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The putative features of the (endo)vascular photothermal response, characterized by laser-induced thermal denaturation of blood and vessel wall constituents, have been elucidated individually, but not simultaneously in dynamic, isolated in vivo systems. A hamster dorsal skin fold model in combination with brightfield/fluorescence intravital microscopy was used to examine the effect of laser pulse duration and blood flow velocity on the size of the thermal coagulum, its attachment behavior, and laser-mediated vasomotion. The size of the coagulum and the extent of vasoconstriction and latent vasodilation were proportional to the laser pulse duration, but pulse duration had no effect on coagulum attachment/dislodgement. Blood flow velocity exhibited no significant effect on the studied parameters. The (endo)vascular photothermal response is governed predominantly by laser energy deposition and to a marginal extent by blood flow velocity.
©2007 Optical Society of America
1. Introduction
The use of lasers in the treatment of vascular pathologies has grown significantly in the last two decades. In ophthalmology, dermatology, and oncology, clinicians have employed different laser systems for the photothermolytic removal of aberrant vasculature [1–8]. The principal mechanism of photothermolytic treatment modalities is based on the photocoagulation of blood, which relies on the conversion of radiant energy to heat by (de)oxyhemoglobin and subsequent heat diffusion that results in thermal denaturation of blood and (peri)vascular tissue. Thermally denatured blood precipitates to form a so-called thermal coagulum; an amorphous clump of coagulated cellular constituents (e.g., membranes and cytosolic proteins) and plasma molecules (e.g., clotting factors and albumin). In therapeutic modalities such as selective photothermolysis [9], laser-induced thermal coagula act as hemostatic plugs through which vascular remodeling is likely mediated [10]. Although the general principles governing endovascular laser-tissue interactions have been described [9–15], the quantitative aspects of laser-induced damage in vivo, including thermal coagulum size, coagulum behavior, and the extent of vasoconstriction have not been measured as a function of laser parameters and rheological conditions. Inasmuch as photothermolysis is frequently used in the clinical setting, a thorough knowledge of endovascular laser-tissue interactions is imperative for treatment optimization.
In this study a hamster dorsal skin fold model and frequency-doubled Nd:YAG laser irradiation were used in conjunction with intravital fluorescence microscopy to examine the effect of laser pulse duration (LPD) and blood flow velocity (BFV) on thermal coagulum size, its attachment to and dislodgement from the vessel wall, and the extent of vasoconstriction and latent vasodilation. A modified fluorescence microscope was optically configured for the simultaneous acquisition of brightfield and fluorescence video images in order to visualize laser-induced thermal coagula and anatomical structures while monitoring blood flow by means of systemically administered fluorescent microspheres.
2. Materials and methods
2.1. Preparation of animals
The animal protocol was approved by the Lille University Hospital Animal Ethics Committee, and all animals were treated in compliance with the Resolution on the use of animals in research (Ministère Français de l’Agriculture et de la Forêt, No. 87-848, Agreement No. 4844). Ninety-five Gold Syrian hamsters (Dépré, Saint Doulchard, France) weighing between 89-113 g were anesthetized by intramuscular injection of ketamine (200 mg/kg), xylazine (10 mg/kg), and buprenorfine (0.03 mg/kg) after brief pre-anesthesia with diethyl ether. The dorsal side of the hamster was shaved and depilated with depilatory cream. The dorsal skin was lifted and placed between two symmetrical stainless steel frames containing a central annular window (Ø = 18 mm) which were secured onto the skin fold by sutures. The skin and adipose tissue covering the target venules (mean Ø = 147±27 μm, 78-233 μm range) were surgically removed from one layer of folded skin in order to improve intravital imaging. The other skin fold layer remained intact. Subsequently, the subclavian vein was exposed for the infusion of fluorescent microspheres. During the experiments vascular spasticity was deterred by continuous irrigation with 0.9% NaCl. After the experimental procedure, the animals were sacrificed by intravenous administration of KCl.
