Krystel Huxlin - US grants

[unreadable] DESCRIPTION (provided by applicant): Laser refractive surgery in the form of photorefractive keratectomy (PRK) and laser-assisted in situ keratomileusis (LASIK) has become an important means of correcting vision in humans. However, even when it is followed by good healing and achieves suitable correction, refractive surgery is often followed by complications that decrease optical benefit. Among these complications is an increase in ocular higher order optical aberrations, especially spherical aberration and coma, and regression of the original lower order correction over time. [unreadable] While recent studies have shed considerable light on the healing response of the cornea to refractive surgery, there is no understanding yet about how this response affects the shape and optical wave aberration structure of the cornea. This is partly due to a lack of appropriate animal models in which to combine such investigations. We have developed an experimental paradigm in which animals are trained to accurately and repeatedly fixate along the optical axis of ophthalmic machines. This allows us to perform optical coherence tomography and wavefront sensing under the same fixation conditions and with the same degree of accuracy and repeatability as in human subjects. These instrument-derived measures can then be correlated with histological studies of corneal biology in the same eyes. Using this animal model, we propose to characterize the cellular substrates of higher order aberrations and regression following PRK by testing the following hypotheses: (1) that corneal wound healing and specifically corneal myofibroblasts, are responsible for a significant proportion of the increase in higher order aberrations after laser refractive surgery, and (2) that regression after laser refractive surgery is primarily due to epithelial hyperplasia induced when the laser ablation shape on the cornea locally [unreadable] exceeds a critical slope. Our goal is to understand cellular events during corneal wound healing in terms of their differential ability to alter corneal shape and change the lower and higher order optical wave aberration properties of the eye. This knowledge is essential for the development of both preventative and therapeutic strategies aimed at controlling the cornea's biological response in order to improve reliability and reduce complication rates after laser refractive surgery, it will also be critical to our successful treatment of highly aberrated eyes such as those resulting from corneal transplantation and refractive surgery. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Corneal scarring is a major cause of decreased visual quality and vision loss worldwide. Scarring follows disruption to normal corneal structure and function, whether from infection, laser refractive surgery, corneal transplantation, ocular trauma (chemical or physical) or corneal dystrophies. There is no suitable means of controlling corneal scaring despite more than 25 years trying to characterize cytokine signaling during wound healing. Our long-term objective is to understand mechanisms of corneal wound healing and our goal is to design effective therapies to treat or prevent corneal scarring. Our overall hypothesis is that peroxisome proliferator activated receptor gamma (PPAR3) ligands prevent corneal fibrosis and can be targeted as a therapy for this condition, with significantly greater efficacy than current clinical treatments or than blocking the activity of single cytokines. Aim 1: Test the hypothesis that PPAR3 ligands inhibit key pro-fibrotic activities of cultured corneal keratocytes. We use immunohistochemistry, proliferation assays, wounding assays, western blots, slot blots and Q-PCR to assess the relative effectiveness of PPAR3 ligands at modulating proliferation, migration, expression of CTGF, 1SMA, Thy-1, collagen I, collagen III, fibronectin and their mRNAs in cultured keratocytes stimulated by optimal doses of TGF2. Aim 2: Test the hypothesis that PPAR3 ligands inhibit key pro-fibrotic activities in cultured corneal keratocytes through both PPAR3-dependent and -independent pathways. We will use both pharmacological and genetic approaches to test our prediction that PPAR3 ligands act both by activating PPAR3 and by inhibiting TGF2-regulated pathways. Knowing the relative strengths of these mechanisms in corneal keratocytes is critical if we are to develop PPAR3 ligands as optimally-targeted therapies for corneal fibrosis. Aim 3: Test the hypothesis that PPAR3 ligands are more efficient inhibitors of fibrosis in PRK-induced corneal wound healing than anti-TGF2 antibodies, steroids or Mitomycin C. We will perform binocular PRK in cats followed by the topical administration of select PPAR3 ligands, anti-TGF2 antibodies, steroids or Mitomycin C. We will use immunohistochemistry to contrast key cellular aspects of the wound healing reaction. We predict that PPAR3 ligands will be associated with lower cell death, faster wound healing, less haze and lower induction of higher-order optical aberrations than use of anti-TGF2 antibodies, steroids or Mitomycin C post-operatively.

