To define the existence of intracellular hydration caused by metabolic derangements in the excised sciatic nerves of diabetic rats quantitatively, relaxation times (T1, T2) and fraction of intracellular water content were measured with 1H-nuclear magnetic resonance (NMR) spectroscopy in normal rats (control group, n = 10), streptozotocin (STZ)-induced (50 mg/kg, i.v.) diabetic rats (DM group, n = 10), STZ-induced diabetic rats treated with an aldose reductase inhibitor (ARI, Epalrestat, 100 mg/kg) (ARI group, n = 8), and STZ-induced diabetic rats treated with insulin (insulin group, n = 4). For selective measurement of intracellular relaxation times, the inversion recovery (IR) method for conventional T1 and Carr-Purcell-Meiboom-Gill method for T2 were used with an aqueous chemical shift reagent, 10 mmol/L dysprosium triethylenetetramine-N,N,N',N",N"',N"'-hexaacetic acid, resulting in distinct separation of intracellular (Schwann cell, axon, endothelial cell, and pericyte) and extracellular waters under the isotonic condition of the rat sciatic nerves. Furthermore, a new method of driven-equilibrium single-pulse observation of T1 (DESPOT) was used for rapid measurement of T1 for the purpose of clinical application on magnetic resonance imaging (MRI). T1 values measured by the DESPOT and IR methods were significantly correlated (p < 0.001). Total and intracellular water contents, sorbitol contents, and relaxation times of the sciatic nerve taken from the DM group were significantly elevated (p < 0.01), while myoinositol (p < 0.01) and extracellular water (p < 0.05) contents were significantly decreased as compared with the control group. Both insulin and ARI treatments significantly improved relaxation times as compared with those in the DM group (p < 0.05-0.01). Relaxation times correlated positively with total water (T1, p < 0.05-0.01; T2, p < 0.01), intracellular water (T1, p < 0.001; T2, p < 0.001), and sorbitol (T1, p < 0.001; T2, p < 0.001) contents of the excised nerve. Sorbitol content correlated positively with total and intracellular water contents (p < 0.01) but negatively with extracellular water content (p < 0.05). These findings indicated that sorbitol itself and/or secondary sodium accumulation caused by an increase in sorbitol may be a major contributor to the increase in intracellular hydration and prolonged relaxation times associated with hyperglycemia, which are reversible with insulin or ARI treatment. It was also suggested that rapid T1 measurement would provide new insights into the pathogenesis of human diabetic neuropathy as a non-invasive evaluation method on MRI.

Kyohei Yasuda

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