cartilage cells caused by cytokine release can only
reach the joint via the blood vessels. In addition to the
well-known therapies for rheumatoid arthritis on the basis of fast-acting non-steroidal anti-inflammatory agents
(NSAID) and long-term suppressive drug therapy with
penicillamine, azathioprine, methotrexate and gold, new
drugs have now been developed that specifically inhibit
angiogenesis only (Oliver et al., 1995). In theory, the influx of inflammatory cells would be prevented or diminished if vascular proliferation could be inhibited, potentially reducing the disease activity of rheumatoid
arthritis.
To assess the primary effects of any new therapy strategy, it is essential to obtain quantitative information on
the number of vessels, but also on the perfusion status of
the vascular network in the synovial tissues during the
arthritic process. Therefore, the aim of this study was to
adapt a methodology that is already available for the
assessment of vascular perfusion of tumour tissue in
joints (Bernsen et al., 1999; Bussink et al., 1998, 1999;
Rijken et al., 1995). This will allow quantitative assessment of the effects of drug therapies on time-specific
changes in vascularity during arthritis.
As a model system we used an arthritic model in the
mouse (van Beuningen et al., 1989; van de Loo et al., 1995;
van den Berg et al., 1981; van Lent et al., 1987). All vessels
were visualized with immunofluorescent staining with a
monoclonal marker 9F1, a specific marker for endothelial
cells (Westphal et al., 1997). The fluorescent dye Hoechst
33342 was used as a marker for perfused vessels (Rijken
et al., 1995; Smith et al., 1988). Hoechst is a small molecule that rapidly binds to the nucleus of all cells, irrespective of their location. Images of Hoechst and 9F1 staining
were analyzed quantitatively by a scoring system that was
programmed to combine the two images (Rijken et al.,
1995).
MATERIALS AND METHODS
Experimental Animals
Twenty-four adult female C57Black6 mice were used in
this experiment. All mice were immunized by subcutaneous injection of an emulsion of 100 ␮g methylated bovine
serum albumin (mBSA) with complete Freund’s adjuvant
in the foreleg and a subcutaneous injection of 100 ␮g of
this emulsion in the back of the mice (Kruijsen et al., 1983;
van den Berg et al., 1981; van Beuningen et al., 1989; van
de Loo et al., 1995). Additionally, an intraperitoneal injection of 1 ml of desiccated Bordetella pertussis was given.
On Day 7, the same procedure was repeated as a boost.
Then, after 21 days, arthritis was induced by a single
intra-articular injection of 60 ␮g mBSA in 6 ␮l saline
through the patellar ligament of the right knee joint. The
left knee joint served as control in this study.
Fixation
Both on Days 4 and 7 after inducing arthritis, 12 animals were killed for routine histology (five animals in each
421
time point) and for vascular quantification (seven animals
in each time point). For routine histology, joints were fixed
by immersion in 0.1 M phosphate-buffered (pH 7.4) 4%
paraformaldehyde, embedded in polymethylmethacrylate
and sectioned (7 ␮M) in the frontal plane. For vascular
analysis animals were injected intravenously in one of the
lateral tail veins with a 0.2 ml solution of phosphatebuffered saline (PBS; pH ⫽ 7.4) containing 0.3 mg Hoechst
33342 (Sigma Chemical Company, St. Louis, MO). One
minute after injection with Hoechst, the mice were killed
by cervical dislocation and both knee joints were quickly
removed (in circa 10 sec), embedded in Tissue Tek Optimal Cutting Temperature (OCT; Miles Diagnostics Division, Elkhart, IN) and snap frozen in isopentane (⫺80°C).
This prevented the Hoechst dye from diffusing too far into
the tissues.
Histological Procedures
The knee joints were sectioned (see Fig. 1 for schematic
representation), without previous decalcification in the
frontal plane into 7 ␮m sections at ⫺22°C on a Bright 3050
cryostat with a tungsten edged knife and mounted on
slides precoated with aminopropyltrietoxysilaan (Sigma).
The sections were air dried for 1 hr and stored at ⫺80°C
until required.
In each knee, three sections were analyzed. The middle
section was the most central section containing synovial
membrane tissue. The other two sections were located at
equal distance (200 ␮m) before and after this central section. After thawing, the sections were fixed in cold acetone
for 10 min and then washed in phosphate-buffered saline
(PBS, pH 7.4) for 5 min. Sections were subsequently incubated at room temperature in a moistened chamber for 45
min with an undiluted monoclonal rat-anti-mouse endothelial cell antibody (9F1, a gift from the Department of
Pathology, University of Nijmegen; Westphal et al., 1997).
