The overall objective of this project is to determine the mechanism(s) underlying the development of painful neuropathy in diabetes. The overall hypothesis is that painful diabetic neuropathy results from sensitization both of the primary afferent neurons and of the spinothalamic neurons that conduct the pain signal. The notion that hyperglycemia causes sensitization of the primary afferent neurons will be tested in the first aim. Sensitization will be measured as an increase in the release of transmitters (substance P, CGRP, glutamate) from the central terminals of primary afferents from streptozotocin treated rats compared with vehicle treated controls by stimulation of spinal cord slices with capsaicin. The ability of hyperglycemia to induce sensitization in vitro will be determined by culturing primary sensory neurons in the presence of high glucose vs normal glucose and testing the capsaicin-stimulated release of transmitters. Subsequent experiments will determine if the mechanism for the sensitization in vitro is a hyperglycemia-induced increase in DAG which in turn activates PKC to increase transmitter release. The ability of gabapentin to reverse the sensitization induced in vivo by hyperglycemia or in vitro by high glucose will be tested. In the second aim the hypothesis that hyperglycemia sensitizes the spinothalamic neurons by increasing their receptors for the transmitters released by the primary afferent neurons will be tested in STZ vs Vehicle treated rats by quantitative autoradiography and immunohistochemistry on slices of spinal cord. Another mechanism for the sensitization of the spinothalamic neurons, that they respond more to stimulation by substance P and glutamate, will be tested on the spinothalamic neurons grown in culture. These neurons will be grown in high or normal glucose and the levels of DAG and IP3 will be measured. It is hypothesized that hyperglycemia causes an increase in DAG in these neurons also and that SP/glutamate cause a greater increase in DAG or in IP3. Overall these experiments will lead to new insights into the mechanisms behind the development of diabetic neuropathy. Since the pain of diabetic neuropathy is difficult to treat, it is important to determine how it develops as a first step in prevention and also as a means to finding ways to treat it after it has developed. If hyperglycemia in vitro reproduces the sensitization caused in vivo on either neuronal level, it will provide an excellent experimental model for examining which agents might be efficacious in treating pain associated with diabetes and/or for examining the mechanism of their action as anagesics in this condition. The studies on gabapentin provide a first example of this approach.
