Ya Hua. M.D. Grant Support

Experimental cerebral hemorrhage: Mechanisms and therapies

Xi G, Keep R, Hua Y
NIH R35 NS 11678601
5/1/2020 - 4/30/2028

Spontaneous intracerebral hemorrhage (ICH) is a common and often fatal stroke subtype. If the patient survives the ictus, the resulting hematoma within brain parenchyma triggers a series of events leading to secondary insults and severe neurological deficits. Although the hematoma in human gradually resolves within several months, restoration of function is graded and usually incomplete. The neurological deficits in ICH patients are permanent and disabling. For the past 20 years, I have been examining the hypothesis that the release of components (e.g., hemoglobin, iron and thrombin) from the hematoma contributes to ICH-induced brain injury. Our work on iron (released from hemoglobin) has led to the current Phase II clinical trial of deferoxamine for ICH (NCT02175225). This proposal continues to examine that underlying hypothesis. Specifically, it will address: 1) the mechanisms of early erythrocyte lysis in the hematoma; 2) the role of CD163 in hemoglobin clearance after ICH; 3) the mechanisms of endogenous hematoma removal; 4) the mechanisms of hydrocephalus development after intraventricular hemorrhage; and 5) combination therapies for ICH. The purpose of our project is to determine mechanisms of brain damage after brain hemorrhage and to develop effective therapies. The long-term goal of our studies is to limit hemorrhagic brain damage in patients.


Peroxiredoxin 2 and intracerebral hemorrhage-induced brain injury

Hua Y, Keep R, Xi G
NIH R21 NS 11239401
6/1/2019 – 5/31/2021

Intracerebral hemorrhage (ICH) is a stroke subtype with high mortality and patients who survive have major neurological deficits. Mass effect and release of hematoma components are two key factors causing brain injury following ICH. Many clinical trials have examined the effect of clot removal, but surgical evacuation has not been shown to be beneficial. Studies have demonstrated that hemolysis in the hematoma occurs the first day after ICH and erythrocyte lysis has a role in ICH-induced brain injury. Peroxiredoxin-2 is the third most abundant protein in red blood cells; peroxiredoxins are key initiators of brain inflammation after stroke. In our preliminary studies, we have also demonstrated: 1) Peroxiredoxin-2 levels are increased in the perihematomal zone after ICH; 2) Intra-caudate injection of peroxiredoxin-2 causes brain swelling, neuronal death and neurological deficits; 3) Conoidin A, an inhibitor of peroxiredoxins, reduces lysed red blood cell-induced brain swelling, neuronal loss and neurological deficits. In this application, therefore, we propose to test the following specific aims: 1) to examine natural history of peroxiredoxin-2 accumulation in the brain in an aged rat model of ICH; 2) to determine whether exogenous/extracellular peroxiredoxin-2 causes brain injury and neurological deficits; 3) to determine the effects of Peroxiredoxin inhibition on ICH- induced brain damage. The purpose of our project is to determine the role of peroxiredoxin-2, a major protein in red blood cells, in ICH-induced brain injury. The long-term goal of our study is to limit hemorrhagic brain damage and to improve functional outcome in patients.


Early hematoma lysis and hemoglobin toxicity in intracerebral hemorrhage

Keep R, Xi G, Hua Y, Xiang J
NIH R01 NS 10674603
3/15/2018 – 2/28/2023

There is much evidence that the hemoglobin released after erythrocyte lysis is a cause of brain injury after cerebral hemorrhage. This may be related to hemoglobin or its degradation products (e.g., iron). How to reduce such Injury is important considering there are no current clinically proven therapies for intracerebral hemorrhage. One mechanism that is involved in limiting hemoglobin toxicity systemically is CD163, a hemoglobin scavenger receptor, which is involved in the cellular uptake of hemoglobin when bound to haptoglobin. However, in cerebral hemorrhage, our recent results, supported by others, indicates that some hemoglobin is released before CD163 and other defense mechanisms are up-regulated in brain (early erythrolysis). In addition, while microglial CD163 may be beneficial in scavenging hemoglobin, CD163 is also up-regulated in neurons and is involved in inducing cell death. The aims of this proposal are, therefore: 1) Determine the mechanisms by which early hemoglobin release from cerebral hematomas occurs and can be reduced, 2) Examine whether CD163 is a therapeutic target in intracerebral hemorrhage, and 3) Determine the mechanisms regulating CD163 in microglia and neurons in order to potentially manipulate those levels independently. These experiments will involve in vivo and in vitro models of intracerebral hemorrhage in rats, mice and pig already established in our laboratories.


