National Institutes of Health grant abstracts
NIH Principal Investigators at Legacy:
Boison, Detlev, PhD
Burgoyne, Claude F., MD
Demirel, Shaban, PhD
Fortune, Brad, OD, PhD
Gardiner, Stuart, PhD
Wang, Lin, MD
PI: Detlev Boison, PhD
R01MH083973 "Adenosine and schizophrenia: mechanisms and therapies"
DESCRIPTION (provided by applicant): Schizophrenia (SZ) is a debilitating mental illness with tremendous human, social and financial costs to society. Unfortunately, existing treatments are unsatisfactory and current development remains stagnant due to poor understanding of the biological bases of the disease. Two perspectives have emphasized disturbances in two neurochemical messengers in the brain -dopamine and glutamate, in relation to disparate SZ- symptoms. This grant will examine a third messenger -adenosine (ADO), as a potential link uniting the dopamine and glutamate hypotheses of SZ. ADO can regulate both dopamine and glutamate neurotransmission via receptors with opposing actions (A1 vs. A2A adenosine receptors). ADO is therefore uniquely positioned as an upstream coordinator/regulator between these two neurotransmitter systems. Hence, ADO-based treatment may be an attractive alternative with dual corrective actions on the glutamate and dopamine systems, thereby achieving effective control over selected SZ symptoms.
Our central hypothesis is that subtle disturbances in adenosinergic neuromodulation can give rise to selected behavioral endophenotypes implicated in SZ; thus corresponding corrective interventions targeting at the ADO system should confer therapeutic potential against such SZ endophenotypes, and thereby validate our hypothesis. Our hypothesis will be tested by three specific aims. First, we will characterize the emergence of selected SZ-related endophenotypes as well as their opposing phenotypes in transgenic mice with either over- or under- expression of brain ADO achieved by genetic manipulation of adenosine kinase. Second, we aim to identify the molecular mechanisms of adenosine-based modulation of dopaminergic and glutamatergic neurotransmission. This will be achieved by behavioral and biochemical examination of A1R and A2AR knockout mice. Third, we aim to dissect the brain regions in which ADO-dopamine interactions and ADO-glutamate interactions contribute to the regulation of specific SZ-related endophenotypes.
To achieve this, ADO will locally be modified by transplantation of ADO-secreting stem cells and by focal infusion of drugs acting on ADO-receptors. The expected outcomes of this project include: (i) the biological validation of a novel neurochemical theory of SZ, and (ii) the feasibility-test of a novel ADO-based strategy to produce behavioral adjustment with therapeutic potential. PUBLIC HEALTH RELEVANCE: Schizophrenia is a devastating mental disorder to the individual and society alike, yet the efficacy of current drug treatment remains poor, and the development of novel drugs is limited to either blocking dopamine or enhancing glutamate neurotransmission. This proposal will examine a novel drug target for schizophrenia-therapy -adenosine: Given adenosine's unique position to interact in parallel with dopamine and glutamate neurotransmission, adenosine-modulating drugs are hypothesized to confer therapeutic potential against multiple SZ symptoms.
PI: Detlev Boison, PhD
R01NS061844 "Astrocyte dysfunction in epileptogenesis: the role of adenosine"
DESCRIPTION (provided by applicant): This grant studies the hypothesis that astrogliosis and resulting dysfunction of adenosine-based neuromodulation is a mechanistic cause for the development of epilepsy (i.e. epileptogenesis). This is of importance, since to date no effective prophylaxis for epilepsy is available. This proposal will explore status epilepticus (SE)- triggered epileptogenesis and dysfunction of the endogenous adenosine-based seizure control system in mice in search for an astrocyte-based mechanism of epileptogenesis and thus may provide a foundation for the development of novel antiepileptogenic therapies. The proposal is based on the following findings: (i) Adenosine kinase (ADK) is the key enzyme for the regulation of adenosine; (ii) In adult brain, ADK is expressed in astrocytes; (iii) Astrogliosis is a hallmark of epileptogenesis; (iv) ADK is over-expressed within epileptic astrogliotic hippocampus; (v) Augmentation of adenosine by implants of adenosine releasing cells prevents kindled seizures. (vi) Transgenic overexpression of ADK increases seizure susceptibility; (vii) Local reduction of ADK in transgenic mice prevents epileptogenesis.
