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Iron chelators, such as the intracellular ferrous chelator 2,2'-bipyridine, are a potential means of ameliorating iron-induced injury after intracerebral hemorrhage (ICH). We evaluated bipyridine against the collagenase and whole-blood ICH models and a simplified model of iron-induced damage involving a striatal injection of FeCl2 in adult rats. First, we assessed whether bipyridine (25 mg/kg beginning 12 h post-ICH and every 12 h for 3 days) would attenuate non-heme iron levels in the brain and lessen behavioral impairments (neurological deficit scale, corner turn test, and horizontal ladder) 7 days after collagenase-induced ICH. Second, we evaluated bipyridine (20 mg/kg beginning 6 h post-ICH and then every 24 h) on edema 3 days after collagenase infusion. Body temperature was continually recorded in a subset of these rats beginning 24 h prior to ICH until euthanasia. Third, bipyridine was administered (as per experiment 2) after whole-blood infusion to examine tissue loss, neuronal degeneration, and behavioral impairments at 7 days post-stroke, as well as body temperature for 3 days post-stroke. Finally, we evaluated whether bipyridine (25 mg/kg given 2 h prior to surgery and then every 12 h for 3 days) lessens tissue loss, neuronal death, and behavioral deficits after striatal FeCl2 injection. Bipyridine caused a significant hypothermic effect (maximum drop to 34.6 °C for 2-5 h after each injection) in both ICH models; however, in all experiments bipyridine-treated rats were indistinguishable from vehicle controls on all other measures (e.g., tissue loss, behavioral impairments, etc.). These results do not support the use of bipyridine against ICH.

Studies treating intracerebral hemorrhage (ICH) with therapeutic hypothermia (TH) have shown inconsistent benefits. We hypothesized that TH's anti-inflammatory effects may be responsible as inflammatory cells are essential for removing degrading erythrocytes. Here, we subjected rats to a collagenase-induced striatal ICH followed by whole-body TH (∼33℃ for 11-72 h) or normothermia. We used X-ray fluorescence imaging to spatially quantify total and peri-hematoma iron three days post-injury. At three and seven days, we measured non-heme iron levels. Finally, hematoma volume was quantified on one, three, and seven days. In the injured hemisphere, total iron levels were elevated ( p < 0.001) with iron increasing in the peri-hematoma region ( p = 0.007). Non-heme iron increased from three to seven days (p < 0.001). TH had no effect on any measure of iron ( p ≥ 0.479). At one and three days, TH did not affect hematoma volume ( p ≥ 0.264); however, at seven days there was a four-fold increase in hematoma volume in 40% of treated animals ( p = 0.032). Thus, even when TH does not interfere with initial increases in total and non-heme iron or its containment, TH can cause re-bleeding post-treatment. This serious complication could partly account for the intermittent protection previously observed. This also raises serious concerns for clinical usage of TH for ICH.

Hyperthermia accelerates and increases ischemic brain damage. Owing to overlapping mechanisms of injury, many assume that hyperthermia also worsens outcome after intracerebral hemorrhage (ICH). However, clinical data do not conclusively prove this, and there is only one animal study examining the impact of hyperthermia. In that study (MacLellan and Colbourne, 2005), several hyperthermia protocols were administered after collagenase-induced ICH in rats; none worsened injury. While the collagenase model is widely used, it differs in important ways from another common model - injecting autologous blood directly into the brain. Thus, we evaluated the impact of immediate hyperthermia (HYP, 39 °C for 3 hr) after a 100-µL infusion of blood into the striatum of rats. This treatment, which markedly increases ischemic damage, was compared to control rats kept normothermic (NOR, 37 °C). Three separate experiments were done to measure: 1) edema at 24 hr, 2) edema at 72 hr, and 3) behavioral impairment and lesion size out to 1 month post-ICH. The HYP treatment did not significantly affect edema at 24 hr, but surprisingly, it modestly reduced edema at 72 hr and partly improved behavioral outcome. However, there were no lasting effects of HYP on behavior (e.g., skilled reaching) or the volume of tissue lost (NOR: 14.0 mm(3) vs. HYP: 14.5 mm(3)). In summary, our findings do not support the common belief that hyperthermia worsens outcome after ICH. Additional research is needed to determine whether more severe or prolonged heating or fever and its cause (e.g., infection) affect morbidity and mortality after ICH.

Background — After an intracerebral hemorrhage (ICH) iron is released from degrading erythrocytes over days, which causes secondary damage by increasing free radicals. Many studies show that iron chelators, such as deferoxamine, lessen injury, but not all studies support this. Hypothesis — The ferrous iron chelator, 2,2 Dipyridyl (DP), will decrease injury after ICH or intraparenchymal FeCl 2 infusion in rats. Experiment 1— Rats were given a collagenase-induced striatal ICH. In experiments 1 and 3, we tested whether behavior was improved (e.g., walking) from administering DP (25mg/kg/day, 12 hours after surgery for 3 days). They were euthanized after 7 days to determine non-heme iron levels in the brain. Experiment 2— Rats were injected with DP (20mg/kg) 6 hours after collagenase infusion and every 24 hours till euthanasia at 3 days for measuring edema. Experiment 3— After injecting FeCl 2 in the striatum rats were given DP (25mg/kg every 12 hr starting 2 hours prior to surgery for 3 days). The volume of tissue loss and Fluoro-jade staining (degenerating neurons) was measured. Results — DP did not improve behavioral or histological outcome or reduce edema in either the collagenase ICH or FeCl 2 model. DP also did not affect parenchymal non-heme iron level. Conclusion — Our data suggests that DP on its own is not an effective strategy for ICH.

Intracerebral hemorrhage (ICH) is often a devastating stroke, and there are no clinically proven neuroprotective treatments. Considerable research points to iron toxicity as a leading contributor to secondary damage after ICH. Iron, released from degraded erythrocytes, catalyzes free radical production, thereby causing cell death in the ensuing days and weeks. Therapeutic hypothermia (TH) is a potential neuroprotective strategy for ICH, but results from animal studies are inconsistent and generally weaker than that found in ischemia models. Thus, we examined whether TH (∼33°C for 24-72 hours) would specifically mitigate iron toxicity, which we modeled by infusing 3.8 μg of FeCl2 in 30 μL of sterile saline into the striatum of rats. Rats were subjected to whole-body cooling beginning 1 hour after FeCl2 infusion. Use of TH reduced (p = 0.025) the small bleed caused by FeCl2 infusion (∼6 μL). However, TH did not lessen FeCl2-induced edema at 24 and 72 hours postinfusion, nor were behavioral impairments (e.g., walking) or brain injury (at 7 and 28 days) attenuated by TH. These results suggest that TH does not directly protect against iron toxicity, which indicates that this is not a means by which TH improves the outlook after ICH.