A longitudinal case series investigating cellular changes to the transplanted cornea using confocal microscopy.

PURPOSE: To perform a longitudinal evaluation of subjects who had undergone penetrating keratoplasty, using slit scanning confocal microscopy. METHODS: In vivo confocal microscopy was used to evaluate the central cornea of four subjects who had recently undergone penetrating keratoplasty. Subjects were examined on four occasions over a 12-month period after surgery. Quantitative and qualitative aspects of corneal morphology were compared against data from normal control subjects. RESULTS: The epithelium varied in appearance between subjects and took at least 12 months to return to a similar arrangement to that seen in normal eyes. Bowman's layer was viewed as an acellular layer immediately after surgery with no evidence of nerve fibres, although nerve components were apparent 12 months after surgery. Stromal nerves were not visible immediately after surgery. One year following penetrating keratoplasty there was evidence of thin nerves running a straight course through the central stroma. Keratocyte density in the anterior and posterior stroma was lower in the transplanted cornea but appeared to remain constant over a period of 1 year. Activated keratocytes were seen in the anterior stroma of all subjects; they appeared to be responsible for significant levels of corneal haze. The time period within which this keratocyte activation occurred varied between individuals. Endothelial cell density decreased at an accelerated rate over the 12-month period. CONCLUSIONS: Confocal microscopy allows cellular changes to be monitored in vivo following penetrating keratoplasty and may assist clinicians in understanding postoperative recovery.     

Confocal microscopy of the normal human cornea.

The slit-scanning confocal microscope is a new clinical paradigm that allows the living human cornea to be viewed at a magnification of 680 x and a lateral resolution of 1 mum. As such, it allows corneal morphology to be inspected at a cellular level. The corneas of both eyes of 119 subjects who were evenly distributed in age from 10-80 years were examined using a Tomey ConfoScan P4 in-vivo slit-scanning real-time confocal microscope (Erlangen, Germany). Good quality representative images of the various corneal layers were selected for detailed qualitative analysis and are displayed here. A grid of corneal layer versus age was constructed from these images; this tool can be used as a normative confocal microscopy reference against which suspected corneal abnormalities can be assessed.     

An evaluation of corneal nerve morphology and function in moderate keratoconus.

PURPOSE: To investigate corneal nerve morphology and corneal sensitivity in keratoconus. METHODS: The central cornea of 13 subjects with keratoconus and 13 age-matched control subjects was assessed using in vivo confocal microscopy and corneal aesthesiometry. RESULTS: Significant differences in corneal nerve fibre density were found between the subjects with keratoconus and the control subjects (keratoconus versus control; 1018.3+/-489.6 microm versus 1820.7+/-789.5 microm; p = 0.006). The mean diameter of nerve fibres in the stroma was found to be greater in subjects with keratoconus compared to control subjects (keratoconus versus control; 10.2+/-4.6 microm versus 5.5+/-1.9 microm; p = 0.007). The orientation of corneal nerve fibres in the subjects with keratoconus appeared to be altered from the predominantly vertical orientation seen in the control subjects. Corneal touch threshold was found to be similar in the two groups, although the subjects with keratoconus using contact lens correction had reduced corneal sensitivity compared to the contact lens-wearing control subjects (keratoconus with contact lenses versus controls with contact lenses; 1.18+/-0.19 g/mm2 versus 0.98+/-0.05 g/mm2; p = 0.03). CONCLUSION: This study reveals significant reductions in nerve density in the keratoconic cornea. The thickened stromal nerve fibres observed in the keratoconic corneas may explain why prominent corneal nerves are often seen using slit lamp biomicroscopy in keratoconic patients.     

Confocal microscopy of the corneas of long-term rigid contact lens wearers.

Slit scanning confocal microscopy (Tomey Confoscan P4) was used to evaluate the central cornea of 22 subjects who had been wearing rigid lenses on a long-term, daily wear basis. Anterior and posterior keratocyte densities appeared unaffected by rigid lens wear (P = 0.10 and 0.34 for anterior and posterior keratocyte densities, respectively). Subjects with a previous history of polymethyl methacrylate (PMMA) lens wear showed a reduction in anterior keratocyte density (AKD) (P < 0.0001) and an increased level of haze in the anterior stroma. This may represent previous hypoxic damage. Endothelial cell density (ECD) was unaffected by rigid lens wear (P = 0.36) although an increase in endothelial polymegethism was evident (P < 0.0001). Subjects who had only worn rigid lenses with no history of PMMA lens wear did not show an increase in endothelial polymegethism (P = 0.10). An increased number of microdot opacities compared to the non-lens wearing eye was apparent (P = 0.05). The number of microdot opacities induced by rigid lenses, as reported in this study, appears to be less than that reported by others in respect of soft lenses.     

