VHIR Seminar led by Daniel De Backer, PhD., from the Dpt of Intensive Care Erasme University Hospital Brussels - Belgium. Abstract: Multiple studies have shown that alterations in microcirculatory perfusion are frequently observed in patients with septic shock. These alterations are characterized by heterogeneity of perfusion with capillaries with stop flow in close vicinity to well perfused capillaries. What are the consequences of these alterations? The presence of stop flow capillaries favours development of zones of tissue hypoxia, even though total perfusion to the organ is preserved. In addition, the heterogeneity in perfusion is associated with inadequate matching of flow to metabolism and is hence less well tolerated by tissues than an homogeneous decrease in perfusion. In patients with septic shock, the severity of the microvascular alterations was associated with development of organ dysfunction and an increase risk of death. Different mechanisms have been implicated in the development of these alterations including loss of communication between vascular segments, impaired endothelial reactivity, alterations in red and white blood cells rheology, alteration in endothelial glycocalyx, platelet aggregation and microthrombosis. In view of the various mechanisms implicated in the development of these alterations, it is unlikely that therapies used in usual hemodynamic resuscitation. Novel therapies should aim at improving the matching of perfusion to metabolism rather than further increasing flow in the already perfused vessels or non selectively dilating microvessels.

1. The Microcirculation
in sepsis
Daniel De Backer
Department of Intensive Care
Erasme University Hospital
Brussels, Belgium

2. • Microcirculatory perfusion is a key
determinant of tissue perfusion.
• Microvascular perfusion is under control of
different mechanisms than systemic
hemodynamics.
• O2 transport is driven at microcirculatory
level by diffusion more than by convection.
TISSUE PERFUSION: Key points

3. Trzeciak et al
Crit Care 9:S20;2005
The density of capillaries is a primary
determinant of tissue oxygenation

4. Saldivar-E et al
AJP 285:H2064;2003
The density of capillaries is a primary
determinant of tissue oxygenation
Adaptation to chronic hypoxia is characterized by an
increased capillary density

5. MICROCIRCULATION
DDB USI
 r4 1
V = P
8 L 
.
. . .
Determinants of microvascular blood flow
Blood flow is adapted to local metabolic
needs through local vasodilation and
upstream changes in vasomotor tone

6. Neural control
Hungerford-JE et al
FASEB 14:197;2000
Perivascular
sympathetic
nerves
10 µm

7. Neural control Beach-JM et al
AJP 275:H1489;1998
Arteriole
Capillaries

8. Microcirculatory alterations in
experimental sepsis

9. Pig
Gut serosa
OPS, 5x probe

10. Pig
Gut serosa (sepsis)
OPS, 5x probe

11. Pig
Liver
OPS, 5x probe

12. Pig
Liver (sepsis)
OPS, 5x probe

13. Sheep
Brain
SDF, 5x probe

14. Sheep
Brain (sepsis)
SDF, 5x probe

15. Hoffmann et al
CCM 32,1011;2004
Hamster skinfold LPS
DECREASED CAPILLARY DENSITY IN
EXPERIMENTAL SEPSIS
ENDOTOXIC SHOCK MODEL

16. ANOVA: in sepsis all times p<0.0001 vs baseline
Evolution of sublingual microcirculation in septic shock
Verdant et al
CCM 37;2875,2009
Sham
Sepsis
Pigs
Cholangitis

17. Evolution of microcirculation in unresuscitated septic shock
Pranskunas A et al
Crit Care 16:R83;2012
Pigs
E Coli infusion

18. MICROVASCULAR ALTERATIONS
IN EXPERIMENTAL SEPSIS
• Decreased capillary density
• Heterogeneity of perfusion

19. HETEROGENEITY OF CAPILLARY PERFUSION IN
EXPERIMENTAL SEPSIS Ellis C et al
AJP 282:H156;2002
rats
SPACE

20. CONTROL
SINUSOIDAL BLOOD FLOW IN ENDOTOXIC SHOCK
After Pannen et al.
AJP 271: H1953; 1996
%
0
10
20
30
0 10 20 30 40 50 >50
Fraction of
perfused
capillaries
ENDOTOXIN
0
10
20
30
40
0 10 20 30 40 50 >50
%
Sinusoidal flow pl/s
Fraction of
perfused
capillaries

