Effect of hemopexin treatment on outcome after intracerebral hemorrhage in mice.

Heme release from hemoglobin may contribute to secondary injury after intracerebral hemorrhage (ICH). The primary endogenous defense against heme toxicity is hemopexin, a 57 kDa glycoprotein that is depleted in the CNS after hemorrhagic stroke. We hypothesized that systemic administration of exogenous hemopexin would reduce perihematomal injury and improve outcome after experimental ICH. Intraperitoneal treatment with purified human plasma hemopexin beginning 2 h after striatal ICH induction and repeated daily for the following two days reduced blood-brain barrier disruption and cell death at 3 days. However, it had no effect on neurological deficits at 4 or 7 days or striatal cell viability at 8 days. Continuous daily hemopexin administration had no effect on striatal heme content at 3 or 7 days, and did not attenuate neurological deficits, inflammatory cell infiltration, or perihematomal cell viability at 8 days. These results suggest that systemic hemopexin treatment reduces early injury after ICH, but this effect is not sustained, perhaps due to an imbalance between striatal tissue heme and hemopexin content at later time points. Future studies should investigate its effect when administered by methods that more efficiently target CNS delivery.

Rapid loss of perihematomal cell viability in the collagenase intracerebral hemorrhage model.

The effective time window of any therapeutic in an experimental stroke model is limited by the rate of injury progression. Intracerebral hemorrhage in rodents is commonly induced by striatal injection of either autologous blood or bacterial collagenase, which digests local blood vessels. During time window studies of the heme oxygenase-1 inducer hemin, which is protective when administered within 1-3 h in both models, the rate of perihematomal injury was directly compared after striatal blood or collagenase injection. Surprisingly, about 80% of the loss of perihematomal cell viability as measured by MTT reduction assay occurred within 6 h of collagenase injection. In contrast, significant viability loss was not observed at this time point after autologous blood injection, but rather it progressed over the subsequent four days to a level similar to that produced by collagenase. Consistent with these observations, systemic hemin therapy reduced blood-brain barrier disruption and perihematomal cell injury when initiated at 6 h after striatal injection of blood but not collagenase. These results indicate that the rate of early cell injury differs markedly in the collagenase and blood injection ICH models, which may contribute to inconsistent results in time window studies. The blood injection model may be more appropriate for prolonged time window studies of a neuroprotective agent.

Protective effect of vitreous against hemoglobin neurotoxicity.

Hemorrhage into the brain parenchyma or subarachnoid space is associated with edema and vascular injury that is likely mediated at least in part by the toxicity of hemoglobin. In contrast, extravascular blood appears to be less neurotoxic when localized to the retina or adjacent vitreous, the gel filling the posterior segment of the eye. In this study, the hypothesis that vitreous protects neurons from hemoglobin toxicity was investigated in a primary cortical cell culture model. Consistent with prior observations, hemoglobin exposure for 24 h resulted in death of most neurons without injury to co-cultured glia. Neuronal loss was reduced in a concentration-dependent fashion by bovine vitreous, with complete protection produced by 3% vitreous solutions. This effect was associated with a reduction in malondialdehyde but an increase in cell iron. At low vitreous concentrations, its ascorbate content was sufficient to account for most neuroprotection, as equivalent concentrations of ascorbate alone had a similar effect. However, other vitreous antioxidants provided significant protection when applied at concentrations present in undiluted vitreous, and prevented all neuronal loss when combined in the absence of ascorbate. These results indicate that vitreous is an antioxidant cocktail that robustly protects neurons from hemoglobin toxicity, and may contribute to the relative resistance of retinal neurons to hemorrhagic injury.

Hemopexin increases the neurotoxicity of hemoglobin when haptoglobin is absent.

