Browsing by Author "Alex, Alpha"

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    Automated Computer-Based Enumeration of Acellular Capillaries for Assessment of Diabetic Retinopathy
    (SPIE, 2020-02) Tuceryan, Mihran; Hemmady, Anish N.; Schebler, Craig; Alex, Alpha; Bhatwadekar, Ashay D.; Computer and Information Science, School of Science
    Diabetic retinopathy (DR) is the most common complications of diabetes; if untreated the DR can lead to a vision loss. The treatment options for DR are limited and the development of newer therapies are of considerable interest. Drug screening for the retinopathy treatment is undertaken using animal models in which the quantification of acellular capillaries (capillary without any cells) is used as a marker to assess the severity of retinopathy and the treatment response. The traditional approach to quantitate acellular capillaries is through manual counting. The purpose of this investigation was to develop an automated technique for the quantitation of acellular capillaries using computer-based image processing algorithms. We developed a custom procedure using the Python, the medial axis transform (MAT) and the connected component algorithm. The program was tested on the retinas of wild-type and diabetic mice and the results were compared to single blind manual counts by two independent investigators. The program successfully identified and enumerated acellular capillaries. The acellular capillary counts were comparable to the traditional manual counting. In conclusion, we developed an automated computer-based program, which can be effectively used for future pharmacological development of treatments for DR. This algorithm will enhance consistency in retinopathy assessment and reduce the time for analysis, thus, contributing substantially towards the development of future pharmacological agents for the treatment of DR.
  • Thumbnail Image
    Item
    Conditional Deletion of Bmal1 Accentuates Microvascular and Macrovascular Injury
    (Elsevier, 2017-06) Bhatwadekar, Ashay D.; Beli, Eleni; Diao, Yanpeng; Chen, Jonathan; Luo, Qianyi; Alex, Alpha; Caballero, Sergio; Dominguez, James M., II; Salazar, Tatiana E.; Busik, Julia V.; Segal, Mark S.; Grant, Maria B.; Ophthalmology, School of Medicine
    The brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (BMAL)-1 constitutes a major transcriptional regulator of the circadian clock. Here, we explored the impact of conditional deletion of Bmal1 in endothelium and hematopoietic cells in murine models of microvascular and macrovascular injury. We used two models of Bmal1fx/fx;Tek-Cre mice, a retinal ischemia/reperfusion model and a neointimal hyperplasia model of the femoral artery. Eyes were enumerated for acellular capillaries and were stained for oxidative damage markers using nitrotyrosine immunohistochemistry. LSK (lineage-negative, stem cell antigen-1-positive, c-Kit-positive) cells were quantified and proliferation assessed. Hematopoiesis is influenced by innervation to the bone marrow, which we assessed using IHC analysis. The number of acellular capillaries increased threefold, and nitrotyrosine staining increased 1.5-fold, in the retinas of Bmal1fx/fx;Tek-Cre mice. The number of LSK cells from the Bmal1fx/fx;Tek-Cre mice decreased by 1.5-fold and was accompanied by a profound decrease in proliferative potential. Bmal1fx/fx;Tek-Cre mice also exhibited evidence of bone marrow denervation, demonstrating a loss of neurofilament-200 staining. Injured femoral arteries showed a 20% increase in neointimal hyperplasia compared with similarly injured wild-type controls. Our study highlights the importance of the circadian clock in maintaining vascular homeostasis and demonstrates that specific deletion of BMAL1 in endothelial and hematopoietic cells results in phenotypic features similar to those of diabetes.
  • Thumbnail Image
    Item
    The Diurnal Rhythm of Insulin Receptor Substrate-1 (IRS-1) and Kir4.1 in Diabetes: Implications for a Clock Gene Bmal1
    (ARVO, 2019-05) Luo, Qianyi; Xiao, Yucheng; Alex, Alpha; Cummins, Theodore R.; Bhatwadekar, Ashay D.; Ophthalmology, School of Medicine
    Purpose: Diabetes leads to the downregulation of the retinal Kir4.1 channels and Müller cell dysfunction. The insulin receptor substrate-1 (IRS-1) is a critical regulator of insulin signaling in Müller cells. Circadian rhythms play an integral role in normal physiology; however, diabetes leads to a circadian dysrhythmia. We hypothesize that diabetes will result in a circadian dysrhythmia of IRS-1 and Kir4.1 and disturbed clock gene function will have a critical role in regulating Kir4.1 channels. Methods: We assessed a diurnal rhythm of retinal IRS-1 and Kir4.1 in db/db mice. The Kir4.1 function was evaluated using a whole-cell recording of Müller cells. The rat Müller cells (rMC-1) were used to undertake in vitro studies using a siRNA. Results: The IRS-1 exhibited a diurnal rhythm in control mice; however, with diabetes, this natural rhythm was lost. The Kir4.1 levels peaked and troughed at times similar to the IRS-1 rhythm. The IRS-1 silencing in the rMC-1 led to a decrease in Kir4.1 and BMAL1. The insulin treatment of retinal explants upregulated Kir4.1 possibly via upregulation of BMAL1 and phosphorylation of IRS-1 and Akt-1. Conclusions: Our studies highlight that IRS-1, by regulating BMAL1, is an important regulator of Kir4.1 in Müller cells and the dysfunctional signaling mediated by IRS-1 may be detrimental to Kir4.1.
  • Thumbnail Image
    Item
    Metformin Corrects Abnormal Circadian Rhythm and Kir4.1 Channels in Diabetes
    (The Association for Research in Vision and Ophthalmology, 2020-06-22) Alex, Alpha; Luo, Qianyi; Mathew, Deepa; Di, Rong; Bhatwadekar, Ashay D.; Ophthalmology, School of Medicine
    Purpose Diabetic retinopathy (DR) is a leading cause of visual impairment. Müller cells in DR are dysfunctional due to downregulation of the inwardly rectifying potassium channel Kir4.1. Metformin, a commonly used oral antidiabetic drug, is known to elicit its action through 5′ adenosine monophosphate-activated protein kinase (AMPK), a cellular metabolic regulator; however, its effect on Kir4.1 channels is unknown. For this study, we hypothesized that metformin treatment would correct circadian rhythm disruption and Kir4.1 channel dysfunction in db/db mice. Methods Metformin was given orally to db/db mice. Wheel-running activity, retinal levels of Kir4.1, and AMPK phosphorylation were determined at study termination. In parallel, rat retinal Müller cell line (rMC-1) cells were treated using metformin and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to assess the effect of AMPK activation on the Kir4.1 channel. Results The wheel-running activity of the db/db mice was improved following the metformin treatment. The Kir4.1 level in Müller cells was corrected after metformin treatment. Metformin treatment led to an upregulation of clock regulatory genes such as melanopsin (Opn4) and aralkylamine N-acetyltransferase (Aanat). In rMC-1 cells, AMPK activation via AICAR and metformin resulted in increased Kir4.1 and intermediate core clock component Bmal-1 protein expression. The silencing of Prkaa1 (gene for AMPKα1) led to decreased Kir4.1 and Bmal-1 protein expression. Conclusions Our findings demonstrate that metformin corrects abnormal circadian rhythm and Kir4.1 channels in db/db mouse a model of type 2 diabetes. Metformin could represent a critical pharmacological agent for preventing Müller cell dysfunction observed in human DR.