Differential vascular response to laser photothermolysis.

Individual blood vessels in the chick chorioallantoic membrane were selectively coagulated through pho tothermolysis, using pulsed laser irradiation at 585 nm. Pulse durations were chosen to be 0.45 ms and 10 ms, which correspond to the thermal relaxation times in blood vessels of 30 Jl.m and 150 Jl.m diaOleter, respec tively. The short pulses, at a light fluence F = 3 Jcm- 2, caused permanent occlusion of vessels of 40 Jl.m diame ter or less, whereas larger caliber vessels (60-120 .um) required F = 4 - 5 Jcm- 2• The long-duration pulses, at F = 7 Jcm- 2, caused coagulation of the larger diameter vessels; the small-caliber vessels and capillaries showed resistance to photothermolysis and required multiple exposures to achieve coagulation. The fluence versus diameter (F versus d) relationship for coagulation was calculated for the two pulse durations. The energy de-T effective. The chick chorioallantoic membrane (CAM) is an established irz vivo model for studying microvascular effects [10]. The CAM vas culature is located in a transparent matrix [11] that allows direct visualization of blood flow as well as real-time observation of pho tothermal effects on blood vessels. such as vessel dilation. constric tion. hemostasis. and rupture. The CAM matrix does not signifi cantly absorb or scatter radiation. Thus. the influence of the pertinent laser parameters (wavelength. pulse duration. and fluence) that affect the CAM vasculature can be studied conveniently. More over. the CAM is a self-contained system that lends itself to mathe matical modeling of optical and thermal effects [12]. Preliminary

Individual blood vessels in the chick chorioallantoic membrane were selectively coagulated through photothermolysis, using pulsed laser irradiation at 585 nm. Pulse durations were chosen to be 0.45 ms and 10 ms, which correspond to the thermal relaxation times in blood vessels of 30 Jl.m and 150 Jl.m diaOleter, respectively. The short pulses, at a light fluence F = 3 Jcm- 2 , caused permanent occlusion of vessels of 40 Jl.m diameter or less, whereas larger caliber vessels (60-120 .um) required F = 4 -5 Jcm-2 • The long-duration pulses, at F = 7 Jcm-2 , caused coagulation of the larger diameter vessels; the small-caliber vessels and capillaries showed resistance to photothermolysis and required multiple exposures to achieve coagulation. The fluence versus diameter (F versus d) relationship for coagulation was calculated for the two pulse durations. The energy de-T he objective of this study is to gain an understanding of the biophysical principles underlying permanent coagulation of blood vessels. through conversion of selectively absorbed radiant energy into thermal energy. Such understanding is of importance in dermatologic laser treatments. such as port wine stains (PWS) [1][2][3][4]. telangiectasias [5.6]. or hemangiomas [7] and may also be of benefit in the treatment of choroidalneovascularization [8.9]. This information is expected to assist physicians in improving laser treatments of vascular lesions. which at the present time are not totally effective.
The chick chorioallantoic membrane (CAM) is an established irz vivo model for studying microvascular effects [10]. The CAM vasculature is located in a transparent matrix [11] that allows direct visualization of blood flow as well as real-time observation of photothermal effects on blood vessels. such as vessel dilation. constriction. hemostasis. and rupture. The CAM matrix does not significantly absorb or scatter radiation. Thus. the influence of the pertinent laser parameters (wavelength. pulse duration. and fluence) that affect the CAM vasculature can be studied conveniently. Moreover. the CAM is a self-contained system that lends itself to mathematical modeling of optical and thermal effects [12]. Preliminary Manuscript received April 20. 1993;revised April 27. 1994; accepted for publication June 3.1994 Abbreviations: CAM. chorioallantoic membrane; Hb. hemoglobin; Hb0 2 • oxyhemoglobin. PWS. port wine stains; RBC. red blood cell. posited in a cylindrical absorber of diameter d by an optical field, incident perpendicular to the vessel, was expressed analytically and compared with the energy required to coagulate a blood vessel of the same lumen diameter. When thermal diffusion is incorporated into the model, our findings can be accounted for quantitatively. This information will be of use for iOlproving the laser treatment of port wine stains and other vasculopathies. A surprising observation was that arterioles were damaged at lower incident energy densities than venules having the same IUOlen diameter, despite the fact that absorbance in oxygen-rich and oxygen-poor blood is the same at 585 nm. Key words: arteries and veins/CAM/hemostasis/pulsed dye laser. ] Invest Dermatol 103: 693 -700, 1994 considerations are presented here for the choice oflaser parameters.
