New Insights Into the Anatomy of the Eyelashes With Regard to Ethnic Differences Against a Clinical Background.

BACKGROUND: To investigate the root depth, root angle, and light and scanning electron microscopical anatomy of human eyelashes relevant to eyelash ablation. METHODS: Eyelash root depth, the angle between eyelash root and skin epithelium, spatial relationship, and scanning electron microscopical features of the eyelashes were studied on 4 upper and 4 lower eyelids of Caucasian (n = 4) and Indian (n = 4) cadaver heads according to a set protocol. RESULTS: There were significant differences in the mean eyelash root depth between Indians (2.3 ± 0.38 mm) and Caucasians (1.9 ± 0.26 mm; p = 0.007), as well as between upper eyelids and lower eyelids (1.9 ± 0.2 mm vs. 1.8 ± 0.1 mm). The mean angle between the lash follicle root and the skin epithelium was 75 ± 11 degrees and similar in both ethnic groups. The eyelash bulb was located close to the tarsal plate and meibomian glands and formed an angle of less than 15 degrees with the eyelash root. Scanning electron microscopy studies revealed that the eyelash bulb was 202 ± 12 µm wide in Indians and 170.6 ± 16.8 µm wide in Caucasian eyelids ( p = 0.08). The eyelashes were placed more closely in Indian eyelids than in Caucasian eyelids ( p = 0.03). The width of the cuticle layer varied between the hair shaft and the inner eyelid segment. CONCLUSIONS: There are differences in eyelash root depth, inter-eyelash distance, and cuticle thickness between Indian and Caucasian eyelids. The oblique orientation of the eyelash root and close proximity of the eyelash bulb to the tarsal plate should be kept in mind while doing the electroepilation procedure.

Suitability of Slaughterhouse-Acquired Pig Eyes as Model Systems for Refractive Ultraviolet and Infrared Femtosecond Laser Research.

PURPOSE: To elucidate whether it is feasible to use porcine eyes from scalded, abattoir-acquired animals for refractive femtosecond laser research. METHODS: An infrared laser (FS 200) and an ultraviolet laser (prototype version) were tested for their applicability on scalded pig eyes. Fifty porcine eyes were divided into two equally-sized groups and assigned to either the infrared or the ultraviolet laser. Both laser groups were comprised of five subgroups of n = 5 eyes each. Group A: non-scalded eyes (negative control); group B: eyes taken from tunnel-scalded animals; group C1: eyes taken from tank-scalded animals without opaque corneal lesion; group C2: eyes taken from animals with opaque corneal lesion; group D: eyes scalded in toto in the laboratory (positive control). In each group the lasers were employed to create a stromal flap. The quality of the laser cuts and the resulting flap beds, as well as of the porcine corneas themselves, was examined by anterior segment optical coherence tomography and scanning electron microscopy. RESULTS: All scalded specimens exhibited substantial corneal swelling, most pronounced in group C2. After ultraviolet laser application, the tank- and tunnel-scalded samples displayed marked irregularities and an increased degree of surface roughness in the flap beds. After infrared laser application, this was only the case in the tank-scalded specimens. CONCLUSION: It is not recommended to use eyes taken from scalded pigs for ultraviolet femtosecond laser experiments. For infrared femtosecond lasers, eyes taken from tunnel-scalded animals may represent an acceptable alternative, if non-scalded eyes are not available.

Impact of polishing system on surface roughness of different ceramic surfaces after various pretreatments and bracket debonding.

