1992 — 1996 |
Nedergaard, Maiken |
R29Activity Code Description: Undocumented code - click on the grant title for more information. |
Cellular Neurobiology of Neuronal Death in Vitro @ New York Medical College |
0.946 |
1995 — 1997 |
Nedergaard, Maiken |
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. |
In Situ Imaging of Neuronal Ca 2+ in Acute Brain Injury @ New York Medical College
DESCRIPTION (Adapted from Applicant's Abstract): Sustained dysregulation of cytosolic calcium concentration may be a proximal common denominator of irreversible neuronal injury. Several lines of evidence support the notion that accumulation of calcium occurs during acute stroke and trauma, as well as in the setting of chronic neurodegenerative processes, such as Alzheimer's and Parkinson's diseases. Delineation of whether substantial increases of cytosolic calcium consistently precede neuronal death, and whether this may be modified by protective maneuvers, is essential for establishing a pathogenic role for calcium. However, it has traditionally been difficult to follow neuronal calcium changes in the intact animal brain, and little information exists on changes in neuronal calcium concentration during brain injury. The objective of this proposal is to quantify the disturbances in cerebral calcium homeostasis that occur during and after ischemia in the living rat brain, and to correlate these changes to neuronal injury on the single cell level. Using laser-activated confocal imaging of intraneuronal calcium, it proposed to test the postulate that late disturbances in calcium homeostasis that follow ischemia mediate neuronal death. This will be performed by asking: 1. Do distinct loci of cellular calcium entry yield differential effects on neuronal viability during and after ischemia? 2. Is the increase of cytosolic calcium necessary or sufficient to promote irreversible neuronal injury? 3. Will earlier determination of neuronal death in vivo improve therapeutic efforts, by allowing therapy to be more precisely directed? To address the latter question, the investigators will employ a new approach that they have developed for the determination of cell viability in vivo, the confocal imaging of nuclear staining with a fluorescent DNA marker, propidium iodide. This approach will permit the mapping of neuronal calcium concentration and viability simultaneously, on the single cell level in vivo. Through this approach, the investigators intend to define the temporal relation of intraneuronal calcium dysregulation and cellular viability to a far more precise degree than current approaches allow. By doing so, it is hoped to provide a definition of the temporal window following cell injury but before loss of viability, during which effective neuroprotective therapy may be directed following traumatic and ischemic brain injury.
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0.928 |
1997 — 2000 |
Nedergaard, Maiken |
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. |
Astrocyte Mediated Propagation of Ischemic Brain Damage @ New York Medical College
DESCRIPTION Two intriguing recent observations in stroke research are that, 1) the outer region of an ischemic infarct, the ischemic penumbra, is potentially salvageable tissue; and 2) waves of infarct-associated spreading depression invade the penumbra, and their frequency and severity correlate with the ultimate extent of structural damage. A key step in understanding why an ischemic infarct expands might be to establish why and how waves of spreading depression are generated within ischemic tissue. The PI has demonstrated that an intact gap junctional network is required for the propagation of spreading depression supporting the notion that spreading depression is an in vivo correlate to gap junction-mediated astrocytic calcium signaling. Imaging of calcium signaling among a population of ischemic astrocytes might therefore provide a powerful tool for analysis of interactions between dying and viable cells. On this basis, the PI postulates that aberrant calcium signaling initiated in dying astrocytes can cause the death of otherwise viable neighbors. Over the last few years the PI's lab has developed a number of methodologies which have allowed: 1) the characterization of gap junctional signaling on a cell-to-cell level 2) the manipulation of the degree of gap junctional coupling by connexin over-expression, 3) influence the resistance of cultured cells to ischemia, by bcl-2 overexpression, 4) the block of the propagation of SD by gap junction blockers, and 5) the evaluation of gap junctional coupling in the live rat. In preliminary studies, it has been established that astrocytic gap junctions remain functional during the process of cell death, that spontaneous calcium activity is initiated during ischemia, and that the extent of injury is a function of the degree of gap junctional coupling. It is now proposed to exploit these advances by applying them to a previously intractable problem, that of the role of signaling across gap junctions in acute brain injury. Experimental questions will include: 1. Are astrocytic gap junctions functional during the process of cell death? 2. Can dying astrocytes cause death of otherwise viable neighboring cells? 3. Is focal ischemia associated with a decrease in gap junctional function intake live rat brain? 4. Will manipulations of junctional conductance affect the outcome of ischemia?
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0.928 |
1999 — 2006 |
Nedergaard, Maiken |
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. |
Control of Astrocytic Signaling @ University of Rochester
DESCRIPTION (provided by applicant): Experimental observations have over the past few years suggested that astrocytes play a more active role in brain function than previously recognized. It is therefore of importance to establish how astrocytes communicate with other cells. In previous observations, we have reported that astrocytic calcium waves are mediated by release of nucleotides and activation of purinergic receptors in neighboring cells. Our intention in this proposal is to identify the mechanism of astrocytic ATP release, as well as to define the cellular events that result in ATP efflux. We first plan to test the hypothesis that efflux of cytosolic ATP is mediated by opening of Cx43 hemichannels. Combining single channel recordings using an inside-out configuration, together with bioluminescence imaging, will enable us to directly establish whether Cx43-hemichannels release ATP. Next we will ask whether the lack of ATP release from neurons and oligodendrocytes reflect their lack of Cx43 expression. We will evaluate the effect of adenoviral transfer of cDNA encoding Cx43 together with an EGFP reporter upon ATP release. These experiments also explore the alternative possibility that the lack of ATP bursting in neurons reflect intrinsic cellular properties, rather than lack of Cx-hemichannels by overexpression of the neuronal gap junction protein, Cx36, in glial cells. We will also ask whether oscillatory calcium increments is a necessary prerequisite and thereby apredictor of ATP burst releases. If so, this approach that will allow us to characterize the cellular events, which result in ATP release. To this end, we will combine bioluminescence detection of ATP with calcium imaging, so as to define calcium dynamic in cells that release ATP. These experimentsoffer the first concurrent visualization of a transmitter and calcium signaling in live cells. Defining the pathways by which astrocytes signal among one another, as well as to their neuronal partners, may provide us an important basis for the directed manipulation of neuronal-glial signaling, both in health and disease.
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1 |
2001 |
Nedergaard, Maiken |
S10Activity Code Description: To make available to institutions with a high concentration of NIH extramural research awards, research instruments which will be used on a shared basis. |
Multi-Photon Confocal Microscope System @ New York Medical College
This proposal requests funds to purchase a multi-photon confocal microscope system integrated with a Ti:sapphire laser, an upright fluorescence microscope, and an electrophysiological set-up. Eleven separate NIH-funded laboratories (from 4 of the 5 basic research departments and 3 clinical departments at New York Medical College and Cornell University Medical College) have proposed experiments using this instrument. These projects include 1) assessing the role of astrocytic calcium signaling in synaptic transmission and ischemic brain injury, 2) evaluating dendritic and axonal calcium spikes in hippocampal and tegmental neurons, 3) visualization of the cell-cell interactions that permit the migration of new neurons generated from the subependyma of the adult brain, as identified by cell-specific, GFP-expressing adenoviral vectors, 4) imaging of GFP-expressing glioma cells during their invasion into host brain, and 5) analysis of apoptosis in several systems, include retina, myocardium, and tumors. These experiments will take advantage of the ability of the two-photon confocal microscope system to 1) image deep into live tissue, 2) image for extended time periods with minimal tissue damage, and 3) focally uncage molecules in small regions inside and outside cells. The multi-photon confocal microscope system will become a core facility at New York Medical College, serving the entire medical science community.
