Thrombosis in the microcirculation

Thrombosis in the microcirculation

Bingmei M. FuDepartment of Biomedical Engineering

The City College of The City University of New York

Microcirculation Lab

ThrombosisNormal blood flow Thrombosis Embolus

large vessel microvessel

30µm

Risk Factors

Thrombosis

Flow Stasis

Heart failure

Prolonged immobilization

Altered coagulability

Increased blood

coagulability

Vessel injurySurgery

Trauma

Thrombosis in microcirculation• Previous studies under conditions

– flow retarded (Nicolaides et al., 1972);

– flow disturbed• secondary flow in branches (Chen et al., 2004);

– vessel injured/damaged by • electrical (Massad et al., 1987; Wong et al., 2000);

• mechanical (oude Egbrink et al., 1988);

• biochemical (Begent et al., 1970);

• PDT, light/dye (Sato et al., 1990; Sasaki et al., 1996; Rucker et al.,2002).

Question 1

Can thrombosis occur in non-injured but bent/stretched microvessels under non-disturbed laminar flow (Re ~0.01) condition?

Question 2

What is the structural mechanism by which PDT induces thrombosis?

Experimental setup

CCD camera

Experimental Design

• Sprague-Dawley rat, 250-300g;• The mesentery gently

arranged on the surface of a polished quartz pillar;

• With a microvessel (post-capillary venule, 20-50 µm) under observation, a rounded-tip glass restraining micropipette used to bend/stretch the microvessel.

Experimental Observations

• In 10-60 min, thrombi formed in 19 out of 61 (~31%) sites in 28 non-injured bent/stretched microvessels;

• all thrombi were initiated from the inner side of the curvature.

outlet

inletmicropipette

(Liu et al, J. Biomech. 2008)

Results

5

10

15

20

25

30

bent angle (degree)

rad

ius

of

vess

el (

μm

) non-thrombosis

thrombosis

0 10 20 30 40 50 60 70 80 90


What are the mechanical factors that initiate thrombosis in these non-injured bent/stretched microvessels?

microvessel diameter 2r = 25 µm (circular)equal perimeters of circular and elliptical cross sections

Ө

2r 2b2a

B

A

B

A180o

B

A

90oθ

Vessel Geometry


Numerical Methods

• Fluent used to solve • Continuity Eq.

• Navier-Stokes Eq. • Element No.:

• 610x103, 770 x103 (90o/180o, circular); • 1.48x106, 1.52 x106 (90o/180o, elliptical);

• µ = 2.5 cp (Levenson et al., 1990), ρ = 1050kg/m3;• Outlet pressure = 10 cmH2O, mean blood velocity = 1 mm/s,

Reynolds No. ~ 0.01;• Convergence criteria: 10-8 of residues.

0μp 2 u

0 u

Circular

Elliptical

Velocity Distributions


A

A

A — A

max

A

A

A — A

max A

A

A — A

max

Circular

Elliptical

Shear Rate Distributions


(Circular)

straight 90o 180o

Shear Rate Distributions along the Curvature


0 1 2 3 4 5 6

0

200

400

600

800

shea

r ra

te (

1/s)

outer sidemiddleinner side

inlet outlet -100 -50 0 50 100

0

200

400

600

800

shea

r ra

te (

1/s)


-100 -50 0 50 100

0

200

400

600

800

shea

r ra

te (

1/s)


0 1 2 3 4 5 6

0

200

400

600

800

shea

r ra

te (

1/s)

outer sidemiddleinner sidemax.

inlet outlet -100 -50 0 50 100

0

200

400

600

800

shea

r ra

te (

1/s)


-100 -50 0 50 100

0

200

400

600

800

shea

r ra

te (

1/s)


Circular

Elliptical

Shear Rate along the Curvature


0 1 2 3 4 5 6

0.5

1.0

1.5

2.0

2.5

3.0

velo

city

(m

m/s

)

