Glutathione peroxidase activity modulates recovery in the injured immature brain anoxic event


Traumatic brain injury (TBI) is the leading cause of death and disability among children 1 – 3. Children less than 4 years of age have poorer motor and cognitive outcomes after TBI than do older children 4 – 6.

To investigate the vulnerability of the immature brain, we developed a murine model of TBI at pnd 21 7 , 8, an age that approximates toddler-aged (2–4 years-old) children in terms of gliogenesis, synaptogenesis, and myelination 9. This injury results in a cortical lesion and hippocampal neuronal loss, both of which continue to increase between 2 weeks and 3 months postinjury 8. Importantly, cognitive dysfunction is delayed in onset and coincides with this prolonged hippocampal neuronal loss 7.Anoxic event

Oxidative stress is a significant component of the injury cascade following TBI. The brain’s antioxidant mechanisms include superoxide dismutase (SOD), which converts free radicals to hydrogen peroxide, and catalase and glutathione peroxidase (gpx), which further metabolize hydrogen peroxide to water and oxygen 10. The young brain is particularly vulnerable to oxidative stress due to its high fatty acid content and proportionately large share of total body oxygen consumption 11. During development, antioxidants are inadequately expressed and do not respond to oxidative stress as robustly as they do in the adult brain, making the immature brain even more susceptible to oxidative stress-induced injury.Anoxic event in the CD1 mouse, SOD1 activity decreases from embryonic (E)18 to pnd 1, and then remains unchanged to pnd 21. GPx activity, on the other hand, rises steeply from E18 to pnd 1, declines sharply by pnd P4, and again from pnd 7 to pnd 14, where it is the same as at pnd 21. Protein levels for both enzymes, however, show a steady increase from el 8 to pnd 21 12. We have shown that in the C57B16 mouse brain, adult levels of gpx are reached bypnd 21 13. These age-dependent differences may impact the response of the immature brain to injury. Whereas SOD overexpression is protective against ischemic injury in the adult brain 14 , 15, SOD overexpression exacerbates hypoxic-ischemic injury in the neonatal brain 16.Anoxic event the latter is thoughtto be due to a failure of downstream antioxidant mechanisms in the immature brain to compensate for increased hydrogen peroxide production 17. While gpx is upregulated in the adult brain in response to TBI, no such upregulation occurs in the injured immature brain 13, suggesting that gpx may be a key factor in the immature brain’s vulnerability to TBI. Such a possibility is reinforced by in vitro and in vivo studies. Overexpression of gpx in immature neurons in vitro is protective against hydrogen peroxide exposure 18 and neonatal mice overexpressing gpx are less susceptible to hypoxic-ischemic injury than wildtype littermates 19.

We hypothesized that overexpression of gpx would be protective against the long-term sequelae of traumatic injury to the immature brain.Anoxic event we demonstrate protection against long-term hippocampal loss associated with an acute reduction in oxidative stress and alterations in long-term behavioral functions, including improved spatial memory.

Overexpression of gpx reduces early oxidative stress but does not alter early hippocampal injury

A 150kd protein carbonyl band was detected in the hippocampus of injured brains with a trend toward differences in genotypes ( figure 2A, table 2, supplemental data). The nitrotyrosine signal remained consistently elevated within the first 24 hours postinjury in WT animals whereas it decreased by 24 hours in the gpxtg group( fig. 2A, B, table 2, supplemental data).

To determine if early reduction of oxidative stress is neuroprotective in the hippocampus, caspase-3 and MAP2 levels were evaluated at 24 hours postinjury.Anoxic event pro-caspase-3 was detected at 32kd while cleaved-caspase-3 was detected at 17 and 19kd. Although pro-caspase-3 tended to decrease after injury in both genotypes, these values were not significantly different from controls ( table 2, supplemental data). Similarly, cleaved-caspase-3, the activated form of caspase-3, and three forms of MAP2 were indistinguishable from sham controls ( table 2, supplemental data).

We next evaluated cell injury, based upon fluoro-jade C and TUNEL staining ( fig. 3 and fig. 4). A large number of cells and their processes in the granule cell layer of the dentate gyrus were fluoro-jade C-positive ( fig. 3 A–C). In contrast, there were qualitatively fewer labeled cells and their processes in CA2/CA3 ( fig. 3 D–F) and few to no cells in CA1 (data not shown).Anoxic event there were no differences between genotypes after injury (p= 0.42 for CA2/CA3 and p= 0.24 for the dentate, unpaired T-test).TUNEL-labeled cells were prominent inthe granule cell layer, particularly within the posterior hippocampus ( supplemental fig. 1 A–E). There was, however, no difference between genotypes.

