Relation between corneal innervation with confocal microscopy and corneal sensitivity with noncontact esthesiometry in patients with dry eye

PURPOSE: An alteration in corneal innervation has been described in dry eye associated with diabetes mellitus, contact lens use, and LASIK. The purpose of this study was to evaluate whether dry eye not related to Sj?gren's syndrome (NSDE) and dry eye related to primary Sj?gren's syndrome (PSDE) are associated with an alteration of the corneal nerves and sensation. METHODS: Twenty-one patients with dry eye (10 NSDE and 11 PSDE) and 20 healthy volunteers were studied. Healthy volunteers were divided into two groups: one younger than 60 years (N<60) and the other 60 years of age or older (N> or =60). The study of the epithelium, stroma, and subbasal corneal nerves was performed with a confocal microscope. Mechanical, chemical, and thermal sensation was evaluated using the Belmonte noncontact esthesiometer. RESULTS: A statistically significant decrease in the number and density of subbasal nerves (P < 0.0001) and the density of superficial epithelial cells (P < 0.0001) was observed in dry eyes. The number and density of subbasal nerves was higher in the N<60 group. A significant decrease was found with respect to mechanical, chemical, and thermal sensitivity (P < 0.0001). Sensibility was better in the healthy eyes. A strong correlation was found between the density of superficial epithelial cells and the nerves and between the number and density of subbasal nerves and sensation (P < 0.001). CONCLUSIONS: The use of confocal microscopy and noncontact esthesiometry allow the detection of the presence of corneal neuropathy in patients with dry eye.     

The cornea in Sjogren's syndrome: an in vivo confocal study

PURPOSE: To analyze the in vivo morphology of corneal cells and nerves in dry eye associated with primary (SSI) and secondary (SSII) Sj?gren's syndrome and to study its relationship with the clinical evaluation. METHODS: Thirty-five patients with SS and 20 age- and gender-matched control subjects were studied. Confocal microscopy was used to investigate corneal thickness, epithelial and stromal cellular density, and subbasal plexus morphology. RESULTS: Corneal central thickness was 514.74 +/- 19.85 microm in the SS group and 550 +/- 21.46 microm in the control group (P < 0.0001, t-test); stromal central thickness was 456.62 +/- 18.05 microm in the SS group and 487.35 +/- 20.40 microm in the control group (P < 0.0001). The density of the superficial epithelial cells in the SSI and SSII groups was 965.40 +/- 96.00 and 999.80 +/- 115.67 cells/mm(2), respectively, and 1488.55 +/- 133.74 cells/mm(2) in the control group (P < 0.001, ANOVA). The number of subbasal nerves was 3.34 +/- 0.76 in the SS group and 5.10 +/- 0.79 in the control group (P < 0.0001, t-test). The average grade of nerve tortuosity was 2.62 +/- 0.94 in the SS group and 1.20 +/- 0.70 in the control group (P < 0.0001). Statistically significant correlations were found between clinical data and confocal microscopy data. CONCLUSIONS: Corneal thickness, cells, and nerves show morphologic changes in patients with dry eye associated with SS. The in vivo confocal study of these alterations may be important in better understanding the complexity of the ocular surface morphofunctional unit and the potentials of therapeutic approaches for the control of the phlogistic process and neuroprotection.     

Corneal innervation and morphology in primary Sjögren's syndrome

PURPOSE: To analyze the in vivo morphology of the different corneal sublayers and corneal nerves in primary Sj?gren's syndrome (SS). METHODS: Ten eyes of 10 patients with primary SS and 10 eyes of 10 sex- and age-matched control subjects were investigated. Diagnosis was based on American-European consensus criteria. In vivo confocal microscopy with through-focusing was used to investigate corneal morphology and to measure corneal sublayer thickness. RESULTS: Epithelial punctate staining with fluorescein was observed in 6 of 10 SS and none of 10 control corneas. In addition, Schirmer I test results were significantly lower in SS. Epithelial thickness did not differ between the SS and control groups. Confocal microscopy revealed patchy alterations or irregularities in surface epithelial cells in 6 of 10 SS corneas, whereas the basal epithelium appeared normal in all corneas. Average corneal thickness was lower in the SS group (515.9 +/- 22.0 micro m) than in the control (547.4 +/- 42.0 micro m; P = 0.050, t-test). Accordingly, the mean intraocular pressure was lower in the SS group (13.9 +/- 2.1 mm Hg) than in the control (16.7 +/- 2.9 mm Hg; P = 0.022). The subbasal nerve plexus and stromal nerve fiber bundles were present in all corneas. No difference was noted in nerve density. However, in 4 of 10 SS eyes, the subbasal nerve plexus showed structures resembling nerve sprouting, suggesting ongoing active neural growth. None of the control corneas exhibited such features. Signs of anterior keratocyte activation were observed in 5 of 10 SS corneas. CONCLUSIONS: In SS, the corneal surface epithelium was irregular and patchy. Anterior keratocytes frequently showed morphologic features of activation. The subbasal nerve fiber bundles revealed abnormal morphology, and the central corneal thickness was reduced by stromal thinning. The findings confirm epithelial, stromal, and neural abnormalities in the corneas of patients with SS.     

