Deferoxamine therapy for intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is a subtype of stroke with very high mortality. Experiments have indicated that clot lysis and iron play an important role in ICH-induced brain injury. Iron overload occurs in the brain after ICH in rats. Intracerebral infusion of iron causes brain edema and neuronal death. Deferoxamine, an iron chelator, is an FDA-approved drug for the treatment of acute iron intoxication and chronic iron overload due to transfusion-dependent anemia. Deferoxamine can rapidly penetrate the blood-brain barrier and accumulate in the brain tissue in significant concentration after systemic administration. We have demonstrated that deferoxamine reduces ICH-induced brain edema, neuronal death, brain atrophy, and neurological deficits. Iron chelation with deferoxamine could be a new therapy for ICH.

Blood-brain barrier function in intracerebral hemorrhage.

In this paper, we review current knowledge on blood-brain barrier (BBB) dysfunction following intracerebral hemorrhage (ICH). BBB disruption is a hallmark of ICH-induced brain injury. Such disruption contributes to edema formation, the influx of leukocytes, and the entry of potentially neuroactive agents into the perihematomal brain, all of which may contribute to brain injury. A range of factors have been implicated in inducing BBB disruption, including inflammatory mediators (e.g., cytokines and chemokines), thrombin, hemoglobin breakdown products, oxidative stress, complement, and matrix metalloproteinases. While there is interaction between some of these mediators, it is probable that prevention of ICH-induced BBB disruption will involve blocking multiple pathways or blocking a common end pathway (e.g., by stabilizing tight junction structure). While the effects of ICH on BBB passive permeability have been extensively examined, effects on other 'barrier' properties (metabolic and transport functions) have been less well-studied. However, recent data suggests that ICH can affect transport and that this may help protect the BBB and the brain. Indeed, it is possible in small bleeds that BBB disruption may be beneficial, and it is only in the presence of larger bleeds that disruption has detrimental effects.

Hyperbaric oxygen preconditioning activates ribosomal protein S6 kinases and reduces brain swelling after intracerebral hemorrhage.

BACKGROUND: New protein synthesis is key to ischemic tolerance induced by preconditioning and ribosomal protein S6 kinases (p70 S6 K) are important enzymes in protein synthesis. Hyperbaric oxygen preconditioning (HBOP) reduces ischemic brain damage. This study investigated if HBOP can activate p70 S6 K and increase new protein synthesis and if HBOP induces brain tolerance against brain swelling after intracerebral hemorrhage (ICH). METHODS: There were two parts of the studies. 1) Rats received five consecutive sessions of HBOP. Twenty-four hours after HBOP, the rats had an ICH and were sacrificed one or three days later for brain edema measurement. 2) Rats received five sessions of HBOP or control pretreatment and were sacrificed for Western blot analysis and immunohistochemistry of activated p70 S6 K and heme oxygenase-1 (HO-1). FINDINGS: Five sessions of HBOP significantly reduced brain edema in the ipsilateral basal ganglia after ICH. Western blot analysis showed that HBOP activated p70 S6 K and increased HO-1 levels in the basal ganglia. Strong activated p70 S6 K immunoreactivity was also found in the basal ganglia. CONCLUSIONS: Our results suggest activation of p70 S6 K may have a role in heat shock protein synthesis after HBOP and may contribute to HBOP-induced brain protection.

The radiographic evolution of a Schmorl's node.

We present a case showing the development of a Schmorl's node from its acute to chronic stage by serial MRI. In contrast to the mature Schmorl's node, imaging characteristics of the acute Schmorl's node have not been well-illustrated, particularly by contrast-enhanced images at various stages of development.

Iron-induced oxidative brain injury after experimental intracerebral hemorrhage.

