The inferocentral whorl region and its directional patterns in the corneal sub-basal nerve plexus: A review.

There has been a growing application of in vivo confocal microscopy (IVCM) in the examination of corneal microstructure, including different corneal layers and corneal nerve fibers in health and in pathological conditions. Corneal nerves forming the sub-basal nerve plexus (SBNP) beneath the corneal basal epithelial cell layer in particular have been intensively researched in health and disease as a marker for corneal neurophysioanatomical and degenerative changes. One intriguing feature in the SBNP that is found inferior to the corneal apex, is a whorl-like pattern (or vortex) of nerves, which represents an anatomical landmark. Evidence has indicated that the architecture of this 'whorl region' is dynamic, changing with time in healthy individuals but also in disease conditions such as in diabetic neuropathy and keratoconus. This review summarizes the known information regarding the characteristics and significance of the whorl region of nerves in the corneal SBNP, as a potential area of high relevance for future disease monitoring and diagnostics.

Comparison of Novel Wide-Field In Vivo Corneal Confocal Microscopy With Skin Biopsy for Assessing Peripheral Neuropathy in Type 2 Diabetes.

Diabetic peripheral neuropathy (DPN) is a serious complication of diabetes, where skin biopsy assessing intraepidermal nerve fiber density (IENFD) plays an important diagnostic role. In vivo confocal microscopy (IVCM) of the corneal subbasal nerve plexus has been proposed as a noninvasive diagnostic modality for DPN. Direct comparisons of skin biopsy and IVCM in controlled cohorts are lacking, as IVCM relies on subjective selection of images depicting only 0.2% of the nerve plexus. We compared these diagnostic modalities in a fixed-age cohort of 41 participants with type 2 diabetes and 36 healthy participants using machine algorithms to create wide-field image mosaics and quantify nerves in an area 37 times the size of prior studies to avoid human bias. In the same participants, and at the same time point, no correlation between IENFD and corneal nerve density was found. Corneal nerve density did not correlate with clinical measures of DPN, including neuropathy symptom and disability scores, nerve conduction studies, or quantitative sensory tests. Our findings indicate that corneal and intraepidermal nerves likely mirror different aspects of nerve degeneration, where only intraepidermal nerves appear to reflect the clinical status of DPN, suggesting that scrutiny is warranted concerning methodologies of studies using corneal nerves to assess DPN. ARTICLE HIGHLIGHTS: Comparison of intraepidermal nerve fiber density with automated wide-field corneal nerve fiber density in participants with type 2 diabetes revealed no correlation between these parameters. Intraepidermal and corneal nerve fibers both detected neurodegeneration in type 2 diabetes, but only intraepidermal nerve fibers were associated with clinical measures of diabetic peripheral neuropathy. A lack of association of corneal nerves with peripheral neuropathy measures suggests that corneal nerve fibers may be a poor biomarker for diabetic peripheral neuropathy.

Schematic sectioning approaches for corneal and retinal surfaces used in ophthalmology and vision-related clinical practice and research.

Schematically, the corneal surface area and other similar surfaces such as the retinal surface and the visual field area have been represented by a circle. While there are different types of schematic sectioning patterns in use, not all patterns are recognized or referred to with their respective appropriate terminology. In scientific communications, as well as in clinical practice, when dealing with corneal or retinal surfaces, it is imperative to have the ability to refer to specific areas with an as high degree of accuracy as possible. The necessity arises in many situations, either when performing tests such as corneal surface staining, corneal sensitivity test, scanning the corneal surface, reporting of the findings related to any specific corneal surface area, or using a sectioning pattern for parts of the retinal surface when locating retinal lesions, or when referring to loci with changes in the visual field. Applying the appropriate geometric terms when any pattern is used for sectioning of surfaces such as cornea or retina, for precise localization and description of the findings or changes with a high degree of accuracy using the correct terminology is a sine qua non. Hence, the idea for this work is to gain an overview of the sectioning methods that are available and in use as methodological guidance in different sectioning patterns related to the corneal, retinal, and visual field.