DESCRIPTION (provided by applicant): Damage to the adult primary visual cortex (V1) causes a loss of conscious vision over the same part of the visual field in both eyes (cortical blindness - CB). This increasingly common cause of permanent disability in older, adult humans is still considered untreatable. Our long-term objective is to define a new paradigm for understanding visual recovery after permanent visual cortex damage. Our goal is to characterize the properties of and signal processing mechanisms that enable visual relearning and recovery in CB. Knowing what mechanisms and brain pathways mediate recovery will allow us to predict the extent to which vision can be recovered, as well as the quality and modality of recovered vision that can be attained in a given individual. We will meet our objective and goal by testing the primary hypothesis that after V1 damage, training-induced relearning in CB fields depends on motion processing for its initiation. This is based on our preliminary findings that motion training in CB fields transfers to static orientation discriminations not normally perceivable in blindsight. However, without the initial motion training, these static discriminations cannot be relearned. While training could work via a variety of mechanisms, our preliminary findings suggest the following alternatives, to be tested here: 1) training stimulates the motion processing complex hMT+ to more effectively process visual information from CB fields, including that needed for static orientation discriminations, 2) training stimulates the motion pathway to reactivate other visual areas (incl. parts of V1, V2, V3, V4, V01) and their pre-existing processing abilities, or 3) training alters readout of information from hMT+/other areas. Aim 1 will use visual psychophysics to test the hypothesis that static orientation relearning depends on learning in the motion pathway, and to measure specificity of learning for trained directions/orientations. Aim 2 will use the perceptual template model (PTM) and psychophysical tests of spatial suppression to test the hypothesis that relearning in CB fields occurs via 1) changes in tuning of basic orientation or direction channels, possibly via changes in spatial suppression within these channels, or 2) that training improves the readout of these channels. Aim 3 will use functional MRI (fMRI) to measure changes in functional anatomy associated with relearning in CB fields. We will test the hypothesis that visual training: 1) alters the blind field's retinotopic representation either in just hMT+ or both in hMT+ and other visual areas (V1, V2, V3, V4, V01); 2) increases direction and/or orientation specificity in just hMT+ or both in hMT+ and V1, V2, V3, V4, V01 or 3) none of the above. Our results will provide critical information about brain pathways and signal processing mechanisms stimulated by training to evoke visual relearning in CB fields. This knowledge is essential theoretically to better understand the type and degree of plasticity possible in damaged, adult visual systems, and to improve our treatment strategies for humans suffering from the disability induced by permanent visual cortical damage.

The Imaging Module continues to evolve to meet the expanding needs of CVS Core users. While histology, microscopy and MRI continue to be intensively performed by CVS members, exciting developments in the area of adaptive optics (AO) imaging at Rochester have created a significant need for AO support. As a result, the Imaging Module now consists of an AO Imaging Facility, a Histology/Microscopy Facility (including confocal imaging) and a Functional/Structural Magnetic Resonance Imaging (MRI) Facility. One highly-qualified, fulltime technician staffs each facility.

