Abstract
Background/Aims:
An objective marker is needed to detect when corneal nerve abnormalities underlie neuropathic corneal pain (NCP), as symptoms often overlap with those of dry eye (DE). This study evaluated microneuroma (MN) frequency in various populations and investigated relationships between MN presence and DE clinical features in individuals with DE symptoms but without a history of refractive surgery, in order to eliminate refractive surgery as a potential confounder of nerve abnormalities.
Methods:
This was a retrospective study that included individuals with and without DE symptoms who underwent a clinical evaluation for DE (symptom surveys and ocular surface evaluation) and in vivo confocal microscopy imaging. DE clinical features (including those suggestive of neuropathic pain) were compared based on MN presence using t-tests, Chi-square analyses, and Pearson’s correlation coefficients with 0.05 alpha level.
Results:
MN frequencies did not significantly differ between individuals with DE symptoms (Dry Eye Questionnaire 5 (DEQ-5) score ≥6) and a history of refractive surgery (n=1/16, 6%), individuals with DE symptoms without a history of refractive surgery (n=26/119, 22%), and individuals without DE symptoms (n=2/18, 11%, p=0.22). Among individuals with DE symptoms without a history of refractive surgery, DE clinical features, including those indicative of NCP (burning sensation and sensitivity to light, wind, and extreme temperatures), did not significantly differ based on MN presence (p>0.05).
Conclusion:
MN frequencies did not significantly differ between individuals with and without DE symptoms. Their presence alone could not distinguish between DE sub-types, including features of NCP in our study population.
Keywords: in vivo confocal microscopy, microneuroma, neuropathic corneal pain, dry eye
INTRODUCTION:
Dry eye (DE) is a common condition with significant morbidity. A challenge in DE is that symptoms of the disease are often discordant with ocular surface findings. One potential explanation for this disconnect is the variable function of nerves along the corneal somatosensory pathway. Specifically, these nerves can appropriately transmit information on the ocular surface environment (i.e. nociceptive pain), or they can become dysfunctional and submit signals inappropriately. The latter scenario can result in neuropathic pain, which is defined as “pain caused by a lesion or disease of the somatosensory nervous system” [1]. Neuropathic pain is generally split into categories (peripheral and central) based on the location of nerve dysfunction. Applied to the cornea, pain can arise at the level of the primary afferent corneal nerves and is thus labeled, neuropathic corneal pain (NCP), or it can arise from second and third order neurons at the levels of the brain stem, thalamus, or cortex. Another challenge in DE management is that symptoms of neuropathic pain overlap with symptoms of DE arising from ocular surface abnormalities. Individuals with both entities report sensations of “dryness”, “discomfort”, and “irritation”. We have found, however, that some symptoms are more suggestive of a neuropathic origin. These include “burning” and evoked pain to wind, light, or extreme temperatures [2]. While individuals with neuropathic pain often have disproportionately severe symptoms in comparison to clinical signs, individuals may also present with co-morbid ocular surface abnormalities [3]. As such, there is a need to identify when nerve dysfunction, including NCP, underlies patient symptoms.
Currently, there are no gold standard tests to confirm the presence of NCP, and thus a clinical diagnosis is derived from a combination of symptom assessments and clinical examination findings. Validated questionnaires such as the Ocular Pain Assessment Survey (OPAS) [4] and Neuropathic Pain Symptom Inventory modified for the eye (NPSI-Eye) [5] are used to subjectively quantify the intensity and quality of ocular pain. Corneal aesthesiometry is used to assess nerve sensitivity, with neuropathic pain associated with abnormal values (both low and high sensitivity) [3]. However, devices such as the Belmonte aesthesiometer are not frequently available in the clinical arena. Persistent ocular pain after the administration of a topical anesthetic, such as proparacaine, suggests a central neuropathic etiology. However, elimination of pain with administration cannot reliably differentiate between a nociceptive or peripheral neuropathic etiology [6]. These diagnostic limitations point to a need for objective methods to diagnose NCP in individuals with DE symptoms.
In vivo confocal microscopy (IVCM) shows promise in this regard. It is a non-invasive and reproducible imaging modality used for high resolution visualization of corneal structures at the cellular level. Multiple studies have characterized the corneal sub-basal nerve plexus (SNP) on IVCM images in individuals with NCP [6–9] and DE [10–12]. One feature that has been proposed as a biomarker of NCP is the presence of microneuromas (MNs), which are believed to represent severed nerve endings [8]. Animal studies have demonstrated that neuromas are common after nerve injury, alter nerve excitability, and are sources of ectopic impulse generation [13]. In one prior study, corneal MNs were reported to occur in 62.5% of individuals with clinically diagnosed NCP (“severe photoallodynia” with “normal slit-lamp examination”, n=16) [8], while in another study by the same group, corneal MNs were reported to be present in 100% of individuals (n=25) with clinically diagnosed NCP under a slightly variable definition (“chief complaint of ocular pain and/or light sensitivity, tear break-up time (TBUT) ≥10 s or Schirmer’s II score >10 mm, and with or without a maximum of trace corneal fluorescent staining”) [9]. However, the significance and extent of MN presence in NCP remains poorly understood.
To address this knowledge gap, we first evaluated MN frequency in individuals with versus without DE symptoms. We then focused on examining relationships between MNs and DE metrics in individuals with DE symptoms but without a history of refractive surgery. We hypothesized that MNs would be more common in individuals with versus without DE symptoms and that MNs would positively correlate with clinical features of “burning” eye pain and evoked pain to wind, light, and extreme temperatures, as these metrics have been previously shown to be suggestive of neuropathic ocular pain [2].