Seasonal variations in presenting symptoms and signs of dry eye disease in Norway.

The study investigated the seasonal variations of presenting symptoms and signs of dry eye disease (DED) in Norway. 652 consecutive DED patients examined between August 2012 and May 2015 in Oslo, Norway, were included. Presenting symptoms and signs were related to the season according to when each patient was examined. Weather report data from the examination day were compared with the presenting symptoms and signs. Oslo's mean seasonal temperatures during spring, summer, fall, and winter were 6.4 °C, 15.6 °C, 9.3 °C, and - 2.1 °C, respectively. Dry eye severity level and self-reported symptoms measured by the Ocular surface disease index questionnaire did not differ between seasons. Schirmer I was lower during summer than in other seasons (P < 0.01). The percentage of patients with a pathological tear meniscus height (< 0.2 mm) was higher during fall (P < 0.01) and lower during winter (P < 0.05) compared to the other seasons. Signs and symptoms of DED generally did not correlate with weather report data, although intraocular pressure was weakly associated with mean daily air temperature (r = - 0.22; P < 0.001). Neither dry eye severity level nor dry eye symptoms differ between seasons in Oslo, Norway. However, some parameters for assessing DED show seasonal variations (Schirmer I and tear meniscus height), which are essential to consider when examining patients with DED.

The relationship between ocular and oral dryness in a cohort from the 65-year-old population in Norway.

In the present study, the relationship between dry eyes and dry mouth was explored in 150 65-year-old subjects randomly selected from the general population in Oslo, Norway. The number of drugs, including xerogenic drugs, and current and previous systemic diseases were recorded. Ocular parameters recorded were the McMonnies Dry Eye Questionnaire, the Ocular Surface Disease Index, the Schirmer I Test, tear film break-up time and ocular surface staining. The oral parameters were xerostomia frequency, Summated Xerostomia Inventory, Clinical Oral Dryness Score, and unstimulated and stimulated whole saliva. The participants with current or previous systemic diseases had significantly more ocular and oral symptoms and significantly more oral clinical findings than the participants without a history of disease. Moreover, correlation and factor analyses demonstrated an association between subjective ocular and oral parameters. A significant correlation between the total number of drugs and the presence of ocular and oral symptoms was also noted. When the participants were categorized based on their ocular symptoms, poorer values were found for the oral parameters among the participants more troubled with dry eyes. The results in the present study call for increased awareness and an interdisciplinary approach in matters related to dry eyes and dry mouth.

Morphology of Meibomian Glands in a 65-Year-Old Norwegian Population without Dry Eye Disease.

Analyses of meibography may help in the diagnosis, prevention, and management of meibomian gland dysfunction (MGD). However, there is currently a paucity of data regarding meibography analyses in the young elderly populations in the Nordic countries. In the current study, meibography of the upper and lower eyelids of 117 65-year-old residents in Oslo, Norway, who did not fulfil the diagnosis of dry eye disease (DED) were analysed. Meibomian gland (MG) dropout and tarsal areas were measured semi-automatically using ImageJ software. The relationship between morphological features of the MGs and clinical dry eye tests was examined. The median percent MG dropout was 26.1% and 40.7% in the upper and lower eyelids, respectively. There was no significant difference between males and females. None of the MG morphological parameters demonstrated significant values in discriminating abnormal dry eye symptom loads or MGD diagnosis from the normal loads. We therefore concluded that moderate MG atrophy was common among the Norwegian population of 65-year-olds without DED and showed no sexual differences. Meibography alone cannot discriminate MGD from non-MGD; thus, both morphological and functional MG tests are necessary when screening for MGD.

Meibomian gland dysfunction is highly prevalent among first-time visitors at a Norwegian dry eye specialist clinic.

