Age is a determinant of leukocyte infiltration and loss of cortical volume after traumatic brain injury.

There is increasing evidence that the inflammatory response differs in the injured developing brain as compared to the adult brain. Here we compared cerebral blood flow and profiled the inflammatory response in mice that had been subjected to traumatic brain injury (TBI) at postnatal day (P)21 or at adulthood. Relative blood flow, determined by laser Doppler, revealed a 30% decrease in flow immediately after injury followed by prominent hyperemia between 7 and 35 days after injury in both age groups. The animals were euthanized at 1-35 days after injury and the brains prepared for the immunolocalization and quantification of CD45-, GR-1-, CD4- and CD8-positive (+) cells. On average, the number of CD45+ leukocytes in the cortex was significantly higher in the P21 as compared to the adult group. A similar trend was seen for GR-1+ granulocytes, whereas no age-related differences were noted for CD4+ and CD8+ cells. While CD45+ and GR-1+ cells in the P21 group remained elevated, relative to shams, over the first 2 weeks after injury, the adult group showed a time course limited to the first 3 days after injury. The loss of ipsilateral cortical volumes at 2 weeks after injury was significantly greater in the adult relative to the P21 group. While the adult group showed no further change in cortical volumes, there was a significant loss of cortical volumes between 2 and 5 weeks after injury in the P21 group, reaching values similar to that of the adult group by 5 weeks after injury. Together, these findings demonstrate age-dependent temporal patterns of leukocyte infiltration and loss of cortical volume after TBI.

Glutathione peroxidase activity modulates recovery in the injured immature brain.

OBJECTIVE: Mice subjected to traumatic brain injury at postnatal day 21 show emerging cognitive deficits that coincide with hippocampal neuronal loss. Here we consider glutathione peroxidase (GPx) activity as a determinant of recovery in the injured immature brain. METHODS: Wild-type and transgenic (GPxTg) mice overexpressing GPx were subjected to traumatic brain injury or sham surgery at postnatal day 21. Animals were killed acutely (3 or 24 hours after injury) to assess oxidative stress and cell injury in the hippocampus or 4 months after injury after behavioral assessments. RESULTS: In the acutely injured brains, a reduction in oxidative stress markers including nitrotyrosine was seen in the injured GPxTg group relative to wild-type control mice. In contrast, cell injury, with marked vulnerability in the dentate gyrus, was apparent despite no differences between genotypes. Magnetic resonance imaging demonstrated an emerging cortical lesion during brain maturation that was also indistinguishable between injured genotypes. Stereological analyses of cortical volumes likewise confirmed no genotypic differences between injured groups. However, behavioral tests beginning 3 months after injury demonstrated improved spatial memory learning in the GPxTg group. Moreover, stereological analysis within hippocampal subregions demonstrated a significantly greater number of neurons within the dentate of the GPx group. INTERPRETATION: Our results implicate GPx in recovery of spatial memory after traumatic brain injury. This recovery may be attributed, in part, to a reduction in early oxidative stress and selective, long-term sparing of neurons in the dentate.

Glutathione peroxidase overexpression does not rescue impaired neurogenesis in the injured immature brain.

Traumatic brain injury (TBI) is a leading cause of disability among young children and is associated with long-term cognitive deficits. These clinical findings have prompted an investigation of the hippocampus in an experimental model of trauma to the developing brain at postnatal day (p21). Previous studies using this model have revealed a progressive loss of neurons in the hippocampus as brain-injured animals mature to young adulthood. Here we determined whether this hippocampal vulnerability is likewise reflected in altered neurogenesis and whether the antioxidant glutathione peroxidase (GPx) modulates neurogenesis during maturation of the injured immature brain. Male transgenic mice that overexpress GPx and wild-type littermates were subjected to controlled cortical impact or sham surgery on p21. At 2 weeks postinjury, the numbers of proliferating cells and immature neurons within the subgranular zone were measured by using Ki-67 and doublecortin, respectively. Bromodeoxyuridine (BrdU) was used to label dividing cells beginning 2 weeks postinjury. Survival (BrdU(+)) and neuronal differentiation (BrdU(+)/NeuN(+)) were then measured 4 weeks later via confocal microscopy. Two-way ANOVA revealed no significant interaction between genotype and injury. Subsequent analysis of the individual effects of injury and genotype, however, showed a significant reduction in subgranular zone proliferation (Ki-67) at 2 weeks postinjury (P = 0.0003) and precursor cell survival (BrdU(+)) at 6 weeks postinjury (P = 0.016) and a trend toward reduced neuronal differentiation (BrdU(+)/NeuN(+)) at 6 weeks postinjury (P = 0.087). Overall, these data demonstrate that traumatic injury to the injured immature brain impairs neurogenesis during maturation and suggest that GPx cannot rescue this reduced neurogenesis.