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0.928 |
2001 — 2004 |
Nedergaard, Maiken |
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. |
Antiapoptotic Actions of Connexin @ University of Rochester
DESCRIPTION (Provided by applicant): The aim of this application is to establish by which mechanisms Cx-proteins improve cellular survival following injury. We have in preliminary observations established that Cx-expression antagonizes cell death indicating that the connexin proteins have death-inhibitory or anti-apoptotic activity. In specific we will ask: Does an adaptive remodeling and reorganization of Cx43 contribute to the high resistance to injury of Cx43 expressing cells? A Cx43-eGFP fusion protein has been stably expressed in C6 cells and time-lapse analysis has revealed that Cx43-eGFP undergo major structural reorganization after injury. We will here test the preposition that Cx43 reorganization represents an adaptive response that improves survival and that the lower resistance of Cx-deficient cells results from their limited ability to initiate the same process after injury on a single cell level. The analysis will be extended to include primary astrocytes transfected with an adenoviral vector encoding Cx43-GFP. By which mechanisms do connexin proteins increase cellular resistance to injury? Is formation of functional gap junction channels a prerequisite for their anti-apoptotic action? Alternatively, do mutant Cx's with deficient channel function also provide injury-resistance indicating that yet undefined actions of Cx.proteins are responsible for the increased survival? We have established several cell lines with stable expression of either wildtype Cx43, or Cx-mutants with deficiencies in either channel function or membrane localization. These clones represent a powerful tool to establish at which level the Cx-proteins antagonize cell death. Is the increase in cellular resistance associated with Cx-expression dictated by the phenotypic transformation? Does loss of cytoskeletal organization increase cellular sensitivity to injury? We will in these studies test the preposition that the Cx-induced phenotypic transformation observed, in part, may contribute to the increased cellular resistance. Does the death-inhibitory activity of Cx require ATP secretion? Do purinergic receptor blockers antagonize the Cx-induced increase in cellular resistance? It is here postulated that ATP secreted from Cx-expressing cells acts as an autocoid differentiation factor that increases the cellular resistance to injury. The effects on cellular resistance of long-term treatment with purinergic-receptor agonist and antagonists will here be analyzed. Defining the pathways, by which connexin proteins increase cellular resistance to injury, may provide a potential new therapeutic target for preventing cell death in acute pathologies as stroke and head trauma.
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1 |
2001 — 2004 |
Nedergaard, Maiken |
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. |
Calcium Signalling Among Non-Neuronal Brain Cells @ New York Medical College
In previous studies, we have reported that calcium waves among gap junction-coupled glia may form the cellular substrate for spreading depression in vivo. Recently, we have noted that calcium waves initiated in astrocytes in slices can propagate to brain endothelial and meningeal cells; all of these cell types express connexin43, which may allow their mutual heterotypic syncytial interaction through homotypic gap junctions. On this basis, we propose to test the hypothesis that astrocytic calcium waves may thereby invade the brain by propagating along the capillary vasculature, as well as through the astrocytic syncytium. These experiments will test the possibility that endothelial calcium waves may follow the venular endothelium to invade the meningeal vasculature, thereby recruiting both meningeal cells and trigeminal sensory afferents. This proposed pathway, by bypassing and traversing the restrictive barrier of the pia limitans, would permit the recruitment of both the meningeal vasculature and its trigeminal sensory afferents into parenchymal waves of spreading depression. We propose here that this scenario might operationally model the initiation of migraine headache in adults. In parallel experiments, we will also follow-up our recent observation of a steroid-induced accentuation of astrocytic calcium signaling, by asking whether calcium signaling among non-neuronal brain cell types may be modulated by gonadal steroids. In particular, we seek to determine whether the cyclical female hormones estrogen and progesterone potentiate signaling from astrocytes to endothelial cells, and if so, whether the likelihood of meningovascular recruitment into a parenchymal calcium wave is thereby increased. This pathway might account for much of the symptomatology of migraine headache, while steroidal accentuation of calcium signaling might account for the cyclicity of migraine occurrence. The long-distance multicellular calcium signaling pathway that we propose, and its attendant hormonal regulation, suggests immediately testable strategies for its abrogation.
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0.928 |
2006 — 2010 |
Nedergaard, Maiken |
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. |
Mediators of Functional Hyperemia and Its Dysregulation in Post-Traumatic Brain @ University of Rochester
DESCRIPTION (provided by applicant): Local increases in blood flow during neural activity form the basis for functional brain imaging, yet the mechanisms of such activity-dependent hyperemia remain poorly defined. Astrocytes, with their extensive fiber arbors, provide an anatomical link between synapses and the microvasculature, and several recent studies have implicated them in the regulation of vasomotor tone. Using 2-photon imaging of anesthetized adult mice, we have found that astrocytic Ca2+ signaling in cortex potently triggers local vasodilatation. Cytosolic Ca2+ was selectively elevated by photolysis of caged Ca2+ in astrocytes, and invariably associated with vasodilation. In this set of studies, we intent to evaluate the relative contribution of astrocytes to functional hyperemia within the parietal sensory cortex, as evoked by whisker stimulation. Several vasoactive compounds, including COX-1 and -2 products, adenosine, NO, and cytochrome P450 vasoactive compounds have previously been implicated in such functional hyperemia. These compounds may affect vascular tone directly by acting on smooth muscle cells, or indirectly through astrocytic intermediaries. By concomitant imaging of Ca2+ and local perfusion, we will first assess the effects of these vasoactive agents upon the astrocytes and vascular tone in vivo, with an emphasis on discriminating which agents operate on astrocytes, which on vasculature downstream of astrocytic stimulation, and which at both loci. We will then use pharmacological approaches to block or enhance Ca2+ signaling in combination with whisker stimulation, so as to establish the relative contribution of astrocytic calcium signaling to functional hyperemia. Next, we will analyze the role of astrocytes in vascular dysregulation following brain injury. Both arterial vasospasm and microvascular vasoconstriction with secondary ischemia are common late complications of brain injury, and their genesis coincides with the appearance of reactive astrocytosis. In addition, we have already noted that following experimental intracerebral hemorrhage, reactive astrocytes display sustained increases in resting Ca2+ and fail to trigger vasodilation. On the basis of these observations, we will assess whether the role of injury-elicited astrocytosis contributes to delayed hypoperfusion following brain injury. This set of studies comprises a concerted effort to define the relationship of glial signaling to vascular tone, in both normal physiology and disease. These experiments promise to yield much information of real clinical utility;the pharmacological modification of vasomotor tone and blood flow may be of critical importance to the treatment of disorders as diverse as acute stroke microvascular angiopathies, trauma, and both subarachnoid and intracerebral hemorrhage.
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1 |
2007 |
Nedergaard, Maiken |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Administrative and Imaging @ University of Rochester |
1 |
2007 — 2011 |
Nedergaard, Maiken |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
The Role of Astrocytes in Ischemic Stroke @ University of Rochester
DESCRIPTION (provided by applicant): Stroke remains the third leading cause of death in the US, behind heart disease and cancer. Each year 750,000 Americans suffer a diagnosed stroke and this number will double by 2050, given the advancing average age of the population, barring further developments in either prevention and/or treatment. The goal of our proposed set of projects is to critically evaluate astrocytes as a potential new therapeutic target in stroke. Over the past few years, a virtual revolution has occurred in our understanding of the cell biology and physiology of astrocytes, and in our understanding of their interactions with neurons in the normal brain. Yet despite their obvious relevance to stroke, the contribution of astrocytes to the process of ischemic infarction has not yet been well-studied. In this application, we propose to use a multimodal approach to define the role of astrocytes in the pathogenesis of ischemic stroke. We shall make use of 2-photon laser scanning microscopy to visualize astrocytes labeled with GFP and Ca2+ sensitive dyes, both in slices and in the intact brain. We will also utilize GFP labeling under control of the astrocyte specific GFAP promoter, to sort astrocytes from animals with experimental stroke, so as to characterize ischemia-induced changes in gene expression. The GFAP promoter will be utilized to knock out expression of HIF-1alpha, the master regulator of ischemia-induced gene expression, and this approach will allow us to define how hypoxia-induced gene expression in astrocytes affect neuronal survival following experimental stroke. The program benefits from the very different training of its investigators, whose expertise spans electrophysiology, stroke models, Ca2+-imaging techniques, fluorescent activated cell sorting, and molecular genetic manipulation. Our hope is that these studies ultimately will result in new treatment options for patients with acute ischemic stroke.