To investigate the prevalence of meibomian gland dysfunction (MGD) in patients presenting with subjective dry eye-related symptoms at their first-time consultation in a Norwegian specialized ocular surface clinic. Additionally, to explore the accuracy of the ocular surface disease index score (OSDI) as an extensively applied tool to assess the severity of dry eye symptoms and MGD diagnosis. Patients with subjective dry eye-related complaints (n = 900) attending the clinic for the first time, from 2012 to 2016, were included in the study. At the baseline, patients completed the OSDI questionnaire. Subsequently, objective clinical tests, including fluorescein break-up time (FBUT), Schirmer-I test, ocular surface staining (OSS), and meibomian gland function assessment using gland expressibility and meibum quality were performed. The association between MGD and its severity in relation to symptom severity defined by OSDI-score was examined. MGD was found in 93.8% of the study group. MGD prevalence was not significantly different between groups based on age (p = 0.302) or sex (p = 0.079). There was a significant association between severity of MGD and dry eye-related symptoms (p = 0.014). OSS was significantly higher in patients with severe symptoms (p = 0.031). Sensitivity and specificity of positive symptom-score (OSDI ≥ 13) for disclosing MGD were 85.5% and 30.4%, respectively. MGD was highly prevalent, not associated with age and sex. OSDI ≥ 13 had high sensitivity and high positive predictive value (PPV), but low specificity and negative predictive value (NPV) for disclosing MGD. This underscores the importance of meibomian gland assessment in patients with dry eye-related symptoms.

TheraPearl Eye Mask and Blephasteam for the treatment of meibomian gland dysfunction: a randomized, comparative clinical trial.

Meibomian gland dysfunction (MGD) is the most common cause of dry eye disease (DED). In this study, we aimed to compare the effects of eyelid warming treatment using either TheraPearl Eye Mask (Bausch & Lomb Inc., New York, USA) or Blephasteam (Spectrum Thea Pharmaceuticals LTD, Macclesfield, UK) in a Norwegian population with mild to moderate MGD-related DED. An open label, randomized comparative trial with seventy patients (49 females, 21 males; mean age 53.6 years). Patients were randomly assigned to treatment with Blephasteam (n = 37) or TheraPearl (n = 33). All received a hyaluronic acid based artificial tear substitute (Hylo-Comod, Ursapharm, Saarbrücken, Germany). Patients were examined at baseline, and at three and six months initiation of treatment. Treatment efficacy was primarily evaluated by fluorescein breakup time (FBUT) and Ocular Surface Disease Index (OSDI) scores. Other outcome measures included ocular surface staining (OSS), Schirmer's test, and meibomian quality and expressibility. Baseline parameter values did not differ between the groups. After six months of treatment, Blephasteam improved FBUT by 3.9 s (p < 0.01) and OSDI by 13.7 (p < 0.01), TheraPearl improved FBUT by 2.6 s (p < 0.01) and OSDI by 12.6 (p < 0.01). No difference between treatments was detected at 6 months (p = 0.11 for FBUT and p = 0.71 for OSDI), nor were there differences in the other tested parameters between the treatment groups. Blephasteam and TheraPearl are equally effective in treating mild to moderate MGD in a Norwegian population after 6-months of treatment.Clinicaltrials.gov ID: NCT03318874; Protocol ID: 2014/1983; First registration: 24/10/2017.

Alterations in meibomian glands in patients treated with intensity-modulated radiotherapy for head and neck cancer.

Patients undergoing intensity-modulated radiotherapy (IMRT) for head and neck cancer may have increased incidence of dry eye disease and the exact mechanism is unclear. The present study aims to assess tear film and meibomian gland (MG) features in patients who received IMRT for head and neck cancer not involving the orbital area. Twenty-seven patients (64.7 ± 9.8 years) and 30 age-matched controls (61.4 ± 11.0 years) underwent a comprehensive dry eye work-up. Compared to the control group, the patients had more lid margin abnormalities, and worse meibum quality. The MG loss, calculated as (tarsal area-MG area)/tarsal area, was higher in the patient group in both the upper (53.0 ± 12.0% vs. 35.1 ± 10.3%, p < 0.001) and lower lids (69.5 ± 12.6% vs. 48.5 ± 12.5%, p < 0.001). In the patient group, more MG loss in the lower lids correlated with worse meibum quality (r = 0.445, p = 0.029). In contrast, there was no significant difference in aqueous tear production level, measured with Schirmer test. Patients treated with IMRT for head and neck cancer seemed to have comparable lacrimal gland function to the controls despite more dry eye symptoms. However, the patients had MG functional and morphological changes, which may present a higher risk for developing dry eye disease.

