Brain edema formation and therapy after intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) accounts for about 10% of all strokes in the United States of America causing a high degree of disability and mortality. There is initial (primary) brain injury due to the mechanical disruption caused by the hematoma. There is then secondary injury, triggered by the initial injury but also the release of various clot-derived factors (e.g., thrombin and hemoglobin). ICH alters brain fluid homeostasis. Apart from the initial hematoma mass, ICH causes blood-brain barrier disruption and parenchymal cell swelling, which result in brain edema and intracranial hypertension affecting patient prognosis. Reducing brain edema is a critical part of post-ICH care. However, there are limited effective treatment methods for reducing perihematomal cerebral edema and intracranial pressure in ICH. This review discusses the mechanisms underlying perihematomal brain edema formation, the effects of sex and age, as well as how edema is resolved. It examines progress in pharmacotherapy, particularly focusing on drugs which have been or are currently being investigated in clinical trials.

Mechanisms of cerebrospinal fluid and brain interstitial fluid production.

Fluid homeostasis is fundamental for brain function with cerebral edema and hydrocephalus both being major neurological conditions. Fluid movement from blood into brain is one crucial element in cerebral fluid homeostasis. Traditionally it has been thought to occur primarily at the choroid plexus (CP) as cerebrospinal fluid (CSF) secretion due to polarized distribution of ion transporters at the CP epithelium. However, there are currently controversies as to the importance of the CP in fluid secretion, just how fluid transport occurs at that epithelium versus other sites, as well as the direction of fluid flow in the cerebral ventricles. The purpose of this review is to evaluate evidence on the movement of fluid from blood to CSF at the CP and the cerebral vasculature and how this differs from other tissues, e.g., how ion transport at the blood-brain barrier as well as the CP may drive fluid flow. It also addresses recent promising data on two potential targets for modulating CP fluid secretion, the Na+/K+/Cl- cotransporter, NKCC1, and the non-selective cation channel, transient receptor potential vanilloid 4 (TRPV4). Finally, it raises the issue that fluid secretion from blood is not constant, changing with disease and during the day. The apparent importance of NKCC1 phosphorylation and TRPV4 activity at the CP in determining fluid movement suggests that such secretion may also vary over short time frames. Such dynamic changes in CP (and potentially blood-brain barrier) function may contribute to some of the controversies over its role in brain fluid secretion.

Novel Approach to Visualize Microglia Death and Proliferation After Intracerebral Hemorrhage in Mice.

BACKGROUND: Microglia are important brain immune cells. However, it is difficult to differentiate microglia from monocyte-derived macrophages. To visualize microglia changes following intracerebral hemorrhage (ICH), we utilized a genetic knock-in mouse line, Tmem119 (transmembrane protein 119)-EGFP (enhanced green fluorescent protein), which expresses EGFP specifically in microglia. METHODS: There were 2 parts in this study. First, autologous blood was injected into the right basal ganglia to model ICH in Tmem119-EGFP mice. Mice were euthanized at 4 hours, days 1, 3, and 7 after ICH. Sham animals were used as controls. Second, Tmem119-EGFP mice were injected with iron or thrombin, factors involved in ICH-induced injury, and were euthanized at 4 hours. Naïve mice were controls. Brains were harvested for histology. RESULTS: The number of perihematomal microglia significantly decreased 1 day after ICH, but markedly increased by days 3 and 7. Microglia death was also induced by intracerebral iron injection while microglia proliferation was found with intracerebral thrombin injection. CONCLUSIONS: Perihematomal microglia death and proliferation after ICH are visualized in vivo with a Tmem119-EGFP transgenic mouse line. Iron and thrombin may contribute to ICH-induced microglia death and proliferation, respectively.

Delayed Minocycline Treatment Ameliorates Hydrocephalus Development and Choroid Plexus Inflammation in Spontaneously Hypertensive Rats.

