Hippocampal damage contributes to cognitive dysfunction after traumatic brain injury (TBI). We previously showed that Fluoro-Jade, a fluorescent stain that labels injured, degenerating brain neurons, quantifies the extent of hippocampal injury after experimental fluid percussion TBI in rats. Coincidentally, we observed that injured neurons in the rat hippocampus also stained with Newport Green, a fluorescent dye specific for free ionic zinc. Here, we show that, regardless of injury severity or therapeutic intervention, the post-TBI population of injured neurons in rat hippocampal subfields CA1, CA3 and dentate gyrus is indistinguishable, both in numbers and anatomical distribution, from the population of neurons containing high levels of zinc. Treatment with lamotrigine, which inhibits presynaptic release of glutamate and presumably zinc that is co-localized with glutamate, reduced numbers of Fluoro-Jade-positive and Newport Green-positive neurons equally as did treatment with nicardipine, which blocks voltage-gated calcium channels through which zinc enters neurons. To confirm using molecular techniques that Fluoro-Jade and Newport Green-positive neurons are equivalent populations, we isolated total RNA from 25 Fluoro-Jade-positive and 25 Newport Green-positive pyramidal neurons obtained by laser capture microdissection (LCM) from the CA3 subfield, linearly amplified the mRNA and used quantitative ribonuclease protection analysis to demonstrate similar expression of mRNA for selected TBI-induced genes. Our data suggest that therapeutic interventions aimed at reducing neurotoxic zinc levels after TBI may reduce hippocampal neuronal injury.

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The authors show, in the first direct comparison of messenger RNA levels in injured and uninjured hippocampal neurons, that injured neurons express lower levels of neuroprotective genes than adjacent uninjured neurons.

Increases of synaptically released zinc and intracellular accumulation of zinc in hippocampal neurons after traumatic or ischemic brain injury is neurotoxic and chelation of zinc has been shown to reduce neurodegeneration. Although our previous studies showed that zinc chelation in traumatically braininjured rats correlated with an increase in whole-brain expression of several neuroprotective genes and reduced numbers of apoptotic neurons, the effect on functional outcome has not been determined, and the question of whether this treatment may actually be clinically relevant has not been answered. In the present study, we show that treatment of TBI rats with the zinc chelator calcium EDTA reduces the numbers of injured, Fluoro-Jade-positive neurons in the rat hippocampus 24 hours after injury but does not improve neurobehavioral outcome (spatial memory deficits) two weeks post-injury. Our data suggest that zinc chelation, despite providing short-term histological neuroprotection, fails to improve long-term functional outcome, perhaps because long-term disruptions in homeostatic levels of zinc adversely influence hippocampus-dependent spatial memory.

Traumatic brain injury (TBI) is a leading cause of death in the elderly and the incidence of mortality and morbidity increases with age. This study tested the hypothesis that, after TBI followed by hemorrhagic hypotension (HH) and resuscitation, cerebral blood flow (CBF) would decrease more in aged compared with young rats. Young adult (4–6 months) and aged (20–24 months) male Sprague-Dawley rats were anesthetized with isoflurane, prepared for parasagittal fluid percussion injury (FPI) and randomly assigned to receive either moderate FPI (2.0 atm) only, moderate FPI + severe HH (40 mm Hg for 45 minutes) followed by return of shed blood, or sham FPI. Intracranial pressure (ICP), CBF, and mean arterial pressure (MAP) were measured and, after twenty-four hours survival, the rats were euthanized and their brains were sectioned and stained with Fluoro-Jade (FJ), a dye that stains injured neurons. After moderate FPI, severe HH and reinfusion of shed blood, MAP and CBF were significantly reduced in the aged group, compared to the young group. Both FPI and FPI + HH groups significantly increased the numbers of FJ-positive neurons in hippocampal cell layers CA1, CA2 and CA3 (p < 0.05 vs Sham) in young and aged rats. Despite differences in post-resuscitation MAP and CBF, there were no differences in the numbers of FJ-positive neurons in aged compared to young rats after FPI, HH and blood resuscitation. Although cerebral hypoperfusion in the aged rats was not associated with increased hippocampal cell injury, the trauma-induced reductions in CBF and post resuscitation blood pressure may have resulted in damage to brain regions that were not examined or neurological or behavioral impairments that were not assessed in this study. Therefore, the maintenance of normal blood pressure and cerebral perfusion would be advisable in the treatment of elderly patients after TBI.