Subsequently the sections were washed in PBS for 5 min
(3 changes), followed by a 30-min incubation with TRITClabeled secondary antibody (goat-anti-rat-TRITC, Jackson
ImmunoResearch Laboratories, Inc., West Grove, PA;
Sanbio; dilution 1:50). After again three washes in PBS,
the sections were incubated for 30 min with the TRITClabeled third antibody (donkey-anti-goat-TRITC, Jackson
ImmunoResearch Lab, Sanbio; 1:50). The second and
third antibodies were diluted in PBS containing 0.5% bovine albumin solution. The wet sections were then shortly
washed in PBS and quickly mounted under a coverslip.
At each time point five mice were fixed in 4% buffered
paraformaldehyde (ph 7.4) for routine histology. Serial
sections were sectioned in the frontal plane and stained
with hematoxylin and eosin and with thionine for assessment of the glycosaminoglycan content of the matrix (Bulstra et al., 1993).
Analysis of the Fluorescent Signals
Immediately after the immunocytochemical procedure,
labeled structures in the joint were visualized with epifluorescence. A filter with 550-nm excitation and 570-nm
emission was used to visualize the TRITC-labeled antibodies, whereas a filter with 365-nm excitation and
420-nm emission nm was used to visualize the Hoechst
33342. A custom-made program was developed to automate all the operations needed for scanning the microscopic slides and analyzing synovial areas. Each slide was
422
BUMA ET AL.
Fig. 1. Summary diagram of frontal view of cross-sectioned femur and ligaments. The left side shows the control situation and the right side the
situation of arthritic joint after one week. Note the synovial swelling. The dotted line indicates the boundary of measured synovial area. F, femur; M,
meniscus; JC, joint capsule; JS, joint space; P, patella; S, synovial membrane.
scanned twice by field movement of the motorized scanning stage (EK 32, Märzhäuzer, Wetzler, Germany) using
the two different filters for scanning the vascular structures and the perfused synovial areas, respectively. The
video signals were digitized, processed and analyzed on a
Macintosh Quadra 650 computer, using the digital imaging application TCL-Image (TNO, Delft, The Netherlands). Before the scanning procedure, a threshold for the
vascular structures was determined for both stain signals.
For each section, composite images were reconstructed
from the individually processed microscopic images: one
with the vascular structures, the other with the perfused
areas. Using the image with the vascular structures, a
contour line was drawn around the synovial area from the
patella to the femur on a new image (see Fig. 1 for location
of synovial area). The lumen of the knee joint was excluded. This area was used as a mask for further image
analysis. After scanning, the coverslips were removed and
the sections were mounted in Fluoritab (Organon
Teknika, Cappel-ICN), and coverslipped again.
right arthritic knee of each mouse, because these results
were not independent. Furthermore, these data did not
meet the requirements for analysis using parametric procedures.
To analyze the difference between the two points of
time, i.e., Day 4 and Day 7, the Mann-Whitney U-test was
used; these data were also non-parametric. P values of less
than 0.05 were considered to represent significance.
RESULTS
Clinical Behavior and Routine Histology
The digital image system measured synovium area
(SA), number of all blood vessels (NBV) and the number of
perfused blood vessels (NpBV). From these data the percentage of perfused vessels of all vessels (perfusion fraction; PF ⫽ NpBV/NBV), the vessel density (VD ⫽ NBV/
SA) and the density of perfused vessels (VDp ⫽ NpBV/SA)
could be calculated.
The gross appearance of the affected joints had not
changed dramatically after induction of mBSA arthritis.
The mice functioned well clinically. Macroscopic examination of the right knee joint exhibited the normal characteristic signs of inflammation, such as swelling and redness.
All micrographs were viewed with the patella on top of
the femur and the tibia below (see Fig. 1 and 2). In control
joints a layer of connective tissue in the synovial membrane (Fig. 2A) covered the surface of the joint cavity
around the femur, tibia and cruciate ligaments.
Four and 7 days after induction of arthritis the joint
cavity was enlarged, the synovial membrane was thickened and both were invaded by massive quantities of
polymorphonuclear lymphocytes (Fig. 2B). The tissues underneath the synovial membrane were filled with massive
numbers of leukocytes. HE sections exhibited an increase
in the number of small blood vessels, which seemed to
penetrate the tissue of the synovial membrane from the
joint capsule (Fig. 2C–E).
