Can Drug Screening Lead to Candidate Therapies for Testing in Diabetic Neuropathy?

A key mechanism of dorsal root ganglia (DRG) neuron injury in high glucose is mitochondrial overload leading to oxidative stress. We screened selected compounds for the ability to prevent hyperglycemia-induced mitochondrial superoxide in primary sensory DRG neurons. Twenty five out of 1,040 compounds decreased both mitochondrial superoxide and subsequent neuronal injury. These data both validate our screening strategy and indicate further mechanistic evaluation of drug hits and related compounds. Such studies may lead to the design of rational therapeutic approaches for this severe complication of diabetes.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63408/1/ars.2007.1815.pd

Design of an RSFQ Control Circuit to Observe MQC on an rf-SQUID

We believe that the best chance to observe macroscopic quantum coherence (MQC) in a rf-SQUID qubit is to use on-chip RSFQ digital circuits for preparing, evolving and reading out the qubit's quantum state. This approach allows experiments to be conducted on a very short time scale (sub-nanosecond) without the use of large bandwidth control lines that would couple environmental degrees of freedom to the qubit thus contributing to its decoherence. In this paper we present our design of a RSFQ digital control circuit for demonstrating MQC in a rf-SQUID. We assess some of the key practical issues in the circuit design including the achievement of the necessary flux bias stability. We present an "active" isolation structure to be used to increase coherence times. The structure decouples the SQUID from external degrees of freedom, and then couples it to the output measurement circuitry when required, all under the active control of RSFQ circuits. Research supported in part by ARO grant # DAAG55-98-1-0367.Comment: 4 pages. More information and publications at http://www.ece.rochester.edu:8080/users/sde/research/publications/index.htm

Hyperlipidemia: a new therapeutic target for diabetic neuropathy

Abstract Emerging data establish dyslipidemia as a significant contributor to the development of diabetic neuropathy. In this review, we discuss how separate metabolic imbalances, including hyperglycemia and hyperlipidemia, converge on mechanisms leading to oxidative stress in dorsal root ganglia (DRG) sensory neurons. We conclude with suggestions for novel therapeutic strategies to prevent or reverse diabetes-induced nerve degeneration.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78728/1/j.1529-8027.2009.00237.x.pd

Short‐term hyperglycemia produces oxidative damage and apoptosis in neurons

Dorsal root ganglia neurons in culture die through programmed cell death when exposed to elevated glucose, providing an in vitro model system for the investigation of the mechanisms leading to diabetic neuropathy. This study examines the time course of programmed cell death induction, regulation of cellular antioxidant capacity, and the protective effects of antioxidants in neurons exposed to hyperglycemia. We demonstrate that the first 2 h of hyperglycemia are sufficient to induce oxidative stress and programmed cell death. Using fluorimetric analysis of reactive oxygen species (ROS) production, in vitro assays of antioxidant enzymes, and immunocytochemical assays of cell death, we demonstrate superoxide formation, inhibition of aconitase, and lipid peroxidation within 1 h of hyperglycemia. These are followed by caspase‐3 activation and DNA fragmentation. Antioxidant potential increases by 3–6 h but is insufficient to protect these neurons. Application of the antioxidant α‐lipoic acid potently prevents glucose‐induced oxidative stress and cell death. This study identifies cellular therapeutic targets to prevent diabetic neuropathy. Since oxidative stress is a common feature of the micro‐ and macrovascular complications of diabetes, the present findings have broad application to the treatment of diabetic patients.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154304/1/fsb2fj042513fje.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154304/2/fsb2fj042513fje-sup-0001.pd