2.2. Blood flow monitoring
Intravenously administered fluorescent polystyrene microspheres (FluoSpheres Red, Ø = 1 μm, λex = 580 ± 15 nm, λem = 605 ± 15 nm, Molecular Probes, Eugene, OR) were used for measuring BFV. The absorption properties of the microspheres were specifically attuned to the absorption properties of blood (i.e., at λex, μa,deoxyHb = 192 cm-1 and μa,oxyHb = 260 cm-1 and at λem, μa,deoxyHb = 62 cm-1and μa,oxyHb = 10 cm-1, Jennifer Barton, personal communication). The high ratio μa,whole blood [λex] /μa,whole blood [λem] confined the optical penetration depth, and thus microsphere excitation, to the upper vessel segment, which allowed the comparison of all visible microspheres and ensured a sufficient contrast between blood and microspheres by the modest absorption-induced attenuation of the emitted light.
The microsphere stock solution was diluted to a concentration of 2.0 × 109 beads/mL with 0.9% NaCl, briefly sonicated, and immediately infused into the systemic circulation (1 mL/kg) through the subclavian vein using a 30 G needle (BD Biosciences, Etten-Leur, the Netherlands).
2.3. Intravital fluorescence microscopy
The animals were placed on a specially engineered platform containing three pins that interlock with holes in the implanted optical chamber so as to minimize motional disturbances during imaging. The platform was designed to fit on the translator stage of a modified intravital fluorescence microscope (10 × objective, NA = 0.30, Eclipse E800, Nikon, Tokyo, Japan). A diagram of the microscopy setup is provided in Fig. 1.
Fig. 1. (a) Experimental setup consisting of a modified intravital fluorescence microscope with mercury lamp epi- and transillumination. Labels (b) and (c) correspond to the respective panels. (b) Left-hand side: mercury lamp epi-illumination (blue beam) was filtered (λex = 560 ± 20 nm) and reflected towards the dorsal skin fold (yellow beam). The emitted light (orange beam) passed through a dichroic mirror (λcutoff = 595 nm) and emission filter (λem = 630 ± 25 nm) and was captured by the CCD video camera. Right-hand side: mercury lamp transillumination (blue beam) enhanced contrast as a result of differences in transmission spectra of thermal coagula, undamaged blood, and (peri)vascular tissue in the wavelength range of the emission filter. (c) The animal was secured to the microscope platform by the implanted optical chamber. The fiber guiding the light from the external mercury lamp was affixed in front of a reflective mirror that was mounted under the dorsal skin flap at a 45° angle with respect to the optical chamber. The 532 nm laser light was focused into the vessel lumen via a mirror in the probe.
The microscope was equipped with a Texas Red filter set (Y2EC-T25, λex = 560 ± 20 nm, λem = 630 ± 25 nm) and a mercury lamp (model 68806, Oriel, Stratford, CT) for excitation of the fluorescent microspheres and epi-illumination of the dorsal skin flap. A fiber-coupled mercury lamp (Karl Storz, Tuttlingen, Germany) was employed for transillumination of the skin fold to modulate contrast. The fiber was coupled to the platform via an SMA connector affixed in front of a reflective mirror that was mounted at a 45° angle with respect to the superiorly secured optical chamber. In this configuration, the 50 nm bandwidth of the emission filter in combination with broadband light transillumination allowed passage of sufficient light to generate brightfield images of the region of interest simultaneously with fluorescence mode for microsphere monitoring. Because the transmission coefficients of photocoagulated blood < undamaged blood < (peri)vascular tissue in the spectral range of the emission filter (605-655 nm) [13], the different structures, i.e., thermal coagula, blood vessels, and perivascular (adipose) tissue could be easily discriminated by virtue of their varying intensities. Images were captured with a Peltier-cooled CCD video camera (model L3C65-06BPV01, E2V Technologies, Chelmsford, UK) at 25 frames per second and a resolution of 720 × 576 pixels. The endovascular events were recorded with a digital video recorder (DVR 30, Sony, Tokyo, Japan) and visualized on a monitor (Trinitron, Sony).