1/4 outer middle1/4 inner

inlet outlet-100 -50 0 50 100

0.5

1.0

1.5

2.0

2.5

3.0

velo

city

(m

m/s

)


-100 -50 0 50 100

0.5

1.0

1.5

2.0

2.5

3.0

velo

city

(m

m/s

)

1/4 outermiddle1/4 inner

0 1 2 3 4 5 6

0.5

1.0

1.5

2.0

2.5

3.0

velo

city

(m

m/s

)


inlet outlet-100 -50 0 50 100

0.5

1.0

1.5

2.0

2.5

3.0

velo

city

(m

m/s

)

1/4 outermiddle1/4 inner

-100 -50 0 50 100

0.5

1.0

1.5

2.0

2.5

3.0

velo

city

(m

m/s

)


Circular

Elliptical

Velocity along the Curvature


0 20 40 60

length m

1000

1010

1020

1030

1040

pre

ssu

re (

Pa)


0 20 40 60

length m

1000

1010

1020

1030

1040

pre

ssu

re (

Pa)


0 20 40 60

length m

1000

1010

1020

1030

1040

pre

ssu

re (

Pa)


Circular

Elliptical

0 20 40 60

length m

1000

1010

1020

1030

1040

pre

ssu

re (

Pa)


0 20 40 60

length m

1000

1010

1020

1030

1040

pre

ssu

re (

Pa)


0 20 40 60

length m)

1000

1010

1020

1030

1040

pre

ssu

re (

Pa)


Pressure along the Curvature


Newtonian & non-Newtonian fluid

Casson Model(Das et al., 1998, 2007)

1/2α

p1/2 )H)(1

μ(μ

21/21/20

1/2c ]/γτ[μμ 1])

H1

1β[(τ α/21/2

0

α = 1.621, β = 0.627 (Das et al., 1998);

μp = 2.5 cP (Levenson et al., 1990).

0

0.5

1

1.5

2

2.5

-12.5 -7.5 -2.5 2.5 7.5 12.5Radial Location (um)

Vel

oci

ty (

mm

/s)

Newtonian

non-Newtonianstraight90o180o

0

100

200

300

400

500

600

700

-12.5 -7.5 -2.5 2.5 7.5 12.5Radial Location (um)

Sh

ea

r R

ate

(1

/s)

Newtonian

non-Newtonian

straight

90o

180o


Summary• Thrombosis occurred in 19 out of 61 sites

(31%) of 28 non-injured bent/stretched microvessels. Thrombi were initiated at the inner side of these microvessels.

• Numerical simulation results showed higher shear stress/rate and higher shear stress/rate gradient at the inner sides of the bent/stretched microvessels, suggesting they were two mechanical factors that initiate thrombi.

Light-dye Treatment

Light-dye treatment (Photodynamic Therapy, PDT):Use of a photosensitizer, activated by a laser of a specific wavelength, to treat tumor and other diseases in the presence of oxygen.

Advantages of PDT

• Applied repeatedly at the same site;

• Selective: photosensitizer can selectively accumulate in the tumor cells;

• Harmless without light illumination;

• Treatment for diseases that surgery is not possible (such as the upper bronchi, the structure cannot be removed surgically).

PDT Induced Thrombosis

• Thrombi induced by light/dye consist primarily of platelets and occasionally of leukocytes in venules (Rumbaut et al., 2004).

• The interaction between blood platelets and vessel wall plays an important role in thrombosis.

glycocalyx

150 nm

Surface Glycocalyx

(Squire et al., 2001)

Molecular Composition of SGL--K+, Na+, Ca++, L-arginine

--Albumin, bFGF, LPL, polycationic peptides

--Choresterol and glycosphingolipids

--Caveolin-1

--K+, Na+, Ca++, L-arginine channels

--Hyaluronic acid

--Heparan sulfates

--Chondroitin sulfates

CD44

Glypican

Syndecans

Sialic Acids

Shedding

C

A BGlycoprotein

(Tarbell and Pahakis, J. Intern. Med , 2006)

Glycocalyx Layer Damage

• Light/dye increases the penetration of macromolecules in the endothelial surface glycocalyx of the vascular wall (Vink & Duling, 1996).

