In vitro models for peripheral nerve regeneration.

The study of peripheral nerve repair and regeneration is particularly relevant in the light of the high clinical incidence of nerve lesions. However, the clinical outcome after nerve lesions is often far from satisfactory and the functional recovery is almost never complete. Therefore, a number of therapeutic approaches are being investigated, ranging from local delivery of trophic factors and other molecules to bioactive biomaterials and complex nerve prostheses. Translation of the new therapeutic approaches to the patient always requires a final pre-clinical step using in vivo animal models. The need to limit as much as possible animal use in biomedical research, however, makes the preliminary use of in vitro models mandatory from an ethical point of view. In this article, the different types of in vitro models available today for the study of peripheral nerve regeneration have been ranked by adopting a three-step stair model based on their increasing ethical impact: (i) cell line-based models, which raise no ethical concern; (ii) primary cell-based models, which have low ethical impact as animal use, although necessary, is limited; and (iii) organotypic ex vivo-based models, which raise moderate ethical concerns as the use of laboratory animals is required although with much lower impact on animal wellbeing in comparison to in vivo models of peripheral nerve regeneration. This article aims to help researchers in selecting the best experimental approach for their scientific goals driven by the 'Three Rs' (3Rs) rules (Replacement, Reduction or Refinement of animal use in research) for scientific research.

Automated tracing of myelinated axons and detection of the nodes of Ranvier in serial images of peripheral nerves.

The development of realistic neuroanatomical models of peripheral nerves for simulation purposes requires the reconstruction of the morphology of the myelinated fibres in the nerve, including their nodes of Ranvier. Currently, this information has to be extracted by semimanual procedures, which severely limit the scalability of the experiments. In this contribution, we propose a supervised machine learning approach for the detailed reconstruction of the geometry of fibres inside a peripheral nerve based on its high-resolution serial section images. Learning from sparse expert annotations, the algorithm traces myelinated axons, even across the nodes of Ranvier. The latter are detected automatically. The approach is based on classifying the myelinated membranes in a supervised fashion, closing the membrane gaps by solving an assignment problem, and classifying the closed gaps for the nodes of Ranvier detection. The algorithm has been validated on two very different datasets: (i) rat vagus nerve subvolume, SBFSEM microscope, 200 × 200 × 200 nm resolution, (ii) rat sensory branch subvolume, confocal microscope, 384 × 384 × 800 nm resolution. For the first dataset, the algorithm correctly reconstructed 88% of the axons (241 out of 273) and achieved 92% accuracy on the task of Ranvier node detection. For the second dataset, the gap closing algorithm correctly closed 96.2% of the gaps, and 55% of axons were reconstructed correctly through the whole volume. On both datasets, training the algorithm on a small data subset and applying it to the full dataset takes a fraction of the time required by the currently used semiautomated protocols. Our software, raw data and ground truth annotations are available at http://hci.iwr.uni-heidelberg.de/Benchmarks/. The development version of the code can be found at https://github.com/RWalecki/ATMA.

Gellan Gum-based luminal fillers for peripheral nerve regeneration: an in vivo study in the rat sciatic nerve repair model.

Peripheral nerve injuries (PNI) resulting in a gap to be bridged between the transected nerve ends are commonly reconstructed with autologous nerve tissue, but there is a need for valuable alternatives. This experimental work considers the innovative use of the biomaterial Gellan Gum (GG) as a luminal filler for nerve guidance channels made from chitosan with a 5% degree of acetylation. The engineered constructs should remodel the structural support given to regenerating axons by the so-called bands of Büngner. Four different GG formulations were produced by combining varying amounts of High-Acyl GG (HA-GG) and Methacrylated GG (MA-GG). The effective porosity of the freeze-dried networks was analysed by SEM and micro-CT 3D reconstructions, while the degradation and swelling abilities were characterized in vitro for up to 30 days. The metabolic activity and viability of immortalized Schwann cells seeded onto the freeze-dried networks were also evaluated. Finally, the developed hydrogel formulations were freeze-dried within the chitosan nerve guides and implanted in a 10 mm rat sciatic nerve defect. Functional and histomorphological analyses after 3, 6, and 12 weeks in vivo revealed that although it did not result in improved nerve regeneration, the NGC25:75 formulations could provide a basis for further development of GG scaffolds as luminal fillers for hollow nerve guidance channels.

Two-component collagen nerve guides support axonal regeneration in the rat peripheral nerve injury model.

Progress in material development has enabled the production of nerve guides that increasingly resemble the characteristics of an autologous nerve graft. In the present study, 20 mm adult rat sciatic nerve defects were bridged with the collagen-based, two-component nerve guide 'Neuromaix', the commercially available NeuraGen® nerve tube or an autologous nerve graft. Neuromaix was able to support structural as well as functional regeneration across this gap. The majority of the axons grew across the scaffold into the distal nerve segment and retrograde tracing confirmed that these axons were of somatosensory and motor origin. Histomorphology revealed that axons regenerating through Neuromaix exhibited reduced myelin sheath thickness, whereas axon diameter and axon density were comparable to those of the autograft. Neuromaix implantation resulted in reinnervation of the gastrocnemius muscle to a level that was not significantly different from that supported by the autograft, as demonstrated by electrophysiology. Our findings show that the use of the Neuromaix scaffold not only allowed axonal regeneration across large nerve gaps, but that the regenerating axons were also able to functionally reinnervate the muscles. These data provide a promising perspective for the first in human application of the materials. Copyright © 2016 John Wiley & Sons, Ltd.

Morphometric parameters of peripheral nerves in calves correlated with conduction velocity.

BACKGROUND: Peripheral nerve injuries are the most frequent neurologic disorder in cattle. So far, no physiologic values have been established for the motor nerve conduction velocity (mNCV) in this precocial species. OBJECTIVES: The electrophysiologic and morphometric reference values of peripheral nerves in calves were determined. It was hypothesized that these parameters would correlate to the high degree of maturity in the first days of life in this species compared to other species. ANIMALS: Twenty-six healthy calves were used in this study. METHODS: The mNCV of the radial and the sciatic/common peroneal nerve was measured in all 26 calves. Nerve biopsies from a group of 6 calves were taken to correlate the obtained electrophysiologic data with morphological parameters. RESULTS: The mean mNCV of the radial nerve was 48.3 ± 10.6 m/s, whereas the mean mNCV of the sciatic/peroneal nerve was with 83.8 ± 5.9 m/s significantly faster (P < .0001). The average fiber diameter was 8.40 ± 2.80 µm (range, 1.98-17.90 µm) and the average g-ratio was 0.61 ± 0.04 SD. CONCLUSION AND CLINICAL IMPORTANCE: The established reference values for mNCV in calves correlate well with the evaluated morphometric parameters. Attributable to their comparably fast mNCV and high fiber diameters, juvenile calves appear to be much more mature individuals than other mammals. Electrophysiologic characterization of peripheral nerve injury now is feasible in this species.