METHODS:
Study Population and Groups:
The study population included individuals (18 years of age and older) seen at the Miami Veterans Administration (VA) Medical Center who underwent an IVCM scan between October 2018 and July 2020. IVCM scanning is performed as part of our routine evaluation for DE symptoms and/or signs as well as for other clinical indications. As we wished to study the clinical relevance of MNs in a general DE population, we excluded individuals with regular contact lens use or a history of viral or fungal keratitis, trigeminal neuralgia, keratopathy due to radiation or chemical exposure, or graft-versus-host disease (GVHD). Additionally, we excluded individuals with anatomic comorbidities that could confound microscopy images, such as corneal scarring or keratoconus. With the above inclusions and exclusions, 153 participants were identified. Charts were reviewed and information on demographics, ocular and systemic comorbidities, surgical history, and medication was collected. The Miami VA institutional review board approved the retrospective examination of patients for this study, which was conducted in accordance with the principles of the Declaration of Helsinki and complied with the requirements of the United States Health Insurance Portability and Accountability Act.
Participants were divided into groups based on the presence (Dry Eye Questionnaire 5 (DEQ-5) score ≥6) or absence (DEQ-5 <6) of DE symptoms. We further grouped those with DE symptoms into those with versus without a history of refractive surgery, as significant and prolonged disruptions in the SNP have been described after refractive surgery [14]. We first compared frequencies of MNs between the three groups. However, our main analysis focused on relationships between MNs and DE metrics in the group of individuals with DE symptoms without history of refractive surgery. We first examined associations between MN presence and DE metrics in the entire group (119 individuals) and then repeated our analyses excluding those with persistent pain after anesthesia (n=30) to remove individuals with potential central nerve abnormalities. Next, we categorized the population based on the presence or absence of clinical criteria suggestive of central, peripheral or mixed neuropathic pain, referred to as “neuropathic features”. This was done to test our hypothesis that individuals with neuropathic features would have a higher frequency of MNs detected on confocal imaging. Neuropathic features included persistent pain measured by NRS score after the application of topical proparacaine, or a total NPSI-Eye Sub-Score ≥25 or a score ≥8 on any of the individual NPSI-E questions. As above, we performed this analysis both including and excluding individuals with persistent pain after anesthesia. Finally, we grouped individuals by whether they had a history of eye surgery (e.g. cataract, glaucoma) and compared IVCM parameters between the groups. The latter analysis was done to ensure that surgical intervention was not a confounder of our results as all ocular surgeries can disrupt corneal nerves, albeit less so when compared to refractive surgery [15].
Clinical Assessment:
Individuals underwent a standard evaluation which included questionnaires regarding symptoms: Dry Eye Questionnaire 5 (DEQ-5, score 0–22) [16], Ocular Surface Disease Index (OSDI, score 0–100) [17], and 4 select questions from the NPSI-Eye [5], regarding intensity of burning pain and evoked pain to wind, light, and heat/cold (each score 0–10, total referred to as NPSI-E Sub-Score).
Ocular surface examinations were performed on all participants. First, Inflammadry tests (Quidel, San Diego, CA) (0–3 scale for each eye: none, mild, moderate, severe) were administered. Next, anterior blepharitis (0–3 scale: none, mild, moderate, severe), eyelid margin vascularity (0–3 scale: none, mild, moderate, severe) and inferior meibomian gland inspissation (0–3 scale: none, 0–1/3 of lid margin, 1/3–2/3 of lid margin, 2/3–3/3 of lid margin) were graded. The inferior palpebral conjunctivae were examined and graded for the presence of papillae (0–2 scale: none, mild, moderate-severe), hyperemia (0–2 scale: none, mild, moderate-severe) and fibrosis (presence or absence). Fluorescein examinations were then performed, assessing for temporal, middle, and nasal conjunctivochalasis (each 0–2 scale: none, mild, moderate-severe), TBUT (3 measurements averaged in each eye), and corneal epithelial cell disruption (NEI grading scale [18], sum total of 15, 0–3 score in each of five areas). An ocular pain assessment (quantified on a numerical rating scale (NRS), score 0–10) [19] was conducted, followed by placement of topical anesthetic with reassessment of pain. Finally, Schirmer’s tests with anesthesia (millimeters of wetting after five minutes) were administered, and meibum quality (0–4 scale: clear, cloudy, granular, toothpaste-like, no meibum extracted) assessed.
Imaging Modality:
Laser IVCM was utilized for qualitative and quantitative evaluation of corneal SNP structure and features after the ocular surface assessment. It was performed using the Rostock Cornea Module of the Heidelberg Retina Tomograph (HRT) III (Heidelberg Engineering, Heidelberg, Germany), which utilizes a 670 nm wavelength Helium-Neon diode laser as the illumination source. Digital en face images (up to 100 per sequence) were recorded using sequence mode at a rate of 30 frames per second. Sequence scans (up to 5 in total) of non-overlapping areas of the central cornea were recorded at variable operator dependent depths but with a target depth of 30–60 μm [10]. The coronal field of view for captured images was 400 μm x 400 μm, with 1–2 μm lateral resolution and 4 μm axial resolution.
Image Capture:
Imaging was performed using the following standardized procedure: One drop of 0.5% proparacaine hydrochloride ophthalmic solution was instilled in the inferior fornix of the study eye for local anesthesia. The patient was properly positioned and instructed to keep eyelids open while fixating on a target light. The minimal necessary amount of refractive index-matching optical coupling gel (Systane Lubricant Eye Gel, Alcon, Fort Worth, TX) was applied to the surface of the 63x objective lens in order to eliminate reflections. The lens was then topped with a disposable sterile plastic cap (Tomo-Cap, Heidelberg Engineering, Heidelberg, Germany), and additional gel was placed on the cap surface. The lubricated lens-cap complex was advanced until contact with the central cornea of the study eye. Maximum image acquisition time was 5 minutes per eye. Upon completion of image capture, the eye was irrigated with balanced salt solution.
Image Selection:
Best-quality representative images were selected for analysis by reviewers masked to the clinical examination findings using the following criteria: 1) in focus SNP in a single plane anterior to Bowman’s layer and posterior to the basal epithelial layer, 2) absence of obvious artifacts, and 3) adequate image contrast allowing visible detection of nerves. For each individual, up to three images of different regions of the SNP were used for analyses, and IVCM parameters were averaged. Blurred, oblique, or incomplete images were excluded from selection for analysis.