Hemopexin (Hpx) binds heme with extraordinary affinity, and after haptoglobin may provide a second line of defense against the toxicity of extracellular hemoglobin (Hb). In this series of experiments, the hypothesis that Hpx protects neurons from Hb neurotoxicity was evaluated in murine primary cultures containing neurons and glial cells. Contrary to hypothesis, Hpx increased neuronal loss due to micromolar concentrations of Hb by 4- to 12-fold, as measured by LDH release assay; conversely, the neurotoxicity of hemin was completely prevented. The endogenous fluorescence of Hpx was quenched by Hb, consistent with transfer of Hb-bound heme to Hpx. This was associated with precipitation of globin chains, as detected by immunostaining and fluorescent Hb labeling. A portion of this precipitate attached firmly to cells and could not be removed by multiple washes. Concomitant treatment with haptoglobin (Hp) prevented globin precipitation and most of the increase in neuronal loss. Hpx weakly attenuated the increase in culture non-heme iron produced by Hb treatment, quantified by ferrozine assay. However, Hb-Hpx toxicity was iron-dependent, and was blocked by deferoxamine and ferrostatin-1. Up-regulation of cell ferritin expression, a primary cell defense against Hb toxicity, was not observed on western blots of culture lysates that had been concomitantly treated with Hpx. These results suggest that Hpx destabilizes Hb in the absence of haptoglobin, leading to globin precipitation and exacerbation of iron-dependent oxidative cell injury. Combined therapy with hemopexin plus haptoglobin may be preferable to hemopexin alone after CNS hemorrhage.

Astrocyte heme oxygenase-1 reduces mortality and improves outcome after collagenase-induced intracerebral hemorrhage.

Pharmacotherapies that increase CNS expression of heme oxygenase-1 (HO-1) and other antioxidant proteins have improved outcome in experimental models of spontaneous intracerebral hemorrhage (ICH). In order to more specifically investigate the relationship between HO-1 and ICH outcome, mice expressing human HO-1 driven by the glial fibrillary acidic protein (GFAP) promoter (GFAP·HMOX1 mice) were tested in a model of in situ parenchymal hemorrhage. Injection of collagenase into the striata of wild-type (WT) mice resulted in a 26.3% mortality rate, with deaths equally distributed between males and females. Mortality was reduced to 4.48% in GFAP·HMOX1 mice. Cell viability in the injected striata of surviving WT mice was reduced by about half at one week and was significantly increased in transgenics; this benefit persisted over a 22day observation period. Cell counts guided by design-based stereology indicated loss of ~40% of neurons in WT hemorrhagic striata at one week, which was decreased by half in transgenics; no significant differences in microglia or astrocyte numbers were observed. Blood-brain barrier disruption and short-term neurological deficits were also mitigated in GFAP·HMOX1 mice, but long-term outcome did not differ from that of WT survivors. These results suggest that astrocyte HO-1 overexpression provides robust neuroprotection after acute intracerebral hemorrhage. Further investigation of drug or genetic therapies that selectively increase astrocyte HO-1 is warranted.

Targeting the Nrf2-Heme Oxygenase-1 Axis after Intracerebral Hemorrhage.

BACKGROUND: Injury to cells adjacent to an intracerebral hemorrhage (ICH) is likely mediated at least in part by toxins released from the hematoma that initiate complex and interacting injury cascades. Pharmacotherapies targeting a single toxin or pathway, even if consistently effective in controlled experimental models, have a high likelihood of failure in a variable clinical setting. Nuclear factor erythroid-2 related factor 2 (Nrf2) regulates the expression of heme oxygenase-1 (HO-1) and multiple other proteins with antioxidant and antiinflammatory effects, and may be a target of interest after ICH. METHODS: Studies that tested the effect of HO and Nrf2 in models relevant to ICH are summarized, with an effort to reconcile conflicting data by consideration of methodological limitations. RESULTS: In vitro studies demonstrated that Nrf2 activators rapidly increased HO-1 expression in astrocytes, and reduced their vulnerability to hemoglobin or hemin. Modulating HO-1 expression via genetic approaches yielded similar results. Systemic treatment with small molecule Nrf2 activators increased HO-1 expression in perivascular cells, particularly astrocytes. When tested in mouse or rat ICH models, Nrf2 activators were consistently protective, improving barrier function and attenuating edema, inflammation, neuronal loss and neurological deficits. These effects were mimicked by selective astrocyte HO-1 overexpression in transgenic mice. CONCLUSION: Systemic treatment with Nrf2 activators after ICH is protective in rodents. Two compounds, dimethyl fumarate and hemin, are currently approved for treatment of multiple sclerosis and acute porphyria, respectively, and have acceptable safety profiles over years of clinical use. Further development of these drugs as ICH therapeutics seems warranted.

