Investigating a clinically actionable BRAF mutation for monitoring low-grade serous ovarian cancer: A case report.

Low-grade serous ovarian cancer (LGSOC) poses a specific clinical challenge due to advanced presentation at diagnosis and the lack of effective systemic treatments. The aim of this study was to use a precision medicine approach to identify clinically actionable mutations in a patient with recurrent LGSOC. Primary, metastatic and recurrence tissue, and blood samples were collected from a stage IV LGSOC patient. Single-gene testing for clinically actionable mutations (BRAF V600, KRAS and NRAS) and subsequent whole-exome sequencing (WES) were performed. Droplet digital PCR was used to evaluate the presence of an identified BRAF D594G mutation in the matched plasma cell-free DNA (cfDNA). No clinically actionable mutations were identified using single-gene testing. WES identified a BRAF D594G mutation in six of seven tumor samples. The patient was commenced on a MEK inhibitor, trametinib, but with minimal clinical response. A newly designed ddPCR assay detected the BRAF alteration in the matched tissues and liquid biopsy cfDNA. The identification and sensitive plasma detection of a common "druggable" target emphasises the impact of precision medicine on the management of rare tumors and its potential contribution to novel monitoring regimens in this field.

Non-contact specular microscopy with Topcon instruments to assess central corneal thickness of healthy human eyes - A 20 year review.

BACKGROUND: The purpose of this review was to evaluate the consistency of central corneal thickness (CCT) values reported with use of Topcon SP-2000 P and SP-3000 P non-contact specular microscopes since their introduction in 1999 with the two microscopes having been commonly used in a wide range of studies. METHODS: As a primary resource, PubMed was used to search for peer-reviewed articles in any language that included CCT values obtained with non-contact specular microscopy reported for humans with nominally healthy corneas. Relevant articles were obtained and any cited publications also checked. RESULTS: A total of 76 articles were identified which reported CCT on different small-to-moderate sized groups of individuals, published between 1999 and 2019. From these, an overall group mean CCT value of 0.525 ± 0.013 mm (median 0.525 mm) can be calculated. An estimated 95 % confidence interval (CI, based on 1.96 SD) would be between 0.500 and 0.550 mm. For the two Topcon models, the group mean ± SD values were 0.529 ± 0.013 mm and 0.517 ± 010 mm respectively. An assessment of the CCT data sets in relation to the reported average age indicated no statistically significant effect (p = 0.289, r = -0.129). Very similar average CCT values were also encountered in 4 other reports where these microscopes were used in large-scale population studies as well as in 2 other reports using the newer Topcon SP-1 P model. CONCLUSIONS: The Topcon stand-alone non-contact specular microscopes have yielded consistent and predictable corneal thickness measures over many years.

On the regional variability of averaged cell area estimates for the human corneal endothelium in relation to the extent of polymegethism.

PURPOSE: To assess variability in the coefficient of variation (COV) in cell area estimates when using different numbers of cells for endothelial morphometry. METHODS: Using non-contact specular microscopy images of the corneal endothelium, 4 sets of 20 cases were selected that included 200 cells and had overall (global) COV values of less than 30 (group 1), 31-40 (group 2), 41-50 (group 3) and over 50% (group 4). Subjects could be normal, or had ophthalmic disease (such as diabetes), a history of rigid or soft contact lens wear or were assessed after cataract surgery. A step-wise analysis was undertaken, 20 cells at a time, of the variability in cell area estimates when using different numbers of cells for the calculations. RESULTS: Variability in the average cell area values was higher if only 20-60 cells were used in the calculations and then tended to decrease. The standard deviation values on these average cell area values and the calculated COV showed the same overall trends and were more than twice as large for endothelia with marked polymegethism. Using more than 100 cells/image in markedly polymegethous endothelia only increased the variability in the calculations. CONCLUSIONS: These analyses indicate that substantial region variability in cell area values can be expected in polymegethous endothelia. The analysis further confirm that using only small numbers of cells (e.g. less than 50/image) in such cases is likely to yield far less reliable estimates of COV.

Giemsa-based cytological assessment of area, shape and nucleus:cytoplasm ratio of goblet cells of rabbit bulbar conjunctiva.

