Prevention of neonatal oxygen-induced brain damage by reduction of intrinsic apoptosis.

Within the last decade, it became clear that oxygen contributes to the pathogenesis of neonatal brain damage, leading to neurocognitive impairment of prematurely born infants in later life. Recently, we have identified a critical role for receptor-mediated neuronal apoptosis in the immature rodent brain. However, the contribution of the intrinsic apoptotic pathway accompanied by activation of caspase-2 under hyperoxic conditions in the neonatal brain still remains elusive. Inhibition of caspases appears a promising strategy for neuroprotection. In order to assess the influence of specific caspases on the developing brain, we applied a recently developed pentapeptide-based group II caspase inhibitor (5-(2,6-difluoro-phenoxy)-3(R,S)-(2(S)-(2(S)-(3-methoxycarbonyl-2(S)-(3-methyl-2(S)-((quinoline-2-carbonyl)-amino)-butyrylamino)propionylamino)3-methylbutyrylamino)propionylamino)-4-oxo-pentanoic acid methyl ester; TRP601). Here, we report that elevated oxygen (hyperoxia) triggers a marked increase in active caspase-2 expression, resulting in an initiation of the intrinsic apoptotic pathway with upregulation of key proteins, namely, cytochrome c, apoptosis protease-activating factor-1, and the caspase-independent protein apoptosis-inducing factor, whereas BH3-interacting domain death agonist and the anti-apoptotic protein B-cell lymphoma-2 are downregulated. These results coincide with an upregulation of caspase-3 activity and marked neurodegeneration. However, single treatment with TRP601 at the beginning of hyperoxia reversed the detrimental effects in this model. Hyperoxia-mediated neurodegeneration is supported by intrinsic apoptosis, suggesting that the development of highly selective caspase inhibitors will represent a potential useful therapeutic strategy in prematurely born infants.

Neurotoxic effects of MDMA (ecstasy) on the developing rodent brain.

The incidence of methamphetamine abuse is particularly high in adolescents and is a common problem among women of childbearing age, leading to an increasing number of children with prenatal exposure. MDMA (3,4-methylenedioxymethamphetamine, ecstasy) is an amphetamine-like stimulant and is known to induce apoptotic damage to fine serotonergic fibers in the adult rat brain. Little is known about toxic effects of MDMA and potential underlying molecular mechanisms in the developing brain. Here, we investigated whether MDMA exposure during the period of rapid brain growth causes neurodegeneration in the developing rat brain. MDMA significantly enhanced neuronal death in the brains of 6-day-old rat pups at a dose of 60 mg/kg, but no significant toxicity was detected at the ages of 14 and 21 days. Brain regions mainly affected were the cortex, septum, thalamus, hypothalamus and the cornu ammonis 1 region. To explore possible molecular mechanisms involved in this neurodegenerative process, we investigated the impact of MDMA on the expression of the neurotrophins brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and nerve growth factor. Neonatal exposure of 6-day-old rats to MDMA triggered a considerable increase in cortical BDNF and NT-3 levels. Moreover, P7 CD1/BDNF knockout mice were noticeably more sensitive to MDMA exposure as compared to their wild-type age-matched littermates. These data suggest that a single injection of MDMA causes neurodegeneration in the neonatal rat brain. The upregulation of BDNF and NT-3 expression may indicate an important compensatory mechanism leading to the survival of neuronal cells in the developing brain.

Acute and long-term proteome changes induced by oxidative stress in the developing brain.

The developing mammalian brain experiences a period of rapid growth during which various otherwise innocuous environmental factors cause widespread apoptotic neuronal death. To gain insight into developmental events influenced by a premature exposure to high oxygen levels and identify proteins engaged in neurodegenerative and reparative processes, we analyzed mouse brain proteome changes at P7, P14 and P35 caused by an exposure to hyperoxia at P6. Changes detected in the brain proteome suggested that hyperoxia leads to oxidative stress and apoptotic neuronal death. These changes were consistent with results of histological and biochemical evaluation of the brains, which revealed widespread apoptotic neuronal death and increased levels of protein carbonyls. Furthermore, we detected changes in proteins involved in synaptic function, cell proliferation and formation of neuronal connections, suggesting interference of oxidative stress with these developmental events. These effects are age-dependent, as they did not occur in mice subjected to hyperoxia in adolescence.

Apoptotic neurodegeneration following trauma is markedly enhanced in the immature brain.

Age dependency of apoptotic neurodegeneration was studied in the developing rat brain after percussion head trauma. In 7-day-old rats, mechanical trauma, applied by means of a weight drop device, was shown to trigger widespread cell death in the hemisphere ipsilateral to the trauma site, which first appeared at 6 hours, peaked at 24 hours, and subsided by 5 days after trauma. Ultrastructurally, degenerating neurons displayed features consistent with apoptosis. A decrease of bcl-2 in conjunction with an increase of c-jun mRNA levels, which were evident at 1 hour after trauma and were accompanied by elevation of CPP 32-like proteolytic activity and oligonucleosomes in vulnerable brain regions, confirmed the apoptotic nature of this process. Severity of trauma-triggered apoptosis in the brains of 3- to 30-day-old rats was age dependent, was highest in 3- and 7-day-old animals, and demonstrated a subsequent rapid decline. Adjusting the mechanical force in accordance with age-specific brain weights revealed a similar vulnerability profile. Thus, apoptotic neurodegeneration contributes in an age-dependent fashion to neuropathological outcome after head trauma, with the immature brain being exceedingly vulnerable. These results help explain unfavorable outcomes of very young pediatric head trauma patients and imply that, in this group, an antiapoptotic regimen may constitute a successful neuroprotective approach.

Modeling pediatric head trauma: mechanisms of degeneration and potential strategies for neuroprotection.

We have developed a model for head trauma in infant rats in an attempt to study mechanisms of neurodegeneration in the developing brain and were able to morphologically characterize two distinct types of brain damage. The first type or primary damage evolved within 4 hrs after trauma and occurred by an excitotoxic mechanism. The second type or secondary damage evolved within 6-24 hrs and occurred by an apoptotic mechanism. Primary damage remained localized to the parietal cortex at the site of impact. Secondary damage affected distant sites such as the cingulate/retrosplenial cortex, subiculum, frontal cortex, thalamus, hippocampal dentate gyrus and striatum. Histological evidence of delayed cell death was preceded by decrease of bcl-2- in conjunction with increase of c-jun-mRNA-levels, already evident at 1 hr after trauma. Increase of CPP32-like activity and elevated concentrations of oligonucleosomes in affected brain regions represented additional findings to indicate that this secondary disseminated degenerative reaction is apoptotic in nature. At the age of 7 days, secondary apoptotic damage was more severe than primary excitotoxic damage, but its severity declined with increasing age. In 7-days-old rats, NMDA antagonists protected against primary excitotoxic damage but increased severity of secondary apoptotic damage whereas the free radical scavenger SPBN, the tumor necrosis factor (TNF) inhibitor pentoxifylline and the antioxidant N-acetylcystein mitigated apoptotic damage. These findings demonstrate that in the developing rat brain apoptosis and not excitotoxicity determines neuropathologic outcome following head trauma. Whereas radical scavengers and TNF-inhibitors may prove useful in treatment of pediatric head trauma, great caution should be applied in regards to the use of NMDA antagonists because of the inherent risk of apoptosis promotion.