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Evidence indicates that the complement system is activated and plays a role in brain injury after intracerebral hemorrhage (ICH). Most studies have focused on the role of C3, C5 and the membrane attack complex. The purpose of this study was to investigate the potential impact of complement C1q, a key upstream component of the classical pathway, on ICH-induced brain injury. Wild-type (WT) and C1qa knock out (KO) mice were compared using an autologous blood injection ICH model. Magnetic resonance imaging (MRI) was performed on days 1, 3 and 7 and brains harvested on days 3 and 7 for immunohistochemistry to examine brain injury mechanisms. WT and C1qa KO mice also received an intracerebral injection of thrombin, a key factor in ICH-induced brain injury. Following MRI scans, brains were harvested for immunohistochemistry on day 1. In comparison to WT mice, C1qa KO mice had reduced hematoma erythrolysis and neutrophil infiltration after ICH. However, they also had delayed hematoma clearance, which was associated with reduced induction of phagocytic multinuclear giant cells, and increased perihematomal neuronal damage. After thrombin injection, C1qa KO mice had smaller lesion volumes, less neuronal loss, reduced neutrophil infiltration, and less BBB damage. C1qa knockout has beneficial and detrimental effects on ICH-induced brain injury mechanisms, but a consistent beneficial effect after thrombin injection. Strategies to balance the roles of C1q after ICH may represent a promising therapeutic direction., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Blood components released by erythrolysis play an important role in secondary brain injury and posthemorrhagic hydrocephalus (PHH) after intraventricular hemorrhage (IVH). The current study examined the impact of N-acetylheparin (NAH), a complement inhibitor, on early erythrolysis, PHH and iron accumulation in aged rats following IVH. This study, on 18-months-old male Fischer 344 rats, was in 3 parts. First, rats had an intracerebroventricular injection of autologous blood (IVH) mixed with NAH or saline, or saline alone. After MRI at four hours, Western blot and immunohistochemistry examined complement activation and electron microscopy choroid plexus and periventricular damage. Second, rats had an IVH with NAH or vehicle, or saline. Rats underwent serial MRI at 4 h and 1 day to assess ventricular volume and erythrolysis. Immunohistochemistry and H&E staining examined secondary brain injury. Third, rats had an IVH with NAH or vehicle. Serial MRIs on day 1 and 28 assessed ventricular volume and iron accumulation. H&E staining and immunofluorescence evaluated choroid plexus phagocytes. Complement activation was found 4 h after IVH, and co-injection of NAH inhibited that activation. NAH administration attenuated erythrolysis, reduced ventricular volume, alleviated periventricular and choroid plexus injury at 4 h and 1 day after IVH. NAH decreased iron accumulation, the number of choroid plexus phagocytes, and attenuated hydrocephalus at 28 days after IVH. Inhibiting complement can reduce early erythrolysis, attenuates hydrocephalus and iron accumulation after IVH in aged animals., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Evidence indicates that erythrocyte-derived iron and inflammation both play a role in intraventricular hemorrhage-induced brain injury including hydrocephalus. Many immune-associated cells, primarily stromal macrophages, reside at the choroid plexus where they are involved in inflammatory responses and antigen presentation. However, whether intraventricular iron impacts those stromal cells is unknown. The aim of this study was to evaluate the relationship between choroid plexus stromal macrophages and iron-induced hydrocephalus in rats and the impact of minocycline and clodronate liposomes on those changes. Aged (18-month-old) and young (3-month-old) male Fischer 344 rats were used to study choroid plexus stromal macrophages. Rats underwent intraventricular iron injection to induce hydrocephalus and treated with either minocycline, a microglia/macrophage inhibitor, or clodronate liposomes, a macrophage depleting agent. Ventricular volume was measured using magnetic resonance imaging, and stromal macrophages were quantified by immunofluorescence staining. We found that stromal macrophages accounted for about 10% of the total choroid plexus cells with more in aged rats. In both aged and young rats, intraventricular iron injection resulted in hydrocephalus and increased stromal macrophage number. Minocycline or clodronate liposomes ameliorated iron-induced hydrocephalus and the increase in stromal macrophages. In conclusion, stromal macrophages account for ~10% of all choroid plexus cells, with more in aged rats. Treatments targeting macrophages (minocycline and clodronate liposomes) are associated with reduced iron-induced hydrocephalus., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