Comparative in vivo high-resolution confocal microscopy of corneal epithelium, sub-basal nerves and stromal cells in mice with and without dry eye after photorefractive keratectomy

BACKGROUND: The present study compares, using a new-generation high-resolution in vivo confocal microscope, the epithelial morphology, sub-basal nerves and stroma in two groups of mice: one exposed to normal conditions (NC) and the other to a desiccating environment (DE), following photorefractive keratectomy (PRK) with mechanical epithelial scraping. METHODS: Twenty-four 4- to 8-week-old female Balb/C mice were used in this study. Twenty mice underwent bilateral corneal epithelial scraping using an electric brush prior to PRK. Then, the mice were divided in two groups: 10 mice were placed in NC. The other 10 mice were exposed to a DE for 2 weeks. Four mice served as controls. Corneas were analysed in vivo using the Rostock Cornea Module of the Heidelberg retina tomograph II. For all eyes, 20 confocal microscopic images of each layer, that is, the superficial and basal corneal epithelium, Bowman's layer, anterior and posterior stroma and the endothelium, were recorded. Epithelial and stromal cell densities and sub-basal and stromal nerves were measured and compared. RESULTS: There was a higher density of superficial epithelial cells in the DE group (693 +/- 148 cells/mm(2) in NC group and 443 +/- 128 cells/mm(2) in DE group; Mann-Whitney U-test; P = 0.05). Higher number of basal cells were observed in the DE group. Its density was 986 +/- 198 cells/mm(2) in NC and 1598 +/- 280 cells/mm(2) in DE group (Mann-Whitney U-test; P < 0.05). Significantly higher number of reflective structures were noted within the stroma without clearly visible nuclei in the DE group compared with the NC eyes. Additionally, higher number of beads, nerve sprouts and higher tortuosity of sub-basal nerves were observed in the DE group. No difference was observed in the endothelial cell density between the groups. CONCLUSION: Exposure of corneas to a DE after PRK with previous mechanical epithelial scraping increases epithelial turnover and is associated with a higher number of reflective structures in the stroma. Additionally increased nerve beading, nerve sprouts and tortuosity of sub-basal nerves were observed in the DE group, possibly directed to repair the alterations observed at the epithelial level.     

Effects of glaucoma medications on corneal endothelium, keratocytes, and subbasal nerves among participants in the ocular hypertension treatment study

PURPOSE: To compare subbasal corneal nerve and keratocyte density and endothelial characteristics of ocular hypertensive patients treated with medications or observation. METHODS: Participants in the Ocular Hypertensive Treatment Study (OHTS) randomized at Mayo Clinic to medication or observation were evaluated with specular microscopy annually for 6 years. Confocal microscopy was performed 78 to 108 months after enrollment. Subbasal nerve density was calculated by manual tracing and digital image analysis. Keratocyte density was determined by manual counting methods. Data were compared using a t test and a rank sum test. RESULTS: After 6 years, corneal endothelial cell density, percent hexagonal cells, and coefficient of variation of cell area for the observation (n = 21) and medication groups (n = 26) were similar (2415 +/- 300 vs. 2331 +/- 239 cells/mm; 63% +/- 11% vs. 65% +/- 10%; and 0.32 +/- 0.07 vs. 0.30 +/- 0.06, respectively). Of 38 participants undergoing confocal examination, the medication group (n = 19) had fewer nerves (3.8 +/- 2.1 vs. 5.9 +/- 2.0 nerves/frame; P = 0.02) and a lower nerve density (5643 +/- 2861 vs. 9314 +/- 3743 mum/mm; P = 0.007) than the observation patients (n = 10). An additional 9 patients in the observation group, who began medication before confocal scanning, had intermediate nerve densities. Full-thickness keratocyte density was similar, with 22,257 +/- 2419 and 23,430 +/- 3285 cell/mm in the observation and medication groups, respectively. CONCLUSIONS: Chronic administration of glaucoma medications causes a decrease in the number and density of corneal subbasal nerve fiber bundles but does not affect keratocyte density or corneal endothelial characteristics.     

