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Non-Peptidergic Nociceptive Neurons Are Essential for Mechanical Inflammatory Hypersensitivity in Mice

Abstract

Small nerve fibers that bind the isolectin B4 (IB4+ C-fibers) are a subpopulation of primary afferent neurons that are involved in nociceptive sensory transduction and do not express the neuropeptides substance P and calcitonin-gene related peptide (CGRP). Several studies have attempted to elucidate the functional role of IB4+-nociceptors in different models of pain. However, a functional characterization of the non-peptidergic nociceptors in mediating mechanical inflammatory hypersensitivity in mice is still lacking. To this end, in the present study, the neurotoxin IB4-Saporin (IB4-Sap) was employed to ablate non-peptidergic C-fibers. Firstly, we showed that intrathecal (i.t.) administration of IB4-Sap in mice depleted non-peptidergic C-fibers, since it decreased the expression of purinoceptor 3 (P2X3) and transient receptor potential cation channel subfamily V member 1 (TRPV1) in the dorsal root ganglia (DRGs) as well as IB4 labelling in the spinal cord. Non-peptidergic C-fibers depletion did not alter the mechanical nociceptive threshold, but it inhibited the mechanical inflammatory hypersensitivity induced by glial cell-derived neurotrophic factor (GDNF), but not nerve growth factor (NGF). Depletion of non-peptidergic C-fibers abrogated mechanical inflammatory hypersensitivity induced by carrageenan. Finally, it was found that the inflammatory mediators PGE2 and epinephrine produced a mechanical inflammatory hypersensitivity that was also blocked by depletion of non-peptidergic C-fibers. These data suggest that IB4-positive nociceptive nerve fibers are not involved in normal mechanical nociception but are sensitised by inflammatory stimuli and play a crucial role in mediating mechanical inflammatory hypersensitivity.

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Abbreviations

ATF-3:

Activating transcription factor 3

AUC:

Area under the curve

BSA:

Bovine serum albumin

cAMP:

Cyclic adenosine monophosphate

CGRP:

Calcitonin-gene related peptide

DRG:

Dorsal root ganglion

EP:

E prostanoid receptor

g:

Grams

GDNF:

Glial cell-derived neurotrophic factor

GFRα-1:

GDNF family receptor alpha 1

GPCR:

G protein-coupled receptor

HCN:

Hyperpolarization-activated cyclic nucleotide-gated

HTMRs:

High-threshold mechanoreceptors

IB4:

Isolectin B4

IB4-Sap:

IB4-saporin

i.pl.:

Intraplantar

i.t.:

Intrathecal

LTMRs:

Low-threshold mechanoreceptors

MCP-1:

Monocyte chemoattractant protein 1

Nav :

Voltage-gated sodium ion channel

NGF:

Nerve growth factor

OCT:

Optimum cutting temperature

PBS:

Phosphate-buffered saline

P2X3 :

Purinoceptor 3

PFA:

Paraformaldehyde

PGE2 :

Prostaglandin E2

PKA:

Protein kinase A

PKCε:

Epsilon isozyme of protein kinase C

Sap:

Unconjugated saporin

SEM:

Standard error of the mean

T-PER:

Tissue protein extraction reagent

TrkA:

Tropomyosin receptor kinase A

TRPV1:

Transient receptor potential cation channel subfamily V member 1

WT:

Wild type

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Acknowledgments

We thank Ieda Regina dos Santos Schivo, Sérgio Roberto Rosa, Eleni Luiza Tamburus Gomes, Elizabete Rosa, and Ana Kátia dos Santos for their excellent technical assistance.

Funding

The research leading to these results received funding from the São Paulo Research Foundation (FAPESP) under grant agreements number 2011/19670–0 (Thematic project) and 2013/08216–2 (Centre for Research in Inflammatory Disease), from the University of São Paulo NAP-DIN under grant agreement number 11.1.21625.01.0 and from a CNPq grant number 485177/2012-9. L.G.P. was supported by doctoral fellowship from FAPESP under grant number 2010/04043-8.

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Pinto, L.G., Souza, G.R., Kusuda, R. et al. Non-Peptidergic Nociceptive Neurons Are Essential for Mechanical Inflammatory Hypersensitivity in Mice. Mol Neurobiol 56, 5715–5728 (2019). https://doi.org/10.1007/s12035-019-1494-5

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Keywords

  • Non-peptidergic C-fibers
  • IB4-saporin
  • Mechanical hypersensitivity
  • Inflammatory pain
  • Nociceptors
  • Mice