Platelet thrombus induced in vivo by filtered light and fluorescent dye in mesenteric microvessels of the rat

X,

Sate ard a N. Ghshima

Division of Biomedical Engineering, Institute of Basic Medical Sciences, . r i'niversity of Tsukuba, Ibaraxz, Japan

(Received

24.70.1984; Accepted in revised by Editor H.M. Frojmovicj

fom

1.5.1984

ABSTRACT

and subsequent throm3as formation intravascular platelet aggregation were ;r*odilc2din the microvasculature by irradiation csinm~ filtered light from a mercury lamp in combination with the intravascular adminThe effects of several factors, such istration 3f a fluorescent dye. dye concentration, and vessel diameter were examas light intensity, ined in v2nules and arterioles of the rat mesentery with respect to the time required for the initiation of thrombus formation; the time to totally occlude the lumen; and the volume of the throzbus formed. It was found that the combined conditions of light intensity and dye concentration at specific values had a critical effect on the rate of piatelet aggregation. In the arterioles and venules examined, the throm3us formation time was prolonged as a function cf increasing vessel diameter. The time required for the initiation of thrombus formation and complete occlusion of the vessel lumen was great2r in arterioles than in venules. This result may suggest the dependence of the kinetics of intravascular thrombus formation on blood floz conditions.

INTRODUCTIOW Intravascular thrombosis is undoubtedly the generator of a wide variety of cardiovascular diseases that have recently attracted growing attention. -Among the various factors which affect thrombus formation, the behavior of platelets, in particular their aggregation, adhesion and thrombogenesis in arteries and arterioles, seems to be of nrime importance. Other physical factors such as blood flow conditions and ~hemorheolo-" aLcal blood properties might

TKey words: Blood floz velccity, fluorescent intrevital. microscopy, .A aanesion, piatelet aggregation, thromb>us volume 319

platelet

'-iwzsls ;l‘iir..%.i

Seventy-three rats of were used and anesihesized femoral muscle (100 q/kg).

the Wistar straLn weighing between 2013 and 500 g with ar. injection of sodiwz pentobarbital into the

Experip.er,talApparatus _An intravital ;r,icroscope-televisior sysre?a developed in our laboratory h schematic diagrazn of throughout the present experiments. (728) 1.1'sused An epi-illunination systera (Vanox, 0lyEpu.s the apparatus is showa in Fig. 1. Tokyo) was utilized to produce thronbi, and a translliu~ination Co. Ltd., Xicroscopic bed. system was used to observe a fiel.d of the microvascuiar Matsuimages observed were recorded by a videotape recorder (VTR, w-3150, through a television caiaera (3/W, iil?i-?i, shita Comunication Co. Ltd., Tokyo) The Xatsushita Comunication Co. Ltd.j and were monitored on a TV screen. .. of the experiiaents was recorded on video tapes by a vmeoti;;;er time course (FOR.A Co. Ltd., Tokyoj. Experimental

Procedures

was perforned, an external jugular vein :;a3 canmAfter a tracneotmy d common carotid ara tube of 1 Fr. size for the dye injection. lated with arterial blood pressure. The cannulated in order to monitor tery was also in the abdominal through a midline incision small intestine was exteriorized sgiutiop. il;al' into a bath and Terfused constantly with a s&iskept at a A nesenteric membrane with microvascular beds was temperature of 3720.3"C.

FIG. i Schematic diagram of intravital microscope-television

system.

placed

on a glass plate, and another plate xas carefully laid on the mesenThe upper plate, veighing 3.4 g, teric sector for microscopic observation. was made of gL2ss of 1 mm thickness and 20 :m in diameter bound to a ring of Microvessels in the mesentery were obstainless steel of 5 mm in height. from a halogen lamp as shor,m in Fig. 1. Xrteriserved by transillumination ales of 20-50 urn in diameter or venules of ZO-SO 'in rcere selected to produce a microthrombus.

