Optical Microscopic Imaging Research On The Relationship Between Structure And Characteristic Of The Peripheral Nerve Bundles

With the development of industry, agriculture, social construction, nerve injury has become a clinical common disease, any part of limbs, trauma, infection, ischemia can lead to oppression and a corresponding neurological deficit. However, duo to the recognition technology is relatively backward and the clinical surgeons lack a clear understanding of fine structure of nerves, it is difficult for surgeons to diagnosis the characteristics of damaged nerve bundles in the operation, thus, patients often do not receive effective treatment of nerve damage. According to statistics, full nerve function recovery after the repair of only is 10% to 25%, resulting in some patients loses the ability to work. At present, the main methods to the identification of peripheral nerve bundles are: electrophysiological method, enzyme histochemistry method, immunohistochemical method and anatomical localization method, but these methods are either low accuracy of identification or the procedure was tedious and time is too long, which limit the application and promotion in the clinical use.Because of its high spatial resolution, non-contact and non-invasive, optical imaging methods are particularly suitable for the identification of nerve bundles. Compared with previous identification methods, optical imaging method does not need staining to visualize the dynamic distribution of fibers and the nerve bundle composition and morphology change after nerve injury, which has a very important significance for the clinical micro-surgery.The main research contents and creative points are as follows:(1) We explored the structures of nerve fibers in ventral and dorsal nerves with a laser scanning confocal microscopy. Thick tissue staining results suggested that nerve fibers have different 3-D structure in ventral and dorsal nerves, and reconstruction from serial sectioning images showed that in ventral nerves the nerve fibers travel in a winding form, while in dorsal nerves, the nerve fibers form in a parallel cable pattern. These structural differences could help surgeons to differentiate ventral and dorsal nerves in peripheral nerve injury repair, and also facilitate scientists to get a deeper understanding about nerve fiber organization. (2) We reported an optical way to identify the sensory and motor nerve bundles, based on the laser scanning infrared DIC microscopy. DIC images with high resolution presented distinct patterns, stripes in the motor nerve bundle, and circles in the sensory nerve bundle, with the recommended cross-section thickness of 40μm. Quantitative analysis further confirmed these by showing much higher stripe/circle ratio in the motor nerve bundle. Accordingly to the significant differences in the structure, models of fiber assembling in the motor and sensory nerve bundles were proposed. The cutting angle of the nerves are considered in the experiments, to ensure the results not influenced by cutting angle of manual operation, motor and sensory nerves were binded parallelly and cut at the same time, the obvious circles pattern in sensory nerves could prove the nerves were vertical with the microtome knife in experiments. Compared to conventional molecular and histochemical staining approaches, this approach cost only 30 minutes, with high accuracy rates for recognitions of the motor and sensory nerves, which may provide an accurate, rapid, label-free way for identifications in surgical peripheral nerve repair.(3) By the method of nonlinear optical imaging, acute isolated spinal nerve bundles form Beagle dogs were studied. Two-photon excited auto-fluorescence signal and back of the second harmonic signal can be simultaneously recorded using 790nm femtosecond pulse laser. In the cross-sectional study of nerve bundles, we confirm the sense of fiber arranged in parallel mode of sensory bundle and stripes distribution of the motor bundles. Krebs's solution was found suitable for preserving the acute isolated nerve bundles in physiological state; this finding will be helpful for the in-vivo clinical research. In the perineurium axial profile study, we found that collagen content in sensory and motor perineurium are significantly different, while the elastin content was not significant different; then collagen fibers in the FFT spectrum and GLCM texture features were analyzed. We investigate the collagen fiber orientation, contrast, angular second moment, correlation and other texture features which are significantly different between sensory and motor nerves.