Statistical Analysis
Fluorescence Signals
In the statistical analysis, the Wilcoxon signed rank test
was used for the data from the left control knee and the
Background staining with 9F1 was rather low (Fig.
3B,F). Controls for method specificity (omission of the first
Quantification of the Perfusion and Vascularity
in the Synovial Area of the Knee Joint
DIGITAL IMAGE ANALYSIS OF MOUSE KNEE JOINT
423
Fig. 2. Thionine- (A) and HE-stained (B–E) stained sections of control (A) and inflamed joints (B–E) 4 (B–D) and 7 (E) days after induction. A: Note
the thin synovial lining (arrows) of joint space (JS). JC, joint capsule. B: Synovial tissues (ST) are swollen and loaded with leukocytes. PT, patella. C–E:
Small blood vessels are found in the thick synovial lining (arrows in D). D: Enlargement of boxed area in C. E: Seven days after induction of arthritis,
the number of vessels containing erythrocytes (arrows) was higher. A, B: ⫻35; C: ⫻100; D: enlargement of C, ⫻170; E: ⫻140.
incubation step) were completely negative. Nuclei of endothelial cells and occasionally cells directly adjacent to
the perfused vessels were stained by the Hoechst marker,
and were thus clearly distinguishable from the rest of the
tissues (Fig. 3A,D,G). Diffusion of the Hoechst marker
outside the endothelial lining was observed only occasionally in a small very small area around the perfused vessels. In all tissues, including muscle (Fig. 3A–C) and med-
424
BUMA ET AL.
ullar tissue of bone (Fig. 3D,E) there was a close match
between vascular 9F1 staining and the perfusion marker
Hoechst.
In control knees relatively few 9F1 positive blood vessels were found in periosteum and tissue directly under
the synovial membrane (Fig. 3I). In some of the areas with
no Hoechst staining, small vessels were stained with 9F1,
and careful comparison of Hoechst images with 9F1 images showed that the larger vessels in particular were
stained by Hoechst (Fig. 3F–H,K).
The strong 9F1-fluorescence signal and the low signal
from the surrounding soft tissue enabled segmentation of
the vessels from the background by the image analysis
system (Fig. 4A,B).
Quantitation of Tissue Changes Associated With
mBSA Arthritis
The measurements exhibited an increase in the area of
the synovial membrane (SA) during arthritis from ca. 0.2
mm2 in control knees to ca. 2.3 mm2 on Day 7 in arthritis
cases (Fig. 5A). On Days 4 and 7, the SA during arthritis
was significantly larger than that in the control knee (Fig.
5A; P ⫽ 0.043 and P ⫽ 0.016, respectively), and the SA
was significantly larger on Day 7 than on Day 4 (P ⫽
0.025). No significant difference was observed between the
control knees.
The number of blood vessels in the area of the synovial
membrane varied between 70 (control) and 1,250 (inflamed joint; Fig. 5B). On Days 4 and 7, the number of
vessels was significantly higher in the SA than in the
controls (Fig. 5B; P ⫽ 0.043 and P ⫽ 0.022, respectively).
Also, significantly more vessels were present on Day 7
than on Day 4 (Fig. 5B; P ⫽ 0.025). Surprisingly, a significant increase in vessel number was also found between
Days 4 and 7 in the control knee (P ⫽ 0.025).
The number of perfused blood vessels (NpBV) in arthritic synovial membrane was always lower than the
total number of vessels (Fig. 5B), again indicating that not
all vessels were perfused, but that they were significantly
larger compared with the number in control knees on the
same day (P ⫽ 0.043, P ⫽ 0.043, respectively). On Day 4
the synovial membrane had significantly less perfused
vessels than on Day 7 (Fig. 5B; P ⫽ 0.025). No differences
were observed between controls on Day 7 and Day 4.
Changes in Vessels and Vascular Perfusion of
the Synovial Membrane Associated With
mBSA Arthritis
On Day 4, the vessel density (VD) in the arthritic knee
was slightly higher than that in the control knee (Fig. 5C;
P ⫽ 0.043). On Day 7, this difference was no longer
present, and the VD was significantly larger than that on
Day 4 (P ⫽ 0.025), but the absolute differences in vessel
density were not very large. The VD of the control knee on
Day 7 was significantly larger than that on Day 4 (P ⫽
0.025). In all measurements, the smallest VD was 250 per
mm2, whereas the largest VD was 675 per mm2 (Fig. 5C).
