© 1999 American Heart Association, Inc.
Original Contributions |
A Bioluminescence Method for the Mapping of Local ATP Concentrations Within the Arterial Wall, With Potential to Assess the In Vivo Situation
From the Wallenberg Laboratory for Cardiovascular Research, Göteborg University, Göteborg, Sweden (M.L., T.B., M.E., O.W.); and the Institute of Physiology & Pathophysiology, Pathophysiology Division, University of Mainz, Mainz, Germany (S.W.).
Correspondence to Dr Tom Björnheden, The Wallenberg Laboratory for Cardiovascular Research, Sahlgrenska University Hospital, S-413 45 Gothenburg, Sweden. E-mail tom.bjornheden{at}wlab.wall.gu.se
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Abstract |
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Abstract—According to the anoxemia theory of atherosclerosis, an imbalance between the demand for and supply of oxygen and nutrients in the arterial wall is a key factor in atherogenesis. However, the energy metabolic state of the arterial tissue in vivo is largely unknown. We applied a bioluminescence method, metabolic imaging, to study local ATP concentrations in cryosections of normal pig and atherosclerotic and normal rabbit aorta. Some vessels were subjected to energy metabolic restrictions by incubation at different oxygen and glucose concentrations and others were rapidly frozen in liquid nitrogen to reflect the in vivo situation. Local ATP concentrations and the ATP distribution at a microscale was dependent on oxygen as well as glucose concentrations during incubation. ATP depletion was seen in the mid media of pig aorta in all incubations, but only at low oxygen concentration without glucose in the media of the thinner rabbit aorta. ATP-depleted zones were seen deep in pig media (>750 µm from the lumen) and in rabbit plaques (>300 µm from the lumen) even at high oxygen (pig 75% O2 and rabbit 21% O2) and glucose concentrations (5.6 mmol/L glucose). This observation probably illustrates an insufficient diffusion of glucose, which highlights the importance of studying the conditions for diffusion not only of oxygen but also of other metabolites in the arterial wall. In rapidly frozen vessels the medial ATP concentration was shown to be 0.6 to 0.8 µmol/g wet weight (both pig and rabbit aorta) and in pig aorta a gradient could be seen indicating higher ATP concentrations at the lumenal side. We propose that metabolic imaging, as applied to snap-frozen tissue, may be used to assess the energy metabolic situation in the arterial wall in vivo. The spatial resolution allows the detection of local variations within the arterial tree. However, steep concentration gradients (eg, near the border of the tissue) will be underestimated. The method may be extended to include determinations of glucose and lactate concentrations and will be used in parallel with an established method to assess hypoxia in the arterial wall in vivo.
Key Words: atherosclerosis • bioluminescence • adenosine triphosphate • hypoxia • metabolic imaging
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Introduction |
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The arterial wall depends on diffusion for its supply of oxygen and nutrients—ie, diffusion from luminal blood to the intima and the inner layers of the media and from adventitial vasa vasorum to the outer layers of the media.1 2 To maintain energy metabolic steady state, a balance must be maintained between oxygen and substrate diffusion and metabolic demand in the tissue. In atherosclerosis, both diffusion distances and oxygen consumption3 4 5 increase. This has been suggested to lead to areas of disturbed metabolism that play a key role in the further development of the lesion. This hypothesis was first proposed by Heuper in 19446 and has since been supported and further developed by other researchers.7 8 9
Arterial wall oxygenation has been the subject of several investigations. Oxygen microelectrodes have been used to measure PO2, in vitro and ex vivo, and decreased PO2 has been shown to occur in the arterial wall of hypertensive,10 11 atherosclerotic,12 and diabetic rabbit models,13 in aged rats,14 and at the carotid bifurcation of unmanipulated dogs.15 However, an adaptive mechanism, proliferation of adventitial vessels, which counteracts hypoxia, has also been demonstrated.16 A method to demonstrate local hypoxia in the arterial wall in vivo has recently been developed in our laboratory.17 With this method, hypoxic areas have been demonstrated in the aortic arch of rabbits with experimental atherosclerosis, lending support to the anoxemia theory.17A