Haptoglobin increases the vulnerability of CD163-expressing neurons to hemoglobin.

Haptoglobin (Hp) binds hemoglobin (Hb) with high affinity and provides the primary defense against its toxicity after intravascular hemolysis. Neurons are exposed to extracellular Hb after CNS hemorrhage, and a therapeutic effect of Hp via Hb sequestration has been hypothesized. In this study, we tested the hypothesis that Hp protects neurons from Hb in primary mixed cortical cell cultures. Treatment with low micromolar concentrations of human Hb for 24 h resulted in loss of 10-20% of neurons without injuring glia. Concomitant treatment with Hp surprisingly increased neuronal loss five-sevenfold, with similar results produced by Hp 1-1 and 2-2 phenotypes. Consistent with a recent in vivo observation, neurons expressed the CD163 receptor for Hb and the Hb-Hp complex in these cultures. Hp reduced overall Hb uptake, directed it away from the astrocyte-rich CD163-negative glial monolayer, and decreased induction of the iron-binding protein ferritin. Hb-Hp complex neuronal toxicity, like that of Hb per se, was iron-dependent and reduced by deferoxamine and 2,2' bipyridyl. These results suggest that Hp increases the vulnerability of CD163+ neurons to Hb by permitting Hb uptake while attenuating the protective response of ferritin induction by glial cells. Cover Image for this issue: doi: 10.1111/jnc.13342.

Targeting heme oxygenase after intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is the primary event in approximately 10% of strokes, and has higher rates of morbidity and mortality than ischemic stroke. Experimental evidence suggests that the toxicity of hemoglobin and its degradation products contributes to secondary injury that may be amenable to therapeutic intervention. Hemin, the oxidized form of heme, accumulates in intracranial hematomas to cytotoxic levels. The rate limiting step of its breakdown is catalyzed by the heme oxygenase (HO) enzymes, which consist of inducible HO-1 and constitutively-expressed HO-2. The effect of these enzymes on perihematomal injury and neurological outcome has been investigated in ICH models using both genetic and pharmacological approaches to alter their expression, with variable results reported. These findings are summarized and reconciled in this review; therapeutic strategies that may optimize HO expression and activity after ICH are described.

Astrocyte overexpression of heme oxygenase-1 improves outcome after intracerebral hemorrhage.

BACKGROUND AND PURPOSE: Heme oxygenase-1 (HO-1) catalyzes the rate-limiting reaction of heme breakdown and may have both antioxidant and pro-oxidant effects. In previous studies, HO-1 overexpression protected astrocytes from heme-mediated injury in vitro. In the present study, we tested the hypothesis that selective astrocyte overexpression of HO-1 improves outcome after intracerebral hemorrhage. METHODS: Male and female transgenic mice overexpressing human HO-1 driven by the GFAP promoter (GFAP.HMOX1) and wild-type controls received striatal injections of autologous blood (25 µL). Blood-brain barrier disruption was assessed by Evans blue assay and striatal cell viability by methylthiazolyldiphenyl-tetrazolium bromide assay. Neurological deficits were quantified by digital analysis of spontaneous cage activity, adhesive removal, and elevated body swing tests. RESULTS: Mortality rate for wild-type mice was 34.8% and was similar for males and females; all GFAP.HMOX1 mice survived. Striatal Evans blue leakage at 24 hours was 23.4±3.2 ng in surviving wild-type mice, compared with 10.9±1.8 ng in transgenics. Perihematomal cell viability was reduced to 61±4% of contralateral at 3 days in wild-type mice, versus 80±4% in transgenics. Focal neurological deficits were significantly reduced and spontaneous cage activity was increased in GFAP.HMOX1 mice. CONCLUSIONS: Selective HO-1 overexpression in astrocytes reduces mortality, blood-brain barrier disruption, perihematomal cell injury, and neurological deficits in an autologous blood injection intracerebral hemorrhage model. Genetic or pharmacological therapies that acutely increase astrocyte HO-1 may be beneficial after intracerebral hemorrhage.