There were no differences in the perfused vessel density
(VDp) on Days 4 and 7 between the control knee and the
arthritic knee (Fig. 5C). Another observation was that the
VDp on Day 7 in the arthritic knee was significantly larger
than that on Day 4 (P ⫽ 0.025). In all measurements the
VDp was in the range of 45–280 per mm2 (Fig. 5C).
The perfusion fraction (PF) in all measurements was in
the range of 0.1– 0.72 (Fig. 5D). The PF in the control
knees on Day 4 was significantly higher than that in the
arthritic knees (P ⫽ 0.043). On Day 7, the difference was
no longer significant. The PF in the control knees was
significantly higher on Day 4 than on Day 7 (P ⫽ 0.025).
DISCUSSION
mBSA-Induced Arthritis Mouse Model
The main purpose of this study was to develop a procedure to quantify the changes in vascular structures during
arthritic inflammation in mouse models. In humans these
changes are difficult to study. Human joints are very
large, which makes it impossible to study all joint structures in one section. Moreover, the specimens are often
incomplete and the soft tissues may be disorientated in
surgically removed specimens (FitzGerald et al., 1991). In
addition, the stages of inflammation in the arthritic process may be very different from one individual to another,
which makes it difficult to demonstrate an effect of vascular inhibitors. Therefore, it is important to use a model
with reproducible tissue changes of the synovial membrane. This requirement was met in the present murine
arthritic model (van de Loo et al., 1995). It has been
described previously that the antigen-induced inflammatory process is highly reproducible in C57Black6 mice (van
Beuningen et al., 1989). The chronicity of this type of
experimental arthritis is dependent on T-cell reactivity
and on the duration of antigen retention in joint tissues
(van Lent et al., 1987). It is also related to age and gender
(van Beuningen et al., 1989). In this experiment the
mBSA-induced arthritis model exhibited highly reproducible swelling of the tissues of the synovial membrane on
Days 4 and 7. Other experiments have shown that between 2 and 8 weeks the tissues of the synovial membrane
will slowly return to their pre-arthritic configuration (van
Beuningen et al., 1989; Buma et al., 2000).
Digital Image Analysis System
The first advantage that also contributed to the success
of our digital image analysis system, was the possibility to
scan entire sections of the synovial membrane, which
made it impossible for the investigator to subjectively
select parts of the synovial membrane. The second advantage is that this procedure not only yields quantitative
data on the total number of vessels, but also allows combinations of this picture with the perfusion marker in the
same area of the synovial membrane. The Hoechst marker
stained nuclei of endothelial cells and some cells located in
very close vicinity to the perfused blood vessels in the
synovial membrane. Perfused vessels could be clearly distinguished from the nonvascularized area of the synovial
membrane. To avoid variation in the diffusion of the
Hoechst marker in the tissues, the time interval between
perfusion and killing the animals must remain constant
between different animals. In this study, the animals were
killed after 1 min, which had proven to be an appropriate
time interval in previous studies using the marker in a
mouse model (Rijken et al., 1995). The scanning procedure, which takes about 20 min, should follow the staining
procedure within a few hours. If not the Hoechst marker
will diffuse away into the tissues, because it is only loosely
bound to the nuclei of stained cells.
Figure 3.
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BUMA ET AL.
Changes in Synovial Membrane Tissue
Associated With mBSA Arthritis
Previous quantitative studies of the human vasculature
of the synovial membrane during inflammation exhibited
an increase in vessel number as well as a change in vessel
morphology in synovial membranes when compared with
clinically unaffected joints (FitzGerald et al., 1991). These
findings conflict with a report by Stevens et al. (1991) that
suggested reduced vascularity with advanced disease. It is
possible that these differences are a reflection of different
stages of disease progression in rheumatoid arthritis, and
they illustrate that it is extremely difficult to evaluate
vascular changes in clinical human material. In this
study, synovial membrane tissues were examined after
two different time intervals during mBSA arthritis.
On Day 4 after induction of arthritis, the swollen subsynovial membrane tissues were avascular (Buma et al.,
2000). An important observation was that in the first
stages of the inflammation process, the area of the synovial membrane (SA) increased significantly. This caused a
situation in which parts of the SA were avascular. The SA
kept on swelling and on Day 7 it was approximately six
times bigger than the size in control knees and one and a
half times bigger than the SA on Day 4 of mBSA arthritis.
In this study we demonstrated an increase in the area of
the synovial membrane, an increased number of vessels
and changes in vessel densities in arthritis. The relatively
low vessel density on Day 4 and the increase in vessel
density on Day 7 as compared with Day 4, strongly suggests that directly after the induction of arthritis, the size
of the synovial area increases rapidly without vessel formation and that after Day 4, vessel formation increases.