Although hypoxia can lead to disturbances per se (hypoxia has been shown to regulate gene expression18 ) the major impact of a low PO2 is probably through effects on energy metabolism. On the other hand, it has been suggested that the energy requirements of the arterial wall may well be provided through anaerobic glycolysis even during extensive hypoxia.19 Because anaerobic glycolysis is less energy-efficient, however, the available diffusion capacity for energy substrates and waste products would be strained and it is possible that glycolysis would not save the arterial wall from such an energy metabolic predicament. So the situation is complex and there is an obvious need to investigate metabolic parameters other than the supply of oxygen to get a comprehensive view of the metabolic situation in the arterial wall. ATP concentrations in arterial tissue have been studied in relation to atherosclerosis. Ragazzi et al20 found no difference in ATP content between homogenates of normal rabbit aorta and rabbit aorta with early atherosclerotic lesions. Heinle21 on the other hand detected an ATP gradient with lower ATP concentrations 50 to 200 µm from lumen in 100- to 200-µm-thick lipid-laden plaques of the rabbit carotid artery. However, the methods that were used in these studies did not allow an assessment of the energy metabolic state of the tissue at a microscale, which is of vital importance because the suggested metabolic disturbances during atherogenesis are most likely local in nature.
A range of methods is available to study local metabolic changes in different organs and tissues. For use in arterial tissue, noninvasive techniques such as positron emission tomography, magnetic resonance spectroscopy, and MRI, however, lack the necessary spatial resolution.
Similarly the resolution is insufficient in the classical Lowry technique,22 whereas the deoxyglucose method according to Sokoloff23 has a high resolution but is only designed to assess glucose uptake.
For this reason we are presenting a bioluminescence method suited to study metabolic parameters in the arterial wall at a microscale. The method, metabolic imaging, allows the demonstration of ATP, glucose, or lactate in tissue cryosections by application of different enzyme solutions that link the studied metabolite to luciferase with subsequent light emission.24 The photons are registered by a photon-counting camera connected to a microscope, and the luminescence intensity in the resulting digital images is proportional to the local concentration of the metabolite (in this article, ATP). The method is fast and easy; and under optimal conditions, the spatial resolution is near or at a cellular level. By using snap-frozen tissue, this analysis will reflect the in vivo metabolic situation.
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Materials and Methods |
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Incubation of Tissue
Animals
Three male New Zealand White rabbits were fed standard rabbit chow with the addition of 1% cholesterol to induce experimental atherosclerosis. The rabbits were anesthetized with 1:1 Ketalar (50 mg/mL; Parke-Davis) and Rompun Vet (20 mg/mL; Bayer AG), 0.35 mL/kg IV, and 0.17 mL/kg every 15 minutes thereafter. The aorta was removed by a technique that has been developed to ensure maximum tissue integrity.25 The major part of the adventitia was dissected away in a bath of oxygenated medium and the aorta was divided into 3- to 4-mm segments. Also, 1 undissected aortic piece was frozen approximately 2 minutes after cardiac arrest.
Two healthy domestic pigs (35 and 45 kg) were anesthetized (thiopental sodium, 25 mg/kg, IV), intubated, and artificially ventilated. The thorax was opened and one piece of the aorta was rapidly cut out and frozen in liquid nitrogen 1 minute after the induction of cardiac arrest (KCl, IV). Another piece of the thoracic aorta to be used for incubation was carefully removed (10 minutes after cardiac arrest) after flushing with oxygenated medium at 37°C during the first 2 minutes. The major part of the adventitia was dissected away, and the tissue was transported to the laboratory for incubation. Dissection and transport was carried out in oxygenated medium at 37°C and required approximately 1 hour.