Image Analysis:
Qualitative image analysis via masked reviewers was used to identify and quantify MNs. MNs were defined as any visible “swelling of injured nerve endings” evidenced by relatively large, diffuse, poorly demarcated but round appearing bright areas on the nerve itself [20]. Examples of MNs detected in our study are provided in Figure 1.
Figure 1: Examples of Microneuromas Observed on IVCM of the Corneal Sub Basal Nerve Plexus.
MNs were defined as any visible “swelling of injured nerve endings” evidenced by relatively large, diffuse, poorly demarcated but round appearing bright areas on the nerve itself.
IVCM = in vivo confocal microscopy
Quantitative image analysis was performed via a validated automated nerve image analysis software (ACCMetrics Corneal Nerve Fiber Analyser V.2, University of Manchester, Manchester, United Kingdom) [21] in order to investigate for associations between MNs and other nerve features. Assessed metrics (“IVCM parameters”) included: Corneal Nerve Fiber Density (CNFD, fibers/mm2) defined as the total number of main nerves per square millimeter, Corneal Nerve Fiber Length (CNFL, mm/mm2) defined as the total length of main nerves and nerve branches per square millimeter, Corneal Nerve Branch Density (CNBD, branches/mm2) defined as the number of branch points on the main nerve fibers per square millimeter, Corneal Total Branch Density (CTBD, branches/mm2) defined as the total number of branch points per square millimeter, Corneal Nerve Fiber Area (CNFA, μm2/mm2) defined as the total nerve fiber area, Corneal Nerve Fiber Width (CNFW, mm/mm2) defined as the average nerve fiber width, and Corneal Nerve Fractal Dimension (CfracDIM) defined as a “novel parameter that measures the structural complexity of corneal nerves”.
Statistical Analyses:
Statistical analyses were performed using SPSS software (SPSS, Chicago, Illinois, USA). Baseline demographics and MN frequencies were evaluated via descriptive statistics (means, standard deviations; proportions). T-tests were used to compare numerical variables between groups (age; DEQ-5, OSDI, NPSI-E scores; ocular surface findings other than presence of fibrosis; corneal IVCM parameters), and Chi-square analyses were used to compare categorical variables (demographics other than age; persistence of pain after anesthesia; burning and sensitivity to wind, light and temperature scores ≥8). Pearson’s correlation coefficients were used to measure the strength of association between numerical variables (MN number; DEQ-5, OSDI, NPSI-E scores; ocular surface findings other than presence of fibrosis; corneal IVCM parameters). Statistical significance was determined by an alpha level of 0.05. Separate power calculations were used for tests of differences in proportions and differences in means. In both cases, accounting for the different sample sizes in the groups with MNs present and MNs absent, we had 80% power to detect a difference with effect size of 0.6, close to a medium effect size (0.5) in the terminology of Cohen [22].
RESULTS:
Demographics of the Study Populations:
Our population was split into three groups: (a) individuals without DE symptoms or history of refractive surgery (mean age 30.5 years ±14.9, 79% male, 58% white), (b) individuals with DE symptoms (DEQ-5 ≥6) and no history of refractive surgery (57.6 years ±15.8, 73% male, 53% white), and (c) individuals with DE symptoms and a history of refractive surgery (53.6 years ±13.9, 76% male, 76% white).
Microneuroma Frequency:
The frequency of MN presence was compared among the three groups (Table 1), but no significant differences in frequencies were found (p=0.22). MN frequency was lowest in individuals with DE symptoms and a history of refractive surgery in the examined eye (6%) and highest in individuals with DE symptoms and no history of refractive surgery in the examined eye (22%).
Table 1:
Frequency of Corneal Sub-Basal Nerve Plexus Microneuromas (MNs) in Our Populations
| DE Symptoms* | n | MN Frequency (%) | P-Value | |
|---|---|---|---|---|
| No | 18 | 11.1 | 0.22 | |
| Yes | No Prior Refractive Surgery | 119 | 21.8 | |
| Prior Refractive Surgery | 16 | 6.3 | ||
n=number in group
DE Symptoms* = Dry Eye Questionnaire 5 (DEQ-5) Score ≥6
Demographics and Co-morbidities in Individuals With and Without Microneuromas:
Our main analysis focused on the 119 individuals with DE symptoms but no history of refractive surgery. 26 (22%) had one or more MNs observed on IVCM. No significant differences were observed when comparing demographics, comorbidities, ocular history, and medication use between those with and without MNs (Table 2).
