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Hemopexin (Hpx) is critical for hemin scavenging after the erythrocyte lysis that occurs following intracerebral hemorrhage (ICH). Low-density lipoprotein receptor-related protein-1 (LRP1, also called CD91) is an important receptor through which the hemin-Hpx complex can undergo endocytosis. This study investigated changes in the hemin-Hpx-CD91 axis in both hematoma and perihematomal tissue in a large animal ICH model. The effect of deferoxamine (DFX) on hemin-Hpx-CD91 was also examined. The study consisted of two parts. First, piglets had an injection of autologous blood into the right frontal lobe of brain and were euthanized from day 1 to day 7. Hematoma and perihematomal tissue of brains were used for hemin assay, immunohistochemistry, and immunofluorescence. Second, piglets with ICH were treated with deferoxamine or vehicle, and were euthanized for hemin measurement and Hpx and CD91 immunohistochemistry. We found that there was an increase of hemin levels within the hematoma and perihematomal brain tissue after ICH. Hpx and CD91-positive cells were present in the clot and perihematomal tissue from day 1. Hpx and CD91 positive cells were Iba1 positive. After DFX therapy, hemin dropped markedly in the hematoma and perihematomal brain tissue. Furthermore, DFX treatment decreased the number of Hpx and CD91 positive cells in and around the hematoma. In conclusion, hemin accumulation occurs in and around the hematoma. Increases in Hpx and CD91 may be important in scavenging that hemin. DFX treatment decreased hemin release from the hematoma and reduced the expression of Hpx and CD91., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Iron from haemolysed blood is implicated in secondary injury after intracerebral haemorrhage. We aimed to assess the safety of the iron chelator deferoxamine mesylate in patients with intracerebral haemorrhage and to establish whether the drug merits investigation in a phase 3 trial., Methods: We did a multicentre, futility-design, randomised, placebo-controlled, double-blind, phase 2 trial at 40 hospitals in Canada and the USA. Adults aged 18-80 years with primary, spontaneous, supratentorial intracerebral haemorrhage were randomly assigned (1:1) to receive deferoxamine mesylate (32 mg/kg per day) or placebo (saline) infusions for 3 consecutive days within 24 h of haemorrhage onset. Randomisation was done via a web-based trial-management system centrally in real time, and treatment allocation was concealed from both participants and investigators. The primary outcome was good clinical outcome, which was defined as a modified Rankin Scale score of 0-2 at day 90. We did a futility analysis: if the 90% upper confidence bound of the absolute risk difference between the two groups in the proportion of participants with a good clinical outcome was less than 12% in favour of deferoxamine mesylate, then to move to a phase 3 efficacy trial would be futile. Primary outcome and safety data were analysed in the modified intention-to-treat population, comprising only participants in whom the study infusions were initiated. This trial is registered with ClinicalTrials.gov, number NCT02175225, and is completed., Findings: We recruited 294 participants between Nov 23, 2014, and Nov 10, 2017. The modified intention-to-treat population consisted of 144 patients assigned to the deferoxamine mesylate group and 147 assigned to the placebo group. At day 90, among patients with available data for the primary outcome, 48 (34%) of 140 participants in the deferoxamine mesylate group, and 47 (33%) of 143 patients in the placebo group, had modified Rankin Scale scores of 0-2 (adjusted absolute risk difference 0·6% [90% upper confidence bound 6·8%]). By day 90, 70 serious adverse events were reported in 39 (27%) of 144 patients in the deferoxamine mesylate group, and 78 serious adverse events were reported in 49 (33%) of 147 patients in the placebo group. Ten (7%) participants in the deferoxamine mesylate and 11 (7%) in the placebo group died. None of the deaths were judged to be treatment related., Interpretation: Deferoxamine mesylate was safe. However, the primary result showed that further study of the efficacy of deferoxamine mesylate with anticipation that the drug would significantly improve the chance of good clinical outcome (ie, mRS score of 0-2) at day 90 would be futile., Funding: US National Institutes of Health and US National Institute of Neurological Disorders and Stroke., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background and Purpose: CD163, a receptor for hemoglobin, is involved in hemoglobin clearance after intracerebral hemorrhage (ICH). In contrast to microglial/macrophage CD163, neuronal CD163 hemoglobin has not been well studied. This study examined the expression of neuronal CD163 in a pig model of ICH and in vitro rat cortical neurons and the impact of deferoxamine on that expression., Methods: There were 2 parts to this study. In the in vivo part, piglets had injection of autologous blood into the right frontal lobe. The time course of CD163 expression and the effect of deferoxamine on the expression of CD163 after ICH were determined in the grey matter. In the in vitro part, the levels of CD163 and neuronal death and the effect of deferoxamine were examined in rat cortical neurons culture treated with hemoglobin., Results: CD163-positive cells were found, and the CD163 protein levels were upregulated in the ipsilateral grey matter after ICH. The CD163 levels peaked at days 1 and 3. The CD163-positive cells were colocated with NeuN-positive, heme oxygenase-2-positive, and terminal deoxynucleatidyl transferase dUTP nick end labeling-positive cells. Deferoxamine treatment attenuated ICH-induced CD163 upregulation and significantly reduced both brain CD163 and hemoglobin levels at day 3. Treating neuronal cultures with hemoglobin for 24 hours resulted in CD163 upregulation and increased cell death. Deferoxamine significantly attenuated the hemoglobin-induced neuronal death and CD163 upregulation., Conclusions: CD163 is expressed in neurons and upregulated after ICH. Deferoxamine reduced ICH-induced CD163 upregulation and brain cell death in vivo and hemoglobin-induced CD163 upregulation and neuronal death in vitro., (© 2017 American Heart Association, Inc.)