Neuroprotective effect of heme oxygenase-2 knockout in the blood injection model of intracerebral hemorrhage.

BACKGROUND: The toxicity of heme breakdown products may contribute to the pathogenesis of intracerebral hemorrhage (ICH). Heme catabolism is catalyzed by the heme oxygenase enzymes. We have previously reported that heme oxygenase-2 (HO-2), the constitutive isoform, protects neurons from hemin in vitro and reduces oxidative stress after striatal blood injection. In order to further evaluate HO-2 as a therapeutic target, we tested the hypothesis that HO-2 gene deletion protects neurons and attenuates behavioral deficits after ICH. FINDINGS: Injection of 20 µl blood into the right striatum of HO-2 wild-type mice resulted in loss of approximately one third of striatal neurons 4-8 days later. Neuronal survival was significantly increased in HO-2 knockout mice at both time points. This was associated with reduced motor deficit as detected by the corner test; however, no differences were detected in spontaneous activity or the adhesive removal or elevated body swing tests. CONCLUSION: HO-2 knockout attenuates perihematomal neuron loss in the blood injection ICH model, but has a weak and variable effect on neurological outcome.

Systemic hemin therapy attenuates blood-brain barrier disruption after intracerebral hemorrhage.

Injury to the blood-brain barrier (BBB) is a key feature of intracerebral hemorrhage (ICH) and may contribute to perihematomal cell injury. Pretreatment with the heme oxygenase (HO)-1 inducer hemin improves barrier function and neurological outcome in experimental models of traumatic and ischemic CNS injury. Since hemin is already in clinical use to treat acute porphyrias, this translational study was designed to test its effect on BBB function when initiated after ICH in two mouse models. At a dose similar to those used in most preconditioning studies (26mg/kg i.p.), post-hemorrhage treatment with hemin reduced parenchymal extravasation of Evans blue by about three-quarters in both the blood injection and collagenase ICH models. Similar efficacy was observed when treatment was begun at 1 or 3h. At the lower dose that is currently in clinical use (4mg/kg beginning at 3h), hemin also improved barrier function in both models, as assessed by both Evans blue and FITC-dextran leakage; however, it was somewhat less potent, reducing Evans blue leakage by about half. This dose was nevertheless sufficient to attenuate striatal cell loss and accelerate neurological recovery. Consistent with prior observations, striatal HO-1 expression was increased by hemin, and was localized to perivascular cells. These results suggest that hemin may be an effective therapy for ICH with a clinically relevant time window. Further study of the repurposing of this old drug seems warranted.

A rapid fluorescent method to quantify neuronal loss after experimental intracerebral hemorrhage.

Neuronal loss in tissue surrounding an intracerebral hemorrhage (ICH) is usually quantified by labor-intensive histological methods that are subject to bias. Fluorescent protein expression has been successfully used as a marker of cell viability in vitro and in retinal studies in vivo, but not in any ICH model to date. The potential of this approach was investigated using transgenic mice that constitutively express the red fluorescent protein variant dTomato in central neurons under the control of the Thy1 promoter. Breeding and growth of these mice were similar to their wild-type counterparts; behavioral phenotyping by digital analysis of home cage video recordings detected no differences. Bright fluorescence was evident in fresh brain samples with minimal background fluorescence, and was reduced in tissue surrounding the hematoma. In order to assess fluorescence loss as an injury marker in a planned study, these mice were crossed with heme oxygenase (HO)-2 knockouts and wild-type controls; striatal hemorrhage was induced by stereotactic injection of collagenase. Fluorescence in hemorrhagic striata was reduced to 86.4±3.9%, 62.2±5.1%, and 58.3±3.0% of contra-lateral on days 1, 4 and 8, respectively, and correlated closely with reduction in striatal cell viability as quantified by MTT assay. HO-2 knockout and wild-type values did not differ significantly. Similar results were observed with stereological cell counts of striatal neurons identified by NeuN immunoreactivity. These results suggest that loss of constitutive dTomato fluorescence is an accurate and efficient marker of neuronal loss in tissue surrounding a striatal hematoma.