Hydrocephalus is a complicated disorder that affects both adult and pediatric populations. The mechanism of hydrocephalus development, especially when there is no mass lesion present causing an obstructive, is poorly understood. Prior studies have demonstrated that spontaneously hypertensive rats (SHRs) develop hydrocephalus by week 7, which was attenuated with minocycline. The aim of this study was to determine sex differences in hydrocephalus development and to examine the effect of minocycline administration after hydrocephalus onset. Male and female Wistar-Kyoto rats (WKYs) and SHRs underwent magnetic resonance imaging at weeks 7 and 9 to determine ventricular volume. Choroid plexus epiplexus cell activation, cognitive deficits, white matter atrophy, and hippocampal neuronal loss were examined at week 9. In the second phase of the experiment, male SHRs (7 weeks old) were treated with either saline or minocycline (20 mg/kg) for 14 days, and similar radiologic, histologic, and behavior tests were performed. Hydrocephalus was present at week 7 and increased at week 9 in both male and female SHRs, which was associated with greater epiplexus cell activation than WKYs. Male SHRs had greater ventricular volume and epiplexus cell activation compared to female SHRs. Minocycline administration improved cognitive function, white matter atrophy, and hippocampal neuronal cell loss. In conclusion, while both male and female SHRs developed hydrocephalus and epiplexus cell activation by week 9, it was more severe in males. Delayed minocycline treatment alleviated hydrocephalus, epiplexus macrophage activation, brain pathology, and cognitive impairment in male SHRs.

Role of Complement Component 3 in Early Erythrolysis in the Hematoma After Experimental Intracerebral Hemorrhage.

Background and Purpose: Early erythrolysis occurs within the hematoma following intracerebral hemorrhage (ICH), and the release of erythrocyte cytoplasmic proteins such as hemoglobin and Prx2 (peroxiredoxin 2) can cause brain injury. Complement activation can induce erythrolysis. This study determined the function of complement component 3 (C3) in erythrolysis in hematoma and brain injury after ICH in mice. Methods: This study has 3 parts. First, ICH was induced in adult male C3-sufficient and deficient mice and animals were euthanized on days 1, 3, 7, and 28 for immunohistochemistry after magnetic resonance imaging and behavioral testing. Second, C3-sufficient and deficient mice with ICH were euthanized on day 1 for Western blot analysis. Third, C3-sufficient mice received injections of PBS and Prx2. Mice underwent both magnetic resonance imaging and behavioral tests on day 1 and were then euthanized. Brains were harvested for immunohistochemistry and Fluoro-Jade C staining. Results: Erythrolysis occurred in the hematoma in C3-sufficient and deficient mice on day 3 following ICH. C3-deficient mice had less erythrolysis, brain swelling, and neuronal degeneration in the acute phase and less brain atrophy in the chronic phase. There were fewer neurological deficits on days 3, 7, and 28 in C3-deficient mice. C3-deficient mice also had less extracellular Prx2 release. Moreover, Prx2 induced brain edema and brain injury and recruited macrophage scavenger receptor-1- and CD4-positive cells following ICH in mice. Conclusions: C3-deficient mice had less severe erythrolysis and brain injury following ICH compared with C3-sufficient mice. Prx2 released after erythrolysis can cause brain damage and neuroinflammation in mice.

Ultra-Early Cerebral Thrombosis Formation After Experimental Subarachnoid Hemorrhage Detected on T2* Magnetic Resonance Imaging.

BACKGROUND AND PURPOSE: The mechanisms of brain damage during ultra-early subarachnoid hemorrhage (SAH) have not been well studied. The current study examined the SAH-induced hyperacute brain damage at 4 hours using magnetic resonance imaging and brain histology in a mouse model. METHODS: SAH was induced by endovascular perforation in adult mice. First, adult male wild-type mice underwent magnetic resonance imaging T2 and T2* 4 hours after an endovascular perforation or a sham operation and were euthanized to assess brain histology. Second, male and female adult lipocalin-2 knockout mice had SAH. All animals underwent magnetic resonance imaging at 4 hours, and the brains were harvested for brain histology. RESULTS: T2* hypointensity vessels were observed in the brain 4 hours after SAH in male wild-type mice. The numbers of T2*-positive vessels were significantly higher in SAH brains than in sham-operated mice. Brain histology showed thrombosis and erythrocyte plugs in the T2*-positive cerebral vessels which may be venules. The number of T2*-positive vessels correlated with SAH grade and the presence of T2 lesions. Brain thrombosis was also accompanied by albumin leakage and neuronal injury. LCN2 deficient male mice had lower numbers of T2*-positive vessels after SAH compared with wild-type male mice. CONCLUSIONS: SAH causes ultra-early brain vessel thrombosis that can be detected by T2* gradient-echo sequence at 4 hours after SAH. LCN2 deficiency decreased the number of T2*-positive vessels.

CD47 blocking antibody accelerates hematoma clearance and alleviates hydrocephalus after experimental intraventricular hemorrhage.

