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.

Introduction

Traumatic brain injury (TBI) injures approximately 1.5 million patients per year in the United States, causes 50,000 deaths (Bruns and Hauser, 2003, Narayan et al., 2002, Thompson et al., 2001) and generates an enormous economic burden in long-term disability (Schiff et al., 2002, Ylvisaker et al., 2003). The long-term disabilities suffered by survivors very often include cognitive deficits, which are attributable in part to damage to the hippocampus, a central locus of learning and memory (Arciniegas et al., 1999, Bigler et al., 1997).

Hippocampal vulnerability to TBI is striking histologically; within anatomically homogeneous populations of hippocampal neurons, injured neurons are scattered among apparently uninjured neurons. In rats subjected to TBI, we observed that injured neurons, identified by Fluoro-Jade (FJ) staining, expressed significantly lower levels of neuroprotective genes than adjacent uninjured neurons (Hellmich et al., 2005b).

The hippocampus contains substantial amounts of sequestered ionic zinc (Frederickson and Danscher, 1988). Intraneuronal cytosolic levels of ionic zinc, while normally low, increase dramatically after excitotoxic injury, presumably in part because zinc is co-released with glutamate from presynaptic glutamatergic nerve terminals (Canzoniero et al., 1999) and also because zinc is released from intraneuronal depots as a consequence of intracellular nitric oxide signaling (Bossy-Wetzel et al., 2004). Zinc neurotoxicity contributes to neurodegeneration and cell death after ischemic brain injury and TBI (Choi and Koh, 1998, Koh, 2001, Lee et al., 2000, Suh et al., 2000, Suh et al., 2006). In rat hippocampal neurons, chelation of zinc with Ca–EDTA significantly reduced both neuronal zinc accumulation and neuronal death (Calderone et al., 2004, Suh et al., 2004). We showed that intracerebroventricularly injected Ca–EDTA significantly increased mRNA levels of neuroprotective genes in the rat brain after TBI (Hellmich et al., 2004).

Cell-impermeant Newport Green (NG), a di-2-picolylamine derivative bound to dichlorofluorescein, is a selective zinc indicator (Sensi et al., 1999, Thompson et al., 2002b) that is considered to be specific for zinc but less sensitive than other zinc indicators (Thompson et al., 2002a, Thompson et al., 2002b). We chose NG to detect neurotoxic zinc accumulation in hippocampal neurons because staining with an ultra-sensitive or cell-permeant indicator could obscure injury-induced neuronal accumulation due to high levels of zinc in presynaptic mossy fibers.

Fluoro-Jade, an anionic fluorochrome considered to be a specific indicator of lethal neuronal injury (Anderson et al., 2005, Duckworth et al., 2005, Schmued et al., 1997), has been used to quantify acute neuronal degeneration after TBI (Anderson et al., 2005). In previous studies, we determined that the number of FJ-positive neurons correlated with the severity of TBI (Hellmich et al., 2005a). We also obtained preliminary evidence that adjacent sections of rat hippocampus showed nearly identical distribution and numbers of FJ-positive and NG-positive neurons, as Suh et al. had previously suggested with TSQ staining and eosinophilic counterstaining (Suh et al., 2000). We therefore hypothesized that acute hippocampal neuronal degeneration after TBI was closely associated with intracellular zinc accumulation. Here, we provide evidence supporting our hypothesis that zinc neurotoxicity is a fundamental mechanism of hippocampal injury after TBI.