_'>L from a 200 I< high pressure In t:le epi-illumination- s-ystem, the l<$s7T? mercury lamp was excited by an FITC filter cIF490, Oiym?us Co. Ltd.) and then This filtered light., refle-+ed 37~ Li a dichroic mirror (0510, Olympus Co. Ltd.). through an objective lens 400-500 nm in wave len_gth irradiated a microvessel The area of the irradiation was adjusted to 3e (x20) as sho;vT in Fig. 1. for each field stop. Five difabout 130 ym in diameter on the focal plane ferent levels of the light intensity, PL, controlled by an aperture stop, were i.e. PL=?O.i, 15.9, 9.2, 6.1 and 2.4 mV/mm2. used in the present experiment; ;;Tereconfirmed before each experiment k.th a power meter (Xode:L These vaiues 404, Spectra Physics Co. Ltd., CA, USA) calibrated with 2 standard radiation At first, the filtered light irradiated the microdetector thermopile$:. vessels. Transillumination from the halogen lam? was subsequently discontkued (Kcbayashi Seiyaku Rogyo Co. after i min and a solution of fluorescein sodium Ltd., Tokyo) s;as then injected through the external jugular vein. The irradiSimultaneation by fiitered light was continued throughout the obsert-ation.

& ,.

me detector :;as caiibrated by t'he ETL

(Eiectro-Technical Laboratcry, Agency of Industrial Science and Technology, ?-Iinistryof International Trade and Industry, Japan) Lrradiance standard lam? rhhieh xas certified by Coniti Consultatif de Photom6trie to have less than 1% uncertainty.

.A wnipa3 of microscoaic imagas &dr$ns23 t'ne co'urse 2: a sicro,. i - exrazpk thrombus formation in the m,icrovascuiature produced by light irradiation in combination 3ith intravascular admG.nistration of fluorescent dy2 is shown in Fig. 2 for a venule of 56 urn in diameter. These photographs were taken consecutively at a light intensity of 20.7 mV/mm2 and a dye concentratior of 50 Figure 2(a) shows a transillumknated Ccrophotograph 41 see after the uglkg. of fluorescent dye and 24 set after the initiation of thrombus appearance the initiation of rormatlon. Figure 2
of the dye on arterial jlood The effects of a single bolus injecting a After the dye injeciion, nressure and pulse rate are shown in Fig. 3. occurred in some runs, and slight decrease in arterial pressure immediately On the other hand, restored with.in one minute. blood pressure was gradually auise rates remained almost constant. The effects of the mean arterial blood pressure on the thrombus formation times are shown in Table 1. Throughout the present study, the kinetic feature of thrombus formation :
FIG. 2 Time course of platelet thronbus formation in a venule with a diameter of 56 ~3: after injectior? of fluorescent dye and transillumination. Se (a> Thronbus 24 see after the initiation of thronbus fornatior,. arrow shoxs t3e directior! of biocd flov. 12i set after ib) Microvessel totaliy occluded by platelet aggregates the initiation of thro;nbus formation.

are mean%SD. 2.=, mean arterial pressure; n, n.umjer of data; ti, initiation iime of thrombus formation; ts, time t0 tC3t2l.l~ 0tC17ddFZ~ Dip internal diameter of microvessels. These data were obtained under the c=r?dition PL= 26 .i nY/rm2 and cf=50 ug/kg. * pccl.005, $ YS.

Valnes

of the fluorescent dye in the observed vessel. initial appearance 911 the data presented in Table i xere obtained under the condition ?~=20,? G;nr,2 and . 1 Cf'50 L&kg. The internal diameters of venules were almost tne sane m got;? Despite the difference in arterial pressures as sho:-;nin Table I, no groups . differences slgcificant xere found with respect to t5-e / thrombus formation times, ti and ts. Effa,cts of Lqght Intensi'iy and Fluorescent A Foxxation Prom the results of Treliminary inzenaiEy ax! the dye concentration

Dye Concentration

on Thro;Ibus

experiments, two factors, i-e. the light were considered as the prime determinants

Th2 effects of the concentration of fluorescein sodium, cf, on tthe thromSUS

fo-rmacion

tix:esf ti and ts, are shox,rilin Table 3. In t&se runs, t'ne iig'n: intensity was ‘kept constant at a value of 20.7 mW/n?m2 and the diameter of venules were in a range of 40-60 pm. Variance analysis also revealed that . 1 three groups of different dye concentration snore d a significant difference in , _ tihe throxbus formation times within a significance level of 1%. Xt tne Lowest concectratlon, iciig/kg, the thrombus formation times, ti and ts, showed muzh larger values than those with higher concentratior;s, i.e. 25 er.d 30 pg/i;g:.