EFFECTS OF METHOTREXATE ON PROLIFERATION OF HUMAN KERATINOCYTES IN VITRO

Normal human keratinocytes, propagated as epithelial outgrowths in vitro, were exposed to different concentrations of methotrexate (MTX) for different periods of time. After a 1-hr exposure, DNA synthesis was inhibited in a reversible manner. No change in the mitotic index was observed. After a 6-hr exposure, both DNA synthesis and mitosis were inhibited, again in a reversible fashion. Prolonged exposure (24 hr) resulted in irreversible mitotic inhibition even when followed by recovery periods of 168 hr. The effective concentrations of MTX in vitro were similar to those described previously in vivo

New Insights into the Mechanisms of Diabetic Neuropathy

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47865/1/11154_2004_Article_5273164.pd

Cell Culture Modeling to Test Therapies Against Hyperglycemia-Mediated Oxidative Stress and Injury

The concept that oxidative stress is a key mediator of nerve injury in diabetes has led us to design therapies that target oxidative stress mechanisms. Using an in vitro model of glucose-treated dorsal root ganglion (DRG) neurons in culture, we can examine both free radical generation, using fluorimetric probes for reactive oxygen species, and cell death via the TUNEL assay. The cell culture system is scaled down to a 96-well plate format, and so is well suited to high-throughput screening. In the present study, we test the ability of three drugs, nicotinamide, allopurinol, and α-lipoic acid, alone and in combination to prevent DRG neuron oxidative stress and cell death. This combination of drugs is currently in clinical trial in type 1 diabetic patients. We demonstrate independent effects on oxidative stress and neuronal survival for the three drugs, and neuronal protection using the three drugs in combination. The data strengthen the rationale for the current clinical trial. In addition, we describe an effective tool for rapid preclinical testing of novel therapies against diabetic neuropathy. Antioxid. Redox Signal. 7, 1494–1506.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63115/1/ars.2005.7.1494.pd

High glucose‐induced oxidative stress and mitochondrial dysfunction in neurons

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154474/1/fsb2fasebj16131027com.pd

The role of endomyocardial biopsy in the management of cardiovascular disease: a scientific statement from the American Heart Association, the American College of Cardiology, and the European Society of Cardiology

The role of endomyocardial biopsy (EMB) in the diagnosis and treatment of adult and pediatric cardiovascular disease remains controversial, and the practice varies widely even among cardiovascular centers of excellence. A need for EMB exists because specific myocardial disorders that have unique prognoses and treatment are seldom diagnosed by noninvasive testing.1 Informed clinical decision making that weighs the risks of EMB against the incremental diagnostic, prognostic, and therapeutic value of the procedure is especially challenging for nonspecialists because the relevant published literature is usually cited according to specific cardiac diseases, which are only diagnosed after EM

Sensory Neurons and Schwann Cells Respond to Oxidative Stress by Increasing Antioxidant Defense Mechanisms

Abstract Elevated blood glucose is a key initiator of mechanisms leading to diabetic neuropathy. Increases in glucose induce acute mitochondrial oxidative stress in dorsal root ganglion (DRG) neurons, the sensory neurons normally affected in diabetic neuropathy, whereas Schwann cells are largely unaffected. We propose that activation of an antioxidant response in DRG neurons would prevent glucose-induced injury. In this study, mild oxidative stress (1 μM H2O2) leads to the activation of the transcription factor Nrf2 and expression of antioxidant (phase II) enzymes. DRG neurons are thus protected from subsequent hyperglycemia-induced injury, as determined by activation of caspase 3 and the TUNEL assay. Schwann cells display high basal antioxidant enzyme expression and respond to hyperglycemia and mild oxidative stress via further increases in these enzymes. The botanical compounds resveratrol and sulforaphane activate the antioxidant response in DRG neurons. Other drugs that protect DRG neurons and block mitochondrial superoxide, identified in a compound screen, have differential ability to activate the antioxidant response. Multiple cellular targets exist for the prevention of hyperglycemic oxidative stress in DRG neurons, and these form the basis for new therapeutic strategies against diabetic neuropathy. Antioxid. Redox Signal. 11, 425-438.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78129/1/ars.2008.2235.pd