Behavioral characterization of neuropathic pain on the glabrous skin areas reinnervated solely by axotomy-regenerative axons after adult rat sciatic nerve crush

Abstract

In cranial and spinal nerve ganglia, both axotomized primary sensory neurons without regeneration (axotomy-nonregenerative neurons) and spared intact primary sensory neurons adjacent to axotomized neurons (axotomy-spared neurons) have been definitely shown to participate in pain transmission in peripheral neuropathic pain states. However, whether axotomized primary sensory neurons with regeneration (axotomy-regenerative neurons) would be integral components of neural circuits underlying peripheral neuropathic pain states remains controversial. In the present study, we utilized an adult rat sciatic nerve crush model to systematically analyze pain behaviors on the glabrous plantar surface of the hindpaw sural nerve skin territories. To the best of our knowledge, our results for the first time showed that heat hyperalgesia, cold allodynia, mechanical allodynia, and mechanical hyperalgesia emerged and persisted on the glabrous sural nerve skin areas after adult rat sciatic nerve crush. Interestingly, mechanical hyperalgesia was sexually dimorphic. Moreover, with our optimized immunofluorescence staining protocol of free-floating thick skin sections for wide-field epifluorescence microscopic imaging, changes in purely regenerative reinnervation on the same skin areas by axotomized primary sensory afferents were shown to be paralleled by those pathological pain behaviors. To our surprise, Protein Gene Product 9.5-immunoreactive nerve fibers with regular and large varicosities ectopically emigrated into the upper dermis of the glabrous sural nerve skin territories after adult rat sciatic nerve crush. Our results indicated that axotomy-regenerative primary sensory neurons could be critical elements in neural circuits underlying peripheral neuropathic pain states. Besides, our results implied that peripheral neuropathic pain transmitted by axotomy-regenerative primary sensory neurons alone might be a new dimension in the clinical therapy of peripheral nerve trauma beyond regeneration.

Antibody Incubation at 37°C Improves Fluorescent Immunolabeling in Free-Floating Thick Tissue Sections

“…For imaging convenience and scalability, we utilized optical clearing-assisted, wide-field epifluorescent imaging and 3-D blind deconvolution, which were shown in previous studies by our group and others to enable high-contrast 3-D visualization of thick tissue samples comparable to confocal imaging (10,12). All of the fluorescently immunolabeled free-floating thick tissue sections were imaged using a fully motorized, Olympus IX-83 widefield (WF) epifluorescence microscope with a DP80 CCD camera under the monochromatic mode controlled by Olympus cellSens Dimension software (Olympus; Tokyo, Japan) (10).…”

“…In brief, after extensive washing with PBS-G (0.3 M glycine in 0.01 M PBS, pH 7.4) at RT, free-floating thick tissue sections were preconditioned for tissue loosening: ( i ) skin: neat DMSO for 15 min to loosen collagen structures in the upper dermis (UD) and dense cellular networks in the epidermis (10); ( ii ) spinal cord: antigen retrieval with sodium citrate solution (0.01 M sodium citrate, 0.05% Tween-20, pH 6.0) at 80C for 45 min to improve antigenic reactivity and antibody penetration (9,11); and ( iii ) sciatic nerve: thermally-assisted delipidation with 0.2 M sodium dodecyl sulfate (SDS) (in PBS, pH 9.0) at 37C for 3 h (7). After extensive washing with PBST (0.2% Tween-20 in 0.01 M PBS, pH 7.4) at RT, thick tissue sections were then permeabilized with PBS-Tx (0.3% Triton X-100 in 0.01 M PBS, pH 7.4): 4 15 min for skin and spinal cords, and 6 15 min for sciatic nerves.…”

“…After extensive washing with PBST, tissue sections were processed for optical clearing with gradient glycerol solutions (diluted in 0.01 M PBS, pH 8.0, 2.5% DABCO, 0.05% Tween-20) as previously described (10,16). Sections were mounted on poly-L-lysine (PLL)-coated slides with the anti-fade mounting medium (2.5% DABCO, 0.05% Tween-20 in 90% glycerol diluted with 0.01 M PBS, pH 8.0) overnight at 4C.…”

“…All of the fluorescently immunolabeled free-floating thick tissue sections were imaged using a fully motorized, Olympus IX-83 widefield (WF) epifluorescence microscope with a DP80 CCD camera under the monochromatic mode controlled by Olympus cellSens Dimension software (Olympus; Tokyo, Japan) (10). For PGP 9.5 immunolabeling of sciatic nerve thick sections, Z-stack images of the same sections were further captured on an Olympus FV1000 confocal microscope for better evaluation of antibody diffusion in sciatic nerve thick sections.…”

“…Previous studies showed that optical clearing-assisted, wide-field epifluorescent imaging and 3-D blind deconvolution allows high-quality 3-D visualization of thick tissue samples comparable to confocal imaging, making 3-D imaging of thick tissue sections simpler and more scalable (10,12). However, due to poor antibody penetration, standard protocols for fluorescent immunolabeling of free-floating thick tissue sections had an unsatisfactory labeling quality (low sensitivity for sparsely or moderately expressed proteins; faint or negative immunolabeling in the center of thick tissue sections and high non-specific binding) and an inadequate labeling efficiency (requiring high antibody concentrations and long antibody incubation times).…”