Dr. Kanchan R Sainani, S16079, Dr. Rohit Shetty
ABSTRACT :
Introduction:The prevalence of dry eye disease (DED) worldwide ranges from 5.5%to 33.7% [1]. Due to its high prevalence it is a public health concern with a significant economic burden. The hallmarks of DED include discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface. In a subset of patients with DED the standard therapeutic strategies fail to alleviate the symptoms [2,3].
Despite the knowledge available on the pathophysiological mechanisms of DED, there is a lack of substantial understanding with relevance to the etiopathology of the symptoms and their association with clinical findings. The source of ocular discomfort or pain in DED cannot solely be explained by tear film metrics suggesting the role of other factors in causation of symptoms. Pain associated with dry eye has been described as neuropathic pain [4-6] and there have been emerging reports regarding dysfunctional ocular somatosensory nerves including the sub-basal nerve plexus in ocular pain [7].
In the current study the association between the severity of dry eye symptoms (pain and/or discomfort), inflammatory markers in tears, corneal dendritic cell density, corneal sub-basal nerve plexus features, was determined.
Purpose: To determine the biological basis of pain and discomfort associated with evaporative dry eye (EDE).
Materials and Methods
Please mention that it is an observational case-control clinical study (or any other) and that clearance was taken from Institutional Review board and that it was cleared by the Ethics Committee and that the study adhered to the “Declaration of Helsinki”.
Study Population. A total of 60 patients who presented to our clinic with symptoms of Dry eyes due to evaporative dry eye disease were included in the evaporative dry eye (EDE) group and 33 healthy volunteer subjects constituted the control group. A thorough medical history was elicited to rule out any other ocular and systemic co-morbidity, following which visual acuity, refraction, detailed slit-lamp examination and fundus evaluation, and DED investigations were performed. All the tests were performed under ambient conditions of temperature and humidity. A hanging drop of 1% fluorescein stain from fluorescein strip (ContaCareOphthalmics and Diagnostics, India) was instilled in the inferior cul-de-sac of the conjunctiva to measure the tear film break-up time (TBUT) in seconds (using a stopwatch) and corneal and conjunctival epithelial staining, if present. Schirmer’s test without anaesthetic was performed using sterile Schirmer’sstrips—Whatmann filter paper (5 × 35-mm2, ContaCareOphthalmics and Diagnostics, India). Schirmer strips were placed in the lower conjunctival sac at the junction of the lateral and middle thirds of the lower eyelid, without instilling anaesthesia. All patients were seated at rest with their eyes closed. Meibomian gland status was examined using infrared meibography (Oculus, Wetzlar, Germany) and was scoring was performed based on the loss of meibomian glands for each eyelid. Patient’s ocular pain or discomfort was graded using ocular surface disease index (OSDI) questionnaire and the total OSDI scores were further classified into discomfort and vision-related subscales. Patients with OSDI scores indicating symptoms of dry eye, normal Schirmer’s test values, and low TBUT were categorized as EDE. The control group included age matched healthy volunteers with Schirmer’s test values > 10 mm and TBUT > 5 seconds and no symptoms of dry eye and other ocular conditions. Exclusion criteria included the use of contact lenses, the presence of drug allergy or ocular or systemic diseases with ocular manifestations such as Sjogren’s syndrome, rheumatoid arthritis, and diabetes mellitus. Patients with disorders involving the lacrimal gland (congenital alacrimia, Steven-Johnson syndrome) and lid disorders including clinically evident meibomian gland dysfunction along with patients using topical medication were also excluded.
Tear samples were collected using Schirmer’s strips by following Schirmer’s test I protocol. Tear analytes were extracted from Schirmer’s strips by cutting them into small pieces, agitation in sterile phosphate buffer solution (PBS) for 2 hours at 48degree Celsius followed by centrifugation.
The levels of various inflammatory proteins in the tears were measured using cytometric bead array (BD CBA Human Soluble Protein Flex Set System, BD Biosciences, Haryana, India) on a flow cytometer (BD FACSCalibur, BD Biosciences). The CBA for this study was designed for simultaneous detection and quantification of interleukin (IL)-1a, IL-1b, IL-2, IL- 4, IL-6, IL-8, IL-9, IL-10, IL-12/IL-23p40, IL-12p70, IL-13, IL-17A, IL-17F, IL-21, neuropeptide-Y, chemokine ligand 2 (CCL2)/monocyte chemotactic protein (MCP)-1, C- X-C motif chemokine 10 (CXCL10)/IP-10, intercellular adhesion molecule 1 (ICAM1), interferon (IFN)- c, and vascular endothelial growth factor (VEGF).