Alterations in corneal sensitivity and nerve morphology in patients with primary Sjögren's syndrome

The aim of the study was to assess subjective symptoms and objective clinical signs of dry eye in relation to corneal nerve morphology and sensitivity in primary Sj?gren's syndrome. Twenty eyes of 20 primary Sj?gren's syndrome patients and ten eyes of 10 healthy age- and sex-matched controls were included in the study. Ocular surface disease index (OSDI) questionnaire and visual analog scales were used to assess subjective symptoms. The mechanical sensitivity of the central cornea was measured using a modified Belmonte non-contact esthesiometer followed by an analysis of corneal nerve morphology using scanning slit confocal microscopy (ConfoScan 3). OSDI symptom scores were high in primary Sj?gren's syndrome patients, compared with controls. Accordingly, the mean corneal detection threshold was low in patients implicating corneal mechanical hypersensitivity (54.5+/-40.1ml/min vs. 85.0+/-24.6ml/min, P=0.036). However, nerve densities were similar, and no correlation was present between corneal sensitivity and nerve density. In contrast, alterations in nerve morphology were found; stromal nerves appeared thicker, and nerve growth cone-like structures were seen in 20% of patients, often associated with dendritic antigen-presenting cells. Sj?gren's syndrome patients presented with corneal mechanical hypersensitivity, although corneal nerve density did not differ from controls. However, alterations in corneal nerve morphology (nerve sprouting and thickened stromal nerves) and an increased amount of antigen-presenting cells, implicating the role of inflammation, were observed. These observations offer an explanation for the corneal mechanical hypersensitivity, or even hyperalgesia often observed in these patients. We hypothesize that patients with primary Sj?gren's syndrome dry eye suffer from neuropathic corneal mechanical hypersensitivity induced by ocular surface inflammation.     

Morphologic alterations of both the stromal and subbasal nerves in the corneas of patients with diabetes

PURPOSE: To evaluate the subbasal and the stromal nerves of the corneas of patients with type 2 diabetes with in vivo confocal microscopy and to compare them with those of nondiabetic patients. METHODS: Thirty-five corneas of patients with type 2 diabetes and 24 corneas of age-matched control subjects were included in the study. Patients with diabetes were further classified with respect to the stage of retinopathy. Subbasal and stromal nerve plexus morphology and thickness were evaluated with in vivo confocal microscopy. Subbasal long nerve fiber (LNF) and total nerve branch (NB) densities were calculated. RESULTS: The mean stromal nerve thickness was significantly higher in patients with diabetes (8.99 +/- 2.32 microm) than that of the control subjects (5.69 +/- 1.49 microm; Mann-Whitney U test; P < 0.001). The proportion of curved stromal nerves in patients with diabetes (45.7%) was also higher than that of normal subjects (20.8%; chi(2), P = 0.05). Subbasal LNF and NB densities were found to significantly lower in the corneas of patients with diabetes (28.3 +/- 10.4 and 39.7 +/- 13.2 nerve/mm(2), respectively) than those of the control subjects (34.1 +/- 5.7 and 58.5 +/- 12.4 nerve/mm(2), respectively; Mann-Whitney, P = 0.012 and P < 0.001). In addition, the subbasal nerve plexus of patients with diabetes appeared significantly thicker and more tortuous than those of the control subjects (Mann-Whitney, P = 0.002 and P = 0.001). CONCLUSION: Both stromal and subbasal nerves appear abnormal in the corneas of patients with diabetes. Patients with proliferative diabetic retinopathy show more pronounced nerve alterations than patients who do not have diabetic retinopathy.     