Goblet cells were visualized in impression cytology specimens from bulbar conjunctiva of the rabbit eye using Giemsa staining. Highly magnified images were used to generate outlines of the goblet cells and their characteristic eccentric nuclei. Using sets of 10 cells from 15 cytology specimens, I found that the longest dimension of the goblet cells averaged 16.7 ± 2.3 µm, the shortest dimension averaged 14.4 ± 1.8 µm and the nucleus averaged 6.3 ± 0.8 µm. The goblet cells were ellipsoid in shape and the longest:shortest cell dimension ratio averaged 1.169 ± 0.091. The goblet cell areas ranged from 108 to 338 µm2 (average 193 ± 50 µm2). The area could be predicted reliably from the longest and shortest dimensions (r2 = 0.903). The areas of goblet cell nuclei were 15-58 µm2 (average 33 ± µm2) and the nucleus:cytoplasm area fraction was predictably greater in smaller goblet cells and less in the larger goblet cells (Spearman correlation = 0.817). The nuclei were estimated to occupy an average of 9.5% of the cell volume. The differences in size, shape and nucleus:cytoplasm ratio may reflect differences in goblet cell maturation.

On the staining of human tissue-cultured (Chang) conjunctival cells with rose bengal and lissamine green.

PURPOSE: To assess the staining of Chang conjunctival epithelial cells with rose bengal and lissamine green. METHODS: A human (Chang) conjunctival epithelial cell line was grown on plastic plates in 10% foetal calf serum-supplemented medium in 5% carbon dioxide-air at 37°C. Cells were examined daily between days 2 and 10 of culture, reaching confluence after 7 days, for overall appearance before and after exposure for 3 min at 37°C to solutions of either rose bengal or lissamine green (dissolved in saline, PBS or balanced salts solution). Measurements of nucleus and cell size were made on some confluent cultures. RESULTS: Chang cells, regardless of the growth state or the vehicle used, stain very intensely with rose bengal, with the nucleus of the cells showing the most notable dye uptake. Cultures showing such intensive staining with rose bengal were consistently non-staining with lissamine green. Nucleus size, based on measurements of the long dimension (NUCLONG) were the same for rose bengal or lissamine green exposed cells (averaging 19.5±2.4 µm and 18.8±2.9 µm, respectively). Cell size at confluence, based on measurements of the long dimension of lissamine green-exposed cells (LONG), averaged 40.5±7.2 µm. The cytoplasm-to-nucleus ratio (based on LONG-NUCLONG/NUCLONG) averaged 1.177±0.406 (i.e. approximately a 1:1 ratio). CONCLUSIONS: Cultured Chang conjunctival cells stain intensely with rose bengal, but the same is not seen with lissamine green. The cell morphology is indicative of flattened epithelial cells.

Assessment of a variable frame (polygonal) method to estimate corneal endothelial cell counts after corneal transplantation.

PURPOSE: To assess the agreement of the 'polygonal' variable frame cell count option on a confocal microscope after keratoplasty, with planimetry as the reference method. METHODS: One hundred clear corneal grafts of 83 patients attending the cornea clinic at Gartnavel General Hospital in Glasgow underwent slit-scanning in vivoconfocal microscopy. Endothelial cell images were assessed with the Nidek Advanced Vision Information System (NAVIS), using the polygonal variable frame and the manual fixed-frame methods. Planimetry was used as the reference. The agreement between methods was assessed by Bland-Altman analysis. RESULTS: Planimetry provided a mean (± SD) endothelial cell density (ECD) of 1348 ± 726 cells/mm(2), a value that was very similar to that found by the polygonal method (1404 ± 784 cells/mm(2)). The fixed-frame method provided lower cell counts with a mean ECD of 1026 ± 610 cells/mm(2) (P<0.001). When compared with the reference ECD, the polygonal method overestimated the ECD only very slightly with a mean difference of 58 cells/mm(2) (limits of agreement, LoA, of -222 and 339 cells/mm(2)). Manual counting underestimated the ECD with a mean difference of -320 cells/mm(2) (LoA -814 and 173 cell/mm(2)). CONCLUSION: Following keratoplasty, endothelial cell counts with the NAVIS polygonal method are in good agreement with planimetry. The 'polygonal' option is proposed as the method of choice for clinical applications with this confocal microscope and a good compromise between reliability and ease of use.

Valproic acid inhibits neurosphere formation by adult subventricular cells by a lithium-sensitive mechanism.