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1 |
2007 |
Nedergaard, Maiken |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Stroke @ University of Rochester |
1 |
2007 — 2011 |
Nedergaard, Maiken |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Astrocytic Ca2+ Signaling in the Ischemic Penumbra @ University of Rochester
The ischemic penumbra was originally defined as peri-infarct tissue with partial reduced blood flow. The blood flow reduction (30-60% of control values) was less severe than in the ischemic core allowing the penumbral tissue to maintain normal transmembrane ionic gradients. The electrical silence of the penumbra was ascribed to the partial reduction in substrate delivery, e.g. the energy supply was sufficient to maintain membrane potential, but not to support synaptic transmission. The fate of astrocytes in the penumbra has received little attention, in part, because the activity of these non-excitable cells only can be analyzed by Ca2+ imaging technique. Combined with recording of local field potential, we can simultaneously monitor local microcirculation, astrocytic Ca2+ signaling, and synaptic activity within the same field. We have observed that astrocytes, as opposed to neurons, are activated and display spontaneous Ca2+ oscillations, as well as propagating Ca2+ waves in the ischemic penumbra. Astrocytic Ca2+ signaling is associated with the release of several neurotransmitters, including glutamate and ATP.-ATP is, in the extracellular space, rapidly degraded to adenosine - anendogenous neuroprotective agent. On the basis of these observations, this proposal will test the proposition that astrocytic Ca2+ signaled- release of ATP, and the latter's rapid degradation to adenosine, mediates the electrical silence of the ischemic penumbra. In Aim 1, we will characterize ATP release in the setting of focal ischemia. Aim 2 intends to define astrocytic Ca2+ signaling in the penumbra, using 2-photon laser scanning microscopy to establish which transmitters (glutamate, ATP) trigger the abnormal Ca2+ signaling by local application of receptor antagonists. Aim 3 will directly image NADH to assess the cellular metabolic responses to reduced blood flow in the ischemic penumbra, and will define the respective contributions of astrocytes and neurons in this regard. Combined imaging of capillary flow and Ca2+ or NADH imaging will allow a correlation between local perfusion and astrocytic Ca2+ signaling or NADH on a single cell level in live animals. For these experiments, we will use of transgenic Thy1-YFP loaded with the astrocyte-specific indicator, sulforhodamine 101 mice. Aim 4 then asks if astrocytic Ca2+ signaling by release of ATP/adenosine reduce synaptic transmission, lower metabolic demands, and thereby increase neuronal survival in the penumbra. In specific, we will define the role of adenosine A1 receptors in synaptic depression in the penumbra. Preliminary observations show that the adenosine A1 receptor antagonist, DPCPX, triggered a robust increase in synaptic activity in the penumbra without a concomitant increase in blood flow. Our expectation is that a more precise mechanistic understanding of the role of astrocytes in this process will ultimately justify the development and assessment of adenosine mimetics and modulators as therapeutic agents in ischemic stroke. PHS 398 (Rev. 09/04) Page 71. Form Page 2 PO1 NS050315 Project 1 Principal Investigator/Program Director (Last, First, Middle): Nedergaard, Maiken Introduction to revised application This is a second revision of our PPG application entitled The role of astrocytes in ischemic stroke, and of my section therein, Astrocytic calcium signaling in the ischemic penumbra. The past submission of this proposal was praised for being highly innovative, and for addressing several fundamentally new areas in stroke research. However, the referees raised concerns regarding: 1) our technical ability to causally link acute ischemic events, e.g. ATP release and Ca2+ signaling, to neuronal death;2) the concept of ATP as an excitatory transmitter, and its potential to trigger neuronal death in ischemia;and, 3) the specificity of P2X receptor antagonists. Based on the reviewers'comments, we have chosen to focus this revised application on acute cellular responses to ischemia, thereby taking maximal advantage of our 2-photon imaging approaches to assessing both intracellular and intercellular signaling events in situ. I have also dropped Aim 3, which correlated ATP release with delayed neuronal injury, and which was viewed as too preliminary by the referees. We have since last application refined the technique of in vivo NADH imaging in the ischemic penumbra. NADH is the principal electron carrier in glycolytic and oxidative metabolism, and is an intrinsic indicator of cellular redox state. Recent developments in 2-photon imaging have improved its spatial resolution substantially, so much so as to resolve subcellular changes in NADH within the ischemic cortex. Another advantage of 2-photon NADH imaging is that we can identify and isolate changes in redox state, in both individual cells and their processes. Use of Thy1-YFP mice, that selectively express YFP in central neurons, combined with loading of the astrocyte specific indicator, sulforhodamine 101, has enabled us to separately assess the respective metabolic responses of neurons and astrocytes within the same field of view. Our preliminary observations have indicated a strong compartmentalization of metabolic responses in the ischemic penumbra. Combined imaging of NADH and capillary flow should therefore enable us to precisely define changes in neuronal and astrocytic redox state, in response to experimentally-defined reductions in blood flow. By imaging NADH and Ca2+, we intend to assess the interdependence of hypoxia-associated NADH and Ca2+ increases. Our collaborator, Frank Kirchhoff, Gottingen, has established a set of spectrally distinct GFAP reporter mice that include GFAP-EGFP, GFAP-AMCyan, and GFAP-mRFP1, which will permit us to directly visualize astrocytes concurrently with NADH and Ca2+ imaging. In light of the upgrade of our 2-photon imaging setup to 3 channel detection, these mice will greatly facilitate the combined analysis of Ca2+, capillary perfusion, and NADH in astrocytes and neurons. By this means, we intend to assess the interaction between Ca2+ signaling and NADH levels among defined cell types within the ischemic penumbra. In our revised aim 3, 1intend to test the hypothesis that astrocytic ATP release comprises a conserved mechanism of cell protection. Our studies so far have suggested that adenosine's neuroprotective effects outweigh P2XR-mediated excitotoxic injury in the cortex. We found that P2X receptor antagonists have little effect upon neuronal injury in the penumbra, whereas adenosine receptor antagonists potently increased ischemic injury after MCA occlusion. ATP is released by astrocytes in response to reduced perfusion in the ischemic penumbra. Besides its activation of local purinergic receptors, it is also rapidly converted to adenosine, which potently inhibits excitatory transmission while lowering cellular energy demands. One of the defining characteristics of the ischemic penumbra is its electrical silence. On that basis, we suspect that neuronal activity is depressed by adenosine;as a corollary to this postulate, we have found that adenosine receptor antagonists increase electrical activity in the ischemic penumbra (Fig. 15). We believe these additions and modifications to our proposal allow us to take better advantage of our available imaging and molecular resources, while expanding both the breadth and rigor of our analysis of the role of astrocytes in stroke. Specific points: Reviewer 2 was unconvinced by the rationale for correlating rates of glycolysis with ATPrelease. We have removed this aim. The validity of the proposed experiments with BAPTA-AM and uncaging was questioned by reviewer2. Chelation of cytosolic Ca2+ with BAPTA is widely used approach to study the functional significance of Ca2+ signaling. BAPTA does efficiently block cytosolic Ca2+ increases, and has low toxicity. We agree with the reviewer that BAPTA-AM and uncaging are unphysiological approaches, but also would like to point out that they are excellent tools to define the impact of Ca2+ signaling. Photolysis of caged Ca2+ is considered state-of- the-art in study of astrocytes and we used this approach in a recent publication (Takano et al., 2006). Few approaches exist that selectively target astrocytes. Use of BAPTA and caged Ca2+ can add important PHS 398/2590 (Rev. 09/04) Page 72~ Continuation Format Page
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1 |
2007 — 2010 |
Nedergaard, Maiken |
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. |
Astrocyte Dysfunction in Epilepsy @ University of Rochester
DESCRIPTION (provided by applicant): Epilepsy is a neurological disorder in which normal brain function is disrupted as a consequence of intensive burst activity from groups of neurons. Synchronized population spikes are key concomitants to seizure, but the phenomenon has remained a paradox because it cannot be explained by any known neuronal synaptic mechanism. Several lines of evidence suggest a key role of glutamate in the pathogenesis of depolarization events, which in turn trigger synchronized firing. The observation that astrocytes release glutamate via a regulated Ca2+ dependent mechanism prompted us to hypothesize that glutamate released by astrocytes plays a causal role in epileptogenesis. Our recent study snowed that chemoconvulsive agents including 4-AP and bicuculline triggered TTX-insensitive paroxysmal depolarization shifts in hippocampal slices which were closely correlated with astrocytic Ca2+ oscillations. Photolysis of caged Ca 2+ in astrocytes, but not in neurons, was sufficient to trigger local depolarization events. Furthermore, agents that blocked astrocytic glutamate release reduced epilepiform activity, with no effect on baseline EEC in adult rats. The next critical step is to expand the analysis to reactive astrocytes in epileptic animals. We propose here to analyze astrocytic Ca2+ signaling in epileptic mice with cranial window using 2-photon imaging concomitant with EEC recordings. We will correlate astrocytic signaling in reactive astrocytes with neuronal firing in epileptic mice. Reactive astrocytes are easily identified in live exposed cortex based upon the intensity of GFP emission in transgenic mice expressing GFP under the GFAP promoter. The hypothesis that the efficacy of antiepileptic drugs is better correlated with the potency by which they reduce astrocytic Ca 2+ signaling and glutamate release, than with their direct effects on synaptic transmission, will be tested. These experiments offer a new conceptual and operational approach to understanding the cellular basis of seizure disorders. If a dysregulation in Ca2+ signaling in reactive astrocytes indeed proves causal in epileptogenesis - as our preliminary data strongly suggest - then the implications of this new perspective to pharmacotherapy could be profound. By more specifically targeting the glial cause of neuronal excitability, we might be able to achieve more specific, less variable and less toxic treatment options for patients with epilepsy.