After a cerebral hemorrhage (intracerebral, subarachnoid, and intraventricular), extravasated blood contributes to both initial brain injury, via physical disruption and mass effect, and secondary injury, through the release of potentially neurotoxic and pro-inflammatory factors such as hemoglobin, iron, and peroxiredoxin-2. Erythrocytes are a major blood component and are a source of such damaging factors. Erythrolysis after cerebral hemorrhage releases potential neurotoxins, contributing to brain injury and edema. Alternatively, erythrocyte phagocytosis via microglia or macrophages may limit the spill of neurotoxins therefore limiting subsequent brain injury. The aim of this review is to discuss the process of phagocytosis of erythrocytes by microglia or macrophages after cerebral hemorrhage, the effect of erythrolysis on brain injury, novel mechanisms of erythrocyte and phagocyte egress from the brain, and exciting new targets in this pathway to attenuate brain injury. Understanding the fate of erythrocytes after cerebral hemorrhage may uncover additional potential interventions for clinical translational research., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Subarachnoid hemorrhage (SAH) is associated with a high incidence of morbidity and mortality, particularly within the first 72 h after aneurysm rupture. We recently found ultra-early cerebral thrombosis, detectable on T2* magnetic resonance imaging (MRI), in a mouse SAH model at 4 h after onset. The current study examined whether such changes also occur in rat at 24 h after SAH, the vessels involved, whether the degree of thrombosis varied with SAH severity and brain injury, and if it differed between male and female rats. Adult Sprague Dawley rats were subjected to an endovascular perforation SAH model or sham surgery and underwent T2 and T2* MRI 24 h later. Following SAH, increased numbers of T2* hypointense vessels were detected on MRI. The number of such vessels correlated with SAH severity, as assessed by MRI-based grading of bleeding. Histologically, thrombotic vessels were found on hematoxylin and eosin staining, had a single layer of smooth muscle cells on alpha-smooth muscle actin immunostaining, and had laminin 2α/fibrinogen double labeling, suggesting venule thrombosis underlies the T2*-positive vessels on MRI. Capillary thrombosis was also detected which may follow the venous thrombosis. In both male and female rats, the number of T2*-positive thrombotic vessels correlated with T2 lesion volume and neurological function, and the number of such vessels was significantly greater in female rats. In summary, this study identified cerebral venous thrombosis 24 h following SAH in rats that could be detected with T2* MRI imaging and may contribute to SAH-induced brain injury., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Intracerebral hemorrhage (ICH) is a stroke subtype with high mortality and severe morbidity. Hemorrhages frequently develop within the white matter, but whether white matter fibers within the hematoma survive after ICH has not been well studied. The current study examines whether white matter fibers persist in the hematoma after ICH, fibers that might be impacted by evacuation, and their relationship to macrophage infiltration in a porcine model. Male piglets had 2.5 ml blood with or without CD47 blocking antibody injected into the right frontal lobe. Brains were harvested from 3 days to 2 months after ICH for brain histology. White matter fibers were detected within the hematoma 3 and 7 days after hemorrhage by brain histology and myelin basic protein immunohistochemistry. White matter still remained in the hematoma cavity at 2 months after ICH. Macrophage scavenger receptor-1 positive macrophages/microglia and heme oxygenase-1 positive cells infiltrated into the hematoma along the intra-hematomal white matter fibers at 3 and 7 days after ICH. Treatment with CD47 blocking antibody enhanced the infiltration of these cells. In conclusion, white matter fibers exist within the hematoma after ICH and macrophages/microglia may use such fibers as a scaffold to infiltrate into the hematoma and aid in hematoma clearance., (© 2020. Springer Science+Business Media, LLC, part of Springer Nature.)

Thrombin is increased in the brain after hemorrhagic and ischemic stroke primarily due to the prothrombin entry from blood either with a hemorrhage or following blood-brain barrier disruption. Increasing evidence indicates that thrombin and its receptors (protease-activated receptors (PARs)) play a major role in brain pathology following ischemic and hemorrhagic stroke (including intracerebral, intraventricular, and subarachnoid hemorrhage). Thrombin and PARs affect brain injury via multiple mechanisms that can be detrimental or protective. The cleavage of prothrombin into thrombin is the key step of hemostasis and thrombosis which takes place in every stroke and subsequent brain injury. The extravascular effects and direct cellular interactions of thrombin are mediated by PARs (PAR-1, PAR-3, and PAR-4) and their downstream signaling in multiple brain cell types. Such effects include inducing blood-brain-barrier disruption, brain edema, neuroinflammation, and neuronal death, although low thrombin concentrations can promote cell survival. Also, thrombin directly links the coagulation system to the immune system by activating interleukin-1α. Such effects of thrombin can result in both short-term brain injury and long-term functional deficits, making extravascular thrombin an understudied therapeutic target for stroke. This review examines the role of thrombin and PARs in brain injury following hemorrhagic and ischemic stroke and the potential treatment strategies which are complicated by their role in both hemostasis and brain.