The mood stabilizer valproic acid (VPA) decreases neural progenitor proliferation and promotes neurogenesis in the adult hippocampus. However, the effects of VPA on progenitor cells in the adult subventricular zone (SVZ) are not as well characterized. Here we report VPA blocks neurosphere formation and inhibits DNA synthesis in cultured NSCs from the SVZ of adult mice. Inhibition of DNA synthesis is associated with the up-regulation of the differentiation transcription factors Egr1 and Neurod1 and down-regulation of transcription factors associated with "stemness". Co-treatment of VPA with the mood stabilizer lithium antagonizes the anti-proliferative effects of VPA on adult NSCs and abolishes VPA activation of Egr1. Co-treatment of VPA with the MEK1/2 inhibitor PD980589 similarly abolishes Egr1 activation consistent with VPA activation and lithium antagonism of MEK-ERK signaling in adult NSCs. However, Western blot reveals VPA significantly suppresses ERK2 phosphorylation in adult NSCs grown in proliferating culture conditions and that lithium co-treatment does not attenuate this effect. Combined the data indicate VPA inhibition of adult NSC proliferation and activation of Egr1 by VPA, along with the antagonism of these effects by lithium, are the effects of cumulative changes in multiple signaling pathways and are not attributable to a common kinase target.

In vivo confocal microscopy of the corneal endothelium: comparison of three morphometry methods after corneal transplantation.

PURPOSE: The purpose of this study was to assess the endothelium of corneal grafts by in vivo confocal microscopy (IVCM), and to evaluate an automated endothelial software system in comparison with a manual cell count and planimetry. PATIENTS AND METHODS: Overall, 40 corneal grafts (20 deep anterior lamellar keratoplasties (DALKs) and 20 penetrating keratoplasties (PKs)) were assessed by scanning-slit IVCM. The endothelial cell density (ECD) was estimated with the automated and the manual cell count method of the instrument's Nidek Advanced Vision Information System (NAVIS) software. The results were compared with planimetry as the reference method, and the agreement was assessed. RESULTS: The mean (±SD) automated ECD was 2278±524 cells/mm(2) (range 1167-3192 cells/mm(2)), whereas the manual cell count method gave significantly lower ECDs with a mean of 1213±677 cells/mm(2) (range 218-2440 cells/mm(2); P<0.001). The manual cell counts were also significantly lower than those by planimetry, with a mean ECD of 1617±813 cells/mm(2) (range 336-2941, P<0.001). Bland-Altman analyses indicated that the limits of agreement (LoA) between the automated and the planimetry method were -671 and +1992 cells/mm(2), whereas they were -1000 and +202 cells/mm(2) when comparing the manual cell counts with planimetry. CONCLUSION: Following keratoplasty, the NAVIS automated method is likely to overestimate endothelial cell counts due to oversegmenting of the cell domains. Automated ECDs are substantially higher than those by the manual counting method or planimetry. The differences are considerably larger post-keratoplasty than for normal corneas, and the methods should not be used interchangeably.

Altered gene expression in the emerging cerebellar primordium of Neurog1-/- mice.

Expression of the basic helix-loop-helix (bHLH) transcription factor Neurogenin1 (Neurog1) coincides with the emergence of the cerebellum and Neurog1-expressing progenitors are fated to become Purkinje cells and later interneurons. However, the gene regulatory functions of Neurog1 in cerebellar development have not been characterized. We performed a genome-wide analysis of gene expression in the cerebellar primordium of E11.5 Neurog1 null (Neurog1-/-) mice to identify the Neurog1 transcriptome in the emerging cerebellum. This screen identified 117 genes differentially enriched in Neurog1-/- versus control sample sets with a high presence of gene sets enriched for functions in nervous system development. Hierarchical clustering revealed complete stratification of differentially expressed genes based on Neurog1 gene deletion status. In silico analysis of promoter regions identifies high probability Neurog1 regulatory (E-box) binding sites in 94 of the 117 differentially expressed genes and Pax6 binding motifs in 25 of these 94 promoters. Our data provide a framework for investigating Neurog1 transcriptional programs in early cerebellar development and suggest functional Neurog1-Pax6 cross-talk in the activation of downstream targets.

Assessment of agreement for assignment of a normal grade to human conjunctival impression cytology samples.

