2007 — 2011 |
Kirov, Sergei A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Activity and Dendritic Structural Rearrangements in the Mature Brain @ Georgia Regents University
DESCRIPTION (provided by applicant): Mature CNS neurons have a significant intrinsic capacity for structural plasticity. This implies that they adapt to acute injury by proliferating new spines, which if consolidated, may rewire existing brain circuitry. In focal ischemia, failure of the Na+/K+ pump caused by depletion of ATP results in the anoxic depolarization (AD) with recurring AD-like peri-infarct depolarizations (PIDs) in the penumbra. The functional collapse of plasma membrane ion selectivity that drives and maintains the propagating AD, causes dramatic neuronal and glial swelling with dendritic beading and spine loss within tens of seconds. Within minutes, recurring PIDs initiate at the edge of the ischemic core, expanding neuronal damage into the penumbra during the next 1-2 days. The immediate goal of the proposed research is to address the role of these maintained depolarizations in evoking acute dendritic injury using in vitro and in vivo ischemia models. We can then test whether injury can be reduced and examine the long-term recovery of dendritic structure in vivo following focal stroke. We have discovered that dendrites become beaded and spines are lost within minutes of the Na+/K+ pump inhibition induced by ouabain or oxygen-glucose deprivation (OGD). We have shown that pump inhibition by cold, ouabain or OGD quickly elicits dendritic beading with spine loss. We have also shown that the intact neuronal membrane at normal resting potential poorly conducts water, resisting acute osmotic stress. A maintained depolarization as during stroke or cold is required to swell neurons and elicit dendritic beading and spine loss. The rapid proliferation of new spines on mature neurons during re-warming reveals an adaptive synaptogenesis in response to acute injury as dendritic structure recovers. It is unclear how long these newly spines persist or whether they are eliminated or stabilized when activated. Therefore the specific aims of this project are: 1) Investigate dynamics of AD-mediated injury and recovery of dendritic structure in acute slices. 2) Assess dynamics of dendritic injury during penumbra recruitment in vivo and during long- term recovery of synaptic circuitry post-stroke. 3) Study the ionic mechanisms underlying dendritic structural changes during the cold-induced depolarization. 4) Determine whether new spines formed on mature neurons are preserved or eliminated upon global synaptic activation. In aims 1 and 4, our synaptic studies will correlate functional data from field recordings with structural data from 2-photon laser scanning microscopy (2PLSM). In aim 2, in vivo dendritic structure during stroke in the core, penumbra and unaffected cortical regions from acute and chronic mouse models will be imaged in real time using 2PLSM. We will directly correlate injury with blood flow and with recurring depolarizations. In aim 3, 2PLSM will be correlated with intracellular recordings to examine dendritic beading and recovery in hippocampal slices. The results will address how neurons are acutely damaged, how they recover and ways to facilitate their recovery.
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1 |
2008 — 2009 |
Kirov, Sergei A |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Neuroprotection in the Human Brain Tissue Model of Stroke @ Georgia Regents University
DESCRIPTION (provided by applicant): During stroke, failure of the sodium-potassium ion pump caused by a depletion of ATP in the ischemic core results in anoxic depolarization (AD) propagating through the stroke focus followed by recurring AD-like peri- infarct depolarizations (PIDs) in the penumbra. The functional collapse of plasma membrane ion selectivity associated with propagating AD, causes dramatic neuronal swelling, dendritic beading and spine loss within seconds with associated glial swelling. In essence, swelling is the initial response and a sign of the acute neuronal damage that follows if ischemia is maintained. Recurring PIDs migrate through regions of compromised blood flow, consuming precious energy supplies and expanding the initial site of infarct during subsequent days. This represents a window of opportunity for therapeutic inhibition of these propagating depolarizations. Reduction of the excitotoxic effects of glutamate release caused by these depolarizations has been a traditional approach to prevent ischemic damage. However, an equally viable rationale representing a different and potentially powerful neuroprotective strategy is to target these upstream rapid depolarizations. Morphological and functional differences exist between the brain of humans and animals. Lower mammals appear to be more amenable to neuroprotection than humans. This may suggest that there may be essential differences between human and lower mammals'brain tissue in response to ischemic injury. Therefore, human brain slices as a model system can provide a missing link between animal models and patients and offer a unique chance to identify and study potentially useful therapeutics before advancing to clinical trials. Here, using live human cortical slices prepared from brain tissue resected during surgeries, we propose to inhibit these propagating depolarizations and associated neuronal and glial swelling to better understand and treat their clinical consequences. The specific aims of this project are: 1) Test candidate therapeutics for efficacy of inhibiting AD in human neocortical slices. 