In Vivo Confocal Microscopy (IVCM).IVCM imaging was performed using Rostock Corneal Module/Heidelberg Retina Tomographll (RCM/HRT ll, Heidelberg Engineering GmBH, Dossenheim, Germany. 0.5% proparacaine drops were used to anaesthetize the cornea prior to the procedure. Study subjects were asked to fixate on a distant target soas to enable examination of the central cornea (MENTION RADIUS/ AREA/ DEFINITION OF CENTRAL CORNEA). The central cornea was scanned in a single area at a desired depth. A drop of 0.5% moxifloxacin was instilled after the procedure. Image acquisition time was approximately 2 minutes per eye, and none of the subjects experienced any visual symptoms or corneal complications as a result of this examination. Both eyes were included for IVCM based investigations in the subjects of EDE cohort, whereas only one eye (right) was included for the control group.
Corneal Sub-basal Nerve Plexus and Dendritic Cell Density Assessments.An experienced masked observer selected five representative IVCM frames for corneal sub-basal nerves and dendritic cells image based analyses. Images of the sub-basal nerve plexus from the center of the cornea were assessed for each subject and for all the images the entire frame of 400 × 400 microns2 was used for analysis. Quantitative analyses of the nerve fibers were performed using Automatic CCMetrics software, version 1.0 (University of Manchester, UK) . The parameters that were quantified included corneal nerve fiber density (CNFD), the total number of major nerves per square millimeter; corneal nerve fiber length (CNFL), the total length of all nerve fibers and branches ( IT SHOULD BE MICRONS PER SQUARE MILIMETER); corneal nerve branch density (CNBD) GIVE DEFINITIONeg number of corneal nerve fibers per square mm, number of branches emanating from major nerve trunks per square millimeter; total branch density (CTBD) PROVIDE DEFINITION, the total number of branch points per square millimeter; the nerve fiber area (CNFA) PROVIDE UNITS and the total nerve fiber area per square millimeter; and the corneal nerve fiber width (CNFW PROVIDE UNITS), the average nerve fiber width per square millimeter[8-12]. Dendritic cells (cells/mm2) were quantified using Cell Count software (Heidelberg Engineering GmbH) by identifying bright individual dendriform structures with cell bodies in each image at the level of basal epithelium or at sub-basal nerve plexus [13]. The images were analyzed by two blinded observers and the average of the values was used for statistical analysis.
Statistical Analysis. All statistical analyses were performed with MedCalcR version 12.5 (MedCalc Software bvba, Belgium) and GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA, USA). Shapiro-Wilk normality test, Spearman correlations analysis and Mann-Whitney test were used for analyses.
Results
Parameters such as TBUT, ocular surface disease index, corneal dendritic cell density (DCD), and corneal sub-basal nerve plexus features were measured and analyzed in controls and patients with EDE. The study subjects were age and gender matched. The ages between control (median 41 years; range 22–78 years) and EDE (median 44.5 years; range 19–73 years) cohort were not significantly different. TBUT was significantly lower in EDE subjects compared to controls. Total OSDI scores including discomfort and vision-related OSDI subscales were observed to be significantly higher in the EDE cohort. An inverse correlation was observed between TBUT with total OSDI score and discomfort and vision-related OSDI subscale.
The levels of cytokines, chemokines, secreted cell adhesion molecules, and pro-angiogenic factor in the tears of control and dry eye patients were studied using cytometric bead array. Of the various inflammatory proteins level quantified IL-1a, IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12/IL-23p40, IL-12p70, IL-17A, IL-17F, CCL2/MCP-1, CXCL10/IP-10, ICAM1, IFNc, and VEGF were in the detectable range in the tears of both the healthy controls and patients. The tear levels of IL-1a, IL-1b, IL-6, IL-8, IL-12/IL-23p40, IL-12p70, CXCL10, and VEGF were not significantly different between the control and patient cohorts. IL-17A, IFNc, ICAM1, MCP1, IL-10, and IL-4 were significantlyhigher in patients with mild dry eye signs but with exaggerated symptoms compared to controls. IL-17F was also markedly higher though not significant (P 1⁄4 0.06) in the tears of these patients. In contrast, neuropeptide-Y and IL-2 were observed to be significantly (P< 0.05) lower in the patient cohort compared with that controls.