Our CENTRAL HYPOTHESIS is that an epileptogenesis triggering event (e.g. SE) induces astrogliosis with resultant regional upregulation of ADK as a necessary component of epileptogenesis and that reconstitution of brain adenosine by stem cell derived brain implants can prevent such epileptogenesis. The model system to address this hypothesis consists of intraamygdaloid application of kainic acid (KA) to initiate epileptogenesis selectively in the CA3 area of the hippocampal formation of mice and to transplant ADK-deficient adenosine releasing embryonic stem (ES) cells into the epileptogenic region. SPECIFIC AIMS: In Aim 1 we will study the causal, temporal, and spatial relations of astrogliosis, upregulation of ADK and seizures in a mouse model of CA3-selective epileptogenesis. In Aim 2 we will use a panel of different Adk-transgenic mice, in which we can molecularly separate cell-type specific functions of ADK expression from astrogliosis, to study both mechanisms independently. In Aim 3 we will use ADK-deficient ES cell-derived intrahippocampal implants in a therapeutic approach to prevent epileptogenesis. The expected outcome of these studies is to define astrocytic ADK as a target for the prevention of epileptic seizures and to translate these findings into a novel stem cell based treatment approach.
PUBLIC HEALTH RELEVANCE: Currently, no therapy is available to prevent the development of epilepsy. This proposal studies a defective function of the brain's own adenosine-based seizure control system as a mechanistic cause for epilepsy and translates these findings into a novel approach to prevent epilepsy by implanting adenosine releasing stem cells.
PI: Detlev Boison, PhD
R01NS084920 "Glycine augmentation therapy for the treatment of epilepsy"
Project Summary/Abstract: The role of glycine homeostasis in epilepsy has largely been neglected, not only in our appreciation of pathophysiological mechanisms, but likewise in therapeutic drug development efforts. This proposal is based on promising preliminary data from our laboratory, which demonstrate an unprecedented anticonvulsant role of glycine augmenting drugs. Specifically, we will evaluate whether glycine augmenting drugs, which are already in clinical development for schizophrenia, could be used for the treatment of seizures in temporal lobe epilepsy (TLE). This grant will fill a critical gap in knowledge and proposes that disruption of glycine homeostasis is implicated in the pathophysiology of TLE and that therapeutic glycine augmentation is a novel pharmacological principle for the treatment of TLE. In hippocampus, glycine is largely regulated by its specific transporter GlyT1, and fulfills a dual role as homeostatic regulator of neuronal excitability by binding to glycine receptors (potentially anticonvulsive) and the glycineB-site of N-methyl-D-aspartate receptors (potentially procognitive).
This goal-oriented proposal will test the CENTRAL HYPOTHESIS that therapeutic glycine augmentation represents a novel strategy for seizure control in temporal lobe epilepsy (TLE). Our preliminary data demonstrate that glycine homeostasis is perturbed in a mouse model of TLE. Further, we demonstrated that engineered mice with conditional disruption of GlyT1 (to increase hippocampal glycine) have increased seizure thresholds, whereas a GlyT1 antagonist robustly suppressed chronic seizures in a mouse model of TLE. In addition, our data document a profound pro-cognitive effect of genetic GlyT1 disruption. Key experimental tools required to test our hypothesis include genetic tools to disrupt GlyT1 function, GlyT1-inhibiting drugs, rodent models of acute seizures and of TLE, and relevant behavioral tests.
Our research goals will be addressed in three Specific Aims: (1) Identify the mechanisms of seizure suppression by glycine. (2) Test the prediction that acute glycine augmentation prevents seizures in rodents. (3) Test the hypothesis that chronic GlyT1 inhibition improves seizures and cognitive function in TLE. Expected outcome and impact: A combination of mechanistic and therapeutic studies will allow us to determine whether GlyT1 antagonists might be useful alternative drugs for the treatment of TLE. We will make novel and innovative use of GlyT1 inhibitors that have already been tested in clinical trials (phase II/III) to treat cognitive symptoms in schizophrenia. Identification and characterization of a novel anticonvulsant role of existing drugs will open new opportunities for clinical translation of therapeutic glycine augmentation as novel pharmacological principle for epilepsy therapy. The expected benefit of therapeutic glycine augmentation is seizure control combined with a pro-cognitive activity, which sets therapeutic glycine augmentation apart from conventional antiepileptic drugs, which tend to be associated with cognitive impairment as prominent side effect.