Macrophage phagocytosis plays an important role in hematoma clearance after intracerebral hemorrhage (ICH). This study examined the characteristics of multinucleated giant cells (MGCs), a group of macrophages with multiple nuclei, in a mouse ICH model. Whether MGCs could be increased by treatment with a CD47 blocking antibody and decreased by treatment with clodronate liposomes were also examined. ICH was induced via autologous blood injection. Male adult C57BL/6 mice in different groups had (1) ICH alone; (2) ICH with anti-CD47 blocking antibody or control IgG; and (3) ICH with anti-CD47 antibody combined with clodronate liposomes or control liposomes. The effect of anti-CD47 antibody on MGC formation was also tested in females. Brains were harvested at days 3 or 7 for brain histology. Many MGCs were found at day 3 post-ICH, but were reduced at day 7. MGCs phagocytosed many red blood cells and were heme oxygenase-1, ferritin, YM-1, and iNOS positive. CD47 blocking antibody injection increased MGC numbers in the peri-hematomal zone and in the hematoma in both sexes. Co-injection of clodronate liposomes depleted MGCs in both the hematoma core and the peri-hematomal area. In conclusion, MGCs represent a macrophage/microglia subtype with strong phagocytosis capacity. MGCs exhibited not only an M2 but also an M1 phenotype and appeared involved in hemoglobin degradation. Anti-CD47 antibody boosted the number of MGCs, which may contribute to enhance hematoma clearance. Understanding the exact roles of MGCs in ICH may reveal novel targets for ICH treatment.

Both experimental studies and surgical clinical trials suggest that hematoma clearance is a therapeutic target in intracerebral hemorrhage (ICH). We have investigated effects of CD47, a "don't eat me" signal expressed on erythrocytes, on hematoma resolution after ICH in young mice. This study expands those findings by examining the effects on a CD47 blocking antibody in aged rats. First, male Fischer 344 rats (18 months old) received an intracaudate injection of 50 μL autologous whole blood or saline. Hematoma features of magnetic resonance imaging (MRI) and neurological deficits were evaluated within 3 days. Second, rats had an intracaudate co-injection of 50 μL autologous blood with either CD47 blocking antibody or IgG. MRI was used to quantify hematoma/iron volume, hemolysis, brain swelling, and atrophy at different time points, behavioral tests to assess neurological deficits, and immunohistochemistry to assess brain injury and neuroinflammation. The CD47 blocking antibody significantly promoted hematoma clearance, attenuated brain swelling, hemolysis, and neuronal loss and increased the number of phagocytic macrophages in and around hematoma 3 days after ICH. Moreover, CD47 blockade reduced neuronal loss, brain atrophy, and neurobehavioral deficits at day 28. These results indicate that a CD47 blocking antibody can accelerate hematoma clearance and alleviate short- and long-term brain injury after ICH in aged rats and that it might be a therapeutic strategy for ICH.

Red blood cell (RBC) lysis within the hematoma causes brain injury following intracerebral hemorrhage. Peroxiredoxin 2 (PRX-2) is the third most abundant protein in RBCs and this study examined the potential role of PRX-2 in inducing brain injury in rats. First, adult male Sprague-Dawley rats had an intracaudate injection of lysed RBCs or saline. Brains were harvested at 1 h to measure PRX-2 levels. Second, rats had an intracaudate injection of either recombinant PRX-2, heat-inactivated PRX-2, or saline. Third, rats had intracaudate co-injection of lysed RBCs with conoidin A, a PRX-2 inhibitor, or vehicle. For the second and third parts of studies, behavioral tests were performed and all rats had magnetic resonance imaging prior to euthanasia for brain immunohistochemistry and Western blotting. We found that brain PRX-2 levels were increased after lysed RBC injection. Intracaudate injection of PRX-2 resulted in blood-brain barrier disruption, brain swelling, neutrophil infiltration, microglia activation, neuronal death, and neurological deficits. Intracerebral injection of lysed RBCs induced brain injury, which was reduced by conoidin A. These results suggest that extracellular PRX-2 released from hematoma can cause brain injury following brain hemorrhage and could be a potential therapeutic target.

