Folie 2 Titel
H. Lagercrantz, T. Ringstedt in Fetal and Neonatal Neurology and Neurosurgery 4th Edn.
Folie 3 Titel
hypoxia
hyperoxia
hypokapnia
infection
metabolic disease
hemorrhage
inflammation
infection
drugs
asphyxia
trauma
term infant
pretrem infant
Neonatal brain injury
Folie 4 Titel
4
Brain development in the preterm infant
Field et al. (2009) Arch Dis Child Fetal Neonatal Ed
time [weeks]
40
Pregnancy
37
~12 % of all life births
23
GA 25 weeks GA 40 weeks
Folie 6 Titel
Periventrikuläre Leukomalazie – diffus und zystisch
Volpe JJ Lancet Neurology 2009, 8:110-124
MRT am Termin
Folie 7 Titel
Periventricular leukomalacia (PVL)
- focal/diffuse white matter damage
- loss of premyelinating oligos (OL)
- hypomyelination
- ventrikulomegaly
Volpe (2001) Neurology of the Newborn
diffuse
• hypoxia/ischemia
• infection/inflammation
key role in the pathogenensis
• hyperoxia
Folie 8 Titel
time unit
6
4
2 %
adult b
rain
“Brain Growth Spurt” – Die Phase des rapiden Hirnwachstums
birth
human (1 unit = 1 month)
rat (1 unit = 1 day)
pig (1 unit = 1 week)
monkey (1 unit = 4 days)
Dobbing and Sands (1979) Early Hum Dev, 3:79-83
• Proliferation
• Differenzierung
• Migration
• Synaptogenese
• Apoptose
Folie 9 Titel
Experimental animal and cell culture models
Preterm brain model
• Hyperoxia
• Hyperoxia/inflammation
• Primary Oligodendrocytes (rat), OLN-93 (cell line)
Term asphyxia model
• Hypoxia-ischemia (HII)
• HII/hypothermia
k/o mice
• synRas
Folie 10 Titel
Experimental animal and cell culture models
Preterm brain model
• Hyperoxia
• Hyperoxia/inflammation
• Primary Oligodendrocytes (rat), OLN-93 (cell line)
Term asphyxia model
• Hypoxia-ischemia (HII)
• HII/hypothermia
k/o mice
• synRas
Folie 11 Titel
•Hess JH: Oxygen unit for premature infants and very young children. Am J Dis Child 47:916, 1934
•Terry TL Extreme prematurity and fibroblastic overgrowth...Am J Ophtalmology 25:203-204, 1942
•Lanman JT. Retrolental fibroplasia and oxygen therapy. J Am Med Assoc 155:223-226, 1954
Folie 12 Titel
Effects of perinatal oxygen increase
O2 25 mmHg
70 mmHg
preterm birth
BPD RPM
Collins MP. (2001) ….. ventilation-related risk factors for cerebral
palsy in low birth weight infants. Pediatr. Res. 50, 712-719.
&
Folie 13 Titel
Oxygen toxicity - experimental animal model
- Wistar rats / C57 BL6 mice (P0 – P35)
- 2 litters with mothers
- 2, 6, 12, 24, 36, 48, 72, 96 hrs
- transcardial perfusion
- tissue preparation
80%O2
Folie 14 Titel
Felderhoff-Mueser et al. (2004) Neurobiol Dis 17:273-282
0 2 6 12 24 48 72 0
25000
50000
75000
100000
125000
150000
175000
***
*** ***
***
**
Time of exposure (h)
Ap
op
toti
c s
co
res
Hyperoxia induces age-dependent apoptosis
P0 P3 P7 P14 0
50000
100000
150000
200000 Normoxia Hyperoxia, 24 h
Postnatal age
Ap
op
toti
c s
co
res
Folie 16 Titel
Dzietko et al. (2008) Ann Neurol
Hyperoxia induces apoptotic signaling pathways
Fas signalling
Fas-Expression after hyperoxia
0 10 20 30 40 50 600
50
100
150
Fas (Tha)
Fas (RSC)
Time after hyperoxia
den
sit
y r
ati
o x
/ß-a
cti
n
C 2 6 12 24 48 72
Fas
C 2 6 12 24 48 720
50
100
150
200
250
*
***
hours of hyperoxiad
en
sit
y r
ati
o
FasR
/ß-a
cti
n (
%)
A
ß-actin
Folie 17 Titel
Hyperoxia-induces proteome changes:
2-dimensional gel-electrophoresis (2-DE)
Proteome changes:
• Apoptosis: Annexin A5, ASK1
• Energy metabolism: Peroxiredoxin 2
• Synaptic function: Synapsin 1
• Neuronal migration: Doublecortin, Neuromodulin
• Neuronal outgrowth: Rho GTPase family
Kaindl et al. (2006) Cell Death Differ, Kaindl et al (2009) Ann Neurol
P6 P7 P14 P35
? ? ?
C57 BL6 mice, O2 , 24 hrs
2-DE
Folie 18 Titel
Developmental stages of oligodendrocytes
A2B5 O4 O1, O4 MBP, O1, O4
NG2 NG2 CNPase CNPase, MAG
Progenitors Pre-OLs Immature-OLs Mature -OLs
Proliferation
Migration Myelination
?
