Nephrol Dial Transplant (2009) 24: 2201–2206
doi: 10.1093/ndt/gfp033
Advance Access publication 10 February 2009
Functional and structural response of arterialized femoral veins
in a rodent AV fistula model
Stephan Langer1 , Christian Heiss2 , Niklas Paulus1 , Nuran Bektas3 , Gottfried Mommertz1 , Zuzanna
Rowinska1 , Ralf Westenfeld2 , Michael J. Jacobs1,4 , Michael Fries5 , Thomas A. Koeppel1 and European
Vascular Center Aachen-Maastricht
1
Department of Vascular Surgery, 2 Division of Cardiology, Pulmonology, and Vascular Medicine, Department of Medicine I,
Institute of Pathology, University Hospital RWTH Aachen, Germany, 4 Department of Vascular Surgery, University Hospital
Maastricht, The Netherlands and 5 Departement of Anesthesiology, University Hospital RWTH Aachen, Germany
3
Correspondence and offprint requests to: Stephan Langer; E-mail: [email protected]
Abstract
Background. Neointimal hyperplasia is considered to be
the major cause of arteriovenous fistula (AVF) failure, resulting in vein wall thickening, stenosis and, ultimately,
occlusion. Ultrasound (US) has been shown to be effective for detecting these morphological changes in patients.
The aim of this study was to develop an experimental AVF
model in the rat that shows typical features of fistula maturation and allows longitudinal monitoring of fistula veins
by high-resolution ultrasound.
Methods. AVFs were created by a handsewn end-to-side
anastomosis between the femoral vein and the femoral
artery in 15 rats. A group of sham-operated animals (n = 3)
served as controls. Time-related functional and morphological AVF characteristics were assessed up to 12 weeks
using ultrasound (15-MHz transducer) and were correlated
to histopathological changes.
Results. All rats survived surgery, and the patency rate was
93%. US showed a 2-fold increase in the fistula vein diameter and mean flow velocity as well as a 4-fold increase
in the intima–media thickness without significant luminal loss. The afferent femoral artery exhibited no change
in intima–media thickness and only minimal adaptive increases in diameter and flow velocity. Histological evaluation confirmed these observations.
Conclusions. Our AVF model in the rat demonstrates maturation effects in fistula veins similar to typical clinical
findings in haemodialysis patients. Noninvasive ultrasound
proved to be a valuable tool for longitudinal in vivo monitoring of the fistulas in this rodent model.
Keywords: AV fistula; maturation; neointimal hyperplasia; rat model;
ultrasound
Introduction
Increasing numbers of patients with end-stage renal disease are surviving worldwide because of improved dialysis
access techniques. Since being introduced in 1966 [1], arteriovenous fistulas (AVFs) have been commonly used as the
vascular access of choice [2]. However, the 1-year patency
rates are ∼63% [3], and recurrent fistula thrombosis is an
important cause of morbidity and mortality among those
patients. Neointimal hyperplasia is considered to be the
major cause of AVF failure, resulting in vein wall thickening, stenosis and, ultimately, occlusion [4]. Whereas excessive wall thickening is an undesired response to arterialized
blood flow, moderate wall thickening is important for ‘fistula maturation’ that makes recurrent dialysis cannulation
principally possible. Morphologically, neointimal hyperplasia is based on a proliferation of smooth muscle cells (SMC)
combined with matrix deposition. Nonetheless, the underlying molecular mechanisms are still poorly understood.
Despite improved surgical and puncture techniques, no
adjuvant strategies such as medical therapies have so far
demonstrated efficacy to preserve fistula patency. A reliable rodent AVF model can be helpful in understanding
and preventing the pathological mechanism of fistula failure. Therefore, a few different central and peripheral AVF
models have been recently described in the mouse [5] and
rat [6–9]. In the past, the analysis of morphological and
functional changes in fistula veins was bound to invasive
techniques. Moreover, the killing of animals was hereby
necessary to perform longitudinal assessments.
Ultrasound, and in particular Doppler ultrasound has
been shown to a valuable tool to investigate the rat vasculature [10]. However, the scientific value of this method
has not yet been demonstrated in an experimental AVF
model. Therefore, the aim of this study is to develop a
novel AVF rat model that shows typical features of fistula
maturation seen in patients and that also allows longitudinal in vivo monitoring of fistula veins by high-resolution
ultrasound.