Background CD47, a glycoprotein on red blood cell membranes, inhibits phagocytosis via interaction with signal regulatory protein α on phagocytes. Our previous research has demonstrated that blocking CD47 accelerates hematoma clearance and reduces brain injury after intracerebral hemorrhage. The current study investigated whether phagocytosis or erythrocyte CD47 impacts hematoma resolution and hydrocephalus development after intraventricular hemorrhage (IVH). Methods Adult (3-month-old) male Fischer 344 rats were intraventricularly injected with 200 µl autologous blood, mixed with either CD47 blocking antibody or isotype IgG, or 200 µl saline as control. In subgroups of CD47 blocking antibody treated rats, clodronate liposomes (to deplete microglia/monocyte-derived macrophages) or control liposomes were co-injected. Magnetic resonance imaging (MRI) was used to evaluate ventricular volume and intraventricular T2* lesion volume (estimating hematoma volume). The brains were harvested after 4 or 72 h for histology to evaluate phagocytosis. Results In adult male rats, CD47 blocking antibody alleviated hydrocephalus development by day 3. In addition, the CD47 blocking antibody reduced intraventricular T2* lesion and T2* non-hypointense lesion size after IVH through day 1 to day 3. Erythrophagocytosis was observed as soon as 4 h after IVH and was enhanced on day 3. Furthermore, intra-hematoma infiltration of CD68, heme oxygenase-1 and ferritin positive phagocytes were upregulated by CD47 blockade by day 3. Clodronate liposomes co-injection caused more severe hydrocephalus and weight loss. Conclusion Blocking CD47 in the hematoma accelerated hematoma clearance and alleviated hemolysis and hydrocephalus development after IVH, suggesting CD47 might be valuable in the future treatment for IVH.

Cerebrospinal Fluid from Aneurysmal Subarachnoid Hemorrhage Patients Leads to Hydrocephalus in Nude Mice.

OBJECTIVE: Our prior studies have found that intracerebroventricular injection of blood components can cause hydrocephalus and choroid plexus epiplexus cell activation in rats. To minimize the cross-species reaction, the current study examines whether intraventricular injection of acellular components of cerebrospinal fluid (CSF) from subarachnoid hemorrhage patients can cause hydrocephalus and epiplexus macrophage activation in nude mice which lack a T cell inflammatory response. METHODS: Adult male nude mice received intraventricular injections of acellular CSF from subarachnoid hemorrhage patients or a control patient. All mice had preoperative magnetic resonance imaging as baseline and postoperative scans at 24 h after CSF injection to determine ventricular volume. Brains were harvested at 24 h for brain histology, immunohistochemistry, and electron microscopy. RESULTS: Intraventricular injection of CSF from two of five subarachnoid hemorrhage patients obtained < 48 h from ictus resulted in ventricular enlargement at 24 h. CSF-related hydrocephalus was associated with activation of epiplexus macrophages and ependymal injury. CONCLUSIONS: Components of the acellular CSF of subarachnoid hemorrhage patients can cause epiplexus macrophage activation, ependymal cell damage, and ventricular enlargement in nude mice. This may serve as a unique model to study mechanisms of hydrocephalus development following subarachnoid hemorrhage.

Hydrocephalus Following Experimental Subarachnoid Hemorrhage in Rats with Different Aerobic Capacity.

Low aerobic capacity is considered to be a risk factor for stroke, while the mechanisms underlying the phenomenon are still unclear. The current study looked into the impacts of different aerobic capacities on early brain injury in a subarachnoid hemorrhage (SAH) model using rats bred for high and low aerobic capacity (high-capacity runners, HCR; low-capacity runners, LCR). SAH was modeled with endovascular perforation in HCR and LCR rats. Twenty-four hours after SAH, the rats underwent behavioral testing and MRI, and were then euthanized. The brains were used to investigate ventricular wall damage, blood-brain barrier breakdown, oxidative stress, and hemoglobin scavenging. The LCR rats had worse SAH grades (p < 0.01), ventricular dilatation (p < 0.01), ventricular wall damage (p < 0.01), and behavioral scores (p < 0.01). The periventricular expression of HO-1 and CD163 was significantly increased in LCR rats (p < 0.01 each). CD163-positive cells were co-localized with HO-1-positive cells. The LCR rats had greater early brain injuries than HCR rats. The LCR rats had more serious SAH and extensive ventricular wall damage that evolved more frequently into hydrocephalus. This may reflect changes in iron handling and neuroinflammation.

