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Current Research Projects

Mechanisms of Brain Edema after Intracerebral Hemorrhage

Investigators: G. Xi, R.F. Keep, Y. Hua
Sponsor: National Institutes of Health 

Goals: The purpose of our project is to investigate the mechanisms involved in thrombin-induced brain edema. If our hypotheses are correct, these experiments may lead to novel methods of treating intracerebral hemorrhage.

Spontaneous 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.

Brain edema plays an important role in the secondary brain injury following ICH. It is the long-term goal of our laboratory to identify the mechanisms involved in brain edema formation following ICH. Our previous studies indicate that edema formation following ICH may involve several phases. An early phase involves the clotting cascade and thrombin production. Although our data indicate that thrombin inhibition is a therapeutic target, concerns over rebleeding may limit the utility of this approach. It is, therefore, important to understand the downstream mechanisms activated by thrombin. Our preliminary studies have found:

We will determine whether:

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Endothelial Preconditioning and Ischemic Brain Injury

Investigators: R.F. Keep, S.R. Ennis, Andjelkovic-Zochowska, J. Xiang
Sponsor: National Institutes of Health

Goals: These experiments should provide information on endogenous defense mechanisms that protect the cerebral endothelium from ischemic injury and may be therapeutic targets; the role of endothelial preconditioning in the effects of IPC on the brain; and the role of endothelial injury in the overall effects of ischemia on the brain.

Ischemic preconditioning has proved to be one of the most effective methods of reducing ischemic brain damage in animal models of stroke. Attention has focused on the mechanisms by which neurons may be protected by such preconditioning. However, we have found that IPC also protects the endothelium, which forms the blood-brain barrier, from ischemic damage. “Ischemic” preconditioning also protects cerebral endothelial cells in vitro from oxygen glucose deprivation induced injury indicating that there can be direct preconditioning of the endothelium. Damage to the cerebral endothelium may potentiate ischemic brain injury in a number of ways; determining mechanisms to reduce endothelial damage is particularly pertinent at the moment considering the role of endothelial injury (hemorrhagic transformation) in limiting the use of tissue plasminogen activator for the treatment of ischemic stroke.

We will examine the mechanisms involved in cerebral endothelial preconditioning both in vivo (rat middle cerebral artery occlusion) and in vitro (primary cultures of rat cerebral microvessel endothelial cells). The in vitro experiments will facilitate exploration of the mechanisms involved in preconditioning while assuring that the preconditioning acts directly on the endothelium. The in vivo experiments will ensure that the mechanisms elucidated in vitro also occur in the whole animal as well as allowing an assessment of the effects of preconditioning on other parameters (such as blood flow, capillary morphology and infarction).

Overall, the research project has three main goals to determine:

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Mechanisms of Thrombin-Induced Tolerance to Brain Injury

Investigators: G. Xi, R.F. Keep, Y. Hua, T. Schallert  
Sponsor: National Institutes of Health

Goals: The purpose of our project is to investigate the mechanisms involved in thrombin preconditioning. An examination of TPC will also help our understanding of ischemic preconditioning since preliminary data suggests that TPC is a component of that phenomenon. TPC seems to be receptor mediated, greatly facilitating analysis compared to ischemic preconditioning. The long-term goal of these studies is to find mechanisms to limit brain injury after ICH.

Intracerebral hemorrhage is a common and often fatal subtype of stroke and produces severe neurologic deficits in survivors. Brain injury after ICH appears to involve several phases, including an early phase involving the clotting cascade and thrombin production and a later phase involving erythrocyte lysis (breakdown) and hemoglobin toxicity. Although high concentrations of thrombin cause brain edema and cell death, low concentrations are neuroprotective. Thus, we have found that prior treatment with a low dose of thrombin attenuates the brain edema induced by thrombin or hemorrhage, and significantly reduces the infarct size in a rat middle cerebral artery occlusion model. We have termed this “phenomenon thrombin preconditioning.” In our recent studies, we have found that TPC not only reduces brain edema induced by high dose thrombin, but also attenuates edema induced by lysed erythrocytes and FeCl2. TPC appear to involve upregulation and increased transferrin, activation of thrombin receptor, HIF-1 transferrin receptor and ferritin levels.