Wide-field mosaics of the corneal subbasal nerve plexus in Parkinson's disease using in vivo confocal microscopy.

In vivo confocal microscopy (IVCM) is a non-invasive imaging technique facilitating real-time acquisition of images from the live cornea and its layers with high resolution (1-2 µm) and high magnification (600 to 800-fold). IVCM is extensively used to examine the cornea at a cellular level, including the subbasal nerve plexus (SBNP). IVCM of the cornea has thus gained intense interest for probing ophthalmic and systemic diseases affecting peripheral nerves. One of the main drawbacks, however, is the small field of view of IVCM, preventing an overview of SBNP architecture and necessitating subjective image sampling of small areas of the SBNP for analysis. Here, we provide a high-quality dataset of the corneal SBNP reconstructed by automated mosaicking, with an average mosaic image size corresponding to 48 individual IVCM fields of view. The mosaic dataset represents a group of 42 individuals with Parkinson's disease (PD) with and without concurrent restless leg syndrome. Additionally, mosaics from a control group (n = 13) without PD are also provided, along with clinical data for all included participants.

The pattern of the inferocentral whorl region of the corneal subbasal nerve plexus is altered with age.

PURPOSE: To describe the pattern of the nerves in the inferocentral whorl region of the human corneal subbasal nerve plexus (SBNP) in health and diseases known to affect the subbasal nerves. METHODS: Laser-scanning in vivo confocal microscopy (IVCM) was used to image the SBNP bilaterally in 91 healthy subjects, 39 subjects with type 2 diabetes mellitus (T2DM), and 43 subjects with Parkinson's disease (PD). Whorl regions were classified according to nerve orientation relative to age and health/disease status. RESULTS: Of 346 examined eyes, 300 (86.7%) had an identifiable whorl pattern. In healthy subjects, a clockwise nerve orientation of the whorl was most common (67.9%), followed by non-rotatory or 'seam' morphology (21.4%), and counterclockwise (10.7%). The clockwise orientation was more prevalent in healthy subjects than in T2DM or PD (P < 0.001). Healthy individuals below 50 years of age had a predominantly clockwise orientation (93.8%) which was reduced to 51.9% in those over 50 years (P < 0.001). Age but not disease status explained whorl orientation in T2DM and PD groups. Moreover, whorl orientation is bilaterally clockwise in the young, but adopts other orientations and becomes asymmetric across eyes with age. Finally, we report reflective 'dot-like' features confined to the whorl region of the subbasal plexus, sometimes appearing in close association with subbasal nerves and present in 84-93% of examined eyes regardless of disease status, eye or sex. CONCLUSION: Subbasal nerves in the inferocentral whorl region are predominantly clockwise in young, healthy corneas. With aging and conditions of T2DM and PD, counterclockwise and non-rotatory configurations increase in prevalence, and bilateral symmetry is lost. Mechanisms regulating these changes warrant further investigation.

Parkinson's disease with restless legs syndrome-an in vivo corneal confocal microscopy study.