We investigated the occurrence of DNA damage in brain after intracerebral hemorrhage (ICH) and the role of iron in such injury. Male Sprague-Dawley rats received an infusion of 100 microL autologous whole blood or 30 microL FeCl2 into the right basal ganglia and were sacrificed 1, 3, or 7 days later. 8-hydroxyl-2'-deoxyguanosine (8-OHdG) was analyzed by immunohistochemistry, while the number of apurinic/apyrimidinic abasic sites (AP sites) was also quantified. 8-OHdG and AP sites are two hallmarks of DNA oxidation. DNA damage was also examined using PANT and TUNEL labeling. Dinitrophenyl (DNP) was measured by Western blot to compare the time course of protein oxidative damage to that of DNA. DNA repair APE/Ref-1 and Ku-proteins were also measured by Western blot. Bipyridine, a ferrous iron chelator, was used to examine the role of iron in ICH-induced oxidative brain injury. An increase in 8-OHdG, AP sites, and DNP levels, and a decrease in APE/Ref-1 and Ku levels were observed. Abundant PANT-positive cells were also observed in the perihematomal area 3 days after ICH. Bipyridine attenuated ICH-induced changes in PANT and DNP. These results suggest that iron-induced oxidation causes DNA damage in brain after ICH and that iron is a therapeutic target for ICH.

Deferoxamine reduces CSF free iron levels following intracerebral hemorrhage.

Iron overload occurs in brain after intracerebral hemorrhage (ICH). Deferoxamine, an iron chelator, attenuates perihematomal edema and oxidative stress in brain after ICH. We investigated the effects of deferoxamine on cerebrospinal fluid (CSF) free iron and brain total iron following ICH. Rats received an infusion of 100-microL autologous whole blood into the right basal ganglia, then were treated with either deferoxamine (100 mg/kg, i.p., administered 2 hours after ICH and then at 12-hour intervals for up to 7 days) or vehicle. The rats were killed at different time points from 1 to 28 days for measurement of free and total iron. Behavioral tests were also performed. Free iron levels in normal rat CSF were very low (1.1 +/- 0.4 micromol). After ICH, CSF free iron levels were increased at all time points. Levels of brain total iron were also increased after ICH (p < 0.05). Deferoxamine given 2 hours after ICH reduced free iron in CSF at all time points. Deferoxamine also reduced ICH-induced neurological deficits (p < 0.05), but did not reduce total brain iron. In conclusion, CSF free iron levels increase after ICH and do not clear for at least 28 days. Deferoxamine reduces free iron levels and improves functional outcome in the rat, indicating that it may be a potential therapeutic agent for ICH patients.

Effects of endogenous and exogenous estrogen on intracerebral hemorrhage-induced brain damage in rats.

The present study examined differences in intracerebral hemorrhage (ICH)-induced brain injury in male and female rats, whether delayed administration of 17beta-estradiol can reduce ICH-induced brain damage, and whether these effects are estrogen receptor (ER)-dependent. Male and female Sprague-Dawley rats received an infusion of 100-microL autologous whole blood into the right basal ganglia. The effects of 1beta-estradiol (5 mg/kg, i.p.) on ICH-induced brain injury were examined by measuring brain edema and neurological deficits 24 hours later. Heme oxygenase-1 (HO-1) was investigated by immuno-analysis. Brain edema was significantly less in female compared to male rats. The ER antagonist ICI182,780 exacerbated ICH-induced brain edema in female but not in male rats, suggesting that ER activation during ICH is protective in female rats. Administration of 17beta-estradiol to male (but not female) rats significantly reduced brain edema, neurological deficits, and ICH-induced increases in brain HO-1 levels when given 2 hours after ICH. This study showed that female rats have less ICH-induced injury than male rats. ER is involved in limiting ICH-induced injury in female rats. ICH-injury in male rats can be reduced by 17beta-estradiol. Since 17beta-estradiol treatment was effective in male rats, it could be a potential therapeutic agent for ICH.

Intracerebral hemorrhage in complement C3-deficient mice.

The complement cascade is activated and contributes to brain damage after intracerebral hemorrhage (ICH). The present study investigated ICH-induced brain damage in complement C3-deficient mice. This study was divided into 2 parts. Male C3-deficient and C3-sufficient mice received an infusion of 30-microl autologous whole blood into the right basal ganglia. In the first part of our study, mice were killed 3 days later for brain water content measurement. Behavioral assessments including forelimb use asymmetry and corner turn tests were also preformed before and after ICH. In the second part of the study, brain heme oxygenase-1 (HO-1) was measured by Western blot analysis and immunohistochemistry 3 days after the infusion. We found that brain water content in the ipsilateral basal ganglia 3 days after ICH was less in C3-deficient mice compared to C3-sufficient mice (p < 0.05). The C3-deficient mice had reduced ICH-induced forelimb use asymmetry deficits compared with C3-sufficient mice (p < 0.05), although there was no significant difference in the corner turn test score. Western blot analysis showed that HO-1 contents were significantly lower in C3-deficient mice (day 3: 2024 +/- 560 vs. 5140 +/- 1151 pixels in the C3-sufficient mice, p < 0.05). We conclude that ICH causes less brain edema and behavioral deficits in complement C3-deficient mice. These results suggest that complement C3 is a key factor contributing to brain injury following ICH.