Subject and methods
All experiments were approved by the local animal humane board
and were performed in accordance with the German legislation on the
C The Author [2009]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
For Permissions, please e-mail: [email protected]
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S. Langer et al.
protection of animals. Female 12-week-old Sprague Dawley rats (250–
300 g) were purchased from a commercial breeder (Charles River Wiga
GmbH, Sulzfeld, Germany). The rats were kept in a climate-controlled
room (21◦ C and 60% relative humidity) with a 12-h cycle of light and
darkness. All animals were housed in normal cages with free access to
water and food.
Experimental groups
A total of 18 animals (3 shams and 15 AVF) were part of the study.
Fifteen rats underwent AVF surgery. Four AVF rats were post-operatively
examined with ultrasound (US) on Day 1, Day 7 (Week 1), Day 14 (Week
2), Day 21 (Week 3), Day 42 (Week 6) and Day 84 (Week 12). At these times
and on Day 140 (Week 20), AVFs and contralateral femoral vessels were
harvested for histological and immunohistological examination. Three
rats underwent invasive haemodynamic assessment on Day 1 and Day 42
(Week 6).
Microsurgical procedure
The procedure was performed on rats anaesthetized by isoflurane inhalation (5% induction and 1.5% maintenance, isoflurane at 1.5 l O2 /min)
thereby allowing spontaneous breathing. Animals were placed in a supine
position on a heating pad (TR-200, Fine Science Tools, Heidelberg, Germany). The entire procedure was performed using a dissection microscope
(M650, Leica Microsystems, Wetzlar, Germany) with 16-fold or 25-fold
optical magnification. Surgical access was conducted via a 3 cm oblique
skin incision of the right groin. After subcutaneous dissection, the femoral
artery and vein were prepared. Following distal vein ligature using silk
8-0, a 1.5 mm longitudinal venous incision was made. After gentle arterial clamping, a corresponding longitudinal femoral artery arteriotomy
was anastomosed to the femoral vein incision in an end to side manner using 10-0 monofilament (Ethilon, Ethicon, Norderstedt, Germany)
running suture (Figure 1). A dilatation of the arterialized vein could be
observed quickly after clamp removal. Finally, the skin was closed with
3-0 continuous suture (Vicryl, Ethicon, Norderstedt, Germany).
High-resolution ultrasound
The afferent femoral artery and fistula vein were visualized with a 15MHz transducer (SONOS 2500, Philips, Amsterdam, The Netherlands).
After identifying the vessel by its characteristic flow pattern, the position
of the probe was optimized to show clear vessel wall/lumen interfaces and
longitudinal B-mode images were recorded. Diameters were measured by
automated analysis software (Brachial Analyzer, Medical Imaging Applications, Iowa City, IA, USA).
Fig. 1. Intraoperative image after fistula creation shows a distinct fistula vein dilatation (25-fold magnification). The middle image illustrates
the reconstruction. The lower image exhibits a longitudinal section (HE
×25) of the entire AVF on Day 84. The intima–media thickness is most
pronounced at the opposite site of the anastomosis (arrow), and the vein
dilatation is obvious. FA = femoral artery, FV = fistula vein, A = anastomosis.
Assessment of haemodynamic parameters
in PBS and incubated with a rat tissue-adsorbed, biotinylated secondary
antimouse antibody (Vector laboratories, Burlingame, CA, USA).
Haemodynamic parameters were assessed invasively by a method similar
to that described by Fries et al. [11]. Briefly, cardiac output was determined
with the transpulmonary thermodilution technique using a thermocouple
microprobe (IT-18, Physitemp Instruments, Clifton, NJ, USA) advanced
into the thoracic aorta via the left femoral artery. A 1 mm flowmeter (1
RB, Transonic Systems, Ithaca, NY, USA) was placed around the fistula
vein for continuous measurement of AVF flow.
Histological and morphometrical procedures
For tissue harvesting, anaesthesia was administered as described above.
The incision in both groins was followed by cardiac puncture to flush
vessels first with a saline solution and then with 4% formaldehyde in
phosphate-buffered saline (PBS, pH 7.4). The AVF in the right groin and
contralateral femoral artery and vein were gently removed and fixed in 4%
formaldehyde in PBS for histological and immunohistological analyses.