21. Increase heterogeneity of renal perfusion in sepsis
Rats
LPS
Legrand et al
ICM 2011

22. EXPERIMENTAL STUDIES IN SEPSIS
Microvascular blood flow alterations are frequent
decreased vascular density
absent or intermittent flow in capillaries
heterogeneity between areas
DDB USI
Branemark et Urbaschek Angiology 18:667;1967
Lam et al. JCI 94: 2077; 1994
Farquhar et al. J Surg Res 61: 190; 1996
Madorin et al CCM 27:394;1999
Ellis et al AJP 282:H156;2002
Verdant et al CCM 37:2875;2009
Secor et al ICM 2010
Different models (LPS, CLP, live bacteria,…)
Various species (rats, mice, hamsters, pigs, sheep…)
Various organs (skin, gut, liver, lung, kidney, heart, brain…)

23. Cardiomyocytes rats
Bateman et al
AJP 293/H448;2007
CTRL LPS
Consequences…

24. 0
10
20
30
40
50
60
70
80
Gut mucosa
SEPSISCTRL
-10
-5
0
5
10
15
p<0.01
%
%
Hemorrhage
p<0.01
Humer et al
JAP 81: 895; 1996
CRITICAL O2ER
CHANGES IN CAPILLARY TRANSIT-TIME HETEROGENEITY
MICROCIRCULATORY ALTERATIONS AND EO2CRIT

25. Potential mechanisms ?

26. PATHOPHYSIOLOGY OF
MICROVASCULAR ALTERATIONS
Triggered by inflammatory mediators
TNF (Vicaut E JCI 87:1537;1991)

27. ENDOTHELIALALTERATIONS
ctrl
5 days
post LPS
Leclers J et al
CCM 28:3672;2000
rabbits
25-35%
vessels

28. Loss of neural control Tyml-K et al
AJP 281:H1397;2001
Change in diameter
and
communication
rate (CR500)
between 500 µm
distant
microvessels
(retrograde
communication)
B: cremaster muscle (mice)
C: Endothelial microlayer

29. Enhanced response to vasoconstrictor substances Pannen B et al
AJP 26:180;2001
CTRL
HEM
CTRL + ET
HEM + ET
Rats

30. 2 µm
Endothelial
cell
Glycocalyx
0.2 µm
GLYCOCALYX ALTERATIONS
van den Berg et al
Circ Res 92:592;2003

31. Marechal X et al
Shock 29:572;2008
Role of glycocalyx in microvascular alterations
Rats, ileum
µm

32. Gupta A et al
AJP 293:F245;2007
LPS promotes rolling and adhesion
in renal microcirculation
Two photons videomicroscopy

33. McCuskey et al
Cardiovasc Res
32:752:1996
Rouleau
formation
Altered Red blood cell deformability

34. Eichelbronner et al
ICM 29:709;2003
ETX promotes adhesion of RBC to endothelium
HUVEC
*

35. Croner et al
Crit Care 10:R15;2006
Microthrombi ?
Rats, CLP
Vasoconstriction
Platelet/WBC interaction with endothelium
Microthrombi not frequent

36. Secor et al
ICM 36:1928; 2010
Mice, CLP

37. De Backer et al
Annals Intensive Care 2011
Potential mechanisms

38. ALSO IN HUMANS ?

41. DDB USI
Vascular density
(all vessels)
++ p <0.01 vs
volunteers
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7n/mm
Volunteers
(10)
Septic patients
(50)
++
De Backer et al
AJRCCM 166:98;2002

42. DDB USI
Percentage of vessels perfused
(large vessels)
60
65
70
75
80
85
90
95
100%
Volunteers
(10)
Septic patients
(50)
MICROCIRCULATORY ALTERATIONS IN SEPTIC PATIENTS
De Backer et al
AJRCCM 166:98;2002

43. DDB USI
0
20
40
60
80
100
Volunteers
(10)
Severe sepsis
(50)
+++
%
+++ p <0.001 vs volunteers
Percentage of vessels perfused
(small vessels)
De Backer et al AJRCCM 166:98-104;2002

44. Emergency department
Trzeciak et al
Ann Em Med 49:1579;2007
N=26
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
Heterogeneity index
CTRL SEPSIS
P<0.01

