IU Indianapolis ScholarWorks :: Browsing by Subject "retina"

Browsing by Subject "retina"

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Analysis of retinal ganglion cell development: from stem cells to synapses
(2018) Ohlemacher, Sarah K.; Meyer, Jason S.
Human pluripotent stem cells (hPSCs) have the ability to self renew indeﬁnitely while maintaining their pluripotency, allowing for the study of virtually any human cell type in a dish. The focus of the current study was the diﬀerentiation of hPSCs to retinal ganglion cells (RGCs), the primary cell type aﬀected in optic neuropathies. hPSCs were induced to become retinal cells using a stepwise diﬀerentiation protocol that allowed for formation of optic vesicle (OV)-like structures. Enrichment of OV like structures allowed for the deﬁnitive identiﬁcation of RGCs. RGCs displayed the proper temporal, spatial, and phenotypic characteristics of RGCs developing in vivo. To test the ability of hPSC-RGCs to serve as a disease model, lines were generated from a patient with an E50K mutation in the Optineurin gene, causative for normal tension primary open angle glaucoma. E50K RGCs displayed signiﬁcantly higher levels of apoptosis compared to a control lines. Apoptosis was reduced with exposure to neuroprotective factors. Lastly, hPSC-derived RGCs were studied for their ability to develop functional features possessed by mature in vivo RGCs. hPSC-derived RGCs displayed a few immature functional features and as such, strategies in which to expedite synaptogenesis using hPSC-derived astrocytes were explored. Astrocyte and RGG co-cultures displayed expedited synaptic and functional maturation, more closely resembling mature in vivo RGCs. Taken together, the results of this study have important implications for the study of RGC development and by extension, the advancement of translational therapies for optic neuropathies.
Blood circulation and aqueous humor flow in the eye : multi-scale modeling and clinical applications
(2016-06-14) Cassani, Simone; Guidoboni, Giovanna; Arciero, Julia Concetta; Harris, Alon
Glaucoma is a multi-factorial ocular disease associated with death of retinal ganglion cells and irreversible vision loss. Many risk factors contribute to glaucomatous damage, including elevated intraocular pressure (IOP), age, genetics, and other diseases such as diabetes and systemic hypertension. Interestingly, alterations in retinal hemodynamics have also been associated with glaucoma. A better understanding of the factors that contribute to these hemodynamic alterations could lead to improved and more appropriate clinical approaches to manage and hopefully treat glaucoma patients. In this thesis, we develop several mathematical models aimed at describing ocular hemodynamics and oxygenation in health and disease. Precisely we describe: (i) a time-dependent mathematical model for the retinal circulation that includes macrocirculation, microcirculation, phenomenological vascular regulation, and the mechanical effect of IOP on the retinal vasculature; (ii) a steady-state mathematical model for the retinal circulation that includes macrocirculation, microcirculation, mechanistic vascular regulation, the effect of IOP on the central retinal artery and central retinal vein, and the transport of oxygen in the retinal tissue using a Krogh cylinder type model; (iii) a steady-state mathematical model for the transport of oxygen in the retinal microcirculation and tissue based on a realistic retinal anatomy; and (iv) a steady-state mathematical model for the production and drainage of aqueous humor (AH). The main objective of this work is to study the relationship between IOP, systemic blood pressure, and the functionality of vascular autoregulation; the transport and exchange of oxygen in the retinal vasculature and tissue; and the production and drainage of AH, that contributes to the level of IOP. The models developed in this thesis predict that (i) the autoregulation plateau occurs for different values of IOP in hypertensive and normotensive patients. Thus, the level of blood pressure and functionality of autoregulation affect the changes in retinal hemodynamics caused by IOP and might explain the inconsistent outcomes of clinical studies; (ii) the metabolic and carbon dioxide mechanisms play a major role in the vascular regulation of the retina. Thus, the impairment of either of these mechanisms could cause ischemic damage to the retinal tissue; (iii) the multi-layer description of transport of oxygen in the retinal tissue accounts for the effect of the inner and outer retina, thereby improving the predictive ability of the model; (iv) a greater reduction in IOP is obtained if topical medications target AH production rather that AH drainage and if IOP-lowering medications are administrated to patients that exhibit a high initial level of IOP. Thus, the effectiveness of IOP-lowering medications depend on a patient’s value of IOP. In conclusion, the results of this thesis demonstrate that the insight provided by mathematical modeling alongside clinical studies can improve the understanding of diseases and potentially contribute to the clinical development of new treatments.