After overnight fixation, the specimens were processed and embedded in paraffin using standard techniques. Longitudinal sections (4 µm)
were stained with haematoxylin-eosin (HE) and Elastic-van Gieson (EVG)
for morphometrical analysis at the site of the anastomosis. After anastomosis site identification, neighbouring intima–media thickness in the
downstream fistula vein was measured from three longitudinal sections
for each AVF using digital microscopy analysis software (Diskus, version 4.30.625, Hilgers, Königswinter, Germany). Furthermore, vascular
calcification was evaluated using von Kossa’s staining as described by
Westenfeld et al. [12]. For smooth muscle α-actin staining, the primary
antibody was clone 1A4 (Dako, Glostrup, Denmark). Slides were washed
Statistics
The data are reported as arithmetic means ± SD. The primary test for
an effect was a test of the interaction in a one-way repeated measure
ANOVA. The family of pair-wise comparisons were conducted using the
Holm-Sidak method. P-values of <0.05 were regarded as significant.
Correlations were Pearson’s r. All analyses were performed with SigmaStat
3.5 (Systat Software Inc., San Jose, CA, USA).
Results
Surgical procedure
AVFs were created in all rats without any procedure-related
complications. The mean surgical time was 57 min (range:
45–73). All animals survived the surgery and lived until
the harvesting time points. The patency rate was 93% at
the time of harvesting. One AVF thrombosis was detected
on Day 7 by ultrasound examination. Immediate surgical
exposure confirmed an AVF thrombosis due to an infected,
false anastomotic aneurysm. During the observation period,
Functional and structural response of arterialized femoral veins in a rodent AV fistula model
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Fig. 2. Representative B-mode ultrasound images (A) and Doppler flow
velocity tracings (B) of the femoral artery (FA) and (C) arteriovenous
fistula (AVF) at 6 weeks. Horizontal marks on the scale bar represent
10 cm/s, vertical 0.5 s.
there were no signs of peripheral ischaemia resulting from
steal or oedema caused by venous congestion.
Sonomorphologic characterization of AVF maturation
Ultrasound investigation was possible in all rats. The mean
time of investigation was 12 min (range: 8–15 min).
Figure 2 shows a representative ultrasound B-mode image
of neighbouring femoral artery and AVF (a) and respective
PW Doppler flow velocity tracings (b and c). The diameter of the fistula vein was 0.17 ± 0.01 mm at baseline
and significantly increased on Day 7 (0.27 ± 0.03 mm,
P = 0.02; Figure 3). The maximum increase was stable
after 2 weeks (0.35 ± 0.03 mm, P = 0.01), whereas the
contralateral control femoral vein diameter remained unchanged throughout the study period (0.15 ± 0.05 mm).
Similarly, the flow velocity significantly increased in AVFs
with maximal velocities reached after 2 weeks and maintained at these velocities thereafter (baseline: 18 ± 4 cm/s;
2 weeks: 34 ± 2 cm/s; P = 0.03). These results were confirmed with a flow probe showing that the fistula volume
doubled within 6 weeks (baseline: 21 ± 11 ml/min, 6 weeks:
42 ± 19 ml/min, P = 0.4). The mean internal diameter of
the afferent femoral artery was 0.12 ± 0.01 mm on Day 1
and rose gradually throughout the study period with maximal significant dilation at 12 weeks (0.20 ± 0.02 mm,
P = 0.01). Whereas the flow velocity of the femoral artery
did not change throughout the first 6 weeks, there was a
significant increase in the femoral artery flow velocity at
12 weeks (baseline: 3.4 ± 0.4 cm/s, 12 weeks: 8.2 ±
2.8 cm/s, P = 0.02). A pronounced thickening of the
intima–media complex was observed in the fistula vein.
Whereas intima–media thickness of the afferent femoral
artery (0.005 ± 0.002 mm) and contralateral femoral artery
(0.006 ± 0.003 mm) remained unchanged throughout the
study period, it increased significantly in the fistula vein,
plateauing after 3 weeks (baseline: 0.003 ± 0.001 mm,
3 weeks: 0.014 ± 0.002 mm, P = 0.02). The mean heart
rate (baseline: 240 ± 46 bpm, 6 weeks: 233 ± 69 bpm,
P = 0.89), mean arterial pressure (baseline: 79 ± 9 mmHg,
6 weeks: 66 ± 15 mmHg, P = 0.31) and cardiac output
Fig. 3. US-based functional and morphometric analysis of time-related
fistula vein (AVF) alterations. AVF creation leads to pronounced significant vasodilation of the AVF (A), increase in flow velocity (B) and
thickening of the intima–media complex reaching a maximum at 3 weeks
(C). The afferent femoral artery (FA) exhibits a less pronounced vasodilation and flow velocity increase, but no intimal thickening. ∗ P < 0.05
versus baseline at 0 week.