45. Alterations of sublingual
microcirculation in
patients with sepsis
• De Backer et al AJRCCM 2002
• Spronk et al Lancet 2002
• Sakr et al CCM 2004
• De Backer et al CCM 2006
• De Backer et al CCM 2006
• Creteur et al ICM 2006
• Boerma et al CCM 2007
• Trzeciak et al Ann Emerg Med 2007
• Sakr et al CCM 2007
• Trzeciak et al ICM 2008
• Boerma et al ICM 2008
• Dubin et al Crit Care 2009
• Buchele et al CCM 2009
• Boerma et al CCM 2010
• Ospina et al ICM 2010
• Spanos et al Shock 2010
• Pottecher et al ICM 2010
• Morelli et al Crit Care 2010
• Ruiz et al Crit Care 2010
• Dubin et al J Crit Care 2010
• Morelli et al ICM 2011
• Edul et al CCM 2012
• Kanvundis et al ICM 2012
• Hernandez et al CCRP 2012
• Pranskunas et al ICM 2013
• ↓ total vascular density
• ↓ perfusion of capillaries
(no flow or intermittent flow)
• Preserved venular perfusion
• Heterogeneity between areas
( close by a few microns)

46. baseline StO2 (%) delta StO2 (%)
slope (%/sec)
0
1
2
3
4
5
6
7
8
sepsis
n = 72
volunteers
n = 18
icu control
n = 18
Slope(%/sec)
*
* p < 0.001 vs volunteers and ICU control
Creteur et al
ICM 2007
Endothelial reacitivity is
impaired in sepsis

47. Alterations of NIRS vasoreactivity test in patients with sepsis
• Girardis et al ICM 2003
• De Blasi et al ICM 2005
• Pareznik et al ICM 2006
• Podbregar et al Crit Care 2007
• Doerschung et al JAP 2007
• Creteur et al ICM 2007
• Skarda et al Shock 2007
• Nanas et al Aenesth Intens Care 2009
• Payen et al Crit Care 2009
• Donati et al Crit Care 2009
• Mesquida et al ICM 2009
• Mozina et al Crit Catre 2010
• Georger et al ICM 2010
• Shapiro et al Crit Care 2011
• Soga T et al Emerg Med J 2013

48. Relationship with outcome ?

49. Severe sepsis (n=252)
Association with outcome De Backer et al
CCM 41:791;2013

50. Edul et al
CCM 2012
Vessel density, proportion of perfused vessels and heterogeneity
but not velocity differ between survivors and non survivors
N=3N=15

51. Severe sepsis (n=252)
Association with outcome De Backer et al
CCM 41:791;2013

52. De Backer et al
CCM 41:791;2013
Severe sepsis (n=252)

53. Doerschug et al
AJP 293:1065;2007
Severe sepsis 24 / volunteers 15

54. Shapiro et al
Crit Care 15 R223; 2011
50 5860
168 pts at emergency dpt

55. 33 pts with septic shock
Trzeciak et al
ICM 34:2210; 2008
• 1st SDF evaluation within 3 hours after EGDT initiation
• 2nd SDF evaluation 3 to 6 hours after EGDT initiation
• SOFA changes between 0 and 24 h

56. DDB USI
EVOLUTION OF MICROCIRCULATORY ALTERATIONS
IN SEPTIC PATIENTS
Death after shock
ANOVA
p<0.01* * *
Sakr et al
CCM 32:1825;2004

57. Top et al
CCM 39:8; 2011
Persistent microcirculatory alterations in pediatric sepsis
N=3N=15

58. 0
1
2
3
4
5
6
7
8
Baseline 24hrs 48 hrs
Nonsurvivors (24/52)
Survivors (28/52)
*
*analysis of variance: p < 0.01
Slope(%/sec)
n = 52 septic patients Creteur et al
ICM 2007
Alterations of NIRS vasoreactivity test in patients with sepsis

59. Vallee F et al
Chest 138:1062;2012
Septic
shock
(n=46)
Ear Lobe PCO2

60. But isn’t it just the consequence of
the altered global hemodynamics ?
Subtitle: could you detect it using hemodynamic
measurements, clinical assessment or biomarkers?