Background and Purpose: Hematoma clearance occurs in the days after intracerebral hemorrhage (ICH) and has not been well studied. In the current study, we examined changes in the hematoma in a piglet ICH model. The effect of deferoxamine on hematoma was also examined., Methods: The ICH model was induced by an injection of autologous blood into the right frontal lobe of piglets. First, a natural time course of hematoma changes ≤7 days was determined. Second, the effect of deferoxamine on hematoma changes was examined. Hemoglobin and membrane attack complex levels in the hematoma were examined by enzyme-linked immunosorbent assay. Immunohistochemistry and Western blotting were used to examine CD47 (a regulator of erythrophagocytosis), CD163 (a hemoglobin scavenger receptor), and heme oxygenase-1 (a heme degradation enzyme) in the clot., Results: After ICH, there was a reduction in red blood cell diameter within the clot with time. This was accompanied by membrane attack complex accumulation and decreased hemoglobin levels. Erythrophagocytosis occurred in the hematoma, and this was associated with reduced clot CD47 levels. Activated macrophages/microglia were CD163 and hemeoxygenase-1 positive, and these accumulated in the clot with time. Deferoxamine treatment attenuated the process of hematoma resolution by reducing member attack complex formation and inhibiting CD47 loss in the clot., Conclusions: These results indicate that membrane attack complex and erythrophagocytosis contribute to hematoma clearance after ICH, which can be altered by deferoxamine treatment., Competing Interests: Potential Conflicts of Interest: We declare that we have no conflict of interest., (© 2016 American Heart Association, Inc.)

