Injured Fluoro-Jade-positive hippocampal neurons contain high levels of zinc after traumatic brain injury

Abstract

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.

Figure 1

Schematic overview of the experimental design for the dose response and gene expression study. We performed neuronal counts of fluorescent cells on 10 representative sections of all experimental rat brains (subjected to sham, mild or moderate injury) that were serially sectioned through the hippocampus and stained for Fluoro-Jade (FJ) or Newport Green (NG). Quantitative ribonuclease protection assay (RPA) analysis was performed using linearly amplified mRNA (from 25 FJ positive neurons in one section and 25 NG positive neurons from the adjacent section in each rat) from a subset of traumatic brain injured rats (n=6).

Figure 2

Representative sets of adjacent cells stained for Fluoro-Jade (A and C) and Newport Green (B and D) in the CA1/2 and CA3 subfields of the rat hippocampus. We counterstained slides for 15 seconds with 1% cresyl violet. Different background levels that varied from experiment to experiment were not adjusted. In adjacent serial sections of injured rat hippocampus, circles indicate identified neurons that appear to be stained by both Fluoro-Jade (E) and Newport Green (F).

Figure 3

Numbers of Fluoro-Jade-positive (injured) and Newport Green-positive (zinc) neurons increased with increasing levels of injury in the CA1, CA3 and dentate gyrus subfields of the rat hippocampus at both 4 and 24 hours post-injury (n=6 per group). The numbers of injured and zinc-positive neurons were not statistically different from each other.

Figure 4

Pretreatment with lamotrigine (lam) or nicardipine (nic) 30 minutes before traumatic brain injury (TBI) significantly reduced numbers of Fluoro-Jade positive and Newport Green positive neurons in both the CA1 (for both lam and nic) and CA3 (lam only) when compared to saline injected TBI rats. * p<0.05

Figure 5

Quantitative ribonuclease protection assay analysis of Bcl-2, Caspase 3 (casp 3), Caspase 9 (casp 9), and heat shock protein 70 (Hsp 70) mRNA (linearly amplified with T7 polymerase) expression in identified Fluoro-Jade positive and Newport Green positive neurons in immediately adjacent sections. We found no statistical difference in gene expression between the two groups of stained neurons. DLU, digital light units.