Abstract
Purpose:
Some dry eye disease (DED) patients have sensitized responses to corneal stimulation, while others experience hypoalgesia. Many patients have normal tear production, suggesting that reduced tears are not always the cause of DED sensory dysfunction. In this study, we show that disruption of lacrimal innervation can produce hypoalgesia without changing basal tear production.
Methods:
Injection of a saporin toxin conjugate into the extraorbital lacrimal gland of male Sprague-Dawley rats was used to disrupt cholinergic innervation to the gland. Tear production was assessed by phenol thread test. Corneal sensory responses to noxious stimuli were assessed using eye wipe behavior. Saporin DED animals were compared to animals treated with atropine to produce aqueous DED.
Results:
Cholinergic innervation and acetylcholine content of the lacrimal gland were significantly reduced in saporin DED animals, yet basal tear production was normal. Saporin DED animals demonstrated normal eye wipe responses to corneal application of capsaicin, but showed hypoalgesia to corneal menthol. Corneal nerve fiber density was normal in saporin DED animals. Atropine-treated animals had reduced tear production but normal responses to ocular stimuli.
Conclusions:
Because only menthol responses were impaired, cold-sensitive corneal afferents appear to be selectively altered in our saporin DED model. Hypoalgesia is not due to reduced tear production, since we did not observe hypoalgesia in an atropine DED model. Corneal fiber density is unaltered in saporin DED animals, suggesting that molecular mechanisms of nociceptive signaling may be impaired. The saporin DED model will be useful for exploring the mechanism underlying corneal hypoalgesia.
Dry eye disease (DED) represents a group of disorders related to disruption of lacrimal function; a primary feature is an altered sensory perception of corneal stimuli. Patients with DED demonstrate either increased or decreased responses to noxious corneal stimulation and sometimes experience spontaneous pain, hyperalgesia, or allodynia.
1–5 Changes in corneal sensory perception in DED have been postulated to be the result of sensitization of corneal sensory fibers due to an aqueous deficit at the ocular surface. Paradoxically, many DED patients do not have dry eyes or overt loss of lacrimal function. Numerous findings support the notion that basal tear production is not a good indicator of corneal sensory dysfunction.
5,6 A recent study found that DED symptoms were significantly associated with nonocular pain and depression, but were not correlated with tear film measurements.
7 In the present study we used two methods to disrupt the tear reflex circuit to determine the effect on sensory responses to noxious corneal stimulation.
Tear production, as well as pain, can be evoked by corneal stimulation. The reflex for tear production involves motor neurons within the superior salivatory nucleus (SSN),
8 which send projections to parasympathetic cholinergic motor neurons in the pterygopalatine ganglion (PPG) that innervate the lacrimal gland and evoke tear production through stimulation of the acini within the gland (
Fig. 1, dotted lines).
9 In contrast, the reflex pathway involving the sensory perception of noxious corneal stimuli involves a pathway from the cornea to the trigeminal dorsal horn to neurons in the parabrachial nuclei
10,11 and higher brain centers (
Fig. 1, solid lines). The motor response to noxious stimulation of the cornea involves stereotypical eye wipe behaviors with the ipsilateral forelimb.
12
We will contrast two models, one that involves pharmacologic blockade of cholinergic receptors to reduce tear production, and a novel rat model of DED that we call saporin DED, which involves denervation of the extraorbital lacrimal gland using a saporin toxin conjugated to a monoclonal antibody that binds to the p75 neurotrophin receptor (p75
NTR).
13 The ribosome-inactivating saporin toxin is injected into the gland where it is taken up by nerves expressing p75
NTR and transported to their cell bodies in the PPG, specifically ablating those nerves (
Fig. 1).