Iron contributes to c-Jun N-terminal kinases (JNK) activation in young rats and white matter injury in piglets after intracerebral hemorrhage (ICH). In the present study, we examined the effect of deferoxamine on ICH-induced white matter injury and JNK activation and in aged rats. Male Fischer 344 rats (18months old) had either an intracaudate injection of 100μl of autologous blood or a needle insertion (sham). The rats were treated with deferoxamine or vehicle with different regimen (dosage, duration and time window). White matter injury and activation of JNK were examined. We found that a dose of DFX should be at more than 10mg/kg for a therapeutic duration more than 2days with a therapeutic time window of 12h to reduce ICH-induced white matter loss at 2months. ICH-induced white matter injury was associated with JNK activation. The protein levels of phosphorylated-JNK (P-JNK) were upregulated at day-1 after ICH and then gradually decreased. P-JNK immunoreactivity was mostly located in white matter bundles. ICH-induced JNK activation was reduced by DFX treatment. This study demonstrated that DFX can reduce ICH-induced JNK activation and white matter damage., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Acute post-traumatic ventricular dilation and hydrocephalus are relatively frequent consequences of traumatic brain injury (TBI). Several recent studies have indicated that high iron levels in brain may relate to hydrocephalus development after intracranial hemorrhage. However, the role of iron in the development of post-traumatic hydrocephalus is still unclear. This study was to determine whether or not iron has a role in hydrocephalus development after TBI. TBI was induced by lateral fluid-percussion in male Sprague-Dawley rats. Some rats had intraventricular injection of iron. Acute hydrocephalus was measured by magnetic resonance T2-weighted imaging and brain hemorrhage was determined by T2* gradient-echo sequence imaging and brain hemoglobin levels. The effect of deferoxamine on TBI-induced hydrocephalus was examined. TBI resulted in acute hydrocephalus at 24 h (lateral ventricle volume: 24.1 ± 3.0 vs. 9.9 ± 0.2 mm(3) in sham group). Intraventricular injection of iron also caused hydrocephalus (25.7 ± 3.4 vs. 9.0 ± 0.6 mm(3) in saline group). Deferoxamine treatment attenuated TBI-induced hydrocephalus and heme oxygenase-1 upregulation. In conclusion, iron may contribute to acute hydrocephalus after TBI.

Background and Purpose: Deferoxamine reduces neuronal death in a piglet model of intracerebral hemorrhage (ICH). This study examined the effect of deferoxamine on perihematomal white matter edema in piglets., Methods: ICH was induced by an injection of autologous blood into the right frontal lobe of piglets. In the first part of study, the time course of edema formation was determined. In the second part, the effects of deferoxamine on ICH-induced white matter edema, tumor necrosis factor α, and receptor-interacting protein kinase 1 were examined., Results: ICH resulted in marked brain edema and increased tumor necrosis factor α and receptor-interacting protein kinase 1 levels in white matter. Systemic treatment with deferoxamine markedly reduced white matter tumor necrosis factor α and receptor-interacting protein kinase 1 levels and attenuated white matter edema after ICH., Conclusions: Deferoxamine reduces white matter edema, tumor necrosis factor α, and receptor-interacting protein kinase 1 levels after ICH in piglets, suggesting deferoxamine is a potential effective therapeutic agent for patients with ICH.

Intracerebral hemorrhage (ICH) is primarily a disease of the elderly. Deferoxamine (DFX), an iron chelator, reduces long-term neurological deficits and brain atrophy after ICH in aged rats. In the present study, we investigated whether DFX can reduce acute ICH-induced neuronal death and whether it affects the endogenous response to ICH (ferritin upregulation and hematoma resolution) in aged rats. Male Fischer 344 rats (18 months old) had an intracaudate injection of 100 μL autologous whole blood into the right basal ganglia and were treated with DFX (100 mg/kg) or vehicle 2 hours post-ICH and then every 12 hours up to 7 days. Rats were euthanized 1, 3, or 7 days later for neuronal death, ferritin and hematoma size measurements. Plasma ferritin levels and behavioral outcome following ICH were also examined. DFX treatment significantly reduced ICH-induced neuronal death and neurological deficits. DFX also suppressed ferritin upregulation in the ipsilateral basal ganglia after ICH and hematoma lysis (hematoma volume at day 7: 13.2±4.9 vs. 3.8±1.2 mm3 in vehicle-treated group, p<0.01). However, effects of DFX on plasma ferritin levels after ICH did not reach significance. In conclusion, DFX reduces neuronal death and neurological deficits after ICH in aged rats. It also affects the endogenous response to ICH.