(baseline: 108 ± 30 ml/min, 6 weeks: 140 ± 40 ml/min,
P = 0.40) remained unaffected.
Histological and morphometrical characterization
The harvested femoral veins of the sham group showed
a normal composition of the intima and media with one
cell layer of endothelium and few SMCs, respectively (HE,
Figure 4A). Endothelial and media cell loss was apparent on Day 1 after surgery at the site of the anastomosis (HE, Figure 5). Corresponding to the ultrasound findings, a continuous increase in intima–media thickness was
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S. Langer et al.
Fig. 4. Histological and immunohistological images of sham femoral vein (A) and artery (B) as well as the fistula vein (C–G). There was a progressive
hyperptrophy in the intima–media thickness from Week 1 (C) to Week 6 (D) to Week 20 (e). SMC proliferation (F) and collagen deposition (G)
obviously contributed to vein wall alteration.
Fig. 5. AVF anastomosis on Day 1. Magnification shows cell death (arrows) in the intima and media layers (HE ×25; HE ×400). FA = femoral
artery, FV = fistula vein, A = anastomosis.
observed at the site of the anastomosis from Day 1 to Day
42. Between the period from Week 6 to Week 20, only
a mild increase in intima–media thickness was noted and
this peaked on Day 140 (HE, Figure 4E). Wall thickening
was inhomogeneously distributed along the fistula veins.
However, this pronounced wall thickening gradually decreased from the site of the anastomosis downstream (HE,
Figure 1). The intima–media thickness was caused by both
Fig. 6. Vein valve calcification after Week 12. FA = femoral artery,
FV = fistula vein, A = anastomosis.
intimal and medial thickening. During fistula maturation,
cells invading the intima that stained positive for α-actin
were observed, which indicate that these were SMC (αactin, Figure 4F). EVG stain showed that AVF maturation was associated with significant collagen deposition
(EVG, Figure 4G). At Week 12, a severe venous valvular
calcification in the fistula vein was observed (von Kossa,
Figure 6).
Functional and structural response of arterialized femoral veins in a rodent AV fistula model
Discussion
The AVF model presented in the current study is characterized by a structural and functional response of fistula veins
that strongly resembles clinical findings. For this purpose,
a limiting of the microsurgical created anastomosis (length
1.5 mm) is essential to prevent a calibre mismatch and a
relevant steal syndrome.
In our study, we could show that high-resolution ultrasound is a feasible and reliable tool for noninvasive longitudinal monitoring in a rodent model of venous arterialization.
Preoperative ultrasound imaging has been proven effective
prior and after AVF creation in patients with end-stage renal
disease [13]. This noninvasive investigation of morphological and haemodynamic parameters has been shown to predict dialysis access function. The expected flow parameters
after 6 weeks in human fistula veins are characterized by a
low resistance biphasic shape, high peak systolic velocities
(PSV) and end-diastolic velocities in about one-half/twothirds of PSV [14].
During our longitudinal rat ultrasound study, we frequently observed similarities to the findings in patients.
Fistula vein dilatation occurs rapidly after AVF creation.
Human studies by Wong et al. [15] and Corpataux et al. [16]
demonstrated that forearm fistula veins dilated by 123%
and 179% within a period of 12 weeks after surgery, respectively. Analogously, we observed an ∼200% increase
in fistula vein diameter. In contrast, this dilatation in rats
was detectable 2 weeks after surgery.
Additional to vein dilatation, we observed an increase
in fistula volume, which is in accordance with Corpateaux
et al.’s human findings in six forearm fistulas. In this study,
it was shown that the venous limb of an AVF is subjected
to high volume flow but low blood-pressure environment
and that the venous wall remodels to an eccentric hypertrophy (constant wall thickness combined with dilatation) due
to higher wall shear stress [16]. In contrast to these findings, we observed a significant increase of intima–media
thickness in our ultrasound investigations. The reason for
the discrepancy remains unclear and needs further investigation. However, the molecular mechanisms that cause
venous dilatation are still not completely understood. It is
very likely that dilatation of the fistula vein results from
increased biomechanical forces such as increased blood
pressure and wall shear stress.
Increased levels of matrix metalloproteinase (MMP) are
a well-known phenomenon in afferent arteries proximal
to AVFs [17]. An increased expression of MMP 2 and 9
was observed in veins that were subjected to arterialization
[7,9], too. This increased MMP activity in arterialized vein
walls may lead to structural changes in the extracellular
matrix composition (e.g. elastin and collagen fibres) that
could explain the diameter increase.