PI: Detlev Boison, PhD
R21NS088024 "Adenosine kinase antisense gene therapy for temporal lobe epilepsy"
Project Summary/Abstract: Temporal lobe epilepsy (TLE) presents as an incapacitating neurological syndrome comprised of seizures and associated comorbidities. Seizures in up to 35% of patients with TLE are refractory to common forms of treatment, which are likewise largely ineffective to control comorbid conditions. In line with the NINDS Epilepsy Research Benchmarks this exploratory research proposal aims to develop an antisense gene therapy vector as a new approach for the targeted treatment of seizures and associated comorbidities in mesial TLE (mTLE). Our proposal is based on the rationale that deficiency of the endogenous anticonvulsant and seizure terminator adenosine (ADO) is a pathological hallmark of the epileptic brain. ADO-levels in the brain are controlled by metabolic clearance through the astrocyte-based enzyme adenosine kinase (ADK), which is pathologically overexpressed in the epileptic brain.
Research from our and other laboratories has convincingly shown that therapeutic ADO augmentation or pharmacological ADK inhibition effectively prevent seizures in at least three different rodent models of mTLE, including seizures that are refractory to common antiepileptic drugs. Despite the proven effectiveness of ADO in seizure control, critical gaps for therapy development remain local specificity to avoid side effects of systemic ADO augmentation, and long-term effectiveness. The overarching goal of our application is (i) to develop, optimize, and characterize a final clinical gene therapy candidate to reduce (not suppress) ADK expression in the epileptic brain through antisense technology and thereby to reinstate near to normal ADO function, and (ii) to provide rigorous efficacy data in two clinically relevant rodent models of mTLE. Availability of a final clinical candidate and proof of effectiveness in relevant disease models are pre-requisites to initiate a pre-pre-IND interaction with the FDA, which is the final expected deliverable of this application.
Our approach is supported by preliminary data showing effectiveness of ADO augmentation in a mouse model of pharmacoresistant mTLE and the availability of antisense constructs to reduce ADK expression in astrocytes. TLE patients with pharmacoresistant seizures and who are candidates for resection surgery are ideal subjects for a mechanism-based gene therapy aimed at down-regulating ADK within the epileptogenic hippocampus for several reasons: (i) identification of ADK as a rational and effective therapeutic target; (ii) defined focal area of epileptogenicity to be targeted by local gene therapy; (iii) confinement of endogenous ADK expression largely to astrocytes minimizes off-target effects of astrocyte-selective therapy; (iv) adverse ADO effects, though unexpected, could be treated with the ADO receptor antagonist caffeine; (v) reversibility (i.e. surgical resection) of gene therapy, if needed. Our Specific Aims are: (1) Develop a clinical candidate gene therapy product to effectively reduce ADK expression in astrocytes. (2) Perform preclinical efficacy and safety tests in two clinically relevant rodent models of mTLE. (3) Prepare regulatory documents for discussion at a pre-pre-IND meeting with the FDA.
PI: Lin Wang, MD
R01EY019939 "Dynamic and Static Autoregulation Impairment in the Optic Nerve Head of Glaucoma"
DESCRIPTION (provided by applicant): Glaucoma is a disease characterized by irreversible damage of optic nerve affecting millions of Americans. Yet details of the underlying disease mechanism are still unclear. While recognizing the crucial role of intraocular pressure (IOP), autoregulation (AR) dysfunction has been proposed as a cause of circulatory aberrations in the optic nerve head (ONH) associated with glaucomatous optic neuropathy. Autoregulation in the normal ONH initiated by an ocular perfusion pressure change contains two phases: an initial dynamic phase (dAR) when vascular smooth muscles dilate and contract to adjust the vascular resistance in an attempt to return blood flow (BF) to its original level; and a later steady-state phase (sAR) when dynamic BF changes have equilibrated to a steady level. Due primarily to methodological limitations, most previous studies in human and experimental glaucoma have assessed only sAR and failed to detect AR dysfunction in the ONH. With a modified laser speckle flowgraphy device (LSFG) and newly established methods for measuring the dAR and sAR, this proposal will test the following central hypothesis: Chronic IOP elevation induces AR dysfunction in the ONH, which importantly contributes to the pathophysiology of glaucomatous ONH damage. This hypothesis will be tested in three Specific Aims. Specific Aim 1: To test the hypothesis that ONH dAR abnormalities develop early in the monkey experimental glaucoma model, that