OBJECTIVE: To assess the level of agreement for assignment of a normal grade for human ocular surface bulbar conjunctival cells as collected by conjunctival impression cytology. METHODS: Suitable images from published articles that included a scale marker were subjected to the same planimetry method to assess cell area, nucleus area, long (L) and short (S) dimensions of the cells and the nucleus. These measures, along with the nucleus-to-cytoplasmic (N/C) ratio, were compared. RESULTS: Area measures on normal grade images indicated a broad unimodal distribution (250-275 µm(2)), and distribution of nucleus area values was heterogeneous, with neither being normally distributed. The inter-sample variance for any particular area value ranged from 11 to 90% (group averages of 49%). The L:S dimension ratio averaged 1.288, consistently showing these cells are not round. N/C values showed a wide range (from 0.151 to 0.655), as did nucleus-to-cytoplasm dimensions (from 0.332 to 0.603). CONCLUSIONS: Current criteria for subjective assignment of a normal grade to bulbar conjunctival cells do not appear to be robust enough to allow for inter-sample comparison. Quantitative measures need to be developed further.

Neurogenin1 expression in cell lineages of the cerebellar cortex in embryonic and postnatal mice.

The basic helix-loop-helix (bHLH) transcription factors Ptf1a and Math1 are necessary for the specification of gamma-aminobutyric acid-ergic and glutamatergic cell lineages in the cerebellum, respectively. Recent evidence suggests cascades of bHLH factor activities drive cell type specificity in Ptf1a(+ve) and Math1(+ve) lineages. In this manuscript, we reveal cell lineages in the cerebellar cortex but not deep cerebellar nuclei express the pro-neural bHLH factor Neurogenin1 (Ngn1). Ngn1 is expressed in ventricular zone progenitors and in newly generated neurons in the caudal cerebellar primordium. In later embryonic and postnatal developmental stages, Ngn1 is expressed in progenitors and in migrating interneurons in the prospective white matter. Transgenic fate-mapping reveals Ngn1 reporter-gene expression in Purkinje cells, multiple inhibitory interneuron cell types, and in unipolar brush cells of the cortex. The data suggest Ngn1 is a component of the bHLH factor code regulating cell type specification in the cerebellar cortex.

Fibre optic spectrophotometry for the in vitro evaluation of ultraviolet radiation (UVR) spectral transmittance of rabbit corneas.

A fibre optic spectrophotometer front-end system for measuring corneas to overcome shortcomings associated with existing instruments was tested. The system allowed prompt measurement postmortem, minimizing beam pathlength to reduce the effects of scatter and unwanted refraction and eliminated optical interfaces and cuvette media. Rabbit corneas were excised immediately postmortem and placed on a detecting fibre optic coupled to an Ocean Optics spectrophotometer and illuminated by a deuterium-halogen source. The compact instrument with its small beam size allowed tissue profiling at test points across the corneal surface and efficient interchange for comparison of different tissues. This simplified system operation allowed rapid tissue altering to study induced changes on transmittance. The corneal transmittance data showed a consistent sharp cut-off at 320 nm in the ultraviolet radiation (UVR) spectrum, which decayed rapidly from postmortem swelling. Inter- and intra-corneal consistency was demonstrated by comparing data from different regions of the same cornea and those from opposite eyes. Changes to the spectra, particularly in the UVB below 300 nm, were evident when the corneal epithelium was removed, indicating that this layer is not the only corneal UVR filter. The new system reduced much of the variability associated with previous methods, as it rapidly measured corneal transmittance postmortem. Data are in broad agreement with published transmittance curves. The removal of the corneal epithelium revealed a substantial stromal contribution to the overall corneal UVR absorption, suggesting that corneas with pathologically or iatrogenically thinned stromas are less effective UVR blockers.

Is manual counting of corneal endothelial cell density in eye banks still acceptable? The French experience.