Microstructural evaluation of the mucin balls and their relations to the corneal surface—Insights by in vivo confocal microscopy

Purpose

The purpose of the current study was to observe and correlate the characteristics of mucin balls to the ocular surface properties, and furthermore, to report the effect of different mucin balls size and number on structural alteration of the anterior cornea.

A pilot study on corneal Langerhans cells in keratoconus

Purpose

To report the density and morphology of cells that are analogous to corneal Langerhans cells and their associations in keratoconus.

Effect of age and contact lens wear on corneal epithelial dendritic cell distribution,density, and morphology

PurposeContact lens wearers aged 15–25 years are at higher risk of corneal inflammation, yet little is known about corneal inflammatory state in this group. Previous investigations show density of corneal epithelial dendritic cells (CEDC) may increase with contact lens wear. However, it is not known how corneal distribution or morphology of CEDC alters with lens wear or whether these markers are affected by age. This study characterised CEDC in adolescent and young adult contact lens wearers to determine effects of contact lens wear and age on CEDC density, distribution and morphology.MethodForty participants (20 contact lens wearers, 20 healthy non-wearers; age 16–36 years; 16M:24F) completed this pilot study. Corneal images were captured using in vivo confocal microscopy (HRTII, Rostock). CEDC were manually counted in a 1 mm² area of the central and mid-peripheral cornea, and ratio of central to midperipheral density was calculated. CEDC morphology and dendrite length were graded. Differences between groups and between regions, and associations with age were examined. Significance was determined at P < 0.05.ResultsA lower ratio of central to mid-peripheral CEDC density was found with younger age (ρ = 0.42, P = 0.01). CEDC morphology was not associated with age or contact lens wear. CEDC density in the mid-peripheral cornea was higher in soft lens wearers than non-wearers (P = 0.04), but central density did not differ. CEDC density and morphology were not significantly different between centre (median density 11 cells/mm², range 0–120) and mid-periphery (10 cells/mm², 0–58).ConclusionDensity, distribution and morphology of CEDC do not differ in established contact lens wearers. A relatively lower density of CEDC in the central cornea of younger patients may allude to a more naive immune status in this group and warrants further study. Decreased central CEDC density identified in orthokeratology lens wear requires confirmation in a larger group.     

The morphology of corneal endothelial cells in long term soft contact lens wearers in Kuala Lumpur

PurposeTo analyse and compare the alterations in corneal endothelium morphology induced by different materials and durations of wearing soft contact lenses (CL) among young adults living in Kuala Lumpur.MethodsHealthy soft CL wearers were invited to participate in this study. Visual acuity (VA) was measured using the Snellen chart, and subjective refraction was performed using cross-cylinder technique. Standard ocular assessments were conducted using a slit lamp biomicroscope and morphology of corneal endothelial cells (endothelial cell density, ECD, coefficient variation, COV, hexagonality, HEX and central corneal thickness, CCT) were evaluated using a non-contact specular microscope. Statistical analysis was conducted using ANOVA and data from the right eye only is included.ResultsA total of 72 subjects (32 SiHy and 40 HCL wearers) and 24 non-CL wearers (control) participated in this study. The gender distribution for study subjects was 13 males and 59 females, with a mean age 22.15 ± 1.84 years old. The mean refraction was −1.86 ± 1.25DS. The duration of wearing CL ranged from 1 to 9 years. Subjects were later divided into 2 groups following duration of CL wear: Group 1 (<5 years) and Group 2 (≥5 years) for analysis purposes. Statistical analysis showed significant alterations in ECD, COV and HEX of CL wearers (p < 0.05), with more changes found in HCL and Group 2 wearers. No significant change was found in CCT.ConclusionThis study concludes that soft CL wear induced alterations in the morphology of corneal endothelial cells. Contact lens material and duration of CL wear (in years) are factors that affect the alterations. Optometrists are recommended to regularly evaluate the morphology of corneal endothelial cells in CL wearers and provide necessary intervention when required.     

Disruption of the tear film by the application of small drops of saline and surfactant.

