神经科学

Enhancing axon regeneration is a major focus of peripheral nerve injury research. Although peripheral axons possess a limited ability to regenerate, their functional recovery is very poor. Various activity-based therapies like exercise, optical stimulation, and electrical stimulation as well as pharmacologic treatments can enhance spontaneous axon regeneration. In this protocol, we use a custom-built cuff to electrically stimulate the whole sciatic nerve for an hour prior to transection and repair. We used a Thy-1-YFP-H mouse to visualize regenerating axon profiles. We compared the regeneration of axons from nerves that were electrically stimulated to nerves that were not stimulated (untreated). Electrically stimulated nerves had longer axon growth than the untreated nerves. We detail how variations of this method can be used to measure acute axon growth.

Peripheral nerve injury (PNI) is common in all walks of life, and the most common PNIs are nerve crush and nerve transection. While optimal functional recovery after crush injury occurs over weeks, functional recovery after nerve transection with microsurgical repair and grafting is poor, and associated with permanent disability. The gold-standard treatment for nerve transection injury is microsurgical tensionless end-to-end suture repair. Since it is unethical to do experimental PNI studies in humans, it is therefore indispensable to have a simple, reliable, and reproducible pre-clinical animal model for successful evaluation of the efficacy of a novel treatment strategy. The objective of this article is two-fold: (A) To present a novel standardized peripheral nerve transection method in mice, using fibrin glue for modeling peripheral nerve transection injury, with reproducible gap distance between the severed nerve ends, and (B) to document the step-wise description of constructing a pressure sensor device for crush injury pressure measurements. We have successfully established a novel nerve transection model in mice using fibrin glue, and demonstrated that this transection method decreases surgical difficulties and variability by avoiding microsurgical manipulations on the nerve, ensuring the reproducibility and reliability of this animal model. Although it is quite impossible to exactly mimic the pathophysiological changes seen in nerve transection with sutures, we hope that the close resemblance of our novel pre-clinical model with gold-standard suturing can be easily reproduced by any lab, and that the data generated by this method significantly contributes to better understanding of nerve pathophysiology, molecular mechanisms of nerve regeneration, and the development of novel strategies for optimal functional recovery. In case of peripheral nerve crush injury, current methods rely on inter-device and operator precision to limit the variation with applied pressure. While the inability to accurately quantify the crush pressure may result in reduced reproducibility between animals and studies, there is no documentation of a pressure monitoring device that can be readily used for real-time pressure measurements. To address this deficit, we constructed a novel portable device comprised of an Arduino UNO microcontroller board and force sensitive resistor (FSR) capable of reporting the real-time pressure applied to a nerve. This novel digital pressure sensor device is cheap, easy to construct and assemble, and we believe that this device will be useful for any lab performing nerve crush injury in rodents.

The thoracic paravertebral sympathetic chain postganglionic neurons (tSPNs) represent the predominant sympathetic control of vascular function in the trunk and upper extremities. tSPNs cluster to form ganglia linked by an interganglionic nerve and receive multisegmental convergent and divergent synaptic input from cholinergic sympathetic preganglionic neurons of the spinal cord (Blackman and Purves, 1969; Lichtman et al., 1980). Studies in the past have focused on cervical and lumbar chain ganglia in multiple species, but few have examined the thoracic chain ganglia, whose location and diminutive size make them less conducive to experimentation. Seminal studies on the integrative properties of preganglionic axonal projections onto tSPNs were performed in guinea pig (Blackman and Purves, 1969; Lichtman et al., 1980), but as mice have become the accepted mammalian genetic model organism, there is need to reproduce and expand on these studies in this smaller model. We describe an ex vivo approach that enables electrophysiological, calcium imaging, and optogenetic assessment of convergence, divergence, and studies on pre- to postganglionic synaptic transmission, as well as whole-cell recordings from individual tSPNs. Preganglionic axonal connections from intact ventral roots and interganglionic nerves across multiple segments can be stimulated to evoke compound action potential responses in individual thoracic ganglia as recorded with suction electrodes. Chemical block of synaptic transmission differentiates spiking of preganglionic axons from synaptically-recruited tSPNs. Further dissection, including removal of the sympathetic chain, enables whole-cell patch clamp recordings from individual tSPNs for characterization of cellular and synaptic properties.

Peripheral nerve injury (PNI) is an excellent model for studying neural responses to injury and elucidating the mechanisms that can facilitate axon regeneration. As such, several animal models have been employed to study regenerative mechanisms after PNI, including Aplysia, zebrafish, rabbits, cats and rodents. This protocol describes how to perform a sciatic nerve injury and repair in mice, one of the most frequently used models to study mechanisms that facilitate recovery after PNI, and that takes advantage of the availability of many genetic models. In this protocol, we describe a method for using fibrin glue to secure the proximal and distal stumps of an injured nerve in close alignment. This method facilitates the alignment of nerve stumps, which aids in regeneration of both sensory and motor axons and allows successful reconnection with peripheral targets.

Although axons in the peripheral nervous system can regenerate, functional recovery after nerve injuries is poor. Activity-based therapies, such as exercise and electrical stimulation, enhance the regeneration of cut peripheral axons. Despite their effectiveness, clinical application of these experimental techniques has been limited. At least part of the basis for this translational barrier has been a lack of information as to the precise mechanism of activity-based therapies on peripheral axon regeneration. To evaluate the requirements for neuron-type specific activation to promote regeneration using these therapies, in the current protocol, we employed optogenetics. Utilizing the advantages of transgenic mouse lines we targeted opsin expression to different neuron types. Using fiber optics we activated those neurons with high temporal specificity as a model of activity-based intervention after nerve injury and to measure functional recovery achieved after such a treatment.

Compared to tissue sectioning techniques, the technique of teasing single nerve fibers provides a better way to understand the structures of myelin sheaths and axons of the peripheral myelinated nerves. This protocol describes a method for preparation of teased single nerve fibers from rat sciatic nerve. In this protocol, fixed nerves are teased into single individual fibers and arranged onto adhesion microscope slides for further immuno-staining.