2.4. Laser-induced (endo)vascular damage
A frequency-doubled Nd:YAG laser (Entertainer, Laser Quantum, Stockport, UK) was used for the induction of (endo)vascular damage. The laser system emits a wavelength of 532 nm, corresponding to a high absorption coefficient of blood [16]. Proper targeting was achieved by mounting the laser on a custom-built xyz-translator stage and guiding the light via a probe, a focusing lens (NA = 0.05), and a mirror onto the target venule. The laser spot (A = 2.3 × 103 ± 0.3 × 103 μm2, measured using photosensitive paper) was elliptically shaped as a result of its angle of incidence. The longitudinal axis of the spot, mean ± SD r1 = 38 ± 2 μm (N = 9), was positioned parallel to the length of the vessel, whereas the lateral axis of the spot, r2 = 20 ± 2 μm, was orthogonal to the length of the vessel. All incident light was focused into the vessel lumen, with pulse energies ranging from 6.7 to 33.6 mJ and radiant exposures from 289 ± 38 to 1444 ± 190 J/cm2 of the 30 and 150 ms pulses, respectively. Pulse duration was regulated with an analog shutter containing a 10% transmission filter to enable aiming of the laser beam prior to irradiation. LPDs were adjusted to 30 (N = 18), 60 (N = 18), 90 (N = 17), 120 (N = 20), and 150 ms (N = 22) at a fixed laser output power of 224 mW. One venule was irradiated per hamster.
2.5. Data analysis
The size of the laser-induced thermal coagulum, its attachment to and dislodgement from the vessel wall, and the extent of vasoconstriction and latent vasodilation were measured as a function of BFV and LPD. BFV was determined by averaging the distance traveled by the 10 fastest microspheres per unit time during a period of 2 min prior to laser irradiation. Each microsphere was traced frame by frame using Adobe Premier Pro and the distance traveled was quantified offline with Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA). For coagulum size measurements, the isolated video frames of laser-induced thermal coagula (first frame after the laser pulse) were manually contoured in Photoshop and the contoured regions quantified for pixel area using SigmaScan Pro 1.5 (Aspire Software, Ashburn, VA). The total pixel area of the thermal coagulum represented an approximation of its size. The duration of coagulum attachment was timed and coagula were considered attached when they did not dislodge within 5 min post-irradiation. The inner diameter of the vessel at the irradiated site was measured prior to and immediately after the laser pulse (to compute vasoconstriction) and at 1 min intervals for a period of 5 min post-irradiation (to compute latent vasodilation). For the vessel diameter measurements, a line was drawn wall-to-wall on the isolated video frame at the site of maximum constriction and its length recorded. The corresponding video frames were isolated with Adobe Premiere Pro and the drawn lines quantified with Photoshop. All vessel diameters were normalized by dividing the diameters after irradiation by the diameters measured prior to irradiation.
Statistical analysis (means, standard deviations, correlation analysis, linear regression analysis, and independent homoscedastic Student’s t-tests) were performed with Statistical Package for Social Sciences 12.0 (SSPS, Chicago, IL). A p-value of ≤ 0.05, designated with (*) throughout the text, was considered statistically significant. A p-value of ≤ 0.01 is designated by (**). Kolmogorov-Smirnov and Shapiro-Wilk tests confirmed the normal distribution of the data sets.
3. Results
3.1. Blood flow velocity distribution
The BFV of the irradiated vessels, measured prior to laser irradiation, ranged from 0.13 mm/s to 1.02 mm/s with a mean ± SD BFV of 0.48 ± 0.21 mm/s (Fig. 2). The mean flow velocity of 0.48 mm/s corresponds to 72 μm/150 ms, where 72 μm approximates the longitudinal diameter of the laser spot. For analysis of thermal coagulum size, coagulum attachment/detachment, vasoconstriction, and latent vasodilation as function of BFV, the BFVs were categorized into three groups: 1) low BFVs: < 0.35 mm/s (N = 30); 2) moderate BFVs: 0.35 ≤ BFV < 0.55 mm/s (N = 32); and 3) high BFVs: ≥ 0.55 mm/s (N = 29) (Fig. 2). Four of the irradiated vessels were excluded from analysis because laser irradiation induced complete vaso-occlusion and/or vessel rupture (1 × 90 ms, 3 × 150 ms). There were no vessels with a high BFV in the 30 ms LPD group.