Background and Purpose: Treatment with the iron chelator, deferoxamine mesylate (DFO), improves neurological recovery in animal models of intracerebral hemorrhage (ICH). We aimed to evaluate the feasibility, safety, and tolerability of varying dose-tiers of DFO in patients with spontaneous ICH, and to determine the maximum tolerated dose to be adopted in future efficacy studies., Methods: This was a multicenter, phase-I, dose-finding study using the Continual Reassessment Method. DFO was administered by intravenous infusion for 3 consecutive days, starting within 18 hours of ICH onset. Subjects underwent repeated clinical assessments through 90 days, and computed tomography neuroimaging pre- and post-drug-administration., Results: Twenty subjects were enrolled onto 5 dose tiers, starting with 7 mg/kg per day and ending with 62 mg/kg per day as the maximum tolerated dose. Median age was 68 years (range, 50-90); 60% were men; and median Glasgow Coma Scale and National Institutes of Health Stroke Scale scores on admission were 15 (5-15) and 9 (0-39), respectively. ICH location was lobar in 40%, deep in 50%, and brain stem in 10%; intraventricular hemorrhage was present in 15%. DFO was discontinued because of adverse events in 2 subjects (10%). Six subjects (30%) experienced 12 serious adverse events, none of which were drug-related. DFO infusions were associated with mild blood-pressure-lowering effects. Fifty percent of patients had modified Rankin scale scores ≤2, and 39% had modified Rankin scale scores of 4 to 6 on day 90; 15% died., Conclusions: Consecutive daily infusions of DFO after ICH are feasible, well-tolerated, and not associated with excessive serious adverse events or mortality. Our findings lay the groundwork for future studies to evaluate the efficacy of DFO in ICH.

Cardiac dysfunction can occur after intracerebral hemorrhage (ICH). This study examined the expression of heat shock proteins (HSPs) in the heart after ICH in aged rats and whether deferoxamine (DFX), an iron chelator, affects HSP expression. Male Fischer 344 rats (18 months old) received an injection of 100 μl autologous blood into the right caudate, whereas sham-operated rats had a needle insertion. The rats were treated with DFX or vehicle at 2 and 6 h after ICH and then every 12 h for 3 days. Rats were killed 3 days after ICH, and the hearts were sampled for Western blot analysis of HSP-27 and HSP-32. Western blotting showed that levels of HSP-32 were reduced in the heart after ICH (p<0.05), and this reduction was normalized by DFX (p<0.05). DFX had no effects on heart HSP-32 levels in sham-operated rats. ICH also resulted in a reduction in HSP-27 (p<0.05), but DFX treatment reduced HSP-27 further (p<0.05). In addition, DFX reduced HSP-27 levels in sham rats. In conclusion, ICH causes HSP-27 and -32 reductions in the heart of aged rats. Deferoxamine treatment has different effects on the expression of HSP-27 and HSP-32.

This study investigated whether deferoxamine (DFX), an iron chelator, reduces cavity size after ICH in aged rats. Aged male Fischer rats (18 months old) had an intracaudate injection of 100 μL autologous blood and were treated with DFX or vehicle. Rats were euthanized at day 56 and brains were perfused for histology and immunohistochemistry. Hematoxylin and eosin staining was used to examine hematoma cavity presence and size. Immunohistochemistry was performed to measure the number of cells positive for ferritin, heme oxygenase-1 (HO-1), glial fibrillary acidic protein (GFAP) and OX-6. Neurological deficits were also examined. In aged rats with ICH, a cavity formed in the caudate in 7 out of 12 vehicle-treated rats and 1 out of 9 DFX-treated rats. DFX treatment significantly reduced the size of the ICH-induced cavity (p<0.05) as well as neurological deficits (p<0.05). DFX also reduced the number of ferritin (p<0.05) and HO-1 (p<0.01) positive cells in the ipsilateral basal ganglia. However, DFX had no effect on brain GFAP and OX-6 immunoreactivity 2 months after ICH.In conclusion, DFX reduces cavity size, neurological deficits, and immunoreactivity for ferritin and HO-1 after ICH in aged rats, supporting the suggestion that DFX may reduce brain injury in ICH patients.

