a Cardiovascular Translational Research Institute, Antwerp, Belgium
b Department of Interventional Cardiology, Middelheim Hospital, Antwerp, Belgium
c Division of Pharmacology, University of Antwerp, Antwerp, Belgium
d Department of Cardiology, Thoraxcenter, University of Rotterdam, Rotterdam, The Netherlands
e Department of Pathology, Middelheim Hospital, Antwerp, Belgium
* Address for correspondence: Stefan Verheye, M.D., Department of Interventional Cardiology, Middelheim Hospital, Lindendreef 1, 2020 Antwerp, Belgium. Tel +32 3 280 32 55; Fax +32 3 230 65 11
E-mail address: stefan.verheye{at}pandora.be
Received 14 August 2003; revised 6 October 2003; accepted 16 October 2003
Abstract
Aim To investigate safety, feasibility, and injurious effect on endothelial cells of a thermography catheter as well as effect of flow on measured temperature in non-obstructive arteries.
Methods and results Safety and feasibility were tested in both rabbit aortas and pig coronary arteries. Evaluation of endothelial damage by the catheter (acute, 7 and 14 days) was performed in pig coronaries using Evans Blue, scanning electron microscopy (SEM) and Factor-VIII antibody and compared with normal arteries and arteries that underwent intravascular ultrasound (IVUS). The effect of flow on temperature heterogeneity was analysed both in vitro and in vivo conditions. All procedures were successful without any adverse events; intra- and inter-operator variability was low. Intracoronary use of the catheter was associated with acute but reversible de-endothelialization, paralleling the findings associated with IVUS use. Changes in flow velocities under physiologic flow conditions did not significantly influence the temperature differences measured both in vitro and in vivo; temperature heterogeneity was more pronounced in absence of flow.
Conclusion Intracoronary thermography using a dedicated catheter is safe and feasible with a similar degree of de-endothelialization as IVUS. Temperature heterogeneity remained unchanged under normal physiologic flow conditions allowing clinical use of thermography.
Key Words: Atherosclerosis Catheters Imaging Plaque IVUS
1. Introduction
There is widespread agreement that early detection and treatment of rupture-prone coronary plaques is required. However, current techniques such as intravascular ultrasound (IVUS) and/or coronary angiography are incapable of detecting such vulnerable plaques. Therefore, newer imaging strategies are being developed trying to identify those plaques.1,2
Atherosclerosis is an inflammatory process characterised by presence of macrophages and lymphocytes.3,4Cascells et al. reported that there is increased temperature heterogeneity in ex vivo atherosclerotic specimen of human carotid arteries.5In vivo temperature heterogeneity was markedly increased in patients presenting with an acute coronary syndrome as opposed to patients having stable angina.6We have recentlyshown that by using a dedicated temperature catheterin an animal model of atherosclerosis, in vivo temperature heterogeneity is determined by plaque composition and more specifically by the total macrophage mass.7Furthermore, temperature heterogeneity in presence of flow appearedto be underestimated in patients with effort angina due to coronary stenosis which was related to cooling of the vessel wall by the blood flow.8Since there are some unknown practical aspects of this technique such as safety, feasibility, effect of the catheter on endothelial cells and the effect of blood flow on wall temperature in non-obstructive lesions, we sought to determine these features associated with the use of a dedicated catheter in order to understand and incorporate its use in a clinical environment.
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2.1. Thermography catheter and pullback-system specifications
The thermography catheter (Thermocore UK Ltd, Guildford, UK) is a 7F-compatible over the wire system that consists of a functional end that can be engaged by retracting a covering sheath.7Briefly, the distal part has four dedicated thermistors at the distal end of four flexible nitinol strips. After engagement, the strips have an expansion width of 9mm ensuring endoluminal surface contact of the vascular wall (Fig. 1A). The thermistors are made of 5k7 bare chips (5 kOhm resistance at 25°C), which are gold metallized with 40awg wires soldered onto it. The thermistor branches demonstrate an angle of 60±5° when the sheath is retracted and the thermistors can perform up to 25 measurements per second; they are delivered with a certified accuracy of 0.006°C. The response time of the thermistors to a small temperature change (15°C) was found to be less than 100ms. Once the catheter is inserted in the vascular system, the thermistors are normalized to a randomly chosen thermistor, and the proximal part of the catheter is then locked onto a dedicated pullback system (ThermosenseTM; Fig. 1B), which by itself is connected to a dedicated thermography console (ThermosenseTM). Pullback at a predefined speed can then be started and recorded.