which are fully described in Materials a/ld Methods.
Wavelength (A) Light anywhere within the visible spectrum may be used to irradiate the transparent CAM blood vessels without encountering absorption in overlying epidermis that normally accompanies irradiation of dermal tissues [13.14]. In accordance with general considerations associated with increased light penetration in tissue at longer wavelengths [1.14-16] the absorption of blood in the yellow-red spectral region around 585 nm was chosen for irradiation. A singular feature of the CAM is the possibiliry to study coagulation patterns in individual arterioles and venules. The primary targets for radiative heating are the red blood cells (RBCs). which undergo morphologic changes following pulsed laser irradiation [ 17]. We seek. therefore. to equalize photothermal effects due to light absorption by the two endogenous chromophores. oxyhemoglobin (Hb0 2 ) and hemoglobin (Hb). present in the RBCs. Equal absorption is achieved at the isosbestic point (A = 585 nm) in the absorption spectra of these two target chromophores. Because of deeper tissue penetration. this wavelength is of clinical interest. even though the absorption coefficient at 585 nm is 50% lower than the absorption peak of Hb0 2 at A = 577 nm [15.16].
Pulse Duration (t p ) The pulse duration governs the spatial confinement of the thermal energy within the targ~ted vessel [1.18].
Ideally. the pulse duration (t p ) should be compatible with the diameter (d) of the vessel and be about equal to the thermal relaxation time (Td) for that dimension (Td = d 2 /16X. where X is the thermal diffusivity). This is defined as the time required for the instantaneous temperature. generated inside the target after exposure to the 0022-202X/94/S07.00 Copyright © 1994 by The Society for Investigative Dermatology. Inc. 693 laser pulse, to decrease by 50% (see Discussion). Taking X = 1.4 X 10-7 m 2 seconds-l , corresponding to water, typical values for 'd are 0.2 mseconds for d = 20 . .um and 4.5 mseconds for d = l00.um. If Ip ~ 'd, heat diffuses outside the vessel during the laser exposure, reducing the target specificity, and can cause extra thermal damage to surrounding tissue. A very short pulse, t ~ 'd, will generate a high-peak intravascular temperature rise, reading to localized explosive vaporization of tissue water, or to photoacoustic transients, which will result in vessel rupture [19]. In such cases, repair mechanisms may revascularize the tissue [1].
Radiant Energy of Pulse per Unit Area, or Fluence (F) For PWS therapy, it is important to know the damage-threshold fluence in hypervascular skin, F,h(l p ), sufficient to effect selective, irreversible thermal injury to vessel wall structures, without causing rupture of the targeted vessel with subsequent hemorrhage. The magnitude of F", (Ip) is difficult to establish by theoretical modeling, because of epidermal melanin absorption, multiple scattering events within the skin, and the fact that blood vessels are located at different dermal depths [16]. In the CAM, scattering and nonspecific absorption are small and measurements are made on identifiable targets. The depth of the targeted vessel in the CAM is of little consequence, provided no other superficial vessel is seen to shield part of the incident beam. Thus, measurements in the CAM will provide single-vessel values of the damage-threshold fluence, Fo(d,I,), which can be used as input in the derivation of the damage-threShold fluence for the composite vasculature, F'h(lp) '

MATERIALS AND METHODS
Laser Parameters A continuous wave (CW) Argon-ion pumped dye laser (model 920, Coherent, Palo Alto, CA) was used for single-vessel irradiation. The laser delivered a maximum power of 1.4 W at 585 nm, as measured with a power meter (model 210, Coherent). The laser beam was transmitted through an 80-Jim core-diameter multimode fiber terminated with an adjustable focusing microlens positioned in a hand piece. The diameter of the beam at the focus was 500 Jim, giving, at maximum, a fluence F = 700l p = 7 )cm-z/pulse. The CW laser was pulsed with a foot-pedalcontrolled mechanical shutter; the pulse duration was preselected at the shortest attainable setting of Ip = 10 ms.
