Henrik Hagberg and Pierre Gressens

Mechanisms of Perinatal Brain Injury

Group Leaders
Professor Henrik Hagberg (Chair in Obstetrics)
Professor Pierre Gressens (Chair in Perinatal Neurology)

Research Team
Dr Catherine I Rousset (Senior Research Associate - MRC funded)
Dr Claire Thornton (Senior Research Associate - MRC funded)
Dr Anton Kichev (Research Associate - AMR funded)
Dr Yasuka Miyakuni (Visiting Researcher - funded from Japan)
Ms Regina Vontell (Research Assistant , PhD Student - MRC funded)
MRes and BSc students

Collaborators
Prof David Carling (MRC and Imperial)
Prof David Edwards (Imperial)
Prof Donna Ferriero (UCSF, San Francisco, USA)
Prof Annemieke Kavelaars and Prof Cobi Heijnen (Utrecht, Holland)
Prof Steven Levison (Newark University, USA)
Prof Carina Mallard (Gothenburg University)
Prof Richard Reynolds (Imperial)
Prof Mary Rutherford (Imperial)
Prof Susan Vannucci (Cornell University, USA)
Dr Etienne Jacotot (Inserm 676, Paris, France)

Research support
Ms Regina Vontell

Background

Cerebral Palsy (CP) is the commonest cause of severe disability in children (2-3/1000 births) and a major cost to sufferers, their families and society; which underlines the need for development of novel treatment and preventive strategies. Perinatal asphyxia and preterm birth are strongly associated with CP and account for about 2/3 of all cases. Furthermore, clinical studies in both preterm and term infants suggest that antenatal exposure to infection/inflammation enhances immature CNS vulnerability to injury and there is a need to understand the underlying mechanisms.

CP results from a variety of insults to the developing brain. Our work focuses on brain injury mechanisms related to hypoxia-ischemia (HI) and inflammation in term and preterm infants.

Brain injury evolves over many hours (secondary brain injury) after HI, and interventions administered in asphyxiated newborns (hypothermia) after HI improves outcome, which provides proof of concept that treatment is a possibility. However, hypothermia only saves one baby in eight and we urgently need to obtain a better understanding of the underlying pathophysiology to improve things further.

Research projects

1.
Our program focuses on mitochondria-targeted protective strategies for limiting injury in the developing brain. Recent work showed that mitochondria have an important role in HI brain injury which turns them into a privileged target for neuroprotection. Our hypothesis is that mitochondrial membrane permeabilization (MP) triggered by the pro-apoptotic protein Bax is a key event in secondary brain injury in the immature.

HI triggers a cascade of injurious events including the release of excitatory amino acids (e.g. glutamate) pro-inflammatory cytokines (e.g. IL-1, IL-18, TNF-α) and chemokines, intracellular Ca²⁺ and accumulation of reactive oxygen species that will contribute to cell death in neurons and oligodendroglial precursors through mitochondrial permeabilisation.

We have found that, in the immature brain, the pro-apoptotic protein Bax plays a critical role by inducing a mitochondrial pathway of cell death. After HI, Bax oligomerises within the outer mitochondrial membrane leading to pore formation and the release of apoptogenic proteins including cytochrome c, AIF, SMAC/Diablo, Endo G, HtrA/Omi. Blockage of caspases (using a broad-spectrum caspase inhibitor) or genetic up-regulation of endogenous inhibitors of apoptosis (IAPs) in combination with genetic inhibition of AIF confer substantial neuronal rescue and reduction of total tissue loss. The Bax inhibitory peptide (BIP) efficiently blocks mitochondrial permeabilisation and caspase-3 activation and reduces neonatal but not adult ischemic brain injury indicating that a Bax-dependent pathway is critical for mitochondrial membrane permeabilisation in the immature brain.

More specifically the research objectives to explore are:

The role of Bax/Bak dependent permeabilization in hypoxia-ischemia
To what extent the up-stream regulation of MP involves cytosolic p53, caspase-2, stress kinase pathways (including JNK/Gadd45β and AMPK) and the mitochondrial fission protein Drp-1
The role of Bax/Bak dependent permeabilisation in inflammatory sensitisation to test the hypothesis that TRAIL death receptors, NFkB-p53 and the ASC protein are involved in enhancement of injury.

As injury in both white and grey matter is common in preterm and term infants, we investigate mechanisms in neurons and oligodendrocyte precursors (OPCs) using in vivo (transgenic mice, siRNA and pharmacological strategies) and in vitro (cell lines, primary cultures, and isolated mitochondria) models. This work will increase understanding of the mechanisms of perinatal brain injury and result in development of neuroprotective strategies to reduce the severe adverse consequences of injury in term and preterm brain.

