Retinal nerve fibre layer thinning associated with diabetic peripheral neuropathy

Aims:  To investigate the relationship between retinal nerve fibre layer thickness and peripheral neuropathy in patients with Type 2 diabetes, particularly in those who are at higher risk of foot ulceration. Methods:  Global and sectoral retinal nerve fibre layer thicknesses were measured at 3.45 mm diameter around the optic nerve head using optical coherence tomography (OCT). The level of neuropathy was assessed in 106 participants (82 with Type 2 diabetes and 24 healthy controls) using the 0–10 neuropathy disability score. Participants were stratified into four neuropathy groups: none (0–2), mild (3–5), moderate (6–8), and severe (9–10). A neuropathy disability score ≥ 6 was used to define those at higher risk of foot ulceration. Multivariable regression analysis was performed to assess the effect of neuropathy disability scores, age, disease duration and retinopathy on RNFL thickness. Results:  Inferior (but not global or other sectoral) retinal nerve fibre layer thinning was associated with higher neuropathy disability scores (P = 0.03). The retinal nerve fibre layer was significantly thinner for the group with neuropathy disability scores ≥ 6 in the inferior quadrant (P < 0.005). Age, duration of disease and retinopathy levels did not significantly influence retinal nerve fibre layer thickness. Control participants did not show any significant differences in thickness measurements from the group with diabetes and no neuropathy (P > 0.24 for global and all sectors). Conclusions:  Inferior quadrant retinal nerve fibre layer thinning is associated with peripheral neuropathy in patients with Type 2 diabetes, and is more pronounced in those at higher risk of foot ulceration.

Retinal and choroidal thickness in myopic anisometropia

PURPOSE: To examine the foveal retinal thickness (RT) and subfoveal choroidal thickness (ChT) between the fellow eyes of myopic anisometropes. METHODS: Twenty-two young (mean age 23 ± 5 years), healthy myopic anisometropes (≥ 1 D spherical equivalent [SEq] anisometropia) without amblyopia or strabismus were recruited. Spectral domain optical coherence tomography (SD-OCT) was used to capture images of the retina and choroid. Customised software was used to register, align and average multiple foveal OCT B-Scan images from each subject in order to enhance image quality. Two independent masked observers then manually determined the RT and ChT at the centre of the fovea from each SD-OCT image, which were then averaged. Axial length was measured using optical low coherence biometry during relaxed accommodation. RESULTS: The mean absolute SEq anisometropia was 1.74 ± 0.95 D and the mean interocular difference in axial length was 0.58 ± 0.41 mm. There was a strong correlation between SEq anisometropia and the interocular difference in axial length (r = 0.90, p < 0.001). Measures of RT and ChT were highly correlated between the two observers (r = 0.99 and 0.97 respectively) and in close agreement (mean inter-observer difference: RT 1.3 ± 2.2 µm, ChT 1.5 ± 13.7 µm). There was no significant difference in RT between the more (218 ± 18 µm) and less myopic eyes (215 ± 18 µm) (p > 0.05). However, the mean subfoveal ChT was significantly thinner in the more myopic eye (252 ± 46 µm) compared to the fellow, less myopic eye (286 ± 58 µm) (p < 0.001). There was a moderate correlation between the interocular difference in ChT and the interocular difference in axial length (r = -0.50, p < 0.01). CONCLUSIONS: Foveal RT was similar between the fellow eyes of myopic anisometropes; however, the subfoveal choroid was significantly thinner in the more myopic (longer) eye of our anisometropic cohort. The interocular difference in ChT correlated with the magnitude of axial anisometropia.

The post-illumination pupil response of melanopsin expressing intrinsically photosensitive retinal ganglion cells in diabetes

Purpose: This study investigates the clinical utility of the melanopsin expressing intrinsically photosensitive retinal ganglion cell (ipRGC) controlled post-illumination pupil response (PIPR) as a novel technique for documenting inner retinal function in patients with Type II diabetes without diabetic retinopathy. Methods: The post-illumination pupil response (PIPR) was measured in seven patients with Type II diabetes, normal retinal nerve fiber thickness and no diabetic retinopathy. A 488 nm and 610 nm, 7.15º diameter stimulus was presented in Maxwellian view to the right eye and the left consensual pupil light reflex was recorded. Results: The group data for the blue PIPR (488 nm) identified a trend of reduced ipRGC function in patients with diabetes with no retinopathy. The transient pupil constriction was lower on average in the diabetic group. The relationship between duration of diabetes and the blue PIPR amplitude was linear, suggesting that ipRGC function decreases with increasing diabetes duration. Conclusion: This is the first report to show that the ipRGC controlled post-illumination pupil response may have clinical applications as a non-invasive technique for determining progression of inner neuroretinal changes in patients with diabetes before they are ophthalmoscopically or anatomically evident. The lower transient pupil constriction amplitude indicates that outer retinal photoreceptor inputs to the pupil light reflex may also be affected in diabetes.