A flashlamp-pumped dye laser (model SPTL-l, Candela, Weyland, MA) was used for multiple-vessel irradiation. The laser was tuned to ;. = 585 nm and delivered pulses of duration t = 0.45 ms. The beam was coupled into a l-mm core-diameter multimod: fiber terminated with a microlens that focused the laser output to a 5-mm diameter circular spot of uniform light intensity. The optical energy exiting the fiber was varied between 0.7 and 1:3), as measured with a calibrated energy meter (Ophir, model lOA-P), glvmg fluences on the CAM membrane ranging between F = 3 and 6 )cm-z/pulse. Preparation of Intact CAMs The protocol for CAM preparation was a modification of a previously described technique [10] . Fertilized eggs (Hyline W36 white leghorn) were washed with 70% alcohol, incubated at 37°C in 60% humidity, and rolled over hourly. On day 3-4 of embryonic development, a hole was drilled in the apex and 2 -3 ml albumin was aspirated from each egg to create a false air sac. On the following day, part of the CAM was exposed by opening a round window of 20-mm diameter in the shell that was covered with a Petri dish. The eggs were placed in a stationary mcubator until the CAM was fully developed and ready for experimentation. On day 10-12, sterile teflon O-rings (6.2 mm inner diameter, 9 mm outer diameter, and 1.4 mm annu lar width) were placed on the surface of the CAM, each demarcating a location where individual blood vessels and capillaries were clearly visible and to which the laser beam was directed. A drop of normal saline was added within the ring area to reduce spurious light reflection and to prevent desiccation of the CAM during the experiment [12]. Outside of the incubator, eggs were kept at 35°C in a heating block filled with glass beads. At the time of irradiation, the CAM was illuminated with a cold white-light fiber-optic source (Volpi, Intralux, model 100 HL) and placed under a stereomicroscope (Olympus, model SZH), equipped with a video camera (Panasonic, model AC-251 0), giving a total magnification of 70X on a color monitor (Sony, model KV-1393R).
Vessel Selection It was convenient to subdivide the extensive microvascular network of the CAM according to the following branching pattern [20]. The capillaries served as a reference and were designated vessels of "order-O." The smallest precapillary vessels (arterioles, a) as well as the smallest postcapillary vessels (venules, v) were assigned "order 1." The  convergence of two order 1 vessels was assigned as an "order 2" vessel and similarly two order 2 vessels formed an "order 3" vessel. Table I presents, for each order, the mean number n of blood vessels per square centimeter, the mean vessel length I (Jim) and diameter d (Jim) in a mature CAM at day 10 [20]. The C~ area, ~iewed at magnification 70X on the monitor during las.er Irradiation (see Fig 1A),. had a dIameter of 3 mm and typically compnsed 1-2 vessels of order 3, SIX of order 2, and approximately 15 of order 1; these were about equally divided between precapillaries (a) and postcapillarles (v). The mean number of vessels in the capillary bed (order 0) was difficult t~ determine at the magnification used, but based on the mean intercapillary distance of 15 Jim, as reported by DeFouw [20], about 100 capillaries were estimated to be in the field of view.

