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The links between neuropathies of the peripheral nervous system (PNS), including Charcot-Marie-Tooth1A and hereditary neuropathy with liability to pressure palsies, and impaired biomechanical and structural integrity of PNS nerves remain poorly understood despite the medical urgency. Here, we present a protocol describing simultaneous structural and biomechanical integrity investigations on isolated nerve fibers, the building blocks of nerves. Nerve fibers are prepared from nerves harvested from wild-type and exemplary PNS neuropathy mouse models. The basic principle of the designed experimental approach is based on the simultaneous combination of atomic force microscopy (AFM) and confocal microscopy. AFM is used to visualize the surface structure of nerve fibers at nano-scale resolution. The simultaneous combination of AFM and confocal microscopy is used to perform biomechanical, structural, and functional integrity measurements at nano- to micro-scale. Isolation of sciatic nerves and subsequent teasing of nerve fibers take ~45 min. Teased fibers can be maintained at 37°C in a culture medium and kept viable for up to 6 h allowing considerable time for all measurements which require 3–4 h. The approach is designed to be widely applicable for nerve fibers from mice of any PNS neuropathy. It can be extended to human nerve biopsies.
Schwann cells (SCs) are of crucial importance for the development, maintenance, and regeneration of the peripheral nervous system (PNS) (Jessen and Mirsky, 2005). They form myelin around peripheral axons, thereby enabling the high-speed propagation of action potentials. In addition to myelin secretion around large and small caliber axons, myelinating SCs, and their associated axons are enwrapped in a continuous basal lamina secreted by SCs which is only 25 nm thick (Thomas, 1963). Different roles have been proposed for this basal lamina, including SC proliferation, survival, migration, and myelination (Colognato et al., 2005; Court et al., 2006; Chernousov et al., 2008; Colognato and Tzvetanova, 2011), but very little is known about its role in the biomechanical support to the nerve fiber. Interestingly, it has been suggested that impaired peripheral nerve biomechanics is directly linked to various neuropathies (Suter and Scherer, 2003; Bai et al., 2010), but the links between nerve biomechanics and neuropathies remain poorly understood. Only a few experiments have described the biomechanical properties of peripheral nerves in vivo (Driscoll et al., 2002; Topp and Boyd, 2012). In some cases, the mechanical measurements were performed on single nerve fibers (Urbanski et al., 2016). However, the fibers were fixed and therefore the physiological interpretation remains debatable (Heredia et al., 2007; Huang et al., 2011; Rosso et al., 2012). To gain a better understanding of the biomechanics of PNS nerves, we developed a novel strategy enabling biomechanical investigation of isolated living native peripheral nerve fibers based on simultaneous combination of AFM and confocal microscopy. Biomechanical, structural, and functional investigations were carried out on isolated nerve fibers from wild-type animals and the neuropathic animal model Pmp22−/−. This knockout model is especially relevant because the peripheral myelin protein 22 (PMP22) is closely connected with several hereditary human PNS neuropathies (Suter and Scherer, 2003; Fledrich et al., 2014), including Charcot-Marie-Tooth1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP). CMT1A and HNPP are associated with mechanical vulnerability and structural remodeling of nerves, in particular myelin and basal lamina of nerve fibers (Amici, 2006; Guo et al., 2014). They are also associated with an increase in the permeability of myelin and disruption of cell junction complexes in the peripheral nerve (Guo et al., 2014). Impaired biomechanical integrity (vulnerability to mechanical compression and lack of recovery) and leakiness are paralleled by severely reduced nerve conduction velocity and are therefore assumed to account for the clinical symptoms (Bai et al., 2010; Guo et al., 2014). The AFM-confocal microscopy setup we present here provides novel insights into understanding the links between nerve fibers biomechanics and neuropathies.
AFM is simultaneously combined with confocal microscopy to investigate native never fibers from multiple structural and biophysical aspects. AFM is applied as a mechanical and structural nano-tool alike. It enables structural investigation of nerve fibers surface at nano-resolution. At the same time, it is used to indent nerve fibers and study their response to the mechanical load. The simultaneous combination with confocal microscopy is used to perform biomechanical, structural and functional integrity measurements at nano- to micro-scale.
The role of biomechanics in diverse biomedical research areas is gaining substantial attention. This includes development, maintenance, differentiation of cells, and tissues as well as diseases (Ingber, 2003; Engler et al., 2006; Wang et al., 2009; Franze et al., 2013). The protocol presented here combines the biophysical nano-approach AFM and confocal microscopy simultaneously to investigate biomechanics of native peripheral nerve fibers from wild-type and neuropathic mice. Hence, this step-by-step protocol can be applied to investigate biomechanical properties of a variety of mouse animal models of peripheral neuropathies such as CMT1A among others (Sereda et al., 1996). In addition, this protocol can be utilized to investigate nerve biomechanics of young animals including rats, extending its applicability to an increased number of PNS diseases (Meyer Zu Hörste and Nave, 2006). Nerves may be investigated in the same way as their individual building blocks. Nerve biopsies from human patients with PNS neuropathies can also be tested. Such investigations will provide valuable information about the biomechanical changes in the peripheral nervous tissue during development and disease. All in all, the protocol targets a broad audience from biophysical, biomedical and clinical research.
The steps in sections 4 and 5a involve the preparation of the AFM-Confocal system for biomechanical and viability investigations. These can be done before step 1 in order to save time (~30 min) between the sample preparation and the measurements.