Table 2:
Descriptive Statistics for Individuals with Dry Eye (DE) Symptoms and No History of Refractive Surgery Grouped by Microneuroma (MN) Presence
| Parameter | MNs Present (n=26) | MNs Absent (n=93) | P-Value | |
|---|---|---|---|---|
| Demographics | ||||
| Age (years), mean, SD | 55.6, 13.8 | 58.7, 15.4 | 0.35 | |
| Male sex, % (n) | 61.5% (16) | 74.2% (69) | 0.21 | |
| Hispanic, % (n) | 34.6% (9) | 19.6% (18) | 0.11 | |
| White, % (n) | 50.0% (13) | 53.3% (49) | 0.77 | |
| Current Smoker, % (n) | 15.4% (4) | 22.6% (21) | 0.43 | |
| Major Depressive Disorder | 30.8% (8) | 37.6% (35) | 0.52 | |
| Post-Traumatic Stress Disorder | 19.2% (5) | 21.5% (20) | 0.80 | |
| Comorbidities, % (n) | ||||
| Hypertension | 57.7% (15) | 59.1% (55) | 0.90 | |
| Diabetes Mellitus | 19.2% (5) | 26.9% (25) | 0.43 | |
| Migraine | 34.6% (9) | 40.9% (38) | 0.57 | |
| Fibromyalgia | 15.4% (4) | 9.7% (9) | 0.41 | |
| Traumatic Brain Injury | 3.8% (1) | 3.2% (3) | 0.88 | |
| Peripheral Neuropathy | 3.8% (1) | 10.8% (10) | 0.28 | |
| Autoimmune Disease* | 11.5% (3) | 7.6% (7) | 0.53 | |
| Sjögren’s Syndrome | 19.2% (5) | 15.2% (14) | 0.62 | |
| Ocular History, % (n) | ||||
| Glaucoma | 0.0% (0) | 10.8% (10) | 0.08 | |
| Cataract Surgery | OD | 7.7% (2) | 21.5% (20) | 0.11 |
| OS | 11.5% (3) | 16.1% (15) | 0.56 | |
| Glaucoma Surgery** | OD | 0.0% (0) | 3.2% (3) | 0.35 |
| OS | 0.0% (0) | 4.3% (4) | 0.28 | |
| Systemic Medications, % (n) | ||||
| Anti-hypertensive | 57.7% (15) | 59.8% (55) | 0.85 | |
| Anti-diabetic | 15.4% (4) | 19.8% (18) | 0.61 | |
| Non-steroidal anti-inflammatory | 30.8% (8) | 43.5% (40) | 0.24 | |
| Acetaminophen | 26.9% (7) | 10.9% (10) | 0.10 | |
| α2γ ligand (gabapentin or pregabalin) | 38.5% (10) | 39.1% (36) | 0.95 | |
| Anti-migraine (triptan) | 11.5% (3) | 12.0% (11) | 0.95 | |
| Anti-depressant (SSRI, SNRI, mirtazapine, TCA) | 50.0% (13) | 44.6% (41) | 0.62 | |
| Doxycycline | 3.8% (1) | 7.6% (7) | 0.50 | |
| Topical Medications, % (n) | ||||
| Artificial Tears | 69.2% (18) | 78.3% (72) | 0.34 | |
| Anti-histamine | 15.4% (4) | 9.8% (9) | 0.42 | |
| Non-steroidal anti-inflammatory | 0.0% (0) | 2.2% (2) | 0.45 | |
| Corticosteroid | 3.8% (1) | 5.4% (5) | 0.75 | |
| Cyclosporine | 38.5% (10) | 33.7% (31) | 0.65 | |
| Lifitegrast | 7.7% (2) | 8.7% (8) | 0.87 | |
| Autologous Serum Tears | 0.0% (0) | 0.0% (0) | -- | |
| Anti-hypertensive | 0.0% (0) | 6.5% (6) | 0.18 | |
SD=standard deviation; n=number in group
Includes autoimmune vasculitides (temporal arteritis, granulomatosis with polyangitis, Behcet’s syndrome), sarcoidosis, rheumatoid or psoriatic arthritis, systemic lupus erythematosus, and psoriasis
Excludes peripheral iridotomy
DE Clinical Profiles and IVCM Parameters in Individuals With and Without Microneuromas:
In a similar manner, no significant differences were noted in DE symptoms (including ones suggestive of neuropathic pain) and signs between individuals with and without MNs (Table 3). Additionally, no significant differences were observed in other IVCM parameters between individuals with and without MNs. Correlations between number of MNs and DE symptoms and signs, as well as correlations between number of MNs and corneal IVCM parameters, were not significant (data not shown, p>0.05 for all).
Table 3:
Dry Eye Symptoms and Signs and IVCM Parameters in Individuals with Dry Eye Symptoms and No History of Refractive Surgery, Grouped by Presence of Microneuromas (MNs)
| Parameter | MNs Present | MNs Absent | P-Value |
|---|---|---|---|
| Symptom | |||
| Persistent Pain After Anesthesia*, % | 24.0% (6/25) | 17.2% (15/87) | 0.45 |
| Burning, % score ≥8 | 26.9% (7/26) | 27.2% (25/92) | 0.98 |
| Wind Sensitivity, % score ≥8 | 15.4% (4/26) | 22.8% (21/92) | 0.41 |
| Light Sensitivity, % score ≥8 | 38.5% (10/26) | 34.1% (31/91) | 0.68 |
| Temperature Sensitivity, % score ≥8 | 11.5% (3/26) | 17.6% (16/91) | 0.46 |
| Total DEQ-5 Score, mean (SD) | 15.7 (4.2) | 15.2 (3.8) | 0.57 |
| Total OSDI Score, mean (SD) | 58.9 (23.4) | 50.9 (25.3) | 0.16 |
| NPSI-E Sub-Score, mean (SD) | 20.0 (12.3) | 19.8 (12.0) | 0.95 |
| Ocular surface finding, mean (SD) | |||
| Inflammadry Score | 1.0 (0.9) | 1.1 (.99) | 0.75 |
| Anterior Blepharitis | 0.5 (0.8) | 0.6 (0.9) | 0.51 |
| Vascularity | 0.9 (1.0) | 0.9 (1.1) | 0.99 |
| Meibomian Gland Inspissation | 0.8 (0.7) | 0.8 (0.7) | 0.60 |
| Temporal Conjunctivochalasis | 0.6 (0.5) | 0.6 (0.6) | 0.75 |
| Middle Conjunctivochalasis | 0.0 (0.0) | 0.1 (0.3) | 0.09 |
| Nasal Conjunctivochalasis | 0.2 (0.4) | 0.3 (0.5) | 0.19 |
| Tear Break Up Time (s) | 5.4 (3.5) | 6.0 (4.0) | 0.45 |
| Total Corneal Staining | 2.3 (3.4) | 2.5 (3.3) | 0.86 |
| Schirmer wetting length (mm/5 min) | 8.3 (6.7) | 10.2 (7.6) | 0.27 |
| Papillae | 0.5 (0.5) | 0.4 (0.5) | 0.77 |
| Fibrosis, % | 0.0% (0/20) | 6.0% (5/84) | -- |
| Hyperemia | 0.6 (0.5) | 0.6 (0.6) | 0.69 |
| Meibum Quality | 1.2 (0.9) | 1.1 (1.1) | 0.85 |
| Corneal IVCM Parameter, mean (SD) | |||
| Nerve Fiber Density (fibers/mm2) | 19.3 (7.4) | 19.0 (9.2) | 0.87 |
| Nerve Fiber Length (mm/mm2) | 12.7 (3.1) | 12.5 (3.8) | 0.81 |
| Nerve Branch Density (branches/mm2) | 22.2 (15.7) | 23.9 (18.5) | 0.68 |
| Total Branch Density (branches/mm2) | 42.2 (28.4) | 40.3 (27.4) | 0.77 |
| Nerve Fiber Area (μm2/mm2) | 0.006 (0.003) | 0.140 (1.296) | 0.60 |
| Nerve Fiber Width (mm/mm2) | 0.022 (0.001) | 0.021 (0.001) | 0.29 |
| Nerve Fractal Dimension | 1.47 (0.04) | 1.46 (0.06) | 0.54 |
SD = standard deviation; DEQ-5 = Dry Eye Questionnaire 5; OSDI = Ocular Surface Disease Index; NPSI-E sub-score = four select questions from the Neuropathic Pain Symptom Inventory modified for the eye; IVCM = in vivo confocal microscopy
NRS pre-anesthesia >0 and NRS post-anesthesia ≥ NRS pre-anesthesia
Similarly, no significant differences were observed after excluding individuals with persistent pain after anesthesia (n=30) (Supplementary Table 1).