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1 |
2009 — 2013 |
Nedergaard, Maiken |
R25Activity Code Description: For support to develop and/or implement a program as it relates to a category in one or more of the areas of education, information, training, technical assistance, coordination, or evaluation. |
Molecular Neurosurgery Training Program (Neurotrap) @ University of Rochester
DESCRIPTION (provided by applicant): Project Description: The objective of a Molecular Neurosurgery Training Program, NEUROTRAP, is to provide a multidisciplinary translational neuroscience research training for Neurosurgeons in training at University of Rochester School of Medicine &Dentistry. The program will prepare Neurosurgical residents for careers as independent investigators by providing funding for one additional year of research training. The neurosurgical resident will, in addition to their research, follow a highly structured mentoring plan that include intensive training in grantmanship, several graduate classes, and submission of multiple applications for extramural support. It is expected that the participants in NEUROTRAP will prepare their first K08 award during their final year of residency. NEUROTRAP takes advantages of the rapidly growing community of academic neurosurgeons at University of Rochester, as well as the unique combination of 6 major neuroscience laboratories These 6 laboratories are actively involved in "cutting-edge" translational research and presently funded by 30 NIH awards (including, 2 K08 awards, 19 R01 awards and one P01 award). Ongoing studies include: 1) novel approaches in experimental models of subarachnoid bleeding;2) the potential of glial progenitors cells (GPCs) for improvement of functional recovery following traumatic brain and spinal cord injury;3) basic mechanisms of deep brain stimulation;4) genomic of malignant gliomas;and 5) neuroprotection strategies in animal models of stroke and neurodegenerative disorders with blood factors based on recombinant or plasma-derived proteins. It is expected that the next generation of Neurosurgeons will be challenged to incorporate molecular therapies and biotechnological advances into treatments based upon multidisciplinary approaches. Since most interventional strategies require Neurosurgeons at the clinical delivery stage, Neurosurgeons actively involved in the developments of evolving therapies and thereby aware of their strengths and limitations are needed. All trainees will be Neurosurgical residents, who will have opportunity to actively participate in all facets of the development and implementation of basic molecular research directed on defining new treatment option for neurological diseases. The Neurosurgical residents will spend 2 years in the laboratory, during which time they will have no clinical duties.
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1 |
2012 — 2016 |
Nedergaard, Maiken |
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. |
Atp as the Instigator of Inflammatory Responses to Spinal Cord Injury @ University of Rochester
Spinal cord injury (SCI) is a devastating condition that has disabled millions of individuals world-wide. Pathophysiologically, its two principal components are the acute tissue damage directly associated with the inciting injury, and a later phase of delayed injury that occurs over the ensuing days. This latter phase is primarily a consequence of inflammation and inflammatory edema, which reduces parenchymal perfusion, thereby resulting in ischemic extension of the initial injury. Remarkably, this delayed inflammatory injury may lead to more structural damage than the initial injury, and typically accounts for the bulk of neurological morbidity in SCI patients. Most contemporary studies have focused on the late effectors of this inflammatory response; few have sought to identify the upstream initiators of this process. This proposal will thus test the novel postulate that the inflammatory response to traumatic SCI is initiated by astrocytic ATP release, which serves to activate local microglia in a purine receptor-dependent fashion; subsequent inflammatory effectors are released in the setting of this secondary microglial response. The proposal is based on our preliminary observations that traumatic SCI is associated with the pathological release of ATP, and that purinergic receptor antagonists effectively reduced inflammation and improved locomotor recovery after SCI. Aim 1 will test the postulate that the dysregulated post-traumatic release of ATP is both necessary and sufficient for microglial activation. These experiments will employ novel transgenic mice that have been engineered to release either abnormally high or low levels of astrocytic ATP in response to SCI: ATP release is attenuated in mice lacking astrocytic connexin hemichannels (Cx43/Cx30 KO), but potentiated in mice with an increased number of astrocytic hemichannels (Cx43 G138R mutation). Aim 2 will attempt to define the pathway intermediates downstream of purinergic activation in the post-traumatic inflammatory response. The effects of genetic and pharmacological manipulations of purinergic signaling on the transcriptional activation of both chemokine and cytokine effectors of SCI will be assessed, so as to define the pathways by which the inflammatory sequelae of purinergic activation are coordinated. Transcriptional changes in microglial gene expression will be analyzed by microarray assessment of FACS-sorted microglia. Aim 3 will then test the idea that microglial inflammatory mediators trigger astrogliosis and hence astroglial scar formation, which in turn provides the nidus for a sustained increase in local ATP release. As such, this Aim will test the possibility that astrocytic ATP release is increased for weeks to months following SCI. Our hypothesis is that the ATP released from reactive astrocytes may drive the further release of pro-inflammatory mediators, thereby enhancing astrogliosis. Together, these experiments promise to fill critical gaps in our understanding of the role of purinergic signaling in SCI, and should permit us to define purine-regulated genes and gene products critical that might permit the therapeutic suppression of delayed spinal cord injury.