Medium
Eagle's MEM with different glucose concentrations (0, 0.056,
0.56, and 5.6 mmol/L) and 10 mmol/L sodium bicarbonate was
used, supplemented with 1% nonessential amino acids and 1% vitamins
(ICN Pharmaceuticals).
Incubation Conditions
In one set of experiments, arterial segments were
exposed to varying degrees of energy metabolic restriction
by incubation at different glucose and oxygen concentrations. (1)
Rabbit A: Glucose, 0.56, 0.056, and 0 mmol/L in medium
equilibrated with 0%, 3%, and 21% O2 plus 5%
CO2 in N2. (2) Rabbits B
and C: Glucose, 5.6 and 0 mmol/L in medium equilibrated with 0%,
1%, 3%, and 21% O2 plus 5%
CO2 in N2. (3) Pig:
Glucose, 5.6 and 0 mmol/L in medium equilibrated with 3%, 10%,
21%, and 75% O2 plus 5%
CO2 in N2. In rabbit A, a
hypoxia marker,
7-(4'-(2-nitroimidazole-1-yl)-butyl)-theophylline (NITP), (0.25
mmol/L; Lancaster Synthesis) was added for the assessment of
arterial wall hypoxia.17 Incubations
were performed at 37°C for 3 hours.
In another set of experiments, the segments were allowed to exhaust the available energy deposits through incubation for varying times. (1) Rabbits B and C: Glucose, 5.6 mmol/L (B) and 0 mmol/L (C) in medium equilibrated with 0% O2 for 30, 60, 90, 120, and 150 minutes. (2) Pig: Glucose, 5.6 mmol/L in medium equilibrated with 1% O2 for 30, 60, 120, and 180 minutes. Incubations were performed at 37°C.
Tissue Preparation
Immediately after incubation or surgical removal, aortic pieces
were frozen in liquid nitrogen and stored at -70°C. All sections,
10 µm, were made on a Tissue-tek II Cryostat at -23°C and
mounted on cover glasses (18x18 mm; 0.13 mm thick). The pig
aorta was fragile, and to avoid tearing of the tissue, only sections
from the part of the vessel where the elastic laminae were
perpendicular to the cutting edge of the knife were used.
To block any intrinsic enzymatic activity, sections were subjected to instant heat fixation on a heating plate at 100°C for 10 minutes and thereafter were stored at -70°C until further use.
From rabbit A, 1 aortic piece for every oxygen concentration (21%, 3%, and 0% O2) was fixed in 4% buffered formaldehyde, pH 7.0. Paraffin sections (5 µm) were transferred to glass slides to be used for immunodetection of the hypoxia marker, NITP.
Metabolic Imaging
To visualize and quantify the distribution of ATP in frozen
sections of the arterial wall, we have applied and modified
a method that is currently used in brain and tumor
research.24 26 27 28 In this method, photons
(bioluminescence) are generated in a quantitative manner when ATP
hydrolyzes in a luciferase-catalyzed reaction. By using a
photon-counting camera mounted to a microscope, it is possible to map
the bioluminescence activity, which is proportional to the local ATP
concentrations within the cryosection.
ATP Standards
ATP standards were produced by mixing 0.5 mL of ATP solution 0
to 13.2 mmol/L in aq dest (Sigma) with 2 g of embedding
medium for frozen tissue specimen (Tissue-tek O.C.T. Compound; Miles
Inc). The mixture was frozen and 10-µm sections were mounted on cover
glasses (18x18 mm; 0.13 mm thick).
Preparation of Enzyme Solution
One gram of desiccated firefly lanterns (Sigma) was
homogenized in 20 mL HEPES-arsenate buffer (0.2 mol/L
HEPES, 0.1 mol/L sodium arsenate, pH 7.6; Sigma). The solution was
ultrasonicated at intervals on ice for 10 minutes and
centrifuged for 10 minutes at 2500 rpm. Twenty microliters of 1
mol/L MgCl2 (Sigma) was added to the supernatant
and the pH was adjusted to 7.6, if necessary. The enzyme solution was
stored in Eppendorf caps at -70°C until further use.