IVCM Parameters in Individuals With and Without Clinical Features of Neuropathic Pain:
Next, individuals were grouped by the presence of clinical features of neuropathic pain. MNs were observed in 16 (25%) individuals with neuropathic features (n=63) and in 10 (18%) individuals without neuropathic features (n=55) (p=0.35). No significant differences in IVCM parameters (fiber density, length, branching) were noted between individuals with and without neuropathic features (Table 4).
Table 4:
IVCM Parameters in Individuals with Dry Eye Symptoms and No History of Refractive Surgery, Grouped by Presence of Neuropathic Features
| Mean (SD) Corneal IVCM Parameter | Neuropathic Features* (n=63) | No Neuropathic Features* (n=55) | P-Value |
|---|---|---|---|
| Microneuroma Frequency, % (n) | 25.4% (16) | 18.2% (10) | 0.35 |
| Nerve Fiber Density (fibers/mm2) | 18.8 (8.4) | 19.5 (9.4) | 0.63 |
| Nerve Fiber Length (mm/mm2) | 12.1 (3.6) | 12.9 (3.8) | 0.20 |
| Nerve Branch Density (branches/mm2) | 21.6 (16.7) | 25.3 (19.0) | 0.26 |
| Total Branch Density (branches/mm2) | 39.4 (26.3) | 41.3 (28.5) | 0.70 |
| Nerve Fiber Area (μm2/mm2) | 0.006 (0.002) | 0.233 (1.685) | 0.29 |
| Nerve Fiber Width (mm/mm2) | 0.021 (0.001) | 0.021 (0.002) | 0.09 |
| Nerve Fractal Dimension | 1.46 (0.04) | 1.46 (0.07) | 0.51 |
IVCM = in vivo confocal microscopy; n=number in group; SD=standard deviation
Neuropathic features = persistent pain after topical anesthetic (NRS pre-anesthesia >0 and NRS post-anesthesia ≥ NRS pre-anesthesia) or score ≥8 on any of the NPSI-Eye questions (burning sensation, sensitivity to wind, light, temperature), or NPSI-E Sub-Score ≥25
A similar pattern emerged when individuals with persistent pain after anesthesia (n=30) were excluded (Supplementary Table 2).
Finally, we separated individuals into those with and without any history of surgery (including cataract and glaucoma surgery) and again did not find differences between the groups in relation to MN frequency (data not shown).
DISCUSSION:
In this study, MNs detected by IVCM were present in individuals with and without DE symptoms, and their presence could not distinguish between DE sub-types, including those with clinical features of neuropathic pain. Overall, the frequency of MNs in our population was lower than that reported previously in other populations that examined MN frequency in the SNP [8 23]. The first study identified 16 individuals with presumed NCP (severe photophobia without clinical signs of ocular surface disease) and reported a MN frequency of 62.5%, while no MNs were found in any of the 16 sex-matched controls [8]. A follow up study by the same group compared another 16 individuals with NCP to 12 age- and sex-matched controls with no ocular surface disease [23] and reported 100% MN frequency vs 0% in the two groups. Of note, DE was listed as an underlying mechanism for NCP in 9 [8] and 3 [23] individuals, despite the absence of active ocular surface disease. Comparisons to our population, however, are difficult as the previous studies included individuals with histories of keratopathy due to refractive surgery (n=11), radiation or ultraviolet exposure (n=5) and herpes zoster ophthalmicus (n=1), which were excluded or not part of our main analyses in our study.
A more recent study by the same group examined MNs in patients with NCP (ocular pain and light sensitivity with TBUT ≥10 s or Schirmer’s II score >10 mm) versus DE disease (DE symptoms of more than 6 months with absence of ocular pain and with 2 or more clinical signs including corneal fluorescent staining ≥1+ (Oxford scale), TBUT <10 seconds, and Schirmer’s II score <10 mm) and age- and sex-matched controls [9]. This study found that MNs were observed in all patients with NCP (n=25) but were not observed in patients with DE disease (n=30) or controls (n=16), reporting 100% specificity and sensitivity of MNs for NCP. History of refractive surgery, radiation or ultraviolet exposure or infectious keratitis were not excluded.
These studies’ findings contrast with ours, in which MN presence was not found to be a reliable marker for NCP. However, our study populations differed depending on inclusion and exclusion criteria, as we sought to focus on the general DE population and focused on individuals without prior refractive surgery, given that refractive surgery causes substantial and prolonged changes to the SNP [14]. Additionally, the NCP populations differed in age (48.3 years ± 3.3 vs. 57.6 years ±15.8) and sex (32% vs. 73% male), though associations between MNs, age, and sex are not well established.