|
1 |
2012 — 2016 |
Nedergaard, Maiken |
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. |
Failure of Metabolite Clearance in a Model of Multi-Lacunar Infarcts @ University of Rochester
DESCRIPTION (provided by applicant): Unlike all other organs, the brain and spinal cord lack lymphatic vessels. Traditional thought has averred that the brain - despite having the highest basal metabolic rate of any organ - can function without such an organized network for the removal of interstitial fluid-borne metabolic waste products. We questioned this position, seeking to define the pathways by which the brain removes the potentially toxic byproducts of cellular activity. Our preliminary analysis, based on in vivo two-photon imaging, shows that low molecular weight tracers delivered to the CSF circulate surprisingly rapidly through the mouse brain, and do so along a defined anatomical route. This consists of a para-arterial inflow path, an intra-parenchymal path of interstitial flow, and a para-venous outflow path. Within the interstitial space, astrocytes support convective fluid currents, as deletion of the astrocytic watr channel AQP4 sharply reduces tracer flow along these routes. Given the continuous movement of fluid along this pathway, and its critical dependence upon astrocytic fluid transport, we propose that this system - which we designate the 'glymphatic system' - subserves a function homologous to the peripheral lymphatic system, and is essential for the clearance of metabolic waste products from the CNS. We will test the provocative hypothesis that cognitive function in an experimental model of vascular dementia in part is suppressed by accumulation of metabolic waste products. This hypothesis is based on the observation that glymphatic transport is sharply reduced in a murine model of multi-lacunar infarcts, which results in widespread trapping of small tracers in the lesioned hemisphere. Aim 1 will use in vivo 2-photon microscopy to assess the spatial dynamics and temporal kinetics of fluorophore-tagged tracer clearance. By systematically comparing the effect of modifications of molecular sizes or surface charge upon tracer clearance, we will define the basic transport properties of the glymphatic system. Aim 2 will extend the preliminary finding that aged mice exhibit a striking decline in glymphatic system function, and evaluate the effect that age-related suppression of arterial wall pulsation and resulting loss of convective inflow along the para-arterial path has on glymphatic function. Aim 3 will extend the observation that intra-parenchymal fluid movement is reduced in a mouse model of multi-lacunar infarcts and evaluate whether aging cause an additional suppression of glymphatic clearance. Aim 4 will take advantage of inducible astrocyte-specific deletion of AQP4 transgenic mice and test the hypothesis that suppressing glymphatic transport in mice with multi-lacunar infarcts will impair their cognitive functions independently of the ischemic injury. To our knowledge, these studies represent the first attempt to systematically define the mechanisms involved in the clearance of metabolic waste products from the brain on a whole-organ level. The proposed studies will provide fundamental new insight into cognitive impairment in vascular dementia, and will likely also improve our understanding of the pathophysiology of brain injury following stroke and head trauma.
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1 |
2012 — 2016 |
Ji, Ru-Rong Nedergaard, Maiken |
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. |
Hemichannels, Astrocytic Release, and Neuropathic Pain
DESCRIPTION (provided by applicant): Pain conditions are a major health problem in the US and lead to medical morbidity and a reduced quality of life for millions of Americans. Chronic neuropathic pain conditions are especially difficult to treat. A largely unaddressed challenge is how the transition from acute pain to chronic neuropathic pain occurs and how to prevent and reverse this transition in patients. Spinal cord synaptic plasticity and long-term potentiation (LTP) have been strongly implicated in chronic neuropathic pain development. Accumulating evidence also points to an important role of glial cells in the pathogenesis of neuropathic pain. Astrocytes are the most abundant cell type in the CNS and maintain the homeostasis of the CNS. It is well established that astrocytic hemichannels such as connexin-43 (Cx43) constitute an important pathway for gliotransmitter release. Although Cx43 was typically thought to regulate gap junction communication between astrocytes, this function could be switched to paracrine signaling via ATP and glutamate release under injury conditions. Our central hypothesis is hemichannels-mediated gliotransmitter release after nerve injury contributes to transition from acute pain to chronic neuropathic pain by modulating spinal cord synaptic plasticity and LTP. We will test our central hypothesis by addressing the following 4 specific aims: Aim 1 will test the hypothesis that spinal nerve injury increases glutamate and ATP release from spinal cord astrocytes; Aim 2 will test the hypothesis that Cx43-mediated astrocytic ATP release plays a chief role in microglia activation and microgliosis in the spinal cord after spinal nerve injury; Aim 3 will determine the role of Cx43-medicated astrocytic gliotransmitter release in spinal cord synaptic plasticity and LTP after nerve injury; Aim 4 will define the role o astrocytic Cx43 in neuropathic pain development and maintenance after nerve injury. This proposal will involve formation of an innovative partnership between Dr. Ji, a pain scientist with expertise in studying neuronal-glial interactions and neural plasticity in neuropathic pain, and Dr Nedergaard with expertise in studying astrocytic ATP and glutamate release after spinal cord injury and stroke. This application will employ a multidisciplinary approach including the use of inducible transgenic mice with genetically modified astrocytes, in vivo imaging of ATP release (bioluminescence) and microglia motility and Ca2+ changes (2-photon) in the spinal cord, behavioral testing of evoked and ongoing neuropathic pain after nerve injury, and ex vivo and in vivo electrophysiology in the spinal cord. The proposed studies will provide a step-by-step analysis of neuron- glia interactions initiated by nerve injury and may comprise an efficient means to prevent and treat chronic pain.
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0.97 |
2012 — 2016 |
Nedergaard, Maiken |
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. |
The Glymphatic System, a New Concept in Glia Biology @ University of Rochester
Virtually unique among somatic tissues, the brain and spinal cord lack a lymphatic system. Despite the high metabolic activity and fragility of neural tissue, there exists no effective understanding of the means by which interstitial fluid and waste products are removed from the CNS. Our preliminary analysis, based on in vivo two- photon imaging, shows that low molecular weight tracers delivered to the CSF circulate surprisingly rapidly through the mouse brain, and do so along a surprising anatomical route. This consists of a para-arterial inflow path, a trans-glial intra-parenchymal path of interstitial flow, and a para-venous outflow path. Within the intra- parenchymal pathway, astrocytes support convective fluid currents through the brain interstitial space, as deletion of the astrocytic water channel AQP4 sharply reduces overall tracer flow along these routes. Given the continuous movement of fluid supported by this pathway, and its critical dependence upon astrocytic water transport, we propose that this system - which we designate here the 'glymphatic system' - subserves a function homologous to the peripheral lymphatic system, and is essential for the clearance of metabolic waste products from the CNS. Aim 1 will use 2-photon in vivo microscopy to assess the spatial dynamics and temporal kinetics of fluorophore-tagged tracer clearance. By systematically comparing the effect of modifications of molecular sizes or surface charge upon tracer clearance, we will define the basic transport properties of the glymphatic system. Aim 2 will extend the preliminary observation that aged mice exhibit a striking decline in glymphatic system function, and evaluate the role of age-related suppression of arterial wall pulsation and resulting reduced convective inflow along the para-arterial path and global glymphatic fucntion. Aim 3 proposes that induced knock-out of either astrocytic AQP4 water channels or gap junctions (Cx43/Cx30) will slow parenchymal convective fluid flow and globally suppress tracer clearance. Aim 4 tests the proposition that suppression of trans-astroglial fluid movement resulting from AQP4 or Cx43/Cx30 deletion will slow clearance of exogenous A¿ and thereby potentiate age-related amyloid plaque formation. We predict that slowing astrocytic parenchymal fluid flow will accelerate paravascular amyloid deposition, which in a feed- forward manner will further reduce the efficiency of clearance of waste products by the glymphatic system. To our knowledge, these studies represent the first attempt to systematically define the mechanisms involved in the clearance of metabolic waste products from the brain on a whole-organ level. Two-photon imaging of through chronic cranial windows will allow imaging of tracer clearance in real time, whereas transgenic mice with inducible deletion of key astroglial membrane proteins will establish the functional role of astrocytes in glymphatic transport. Combined, these studies will provide fundamental insight into the mechanisms contributing to age-related accumulation of neurotoxic metabolic waste products and define novel, and likely highly important, functional properties of astrocytes.
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1 |
2013 — 2015 |
Nedergaard, Maiken |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Eager: Collaborative Research: Non-Local Cortical Computation and Enhanced Learning With Astrocytes @ University of Rochester
The brain is composed of two major cell types: Neurons and glial cells. Glial cells are traditionally regarded as the brain's supportive cells. However, many lines of work over the past decade have documented that glial cells may also participate in complex neural processes and thereby comprise an integral element of higher cognitive function, such as working memory, learning, and sleep. Other lines of work have shown that human astrocytes are larger and structurally more complex than astrocytes in the rodent brain. In support of this concept, transplantation of human glial cells into mice resulted in generation of mice that were faster learners and performed better on memory tests. However, existing computational modeling techniques employed for understanding the processes involved in learning and memory do not include glial cells,. The aim of the proposed research is to: 1) Develop computational modeling techniques that incorporate glial cells. 2) Use these novel computational modeling techniques to make predictions regarding the role of glial cells in learning and memory. 3) Test the predictions using a combination of patch clamping and Ca2+ imaging. 4) Use the data collected to continuously refine the computational modeling techniques. The broader impact of this proposal will be to further the scientific understanding of underappreciated, yet essential substrates of learning and memory. Including glial cells in addition to neurons in modeling approaches additionally carries the hope of increasing computational power and processing capabilities of adaptive learning technology, in addition to improving the performance of bio-integrated prostheses for individuals with impaired learning or other debilitating neurological disorders.