Measurement Setup
The setup for measurement is displayed schematically in Figure 1
. We used an Axiovert 135 M inverted
microscope (Carl Zeiss) together with a Hamamatsu photon-counting
camera (Intensifier head, C4484 to 04; Relay lens, A2098; CCD C5405)
connected to a Hamamatsu Argus 20 Image Processor (Hamamatsu Photonics
K.K.). The camera was mounted underneath to get a straight and short
optical path. After integration was completed, the images were stored
and further analyzed using a KS 400 image analyzing system
(Carl Zeiss).
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Measurement Technique
Before starting the measurement, the cover glass with the frozen
section was dried on a heating plate at 100°C for 10 seconds to avoid
condensation of water. The section was put on the microscope stage at
23±1°C and the focus was adjusted. The enzyme solution was applied
to the section from above via a 1-mm hole in a small dark box covering
the specimen. The photon integration was started immediately and
allowed to continue for 25 seconds. The resulting digital images were
displayed with a color-coded intensity scale to illustrate the
differences in intensity more clearly.
Image Analysis
For each incubation, 4 or 5 bioluminescence registrations were
made on consecutive cryosections (1 registration/section). For every
registration, 2 images were stored: 1 light microscopic image of the
cryosection and 1 bioluminescence image.
The bioluminescence and the light microscopic images were obtained
under identical conditions except for the mode of registration. This
means that contours of arterial structures could be
delineated and used to assess bioluminescence activity in selected
areas of the section. In the bioluminescence image, light intensity was
translated to gray values (0–255) per surface area
(pixel2) by the KS 400 software, and these values
could, in turn, be translated to ATP concentration using the
calibration curve obtained from the ATP standards (Figure 2).
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In the atherosclerotic rabbit aorta ATP concentration was assessed in plaques, media under plaques, and in media in which no plaque was present. In pig aorta, the average ATP concentration was assessed in the media and the ATP distribution profiles were registered.
Hypoxia Assessment With NITP
NITP, a nitroimidazole derivative with a theophylline ligand,
was originally developed by Hodgkiss et al29 to study
hypoxia in tumors. At this laboratory the compound has been
applied to assess arterial wall hypoxia in
vitro17 and in vivo.27a NITP is taken up
intracellularly and during hypoxia; the theophylline
residue is trapped in the cell and may be detected by immunological
methods.
Immunohistochemistry
Immunofluorescence was used to assess
hypoxia in the arterial wall using NITP as a
marker. This technique has been described elsewhere.17
Microscopy
Immunofluorescence was studied on an
Axiovert microscope using filter set 16 (BP 485/20, FT 510, LP 520;
Carl Zeiss). Images for publication were registered with a color video
camera (Sony 3CCD, DXC-930P; Sony) in an integrating mode using the KS
400 image analysis software (Carl Zeiss).
Statistics
The student's t test for unpaired data was used when
2 groups were compared; P<0.05 was considered significant.
Measurement values are expressed as mean±SD.
Establishment of Experimental Conditions
Initiation of Luminescence Reaction
In our preliminary experiments, we used a procedure as
originally described by Mueller-Klieser and Walenta.24
They initiated the luminescence reaction by placing the cover glass
with the tissue section upside down on a small plexiglas well filled
with enzyme solution. With this approach, there was a delay (
30s)
until the section was in focus under the microscope and integration
(=bioluminescence registration) could be initiated. Because of this
delay, diffusion of ATP from the tissue resulted in blurred edges. This
problem has been pointed out by the authors24 27 28 who
suggested that the measurement temperature could be lowered or gelatin
added to the enzyme cocktail to reduce diffusion. So far we have
achieved the best results when the enzyme solution is applied on the
section from a hole on top of the small dark box. This makes it
possible to start integration at the same time as the bioluminescence
reaction starts.
