Donald G. Stein - US grants

The projects described in this proposal are designed to evaluate the role of fetal brain tissue transplants in mediating functional recovery from traumatic injuries to the central nervous system. Using behavioral, anatomical and electrophysiological techniques, this project will examine whether: (a) the effectiveness of transplants in promoting behavioral recovery varies as a function of the time between the initial brain injury and the introduction of the fetal tissue into the host brain; (b) "recovered" behaviors are different than those used by intact animals--(i.e., is there restitution or substitution of behavioral functions induced by transplant surgery?); (c) the effectiveness of fetal brain tissue transplants can be enhanced by incubation in neurotrophic substances prior to implantation in the damaged host brain; (d) specificity of tissue between fetal tissue and host is an essential condition for obtaining functional recovery (e.g., can embryonic frontal cortex assist in facilitating behavioral recovery following occipital cortex lesions?); (e) interspecies transplants are effective in promoting functional recovery (e.g.,, can embryonic cortical tissue taken from the hamster be used to reduce lesion-induced, cognitive deficits in adult rats?); (f) specific anatomical connections formed between the host brain and the transplant material are necessary to mediate functional recovery. Although there are a number of new and exciting approaches to the study of neural plasticity, it is surprising how few are concerned with detailed analysis of the behavioral consequences of injury-induced changes in C.N.S. functions. Yet, understanding how the nervous system mediates, or can be induced to mediate, behavioral and functional recovery is the key issue for those faced with the prospect of treating the more than 400,000 patients who suffer severe C.N.S. injuries in this country each year. Any attempts to develop specific, effective and long-lasting treatments for the victims of brain and spinal cord injuries, should be given some attention. This proposal addresses the question of whether fetal brain cells can serve as one form of therapy for severe brain dysfunctions. Careful behavioral analyses, using complex learning tasks are followed by detailed electrophysiological and anatomical assays of the transplant effects. The long-term goals of this research are to find the means to alleviate some of the suffering caused by C.N.S. injuries and restore the patient to a more normal life.

DESCRIPTION: The long-term goal of this revised research proposal is to determine the specific mechanisms by which progesterone mediates neuronal rescue and recovery of function in traumatic brain injury (TBI); the major cause of death in young adults under the age of 35. These findings will be employed in the evaluation of progesterone as a safe and effective treatment for TBI. Progesterone has recently been classified as a neurosteroid because it is also synthesized in astrocytes and oligodendrocytes in the brains of both males and female, where it may play other roles than that of a sex hormone. Previous research has already shown that systemic injection of progesterone in laboratory rats can reduce some of the neuropathological consequences of TBI and enhance behavioral recovery of function. Progesterone treatments reduce cerebral edema dramatically in both males and females when administered within 24 hours of the injury. This is one type of protective effect that can lead to reduced neuronal death and improved functional outcomes. The primary goal of the current proposal is to understand better the physiological mechanisms by which progesterone mediates its neuroprotective actions. We propose a series of 4 Aims using both in vitro and in vivo models of neural injury to determine: (1) if progesterone exerts its effects via its specific receptors. This will be studied by co-administering the hormone with ORG31710, a known progesterone receptor antagonist, or substituting progesterone with an agonist that cannot be metabolized (R5020) in both in vivo and in vitro models of TBI; (2) if, in an in vitro model of injury, progesterone's short-term neuroprotective effects are mediated specifically via sigma receptors; (3) if progesterone will prevent loss of mitochondrial function by reducing oxidative stress initiated in both in vivo and in vitro models of TBI; and (4) if progesterone will reduce the destructive aspects of the inflammatory immune reaction that occurs after TBI. The combination of in vivo and in vitro models will provide parallel evaluation of the mechanisms of progesterone's action. The research proposed here will help to determine whether progesterone can be used as a low-cost, safe and effective therapeutic agent in the acute stages of CNS injury. Furthermore, the detailed assessment of progesterone's mechanisms of action will provide a foundation for the intelligent design of artificial therapeutic pharmaceuticals.