Key words: perinatal brain injury, cell death, mitochondria, hypoxia-ischemia, inflammation, neuroprotection strategies.

2.
The second project aims at identifying genes of vulnerability / protection of the perinatal brain towards hypoxic-ischemic, excitotoxic and/or inflammatory insults. Our hypothesis is that different strains of rodents will display different sensibility to brain damage using well-characterized models of perinatal brain damage. The comparison of the genome of susceptible and resistant strains should allow to identify potential loci and/or genes of vulnerability and/or resistance.

This project will be based on the combination of well-established animal models of perinatal brain damage including i) hypoxic-ischemic brain damage; ii) excitotoxic brain damage; iii) inflammation-induced brain damage; combined inflammatory and excitotoxic brain damage.

The same models will be then used to validate the candidate genes, using a combination of histological, immunohistochemical, molecular biology, imaging (MRI) and behavioural techniques.

This project will be done in collaboration with Tim Aitman’s group.

Key words : perinatal brain injury, susceptibility, resistance, vulnerability, gene, rodent.

References.

Gupta SK, Mishra R, Kusum S, Spedding M, Meiri KF, Gressens P, Mani S . GAP-43 is essential for the neuroprotective effects of BDNF and positive AMPA receptor modulator S18986. Cell Death Differ 16: 624-637, 2009

Wang X, Carlsson Y, Basso E, Zhu C, Rousset CI, Rasola A, Johansson BR, Blomgren K, Mallard C,Bernardi P,Forte MA, Hagberg H: Developmental shift of cyclophilin D contribution to hypoxic-ischemic brain injury. J Neurosci 29:2588-96, 2009

Fontaine RH, Cases O, Lelievre V, Mesples B, Renauld JC, Loron G, Degos V, Dournaud P, Baud O, Gressens P. IL-9/IL-9 receptor signalling selectively protects cortical neurons against developmental apoptosis. Cell Death Differ, 15: 1542-1552, 2008

C Zhu, X Wang, J Deinum, Z Huang, J Gao, N Modjtahedi, MR. Neagu, M Nilsson, P S. Eriksson, H Hagberg, J Luban, G Kroemer, K Blomgren: Cyclophilin A participates in the nuclear translocation of apoptosis inducing factor in neurons after cerebral hypoxia-ischemia. J Exp Med 204:1741-8, 2007.

Rangon CM, Fortes S, Lelièvre V, Leroux P, Plaisant F, Joubert C, Lanfumey L, Cohen-Salmon C*, Gressens P*. Chronic mild stress during gestation worsens neonatal brain lesions in mice. J. Neurosci 27: 7532-7540,2007

Gustavsson M, Mallard C, Vannucci SJ, Wilson MA, Johston MV, Hagberg H: Vascular response to hypoxic preconditioning in the immature brain. J Cereb Blood Flow Metab 27:928-38, 2007.

Medja F, Lelièvre V, Fontaine RH, Lebas F, Leroux P, Ouimet T, Saria A, Rougeot C; Dournaud P, Gressens P. Thiorphan, a neutral endopeptidase inhibitor used for diarrhea, is neuroprotective in newborn mice. Brain, 129: 3209-3223, 2006

Zhu C, Xu F, Wang X, Shibata M, Uchiyama Y, Blomgren K, Hagberg H: Different apoptotic mechanisms are activated in male and female brains after neonatal hypoxia-ischaemia. J Neurochem 96:1016-27, 2006.

Blomgren K, Hagberg H: Free radicals, mitochondria, and hypoxia-ischemia in the developing brain. Free Radic Biol Med 40:388-97, 2006.

Husson I, Mesplès B, Bac P, Vamecq J, Evrard P, Gressens P. Melatoninergic neuroprotection of the murine periventricular white matter against neonatal excitotoxic challenge. Ann Neurol, 51: 82-92, 2002

Dommergues MA, Patkai J, Renauld JC, Evrard P, Gressens P. Pro-inflammatory cytokines and IL-9 exacerbate excitotoxic lesions of the newborn murine neopallium. Ann Neurol 47 : 54-63, 2000

Gressens P, Marret S, Hill JM, Brenneman DE, Gozes I, Fridkin I, Evrard P. Vasoactive intestinal peptide prevents excitotoxic hypoxic-like cell death in the murine developing brain. J Clin Invest, 100: 390-397, 1997