Hemopexin decreases hemin accumulation and catabolism by neural cells.

Hemopexin is a serum, CSF, and neuronal protein that is protective after experimental stroke. Its efficacy in the latter has been linked to increased expression and activity of heme oxygenase (HO)-1, suggesting that it facilitates heme degradation and subsequent release of cytoprotective biliverdin and carbon monoxide. In this study, the effect of hemopexin on the rate of hemin breakdown by CNS cells was investigated in established in vitro models. Equimolar hemopexin decreased hemin breakdown, as assessed by gas chromatography, by 60-75% in primary cultures of murine neurons and glia. Extracellular hemopexin reduced cell accumulation of 55Fe-hemin by over 90%, while increasing hemin export or extraction from membranes by fourfold. This was associated with significant reduction in HO-1 expression and neuroprotection. In a cell-free system, hemin breakdown by recombinant HO-1 was reduced over 80% by hemopexin; in contrast, albumin and two other heme-binding proteins had no effect. Although hemopexin was detected on immunoblots of cortical lysates from adult mice, hemopexin knockout per se did not alter HO activity in cortical cells treated with hemin. These results demonstrate that hemopexin decreases the accumulation and catabolism of exogenous hemin by neural cells. Its beneficial effect in stroke models is unlikely to be mediated by increased production of cytoprotective heme breakdown products.

Effect of iron chelators on methemoglobin and thrombin preconditioning.

Cell loss immediately adjacent to an intracerebral hemorrhage may be mediated in part by the toxicities of extracellular hemoglobin (Hb) and thrombin. However, at low concentrations, these proteins induce tolerance to hemin and iron that may limit further peri-hematomal injury as erythrocyte lysis progresses. The mechanisms mediating these preconditioning effects have not been completely defined, but increased expression of both heme oxygenase (HO)-1 and iron binding proteins likely contributes. In the present study, we hypothesized that iron chelator therapy would attenuate this protective response. Pretreatment of cortical glial cultures (> 90 % GFAP+) with 3 µM methemoglobin (metHb) or 5 units/ml thrombin for 24 h was nontoxic per se, and increased HO-1 and ferritin expression. When challenged with a toxic concentration of hemin, the increase in cellular redox-active iron was attenuated in preconditioned cultures and cell survival was increased. However, if cultures were pretreated with metHb or thrombin plus deferoxamine or 2,2'-bipyridyl, ferritin induction was prevented and cellular redox-active iron increased with hemin treatment. Preconditioning-mediated cytoprotection was consistently reduced by deferoxamine, while 2,2'-bipyridyl had a variable effect. Neither chelator altered HO-1 expression. A cytoprotective response was preserved when chelator therapy was limited to 11 hours of the 24 h preconditioning interval. These results suggest a potentially deleterious effect of continuous iron chelator therapy after ICH. Intermittent therapy may remove peri-hematomal iron without negating the benefits of exposure to low concentrations of Hb or thrombin.

Iron accumulation and neurotoxicity in cortical cultures treated with holotransferrin.