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1 |
2013 — 2017 |
Goldman, Steven Alan [⬀] Nedergaard, Maiken |
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. |
A Humanized Mouse Model of Astrocytic Pathology in Schizophrenia @ University of Rochester
DESCRIPTION (provided by applicant): Human evolution has been accompanied by a diversification of astrocytic phenotype and function, that has contributed to species-specific aspects of both human brain function and disease. As such, the development of human astrocytic complexity has paralleled the appearance in evolution of psychiatric disorders unique to humans - the schizophrenias in particular. Yet despite this correlative suggestion that astrocytic pathology might contribute to the disordered thought of schizophrenia, the role of astroglial pathology in its pathogenesis has been difficult to study, in part because of the lack o animal models of human glial pathophysiology. We propose to overcome this limitation, using a new model of human glial-chimeric mouse brains that we have developed, paired with our newly-developed protocols for efficiently and reliably generating astrocytes from patient-derived human induced pluripotential cells (hiPSCs). Using mice neonatally engrafted with glial progenitor cells (GPCs) derived from hiPSCs generated from schizophrenic patients, we will assess the specific contributions of schizophrenic patient-derived astrocytes to disease pathogenesis. In these human glial chimeric mouse brains, the vast majority of resident glia are replaced by human GPC-derived astrocytes and their progenitors, allowing human glial physiology, gene expression, and effects on neural function to be assessed in live adult mice. By pairing this chimerization approach with protocols that we have developed for both generating and purifying GPCs and astrocytes from hiPSCs, and using hiPSC lines produced from patients with juvenile-onset schizophrenia, we will produce mice whose resident glia are largely derived from patients with schizophrenia. In Aim 1, we will assess the relative effects of these schizophrenia-associated astrocytes upon glial syncytial transmission within the cortices of the chimeric mice. In Aim 2, we will next assess the synaptic plasticity of the chimeric mice, as well as the effects of schizophrenia-derived glial chimerization upon their behavioral phenotype and responses to pharmacological stressors. In Aim 3, we will sort engrafted astroglia from the brains into which they have integrated, so as to assess the gene expression patterns of schizophrenic iPSC-derived astrocytes, relative to those of normal hiPSC-derived glia. By means of this multimodal approach, we hope to define the disease-specific effects, gene expression patterns, and paracrine toxicities of schizophrenic hiPSC-derived astrocytes relative to normal hiPSC-derived glia. These diverse lines of investigation should provide us great insight into the species- and cell type-specific roles of human astrocytes in the pathogenesis of schizophrenia. At the same time, by providing a new human glial chimeric model system, new cellular reagents in the form of schizophrenic patient-derived astrocytes, and new gene expression databases covering schizophrenic hiPSC-derived astrocytes, this project should allow us to make available to the field a broad and exciting new set of tools, capabilities and databases. Together, these should greatly accelerate our understanding of human glial dysfunction in the pathogenesis of schizophrenia.
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1 |
2014 — 2018 |
Benveniste, Helene D (co-PI) [⬀] Nedergaard, Maiken |
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. |
Glymphatic Function in a Transgenic Rat Model of Alzheimer's Disease @ University of Rochester
DESCRIPTION (provided by applicant): All neurodegenerative diseases, including Alzheimer's disease (AD) are associated with the accumulation of misfolded protein aggregates. The brain lacks the lymphatic drainage system that peripheral tissues rely on for macroscopic waste removal; however we recently discovered a brain-wide system that subserves this role. We named it the 'glymphatic' pathway, because it is dependent on aquaporin 4 (AQP4) water channels expressed in a highly polarized pattern on astroglial processes surrounding blood vessels. As much as 60% of soluble A¿ proteins are cleared from the interstitial space along the glymphatic pathway and clearance is sharply reduced in a murine AD model [expressing mutant human amyloid precursor protein (APPsw) and presenilin 1(PS E9)] when compared to age-matched wildtype mice. The proposed studies will utilize the first rodent AD model-transgenic APPsw/PS E9 rats-that replicates all the hallmarks of AD in humans, including amylodosis, reactive astro- and microgliosis, and progressive neuronal loss. HYPOTHESES: (1) Dysfunction of the glymphatic system in young, middle aged and old rats caused by oxidative stress and mislocation of AQP4 contributes to vascular amyloid deposition in APPsw/PS E9 rats. (2) Glymphatic transport can be quantified brain-wide using clinically relevant magnetic resonance imaging (MRI) in live rodents by minimally invasive lumbar administration of contrast agents. (3) Young (6 months) and middle-aged (16 months) APPsw/PS E9 rats that by MRI imaging are identified as those that exhibit the most severe decline in glymphatic clearance are at higher risk of developing AD pathology, detected as cognitive decline, and oxidative stress and amyloidosis, when they reach old age (26 months). Aim 1: Using CSF tracers and optical imaging, we will correlate glymphatic decline locally with the severity oxidative stress, astro- an microgliosis, loss of polarized perivascular AQP4, amyloid burden, and neuronal loss as a function of age in APPsw/PS E9 and wildtype rats. Aim 2: Establish a clinically relevant rodent imaging platform for evaluation of glymphatic pathway function using minimally invasive techniques using clinically relevant magnetic resonance imaging (MRI) combined with minimally invasive administration of paramagnetic contrast via lumbar intrathecal space. Aim 3: Using the glymphatic diagnostic MRI test developed in Aim 2, we will test the hypothesis that glymphatic pathway dysfunction in 6 and 16 months old APPsw/PS E9 rats predicts the severity of cognitive decline and amyloid burden in the same rats at 26 months. These studies will take advantage of the known interanimal variability of disease progression in APPsw/PS E9 rats to define whether failure of glymphatic function contributes to AD pathology. These studies present the first attempt to apply MRI imaging to track glymphatic dysfunction in normal aging and in AD. Clinical translation of the MRI imaging platform may allow similar questions to be asked in humans and permit tracking of interventional therapeutic approaches intended to slow AD progression.
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1 |
2014 — 2015 |
Benveniste, Helene D [⬀] Nedergaard, Maiken |
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.) |
Metabolic Profiling of Neonatal Anesthesia Toxicity @ State University New York Stony Brook
DESCRIPTION (provided by applicant): Emerging evidence has led to questions about the safety of anesthetic agents used in routine clinical practice, especially with regard to the use of general anesthesia in infancy. A wealth of data shows neuronal demise in response to general anesthesia in the very young brain of rodents and non-human primates. Thus far, parallel evidence in humans has been limited to retrospective studies correlating repetitive anesthesia exposures during early childhood. These studies have documented learning difficulties later in life as well as abnormal behavior and psychosocial issues. There is an urgent need to establish whether or not exposure to general anesthesia in the very young human brain interferes with normal brain development. We propose to implement a clinically relevant, non-invasive imaging technology - proton magnetic resonance spectroscopy (1HMRS) - that has the ability to track apoptosis, neurodegeneration, inflammation and metabolic status in the young brain, and use this approach in combination with traditional behavioral and histological techniques to provide a direct linkage between anesthesia exposure, brain defects and long-term behavioral effects. The proposed studies are based on our novel preliminary findings that very young rodents exposed to sevoflurane anesthesia on post-natal day 7 show 1) abnormalities in brain maturation determined by changes in the neuronal marker N-acetyl-aspartate (NAA), 2) inflammatory changes including reactive gliosis and changes in levels of choline metabolites, and 3) that these metabolite changes tracked non-invasively by 1HMRS are associated with increased apoptosis. Specifically, we propose an innovative hypothesis that metabolic profiling by 1HMRS can gauge anesthesia-induced neurotoxicity in the young brain over time. We further seek to establish whether repeated exposures to inhalational anesthetics (sevoflurane) at the time of peak synaptogenesis in the developing rodent brain result in severe cerebral metabolic defects including impediment of brain maturation reflected by age-dependent changes in the concentration of [NAA] and long-lasting reactive gliosis reflected by changes in choline and/or myo-inositol. The specific aims are: 1) to evaluate metabolic profiles in the neonatal brain in 13, 20 and 90 day old rat pups with single or repetitive exposure to sevoflurane compared to non- exposed age-matched rat pups evaluated at the same time points, and 2) to evaluate whether cerebral metabolic defects impair cognitive functions and trigger neuronal loss and long-lasting activation of astrocytes and microglia after a single or repetitive exposure to sevoflurane at the time of peak synaptogenesis. Behavioral data in the neonatal rats exposed to single or repetitive sevoflurane anesthesia in Aim 1 will be collected at 30 to 80 days, to document the effect of neonatal sevoflurane exposure on complex brain function. The same rats will be evaluated for neuronal loss and reactive astro- and microgliosis at 3 months and compared to non-exposed rats. To our knowledge the proposed studies constitute the first attempt to develop a clinically relevant diagnostic test to assess anesthesia induced neurotoxicity in the young brain.
