Efficacy of autologous serum tears for treatment of neuropathic corneal pain.

This was a retrospective case-control study. Sixteen patients suffering from severe NCP and no current ocular surface disease were compared to 12 controls. In vivo confocal microscopy (IVCM) (HRT3/RCM; Heidelberg, Germany) of the central corneas was performed bilaterally. Change in pain severity (scale of 0-10), corneal nerve density, tortuosity, reflectivity and presence of beading and microneuromas before and after treatment were recorded.

MATERIALS AND METHODS Design and Patients

This study is a retrospective cohort study with a comparison control group. It was conducted at the Massachusetts Eye & Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts. Sample size calculation was performed, assuming pain severity as the primary endpoint. Assuming an alpha of 0.05 and beta of 0.9, and assuming a baseline pain score of 8 with standard deviation of 1, to detect a 2 point reduction in the pain score (e.g., from 8 to 6), 6 patients would be needed at baseline and post-intervention. We then performed an additional sample size calculation assuming total nerve length as the primary end point. Assuming an alpha of 0.05 and beta of 0.9, and assuming a baseline total nerve length of 11,000 μm with standard deviation of 1,500 μm, to detect a 3,000 μm improvement in the total nerve length (e.g., from 11,000 to 14,000 μm), 6 patients would be needed at baseline and post-intervention. Thus, we determined that we needed data for a minimum of 6 patients pre and post-intervention in order for the study to be appropriately powered. We included 16 patients with chief complaints of severe corneal pain (greater or equal than 7/10), refractory to all previous treatment, including frequent lubrication with artificial tears, topical steroid therapy and other anti-inflammatory therapies, without any relief in symptoms. Autologous serum tears 20%, were added for treatment of all patients for the purpose of nerve regeneration to help alleviate pain. All consecutive patients with NCP (pain≥7) who were treated with AST within our study time frame (October 2012 to March 2014; 15 months) were included. The visual analogue scale was used for pain grading; the pain was documented from 0-10, 10 being the worst pain. These patients had an absence of ocular surface disease on slit-lamp examination, including lack of staining with vital dye stains and normal tear break-up time at the time of baseline examination. Exclusion criteria included any other pathology that could cause symptoms of ocular pain, including corneal infections, abrasions, recurrent erosion syndrome, iridocyclitis or uveitis. The patients were identified by one observer and verified by the senior author. All the clinical examination was carried out by the senior observer. (P.H.). These patients were compared to 12 age- and sex-matched controls. These controls were chosen from an existing normative database. In this data registry, subjects are enrolled prospectively. Only subjects without any ocular history or ocular signs on examination are included. These controls were confirmed to have no active ocular surface signs i.e. Schirmer’s test, tear breakup time were within normal limits and there was no vital dye staining of the surface. The Institutional Review Board/ Ethics Committee approved the protocol. We ensured compliance with the Health Insurance Portability and Accountability Act (HIPAA) and adherence to the tenets of the Declaration of Helsinki. Clinical Chart Review We conducted a thorough chart review and recorded clinical parameters at two time points – before treatment with autologous serum tears and post treatment. After starting AST therapy, the patients were followed and severity of patient-reported pain was recorded and IVCM was conducted in all subsequent visits. The quantitative IVCM analysis time point post-treatment was chosen at the time of maximum improvement in patient-reported symptoms. The parameters recorded included patient reported symptoms, specifically corneal pain, pain severity, description of effect of pain on the activities of daily living, and previous ophthalmic history. Slit-lamp bio-microscopic findings and corneal fluorescein staining, the treatment regimen and duration of treatment were also recorded. In Vivo Confocal Microscopy and Image Analysis Laser IVCM (Heidelberg Retina Tomograph 3 with the Rostock Cornea Module [HRT3/RCM], Heidelberg Engineering GmbH, Heidelberg, Germany) was conducted on central corneas of all patients and controls, bilaterally (C.C.), as previously described. 