Iron, minocycline and brain injury after intracerebral hemorrhage

Xi G, Hua Y, Keep RF
NIH R01 NS09092505
5/1/2015 – 3/30/2021

Spontaneous intracerebral hemorrhage (ICH) is a common and often fatal stroke subtype. If the patient survives the ictus, the resulting hematoma within brain parenchyma triggers a series of events leading to secondary insults and severe neurological deficits. Although the hematoma in human gradually resolves within several months, restoration of function is graded and usually incomplete. The neurological deficits in ICH patients are permanent and disabling. Iron overload and oxidative stress contribute to brain damage after ICH. Both iron chelators and free radical scavengers can reduce ICH-induced brain injury in animals. minocycline, a second-generation tetracycline-based molecule, is a potent inhibitor of microglia activation. It is a highly lipophilic compound and penetrates the brain-blood barrier easily. Minocycline can chelate iron and a recent study has shown that minocycline attenuates Iron neurotoxicity in cortical neuronal culture by chelating iron. In our preliminary study, we also have demonstrated: 1) brain iron overload occurs in a rat model of ICH and minocycline reduces brain non-heme iron levels following ICH; 2) Co-injection of minocycline rather than macrophage/microglia inhibitory factor with Iron abolishes iron-induced brain edema in young rats; 3) minocycline reduces brainedema, brain atrophy and neurological deficits after ICH in young rats; 4) Levels of serum total iron are increased after ICH, which is reduced by systemic use of minocycline. However, major gaps in our knowledge regarding minocycline and ICH need to be filled. For example, it is not clear whether minocycline attenuates ICH-induced Iron overload and brain injury in a dose dependent manner, whether minocycline can reduce brain injury in aged ICH animals (ICH is primarily a disease of the elderly), and whether minocycline reduces white matter damage after ICH. In this application, therefore, we propose to test the following specific aims: 1) To determine whether minocycline acts as a combination therapy reducing ICH-induced brain injury via iron chelation and microglial inhibition in aged rats; 2) To determine whether minocycline reduces brain Ironoverload and brain injury after intracerebral hemorrhage in aged rats; 3) To determine whether minocycline reduces ICH-induced brain iron overload and brain damage in pigs. Data from the proposed studies are very useful for developing a minocycline-ICH trial. The purpose of our project is to determine whether minocycline reduces brain Ironoverload and ICH-induced brain damage in aged rats and pigs. The long-term goal of our studies is to limit hemorrhagic brain damage in patients.


Targeting CD47 to aid in clearing intracerebral hemorrhage

Xi G, Hua Y, Keep RF
NIH R01 NS09691705
5/1/2016 – 3/31/2021

Spontaneous intracerebral hemorrhage (ICH) is a common and often fatal stroke subtype. If the patient survives the ictus, the resulting hematoma within the brain parenchyma triggers a series of events leading to secondary insults and severe neurological deficits. After an ICH, lysis of erythrocytes in the hematoma and the mass itself result in brain swelling, neuronal death and neurological deficits. The hematoma can be removed by surgery or naturally by microglia/macrophages. Our previous studies demonstrated that surgical clot removal with tissue plasminogen activator (tPA) reduces acute perihematomal edema in pigs. Recent studies found that enhancing microglia/macrophage-mediated hematoma clearance reduces ICH-induced brain edema and improves functional outcome. CD47, also called integrin-associated protein, is expressed on erythrocytes and other cells regulating target cell phagocytosis. The role of CD47 in ICH has not been well studied. in preliminary studies, we found: 1) CD47 is expressed in rodent and pig erythrocytes; 2) Erythrophagocytosis occurs in the perihematomal area and in the clot in both mouse and pig ICH models; 3) Hematomaclearance is faster when the ICH is formed using CD47 knockout blood in Mice; 4) the injection of CD47 knockout blood causes less brain swelling and neurological deficits than wild-type blood; and 5) co-injection of clodronate liposomes depletes M2 microglia/macrophages and causes more brain swelling and less hematoma clearance. These results suggest that CD47 inhibition is a target for reducing ICH-induced brain Injury. Recently, there has been considerable interest in the cancer field in targeting CD47 with blocking antibodies to enhance phagocytosis and that approach is currently in clinical trial. in this application, we propose to test the following specific aims: 1) to examine whether CD47 levels in the hematoma are associated with Infiltration of macrophages/microglia into the clot and clot clearance; 2) To examine whether modifying erythrocyte CD47 levels in the clot or blocking CD47 will affect hematoma clearance and ICH-induced brain Injury in aged mice and in pigs; and 3) to determine whether blocking CD47 plus surgical clot removal with tPA will significantly reduce ICH-induced white matter injury. The long-term goal of our studies is to limit brain damage following ICH.

Principal Investigator

Ya Hua, M.D.

Research Professor, Neurosurgery
734-764-1207