Tear Proteins Altered in Patients with Persistent Eye Pain after Refractive Surgery: Biomarker Candidate Discovery.

Some patients develop persistent eye pain after refractive surgery, but factors that cause or sustain pain are unknown. We tested whether tear proteins of patients with pain 3 months after surgery differ from those of patients without pain. Patients undergoing refractive surgery (laser in situ keratomileusis or photorefractive keratectomy ) were recruited from 2 clinics, and tears were collected 3 months after surgery. Participants rated their eye pain using a numerical rating scale (NRS, 0-10; no pain-worst pain) at baseline, 1 day, and 3 months after surgery. Using tandem mass tag proteomic analysis, we examined tears from patients with pain [NRS ≥ 3 at 3 months (n = 16)] and patients with no pain [NRS ≤ 1 at 3 months (n = 32)] after surgery. A subset of proteins (83 of 2748 detected, 3.0%) were associated with pain 3 months after surgery. High-dimensional statistical models showed that the magnitude of differential expression was not the only important factor in classifying tear samples from pain patients. Models utilizing 3 or 4 proteins had better classification performance than single proteins and represented differences in both directions (higher or lower in pain). Thus, patterns of protein differences may serve as biomarkers of postsurgical eye pain as well as potential therapeutic targets.

Perineuronal nets in the rat medial prefrontal cortex alter hippocampal-prefrontal oscillations and reshape cocaine self-administration memories.

The medial prefrontal cortex (mPFC) is a major contributor to relapse to cocaine in humans and to reinstatement behavior in rodent models of cocaine use disorder. Output from the mPFC is modulated by parvalbumin (PV)-containing fast-spiking interneurons, the majority of which are surrounded by perineuronal nets (PNNs). Here we tested whether chondroitinase ABC (ABC)- mediated removal of PNNs prevented the acquisition or reconsolidation of a cocaine self-administration memory. ABC injections into the dorsal mPFC prior to training attenuated the acquisition of cocaine self-administration. Also, ABC given 3 days prior to but not 1 hr after memory reactivation blocked cue-induced reinstatement. However, reduced reinstatement was present only in rats given a novel reactivation contingency, suggesting that PNNs are required for the updating of a familiar memory. In naive rats, ABC injections into mPFC did not alter excitatory or inhibitory puncta on PV cells but reduced PV intensity. Whole-cell recordings revealed a greater inter-spike interval 1 hr after ABC, but not 3 days later. In vivo recordings from the mPFC and dorsal hippocampus (dHIP) during novel memory reactivation revealed that ABC in the mPFC prevented reward-associated increases in beta and gamma activity as well as phase-amplitude coupling between the dHIP and mPFC. Together, our findings show that PNN removal attenuates the acquisition of cocaine self-administration memories and disrupts reconsolidation of the original memory when combined with a novel reactivation session. Further, reduced dHIP/mPFC coupling after PNN removal may serve as a key biomarker for how to disrupt reconsolidation of cocaine memories and reduce relapse.

Ischemia-reperfusion myocardial infarction induces remodeling of left cardiac-projecting stellate ganglia neurons.

Neurons in the stellate ganglion (SG) provide sympathetic innervation to the heart, brown adipose tissue (BAT), and other organs. Sympathetic innervation to the heart becomes hyperactive following myocardial infarction (MI). The impact of MI on the morphology of cardiac sympathetic neurons is not known, but we hypothesized that MI would stimulate increased cell and dendritic tree size in cardiac neurons. In this study, we examined the effects of ischemia-reperfusion MI on sympathetic neurons using dual retrograde tracing methods to allow detailed characterization of cardiac- and BAT-projecting neurons. Different fluorescently conjugated cholera toxin subunit B (CTb) tracers were injected into the pericardium and the interscapular BAT pads, respectively. Experimental animals received a 45-min occlusion of the left anterior descending coronary artery and controls received sham surgery. One week later, hearts were collected for assessment of MI infarct and SGs were collected for morphological or electrophysiological analysis. Cardiac-projecting SG neurons from MI mice had smaller cell bodies and shorter dendritic trees compared with sham animals, specifically on the left side ipsilateral to the MI. BAT-projecting neurons were not altered by MI, demonstrating the subpopulation specificity of the response. The normal size and distribution differences between BAT- and cardiac-projecting stellate ganglion neurons were not altered by MI. Patch-clamp recordings from cardiac-projecting left SG neurons revealed increased spontaneous excitatory postsynaptic currents despite the decrease in cell and dendritic tree size. Thus, increased dendritic tree size does not contribute to the enhanced sympathetic neural activity seen after MI.NEW & NOTEWORTHY Myocardial infarction (MI) causes structural and functional changes specifically in stellate ganglion neurons that project to the heart, but not in cells that project to brown adipose fat tissue.