(VZ /SVZ)
Folie 21 Titel
Hyperoxia-induced caspase-3 activation
activated caspase-3
80% O2
80% O2
+
caspase-
inhibitor
Gerstner et al. (2008) J Neurosci 28:1236-45
Folie 23 Titel
Estrogen receptors on oligodendrocytes
ER-α
Pre Mature
ER-α
β-actin
Pre OLs
ER-β
β-actin
Pre Mature
Folie 24 Titel
MBP staining
P11
21% O2
80% O2 17ß estradiol/80% O2
17ß Estradiol protects white matter
Folie 25 Titel
Tiermodell
Noxen:
Tag 3: Endotoxin 0,25 mg/kg i.p.
Tag 5: Hyperoxie 80% O2 für 24 h
Endpunkte:
Tag 6: Neurale Apoptose
Tag 11: Myelinisierung
LPS
Folie 27 Titel
Experimental animal and cell culture models
Preterm brain model
• Hyperoxia
• Hyperoxia/inflammation
• Primary Oligodendrocytes (rat), OLN-93 (cell line)
Term asphyxia model
• Hypoxia-ischemia (HII)
• HII/hypothermia
k/o mice
• synRas
Folie 28 Titel
Therapeutic hypothermia
• early start (<6h after insult) – „time is brain“
• temperature 33-34°C (rectal); for 72hrs
• controlled rewarming (max. 0,5°C/h)
Folie 29 Titel
0.1 1 10
Combined
CoolCap
NICHD
TOBY effect of hypothermia on
combined rate of death
or handicap at the age
of 18 months
Edwards AD, BMJ 2010
Folie 30 Titel
Therapeutic hypothermia can be offered for infants who meet the criteria of
published trials provided that the infrastructure and trained personnel to
perform hypothermia are in place.
American Heart Association guidelines for cardiopulmonary resuscitation and emergency
cardiovascular care, part 15: neonatal resuscitation. Circulation 2010
National Institute for Health and Clinical Excellence. Therapeutic hypothermia with intracorporeal
temperature monitoring for hypoxic perinatal brain injury: interventional procedure guidance
www.nice.org.uk/nicemedia/live/11315/48809/48809.pdf
British Association of Perinatal Medicine position statement on therapeutic cooling for neonatal
encephalopathy
www.bapm.org/publications/documents/guidelines/Position_Statement_Therape
utic_Cooling_Neonatal_Encephalopathy_July%202010.pdf
Folie 31 Titel
Experimentelles Modell der neonatalen Asphyxie
P14 Wistar rats/C57BL6
- ligation of the right A. carotis communis
- 65 minutes 10 % oxygen
- 2 hrs later start hypothermia for 10 hrs
Rice and Vannucci (1981) Ann Neurol; 9:131-141
Folie 32 Titel
Yager J, Towfighi J, Vannucci RC
Influence of mild hypothermia on
hypoxic-ischemic
brain damage in the immature rat.
Pediatr Res. 1993; 34:525-529
Thoresen M et al.
Mild hypothermia after severe
transient hypoxia-ischemia ameliorates
delayed cerebral energy failure in the
newborn piglet.
Pediatr Res. 1995 ; 37:667-670
Therapeutic hypothermia
Folie 33 Titel
Zeitliche Dynamik der Asphyxie
Therapeutisches Fenster
Primäre Phase 0-12 h:
Primäres Energieversagen und
Erholungsphase
Sekundäre Phase 6-48 h:
Sekundäres Energieversagen
(Hauptschädigungsphase)
Folie 34 Titel
Experimental animal and cell culture models
Preterm brain model
• Hyperoxia
• Hyperoxia/inflammation
• Primary Oligodendrocytes (rat), OLN-93 (cell line)
Term asphyxia model
• Hypoxia-ischemia (HII)
• HII/hypothermia
k/o mice
• synRas
Folie 35 Titel
Mice overexpressing RAS
Hyperoxia
Ras-GTP (Apoptose)
synRas WT 0
10000
20000
30000
40000
Ap
op
toti
c
ce
lls
*
Felderhoff-Mueser et al. (2004) Neurobiol Dis
Ras-GTP ?
Ras-GTP in OLN
Folie 36 Titel
• histology/immunohistochemistry
• fluoreszenzmicroscopy
• western blotting
• PCR
• Elisa
• zymography
• neurodevelopmental testing
• Facs
• confocal microscopy with O2 Chamber
• stereology (in Kooperation)
Methods
Folie 37 Titel
Dept. of Neonatology und Ped. Neurology,
Charité Berlin
Felix Brehmer
Christoph Bührer
Angela Kaindl
Children’s Hospital Boston
Harvard Medical School
Paul A. Rosenberg
Joseph J. Volpe
www.NEOBRAIN.eu
EU 6th framework
BMBF, DFG
Schering Stiftung
Sonnenfeld Stiftung
Stiftung Universitätsmedizin,
IFORES Program Essen,
Mercator Stiftung
INSERM, Paris
Pierre Gressens
Neonatal Neuroscience Lab
Division of Pediatrics I
University Hospital Essen
Ivo Bendix
Ralf Herrmann
Matthias Keller
Karina Kempe
Sebastian Prager
Gerald Schlager
Katja Strasser
Karla Drommelschmidt
University Medical Center
Utrecht
Cobi Heijnen
Annemieke Kavelaars
Dept. of Pediatric Cardiology,
Justus-Liebig University of Giessen
Bettina Gerstner
Thank you !