Integration Conditions
A too-long integration time will lead to increased diffusion and
impaired resolution, whereas a short integration time will give a weak
luminescence intensity, especially, of course, in sections where the
ATP content is low. With a high sensitivity setting of the
photon-counting camera it is possible to detect a weaker luminescence
signal or to use a shorter integration time, which reduces the
influence of diffusion. At the same time, however, more pixels are
activated by every photon, leading to a decreased resolution.
In preliminary experiments, we found that an integration time of 25
seconds and a sensitivity setting of 8.0 (maximum 10.0) was optimal for
our purposes and these settings were used henceforth.
The amount of background activity, stray light and instrumental background, was investigated in measurements using saline instead of enzyme solution. The background activity was very low compared with the registered activity in rabbit and pig arteries.
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Results |
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Metabolic Imaging
ATP Standard
ATP-dependent bioluminescence was registered in ATP standard preparations (8 different ATP concentrations) under the same conditions (temperature, section thickness, integration time, and sensitivity level) as during the experiments. Figure 2
shows the average
bioluminescence (=light) intensity ±SD (gray
value/pixel2) as a function of ATP-content (n=6
to 7 for all ATP concentrations) in standard preparations.
Pig Aorta
Effect of Varying Glucose and Oxygen Concentrations on the ATP
Content in Pig Aorta
Pieces of pig aorta were incubated for 3 hours at 37°C at
different O2 concentrations (3%, 10%, 21% and
75%) in medium with 0 or 5.6 mmol/L glucose. The ATP
concentration in the sections varied from <0.1 µmol/g wet
weight (ww) (minimum detection level) to 1.2 to 1.6
µmol/g ww (maximum readout level at our settings).
The ATP distribution within the arterial wall showed a
distinct pattern with the highest concentration adjacent to the
incubation medium and a zone in the central part of the media where the
ATP content was minimal (ie, <0.1 µmol/g ww). This zone was
wider in the hypoxic and glucose depleted incubations (Figure 3), and thus it was closer to the lumen
(Table 1).
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For all oxygen concentrations, the average ATP concentration in the
media was significantly higher in the presence of than in the absence
of glucose. Also, the ATP content was higher at higher oxygen
concentrations. However, no difference could be seen between the 21%
and 75% O2 incubation in the presence of glucose
(Figure 3).
Incubation of Pig Aorta at an O2Concentration of 1% for Different Time Periods
Pieces of pig aorta were incubated in medium with 5.6 mmol/L
glucose at 1% O2 concentration for 30, 60, 120,
and 180 minutes. The ATP distribution profiles in the
arterial wall at the different incubation times is
illustrated in Figure 4. In the piece
incubated for 30 minutes, a decreasing gradient of 0.05 µmol
ATP/g ww/100 µm from lumen to midmedia (400 to 500 µm
from lumen) was followed by a similar increase toward the adventitia.
After >30 minutes, ATP depletion was detected. The depletion was
confined to the midmedia and surrounded by zones with higher ATP
concentrations after 60 and 120 minutes. After 180 minutes, only a rim
(200 µm) of detectable ATP (0.1 to 0.2 µmol/g) was
seen along the luminal circumference.
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Rapidly Frozen Pig Aorta
To get an estimation of the ATP distribution in vivo, pieces of
thoracic aorta were frozen 1 minute after cardiac arrest (Figure 5). These pieces showed an average
ATP concentration in the media of 0.70±0.04 µmol/g ww (Figure 5). The concentration profile (Figure 5B) indicated that
ATP levels were higher close to the lumen, showed a gradual decline
toward the midmedia (0.04 µmol · g-1 ·
100 µm-1), and then leveled off toward the
adventitia.