The effect of age on the corneal subbasal nerve plexus

PURPOSE: To measure subbasal nerve density and orientation in normal human corneas across a broad age range. METHODS: Sixty-five normal corneas of 65 subjects were examined by using tandem scanning confocal microscopy. Ages of subjects ranged from 15 to 79 years (mean 46 +/- 19 years), with 5 subjects from each hemidecade. Subbasal nerve fiber bundles appeared as bright, well-defined linear structures in confocal images of the central cornea. Images from 3 to 8 scans per eye (mean 4.6 +/- 1.8 scans) were randomly presented to a masked observer for analysis. The mean subbasal nerve density (total nerve length [microm] within a confocal image [area = 0.166 mm]), the mean nerve number per confocal scan, and the mean nerve orientation were determined by using a custom software program. Correlations between age and nerve density and age and nerve orientation were assessed by using Pearson correlation coefficients. RESULTS: The subbasal nerve plexus was visible in the central cornea of all subjects. The mean subbasal nerve density was 8404 +/- 2012 microm/mm (range 4735 to 14,018 microm/mm). The mean subbasal nerve number was 4.6 +/- 1.6 nerves (range 1 to 8 nerves). The mean subbasal nerve orientation was 94 +/- 16 degrees (range 58 to 146 degrees). There was no correlation between age and subbasal nerve density (r = 0.21, P = 0.09) or between age and subbasal nerve orientation (r = -0.19, P = 0.12). CONCLUSION: The density and orientation of the subbasal nerve plexus in the central human cornea does not change with age.     

Recovery of corneal subbasal nerve density after PRK and LASIK

PURPOSE: To measure and compare the return of corneal innervation up to 5 years after photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK). DESIGN: Prospective, nonrandomized clinical trial. METHODS: Eighteen eyes of 12 patients received PRK to correct a mean refractive error of -3.73 +/- 1.30 diopters, and 16 eyes of 11 patients received LASIK to correct a mean refractive error of -6.56 +/- 2.44 diopters. Corneas were examined by confocal microscopy before and at 1, 2, 3, and 5 years after the procedures. Subbasal nerve fiber bundles were measured to determine density (visible length of nerve/frame area) and expressed as micrometers per square millimeters. Differences were compared by Friedman's test and adjusted for multiple comparisons by the Student-Newman-Keuls procedure. RESULTS: After PRK, mean subbasal nerve density was reduced by 59% at 1 year (2764 +/- 1321 microm/mm(2) [+/-SD]) when compared with preoperative (6786 +/- 1948 microm/mm(2); P < .001). By 2 years, subbasal nerve density (6242 +/- 1763 microm/mm(2)) was not significantly different from density before PRK and remained unchanged to 5 years (5903 +/- 3086 microm/mm(2)). After LASIK, subbasal nerve density was reduced by 51%, 35%, and 34% at 1, 2, and 3 years, respectively (P < .001). By 5 years, subbasal nerves had returned to densities (4441 +/- 2819 microm/mm(2)) that were not significantly different from densities before LASIK (5589 +/- 2436 microm/mm(2)). CONCLUSION: Corneal subbasal nerve density does not recover to near preoperative densities until 5 years after LASIK, as compared with 2 years after PRK.     

Tear lipocalin and lysozyme in Sj?gren and non-Sj?gren dry eye

PURPOSE: To evaluate the concentration of tear lipocalin, lysozyme, and total protein in Sj?gren's Syndrome (SS), non-Sj?gren's keratoconjunctivitis sicca (KCS), and non-dry-eyed (NDE) individuals. METHODS: Seventy-six subjects were recruited for this study: 25 SS subjects; 25 KCS subjects, and 26 NDE individuals. Symptoms were measured with a visual analogue scale. Tear flow was measured by the Schirmer I test without anesthesia. Tears were collected using an eye wash technique. Total tear protein was quantified using the DC Protein Assay Kit. Tear lipocalin and lysozyme were quantified via Western blotting performed on a Phast System. RESULTS: By definition, the SS and KCS groups both had significantly lower mean Schirmer scores (5.12 +/- 5.96 mm and 7.84 +/- 7.35 mm) compared with the NDE group (23.83 +/- 7.85 mm; p < 0.0001). There was no difference in mean Schirmer scores between SS and KCS groups (p = 0.19). The tear film of the SS group was characterized by significantly reduced (p < 0.0001) total protein and lipocalin concentrations compared with both KCS and NDE groups. No difference between the KCS and NDE groups was found in total protein (p = 0.92) or lipocalin (p = 0.19) concentration. In contrast, the concentration of tear film lysozyme was found to be statistically similar in all three groups examined. No statistically significant correlation was found in any group between mean Schirmer values compared with total protein, lipocalin or lysozyme concentration. CONCLUSION: Our data demonstrate a biochemical distinction between the Sj?gren's group compared with both KCS and control groups, in that both tear lipocalin and total tear protein were significantly reduced. Although correlations were not found between protein measurements and tear flow, a combination of tests including Schirmer I and quantitation of tear film biomarkers may allow for the identification of SS patients without the need for invasive testing.     