16 Equipped with a 63× objective immersion lens with a numerical aperture of 0.9 (Olympus, Tokyo, Japan), this microscope uses a 670-nm red wavelength diode laser source to produce an image representing a coronal section of the cornea of 400 × 400 μm (horizontal x vertical). Digital images are recorded at of 30 frames/s. Adjacent images are separated by 1 μm, with a lateral resolution of 1 μm/pixel. To perform this procedure, both eyes were topically anesthetized using 0.5% proparacaine hydrochloride (Alcaine; Alcon, Fort Worth, TX). This was followed by administration of a drop of hydroxypropyl methylcellulose 2.5% (GenTeal gel, Novartis Ophthalmics, East Hanover, NJ) to improve the optical coupling with the cornea module of the microscope. The cornea module was mounted with a disposable, sterile polymethylmethacrylate cap (Tomo-Cap; Heidelberg Engineering GmbH), filled with a layer of hydroxypropyl methylcellulose 2.5% (GenTeal gel; Novartis Ophthalmics), gel was also applied to the surface of the cap. The equipment is manually advanced until the gel on the cap comes in contact with the surface of the central cornea. Out of a total of six to eight sequence scans performed on the full thickness of the central cornea, resulting in a total of 50-100 images of the corneal subbasal, a masked observer chose three images most representative of the subbasal nerve plexus. The subbasal plexus is seen in subepithelial area, immediately at or posterior to the basal epithelial layer and anterior to the Bowman's layer, typically at a depth of 50 to 80 μm. The criteria to select the images were the best focused images, in a single layer, without folds and a good contrast. Two masked observers (S.A.; A.K.) then evaluated the confocal images for morphology and density of the subbasal plexus. In case of any discrepancy, the images were analyzed by a third observer. The quantitative parameters considered were number and length of main nerves, nerve branches and total nerves. Presence of beading, microneuromas, tortuosity and reflectivity were used as qualitative parameters. Microneuromas: thickening of nerves at point of injury and were identified as stumps of a nerve fiber on confocal images; and beading: discrete small beads along the length of the nerves. We performed quantitative nerve analysis using the semi-automated tracing program NeuronJ ( http://www.imagescience.org/meijering/software/neuronj/ ), 30 a plug-in for ImageJ software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/http://rsb.info.nih.gov/ij/ ) as previously described. 16 - 18 We quantified all quantitative data as density (μm/mm 2 ) ± SEM. Qualitatively, we classified tortuosity and reflectivity in four grades, according to a grading scale reported by Oliveira-Soto and Efron. 31 We reported microneuromas and beading based on presence or absence of findings ( Figure 1 ) as previously described. 6 Preparation and Administration of Autologous Serum Tears The compounding pharmacy at our institution prepared the AST in the following manner: After centrifuging blood for 10 minutes at 3000 rpm, the serum was separated in a sterile manner and filtered using MILLLEX – HP PES 5 micron low protein binding filter. After diluting to 20% using sterile saline solution, the final preparation was aliquoted into 5 ml bottles and kept frozen at −20°C until ready for use, for a maximum of 3 months. Expiration at 4°C was 2 weeks (unopened) and 1 week (after opening). We instructed the patients to keep the bottles under refrigeration and to use the provided amber colored sleeve to protect from light. Patients used AST topically 8 times daily. Statistical Analysis: We performed statistical analysis with statistical analysis software in Microsoft Excel 2010 (Analysis Toolpak). We used Student's t -test and analysis of variance (ANOVA) to compare the different groups and paired t -test was used to compare pre- and post-treatment groups. P values less than 0.05 were considered statistically significant. Normality of data was tested using the Shapiro-Wilk test. Literature Search: We performed a systematic search of all relevant publications between 1966 and 2018, from PubMed, PubMed Central, MEDLINE (National Library of Medicine), Cochrane Database and OVID. Search terms included the following: pain, neuropathic pain, peripheral pain, central pain, keratoneuralgia, corneal neuropathic pain, corneal neuropathy, corneal neuralgia, pain post refractive surgery, ocular surface disease, dry eye, ocular discomfort, allodynia, photoallodynia, confocal microscopy. We considered all systematic reviews, meta-analyses, randomized controlled trials (RCTs), retrospective studies, case series, and case reports. Studies were evaluated according to the Oxford Centre for Evidence-Based Medicine levels of evidence.