The effects of disrupting the tear film by placing 5 or 10 microl drops of saline and mild surfactant solution (SS) directly onto the tear films of 10 subjects were observed and are described here. Disruption of the tear film was only transient, with restoration of the film occurring within about two to three blinks for saline drops, and four to six blinks for SS drops. When a drop of SS was placed on a cornea, two types of black spots/areas may be observed: black areas apparently similar to those observed in the TBUT test, and black areas forming outside the border of the pattern of the spreading SS drop. In general, staining of the black areas formed in the fluorescein-stained tears did not occur even when blinking was inhibited for up to 13 s. Stained areas on the corneal surface did not have a tendency to form black spots first; the tear film over these areas did not appear to be less stable than on other areas of the corneal surface.     

A physiological perspective on the swelling properties of the mammalian corneal stroma.

PURPOSE: The present studies were designed to assess whether measurement of corneal stroma swelling in the laboratory, especially in non-physiological solutions, was associated with a measurable effect on the keratocytes. METHODS: Complete corneal stroma preparations were made from quality- and age-selected recent post-mortem cattle eyes. These were either assessed immediately or incubated in three different solutions, namely a balanced salts solution with glucose (BSSG), isotonic phosphate buffered saline (PBS) or pure water. Incubations were carried out at 37 degrees C for 9h, and repeated measures of wet mass made so that the rates and extent of swelling could be determined. After incubation, an aqueous extract was made of the stroma for measurements of the levels the enzymes lactate dehydrogenase (LDH), aldehyde dehydrogenase (ALDH) and N-acetyl-beta-glucosaminidase. RESULTS: The initial rates of swelling were lowest in BSSG, marginally faster in PBS and much faster in water. The secondary rates of swelling showed the same sequence being 10.0%/h in BSSG, 14.8%/h in PBS and 34.2%/h in water. Compared to non-incubated preparations, reductions in all three enzyme activities occurred. For LDH, these were 15% with BSSG, 40% in PBS and 80% with water. Similar results were seen with ALDH activity when comparing the three incubation solutions, while incubation in BSSG also resulted in a substantial (40%) reduction in N-acetyl-glucosaminidase activity. CONCLUSIONS: When immersed in an isotonic BSSG with added glucose at 37 degrees C, the swelling of a complete bovine corneal stroma is much less than smaller pieces of stroma, and also slightly less than if isotonic PBS was used. With the use of BSSG, little or no change in cytoplasmic enzyme activities occurred, but measurable decreases were noted with PBS and very substantial decreases when water was used, indicating a toxic effect on the keratocytes. The observation that substantial decreases in a lysosomal enzyme activity could occur even with the use of BSSG indicate substantial stress is imposed on the stroma during these types of experiments. Notwithstanding, the data collectively indicate that the keratocyte cells within the collagen matrix of the stroma can be substantially damaged and this needs to be taken into account in future experiments on the true physiology of the corneal stroma.     

Non-contact laser-scanning confocal microscopy of the human cornea in vivo

PurposeTo investigate the utility of using non-contact laser-scanning confocal microscopy (NC-LSCM), compared with the more conventional contact laser-scanning confocal microscopy (C-LSCM), for examining corneal substructures in vivo.MethodsAn attempt was made to capture representative images from the tear film and all layers of the cornea of a healthy, 35 year old female, using both NC-LSCM and C-LSCM, on separate days.ResultsUsing NC-LSCM, good quality images were obtained of the tear film, stroma, and a section of endothelium, but the corneal depth of the images of these various substructures could not be ascertained. Using C-LSCM, good quality, full-field images were obtained of the epithelium, subbasal nerve plexus, stroma, and endothelium, and the corneal depth of each of the captured images could be ascertained.ConclusionsNC-LSCM may find general use for clinical examination of the tear film, stroma and endothelium, with the caveat that the depth of stromal images cannot be determined when using this technique. This technique also facilitates image capture of oblique sections of multiple corneal layers. The inability to clearly and consistently image thin corneal substructures – such as the tear film, subbasal nerve plexus and endothelium – is a key limitation of NC-LSCM.     

Does a two-year period of orthokeratology lead to changes in the endothelial morphology of children?

Purpose

To compare changes in endothelial morphology in the central and superior cornea in subjects wearing single-vision spectacles and orthokeratology lenses over two years.

