Candace L. Floyd, Ph.D. - US grants

DESCRIPTION (provided by applicant): The goal of the current proposal is to evaluate the hypothesis that traumatic brain injury alters intercellular communication between astrocytes, a potentially crucial and often overlooked aspect of brain processing. Astrocytes were once viewed as passive cells, but are now characterized as active contributors to signal processing in the brain. Yet, the effect of traumatic injury on calcium-mediated astrocyte intercellular signaling has not been evaluated. Therefore, the proposal evaluates the hypothesis that traumatic injury alters intercellular calcium signaling between astrocytes in the glial syncytium using the following specific aims: Specific Aim 1: Injury of cortical astrocytes alters origination and propagation of intercellular calcium waves. We will compare the velocity and distance of intercellular calcium waves in injured astrocytes to that of uninjured astrocytes. (Method: Use quantitative fluorescent microscopy to evaluate intercellular calcium waves). Specific Aim 2: Injury alters gap junction coupling. We will compare fluorescent dye transfer through gap junctions, expression of Connexin 43 protein, and phosphorylation of Connexin 43 protein between uninjured and injured astrocytes. (Method: Use lucifer yellow transfer to measure coupling of gap junctions; use Western Blots to measure expression and phosphorylation of Connexin 43 protein). Specific Aim 3: Intercellular calcium signaling will be maintained in astrocytes by pharmacological manipulation of IP3-mediated intracellular calcium signaling. (Method: Use agonists and antagonists of elements in the signaling pathway to determine if alterations in intercellular calcium waves can be attenuated). These proposed experiments evaluate, for the first time, the effect of traumatic brain injury on the intricate and extensive calcium signaling network among astrocytes. Aim #1 directly examines the effects of mechanical injury on intercellular calcium signaling in astrocytes. Aim #2 examines the potential mechanisms (i.e. gap junctions) involved in injury-induced alterations in intercellular calcium signaling. Aim #3 examines potential therapeutic interventions which could restore intercellular signaling. The results of the proposed experiments may provide not only new insights into the pathophysiology of TBI, but also potentially lead to the development of novel therapeutic approaches for the treatment of the traumatically injured brain.

[unreadable] DESCRIPTION (provided by applicant): With the continuing controversy over the clinical efficiency and use of methylprednisolone after spinal cord injury (SCI), interest is renewed is discovering new therapeutic targets for neuroprotection. Recently, 17[unreadable]-estradiol was found to be beneficial in SCI which suggests that estrogen receptors (ERs) could be therapeutic targets. Estrogen binds with equal affinity to two subtypes of the ER, the classical ERa and the more recently discovered ERa. However, which subtype is necessary for neuroprotective effects remains controversial and has not been investigated in SCI. Also, no subtype selective antagonists are available to tease apart specific effects. A clue that selective ERp activation may confer protection is SCI comes from studies of plant-derived estrogens, or phytoestrogens. Genistein, a phytoestrogen from soy and a preferential ERp agonist, dose-dependently confers protection in models of neuronal injury and cardiac ischemia. We hypothesize that selective activation of the ER[unreadable] by genistein will produce significant protection in SCI. [unreadable] [unreadable] We will test this hypothesis, in aim 1, by administering either a low, medium or high dose of genistein 30 minutes after a moderate thoracic spinal cord injury in rats. An additional group will receive co-administration of genistein and the ER antagonist ICI 182,780. At seven days post-SCI, we will evaluate acute injury markers including: cell death, ER expression, and expression of apoptosis related proteins bcl-2, bax, and activated caspase-3. In aim 2, we will administer genistein with and without the ER antagonist ICI 182,780 and evaluate sub-acute markers of secondary injury including locomotor impairment, white matter sparing, lesion volume, and lower urinary tract function. [unreadable] [unreadable] The experiments in this proposal explore the clinically relevant possibility that preferentially targeting the non-feminizing ER[unreadable] is neuroprotective in SCI by evaluating the neuroprotective potential of a natural, plant- derived estrogen, genistein. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) affects approximately 1.7 million people in the United States every year and it is estimated that up to 75% of these injuries are classified as mild TBI. However, the word mild is an inadequate description as mild TBI (mTBI) is typically accompanied acutely by significant deficits in cognition that often manifest as long-term alterations in learning and memory, attention, and emotional control. Despite the high incidence and often lasting impact of mild TBI, the factors that induce long-term cognitive deficits remain unknown. Consequently, the goal of this project is to identify alterations in neuronal function that may underlie long-term deficits caused by mTBI. Based on our preliminary data, we hypothesize that mTBI causes structural and functional alterations in the dentate gyrus that contribute to lasting cognitive deficits. A principal function of the dentate gyrus is to restrict the flow of neural activity through the hippocampus. This gating function is essential for propagating sparse representation of cortical sensory signals to downstream pyramidal cells and is achieved by strong local inhibitory circuitry. Furthermore, the dentate gyrus is one of two brain regions where neural progenitor cells continuously generate newborn neurons. Our preliminary data indicate that a single episode of mTBI acutely disrupts the balance of inhibition and excitation and is followed by a robust enhancement in neurogenesis that persists for months. We propose that the transient breakdown of the dentate gate leads to activity-induced enhanced neurogenesis and predict that mTBI-induced neurogenesis has long-term detrimental effects on dentate function that contribute to lasing cognitive impairments. Using a clinically-relevant mouse model of mTBI, we will evaluate our hypothesis using three specific aims. First, we will determine how mTBI alters the gating function of the dentate gyrus using electrophysiological techniques in hippocampal slices and corroborate these findings in vivo after mTBI. In the second aim, we will use transgenic reporter mice to determine how mTBI alters the structural and functional properties of mTBI-induced new neurons. We will evaluate how newly generated cells integrate into the circuitry of the dentate gyrus and affect the gating function. In the third aim, we will test the hypothesis that dentate alterations contribute to cognitive impairments. At time points when dentate abnormalities are present, mice that received mTBI will be evaluated in a variety of well-established behavioral paradigms to test learning, memory, attention and emotional control. We will also test whether manipulating gating and neurogenesis are sufficient to recapitulate and block the behavioral impairments caused by mTBI. The successful completion of this project will elucidate a potential mechanism for cognitive deficits after mTBI as well as identify novel targets for treating the most common form of brain injury. PUBLIC HEALTH RELEVANCE: Traumatic brain injury (TBI) affects approximately 1.7 million people in the United States every year and it is estimated that up to 75% of these injuries are classified as mild TBI. However, the word mild is an inadequate description as mild TBI (mTBI) is typically accompanied acutely by significant deficits in cognition that often manifest as long-term alterations in learning and memory, attention, and emotional control. The research proposed here will elucidate a potential mechanism for cognitive deficits after mTBI as well as identify novel targets for treating this most common form of brain injury.