TAZLE 3 illlCc.Iof Dye Concentration on Thrombus Formation x=---c

50

25 10

13 5 5

1224 1825 147i65

139535 221258 2498?104 j

in Venuies

li3+i6 1001irlr; 113220

5225 4927 55+5

Vaiues are mean?SD. Tfiese data were obtained for vennles of 40-50 urn i;? internal diameter at pi=20.7 ixvlm2.

T32

effects

05

t,i.2 v23s21

diaa2ter

art

th2

thronbus

f=ms:ioc

t&es,

ti

examLined under th2 condition P~=20.7 mZ.jmm2 and cf=5C ;~g/'kg!nsing a r.iiderang2 of vessel diameters between I;h2 j-2SultS 2i ad 76 im. obtained vessels are classified into four riitn venuies are sh.o;ii? i.L 7 Table 4, in r&i& groups according to their diameier range. Groups I, II, III and IV correspond co ih2 V2SS2l.S C3f 20-30 'Lim, 30-40 urn, 40-60 urn, and 6C-80 ~:r?.in di.ameter The results show the tend2ncy of increasing thrombus formation respectively. times, ti and ts, -with increasing vessel diameter. Variance analysis revealed thai t'ne four groups of different vessel diameters S'nowed signifieani differences in the time, ts, but no significant difference in the time, ti+ Statistically significant increases in ts values ~i2r2 fci;nd 'Djr as2 of the t-test in Groups III and IV compared with Groups I and II. s-_.d

Es,

:;ere

Sffeet of Vessel Diameter

Groups

I II III IV

Di, '4 m

2422 3553 5225 5925

n

5 5 13 5

on Thrombus

Formation

in Venul2s

ti, set

ts, set

p. 1' mmtig

1024 1325 1224 1424

70211 81+x 139235 159+41

122214 105i20 113?15 I.16129

These data were obtained Values are mea&SD. mWfmm2 and cf=50 ug/kg.

under the condition ?I=20.7

the results of similar e_xperiments performed in arteTable 5 summarizes in w'2ich the vessels are also classified into three groups according riol2s, It was found t'nat the values of th2 thrombus formation times, ti to diameter. for arterioles with larger diameters. A and t,, were significantly larger comparison of Tables 4 and 5 s'hons that the platelet aggregation in the arteThe riol2s is m*uch slower than chat in venules of almost the same diameter. difference in platelet beha-vior between the venules and arterioles could be as well as the attributed to the difference of the structure of endothelium effect of blood flow velocity or shear rate. The blood flow velocity in arterioles was reported to be about five times higher than that in venules of the same diameter in the rat mesentery (9). Time Course Changes of Thronbus

Formation

the tnromous 'began to grow from both sides of the vessel ~a11 as sho:?i in Fig. clTi. -T:.y, in formed in th e microvessel is schematically 2, The throclb~~s &lich t-c2 dynamic COUPY2 Of thrOdw3 fornation, time In order to analyze Fig. 4. course changes of thronbus configurations were assessed in terms of the ratios of the lengths, L/Lo, the cross-sectional areas, hi_%,, and the volumes of the :hromilcus,VjV,, where Lo ariasthe thrombus lsngth along the endothelium at ths . ? vzs completelY stopped, _ko iias the C~oSs-se~tronai tl.me When itie blood flOG .i area of the vessel lumen and V, was equal to A, x Lo. The results are shi?xn ThGlsa in Fig. 5(a) through Fig, 5(c) on semilogarithmic coordinates. I_ i data . ' the venules snoxn represent some typical examples taken fr32 several runs wltn in Table 4. Coqarison of Figs. j(3), 5(b) and 5(c) sho:lis2 characteristic formation as follows: the thrombus lsngch is found growti: pattern of +iiromSus i_ to increase almost linearly with a relatively rapid gr0wth in the earlier

blood :low

I

FIG. 4 Schematic diagram of a microvessel and the throm3us formed. Since a 10ngit:Adinal section shows that both sides Of the thrombus are slightiy different, the length, L, the area, A, and the volux, V, are calculated by averaging the hatched section 2s follows: L=(L1-%2)/2, A=(Di2-D2)/4, wh2re Di is the internal diameter Of the and D is the narrowest vessel b2tr22I;. t'he tW0 longitudinal distance of the nhrombus. V=(Vl+V2)/2, sections V,=hr,S, (n=l,Z), where rn is the distance from the axis of qzrmetry to the gravity center of the longitudinal section of the thrombus and S, is the area of the longitudinal section, The coordinate of the gravity center, was calculated by the (xcn. Ycm), follo~~ing eq:-lar-ions;Xcm=l/Sn*'Sn"dSn and Ytm=l/Sn',~Sn':.dSn_

time.

