Effects of PMA (PHORBOL-12-MYRISTATE-13-ACETATE) on the Developing Rodent Brain.

Perinatal infections have a negative impact on brain development. However, the underlying mechanisms leading to neurological impairment are not completely understood and reliable models of inflammation are urgently needed. Using phorbol-myristate-acetate as an activator of inflammation, we investigated the effect on the developing rodent brain. Neonatal rats and mice deficient in IL-18 or IRAK-4 were exposed to PMA. Brains were assessed for regulation of pro- and anti-inflammatory cytokines and cell death 24 hrs, 7 and 14 days after treatment. PMA induced an inflammatory response and caused widespread neurodegeneration in the brains of 3- and 7-day-old rats. In contrast, 14-day-old rats were resistant to the neurotoxic effect of PMA. Histological evaluation at the age of 14 and 21 days revealed a destruction of the cortical microstructure with decreased numerical density of neuronal cells. Mice deficient in IL-18 or IRAK-4 were protected against PMA induced brain injury. PMA treatment during a vulnerable period can alter brain development. IL-18 and IRAK-4 appear to be important for the development of PMA induced injury.

A critical role for Fas/CD-95 dependent signaling pathways in the pathogenesis of hyperoxia-induced brain injury.

OBJECTIVE: Prematurely born infants are at risk for development of neurocognitive impairment in later life. Oxygen treatment has been recently identified as a trigger of neuronal and oligodendrocyte apoptosis in the developing rodent brain. We investigated the role of the Fas death receptor pathway in oxygen-triggered developmental brain injury. METHODS: Six-day-old Wistar rats were exposed to 80% oxygen for various periods (2, 6, 12, 24, 48, and 72 hours), and mice deficient in either Fas (B6.MRL-Tnfrsf6(lpr)) or Fas ligand (B6Smn.C3-Fasl(gld)) and control mice (C57BL/6J) were exposed to 80% oxygen for 24 hours. Polymerase chain reaction, Western blotting, and caspase activity assays of thalamus and cortex tissue were performed. RESULTS: Fas and Fas ligand messenger RNA and protein were upregulated. Furthermore, hyperoxia resulted in induction of downstream signaling events of Fas, such as Fas-associated death domain (FADD), the long and short form of FADD-like interleukin-1beta-converting enzyme (FLICE) inhibitory protein (FLIP-L, FLIP-S), and cleavage of caspase-8 and caspase-3. Injection of a selective caspase-8 inhibitor (TRP801, 1mg/kg) at the beginning of hyperoxia blocked subsequent caspase-3 cleavage in this model. B6.MRL-Tnfrsf6(lpr) mice were protected against oxygen-mediated injury, confirming Fas involvement in hyperoxia-induced cell death. Mice deficient in Fas ligand did not differ from control animals in the amount of cell death. INTERPRETATION: We conclude that neonatal hyperoxia triggers Fas receptor and its downstream signaling events in a Fas ligand-independent fashion. Lack of functional Fas receptors and selective pharmacological inhibition of caspase-8 prevents activation of caspase-3 and provides significant neuroprotection.

Maturation-dependent oligodendrocyte apoptosis caused by hyperoxia.

In the immature human brain, periventricular leukomalacia (PVL) is the predominant white matter injury underlying the development of cerebral palsy. PVL has its peak incidence during a well-defined period in human brain development (23-32 weeks postconceptional age) characterized by extensive oligodendrocyte migration and maturation. We hypothesized that the dramatic rise of oxygen tissue tension associated with mammalian birth and additional oxygen exposure of the preterm infant during intensive care may be harmful to immature oligodendrocytes (OLs). We therefore investigated the effects of hyperoxia on rat oligodendroglia cells in vitro and in vivo. Immature OLs (OLN-93), their progenitors [preoligodendrocytes (pre-OL)], and mature OLs were subjected to 80% hyperoxia (24-96 hr). Flow cytometry was used to assess cell death. Cell viability was measured by metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT). In addition, 6-day-old rat pups were subjected to 80% oxygen (24 hr) and then sacrificed, and their brains were processed for immunfluorescence staining. Apoptosis was detected at various stages (annexin-V, activated caspase-3) after 24-48 hr of incubation in 80% oxygen in pre- and immature OLs. Mature OLs were resistant to oxygen exposure. These results were confirmed by MTT assay. This cell death was blocked by administration of the pan-caspase inhibitor zVAD-fmk. Degeneration of OLs was confirmed in 7-day-old rat brains by positive staining for activated caspase-3. Hyperoxia triggers maturation-dependent apoptosis in immature and pre-OLs and involves caspase activation. This mechanism may be relevant to the white matter injury observed in infants born preterm.

Caspase-1-processed interleukins in hyperoxia-induced cell death in the developing brain.

Infants born prematurely may develop neurocognitive deficits without an obvious cause. Oxygen, which is widely used in neonatal medicine, constitutes one possible contributing neurotoxic factor, because it can trigger neuronal apoptosis in the developing brain of rodents. We hypothesized that two caspase-1-processed cytokines, interleukin (IL)-1beta and IL-18, are involved in oxygen-induced neuronal cell death. Six-day-old Wistar rats or C57/BL6 mice were exposed to 80% oxygen for various time periods (2, 6, 12, 24, and 48 hours). Neuronal cell death in the brain, as assessed by Fluoro-Jade B and silver staining, peaked at 12 to 24 hours and was preceded by a marked increase in mRNA and protein levels of caspase 1, IL-1beta, IL-18, and IL-18 receptor alpha (IL-18Ralpha). Intraperitoneal injection of recombinant human IL-18-binding protein, a specific inhibitor of IL-18, attenuated hyperoxic brain injury. Mice deficient in IL-1 receptor-associated kinase 4 (IRAK-4), which is pivotal for both IL-1beta and IL-18 signal transduction, were protected against oxygen-mediated neurotoxicity. These findings causally link IL-1beta and IL-18 to hyperoxia-induced cell death in the immature brain. These cytokines might serve as useful targets for therapeutic approaches aimed at preserving neuronal function in the immature brain, which is exquisitely sensitive to a variety of iatrogenic measures including oxygen.