? DESCRIPTION (provided by applicant): Corneal nerves are important for the health of the cornea, as well as for protecting the eye from outside elements. These nerves are predominantly nociceptive, coding discomfort and pain in response to mechanical stimulation, temperature change and/or chemical stimulation. Disease, infection and ocular surgery can all damage corneal nerves, with long-term consequences in terms of pain, dry eye, recurrent erosions, opacity and even blindness. Yet, there are no effective therapies in clinical practice fo treating nerve dysfunction in the will use our cat model of corneal wound healing after photorefractive keratectomy (PRK) and a combination of in vivo and in vitro approaches to study the basic mechanisms controlling adult, corneal nerve regeneration post-injury. Our preliminary data show clear abnormalities of re-innervation in different nerve layers (stroma, sub-basal plexus, epithelium) and major modulation of nerve regeneration by topical anti-fibrotics. These data suggest an inhibitory influence of myofibroblasts on regenerating nerves and/or their associated glia (non-myelinating Schwann cells - NMSCs), leading us to propose the following central hypothesis for this renewal application: myofibroblast transformation that occurs in response to large corneal wounds directly inhibits nerve regeneration. Thus, blocking myofibroblast differentiation during the early wound healing response is critical for restoring normal corneal innervation to the epithelium and stroma. We will test this hypothesis by: Aim 1 - assessing the impact of myofibroblast differentiation on corneal nerve regeneration and NMSCs after PRK; Aim 2 - assessing the effect of blocking myofibroblast differentiation on corneal nerve regeneration and NMSCs after PRK; Aim 3 - context of corneal wounds. The proposed experiments assessing the long-term impact of abnormal corneal re-innervation on corneal optics; and Aim 4 - characterizing the interactions between corneal fibroblasts, myofibroblasts and sensory neurons in vitro and testing the hypothesis that myofibroblasts inhibit neurite outgrowth via Sema3A. The proposed, systematic characterization of nerve regeneration, nerve-myofibroblast interactions and their molecular mechanisms during wound healing are critical for the development of new therapeutic strategies to treat corneal wounds with an eye to promoting optimal nerve regeneration and ensuring long-term health of the ocular surface.

In adult humans, damage to the primary visual cortex (V1) and the associated contralateral, homonymous loss of conscious vision in both eyes (cortical blindness?CB) affects ~1% of the population >49 years of age. CB is considered to be clinically intractable and permanent, with no accepted, standardized rehabilitation strategies. A critical barrier to progress in treating CB is the assumption by many in the clinical and scientific communities that the damaged, adult visual system cannot recover functionally. However, in the last decade, work by several teams worldwide, including ours, has identified one method that can reliably recover vision in chronic CB patients: visual training to detect and discriminate single stimuli presented repeatedly, in a gaze contingent manner, at single locations in the blind field. While a definite step forward, training is difficult, requires weeks-to-months of daily computer work to elicit measurable improvements and the recovery attained is incomplete and location specific. Moreover, the factors limiting full vision restoration are only partially understood. The current proposal builds on our prior work to test specific hypotheses about the processing limitations that underlie residual visual deficits, with the goal of overcoming them. Our data suggest that the deficits observed after visual training in CB are due to abnormally high internal processing noise and broader-than-normal tuning for ?features? such as motion direction. Attention cueing either to stimulus features (feature-based attention, FBA) or visual field locations (spatial attention, SA) has been shown to enhance visual processing by decreasing internal noise and/or improving direction tuning. Spatial cueing can also enable and generalize learning across locations. Motivated by these established properties of attention, our goal here is to test the hypothesis that attention will increase the effectiveness of training for features and across locations in CB fields, restoring vision to a greater degree, faster and at multiple sites simultaneously. To this end, Aim 1 will test the hypothesis that feature-based attention during training can overcome fine discrimination deficits in CB by sharpening direction tuning and increasing the gain of spared visual circuits. Aim 2 will test the hypothesis that spatial attention can potentiate visual recovery at multiple blind field locations simultaneously via a spatially distributed multiplicative increase in gain of the tuned population's response. If successful, our proposal will address 3 key questions in the context of CB: (1) whether normal, fine visual discriminations can ever be recovered without an intact V1; (2) what type of training can most effectively induce the necessary signal processing changes; and (3) whether recovery can be attained at multiple locations simultaneously. Comparing and contrasting different types of attention will allow us to gain insights into mechanistic changes that underlie effective vision recovery in CB. This in turn, is important both neuro-scientifically, and for devising more realistic treatment and outcome expectations for this patient population.

