Methods of peripheral nerve tissue preparation for second harmonic generation imaging of collagen fibers.

Second harmonic generation (SHG) imaging of the peripheral nerve using multi-photon microscopy is a novel technique with little documentation. It affords the significant possibility of non-destructive imaging of internal nerve anatomy. The nature of nerve tissue, especially its size and viscoelastic properties, present special challenges for microscopy. While nerves are under an innate in situ strain, they retract once dissected, thus distorting microscopic structure. The challenge is to preserve the nerve in its natural strain range to obtain images that most truly reveal its structure. This study examined backscattered SHG images of rat median nerve prepared by several different methods to compare image quality and content. Nerve segments were fixed under strained (constant load or length) and unstrained conditions and imaged as whole nerve as well as plastic (methyl methacrylate) and paraffin embedded sections. These were tested for optimal excitation wavelength, quantitative image contrast, and overall quality. Root mean squared (RMS) contrast proved to be a reliable measure of the level of image contrast perceived by eye. We concluded that images obtained from tissue sections (plastic and paraffin) provided the most accurate and revealing SHG images of peripheral nerve structure. Removing the embedding material prior to imaging significantly improved image quality. Optimal excitation wavelengths were consistent regardless of the preparation method.

Multiple exposures to unloading decrease bone's responsivity but compound skeletal losses in C57BL/6 mice.

A single exposure to mechanical unloading can result in significant bone loss, but the consequences of multiple exposures are largely unknown. Within a 18-wk period, adult C57BL/6 male mice were exposed to 2 wk of hindlimb unloading (HLU) followed by 4 wk of reambulation (RA) once (1x-HLU), twice (2x-HLU), or three times (3x-HLU), or served as ambulatory age-matched controls. In vivo µCT longitudinally tracked changes in trabecular and cortical compartments of the femur. Normally ambulating control mice experienced significant age-related loss in trabecular bone volume fraction throughout the course of the experiment. This loss was compounded by HLU with 2x- and 3x-HLU mice experiencing a 27% and 24% greater reduction in trabecular bone and a 60% and 63% inhibition of age-related trabecular thickening. The recovery of cortical bone was also incomplete during each 4-wk RA period and, at completion of the experiment, cortical area in 3x-HLU mice was 5% smaller than in control and 1x-HLU. When eliminating age as a confounding variable, comparison between individual HLU/RA cycles showed that the magnitude of the response diminished during subsequent exposures. The extent of trabecular thinning in mice unloaded for the first time was 1.6-fold greater than the second time and nearly twofold greater than the third time. Similarly, the increase in trabecular thickness during the first RA cycle was twofold greater than during the second and third RA cycle. Together, our data demonstrate that even though multiple exposures to mechanical unloading are more detrimental than a single unloading period, bone's mechanosensitivity is reduced with consecutive unloading/reambulation cycles.

Detecting structural and inflammatory response after in vivo stretch injury in the rat median nerve via second harmonic generation.

BACKGROUND: Second Harmonic Generation (SHG) microscopy is a promising method for visualizing the collagenous structure of peripheral nerves. Assessing collagen continuity and damage after a stretch injury provides inferential insight into the level of axonal damage present. NEW METHODS: This study utilizes SHG microscopy after a calibrated in vivo stretch injury of rat median nerves to evaluate collagen continuity at several time points throughout the recovery process. Endoneurial collagen was qualitatively assessed in nerves that were subjected to low strain (LS) and high strain (HS) injuries using SHG microscopy, conventional histology, and immunohistochemistry. RESULTS: Following an in vivo stretch injury, both LS and HS damaged nerves exhibit signs of structural collagen damage in comparison with sham control nerves (SC). Furthermore, LS nerves exhibit signs of full regeneration while HS nerves exhibited signs of only partial regeneration with lasting damage and intra-neural scar formation. COMPARISON WITH EXISTING METHODS: SHG observations of structural changes and inflammatory response due to stretch injury were validated upon comparison with conventional histological methods CONCLUSIONS: We propose that SHG microscopy can be utilized to visualize significant structural artifacts in sectioned median nerves following in vivo stretch injury. Based on the findings in this study, we believe that the in vivo application of SHG microscopy should be further investigated as a means for real-time, intra-operative, quantitative assessment of nerve damage.

Extending Rest between Unloading Cycles Does Not Enhance Bone's Long-Term Recovery.

PURPOSE: Multiple exposures to unloading are overall more deleterious to the skeleton than is single exposure, although the rate of bone loss may diminish during multiple exposures. Here, we determined whether extending the reambulation (RA) period from 3 wk to 9 wk will mitigate bone loss during three distinct 3-wk hindlimb unloading (HLU) periods and enhance long-term recovery in skeletally mature, genetically heterogeneous mice. METHODS: Female adult mice (4 months old) were subjected to three cycles of 3-wk unloading with 3-wk or 9-wk RA periods in between. Mice were terminated 46 wk after initiation of the study. Outcome measures for the distal femur were determined from multiple in vivo micro-computed tomography scans and finite-element modeling. RESULTS: Tripling RA duration enhanced trabecular bone recovery in between HLU periods but also increased the rate of loss of bone volume fraction (bone volume/tissue volume) and metaphyseal stiffness during subsequent HLU periods. With shorter RA periods, the magnitude of bone loss decreased by the second HLU period, whereas this decrease was delayed with longer RA periods. RA duration did not affect long-term recovery 46 wk after the start of the experimental protocol, as both HLU groups had similar levels of bone volume/tissue volume, cortical area, and stiffness. Individual cage activity levels were unrelated to the magnitude of bone loss during HLU or bone recovery during RA. CONCLUSIONS: These data suggest that extending recovery duration between periods of unloading may provide temporary benefits but is an ineffective long-term strategy for combating the devastation of trabecular morphology and mechanics, as temporarily enhanced recovery is largely cancelled out by greater susceptibility to unloading. They also emphasize that cortical bone is more amenable to long-term recovery than is trabecular bone.