Abstract
Immunohistochemical characterization of primary afferent fibers (intact or after nerve damage) is traditionally performed in thin sections from dorsal root ganglia (DRGs) or in teased fibers, as light scattering in whole-mounts compromises visualization. These procedures are time-consuming, require specific equipment and advanced experimental skills. Lipid-clearing techniques are increasing in popularity, but they have never been used for the peripheral nervous system. We established a modified, inexpensive clearing method based on lipid-removal protocols to make transparent peripheral nerve tissue (inCLARITY). We compared retrograde-labeling and free-floating immunostaining with cryo-sections. Confocal microscopy on whole-mount transparent DRGs showed neurons marked with retrograde tracers applied to experimental neuromas (Retrobeads, Fluoro-ruby, Fluoro-emerald, DiI, and Fluoro-gold). After immunostaining with calcitonin gene-related peptide (peptidergic) or isolectin IB4 (non-peptidergic), nociceptors were visualized. Immunostaining in transparent whole-mount nerves allows simultaneous evaluation of the axotomized branches containing the neuroma and neighboring intact branches as they can be mounted preserving their anatomical disposition and fiber integrity. The goal of our study was to optimize CLARITY for its application in peripheral nerve tissues. The protocol is compatible with the use of retrograde tracers and improves immunostaining outcomes when compared to classical cryo-sectioning, as lack of lipids maximizes antibody penetration within the tissue.
Highlights
Peripheral afferents are pseudounipolar neurons which cell bodies give rise to a central axon that goes into the central nervous system and a peripheral axon that ends in the skin, muscles or visceral organs among others
Immunohistochemistry (IHC) allows to classify functional populations of unmyelinated pain C-fibers in DRGs or TGs based on the presence of calcitonin-gene related peptide (CGRP) or substance P (SP) and
We have shown its suitability for the study of the alterations observed in the peripheral nervous system upon damage, but we believe it might be adapted for other experimental contexts
Summary
Peripheral afferents are pseudounipolar neurons which cell bodies (located in the dorsal root ganglia -DRG- or trigeminal ganglia -TG-) give rise to a central axon that goes into the central nervous system and a peripheral axon that ends in the skin, muscles or visceral organs among others As they convey sensory information, any lesion or disease affecting these neurons lead to the perception of abnormal sensations (typically spontaneous and/or evoked pains) termed neuropathic pain. Recent advances in the treatment of neuropathic pain are based on patient’s “sensory-abnormalities” rather than the etiology of the neuropathy[8,9] Such a mechanism-based approach requires the identification and characterization of each of the peripheral fibers involved. The introduction of these approaches in research laboratories is still scarce due to their high cost, the complexity of published protocols or possible negative impact on the fluorescence signal
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