BMP Pathway and Reactive Retinal Gliosis
(2013-03-06) Dharmarajan, Subramanian; Belecky-Adams, Teri; Skalnik, David Gordon; Zhang, Xin; Atkinson, Simon
Reactive gliosis is known to have a beneficial and a degenerative effect following injury to neurons. Although many factors have been implicated in reactive gliosis, their role in regulating this change is still unclear. We investigated the role of bone morphogenetic proteins in reactive gliosis in vivo and in vitro. In vivo, IHC analysis indicated reactive gliosis in the 6 week Ins2Akita mouse and WPK rat retinas. Expression of BMP7 was upregulated in these models, leading to an increase in the phosphorylation of downstream SMAD1. In vitro, treatment of murine retinal astrocyte cells with a strong oxidizing agent such as sodium peroxynitrite regulated RNA levels of various markers, including GFAP, CSPGs, MMPs and TIMPs. BMP7 treatment also regulated RNA levels to a similar extent, suggesting reactive gliosis. Treatment with high glucose DMEM and BMP4, however, did not elicit increase in levels to a similar degree. Increase in SMAD levels and downstream targets of SMAD signaling such as ID1, ID3 and MSX2 was also observed following treatment with sodium peroxynitrite in vitro and in the 6 week Ins2Akita mouse retinas in vivo. These data concur with previously established data which show an increase in BMP7 levels following injury. It also demonstrates a role for BMP7 in gliosis following disease. Further, it suggests SMAD signaling to play a role in initiating reactivity in astrocytes as well as in remodeling the extracellular matrix following injury and in a disease condition.
Circadian Rhythm Disruption Results in Visual Dysfunction
(Cold Spring Harbor Laboratory Press, 2020) Mathew, Deepa; Bhatwadekar, Ashay D.; Ophthalmology, School of Medicine
Circadian rhythm disruption (CRD) contributes to the development of multiple metabolic and neurodegenerative diseases. However, its effect on vision is not understood. We evaluated the impact of CRD on retinal morphology, physiology, and vision after housing mice in a disruption inducing shorter light/dark cycle (L10:D10). Interestingly, the mice under L10:D10 exhibited three different entrainment behaviors; ‘entrained’, ‘freerunning’, and ‘zigzagging.’ These behavior groups under CRD exhibited reduced visual acuity, retinal thinning, and a decrease in the number of rod photoreceptors. Intriguingly, the electroretinogram response was decreased only in the mice exhibiting ‘entrained’ behavior. The retinal proteome showed distinct changes with each entrainment behavior. These results demonstrate that CRD leads to photoreceptor degeneration and visual dysfunction. We uniquely show the effect of entrainment behavior on retinal protein composition and physiology. Our data has broader implications in understanding and mitigating the effect of CRD on vision health.
Computer-based Quantification of Acellular Capillaries to Assess Experimental Diabetic Retinopathy
(Office of the Vice Chancellor for Research, 2016-04-08) Hemmady, Anish; Tuceryan, Mihran; Bhatwadekar, Ashay
Diabetic retinopathy (DR) is a disease of small blood vessels in the retina. The increase in the number of acellular capillaries is used as a marker to assess the severity of DR. The traditional approach for identifying acellular capillaries is manual counting of the capillaries either directly under the microscope or using the captured images. However, these methods are cumbersome and often involve inconsistencies among researchers. The purpose of this study is to reduce discrepancies in the enumeration of acellular capillaries using computer-based image processing algorithms. The retinas of control and diabetic mice were processed using trypsin digestion. The high resolution png format images of retinal quadrants were prepared from the trypsin digested retina. The computer programming was performed using the Python language along with open source packages such as OpenCv, Python Imaging Library (PIL), NumPy (Numerical Python) and SciPy. The images initially corrected for a Gaussian Blur and a Median blur to remove noise followed by the histogram based image segmentation. After image segmentation, a binary image was generated based on a histogram analysis. The segmentation threshold for binary image was determined and the medial axis transform (MAT) algorithm was applied to the binary image. The MAT representation was used to skeletonize the blood vessels and to detect branches and branch-points in those blood vessels. As part of the MAT computation, the distances from the skeleton to the vessel boundaries are encoded. The thin capilleries, i.e., acellular capilleries, were identified using a threshold on this distance which encodes the thickness of the vessel. Finally, acellular capillaries were counted by connected component algorithm. In conclusion, we have designed an automated computer-based system to enumerate the acellular capillaries. This computer-based automated system will help to maintain consistency in retinopathy assessment and may reduce time for analysis.
