Richard Keep, Ph.D. Grant Support

Phenomenon of ultra-early erythrolysis in intracranial hemorrhage in humans on MRI

Pandey AS, Chaudhary N, Xi G, Keep R
NIH R21 NS 10466302
9/30/2018 – 8/31/2021

Intracerebral hemorrhage is a subtype of hemorrhagic stroke that has devastating consequences when it occurs in humans. Broadly there are two mechanisms of neuronal damage that occurs in the event of an ICH. The early brain injury is secondary to the hematoma itself and early erythrolysis within 24 hours of the injury. Delayed brain injury is mediated by various pathways related to the scavenging of the hematoma by the macrophage system. There is reasonable animal model data that validates pathomechanisms of delayed neurotoxicity following an ICH. Ultra-early erythrolysis is a novel phenomenon that has been observed in a recent animal ICH model study by the authors. In this rat model there is good correlation of early erythrolysis with hypo or isointensity on T2* sequences within the hematoma within first 24 hours after an ICH and increased tissue iron levels in the perihematomal brain tissue on histology. There is no such correlative phenomenon that has been documented in the human population. The proposed study will attempt to confirm or refute the presence of the phenomenon of ultra-early erythrolysis and mediated neuronal injury due to increased iron levels in the periphery of the hematoma based on MRI. Various MRI parameters like volume of T2* iso/non-hypointensity within the hematoma, R2* value in the periphery of the hematoma and volume of edema on FLAIR sequence will be measured on day 1, day 14 and day 30 to evaluate the presence of the phenomenon of ultra-early erythrolysis in ICH in human subjects, not only in the first 24 hrs but also up to 30 days following the hematoma.


Experimental Cerebral Hemorrhage: Mechanisms and Therapies

Xi G, Keep R, Hua Y
NIH 1R35NS116786-01
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

Hua Y, Keep R, Xi G
NIH R21 NS11239401
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.


UM Clinical Neuroscientist Training Program

Albin R, Keep RF
NIH R25 NS089450
7/15/2019 – 6/30/2024

There is a marked gap between our ability to treat neurologic diseases and our rapidly increasing understanding of normal nervous system function, disease pathogenesis, and disease pathophysiology. Improving treatment of neurologic diseases requires considerably improved integration of burgeoning basic neuroscience with clinical practice across the translational spectrum from basic discovery through translational and clinical research. An obstacle to improved integration of disease-oriented neuroscience research activities and more efficient translation is a deficit of appropriately trained clinician-neuroscientists. American medical schools graduate significant numbers of physicians with substantial research experience, including MD/PhDs and individuals with significant experience with and formal training in clinical research. The traditional clinical training structure, however, impedes the ability of these talented and well trained individuals to efficiently initiate productive, independent research careers. We propose continuation of a residency-fellowship based training program integrating residency-fellowship based clinical training with mentored research training to move talented and experienced trainees in Neurology, Neurosurgery, and Neuropathology to the initial stage of an independent career. The University of Michigan Clinical Neuroscientist Training Program (UMCNTP) prepares talented clinician-neuroscientists for independent research careers across the full spectrum of disease-oriented neuroscience research. The UMCNTP is an integrated residency-fellowship program preparing talented fledgling clinician-neuroscientists for successful applications for initial independent career support. The UMCNTP melds productive mentored research experience under the guidance of experienced senior investigators with focused didactic and hands-on career training to prepare UMCNTP trainees for successful career development applications such as K08, K23, VA CDA, or equivalents. Our residency programs matriculate talented individuals without significant prior research experience. For those individuals, the UMCNTP offers an integrated residency-fellowship-PhD training program in Neuroscience and related disciplines. The UMCNTP features a strong roster of mentors in the Depts. of Neurology, Neurosurgery, and Pathology, a training program based on highly successful prior experiences with training clinician-neuroscientists, utilizes excellent career development resources available at the University of Michigan, and draws on the great diversity and general excellence of the Neuroscience research community of the University of Michigan.


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.