Early hemolysis occurs in the hematoma within 24 h in rat model of intracerebral hemorrhage (ICH). The present study investigated the prevalence of early hemolysis in ICH patients using MRI and the relationship between early hemolysis and perihematomal edema. Thirty ICH patients were prospectively enrolled within 24 h of onset. All patients had cranial CT on admission. Cranial MRI with T2 FLAIR-weighted imaging and T2*-weighted imaging were undertaken at days 1 and 14. The evolution of a non-hypointense lesion on T2*-weighted images and the relationship between the volume of that non-hypointense lesion and perihematomal edema volume were investigated. MRI images of 15 patients were analyzed. The median hematoma volume was 16.3 ml on admission. All patients underwent a baseline MRI within 24 h of ICH onset and showed a non-hypointense lesion within the hematoma on T2*-weighted images. The volume of non-hypointense lesion on T2*-weighted image was 6.0 (8.9) ml at day 1 and 8.6 (17.3) ml at day 14. The absolute perihematomal edema volume was 16.0 (17.9) ml and 24.8 (27.5) ml at days 1 and 14, respectively. There was a linear correlation between non-hypointense T2* lesion and perihematomal edema volume at day 1 and day 14 (p < 0.01). Early hemolysis in the hematoma occurs in humans and contributes to the development of perihematomal edema.

Ceruloplasmin (CP) is an essential ferroxidase that is involved in maintaining iron homeostasis by oxidizing toxic ferrous iron (Fe 2+ ) to less-toxic ferric iron (Fe 3+ ). CP has been well studied in many neurodegenerative diseases, but there has not been an in-depth investigation in intracerebral hemorrhage (ICH). This research investigated brain CP expression in rats after ICH and the effect of CP on Fe 2+ -induced brain injury. This study had two parts: first, rats had injection of autologous blood into the right basal ganglia and the time course of CP expression in the brain examined (protein and mRNA). Second, rats had an injection of either Fe 2+ in saline, Fe 2+ plus CP in saline, or saline alone into the right basal ganglia. All rats in the second part had T2-weighted magnetic resonance imaging, and behavioral tests before the brains were harvested for immunohistochemistry and Western blotting. We found that CP was expressed on neurons and astrocytes in both cortex and basal ganglia after ICH. The time course showed that ICH induced CP expression increased from 4 h to 7 days, peaking at day 3. Whether the brain itself can produce CP was confirmed by RT-PCR. Exogenous CP reduced Fe 2+ -induced T2 lesions, blood-brain barrier disruption, brain cell death, and neurological deficits. These results suggest a role of CP in potentially reducing ICH-induced brain injury.

A number of mechanisms have been proposed for the early brain injury after subarachnoid hemorrhage (SAH). In this study, we investigated the radiographic characteristics and influence of gender on early brain injury after experimental SAH. SAH was induced by endovascular perforation in male and female rats. Magnetic resonance imaging was performed in a 7.0-T Varian MR scanner at 24 h after SAH. The occurrence and size of T2 lesions, ventricular dilation, and white matter injury (WMI) were determined on T2-weighted images (T2WI). The effects of SAH on heme oxygenase-1 and fibrin/fibrinogen were examined by Western blotting and immunohistochemistry. SAH severity was assessed using a MRI grading system, and neurological function was evaluated according to a modified Garcia's scoring system. T2 hyperintensity areas and enlarged ventricles were observed in T2WI coronal sections 24 h after SAH. The overall incidence of T2 lesions, WMI, and hydrocephalus was 54, 20, and 63%, respectively. Female rats had a higher incidence of T2 hyperintensity lesions and hydrocephalus, as well as larger T2 lesion volumes and higher average ventricular volume. SAH rats graded at 3-4 (our previously validated MRI grading scale) had larger T2 lesion volumes, more hydrocephalus, and worse neurological function compared with those graded at 0-2. In conclusion, T2 lesion, WMI, and hydrocephalus were the most prevalent MRI characteristics 24 h after experimental SAH. The T2 lesion area matched with fibrinogen/fibrin positive staining in the acute phase of SAH. SAH induced more severe brain injury in females compared to males in the acute phase of SAH.