OBJECTIVE: Evaluating various polishing methods after bracket debonding and excessive attachment material removal for different ceramics and pretreatments. MATERIAL AND METHODS: Zirconia (ZrO2), leucite (LEU) and lithium disilicate (LiSi) specimens were pretreated with a) silica coated alumina particles (CoJet); LEU and LiSi additionally with b) hydrofluoric acid (HF), c) Monobond Etch&Prime (MEP), d) silicium carbide grinder (SiC) before bracket bonding, shearing off, ARI evaluation, excessive attachment material removal and polishing with i) Sof-Lex Discs (Soflex), ii) polishing paste (Paste), iii) polishing set (Set). Before/after polishing surface roughness (Ra) was measured with a profilometer. Martens hardness parameter were also assessed. RESULTS: Irrespective of pretreatment Ra of LEU increased the most, followed by LiSi and ZrO2 (p < 0.001, SiC: p = 0.012), in accordance with the measured Martens hardness parameter. CoJet/SiC caused greater roughness as HF/MEP (p < 0.001). The ZrO2 surface was rougher after polishing with Paste/Set (p < 0.001; p = 0.047). Ra improved in the LEU/CoJet, LEU/SiC and LiSi/SiC groups with Soflex/Set (p < 0.001), in the LiSi/CoJet and LEU/HF groups by Soflex (p = 0.003, p < 0.001) and worsened by Paste (p = 0.017, p < 0.001). Polishing of HF or MEP pretreated LiSi with Set increased Ra (p = 0.001, p < 0.001), so did Paste in the LEU/MEP group (p < 0.001). CONCLUSIONS: Paste couldn't improve the surfaces. Soflex was the only method decreasing Ra on rough surfaces and not causing roughness worsening. Polishing of LEU/LiSi after MEP, LEU after HF pretreatment doesn´t seem to have any benefit. CLINICAL RELEVANCE: To avoid long-term damage to ceramic restorations, special attention should be paid to the polishing method after orthodontic treatment.

Impact of different pretreatments and attachment materials on shear bond strength of indirectly bonded brackets using CAD/CAM transfer trays to monolithic zirconia.

OBJECTIVE: Investigating the impact of different pretreatment methods, attachment materials and aging regimens on shear bond strength (SBS) between zirconia and indirectly bonded brackets using CAD/CAM transfer trays. METHODS: Zirconia substrates were conditioned with silica coated alumina (CoJet) and a) Clearfil Ceramic Primer Plus (CF), b) RelyX Ceramic Primer (RXP), c) Futurabond U (FU). Brackets were virtually placed, transfer tray designed (OnyxCeph) and 3D-printed for indirect bonding with a) Transbond LV (TBL), b) Nexus NX3 (NX3), c) Maximum Cure (MC). SBS testing was performed with a universal testing machine after 24 h, 500 thermal cycles, 90 d. Directly bonded brackets to human enamel using Transbond XT Adhesive served as control. The adhesive remnant index (ARI) was evaluated. Data was analyzed with Shapiro-Wilk, Kruskal-Wallis and Dunn's post-hoc test with Bonferroni correction, Chi2 test (p < 0.05), and the Weibull modulus was calculated. RESULTS: SBS ranged from 0.1 to 15.5 MPa and were influenced mostly by the attachment material. NX3 generally showed the highest values (9.5-15.8 MPa). Initially RXP/TBL and FU/TBL presented the lowest values (4.3/4.8 MPa). Aging regimens reduced SBS of MC irrespective of pretreatment, after 90 d values ranged from 0.1 to 0.9 MPa. ARI 1 was predominant in all MC groups and FU/NX3, 2 and 3 in the other groups. Weibull moduli ranged between 0.15 (MC/RXP/500 TC) and 6.24 (NX3/RXP/500 TC). SIGNIFICANCE: MC seems not to be suitable for indirect bonding using CAD/CAM transfer trays to zirconia. NX3 showed similar SBS values compared to the control, TBL lower.

Urea and ocular surface: Synthesis, secretion and its role in tear film homeostasis.

Urea has been detected in the tear film, aqueous humor, and vitreous of the eye. While most of the urea in the aqueous humor and vitreous is considered to be an ultrafiltrate from the blood vessels, the presence of urea transporters and urea-synthesizing enzymes in the lacrimal gland, meibomian glands, conjunctiva, and cornea suggests ureagenesis occurring at the ocular surface. This review summarizes the distribution and function of urea transporters, urea and its synthesizing enzymes at the ocular surface to analyze their role in the tear film homeostasis. Urea transporters (UT)-A- and UT-B-as well as the enzymes arginase I, II, and agmatinase are located at the ocular surface. Urea concentration on the ocular surface is influenced by blood urea concentration, the amount of urea released by the tear fluid, tear evaporation, and arginase concentration in the tears. There are conflicting reports on the relationship between tear and plasma urea levels though a linear correlation exists between their levels. Urea protects the ocular surface from osmotic stress and is thought to maintain a lipid-water interface in the lamellar phase of the tear film. The reduction of urea levels in the tears of patients with evaporative dry eye suggests its possible role in tear film stability. Other than mitigating osmotic stress, urea has hydrating properties as well. Animal studies have demonstrated the healing effects of urea on the corneal epithelium. Future studies examining the variations in urea content in tears from different ocular surfaces, at different times of day, and under different environmental conditions would further solidify the role of urea in tear film stability.

