Chelating Intracellularly Accumulated Zinc Decreased Ischemic Brain Injury Through Reducing

Zinc (Zn) is the second most abundant transition metal in the human body. It is an essential cofactor for many enzymes and transcription factors. In addition, it has roles in neuronal synaptic transmission because high concentrations of Zn are accumulated in many synaptic vesicles within the central nervous system in response to presynaptic activation. Zn influx from the extracellular space is a well-demonstrated inducer of injury in cultured neurons. Recent reports have indicated that Zn plays a major role in the mechanism of brain injury following ischemic stroke. Microdialysis studies have confirmed accumulation of extracellular Zn in focal and global ischemia models. Zn at high concentration has been consistently shown to be a critical mediator of neuronal death associated with experimental global ischemia. In contrast, the situation with focal ischemia is not as clear. Both neurotoxic and neuroprotective capabilities of zinc have been reported for experimentally induced focal ischemia. The mechanisms of zinc’s action are also not well understood. In focal ischemia, EDTA calcium has been used to inhibit the brain’s zinc in vivo. However, EDTA calcium is not permeable to the neuronal cell membrane. It reduces only extracellular but not intracellular zinc. Apart from vesicular zinc, ≈90% of the total brain Zn is bound to endogenous proteins. This binding is reversible because oxidative stress has been found to be a key regulator of intracellular zinc homeostasis by interfering with zinc binding to metallothioneins. Such intracellular release has been demonstrated in cultured neurons and ZnT3 knockout animals. Thus, EDTA calcium is not suitable for inhibiting both intracellular and extracellular zinc in the brain. In contrast, N,N,Nʹ,Nʹ-tetrakis(2-pyridylmethyl) Background and Purpose—Zinc has been reported to possess both neurotoxic and neuroprotective capabilities. The effects of elevated intracellular zinc accumulation following transient focal cerebral ischemia remain to be fully elucidated. Here, we investigated whether removing zinc with the membrane-permeable zinc chelator, N,N,Nʹ,Nʹ-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), would decrease the intracellular levels of zinc in the ischemic tissue, leading to reduced brain damage and improved neurological outcomes. Methods—Rats were pretreated with TPEN or vehicle before or after a 90-minute middle cerebral artery occlusion. Cerebral infarct volume, neurological functions, neuronal apoptosis, poly(ADP-ribose) polymerase activity, and cytosolic labile zinc were assessed after ischemia and reperfusion. Results—Cerebral ischemia caused a dramatic cytosolic labile zinc accumulation in the ischemic tissue, which was decreased markedly by TPEN (15 mg/kg) pretreatment. Chelating zinc lead to reduced infarct volume compared with vehicle-treated middle cerebral artery occlusion rats, accompanied by much improved neurological assessment and motor function, which were sustained for 14 days after reperfusion. We also determined that reducing zinc accumulation rescued neurons from ischemia-induced apoptotic death by reducing poly(ADP-ribose) polymerase-1 activation. Conclusions—Ischemia-induced high accumulation of intracellular zinc significantly contributed to ischemic brain damage through promotion of neuronal apoptotic death. Removing zinc may be an effective and novel approach to reduce ischemic brain injury. (Stroke. 2014;45:1139-1147.)