In addition to differing populations, discrepancies in MN frequency among reports are likely due to multiple other factors including definitions of NCP and DE disease, types of IVCM, and the subjective nature of discerning MNs. Definitions for MNs are varied in the literature and include “stumps of severed nerves identified as abrupt endings of a nerve fiber” [8], “irregularly shaped, terminal enlargements of sub-basal nerve ending (s) with variable hyperreflectivity” [9], “thickening of nerves at point of injury identified as stumps of a nerve fiber” [23], “bulges, varicosities, tangles, and/or hyper-reflective sites” [24], “beadings and nerve sprouts” [25] and “engorged abrupt endings of the sub-basal nerves” [26]. Our study utilized the most commonly used definition for MNs, as “swelling of injured nerve endings” [20].
Regardless of the criteria used to identify them, MNs are believed to represent areas of neuro-regeneration at an area of localized nerve damage [27]. Damage can occur due to axonal injury (trauma, surgery), viral infection (herpes), and systemic disease (diabetes) [28]. MNs are believed to be the site of spontaneous and ectopic discharge leading to spontaneous pain, hyperalgesia, and allodynia [20 23 28 29]. These claims are supported by animal models [13]. For example, one study of spinal nerve injury (via ligation of the L5 and L6 ventral rami and transection of the L5 dorsal ramus and root) in rats recorded ectopic discharge patterns from L5 dorsal rootlets after injury. The presence of corresponding allodynia in these animals was suggested by foot withdrawal to an innocuous mechanical stimuli [30]. Furthermore, spontaneous activity may occur in intact adjacent nociceptive neurons with no obvious injury [31], as evidenced by another study of rats that underwent ligation and transection of the L5 spinal root. In this study, spontaneous action potentials were recorded from uninjured nociceptive afferents from the L4 spinal nerve up to one week after L5 transection [31]. Thus, it is biologically plausible that spontaneous discharges causing the sensation of pain in NCP come both from MNs and from adjacent, normal appearing fibers. Thus, we expected to observe positive associations between MN presence and clinical features of hyperalgesia and allodynia. Our findings, however, did not demonstrate MN presence to be a reliable indicator of DE sub-type, including individuals with symptoms suggestive of neuropathic pain, in a population of individuals with DE symptoms but without a history of refractive surgery. Our findings were robust even when we excluded individuals suspected of having central pain. This suggests that in our DE population, MNs were not a differentiating feature of NCP. In fact, they were also seen in young individuals without DE symptoms.
Thus, the question is raised as to whether the hyper-reflective foci characterized as MNs are always representative of a pathological state [24]. An alternative hypothesis is that these hyper-reflective structures seen on IVCM and potentially characterized as MNs are actually normal structures that represent physiological phenomena [24]. Data to support this hypothesis comes from one group that used acetylcholinesterase staining, Nanozoomer microphotography, scanning and transmission electron microscopy, and IVCM to demonstrate nerve morphology in human donor corneas with no reported pathology [32]. They found that as stromal nerves entered the epithelium, they formed bulb-like structures where associated Schwann cells, endoneural fibroblasts, and matrix proteins were shed [32]. These bulb-like structures exited at entry points where the stromal nerves penetrated the epithelial basement membrane and either terminated or continued through to the epithelium, branching into thinner nerve fibers and producing characteristic hyper-reflective structures observed on IVCM [32]. These non-pathological formations, termed “corneal stromal-epithelial nerve penetration sites (CSENPS) [24], are similar in appearance to structures that have been previously described as MNs by others [23 33], thereby questioning the biological relevance of MNs visualized on IVCM.
Our findings must be interpreted in light of the study limitations, which included a defined population with few older individuals without DE symptoms and limited sample size due to available clinical data. Furthermore, there are limitations inherent in IVCM, including a small field of view and a static image, which does not capture the full dynamic status of nerves. Additionally, there are limitations in the software, as it does not assess nerve features such as beading, reflectivity, and tortuosity. Finally, assessment of MNs is subjective and currently not standardized in the field. Built-in software that assesses for presence and number of MNs, as well as additional nerve features like beading, reflectivity, and tortuosity, would enhance the quality of current and future studies utilizing IVCM. It is also important to recognize that corneal morphology detectable on IVCM can change with age [34], so the production and utilization of age-related nomographs would be beneficial in interpretation of future findings.
Despite these limitations, our study provides insight into the relationship between MNs and DE metrics in a non-refractive surgery population, a clinically relevant population in which neuropathic etiology is difficult to determine. This is because neuropathic ocular pain can occur in the absence of ocular surface abnormalities [3] or co-exist with diseases associated with nociceptive pain (e.g. Sjogren’s syndrome [35]). In our population, we did not find MN presence to distinguish between DE sub-types, including those suggestive of NCP. As such, while MNs are likely an important clinical sign, our work suggests that their presence cannot be used in isolation to determine a neuropathic etiology of DE symptoms in our population based on our current definitions. More work is needed to evaluate the diagnostic utility of MNs among different populations.
Supplementary Material
Synopsis:
This retrospective study identified corneal microneuromas in individuals with and without dry eye symptoms using in vivo confocal microscopy. Microneuroma presence was not found to be associated with clinical features suggestive of neuropathic corneal pain in our study population.
ACKNOWLEDGEMENTS:
Francisco M. and Ramon D. for image acquisition.
Funding:
Supported by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Clinical Sciences R&D (CSRD) I01 CX002015 (Dr. Galor) and Biomedical Laboratory R&D (BLRD) Service I01 BX004893 (Dr. Galor), Department of Defense Gulf War Illness Research Program (GWIRP) W81XWH-20-1-0579 (Dr. Galor) and Vision Research Program (VRP) W81XWH-20-1-0820 (Dr. Galor), National Eye Institute R01EY026174 (Dr. Galor) and R61EY032468 (Dr. Galor), NIH Center Core Grant P30EY014801 (institutional) and Research to Prevent Blindness Unrestricted Grant (institutional).