Prx2 (Peroxiredoxin 2) as a Cause of Hydrocephalus After Intraventricular Hemorrhage.

Background and Purpose- Our recent study demonstrated that release of Prx2 (peroxiredoxin 2) from red blood cells (RBCs) is involved in the inflammatory response and brain injury after intracerebral hemorrhage. The current study investigated the role of extracellular Prx2 in hydrocephalus development after experimental intraventricular hemorrhage. Methods- There were 4 parts in this study. First, Sprague-Dawley rats received an intraventricular injection of lysed RBC or saline and were euthanized at 1 hour for Prx2 measurements. Second, rats received an intraventricular injection of Prx2, deactivated Prx2, or saline. Third, lysed RBC was coinjected with conoidin A, a Prx2 inhibitor, or vehicle. Fourth, rats received Prx2 injection and were treated with minocycline or saline (i.p.). The effects of Prx2 and the inhibitors were examined using magnetic resonance imaging assessing ventriculomegaly, histology assessing ventricular wall damage, and immunohistochemistry to assess inflammation, particularly at the choroid plexus. Results- Intraventricular injection of lysed RBC resulted in increased brain Prx2 and hydrocephalus. Intraventricular injection of Prx2 alone caused hydrocephalus, ventricular wall damage, activation of choroid plexus epiplexus cells (macrophages), and an accumulation of neutrophils. Conoidin A attenuated lysed RBC-induced injury. Systemic minocycline treatment reduced the epiplexus cell activation and hydrocephalus induced by Prx2. Conclusions- Prx2 contributed to the intraventricular hemorrhage-induced hydrocephalus, probably by inducing inflammatory responses in choroid plexus and ventricular wall damage.

Endothelial Targets in Stroke: Translating Animal Models to Human.

Cerebral ischemia (stroke) induces injury to the cerebral endothelium that may contribute to parenchymal injury and worsen outcome. This review focuses on current preclinical studies examining how to prevent ischemia-induced endothelial dysfunction. It particularly focuses on targets at the endothelium itself. Those include endothelial tight junctions, transcytosis, endothelial cell death, and adhesion molecule expression. It also examines how such studies are being translated to the clinic, especially as adjunct therapies for preventing intracerebral hemorrhage during reperfusion of the ischemic brain. Identification of endothelial targets may prove valuable in a search for combination therapies that would specifically protect different cell types in ischemia.

Enhancement of Hematoma Clearance With CD47 Blocking Antibody in Experimental Intracerebral Hemorrhage.

Background and Purpose- Our previous studies found that erythrocyte CD47 has a role in regulating hematoma resolution following experimental intracerebral hemorrhage (ICH). The current study examined whether or not a CD47 blocking antibody enhances hematoma clearance in a mouse ICH. Methods- ICH was induced by intracaudate injection of autologous blood in adult C57BL/6 mice. Mice had an ICH or ICH with CD47 blocking antibody or IgG coinjection. In subgroups of CD47 blocking antibody-treated mice, clodronate (to deplete microglia/macrophages) or control liposomes were coinjected. The effects of CD47 blocking antibody on ICH-induced brain injury were also tested in both males and females. Mice had magnetic resonance imaging to examine clot volume, iron deposition, brain swelling, and brain tissue loss. Behavioral tests were performed in all mice, and brains were harvested for brain immunohistochemistry. Results- In male mice, CD47 blocking antibody speeded up hematoma/iron clearance by macrophages/microglia and reduced ICH-induced brain swelling, neuronal loss, and neurological deficits. In contrast, clodronate liposome-induced microglia/macrophage depletion caused more severe brain swelling, neuronal loss, and functional deficits. In addition, similar injury severity in males and females was found in IgG control group and CD47 blocking antibody was also effective in females. Conclusions- Blocking CD47 in the hematoma speeded hematoma clearance and reduced brain injury after ICH suggesting it could be a treatment for ICH patients with surgical clot removal.

Complement Inhibition Attenuates Early Erythrolysis in the Hematoma and Brain Injury in Aged Rats.

