The neurotoxic cascade in the ischemic penumbra

Radiopharmacie, Service de Médecine Nucléaire,
In vivo imaging of experimental stroke :
animal models, PET and SPECT radiotracers
Part 1 : animal models of stroke
Part 2 : major processes and mediators involved in stroke
PET and SPECT imaging of stroke
P. PISANO, PU/PH
B. GUILLET, MCU, Assistant

valence and classification of acute strokes
Third leading cause of death
Leading cause of permanent disability
No in vitro methods
Acute stroke
Hemorrhagic stroke
Ischemic stroke 85%
arachnoïd
morrhage
(SAH)
Intra-cerebral
Hemorrhage
(ICH)
Extra-cranial
embolism
Intra-cran
thrombos

w to reproduce human thromboembolic stroke?

al of experimental ischemic stroke
reduction of cerebral blood flow (CBF)
reduction of oxygen and glucose supply
to brain tissue
Size infarction predicted by
intensity
duration
of the CBF reduction
100 %
CBF Flow
50 %
25 %
probability for infarction < 5%
probability for infarction > 95%

BF reduction in focal ischemic strokes
cal ischemia = reduction in blood flow to a very distinct specific brain regio
No blood flow
the very central core of
the ischemia,
ut collateral circulation)
from Dinargl, 1999,
Gradient of blood flow from the inner core
reaching out to the limits of the ischemic area :

cal ischemic stroke by MCAO
Willis circle :
carotid and vertebrobasilar
arterial system connected
Occlusion of one of the major cerebral
blood vessel middle cerebral artery (MCA
X
Proximal
Distal
X X

nesthesia Protocol and Monitoring
Anesthesia:
intubation and mechanical ventilation
sevoflurane 1.2 MAC
blood arterial pressure
Monitoring:
temperature
sevoflurane, O 2 et CO 2
blood gases
glycemiae
Measurement and control of physiological parameters so that
infarction size or cell injury can be reproducible and consistent.

urgical protocol without craniotomy
1h MCAO
reperfusio
A. Cérébrale
Moyenne
A. Ptérygo-Pallatine
A. Carotide Externe
A. Carotide interne
A. Carotide Commune

utcome measurement of injury
eurological score (Longa et al., 1989).
Neurobehavioral assessment
S1
otarod
S2
0
1
2
3
4
no neurolocal deficit
forelimb flexion
unilateral circling
swinging of the body on the left side
motionless
Brain infarct volume calculation
mild deficit
moderate deficit
severe deficit
unconciousness
Swanson et al. JCBFM 1990
h= inter-slice
distance

markers initially developed to explore : - tumor hypoxia
- myocardial
ischemia
Part 2 : neurotoxic cascade of
ischemia
PET and SPECT imaging of penumbra

neurotoxic cascade in the ischemic penumbra
De Keyser et al., 1999
15
-O, 18 FDG, 18 F-MISO,
11
C-FMZ, 18F-CPFPX
PLA2
BBB disruption, endothelial disturbances
Cell infiltration
Cell necrosis
Tissue remodeling, angiogenesis,

T imaging of reduced CBF Phelps, Mol. Imaging, 2004
Initially, limited exclusively to the determination of :
Cerebral blood flow (CBF, 15O-H2O)
Oxygen extraction fraction (15O-O2)
Glucose metabolism (18FDG)
In penumbra, oxygen extraction fraction (OEF) is increased and neurons
are functionally disturbed but still viable

ET imaging of hypoxic cells with nitroimidazoles
18F-fluoromisonidazole (18FMISO)
FMISO
Nitroréductase
Radical anion
Normoxia
Diffuses
Hypoxia
Irreversibly bound to

ET imaging of hypoxic cells
Read et al., Neurology. 19
18
F-fluoromisonidazole (18FMISO) in humans
22h post-infarct
10d post-infarct
MISO binding is indicative of :
final CT infarct area
- ongoing tissue hypoxia
- not merely recent tissue inju

ET imaging of hypoxic cells
Ito et al., Acad Radiol 200
68
Ga-labeled metronidazole in a breast tumor-bearing r

PECT imaging of hypoxic cells
Song et al., (Stroke 2003)
99m
Tc-metronidazole in patients with ischemic stroke
EC-MN
99m
Tc-Neurolite®
99m
Tc-EC-MN

T imaging of hypoxic cells by down-regulated receptors
11
C-flumazenil in cats
Heiss et al., Stroke 19
C I 1h 4h
Defect in FMZ binding predicts the size of the final infarct whereas

T imaging of hypoxic cells by down-regulated receptors
adenosine A(1) receptors
Tadashi et al., J Nucl Med 20
• [1-methyl-( 11 )C]8-dicyclopropylmethyl-1-methyl-3-propylxanthine (MPD
•
18
F-CPFPX (8-cyclopentyl-3-(3 fluoropropyl)-1-propylxanthine)
-CPFPX
Degree of decrease
in A1R ligand binding is a sensitive
predictor of severe ischemic insult
Transient MCAO in cats

neurotoxic cascade in the ischemic penumbra
De Keyser et al., 1999
-Annexin
PLA2
BBB disruption, endothelial disturbances
Cell infiltration
Cell necrosis
Tissue remodeling, angiogenesis,

apoptotic cells/necrotic cells = 1:1
aging of apoptotic cell death
ll death 4h after 2h MCAO
apoptotic cells/necrotic cells = 9:1
Charriaut-Marlangue C. et al., JCBFM, 19
Apoptosis
Necrosis

aging of apoptotic cell death
Annexin V
- 36-kDa protein
- high affinity to phosphatidylserine at the
surface of apoptotic cel
- detection of the earliest stages of apoptosis
SPECT
– 99mTc-HYNIC-annexin V
(hydrazinonicotinamide
derivatized annexin V)
– novel form of annexin V :
PET
– 18F-Annexin V
– 124I-annexin V