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Rabbit Aorta
The extent of atherosclerotic involvement varied between the
different rabbits with the maximal intima:media ratio ranging from
approximately 3.5 in rabbit A to 2.5 in rabbit B and 1.0 in rabbit C.
In cross-sections of aorta from rabbits A and B, 50% to 100% of the
circumference showed atherosclerotic involvement, whereas in rabbit C,
only 0% to 15% was involved.
Effect of Varying Glucose and Oxygen Concentrations on the ATP
Content in Atherosclerotic Rabbit Aorta
Pieces of atherosclerotic rabbit aorta were incubated for 3 hours
at 37°C at different O2 concentrations (21%,
3%, 1%, and 0%) in medium with 5.6, 0.56, 0.056, or 0 mmol/L
glucose. The ATP concentration within the sections varied from
<0.1 µmol/g ww (minimum detection level) to 1.2 to 1.6
µmol/g ww (maximum readout level at our settings).
Media Without Plaque
The ATP distribution was homogeneous except in
segments exposed to the most severe energy metabolic
restrictions (0% and 1% O2, no glucose), where
an ATP-depleted zone was observed in the media (Figure 6F and H). At 1%
O2, the distance from lumen to this zone was
100 µm (Table 2).
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The average ATP concentration was higher in the presence than in the
absence of glucose (significantly different for all
PO2). Also, the average ATP concentration seemed
to be higher at higher PO2 in the presence of, as
well as in the absence of, glucose ranging from 0.76±0.12
µmol/g ww (21% O2, with glucose) to background
level (0% O2, without glucose) (Figure 6).
Absolute ATP concentrations were not calculated for rabbit A, because the measurements were made before a reliable ATP standard had been developed. However, the pattern of ATP distribution agreed with the other measurements.
Media with Plaque
In those segments where plaques + media were >5 to 600 µm
thick (intima:media 1), the ATP distribution was characterized by a
central zone with minimal ATP concentration (<0.1 µmol/g ww)
and a higher concentration toward the adventitial and lumenal surfaces
(Figure 6A–E and G). The ATP-depleted zone was mainly located
in the deeper parts of the lesion, and the distance from the lumen was
greater at lower PO2 and in the absence of
glucose, except for at the highest oxygen concentration (21%
O2) (Table 2). Furthermore, in areas close
to the incubation medium, the concentration of ATP seemed to be lower
in the plaque than in the media (Figure 6A to 6E and 6G). In
segments with only limited atherosclerotic involvement an ATP-depleted
zone was only seen at low PO2 and without
glucose.
In most incubations, the ATP concentration in the media under plaques
was similar to that in the media where no plaques were present (ie,
about 0.5 to 0.7 µmol/g ww). However in incubations exposed to
severe energy metabolic restriction (1% and 3%
O2, without glucose), the ATP concentration was
significantly lower in media under plaques (Figure 7).
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Incubation of Atherosclerotic Rabbit Aorta at an
O2 Concentration of 0% for Different Time
Periods
Pieces of rabbit aorta were incubated in medium with 0 or 5.6
mmol glucose/L at 0% O2 concentration for 0 (ie,
frozen immediately before incubation), 30, 60, 90, 120, and 150
minutes. In glucose-free medium, the ATP concentration in the media had
declined by 40% (P<0.05) after 30 minutes and was 0 after
60 minutes. With 5.6 mmol/L glucose present, there was a
steady decline to 40% of the original value toward the end of the
150 minutes of incubation (Figure 8).
Plaques were only present in the aortic pieces incubated in
glucose-free medium and they were already ATP-depleted after 30 minutes
of incubation.
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Rapidly Frozen Rabbit Aorta
In rapidly frozen rabbit aorta the ATP concentration was
0.65±0.20 µmol/g ww in the media and 0.55±0.18 µmol/g
ww in the plaques. The ATP distribution was homogenous in the media as
well as in the plaques. These measurements were carried out on tissue
with moderate atherosclerotic involvement (intima:media ratio
1.0).