Fig. 2. Blood flow velocity (BFV) distribution of the irradiated venules, measured prior to laser irradiation. BFV category 0.1 corresponds to a BFV range of 0.05 ≤ BFV < 0.15 mm/s; BFV category 0.2 corresponds to a range of 0.15 ≤ BFV < 0.25 mm/s; etc. The color of the bars indicates the grouping of BFVs for the quantification of coagulum size, coagulum attachment/detachment, vasoconstriction, and latent vasodilation.
3.2. Thermal coagulum size
Figure 3 shows the mean sizes of laser-induced coagula as a function of BFV and LPD. With the exception of the 60 ms LPD group (Pearson’s r = -0.678**), no correlation was found between coagulum size and BFV. Moreover, the intragroup variability in r-values underscores the overall random nature of this relationship. For example, the coagulum size in the 60 ms LPD group decreased at higher BFVs (negative r-value), whereas this trend was opposite for the 150 ms laser pulses (positive r-value) and absent for the 90 ms LPD group (r-value close to 0).
Fig. 3. Laser-induced coagulum size plotted as a function of blood flow velocity (BFV) and laser pulse duration (LPD). The mean coagulum sizes exhibit an increasing trend with longer LPDs, but seem to be independent of BFV.
To analyze coagulum size as a function of LPD, the mean coagulum sizes for each LPD group were calculated (blue bars). The mean coagulum size exhibited an increasing trend with longer LPDs (Fig. 3, R2 = 0.88 and r = 0.524**). The coagula produced by a 30 ms laser pulse were significantly smaller (**) than the coagula produced at longer LPDs. An example of the size differences is provided in Fig. 4 (movie), where a 30 ms LPD-induced coagulum is juxtaposed to a thermal coagulum produced by a 150 ms pulse. Significant differences were also found between the mean coagulum size of the 60 and 150 ms (*) and the 90 and 150 ms (*) LPD groups.
Fig. 4. (2.7 MB) Movie: Laser pulse duration-dependent thermal coagulum size (10.0 MB version). Thermal coagulum size induced by 30 ms irradiation (a) (4.6 × 103 μm2) was significantly smaller than the coagulum size induced by 150 ms irradiation (b) (10.4 × 103 μm2). Scale bar = 100 μm.
3.3. Coagulum attachment
The percentage of thermal coagula that remained attached to the vessel wall during a period of 5 min was calculated as a function of BFV and LPD. Coagulum attachment was independent of both BFV and LPD, since no statistical pattern could be found for either parameter. This is exemplified in Fig. 5 (movie), where a 6.5 × 103 μm2 coagulum induced by a 30 ms pulse remains attached at moderate BFV (0.5 mm/s) and a 16.9 × 103 μm2 coagulum induced by a 120 ms pulse dislodges at low BFV (0.2 mm/s). Of all coagula 37.9% dislodged, mostly within 0-10 s post-irradiation. Only a few coagula did not initially attach at all and were directly translocated downstream. All attached coagula exhibited gradual volume reduction, possibly due to shear stress-induced fragmentation.
Fig. 5. (2.8 MB) Movie: Randomness of coagulum attachment and dislodgement (13.6 MB version). In the first example (a) a coagulum (6.5 × 103 μm2) induced by a 30 ms laser pulse attaches at 0.5 mm/s blood flow velocity (BFV), whereas in (b) a coagulum (16.9 × 103 μm2) produced by a 120 ms pulse dislodges at a 0.2 mm/s BFV. Scale bar = 100 μm.
3.4. Vasoconstriction and latent vasodilation
The mean normalized vessel diameters following a laser pulse are shown in Fig. 6. The diagram only contains the results for the 30 and 150 ms LPD groups, inasmuch as the results for the 60, 90, and 120 ms LPD groups follow a similar pattern. Vasoconstriction occurred during the laser pulse, which manifested itself in both lateral and axial shrinkage of the vascular tube (Fig. 7, movie), and was ensued by a gradual dilation of the constricted vessel segment. However, only lateral constriction was measured.