ECT imaging in rats submitted to
2 h left MCAO followed by
SPECT imaging in rabbits sufferin
from neonatal hypoxic brain injur
ECT imaging of apoptosis
99mTc-HYNIC-annexin V
Mari et al., Eur J Nucl Med Mol Imaging 2004
D’Arceuil et al., Stroke 2000

T imaging of apoptosis
PET annexin V
Radiolabeled annexin V PET imaging of liver apoptosis
Control
Cycloheximide
treated rats
Untreated mice
anti-Fas mAb-treated
mice
124
I- Annexin V
18
F- Annexin V

neurotoxic cascade in the ischemic penumbra
De Keyser et al., 1999
11
C-PJ34
PLA2
BBB disruption, endothelial disturbances
Cell infiltration
Cell necrosis
Tissue remodeling, angiogenesis,

T imaging of free radical-induced necrosis
11
C-PJ34 : phenanthridinone derivative which
binds to PARP
PARP-1 is up-regulated during cell necrosis
streptozotocin-induced pancreas necrosis
Control
No staining was observed in control rats.
STZ treated
Accumulation of 11 C-PJ34 is shown in STZtreated
rats as dark brown staining localized
in the nuclei of islets in the rat pancreas

neurotoxic cascade in the ischemic penumbra
De Keyser et al., 1999
11
C-octanoate
11
C-PK11195
PLA2
BBB disruption, endothelial disturbances
Cell infiltration
Cell necrosis
Tissue remodeling, angiogenesis,

aging of microglia infiltration after ischemia–reperfusion
RICHARD B. BANATI GLIA (2002)
peripheral
benzodiazepine
receptors

T Imaging of activated microglia infiltration
Sette et al. Stroke,199
11
C-PK11195
11C-PK11195 binding after focal ischemia (MCAO) in the anesthetized baboon
11
C-flumazenil

T imaging of during MCAO-induced astrocytes infiltration
Kuge et al., Ann.Nucl.Med, 2
11
C-octanoate uptake in a dog 24h after MCAO

neurotoxic cascade in the ischemic penumbra
Dimeric RGD peptide
Monomeric RGD peptide
a 5 ß 3 integrin receptors
VEGF receptors
PLA2
BBB disruption, endothelial disturbances
Cell infiltration
Cell necrosis
Tissue remodeling, angiogenesis,

T imaging of integrin receptors
18
F-FB-E[c(RGDyK)]2 dimeric ligand of avß3 integrin receptor
inoculation of U87MG human glioblastoma cells into mouse forebrai
Tumor to contralateral background ratio = 9.5 +/- 0.8
Chen et al., Mol Imaging Biol, 2006

ECT imaging of integrin receptors
Meoli et al., J Clin Invest. 200
111In-RP748 : quinolone targeted at avß3 integrin receptor
111In-RP748, a
gs with chronic myocardial infarction
increased myocardial uptake (red) in the
region of injury-induced angiogenes
99m
Tc-MIBI
111
In-RP748

aging of VEGF receptors
VEGF is an angiogenic protein secreted in response to
hypoxia that binds to VEGF receptors Flt-1 and KDR
overexpressed by ischemic microvasculature.

aging of VEGF receptors
111In-labeled VEGF
Lu et al., Circulation 2003
Ischemic hindlimb (rabbit)
111
In-labeled VEGF accumulation in
ischemic hindlimb
124I radiolabeled mAb against VEGF Collingridge et al., Cancer Res. 200
Binds to angiogenic areas in tumor-bearing mice

ture directions of stroke PET and SPECT imaging
Imaging hypoxic cells with reporter genes
Acycloguanosine Pyrimidine derivatives
HSV-tk1
HIF-1

Conclusion
Most promising PET and SPECT radiotracers for
stroke : angiogenesis radiotracers developed for
tumor hypoxia
Transferability from tumor to brain hypoxia :
pharmacokinetic considerations
PET and SPECT tracking of stem cell therapy of
stroke by reporter genes transfected inside the cells.
– Neural stem cells
124I
– Endothelial progenitors

Leucocytes infiltration
after ischemia–reperfusion

perimental models and Animal species
enetically homogeneous
ophisticated neurosensory and motor behavior measurements
as outcome measures of injury from ischemia
mall animals : lissencephalic brains
Focal cerebral ischemia (transient or permanent) :
non human primate, dog, cat,
rabbit, guinea pig, rat, mouse.
Global cerebral ischemia
- complete (transient) :
- incomplete (transient or premanent) :
non human primate, dog, cat, rat,
mouse, gerbil, sheep
dog, cat, pig, rat, mouse, gerbil,
sheep.
Multifocalcerebral ischemia :
Rabbit, rat.
Intracerebelar hemorrhage :
Subarachnoid hemorrhage and vasospasm :
non human primate, dog, cat, rat,
pig, rabbit.
non human primate, dog, cat, rat,
mouse, pig, rabbit.
(from Traytsman, ILAR J. 2003; Andaluz et al., Neurosurg Clin N Am 2