At present, there are no clinically effective treatments for traumatic brain injury (TBI). The long-term objective of this research is to develop such a therapy that will enhance morphological and behavioral recovery of function. TBI triggers a cascade of dramatic, biochemical events leading to primary and secondary neuronal loss and dysfunction. Many of these events, such as disruption of the blood brain barrier, excitotoxicity, ischemia, oxidative stress, glial activation and the inflammatory immune response, contribute to an increase in cerebral edema, which is often severely disabling or fatal to patients. There is growing experimental evidence that, in laboratory animals, progesterone and its metabolites are safe and effective in providing neuroprotection and in reducing cerebral edema after TBI. How these neurosteroids act specifically in the central nervous system to enhance recovery of function is not yet completely understood, and this gap in our knowledge limits interest in proceeding to clinical trials. The focus of the proposed research is to extend our knowledge of how progesterone and its metabolites enhance neuronal repair and recovery of function in the damaged nervous system by controlling the events causing cerebral edema and neuronal death. We will use a model of brain injury that creates controlled contusions of the frontal cortex in rodents. We will then measure the effects of progesterone, allopregnanolone and epiallopregnanolone treatments on behavioral recovery, cytokine expression, and oxidative stress following TBI. Four projects are proposed: (1) determines whether the progesterone-related metabolites, allopregnanolone and epiallopregnanolone, have effects similar to progesterone in promoting neuroprotection and behavioral recovery after TBI; (2) examines whether the metabolites are as effective as progesterone in reducing post-injury inflammatory signals; (3) explores whether these progesterone-related hormones modulate the temporal and spatial distribution of inflammatory cells; (4) investigates whether reducing these inflammatory signals results in the decrease of oxidative stress and neural cell death.

[unreadable] DESCRIPTION (provided by applicant): Over a million cases of traumatic brain injury (TBI) occur each year, yet at present there are no clinically effective treatments to prevent neuronal loss and enhance behavioral functions. An examination of the CRISP database (1972-2004) found only two NIH-sponsored grants addressing TBI in the elderly! What is clearly needed is a safe, easy-to-administer agent that can promote morphological and behavioral recovery in brain and spinal cord injuries across the life span. A promising agent currently under test at Emory University in a phase I/II clinical trial for moderate to severe blunt head trauma, is the neurosteroid progesterone. In our previous research, of which this revised proposal is an extension, our laboratory showed that a short course of post-injury progesterone injections in rats could reduce cerebral edema, enhance neuronal sparing, and improve cognitive, sensory and motor functions in rats with bilateral contusions of the medial frontal cortex. Although progesterone was deemed effective enough to warrant clinical testing, there is still much to learn about how it, and its related precursors and metabolites, affect functional and morphological recovery in old animals. The bulk of TBI research focuses on children and young adults, but it is also a substantive issue for the elderly, who are often seriously brain-injured by falls and accidents! Our specific aims here are: (1) Using behavioral assays in a dose-response paradigm, we will examine the functional effects of progesterone treatments in senescent and young adult male and female laboratory rats. (2) Having determined that progesterone reduces the expression of pro-inflammatory genes, we will use immunocytochemical (ICC) and molecular biological techniques to investigate how this steroid affects the level of inflammatory proteins made by the genes, and how the reduction of these substances affects edema and immune cell invasion after TBI in both adult and old animals. (3) Because synthetic forms of progesterone are widely available for human use and are often interchanged for natural progesterone (nPROG) in clinical practice, we will compare the effectiveness and mechanisms of action of nPROG with medroxyprogesterone acetate (MPA), a synthetic molecule which has receptor and cellular actions that can be different from those of progesterone itself. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Dozens of phase II and III clinical trials for traumatic brain injury (TBI) therapies have failed. One reason cited is that the complex mechanisms and injury cascades associated with different types of TBI are not being addressed by drugs with limited modes of action. Progesterone (PROG) has been shown to be a safe and effective treatment for TBI. A phase I/II clinical trial using PROG to treat moderate to severe brain injury found that mortality from severe injury was reduced almost 60% and there was better functional outcome. This trial was replicated with an additional 159 patients in an independent study from abroad. The PIs'current research is seeking even better results by combining PROG with other neuroprotective agents. A logical first step is to examine the conditions that might limit PROG's beneficial effects in a clinical setting. Vitamin D hormone (VDH) is an FDA-approved molecule whose neuroprotective and neurotrophic actions are increasingly being recognized. The PIs'recent research suggests that, for older subjects, VDH deficiency (VDHdef) reduces the beneficial effects of PROG. It has been reported that well over half of older adults are VDHdef, a condition which could exacerbate the outcome of any TBI, and the elderly population is disproportionately subject to TBI. Further, about 30-35% of the American public has been reported to be VDHdef, so patients of any age, including children, presenting with a TBI could be more at risk and have less favorable outcome if they are also VDH deficient. Thus something as simple as VDH could enhance the benefit of PROG treatment as a neuroprotective agent. Both PROG and VDH are natural hormones, synthesized in both males and females, and have distinct, pleiotropic modes of action in CNS repair. These properties make the preclinical testing of combined PROG and VDH a compelling approach to consider for later evaluation and testing in a clinical TBI trial. First, the PIs will determine the optimal dose for combination treatment to enhance PROG's effects on post- traumatic recovery from neurological deficits and resolution of lesion size in male and female rats. Second, they will see whether PROG's effects are compromised in a co-morbid VDH deficient state in old animals and whether supplementing VDH is necessary to promote optimal benefit. Third, they will establish a correlation, if any, between PROG and VDH levels, functional recovery, and serum levels of surrogate serum biomarkers for TBI. To measure the severity of the injury and predict extent of repair, the time course of changes in neurologic outcome and serum levels of TBI markers will be evaluated. Fourth, the PIs will identify mechanisms that contribute to better neuroprotection by combination of PROG with VDH. The PIs will analyze the gene products most significantly changed in response to combination treatment or either agent alone, and most related to brain injury and repair. PUBLIC HEALTH RELEVANCE: Progesterone (PROG) has been shown to be a safe and effective treatment for moderate and severe traumatic brain Injury. There is evidence that its effectiveness can be increased by combining it with Vitamin D hormone (VDH), especially in the elderly. The fact that both PROG and VDH have high safety profiles, act on many different injury and pathological mechanisms, and are easy to administer and inexpensive, make this combination an obvious approach to develop what may become the first successful treatment for traumatic brain injury.

