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 alpha-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.
Vincent
Andrea M
AM
Department of Neurology, University of Michigan, Ann Arbor, MI 48109, USA. andreav@umich.edu
Stevens
Martin J
MJ
Backus
Carey
C
McLean
Lisa L
LL
Feldman
Eva L
EL
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D018929:Cell Culture Techniques; D016923:Cell Death; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005727:Ganglia, Spinal / Q000166:cytology / Q000187:drug effects / Q000293:injuries; D005947:Glucose / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006943:Hyperglycemia / Q000378:metabolism* / Q000473:pathology* / Q000517:prevention & control; D015227:Lipid Peroxidation; D008954:Models, Biological; D009474:Neurons / Q000166:cytology / Q000187:drug effects; D009536:Niacinamide / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D008063:Thioctic Acid / Q000494:pharmacology
D000975:Antioxidants; D017382:Reactive Oxygen Species; D009536:Niacinamide; D008063:Thioctic Acid; D006861:Hydrogen Peroxide; D005947:Glucose