This was confirms previous preliminary findings in that
blood vessels sprouted, particularly between Days 4 and 7
(Buma et al., 2000). The relative avascularity of the synovial membrane during the first few days after the induction of arthritis might induce hypoxia, which is a powerful
inducer of angiogenesis (Blake et al., 1989; Fava et al.,
1994; Paleolog, 1996; Stevens et al., 1991).
The relatively low number of perfused vessels on Day 4
indicates that the newly formed vessels were initially
non-functional. This is in agreement with our observation
that sprouting vessels end blindly in the synovial tissue,
making actual flow impossible (Buma et al., 2000). The
perfused vessel density (VDp) on Day 7 was again comparable to that of the control knee on day seven, indicating
that after a few days, newly formed vessels become functional. So it might be speculated that there is in synovial
tissue a fixed perfusion rate of vessels that is independent
of vessel density.
Fig. 3. 9F1 staining (B,F) Hoechst staining (A,D,G) and double photographs showing co-localization of 9F1 and Hoechst (C,E,H,I,K). A–C:
Same location of muscle (M) of mouse. Note the close overlap between
9F1 (B,C) and Hoechst (A,C) in endothelial cells (arrows). Hoechst staining is only found in the nuclei of the endothelial cells (⫻120). D,E: Bone
marrow (BM) of tibia. Close overlap between Hoechst (D) and 9F1
staining (E) (⫻180). F–H: 9F1, Hoechst and the combination in arthritic
synovial membrane four days after induction (S). Note that some of the
small 9F1-positive vessels are not stained by Hoechst (arrow) (⫻200).
I,K: Control joint (I) and synovial membrane seven days after induction of
arthritis (K). The arrow is pointed at a vessel not stained by Hoechst. BV,
blood vessel; BM, bone marrow; C, capsule of joint; JS, joint space; M,
muscle; P, periosteum; S, synovial membrane (⫻60).
Fig. 4. Digitized images of control joint (A) and joint seven days after
induction of arthritis (B). JS, joint space; P, periosteum; PA, patella; S,
synovial membrane.
An interesting observation was the increase in NBV and
the VD in the control knee on Day 7 compared with Day 4.
We do not know exactly what adaptive mechanism is
involved in the increase in NBV and VD. Firstly, circulating inflammatory mediators released from the inflamed
knee joint could have a more general effect on vascularity
in the whole animal. This seems very plausible in the light
of the observed proliferative effect of AIA induced arthritis
on vessels outside the joint in the inflamed joint (unpublished observations). A second explanation could be that
the AIA in the inflamed joint induced a changed load
distribution in the contra lateral joint. This might have
induced an adaptive response in the vessel density. This
does not seem very plausible, however, because a changed
walking pattern after induction of AIO was never ob-
Fig. 5. Quantitative data of synovial area (A), number of vessels and number of perfused vessels (B), the vessel density in the synovial area (C)
and the perfusion fraction (D).
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BUMA ET AL.
served. Irrespective of the mechanism involved, because
the number of perfused vessels and the synovial area did
not change, we speculate that no further vascular capacity
was needed and that angiogenesis of non-functional vessels took place.
Finally, we think that the measurements will be highly
reproducible. In a pilot experiment we analyzed Day 7
arthritis. All parameters in the experiment exhibited the
same significant differences between arthritic and control
joints, indicating that the arthritis model in the mouse is
highly reproducible. In similar studies of vascularization
of tumour tissue the variations of the measured parameters were relatively limited (Rijken et al., 1995). Therefore, we think that this procedure yields very accurate
measurements of the relationships between tissue
changes during disease and the concomitant changes in
vascularity. Moreover, this procedure may also prove very
useful in other fields of research in which vascularity
plays an important role. Important fields in orthopaedic
research are studies related to avascular necrosis (Simank
et al., 1997), tissue engineering studies of different parts
of the locomotion system (Germain et al., 2000) and to
follow revascularization of bone grafts and other substitutes (Kirschner et al., 1999).
In conclusion, the Hoechst perfusion combined with
immunocytochemistry allows an efficient quantitation
of total vascularity and of functional vessels during
arthritis. The procedure described here should allow us
not only to quantify the effects of inhibitors of vascular
sprouting on the vascularity but also to assess the effects on the arthritic processes in our mouse models.
Moreover, there are clear applications of this procedure
in the study of other bone and joint diseases in animal
models.
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