Differentiation and Three-dimensional Organization of Retinal Ganglion Cells using Human Induced Pluripotent Stem Cells
(Office of the Vice Chancellor for Research, 2015-04-17) Ho-A-Lim, Kimberly T.; Ohlemacher, Sarah K.; Meyer, Jason S.
Retinal Ganglion Cells (RGCs) are a type of neuron which function to relay visual messages between the retina and brain, and are characterized by their long axons which form part of the optic nerve. Dysfunction in this communication pathway is highly implicated in degenerative blinding disorders such as glaucoma. Unique applications using human induced pluripotent stem cells (hiPSCs) offer the ability to model human diseases, and potentially develop novel therapeutic approaches to rescue or replace damaged cells. In order to better understand the progression of degenerative eye diseases, a remaining challenge is to precisely identify the sequence of events which contribute to the diseased state, and how their features differ from non-diseased cells. Efforts were therefore undertaken to visually document the maturation of RGCs by analyzing their morphology and three-dimension organization at varying stages of development. Induced retinal cells were harvested at six different stages of development and fixed in 4% paraformaldehyde (PFA) solution to arrest their development. Cells were then cryoprotected in combinations of sucrose and Optimal Cutting Temperature (OCT) solutions, and frozen using powered dry ice. Following cryostat sectioning, samples were subject to immunocytochemistry staining to visualize for retinal-like organization of cells. Preliminary results have indicated the presence of the RGC marker Brn3, as well as markers for other retinal cell types. Future tests intend to characterize these retinal cell types according to their morphology and three-dimensional organization.
The Direct Reprogramming of Somatic Cells: Establishment of a Novel System for Photoreceptor Derivation
(2013-08-22) Steward, Melissa Mary; Meyer, Jason S.; Dai, Guoli; Randall, Stephen Karl, 1953-; Atkinson, Simon
Photoreceptors are a class of sensory neuronal cells that are deleteriously affected in many disorders and injuries of the visual system. Significant injury or loss of these cells often results in a partial or complete loss of vision. While previous studies have determined many necessary components of the gene regulatory network governing the establishment, development, and maintenance of these cells, the necessary and sufficient profile and timecourse of gene expression and/or silencing has yet to be elucidated. Arduous protocols do exist to derive photoreceptors in vitro utilizing pluripotent stem cells, but only recently have been able to yield cells that are disease- and/or patient-specific. The discovery that mammalian somatic cells can be directly reprogrammed to another terminally-differentiated cell phenotype has inspired an explosion of research demonstrating the successful genetic reprogramming of one cell type to another, a process which is typically both more timely and efficient than those used to derive the same cells from pluripotent stem cell sources. Therefore, the emphasis of this study was to establish a novel system to be used to determine a minimal transcriptional network capable of directly reprogramming mouse embryonic fibroblasts (MEFs) to rod photoreceptors. The tools, assays, and experimental design chosen and established herein were designed and characterized to facilitate this determination, and preliminary data demonstrated the utility of this approach for accomplishing this aim.
Ferrochelatase regulates retinal neovascularization
(Wiley, 2020) Pran Babu, Sardar Pasha Sheik; White, Darcy; Corson, Timothy W.; Ophthalmology, School of Medicine
Ferrochelatase (FECH) is the terminal enzyme in heme biosynthesis. We previously showed that FECH is required for endothelial cell growth in vitro and choroidal neovascularization in vivo. But FECH has not been explored in retinal neovascularization, which underlies diseases like proliferative diabetic retinopathy and retinopathy of prematurity. Here, we investigated the inhibition of FECH using genetic and chemical approaches in the oxygen-induced retinopathy (OIR) mouse model. In OIR mice, FECH expression is upregulated and co-localized with neovascular tufts. Partial loss-of-function Fechm1Pas mutant mice showed reduced retinal neovascularization and endothelial cell proliferation in OIR. An intravitreal injection of the FECH inhibitor N-methyl protoporphyrin had similar effects. Griseofulvin is an antifungal drug that inhibits FECH as an off-target effect. Strikingly, intravitreal griseofulvin decreased both pathological tuft formation and areas of vasoobliteration compared to vehicle, suggesting potential as a FECH-targeting therapy. Ocular toxicity studies revealed that intravitreal injection of griseofulvin in adult mice does not disrupt retinal vasculature, function, or morphology. In sum, mutation and chemical inhibition of Fech reduces retinal neovascularization and promotes physiological angiogenesis, suggesting a dual effect on vascular repair upon FECH inhibition, without ocular toxicity. These findings suggest that FECH inhibitors could be repurposed to treat retinal neovascularization.