• Disruption of the glycocalyx would result in adhesion of platelets and blood cells to the vessel wall (Mulivor & Lipowsky, 2002).

Hypotheses

PDT disrupts the endothelial surface glycocalyx

increase microvessel permeability to water and solutes

platelet and blood cells binding to the endothelium and induces thrombi.

Experimental Setup

Xenon Laser (495 nm)

VCR

Laser Spot

CCD camera

Experimental Design• Sprague-Dawley rat, 250-300g;

• Laser: Xenon laser, 495 nm, intensity 0.37 and 0.70 mW/mm2;

• NaF: 50 mg/kg body wt., injected from the carotid artery;

• With a microvessel (post-capillary venule, 20-50 µm) under observation, NaF was injected and the laser was turned on simultaneously.

Thrombosis by Light/dye

20µm

Thrombus Growth Rate

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20 25 30 35

No

rma

lize

d a

rea

oc

cu

pie

d b

y t

hro

mb

i

time (min)

Series2

Series1

0.70 mW/mm2 (n=9)

0.37 mW/mm2 (n=8)

29.3 ± 2.215.5 ± 1.8

2.5

3.8

(Liu et al., BMMB, 2010)

Technique of Lp Measurement (Curry, 1984)

dL/dt

pdtdL

Lr

Lp 0

12

techniqueLandis

2r

Blocker

L0

Micropipette

Marker Cell

Lp Measurement

30µm

Lp Change under Light/Dye Treatment

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

-15 -10 -5 0 5 10 15 20 25 30 35 40 45 50 55

Lp

/ m

ea

n L

p o

f b

as

elin

e

time (min)

BSA-laser/NaF-laser off/NaF (n=11)

BSA-laser-laser off (n=4)

BSA-NaF-NaF (n=4)

BSA-BSA-BSA (n=4)

NaF + laser or laser onlyor NaF onlyor BSA

Baseline (1% BSA)

NaF + laser of f or laser of for NaF onlyor BSA

*#


Early Change of Lp under Light/Dye Treatment

0

0.5

1

1.5

2

2.5

3

-6 -4 -2 0 2 4 6 8 10 12 14

Lp

/mea

n L

p o

f b

asel

ine

time (min)

n = 6

Baseline (1% BSA)

NaF + laser

*


Results

Lp baseline (mean±SE)

10-7 cm/s/cmH2O

n=11

Lp PDT (mean±SE)

10-7 cm/s/cmH2O

n = 11

Lp PDT / Lpbaseline

(mean±SE)

0.98±0.08(range: 0.69-1.52)

2.07±0.21 (range: 1.11-3.16) 2.2±0.2


Technique of P MeasurementDye side

Washout side

200 µm

400 µ

m

Measu

rin

g

Win

dow

2

1

00

r

dt

dI

IP f

f

(Fu et al., 2005)

0

0.2

0.4

0.6

0.8

1

1.2

Time

Flu

ore

scen

ce In

ten

sit

y

∆Ifo

10 seconds

(dI/dt)0

0

dt

dIslope f

P Measurement

P to albumin

0

1

2

3

4

5

6

-15 -10 -5 0 5 10 15 20 25 30 35

time (min)

P /

mea

n P

of

bas

elin

e

Palbumin, n = 7

baseline (1% BSA)

NaF + laser

*


Early Change of P under Light/dye Treatment

0.0

1.0

2.0

3.0

4.0

5.0

6.0

-6 -4 -2 0 2 4 6 8 10 12 14

P /

mea

n P

of

bas

elin

e

time (min)

Palbumin, n=6

Baseline (1% BSA)

NaF + laser

*


Results

P baseline (mean±SE)

10-6 cm/sn=7

PPDT (mean±SE)

10-6 cm/sn = 7

PPDT / Pbaseline

(mean±SE)