This suggests that thrombin release during an ICH might induce protective mechanisms against factors released upon clot lysis. This data has led us to the following specific aims:

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Thrombin-mediated Ischemic Brain Injury

Investigators: Guohua Xi, M.D.
Sponsor: American Heart Association Grant-in-Aid

Goals: The research project seeks to investigate the role of thrombin in brain injury after ischemic stroke. The long-term goal of our studies is to identify pathophysiologic mechanisms underlying brain injury from stroke, so that effective therapies can be developed to prevent secondary injury.

Every year, cerebral ischemia affects about 600,000 people in the United States. We will determine the effects of thrombin production on ischemic brain injury. Clarification of the mechanisms of brain injury after cerebral ischemia will help develop new therapeutic strategies for stroke.

Stroke ranks high as a cause of death and significant neurologic impairment with no effective treatment except thrombolytic recanalization with tissue plasminogen activator within three hours. Many different mechanisms have been implicated in secondary ischemic brain injury. Recent studies and our preliminary data indicate that thrombin is an important factor in ischemic injury. Thrombin receptors are upregulated in brain slices after oxygen-glucose deprivation. Hirudin, a thrombin inhibitor, reduces infarct volume in gerbils. In addition, our preliminary studies indicate that thrombin activity is increased in the ischemic core with an increase of expression of prothrombin mRNA. Furthermore, anti-thrombin treatment attenuates neurological deficits after cerebral ischemia with reperfusion, whereas exogenous thrombin exacerbates ischemic injury. After ischemia, the cerebral vasculature becomes permeable to plasma protein within several hours. Blood-brain barrier disruption then allows large molecules such as prothrombin entry into the brain parenchyma which may cause secondary damage following ischemia. In addition, prothrombin is also generated within the brain itself.

The specific aims of this project are:

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Delayed Neurodegeneration After Intracerebral Hemorrhage

Investigators: G. Xi, R.F. Keep, Y. Hua
Sponsor: National Institutes of Health

Goals: The purpose of our project is to investigate the mechanisms of delayed neurodegeneration after ICH. The long-term goal of our studies is to limit hemorrhagic brain injury. If our hypotheses are correct, these experiments may lead to novel therapies for intracerebral hemorrhage by either limiting iron overload or attenuating oxidative brain injury.

Spontaneous 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.

To understand the underlying mechanisms of ICH-induced brain injury and to evaluate therapeutic interventions a number of animal models of ICH have been developed. A reproducible rat ICH model, involving infusion of autologous blood into the caudate, has been used extensively to study mechanisms of brain injury and, in particular, early edema formation. It has been difficult to find correlates of the long-term effects of human ICH. Recently, however, we have developed behavioral tests that can detect prolonged neurological deficits and we, and others, have found that there is delayed brain atrophy in animal models of ICH. The mechanisms involved in this prolonged and delayed brain injury after ICH are as yet unknown, but our preliminary data suggest a role for iron overload and oxidative stress.

We propose to test the following hypotheses:

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Does Surgical Debulking of Pituitary Adenomas Improve Responsiveness to Octreotide Long-acting Release in the Treatment of Acromegaly

Investigator: W.F. Chandler 
Sponsor:  Novartis

Goals: Until now, no randomized studies have been done to assess whether this surgical debulking improves the efficacy of therapy with medical therapy, including somatostatin analogs. In this study, we will investigate whether surgical debulking increases the efficacy of a long-acting depot somatostatin preparation, Sandostatin LAR Depot, in achieving biochemical normalization.

Acromegaly, a rare, debilitating disease associated with increased morbidity and mortality, is caused usually by a growth hormone-secreting pituitary tumor. Excess GH leads, in turn, to elevated levels of insulin-like growth factor I (IGF-I), which is responsible for many of the biological manifestations of the disease.

The goals of treatment of acromegaly include amelioration of the signs and symptoms of the disease, reduction of tumor mass, preservation of pituitary function, and normalization of GH and IGF-I. None of the currently available treatment options, including surgical removal of the tumor, radiation therapy of the pituitary gland and drug therapy, are fully effective in curing patients. Conventional radiation therapy takes more than 10 years to lower the GH level, surgery provides a permanent cure for about 40 percent of subjects, primary medical therapy with somatostatin analogs results in IGF-I normalization in about 60 percent of patients and dopaminergic agonists are effective in less than 20 percent of individuals. Thus, a significant number of individuals have persistently elevated GH and IGF-I level with any single treatment modality.