Small fiber neuropathy (SFN) has been suggested as a trigger of restless legs syndrome (RLS). An increased prevalence of peripheral neuropathy has been demonstrated in Parkinson's disease (PD). We aimed to investigate, in a cross-sectional manner, whether SFN is overrepresented in PD patients with concurrent RLS relative to PD patients without RLS, using in vivo corneal confocal microscopy (IVCCM) and quantitative sensory testing (QST) as part of small fiber assessment. Study participants comprised of age- and sex-matched PD patients with (n = 21) and without RLS (n = 21), and controls (n = 13). Diagnosis of RLS was consolidated with the sensory suggested immobilization test. Assessments included nerve conduction studies (NCS), Utah Early Neuropathy Scale (UENS), QST, and IVCCM, with automated determination of corneal nerve fiber length (CNFL) and branch density (CNBD) from wide-area mosaics of the subbasal nerve plexus. Plasma neurofilament light (p-NfL) was determined as a measure of axonal degeneration. No significant differences were found between groups when comparing CNFL (p = 0.81), CNBD (p = 0.92), NCS (p = 0.82), and QST (minimum p = 0.54). UENS scores, however, differed significantly (p = 0.001), with post-hoc pairwise testing revealing higher scores in both PD groups relative to controls (p = 0.018 and p = 0.001). Analysis of all PD patients (n = 42) revealed a correlation between the duration of L-dopa therapy and CNBD (ρ = -0.36, p = 0.022), and p-NfL correlated with UENS (ρ = 0.35, p = 0.026) and NCS (ρ = -0.51, p = 0.001). Small and large fiber neuropathy do not appear to be associated with RLS in PD. Whether peripheral small and/or large fiber pathology associates with central neurodegeneration in PD merits further longitudinal studies.

Sex and age differences in symptoms and signs of dry eye disease in a Norwegian cohort of patients.

PURPOSE: To investigate sex and age differences in symptoms and signs in a Norwegian clinic-based cohort of patients with dry eye disease (DED). METHODS: Visitors at the Norwegian Dry Eye Clinic were examined using Ocular Surface Disease Index (OSDI) questionnaire score, tear osmolarity, tear break-up time (TFBUT), ocular surface staining, corneal sensitivity, Schirmer I test, and meibum expressibility (ME) and quality (MQ). A diagnosis of DED was made by an ophthalmologist based on symptoms and signs, and only DED patients were enrolled in the study: 1823 patients (338 males; mean age 51.2 ± 16.2 years; 1485 females; mean age 52.5 ± 16.0 years). The patients were divided into age subgroups: 20-39 years, 40-59 years and ≥60 years. Sex differences in the aforementioned tests were analyzed. Values were reported as mean ± standard deviation (SD), and intergroup comparisons were performed using Mann-Whitney U test. Multiple regression was used to analyze sex and age influences on symptoms and signs. RESULTS: When patients of all ages were analyzed, females had increased osmolarity, shorter TFBUT, reduced MQ and ME and higher corneal sensitivity. OSDI, Schirmer I test, ocular surface staining and corneal staining were not significantly different between the sexes. Only with TFBUT and ME were the sex difference present in all age subgroups. Multiple regression showed that all parameters were influenced by either sex or age, but only TFBUT and ME were influenced by both sex and age. (all p < 0.05). CONCLUSIONS: Sex and age differences in dry eye were most consistent in TFBUT and ME, that indicate differences in meibomian gland functionality. Sex and age subgroup stratification is important in future studies investigating DED in other populations.

Region of interest and directional analysis of subbasal nerves in wide-area corneal nerve plexus mosaics in type 2 diabetes mellitus.

In vivo confocal microscopy (IVCM) imaging of the corneal subbasal nerve plexus (SBNP) is a clinical imaging modality gaining popularity for the diagnosis and follow-up of corneal neuropathy in various conditions such as diabetes mellitus. There remain, however, major limitations to the method, hindering its widespread clinical use. Finding the same exact area of the central cornea to standardize image acquisition is difficult without a reference point. Alternatively, creating wide-area mosaics of the SBNP is resource-intensive and has not yet been developed for routine clinical use. Here, we investigated whether IVCM analysis of the corneal SBNP in a predetermined, anatomically standardized region of interest (ROI) could be applied as an equivalent substitution for wide-area SBNP mosaic generation and analysis. Furthermore, we investigated nerve patterns outside the central corneal region for a possible relationship to type 2 diabetes mellitus status using a publicly available dataset. We found that corneal nerve fibre length density (CNFL) based on the ROI underestimated the mosaic-based CNFL by an average of 34% in 90% of cases (150 eyes), and did not exhibit a significant reduction with diabetes, as seen in the full SBNP. Outside the central cornea, nerve orientation differed depending on the anatomic region (left, central or right superior plexus, P < 0.001). Moreover, in long-term type 2 diabetes mellitus (≥ 10 years, 28 subjects), nerve density in the left superior sector of the SBNP was decreased (P < 0.001) while that in the central superior SBNP increased (P = 0.01) relative to 35 age-matched healthy subjects with normal glucose tolerance. These results indicate that subbasal nerve degeneration in type 2 diabetes mellitus can vary according to anatomic location, and regions with potential diagnostic value outside the central SBNP may warrant further investigation.