Irra~iati~)D. Procedur~s
The long-pulse laser was used for precise microspot irradIation of mdlvldual target vessels of a given type (a or v) and order (1, 2, or 3). Focusing adjustment and diameters of specific vessels to be irradiated were ascertained in siw by videotaping the field of view with the aiming beam in place and comparing it with the 1.4-mm annular width of the teflon ring (see Fig 1). Laser exposures were performed under standard conditions: Ip = 10 mseconds, spot size 200 Jim, and F = 7 )cm-2 • Each vessel was exposed three times at the same site, keeping the time interval b.etween sequenti~l exposures at 30 seconds, so that the subsequent irradiation mteracted With a vessel that had cooled down to ambient temperature and in which ~he exposed blood had been replaced. Repeated exposures caused cumulatIve thermal damage to the vessel wall, eventually leading to occlusion or to hemorrhage (when the exposures were stopped). The short-pulse laser was used to irradiate a field of vessels located inside a teflon ring on the CAM (see Fig 2). The laser pulse irradiance was increased, from a subthreshold fluence F = 3 jcm-Z to F = 6 ]cm-2 , in increments of 0.5 )cm-z . Each field was exposed to three laser pulses, at 30-second inter-vals~ unless hemorrhage occurred in an order 1 or higher-order vessel followmg the first or second exposure-at which point irradiation was stopped. When irradiating additional fields in the same CAM, care was taken to assure that the arterial and venous trees were not compromised by the previous exposures in an adjacent field. . After laser irradiation: the eggs were covered and returned to a stationary I.l1cubator. Selected ~pecllnens (among those that had not undergone massive hemorrhage) were I.l1spected 24 h later for re-perfusion of the vessels.
Cill-square tests u~l.l1~ stepwise logistic regression analysis [21] was used to assess the statIstIcal sIgnificance of vessel type (arteriole versus venule), vessel order (1 versus 2 and 3; 1 and 2 versus 3), and energy level (for the short-pulse laser only). The two dependent variables analyzed were the number of exposures to any vessel damage (grade> 0) and the number of exposures to moderate or severe damage (grade > 1). When occlusion or hemorrhage ?ccu~red after. 1 or 2 pulses, irradiation was stopped. For the short-pulse IrradiatIOn, tillS resulted m some vessels III the same exposure field not being graded at all three exposures.

RESULTS
Long-Pulse Laser The £1uence F = 7 jcm-2 was marginally sufficient to cause injury and vessel damage (mostly grade 1) was observed to occuronl.y in the beam center (spot size = 200 .um). Order 1 vessels had Significantly less damage compared to higher-order vessels (p = 0.0001). Forty-three percent of order 1 vessels were damaged after a single pulse compared to 75% for vessels of order 2 or 3; this differential was preserved after additional exposures ( Fig  Figure 1. Video micrographs of a typical irra~ation procedure with the long-pulse laser (tp = 10 ms~cond). The last ~ortion of the digital display in the lower left corner of each panel gives the rul1l1mg time of the vIdeo 111 h : nun: seconds. A: Low-power magruficatlOn vIew of nng placement on the area of the CAM selected for treatment. Bar, 2 mm. B: High-power magnification view of an order 2 arteriole selected for irradiation. ArrolV, aiming beam over the target area. Bar, 1 mm. C: Target area of the arteriole in B immediately following laser .exposure with a single pulse. of laser light  . Arrow, area irradiated that shows "blanching" of the RBCs wlth1l1 the vessel. D: Target area of the artenole 111 B 7 seconds after the tiurd exposure to laser hght. Solid arrow, irradiation point at which a thrombus has formed , blocking the vessel and causing the vessel to begin to "empty" between the ope/J arrolVS.
3). Arterioles were significantly more vulnerable to moderate or severe damage than venules (p = 0.001). Fifteen of89 (17%) arterioles sustained moderate or severe damage after three exposures. By contrast, only three of 1 02 (3%) of the venules sustained comparable damage.