0.84±0.09 3.46±0.53 4.1±0.7


Comparison of Permeability Change w. & w/o. Blood Cells

Lp P to albumin

0

0.5

1

1.5

2

2.5

3

3.5

4

-15 -10 -5 0 5 10 15 20 25 30 35 40

Lp

/mea

n L

p o

f b

asel

ine

time (min)

no blood cells (n=11)

After initiation (n=9)

After 50-75% occlusion (n=6)

NaF + laserBaseline

0.0

1.0

2.0

3.0

4.0

5.0

6.0

-15 -10 -5 0 5 10 15 20 25 30 35 40

P /

mea

n P

of

bas

elin

e

time (min)

No blood cells (n=7)

After initiation (n=6)

Baseline NaF + laserBaseline NaF + laser


What are the most likely structural mechanisms by which light-dye treatment induced microvascular hyperpermeability and thrombosis?

Model Geometry

2 D

Y

Lf

Tis

sue

sid

e

Jun

ctio

n

stra

nd

2d

L

XOLu

men

sid

e

2aΔ

Su

rfac

e gl

ycoc

alyx

(revised from Fu et al., 1994)

Y

X

Z

Lf

L

2DLumen side

Tissue side

2B

2d

Model Predictions

0

2

4

6

8

10

12

-0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1

P/P

co

ntr

ol

Lp

/Lp

co

ntr

ol

Lf/Lfcontrol

Lp

Palbumin

0

2

4

6

8

10

12

14

16

18

0 1 2 3 4 5

P/P

co

ntr

ol

Lp

/Lp

co

ntr

ol

B/Bcontrol

Lp

Palbumin

0

1

2

3

4

5

0 2 4 6 8 10 12

P/P

co

ntr

ol

Lp

/Lp

co

ntr

ol

d/dcontrol

Lp

Palbumin

0

1

2

3

4

5

0 2 4 6 8 10

P/P

co

ntr

ol

Lp

/Lp

co

ntr

ol

Dcontrol/D

Lp

Palbumin


Model Predictions and Exp. Results

0

1

2

3

4

5

6

7

8

9

10

-0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1

Lf/Lfcontrol

P/P

co

ntr

ol

Lp

/Lp c

on

tro

l

Lp experiment

Lp model (Lf = 150nm)


Palbumin experiment

Palbumin model (Lf = 150nm)


0.08

Model Predictions and Exp. Results

0

1

2

3

4

5

6

7

8

9

10

-0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1

Lf/Lfcontrol

P/P

co

ntr

ol

Lp

/Lp c

on

tro

l

Lp experiment



Palbumin experiment



0.14

Growth Rate vs. Permeability

0

1

2

3

4

5

6

-0.02

0.02

0.06

0.1

0.14

0.18

0.22

0.26

0.3

-5 5 15 25 35

Lp

/me

an

Lp

of

ba

se

line

P/m

ea

n P

of

ba

se

line

No

rmal

ized

thro

mb

us

gro

wth

rat

e (1

/min

)

time (min)

thrombus growth rate (n=8)P/mean P of baseline (n=7)Lp/mean Lp of baseline (n=11)

Summary• Light/dye treatment with 0.37mW/mm2

induced thrombosis in 3.8 min, complete occlusion at ~29 min.

• This power gradually increased Lp and Palbumin to a plateau in 3 – 5 min by 2.2-fold and 4.1-fold respectively.

• Our model predictions indicated that Lp and P increase under light/dye treatment was most likely due to 86% - 92% diminishment of the endothelial surface glycocalyx.

Summary

• Increased Lp would increase the radial fluid flow and enable more platelets and leukocytes move towards the vessel wall.

• Degradation of glycocalyx layer exposes endothelium and increases the binding of platelets and other blood cells to the endothelial cells, therefore induces thrombosis.

Acknowledgements

Dr. Qin LiuDavid MircMin Zeng

NSF CBET- 0133775 and 0754158 CUNY Graduate Fellowship

Thank you!

Thrombosis in the microcirculation

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