The effect of subarachnoid hemoglobin on neuroglial cells contributing to early brain injury is unclear. Several intracerebral hemorrhage studies indicated that pathological iron deposition in the brain contributes to secondary brain injury. Therefore, the purpose of this study was to investigate the relationship between iron and neuroglial cell changes following SAH, and examine the effect of deferoxamine (DFX). SAH was induced in male Sprague-Dawley rats (n = 56) using an endovascular perforation technique. Animals were treated with DFX (100 mg/kg) or vehicle for 3 days. Rats were sacrificed at 6 h, days 1 and 3 to determine non-heme iron and heme oxygenase (HO)-1 expression using Western blot and immunohistochemistry analysis. To assess neuronal cell death, Fluoro-Jade- and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) stainings were performed. Marked HO-1 upregulation at day 3 (P < 0.01) was accompanied by elevated non-heme iron (P < 0.01) and ferritin levels (P < 0.01). DFX treatment reduced brain non-heme iron concentration, ferritin expression and neuronal cell death at day 3 (P < 0.01) following SAH. These results suggest that excessive hemoglobin and iron overload play an important role in early brain injury following SAH. Acute treatment with DFX significantly ameliorates neuronal cell death and may be a potential therapeutic agent for SAH.

The purpose of this study was to investigate hemoglobin and iron handling after subarachnoid hemorrhage (SAH), examine the relationship between iron and neuroglial cell changes, and determine whether deferoxamine (DFX) can reduce SAH-induced injury. The SAH was induced in Sprague-Dawley rats (n=110) using an endovascular perforation technique. Animals were treated with DFX (100 mg/kg) or vehicle 2 and 6 hours after SAH induction followed by every 12 hours for 3 days. Rats were killed at 6 hours, Days 1 and 3 to determine nonheme iron and examine iron-handling proteins using Western blot and immunohistochemistry. 8-Hydroxyl-2'-deoxyguanosine and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining were performed to assess oxidative DNA damage and neuronal cell death. After SAH, marked heme-oxygenase-1 (HO-1) upregulation at Day 3 (P<0.01) was accompanied by elevated nonheme iron (P<0.01), transferrin (Tf) (P<0.01), Tf receptor (P<0.05), and ferritin levels (P<0.01). Deferoxamine treatment reduced SAH-induced mortality (12% versus 29%, P<0.05), brain nonheme iron concentration, iron-handling protein expression, oxidative stress, and neuronal cell death at Day 3 (P<0.01) after SAH. These results suggest that iron overload in the acute phase of SAH causes oxidative injury leading to neuronal cell death. Deferoxamine effectively reduced oxidative stress and neuronal cell death, and may be a potential therapeutic agent for SAH.

Background and Purpose: Deferoxamine (DFX) reduces brain edema, neurological deficits, and brain atrophy after intracerebral hemorrhage (ICH) in aged and young rats. Our previous study found that 50 mg/kg is an effective dose in aged rats. In the present study, we explored potential therapeutic time windows and optimal therapeutic durations., Methods: Aged male Fischer 344 rats (18 months old) sustained an intracaudate injection of 100 microL autologous whole blood, followed by intramuscular DFX or vehicle beginning at different time points, or continuing for different durations. Subgroups of rats were euthanized at day 3 for brain edema measurement and day 56 for brain atrophy determination. Behavioral tests were performed on days 1, 28, and 56 after ICH., Results: Systemic administration of DFX, when begun within 12 hours after ICH, reduced brain edema. DFX treatment started 2 hours after ICH and administered for >or=7 days attenuated ICH-induced ventricle enlargement, caudate atrophy, and neurological deficits. DFX attenuated ICH-induced brain atrophy and neurological deficits without detectable side effects when begun within 24 hours and administered for 7 days., Conclusions: To the extent that these results can be translated to humans, the therapeutic time window and the optimal duration for DFX in this aged rat model of ICH may provide useful information for an ongoing DFX-ICH clinical trial.