Immunohistochemical detection of urea transporter-A in the tear-producing part of the lacrimal system.

OBJECTIVE: Urea constitutes a physiological and presumably well-regulated constituent of tear fluid. Its lacrimal concentration is significantly decreased in dry eye disease. Urea homeostasis within the tear fluid may also depend on the expression of urea transporters. The present study reports on the expression patterns of urea transporter A (UT-A) in the cells and tissues of the ocular surface and the lacrimal glands. METHODS: UT-A immunohistochemistry was performed on 5 µm paraffin sections of paraformaldehyde-fixed human, porcine, and murine corneas, eyelids, and lacrimal glands (n = 5 each). RESULTS: UT-A immunostaining was largely comparable in all three species. UT-A signals were detected in the corneal epithelium and endothelium, in the conjunctival epithelium, in the acinar cells and excretory ducts of the lacrimal gland, Meibomian gland, and in the glands of Moll and Zeis. The Meibomian glands and the glands of Zeis exhibited a marked UT-A-positive staining in the basal cells of the alveolar epithelia and in the ductal epithelia. CONCLUSION: UT-A shows comparable expression patterns to UT-B (previous study) at the ocular surface and in the lacrimal glands, as determined by immunohistochemistry. The presence of both urea transporters in the lacrimal functional unit suggests that they are essential for the normal function of the lacrimal system and the integrity of the tear film. Potential alterations in urea transporter expression might be associated with the significant reduction of urea found in the tear fluid of dry eye patients. They may thus play an important role in the pathogenesis of dry eye disease.

Urea transporter-B expression on the ocular surface and in the lacrimal glands.

OBJECTIVE: Urea is a component of tear fluid showing a significantly decreased concentration in dry eye disease. The urea content of tear fluid may depend on urea transporters. The purpose of this study was to examine the expression of urea transporter B (UT-B) at the ocular surface and in the lacrimal glands. METHODS: UT-B protein and mRNA expression was investigated in human, porcine, and murine samples. Immunohistochemical staining for UT-B was performed on paraffin sections of human, porcine, and murine corneas, eyelids, and lacrimal glands (n = 5 each). Reverse transcriptase polymerase chain reaction was conducted to detect UT-B mRNA in human and murine cornea, conjunctiva, Meibomian gland, and lacrimal gland (n = 5 each). RESULTS: UT-B protein expression was comparable in all three species. It was found in the corneal epithelium and endothelium, in the conjunctival epithelium, in the end pieces and excretory ducts of the lacrimal gland, Meibomian gland, and in the glands of Moll and Zeis. The glands of Zeis and the Meibomian glands showed intense UT-B signals in the basal layers of the alveolar epithelia and in the cells of the ductal epithelia. UT-B mRNA was detected in all samples analyzed. CONCLUSION: UT-B is expressed by the cells and tissues of the ocular surface and in the lacrimal glands. Potential changes in urea transporter expression might have implications for the pathogenesis of dry eye disease. Since comparable results were obtained for all species investigated, the presented findings may open the door for DED-relevant experimentation on porcine and murine model systems.

Orthodontic bonding to silicate ceramics: impact of different pretreatment methods on shear bond strength between ceramic restorations and ceramic brackets.

OBJECTIVE: The study aims to investigate the shear bond strength (SBS) between silicate ceramic restorations and ceramic brackets after different pretreatments and aging methods. MATERIAL AND METHODS: Leucite (LEU) and lithium disilicate (LiSi) specimens were pretreated with (i) 4% hydrofluoric acid + silane (HF), (ii) Monobond Etch&Prime (MEP), (iii) silicatization + silane (CoJet), and (iv) SiC grinder + silane (SiC). Molars etched (phosphoric acid) and conditioned acted as comparison group. SBS was measured after 24 h (distilled water, 37 °C), 500 × thermocycling (5/55 °C), and 90 days (distilled water, 37 °C). Data was analyzed using Shapiro-Wilk, Kruskal-Wallis with Dunn's post hoc test and Bonferroni correction, Mann-Whitney U, and Chi2 test (p < 0.05). The adhesive remnant index (ARI) was determined. RESULTS: LEU pretreated with MEP showed lower SBS than pretreated with HF, CoJet, or SiC. LiSi pretreated with MEP resulted in lower initial SBS than pretreated with HF or SiC. After thermocycling, pretreatment using MEP led to lower SBS than with CoJet. Within LiSi group, after 90 days, the pretreatment using SiC resulted in lowest SBS values. After HF and MEP pretreatment, LEU showed lower initial SBS than LiSi. After 90 days of water storage, within specimens pretreated using CoJet or SiC showed LEU higher SBS than LiSi. Enamel presented higher or comparable SBS values to LEU and LiSi. With exception of MEP pretreatment, ARI 3 was predominantly observed, regardless the substrate, pretreatment, and aging level. CONCLUSIONS: MEP pretreatment presented the lowest SBS values, regardless the silicate ceramic and aging level. Further research is necessary. CLINICAL RELEVANCE: There is no need for intraoral application of HF for orthodontic treatment.