Imaging and quantifying ganglion cells and other transparent neurons in the living human retina
(National Academy of Sciences, 2017-11-28) Liu, Zhuolin; Kurokawa, Kazuhiro; Zhang, Furu; Lee, John J.; Miller, Donald T.; Engineering Technology, School of Engineering and Technology
Ganglion cells are the primary building block of retinal neural circuitry, but have been elusive to observe and quantify in the living human eye. Here, we show a light microscopy modality that reveals not only the somas of these cells, but also their 3D packing geometry, primary subtypes, and spatial projection to other neurons. The method provides a glimpse of the rich tapestry of neurons, glia, and blood vessels that compose the retina, thus exposing the anatomical substrate for neural processing of visual information. Clinically, high-resolution images of retinal neurons in living eyes hold promise for improved diagnosis and assessing treatment of ganglion cell and other neuron loss in retinal disease., Ganglion cells (GCs) are fundamental to retinal neural circuitry, processing photoreceptor signals for transmission to the brain via their axons. However, much remains unknown about their role in vision and their vulnerability to disease leading to blindness. A major bottleneck has been our inability to observe GCs and their degeneration in the living human eye. Despite two decades of development of optical technologies to image cells in the living human retina, GCs remain elusive due to their high optical translucency. Failure of conventional imaging—using predominately singly scattered light—to reveal GCs has led to a focus on multiply-scattered, fluorescence, two-photon, and phase imaging techniques to enhance GC contrast. Here, we show that singly scattered light actually carries substantial information that reveals GC somas, axons, and other retinal neurons and permits their quantitative analysis. We perform morphometry on GC layer somas, including projection of GCs onto photoreceptors and identification of the primary GC subtypes, even beneath nerve fibers. We obtained singly scattered images by: (i) marrying adaptive optics to optical coherence tomography to avoid optical blurring of the eye; (ii) performing 3D subcellular image registration to avoid motion blur; and (iii) using organelle motility inside somas as an intrinsic contrast agent. Moreover, through-focus imaging offers the potential to spatially map individual GCs to underlying amacrine, bipolar, horizontal, photoreceptor, and retinal pigment epithelium cells, thus exposing the anatomical substrate for neural processing of visual information. This imaging modality is also a tool for improving clinical diagnosis and assessing treatment of retinal disease.
Predicting retinal tissue oxygenation using an image-based theoretical model
(Elsevier, 2018-11) Fry, Brendan C.; Coburn, Ehren Brant; Whiteman, Spencer; Harris, Alon; Siesky, Brent; Arciero, Julia; Mathematical Sciences, School of Science
Impaired oxygen delivery and tissue perfusion have been identified as significant factors that contribute to the loss of retinal ganglion cells in glaucoma patients. This study predicts retinal blood and tissue oxygenation using a theoretical model of the retinal vasculature based on confocal microscopy images of the mouse retina. These images reveal a complex and heterogeneous geometry of vessels that are distributed non-uniformly into multiple distinct retinal layers at varying depths. Predicting oxygen delivery and distribution in this irregular arrangement of retinal microvessels requires the use of an efficient theoretical model. The model employed in this work utilizes numerical methods based on a Green's function approach to simulate the spatial distribution of oxygen levels in a network of retinal blood vessels and the tissue surrounding them. Model simulations also predict the blood flow rates and pressures in each of the microvessels throughout the entire network. As expected, the model predicts that average vessel PO2 decreases as oxygen demand is increased. However, the standard deviation of PO2 in the vessels nearly doubles as oxygen demand is increased from 1 to 8 cm3 O2/100 cm3/min, indicating a very wide spread in the predicted PO2 levels, suggesting that average PO2 is not a sufficient indicator of oxygenation in a heterogeneous vascular network. Ultimately, the development of this mathematical model will help to elucidate the important factors associated with blood flow and metabolism that contribute to the vision loss characteristic of glaucoma.

Browsing by Subject "retina"

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