Temporal redistribution of cap and residual stromal thickness after SMILE.

PURPOSE: To investigate corneal sublayer alterations during the postoperative period after small-incision lenticule extraction (SMILE). SETTING: Synslaser clinic, Oslo, Norway. STUDY DESIGN: Retrospective. METHODS: Patients who underwent SMILE for treating myopia were included. The thicknesses of the corneal epithelium, cap, stromal part of the cap (StromaCap), residual stromal bed (StromaRes), and total stroma (StromaTot) were measured using spectral-domain optical coherence tomography at 1 day, 1 week, 1 month, 3 months, and 6 months postoperatively. Postoperative changes in the corneal sublayer thicknesses were analyzed and correlated with changes in spherical equivalence and anterior and posterior keratometry (K). RESULTS: The study was based on analyses of the right eyes of 51 patients. From 1 day to 6 months postoperatively, the corneal epithelium, cap, StromaCap, StromaRes, and StromaTot thicknesses increased from 54.4 ± 4.0 µm to 57.3 ± 5.2 µm; 137.1 ± 5.5 µm to 140.3 ± 5.1 µm; 82.7 ± 5.9 µm to 82.8 ± 6.3 µm; 375.0 ± 40.8 µm to 381.4 ± 30.6 µm; and 457.6 ± 41.1 µm to 462.1 ± 36.7 µm, respectively. Between 1 month and 6 months postoperatively, the increase in anterior K correlated significantly with the thickening of the cap (r = 0.37, P = .03) and the stromal component of the cap (r = 0.36, P = .04) within the central cornea. CONCLUSIONS: The post-SMILE remodeling behavior between the anterior (StromaCap) and posterior (StromaRes) stroma were dissimilar. There was a significant correlation between changes in anterior K and the central cap and the stromal component of the cap. This might be because of biomechanical changes, tissue remodeling, and wound healing or a combination of some or all of the aforementioned processes.

Dendritic cell maturation in the corneal epithelium with onset of type 2 diabetes is associated with tumor necrosis factor receptor superfamily member 9.

Type 2 diabetes mellitus is characterized by a low-grade inflammation; however, mechanisms leading to this inflammation in specific tissues are not well understood. The eye can be affected by diabetes; thus, we hypothesized that inflammatory changes in the eye may parallel the inflammation that develops with diabetes. Here, we developed a non-invasive means to monitor the status of inflammatory dendritic cell (DC) subsets in the corneal epithelium as a potential biomarker for the onset of inflammation in type 2 diabetes. In an age-matched cohort of 81 individuals with normal and impaired glucose tolerance and type 2 diabetes, DCs were quantified from wide-area maps of the corneal epithelial sub-basal plexus, obtained using clinical in vivo confocal microscopy (IVCM). With the onset of diabetes, the proportion of mature, antigen-presenting DCs increased and became organized in clusters. Out of 92 plasma proteins analysed in the cohort, tumor necrosis factor receptor super family member 9 (TNFRSF9) was associated with the observed maturation of DCs from an immature to mature antigen-presenting phenotype. A low-grade ocular surface inflammation observed in this study, where resident immature dendritic cells are transformed into mature antigen-presenting cells in the corneal epithelium, is a process putatively associated with TNFRSF9 signalling and may occur early in the development of type 2 diabetes. IVCM enables this process to be monitored non-invasively in the eye.

Wide-field corneal subbasal nerve plexus mosaics in age-controlled healthy and type 2 diabetes populations.