AIM: To examine the differences in manual endothelial cell counting methods in French eye banks and to analyse whether these differences could explain some substantial discrepancies observed in endothelial cell density (ECD) for corneas made available for transplant. METHODS: A questionnaire was sent to the 22 eye banks asking for details of the technical features of the light microscopes used, the microscope calibration, strategy for cell counting, the technical staff, and the method of presenting endothelial data. RESULTS: All eye banks responded and 91% (20/22) used only manual counting methods, in real time, directly through a microscope, and 62 different technicians, with varying experience, were involved in such counting. Counting of cells within the borders of a grid that were in contact with two adjacent borders was the most common method (17/22, 77%). Of the eight banks (8/22, 36%) that did not calibrate their microscopes, six reported the highest ECD values. Of the 14 others (64%), six applied a "magnification correcting factor" to the initial cell counts. In five of these cases, the corrected ECD was lower than estimated on initial count. Most of the banks (12/22, 55%) counted 100 cells or less in one to six non-adjacent zones of the mosaic. 14 of the banks (14/22, 64%) also graded cell polymegethism while seven (7/22, 32%) also graded pleomorphism ("hexagonality"). CONCLUSIONS: Lack of microscope calibration appears to be the leading cause of variance in ECD estimates in French eye banks. Other factors such as differences in counting strategy, the evaluation of smaller numbers of cells, and the different extent of experience of the technicians may also contribute to intraobserver and interobserver variability. Further comparative studies, including cross checking and the outcome of repeated counts from manual methods, are clearly needed with cross calibration to a computer based image archiving and analysis system.

A semi-automated assessment of cell size and shape in monolayers, with optional adjustment for the cell-cell border width-application to human corneal endothelium.

Measurements of large numbers of feature sizes within defined domains (e.g. cell areas within cell-cell borders) can be a time-consuming activity, but automation that includes defining such domains has not be proven to be very reliable. Other alternatives are therefore needed, and the goal of the present studies was both to develop a semi-automated (interactive) measurement system for cell areas and to carefully compare the output to that obtained using a manual digitiser pad method. A particular interest was in the contribution made by the cell-cell border zones. Non-contact specular micrographs of central corneal endothelium were obtained from 20 white male adults, aged 40-60 years. An overlay of the endothelial image was generated manually, from which the areas of around 200 cells were measured manually with a digitiser pad and also by a computer-assisted scanning method. The pad data was typed into a spread sheet along with details of the number of cell apices (sides). The computerised analysis identified borders of the same cells on the overlay, reduced these borders to a minimum, and then assessed cell area by the pixel count along with the number of neighbouring cells (to give cell sides data). The average cell area was 393 +/- 28 and 422 +/- 29 microm(2) (mean+/-SD) by the digitiser pad and computer-based methods, respectively. The average areas for each cell type were 153, 270, 392, 519 and 685 microm(2) for 4-, 5-, 6-, 7- and 8-sided cells, respectively. Assessment of the relationship between cell area and the number of cell sides (area-side relationships) showed a highly significant and positive correlation (P<0.001; r(2)=0.865). Comparing the two methods, the average cell area was 7.5% higher in the computer scan method, and this is attributed to the fact that the contribution made by the cell borders (the para-cellular space) had been essentially eliminated. A proportional correction factor can be applied to add back the cell borders/intercellular space to the computerised output, and examples are given based on using the average data from digitiser pad for each cell type. In conclusion, a computer assisted method has been developed to simultaneously provide data on the variance in cell areas (polymegethism) and cell shape (pleomorphism) from overlays of 200 cells from human corneal endothelial images, with the cell border zone corrected to allow for a finite para-cellular space.

Morphometry of the corneal endothelium in glassblowers compared to non-glassblowers.

Acute exposure to high levels of IR radiation (IRR) can damage the endothelial cell layer of the human cornea, but the impact of chronic lower-dose exposure has not been assessed objectively. The corneal endothelium of 10 individuals who had occupational exposure to IRR as glassblowers (average 16 years) was examined by photo-slitlamp biomicroscopy, the endothelial mosaic assessed by morphometry, and compared to 10 non-glassblowers (controls). The analyses reveal the glassblowers to have higher than expected endothelial cell density (ECD, average 3371+/-304 cells/mm(2) compared to a control value of 3072+/-198 cells/mm(2)), a higher variance in cell area, and a lower percentage of the most-prevalent cell type, i.e. the six-sided cells (average 52.0+/-12.2%, compared to controls of 64.1+/-6.6%). Analyses of the sizes of different cell types (four-, five-, six-sided, etc.) indicate that the cells in both groups are proportionately larger as the number of sides increases, but that this area-side relationship is different in the glassblowers, who had both smaller and larger cells compared to controls. Two other cases had even higher cell density values (>5000 cells/mm(2)) and <50% six-sided cells. Occupational exposure to a mixture of IRR, perhaps some UVR as well as thermal convection effects, can apparently result in morphological changes in the human corneal endothelium. These may be the result of IRR-stimulated cell division.