Systemic zinc protoporphyrin administration reduces intracerebral hemorrhage-induced brain injury.

Hemoglobin degradation products result in brain injury after intracerebral hemorrhage (ICH). Recent studies found that intracerebral infusion of heme oxygenase inhibitors reduces hemoglobin- and ICH-induced brain edema in rats and pigs. The present study examined whether systemic use of zinc protoporphyrin (ZnPP), a heme oxygenase inhibitor, can attenuate brain edema, behavioral deficits, and brain atrophy following ICH. All rats had intracerebral infusion of 100-microL autologous blood. ZnPP (1 nmol/hour/rat) or vehicle was given immediately or 6 hours following ICH. ZnPP was delivered intraperitoneally up to 14 days through an osmotic mini-pump. Rats were killed at day 3 and day 28 after ICH for brain edema and brain atrophy measurements, respectively. Behavioral tests were performed. We found that ZnPP attenuated brain edema in animals sacrificed 3 days after ICH (p < 0.05). ZnPP also reduced ICH-induced caudate atrophy (p < 0.05) and ventricular enlargement (p < 0.05). In addition, ZnPP given immediately or 6 hours after ICH improved neurological deficits (p < 0.05). In conclusion, systemic zinc protoporphyrin treatment started at 0 or 6 hours after ICH reduced brain edema, neurological deficits, and brain atrophy after ICH. These results indicate that heme oxygenase may be a new target for ICH therapeutics.

Presentation of intracerebral haemorrhage in a community.

BACKGROUND: Studies on intracerebral haemorrhage (ICH) from tertiary care centres may not be an accurate representation of the true spectrum of disease presentation. OBJECTIVE: To describe the clinical and imaging presentation of ICH in a community devoid of the referral bias of an academic medical centre; and to investigate factors associated with lower Glasgow coma scale (GCS) score at presentation, as GCS is crucial to early clinical decision making. METHODS: The study formed part of the BASIC project (Brain Attack Surveillance in Corpus Christi), a population based stroke surveillance study in a bi-ethnic Texas community. Cases of first non-traumatic ICH were identified from years 2000 to 2003, using active and passive surveillance. Clinical data were collected from medical records by trained abstractors, and all computed tomography (CT) scans were reviewed by a study physician. Multivariable linear regression was used to identify clinical and CT predictors of a lower GCS score. RESULTS: 260 cases of non-traumatic ICH were identified. Median ICH volume was 11 ml (interquartile range 3 to 36) with hydrocephalus noted in 45%. Median initial GCS score was 12.5 (7 to 15). Hydrocephalus score (p = 0.0014), ambient cistern effacement (p = 0.0002), ICH volume (p = 0.014), and female sex (p = 0.024) were independently associated with lower GCS score at presentation, adjusting for other variables. CONCLUSIONS: ICH has a wide range of severity at presentation. Hydrocephalus is a potentially reversible cause of a lower GCS score. Since early withdrawal of care decisions are often based on initial GCS, recognition of the important influence of hydrocephalus on GCS is warranted before withdrawal of care decisions are made.

The role of thrombin in gliomas.