Short-Pulse Laser Order 1 vessels were most vulnerable to damage; vessels of order 3 were most resistant. Arterioles were more vulnerable than venules and higher energy exposures resulted in more damage. Upon multiple exposures, i1-Uury first occurred to the capillary system, next to arterioles a1, and then to vessels a2 and v1, as was observed on the video screen (Fig 2). All P values were less than 0.0001. Figure 4a,b shows the percentage of arterioles and venules with any (grade> 0) damage and with moderate or severe (grade> 1) damage, respectively, after one, two, and three exposures. Notably, nearly all arterioles of order 1 are damaged after one single exposure, whereas arterioles of order 3 have a similar damage profile as venules of order 2. A general comparison of thermal damage may be made between arterioles of "order n" and venules of "order n -1." Figure 4a,b presents cumulative results obtained for a total of 688 vessels, irradiated with fluences distributed among three fluence ranges: F = 3-4 ]cm-2 (247 vessels), F = [4][5]cm-2 (257 vessels), and F = 5 -6 ]cm-2 (184 vessels). Figure 4',d shows the effect of fluence on vessel (arterioles and venules) damage.
All damage to capillaries (less than order 1) was graded as either moderate or severe. Of 159 capillaries, 49 (31 %) were damaged after one pulse, six additional capillaries (4%) were damaged on the second exposure, and two (1 %) were damaged on the third exposure; 102 (64%) were not damaged. Laser fluence was not statistically correlated with capillary damage.

DISCUSSION
Absorption by a Cylindrical Vessel in a Uniform Optical Field Consider a cylindrically shaped blood vessel, with outer diameter d and inner (lumen) diameter d;, lying along the y-direction and exposed over length 1 to a pulsed, collimated light beam propagating in the z-direction as illustrated in Fig 5. It is assumed that the diameter of the beam (and thus I) is larger than d. Thermal injury of the vessel wall occurs through heat conducted from the RBCs that have absorbed incoming light. To a first approximation, the energy q required to induce thermal coagulation of blood in a vessel of unit length is given as (1 ) Here p is the mass density (gcm- 3 ) and c is the specific heat of blood (4.2 ]g-lK-l) taken to be equal to the corresponding values for water; T; and T f denote, respectively, the temperature before (35 0 C) and immediately after the laser pulse.
The critical temperature T J at the vessel wall required for thermal denaturation of the tissue proteins, causing permanent occlusion of blood vessels, is expected to be higher than the T J required for thermal denaturation of RBCs or circulating proteins, which may cause transient microvascular hemostasis (T J = 70 0 C) due to coagu- In B -D the entire visible field was irradiated. All panels are at the same magnification. A: The target field pre-irradiation shows an arterial "tree" (a) and a venous "tree" (v), each having a main vessel of order 2 with numerous order 1 branches interdigitating with one another. Vessels 1-3 connect the venous system with the arterial system through the common capillary bed. Vessel 4 connects th e venous system to an arterial "tree" located outside the irradiation field. Bar, 1 mm. B: Target field 5 seconds after the first irradiation pulse (5 Jcm-2 ). The order 1 arterioles are becoming blocked by small thrombi (arrows) . The order 2 arterioles as well as the order 1 and 2 venules remain open. C: Target field 5 seconds after the second irradiation pulse (5 Jcm-2 ). The order 1 arterioles remain blocked and thrombi begin to form at the bifurcation point between orders 1 and 2 (solid arrows). The vessel between the clots becomes empty (ope" arrow). Venules 1,2, and 3 have emptied due to lack offlow through the cap illary bed fed by the arterioles. The wall of the empty arteriole 1 remains visible. Vessel 4 (fed from outside the field) and the order 2 venule (also fed from outside) remain open. D: Target field 5 seconds after the third irradiation (5 Jcm-2 ). The arterial "tree" is completely thrombosed. The order 1 vennles fed from the blocked arterioles are empty. The order 1 and 2 venules fed from outside remain open. lation of vessel content [13,22]. In the sample calculations of q presented in Table II we have assumed T J = 90°C. When considering long-pulse microspot irradiation, values of q in Table II should be corrected for the cooling effect of blood flow in the axial direction during the time tp [23]. Measured flow velocities (v) in the CAM vessels ranged from v = 0.7 mm' seconds-t when d = 40).Lm to v = 1.4 mmseconds-t when d = 100).Lm [12]. For example, during tp = 10 mseconds, the blood flow in a vessel of 1= 200).Lm and d = 100).Lm replaces about tv/I = 7% of the total volume in the exposed part of the vessel anJ q is correspondingly larger (neglected in Table II). Convective heat loss by blood perfusion is even less important with short-pulse exposure since tp is 20 times shorter and I is generally much larger than in the long-pulse microspot exposure. Spotsize effects following pulsed irradiation, as observed in highly scattering skin tissue [24], are probably not important in the nonscattering CAM matrix. This is expected to be true even for the widely different spotsizes employed in this study. A definitive answer must await pathologic analysis of damaged vessels. Such a study is envisaged by us, in conjunction with measurement of the temporal evolution of temperatures in individual vessels during and following irradiation.