2) Evaluate at the cellular level the neuroprotective effect of candidate therapeutics during onset and recovery from AD-mediated injury in human neocortical slices. AD will be induced by oxygen-glucose deprivation, an in vitro ischemia model, or by application of ouabain, a sodium- potassium pump blocker. In aim 1, changes in light transmittance (LT) will track AD and associated slice injury in time and space. In aim 2, cellular dynamics of neurons and astrocytes will be imaged in real time with 2- photon laser scanning microscopy (2PLSM). We will directly correlate structural data from 2PLSM and changes in LT with functional data from field recordings with and without a drug treatment. As a result, we anticipate developing a valuable model for reliable testing and the preliminary screening of candidate therapeutic drugs. PUBLIC HEALTH RELEVANCE: Project Narrative Stroke is the third leading cause of death in industrialized countries and the main source of adult disability. Recent experimental and clinical data imply that the spread of a propagating wave of anoxic depolarization defines the ischemic core and recurring waves of peri-infarct depolarizations expand a cerebral infarct contributing to secondary injury in patients with stroke and traumatic brain injury. Choosing these depolarizing events as targets for therapeutic intervention, this exploratory project aims to develop an assay using human neocortical slices for the preliminary screening of candidate stroke therapeutics before advancing to clinical trial.
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2013 — 2021 |
Kirov, Sergei A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Synaptic Circuitry in Stroke @ Georgia Regents University
DESCRIPTION (provided by applicant): Spreading depolarizations (SDs) are waves of sustained near-complete neuronal and glial depolarization that actively propagate a collapse of ion gradients through the brain with associated dramatic neuronal and glial swelling that entails cytotoxic edema. In a healthy brain, neuronal activity can be present until the onset of SD which triggers spreading depression (silencing) of activity that translates into migraine aura. Recovery of ion gradients depends on the sufficient sodium pump activity which is energy-dependent. Yet, even in normal cortex SD could cause a shortage of energy supply suggesting that even normoxic SD might pose a threat in healthy cortex. Indeed, mild ischemia induced by normoxic SD could result in migrainous stroke. In stroke and trauma patients SDs are known to exacerbate tissue damage in the at-risk cortical territory supporting the view that SD may be an important mechanistic endpoint in clinical studies. We have shown that in the penumbra SDs result in rapid dendritic beading which is reversible, but signals leading to neuronal death could be initiated during this time. Hence, dendritic beading is the hallmark of neuronal injury. Once the energy demands for recovery of penumbral dendrites are no longer met by the diminishing blood flow, SD irreversibly injures dendrites and spines are lost signifying acute damage to synaptic circuitry. Importantly, we have shown that SD-induced dendritic injury in the penumbra could be stopped pharmacologically. We have also shown that persistent astroglial swelling is initiated and exacerbated during SD in brain tissue with moderate to severe energy deficits, likely disrupting astroglial maintenance of normal homeostatic function and thus their ability to support neurons. SD-induced cytotoxic edema contributes to stroke injury. Mammalian pyramidal neurons lack functional aquaporins, thus the molecular pathways by which they accumulate osmotically obligated water and rapidly swell during SD is unknown. Bulk water influx could occur through large-pore pannexin hemichannels opened by SD. Transporters may also be responsible for water accumulation as well as recovery. The specific aims are: 1) To test the hypothesis that SD-inflicted dendritic structural rearrangements in naive healthy neocortex depend on the degree of the transient tissue hypoxia imposed by SD. 2) To test the hypothesis that SD-inflicted dendritic injury is aggravated in tissue with selectively impaired glial metabolism. 3) To examine possible routes of rapid water entry during SD-induced dendritic beading. 4) To test the hypothesis that SD is the mechanism implicated in rapid synapses disruption and loss. To achieve these aims we will combine in vivo 2-photon laser scanning microscopy of fluorescent neurons, astrocytes and blood flow in adult mouse somatosensory cortex with other sophisticated in vivo approaches such as laser speckle and intrinsic optical signal imaging while simultaneously monitoring occurrence of SD. Quantitative serial section electron microscopy analyses will be used to reveal SD-induced injury at the level of single synapses. The results will bring new insight to the development and recovery from acute injury of synaptic circuits in stroke.
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0.931 |