Hypoxia Assessment With NITP
Rabbit Aorta
Sections from the pieces of aorta (rabbit A) with experimental
atherosclerosis that had been incubated at 0%
O2 for 3 hours in NITP-containing medium with
different glucose concentrations (0, 0.056, and 0.56 mmol
glucose/L) exhibited an extensive immunoreaction throughout the
endothelium, plaques, media, and adventitial layer
(Figure 9C).
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In the sections incubated at 3% O2
concentration, immunoreaction was seen in the plaques and in a narrow
band in the media ( 50 to 100 µm thick and situated
150 µm from the lumen). This zone was more extensive in the
media under the plaques (Figure 9B). There was also
immunoreaction in the foam cells of the plaques, in the
endothelium, and in fat cells in the adventitia.
Finally, in the sections incubated at 21% O2
concentration, the immunoreaction was weak and confined to
endothelial cells, fat cells of the adventitia, and
foam cells in the central parts of some, but not all plaques. The
plaques where the foam cells showed immunoreaction were frequently, but
not always, thicker than the ones where no reaction could be detected
(Figure 9A).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present paper we have developed a bioluminescence methodology for the study of local metabolic changes in arterial tissue. To get a wide range of metabolic states the methodology was tested both on rapidly frozen sections and on sections that had been subjected to varying degrees of energy metabolic restriction. Sections were obtained from nondiseased pig aorta as well as from rabbit aorta with experimental atherosclerosis and from normal rabbit aorta.
Our data lend themselves to 2 principally different discussions. First, we propose that the methodology is well adapted to demonstrate local variations of the ATP concentration in the arterial wall. Second, our in vitro data add insights into the inhomogeneity of energy metabolism of the arterial wall in vitro.
Methodology
In an excess of oxygen and substrate the oxidation of luciferin
produces photons at a rate that is proportional to the ATP
concentration. Consequently, we found that within the lower range, the
light intensity increased with increasing ATP concentration in an
almost linear fashion (Figure 2). For higher ATP levels, the
standard curve deviated, probably reflecting a relative shortage of
substrate or oxygen. However, in the present application, the light
intensity fell within the lower 60% of the range of the standard
curve. The lowest detectable ATP level was about 0.1 µmol/g ww
and the background signal was negligible. The sensitivity of the photon
detection system was set below its maximum and it seems likely that ATP
levels of even <0.1 µmol/g ww could be measured. This would
require an increased signal enhancement, but was not specifically
tested in the present study.
The major part of the variability of the standard curve most likely reflects variations in thickness between consecutive cryosections because the intensity of the emitted light is proportional not only to the ATP concentration but also to the thickness of the section. It should be pointed out, however, that the strength of the presented method resides in the detection of local variations of ATP concentrations within single sections and not in the determination of absolute ATP levels with high precision.
The spatial resolution of the bioluminescence image improves with time
as increasing numbers of photons are collected during the formation of
the image. At the same time, however, the resolution deteriorates
because of diffusion of the ATP molecules in the section. Our
measurement settings were chosen to give the best possible compromise
under the prevailing circumstances, but other experimental designs may
require modifications in the measurement setup. The resulting
resolution is illustrated in a semiquantitative way in Figures 3, 5A, 6, 7, and 10, where zones with a width down to
100 to 200 µm may be discriminated. In Figures 4A to 4D
and 5B, ATP concentration is plotted versus distance from the
lumen in pig aortic sections. Peak levels of ATP close to the lumen and
to the adventitia stand out against the lower levels in the mid media
and concentration gradients are seen between these extremes. Diffusion
of ATP from the tissue during bioluminescence registration impairs the
resolution of the method.24 27 28 Because of this
substrate diffusion, concentration differences between closely situated
areas will be slightly reduced in the metabolic imaging
registration compared with the actual tissue. The error is most
pronounced at steep concentration gradients, which is most clearly
illustrated at the tissue borders where a gradient of 0.3
µmol/g ww/100 µm is seen and the gradient, in fact, should be
infinitely steep (Figures 4A to 4D and 5B).