FIG.

t

iminl

7

Time course changes of the thromjus volume with different icternal diameters.

for

venules

ar.d arterioles

CISCCSSION We confirmed that oilr ~2% experimcztal model (10) 50r inducizzg platelet adhesion and aggregation in the living microvasculature possessed a2 escelleilt reproducibility. Thus it is expected to provide a powerful and quantitative tool for studying the dynamics of in vivo platelet thrombogenesis. Eowever, the exact mechanism of thrombus formation in the present model is still unkno>m at present. Rosenblum (11) deduced that the mechanism of the induced platelet aggregation by iight irradiation under the presence of intraluminal fluorescent dye was similar to that of the technique (17) employing a laser 3eam irradiation intravascular with of a colored i.e. injection Evans blue. Kovdcs et al. (12) suggested that the dse absor3ed the laser converted it to heat, and caused a "microburn" injury of the luminal energy, site of the vessel Fall. It has been thought by these researchers that the on the endothelium because it was injured by the heat, platelets aggregated by electron microscopic studies that the Rosenblum et al. (6) also verified endothelium of the vessel wall as well as the red cells were damaged in their in the experiments using laser stimulation. experiments as was the case The intensity of the laser beam used by Kov&s et al. cas as high as 5.7~10' mX/ than the intensities used in the mm2, or about 3000 to ii000 times greater present study and that of Rosenblum. It is believed Yhat the endothelial cells at these higher intensities. of microvessels may be damaged by the microburn It is unbelievable, however, that the fluorescent dye can liberate enough heat by iight stimulation as to induce a microburn in our experiments as well as in factors other than heat must be considered as Rosenblum's (13). Therefore, the causes of the induced endothelial injury. Free radicals such as superoxide Rosenblum et al. (14) recently shoved that may be one of the possible causes. the hydroxyl radical scavengers f dimethyl sulfoxide (IXGD> and glyceroi, were effective inhibitors of plateiet aggregation in the same in vivo eFerimenta1 model .using the pial arteriole of a mouse. They pointed out tvo possibilities material,

It uas shown in Tables 4 and 5 that the throm3us formation tim2s, tzi and and arterioles. Generally, r.he t,, depend on vessel diameters in both V2nules In fact, it 3lood flow velocity is higher in vsssels with a larger diameter. wa 3 reported that the mean value of ihft center line blood E1ox veloci:y inf:iow and that the blood creased almost linearly s*?ith the vessel diameter, is five times greater than that in venules within t'ne velocity in arterioies rat mesenteric DliC?ZOVaSCUiZLtUT2 (9), T'nerefor2, it seems necessary to examine wh2tie~ I i the dependence of the thrombus formation times on the vessel diam2cer was affected by the blood flax velocity or by some other factor, Based on t'ne results shown in Tables 4 and 5, the mean radial growth rate of the thronjus was estimated from the values of the vessel radius and the time required for In venules, total occlusion. the growth rates were almost the same, being Thus, 0.17 to 0.22 um/sec, it could be easily understood that longer times vere needed for the larger vessels to become occluded. On the other hand, in arterioles, the grcwth rates were 0.17 umisec far Group I, 0.03 F;m/sec for The growth rates were significantly Group II and 0.02 gm/sec for Group III. lotier in Groups II and III. In these cases, it >Jas suggested that the throm'bns growth might be disturbed by the higher flow velocity.

It was also pointed out that the difference in the behavior of thrombus formation between venules and arterioles might be attributed to the difference in the structlure of the endotheiium as well as the effect of the blood flow velocity Or shear rate. It can not be concluded, at present, <.i:Iich factor is nore importent for the initiation of platele t aggregation in normal physiological ccnditions. The effects of shear rate or shear stress on platelet aggregation studied in vivo and in vitro (3,16,17) still remains controversial by enhancing the release reac(18); the fluid shear may promote aggregation t;ion of serotonin and .ADP (19,20), or by enhancing the over211 arrival of platelet to the surface (17). Conversely th2 shear stress may directly inhiSit the formation of platelet-platelet bonds or decrease platelet sensitivity to &ADP (19). Judging from our results in Tables 4 and 5 showing that the rate of throm3us formation in arterioles was slower than in vemles, it might be concluded that the higher blood fio:~ depressed the platelet throinbus forr?.ation within the velocity range studied. Rosenblum et al. (21) also reported that the tine to initiate aggregation was significantly prolonged for the cerebral

The

ly, tiie g~xive

z_et;>&

in

&sc:i’yed

e:qe-ciinental

proced-;re Secondly,

this pa?er has the foll?iZ.ng azvaltages ~

repr&:ci’sle is siqjiril.e an-i easily thro&us our method quastifies

yitb.