Temporal summation of intrinsically photosensitive retinal ganglion cell (ipRGC) contributions to the human pupil.

Purpose: IpRGCs mediate non-image forming functions including photoentrainment and the pupil light reflex (PLR). Temporal summation increases visual sensitivity and decreases temporal resolution for image forming vision, but the summation properties of nonimage forming vision are unknown. We investigated the temporal summation of inner (ipRGC) and outer (rod/cone) retinal inputs to the PLR. Method: The consensual PLR of the left eye was measured in six participants with normal vision using a Maxwellian view infrared pupillometer. Temporal summation was investigated using a double-pulse protocol (100 ms stimulus pairs; 0–1024 ms inter-stimulus interval, ISI) presented to the dilated fellow right eye (Tropicamide 1%). Stimulus lights (blue λmax = 460 nm; red λmax = 638 nm) biased activity to inneror outer retinal inputs to non-image forming vision. Temporal summation was measured suprathreshold (15.2 log photons.cm−2.s−1 at the cornea) and subthreshold (11.4 log photons.cm−2.s−1 at the cornea). Results: RM-ANOVAs showed the suprathreshold and subthreshold 6 second post illumination pupil response (PIPR: expressed as percentage baseline diameter) did not significantly vary for red or blue stimuli (p > .05). The PIPR for a subthreshold red 16 ms double-pulse control condition did not significantly differ with ISI (p > .05). The maximum constriction amplitude for red and blue 100 ms double- pulse stimuli did not significantly vary with ISI (p > .05). Conclusion: The non-significant changes in suprathreshold PIPR and subthreshold maximum pupil constriction indicate that inner retinal ipRGC inputs and outer retinal photoreceptor inputs to the PLR do not show temporal summation. The results suggest a fundamental difference between the temporal summation characteristics of image forming and non-image forming vision.

The cultivation of human retinal pigment epithelial cells on Bombyx mori silk fibroin

We have presently evaluated membranes prepared from Bombyx mori silk fibroin (BMSF), for their potential use as a prosthetic Bruch’s membrane and carrier substrate for human retinal pigment epithelial (RPE) cell transplantation. Porous BMSF membranes measuring 3 μm in thickness were prepared from aqueous solutions (3% w/v) containing poly(ethylene oxide) (0.09%). The permeability coefficient for membranes was between 3 and 9 × 10-5 cm/s by using Allura red or 70 kDa FITC-dextran respectively. Average pore size (± sd) was 4.9 ± 2.3 µm and 2.9 ± 1.5 µm for upper and lower membrane surfaces respectively. Optimal attachment of ARPE-19 cells to BMSF membrane was achieved by pre-coating with vitronectin (1 µg/mL). ARPE-19 cultures maintained in low serum on BMSF membranes for approximately 8 weeks, developed a cobble-stoned morphology accompanied by a cortical distribution of F-actin and ZO-1. Similar results were obtained using primary cultures of human RPE cells, but cultures took noticeably longer to establish on BMSF compared with tissue culture plastic. These findings encourage further studies of BMSF as a substrate for RPE cell transplantation.

Retinal and choroidal thickness in myopic anisometropia

Purpose: To compare the retinal thickness (RT) and choroidal thickness (ChT) between the fellow eyes of non-amblyopic myopic anisometropes. Methods: The eyes of 22 non-amblyopic myopic anisometropes (1 D spherical equivalent refraction [SER] anisometropia) were examined using spectral domain optical coherence tomography (SD-OCT). Customised software was used to register, align and average multiple foveal OCT B-Scan images from each subject in order to enhance image quality. Two independent masked observers manually determined the RT and ChT from each SD-OCT image up to 2.5 mm nasal and temporal to the fovea. Axial length (AXL) was measured using optical low coherence biometry during relaxed accommodation. Results: The mean SER anisometropia was 1.74 ± 0.95 D and the mean interocular AXL difference was 0.58 ± 0.41 mm. There was no significant difference in foveal RT between the fellow eyes (P > 0.05). Mean subfoveal ChT was significantly thinner in the more myopic eye (252 ± 46 μm compared to the fellow, less myopic eye (286 ± 58 μm) (P < 0.001). There was a moderate correlation between the interocular difference in subfoveal ChT and the interocular difference in AXL (r = -0.50, P < 0.01). Asian anisometropes displayed more regionally symmetrical (nasal-temporal)interocular differences in ChT profile compared to Caucasians. Conclusions: RT was similar between the fellow eyes of myopic anisometropes; however, the subfoveal choroid was significantly thinner in the more myopic (longer) eye of this anisometropic cohort. The interocular asymmetry in ChT correlated with the interocular difference in AXL.