DESCRIPTION (provided by applicant): Dozens of phase II and III clinical trials for traumatic brain injury (TBI) therapies have failed. One reason cited is that the complex mechanisms and injury cascades associated with different types of TBI are not being addressed by drugs with limited modes of action. Progesterone (PROG) has been shown to be a safe and effective treatment for TBI. A phase I/II clinical trial using PROG to treat moderate to severe brain injury found that mortality from severe injury was reduced almost 60% and there was better functional outcome. This trial was replicated with an additional 159 patients in an independent study from abroad. The PIs'current research is seeking even better results by combining PROG with other neuroprotective agents. A logical first step is to examine the conditions that might limit PROG's beneficial effects in a clinical setting. Vitamin D hormone (VDH) is an FDA-approved molecule whose neuroprotective and neurotrophic actions are increasingly being recognized. The PIs'recent research suggests that, for older subjects, VDH deficiency (VDHdef) reduces the beneficial effects of PROG. It has been reported that well over half of older adults are VDHdef, a condition which could exacerbate the outcome of any TBI, and the elderly population is disproportionately subject to TBI. Further, about 30-35% of the American public has been reported to be VDHdef, so patients of any age, including children, presenting with a TBI could be more at risk and have less favorable outcome if they are also VDH deficient. Thus something as simple as VDH could enhance the benefit of PROG treatment as a neuroprotective agent. Both PROG and VDH are natural hormones, synthesized in both males and females, and have distinct, pleiotropic modes of action in CNS repair. These properties make the preclinical testing of combined PROG and VDH a compelling approach to consider for later evaluation and testing in a clinical TBI trial. First, the PIs will determine the optimal dose for combination treatment to enhance PROG's effects on post- traumatic recovery from neurological deficits and resolution of lesion size in male and female rats. Second, they will see whether PROG's effects are compromised in a co-morbid VDH deficient state in old animals and whether supplementing VDH is necessary to promote optimal benefit. Third, they will establish a correlation, if any, between PROG and VDH levels, functional recovery, and serum levels of surrogate serum biomarkers for TBI. To measure the severity of the injury and predict extent of repair, the time course of changes in neurologic outcome and serum levels of TBI markers will be evaluated. Fourth, the PIs will identify mechanisms that contribute to better neuroprotection by combination of PROG with VDH. The PIs will analyze the gene products most significantly changed in response to combination treatment or either agent alone, and most related to brain injury and repair. PUBLIC HEALTH RELEVANCE: Progesterone (PROG) has been shown to be a safe and effective treatment for moderate and severe traumatic brain Injury. There is evidence that its effectiveness can be increased by combining it with Vitamin D hormone (VDH), especially in the elderly. The fact that both PROG and VDH have high safety profiles, act on many different injury and pathological mechanisms, and are easy to administer and inexpensive, make this combination an obvious approach to develop what may become the first successful treatment for traumatic brain injury.