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Energy Metabolism of the Arterial Wall
Aortic tissue from a nonmanipulated pig and from rabbits with
experimental atherosclerosis was incubated with or
without glucose at between 0% and 75% oxygen concentration for 3
hours. Except in the tissue exposed to the lowest oxygen and glucose
concentrations, the lumenal ATP content was well above zero, indicating
a satisfactory energy metabolic situation. Beyond a certain
depth, however, no ATP could be demonstrated. With decreasing
PO2 and glucose concentration, this ATP-depleted
zone became wider and, in consequence, the zone with adequate ATP
production became more shallow. In this series of experiments
and in previously published identical experiments,17 zones
with severe hypoxia (<2 to 3 mm Hg) were shown to
increase in width as PO2 was decreased during
incubation.
In Figure 10 the type of information that may be derived from
parallel determinations of ATP and oxygen concentrations is
illustrated. In this figure, the extent of ATP-depleted zones is
compared with the distribution of hypoxic areas in tissue incubated at
different occasions but under identical conditions. Such comparisons
make it possible to demonstrate areas within the arterial
wall where severe hypoxia coincides with an ATP content close
to zero (blue zones) as well as the opposite—ie,
oxygenated zones with adequate ATP concentration (red
zones). Furthermore, such comparisons could also help to demonstrate
areas (violet zones, yellow arrows) with adequate energy
production despite severe hypoxia. Most likely, these
areas would reflect zones with adequate anaerobic
glycolysis. The observation that ATP production seemed to be
inadequate deep in these zones could indicate an insufficient provision
of glucose locally, which highlights the importance of studying the
conditions for diffusion not only of oxygen, but also of glucose and
other metabolites in the arterial wall. This point is
emphasized further by the finding that, in very abluminal parts of pig
media (>750 µm) and rabbit lesion (>300 µm), ATP
production seemed to be insufficient despite adequate
oxygenation and high glucose concentration (5.6
mmol/L) in the surrounding medium (Figure 10, black
arrows).
To assess the ATP distribution in vivo, metabolic imaging
was performed on snap-frozen tissue. These studies suggested medial ATP
concentrations of 0.6 to 0.8 µmol/g ww In pig aorta, ATP
concentrations were higher close to the lumen and decreased
0.04 µmol/g ww/100 µm toward the mid media (Figure 5). These ATP concentrations were in agreement with previously
reported values for vascular smooth muscle measured with established
methods.30 However, studies must be performed on larger
groups of animals to get reliable in vivo results with the
presented method.
Conclusions
We have designed a method to study local ATP concentrations in the
arterial wall at a microscale. Parallel assessments of
local hypoxia were also made. Our observations during the
development of the methodology were in agreement with certain
established aspects of the interaction between the availability of
substrates and ATP production in tissue. The feasibility to
illustrate the dynamic relationship between these
parameters at a microscale was demonstrated.
Our in vitro data pointed to the possibility that, during hypoxia, the mere diffusion of glucose might not be sufficient to maintain energy production via glycolysis in the inner media. It was also suggested that ATP-depleted areas may exist in the central parts of atherosclerotic lesions even after incubation under metabolically favorable conditions.
These are 2 examples of aspects on arterial wall metabolism with relevance for the atherosclerotic process that may be studied with the presented technique. Also, the method may be extended to study other metabolites, glucose and lactate, as well. We conclude that metabolic imaging is a potentially powerful tool in the study of alterations in the arterial wall metabolism.
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Acknowledgments |
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This study was supported by the Swedish Heart and Lung Foundation (project numbers 51009, 61007, and 61510).
Received May 19, 1998; accepted September 3, 1998.
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References |
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Wolinsky H, Glagov S. Nature of species
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