they precede sAR and bBF alterations and that they progress in parallel with clinical measures of ONH and RNFL structural disruption. Specific Aim 2: To test the hypothesis that the ONH AR abnormalities occurring in the monkey model of experimental glaucoma are a primary result of exposure to chronic IOP elevation rather than a secondary result of neurodegeneration. Specifically, we will test the prediction that AR abnormalities will not develop in two alternative, non-IOP-related, axonal injury models - optic nerve transection and intra-retinal laser axotomy. Specific Aim 3: the ONH tissues obtained from the animals studied in Specific Aims 1 and 2 will be used to carry out two postmortem histological studies: 3A) To assess regional BF in the monkey ONH using a state-of-the-art microsphere method and compare these measurements with the LSFG bBF estimates obtained immediately prior to sacrifice; and 3B) To test the hypothesis that AR dysfunction detected in vivo by LSFG is associated with derangement of the relationship between ONH astrocytes, lamina cribrosacytes and the blood vessels within the underlying laminar beams and peripapillary scleral beam insertions. In a follow up R01 proposal we expect to extend our investigation in: 1) clinical application by modifying the current techniques of dAR analysis to noninvasively so as to elicit a controlled ONH dAR response. 2) Characterization of ONH astrocytes and lamina cribrosacyte role in ONH AR using 3D electron microscopic reconstructions and fresh ONH tissue slice preparations. 3) To test the hypothesis of age-related alterations between astrocytes and capillary endothelial/pericytes in AR.
PUBLIC HEALTH RELEVANCE: This project seeks evidence of autoregulation (AR) dysfunction in the optic nerve head and its role in pathogenesis of glaucoma as well as to develop and assess a method for its clinical detection. These developments will make the detection of AR dysfunction in human ocular hypertensive and glaucoma patients a credible goal and the presence of AR dysfunction a therapeutic target for the treatment of the disease.
PI: Claude Burgoyne, MD
R01EY011610 "IOP-related force and failure in the optic nerve head"
DESCRIPTION (provided by applicant): In glaucoma, there is no science to predict what level of intraocular pressure (IOP) will be safe for a given optic nerve head (ONH). The goal of this project is to identify the clinically important components of optic nerve head (ONH) susceptibility to glaucomatous damage using basic engineering principles.
The proposed biomechanical studies test the following hypotheses within high-resolution, digital, three dimensional (3D) reconstructions (1.5 x 1.5 x 1.5 urn voxel) and finite element models (FE Models) of the normal and glaucomatous ONH: 1) The distribution of stress (force/cross-sectional area) and strain (local deformation) within the lamina cribrosa and peripapillary scleral of the Normal ONH predicts the sites of connective tissue damage in Early Experimental Glaucoma; 2) At all levels of IOP, strains within the remaining connective tissues of Early and Moderate but not Severe Glaucoma eyes are higher than in Normal eyes; 3) A predictable pattern of fixed (permanent) deformation of the ONH connective tissues underlies the onset and progression of glaucomatous cupping in Early, Moderate and Severe Glaucoma but is not present in the optic neuropathies of Early and Late ONH Ischemia and Optic Nerve Transection; 4) the ONH connective tissues of Old eyes are hardened compared to those of Young eyes and this difference in ONH connective tissue stiffness affects the clinical behavior of the Aged, Glaucomatous ONH.
The Specific Aims and Objectives are: 1) To expand our continuum and micro FE Modeling of the ONH tissues to Severe Glaucoma and to the Young and Very Old Normal ONH; 2) To perform 3D histomorphometry within the digital 3D geometries of Normal Young and Very Old, Early, Moderate and Severe Glaucomatous, Early and Late Ischemic and Optic Nerve Transection ONH so as to characterize and compare the 3D patterns of connective tissue architecture and damage; 4) To test the hypothesis that the ONH connective tissues of the Aged ONH are less susceptible to deformation than those of the Young ONH and that this difference in susceptibility affects the clinical appearance and behavior of the Aged ONH at the onset of glaucomatous damage as seperately defined by structural and functional criteria. The methodology includes longitudinal ONH surface change detection using Heidelburg Retinal Tomographic Change Analysis; multifocal Electroretinogram (mfERG) testing; high-resolution, 3D reconstruction and 3D histomorphetry of the ONH neural and connective tissues; automated optic nerve axon counting; 3D material properties testing of intact posterior scleral shells; and continuum and micro FE modeling of the ONH neural and connective tissues.