Usability of abattoir-acquired pig eyes for refractive excimer laser research.

The purpose of this study was to elucidate, under which conditions abattoir-acquired pig eyes are suitable for refractive excimer laser experiments. Porcine eyes from tunnel-scalded (n = 5) and tank-scalded (n = 10) pigs were compared to unscalded eyes (n = 5) and to eyes scalded in the laboratory (n = 5). The corneal epithelium was removed before an excimer laser was used to perform a - 8.0 D photoablation. Corneal thickness was measured by optical coherence topography before and after photoablation. The ablation depth was determined with a contour measuring station, the morphology of the ablated areas was characterized by scanning electron microscopy and white-light profilometry. The scalded eyes showed an increase in corneal swelling which gained statistical significance in tank-scalded eyes showing a wedge-shaped opaque stromal lesion in the nasal corneal quadrant. A measurable deterioration of photoablation was only found in tank-scalded eyes that exhibited the opaque lesion. Ablated area morphology was smooth and regular in the unscalded and tunnel-scalded eyes. The tank-scalded eyes showed conspicuous wrinkles. While unscalded eyes should always be preferred for excimer laser laboratory experiments, the data suggest that the use of tunnel-scalded eyes may also be acceptable and should be chosen over tank-scalded eyes.

Curvature-dependent shear bond strength of different attachment materials for orthodontic lingual indirect bonding.

To evaluate the shear bond strength (SBS) of different attachment materials used for lingual bonding, the influence of artificial aging and the radii of curvature of the enamel surface on SBS, 192 third molars were photographed to determine the radius of curvature of the oral surface. After phosphoric acid etching a cylindrical test piece was bonded to the oral enamel using a mold that was filled with a chemically curing (Maximum Cure, Transbond IDB Premix) or a dual-curing (Nexus NX3, RelyX Unicem2) attachment material. SBS was tested after 24 h, 500 thermal cycles or 90 days at 37 °C with a universal testing machine. Computed tomography scans were performed to determine the bonded surface and calculate SBS. Values ranged from 8.3 to 20.9 MPa. RelyX Unicem2 showed the highest SBS values at baseline, 500 thermal cycles and after 90 days (p < 0.001). Ninety days of wet storage significantly reduced SBS of Maximum Cure (p = 0.028). The radius of curvature correlated positively with SBS (rs = 0.204, p = 0.005). The SBS of all attachment materials was sufficient for clinical use, even after artificial aging. RelyX Unicem2 showed almost twice as high SBS values as the other attachment materials.

Prevalence and characteristics of test anxiety in first year anatomy students.

BACKGROUND: Test anxiety is a common phenomenon at universities with the potential to impair academic performance and student well-being. This study was conducted to investigate prevalence, characteristics, and development of the test anxiety categories "emotionality", "worry", "interference", and "lack of confidence" among first year medical students. METHODS: Overall, 625 freshman medical students were enrolled. They were recruited from the participants of a first semester anatomy course. The participants were assessed four times with a validated psychological test anxiety questionnaire (Prüfungsangstfragebogen, PAF). The first assessment was conducted at the commencement of the first semester. All further assessments were performed two days prior to each of three mandatory oral anatomy tests in the course of one half year. Prevalence rates as well as mean global and subscale scores of the test anxiety dimensions "emotionality", "worry", "interference" and "lack of confidence" were determined and compared between assessments. RESULTS: Approximately 50% of the study participants showed pronounced test anxiety in at least one dimension over the observation period. Only about 10% were considered test anxiety-positive according to the global PAF scale. Worry showed the highest (up to 48%) and interference the lowest (≈5%) prevalence rates. Emotionality had a stable prevalence of approximately 17%, whereas lack of confidence showed a rising trend over the observation period from 15.2% up to 24.0%. CONCLUSION: Test anxiety is substantially more prevalent among medical students as commonly reported and deserves more detailed, dimension-specific exploration in future. Especially worry and lack of confidence give reason for concern, demanding further investigation.