It is generally assumed (and supported by the limited available data) that individuals with lower initial GH levels are more responsive to medical therapy. To optimize utilization of combination therapy, it is necessary to determine whether lowering GH with surgery will render non-curved patients more responsive to medical therapy.

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Effect of Spinal Cord Stimulation on Cerebral Blood Flow

Investigators: O. Sagher 
Sponsor: National Institutes of Health 

Goals: We will utilize an in vivo model of SCS and CBF measurement in rats in order to obtain information about the magnitude, time course and spatial characteristics of CBF changes induced by SCS. Using this model, we will also investigate the effects of SCS on stroke induced by focal cerebral ischemia. An understanding of the mechanisms involved in the cerebrovascular effects of SCS has direct ramifications in the treatment of conditions where cerebral blood flow is compromised, such as stroke, cerebral vasospasm and traumatic brain injury.

Spinal cord stimulation, a widely used modality for the treatment of chronic pain, is also known to have a significant vasodilatory effect on peripheral vasculature. Interest in the vascular effects of SCS has resulted in its use to treat both peripheral limb ischemia and angina. Similar effects of SCS on cerebral blood flow also have been suggested. The significance, character and underlying mechanisms of the cerebrovascular effects of SCS, however, remain unclear.

Our study delineates the cerebrovascular effects of SCS in an animal model and examines the mechanism(s) that may be involved. We also will examine the feasibility of using SCS in the prevention of cerebral infarction. Specifically, we will test the following central hypotheses:

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Role of PEPT2 in Peptide/Mimetic Disposition-Dynamics

Investigators:  D. Smith, F.C. Brosius, R.F. Keep
Sponsor:  National Institutes of Health

Goals: By combining membrane, cellular, immunolocalization and whole animal studies in wild type and PEPT2 null mice, the studies will greatly advance our understanding of the in vivo role, significance and vectorial transport of peptide substrates and drugs by PEPT2 (as opposed to PEPT1, PHT1, PHT2 and other potential transporters). These studies may also provide rare insight into the variability of peptide/mimetic kinetics and response in those human subjects with genetic polymorphisms (e.g., hPEPT2 R57H). Finally, the studies may have important implications for the design, delivery and targeting of peptide-based pharmaceuticals to the brain for severe CNS disorders (e.g., Alzheimer’s disease, AIDS dementia, stroke, epilepsy, schizophrenia, infection and cancer).

Cellular, molecular and physiological studies have made major contributions toward a mechanistic understanding of PEPT2 structure, function and localization. However, these experimental approaches are often limited by an in vitro design and lack of blood supply, overlapping substrate specificities and contribution of multiple transport systems, some of which are unknown at the time of study. As a result, it is difficult, if not impossible, to define the function of a single specific gene product and its significance in relation to other possible proteins that are present in the tissue or organ of interest. Our laboratory has recently developed PEPT2-deficient mice, a unique resource in which to validate in vitro mechanistic studies with in vivo whole animal experiments. With this in mind, the long-term objectives of this competing renewal application are to define the physiological and pharmacological roles and relevance of PEPT2, a nutrient and drug transporter.

Our working hypothesis is that PEPT2 is the primary transporter responsible for the disposition and dynamics of peptides and peptide-like drugs within the body, particularly the brain and kidney. To test this hypothesis, the following specific aims are proposed: 

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Stem Cell-Based Gene Transfer for Hurler's Disease

Investigator: J. Heth
Sponsor:  William and Ella Owens Medical Research Foundation  

Goals: This research proposes to explore two methods to improve distribution of the gene and the enzyme within the central nervous system to treat Hurler’s disease.

Hurler’s disease is a prototypical lysosomal storage disease. Patients inherit the syndrome in autosomal recessive fashion and lack the enzyme -L-iduronidase. A reproducible, durable, low-risk method to treat the CNS in Hurler’s disease and the LSDs has not been found. One of the primary difficulties lies in distributing either the enzyme or its gene within the CNS. Stem cells, viral-mediated gene transfer and the peripheral administration of recombinant iduronidase show promise in the treatment of Hurler’s disease.

