Select noxious stimuli induce changes on corneal nerve morphology

Abstract

The surface of the cornea contains the highest density of nociceptive nerves of any tissue in the body. These nerves are responsive to a variety of modalities of noxious stimuli and can signal pain even when activated by low threshold stimulation. Injury of corneal nerves can lead to altered nerve morphology, including neuropathic changes which can be associated with chronic pain. Emerging technologies that allow imaging of corneal nerves in vivo are spawning questions regarding the relationship between corneal nerve density, morphology, and function. We tested whether noxious stimulation of the corneal surface can alter nerve morphology and neurochemistry. We used concentrations of menthol, capsaicin, and hypertonic saline that evoked comparable levels of nocifensive eye wipe behaviors when applied to the ocular surface of an awake rat. Animals were sacrificed and corneal nerves were examined using immunocytochemistry and three-dimensional volumetric analyses. We found that menthol and capsaicin both caused a significant reduction in corneal nerve density as detected with β-tubulin immunoreactivity 2 hr after stimulation. Hypertonic saline did not reduce nerve density, but did cause qualitative changes in nerves including enlarged varicosities that were also seen following capsaicin and menthol stimulation. All three types of noxious stimuli caused a depletion of CGRP from corneal nerves, indicating that all modalities of noxious stimuli evoked peptide release. Our findings suggest that studies aimed at understanding the relationship between corneal nerve morphology and chronic disease may also need to consider the effects of acute stimulation on corneal nerve morphology.

Differential response of pineal microglia to surgical versus pharmacological stimuli

“…The mouse monoclonal anti‐neuronal class III β–tubulin (Tuj1; BioLegend, code #801201 and 801,202, RRID: http://scicrunch.org/resolver/AB_10063408; Covance, code #MMS‐435P, RRID: http://scicrunch.org/resolver/AB_2313773) is a well‐characterized antibody that was generated using microtubules from rat brain (see manufacturer's technical information). This antibody recognizes a single ∼50 kDa protein band on WBs (Benitez, Castro, Patterson, Muñoz, & Seltzer, ), and intact and degenerated nerve fibers by IHC (Hegarty, Hermes, Yang, & Aicher, ). We used this anti‐Tuj1 antibody to stain nerve fibers in the postnatal pineal gland (Ibañez Rodriguez et al, ; Yu et al, ).…”

Acute hyperalgesia and delayed dry eye after corneal abrasion injury

“… 1 , 12 , 40 Two observers counted the number of ipsilateral eye wipes for 3 minutes after menthol stimulation. 24 Rats were habituated to handling the testing apparatus and testing room for 40 minutes per day for 2 days before nociceptive testing. Each rat received a single corneal menthol application to avoid sensitization; thus, separate groups were used for 24-hour and 1-week assessments.…”

“…Rats were overdosed with sodium pentobarbital (150 mg/kg) and perfused transcardially through the ascending aorta with 10 mL of heparinized saline (1000 units/mL) followed by 600 mL of 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. 24 , 27 The eyes were enucleated immediately after perfusion and placed in PB. Using a 5 mm corneal trephine (Ambler Surgical, Exton, PA), the central corneas were removed and placed in fresh PB at 4°C until immunoprocessing.…”

“…Whole-mount corneas were processed free-floating, whereas TG were processed on slides as previously described. 1 , 23 , 24 Tissue was incubated in 1% sodium borohydride solution for 30 minutes before primary antibody incubation. Corneas were incubated for 3 nights, and TG sections were incubated for 2 nights at 4°C in primary antibody cocktails in 0.25% Triton X-100 (Sigma-Aldrich, St. Louis, MO) and 0.1% bovine serum albumin (Sigma) in 0.1 M Tris-buffered saline pH 7.6 (Table 1 ).…”

Lacrimal Gland Denervation Alters Tear Protein Composition and Impairs Ipsilateral Eye Closures and Corneal Nociception

“…Awake rats were lightly restrained while 10 μL 50 mM menthol (Sigma-Aldrich, St. Louis, MO, USA) was applied to the ocular surface. 12 Ipsilateral eye wipes with the forelimb were counted for 3 minutes. Facial grooming behaviors or hind paw scratches were not included, as these are not validated measures of eye pain.…”