Erythrolysis occurs in the clot after intracerebral hemorrhage (ICH), and the release of hemoglobin causes brain injury, but it is unclear when such lysis occurs. The present study examined early erythrolysis in rats. ICH rats had an intracaudate injection of 100 μl autologous blood, and sham rats had a needle insertion. All rats had T2 and T2* magnetic response imaging (MRI) scanning, and brains were used for histology and CD163 (a hemoglobin scavenger receptor) and DARPP-32 (a neuronal marker) immunohistochemistry. There was marked heterogeneity within the hematoma on T2* MRI, with a hyperintense or isointense core and a hypointense periphery. Hematoxylin and eosin staining in the same animals showed significant erythrolysis in the core with the formation of erythrocyte ghosts. The degree of erythrolysis correlated with the severity of perihematomal neuronal loss. Perihematomal CD163 was increased by day 1 after ICH and may be involved in clearing hemoglobin caused by early hemolysis. Furthermore, ICH resulted in more severe erythrolysis, neuronal loss, and perihematomal CD163 upregulation in spontaneously hypertensive rats compared to Wistar-Kyoto rats. In conclusion, T2*MRI-detectable early erythrolysis occurred in the clot after ICH and activated CD163. Hypertension is associated with enhanced erythrolysis in the hematoma.

Polarized microglia play a dual (beneficial/detrimental) role in neurological diseases. However, the status and the factors that modulate microglia polarization in intracerebral hemorrhage (ICH) remain unclear. In the present study, we investigated the role of protease-activated receptor-1 (PAR-1, a thrombin receptor) in ICH-induced microglia polarization in mice. Male wild-type (WT) and PAR-1 knockout (PAR-1 KO) mice received an infusion of 30-μL autologous blood or saline into the right basal ganglia. Mice were euthanized at different time points and the brains were used for Western blotting and immunohistochemistry. Some mice had magnetic resonance imaging. We found that ICH induced microglia activation and polarization. M1 phenotypic markers were markedly increased and reached a peak as early as 4 h, remained high at 3 days and decreased 7 days after ICH. M2 phenotypic markers were upregulated later than M1 markers reaching a peak at day 1 and declining by day 7 after ICH. PAR-1 was upregulated after ICH and expressed in the neurons and microglia. ICH induced less brain swelling and neuronal death in PAR-1 KO mice, and this was associated with less M1 polarization and reduced proinflammatory cytokine levels in the brain. In conclusion, these results suggest that polarized microglia occur dynamically after ICH and that PAR-1 plays a role in the microglia activation and polarization., Competing Interests: Shu Wan, Yingying Cheng, Hang Jin, Dewei Guo, Ya Hua, Richard F. Keep, and Guohua Xi declare that they have no conflict of interest.

Sphingolipids are a series of cell membrane-derived lipids which act as signaling molecules and play a critical role in cell death and survival, proliferation, recognition, and migration. Sphingosine-1-phosphate acts as a key signaling molecule and regulates lymphocyte trafficking, glial cell activation, vasoconstriction, endothelial barrier function, and neuronal death pathways which plays a critical role in numerous neurological conditions. Stroke is a second leading cause of death all over the world and effective therapies are still in great demand, including ischemic stroke and hemorrhagic stroke as well as poststroke repair. Significantly, sphingolipid activities change after stroke and correlate with stroke outcome, which has promoted efforts to testify whether the sphingolipid pathway could be a novel therapeutic target in stroke. The sphingolipid metabolic pathway, the connection between the pathway and stroke, as well as therapeutic interventions to manipulate the pathway to reduce stroke-induced brain injury are discussed in this review., Competing Interests: Na Sun, Richard F. Keep, Ya Hua, and Guohua Xi declare that they have no conflict of interest.

Early brain injury (EBI) plays a significant role in poor outcomes for subarachnoid hemorrhage (SAH) patients. Further investigations are required to characterize the cellular metabolic and related histological changes that may contribute to EBI following SAH. We investigated the image patterns of 18-fluorodeoxyglucose positron emission tomography-computed tomography ((18)FDG PET-CT) during EBI and correlated histopathological changes utilizing a rat SAH model. SAH was induced in six adult male Sprague-Dawley rats by endovascular perforation, and animals were randomly assigned to receive (18)FDG PET-CT imaging at either 3 or 12 h post-procedure. Mean (18)FDG standard uptake value (SUV) of the brain was calculated. Animals were euthanized 48 h post-procedure, and brain samples were used for heme oxygenase-1 (HO-1) and dopamine- and cAMP-regulated phosphoprotein (DARPP-32) Mr 32 kDa immunohistochemistry. Rats within the SAH group had higher mean whole brain (18)FDG SUV (2.349 ± 0.376 g/ml in the 3-h group and 2.453 ± 0.495 g/ml in the 12-h group) compared to that of sham (n = 3; mean SUV = 2.030 ± 0.247 g/ml; P < 0.05) or control groups (n = 3; mean SUV = 1.800 ± 0.484 g/ml; P < 0.05). Whole brain (18)FDG SUV did not vary significantly between rats imaged at 3 h vs. those imaged at 12 h post-SAH (P > 0.05). Regions of decreasing SUV in SAH rats correlated with neuronal death and increased expression of HO-1. Higher (18)FDG PET SUV was evident in rats post-SAH compared to sham and control groups. Regions of decreasing SUV in SAH rats correlated with neuronal death and increased HO-1 expression as evaluated by histopathology.

