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BACKGROUND: Satellite glial cells (SGCs) envelope the neuronal somas in the dorsal root ganglia (DRG) and are believed to provide important neuronal support. Animal models of peripheral nerve injury, diabetes or chemotherapy all demonstrate activation of SGCs, suggesting important physiological roles for SGCs in various states of peripheral neuropathy. However, the biology of these glial cells is only poorly characterized under normal as well as pathological conditions due to suboptimal isolation methods.NEW METHOD: The method presented here allows complete dissociation and isolation of highly pure SGCs from rat DRGs by fluorescence-activated cell sorting (FACS) using SGC-specific antibodies. The method further allows purification of high-quality RNA from the fixed and permeabilized cells.RESULTS: The purified RNA shows very little degradation, demonstrated by RNA integrity number (RIN) analysis with an average value of 8. The purified RNA, therefore, lends itself very well to downstream applications such as qPCR and transcriptome analysis.COMPARISON WITH EXISTING METHODS: Primary SGC cultures have previously been established for in vitro studies. Unfortunately, SGCs quickly change morphology and gene expression in vitro, complicating biologically meaningful interpretation of the obtained results. In contrast, this method allows the investigation of SGC gene regulation in vivo by isolation of high-quality RNA.CONCLUSIONS: This method enables investigation of SGC transcriptional response in vivo by isolation and analysis of mRNA expression, allowing a more detailed investigation of SGC biology under normal as well as pathological conditions.

Quantitative estimation of myelinated nerve fiber number, together with fiber size parameters, is one of the most important tools for nerve regeneration research. In this study we used a design-based stereological method to evaluate the regenerative process in two experimental paradigms: crush injury and autograft repair. Samples were embedded in resin and morphometric counting and measurements were performed using both light and electron microscopes. Results show a significant difference in myelinated fiber number estimation between light and electron microscopes, especially after autograft repair; light microscope significantly underestimates the number of fibers because of the large number of very small axons that can be detected only in electron microscope. The analysis of the size parameters also shows a higher number of small fibers in electron microscopic analysis, especially in regenerated nerves. This comparative study shows that the integration of data obtained in light microscope with those obtained in electron microscope is necessary in revealing very small myelinated fibers that cannot be detected otherwise. Moreover, the difference in the estimation of total number of myelinated fibers between light and electron microscopes must be considered in data analysis to ensure accurate interpretation of the results.

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

The Neuregulin/ErbB system plays an important role in the peripheral nervous system, under both normal and pathological conditions. We previously demonstrated that expression of soluble ecto-ErbB4, the released extracellular fragment of the ErbB4 receptor, stimulated glial cell migration in vitro. In this study we examined the possibility of manipulating this system in vivo in order to improve injured peripheral nerve regeneration. Transected rat median nerves of adult female Wistar rats were repaired with a 10-mm-long graft made by muscle-in-vein combined nerve guide previously transduced with either the adeno-associated viral (AAV) vector AAV2-LacZ or AAV2-ecto-ErbB4. Autologous nerve grafts were used as control. Both stereological and functional analyses were performed to assess nerve regeneration. Data show that delivery of soluble ecto-ErbB4 by gene transfer in the muscle-in-vein combined nerve guide has a positive effect on fiber maturation, suggesting that it could represent a potential tool for improving peripheral nerve regeneration.

Antibodies are valued reagents in most modern laboratories and indispensable for many experiments. They can be utilized in a wide range of experimental setups designed to elucidate various scientific questions. In our laboratory, antibodies are used to investigate protein localization, quantity and phosphorylation state, and furthermore to identify various cell types as cell markers or to block protein function.

Sciatic nerve crush injury in rat animal model is one of the most common experimental models used in regenerative research. However, the availability of transgenic mouse for nerve regeneration studies is constantly increasing and, therefore, the shift from rat model to mouse model is, in some cases, necessary. Moreover, since most of the human nerve lesions occur in the upper limb, it is also advantageous to shift from sciatic nerve to median nerve. In this study we described an experimental model which involves lesions of the median nerve in the mouse. Data showed that the finger flexor muscle contraction strength, assessed to evaluate the motor function recovery, and reached values not different from the control already 20 days after injury. The degree of nerve regeneration evaluated with stereological methods in light microscopy showed that, 25 days after injury, the number of regenerated myelinated fibers was comparable to the control, but they were smaller with a thinner myelin thickness. Stereological analysis made in electron microscopy confirmed these results, although the total number of fibers quantified was significantly higher compared to light microscopy analysis, due to the very small size of some fibers that can be detected only in electron microscopy.