Identification of GABA receptors in chick retinal pigment epithelium

Purpose: The retinal pigment epithelium (RPE) is a multifunctional, monolayer of cells located between the neural retina and the choroicapillaris. γ-Aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the retina and GABA receptors are known to be present in chick retina, sclera and cornea. There is a report of genes involved in GABA receptor signaling being expressed in human RPE, however, whether GABA receptors are present in chick RPE is unknown. Methods: Real time PCR and western blot were used to determine the expression of GABA receptors (alpha1 GABAA, GABABR2, and rho1 GABAC receptors) in isolated chicken RPE. Immunofluorescence using antibodies against one of the GABA receptor sub-types was used to determine receptor localization. Results: Both real-time PCR and western blot demonstrated that alpha1 GABAA, GABABR2 and rho1 GABAC receptors were expressed in isolated chick RPE. Immunofluorescence further demonstrated that GABA receptors were localized to the cell membrane and plasma of RPE cells. Conclusions: Alpha1 GABAA, GABABR2 and rho1 GABAC receptors were expressed in chick RPE. The purpose of the GABA receptors within the RPE remains to be explored.

Exploring retinal markers of diabetic neuropathy

Purpose To investigate the application of retinal nerve fibre layer (RNFL) thickness as a marker for severity of diabetic peripheral neuropathy (DPN) in people with Type 2 diabetes. Methods This was a cross-sectional study whereby 61 participants (mean age 61 [41-75 years], mean duration of diabetes 14 [1-40 years], 70% male) with Type 2 diabetes and DPN underwent optical coherence tomography (OCT) scans. Global and 4 quadrant (TSNI) RNFL thicknesses were measured at 3.45mm around the optic nerve head of one eye. Neuropathy disability score (NDS) was used to assess the severity of DPN on a 0 to 10 scale. Participants were divided into three age-matched groups representing mild (NDS=3-5), moderate (NDS=6-8) and severe (NDS=9-10) neuropathy. Two regression models were fitted for statistical analysis: 1) NDS scores as co-variate for global and quadrant RNFL thicknesses, 2) NDS groups as a factor for global RNFL thickness only. Results Mean (SD) RNFL thickness (µm) was 103(9) for mild neuropathy (n=34), 101(10) for moderate neuropathy (n=16) and 95(13) in the group with severe neuropathy (n=11). Global RNFL thickness and NDS scores were statistically significantly related (b=-1.20, p=0.048). When neuropathy was assessed across groups, a trend of thinner mean RNFL thickness was observed with increasing severity of neuropathy; however, this result was not statistically significant (F=2.86, p=0.065). TSNI quadrant analysis showed that mean RNFL thickness reduction in the inferior quadrant was 2.55 µm per 1 unit increase in NDS score (p=0.005). However, the regression coefficients were not statistically significant for RNFL thickness in the superior (b=-1.0, p=0.271), temporal (b=-0.90, p=0.238) and nasal (b=-0.99, p=0.205) quadrants. Conclusions RNFL thickness was reduced with increasing severity of DPN and the effect was most evident in the inferior quadrant. Measuring RNFL thickness using OCT may prove to be a useful, non-invasive technique for identifying severity of DPN and may also provide additional insight into common mechanisms for peripheral neuropathy and RNFL damage.

Retinal nerve fibre layer thickness is associated with higher risk of foot ulceration in diabetic peripheral neuropathy