61. Cardiac indexMean arterial pressure
LactateSvO2
mmHg L/min.M²
% mEq/L
40
50
60
70
80
90
100
1
2
3
4
5
6
7
20
30
40
50
60
70
80
90
Septic shockSevere sepsis
p<0.01
0
2
4
6
8
10
12
Septic shockSevere sepsis
DDB USI
MICROCIRCULATORY ALTERATIONS IN SEPTIC PATIENTS
De Backer et al AJRCCM 166:98-104;2002

62. De Backer et al
CCM 41:791;2013
Severe sepsis (n=252)
Association with systemic variables ?

63. De Backer et al
CCM 41:791;2013
Early severe sepsis (n=204)
Association with systemic variables ?

64. Primary event or adaptive phenomenon ?

65. Ellis C et al
AJP 282:H156;2002
In perfused capillaries, O2 extraction is INCREASED in sepsis
Single perfused capillary

66. Cardiomyocytes
rats
Bateman et al
AJP 293/H448;2007
During LPS, ICAM-1
expression and HIF gene
induction are heterogeneous,
and inversely related to flow.

67. Microcirculatory
alterations are
associated with renal
impairment
Wu L et al
AJP 292:F261; 2007

68. Alteration in redox potential are proportional
to microcirculatory alterations
Wu L et al
AJP 292:F261; 2007
Mice / LPS
Peritubular capillaries

69. Microcirculatory alterations are associated
with renal hypoxia (co-localized with NADH)
Wu L et al
AJP 292:F261; 2007
CTRL
LPS
Peritubular capillaries
Mice
NADHHypoxia

70. Fink T et al
Shock 2013
Liver microvascular perfusion and
redox state are inversely related
Rats / Fecal peritonitis / pretreatment / absence of shock

71. Brain microcirculation alteration precedes
the loss of function
Rosengarten B et al
Crit Care 13:R139;2009
Rats / LPS

72. R
2
= 0,80
0
20
40
60
80
100
20 40 60 80
Baseline
% well perfused capillaries
PslCO2gap
=> Microcirculatory alterations are primary events rather than
secondary to altered cellular metabolism.
Creteur et al
ICM 32:516;2006
Expected
(flow adapted to metabolism)
Stagnation of waste products

73. b-adrenoceptor stimulation improved liver
microvascular perfusion and redox state
Rats / Fecal peritonitis
Fink T et al
Shock 2013
Sham Sepsis DobuSham Sepsis Dobu

74. Change in Lactate
%
Change in capillary perfusion
mEq/L
DOBU 5 mcg/kg.min
-1
-0,8
-0,6
-0,4
-0,2
0
0,2
0,4
0 10 20 30 40 50
De Backer et al
CCM 34:403;2006

75. Effects of fluids

76. Influence of timing of fluid resuscitation
Rats
LPS
Legrand et al
ICM 37:1534; 2011

77. 20
30
40
50
60
70
80
90
100
Baseline Fluids
Late stage
(>48h)
N=23
Early stage
(<24h)
N=37
Proportion of perfused small vessels
%
$ p<0.01 fluids vs baseline and + p<0.01 late vs early
+
$
Microvascular effects of fluid challenge in patients with septic shock
Ospina et al
ICM 35:949;2010

78. Sensitivity Specificity Pos pred
val
Neg pred
val
Correct
classifica
tion
MAP 0.55 0.50 0.42 0.64 0.52
CI 0.42 0.68 0.42 0.68 0.58
DPP 0.50 0.56 0.42 0.64 0.53
The microvascular response to fluids cannot be
predicted by global hemodynamic changes
(including indices of fluid responsiveness) !
Ospina et al
ICM 35:949;2010

79. 20
30
40
50
60
70
80
90
100
Late, RL
Late, alb
Early, RL
Early, alb
Baseline After fluids
Proportion of perfused vessels
$ p<0.01 fluids vs baseline Coll vs cryst p = NS
%
$ $
The time of administration but not the type of fluid
influenced the microvascular response
Ospina et al
ICM 35:949;2010

80. Pottecher et al
ICM 2010

81. Futier E et al
Crit Care 15:R214;2011
High risk surgery (n=42)
Fluids improve microvascular reactivity
(NIRS ascending slope)