GPx and evolution of the lesion

There were notable individual variations in apparent size of the injury by MRI (WT, 6.1±3.1% and gpxtg, 5.1±2.2% of ipsilateral brain pixels at 1 day), but no significant differences between genotypes. At 1 day the injured brain was manifest as a heterogenous hyperintense region on both T2W and DW images(supplemental fig. 2A). T2 hyperintense regions exhibited 45±9% greater signal intensity than contralateral brain, consistent with substantial vasogenic edema.Anoxic event ADC values of the lesion were 4.56±0.21×10 −4 and 4.63±0.27×10 −4 mm 2/second for WT and gpxtg mice, respectively, consistent with cytotoxic edema, while the contralateral brain ADC was 7.36±0.18×10 −4 mm 2/second. GdDTPA-BMA caused an increase in signal by 4±2% in the contralateral brain that did not increase thereafter. The injured brain, however, exhibited a steady increase in signal intensity over 30 minutes after contrast administration ( supplemental fig. 2B) with slopes of 1.65±0.67 and 1.0±0.36 hours −1 for WT and gpxtg animals, respectively, consistent with substantial barrier leakage of the contrast agent.

At 7 days postinjury, the lesion was more complex, with a hypointense blood clot adjacent to hyperintense injury on T2WI.Anoxic event the lesion area was hypointense on DWI, with ADC values enlarged to 19.2±6.4×10 −4 mm 2/second consistent with partial liquifaction. Contrast uptake was reduced to 0.92±0.63 hours −1. At 14 and 28 days postinjury, the lesion exhibited more homogeneous hyperintensity on T2WI and hypointensity on DWI compared to 7 days postinjury. ADC values increased to 23.4±2.2×10 −4 mm 2/second, consistent with more mature liquifaction, typically extending from the lateral ventricle to the cortical surface and surrounding CSF space. The liquefied region showed little contrast uptake (0.38±0.18 hours −1).

GPx is a determinant of behavioral outcome after injury

GPxTg animals spent more time in the center of the open field ( supplemental fig. 3A), suggesting that they are less anxious compared to WT littermates 27.Anoxic event total distance traveled in the open field did not differ among groups, suggesting no differences in exploratory activity ( supplemental fig. 3B). Within the gpxtg group, injured mice entered the center less ( supplemental fig. 3C) and traveled shorter distances while in the center ( supplemental fig. 3D), suggesting that TBI has a larger effect on these measures of anxiety in gpxtg group.

All groups exhibited improvement with training on the rotorod ( supplemental fig 4A). There was no effect of genotype or injury. However, the difference between performance in the first and final trials revealed that injured gpxtg mice showed less improvement ( supplemental fig. 4B) than sham-gpxtg mice, suggesting that gpx overexpression actually impairs sensorimotor learning after injury.Anoxic event

Spatial and non-spatial memory was assessed using the morris water maze test. There were no differences between groups in swim speeds during the visible session (data not shown). In the visible sessions, there was anoverall group difference with the gpxtg injured animals showing higher latencies to locate the platform than the WT and gpxtg shams. However, in the hidden sessions, there was also an overall group difference with the WT injured group showing higher latencies to locate the platform than the WT and gpxtg shams ( fig. 4A). Cumulative distances to locate the platform showed a similar pattern ( fig. 4B). Following hidden platform training, WT injured animals showed no spatial bias in the first probe trial (platform removed), while all 3 other groups showed spatial memory retention in the first probe trial and searched significantly longer in the quadrant that previously contained the platform (target quadrant) than in any other quadrant ( fig. 4C).Anoxic event collectively, these data show that TBI results in long-term non-spatial and spatial memory deficits. Overexpressionof gpx worsens the deficit in non-spatial memory. However, the deficit in spatial memory is rescued by gpx overexpression.

Role of gpx after brain injury

We focused on the hippocampus, a structure known to be vulnerable after traumatic injury to the immature brain 8.In many injury models, the neonatal hippocampus sustains more severe injury than the thalamusand cortex 29 – 31. The hippocampus has higher basal levels of fas expression than either the thalamus or cortex and, the hippocampus has minimal baseline expression of [fas-associated death-domain-like IL-1 beta-converting enzyme]-inhibitory protein (FLIP) relative to cortical and thalamic samples.Anoxic event after hypoxic ischemic injury, there is some up-regulation of fas and FLIP in all regions 30. The importance of reactive oxygen species in fas death receptor signaling has been verified in studies showing enhanced antioxidant status protects against fas death receptor mediated cell death 32.