2.2. Data acquisition and processing
The thermistors are measuring resistance changes induced by changes in temperature. The latterchanges are transformed into voltage changes via a Wheatstone bridge and recorded by the ThermosenseTM Console that allows the data to be displayed in real-time (Fig. 1C).
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In order to assess the in vivo validity of the catheter measurements, intra- and inter-operator variability was tested innormocholesterolaemic male New-Zealand White rabbits (n=6; 4.0±0.4kg). Briefly, the marginal ear vein was canulated and the rabbit was anesthetized with sodium pentobarbital (30mg/kg i.v.). After shaving the groin, the femoral artery was dissected and a 6F sheath was introduced. Under fluoroscopy, a 0.014inch guide wire (Boston Scientific) was positioned in the aortic arch prior to thermography of the descending aorta. A 30mm segment in the descending thoracic aorta (between 4th and 7th rib) was chosen and operator A performed two consecutive pullback measurements, blinded to the temperature data output. Then, operator B performed the same measurement on the same aortic segment blinded to the previously obtained temperature data.
Safety and feasibility studies were further expanded in normal coronary arteries (n=24) of 12 juvenile domestic pigs (Sus scrofa). Animals received ketamine (20mg/kg) and midazolam (3mg/kg) prior to intubation. The animals were then mechanically ventilated after sedation with Thiopental (1015mg/kg); Isoflurane (12.5% volume) given as needed for anaesthetic maintenance. A similar approach, this time using a 7F sheath via the carotid artery was used. After positioning the guiding catheter in the ostium of the left coronary artery, intravenous heparin (5000IU) and aspirin (300mg) were administered prior to placement of the intracoronary guide wire. Afterwards, scanning of the proximal segments (6cm) of each coronary artery (both left anterior descending and left circumflex artery) using the thermography catheter was performed.
2.5. Effect on endothelium
To evaluate the acute and sub-acute effects of the catheter on the coronary endothelium, we performed thermography in four coronary arterial segments of four pigs and compared the effects to a similar pullback by IVUS (CVIS, Boston Scientific) in four other coronary arterial segments of the same four pigs; the non-analysed arteries served as controls. Immediately after angiography, the catheters were advanced into the coronary artery, their position was recorded, and a motorised pullback was performed. In two animals, 150 to 200ml Evans Blue (0.3% in saline) were infused immediately after the procedure, directly into the coronary circulation following a saline flush. After completion of the Evans Blue-infusions, the coronary arteries were flushed with approximately 300ml saline before pressure fixation in situ (approximately 100mmHg) with 500ml electron microscope-fixative (4% buffered paraformaldehyde and 2%glutaraldehyde).9The heart was excised and vessels were prepared for further analysis.
The remaining animals were allowed to recover from anaesthesia and returned to the animal care facilities for 7 days during which 300mg of aspirin was administered daily until restudy including Evans Blue and scanning electron microscopy (SEM).
The excised vessels were opened longitudinally and evaluated under a dissection microscope for penetration of the blue dye. Macroscopic images of the arteries were documented in digital format. All arteries were processed for, and analysed using SEM using routine techniques.9
Endothelial assessment using immunohistochemical techniques was performed in six animals at three different time points (n=2 acute, 7 and 14 days). These animals underwent intravascular thermography in a segment of a coronary artery. At each time point, animals were euthanized and vessels were prepared for immunohistochemical staining by using the indirect peroxidase antibody conjugate technique (Factor-VIII antibody, dilution1/250, Binding Site). Sections were incubated with a goat anti-mouse peroxidase antibody (Jackson Laboratory) for 45min. The polyclonal sheep anti-Factor-VIII antibody was visualized by a pig anti-sheep peroxidase. Two experienced operators blinded to the invasive strategy performed histologic assessment of the endothelium. Four sections of each scanned arterial segment were analysed for circumferential lack ofendothelial cells and presence of thrombus.