Neurocritical care complications and interventions influence the outcome in aneurysmal subarachnoid hemorrhage.

BACKGROUND: This observational study was performed to show the impact of complications and interventions during neurocritical care on the outcome after aneurysmal subarachnoid hemorrhage (SAH). METHODS: We analyzed 203 cases treated for ruptured intracranial aneurysms, which were classified regarding clinical outcome after one year according to the modified Rankin Scale (mRS). We reviewed the data with reference to the occurrence of typical complications and interventions in neurocritical care units. RESULTS: Decompressive craniectomy (odds ratio 21.77 / 6.17 ; p < 0.0001 / p = 0.013), sepsis (odds ratio 14.67 / 6.08 ; p = 0.037 / 0.033) and hydrocephalus (odds ratio 3.71 / 6.46 ; p = 0.010 / 0.00095) were significant predictors for poor outcome and death after one year beside "World Federation of Neurosurgical Societies" (WFNS) grade (odds ratio 3.86 / 4.67 ; p < 0.0001 / p < 0.0001) and age (odds ratio 1.06 / 1.10 ; p = 0.0030 / p < 0.0001) in our multivariate analysis (binary logistic regression model). CONCLUSIONS: In summary, decompressive craniectomy, sepsis and hydrocephalus significantly influence the outcome and occurrence of death after aneurysmal SAH.

Feasibility of Clinical Hypnosis for Test Anxiety in First-Year Medical Students.

The purpose of this pilot study was to characterize test anxiety among first-year medical students at the University of Erlangen-Nürnberg and to test clinical hypnosis as a possible intervention. The "Prüfungsangstfragebogen," a shortened and revised German version of the Test Anxiety Inventory, was used to estimate test anxiety. It was administered 3 times: at the commencement of winter term 2017/18 and 2 days prior to each of 2 mandatory oral anatomy exams. The test-anxiety categories emotionality, worry, interference, and lack of confidence were evaluated. The effect of clinical hypnosis on test anxiety was estimated. The global test anxiety and lack of confidence scores were reduced significantly over time in the group that underwent clinical hypnosis but not in the control group. Direct comparisons of hypnosis vs. control group yielded no statistically significant differences in the test anxiety scores.

Ansa cervicalis - A new classification approach.

Normally, the inferior root of Ansa cervicalis passes around the internal jugular vein and runs in an anterior direction to meet the superior root ventral to the common carotid artery. However, anatomical variants of the Ansa cervicalis are as yet not well investigated and understood. To close this gap the present study was undertaken. The Ansa cervicalis was examined in 54 human formalin-fixed cadavers and preparations of the head and neck by conventional dissection. In 66% of the specimens the Ansa cervicalis displayed the typical course that was classified as "internal type" (located medial to the internal jugular vein inside the carotid sheath). The remaining 34% pertained to the "external type" of the Ansa cervicalis (lateral to the internal jugular vein). The distance of the Ansa cervicalis relative to the superior margin of the thyroid cartilage was measured in every specimen. The external type Ansa cervicalis was located significantly lower than the internal type relative to the superior margin of thyroid cartilage. Regarding its location relative to the internal jugular vein four variants of combinations of the external and internal types of Ansa cervicalis on the right and left sides were distinguished. Based on their distance from the superior margin of the thyroid cartilage three types of Ansa cervicalis were defined.

Interface morphology and gas production by a refractive 347 nm ultraviolet femtosecond laser: Comparison with established laser systems.