Brain iron overload has a key role in brain injury after intracerebral hemorrhage (ICH). Our recent study demonstrated that ICH-induced brain injury was greater in low capacity runner (LCR) than in high capacity runner (HCR) rats. The present study examines whether iron-induced brain injury differs between LCRs and HCRs. Adult male LCR and HCR rats had an intracaudate injection of iron or saline. Rats were euthanized at 2 and at 24 h after T2 magnetic resonance imaging, and the brains were used for immunostaining and Western blotting. LCRs had more hemispheric swelling, T2 lesion volumes, blood-brain barrier disruption, and neuronal death at 24 h after iron injection (p < 0.05). Many propidium iodide (PI)-positive cells, indicative of necrotic cell death, were observed in the ipsilateral basal ganglia of both HCRs and LCRs at 2 h after iron injection. PI fluorescence intensity was higher in LCRs than in HCRs. In addition, membrane attack complex (MAC) expression was increased at 2 h after iron injection and was higher in LCRs than in HCRs. The PI-positive cells co-localized with MAC-positive cells in the ipsilateral basal ganglia. Iron induces more severe necrotic brain cell death, brain swelling, and blood-brain barrier disruption in LCR rats, which may be related with complement activation and MAC formation.

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.

Thrombin causes blood-brain barrier disruption, and this study examined whether thrombin can cause brain hemorrhage through protease-activated receptor-1 (PAR-1). Male wild type and PAR-1 knockout mice had an intracerebral injection of thrombin or saline. Mice then underwent serial T2 magnetic resonance imaging and were euthanized for brain hemoglobin, iron, and interleukin-1β measurements. Thrombin caused massive T2 lesions and brain hemorrhage in wild type mice. These effects were markedly reduced in PAR-1 knockout mice. Thrombin also increased brain interleukin-1β, and this was absent in PAR-1 knockout mice. In conclusion, thrombin increases interleukin-1β levels and induces intracerebral hemorrhage through PAR-1 activation.

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.

Diverse preconditioning (PC) stimuli protect against a wide variety of neuronal insults in animal models, engendering enthusiasm that PC could be used to protect the brain clinically. Candidate clinical applications include cardiac and vascular surgery, after subarachnoid hemorrhage, and prior to conditions in which acute neuronal injury is anticipated. However, disappointments in clinical validation of multiple neuroprotectants suggest potential problems translating animal data into successful human therapies. Thus, despite strong promise of preclinical PC studies, caution should be maintained in translating these findings into clinical applications. The Stroke Therapy Academic Industry Roundtable (STAIR) working group and the National institute of Neurological Diseases and Stroke (NINDS) proposed working guidelines to improve the utility of preclinical studies that form the foundation of therapies for neurological disease. Here, we review the applicability of these consensus criteria to preconditioning studies and discuss additional considerations for PC studies. We propose that special attention should be paid to several areas, including 1) safety and dosage of PC treatments; 2) meticulously matching preclinical modeling to the human condition to be tested; and 3) timing of both the initiation and discontinuation of the PC stimulus relative to injury ictus.

Quantification of acute brain injury in basal ganglia is essential for mechanistic and therapeutic studies in experimental intracerebral hemorrhage (ICH). Using conventional counting of degenerating cells based on morphological or immunohistochemical criteria, it is hard to define the boundary of the whole lesion area. Dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa (DARPP-32) is a cytosolic protein highly enriched in medium-sized spiny neurons of the striatum. We developed new methods for quantifying lesion area by detecting the difference of the DARPP-32 negative area and the hematoma clot, and by measuring DARPP-32 protein level for semi-qualification in rat model of ICH. We found that DARPP-32 negative area around hematoma was present at day-1, peaked at day-3, and decreased at day-14 after ICH, a time course paralleled by DARPP-32 Western blots. The DARPP-32 negative area matched well with the necrotic area determined using propidium iodide. Treatment with an iron chelator, deferoxamine, attenuated the ICH-induced reduction in DARPP-32 protein levels. These results suggest that DARPP-32 is a simple and quantifiable indicator of ICH-induced neuronal death in basal ganglia.