BACKGROUND: In a previous study we found that intracerebral infusion of argatroban, a specific thrombin inhibitor, reduces brain edema and neurologic deficits in a C6 glioma model. OBJECTIVES: To examine the role of thrombin in gliomas and whether systemic argatroban administration can reduce glioma mass and neurologic deficits and extend survival time in C6 and F98 gliomas. METHODS: The presence of thrombin in human glioblastoma samples and rat C6 glioma cells (in vitro and in vivo) was assessed using immunohistochemistry. The effect of thrombin on C6 cell proliferation in vitro was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. The role of thrombin in vivo was assessed in rat C6 and F98 glioma cell models using argatroban, a thrombin inhibitor. The effects of argatroban on tumor mass, neurologic deficits and survival time were investigated. RESULTS: Thrombin immunoreactivity was found in cultured rat C6 glioma cells and human glioblastomas. Thrombin induced C6 cell proliferation in vitro. In C6 glioma, argatroban reduced glioma mass (P < 0.05) and neurologic deficits (P < 0.05) at day 9. In F98 glioma, argatroban prolonged survival time (P < 0.05). CONCLUSION: These results suggest that thrombin plays an important role in glioma growth. Thrombin may be a new therapeutic target for gliomas.

Complement inhibition attenuates brain edema and neurological deficits induced by thrombin.

The present study examined whether thrombin activates the complement cascade in the brain and whether N-acetylheparin, an inhibitor of complement activation, attenuates brain injury induced by thrombin. There were three sets of studies. In the first set, rats had an intracerebral infusion of either five-unit thrombin or a needle insertion. Brains were sampled at 24 hours for Western blot analysis and immuno-histochemistry. In the second set, rats received either five-unit thrombin+saline, five-unit thrombin+25 microg N-acetylheparin or five-unit thrombin+100 microg N-acetylheparin infusion. Brains were sampled 24 hours later for water content measurement. In the third set, rats received either five-unit thrombin+saline or five-unit thrombin+ 100 microg N-acetylheparin. Behavioral tests sensitive to unilateral striatal damage were carried out for two weeks. Western blotting demonstrated that complement C9 and clusterin levels increase 24 hours after thrombin infusion (P < 0.01). Both C9 and clusterin positive cells were found around the injection site. High-dose (100-microg) but not low-dose (25-microg) N-acetylheparin attenuated thrombin-induced brain edema (81.5 +/- 0.4% vs. 83.7 +/- 0.3% in the vehicle, P < 0.05). Behavior was also significantly improved by N-acetylheparin (P < 0.05). In conclusion, thrombin-induced edema formation and neurological deficits were both reduced by N-acetylheparin. This suggests that inhibition may be a novel treatment for the thrombin-induced brain injury that occurs in intracerebral hemorrhage.

Systemic use of argatroban reduces tumor mass, attenuates neurological deficits and prolongs survival time in rat glioma models.

Our previous studies showed that intracerebral infusion of argatroban, a specific thrombin inhibitor, reduces brain edema and neurological deficits in a C6 glioma model. The present study investigated whether systemic argatroban administration can reduce glioma mass and neurological deficits and extend survival time in C6 and F98 gliomas. Rat C6 or F98 glioma cells were infused into the right caudate of adult male Fischer 344 rats. Osmotic minipump loaded with argatroban (0.3 mg/hour) or vehicle was implanted into abdomen immediately after glioma implantation. Tumor mass was determined at day 9. Over the period of the experiment, the animals underwent behavioral testing (forelimb placing and forelimb use asymmetry). In addition, survival time was tested in the F98 glioma model. In C6 glioma, argatroban reduced glioma mass (p < 0.05) and neurological deficits (p < 0.05) at day 9. In F98 glioma, agratroban prolonged the survival time (p < 0.05) and reduced the body weight loss (84 +/- 15 gram vs. 99 +/- 2 gram in the vehicle group, P < 0.05). In conclusion, systemic use of argatroban reduced tumor mass and neurological deficits, and prolonged survival time. These results suggest that thrombin plays a key role in glioma growth and thrombin inhibition with argatroban may be a novel treatment for gliomas.

Intracerebral hemorrhage induces edema and oxidative stress and alters N-methyl-D-aspartate receptor subunits expression.