PI: Claude Burgoyne, MD
R01EY021281 "Optic nerve head SDOCT imaging in glaucoma"
DESCRIPTION (provided by applicant): The primary goal of this project is to test three hypotheses regarding glaucomatous damage to the visual system. First, that clinically detectable neural, glial and connective tissue alterations occur deep in the optic nerve head (ONH) at a very early stage in the pathophysiology of glaucomatous damage to the visual system. Second, that the location and magnitude of the earliest of these ONH changes, detectable in vivo by spectral domain optical coherence tomography (SDOCT), predict the specific locations of subsequent alterations of the peripapillary retinal nerve fiber layer (RNFL) and orbital optic nerve axon loss. Third, that ONH connective tissue structural stiffness is altered both by age and glaucomatous damage and that it underlies the clinical appearance of the glaucomatous optic disc, specifically by influencing the "depth" of glaucomatous ONH structural change or "cupping". Until now, all animal models of glaucoma have been studied in isolation from human glaucoma. A second goal is to demonstrate that our hypotheses and techniques have evolved to a point where both can be simultaneously tested in monkeys (Specific Aim 1) and humans (Specific Aim 2). Aim 1 is to characterize the onset and progression of SDOCT ONH structural change within pre- and post- laser SDOCT ONH data sets from both eyes of 70 unilateral experimental glaucoma (EG) monkeys. Aim 2 is to characterize the onset and progression of ONH structural change within longitudinal SDOCT ONH data sets from 250 human ocular hypertensive and early glaucoma patients. The methodology includes: longitudinal Heidelberg Spectralis 870 and 1060 nm SDOCT ONH image acquisition in monkeys (870 nm only in humans); their visualization, delineation and quantification within custom Multiview software; and in monkeys only, post- mortem 3D histomorphometric ONH reconstruction and quantification, co-localized, eye-specific comparison of SDOCT ONH and 3D histomorphometric reconstructions and regionally-aligned orbital optic nerve axon damage map generation using our custom Axonmaster axon counting software. The expected outcomes are: 1) deep ONH structural change will occur before and predict subsequent ONH surface and RNFL change during the onset and progression of glaucomatous damage in both monkey and human eyes; 2) early ONH structural change will co-localize to orbital optic nerve axon loss in monkey eyes and precede RNFL alterations in both monkey and human hypertensive eyes; 3) in monkeys, younger ONHs will be "more compliant" and older ONHs will be "stiffer" when normal, and both will demonstrate transient hypercompliance followed by progressive stiffening as glaucomatous damage progresses; 4) younger monkey and human ONHs will demonstrate a "deeper" form of "cupping" than older ONHs; 5) in younger compared to older eyes, the onset and progression of structural change ("cupping") will include a larger connective tissue component; and 6) 1060 nm SDOCT imaging will improve visualization of deep monkey ONH imaging targets compared to the existing clinical standard (870 nm imaging).
PUBLIC HEALTH RELEVANCE: The clinical detection of the onset and progression of glaucomatous damage to the optic nerve head (ONH) is central to the care of every glaucoma patient. We propose to use 870 nm and 1060 nm Heidelberg Spectralis Spectral Domain Optical Coherence Tomography (SDOCT) to characterize the onset and progression of ONH structural change within pre and post- laser SDOCT ONH data sets from both eyes of 70 unilateral experimental glaucoma (EG) monkeys and 250 ocular hypertensive and early glaucoma patients. In this project we will translate 11 years of NIH-funded, post-mortem monkey work to an in-vivo imaging modality that will be shown to have important and novel clinical care applications in humans.