Nonheme iron accumulates in CNS tissue after ischemic and hemorrhagic insults and may contribute to cell loss. The source of this iron has not been precisely defined. After blood-brain barrier disruption, CNS cells may be exposed to plasma concentrations of transferrin-bound iron (TBI), which exceed that in the CSF by over 50-fold. In this study, the hypothesis that these concentrations of TBI produce cell iron accumulation and neurotoxicity was tested in primary cortical cultures. Treatment with 0.5-3mg/ml holotransferrin for 24h resulted in the loss of 20-40% of neurons, associated with increases in malondialdehyde, ferritin, heme oxygenase-1, and iron; transferrin receptor-1 expression was reduced by about 50%. Deferoxamine, 2,2'-bipyridyl, Trolox, and ascorbate prevented all injury, but apotransferrin was ineffective. Cell TBI accumulation was significantly reduced by deferoxamine, 2,2'-bipyridyl, and apotransferrin, but not by ascorbate or Trolox. After treatment with (55)Fe-transferrin, approximately 40% of cell iron was exported within 16h. Net export was increased by deferoxamine and 2,2'-bipyridyl, but not by apotransferrin. These results suggest that downregulation of transferrin receptor-1 expression is insufficient to prevent iron-mediated death when neurons are exposed to plasma concentrations of TBI. Chelator therapy may be beneficial for acute CNS injuries associated with loss of blood-brain barrier integrity.

Apotransferrin protects cortical neurons from hemoglobin toxicity.

The protective effect of iron chelators in experimental models of intracerebral hemorrhage suggests that nonheme iron may contribute to injury to perihematomal cells. Therapy with high affinity iron chelators is limited by their toxicity, which may be due in part to sequestration of metals in an inaccessible complex. Transferrin is unique in chelating iron with very high affinity while delivering it to cells as needed via receptor-mediated endocytosis. However, its efficacy against iron-mediated neuronal injury has never been described, and was therefore evaluated in this study using an established cell culture model of hemoglobin neurotoxicity. At concentrations similar to that of CSF transferrin (50-100 micrograms/ml), both iron-saturated holotransferrin and apotransferrin were nontoxic per se. Overnight exposure to 3 µM purified human hemoglobin in serum-free culture medium resulted in death, as measured by lactate dehydrogenase release assay, of about three-quarters of neurons. Significant increases in culture iron, malondialdehyde, protein carbonyls, ferritin and heme oxygenase-1 were also observed. Holotransferrin had no effect on these parameters, but all were attenuated by 50-100 micrograms/ml apotransferrin. The effect of apotransferrin was very similar to that of deferoxamine at a concentration that provided equivalent iron binding capacity, and was not antagonized by concomitant treatment with holotransferrin. Transferrin receptor-1 expression was localized to neurons and was not altered by hemoglobin or transferrin treatment. These results suggest that apotransferrin may mitigate the neurotoxicity of hemoglobin after intracerebral hemorrhage. Increasing its concentration in perihematomal tissue may be beneficial.

Increased striatal injury and behavioral deficits after intracerebral hemorrhage in hemopexin knockout mice.

OBJECT: Heme toxicity may contribute to the pathogenesis of intracerebral hemorrhage (ICH). The primary defense against extracellular heme is provided by hemopexin, a serum and neuronal glycoprotein that binds it with very high affinity and mitigates its prooxidant effect. In the present study, the authors tested the hypothesis that hemopexin knockout mice would sustain more injury after experimental ICH than their wild-type counterparts. METHODS: Striatal ICH was induced by the stereotactic injection of bacterial collagenase or autologous blood. Three days later, striatal protein oxidation was assessed via carbonyl assay. Cell viability was quantified at 8-9 days by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Behavioral deficits were detected with high-resolution digital analysis of 6-hour home cage video recordings and standard testing. RESULTS: Perihematomal protein oxidation was increased in wild-type collagenase-injected striata by approximately 2.1-fold, as compared with contralateral striata; protein carbonyls were increased 3-fold in knockout mice. Striatal cell viability was reduced by collagenase injection in wild-type mice to 52.9 ± 6.5% of that in the contralateral striata, and to 31.1 ± 3.7% of that in the contralateral striata in knockout mice; similar results were obtained after blood injection. Digital analysis of 6-hour video recordings demonstrated an activity deficit in both models that was significantly exacerbated at 8 days in knockout mice. Striatal heme content 9 days after blood injection was increased approximately 2.7-fold in knockouts as compared with wild-type mice. CONCLUSIONS: These results suggest that hemopexin has a protective effect against hemorrhagic CNS injuries. Hemopexin deficiency, which is often associated with sickle cell disease, may worsen outcome after ICH.

