Faculty Bibliography

PURPOSE/UNASSIGNED:Keratoconus (KC) is a common eye disease characterized by progressive corneal thinning and steepening. Despite multiple treatment options, there is no definitive cure for KC. Previously we identified loss and dysregulation of nuclear factor erythroid 2-related factor 2 (NRF2) mediated antioxidant functions in stromal cells and extracellular matrix (ECM) in KC. Here we used tear fluid samples and cell culture models to investigate oxidative stress in KC. METHODS/UNASSIGNED:Primary human KC and donor (DN) stromal fibroblasts were exposed to hydrogen peroxide (H2O2) to induce oxidative stress and treated with sulforaphane (SFN) for antioxidant rescue. The fibroblasts were then assessed for NRF2 activation and apoptosis by measuring TXNRD1, HMOX1, NRF2, and GPX3 expression and caspase-3/7 activity. ML385 was used to inhibit NRF2 functions in DN fibroblast cultures followed by measurements of cell death (Caspase 3/7), proliferation (BrdU and Ki-67 labeling) and ECM deposition by immunohistology. Oxidative stress was directly assessed in KC and non-KC subjects by measuring malondialdehyde (MDA) and glutathione peroxidase 3 (GPX3) levels in the tear fluid. RESULTS/UNASSIGNED:H2O2-stressed KC fibroblasts displayed increased apoptosis and suboptimal NRF2 activation, which could be rescued with SFN. Conversely, DN fibroblasts treated with ML385 elicited KC-like cellular phenotypes, including decreased antioxidant response, reduced cell growth, myofibroblastic changes and poor ECM deposition. Compared to unaffected controls, KC patient tear fluid exhibited elevated levels of GPX3 and MDA, a byproduct of lipid peroxidation. CONCLUSIONS/UNASSIGNED:NRF2-mediated anti-oxidative functions are dysregulated in KC. In the future SFN antioxidant treatments may be therapeutic in KC, while MDA and GPX3 may lead to promising biomarkers for diagnosis and severity predictions.

PURPOSE/OBJECTIVE:To report on the presentation, treatment, and visual outcome of stromal keratitis (SK) in the Zoster Eye Disease Study (ZEDS). DESIGN/METHODS:Secondary analysis of SK endpoint of randomized clinical trial. SUBJECTS/METHODS:Herpes Zoster Ophthalmicus (HZO) patients were randomized in a double-masked clinical trial of oral valacyclovir 1g daily or placebo for 1 year. They were followed prospectively every 3 months for 18 months for endpoints of SK, iritis (IR), endothelial keratitis (EK), or dendritiform epithelial keratitis (DEK). METHODS:Presentation of recurrent, new, or worsening SK was evaluated retrospectively by treatment assignment, randomization strata, and use of topical steroids. Investigators had been allowed discretionary treatment of endpoints including open label valacyclovir and topical steroids. Visual outcome and treatment with open label oral valacyclovir and topical steroids were evaluated. MAIN OUTCOME MEASURES/METHODS:Use of open label valacyclovir and topical steroid treatment of recurrent, new, or worsening SK, and visual acuity at 12 months. RESULTS:Recurrent, new, or worsening SK occurred in 105/527(20%) participants. Randomization group was not associated with this complication. Mean best corrected visual acuity at enrollment was logMAR 0.10±0.14 with no difference at 1 year, logMAR 0.13±0.2, and no difference between valacyclovir and placebo groups at enrollment or at 1 year. Among the 105 instances of SK, 79(75%) were recognized at scheduled study visits rather than at episodic visits. In only 11/105(10%) of recurrent, new, or worsening SK, did masked investigators opt to treat with open label oral antiviral. At the time of SK complication, 52/105(50%) were on topical steroid, but 47/52(90%) on topical steroids were using 1x daily or less, 21/47(45%) high potency and 26/47(55%) low potency (p=0.47). Of 48/105(47%) on no topical steroids at recurrent, new, or worsening SK, 18/48(38%) had discontinued steroids in the prior 3 months. 38/48(75%) on no topical steroids at complication SK were subsequently treated with high potency steroids 2x daily or more. Of 26/52(50%) on low potency steroids at complication SK, 23/26(88%) were treated with increase in frequency only. CONCLUSIONS:Individuals with ocular complications of HZO who develop SK generally maintain very good vision without use of oral antiviral therapy when monitored closely and SK is recognized and treated. Low potency topical steroids should be considered for treatment and ongoing suppression of SK in HZO.

In a dynamic environment, sensory systems must filter out irrelevant information to construct a stable percept. Animals who rely on smell need to identify and discriminate odors despite fluctuations in concentration, yet odor receptor activation is strongly concentration dependent. Here we explored how odor signals are transformed within the mouse olfactory bulb (OB) by developing an all-optical approach to identify the connectivity between odor receptor channels (glomeruli) and the mitral and tufted cells (MTCs), while monitoring their odor responses. We found that the glomeruli and MTCs activated earliest in a sniff robustly represented odor identity across concentrations, whereas MTCs connected to later activated glomeruli were concentration dependent. Furthermore, probing the responsiveness of MTCs to glomerular input found a short temporal window of excitability at a sniff's onset, followed by prolonged odor-evoked inhibition. Our findings demonstrate, in awake animals, that the OB implements a rapid temporal filter, which is responsible for stabilizing identity across concentrations while decorrelating responses between odors.