A pilot study investigating the effect of extended contact lens wear on limbal and central corneal morphology

PurposeTo investigate the effect of long-term extended soft contact lens wear on limbal and central corneal cell morphology, and limbal architecture.MethodsEach participant attended a study visit involving in vivo confocal microscopy of central corneal and limbal epithelium. Scans were graded by five masked graders for three features: central epithelial irregularity, limbal epithelial irregularity and the prominence of palisades of Vogt. The variability of grades between different graders and the difference of grades between extended wearers and daily soft/non-contact lens wearers were assessed.ResultsNineteen participants (9 extended soft contact lens wearers and 10 daily soft/non-contact lens wearers) aged 31–65 years were enrolled in this study. Scans from 37 eyes were included in the analysis. Agreement between graders for each feature was moderate to good with inter class correlation >0.7. While there were no significant differences in central epithelial cell irregularity (p = 0.527) and the prominence of palisade of Vogt (p = 0.182) between extended or daily soft/non-contact lens wearers, limbal epithelial cell irregularity showed a trend with increased irregularity in extended soft contact lens wearers (p = 0.091).ConclusionsWhile no differences in limbal cell morphology and structure or central epithelial cell was found in this subjective grading study of extended wearers compared to daily soft/non-contact lens wearers, further studies using a larger sample size or a longitudinal study design are warranted.     

Dose-response of the cultured bovine lens to butyl, methyl and propyl parabens

Pre-screening of cosmetic ingredients is vital for consumer safety. Previous in vivo techniques, such as the Draize test, have proved to be unreliable in predicting ocular irritancy and therefore there is a need for alternate testing methodologies. One such test is the scanning laser in vitro assay system which quantifies irritancy based on the focusing ability of the cultured bovine lens. In combination with confocal microscopy, a more thorough documentation of ocular irritancy can be achieved. This study investigates the response of cultured bovine lenses over time to butyl, methyl and propyl parabens, which are common antimicrobial agents found in cosmetic and ophthalmic products. The focusing ability of the lens was measured with an automated laser scanner over a period of 96 h. At 120 h post-treatment, the lenses were analysed by using a confocal laser scanning microscope to determine the characteristics of nuclei, and the morphology and distribution of mitochondria within the lenses. Irritancy to the three parabens was investigated at both an optical and cellular level. Each of the parabens was tested at 0.002% and 0.2%, where the 0.2% butyl paraben was found to be the most irritating.     

Microstructure of Feta Cheese Made Using Different Cultures as Determined by Confocal Scanning Laser Microscopy

ABSTRACT: The objective of this work was to observe the microstructure of feta cheese using confocal scanning laser microscopy. Low fat cheese and cheese containing capsule-producing cultures were made. The protein network was observed using the reflectance mode of the confocal microscope. Fat was stained by Nile red dye diluted with whey. More even distribution of fat with a larger number of smaller globules was observed in cheese made with noncapsule-forming culture compared to that made with the capsule-forming culture. Nonfat cheese had compact structure when made with noncapsule-forming culture which contrasted with the open structure observed in cheese made with the capsule-forming culture     

The effect of the duration of diabetes on dry eye and corneal nerves

PurposeTo investigate the relationship between the duration of type 2 diabetes mellitus (DM) and the ocular surface, and to address the question of why some people with lengthy DM duration are asymptomatic, whereas some people with shorter DM duration have pain or discomfort in their eyes.MethodsEighty-seven eyes of 87 subjects with different durations of DM and 49 eyes of 49 subjects without DM underwent Schirmer I test, tear film break-up time, sodium fluorescein staining and tear meniscus height (TMH) measurement, and completed the Standardized Patient Evaluation of Eye Dryness (SPEED) questionnaire. Corneal structure and function were assessed with in vivo confocal corneal microscopy and with a corneal sensitivity esthesiometer. Both corneal nerve fiber length and inferior whorl length (IWL) were assessed as indices for neural structure. Age and gender were matched between groups. HbA1c levels >7.8% and proliferative diabetic retinopathy were exclusion criteria.ResultsIn the DM group, compared with the non-DM group, the SPEED score was significantly higher (p = 0.013), and corneal sensitivity and IWL were lower (p < 0.001). Schirmer I test, corneal sensitivity and IWL differed significantly between the group with DM duration >10 years and the non-DM (control) group (p = 0.021, p < 0.001, p < 0.001, respectively). Schirmer I test and IWL were significantly lower in the group with DM >10 years than in the group with DM ≤10 years (p = 0.023, p < 0.001, respectively). Corneal sensitivity was positively correlated with IWL regardless of diabetes status.ConclusionsThe lower SPEED score and asymptomatic feeling in people with a longer DM duration may be explained by the decreased IWL and reduced sensitivity.     