Background and Purpose- Early erythrolysis in the hematoma contributes to brain injury after intracerebral hemorrhage (ICH). This study investigated the effects of N-acetylheparin, a complement inhibitor, and aurin tricarboxylic acid, a membrane attack complex inhibitor, on early erythrolysis, brain iron deposition, and brain injury in aged rats. Methods- There were 3 parts in the study. First, aged (18 months old) male Fischer 344 rats had an ICH. The time course of erythrolysis in the hematoma was determined by T2* weighted magnetic resonance imaging, and the expression of CD163 was examined. Second, aged rats had an ICH with N-acetylheparin or vehicle. Rats were euthanized at days 1, 3, and 28 after magnetic resonance imaging (T2-, T2*-weighted, and T2* array) and behavioral tests. Brains were used for immunohistochemistry. Third, aged rats had an ICH with avaurin tricarboxylic acid or vehicle. The rats had magnetic resonance imaging and behavioral tests and were euthanized at day 3. Brains were used for immunohistochemistry. Results- Early erythrolysis occurred within the clot in aged F344 rats. There were increased numbers of CD163-positive cells after ICH. Almost all perihematomal CD163-positive cells were microglia/macrophages, while positive neurons were found more distant from the hematoma. Coinjection of N-acetylheparin attenuated erythrolysis, iron accumulation, CD163 expression, microglia activation, brain swelling, and neuronal death in the acute phase, as well as reducing brain atrophy and neurological deficits in the chronic phase. Coinjection of aurin tricarboxylic acid also reduced erythrolysis and ICH-induced brain injury. Conclusions- Inhibiting complement activation resulted in less erythrolysis and brain injury after ICH.

Minocycline attenuates brain injury and iron overload after intracerebral hemorrhage in aged female rats.

Brain iron overload is involved in brain injury after intracerebral hemorrhage (ICH). There is evidence that systemic administration of minocycline reduces brain iron level and improves neurological outcome in experimental models of hemorrhagic and ischemic stroke. However, there is evidence in cerebral ischemia that minocycline is not protective in aged female animals. Since most ICH research has used male models, this study was designed to provide an overall view of ICH-induced iron deposits at different time points (1 to 28 days) in aged (18-month old) female Fischer 344 rat ICH model and to investigate the neuroprotective effects of minocycline in those rats. According to our previous studies, we used the following dosing regimen (20 mg/kg, i.p. at 2 and 12 h after ICH onset followed by 10 mg/kg, i.p., twice a day up to 7 days). T2-, T2⁎-weighted and T2⁎ array MRI was performed at 1, 3, 7 and 28 days to measure brain iron content, ventricle volume, lesion volume and brain swelling. Immunohistochemistry was used to examine changes in iron handling proteins, neuronal loss and microglial activation. Behavioral testing was used to assess neurological deficits. In aged female rats, ICH induced long-term perihematomal iron overload with upregulated iron handling proteins, neuroinflammation, brain atrophy, neuronal loss and neurological deficits. Minocycline significantly reduced ICH-induced perihematomal iron overload and iron handling proteins. It further reduced brain swelling, neuroinflammation, neuronal loss, delayed brain atrophy and neurological deficits. These effects may be linked to the role of minocycline as an iron chelator as well as an inhibitor of neuroinflammation.

Minocycline Effects on Intracerebral Hemorrhage-Induced Iron Overload in Aged Rats: Brain Iron Quantification With Magnetic Resonance Imaging.

BACKGROUND AND PURPOSE: Brain iron overload is a key factor causing brain injury after intracerebral hemorrhage (ICH). This study quantified brain iron levels after ICH with magnetic resonance imaging R2* mapping. The effect of minocycline on iron overload and ICH-induced brain injury in aged rats was also determined. METHODS: Aged (18 months old) male Fischer 344 rats had an intracerebral injection of autologous blood or saline, and brain iron levels were measured by magnetic resonance imaging R2* mapping. Some ICH rats were treated with minocycline or vehicle. The rats were euthanized at days 7 and 28 after ICH, and brains were used for immunohistochemistry and Western blot analyses. Magnetic resonance imaging (T2-weighted, T2* gradient-echo, and R2* mapping) sequences were performed at different time points. RESULTS: ICH-induced brain iron overload in the perihematomal area could be quantified by R2* mapping. Minocycline treatment reduced brain iron accumulation, T2* lesion volume, iron-handling protein upregulation, neuronal cell death, and neurological deficits (P<0.05). CONCLUSIONS: Magnetic resonance imaging R2* mapping is a reliable and noninvasive method, which can quantitatively measure brain iron levels after ICH. Minocycline reduced ICH-related perihematomal iron accumulation and brain injury in aged rats.

CD163 Expression in Neurons After Experimental Intracerebral Hemorrhage.

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

Role of Lipocalin-2 in Thrombin-Induced Brain Injury.