PI: Shaban Demirel, PhD
R01EY019674 "Predicting the rate of progression in glaucoma"
DESCRIPTION (provided by applicant): Glaucoma is one of the leading causes of treatable blindness in the world. However, the manner and rate of disease progression varies markedly between patients, and so predicting future progression and hence the prognosis for an individual is challenging. Much of the previous work in this field has concentrated on determining whether a patient with certain risk factors will develop glaucoma. There has been less attention paid to the likelihood that a patient will suffer significant loss of vision as a result of their glaucoma, or how soon vision loss may manifest. The overall goal of this project is to improve the ability to predict the future course of an individual's disease, and so enable the clinician to adapt their management strategy accordingly. Better knowledge of glaucomatous pathophysiology will also be gained, with meaningful implications for developing new methods of diagnosis and treatment strategies. The primary tool used in this project will be longitudinal data collected biannually from over 250 subjects with early or suspected glaucoma, and 50 normal subjects. For the majority of the glaucoma subjects, up to ten years of prior information is available and will also be used. The first part of the project aims to improve understanding of the structure-function relationship in glaucoma. Three non-competing hypotheses will be tested that seek to explain the weak observed correlation between functional change (to the patient's visual field) and structural change (to the optic nerve head and/or retinal nerve fiber layer). The first experiment will determine the true underlying correlation after taking into account the variability inherent in the testing procedures. The second experiment will compare changes to the optic nerve head surface topography against subsurface changes and changes to the retinal nerve fiber layer, to determine whether there is temporal dissociation caused by surface change not driven by a loss of neural tissue. The third experiment will use a sampling-limited test of retinal ganglion cell density, to determine whether there is temporal dissociation caused by dysfunctional retinal ganglion cells that are still present in the retina but have altered response characteristics. Results from these three experiments will provide new insights into the disease process, with possible implications for future development of testing and treatment strategies. The second part of the project aims to use this new information and advanced analysis techniques (including regression trees) to better predict the future rate of functional change on a per-eye basis. First, subsequent change will be predicted based on test results at one time point. Second, improved predictions will be made when a series of test results are available. The fundamental motivation for this project is to enable a clinician to prevent severe visual disability or blindness in an ocular hypertensive or glaucoma patient, by identifying a rapid progression rate or a high likelihood for rapid progression at the earliest stages of the disease.
PI: Brad Fortune, OD, PhD
R01EY019327 "Axonal Cytoskeletal Changes in Experimental Glaucoma"
DESCRIPTION (provided by applicant): This project proposes that prior to retinal ganglion cell (RGC) death in glaucoma, and before permanent loss of vision, there exists a stage of RGC dysfunction characterized by degradation of axonal microtubules (MTs). Emerging evidence suggests that MT degradation can occur initially without substantial changes in axonal caliber. Therefore, it is proposed that early stage RGC dysfunction involving MT degradation should be preferentially detectable by scanning laser polarimetry (SLP) of the retinal nerve fiber layer (RNFL) prior to changes in RNFL thickness. This is because the fundamental optical principle of SLP is based on detecting phase retardance of polarized light, which is due to the optical property birefringence produced in the RNFL by the long, thin cylindrical MTs. Preliminary studies demonstrate that RNFL retardance declines prior to, and faster than RNFL thickness in several different experimental models of RGC injury, including experimental glaucoma (EG). Clinical detection of axonal MT disruption by SLP, in the absence of RNFL thickness changes, might represent an early and potentially reversible phase of glaucomatous damage and provide a clinically detectable marker for therapeutic adjustment. Thus the central hypothesis of this proposal is that disruption of MTs within the axons of the peripapillary RNFL is an early indicator of glaucomatous damage, preceding both changes in axonal caliber and physical loss of those axons. Predictions arising from this hypothesis are tested in three Specific Aims using a non-human primate (NHP) model of EG. Specific Aim 1: To test the prediction that peripapillary RNFL retardance will decline prior to RNFL thickness changes measured by spectral domain optical coherence tomography (sd-OCT) and prior to optic nerve head (ONH) surface changes measured by confocal scanning laser tomography (CSLT) in NHP eyes with EG; Specific Aim 2: To test the predictions that histological evidence of peripapillary RNFL MT disruption will be more pronounced than histologically-defined RNFL thickness changes and retrobulbar optic nerve axon loss; Specific Aim 3: To test the prediction that RGC functional abnormalities are associated with the intermediate stage of RGC degeneration characterized by abnormal axonal MTs. To achieve these Aims, EG will be induced via laser photocoagulation of the trabecular meshwork to cause moderate, unilateral chronic IOP elevation in 24 NHPs. Weekly measurements of peripapillary RNFL retardance, RNFL thickness and ONH surface topography will be made in both eyes of each NHP using SLP, sd-OCT and CSLT, respectively, during a 4-week pre-laser baseline period and for up to 8 months after onset of EG (Aim 1). For each parameter, statistically significant change is defined as any change exceeding the baseline intersession variability for each individual eye, twice confirmed. Once each animal progresses to its endpoint, it is sacrificed for histological data collection and analysis (Aim 2). During each week of in vivo structural testing for Aim 1, RGC function will also be assessed in both eyes using three proven forms of electroretinography (Aim 3). PUBLIC HEALTH RELEVANCE: Glaucoma is one of the most common causes of blindness in the United States and around the world. It is a chronic disease with no known cure, but prospective longitudinal trials have found that treatment to lower intraocular pressure decreases the rate of progressive vision loss. Thus, early diagnosis enables timely therapeutic intervention and reduces the overall impact of glaucoma. However, achieving a timely diagnosis requires clinical detection of the onset and progression of glaucomatous damage to the optic nerve head (ONH) and retinal nerve fiber layer (RNFL), which remain a central challenge in the clinical care of every glaucoma patient. This project evaluates and advances clinical tools for detecting early damage and progression of glaucoma.