Purpose To evaluate the association between retinal nerve fibre layer (RNFL) thickness and diabetic peripheral neuropathy in people with type 2 diabetes, and specifically those at higher risk of foot ulceration. Methods RNFL thicknesses was measured globally and in four quadrants (temporal, superior, nasal and inferior) at 3.45 mm diameter around the optic nerve head using optical coherence tomography (OCT). Severity of neuropathy was assessed using the Neuropathy Disability Score (NDS). Eighty-two participants with type 2 diabetes were stratified according to NDS scores (0-10) as: none, mild, moderate, and severe neuropathy. A control group was additionally included (n=17). Individuals with NDS≥ 6 (moderate and severe neuropathy) have been shown to be at higher risk of foot ulceration. A linear regression model was used to determine the association between RNFL and severity of neuropathy. Age, disease duration and diabetic retinopathy levels were fitted in the models. Independent t-test was employed for comparison between controls and the group without neuropathy, as well as for comparison between groups with higher and lower risk of foot ulceration. Analysis of variance was used to compare across all NDS groups. Results RNFL thickness was significantly associated with NDS in the inferior quadrant (b= -1.46, p=0.03). RNFL thicknesses globally and in superior, temporal and nasal quadrants did not show significant associations with NDS (all p>0.51). These findings were independent of the effect of age, disease duration and retinopathy. RNFL was thinner for the group with NDS ≥ 6 in all quadrants but was significant only inferiorly (p<0.005). RNFL for control participants was not significantly different from the group with diabetes and no neuropathy (superior p=0.07, global and all other quadrants: p>0.23). Mean RNFL thickness was not significantly different between the four NDS groups globally and in all quadrants (p=0.08 for inferior, P>0.14 for all other comparisons). Conclusions Retinal nerve fibre layer thinning is associated with neuropathy in people with type 2 diabetes. This relationship is strongest in the inferior retina and in individuals at higher risk of foot ulceration.

Characterization and novel activation of 72-kDa metalloproteinase in retinal interphotoreceptor matrix and Y-79 cell culture medium

Analysis of bovine interphotoreceptor matrix and conditioned medium from human Y-79 retinoblastoma cells by gelatin SDS-PAGE zymography reveals abundant activity of a 72-kDa M(r) gelatinase. The 72-kDa gelatinase from either source is inhibited by EDTA but not aprotinin or NEM, indicating that it is a metalloproteinase (MMP). The 72-kDa MMP is converted to a 62-kDa species with APMA treatment after gelatin sepharose affinity purification typical of previously described gelatinase MMP-2. The latent 72-kDa gelatinase from either bovine IPM or Y-79 media autoactivates without APMA in the presence of calcium and zinc after 72 hr at 37°C, producing a fully active mixture of proteinase species, 50 (48 in Y-79 medium), 38 and 35 kDa in size. The presence of inhibitory activity was examined in both whole bovine IPM and IPM fractions separated by SDS-PAGE. Whole IPM inhibited gelatinolytic activity of autoactivated Y-79-derived MMP in a dose-dependent manner. Inhibitory activities are observed in two protein fractions of 27-42 and 20-25 kDa. Western blots using antibodies to human tissue inhibitor of metalloproteinase 1 and 2 (TIMP-1 and -2) reveal the presence of two TIMP-1-like proteins at 21 and 29 kDa in inhibitory fractions of the bovine IPM. TIMP-2 was not detected in the inhibitory IPM fractions, consistent with the observed autoactivation of bovine IPM 72-kDa gelatinase. Potential roles for this IPM MMP-TIMP system include physiologic remodelling of the neural retina-RPE cell interface and digestion of shed rod outer segment as well as pathological processes such as retinal detachment, PE cell migration, neovascularization and tumor progression. Cultured Y-79 cells appear to be a good model for studying the production and regulation of this proteinase system.

Scleral matrix metalloproteinases, serine proteinase activity and hydrational capacity are increased in myopia induced by retinal image degradation

In the avian model of myopia, retinal image degradation quickly leads to ocular enlargement. We now give evidence that regionally specific changes in ocular size are correlated with both biomechanical indices of scleral remodeling, e.g. hydration capacity and with biochemical changes in proteinase activities. The latter include a 72 kDa matrix metalloproteinase (putatively MMP-2), other gelatin-binding MMPs, an acid pH MMP and a serine protease. Specifically, we have found that increases in scleral hydrational capacity parallel increases in collagen degrading activities. Gelatin zymography reveals that eyes with 7 days of retinal image degradation have elevated levels (1.4-fold) of gelatinolytic activities at 72 and 67 kDa M(r) in equatorial and posterior pole regions of the sclera while, after 14 days of treatment, increases are no longer apparent. Lower M(r) zymographic activities at 50, 46 and 37 kDa M(r) are collectively increased in eyes treated for both 7 and 14 days (1.4- and 2.4-fold respectively) in the equator and posterior pole areas of enlarging eyes. Western blot analyses of scleral extracts with an antibody to human MMP-2 reveals immunoreactive bands at 65, 30 and 25 kDa. Zymograms incubated under slightly acidic conditions reveal that, in enlarging eyes, MMP activities at 25 and 28 kDa M(r) are increased in scleral equator and posterior pole (1.6- and 4.5-fold respectively). A TIMP-like protein is also identified in sclera and cornea by Western blot analysis. Finally, retinal-image degradation also increases (~2.6-fold) the activity of a 23.5 kDa serine proteinase in limbus, equator and posterior pole sclera that is inhibited by aprotinin and soybean trypsin inhibitor. Taken together, these results indicate that eye growth induced by retinal-image degradation involves increases in the activities of multiple scleral proteinases that could modify the biomechanical properties of scleral structural components and contribute to tissue remodeling and growth.