Ocular Pain after Refractive Surgery: Interim Analysis of Frequency and Risk Factors.

PURPOSE: To examine the frequency and risk factors for ocular pain after laser assisted in situ keratomileusis (LASIK) and photorefractive keratectomy (PRK). DESIGN: Prospective study of individuals undergoing refractive surgery at 2 different centers. PARTICIPANTS: One hundred nine individuals undergoing refractive surgery: 87% LASIK and 13% PRK. METHODS: Participants rated ocular pain on a numerical rating scale (NRS) of 0 to 10 before surgery and 1 day, 3 months, and 6 months after surgery. A clinical examination focused on ocular surface health was performed 3 and 6 months after surgery. Persistent ocular pain was defined as an NRS score of 3 or more at both 3 and 6 months after surgery (patients), and this group was compared with individuals with NRS scores of < 3 at both time points (control participants). MAIN OUTCOME MEASURES: Individuals with persistent ocular pain after refractive surgery. RESULTS: The 109 patients who underwent refractive surgery were followed up for 6 months after surgery. Mean age was 34 ± 8 years (range, 23-57 years); 62% self-identified as female, 81% as White, and 33% as Hispanic. Eight patients (7%) reported ocular pain (NRS score ≥ 3) before surgery, with the frequency of ocular pain increasing after surgery to 23% (n = 25) at 3 months and 24% (n = 26) at 6 months. Twelve patients (11%) reported an NRS score of 3 or more at both time points and constituted the persistent pain group. Factors that predicted persistent pain after surgery in a multivariable analysis were (1) ocular pain before surgery predicated persistent pain after surgery (odds ratio [OR], 1.87; 95% confidence interval [CI], 1.06-3.31), (2) symptom report of depression before surgery (Patient Health Questionnaire-9: OR, 1.3; 95% CI, 1.1-1.6; P = 0.01), (3) use of an oral antiallergy medication before surgery (OR, 13.6; 95% CI, 2.1-89.3; P = 0.007), and (4) pain intensity day 1 after surgery (OR, 1.6; 95% CI, 1.2-2.2; P = 0.005). There were no significant associations between ocular surface signs of tear dysfunction and ocular pain, P > 0.05 for all ocular surface signs. Most individuals (> 90%) were completely or somewhat satisfied with their vision at 3 and 6 months. CONCLUSIONS: Eleven percent of individuals reported persistent ocular pain after refractive surgery, with several preoperative and perioperative factors predicting pain after surgery. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Experimental design considerations for studies of human tear proteins.

PURPOSE: Human tears contain abundant, diverse sets of proteins that may serve as biomarkers of ocular surface health. There is a need for reproducible methods that consider multiple factors influencing the tear proteome, in addition to the variable of interest. Here we examined a workflow for proteomic analysis of tear proteins without the need to pool tear samples from multiple individuals, thus allowing for analyses based on individual factors, and increasing opportunities for protein biomarker discovery. METHODS: Tears were collected by Schirmer strip following topical ocular anesthetic application then individually stored at -80 °C prior to processing for proteomics. Tear proteins were extracted from Schirmer strips, digested using suspension trapping spin columns (S-Trap), and labeled with high multiplicity tandem mass tags (TMT). Peptide digests were then extensively fractionated by two-dimensional chromatography and analyzed by mass spectrometry to identify and measure changes in protein abundance in each sample. Analysis of select samples was performed to test protocols and to compare the impact of clinically relevant parameters. To facilitate comparison of separate TMT experiments, common pool samples were included in each TMT instrument run and internal reference scaling (IRS) was performed. RESULTS: Differences in subsets of tear proteins were noted for: geographic site of tear collection, contact lens use, and differences in tear fluid volume among individuals. CONCLUSION: These findings demonstrate that proteomic analysis of human tear proteins can be performed without the need to pool samples, and that development of analytic workflows must consider factors that may affect outcomes in studies focused on diverse clinical samples.