Conjunctival surface changes in patients with Sjogren's syndrome: a transmission electron microscopy study

PURPOSE: To demonstrate the ultrastructural appearance of the conjunctival surface epithelium in patients with Sj?gren's syndrome (SS) compared with normal subjects. METHODS: Conjunctival tissue specimens from seven normal subjects and eight patients with SS were obtained by bulbar conjunctival biopsy and examined by transmission electron microscopy. RESULTS: The average number of microvilli per 8.3 microm epithelial length was significantly lower in the SS group than that in the control group (19.6 +/- 2.5 vs. 28.0 +/- 3.4, P < 0.0001). The microvillus height (0.539 +/- 0.151 microm) and height-width ratio (1.825 +/- 0.549) in the conjunctival epithelium in the SS group were significantly lower than those (height: 0.946 +/- 0.117 microm, P < 0.001; and height-width ratio: 3.717 +/- 0.696, P < 0.0001) in normal individuals. The microvilli in the SS group were wider than those in the control group (P = 0.003). Furthermore, the average number of secretory vesicles (per 8.3 microm epithelial length) in the apical conjunctival epithelial cell was significantly reduced in the SS group (16.4 +/- 6.8 vesicles), compared with the control group (34.7 +/- 1.2 vesicles, P = 0.003). In addition, although the ocular surface glycocalyx (OSG) was always present in control subjects, it was not detectable in all but one of the SS conjunctival specimens. CONCLUSIONS: The ultrastructural morphology of the apical conjunctival epithelium is altered in patients with SS. The findings suggest that an intact OSG may play a key role in the maintenance of a healthy ocular surface, possibly by preventing abrasive influences on the apical epithelial cells.     

Corneal nerves alterations in various types of systemic polyneuropathy, identified by in vivo confocal microscopy

BACKGROUND: In vivo confocal microscopy (IVCM) is a newly developed application to assess corneal nerve morphology. The purpose of the study is to evaluate the role of IVCM in the assessment of various types of polyneuropathy, and to define alterations of corneal nerves in such conditions. PATIENTS AND METHODS: Eighteen patients with various types of polyneuropathy were characterized by clinical neurological and ophthalmic examinations, as well as by electroneuromyography (ENMG). Full thickness IVCM of corneal nerves was carried out on all patients and 15 age-matched eyes using Heidelberg Retina Tomograph II (HRT II). The subbasal nerve plexus were statistically analysed regarding long nerve fiber density, nerve branch density, nerve thickness, nerve bead number and nerve tortuosity. RESULTS: In subbasal nerve plexus, the following three parameters were significantly reduced in patients with polyneuropathy compared to controls: long nerve fibre density (p < 0.01), nerve branch density (p < 0.001), and nerve bead number (p = 0.001). In addition, the average grade of nerve tortuosity was 2.87 +/- 0.97 in the polyneuropathic group and 1.17 +/- 0.68 in the control group (p < 0.0001). CONCLUSIONS: IVCM allows a non-invasive, in vivo study of corneal nerves with high resolution. It therefore appears invaluable in clinical investigations. IVCM appears to be valuable in a large variety of polyneuropathic conditions.     