A dense nerve plexus in the clear outer window of the eye, the cornea, can be imaged in vivo to enable non-invasive monitoring of peripheral nerve degeneration in diabetes. However, a limited field of view of corneal nerves, operator-dependent image quality, and subjective image sampling methods have led to difficulty in establishing robust diagnostic measures relating to the progression of diabetes and its complications. Here, we use machine-based algorithms to provide wide-area mosaics of the cornea's subbasal nerve plexus (SBP) also accounting for depth (axial) fluctuation of the plexus. Degradation of the SBP with age has been mitigated as a confounding factor by providing a dataset comprising healthy and type 2 diabetes subjects of the same age. To maximize reuse, the dataset includes bilateral eye data, associated clinical parameters, and machine-generated SBP nerve density values obtained through automatic segmentation and nerve tracing algorithms. The dataset can be used to examine nerve degradation patterns to develop tools to non-invasively monitor diabetes progression while avoiding narrow-field imaging and image selection biases.

Reduced Corneal Nerve Fiber Density in Type 2 Diabetes by Wide-Area Mosaic Analysis.

Purpose: To determine if corneal subbasal nerve plexus (SBP) parameters derived from wide-area depth-corrected mosaic images are associated with type 2 diabetes. Methods: One hundred sixty-three mosaics were produced from eyes of 82 subjects by laser-scanning in vivo confocal microscopy (IVCM). Subjects were of the same age, without (43 subjects) or with type 2 diabetes (39 subjects). Mosaic corneal nerve fiber length density (mCNFL) and apical whorl corneal nerve fiber length density (wCNFL) were quantified and related to the presence and duration of diabetes (short duration < 10 years and long duration ≥ 10 years). Results: In mosaics with a mean size of 6 mm2 in subjects aged 69.1 ± 1.2 years, mCNFL in type 2 diabetes was reduced relative to nondiabetic subjects (13.1 ± 4.2 vs. 15.0 ± 3.2 mm/mm2, P = 0.018). Also reduced relative to nondiabetic subjects was mCNFL in both short-duration (14.0 ± 4.0 mm/mm2, 3.2 ± 3.9 years since diagnosis) and long-duration diabetes (12.7 ± 4.2 mm/mm2, 15.4 ± 4.2 years since diagnosis; ANOVA P = 0.023). Lower mCNFL was associated with presence of diabetes (P = 0.032) and increased hemoglobin A1c (HbA1c) levels (P = 0.047). By contrast, wCNFL was unaffected by diabetes or HbA1c (P > 0.05). Global SBP patterns revealed marked degeneration of secondary nerve fiber branches outside the whorl region in long-duration diabetes. Conclusions: Wide-area mosaic images provide reference values for mCNFL and wCNFL and reveal a progressive degeneration of the SBP with increasing duration of type 2 diabetes.

Tissue Harvesting Site and Culture Medium Affect Attachment, Growth, and Phenotype of Ex Vivo Expanded Oral Mucosal Epithelial Cells.

Transplantation of cultured oral mucosal epithelial cells (OMECs) is a promising treatment strategy for limbal stem cell deficiency. In order to improve the culture method, we investigated the effects of four culture media and tissue harvesting sites on explant attachment, growth, and phenotype of OMECs cultured from Sprague-Dawley rats. Neither choice of media or harvesting site impacted the ability of the explants to attach to the culture well. Dulbecco's modified Eagle's medium/Ham's F12 (DMEM) and Roswell Park Memorial Institute 1640 medium (RPMI) supported the largest cellular outgrowth. Fold outgrowth was superior from LL explants compared to explants from the buccal mucosa (BM), HP, and transition zone of the lower lip (TZ) after six-day culture. Putative stem cell markers were detected in cultures grown in DMEM and RPMI. In DMEM, cells from TZ showed higher colony-forming efficiency than LL, BM, and HP. In contrast to RPMI, DMEM both expressed the putative stem cell marker Bmi-1 and yielded cell colonies. Our data suggest that OMECs from LL and TZ cultured in DMEM give rise to undifferentiated cells with high growth capacity, and hence are the most promising for treatment of limbal stem cell deficiency.