IVCM investigations revealed the presence of corneal dendritic cells (DCs) in EDE. Image based analyses revealed a significant increase in corneal dendritic cell (DC) density and subsets (DCs with and without dendritic processes) in the eyes of EDE patients compared to controls. Number of major nerves and nerve fiber width were significantly lower in EDE patients with moderate-to-severe OSDI score compared to controls. OSDI score, specifically pain or discomfort-related subscale, exhibited a positive correlation with total corneal DC density, as well as density of DCs with and without dendritic process in EDE patients subscale and corneal dendritic cell density in EDE patients. A significant association between the corneal DC density (total, with and without dendritic process) and various sub-basal nerve plexus features in EDE cohort was also observed.
Discussion
The persistence of ocular pain and discomfort in a subset of patients with DED following standard therapeutic strategies as well as the lack of tear film metrics to predict this population poses a major challenge in the management of DED. It is therefore imperative to identify diagnostic modalities that can accurately predict patients whose symptoms may not resolve with conventional therapy or may require additional dietary or environmental interventions along with topical therapy to ensure a favourable prognosis.
IL-17, well known for its pathologic role in inflammatory disorders is involved in nociception by mediating mechanical allodynia by altering the expression of neuronal TRPV4 (MENTION FULL FORM OF TRPV) channels essential for transduction of pain stimulus[14,15]was found to be higher in the tears of patients. Furthermore, a clinical trial (NCT01250171, www.clinicaltrials.gov/ct2/show/ NCT01250171) reports a decrease in OSDI score in dry eye patient cohort on IL-17A blockers compared to IL-1b blockers or placebo controls. Similarly, an increased IFNc, MCP1 and ICAM1 observed in the patients could exacerbate the ocular symptoms, as they are reported to mediate pain.[16–18]Increased anti-inflammatory analgesic cytokines, IL- 4, and IL-10 observed in the patients could be a compensatory mechanism to counter the pro-nociceptive effects. However, it should be noted that IL-4 can stimulate ICAM1 expressionthat was also found to be elevated in patients. IL-2 known for reducing chronic neuropathic painwas found to be significantly reduced in the tears of patients, indicating a loss of the potential anti-nociceptive role of IL-2. Pain symptoms or hyperalgesia observed could be due to the direct effects of these inflammatory mediators which act by decreasing the sensory nerve thresholds in the ocular surface.
IVCM used to study architecture of the cornea in dry eye and other ocular conditions can provide additional predictive information such as corneal DCD and SBNP (MENTION FULLFORM OF SBNP)features which are altered in DED. In our study, we observed a significant association between OSDI scores, especially the discomfort subscale, with corneal DCD. Despite the absence of correlation between the decreased SBNP features and OSDI in EDE patients, we did observe a significant decrease in a subset of EDE patients with moderate-to-severe OSDI.
In our current study we have observed a significant decrease in various nerve features in EDE patients with moderate-to-severe symptoms, thus suggesting the use of corneal nerve morphological features as a predictor of the presence of pain in EDE patients. Neuropathic pain such as dysesthesias and hyperalgesia in dry eye patients can be due to either peripheral sensitization of neurons or damage to free nerve endings that interdigitate between superficial epithelial cells and are exposed to environmental and/or inflammatory stimuli. The presence of inflammation has also been found to directly and indirectly affect the structure and function of peripheral nerves resulting in altered nociception [19]. On the other hand excited nerve fibers can secrete neuropeptides which in turn trigger a neurogenic inflammatory response.
Dendritic cells play a role in immunomodulation and in antigen presentation and may influence pain pathways through their effect on T helper cells.In our study the significant increase in the corneal dendritic cells observed in EDE patients was found to have positive association with the OSDI discomfort-related subscale scores and not vision-related OSDI scores. The current study also reports a differential association between corneal dendritic cells and SBNP features in EDE. Tuisku et al. demonstrated altered stromal corneal nerves and the presence of increased antigen presenting cells in patients with dry eye. They proposed that these changes were responsible for dysesthesia experienced by the patient in dry eye disease. In their study, however, they did not describe association between the dendritic cell density and changes in the corneal nerves [20]. We propose that an increase in inflammatory cells and the associated changes in sub-basal nerve plexus may be responsible for ocular discomfort experienced by patients in our cohort. Furthermore, an increase in the number of dendritic cells in close proximity to the sub-basal nerves was observed in patients with severe symptoms. Whether DC- mediated inflammatory or physical irritation of the nerve or changes in nerve physiology are responsible for pain in these patients needs to be determined. Therefore, this incidental observation warrants further investigation.
Conclusion: Altered pain mediators like neuropeptides and cytokines (molecular) and high corneal dendritic cell density or DCD (cellular) may underlie exaggerated nociception inevaporative dry eye ( EDE).
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