Resveratrol increases tear production and ocular pain after corneal abrasion in male, but not female, rats using a photorefractive keratectomy model.

Photorefractive keratectomy (PRK) is an alternative to LASIK and can cause intense acute pain that is often not relieved by standard treatments. To assess potential therapeutics for this type of acute pain, appropriate preclinical models are needed. We describe a preclinical corneal abrasion rat model that simulates the initial stages of PRK surgery and demonstrates similar pain and tear dysfunction as seen clinically. We used both behavioral and homeostatic assays to determine the therapeutic potential of resveratrol on pain and tear production. Studies were conducted in male and female Sprague-Dawley rats. Heptanol was applied to one eye and the superficial corneal epithelium was removed, mimicking the abrasion used in PRK. Spontaneous pain was assessed with orbital tightening (OT) scores for 7 days. Topical resveratrol increased OT scores sex-specifically in abraded males, but not females, at 72 h and 1 week after abrasion. Resveratrol increased tear production in abraded males, with no effect in abraded females. There was no correlation between OT score at 1 week and tear production measurements, demonstrating no relationship between spontaneous ocular pain and tear dysfunction in this model. These findings demonstrate the usefulness of our corneal abrasion preclinical PRK model for the assessment of ocular pain therapeutics and indicate that topical resveratrol may not be useful for managing PRK-induced pain.

Distinct morphology of cardiac- and brown adipose tissue-projecting neurons in the stellate ganglia of mice.

Sympathetic neurons that innervate the heart are located primarily in the stellate ganglia (SG), which also contains neurons that project to brown adipose tissue (BAT). These studies were designed to examine the morphology of these two populations (cardiac- and BAT-projecting) and their target connectivity. We examined SG neurons in C57BL/6J mice following injections of the retrograde tracer cholera toxin B (CTb) conjugated to Alexa Fluor 488 and Alexa Fluor 555, into cardiac tissue and intrascapular BAT. BAT-projecting SG neurons were widely dispersed in SG, while cardiac-projecting SG neurons were localized primarily near the inferior cardiac nerve base. SG neurons were not dual-labeled, suggesting that sympathetic innervation is specific to the heart and BAT, supporting the idea of "labeled lines" of efferents. Morphologically, cardiac-projecting SG somata had more volume and were less abundant than BAT-projecting neurons using our tracer-labeling paradigm. We found a positive correlation between the number of primary dendrites per neuron and soma volume in cardiac-projecting SG neurons, though not in BAT-projecting neurons. In both SG subpopulations, the number of cholinergic inputs marked with vesicular acetylcholine transporter (VAChT) puncta contacting the soma was positively correlated to soma volume, suggesting scaling of inputs across a range of neuronal sizes. In separate studies using dual tracing from left and right BAT, we found that BAT-projecting SG neurons were located predominately ipsilateral to the injection, but a small subset of SG neurons project bilaterally to BAT. This tracing approach will allow the assessment of cell-specific mechanisms of plasticity within subpopulations of SG neurons.

Nicotinamide Riboside Alleviates Corneal and Somatic Hypersensitivity Induced by Paclitaxel in Male Rats.