Traumatic injury to the immature brain results in progressive neuronal loss, hyperactivity and delayed cognitive impairments.

The immature brain may be particularly vulnerable to injury during critical periods of development. To address the biologic basis for this vulnerability, mice were subjected to traumatic brain injury at postnatal day 21, a time point that approximates that of the toddler-aged child. After motor and cognitive testing at either 2 weeks (juveniles) or 3 months (adults) after injury, animals were euthanized and the brains prepared for quantitative histologic assessment. Brain-injured mice exhibited hyperactivity and age-dependent anxiolysis. Cortical lesion volume and subcortical neuronal loss were greater in brain-injured adults than in juveniles. Importantly, cognitive decline was delayed in onset and coincided with loss of neurons in the hippocampus. Our findings demonstrate that trauma to the developing brain results in a prolonged period of pathogenesis in both cortical and subcortical structures. Behavioral changes are a likely consequence of regional-specific neuronal degeneration.

Traumatic injury to the immature brain: inflammation, oxidative injury, and iron-mediated damage as potential therapeutic targets.

Traumatic brain injury (TBI) is the leading cause of morbidity and mortality among children and both clinical and experimental data reveal that the immature brain is unique in its response and vulnerability to TBI compared to the adult brain. Current therapies for pediatric TBI focus on physiologic derangements and are based primarily on adult data. However, it is now evident that secondary biochemical perturbations play an important role in the pathobiology of pediatric TBI and may provide specific therapeutic targets for the treatment of the head-injured child. In this review, we discuss three specific components of the secondary pathogenesis of pediatric TBI-- inflammation, oxidative injury, and iron-induced damage-- and potential therapeutic strategies associated with each. The inflammatory response in the immature brain is more robust than in the adult and characterized by greater disruption of the blood-brain barrier and elaboration of cytokines. The immature brain also has a muted response to oxidative stress compared to the adult due to inadequate expression of certain antioxidant molecules. In addition, the developing brain is less able to detoxify free iron after TBI-induced hemorrhage and cell death. These processes thus provide potential therapeutic targets that may be tailored to pediatric TBI, including anti-inflammatory agents such as minocycline, antioxidants such as glutathione peroxidase, and the iron chelator deferoxamine.

Overexpression of glutathione peroxidase protects immature murine neurons from oxidative stress.

Neuronal enzyme systems involved in free radical detoxification are developmentally regulated such that intracellular glutathione peroxidase (GPx-1) activity is low in the newborn mouse brain. We hypothesized that neurons expressing a higher level of GPx-1 will be more resistant to hydrogen peroxide (H(2)O(2)) exposure. We show a dose-dependent protection against H(2)O(2) in primary neuronal cultures from fetuses overexpressing human GPx-1 compared to wild types of the same genetic background. Exogenous antioxidants completely protected neurons, even at extremely high H(2)O(2 )concentrations and regardless of the genotype. Specific depletion of glutathione with buthionine sulfoximine increased cell death in transgenic cultures exposed to 200 microM H(2)O(2), reducing protection afforded by increased GPx-1 activity. Increased GPx-1 expression in immature cortical neurons confers protection from oxidative stress, but availability of reducing equivalents determines susceptibility to oxidative cell death.

Heme regulation in traumatic brain injury: relevance to the adult and developing brain.

Intracranial bleeding is one of the most prominent aspects in the clinical diagnosis and prognosis of traumatic brain injury (TBI). Substantial amounts of blood products, such as heme, are released because of traumatic subarachnoid hemorrhages, intraparenchymal contusions, and hematomas. Despite this, surprisingly few studies have directly addressed the role of blood products, in particular heme, in the setting of TBI. Heme is degraded by heme oxygenase (HO) into three highly bioactive products: iron, bilirubin, and carbon monoxide. The HO isozymes, in particular HO-1 and HO-2, exhibit significantly different expression patterns and appear to have specific roles after injury. Developmentally, differences between the adult and immature brain have implications for endogenous protection from oxidative stress. The aim of this paper is to review recent advances in the understanding of heme regulation and metabolism after brain injury and its specific relevance to the developing brain. These findings suggest novel clinical therapeutic options for further translational study.