13 There are a variety of conditions in which lacrimal gland nerves are damaged by either an immune response or chemical exposure, and thus we felt that specifically damaging the nerves in the lacrimal gland might yield a scientifically interesting DED model by disrupting the lacrimal reflex pathway. To assess change in corneal nociception, we test responses to ocular stimuli that activate specific classes of nociceptors. Capsaicin activates sensory fibers that contain TRPV1, which is also responsive to noxious heat stimulation,
14 and is present in corneal polymodal nociceptors.
15 Menthol activates cold-sensitive fibers,
16 which represent a smaller but distinct population of corneal nociceptors that have been implicated in dry eye mechanisms.
17 Since responses to capsaicin and menthol are mediated by distinct molecular mechanisms, it is important to assess nociceptive responses to stimuli representing distinct modalities.
Methyl Atropine Model.
Saporin Model.
Rats were deeply anesthetized with vaporized isoflurane in oxygen (5% for induction, 2%–3% maintenance), and the left extraorbital lacrimal gland was isolated. 192-IgG-saporin (5 μL, 0.5 μg/μL; Advanced Targeting Systems, San Diego, CA, USA) was microinjected into the gland through a glass pipette. Trypan blue was included in the solution to monitor the spread of the injectate, and cotton swabs were used to prevent spread of the saporin toxin to other tissues. The incision was closed with 3-0 monocryl suture (Ethicon, Cornelia, GA, USA) and covered with anesthetic ointment. The rats were returned to their home cage and monitored during recovery from anesthesia. Control rats were injected with vehicle (phosphate-buffered saline [PBS]) into the gland.
Inflammation Model.
Fresh, whole extraorbital lacrimal glands were excised, weighed, and immediately transferred to prechilled tubes on dry ice and stored at −80°C until processed. Acetylcholine was measured by isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) using a Shimadzu Prominence UPLC with a Scherzo SS-C18 50 × 2-mm 3 μm column interfaced to a 4000 Q TRAP mass spectrometer (Applied Biosystems, Foster City, CA, USA) with an electrospray ionization source in positive mode. Multiple reaction monitoring was used monitoring the [M+H]+ transitions of m/z 146 → 87 and m/z 150 → 91 for acetylcholine and d4-acetylcholine internal standard, respectively. Standards containing 1 to 200 ng/mL acetylcholine in 0.1 M perchloric acid containing 10 ng/mL d4-acetylcholine were prepared for each sample set.
Statistical analyses were performed using SigmaPlot 12.0 software (Systat Software, Inc., San Jose, CA, USA).
A one-way ANOVA with Holm-Sidak post hoc test was used to compare weights of left and right extraorbital lacrimal glands from saporin and control animals. The same test was used to compare acetylcholine (ACh) levels in saporin and control animals. This analysis allowed us to not only verify effectiveness of saporin lesions, but also determine if there were compensatory responses in the contralateral gland. An independent samples t-test was used to compare the mean area fractions of nerve fibers innervating the saporin-injected and naïve extraorbital lacrimal glands, as well as corneal fiber densities between saporin and control animals. This test was also used to compare the mean number of stimulus-evoked eye wipes of the saporin DED and MA DED models compared to controls. Paired t-tests were used for within-animal comparisons of phenol thread measurements taken prior to treatment (baseline) and at the endpoint of each DED model. We used a Kruskal-Wallis one-way ANOVA on ranks with Dunn's post hoc test to compare percent changes in phenol thread measurements among control, saporin, and MA DED rats. In all cases, a P value less than 0.05 was considered significant.
Saporin Toxin-Induced Denervation Reduced Lacrimal Gland Weight, ACh Content, and Innervation
Dose-Dependent Eye Wipe Behaviors Evoked by Corneal Stimulation With Capsaicin and Menthol
The authors thank Dennis Koop and Jenny Luo of the Oregon Health & Science University Bioanalytical Shared Resources/Pharmacokinetics Core for their assistance with the acetylcholine assay.
Supported by National Institutes of Health Grants DE12640 (SAA, DMH, SMH) and P30 NS061800.
Disclosure: S.A. Aicher, None; S.M. Hermes, None; D.M. Hegarty, None