82. Futier E et al
Crit Care 15:R214;2011
High risk surgery (n=42)
Relationship with
systemic response

83. Vallée et al
Chest 138:1062;2010
Ear lobe PCO2
Relationship with systemic response

84. Effects of RBC transfusions

85. EFFECTS OF RED BLOOD CELL TRANSFUSIONS
N=35 RBCBaseline
Capillary density
7
6
5
4
3
2
Sakr et al
CCM 35:1639;2007

86. Baseline capillary perfusion, %
Deltacapillaryperfusion,%
EFFECTS OF RED BLOOD CELL TRANSFUSIONS
N=35
Sakr et al
CCM 35:1639;2007

87. Vasoactive agents ?

88. Secchi et al
Journ Crit Care
12:137;1997
Rats, gut villi
Sham
LPS
LPS + Dobu 2.5
Dobutamine in experimental sepsis

89. Secchi et al
CCM 29:597;2001
Rats, liver sinusoids
Dobutamine in experimental sepsis

90. b-adrenoceptor stimulation improved
liver microvascular perfusion
Rats / Fecal peritonitis / pretreatment / absence of shock
Fink T et al
Shock 2013

91. Capillary Perfusion
Dobutamine 5 mcg/kg.min De Backer et al
CCM 34:403;2006
21 patients in septic shock

92. Change in Lactate
%
Change in capillary perfusion
mEq/L
DOBU 5 mcg/kg.min
-1
-0,8
-0,6
-0,4
-0,2
0
0,2
0,4
0 10 20 30 40 50
De Backer et al
CCM 34:403;2006

93. Beta-adrenergic stimulation blunt PAF mediated
leucocyte adhesion and rolling
HUVEC

94. Red blood cell White blood cell/platelet
Arterioles Capillaries
BETA
(vasodilatation)
BETA
(WBC / PLT)
Decrease rolling et
adhesion
Effects of adrenergic agents on the microcirculation

95. Vasopressor agents ?

96. Influence of blood pressure on
microvascular perfusion ?

97. Cerebral microcirculation
Sheep, peritonitis
Taccone FS et al
Crit Care 14 R140; 2010

98. Cerebral microcirculation and metabolism
Sheep, peritonitis
Taccone FS et al
CCM (in press)

99. De Backer et al
CCM 41:791;2013
Severe sepsis (n=252)
No clear cut-off….

100. Blood pressure targets
Impact of vasopressor agents

101. 0
20
40
60
80
100
120
MAP (mmHg)
Vasodilation
Ctrl
Vasoconstiction

102. Impact of vasopressors on the microcirculation
(Norepinephrine vs Vasopressine)
Hamster, control condition
Friesenecker et al
Crit Care 10:R75;2006

103. Maier-S et al
BJA 2009
Phenylephrine impairs microvascular perfusion in CPB

104. Impact of vasopressors on the microcirculation
Rats, LPS, gut muscularis
Nacul F et al
Anesth Analg
110:447;2010
No fluids
Doses:
NE 5 mcg/kg.min
EPI 5 mcg/kg.min
PHE 10 mcg/kg.min
DOPA 20 mcg/kg.min
Dobu 12 mcg.kg.min
Normotensive sepsis

105. Red blood cell White blood cell/platelet
Arterioles Capillaries
BETA
(vasodilatation)
BETA
(WBC / PLT)
Decrease rolling et
adhesion
Effects of adrenergic agents on the microcirculation
ALPHA
(vasoconstriction)

106. Flow
Mean arterial pressure
Kidney
Muscle
Liver
Brain / heart
Different vascular beds have different vascular resistances
Gut

107. DDB USI
Does correction of hypotension result
in an improved tissue perfusion ?

108. Impact of vasopressors on the microcirculation
(Norepinephrine vs Vasopressin)
Rats, LPS
Baseline value
Shock value
Nakajima et al
CCM 34:1847;2006
MAP 46 71 70 mmHg

109. Fries et al
CCM 36:1886; 2008
Norepinephrine
improved slightly
microvascular
perfusion but not
µPO2
Rats / CLP
Buccal mucosa