Ischemic preconditioning (IPC) provides protection against subsequent severe ischemic injury. A recent study found that cerebral IPC prolongs bleeding time. In this study, we examined whether IPC protects against intra-cerebral hemorrhage (ICH)-induced brain edema formation and whether IPC affects blood coagulation. There were three sets of experiments in this study. In the first set, male Sprague-Dawley rats were preconditioned with either 15 min of left middle cerebral artery occlusion, an IPC stimulus, or a sham operation. Three days later, rats received an infusion of autologous whole blood in the ipsilateral or contralateral caudate. Rats were killed 24 h later for brain water content measurement. In the second set, rats underwent 15 min of IPC or a sham operation. Three days later, rats were used for bleeding and thrombin clotting time tests. In the third set, the levels of p44/42 mitogen-activated protein kinases (MAPKs), heme oxygenase-1 (HO-1), transferrin (Tf), and transferrin receptor (TfR) in the brain 24 or 72 h after IPC were examined. We found that IPC reduced ICH-induced brain edema when blood was injected into the ipsilateral caudate but it did not when blood was injected into the contralateral caudate. IPC resulted in prolongation of bleeding time and thrombin clotting time. IPC also induced the activation of p44/42 MAPKs and upregulation of HO-1, Tf, and TfR levels in the ipsilateral caudate. These results suggest that IPC protects against ICH-induced brain edema formation and decreases blood coagulation. The protection of IPC against ICH is mainly due to local factors in the brain and may be related to activation of p44/42 MAPKs and upregulation of HO-1, Tf, and TfR.

Intraventricular hemorrhage (IVH) is a cause of significant morbidity and mortality and is an independent predictor of a worse outcome in intracerebral hemorrhage (ICH) and germinal matrix hemorrhage (GMH). IVH may result in both injuries to the brain as well as hydrocephalus. This paper reviews evidence on the mechanisms and potential treatments for IVH-induced hydrocephalus. One frequently cited theory to explain hydrocephalus after IVH involves obliteration of the arachnoid villi by microthrombi with subsequent inflammation and fibrosis causing CSF outflow obstruction. Although there is some evidence to support this theory, there may be other mechanisms involved, which contribute to the development of hydrocephalus. It is also unclear whether the causes of acute and chronic hydrocephalus after hemorrhage occur via different mechanisms; mechanical obstruction by blood in the former, and inflammation and fibrosis in the latter. Management of IVH and strategies for prevention of brain injury and hydrocephalus are areas requiring further study. A better understanding of the pathogenesis of hydrocephalus after IVH, may lead to improved strategies to prevent and treat post-hemorrhagic hydrocephalus.

Brain edema is a major contributor to poor outcome following ischemic and hemorrhagic stroke. In animal models, edema has historically been quantified as a change in % brain water content (water content/wet weight). As described in this communication, this number can be misleading, as 'small' changes in % brain water content actually reflect much bigger changes in brain swelling. Using either water content, expressed as g/g dry weight, or a measure of brain swelling, better reflect the impact of edema after stroke and brain injury.

Brain microbleeds often occur in Alzheimer's disease patients. Our previous studies have demonstrated that iron contributes to brain injury following intracerebral hemorrhage. This study investigated the effect of iron on amyloid β (Aβ)-mediated brain injury. There were two parts to this study. In first part, rats received an intracaudate injection of saline, iron, Aβ 25-35 or iron+Aβ 25-35. In the second part, rats received intracaudate injection of iron+Aβ and were treated with saline or cystamine, an inhibitor of transglutaminase. Rats were killed after 24 hours for brain edema measurement. DNA damage, neuronal death and tissue-type transglutaminase (tTG) expression were also examined. We found that brain water content in the ipsilateral caudate was higher (p<0.05) in rats injected with iron+Aβ than with iron, Aβ or saline. Combined iron+Aβ injection also resulted in more severe DNA damage (both single- and double-strand; p<0.01) and more Fluoro-Jade C staining (p<0.05). Expression of tTG increased markedly in the iron+Aβ group (p<0.05) and treatment with a tTG inhibitor reduced brain edema (p<0.05) and reduced degenerating neurons (124±25 vs. 249±50/mm(2) in vehicle-treated group, p<0.05). These results suggest that increased brain iron from microbleeds may exaggerate brain Aβ toxicity and that tTG is involved in the enhanced toxicity.