Fig. 6. Mean normalized vessel diameters before irradiation and at 0, 1, 2, 3, 4, and 5 min after irradiation for the 30 and 150 ms laser pulse duration (LPD) groups. Blood flow velocity (BFV) data is discounted since no correlation was found between vasoconstriction and latent vasodilation as a function of BFV. The normalized vessel diameters measured directly after irradiation decreased with longer LPDs. Within 5 min post-irradiation, vessel diameters expanded over time for each LPD group; the rate of expansion was positively correlated to the degree of laser-induced vasoconstriction (Fig. 9).
Fig. 7. (2.9 MB) Movie: Laser-induced axial vasoconstriction (15.1 MB version). Two microspheres that are attached to the vessel wall ((a), arrow) are ‘pulled’ towards the irradiation site during the 150 ms laser pulse, demonstrating that vasoconstriction also occurs along the longitudinal axis ((b), arrows, indicating pre- (grey) and post-irradiation (black) microsphere positions). Scale bar = 100 μm.
No correlation existed between vasoconstriction/latent vasodilation and BFV. The mean vessel diameters measured directly after irradiation decreased with larger LPDs (r = -0.600**), suggesting that the extent of vasoconstriction is proportional to the LPD, albeit in a non-linear fashion (R2 = 0.91). The levels of vasoconstriction produced at 30 ms differed statistically from the higher LPD groups (**). A statistical difference also existed between the 60 and 120 ms (*), 60 and 150 ms (**), and 90 and 150 ms LPDs (*). The mean vessel diameters gradually expanded during a 5 min time span after the laser pulse, but never reached their original diameter (Fig. 8, movie). The vessel diameters in the 30, 60, 90, 120, and 150 ms LPD groups restored to 79%, 70%, 69%, 65%, and 62% on average, respectively (R2 = 0.94). Latent vasodilation after 5 min exhibited a weak negative correlation with LPD (r = -0.433**). Vasodilation in the 30 ms LPD group differed statistically from the extent of vasodilation after 5 min in the higher LPDs (* or **). Also, a significant difference existed between the 60 and 150 ms LPD groups (*), but no difference was found between the 90, 120, and 150 ms LPD groups. Moreover, the extent of latent vasodilation after 5 min correlated strongly with the degree of vasoconstriction (r = 0.767**). The most profound mean dilation occurred in the first minute after the laser pulse (**) (Fig. 9), which correlated weakly to the LPD (r = 0.415**). However, statistical differences in dilation rates (defined as [(Ø t - Ø t-1) / Ø t], where t is time after irradiation in min) during the first min following a laser pulse were only found between 30 ms LPD and the 90, 120, and 150 ms LPD groups (**).
Fig. 8. (2.9 MB) Movie: Laser-induced vasoconstriction and latent vasodilation (8.5 MB version). Vessel diameter measurements were performed prior to irradiation, directly after the laser pulse, and every min up to 5 min post-irradiation. During a 30 ms laser pulse, the vessel constricts from 150 μm to 51 μm ((a), arrow) and then dilates during a period of 5 min to a diameter of 62 μm ((b), arrow). Scale bar = 100 μm.
Fig. 9. Mean rate of latent vasodilation expressed as a percentage, plotted as a function of time (min) after laser irradiation. For the calculation of the mean latent vasodilation, the pulse durations (30, 60, 90, 120, and 150 ms) were grouped per time interval. The rate of vasodilation was calculated as [(Øt - Øt-1) / Øt], where Ø is the vessel diameter and t is time (min) after laser irradiation, and expressed as a percentage.
4. Discussion
Endovascular laser-tissue interactions are defined by 1) a photothermal response, which results in thermal damage to blood and (peri)vascular tissue, and 2) a hemodynamic response, which is consequential to the photothermal effects and manifests itself by thrombosis at the site of laser-induced vascular damage [10,17]. Suthamjariya et al. provided an elaborate account of the photothermal response in vivo during the laser pulse at different laser parameters [14], whereas our group has qualitatively examined the laser-induced events that occur in vivo mainly after irradiation [12,15,17].