Bessel beams are commonly used in two-photon microscopy to extend the depth of field and thereby achieve functional volumetric imaging of the living brain. In practice, this approach suffers from background signals and limited lateral resolution due to the Bessel beam's strong side lobes. We introduce and demonstrate a new approach to side lobe suppression based on non-degenerate two-photon excitation, in which dual wavelength illumination produces an imaging point-spread function that is the product of the two coaxial Bessel beams. This technique can reduce the main side lobe intensity of a Bessel beam by 50% or more. We illustrate the approach conceptually with an analytical paraxial model and use detailed physical simulation to show that the approach is effective in the presence of the symmetry-breaking aberrations that amplify side lobes in high NA systems. We experimentally demonstrated the technique using a refractive axicon and the pump and tunable beams of a femtosecond laser. This work establishes non-degenerate two-photon excitation as a practical and broadly applicable strategy for improving point spread-function quality in high-resolution volumetric microscopy.

Cerebrospinal fluid (CSF), partly driven by sensory stimulation, is crucial for maintaining homeostasis and clearing metabolic waste. Whether such stimulus-driven CSF flow is disrupted in age-related neurodegenerative diseases of the visual system remains unclear. This study examined the CSF flow during visual stimulation in glaucoma patients and healthy older adults using functional magnetic resonance imaging. In glaucoma, CSF inflow becomes progressively decoupled from the visually evoked blood-oxygenation-level-dependent (BOLD) response. Specifically, the characteristic stimulus-locked CSF patterns, which decrease after stimulus onset and increase after offset, diminish with disease severity. Mediation analysis suggests this flattened CSF pattern is driven by a flatter ascending BOLD slope, leading to a shallower CSF trough and a reduced post-stimulus surge. These results indicate that glaucoma-related functional impairments contribute to downstream alterations in CSF dynamics. Overall, this study provides insight into how glaucoma disrupts visually driven CSF inflow and highlights in vivo biomarkers for monitoring CSF dynamics.

Monitoring intracellular calcium is central to understanding cell signaling across nearly all cell types and organisms. Fluorescent genetically encoded calcium indicators (GECIs) remain the standard tools for in vivo calcium imaging, but require intense excitation light, leading to photobleaching, background autofluorescence and phototoxicity. Bioluminescent GECIs, which generate light enzymatically, eliminate these artifacts but have been constrained by low dynamic range and suboptimal calcium affinities. Here we show that CaBLAM ('calcium bioluminescence activity monitor'), an engineered bioluminescent calcium indicator, achieves an order-of-magnitude improvement in signal contrast and a tunable affinity matched to physiological cytosolic calcium. CaBLAM enables single-cell and subcellular activity imaging at video frame rates in cultured neurons and sustained imaging over hours in awake, behaving animals. These capabilities establish CaBLAM as a robust and general alternative to fluorescent GECIs, extending calcium imaging to regimes where excitation light is undesirable or infeasible.

Precision-targeted ultrasonic neuromodulation offers immense potential for studying brain function and treating neurological diseases. Yet, its application has been limited by challenges in achieving precise spatio-temporal control and monitoring of ultrasound effects on brain circuits. Here we show that transcranial ultrasound elicits direct and highly focal responses, which can be dynamically steered at spatio-temporal scales relevant for neural function. Furthermore, holographic transcranial ultrasound stimulation allows direct control of the stimulated volume and actively modulates local and mid-range network projections, effectively lowering the activation threshold by an order of magnitude. To better understand this previously unexplored excitability regime not fully explained by the conventional pressure-frequency dyad, we developed a dual modelling framework, where both an empirical and a mechanistic model were constructed to capture the intricacies of holographic transcranial ultrasound stimulation. These models achieve qualitative agreement with our experimental results, suggesting that these findings are predominantly driven by putative network interactions. Our results bring insight on the complex interaction mechanisms of ultrasound with neural tissue and highlight its potential for the noninvasive interfacing of distributed brain networks.

Glaucoma is a chronic neurodegenerative disease of the visual system, and treatment is targeted toward lowering intraocular pressure. However, some patients fail to respond to treatment and their intraocular pressure levels remain high, risking continuous vision loss. Explainable machine learning provides a mechanism for both individual prognostication and the identification of factors associated with treatment outcome. Here, we used explainable machine learning to predict intraocular pressure for glaucoma patients receiving medication treatment. We accessed the UK Biobank to obtain information on 290 eyes from 161 participants who reported a diagnosis of glaucoma and were receiving treatment. Features were divided into three distinct datasets containing demographic data only, physiometabolic parameters and medication prescription data, and all data combined. We evaluated five machine learning techniques for each feature set in terms of their ability to predict intraocular pressure at a follow-up visit in a classification task. We then calculated SHapley Additive exPlanation (SHAP) values for the best performing model to determine feature importance, stability, and interactions. We found that eXtreme Gradient Boosting (XGBoost) outperformed all other models when trained and tested on the combined feature set with an area under receiver operating characteristic curve (AUC) of 0.708. Insulin-like growth factor 1 (IGF-1), low-density lipoprotein (LDL), and lymphocyte count ranked as the three most important features for this model. LDL and IGF-1 exhibited a low degree of global variability in contribution to the model output across all cross-validation repeats. SHAP values demonstrated the strongest interactions being between LDL and IGF-1. In summary, our studies indicated the importance of blood LDL and IGF-1 in contributing to the outcomes of intraocular pressure lowering treatment and demonstrated the ability of XGBoost to predict these outcomes.