高压射流磨处理对燕麦浆稳定性的影响

目的：研究高压射流磨对燕麦浆稳定性的影响及机制,为全谷物饮料加工提供依据。方法：采用不同压力高压射流磨对燕麦浆进行处理,比较贮藏期（30 d）的形态、不稳定指数、粒径、流变特性变化,并对微观形貌（光学显微镜、激光共聚焦扫描显微镜、扫描电子显微镜）、可溶性成分含量（可溶性固形物、可溶性蛋白、可溶性膳食纤维）进行分析。结果：高压射流磨处理使燕麦浆的粒径、不稳定指数、表观黏度逐渐减小,并能减缓淀粉的老化和颗粒的聚集。高压射流磨能均化蛋白和油脂,破坏细胞壁组织纤维,使更多可溶性物质溶出,并在颗粒内部产生孔腔,增加水合能力,提高体系总体稳定性。结论：高压射流磨技术可提高全谷物产品贮藏稳定性,延长货架期。     

On the in vivo assessment of goblet cells of the human bulbar conjunctiva by confocal microscopy – A review

BackgroundIn vivo confocal microscopy (IVCM) has been used for over 10 years to assess the goblet cell density (GCD) within the human conjunctiva, but the reported values have been variable with no obvious indications as to why.MethodsFrom publications between 2008 and 2019, representative GCD values were extracted, as well as on the image sampling strategy used.ResultsAverage GCD values for any particular group of individuals ranged from 7 to 979 goblet cells / sq. mm, and with one notable outlier removed, an overall group-mean value for GCD (+/− SD) from single site locations was 207 +/− 143 goblet cells / sq. mm from 15 data sets for those usually designated as control subjects, with a value of 190 +/− 161 goblet cells / sq. mm calculated from 20 single site data sets from other (patient) groups. An overall analysis indicated that the reported average values for GCD from different groups of individuals increased according to the number of images assessed / individual (Spearman rho = 0.304), on the number of individuals evaluated to generate an averaged value for each group (rho = 0.367), and the total number of images assessed (rho = 0.346, multivariate analysis partial r = greater or = to 0.522).ConclusionsIn the use of confocal microscopy to assess the number of goblet cells in the human bulbar conjunctiva, the substantial differences reported appear to be linked to the protocols used for image selection, and some type of standardization needs to be developed.     

The impact of lens care solutions on corneal epithelial changes during daily silicone hydrogel contact lens wear as measured by in vivo confocal microscopy

Purpose

To assess corneal epithelial microstructure via confocal microscopy and determine if cellular changes are associated with lens care solutions during daily wear of silicone hydrogel contact lenses.

Effects of variation in the palm stearin: Palm olein ratio on the crystallisation of a low-trans shortening

Changes in the rheological properties during crystallisation and in the crystal size and morphology of blends containing rapeseed oil with varying percentages of palm stearin (POs) and palm olein (POf) have been studied. The crystals formed from all three blends were studied by confocal laser scanning microscopy, light microscopy and environmental scanning electron microscopy, which revealed the development of clusters of 3–5 individual elementary “spherulites” in the early stages of crystallisation. The saturated triacylglycerol content of the solid crystals separated at the onset of crystallisation was much greater than that in the total fat. Fat blends with a higher content of palm stearin had a more rapid nucleation rate when observed by light microscopy, and this caused an earlier change in the rheological properties of the fat during crystallisation. Using a low torque amplitude (0.005 Pa, which was within the linear viscoelastic region of all samples studied) and a frequency of 1 Hz, the viscoelastic properties of melted fat during cooling were studied. All samples, prior to crystallisation, showed weak viscoelastic liquid behaviour (G″, loss modulus >G′, storage modulus). After crystallisation, a more “solid like” behaviour was observed (G′ similar to or greater than G″). The blend having the highest concentration of POs was found to have the earliest onset of crystallisation (27% w/w POs; 12 mins, 22% w/w POs; 13.5 mins, 17% w/w POs, 15 mins, respectively). However, there were no significant differences in the time to the point when G′ became greater than G′ among the three blends.