In this study, we found that collagenase-induced ICH in mice results in iron overload, oxidative damage, glial activation, and neutrophil infiltration, consequences that may contribute to secondary brain damage. Using this model, we have confirmed the potential neuroprotective properties of the iron chelator DFX in 12-month-old mice with collagenase-induced ICH. We showed that DFX decreased iron accumulation and neuronal death, attenuated production of ROS, and reduced microglial activation and neutrophil infiltration. Although DFX treatment improved neurologic function, it did not reduce brain injury volume, edema, or swelling. These findings, collectively, provide novel evidence that iron toxicity contributes to collagenase-induced hemorrhagic brain injury and that reducing iron accumulation might reduce neuronal death and improve functional outcome after ICH.
It has been suggested that ICH-associated secondary brain injury is mediated by the generation of ferrous iron during heme degradation. Iron is toxic to neurons because it catalyzes the Fenton reaction, which produces highly reactive hydroxyl radicals that lead to oxidative stress and cell death (Gaasch et al, 2007
). Moreover, iron could induce neuronal death even after it has bound to ferritin because it can be locally released in its ferrous form under the acidic conditions that follow stroke (Bishop and Robinson, 2001
). Iron-mediated neurotoxicity and the benefits of DFX have been reported in a whole-blood model in rats and piglets (Gu et al, 2009
; Okauchi et al, 2009
; Song et al, 2007
), but we are the first to show iron accumulation after collagenase-induced ICH in mice. Consistent with the results obtained from the whole-blood model in piglets (Gu et al, 2009
), we showed in this study that iron chelation with DFX decreases iron accumulation and neuronal death after collagenase-induced ICH in mice.
It is well known that ROS are produced during normal oxidative metabolism, but high ROS levels can damage neurons and cause death. Abnormal iron overload is believed to participate in the induction of toxic ROS. Substantial evidence indicates that ROS are critical to the oxidative brain damage that occurs after ICH (Wang and Doré, 2007b
). After ICH, the extracellular spaces of the brain are exposed to high concentrations of hemoglobin and its breakdown products. The fact that iron chelators and free radical scavengers can block hemoglobin-induced neurotoxicity (Wang et al, 2002
) suggests that iron and iron-mediated oxidative stress contribute to hemoglobin-induced neurotoxicity. Indeed, high levels of oxidative stress, as measured by protein carbonyl formation or increased ethidium (oxidized hydroethidine), have been shown after intrastriatal injection of blood or collagenase (Qu et al, 2007
; Wang and Tsirka, 2005a
). Neurons in the hemorrhagic brain show a decreased ability to respond to oxidative stress (Wang and Doré, 2007a
), particularly with regard to their low levels of glutathione and glutathione peroxidase. Consequently, excess iron released during heme degradation after ICH may predispose neurons to iron-induced oxidative stress. Deferoxamine binds ferric iron and prevents the formation of hydroxyl radicals through the Fenton reaction. Moreover, DFX reduces hemoglobin-induced brain Na+
ATPase inhibition and neuronal toxicity (Song et al, 2007
; Wan et al, 2006
). In our study, ROS production measured by increased ethidium was markedly increased in the hemorrhagic striatum 1 day after ICH. Deferoxamine attenuated ROS production, further supporting the idea that iron toxicity underlies the increase in oxidative damage after ICH.
In addition to iron-mediated ROS production, inflammation has also been shown to be associated with neuronal death (Wang, 2010
; Wang and Doré, 2007b
). We along with others have shown that activated microglia and astrocytes and infiltrating neutrophils are present in the hemorrhagic striatum early after ICH (Wang and Doré, 2007b
). Increasing evidence supports the premise that activated microglia/macrophages and infiltrating neutrophils are the major sources of proinflammatory mediators (Wang, 2010
; Wang and Doré, 2007b
). Consistent with this notion, inhibition of microglial activation before or after ICH decreased neuronal death and improved neurologic function (Wang et al, 2003
; Wang and Tsirka, 2005b
). We showed for the first time that DFX reduced the number of activated microglia/macrophages and infiltrating neutrophils but not of astrocytes 3 days after ICH. Although the underlying mechanisms are not clear, microglia have a larger capacity to take up free iron than do neurons and astrocytes (Bishop et al, 2010
). Iron accumulation in microglia might stimulate the activation of these cells in the hemorrhagic brain. Deferoxamine may suppress inflammation by reducing iron-mediated oxidative damage or by blocking the signals that activate microglia and recruit neutrophils. Astrocytes provide little iron storage (Zecca et al, 2004
) and hence are highly resistant to iron-induced toxicity (Kress et al, 2002
Studies have shown that DFX improves neurologic function in rats subjected to the whole-blood ICH model (Okauchi et al, 2009
). Consistent with these studies, we found that DFX improved neurologic function after a 3-day treatment when begun 6
hours after ICH. However, the improvement was relatively modest. Furthermore, DFX did not reduce lesion volume, edema, or swelling, results consistent with those reported in the rat collagenase model (Warkentin et al, 2010
). In addition, we found that DFX slightly enhanced weight loss. Although the underlying mechanisms are not clear, DFX produces adverse effects in the digestive system that include abdominal discomfort and nausea (Okauchi et al, 2010
). Others have not reported weight loss in rats exposed to the whole-blood or collagenase model (Okauchi et al, 2009
; Warkentin et al, 2010
), but the discordant findings may be explained by differences in animal models and species and the age of the animals studied.
In conclusion, systemic administration of DFX decreased perihematomal iron accumulation and neuronal death, attenuated production of ROS, and reduced microglial activation and neutrophil infiltration. Although DFX treatment did not reduce lesion volume, brain edema, or brain swelling, it modestly improved neurologic function. These findings provide novel evidence that iron chelation with DFX could offer some benefit to patients with ICH.