Consideration of three types of spontaneous eyeblink activity in normal humans: during reading and video display terminal use, in primary gaze, and while in conversation.

PURPOSE: To consider the conditions under which the so-called spontaneous (endogenous) eyeblink activity has been assessed by different investigators over the last 75 years and to consider the most appropriate terminology. The reason for this analysis was to try to identify why such different values for the spontaneous eyeblink rate (SEBR) have been reported. METHODS: A retrospective evaluation of published articles was carried out to identify whether the SEBR assessments were consistently dependent on the activity of the subjects while spontaneous eyeblink activity was being investigated. These were compared with assessments of SEBR in young adult subjects under equivalent conditions. RESULTS: Assessments of spontaneous eyeblink activity revealed that SEBR in a reading posture is lower than that in primary gaze, and SEBR is higher when subjects are in conversation. Such differences are also generally found in literature reports, especially if the primary gaze assessment is carried in silence. Electrophysiological measures of SEBR yield slightly higher values compared with observational techniques. Statistical analysis (with calculation of 95% confidence interval values) indicate that reading-SEBR should be between 1.4 and 14.4 eyeblinks/min, primary gaze-SEBR between 8.0 and 21.0 eyeblinks/min and conversational-SEBR between 10.5 and 32.5 eyeblinks/min for normal adults. CONCLUSIONS: It is inappropriate to simply state a value for spontaneous eyeblink rate because it is so dependent on experimental conditions. It is further proposed that reports of SEBR measures should be prefixed by these experimental conditions, namely reading-SEBR, primary gaze-SEBR (in silence) and conversational-SEBR.

Video-assessment of tear meniscus height in elderly Caucasians and its relationship to the exposed ocular surface.

PURPOSE: An adequate volume of tears is considered essential to prevent desiccation of the exposed ocular surface, especially in elderly individuals. Few assessments of tear meniscus height (TMH) have however been reported for elderly individuals. METHODS: Close-up images of the lower marginal tear strip were obtained by videography over about 30 s in 56 elderly individuals (38 women, 18 men; 71 +/- 5 years of age) without significant eye disease and having a functional lacrimal system (Schirmer test). From such an "en face" view, the vertical height of the tear meniscus (TMH) at the lower eyelid was measured from stopped video frames. Firstly, TMH was assessed at the mid-point of the eyelid from five frames separated by 5 s; these values were averaged to give a time-averaged assessment of TMH (a tTMH value). Secondly, a position-averaged method (pTMH) was used in which TMH was assessed at 5 locations along the eyelid margin from a single frame that included several mm of marginal tear strip, and an average value calculated. The videographs were also used to assess palpebral aperture and the exposed ocular surface, a 1 min Schirmer test measure (closed eye) was also made, and keratometry data was available for most subjects. RESULTS: A wide range of average values was encountered for both tTMH (range 0.057 to 0.271 mm) and pTMH (0.031 mm to 0.325 mm). The group-averaged tTMH value was 0.172 +/- 0.047 mm (median 0.164 mm) while the group-averaged pTMH value was 0.171 +/- 0.058 mm (median 0.168 mm), with no difference being found between the two methods (p = 0.84, paired t-test). With either method, the apparent TMH value was greater when the exposed ocular surface value (average 1.68 +/- 0.51 cm(2)) was lower and vice versa, and was statistically-significant for the tTMH measures (p = 0.02, r = 0.32), but not the pTMH measures (p = 0.18, r = 0.18). Neither TMH measure was related to the Schirmer test values or to central corneal curvature. CONCLUSIONS: The results indicate that healthy elderly individuals can be expected to have a measurable lower marginal tear strip, and that objective assessment of this may be a useful alternative to the Schirmer test. A possible association was found between the measures of TMH and the palpebral aperture height (and thus the exposed ocular surface), and so might need to be considered when TMH is assessed.

A descriptive and quantitative study of the keratocytes of the corneal stroma of albino rabbits using transmission electron microscopy.