Carbonic anhydrase-1 (CA-1) is a metalloenzyme present at high concentrations in erythrocytes. Our previous studies showed that erythrocyte lysis contributes to brain edema formation after intracerebral hemorrhage (ICH) and a recent study indicates that CA-1 can cause blood-brain barrier disruption. The present study investigated the role of CA-1 in ICH-induced brain injury.There were three groups in the study. In the first, adult male Sprague-Dawley rats received 100 μl autologous blood injection into the right caudate. Sham rats had a needle insertion. Rat brains were used for brain CA-1 level determination. In the second group, rats received an intracaudate injection of either 50 μl CA-1 (1 μg/μl) or saline. Brain water content, microglia activation and neuronal death (Fluoro-Jade C staining) were examined 24 hours later. In the third group, acetazolamide (AZA, 5 μl, 1 mM), an inhibitor of carbonic anhydrases, or vehicle was co-injected with 100 μl blood. Brain water content, neuronal death and behavioral deficits were measured. We found that CA-I levels were elevated in the ipsilateral basal ganglia at 24 hours after ICH. Intracaudate injection of CA-1 induced brain edema (79.0 ± 0.6 vs. 78.0±0.2% in saline group, p<0.01), microglia activation and neuronal death (p<0.01) at 24 hours. AZA, an inhibitor of CA, reduced ICH-induced brain water content (79.3 ± 0.7 vs. 81.0 ± 1.0% in the vehicle-treated group, p<0.05), neuronal death and improved functional outcome (p<0.05).These results suggest that CA-1 from erythrocyte lysis contributes to brain injury after ICH.

Our recent study has demonstrated that hemoglobin (Hb) is present in cerebral neurons and neuronal Hb is inducible after cerebral ischemia. In the present study, we examined the effects of intracerebral hemorrhage (ICH) on the mRNA levels of the α-globin (HbA) and the β-globin (HbB) components of Hb and Hb protein in the brain in vivo and in vitro. In vivo, male Sprague-Dawley rats received either a needle insertion (sham) or an infusion of autologous whole blood into the basal ganglia and were killed at different time points. In vitro, cultured rat brain cells were used for HbA, HbB and Hb determination. Cultured neurons were exposed to 50 or 100 μM hemin for 24 h. Some neurons also were treated with deferoxamine, an iron chelator, or vehicle. Levels of HbA and HbB, Hb and hemopexin, a transporter of heme, were measured. We found that HbA, HbB and Hb are primarily expressed in neurons, with much lower expression in astrocytes and microglia. HbA, HbB and Hb expression in the perihematomal zone was increased after ICH and Hb was localized in neurons and glia. Hemin increased HbA, HbB and hemopexin mRNA levels in cultured neurons. Deferoxamine reduced hemin-induced neuronal Hb expression. ICH increased HbA and HbB expression in the brain, which may potentially serve to buffer the heme released during clot resolution.

Background and Purpose: To examine the utility of magnetic resonance imaging (MRI) T2* sequences as a measure of iron overload in the brain following intracerebral hemorrhage (ICH)., Methods: We examined the time course of T2* changes in the brain around intracerebral hemorrhages in a series of patients. We also performed a series of experiments in an animal model of ICH, examining the time course of T2* changes along with correlation of these changes with histological markers of ferric iron deposition., Results: We found that T2* changes in the brain occur with increasing intensity and spatial distribution over a three month period. Experimental ICH in the rat model induces similar changes, and these changes correlate tightly with histological markers of ferric iron deposition., Conclusions: MRI T2* changes after ICH can be used to measure the degree of iron overload in the brain. The T2* sequence may be useful as a measure of interventions aimed at reducing ICH-related brain injury by reducing iron deposition.

Preconditioning (PC) describes a phenomenon whereby a sub-injury inducing stress can protect against a later injurious stress. Great strides have been made in identifying the mechanisms of PC-induced protection in animal models of brain injury. While these may help elucidate potential therapeutic targets, there are questions over the clinical utility of cerebral PC, primarily because of questions over the need to give the PC stimulus prior to the injury, narrow therapeutic windows and safety. The object of this review is to address the question of whether there may indeed be a clinical use for cerebral PC and to discuss the deficiencies in our knowledge of PC that may hamper such clinical translation.