PURPOSE: To evaluate gas production and interface quality after refractive lenticule creation. A new ultraviolet (UV) 347 nm femtosecond laser was compared with the FS200 and the Visumax lasers. SETTING: Department of Anatomy II, University of Erlangen-Nürnberg, Erlangen, Germany. DESIGN: Experimental study. METHODS: Refractive lenticules were created in 52 porcine corneas with the UV laser (n = 20), the Visumax laser (n = 20), and a modified FS200 laser (n = 12). Twelve corneas in each laser group were fixed immediately after lenticule creation to entrap the gas produced. Lenticule extraction was omitted. From 16 other corneas (UV and Visumax lasers; n = 8 each) -5.0 diopter (D) and -10.0 D lenticules were extracted. On lenticule extraction, a flap was created and lifted. In 12 additional corneas, a flap was cut using the FS200 laser and lifted afterward; laser in situ keratomileusis was performed on 8 of these samples. The corneas were processed for light microscopy (gas bubble 2-dimensional morphometry) and scanning electron microscopy (interface quality). RESULTS: The Visumax generated approximately one half as much gas as the FS200. The UV laser undercut the Visumax by another 50%. The interfaces created by the FS200 appeared smooth and without irregularities. The optical zone margin was always conspicuous in the Visumax samples but hardly discernible in the UV specimens. Slight irregularities and very small lenticule pieces were found occasionally in both groups. The UV interfaces appeared slightly rougher than the Visumax interfaces. CONCLUSIONS: Ultraviolet laser lenticule extraction seems promising. The interface quality was similar to that of the clinical Visumax system while gas production was reduced significantly.

Impact of Comorbidities and Smoking on the Outcome in Aneurysmal Subarachnoid Hemorrhage.

The intention of this observational study is to show the significant impact of comorbidities and smoking on the outcome in aneurysmal subarachnoid hemorrhage (SAH). During this observational study 203 cases of treatment of ruptured intracranial aneurysms were analyzed. We examined and classified prospectively the 12 month outcome according to the modified Rankin Scale (mRS) considering retrospectively a history of smoking and investigated prospectively the occurrence of early and delayed cerebral ischemia between 2012 and 2017. Using logistic regression methods, we revealed smoking (odds ratio 0.21; p = 0.0031) and hypertension (odds ratio 0.18; p = 0.0019) to be predictors for a good clinical outcome (mRS 0-2). Age (odds ratio 1.05; p = 0.0092), WFNS Grade (odds ratio 6.28; p < 0.0001), early cerebral ischemia (ECI) (odds ratio 10.06; p < 0.00032) and delayed cerebral ischemia (DCI) (odds ratio 4.03; p = 0.017) were detected as predictors for a poor clinical outcome. Significant associations of occurrence of death with hypertension (odds ratio 0.12; p < 0.0001), smoking (odds ratio 0.31; p = 0.048), WFNS grade (odds ratio 3.23; p < 0.0001) and age (odds ratio 1.09; p < 0.0001), but not with ECI (p = 0.29) and DCI (p = 0.62) were found. Smoking and hypertension seem to be predictors for a good clinical outcome after aneurysmal SAH.

SFTA3 - a novel surfactant protein of the ocular surface and its role in corneal wound healing and tear film surface tension.

The study aimed to characterize the expression and function of SFTA3 at the ocular surface and in tears. Ocular tissues, conjunctival (HCjE) and human corneal (HCE) epithelial cell lines as well as tearfilm of patients suffering from different forms of dry eye disease (DED) were analyzed by means of RT-PCR, western blot, immunohistochemistry, and ELISA. A possible role of recombinant SFTA3 in corneal wound healing was investigated performing in vitro scratch assays. Tear film regulatory properties were analyzed with the spinning drop method and the regulation of SFTA3 transcripts was studied in HCE and HCjE after incubation with proinflammatory cytokines as well as typical ocular pathogens by real-time RT-PCR and ELISA. The results reveal that human ocular tissue as well as tears of healthy volunteers express SFTA3 whereas tears from patients with DED showed significantly increased SFTA3 levels. In vitro wounding of HCE cell cultures that had been treated with recombinant SFTA3 demonstrated a significantly increased wound closure rate and rSFTA3 reduced the surface tension of tear fluid. The results indicate that SFTA3 at the ocular surface seemed to be involved in wound healing and the reduction of surface tension.

Differentiation Patterns of Immortalized Human Meibomian Gland Epithelial Cells in Three-Dimensional Culture.