PI: Brad Fortune, OD, PhD
1R21EY021311 "Imaging retinal astrocytes, ganglion cells and axonal transport in vivo"
DESCRIPTION (provided by applicant): Astrocytes are a major class of glia in the vertebrate retina. They are located primarily within the innermost retinal layers; their processes surround retinal ganglion cell (RGC) axons and axon bundles as well as all blood vessels. Because of this anatomical relationship, and a variety of physiological evidence, astrocytes are thought to have a major role in the mechanisms of retinal blood flow autoregulation, i.e. the maintenance of nearly constant blood flow in response to variations of ocular perfusion pressure. Astrocytes are also thought to play an important role in the pathophysiology of many ocular diseases by responding to a variety of insults such as ischemia, increased intraocular pressure and neuronal degeneration in a manner that has been characterized as gliosis. Hence, the ability to image astrocytes in vivo could help to elucidate aspects of disease pathophysiology. Similarly, there is evidence to suggest that RGC axonal cytoskeletal components, specifically microtubules, are disrupted during the earliest stages of response to experimental injuries such as axotomy and experimental glaucoma. This disruption is significant because microtubules are the "tracks" upon which axonal transport is driven. Thus, if microtubule abnormalities develop early in response to injury, the resultant axonal transport disruption could exacerbate the injury and inhibit protective or rescue responses from achieving full potential. The overall goal of this R21 project is to develop the methods for imaging retinal astrocytes, RGCs, their axons and axonal transport in vivo. The specific objectives are as follows: Specific Aim 1: To establish methodologies for in vivo visualization of retinal astrocytes, RGCs, their axons and active axonal transport in the rat eye. To evaluate the optimal concentration, follow-up duration and persistence of in vivo markers as well as perform histopathological studies to corroborate in vivo observations. Specific Aim 2: To evaluate potential toxicity of in vivo astrocyte markers and axonal transport tracers using sensitive measures of retinal function (electroretinography, ERG) and retinal structure (spectral domain optical coherence tomography, SDOCT), so as to assess potential for use in primate experimental models. Specific Aim 3: To evaluate the sensitivity of our newly developed methods by comparing the impact of four unilateral experimental injury models (intravitreal injection of nocodazole/colchicine to disrupt axonal microtubules and inhibit active axonal transport; acute elevation of intraocular pressure; chronic elevation of intraocular pressure; and optic nerve crush) with results obtained in bilateral control eyes. The novel methods developed in this proposal will make possible in future proposals, studies about the onset of astrocyte abnormalities and RGC axonal transport abnormalities and comparisons of those phenomena to the course of RGC and axonal degeneration in experimental models of RGC injury.
PUBLIC HEALTH RELEVANCE: Glaucoma is one of the most common causes of blindness in the United States and around the world. It is a chronic disease with no known cure. Though prospective longitudinal trials have found that treatment to lower intraocular pressure decreases the rate of progressive vision loss, some individuals continue to lose vision despite successful therapy to lower their intraocular pressure. Thus, a more thorough understanding of the events leading to damage and vision loss in glaucoma is required. The goal of this project is to develop methods for evaluating two groups of cells and aspects of their function in the living eye using specialized imaging techniques.