Role of S100a8/A9 in blood brain barrier dysfunction after sepsis

Singer BH, Andjelkovic-Zochowska AV, Segal BM, Keep RF
NIH K08 NS10105403
4/1/2017 – 3/31/2022

This proposal describes a five-year career development program designed to lead the PI to a career as an independent clinician scientist studying the intersection of medical critical illness, neuroimmunology, and vascular biology. Research plan: Long term brain dysfunction, including cognitive and affective disorders, is common among the 1.3 million patients who survive critical illness every year in the United States. While the population impact of brain dysfunction after critical medical illness is similar to morbidity associated with stroke and is an area of growing scientific interest, few studies investigate the mechanism of these changes, and there is little scientific basis for the rational design of treatments. The applicant has found that in a mouse model of sepsis, systemic illness results in long lasting neuroinflammation with infiltration of inflammatory cells, changes in resident microglial gene expression, and blood brain barrier dysfunction. In this proposal, the applicant will investigate the role of an endogenous danger signal, the protein S100A8/A9, in sustaining neuroinflammation and blood brain barrier dysfunction in a mouse model of sepsis, and determine if blockade of S100A8/A9 signaling ameliorates these effects. He will study the role of S100A8/A9 both in an in vitro model system and in vivo using a combination of pharmacologic and genetic approaches. These questions will also be extended to post mortem studies of neuropathology and gene expression in the brains of patients with sepsis. Applicant: The applicant holds M.D. and Ph.D. degrees and has completed specialty training in Internal Medicine and Pulmonary and Critical Care Medicine. He has previous experience in neuroscience research and using mouse models to study normal brain physiology and disease. The career development plan includes a period of mentored research aimed at developing new knowledge in both immunology and vascular biology that will greatly enhance his existing training and allow him to develop as an independent investigator in studying an intrinsically interdisciplinary problem in translational neuroscience. The training will include learning research techniques and acquiring scientific knowledge in the laboratories of and through meetings with the mentors and key collaborators, as well as didactic training, seminars, lab meetings, topic focused working groups, and national meetings. The training plan includes didactic training in grant writing and responsible conduct of research. The research environment provides intellectual interaction with investigators from neuroscience, immunology and vascular biology, as well as basic and clinical/translational scientists. Technology for advanced imaging, gene expression, and immunophenotyping is available. These opportunities will allow the applicant to be guided in developing both powerful investigative techniques and the intellectual tools for an independent career in a clinically important but underserved field of study.


Claudin expression profiles and blood brain barrier in aging

Andjelkovic-Zochowska AV, Stamatovic S, Keep RF
NIH RF1 AG057928
9/15/2017 – 6/30/2022

An accumulating body of evidence suggests that cerebral “microvasculature” disease increases with advancing age and is associated with lacunar stroke, leukoaraiosis, and vascular dementia and Alzheimer disease. The increased blood brain barrier (BBB) permeability/leakage was consider as a consequence of ongoing processes like inflammation, atherosclerosis, and lack of vaso-autoregulation or microthrombosis, although that several recent clinical MRI studies indicate that BBB leakage could be a primary reason for the development of vascular/brain parenchymal injury during aging. The specific alterations in brain endothelial barrier components may ultimately lead to vascular hyperpermeability, extravasation of plasma components and inflammatory response in the brain parenchyma. Although significant effort has been made in defining the gene mutations and risk factors involved in microvascular alteration in vascular dementia and Alzheimer disease, the intra- and intercellular pathogenic mechanisms responsible for vascular hyperpermeability are still largely unknown. The proposed study is designed to elucidate critical molecular events in maintaining the integrity of the brain endothelial barrier and how these are altered during vascular aging. It will highlight the structural alteration in major regulator of BBB permeability –Tight junction complex and how interaction between signaling molecules and structural proteins claudins (Claudin1,-5 and -12), affect the organization and stability of brain endothelial tight junctional complex during the process of vascular aging. Specifically, the following objectives will be evaluated: a) the contribution of claudin-1 and claudin-12 to BBB hyperpermeability during aging, b) the effect of claudin-1 and claudin-12 on claudin-5 expression and function during brain endothelial cell ‘aging’c) the role of Sirt-1 in modifying claudin-1 and claudin-12 expression in aging. Collectively, these studies will provide new information related to the mechanisms involved in maintaining the brain endothelial barrier that is relevant not only to aging but also to multiple disease states. Hopefully, this will help to elucidate novel therapeutic strategies to restore vascular hyperpermeability.