长期使用前列腺素类滴眼液对角膜结构与知觉的影响

目的 探讨青光眼患者长期使用前列腺素类滴眼液对角膜结构与知觉的影响。方法 横断面对照研究。收集2011年8月至2012年4月期间在复旦大学附属眼耳鼻喉科医院就诊的原发性开角型青光眼患者20例（40眼）,分为2组：A组为使用前列腺素类滴眼液1年以上且半年内用药方案未改变患者10例（20眼）;B组为初诊未用药患者10例（20眼）。所有患者使用Cochet & Bonnet角膜知觉仪测量角膜知觉,使用激光活体共聚焦显微镜观察角膜上皮下神经纤维及上皮、基质、内皮细胞形态,记录并分析图像。组间采用双眼设计模型进行方差分析,角膜知觉与上皮下神经、炎症细胞、朗格罕细胞等的关系进行Spearman相关分析。结果 与B组比,A组角膜上皮基底层细胞密度明显较高[（7009±638）个/ mm² vs. （6745±482）个/ mm²;F=5.22,P<0.05],上皮下神经纤维分支明显较少[（17.44±2.56）mm/ mm² vs. （19.54±5.02）mm/ mm²;F=8.22,P<0.05]、神经扭曲度较高[（1.71±0.46） vs. （1.42±0.43）;F=7.29,P<0.05],角膜基质细胞激活态分级明显较高[（2.35±0.81） vs. （1.25±0.44）;F=59.67,P<0.01],角膜知觉明显较低[（54.25±6.74）mm vs. （59.50±1.54）mm;F=12.08,P<0.01]。角膜上皮下神经纤维密度及反光度、朗格罕细胞及炎症细胞浸润量、角膜内皮细胞形态未见明显改变。角膜知觉与角膜上皮下神经纤维密度呈正相关（r=0.379,P<0.05）,与角膜上皮下神经分支数、扭曲度、反光度及炎症细胞、朗格罕细胞浸润无明显相关性。结论 长期使用前列腺素类滴眼液可引起角膜深达基质层细胞的形态学变化和角膜知觉下降,知觉下降与角膜上皮下神经纤维密度减少有关。     

Confocal microscopy in vivo in corneas of long-term contact lens wearers

PURPOSE: To compare keratocyte density, stromal backscatter, epithelial thickness, and corneal sensitivity between corneas of long-term contact lens wearers and those of non-contact lens wearers. METHODS: Twenty corneas of 20 daily contact lens wearers (>10 years' duration) and 20 corneas of 20 age-matched (+/-5 years) control subjects who had never worn contact lenses, were examined by confocal microscopy in vivo. The contact lens wearers removed their lenses 12 to 24 hours before the examination. Full-thickness images were recorded from the central and temporal cornea, and bright objects (keratocyte nuclei) in images were manually counted to calculate keratocyte density. Stromal intensity (backscatter) was measured by calculating the mean grayscale value (corrected for camera and light source variations) from the center of stromal images. Epithelial thickness was determined from the distance between images of the surface epithelium and subbasal nerve plexus. Central corneal sensitivity was measured by Cochet-Bonnet esthesiometry and correlated with the number of nerve fiber bundles in the subbasal nerve plexus. RESULTS: Full-thickness central and temporal keratocyte densities in contact lens wearers were 22,122 +/- 2,676 cells/mm(3) (mean +/- SD) and 20,731 +/- 2,627 cells/mm(3), respectively, and were not significantly different from central and temporal keratocyte densities in control subjects (P = 0.29). The minimum detectable difference in cell density was 11% (2346 cells/mm(3) and 2235 cells/mm(3) in central and temporal stroma, respectively). Temporal epithelial thickness was 46.3 +/- 4.7 microm in contact lens wearers and 50.9 +/- 4.7 microm in control subjects (P = 0.02). Central epithelial thickness and stromal backscatter did not differ between contact lens wearers and control subjects (P > 0.05). Corneal sensitivity was lower in contact lens wearers than it was in control subjects (P = 0.05) and did not correlate with the number of nerve fiber bundles in the subbasal nerve plexus. CONCLUSIONS: Long-term daily contact lens wear and its associated stromal hypoxia and acidosis have no demonstrable effect on keratocyte density. The temporal epithelium is thinner in corneas of long-term contact lens wearers than in control subjects. Decreased corneal sensitivity in contact lens wearers is not accompanied by decreased nerve fiber bundle density.     

Corneal reinnervation after LASIK: prospective 3-year longitudinal study

PURPOSE: To measure the return of innervation to the cornea during 3 years after LASIK. METHODS: Seventeen corneas of 11 patients who had undergone LASIK to correct myopia from -2.0 D to -11.0 D were examined by confocal microscopy before surgery, and at 1, 3, 6, 12, 24, and 36 months after surgery. In all available scans, the number of nerve fiber bundles and their density (visible length of nerve per frame area), orientation (mean angle), and depth in the cornea were measured. RESULTS: The number and density of subbasal nerves decreased >90% in the first month after LASIK. By 6 months these nerves began to recover, and by 2 years they reached densities not significantly different from those before LASIK. Between 2 and 3 years they decreased again, so that at 3 years the numbers remained <60% of the pre-LASIK numbers (P <0.001). In the stromal flap most nerve fiber bundles were also lost after LASIK, and these began recovering by the third month, but by the third year they did not reach their original numbers (P <0.001). In the stromal bed (posterior to the LASIK flap interface), there were no significant changes in nerve number or density. As the subbasal nerves returned, their mean orientation did not change from the predominantly vertical orientation before LASIK. Nerve orientation in the stromal flap and the stromal bed also did not change. CONCLUSIONS: Both subbasal and stromal corneal nerves in LASIK flaps recover slowly and do not return to preoperative densities by 3 years after LASIK. The numbers of subbasal nerves appear to decrease between 2 and 3 years after LASIK. The orientation of the regenerated subbasal nerves remains predominantly vertical.     