Diabetic peripheral neuropathy and retinal tissue thickness

Using retinal imaging, the nature and extent of compromise of retinal structural integrity has been characterized in individuals suffering from diabetic peripheral neuropathy. These findings extend our understanding of the pathological processes involved in diabetic neuropathy and offer novel ophthalmic approaches to the diagnosis and monitoring of this debilitating condition.

Melanopsin expressing intrinsically photosensitive Retinal Ganglion cells in retinal disease

Melanopsin containing intrinsically photosensitive Retinal Ganglion Cells (ipRGCs) are a class of photoreceptors with established roles in non-image forming processes. Their contributions to image forming vision may include the estimation of brightness. Animal models have been central for understanding the physiological mechanisms of ipRGC function and there is evidence of conservation of function across species. ipRGCs can be divided into 5 ganglion cell subtypes that show morphological and functional diversity. Research in humans has established that ipRGCs signal environmental irradiance to entrain the central body clock to the solar day for regulating circadian processes and sleep. In addition, ipRGCs mediate the pupil light reflex (PLR), making the PLR a readily accessible behavioural marker of ipRGC activity. Less is known about ipRGC function in retinal and optic nerve disease, with emerging research providing insight into their function in diabetes, retinitis pigmentosa, glaucoma and hereditary optic neuropathy. We briefly review the anatomical distributions, projections and basic physiological mechanisms of ipRGCs, their proposed and known functions in animals and humans with and without eye disease. We introduce a paradigm for differentiating inner and outer retinal inputs to the pupillary control pathway in retinal disease and apply this paradigm to patients with age-related macular degeneration (AMD). In these cases of patients with AMD, we provide the initial evidence that ipRGC function is altered, and that the dysfunction is more pronounced in advanced disease. Our perspective is that with refined pupillometry paradigms, the pupil light reflex can be extended to AMD assessment as a tool for the measurement of inner and outer retinal dysfunction.

Exploring Retinal Markers of Diabetic Neuropathy

Purpose : To investigate the application of retinal nerve fibre layer (RNFL) thickness as a marker for severity of diabetic peripheral neuropathy (DPN) in people with Type 2 diabetes. Methods : This was a cross-sectional study whereby 61 participants (mean age 61 [41-75 years], mean duration of diabetes 14 [1-40 years], 70% male) with Type 2 diabetes and DPN underwent optical coherence tomography (OCT) scans. Global and 4 quadrant (TSNI) RNFL thicknesses were measured at 3.45mm around the optic nerve head of one eye. Neuropathy disability score (NDS) was used to assess the severity of DPN on a 0 to 10 scale. Participants were divided into three age-matched groups representing mild (NDS=3-5), moderate (NDS=6-8) and severe (NDS=9-10) neuropathy. Two regression models were fitted for statistical analysis: 1) NDS scores as co-variate for global and quadrant RNFL thicknesses, 2) NDS groups as a factor for global RNFL thickness only. Results : Mean (SD) RNFL thickness (µm) was 103(9) for mild neuropathy (n=34), 101(10) for moderate neuropathy (n=16) and 95(13) in the group with severe neuropathy (n=11). Global RNFL thickness and NDS scores were statistically significantly related (b=-1.20, p=0.048). When neuropathy was assessed across groups, a trend of thinner mean RNFL thickness was observed with increasing severity of neuropathy; however, this result was not statistically significant (F=2.86, p=0.065). TSNI quadrant analysis showed that mean RNFL thickness reduction in the inferior quadrant was 2.55 µm per 1 unit increase in NDS score (p=0.005). However, the regression coefficients were not statistically significant for RNFL thickness in the superior (b=-1.0, p=0.271), temporal (b=-0.90, p=0.238) and nasal (b=-0.99, p=0.205) quadrants. Conclusions : RNFL thickness was reduced with increasing severity of DPN and the effect was most evident in the inferior quadrant. Measuring RNFL thickness using OCT may prove to be a useful, non-invasive technique for identifying severity of DPN and may also provide additional insight into common mechanisms for peripheral neuropathy and RNFL damage.