4.1. Laser-induced (endo) vascular photothermal response
The photothermal response can be subcategorized into two fundamental effects: 1a) endovascular effects, such as denaturation and aggregation of plasma proteins, formation of a thermal coagulum, and coagulum attachment/dislodgement; and 1b) (peri)vascular effects, including thermal coagulation and constriction of the vessel wall, heat diffusion-mediated denaturation of matrix proteins and consequent shrinkage of perivascular tissue, and vessel wall rupture in cases of rapid and/or excessive heat build-up. To describe the events that occur during the formation of a thermal coagulum, a tri-phasic process has been described by Black and Barton [13,18,19], consisting of a heating phase (T < 70 °C), a primary coagulation phase (T ≥ 70 °C, during irradiation), and a secondary coagulation phase (T ≥ 70 °C, post-irradiation). During the heating phase plasma proteins such as albumin, which have a lower thermal denaturation threshold than lipoproteins or cell membranes [20], denature and precipitate. These aggregates are optically translucent when observed with 532 nm darkfield orthogonal polarized spectral imaging [12], suggesting that they are devoid of (de)oxyhemoglobin-containing cells. In the subsequent primary coagulation phase, red blood cells transform from biconcave disks to spherical cells (spherocytes), which rupture and aggregate to form a thermal coagulum. Post-irradiation heat diffusion causes an expansion of the thermal coagulum by the addition of proximal red blood cells/spherocytes undergoing the same processes during the secondary coagulation phase. Heat diffusion into collagen-rich tissue is responsible for the laser-induced (peri)vascular effects, as has been corroborated by numerous studies on structural and mechanical alterations of collagen subjected to excessive hyperthermia [21–27] as well as by histological examination of laser-treated port wine stains [28,29]. Thermally-induced breakage of intramolecular (hydrogen and disulfide) bonds leads to the transformation of the collagen’s triple helix molecular structure into a random coil. This results in an overall shrinkage of the collagenous tissue [21–27], which, in case of (peri)vascular collagen, likely contributes to the local constriction of the vascular tube or even complete vessel closure. In addition, chemical interactions between the thermal coagulum and vessel wall constituents might be thermally-mediated during both coagulation phases and could be associated with coagulum attachment to the intima.
The aim of this study was to further investigate the photothermal response in an isolated, dynamic in vivo system in order to elucidate additional features of (endo)vascular laser-tissue interactions that may also bear clinical relevance. First, the role of BFV was studied since blood flow is responsible for heat convection and creates hemodynamic pressure on the thermal coagulum and constricted vascular segment. It was therefore expected that high pre-irradiation BFVs would limit vasoconstriction and enhance coagulum dislodgement and latent vasodilation. Secondly, the photothermal effects were examined at varying LPDs. During the heating- and primary coagulation phase, the radiant energy from the laser pulse is converted to heat that diffuses from the nucleation site, creating an isotropic Gaussian temperature profile which broadens as a function of time (notwithstanding the complex influence of convection and the changing structural and optical properties of irradiated tissue) [11]. The extent of the isotropic broadening is theoretically proportional to the deposited energy and hence to the laser power and LPD. Increasing LPDs at a constant laser power should therefore coincide with a three-dimensional expansion of supracritical isotherms (≥ 70 °C), which in turn should result in larger thermal coagula, an increased probability of coagulum attachment (due to a larger contact area with the intima), and a greater degree of vasoconstriction.
The experimental results have shown that all measured parameters (coagulum size, coagulum attachment/dislodgement, vasoconstriction, and latent vasodilation) were independent of pre-irradiation BFV but correlated positively to the LPD, albeit to a limited extent. Coagulum size and the extent of vasoconstriction increased proportionally to the LPD. A positive correlation was found between the extent of vasoconstriction and latent vasodilation across all LPD groups, with the rate of latent vasodilation being most pronounced in the first minute after laser irradiation. No relationship was found between coagulum attachment and either BFV or LPD.