Purpose: Patients receiving chemotherapy may experience ocular discomfort and dry eye-like symptoms; the latter may be neuropathic in nature. This study assessed corneal and somatic hypersensitivity in male rats treated with paclitaxel and whether it was relieved by nicotinamide riboside (NR). Methods: Corneal sensitivity to tactile and chemical stimulation, basal tear production, and sensitivity of the hindpaw to tactile and cool stimuli were assessed before and after paclitaxel in the absence and presence of sustained treatment with 500 mg/kg per os NR. Corneal nerve density and hindpaw intraepidermal nerve fiber (IENF) density were also examined. Results: Paclitaxel-treated rats developed corneal hypersensitivity to tactile stimuli, enhanced sensitivity to capsaicin but not hyperosmolar saline, and increased basal tear production. Corneal nerve density visualized with anti-ß-tubulin or calcitonin gene-related peptide (CGRP) was unaffected. Paclitaxel induced tactile and cool hypersensitivity of the hindpaw and a loss of nonpeptidergic hindpaw IENFs visualized with anti-protein gene product (PGP) 9.5 and CGRP. NR reversed tactile hypersensitivity of the cornea without suppressing tear production or chemosensitivity; it did not alter corneal afferent density. NR also reversed tactile and cool hypersensitivity of the hindpaw without reversing the loss of hindpaw IENFs. Conclusions: These findings suggest that paclitaxel may be a good translational model for chemotherapy-induced ocular discomfort and that NR may be useful for its relief. The ability of NR to relieve somatic tactile hypersensitivity independent of changes in sensory nerve innervation suggests that reversal of terminal arbor degeneration is not critical to the actions of NR.

Dedicated C-fiber vagal sensory afferent pathways to the paraventricular nucleus of the hypothalamus.

The nucleus of the solitary tract (NTS) receives viscerosensory information from the vagus nerve to regulate diverse homeostatic reflex functions. The NTS projects to a wide network of other brain regions, including the paraventricular nucleus of the hypothalamus (PVN). Here we examined the synaptic characteristics of primary afferent pathways to PVN-projecting NTS neurons in rat brainstem slices.Expression of the Transient Receptor Potential Vanilloid receptor (TRPV1+ ) distinguishes C-fiber afferents within the solitary tract (ST) from A-fibers (TRPV1-). We used resiniferatoxin (RTX), a TRPV1 agonist, to differentiate the two. The variability in the latency (jitter) of evoked excitatory postsynaptic currents (ST-EPSCs) distinguished monosynaptic from polysynaptic ST-EPSCs. Rhodamine injected into PVN was retrogradely transported to identify PVN-projecting NTS neurons within brainstem slices. Graded shocks to the ST elicited all-or-none EPSCs in rhodamine-positive NTS neurons with latencies that had either low jitter (<200 µs - monosynaptic), high jitter (>200 µs - polysynaptic inputs) or both. RTX blocked ST-evoked TRPV1 + EPSCs whether mono- or polysynaptic. Most PVN-projecting NTS neurons (17/21 neurons) had at least one input polysynaptically connected to the ST. Compared to unlabeled NTS neurons, PVN-projecting NTS neurons were more likely to receive indirect inputs and be higher order. Surprisingly, sEPSC rates for PVN-projecting neurons were double that of unlabeled NTS neurons. The ST synaptic responses for PVN-projecting NTS neurons were either all TRPV1+ or all TRPV1-, including neurons that received both direct and indirect inputs. Overall, PVN-projecting NTS neurons received direct and indirect vagal afferent information with strict segregation regarding TRPV1 expression.

Diurnal changes in perineuronal nets and parvalbumin neurons in the rat medial prefrontal cortex.

Perineuronal nets (PNNs) surrounding fast-spiking, parvalbumin (PV) interneurons provide excitatory:inhibitory balance, which is impaired in several disorders associated with altered diurnal rhythms, yet few studies have examined diurnal rhythms of PNNs or PV cells. We measured the intensity and number of PV cells and PNNs labeled with Wisteria floribunda agglutinin (WFA) and also the oxidative stress marker 8-oxo-deoxyguanosine (8-oxo-dG) in rat prelimbic medial prefrontal cortex (mPFC) at Zeitgeber times (ZT) ZT0 (lights-on, inactive phase), ZT6 (mid-inactive phase), ZT12 (lights-off, active phase), and ZT18 (mid-active phase). Relative to ZT0, the intensities of PNN and PV labeling were increased in the dark (active) phase compared with the light (inactive) phase. The intensity of 8-oxo-dG was decreased from ZT0 at all times (ZT6,12,18). We also measured GAD 65/67 and vGLUT1 puncta apposed to PV cells with and without PNNs. There were more excitatory puncta on PV cells with PNNs at ZT18 vs. ZT6, but no changes in PV cells without PNNs and no changes in inhibitory puncta. Whole-cell slice recordings in fast-spiking (PV) cells with PNNs showed an increased ratio of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor:N-methyl-D-aspartate receptor (AMPA: NMDA) at ZT18 vs. ZT6. The number of PV cells and PV/PNN cells containing orthodenticle homeobox 2 (OTX2), which maintains PNNs, showed a strong trend toward an increase from ZT6 to ZT18. Diurnal fluctuations in PNNs and PV cells are expected to alter cortical excitatory:inhibitory balance and provide new insights into treatments for diseases impacted by disturbances in sleep and circadian rhythms.

