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Experimentelle Modelle in der Neonatologie Ursula Felderhoff-Müser Klinik für Kinderheilkunde 1

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Experimentelle Modelle in der Neonatologie

Ursula Felderhoff-Müser

Klinik für Kinderheilkunde 1

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 5 Titel

Cognitive outcome of ELBWs at 6 years

Marlow et al. (2005) N Engl J Med 352:9-19

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 15 Titel

Oxygen-induced apoptotic cell death (P7)

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 19 Titel

Primäre Mixglia-Zellkultur

Pre-OL

Astrocyte

Folie 20 Titel

80%O2 PreOL

80%O2 MatOL

Maturity-dependent apoptotic death of OLs

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 22 Titel

Sudden drop of estrogen after birth

17β-estradiol

Preterm birth

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 26 Titel

IHC CEACAM1/CNPase

CNPase: marker for oligodendrocytes

Prager et al. in preparation

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 !