Hemorrhagic transformation (HT) is a major factor limiting the use of tissue plasminogen activator (tPA) for stroke patients. This study examined the role of deferoxamine (DFX) in brain injury and HT in a rat model of transient focal ischemia with hyperglycemia. Rats received an injection of 50% glucose (6 mL/kg, i.p.) 15 min before undergoing transient middle cerebral artery occlusion (tMCAO; 2 h occlusion) with reperfusion. Rats were treated with DFX (100 mg/ kg, i.m.) or vehicle immediately after tMCAO. Rats were killed at 4, 8 and 24 h later and used for brain edema, blood-brain barrier permeability, hemorrhage volume, hemoglobin content, and infarct volume measurements. Mortality rate was also evaluated. DFX treatment reduced mortality at 24 h (4% vs. 24% in the vehicle-treated group, p<0.05). DFX also reduced infarct volume (85.1+/-56.3 vs. 164.3+/-93.4 mm(3) in vehicle, p<0.05) and swelling in the basal ganglia (p<0.05) 24 h after tMCAO. The total hemorrhage volume in the ipsilateral hemisphere at 8 h post tMCAO was less in DFX-treated animals (p<0.05). However, blood-brain barrier permeability was same in DFX- and vehicle-treated groups. DFX attenuates death rate, hemorrhagic transformation, infarct volume, and brain swelling in a rat transient focal ischemia with hyperglycemia model, suggesting that DFX could be potential treatment to reduce the hemorrhagic transformation for stroke patients.

Background and Purpose: Deferoxamine (DFX) reduces brain edema, neuronal death, and neurological deficits after intracerebral hemorrhage (ICH) in young rats. In the present study, we investigated whether DFX is effective on brain injury after ICH in aged rats and examined dose dependency., Methods: Male Fischer 344 rats (18 months old) had an intracaudate injection of 100 microL autologous whole blood and were treated with different doses of DFX (10, 50, and 100 mg/kg) or vehicle 2 and 6 hours post-ICH and then every 12 hours up to 7 days. Rats were euthanized at Day 3 for brain edema determination and Day 56 for brain atrophy measurement. Behavioral tests were performed during the experiments., Results: All 3 doses of DFX attenuated perihematomal brain edema at 3 days (eg, at dose 50 mg/kg, 80.4+/-0.5 versus 81.6+/-0.9% in the vehicle-treated group, P<0.01). Fifty and 100 mg/kg DFX also reduced ICH-induced ventricle enlargement, caudate atrophy, and ICH-induced neurological deficits in aged rats. However, although 10 mg/kg DFX reduced ventricle enlargement and forelimb-placing deficits, it did not reduce caudate atrophy and corner turn deficits., Conclusions: These results indicate that DFX can reduce ICH-induced brain injury in aged as well as young rats and that a dose >10 mg/kg is the optimal dose of DFX in this model.

Background and Purpose: There is an urgent need to develop a model in which to study the mechanism of intracerebral hemorrhage-induced neuronal death in vivo., Methods: This study was divided into 2 parts: (1) Rats received either an infusion of hemoglobin, ferrous iron, or saline into the right hippocampus; (2) Rats had an infusion of hemoglobin and then were treated with either deferoxamine or vehicle. Rats were killed for hippocampus size, DNA damage, and neuronal death measurements., Results: Compared with saline, hemoglobin or iron injection caused hippocampal neuronal death. Systemic use of deferoxamine reduced hemoglobin-induced DNA damage, hippocampal neuronal death, and atrophy., Conclusions: This article demonstrates a new model and indicates that iron has a key role in hemoglobin-induced neuronal death.