Diabetes and corneal cell densities in humans by in vivo confocal microscopy

PURPOSE: Diabetes is accompanied by an increased autofluorescence of the cornea, probably because of accumulation of advanced glycation end products (AGEs). The pathogenic mechanism is still unknown. This study aimed to quantify differences in corneal cell densities between diabetic patients and healthy controls. METHODS: The left cornea of 15 patients with non-insulin-dependent diabetes mellitus (NIDDM) with level of retinopathy 20 according to the Early Treatment of Diabetic Retinopathy Study (ETDRS) and of 15 healthy controls were examined by noninvasive in vivo confocal microscopy in an observational prospective study. The cell densities in 6 corneal layers were determined along the optical axis of the cornea by using stereologic methods. RESULTS: The average cell density per unit area in the superficial and basal epithelium and the endothelial layer was 725 +/- 171, 5950 +/- 653, and 2690 +/- 302 cells/mm in controls and 815 +/- 260, 5060 +/- 301, and 2660 +/- 364 cells/mm in diabetic patients. The cell density per unit volume in the anterior, mid-, and posterior stroma was 26,300 +/- 4090, 19,390 +/- 3120, and 25,700 +/- 3260 cells/mm in controls and 27,560 +/- 3880, 21,930 +/- 2110, and 25,790 +/- 3090 cells/mm in patients with diabetes. In both groups, the density in the midstroma was significantly lower than in both the anterior stroma and the posterior stroma (P < 0.02). The cell density in the basal layer of diabetic patients was significantly lower than in healthy controls (-15.0%, P < 0.0004). In the other layers, no significant differences between both groups (P > 0.07) were observed. CONCLUSIONS: The lower basal cell density found in patients with diabetes may result from a combination of different mechanisms including decreased innervation at the subbasal nerve plexus, basement membrane alterations, and higher turnover rate in basal epithelial cells. The lower cell density in the midstroma of diabetic patients and healthy controls may be attributed in part to differences in oxygen concentration in the stromal layers (depths). Changes in cellular density did not seem to be responsible for the increased autofluorescence in diabetes.     

Laser scanning in vivo confocal microscopy of the normal human corneoscleral limbus

PURPOSE: To elucidate the structure of the human corneoscleral limbus by in vivo laser scanning confocal microscopy and to correlate limbal epithelial dimensions and density with the central epithelium and in relation to age. METHODS: Fifty adult subjects were recruited into one of two age groups: younger (age<45 years) and older (age>or=45 years). Fifty left eyes of these 50 healthy subjects were examined by laser scanning in vivo confocal microscopy, to assess the basal epithelium of the central cornea and inferior limbus. Mean epithelial cell diameter, area, and density were calculated for the central basal epithelium, limbus-corneal basal epithelium, and limbus-palisade epithelium. RESULTS: Data were analyzed in relation to the two age groups, group A, 30+/-6 years (n=25; mean+/-SD), and group B, 60+/-11 years (n=25; P<0.01). Mean epithelial density in the limbus-cornea and limbus-palisade regions decreased significantly with age: limbus-cornea group A=7253+/-1077 cells/mm2 group B=6614+/-987 cells/mm2, P=0.03; limbus palisade group A=5409+/-799 cells/mm2, group B=5055+/-722 cells/mm2, P=0.03). Central corneal epithelial density did not change with age: group A=6162+/-503 cells/mm2, group B=6362+/-614 cells/mm2, P=0.08. Mean epithelial density was greatest at the limbus-cornea (7010+/-1081 cells/mm2) and lowest at the limbus-palisades (5289+/-847 cells/mm2). The mean width of palisade ridges was 25.0+/-6.3 microm. CONCLUSIONS: This is the first study to image clearly the living human corneal limbus by laser scanning in vivo confocal microscopy and to demonstrate quantitative changes in the basal epithelium with age.