Cocaine memory reactivation induces functional adaptations within parvalbumin interneurons in the rat medial prefrontal cortex.

Substance use disorder is a complex disease created in part by maladaptive learning and memory mechanisms following repeated drug use. Exposure to drug-associated stimuli engages prefrontal cortex circuits, and dysfunction of the medial prefrontal cortex (mPFC) is thought to underlie drug-seeking behaviors. Growing evidence supports a role for parvalbumin containing fast-spiking interneurons (FSI) in modulating prefrontal cortical microcircuit activity by influencing the balance of excitation and inhibition, which can influence learning and memory processes. Most parvalbumin FSIs within layer V of the prelimbic mPFC are surrounded by specialized extracellular matrix structures called perineuronal nets (PNN). Previous work by our group found that cocaine exposure altered PNN-surrounded FSI function, and pharmacological removal of PNNs reduced cocaine-seeking behavior. However, the role of FSIs and associated constituents (parvalbumin and PNNs) in cocaine-related memories was not previously explored and is still unknown. Here, we found that reactivation of a cocaine conditioned place preference memory produced changes in cortical PNN-surrounded parvalbumin FSIs, including decreased parvalbumin intensity, increased parvalbumin cell axis diameter, decreased intrinsic excitability, and increased excitatory synaptic input. Further investigation of intrinsic properties revealed changes in the interspike interval, membrane capacitance, and afterhyperpolarization recovery time. Changes in these specific properties suggest an increase in potassium-mediated currents, which was validated with additional electrophysiological analysis. Collectively, our results indicate that cocaine memory reactivation induces functional adaptations in PNN-surrounded parvalbumin neurons, which likely alters cortical output to promote cocaine-seeking behavior.

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

Purpose: To evaluate spontaneous and evoked ocular sensory responses in rats after denervation of the lacrimal gland, as well as protein changes in tears that may mediate functional changes. Methods: Sprague-Dawley rats served as subjects. The left lacrimal gland was partially denervated with saporin toxin conjugated to p75. Unilateral and bilateral eye closures (winks and blinks) and grooming behaviors were measured weekly. Nociceptive responses were evoked by ocular application of menthol; tear production was assessed using the phenol thread test. Relative changes in tear protein abundances were measured using a Tandem Mass Tagging approach. Results: Denervation of the lacrimal gland reduced eye closure behavior, particularly in the ipsilateral eye, and eye wipe responses to noxious menthol were also reduced. Tear volume did not change, but tear protein composition was altered. Proteins implicated in the structural integrity of epithelial cells and in protective functions were reduced by lacrimal denervation, including keratins, serotransferrin, and beta-defensin. Other proteins that may modulate TRPM8 channels and alter sensory neuronal function were reduced, including arachidonate 15-lipoxygenase B. A low-abundance protein that responds to oxidative stress and injury, proteasome subunit beta type 10, was upregulated in denervated rats. Conclusions: Denervation of the lacrimal gland causes long-lasting hypoalgesia, impairs the blink response, and alters tear proteins. Tear proteins were altered without changing tear volume. We speculate that impaired TRPM8 function in corneal sensory nerves may contribute to ocular hypoalgesia, supporting growing evidence that this transduction molecule is important for both nociceptive and spontaneous blinking behaviors.