Object: Previous studies undertaken by the authors have indicated that iron accumulation and oxidative stress in the brain contribute to secondary brain damage after intracerebral hemorrhage (ICH). In the present study the authors investigate whether deferoxamine, an iron chelator, can reduce ICH-induced brain injury., Methods: Male Sprague-Dawley rats each received an infusion of 100 microl of autologous whole blood into the right basal ganglia and were killed 1, 3, or 7 days later. Iron distribution was examined histochemically (enhanced Perls reaction). The effects of deferoxamine on ICH-induced brain injury were examined by measuring brain edema and neurological deficits. Immunohistochemical analysis was performed to investigate 8-hydroxyl-2'-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage, and Western blot analysis was performed to measure the amount of apurinic/apyrimidinic endonuclease/redox effector factor-1 (APE/Ref-1), a repair mechanism for DNA oxidative damage. Iron accumulation was observed in the perihematomal zone from 1 day after ICH. Deferoxamine attenuated brain edema, neurological deficits, and ICH-induced changes in 8-OHdG and APE/Ref-1., Conclusions: Deferoxamine and other iron chelators may be potential therapeutic agents for ICH. They may act by reducing the oxidative stress caused by the release of iron from the hematoma.

Object: In the authors' previous studies they found that brain iron accumulation and oxidative stress contribute to secondary brain damage after intracerebral hemorrhage (ICH). In the present study they investigated whether deferoxamine, an iron chelator, can reduce ICH-induced brain injury., Methods: Male Sprague-Dawley rats received an infusion of 100 microl of autologous whole blood into the right basal ganglia and were killed 1, 3, or 7 days thereafter. Iron distribution was examined histochemically (enhanced Perl reaction). The effects of deferoxamine on ICH-induced brain injury were examined by measuring brain edema and neurological deficits. Apurinic/apyrimidinic endonuclease/redox effector factor-1 (APE/Ref-1), a repair mechanism for DNA oxidative damage, was quantitated by Western blot analysis. Iron accumulation was observed in the perihematoma zone beginning 1 day after ICH. Deferoxamine attenuated brain edema, neurological deficits, and ICH-induced changes in APE/Ref-1., Conclusions: Deferoxamine and other iron chelators may be potential therapeutic agents for treating ICH. They may act by reducing the oxidative stress caused by the release of iron from the hematoma.

Abstract: Brain iron overload has a detrimental role in brain injury after intracerebral hemorrhage (ICH). Lipocalin 2 (LCN2), a siderophore-binding protein, is involved in cellular iron transport. The present study investigated changes in LCN2 expression after ICH and the role of iron in those changes. Male Sprague-Dawley rats had an intracaudate injection of autologous blood (ICH) or iron. Control rats received a needle insertion or saline injection. Some ICH animals were treated with either vehicle or deferoxamine, an iron chelator. Brain LCN2 expression was determined by Western blot analysis and immunohistochemistry. Real-time PCR was also used to confirm brain LCN2 mRNA expression. The number of LCN2 positive cells was markedly increased in the ipsilateral basal ganglia and cortex after ICH and most LCN2 positive cells were astrocytes. Western blots showed that brain LCN2 levels were higher at days 1, 3 and 7 in the ipsilateral hemisphere after ICH (70 to 80 fold higher than contralateral hemisphere or sham-operated rats at 3 days), and declined to lower levels at day 14. Iron, but not saline injection also caused brain LCN2 upregulation (a more than 100-fold increase). In addition, systemic treatment of deferoxamine reduced ICH-induced LCN2 upregulation (p<0.05). These results suggest that iron has a role in brain LCN2 upregulation following ICH. LCN2 upregulation after ICH may be part of the response to clear iron released from the hematoma during clot resolution. [Copyright &y& Elsevier]