Neuropathic animal models, Pmp22−/−, exhibit biomechanical vulnerability and are prone to conduction block when exposed to compression (Bai et al., 2010). We anticipate significant differences in biomechanical, structural and functional integrity between intact and neuropathic nerve fibers. Representative AFM-Confocal images for biomechanical, functional, and structural integrity investigations on myelinated nerve fibers from intact and Pmp22−/− mice are shown in Figures Figures4,4, ,5.5. Nerve fibers are kept in a medium containing 70 kDa dextran. They are then exposed to incremental loading forces and their response is tested. The high molecular weight dextran is excluded from axonal entry unless fibers lose their biomechanical, structural or functional integrity.
The basal lamina of nerve fibers from Pmp22−/− mice is assumed to be loose (Amici, 2006) and we therefore anticipate marked differences in the surface architecture of Pmp22−/− compared to intact fibers. As a matter of fact, Pmp22−/− nerve fibers exhibit a basal lamina arrangement pattern (parallel) which is significantly different from the pattern in intact fibers (interwoven) (Figure (Figure5).5). The basal lamina is crucial for mechanoprotection of nerve fibers. Hence, we expect that alteration in its structure will impair the biomechanical integrity of nerve fibers. Indeed, chemical digestion of collagen from the basal lamina is paralleled by high vulnerability to mechanical compression (Figure (Figure55).
Fully elastic materials strain when stretched and rapidly restore their original shape once the stress is removed. Viscoelastic materials possess both elastic and viscosity properties and exhibit time-dependent strain rate. The elastic Young's modulus provides information about stiffness and elasticity and can be derived from force-deformation curves obtained with AFM (Figure (Figure44 and “Point 7: Data interpretation and analysis”). Likewise, the elastic/viscoelastic behavior of fibers can be derived from the force-deformation curves upon fibers indentation (Figure (Figure44).
We anticipate no full elastic behavior of nerve fibers. We also anticipate significantly lower elastic modulus of Pmp22−/− compared to intact fibers. Indeed, the apparent overall elasticity of myelinated nerve fibers from intact mice (31.4 ± 8.8 kPa, N = 11) is significantly higher compared to Pmp22−/− (15.6 ± 5.2 kPa, N = 11) fibers (Rosso et al., 2014). Viscoelasticity presents as an hysteresis loop in force-deformation curves; approach and retract curves are non-linear and do not coincide. Intact nerve fibers show viscoelastic behavior whereas (Figure (Figure4)4) Pmp22−/− are plastic and thus strain irreversibly when exposed to stress (Rosso et al., 2014).
This is the average time necessary for nerve fibers to recover after force removal. We expect either a lack or slowdown of recovery time after compression in neuropathic nerve fibers. Based on the analysis of force-indentation curves, it takes intact nerve fibers 2–6 s to fully recover after removal of the force (Figure (Figure5A,5A, right image). In contrast, nerve fibers from Pmp22−/− mice fail to recover (Figure (Figure5C).5C). Digestion of collagen from the basal lamina of intact fibers deprives them of the ability to recover (Figure (Figure5C5C).
Functional, structural, and biomechanical integrity is crucial for high conduction velocity of myelinated nerve function. We therefore anticipate mechanical resilience of intact myelinated nerve fibers. As a matter of fact, exposure of intact myelinated nerve fibers to substantial mechanical forces in incremental steps to the extent of squeezing fail to inflict structural or functional damage upon them. The sharp and stiff AFM tip can be likened to a needle exerting remarkable local pressure while mechanically probing the fibers, and yet the resulting pressure is resisted. Not only the axon caliber but also the tightness of myelin which is reinforced by the correct assembly of compact myelin proteins such as PMP22 is critical to ensure the high conduction velocity (Bai et al., 2010). Increase in nerve fiber permeability across the myelin sheath in paranodal regions has been shown to severely reduce the nerve conduction velocity (Guo et al., 2014). Therefore, structural, functional, and biomechanical integrity of intact nerve fibers naturally prevents leakiness. We anticipate leakiness to naturally excluded large macromolecules, 70 kDa FITC-dextran, in nerve fibers lacking biomechanical integrity. Consistently, following digestion of collagens from the mechanoprotective basal lamina in nerve fibers from intact mice, leakiness is visualized as dextran entry into axons in combined AFM-Confocal images (Rosso et al., 2014).
Lack of mechanical resilience is detectable as “breakthrough events” in force-deformation curves. These events represent the disruption, softening and destabilization of nerve fibers when mechanically probed by the sharp AFM tip during sample indentation. They can be visualized in the approach curves as “jagged zones.” The zones are an indicator of nerve fiber vulnerability to compression or impaired mechanical resilience (Rosso et al., 2014).
VS and GR designed the experiments, analyzed data, and wrote up the manuscript paper. GR carried out all the experiments, analyzed data, and prepared the figures. PY and IL contributed to the design of the experiments and the paper, and were actively involved in the analysis of the data and provided essential inputs.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This research was supported by funds from the DAAD (PhD-Scholarship), Innovative Medizinische Forschung (IMF I-LI211304, SH111208, and SH111511), Deutsche Forschungsgemeinschaft (SH 167/6-1), and Cluster of Excellence (EXC 1003-CIM). We thank Sandra Goebbels (Max Planck Institute for Experimental Medicine, Göttingen) and Ueli Suter (ETH Zürich) for the kind provision of PMP22 knockout mice.
The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fnmol.2017.00277/full#supplementary-material