BACKGROUND AND PURPOSE: Thrombin and lipocalin-2 (LCN2) contribute to intracerebral hemorrhage-induced brain injury. Thrombin-induced brain damage is partially through a thrombin receptor, protease-activated receptor-1. LCN2 is involved in cellular iron transport and neuroinflammation. This study investigated the role of LCN2 in thrombin-induced brain injury. METHODS: There were 3 parts in this study. First, male adult C57BL/6 wild-type or LCN2 knockout (LCN2 KO) mice had an intracaudate injection of thrombin (0.4 U) or saline. Second, LCN2 KO mice had an injection of thrombin (0.4 U) with recombinant mouse LCN2 protein (1 µg) into the right caudate. Third, protease-activated receptor-1 KO or wild-type mice had an intracaudate injection of thrombin or saline. All mice had T2-weighted magnetic resonance imaging and behavioral tests. Brains were used for histology, immunohistochemistry, and Western blotting. RESULTS: Intracerebral thrombin injection caused LCN2 upregulation and brain injury in mice. Thrombin-induced brain swelling, blood-brain barrier disruption, neuronal death, and neurological deficits were markedly less in LCN2 KO mice (P<0.05) and were exacerbated by exogenous LCN2 coinjection. In addition, thrombin injection resulted in less LCN2 expression and brain injury in protease-activated receptor-1 KO mice. CONCLUSIONS: Thrombin upregulates LCN2 through protease-activated receptor-1 activation and causes brain damage.

Role of Erythrocyte CD47 in Intracerebral Hematoma Clearance.

BACKGROUND AND PURPOSE: Enhancing hematoma clearance through phagocytosis may reduce brain injury after intracerebral hemorrhage. In the current study, we investigated the role of cluster of differentiation 47 (CD47) in regulating erythrophagocytosis and brain injury after intracerebral hemorrhage in nude mice. METHODS: This study was in 2 parts. First, male adult nude mice had an intracaudate injection of 30 µL saline, blood from male adult wild-type (WT) mice, or blood from CD47 knockout mice. Second, mice had an intracaudate injection of 30 µL CD47 knockout blood with clodronate or control liposomes. Clodronate liposomes were also tested in saline-injected mice. All mice then had magnetic resonance imaging to measure hematoma size and brain swelling. Brains were used for immunohistochemistry and Western blot. RESULTS: Erythrophagocytosis occurred in and around the hematoma. Injection of CD47 knockout blood resulted in quicker clot resolution, less brain swelling, and less neurological deficits compared with wild-type blood. Higher brain heme oxygenase-1 levels and more microglial activation (mostly M2 polarized microglia) at day 3 were found after CD47 knockout blood injection. Co-injection of clodronate liposomes, to deplete phagocytes, caused more severe brain swelling and less clot resolution. CONCLUSIONS: These results indicated that CD47 has a key role in hematoma clearance after intracerebral hemorrhage.

Hematoma Changes During Clot Resolution After Experimental Intracerebral Hemorrhage.

BACKGROUND AND PURPOSE: Hematoma clearance occurs in the days after intracerebral hemorrhage (ICH) and has not been well studied. In the current study, we examined changes in the hematoma in a piglet ICH model. The effect of deferoxamine on hematoma was also examined. METHODS: The ICH model was induced by an injection of autologous blood into the right frontal lobe of piglets. First, a natural time course of hematoma changes ≤7 days was determined. Second, the effect of deferoxamine on hematoma changes was examined. Hemoglobin and membrane attack complex levels in the hematoma were examined by enzyme-linked immunosorbent assay. Immunohistochemistry and Western blotting were used to examine CD47 (a regulator of erythrophagocytosis), CD163 (a hemoglobin scavenger receptor), and heme oxygenase-1 (a heme degradation enzyme) in the clot. RESULTS: After ICH, there was a reduction in red blood cell diameter within the clot with time. This was accompanied by membrane attack complex accumulation and decreased hemoglobin levels. Erythrophagocytosis occurred in the hematoma, and this was associated with reduced clot CD47 levels. Activated macrophages/microglia were CD163 and hemeoxygenase-1 positive, and these accumulated in the clot with time. Deferoxamine treatment attenuated the process of hematoma resolution by reducing member attack complex formation and inhibiting CD47 loss in the clot. CONCLUSIONS: These results indicate that membrane attack complex and erythrophagocytosis contribute to hematoma clearance after ICH, which can be altered by deferoxamine treatment.