Intracerebral hemorrhage is primarily a disease of the elderly and it is frequently accompanied by intraventricular hemorrhage (IVH) which can lead to posthemorrhagic hydrocephalus and poor prognosis. Red blood cell iron has been implicated in brain injury after cerebral hemorrhage. The current study examined using T2* magnetic resonance imaging (MRI) to detect periventricular iron deposition after IVH and investigated the effects of minocycline on hydrocephalus in an aged rat IVH model. It had three parts. In part 1, male aged rats received a 200 μl injection of saline or autologous blood into the lateral ventricle and were euthanized at day 14. In parts 2 and 3, aged IVH rats were treated with vehicle or minocycline and euthanized at day 7 or 14. Rats underwent MRI to quantify hydrocephalus and iron deposition followed by brain histology and immunohistochemistry. Periventricular iron overload was found after IVH using T2* MRI and confirmed by histology. IVH also caused ventricular wall damage and increased the number of CD68(+) choroid plexus epiplexus cells. Minocycline administration reduced iron deposition and ventricular volume at days 7 and 14 after IVH, as well as ventricle wall damage and epiplexus cell activation. In summary, IVH-induced hydrocephalus is associated with periventricular iron deposition, ependymal damage and choroid plexus epiplexus cell activation in aged rats. Minocycline attenuated those effects and might be a potential treatment for posthemorrhagic hydrocephalus in the elderly. • Periventricular iron overload after intraventricular hemorrhage (IVH) can be detected by T2* magnetic resonance imaging. • Minocycline significantly reduced periventricular iron accumulation and hydrocephalus after IVH in aged rats. • Minocycline decreased IVH-induced ventricular wall damage and choroid plexus epiplexus cell activation in aged rats. [ABSTRACT FROM AUTHOR]

Neuronal degeneration following neonatal intraventricular hemorrhage (IVH) is incompletely understood. Understanding the mechanisms of degeneration and cell loss may point toward specific treatments to limit injury. We evaluated the role of hemoglobin (Hb) in cell death after intraventricular injection in neonatal rats. Hb was injected into the right lateral ventricle of post-natal day 7 rats. Rats exposed to anesthesia were used for controls. The CA-1 region of the hippocampus was analyzed via immunohistochemistry, hematoxylin and eosin (H&E) staining, Fluoro-Jade C staining, Western blots, and double-labeling stains. Compared to controls, intraventricular injection of Hb decreased hippocampal volume (27% decrease; p <0.05), induced neuronal loss (31% loss; p <0.01), and increased neuronal degeneration (2.7 fold increase; p <0.01), which were all significantly reduced with the iron chelator, deferoxamine. Hb upregulated p-JNK (1.8 fold increase; p <0.05) and increased expression of the Hb/haptoglobin endocytotic receptor CD163 in neurons in vivo and in vitro (cultured cortical neurons). Hb induced expression of the CD163 receptor, which co-localized with p-JNK in hippocampal neurons, suggesting a potential pathway by which Hb enters the neuron to result in cell death. There were no differences in neuronal loss or degenerating neurons in Hb-injected animals that developed hydrocephalus versus those that did not. Intraventricular injection of Hb causes hippocampal neuronal degeneration and cell loss and increases brain p-JNK levels. p-JNK co-localized with the Hb/haptoglobin receptor CD163, suggesting a novel pathway by which Hb enters the neuron after IVH to result in cell death. [ABSTRACT FROM AUTHOR]

Abstract: Iron accumulates in the brain and contributes to brain injury after intracerebral hemorrhage (ICH). The c-Jun-N-terminal kinase (JNK) signaling pathway mediates cell death after ischemic stroke, however, the involvement of JNK in ICH is not well known. This study investigated whether the JNK signaling pathway is activated by iron after ICH. Male Sprague–Dawley rats received an infusion of autologous whole blood (as a model of ICH) or ferrous iron into the right basal ganglia and control rats had an infusion of saline. Some ICH rats were treated with either deferoxamine (DFX), an iron chelator, or vehicle. Activation of JNK was measured by Western blot analysis and immunohistochemistry. Free iron in cerebrospinal fluid (CSF) and behavioral outcomes following ICH were also examined. We found that activated JNK in the brain were increased after ICH, and an intracerebral infusion of ferrous iron also upregulated brain activated JNK. Free iron accumulated in CSF and systemic administration of DFX after ICH reduces free iron contents in CSF, suppresses JNK activation and improves ICH-induced neurological deficits. Our results demonstrated that the JNK signaling pathway is activated after ICH and iron may contribute to this activation. DFX reduces free iron levels and attenuates activation of JNK suggesting iron chelation may be useful therapy for ICH patients. [Copyright &y& Elsevier]