Purpose: To establish a simplified three-dimensional (3D) meibomian gland culture model using a meibomian gland epithelial cell (HMGEC) line that might be a useful tool to gain deeper insights into meibomian gland dysfunction. For this purpose, 3D differentiation patterns and growth characteristics of HMGECs were studied on various membranes/scaffolds as well as in hanging drops. Methods: Several types of inserts consisting of different materials (Millicell-HA, Millicell-PCF, ThinCert, and Alvetex) as well as hanging drop culture were analyzed. Culture conditions were optimized employing exposure to air (air-lift) and different cell culture media for a maximum of 28 days. To characterize cell differentiation in the developed 3D model, the expression pattern of cytokeratins was investigated by immunohistochemistry. Sudan III staining was performed for detection of lipid formation and transmission electron microscopy (TEM) was used for ultrastructural analysis. Results: Only Alvetex scaffolds and the hanging drop method revealed satisfactory results with regard to 3D culture. Continuous use of proliferation medium (serum-free keratinocyte medium containing epidermal growth factor and bovine pituitary extract) and air-lift were important steps for HMGEC differentiation in 3D culture. However, HMGECs only reached a differentiating state and never became mature or hypermature. When cultured in hanging drops, HMGECs showed serum-induced keratinization processes. Conclusions: HMGECs have the capability to differentiate in a long-term 3D culture, especially when adapted to an air-rich environment. However, even in the 3D format, HMGECs only reach a state of differentiating meibocytes.

Quantification of surfactant proteins in tears of patients suffering from dry eye disease compared to healthy subjects.

PURPOSE: To quantify and compare the amounts of surfactant proteins SP-A, SP-B, SP-C and SP-D in the tear fluid collected from patients with dry eye syndrome and from individuals with a healthy ocular surface. METHODS: Schirmer strips were used to collect tear fluid from both eyes of 241 volunteers (99 men, 142 women; age range: 18-87 years). Dry eye syndrome was diagnosed by ophthalmologists in 125 patients, whereas the healthy control group comprised 116 individuals. The total protein concentration was determined via Bradford assay. The relative concentration of surfactant proteins SP-A through -D was measured by enzyme-linked immuno-sorbent assay (ELISA). RESULTS: The mean relative concentrations of SP-A, SP-C and SP-D were significantly higher in the dry eye group as compared to the healthy controls (p<0.05, one-way ANOVA). SP-B was also detected at a higher concentration in the dry eye group, but the difference to the control group was not statistically significant. CONCLUSIONS: The upregulation of SP-A and SP-D in the dry eye group is probably related to these proteins' known antimicrobial and immunomodulatory effects at the ocular surface. It may represent a pathophysiological response to the inflammatory condition of the ocular surface in dry eye. The upregulation of SP-B and SP-C may represent an effort of the lacrimal system to reduce surface tension and thus to counteract the increased tendency of the tear film to tear in dry eye.

Wound healing in rabbit corneas after flapless refractive lenticule extraction with a 345 nm ultraviolet femtosecond laser.

PURPOSE: To characterize corneal wound healing in a rabbit model after flapless refractive lenticule extraction with a 345 nm ultraviolet femtosecond laser. SETTING: Departments of Ophthalmology and Anatomy II, University of Erlangen-Nürnberg and Wavelight GmbH, Erlangen, Germany. DESIGN: Experimental study. METHODS: Flapless refractive lenticule extraction was performed in 1 eye each of 20 New Zealand white rabbits (-5.0 diopters). Groups of 4 animals were euthanized after 48 hours, 1 week, 2 weeks, 4 weeks, and 3 months, respectively. Corneal samples were prepared for histology and fluorescence microscopy. To assess corneal cell death, proliferation, and myofibroblastic transdifferentiation, terminal uridine deoxynucleotidyl nick end-labeling (TUNEL) assay as well as immunostaining for Ki67 and α-smooth muscle actin (αSMA) were performed on sagittal cryosections. RESULTS: Histology revealed a zone of keratocyte depletion with a thickness of approximately 50 µm around the extraction site. At 48 hours, pronounced TUNEL staining of keratocytes was detected around the interface (159.9 cells/mm ±â€¯18.4 [SD]), which steadily decreased to 74.9 ±â€¯19.8 cells/mm at 1 week and 5.7 ±â€¯4.8 cells/mm at 2 weeks. Ki67 staining of keratocytes was evident at 48 hours (10.0 ±â€¯3.8 cells/mm), which then decreased at 1 week (5.2 ±â€¯1.7 cells/mm) and 2 weeks (0.4 ±â€¯0.5 cells/mm). From 4 weeks onward, no TUNEL or Ki67 staining was detected. The corneal stroma was αSMA-negative at all timepoints. CONCLUSION: Application of the 345 nm laser showed no signs of problematic repair processes in the cornea, which supports the initiation of the clinical phase.