Abbreviations:
- CfracDIM
corneal nerve fractal dimension
- CNBD
corneal nerve branch density
- CNFA
corneal nerve fiber area
- CNFD
corneal nerve fiber density
- CNFL
corneal nerve fiber length
- CNFW
corneal nerve fiber width
- CTBD
corneal total branch density
- DE
dry eye
- DEQ-5
Dry Eye Questionnaire 5
- IVCM
in vivo confocal microscopy
- MN
microneuroma
- NCP
neuropathic corneal pain
- NPSI-Eye
Neuropathic Pain Symptom Inventory modified for the eye
- NRS
numerical rating score
- OSDI
Ocular Surface Disease Index
- SNP
sub-basal nerve plexus
Footnotes
Publisher's Disclaimer: Disclaimer: The views expressed in this work are not an official position of the Veterans Health Administration.
Declarations of Interest: None.
REFERENCES
- 1.IASP terminology. http://www.iasp-pain.org/Education/Content.aspx?ItemNumber=1698. Accessed January 2019.
- 2.Crane AM, Levitt RC, Felix ER, Sarantopoulos KD, McClellan AL, Galor A. Patients with more severe symptoms of neuropathic ocular pain report more frequent and severe chronic overlapping pain conditions and psychiatric disease. Br J Ophthalmol 2017;101(2):227–31 doi: 10.1136/bjophthalmol-2015-308214[published Online First: Epub Date]|. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Galor A, Moein HR, Lee C, et al. Neuropathic pain and dry eye. Ocul Surf 2018;16(1):31–44 doi: 10.1016/j.jtos.2017.10.001[published Online First: Epub Date]|. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Qazi Y, Hurwitz S, Khan S, Jurkunas UV, Dana R, Hamrah P. Validity and Reliability of a Novel Ocular Pain Assessment Survey (OPAS) in Quantifying and Monitoring Corneal and Ocular Surface Pain. Ophthalmology 2016;123(7):1458–68 doi: 10.1016/j.ophtha.2016.03.006[published Online First: Epub Date]|. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Farhangi M, Feuer W, Galor A, et al. Modification of the Neuropathic Pain Symptom Inventory for use in eye pain (NPSI-Eye). Pain 2019;160(7):1541–50 doi: 10.1097/j.pain.0000000000001552[published Online First: Epub Date]|. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Ross AR, Al-Aqaba MA, Almaazmi A, et al. Clinical and in vivo confocal microscopic features of neuropathic corneal pain. Br J Ophthalmol 2019. doi: 10.1136/bjophthalmol-2019-314799[published Online First: Epub Date]|. [DOI] [PubMed] [Google Scholar]
- 7.Theophanous C, Jacobs DS, Hamrah P. Corneal Neuralgia after LASIK. Optom Vis Sci 2015;92(9):e233–40 doi: 10.1097/OPX.0000000000000652[published Online First: Epub Date]|. [DOI] [PubMed] [Google Scholar]
- 8.Aggarwal S, Kheirkhah A, Cavalcanti BM, et al. Autologous Serum Tears for Treatment of Photoallodynia in Patients with Corneal Neuropathy: Efficacy and Evaluation with In Vivo Confocal Microscopy. Ocul Surf 2015;13(3):250–62 doi: 10.1016/j.jtos.2015.01.005[published Online First: Epub Date]|. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Moein HR, Akhlaq A, Dieckmann G, et al. Visualization of microneuromas by using in vivo confocal microscopy: An objective biomarker for the diagnosis of neuropathic corneal pain? Ocul Surf 2020;18(4):651–56 doi: 10.1016/j.jtos.2020.07.004[published Online First: Epub Date]|. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Shetty R, Sethu S, Deshmukh R, et al. Corneal Dendritic Cell Density Is Associated with Subbasal Nerve Plexus Features, Ocular Surface Disease Index, and Serum Vitamin D in Evaporative Dry Eye Disease. Biomed Res Int 2016;2016:4369750 doi: 10.1155/2016/4369750[published Online First: Epub Date]|. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Kheirkhah A, Qazi Y, Arnoldner MA, Suri K, Dana R. In Vivo Confocal Microscopy in Dry Eye Disease Associated With Chronic Graft-Versus-Host Disease. Investigative ophthalmology & visual science 2016;57(11):4686–91 doi: 10.1167/iovs.16-20013[published Online First: Epub Date]|. [DOI] [PubMed] [Google Scholar]
- 12.Giannaccare G, Pellegrini M, Sebastiani S, Moscardelli F, Versura P, Campos EC. In vivo confocal microscopy morphometric analysis of corneal subbasal nerve plexus in dry eye disease using newly developed fully automated system. Graefes Arch Clin Exp Ophthalmol 2019;257(3):583–89 doi: 10.1007/s00417-018-04225-7[published Online First: Epub Date]|. [DOI] [PubMed] [Google Scholar]
- 13.Matzner O, Devor M. Hyperexcitability at sites of nerve injury depends on voltage-sensitive Na+ channels. J Neurophysiol 1994;72(1):349–59 doi: 10.1152/jn.1994.72.1.349[published Online First: Epub Date]|. [DOI] [PubMed] [Google Scholar]
- 14.Murueta-Goyena A, Canadas P. Visual outcomes and management after corneal refractive surgery: A review. J Optom 2018;11(2):121–29 doi: 10.1016/j.optom.2017.09.002[published Online First: Epub Date]|. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Lum E, Corbett MC, Murphy PJ. Corneal Sensitivity After Ocular Surgery. Eye Contact Lens 2019;45(4):226–37 doi: 10.1097/ICL.0000000000000543[published Online First: Epub Date]|. [DOI] [PubMed] [Google Scholar]