DESCRIPTION (provided by applicant): To date, clinical trials for the treatment of ischemic stroke after it has occurred have been disappointing. However, in a recently completed Phase lla, single-center, randomized, double-blind trial in patients with moderate to severe traumatic brain injury (TBI), progesterone (PROG), given intravenously for three days after injury, reduced mortality by almost 60% and significantly improved functional outcome at 30 days survival compared to patients given state-of-the-art treatment and placebo. Preliminary data with PROG in the treatment of stroke are promising, but before PROG can be tested in a clinical trial for stroke, more animal studies are needed to determine whether dose response relationships and window of treatment opportunity differ between stroke and TBI. We will establish the safety, dosing parameters, and effects on physiological and functional outcomes of PROG in the target class of animal subjects. We will then apply for, and gain, FDA approval to use PROG in a clinical trial for the treatment of ischemic stroke. In the present proposal we will use both transient and permanent cerebral ischemic stroke models in both female and male rats. Our Primary goal will be to determine whether PROG will work effectively in middle-aged male and female rats (about 15 months), which are better models of the middle-aged population more at risk for stroke than younger counterparts. Second, we will collaborate with three laboratories known for their animal stroke research to model a randomized, double-blind multi-laboratory trial in rats. Third, we will examine long- term functional outcomes after the injury and treatment to see whether PROG's effects are enduring. Fourth, we will extend our treatment parameters to stroke-prone rats, so our stroke models are more characteristic of the risk factors seen in humans. Fifth, we will use these data to apply to the FDA for an IND to test PROG in human stroke patients. We believe the U01 cooperative agreement for translational research is an ideal way for our laboratory and those of our collaborators to provide the translational data needed to move to clinical testing of PROG as a treatment for stroke. RELEVANCE: Stroke remains a major public health problem worldwide. Aside from tPA, of the more than 700 drugs studied, none have proven effective in clinical trial. PROG has been shown to be safe and effective in clinical TBI and we now need to determine whether it can be as effective in the clinical treatment of ischemic stroke. The studies proposed here move us closer to that possibility.

DESCRIPTION (provided by applicant): Dozens of phase II and III clinical trials for traumatic brain injury (TBI) therapies have failed. One reason cited is that the complex mechanisms and injury cascades associated with different types of TBI are not being addressed by drugs with limited modes of action. Progesterone (PROG) has been shown to be a safe and effective treatment for TBI. A phase I/II clinical trial using PROG to treat moderate to severe brain injury found that mortality from severe injury was reduced almost 60% and there was better functional outcome. This trial was replicated with an additional 159 patients in an independent study from abroad. The PIs' current research is seeking even better results by combining PROG with other neuroprotective agents. A logical first step is to examine the conditions that might limit PROG's beneficial effects in a clinical setting. Vitamin D hormone (VDH) is an FDA-approved molecule whose neuroprotective and neurotrophic actions are increasingly being recognized. The PIs' recent research suggests that, for older subjects, VDH deficiency (VDHdef) reduces the beneficial effects of PROG. It has been reported that well over half of older adults are VDHdef, a condition which could exacerbate the outcome of any TBI, and the elderly population is disproportionately subject to TBI. Further, about 30-35% of the American public has been reported to be VDHdef, so patients of any age, including children, presenting with a TBI could be more at risk and have less favorable outcome if they are also VDH deficient. Thus something as simple as VDH could enhance the benefit of PROG treatment as a neuroprotective agent. Both PROG and VDH are natural hormones, synthesized in both males and females, and have distinct, pleiotropic modes of action in CNS repair. These properties make the preclinical testing of combined PROG and VDH a compelling approach to consider for later evaluation and testing in a clinical TBI trial. First, the PIs will determine the optimal dose for combination treatment to enhance PROG's effects on post- traumatic recovery from neurological deficits and resolution of lesion size in male and female rats. Second, they will see whether PROG's effects are compromised in a co-morbid VDH deficient state in old animals and whether supplementing VDH is necessary to promote optimal benefit. Third, they will establish a correlation, if any, between PROG and VDH levels, functional recovery, and serum levels of surrogate serum biomarkers for TBI. To measure the severity of the injury and predict extent of repair, the time course of changes in neurologic outcome and serum levels of TBI markers will be evaluated. Fourth, the PIs will identify mechanisms that contribute to better neuroprotection by combination of PROG with VDH. The PIs will analyze the gene products most significantly changed in response to combination treatment or either agent alone, and most related to brain injury and repair. PUBLIC HEALTH RELEVANCE: Progesterone (PROG) has been shown to be a safe and effective treatment for moderate and severe traumatic brain Injury. There is evidence that its effectiveness can be increased by combining it with Vitamin D hormone (VDH), especially in the elderly. The fact that both PROG and VDH have high safety profiles, act on many different injury and pathological mechanisms, and are easy to administer and inexpensive, make this combination an obvious approach to develop what may become the first successful treatment for traumatic brain injury.