2.6. Effect of flow
To investigate the potential influence of blood flow on temperature changes, we performed additional in vitro and in vivo tests in an experimental set up. For the in vitro experiment, we used a water bath in which the water was maintained at a constant temperature of 37°C (Fig. 2). Inside the bath, a 5mm diameter aluminium tubular section wound with a heater coil, potted in resin was inserted to create an artificial change (lesion). The lesion temperature was controlled to maintain a surface temperature difference (measured for zero flow) of 1°C. The flow rate was controlled through the lesion to achieve flow velocities of between 0 and 40cm/s. The upper values resemble velocities within the human coronary artery system. Pullbacks were performed to determine the measured temperature profile of the lesion.
To evaluate the effects of flow in vivo, we changed the flow in the infrarenal denuded aorta of rabbits that were fed a high cholesterol (2%) diet for 2 months, by inflating a balloon (5.0 Maxxum, Boston Scientific, USA) upstream of the region of interest. Velocity was measured by an intravascular Doppler wire (Flowmap®, Cardiometrics, USA) located downstream of the inflated balloon. In each animal, zero flow was induced and a pull back was performed. The highest, median and lowest temperature difference were then identified. Afterwards five flow steps (09, 1019, 2029, 3039 and 4049cm/s) in each of the animals were induced and changes in temperature difference at the previously defined locations were analysed.
2.7. Analysis and statistics
Data are given as mean±standard deviation (SD). Intra- and inter-observer variability was evaluated using Bland and Altman plots. To investigate the effect of flow, a regression analysis using an exponential fit was applied. The SPSS 10.0 software package was used for all analyses. A P-value <0.05 was considered significant.
3. Results
3.1. System stability
Measurements in a temperature controlled water bath with and without flow are shown in Table 1. The measurements performed with the catheter position stationary and with no flow past the sensors showed ±0.01°C variation. With flow and movement, the maximum temperature variation was 0.03°C.
3.2. In vivo and ex vivo experiments
All procedures were completed successfully, i.e. at no point during or after the procedure any adverse event (death, stroke, infection, allergic reaction or misbehaviour) occurred in any of the rabbits or pigs. Intracoronary spasm of pig coronary arteries was seen in the first two measurements, which were reversible with intracoronary administration of nitrates and did not occur anymore in any of the following animals after prior intracoronary administration of nitrates.
Intra-operator and inter-operator variability was assessed in the descending thoracic aorta of normocholesterolaemic rabbits by using Bland and Altman plots, i.e. of the difference (secondfirst pullback, Fig. 3A and operator Boperator A, Fig. 3B) against their temperature mean. The coefficients of repeatability (1.96xSD of the differences between the measurements) were 0.37 and 0.21 respectively.
3.3. Effect on endothelium
In acute experiments, control arteries showed a normal endothelium or only minor changes with small spots of Evans Blue coloration (Fig. 4A). These minor changes were characterised on SEM by raised nuclei and some surface folds (Fig. 4B). Evans Blue staining of arteries that underwent intracoronary thermography revealed intense coloration of the artery wall in clear lines that seemed to demarcate the pullback line of the thermistors (Fig. 4C). SEM showed endothelial denudation at the site of Evans Blue staining with occasionally adherence of leucocytes and platelets to the denuded surface. In areas of marked blue staining, correlating with the catheter tip, SEM showed the presence of islands of endothelium (Fig. 4D). In another coronary artery of the same pig, the IVUS catheter induced a similar degree of acute de-endothelialization (Fig. 4E). The areas stained following IVUS catheter injury also showed extensive denudation with patches of endothelium remaining (Fig. 4F).
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Intravascular ultrasound catheter injury also showed deep Evans Blue staining of the arterial wall (Fig. 4I). The injury inflicted by both the guiding- (ostium of the vessel) and IVUS-catheter, as visualized by the Evans Blue coloration, was characterized by denuded patches. Areas with less intense Evans Blue staining showed re-grown endothelium with dysfunctional cell-cell contact (Fig. 4J).
