Catheter confocal fluorescence imaging and functional magnetic resonance imaging of local and systems level recovery in the regenerating rodent sciatic nerve

The goal of the present work was to develop minimally invasive imaging techniques to monitor local regeneration of peripheral nerves and to determine the extent of return to function of brain cortical regions associated with that nerve. The sciatic nerve crush model was applied to Sprague-Dawley rats and conventional histological staining for myelin, axons and cell architecture was carried out, as well as traditional behavioral testing, to verify that nerve regeneration was occurring. The rate of sciatic nerve regeneration was measured by determining the distance a lipophilic, fluorescence probe (DiO) would move along the nerve's membrane following a direct injection into the sciatic nerve. This movement was monitored using a catheter based, confocal fluorescence microscope. Two to five days after the crush, the dye moved 1.4 + 0.6 mm/day, as compared to a distance of 5.3 + 0.5 mm/day in the normal nerve. Between 9 and 13 days following the crush, the distance the dye moved increases to 5.5 + 0.5 mm/day, similar to the control, and by 15 days following the crush, the distance increased to 6.5 + 0.9 mm/day. Functional Magnetic Resonance Imaging (fMRI) measurements were performed on alpha-chloralose anesthetized rats to monitor the return of somatosensory cortical functions, which were activated by the stimulation of the lesioned peripheral nerve. fMRI results showed the return of cortical activation around 15 days following the crush procedure. However, the somatosensory cortical region activated by stimulating the crushed hindpaw was significantly smaller in extent than the intact hindpaw stimulation. These findings demonstrate that fluorescence imaging and fMRI can integrate local and system level correlates of nerve regeneration in a non-destructive manner, thus enabling serial imaging of individual animals.