Neuroprotection after perinatal asphyxia: mitochondrial and inflammatory targets

Abstract

Neonatal hypoxia-ischemia (HI) is a severe condition and defined as a period of insufficient blood gas exchange before, during or early after birth. It is one of the primary causes of neonatal mortality. In this thesis we study the contribution of apoptotic cell death and inflammation to the development of HI brain damage in neonatal rodent models.

In the first part of this thesis, we demonstrate the potent neuroprotective effects of three small peptides (D-JNKi, Sabkim1, TAT-P7-pi) that prevent mitochondrial outer membrane permeabilization after neonatal HI by targeting the MAP kinase c-Jun N-terminal kinase (JNK) and the tumor suppressor p53, two proteins that control the apoptotic balance at the mitochondrial membrane.

Inhibition of mitochondrial JNK activation by D-JNKi protected the neonatal brain after HI by preserving mitochondrial integrity and reducing apoptotic cell death. Upregulation of mitochondrial anti-apoptotic proteins was crucial for maintaining neuroprotection after D-JNKi treatment after the initial protective effects on mitochondrial integrity. The importance of mitochondrial JNK activation was further underlined by the fact that treatment with SabKIM1, a peptide that prevents JNK binding to the mitochondrial scaffold Sab, also protected the brain after HI. Furthermore JNK can induce apoptosis via phosphorylation of p53, which results in an increased stability and half-life of p53. We describe for the first time that treatment with TAT-P7-pi, a peptide that prevents binding of JNK to p53, resulted in a profound reduction in infarct volume and improvement in sensorimotor and cognitive function after neonatal HI. We emphasize the importance of early protection of the mitochondria to convey efficient long-term neuroprotection after neonatal HI.

In the second part of this thesis we focused on the role of inflammation in the development of neonatal HI brain damage. We demonstrate that neonatal HI induces an early cerebral inflammatory response with polarization of microglia/macrophages towards a pro-inflammatory M1 and later to anti-inflammatory M2 phenotype. Cerebral HI reduces the peripheral hepatic inflammatory response, except from a potent upregulation of hepatic CINC-1 expression. Furthermore, we show that a systemic inflammation induced by LPS aggravates the severity of HI-induced brain damage. LPS-sensitized HI results in a gradual increase of neuronal damage, with strong effects on white matter integrity and an increased and prolonged cerebral inflammatory response. Systemic inflammation not only exacerbates HI-induced brain damage, but fundamentally changes some molecular pathways involved in the development of HI brain damage. When HI was preceded by LPS, cell death was mainly regulated via the extrinsic instead of the intrinsic apoptotic pathway. We further illustrate the clinical relevance of these changes by demonstrating that two important treatment strategies (D-JNKi and etanercept) in the neonatal HI model lose their neuroprotective effect when inflammation precedes HI. We emphasize that clinicians and scientists have to be aware of the fact that treatment strategies might not be effective or require modifications in therapeutic window, dose or combination of different treatments to achieve efficacious neuroprotection when neonates face a HI insult in combination with inflammation.