Accelerated hemolysis and neurotoxicity in neuron-glia-blood clot co-cultures.

A growing body of experimental evidence suggests that an intracerebral hematoma is toxic to neighboring cells. However, injury mechanisms remain largely undefined, due in part to conflicting results from in vivo studies. In order to investigate blood toxicity in a more controlled environment, murine clots were co-cultured on porous membrane inserts with primary neurons and glia. Erythrocyte lysis was apparent within 48 h, but was reduced by almost 80% in cultures lacking neurons, and by over 90% in the absence of both neurons and glial cells. By 72 h, most released hemoglobin had oxidized to methemoglobin or its hemichrome degradation products. At this time point, approximately 50% of neurons were non-viable, as detected by propidium iodide staining; glia were not injured. Deferoxamine, Trolox and the NMDA receptor antagonist MK-801 prevented most neuronal death, but had no effect on hemolysis at neuroprotective concentrations. The 27-fold increase in culture malondialdehyde and 5.8-fold increase in heme oxygenase-1 expression were also attenuated by deferoxamine and Trolox, but not by MK-801. These results suggest that hemoglobin release from clotted blood is accelerated by adjacent neurons and glia. Subsequent neurotoxicity is mediated by both iron-dependent and excitotoxic injury pathways.

Iron regulatory protein-2 knockout increases perihematomal ferritin expression and cell viability after intracerebral hemorrhage.

Iron is deposited in perihematomal tissue after an intracerebral hemorrhage (ICH), and may contribute to oxidative injury. Cell culture studies have demonstrated that enhancing ferritin expression by targeting iron regulatory protein (IRP) binding activity reduces cellular vulnerability to iron and hemoglobin. In order to assess the therapeutic potential of this approach after striatal ICH, the effect of IRP1 or IRP2 gene knockout on ferritin expression and injury was quantified. Striatal ferritin in IRP1 knockout mice was similar to that in wild-type controls 3 days after stereotactic injection of artificial CSF or autologous blood. Corresponding levels in IRP2 knockouts were increased by 11-fold and 8.4-fold, respectively, compared with wild-type. Protein carbonylation, a sensitive marker of hemoglobin neurotoxicity, was increased by 2.4-fold in blood-injected wild-type striata, was not altered by IRP1 knockout, but was reduced by approximately 60% by IRP2 knockout. Perihematomal cell viability in wild-type mice, assessed by MTT assay, was approximately half of that in contralateral striata at 3 days, and was significantly increased in IRP2 knockouts but not in IRP1 knockouts. Protection was also observed when hemorrhage was induced by collagenase injection. These results suggest that IRP2 binding activity reduces ferritin expression in the striatum after ICH, preventing an optimal response to elevated local iron concentrations. IRP2 binding activity may be a novel therapeutic target after hemorrhagic CNS injuries.

Minocycline attenuates iron neurotoxicity in cortical cell cultures.

Iron neurotoxicity may contribute to the pathogenesis of intracerebral hemorrhage (ICH). The tetracycline derivative minocycline is protective in ICH models, due putatively to inhibition of microglial activation. Although minocycline also chelates iron, its effect on iron neurotoxicity has not been reported, and was examined in this study. Cortical cultures treated with 10 microM ferrous sulfate for 24h sustained loss of most neurons and an increase in malondialdehyde. Minocycline prevented this injury, with near-complete protection at 30 microM. Two other inhibitors of microglial activation, doxycycline and macrophage/microglia inhibitory factor, were ineffective. Oxidation of isolated culture membranes by iron was also inhibited by minocycline. Consistent with prior observations, minocycline chelated iron in a siderophore colorometric assay; at concentrations less than 100 microM, its activity exceeded that of deferoxamine. These results suggest that attenuation of iron neurotoxicity may contribute to the beneficial effect of minocycline in hemorrhagic stroke and other CNS injury models.