Protein nitration remained elevated at 24 hours post injury in the WT group. This finding contrasts that of the traumatized, adult brain, where protein nitration returns to baseline by 24 hours 33. Although this difference may be related to the age of the animals at the time of injury, it may also be due to region-specific variations in protein nitration in the injured brain. Whereas deng et al. Measured protein nitration in the injured cortex, the site of maximal mechanical damage, our measurements were from the ipsilateral hippocampus.Anoxic event although each of these regions shows evidence of damage, the underlying mechanisms and time course for cell injury may be quite different and as such may translate to differences in the temporal course of the nitrotyrosine signal. A second consideration relates to the instability of modified proteins due to their accelerated degradation by the proteosome, as shown in vitro by souza et al. How this instability might translate to in vivo studies is not clear. Although protein nitration in adult brain is reduced at 24 hours after injury there is a significant rise thereafter at 48 hours 33. Thus, although modified proteins may be unstable after injury, there are likely other determinants in vivo that govern temporal pattern of their instability.Anoxic event

Overexpression of gpx resulted in a reduction in protein nitration at 24 hours postinjury. Despite this, there were no improvements in acute molecular and histologic measures of cell damage. While we have considered the possibility that western blots may not be robust enough to detect genotypic differences 34, subsequent anatomical studies, which offer greater sensitivity, likewise revealed no genotypic differences. One plausible explanation for these early negative findings may be related to the timing of reduction of oxidative stress. A reduction in oxidative stress in the gpxtg group was not apparent until 1 day postinjury. Thus, any benefit afforded by the reduction in oxidative stress may have been masked by a population of dying neurons that evolved within hours after injury.Anoxic event

In the injured adult brain, oxidative stress is not limited to the acutely injured brain but rather extends into the subacute period 33 , 35. It is conceivable that the immature brain likewise shows an extended period of oxidative stress with concomitant cell loss. Although we did not evaluate subacute cell loss, we do show long-term higher numbers of neurons in the dentate of the hippocampus of brain-injured gpxtg relative to WT animals.

MRI has been employed to longitudinally analyze brain injury in many studies 36 – 38, but few have analyzed damage beyond 2 weeks postinjury. This is the first study to use MRI to assess brain edema and to evaluate progression of the lesion over time in this model of TBI.Anoxic event

In general, the evolution of the cortical lesion, defined by MRI, was similar between genotypes. Stereologic analyses revealed a significant loss in cortical volume after injury but no genotypic differences, further validated the MRI findings. Collectively, these data suggest that gpx is not a determinant of the evolution of the cortical lesion. Loss of cortical volume after injury may reflect both aberrant developmental processes as well as ongoing secondary pathogenesis. That gpx failed to rescue this phenotype may reflect insensitivity of either or both processes to increased gpx activity.

GPx and behavior after TBI

Here we show that gpxtg animals show a complex response to injury that includes increased anxiety as well an improvement in spatial memory.Anoxic event anxiety disorders have been reported after pediatric TBI 39. Multiple studies have implicated the prefrontal cortex and several subcortical regions, including the amygdala and retrosplenial cortex in anxiety disorders 40 , 41. Antioxidant enzymes have been linked to anxiety regulation through genetic analysis 42. One such antioxidant enzyme, glutathione reductase, is responsible for replenishing the stores of glutathione for use by gpx. The associations of oxidative stress, antioxidant enzymes, and gpx in particular with anxiety, are clearly complicated and warrant further study.

We have previously reported that traumatic injury to the immature brain results in delayed deficits in hippocampal-dependent spatial memory 7.Anoxic event here we show that overexpression of gpx partially rescues this phenotype. The mechanism underlying this finding remains unclear. Hippocampal-dependent spatial memory is mediated in part through interactions between the dentate gyrus and CA3 43. Cognitive impairment may thus arise as a consequence of progressive loss of neurons in the hippocampus during maturation 7. There are several scenarios that might explain the higher numbers of neurons in the dentate of brain-injured gpx animals relative to wts. Overexpression of gpx may confer protection to this population, shown to be vulnerable, and as such improve cognitive outcome. It is also conceivable that overexpression of gpx influences ongoing neurogenesis which serves to replenish the neuronal population in the dentate.Anoxic event further studies are needed to explore each of these possibilities.