110. Correction of hypotension improves
microvascular reactivity (NIRS)
MAP 54 => 77 mmHg
Georger et al
ICM 36:1882;2010

111. DDB USI
What is the optimal blood pressure
target for the microcirculation ?

112. Jhanji et al
CCM 37:1961;2009
p<0.05
p=0.45
N=16
Impact of MAP/NE on microvascular perfusion

113. Dubin et al
Crit Care 2009
N=20
Impact of MAP/NE on microvascular perfusion

114. Thooft et al
Crit Care 2011
Impact of MAP/NE on microvascular perfusion
Density of perfused small vessels
65 (baseline) 75 85 65
6
8
10
12
level of mean arterial pressure, mmHg
FCDsmallvessels,n/mm
*
*

115. Thooft et al
Crit Care 2011
Impact of MAP/NE on microvascular perfusion
65 (baseline) 75 85 65
0
100
200
300
400
500
* *
level of mean arterial pressure, mmHg
AscendingSlope%/min
* *

116. • Vasopressor agents have a dual effect on the
microcirculation: on the one hand vasopressors
decrease microvascular perfusion by constriction of
precapillary sphincters.
• On the other hand, achievement of a minimal
perfusion pressure is needed to preserve organ blood
flow and microcirculatory perfusion.
• Optimal pressure targets are variable and should
be individualized.
Vasopressors and the microcirculation

117. Vasodilatory agents ?

118. 65
70
75
80
85
90
95
100
BASE
Proportion of perfused vessels
(all vessels)
++ p <0.01 vs base
TOPICAL ACETYLCHOLINE
(10-2 M)
Patients with septic shock (n = 11)
%
DDB USI
MICROCIRCULATORY ALTERATIONS IN SEPTIC PATIENTS
De Backer et al
AJRCCM 166:98;2002

119. Spronk et al
Lancet 360:1395;2002
Effects of nitroglycerin
8 pts with septic shock

120. Boerma E et al
CCM 38:93-100;2010
Effects of nitroglycerin
70 pts with severe sepsis

121. Effects of nitroglycerin
70 pts with severe sepsis
But can normal be more normal than normal ?
Boerma E et al
CCM 38:93-100;2010

122. Proportion of Perfused Small Vessels
BA
SELIN
E
BEFO
R
E
D
R
U
G
SH
O
C
K
N
E
1H
N
E
2H
0
20
40
60
80
100
ENALAPRILAT
PLACEBO
Enalaprilat
Placebo
n=8
n=8
n=8
n=8
n=6
n=7
n=7
n=7
n=6
n=7
* p<0.05 vs baseline and before drug
p=0.83 for trend in enalaprilat group
p=0.006 for trend in placebo group
p=0.48 for group/time interaction
* *
PPVsmallvessels,%
ACE inhibitors?
Salgado D et al
Shock 2011
Sheep
CLP
But no impact on
• outcome
• organ function

123. Magnesium sulfate?
Pranskunas A et al
BMC Anesth 11:12; 2011
14 pts severe sepsis

124. Modulation of endothelial function ?

125. Vitamin C
Tyml K et al
CCM 33,1823;2005
Rat / muscle
CLP
Ascorbate 7.6g/100g BW
1h or 24h post CLP

126. Vitamin C
The effect is related to endothelial NOS
Tyml K et al
CCM 2008
Mice / muscle
Feces in peritoneum
Ascorbate 10-200 mg/kg
6h post peritonitis

127. BH4 (tetrahydrobiopterin)
Effect on NO metabolism at endothelial level
Schmidt S and Alp N
Clinical Science
113: 47; 1997

128. He X et al
CCM 20:2833; 2012
Sheep
CLP
BH4 (tetrahydrobiopterin)

129. He X et al
CCM 20:2833; 2012
Sheep
CLP
BH4 (tetrahydrobiopterin)
Improved
• outcome
• organ function

130. • Multiple experimental and clinical studies suggest
that microvascular alterations play a key role in the
pathophysiology of sepsis and in the development of
sepsis-induced organ failure.
CONCLUSIONS
• These alterations are due to several factors
(endothelial dysfunction, interaction with circulating
cells) that make unlikely that classical hemodynamic
resuscitation can be effective in restoring an adequate
microcirculation.
• Modulation of endothelial NO synthase seems
promizing.