- 16.Chalmers RL, Begley CG, Caffery B. Validation of the 5-Item Dry Eye Questionnaire (DEQ-5): Discrimination across self-assessed severity and aqueous tear deficient dry eye diagnoses. Cont Lens Anterior Eye 2010;33(2):55–60 doi: 10.1016/j.clae.2009.12.010[published Online First: Epub Date]|. [DOI] [PubMed] [Google Scholar]
- 17.Schiffman RM, Christianson MD, Jacobsen G, Hirsch JD, Reis BL. Reliability and validity of the Ocular Surface Disease Index. Arch Ophthalmol 2000;118(5):615–21 doi: 10.1001/archopht.118.5.615[published Online First: Epub Date]|. [DOI] [PubMed] [Google Scholar]
- 18.Lemp MA. Report of the National Eye Institute/Industry workshop on Clinical Trials in Dry Eyes. CLAO J 1995;21(4):221–32 [PubMed] [Google Scholar]
- 19.Ferreira-Valente MA, Pais-Ribeiro JL, Jensen MP. Validity of four pain intensity rating scales. Pain 2011;152(10):2399–404 doi: 10.1016/j.pain.2011.07.005[published Online First: Epub Date]|. [DOI] [PubMed] [Google Scholar]
- 20.Cruzat A, Qazi Y, Hamrah P. In Vivo Confocal Microscopy of Corneal Nerves in Health and Disease. Ocul Surf 2017;15(1):15–47 doi: 10.1016/j.jtos.2016.09.004[published Online First: Epub Date]|. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Petropoulos IN, Alam U, Fadavi H, et al. Rapid automated diagnosis of diabetic peripheral neuropathy with in vivo corneal confocal microscopy. Investigative ophthalmology & visual science 2014;55(4):2071–8 doi: 10.1167/iovs.13-13787[published Online First: Epub Date]|. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Cohen J. Statistical Power analysis for the behavioral science. In: Cohen J, ed. Statistical Power analysis for the behavioral science. Second ed. New Jersey: Lawrence Erlbaum associates, 1988. [Google Scholar]
- 23.Aggarwal S, Colon C, Kheirkhah A, Hamrah P. Efficacy of autologous serum tears for treatment of neuropathic corneal pain. Ocul Surf 2019;17(3):532–39 doi: 10.1016/j.jtos.2019.01.009[published Online First: Epub Date]|. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Stepp MA, Pal-Ghosh S, Downie LE, et al. Corneal Epithelial “Neuromas”: A Case of Mistaken Identity? Cornea 2020. doi: 10.1097/ICO.0000000000002294[published Online First: Epub Date]|. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Benitez-Del-Castillo JM, Acosta MC, Wassfi MA, et al. Relation between corneal innervation with confocal microscopy and corneal sensitivity with noncontact esthesiometry in patients with dry eye. Investigative ophthalmology & visual science 2007;48(1):173–81 doi: 10.1167/iovs.06-0127[published Online First: Epub Date]|. [DOI] [PubMed] [Google Scholar]
- 26.Morkin MI, Hamrah P. Efficacy of self-retained cryopreserved amniotic membrane for treatment of neuropathic corneal pain. Ocul Surf 2018;16(1):132–38 doi: 10.1016/j.jtos.2017.10.003[published Online First: Epub Date]|. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Kalangara JP, Galor A, Levitt RC, Felix ER, Alegret R, Sarantopoulos CD. Burning Eye Syndrome: Do Neuropathic Pain Mechanisms Underlie Chronic Dry Eye? Pain Med 2016;17(4):746–55 doi: 10.1093/pm/pnv070[published Online First: Epub Date]|. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Rosenthal P, Baran I, Jacobs DS. Corneal pain without stain: is it real? Ocul Surf 2009;7(1):28–40 doi: 10.1016/s1542-0124(12)70290-2[published Online First: Epub Date]|. [DOI] [PubMed] [Google Scholar]
- 29.Belmonte C, Acosta MC, Gallar J. Neural basis of sensation in intact and injured corneas. Exp Eye Res 2004;78(3):513–25 doi: 10.1016/j.exer.2003.09.023[published Online First: Epub Date]|. [DOI] [PubMed] [Google Scholar]
- 30.Han HC, Lee DH, Chung JM. Characteristics of ectopic discharges in a rat neuropathic pain model. Pain 2000;84(2–3):253–61 doi: 10.1016/s0304-3959(99)00219-5[published Online First: Epub Date]|. [DOI] [PubMed] [Google Scholar]
- 31.Wu G, Ringkamp M, Hartke TV, et al. Early onset of spontaneous activity in uninjured C-fiber nociceptors after injury to neighboring nerve fibers. J Neurosci 2001;21(8):RC140. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Al-Aqaba MA, Dhillon VK, Mohammed I, Said DG, Dua HS. Corneal nerves in health and disease. Prog Retin Eye Res 2019;73:100762 doi: 10.1016/j.preteyeres.2019.05.003[published Online First: Epub Date]|. [DOI] [PubMed] [Google Scholar]
- 33.Dieckmann G, Goyal S, Hamrah P. Neuropathic Corneal Pain: Approaches for Management. Ophthalmology 2017;124(11S):S34–S47 doi: 10.1016/j.ophtha.2017.08.004[published Online First: Epub Date]|. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Germundsson J, Karanis G, Fagerholm P, Lagali N. Age-related thinning of Bowman’s layer in the human cornea in vivo. Investigative ophthalmology & visual science 2013;54(9):6143–9 doi: 10.1167/iovs.13-12535[published Online First: Epub Date]|. [DOI] [PubMed] [Google Scholar]
- 35.Tuisku IS, Konttinen YT, Konttinen LM, Tervo TM. Alterations in corneal sensitivity and nerve morphology in patients with primary Sjogren’s syndrome. Exp Eye Res 2008;86(6):879–85 doi: 10.1016/j.exer.2008.03.002[published Online First: Epub Date]|. [DOI] [PubMed] [Google Scholar]
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