Intracerebral hemorrhage (ICH) induces brain edema formation via a variety of mechanisms including toxicity due to thrombin and erythrocyte lysis. However, the roles of oxidative damage and excitotoxicity have not been fully elucidated and they are examined in this rat ICH study. Adult male Sprague-Dawley rats received an intracaudate injection of 100 microl autologous whole blood and 5 U of thrombin. Rats were sacrificed at 1 hour, 1 and 3 days, and then the brains processed using Western blotting to quantify N-methyl-D-aspartate receptor (NR) subunit expression. At 3 days, animals were also sacrificed for assessment of protein oxidation using Western blot analysis for dinitrophenyl (DNP) and brain water content. Compared to the contralateral side, ipsilateral basal ganglia NR1 and NR2A subunit expression transiently increased at 1 hour after ICH and thrombin injection. From 24 hours there was a marked down-regulation. At 3 days, marked edema and DNP up-regulation were observed in ICH and thrombin injection groups. The present NR expression up-regulation at 1 hour may reflect the acute cell response after ICH. The down-regulation of NR subunits and upregulation of DNP may be associated with cell damage, towards which thrombin may contribute.

Aging enhances intracerebral hemorrhage-induced brain injury in rats.

Age is an important factor affecting oxidative stress and plasticity after brain injury. The present study investigated the effects of aging on brain injury after intracerebral hemorrhage (ICH). Aging (18-month) and young (3-month) male Sprague-Dawley rats received an intracerebral infusion of 100-microl autologous blood. Age-related changes in brain edema and neurological deficits were examined and heat shock protein 27 (HSP27) and heat shock protein 32 (HSP32) levels were determined by Western blotting. Perihematomal brain swelling was more severe in aged rats compared to young rats at three days after ICH (P < 0.05). The behavioral tests used were forelimb placing test and forelimb use asymmetry test. There were more severe neurological deficits and a slower recovery in aged rats compared to those in young rats after ICH (P < 0.05). In addition, perihematomal HSP27 and HSP32 protein levels were higher (p < 0.05) in aged rats. In conclusion, ICH causes more severe brain swelling and neurological deficits in aged rats. Clarification of the mechanisms of brain injury after ICH in the aging brain should help develop new therapeutic strategies for hemorrhagic brain injury.

Thrombin preconditioning upregulates transferrin and transferrin receptor and reduces brain edema induced by lysed red blood cells.

Pretreatment with a low dose of thrombin reduces brain edema after both hemorrhagic and ischemic stroke. We call this phenomenon thrombin preconditioning (TPC) or thrombin-induced brain tolerance. The present study examines whether TPC can attenuate the brain edema induced by lysed red blood cells (RBCs) to determine whether thrombin production early in an intracerebral hemorrhage (ICH) might alter potentially injurious events associated with clot resolution. It also examines whether TPC might be protective by altering iron handling within the brain, particularly through modulating transferrin (Tf) and transferrin receptor (TfR) levels. Brain edema was measured by wet/dry weight. Western blot analysis and immunohistochemistry were used for Tf and TfR measurements. We found that TPC reduces lysed RBC-induced brain edema and upregulates both Tf and TfR levels in the brain. Thrombin formation after an ICH may be part of a signaling cascade that acts to limit potentially injurious events associated with clot resolution through altering iron-handling proteins.

Overexpression of interleukin-1 receptor antagonist reduces brain edema induced by intracerebral hemorrhage and thrombin.

Recent studies indicate that inflammatory reaction occurs around hematoma after intracerebral hemorrhage (ICH). In this study the authors examine the hypothesis that overexpression of IL-1ra in the brain attenuate brain edema formation after ICH. Adenoviruses expressing IL-1ra (Ad.RSVIL-1ra) or LacZ (Ad.RSVLacZ) or saline were injected into the lateral ventricle. On the fifth day after virus injection, 100 microl of autologous blood or 5 U thrombin was infused into the right basal ganglia. Rats with ICH were killed 24 or 72 hours later for measurement of brain water content. Thrombin-treated rats were killed 24 hours later for edema measurements and an assessment of polymorphonuclear leukocyte (PMNL) infiltration by myeloperoxidase (MPO) assay. Compared with control groups, Ad.RSVIL-1ra treated rats had less brain edema formation in the ipsilateral basal ganglia 3 days after ICH (81.5 +/- 0.3% compared with 83.4 +/- 0.4% and 83.3 +/- 0.5% in control animals). Ad.RSVIL-1ra treated rats had also less brain edema following thrombin injection. The reduction of brain edema induced by thrombin was involved in the reduction of PMNL infiltration in basal ganglia, as assessed by MPO assay. Adenovirus-mediated overexpression of IL-1ra attenuated brain edema formation following ICH, perhaps by reduction of thrombin-induced brain inflammation.