MEASUREMENT OF INTRARETINAL NITRIC OXIDE IN EARLY DIABETIC RETINOPATHY

Diabetic retinopathy (DR) is the most frequent cause of new cases of blindness among adults aged 20-74 years. Nearly all patients with Type 1 diabetes and greater than 60% of patients with Type 2 diabetes will develop retinopathy within the rst two decades of the disease. Nitric oxide (NO) has been shown to play a role in the progression of DR, contributing to neuronal dysfunction and the breakdown of the blood-retina barrier early stages of the disease. The objective of the current study was to investigate the changes in intraretinal NO levels in early DR. To accomplish this, a dual NO/electroretinogram (ERG) electrode was developed to make the rst direct measurements of NO concentration throughout the in vivo retina. These electrodes were validated in an in vivo animal model by comparing control recordings to those taken after injection of the broad spectrum nitric oxide synthase (NOS) inhibitor L-NG-Nitroarginine methyl ester (L-NAME). Control NO pro les showed high levels of NO in the photoreceptor layer with localized areas of increased NO in the amacrine/ganglion cell layer. L-NAME NO pro les showed substantially reduced NO in the retina, indicating that the electrodes were measuring actual NO. The electrodes were then used to record NO pro les from the retinas of rats made diabetic with streptozotocin (STZ). The recordings were obtained three weeks after injection of STZ. Blood glucose levels were also monitored in order to correlate the blood glucose level with intraretinal NO concentration. It was found that mild diabetic rats (blood glucose 250-400 mg/dL) had higherthan- control levels of NO throughout their retinas. Severe diabetics (500-600 mg/dL) had lower-than-control levels, while moderate diabetics (400-500 mg/dL) did not have signi cantly di erent NO levels than controls. The NO pro les from the severe diabetics were very similar to L-NAME pro les, indicating that NOS production may be abnormal in severe diabetics. It was also found that intraretinal NO concentration was inversely correlated with the blood glucose of diabetic rats. To determine if the changes in NO seen in diabetic rats were due to direct tissue exposure to high glucose, NO pro les were also recorded from rats acutely injected with glucose solution to achieve similar levels of hyperglycemia. No changes in NO levels were seen in the retinas of these acute hyperglycemic rats, indicating that there are other factors besides high glucose contributing to the NO changes in DR. The results show that there is not a simple increase in NO as severity of diabetes increases and highlight the importance of being able to make measurements of bioavailable NO in retinal tissue. The electrodes were able to detect clear di erences in experimental DR, indicating their utility in investigating NO changes in the early stages of the disease. Future work with the electrodes needs to be performed to investigate the mechanisms of NO changes in DR in order to develop potential treatments which could mitigate the damage at an early stage before vision loss occurs.M.S. in Biomedical Engineering, December 201