Immunohistochemical staining of the endothelial cells for Factor-VIII showed partial de-endothelialization in the thermography-interrogated segments immediately after the procedure (Fig. 5AB); small mural thrombi were found in a small number of sections (Fig. 5C) and were absent in control arteries (Fig. 5D). At 7 and 14 days, endothelial cells were visualized circumferentially in both control un-scanned and thermography-scanned pig coronary arteries (Fig. 5E and F) and mural thrombi were absent.
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We demonstrated that intracoronary thermography using a dedicated catheter in an experimental setting is safe and feasible. Intracoronary thermography was associated with an initial and partial de-endothelialization, which normalised within 2 weeks and paralleled the findings observed with an intravascular ultrasound catheter. Finally, temperature heterogeneity was minimally influenced under normal flow conditions but was more pronounced in absence of flow.
Detection of vulnerable plaque is becoming increasingly important since the majority of acute coronary syndromes with subsequent and oftenunpredicted adverse events arises from rupture of such plaques. Since these plaques or associated with an increase in inflammatory cells, especially at sites of ruptures,10thermography may therefore help in determining these plaques. It is suspected that inflammatory cells may be associated with increased temperature perhaps due to increased cellular metabolism.5,7,11Intracoronary thermography may therefore serve as a tool to determine these patients who are or might be at increased risk by characterizing these arteries showing increased temperature heterogeneity by means of a dedicated intravascular thermography catheter.
We demonstrated that intravascular thermography is at least as safe and feasible as IVUS, a widely applied technique of which an increase in the incidence of adverse events related to the technique has not been reported.12,13In a multicentre survey of 2207 examinations, IVUS was associated with a minor acute clinical risk.13Vessel coronary spasm was the most frequent event occurring during IVUS. We have seen some spasm during intracoronary thermography in pigs, which was completely reversible after administration of nitrates. However, by administering nitrates prior to thermography, spasms remained absent. There is no published data on endothelial damage caused by or associated with IVUS; however, we have shown that there is de-endothelialization after passage of an IVUS catheter. Thermography did also cause some initial and partial de-endothelialization but only to a same degree as IVUS did. Our histology findings at 14 days strongly suggest that repair of the endothelium occurred within 714 days after intracoronary thermography and ultrasound since staining for endothelial cells was complete on all samples. In addition, early and late events were absent and all vessels analysed were patent and free of intracoronary or mural thrombi at 7 and 14 days.
Today, there is only sparse information on the amount of temperature heterogeneity. In the landmark report of Cascells et al., temperature variations in ex vivo atherectomy specimen up to 2.2°C were documented.5The degree of heterogeneity was less pronounced in in vivo settings.6,7One might suspect that flow may play an important role since flow along the vascular wall may cause a cooling effect due to dissipation at the inflamed site and therefore decrease the temperature. Recently, Stefanadis et al. have shown that there is indeed a cooling effect of the blood flow on temperature heterogeneity in human stable plaques.8However, these findings were observed in patients with stable angina having mean diameter stenoses of 73%. Even in lesions showing no baseline temperature heterogeneity, there was an increase of 76% by blocking flow, which may suggest that other energy sources (i.e. myocardium) may be responsible for increased temperature in circumstances of absence of flow. It remains to be seen whether these observations can be extrapolated to vulnerable plaque lesions for which this technique has originally been designed for. Stable lesions are known to have less inflammatory cells and more smooth muscle cells with collagen fibres, which are less likely to show an increased temperature.7Our in vitro and in vivo experiments illustrate that temperature heterogeneity is not influenced by flow variation in the physiologic range. Although temperature heterogeneity is more pronounced in absence of flow, it remains unclear what the value of reduction or stop in flow means in a clinical setting of vulnerable plaque. Perhaps absence of flow in a clinical setting may lead to false positive temperature heterogeneity due to other energy expenditure unrelated to the vulnerable plaque.
In conclusion, intracoronary thermography using this dedicated catheter is a safe and feasible technique with a similar degree of de-endothelialization as the frequently used IVUS catheter. Temperature heterogeneity remained unchanged under normal flow conditions but was more pronounced in absence of flow. Intracoronary thermography may therefore be proposed as a valuable clinical tool for assessing the degree and meaning of temperature heterogeneity in human atheroscleroticarteries.
References