Twenty-eight faculty of the Center for Visual Science (CVS) at the University of Rochester request renewal of support for a pre-doctoral and postdoctoral training program that emphasizes four broadly defined areas of vision research: (1) Advanced optical technology for vision correction and retinal imaging, (2), cell biology of the normal and diseased eye, (3) neural mechanisms of vision and (4) vision in behavior. Training faculty have extensive cross-campus and cross-department collaborations, a strong record of mentoring and high research funding levels. Training is interdisciplinary, drawing particularly on the unique technical and intellectual resources of CVS. It covers a broad range of basic and clinical problems in vision but emphasizes approaches that link visual performance to underlying biological mechanisms. The program has a strong record of trainee productivity, PhD student retention and fast average time to PhD. Similarly, postdoctoral trainees overwhelmingly pursue scientific careers, with significant numbers entering tenure-track positions in academia. Trainees have a good record of subsequent individual training and research funding, as well as successful research and research-related career paths. The program also has a good record of recruiting, training and career placement for trainees from under-represented minority groups. For our renewal application, we request support for 5 pre-doctoral trainees per year, who will generally enter the program through the Departments of Brain and Cognitive Science, Biomedical Engineering, the Neuroscience Graduate Program and the Institute of Optics. Students will take core courses plus advanced seminars in visual science, augmented by courses from the departments through which they entered the program. Concurrently with course work, students complete research projects in CVS preceptor labs. Finally, we also request support for two postdoctoral fellows per year. Postdoctoral training has a heavy emphasis on research performance, productivity and communication. All trainees take part in topical workshops, special topics seminars, regular colloquia, research talk series, the CVS retreat, and the biannual CVS Symposium.

In adulthood, stroke damage to the primary visual cortex (V1) causes a large, contralateral loss of conscious vision referred to as hemianopia or cortical blindness (CB). Although this condition affects up to ½ million new cases each year in the US alone, there is a total lack of accepted vision restoration therapies ? in marked contrast with early-onset physical therapies prescribed to those with motor cortex damage. Two decades of work in chronic CB patients, whose deficits are deemed stable, permanent and thus amenable to scientific study, have generated one method consistently able to recover vision after long-standing V1 damage: gaze- contingent visual training to detect or discriminate stimuli in the blind field. Over the last 2 grant periods, we have taken clear leadership in the field, providing hope that an effective therapy for CB may finally be on the horizon. However, while characterizing training-induced recovery and its underlying mechanisms, we also found that recovery in chronic CB requires months of daily training and the vision restored is low-contrast, coarse, impaired by excessive internal processing noise and restricted to the blind field perimeter. Accumulating evidence suggests that these limitations may occur because chronic patients have lost a substantial portion of neurons that contribute to vision fundamentals not only in V1, but through retrograde degeneration, in the dorsal lateral geniculate nucleus (dLGN) and retina. Our new pilot data show subacute CB patients <6 months post-stroke to lack significant signs of degeneration, and more than half of subacutes tested retained visual discrimination abilities in their blind field, which disappeared by the start of the chronic period (6 months post-stroke). Moreover, when training was administered to subacutes, they recovered the same discrimination abilities as chronics, but much faster, and with recovery extending deeper into their blind field. These data form a strong premise for testing the hypothesis that substantial differences in plastic potential between subacute and chronic V1-stroke visual systems can be exploited to maximize visual restoration in CB, and that the extent of recovery attainable is limited by the amount of retrograde degeneration sustained. We now propose to: (Aim 1) assess how visual performance relates to structural evidence of retrograde degeneration in the subacute period post-V1-stroke. We will then (Aim 2) assess the impact of subacute training on blind-field functions, the progression of retrograde degeneration and the continued potential for training-induced recovery in the chronic period. Finally, we will (Aim 3) contrast mechanistic substrates of perceptual learning in subacute & chronic CB. All in all, the work proposed is unique in the field, which it stands to advance significantly by generating entirely new knowledge and understanding of the change in visual plastic potential with time in the early period after permanent V1 damage in humans. This knowledge is important both neuro-scientifically, and for devising more effective treatment and realistic outcome expectations for this growing patient population.