|
0.93 |
2014 — 2018 |
Nedergaard, Maiken |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Alexander's Accentuation by Failure of Glymphatic Clearance @ University of Wisconsin-Madison
ABSTRACT Alexander's disease (AxD) is a fatal leukodystrophy caused by mutation in the gene encoding glial fibrillary acidic protein (GFAP). Pathologically, AxD is characterized by reactive gliosis, upregulation of GFAP, and accumulation of Rosenthal fibers. AxD is associated with early and progressive loss of neurons and oligodendrocytes. Understanding how an astrocyte specific genetic defect of a non-essential intermediary filament, can so severely influence neuronal function, is critical to developing therapeutic strategies. We believe that the discovery of a brain-wide interstitial clearance system in CNS - the glymphatic system - driven by astrocytes and dependent upon the astrocytic water channel, AQP4, could begin to address this question. This novel clearance path subserves a function homologous to the lymphatics, which in other organs is essential for the clearance of extracellular proteins. We propose that glymphatic flow comprises an important pathway by which GFAP and its proteolytic products can be cleared, but that the formation of Rosenthal fibers, or the toxic effects of GFAP oligomers, and consequent disruption of astroglial cytoarchitecture in AxD result in the mislocation of AQP4, and the consequent failure of glymphatic flow. This in turn leads to reactive changes in affected astroglia, which further increase the transcription of mutated GFAP in a feed-forward process that accelerates both Rosenthal fiber accumulation, and effectively abrogates glymphatic flow, inexorably extending AxD pathology to all cell types and regions within the affected brain. Aim 1 will be to evaluate the contribution of the glymphatic system to the physiological clearance of soluble GFAP in wild type mice using several alternative approaches, including in vivo 2-photon imaging, ex vivo mapping of fluorescently-tagged GFAP distribution at defined time-points, quantitative measurement of radiolabeled GFAP clearance, microdialysis combined with ELISA detecting of endogenous GFAP, immunohistochemistry and Western blot. Aim 2 will characterize the impact of AxD in Gfap+/R236H mice on glymphatic GFAP clearance and manipulate glymphatic clearance by inducible, astrocyte-specific deletion of AQP4 in juvenile AxD transgenic mice. Aim 3 will test the hypothesis that increasing glymphatic clearance in Gfap+/R236H mice by inhibition of inducible nitric oxide synthase (iNOS) will reduce Rosenthal fiber burden and delay age-related impairment of cognitive function. To our knowledge, these studies represent the first analysis of GFAP clearance on an organ level and the first systematic analysis of how AxD affects this clearance pathway. We hope that the experiments will provide fundamentally new insights into the role of glymphatic GFAP clearance on reactive gliosis and Rosenthal fiber burden and thereby define novel targets for treatment of this grave disease.
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0.939 |
2016 |
Benveniste, Helene D [⬀] Nedergaard, Maiken Tannenbaum, Allen R. (co-PI) [⬀] Van Nostrand, William E. (co-PI) [⬀] |
RF1Activity Code Description: To support a discrete, specific, circumscribed project to be performed by the named investigator(s) in an area representing specific interest and competencies based on the mission of the agency, using standard peer review criteria. This is the multi-year funded equivalent of the R01 but can be used also for multi-year funding of other research project grants such as R03, R21 as appropriate. |
Characterizing the Glymphatic Peri-Vascular Connectome and Its Disruption in Ad
We are proposing a novel approach to diagnosing early Alzheimer?s disease (AD) and predicting progression via a robust biomarker that captures ?glymphatic? pathway transport on a systems level. The glymphatic pathway is a brain-wide system, which was recently discovered to function as a clearance pathway for toxic brain waste proteins including soluble amyloid beta (A?) and tau similarly to the classical body-wide lymphatic system. As such, the glymphatic pathway comprises a previously overlooked and unique compartment of the brain vasculature, the peri-vascular space wherein cerebrospinal fluid (CSF) is flowing and streaming into the brain interstitial fluid (ISF) space thereby forcing waste solutes out of the brain. Except for rare familial AD, where excessive A? production and deposition in the brain clearly drives cognitive decline, there is limited evidence in the more common sporadic AD that cerebral A? accumulation is the result of A??overproduction. In fact, emerging evidence suggests that parenchymal A? accumulation in sporadic AD is driven by reduced A? clearance. The glymphatic pathway is thus a prime candidate for linking disruptive clearance of A? to AD, and we will use this opportunity to develop new tools and computational analysis aimed explicitly at capturing global glymphatic pathway function and serve as a novel diagnostic AD biomarker. Currently there is no method available to capture all of the intricate and dynamic components of glymphatic transport, in particular, parenchymal transport and clearance pathways. We propose to integrate imaging techniques and develop novel computational analysis including optimal mass transport to characterize the glymphatic pathway as a brain-wide dynamic ?unit?. The ultimate goal of the proposed investigation is to apply the glymphatic biomarker and track its disruption in progressing vascular and parenchymal amyloid pathologies. The proposed studies are based on novel preliminary findings that 1) glymphatic transport can be visualized as an integrative system through perivascular and interstitial spaces; 2) that state dependent changes induced by specific anesthetic regimens which dramatically affect the glymphatic transport can be captured by optimal mass transport analysis; and 3) a new transgenic rat model of cerebral amyloid angiopathy (rTg-SwDI) which will be used for specific hypothesis testing against the transgenic rat AD model (rTgF344-AD36) in the proposed studies. The specific aims are the following: (1) To develop biomarkers to visualize and functionally quantify macroscopic, glymphatic transport based on computational analysis of MRI and macroscopic optical imaging of CSF tracers in normal young (3 month old) rats and (2) to determine how and when normal aging and specific AD-like cerebral vascular and parenchymal amyloid pathologies influence glymphatic transport in the brain using the computational pipeline developed in SA1. Successful completion of the proposed highly innovative experiments will yield an entirely new and promising biomarker to track reduced Aß clearance via the glymphatic pathway which is key to the propagation of CAA and AD.