4.2. Blood flow velocity
The absence of a relationship between the BFV and the measured parameters may result from transiently altered hemodynamics and vasoconstriction during the laser pulse, neutralizing any influence of convection. Figure 10(a) (movie) shows that a microsphere flowing downstream of the irradiation site accelerates during laser irradiation. This is likely caused by the increasing endoluminal temperature during the heating phase, which leads to a rapid expansion of the irradiated blood volume. In addition, the locally constricting vessel segment could ‘squeeze’ the (photocoagulated) blood away from the irradiation zone during the primary coagulation phase. During these events blood is translocated away from the irradiation site, both upstream and downstream. Furthermore, the transiently attached coagulum and the local vasoconstriction may jointly cause temporary hemostasis in the irradiated vessel segment until the coagulum dislodges (Fig. 9(b)). Naturally, if blood flow during the laser pulse is distorted, then no (or perhaps a limited) relation exists between the pre-irradiation BFV and the laser-induced thermal coagulum size and the extent of vasoconstriction. It should be noted that post-irradiation events such as coagulum dislodgement and vasodilation could still be influenced by the (possibly altered) BFV through heat convection and hemodynamic pressure on the coagulum and the constricted vessel segment. Also, BFV may have a more measurable impact on coagulum dynamics at LPDs shorter than 30 ms due to less extensive photothermal effects.
Fig. 10. (2.0 MB) Movie: Altered hemodynamics during laser irradiation (14.2 MB version). Microspheres flowing downstream of the irradiation site accelerate during the laser pulse ((a), arrow). Directly after laser irradiation, stasis of microspheres coincides with cessation of blood flow (b). After a few seconds, the attached coagulum dislodges and blood flow is restored. Scale bar = 100 μm.
4.3. Laser pulse duration
The LPD, in contrast, was shown to be a considerable factor in the photothermal response. Thermal coagula and vasoconstriction were observed in all irradiated vessels at all LPDs, indicating that even during the short (30 ms) laser pulses blood and (peri)vascular tissue were supracritically heated. With a positive correlation between LPD and thermal coagulum size it could therefore be concluded that at longer pulse durations (60, 90, 120, and 150 ms), isotropic broadening of the thermal damage zone is achieved due to more pronounced primary- and secondary coagulation effects. Additionally, thermal denaturation is a rate process, meaning that the extent of laser-induced tissue damage is a time- and temperature-dependent phenomenon [23]. Hence, longer LPDs lower the critical temperature threshold for denaturation and therefore contributed to a more profound damage profile.
4.4. Latent vasodilation
Constricted vessel segments exhibited latent dilation during a period of 5 min after the laser pulse, which was prevalent in all LPD groups. Mechanistically, the vasodilation observed in our study differed from the dilation found by Verkruysse et al. [15], which occurred in the absence of prior vasoconstriction and within ms after the laser pulse. The rate at which vasodilation occurred increased with the extent of constriction (and thus with LPD), suggesting that an elastic force on and/or in the constricted vascular tube may be causing the latent dilation. This force may originate from an increased tension imposed by thermally unaffected perivascular tissue, slightly stretched due to the local constriction of the vessel and directly adjacent tissue, thereby ‘pulling’ the tube segment back towards its original shape [25–27]. The rapid dilation rate in the first min following laser irradiation (Fig. 9) suggests that the tension is quickly redistributed over the stretched tissue volume until a state of tensile equilibrium has been achieved, as is deducible from the asymptotic decay of the curve. Figure 7 shows that shrinkage of (peri)vascular tissue occurred in both lateral and axial direction. Latent vasodilation may therefore be even more pronounced in situations where the irradiated vasculature is entirely tissue-embedded as the elastic forces will be distributed more isotropically. Consequently, spatial confinement of thermal damage may decrease the latent vasodilation rate inasmuch as the elastic tension caused by contraction of the vascular tube will be distributed over a greater volume of malleable, non-affected perivascular tissue. Another possible contribution to the dilation is the increased shear stress in the constricted segment, resulting from the local reduction of vessel diameter in combination with an unaltered blood flow (shear stress is inversely proportional to the third power of the vascular diameter). Nevertheless, it should be stressed that biochemical signaling pathways and neural modulation could play a role in both vasoconstriction and the latent vasodilation.