Cocaine Exposure Modulates Perineuronal Nets and Synaptic Excitability of Fast-Spiking Interneurons in the Medial Prefrontal Cortex.

We previously reported that perineuronal nets (PNNs) are required for cocaine-associated memories. Perineuronal nets are extracellular matrix that primarily surrounds parvalbumin (PV)-containing, GABAergic fast-spiking interneurons (FSIs) in the medial prefrontal cortex (mPFC). Here we measured the impact of acute (1 d) or repeated (5 d) cocaine exposure on PNNs and PV cells within the prelimbic and infralimbic regions of the mPFC. Adult rats were exposed to 1 or 5 d of cocaine and stained for PNNs (using Wisteria floribunda agglutinin) and PV intensity 2 or 24 h later. In the prelimbic and infralimbic PFC, PNN staining intensity decreased 2 h after 1 d of cocaine exposure but increased after 5 d of cocaine exposure. Cocaine also produced changes in PV intensity, which generally lagged behind that of PNNs. In the prelimbic PFC, both 1 and 5 d of cocaine exposure increased GAD65/67 puncta near PNN-surrounded PV cells, with an increase in the GAD65/67-to-VGluT1 puncta ratio after 5 d of cocaine exposure. In the prelimbic PFC, slice electrophysiology studies in FSIs surrounded by PNNs revealed that both 1 and 5 d of cocaine exposure reduced the number of action potentials 2 h later. Synaptic changes demonstrated that 5 d of cocaine exposure increased the inhibition of FSIs, potentially reducing the inhibition of pyramidal neurons and contributing to their hyperexcitability during relapse behavior. These early and rapid responses to cocaine may alter the network stability of PV FSIs that partially mediate the persistent and chronic nature of drug addiction.

Acute hyperalgesia and delayed dry eye after corneal abrasion injury.

INTRODUCTION: Corneal nerves mediate pain from the ocular surface, lacrimation, and blinking, all of which protect corneal surface homeostasis and help preserve vision. Because pain, lacrimation and blinking are rarely assessed at the same time, it is not known whether these responses and their underlying mechanisms have similar temporal dynamics after acute corneal injury. METHODS: We examined changes in corneal nerve density, evoked and spontaneous pain, and ocular homeostasis in Sprague-Dawley male rats after a superficial epithelial injury with heptanol. We also measured changes in calcitonin gene-related peptide (CGRP), which has been implicated in both pain and epithelial repair. RESULTS: Hyperalgesia was seen 24 hours after abrasion injury, while basal tear production was normal. One week after abrasion injury, pain responses had returned to baseline levels and dry eye symptoms emerged. There was no correlation between epithelial nerve density and pain responses. Expression of both ATF3 (a nerve injury marker) and CGRP increased in trigeminal ganglia 24 hours after injury when hyperalgesia was seen, and returned to normal one week later when pain behavior was normal. These molecular changes were absent in the contralateral ganglion, despite reductions in corneal epithelial nerve density in the uninjured eye. By contrast, CGRP was upregulated in peripheral corneal endings 1 week after injury, when dry eye symptoms emerged. CONCLUSION: Our results demonstrate dynamic trafficking of CGRP within trigeminal sensory nerves following corneal injury, with elevations in the ganglion correlated with pain behaviors and elevations in peripheral endings correlated with dry eye symptoms.

Select noxious stimuli induce changes on corneal nerve morphology.

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 ß-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.

Denervation of the Lacrimal Gland Leads to Corneal Hypoalgesia in a Novel Rat Model of Aqueous Dry Eye Disease.

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.

Capsaicin-responsive corneal afferents do not contain TRPV1 at their central terminals in trigeminal nucleus caudalis in rats.