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0.97 |
2016 — 2020 |
Nedergaard, Maiken |
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. |
Para-Vascular Basis of Small Vessel Disease @ University of Rochester
Abstract Among adult dementias, a large proportion are either due to or associated with small vessel disease (SVD) of the brain. The incidence and prevalence of SVD, which may be anticipated by aging, hypertension and diabetes, is on the rise, and causally linked to the rising incidence of dementia and age-related neurological disability. Despite the fact that diagnostic MRI can track SVD progression during a premanifest period and potential therapeutic window of years to decades, no treatments are yet available. The pathophysiology of SVD is presently poorly understood. A widening of perivascular spaces (PVS) and white matter hyperintensities on neuroimaging are strongly associated with SVD. The perivascular expansion in SVD appears to correspond structurally with the perivascular spaces of the glymphatic system, our recently described system of glial-mediated lymphatic-like convective flow that directs interstitial fluid fluxes in the brain. The glymphatic system is analogous to the lymphatic system in peripheral tissues, which clears excess interstitial fluid and waste products from the brain. Glymphatic fluid transport pathways circulate cerebrospinal fluid (CSF) which exchanges with interstitial fluid (ISF), and relies on the aquaporin-4 (AQP4)-defined water channels in astrocytic endfeet to achieve parenchymal entry. Astrocytic endfeet effectively enclose the vasculature and thereby create a network of interconnected donut-shaped tunnels around the brain's arteries, capillaries, and veins. The existence of an astrocyte-enclosed perivascular space is recognized as a unique anatomical feature of the CNS, but its functional importance has only recently become apparent. Since a hallmark feature of SVD is the structural remodeling of the perivascular space, we here propose to ask, based on a compelling set of preliminary observations, if an increase of glymphatic fluid exchange plays an essential role in SVD pathobiology. The proposed studies will address the following questions: Aim 1 will map glymphatic activity in several experimental rodent SVD models, including CADASIL, hypertensive rats and mice (SHR and BPH/2J mice). Aim 2 will use MRI to assess the roles of blood pressure fluctuations, glymphatic influx or efflux, and BBB permeability on glymphatic transport and pathological fluid accumulation in SHR and controls. Aim 3 will test the hypothesis that interventions which promote normal glymphatic function will slow myelin loss and the cognitive impairment in SVD. The experiments will directly correlate the benefits of anti-hypertensive treatment, exercise, and insomnia treatment with improvements in glymphatic transport. To our knowledge, this application constitutes the first formal study focused on glymphatic functions and the perivascular space in SVD. The proposed studies will address key gaps in our understanding of SVD. Our hope is to provide novel mechanistic insight into the pathological events that leads to myelin loss in SVD.
|
1 |
2017 |
Nedergaard, Maiken |
RF1Activity Code Description: To support a discrete, specific, circumscribed project to be performed by the named investigator(s) in an area representing specific interest and competencies based on the mission of the agency, using standard peer review criteria. This is the multi-year funded equivalent of the R01 but can be used also for multi-year funding of other research project grants such as R03, R21 as appropriate. |
Age and Ad Related Bottlenecks in Glymphatic-Lymphatic Waste Transport @ University of Rochester
Abstract All neurodegenerative diseases, including Alzheimer?s disease (AD), are associated with the accumulation of misfolded protein aggregates. This proposal is rooted in the discovery of the brain?s glymphatic system, a brain- wide perivascular fluid transport system analogous to the lymphatic system in peripheral tissues, which clears protein waste products. Since a hallmark feature of aging and AD is a sharp reduction in glymphatic amyloid-? and tau clearance, we propose that degeneration of the glymphatic-lymphatic drainage plays an essential role in the pathogenesis of Alzheimer?s. Aim 1 will be to define, for the first time, the major efflux pathways of the glymphatic-lymphatic system based on uDISCO clearing, which renders whole rodent bodies transparent. The advantage of uDISCO clearing is that the efflux paths can be visualized without removing the brain from the skull ? a procedure that necessarily severs the glymphatic-lymphatic connections. Fluid-dynamic modeling based on real-time imaging of fluorescent tracers will provide a global map of the glymphatic-lymphatic flow patterns and their driving forces. Aim 2 will be to determine why glymphatic-lymphatic fluid transport is sharply reduced in aging and AD. The experiments will focus on identifying the bottlenecks that hamper clearance of amyloid-? and tau in aging and AD. Also, the distribution of immune cells (microglia, T-cells, and more) in CNS and meningeal lymphatic vessels will be mapped as a function of aging and AD after uDISCO clearing. Aim 3 will be to determine whether improving the quality of sleep delays aging and AD-induced degeneration of the glymphatic-lymphatic system. Non- pharmacological improvement of sleep architecture will test whether the long-term benefit of improved sleep quality is a result of delaying age-induced and AD-induced degeneration of glymphatic-lymphatic amyloid-? and tau clearance. We will test whether insomnia treatments, including non-benzodiazepine receptor agonists, GABAB receptor agonists, or sedating antidepressants, improve ? or perhaps worsen ? AD progression. To our knowledge, this proposal constitutes the first systematic analysis of the efflux pathways of the glymphatic- lymphatic system. The proposed research leverages very recent developments in tissue clearing and light-sheet microscopy techniques. The studies will address key gaps in our understanding of the glymphatic-lymphatic system, and will use state-of the art approaches to investigate what we believe is one of the most fundamental unexplored avenues ? and opportunities ? in CNS pathophysiology. The team of investigators has broad and complementary relevant expertise, including analysis of the glymphatic system, invention of the whole body uDISCO clearing, and fluid-dynamic analysis and modeling. Our goal is to create a complete map of the glymphatic-lymphatic connections in young, middle aged, and old wildtype and APP/PS1 mice and to identify age-related and AD-related bottlenecks in the clearance of amyloid-? and tau.
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2021 |
Nedergaard, Maiken |
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. |
The Glymphatic System At the Crossroad of Integrative Health Approaches Inchronic Pain @ University of Rochester
Abstract: The glymphatic system is a network of perivascular spaces that function as a waste clearance system, analogous to the peripheral lymphatic system. Reduced glymphatic function has been a hallmark observation in aging as well as models of Alzheimer's disease, diabetes, hypertension, traumatic brain injury, excess alcohol intake, and chronic unpredictable stress. Preliminary data shows that acute and chronic pain, and one night of light all suppressed glymphatic function. This application will use the murine sparse nerve injury (SNI) model to understand how the brain responds to chronic neuropathic pain. Sleep complaints are prevalent in chronic pain patients, and chronic sleep restriction increases pain sensitivity in mice. Norepinephrine (NE), which disrupts sleep and is released in stressful conditions, suppresses glymphatic function. We hypothesize that increase NE levels in SNI reduce glymphatic function, triggering cytokine accumulation, neuronal excitability, sleep disruption and pain sensitization in a feedforward loop (Aim 1). Traditional analgesics have been shown to relieve pain in models of chronic pain. Our preliminary data show that the same agents restore glymphatic function in SNI mice with no effect on glymphatic functions in control mice. We hypothesize that reducing the severity of pain via analgesia improves glymphatic function by reducing NE levels, which in turn reduces cytokine accumulation and excitability and improves sleep quality (Aim 2.1). Yet, efficacy of modern pharmacology is variable in the patient population, suggesting that while modulation of neural pathways is partially effective, pathology remains. We hypothesize that neuropathic pain induces a CNS maladaptive response involving reduced glymphatic flow, inflammation and waste accumulation. Because both natural and mind-body interventions target multiple facets of glymphatic disruption (sleep, inflammation, cardiovascular disease), we hypothesize that natural supplements (melatonin and eicosapentaenoic acid (an ?-3 fatty acid)) and mind-body interventions (voluntary exercise, improved sleep, and acupuncture) will improve glymphatic disruption in chronic pain (Aims 2.2 and 2.3). The timing of treatment is critical, because the circadian system is integrated into every process in the body including the glymphatic system, the immune system, and chronic pain. We propose that targeting therapeutics to reinforce the rhythm in glymphatic function and clearance will optimize the effect of treatment which can be quantified as an additional decrease in cytokine accumulation and hyperalgesia in SNI (Aim 3). We will time sleep improvements via increased temperature, voluntary exercise, melatonin, and acupuncture, to the endogenous rhythm of CSF distribution - high glymphatic clearance during rest, and low during wakefulness. Aim 3 is unique in that it tests whether efficacy of mind-body therapies, in improving glymphatic function and reducing pain sensitivity, can change based on when during the day they are administered. Overall, this application aims to define whether glymphatic activity may serve as a target for complementary therapeutic approaches and also as a biomarker establishing the efficacy of treatment.
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