--;rszan ixe:<-

formation I2 a apparatus. in ve.:o 3easuyemeats soxethLng have Ghic'h conventional reproaucible manner, The present model seems applica3le to sev2ral artificial + been able to do, r.":. conditions. rz 5s situatilfns eXpeCied to be to physiological as well 2s suci", 2s ev2luating the effects a;;r?lio.able to several pharmcological studies .of 9-T Ii* :developed ~hysiologlca: studies antithrozbotic agents, and also to ,._\>7-;

the platelet behavior in microvessels axd the mechanism of investigating drawback of the a major platelet throzb:is forzatioi-,. On the other hand, in a single animal is unrepeata3ie present model is that the observation because the flnorescent dye must be injected intravascuiarl;:.

fomation were ggregation and consequent thro+us Intravascular plaCelet ii induced with excellent reproducibiliry in microvessels by filtered light irrafluorescent administration of a with intravascular in combination diation depended on both the light of thrombus formation The kinetic behavior dye. of thr.orr;.~bus formation 5ias and the rate the dye Concentration, intensity and model controllable throug'n these two factors. it has been snoLx in the present the tize to totally OcClude formation, that the initiation tis.e of thrombus Bight be used as volcze the growth rate of the throx3us the vessel luner, and quantitative

cal

parameters

The authors assistance.

wish

to

estimate

to acknowledge

the

thrombus

Satsuki

fomatim

1iomats.u for

process,

her

excellent

techni-

?.

3.

:~p&-s _Y

)

7in

2.

cBacE, P. Laser-induced thrombosis test suitable for screening studies. :_licrcvaec*Res, 18, 403-512, 1979.

and

pharmacological 5.

ROSEKBL?_X~ 5;~I = and EL-SABBAN, F. Blateiet aggregation in the cerebral Circ. Res. -9 $0 320 mlcrocirc~uiatlon, Effect of aspirin and other agents. -328, 1977+

7.

:G. a:id SATO, ii, Blood flosvnetry in the microcirculation sure-flov in the microvessels 3f rat nesencery--, relationships ?Ied.Eiec*Biol, “ing. 17, 120-136, 1979.

8.

iXsHI:~%4*I‘, and SATO, $1. flow velocity. A_~~~j_ology

9.

OHSH;XA,

Effect of pentosifylline 752-763, 1381,

on microvascular

blood

32,

and WAYL_kX~, 9 HASEGAZ'b, X. .* of the rat mesentery.

vessels

--PresJap.J.

Erythrocyte flOi< J.Jap.Coll.Angiol.

velocity in the 20, 3-9, 1380.

ml.cro .

-

10.

SlTO, M. and OHSHIM, ?;. Quantitative analysis of the growth of platelet microemboli induced in vivo by ultraviolet light and fluorescent dye (meeting abstract). Biorheology 2, 379, 1982.

Il.

ROSENBLLX, W.I. Fluorescence induced in platelet aggregates as a guide to luminai contours in the presence of platelet aggregation. Yicrovasc.Res.l5_, 103-106, 1978.

12.

KGVdCS

ideal He-Se

f

I. B. , TICYI-SEBES, A., TROHBITk, K. and G6Rk, energy-absorbing material to prduce intravascular gas laser. W_crovasc.Res. 2, 107-124, 1975.

P. Evans blue: An xicroinjury

by

13.

BOWEN, E.J. and WOKES, F. Fluorescen.ce of Solutions. Tronto: Longmans, Green and Co., 1953.

14 *

ROSZNBLCI, 57. I. and EL-SABB_AX, F. Dimethyl sulfoxide (DISC) and glycerol, hydroxyl radical scavengers, impair piatelet aggregation within and eliminate the accompanying vasodilation of, injured mouse pial arterioles. Stroke 13, 35-39, 1982. ~-

15.

SATO, M.

and OHSHI%,

X.

Basic study on

the mechanism

London-Xew York-

of intravascular