In addition to dilatation, we noticed a significant increase in intima–media thickness in fistula veins in the Bmode images. Intima–media thickness in human AVFs is a
frequent phenomenon in the fistula vein [17], and mostly
intima–media thickness is located in the anastomotic area as
shown in angiographically evaluated AVF occlusions [18].
In our longitudinal sections, we could clearly verify intima–
media thickness in the fistula vein. Importantly, it was most
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pronounced immediately downstream of the anastomosis
and in the anastomosis itself. Our data are consistent with
those of Chan et al. who showed varying degrees of intima–
media thickness and thickening of the venous wall in a rat
AVF femoral loop model [7]. Furthermore, our structural
analysis showed that neointimal hyperplasia formation was
composed by proliferating SMC and collagen matrix deposition. Similar findings were described by Lin et al. in
an AVF rat model, where a microsurgical anastomosis was
created between the lateral and the ventral artery of the rat
tail [8].
There is increasing evidence that neointimal hyperplasia
formation in arterialized veins is triggered by endothelial
cell dysfunction or damage. Using a mouse model for vein
grafting, Xu et al. could demonstrate that early cell death
due to apoptosis and necrosis occurs shortly after vein arterialization and is followed by endothelial progenitor cell repair [19]. In our study, we could demonstrate an endothelial
and media cell loss predominantly at the site of the anastomosis as early as 1 day after surgery. Since neointimal hyperplasia was most prominent in the perianastomotic area,
our observations suggest that early endothelial cell death
may represent a key event for a pathological remodelling
of the proximal fistula vein. In addition to alterations of the
arterialized veins, we could also observe an ∼40% increase
in diameter of the afferent femoral artery within 3 weeks
after fistula creation, which is in accordance with human
dialysis access findings [16]. Nevertheless, the underlying
physiological mechanisms that induce arterial dilatation are
still not completely understood. There is evidence, however,
that longitudinal wall shear stress and circumferential deformation also represent substantial mechanical factors that
influence vascular dilatation and remodelling [20].
These structural alterations in both arteries and veins of
the AVF were accompanied by changes in fistula haemodynamics that could be detected by high-resolution ultrasound, flow measurements and cardiac output assessment.
Although a strong correlation between shunt volume and
cardiac output exists [21], the increasing fistula volume in
our study was only associated with a mild increase of cardiac output within 6 weeks after AVF surgery. Therefore,
increased fistula flow in our AVF model primarily resulted
from reduced venous resistance. The significance of this
finding, however, might be restricted by this relatively short
observation period of 6 weeks. Thus, it cannot be excluded
that fistula flow increases beyond that observation time and
causes a further increase in cardiac output.
Finally, we could observe severe calcification of venous
valves in fistula veins 12 weeks following surgery. Although vascular calcification predominantly affects arterial
vessels, calcification of arterialized vein grafts and of AVFs
in haemodialysis patients is a well-known phenomenon,
although the underlying pathomechanisms have not been
completely elucidated [22]. Vascular calcification does not
merely consist in passive deposition of calcium phosphate
crystals; it is now clear that it is a well-organized process that involves cell activity and specific protein synthesis [23]. Interestingly, systemic effects of uraemia or other
metabolic disorders can be excluded in our study. Therefore, it is likely that calcification occurred as a pathological response of the fistula vein to the arterialization. This
2206
finding suggests a role for local calcification regulators and
needs further investigation. In addition, future research has
to focus on a possible association between factors leading to
venous calcification and fistula vein thrombosis/neointimal
hyperplasia.
In summary, our rodent model proved excellent comparability to the clinical findings in patients with AVF.
The morphological and functional response of fistula veins
was characterized by a continuous increase in intima–media
thickness and mean flow velocity. The vessel diameter rose
in both, the femoral artery and femoral vein. Importantly,
assessment of these parameters by high-resolution ultrasound proved to be a practicable and reliable tool for longitudinal in vivo monitoring of AVF function and structure.
On the basis of this model, noninvasive in vivo monitoring
of the fistulas will allow investigating systemic effects, such
as chronic renal failure on fistula maturation and haemodynamics. Moreover, it will also be possible to evaluate new
therapeutic approaches that prevent AVF failure.
Acknowledgements. The study was funded by the research promotion
program ‘START’ of the Medical faculty of the RWTH Aachen University
(Title: ‘Die Arterialisation venöser Gefäße’).
Conflict of interest statement. None declared.
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Received for publication: 5.12.08; Accepted in revised form: 15.1.09