4.5. Coagulum/vessel wall interactions
A striking result was the complete lack of correlation between coagulum attachment/dislodgement and all the parameters, e.g., BFV, LPD, coagulum size, and vasoconstriction. Coagulum attachment has been examined histologically in a 577 nm pulsed dye laser-treated port wine stain biopsy, where photocoagulated red blood cells appeared to be affixed to a relatively intact and morphologically distinguishable endothelial cell [30, also shown in 10]. These findings, in addition to the yet inexplicable random character of coagulum behavior, underscore the rather obscure nature of physiological and biochemical phenomena that arise during endovascular laser-tissue interactions. Namely, the thermodynamic implications of the primary and secondary coagulation phases appear to be cell type-specific, and the thermal coagulum-endothelial layer interactions are complex and probably governed by a multitude of variables. With respect to the former, it is difficult to rationalize why endothelial cells, positioned only a few Ångströms from the nucleation site that has unequivocally passed through the primary and secondary coagulation phases, do not exhibit substantial heat diffusion-induced swelling and consequent cell rupture. The endothelial cells appear to retain their general morphological features which have been completely lost by the thermal coagulum-embedded red blood cells. Upon closer examination, the margins of the photocoagulated red blood cells are not in all instances in direct contact with the endothelial cell membrane as evidenced by the electron-lucent void between these structures. This, in conjunction with the discrete endothelial cell-thermal coagulum interface, is why we postulated in [10] that thermal coagulum-endothelial cell interactions are likely due to heat-mediated chemical affixation of blood cell/endothelial cell membrane (glyco)proteins. At supracritical intraluminal temperatures, these proteins undergo denaturation, during which charged moieties or atoms capable of electrostatic bonding become available for intermolecular interactions. When the denatured proteins of two different membranes come in close proximity, such as during simultaneously occurring vasoconstriction and thermal coagulum formation, they can cross-link rather than revert to their original or slightly altered 3D configuration [31], thereby covalently (S-S bridges) or electrostatically (e.g., H-bonding) tethering the thermal coagulum to the endothelial layer. Involvement of the cell membrane in the attachment process cannot be dismissed given the protein/polypeptide kinetics in a thermotropically-disturbed phospholipid bilayer/aqueous system [32,33]. Following this hypothesis, one would expect that the percentage of attached coagula would increase with LPD as a result of a larger adhesion area of the thermal coagulum, and would decrease at higher BFVs since hemodynamic pressure on the coagulum increases with coagulum size and BFV during and after irradiation. However, the results suggest that factors other than the ones studied here, perhaps acting in concert with LPD and BFV, are responsible for coagulum behavior. More in-depth studies on in vivo coagulum behavior are warranted.
5. Conclusions
The expanded model of the laser-induced (endo)vascular photothermal response might serve to improve the laser-treatment of numerous vascular pathologies. As was expected, LPD was shown to be an important treatment parameter, since both coagulum size and extent of vasoconstriction increased with pulse duration. However, the increased vasoconstriction reflects a decreased spatial confinement of the thermal damage due to excessive heat diffusion, leading to thermal denaturation of non-target tissue. The resulting latent vasodilation underscores the necessity of confining thermal damage to the vascular tube, inasmuch as dilation (and thus reperfusion) may impair proper vascular remodeling [10]. However, the precise (patho)physiological implications of vasodilation are difficult to deduce from this study. In addition, the mechanisms of coagulum attachment should be studied explicitly, since the formation of thermal coagula and their interaction with the intima are paramount for selective photothermolytic therapies and could serve as a basis for site-specific pharmaco-laser therapy, as proposed in [10] and [17].
Acknowledgments
This work was supported by the Technological Collaboration Grant (TSGE 1048) of the Dutch Ministry of Economic Affairs. M.H. was partially supported by a personal grant from Novo Nordisk Farma, thanks to Peter Gisberts and Pim Tijburg.
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