Ceruloplasmin is a key regulator in iron metabolism and its loss has been shown to cause age-dependent iron dysregulation in humans and animal models 
. We found that total iron levels in the cerebral cortex and striatum of young CpKO mice were significantly lower than WT mice. This iron deficit was concurrent with decreased levels of BDNF mRNA and protein. The extent of brain damage caused by MCAO reperfusion was significantly greater in the CpKO mice compared to WT mice, as a result of the expansion of the damage in the ischemic penumbra region of the cerebral cortex. These findings suggest that lack of Cp can cause a change in brain iron and BDNF levels, which normally play important roles in protecting neurons against ischemic brain injury.
Previous studies of other lines of CpKO mice have shown that iron levels increase in the brain stem, cerebellum and spinal cord as they age 
. However, levels of iron in striatum and cerebral cortex, from young CpKO mice have not been reported in the latter studies. The larger infarct volume we observed in the CpKO mice after MCAO, suggests that lack of Cp may affect the vulnerability of cortical and striatal neurons. While the present study is the first to examine the effects of ceruloplasmin deficiency on the vulnerability of the brain to ischemic stroke, Rathore et al. 
demonstrated that young CpKO mice show increased secondary damage coupled with oxidative damage and a decrease in functional recovery after spinal cord injury compared to WT animals. Previous evidence has also shown enhanced levels of oxidative stress markers in the brain of patients with aceruloplasminemia 
, as well as in the hippocampus of CpKO mouse compared to WT following 4 weeks of rotenone treatment 
. However, in our experimental model we found no evidence of increased lipid peroxidation or astrocyte reactivity the in cortex or striatum of young CpKO mice compared to WT mice measured 24 hours after the insult. We also did not observe increased proinflammatory cytokine levels 3 hours after the insult. These findings suggest that Cp deficiency and reduced iron levels do not alter levels of membrane-associated oxidative stress or generation of proinflammatory cytokines by glial cells. However, we cannot exclude the possibility that Cp and/or iron levels modify oxidative stress and inflammatory processes during the later post-stroke period.
The majority of the literature on cerebral ischemia and iron indicate that increased levels of iron contributing to the exacerbation of infarct volume or functional recovery. Elevated iron levels may contribute to ischemic brain injury by promoting the production of hydroxyl radicals and consequent lipid peroxidation 
. Human studies have also shown that increased iron stores are associated with a worse outcome after stroke 
. Studies involving iron chelators have demonstrated reduced infarct size with both pre and post 
. The mechanisms in which chelators promote protection though, may not be due just to their reduction of free iron but to other factors such as HIF activation 
or reduction of brain edema 
. Toxicity from iron during ischemia likely arises from liberation of iron from high-molecular weight storage proteins 
. We found decreased concentrations of iron in the cortex and striatum of CpKO mice in conjunction with increased infarct volumes. This counterintuitive result may be due to the particular importance of iron in the brain. While high levels of iron may be toxic, it is also true that abnormally low levels of iron will make cells more sensitive to insults by impairing normal brain functions which rely on adequate iron supply, such as ATP production, regulation of cellular energy, myelination and neurotransmitter production 
. Interestingly, others have shown that increases in infarct volume after MCAO in rats on high iron diets were not associated with alterations in brain iron levels 
. Consistently, aggressive iron chelator treatment in rats was not able to reduce infarct volume following ischemia 
. This supports our hypothesis that reduced brain iron may increase neuronal vulnerability.
Previous studies have demonstrated that BDNF can protect neurons against cerebral ischemic damage in animal models 
and against more specific insults relevant to ischemic stroke including glucose deprivation, excitotoxicity and oxidative stressors 
. BDNF is known to promote the plasticity and survival of neurons, playing key roles in adaptive responses of the brain to environmental challenges 
. Studies in rats have shown that cerebral ischemia can differently affect BDNF levels in the core, where a decrease occurs, and penumbra areas where an increase occurs 
, supporting a role for protection by BDNF. Indeed studies in both mice and rats have demonstrated that administration of exogenous BDNF can promote a reduction in infarct volume and functional recovery after cerebral ischemia 
. Similarly, studies using interventions known to up regulate basal BDNF levels, such as dietary restriction, enriched environment or exercise, have shown decreased infarct volumes following MCAO 
. Conversely decreasing BDNF levels or attenuating its effects following cerebral ischemia diminishes recovery of function 
. BDNF has been shown to exert anti-apoptotic and neuroplastic properties, as well as to enhance neuro- and angio-genesis. There is also evidence that BDNF levels can modulate cortical excitability with lower levels of BDNF enhancing excitability 
. More specifically, lack of BDNF transcript IV has been show to cause impairment in cortical inhibitory signaling 
. Our finding of reduced BDNF transcript levels, including transcript IV, in the CpKO mice suggests a mechanism whereby perturbed cellular iron metabolism in brain cells results in reduced BDNF levels in neurons which renders them vulnerable to ischemic injury. Indeed, we found that chelation of cellular iron in results in a reduction in BDNF mRNA levels in cultured neural cells. The lack of changes in BDNF promoter-driven luciferase activity following deferoxamine, suggests that the reduction in BDNF mRNA is likely the result of decreased mRNA stability. Notably, iron has been shown to play a role in the regulation of transcript stability in various experimental models by modifying the ability of iron-responsive proteins to bind to specific stabilizing/destabilizing sequences at the 3′ and/or 5′ of mRNA untranslated regions 
Because aceruloplasminemia's most striking symptoms manifest late in life, studies looking at iron brain levels in pre-symptomatic young patients, or in young rodent models of aceruloplasminemia may provide novel insight into the roles of cellular iron metabolism in developmental neuroplasticity and disease vulnerability. Our data clearly show that the lack of Cp leads to an early-on reduction in total brain iron and BDNF levels, resulting in increased vulnerability of neurons to ischemic injury .