Connexin-43 isoform Cx43-20kDA in cerebral cavernous malformation type 3

Andjelkovic-Zochowska AV, Stamatovic S, Keep RF
NIH R21 NS111205
4/1/2019 – 3/31/2021

Brain vascular malformations and blood-brain barrier defects represent important substrates for developing stroke, epilepsy and other neuropathological diseases. The most common type of brain malformation closely associated with stroke is the cerebral cavernous malformations (CCMs), which affect approximately 0.5% of population. Recognized as inherited and sporadic, CCMs are characterized as a single or multi- cluster of enlarged capillary-like channels with a single layer of endothelium and without intervening brain parenchyma. There are specific alterations in the brain endothelial barrier components that ultimately lead to vascular hyperpermeability, extravasation of red cells and an inflammatory response in brain parenchyma. Although significant progress has been made in defining the genes mutations involved in the inherited CCM3 form, the intra- and intercellular pathogenic mechanisms responsible for vascular hyperpermeability are still largely unknown. The proposed study is designed to elucidate critical molecular events involved in impairment of the brain endothelial barrier in CCM3 and CCm3 lesion progression. It will highlight the role of connexin 43 isoform (Cx43- 20kDa), one of the protein identify by our screening analysis to be highly expressed in condition of loss of CCM3 function in brain endothelial cells in driving the brain endothelial and barrier injury in CCM3. Specifically, it will be evaluated a) the role of Cx43-20kDa in generating gap junctions and hemi-channels in brain endothelial cells that will facilitate injury spread in CCM3-KD conditions and b) the benefits of blocking Cx43-20kDa and gap junction on CCM3 lesion progression. Collectively, these studies will provide new information related to the mechanisms of maintenance of brain endothelial barrier that is relevant also to multiple disease states and will, hopefully, elucidate novel therapeutic strategies to restore the vascular hyperpermeability.


Connexin-43 and hemorrhagic complication in cerebral amyloid angiopathy

Andjelkovic-Zochowska AV, Stamatovic S, Keep RF
NIH RF1 AG064957
8/1/2019 – 4/30/2024

An accumulating body of evidence suggests that cerebral small vessel disease (CSVD) increases with advancing age and is associated with lacunar stroke, leukoaraiosis, and vascular dementia and Alzheimer disease. CSVD poses one of the major challenges in the aging population, due to high morbidity, related to recurrent ischemic stroke and cognitive disturbance. Recognized as hereditary or idiopathic cases, CSVD is histologically characterized by segmental arteriolar disorganization, perivascular lesions, focal inflammation, and/or microartheroma appearance. One of the common sporadic form of CSVD is Cerebral amyloid angiopathy (CAA) predominantly present in the cerebral cortex, leptomeninges, and gray–white matter junction and characterized by the progressive deposition of amyloid β peptide (Aβ) within walls of cerebral arteries, arterioles and capillaries and causing of micro-intracerebral hemorrhage, ischemic and hemorrhagic stroke, and cognitive dysfunction in elderly patients. The pathological substrate for progressive CAA is indicated to be blood brain barrier dysfunction, which is manifest as vascular hyperpermeability, extravasation of plasma components and inflammatory response in the brain parenchyma. Although significant effort has been made in defining the gene mutations and risk factors involved in CAA the intra- and intercellular pathogenic mechanisms responsible for vascular injury are still largely unknown. The proposed study is designed to elucidate critical molecular events in endothelial dysfunction associated CAA. It will highlight how gap junction protein Connexin-43 and its isoform Cx43-20, highly expressed at brain endothelial cells in presence of Aβ, lead to brain endothelial barrier remodeling and facilitating the vascular injury in CAA vasculopathy. Specifically, the following objectives will be evaluated: a) the role of Cx43 and Cx43-20 in brain endothelial barrier dysfunction and microhemorragy in CAA vasculopathy, b) how Cx43-20 cause the brain barrier dysfunction in CAA vasculopathy, c) the effect of amyloid beta on Cx43-20 expression and function in CAA vasculopathy d) how the targeting CX43-20 may improve vascular stability and prevent CAA induced micro- and macrohemorrage. Collectively, these studies will provide new information related to the mechanisms involved in maintaining the brain endothelial barrier that is relevant not only to CAA but also to multiple disease states. Hopefully, this will help to elucidate novel therapeutic strategies to restore vascular hyperpermeability.

Principal Investigator

Richard Keep, Ph.D.

Director, Crosby Neurosurgical Laboratories
Associate Chair for Research, Neurosurgery
Crosby-Kahn Collegiate Professor of Neurosurgery and Neuroanatomy
Professor, Molecular and Integrative Physiology
734-764-5128