When we neglect light scattering and also reflections at the air/ CAM and the CAM/vessel interfaces, the energy per pulse, ~, deposited in a blood vessel due to absorption from a uniform optical field, can be expressed analytically. In a cylindrical vessel of lumen diameter d; and length I the net absorbed energy is Jo (2)
Here d~(I') denotes the incremental amount of optical energy absorbed in the exposed lumen during dl' at a time I'; dQ (t,I' ) denotes the corresponding thermal energy after the time interval (I -I') . The thermal energy remaining in the vessel at time I is found by integrating Eq. (4)  and for short-pulse irradiation (Ip = 0.45 mseconds and F = 3 ]cm-2 ). For the two curves inFig 7, a striking feature is the different dependence of tl T on the vessel diameter d. The long-pulse exposure causes a monotonic temperature rise with d over the given range d < 130 11m. At larger d, the temperature rise will reach a maximum and, eventually, decrease as 1/ d. In contrast, the temper-VASCULAR RESPONSE TO LASER PHOTOTHERMOLYSIS 697 ature rise due to the short-pulse exposure reaches its maximum at a smaller diameter. Consequently, for a critical temperature T J = 90° C (!:l. T = 55 ° C) , Fig 7 indicates t hat the short-pulse exposure at F = 3 ]cm-2 affects predominantly small diameter vessels (of order 1 and 2), whereas the long-pulse exposure will damage larger diameter vessels. Figure 7 may also be used to interpret vessel damage reported in the hamster cheek pouch microvasculature at two temperatures Ti = 34°C and Ti = 8°C (Fig 2 in [27]). A flu ence increase of M = 1. 4 ] cm-2 was required to obtain an identical (72%) " percentage of vessels showing any visible change in response to a single laser pulse" delivered from a short-pulse Candela laser similar to the one used in the present study. T he ordinate in Fig 7 scales linearly with flu ence [cf. Eqs. (3) and (5) where d~/d V is the absorbed energy density (Jcm-J ) and the other parameters are as defin ed in Eqs. (1) and (2). Upon substituting z (x), as defined in Eq. (7) (see Fig 5), into Eq. (6) the spatial distribution of the temperature rise in the lumen is obtained. In Fig 8, the cross sections of vessels of order 1 2 and 3 with (see Table I) d ~ 50, 80, and 110 11m, respectively, ~r~ di agrammed. InSide each diagram are plotted t he isotherm curves (!:l.T = 55°C) for F = 3 and 5 ]cm-2. T h e curves define the transition zO I!es between damage? (1» 90°C) and non-damaged (T J < 90 C) lumen volume. It IS eVident that smaller caliber vessels are more easil~ co.ag ul ated than larger ones. According to t he hemod ynalllic cntenon, total occlusion occurs w hen at least 61 % of the lumen has coagulated, i.e., the critical temperature T J = Ti + 55 K = 90°C reaches halfway through the vessel diameter [16].