We examined the substrates for ocular nociception in adult male Sprague-Dawley rats. Capsaicin application to the ocular surface in awake rats evoked nocifensive responses and suppressed spontaneous grooming responses. Thus, peripheral capsaicin was able to activate the central pathways encoding ocular nociception. Our capsaicin stimulus evoked c-Fos expression in a select population of neurons within rostral trigeminal nucleus caudalis in anesthetized rats. These activated neurons also received direct contacts from corneal afferent fibers traced with cholera toxin B from the corneal surface. However, the central terminals of the corneal afferents that contacted capsaicin-activated trigeminal neurons did not contain TRPV1. To determine if TRPV1 expression had been altered by capsaicin stimulation, we examined TRPV1 content of corneal afferents in animals that did not receive capsaicin stimulation. These studies confirmed that while TRPV1 was present in 30% of CTb-labeled corneal afferent neurons within the trigeminal ganglion, TRPV1 was only detected in 2% of the central terminals of these corneal afferents within the trigeminal nucleus caudalis. Other TRP channels were also present in low proportions of central corneal afferent terminals in unstimulated animals (TRPM8, 2%; TRPA1, 10%). These findings indicate that a pathway from the cornea to rostral trigeminal nucleus caudalis is involved in corneal nociceptive transmission, but that central TRP channel expression is unrelated to the type of stimulus transduced by the peripheral nociceptive endings.

Physiological temperatures drive glutamate release onto trigeminal superficial dorsal horn neurons.

Trigeminal sensory afferent fibers terminating in nucleus caudalis (Vc) relay sensory information from craniofacial regions to the brain and are known to express transient receptor potential (TRP) ion channels. TRP channels are activated by H(+), thermal, and chemical stimuli. The present study investigated the relationships among the spontaneous release of glutamate, temperature, and TRPV1 localization at synapses in the Vc. Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded from Vc neurons (n = 151) in horizontal brain-stem slices obtained from Sprague-Dawley rats. Neurons had basal sEPSC rates that fell into two distinct frequency categories: High (≥10 Hz) or Low (<10 Hz) at 35°C. Of all recorded neurons, those with High basal release rates (67%) at near-physiological temperatures greatly reduced their sEPSC rate when cooled to 30°C without amplitude changes. Such responses persisted during blockade of action potentials indicating that the High rate of glutamate release arises from presynaptic thermal mechanisms. Neurons with Low basal frequencies (33%) showed minor thermal changes in sEPSC rate that were abolished after addition of TTX, suggesting these responses were indirect and required local circuits. Activation of TRPV1 with capsaicin (100 nM) increased miniature EPSC (mEPSC) frequency in 70% of neurons, but half of these neurons had Low basal mEPSC rates and no temperature sensitivity. Our evidence indicates that normal temperatures (35-37°C) drive spontaneous excitatory synaptic activity within superficial Vc by a mechanism independent of presynaptic action potentials. Thus thermally sensitive inputs on superficial Vc neurons may tonically activate these neurons without afferent stimulation.

Corneal pain activates a trigemino-parabrachial pathway in rats.

Corneal pain is mediated by primary afferent fibers projecting to the dorsal horn of the medulla, specifically the trigeminal nucleus caudalis. In contrast to reflex responses, the conscious perception of pain requires transmission of neural activity to higher brain centers. Ascending pain transmission is mediated primarily by pathways to either the thalamus or parabrachial nuclei. We previously showed that some corneal afferent fibers preferentially contact parabrachial-projecting neurons in the rostral pole of the trigeminal nucleus caudalis, but the role of these projection neurons in transmitting noxious information from the cornea has not been established. In the present study, we show that noxious stimulation of the corneal surface activates neurons in the rostral pole of the nucleus caudalis, including parabrachially projecting neurons that receive direct input from corneal afferent fibers. We used immunocytochemical detection of c-Fos protein as an index of neuronal activation after noxious ocular stimulation. Animals had previously received injections of a retrograde tracer into either thalamic or parabrachial nuclei to identify projection neurons in the trigeminal dorsal horn. Noxious stimulation of the cornea induced c-Fos in neurons sending projections to parabrachial nuclei, but not thalamic nuclei. We also confirmed that corneal afferent fibers identified with cholera toxin B preferentially target trigeminal dorsal horn neurons projecting to the parabrachial nucleus. The parabrachial region sends ascending projections to brain regions involved in emotional and homeostatic responses. Activation of the ascending parabrachial system may explain the extraordinary salience of stimulation of corneal nociceptors.