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Placed Beacons for Time Reversal Aberration Correction

Investigators: O.D. Kripfgans, O. Sagher
Sponsor:  National Institutes of Health 

Goals: We propose to create a system for highly selective transcranial tissue ablation and coagulation by combining the techniques of time reversal acoustics and acoustic droplet vaporization. Upon positive outcome of the in vivo section, we will design a clinical study for human application of this methodology.

The project is subdivided into an R21 phase, where we create the necessary fundamental apparatus and techniques, and an R33 phase where we apply the apparatus and techniques to eventually perform in vivo, transcranial tissue ablation with a sharply focused ultrasound beam.

The crucial hardware component in the R21 phase is an efficient chaotic reverberant cavity that approaches the capabilities of a full, 2-D array, at a much-reduced cost and with less complexity and fragility. It will be driven by a TR-system operating with point beacons to overcome any aberrators between the CRC and the beacon. Acoustically vaporized droplets will yield approximately 30- to 60-micron-diameter gas bubbles that will be used as point beacons for refocusing. Development of a library of focused beams for the CRC will allow us to scan and image in an unknown/unaberrated 3-D space.

In the R33 phase of this project, we will extend the CRC's frequency range to include 100 kHz to 5 MHz. This will allow us to drive bubbles in a nonlinear manner and enhance bubble detection and scattering. Moreover, higher frequencies will allow for easier vaporization but are more subject for higher insertion loss and attenuation of incident waves. Possibly, higher frequencies also will result in better spatial resolution after aberration correction. Phantoms with spatially distributed droplets/gas bubbles will be used to demonstrate an iterative focusing technique to identify and utilize multiple beacons individually.

From this ability, we will then analyze the system's capability to perform dynamic focusing (both with regard to location and focal zone shape). Finally, the system will be analyzed for its (aberration correction) performance through an ex vivo human skull cap in conjunction with a tissue mimicking US phantom, ex vivo rabbit brain and in vivo rabbit brain with the rabbit's skull cap removed. The pressure threshold for the onset of lesions will be determined after aberration correction.

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Carotid Occlusion Surgery Study

Investigator: B.G. Thompson, M.D.
Sponsor:  National Institutes of Health

Goal: This clinical trial will require an intensive and continuing effort to identify and enroll 930 clinically eligible subjects and will require the participation of qualified neurosurgeons, clinical neurologists, PET investigators and trained clinical coordinators.

Participation in this trial will test the hypothesis that surgical anastomosis of the superficial temporal artery to the middle cerebral artery when added to the best medical therapy can reduce by 40 percent – despite perioperative stroke and death – subsequent ipsilateral ischemic stroke (fatal and non-fatal) at two years in subjects with recently (within 120 days) symptomatic internal carotid artery occlusion, and ipsilateral increased oxygen extraction fraction as measured by positron emission tomography. This hypothesis will be tested by conducting a randomized, non-blinded, controlled trial in 372 patients randomized equally to surgical or non-surgical treatment. 

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Intracerebral Hemorrhage in Aging Rats

Investigator: Y. Hua 
Sponsor:  American Heart Association 

Goal: Clarification of the mechanisms of brain injury after ICH in the aging brain should help develop new therapeutic strategies for hemorrhagic brain injury. It also may give insight into how aging may affect other forms of brain injury.

Intracerebral hemorrhage is a common and often fatal subtype of stroke and produces severe neurological deficits in survivors. Edema following ICH contributes to these outcomes causing both acute herniation-related death and long-term neurological deficits. To understand the underlying mechanism of ICH-induced brain injury and to evaluate therapeutic interventions, a number of animal models of ICH have been developed. A reproducible rat ICH model, involving infusion of autologous blood into the caudate, has been used extensively to study mechanisms of brain injury and neurological deficits.

ICH is mostly a disease of the elderly, but most current experimental ICH models have used young animals. Age is an important factor affecting brain injury in cerebral ischemia, which may also affect brain injury after ICH. Our previous studies that complement system activated and system depletion complement system reduce the brain edemafter ICH. Our preliminary data showed that microglia activation, brain edema and neurological deficits were greater in the 18-month-old rats than those in the three-month-old rats. Microglia plays important role in activation of complement by synthesis and release complement components. It is the long-term goal of our laboratory to study the mechanisms of brain injury after ICH.