The present morphometric study was designed to assess the dimensions and shape of keratocytes and their nuclei by transmission electron microscopy, and to assess these features in relation to the stromal lamellae. Corneas from 10 albino rabbits were fixed in 2% glutaraldehyde in cacodylate buffer (pH 7.4, 300 mOsm/kg) and embedded in Spurr's epoxy resin. Both transverse and coronal thin sections through the corneal stroma were prepared. The stromal lamellae had an average thickness of 2.45+/-1.15 microm. The average cell thickness of the keratocytes was 1.34+/-0.46 microm (range 0.49-4.76 microm), with the apparent cell thickness being related to the average anterior-posterior thickness of the adjacent lamellae (r = 0.424, P = 0.001)). The relative length and thickness of the cell nucleus, in transverse section, was measured to be 0.65+/-0.13 and 0.76+/-0.10 of the cell body section respectively. As assessed by planimetry, the area of the keratocyte cell body viewed in coronal section was 292+/-118 microm2, with a nucleus-to-cytoplasm ratio of 0.437+/-0.295. The electron micrographs confirmed the presence of gap junctions between keratocyte cell processes, and the occasional presence of centrioles in the cells. Some keratocyte processes were observed to extend from one face of the lamellae to the other, suggesting anterior-to-posterior cell communication. These studies indicate that the keratocyte cell thickness is influenced by the physical pressure exerted by adjacent stromal lamellae. The cell nucleus, while a dominant feature in transverse section, has a normal size in relation to the cell cytoplasm when viewed in coronal section.

Influence of chronic alcohol abuse and ensuing forced abstinence on static subjective accommodation function in humans.

PURPOSE: Acute alcohol ingestion can change accommodation, but the long term effects of sustained alcohol consumption on accommodative function have not been studied in detail. This study was thus undertaken on individuals with a history of alcohol abuse. METHODS: Thirty-seven male individuals aged 25-56 years (average 40 years) from an alcohol rehabilitation centre in Inverness, Scotland, were assessed on admission and after a week of forced abstinence. The results were compared to a paired age-matched set of control male subjects. The static amplitude of accommodation was measured by an RAF rule, and the pupil size measured with a pupil gauge. RESULTS: On admission, the group mean measured amplitude of accommodation was 4.7 +/- 2.2 D (mean +/- SD). These values for the alcoholics were lower than age-matched controls (of 5.9 +/- 2.9 D). The slope of the age-dependent decline in RAF rule accommodation measures was significantly smaller for the alcoholics compared to controls (at 0.215 +/- 0.027 D/year versus 0.332 +/- 0.015 D/year, respectively; p < 0.001), with the younger alcoholics showing a greater impairment. Following abstinence, there was no measurable change in accommodation measured, indicating the lower amplitude in the alcoholics was not attributable to circulatory alcohol levels. The resting pupil diameter in the alcoholics was 4.37 +/- 0.63 mm compared to the controls of 3.97 +/- 0.75 mm, with a higher incidence of small pupils (< or = 3 mm) in the controls. CONCLUSIONS: The results indicate that chronic alcohol use can adversely affect subjective static accommodation, especially in younger alcoholics, as well as cause slight mydriasis.

Changes in hydration, protein and proteoglycan composition of the collagen-keratocyte matrix of the bovine corneal stroma ex vivo in a bicarbonate-mixed salts solution, compared to other solutions.

Many solutions have been used to investigate the swelling properties of the mammalian corneal stroma but few of the solutions resemble the expected extracellular matrix fluid of the corneal stroma, and little information is available on whether incubation ex vivo causes significant changes in the gross composition of the stroma. From quality-selected recent post-mortem eyes of adult cattle, stroma preparations were cut from the central part of the cornea. The time-dependent changes in wet mass were assessed over 9 h at 37 degrees C, and the preparations then dried. Various solutions of known pH (6.88-8.32) and osmolality (<50-327 mosmol/kg) were used, and were assayed for protein and proteoglycan after the incubation. The rates and extent of stromal swelling were lowest in a glucose-supplemented mixed salts solution containing 35 mM bicarbonate (0.5% CO2) solution, marginally greater in a mixed salts solution containing 35 mM bicarbonate (5% CO2) or similar non-bicarbonate mixed salts solutions (including BSS), and progressively greater in phosphate-buffered saline (PBS), various phosphate buffers (10-67 mM) and saline solutions (0.025-1%), and greatest in water. The initial rates of swelling ranged from 44 to 451 mg/h and the secondary rates from 9 to 106 mg/h. In all solutions, protein and proteoglycans were detected, but these ranged from around 1 to 10% of the samples with the bicarbonate-buffered solutions, to around 30% with the use of some phosphate buffers or saline.