PI: Stuart Gardiner, PhD
1R01EY020922 "Functional Testing for Glaucoma"
DESCRIPTION (provided by applicant): Glaucoma is a leading cause of blindness both in the US and worldwide. The long-term purpose of this project is to improve functional testing in glaucoma. Assessment and follow-up of patients currently relies on automated perimetry to provide functional testing of the visual field. However, the ability to assess progression and/or response to treatment using perimetry is hampered by high variability, especially in areas of moderate or severe glaucomatous damage. Recent findings by our laboratory and others have advanced our understanding of perimetry by challenging key assumptions about the test. This proposal aims to use these advances to explain and reduce the test variability. This will improve the accuracy, efficiency and utility of current functional testing, giving immediate impact in both research and clinical settings, and laying groundwork for the next generation of instruments and algorithms. The first Specific Aim is to produce an accurate and physiologically justified measure of the Effective Dynamic Range (EDR) of perimetry. It is postulated that the very high contrast stimuli used by perimetry in glaucomatous defects saturate the response of the visual system. The resultant nonlinearity in the contrast-response function would cause the detection probability to asymptote below 100%, explaining the high variability in sensitivities in damaged areas. The limit of the EDR will be defined as the contrast beyond which response linearity cannot be assumed. This will be measured by collecting frequency-of-seeing curves in subjects with moderate or advanced glaucoma. The same technique will be used to determine whether the EDR is extended by use of an increased stimulus size. The second Specific Aim is to derive and test a spatial filter to reduce the variability. This will be the first filter to be based both on sensitivities at other locations in the visual field and on the structure of the optic nerve head. The third Specific Aim is to assess the potential utility of using a linear scale for sensitivity, rather than the current logarithmic decibel scale. First, an efficient linear-scaled thresholding algorithm will be derived and tested, to determine whether it will reduce variability both between tests and in the structure-function relation. Second, linear-scaled global indices of the central visual field will be examined, to determine whether they offer improved prognostic value compared with current decibel-scaled indices when assessing progression. The three aims are complementary. It is anticipated that by combining these aims, variability in perimetry will be better understood, and significantly reduced. Such an improvement in a test as commonly performed as perimetry will significantly impact future clinical practice.
PI: Lin Wang, MD, PhD
R21EY024432 "Astrocyte-mediated blood flow autoregulation as a disease mechanism in glaucoma"
Compromised blood flow autoregulation has been proposed as one of important pathological mechanisms contributing to decreased BF and retinal ganglion cell damage in glaucoma. Within this paradigm, blood flow (BF) in the eye, particularly in the optic nerve head, can no longer be held within a normal range when intraocular pressure (IOP) and/or blood pressure (BP) fluctuates. As evidenced in clinical and laboratory experiments, such hemodynamic changes result in a blood flow decrease within the ocular tissues and associated ganglion cell damage measured by retinal nerve fiber layer thickness and functional loss. Thus, early intervention targeting specific pathways leading to autoregulation dysfunction could mitigate the hemodynamic changes and protect retinal ganglion cells from damage in glaucoma.
However, the current knowledge of ocular BF autoregulation associated with the mechanism of the disease is insufficient to interpret many of the phenomena observed in clinics and in experiments. Astrocytes are essential to couple neuronal activities with blood flow (known as neurovascular coupling) in both brain and retina in a feed forward mechanism. A line of studies have suggested that astrocyte is also an essential cellular element involved in blood flow autoregulation. This has clinical implications because identification of the astrocyte role in BF autoregulation is likely to lead to a novel pathological mechanism of hemodynamic change and subsequently innovative pharmacological interventions for glaucoma. This study proposes to develop new techniques to examine peri-arterial astrocyte bioactivities and vascular response in relation to intra and extravascular pressure alteration.
This initiative will lead future studies by utilizing these techniques to examine hypothesized mechanisms of BF autoregulation in normal and in glaucomatous eyes. In Specific Aim 1: (a) To develop and validate a system to set and manipulate both intravascular pressure (simulating BP) and extravascular pressure (simulating IOP) to different level and, simultaneously, image calcium signals (labeled with Fluo-4AM) in peri-arterial astrocytes and vessel diameter (labeled with Dil). (b) Compare the calcium signals within the peri-arterial astrocytes and vessel diameter changes against the magnitude of mechanical stimuli induced by changes in intra- and/ or extravascular pressure in isolated pig retina. In Specific Aim 2: To develop a novel in vivo calcium imaging technique to monitor calcium changes within peri-arterial astrocytes while BP is changed. This will be achieved by intravitreal injection of a calcium indicator (Fluo-4AM). Utilizing fluorescence mode in confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography, the rat fundus is imaged continuously to monitor the calcium signals in peri-arterial astrocytes and vessel diameter, whilst the rat BP is deterministically increased and decreased. Proving the involvement of astrocytes in BF autoregulation, identification of specific targets for therapeutic intervention may protect the ocular tissue from ischemic insult and subsequent progressive RGC damage in human glaucoma.