The study will:

Stroke ranks third as a cause of death and affects about 600,000 people each year in the U.S. Stroke is also a major cause of mental and physical disability with approximately 4.4 million stroke victims; most of these patients are elderly people.

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The Medtronic Sofamor Danek Spine Fellowship

Investigator: F. La Marca, M.D. 
Sponsor:  Medtronic  

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Obesity and Hypertension: The Role of 5HT2C Receptors

Investigators: Richard Keep, Ph.D., Steven R. Ennis Ph.D., Roger Grekin, M.D.,
Sponsor: National Institutes of Health 

Hypertension related to obesity is a major cause of morbidity and mortality in the U.S. A better understanding of the basic mechanisms linking these two factors will have major therapeutic relevance.

Acute intracerebroventricular infusions of 5HT2 receptor agonists activate the sympathetic nervous system and increase blood pressure. Preliminary data indicate that activation of a subset of the 5HT2 receptors in the brain, the 5HT2C receptors, is a contributory factor to deoxycorticosterone-salt induced hypertension. The central 5HT2C receptors are also involved in reducing food intake and body weight, 5HT2C receptor knock-out mice becoming obese. This suggests that 5HT2C receptor activation could be a link between obesity and hypertension. Specifically, we will examine the hypothesis that obesity causes 5HT2C receptor activation to reduce body weight but that chronic activation of this system causes hypertension.

This overall hypothesis is supported by preliminary data that hypertension induced by intracerebroventricular infusions of leptin, a hormone involved in reducing body weight, is blocked by 5HT2C receptor antagonists. Leptin is secreted by adipose tissue, is transported into brain at the blood-brain and blood-CSF barriers and has hypothalamic actions. The choroid plexus (the site of the blood-CSF barrier) have the highest densities of leptin and 5HT2C receptors suggesting that the choroid plexus may be one site of interaction between leptin and 5HT. We will examine whether 5HT2C receptor activation modulates blood to brain leptin transport as a feedback mechanism.

This project has four specific aims:

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Blood-Brain Barrier Transport and Ischemic Brain Injury

Investigators: Richard F. Keep, Ph.D., Steven R. Ennis, Ph.D.
Sponsor: National Institutes of Health

Because of its juxtaposition to blood, the cerebral endothelium (which forms the blood-brain barrier) has been thought to be relatively resistant to the effects of cerebral ischemia. However, examination of taurine, glutamine and myo-inositol influx into brain (all Na+-dependent processes) indicate a marked early (< 1 hour) reduction in transport during focal cerebral ischemia suggesting that endothelial cell injury could play a role in primary, rather than secondary, ischemic brain damage. This may be particularly the case if efflux from as well as influx into brain are affected since those efflux systems are involved in controlling the concentration of potentially toxic factors in the brain extracellular space. This research project, therefore, has two major goals:

The cerebral volume of distribution reached by [3H]vinblastine (a P-glycoprotein substrate) and p-[3H] aminohippuric acid (PAH, an organic acid transporter substrate) will be determined following middle cerebral artery occlusion in rat and mouse (Specific Aim 1). Whether an increased volume of distribution with ischemia reflects a change in influx or an alteration in efflux at the BBB will then be determined, the latter by examining the effect of cerebral ischemia in the absence of BBB P-glycoprotein (the mdr1a knock out mouse) or during probenecid-induced inhibition of organic acid transport. Specific Aim 2 will examine the mechanism by which ischemia inhibits efflux, by examining PAH, L-glutamate and methyl aminoisobutyric acid efflux (an A-System amino acid transporter substrate) uptake into choroid plexus using ventriculo-cisternal perfusion. Specific Aim 3 will determine the effect of altering specific transporters at the BBB on ischemic brain injury and will examine whether drugs known to ameliorate the effect of reperfusion on BBB disruption actually have these effects by altering transport during ischemia.

As well as determining whether early BBB dysfunction should be an alternate therapeutic target early during cerebral ischemia, the finding that there is an inhibition of energy-dependent efflux at the BBB during ischemia has major implications for drug delivery to the injured brain. P-glycoprotein and the organic acid transporter both play a major role in limiting the access of some drugs to the brain.

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