Also,. we note that at the lowest flu ence (F = 3 ]cm-2) only the small diameter vessels show th rombosis (>6 1 % of lumen has coagul ated) , whereas at F = 5 ] cm-2 the small and medium caliber vessels will have coagul ated completel y and the larger vessels of order 3 will show partial occlusion. This behav ior is borne out by the results for the short-pulse laser presented in Fig 4. Long-Pulse Photothermolysis For the long-pul se exposure the results given above must be modified to include the full effects of thermal diffusion. The cooling effect due to rad ial th ermal diffusion at the vessel periphery will cause a nonuniform spatial distribution of temperature within the vessel, with a peak temperature in the center of the vessel (displaced somewh at toward the upper part facing the light beam) and lower temp eratures at the periphery of the vessel [28] . This situation has been modeled analytically [1 3] and numericall y [16,28 -30] but is beyond the scope of this discussion.
Our main aim has been to present analytical results that provide direct answers for single vessel exposure.
Arterial and Venous Response One of th e more salient phenomena observed in the present study was the higher vulnerability for thermal iI~ury of arterioles as compared to venules. This occurred for the three vessel calibers considered and for both shortpulse and long-pulse exposures. In the CAM, arterial (oxygen poor) and venous (oxygen rich) blood possess eq ual light absorbance at 585 11m; thus, vessels of the sa me lumen diameter are expected to undergo similar thermal stress. In a study of prolonged steady-state · ----------------t-----------------· · . . · . . · . . · · . . · · · · . . · · ·· ·· · . . · . . · :t . . · . . , · : · · · · ·· ·· · · ·· · · . . · · . . · · ... . . .  to arterioles and venules of orders 1, 2, and 3. B : Probability of moderate or severe damage (grade 2 or 3) to arterioles and venules of order 1, 2, and 3. C: Probability of damage to all vessel types (arterio les or venules of any order) versus incident laser flu ence. D: As C for moderate or severe vessel damage. 10soCsecond-l ) and intraluminall y. In this case, the venous-arterial lumen difference may assume significance. However, in the CAM the difference in vascular cross section is generally small. If we take as typical wall thicknesses 0.06d and 0 .035d for arterioles and venules, respectively, the ratio for the lumen volume of arterioles and venules for two vessels with the same outer diameter will be (0.88/ 0.93)2 = 0.9. This ratio is too close to unity to explain our observations. Moreover, contrary to the observations, the long-pulse laser would then be expected (see Fig 7) to damage preferentially the venules, whereas the short-pulse laser would preferentially damage the arterioles. Another point of difference is the platelet aggregation initiated by the chain of biochemical reactions triggered by thermal trauma, which is different in arterioles and venules. This seems to be consistent with reports of PDT-induced vasoconstriction, where it was shown that 90% of the arterioles were affected by photochemical injury versus 70% of the venules [33] . However, on the time scale of photothermolysis (t = 10 mseconds) no platelet aggregation is expected to ~ccur in real time.
. . Finally, It should be noted that coagulated blood emboli, consisting of agglutinated damaged RBCs [17], can be transported downstream in venules, but not in arterioles because they get blocked in the capillaries. This difference in permanent clotting is a component in our interpretation for the lower thres hold for arteriolar photothermolysis.
In conclusion, this study described the first controlled experiment of the relationship between laser fluence and vessel diameter in the photo thermolysis of single blood vessels in I/ivo. Small-caliber vessels are relatively spared when using long-duration exposures. Arterioles were found to be significantly more sensitive to thermal damage than venules of comparable size. The observations of damage-threshold fluence versus vessel diameter were quantified for two widely different pulse durations. They were interpreted using a theoretical model that has applicability to a number of vasculopathies. On the basis of our results it would appear that a pulse duration between 1 and 5 mseconds might be better suited to treat PWS than the two pulse durations used in this study. However, individual variations in the diameter of the ectatic venules in PWS (depending on age of patients) and their depths in the dermis [34) prevent specifying any generally applicable optimal pulselength. !I,d