Corneal Imaging and Densitometry Measurements in Juvenile and Adult Keratoconus Patients to Evaluate Disease Progression and Treatment Effects After Corneal Cross-Linking.

PURPOSE: In this study densitometry software for the Oculus Pentacam was used to investigate the treatment outcomes of corneal cross linking (CXL) in adult and juvenile keratoconus (KCN) patients. Densitometry measurements were taken before and after treatment and followed up for one year. METHODS: A comparative study was carried out at Manchester Royal Eye Hospital. Corneal densitometry measurements collected before and after CXL treatment for 32 eyes from KC patients, aged between 12 and 39, were divided to 2 groups 13-18 years (juvenile group) and 19-39 years (adult group) and analysed and compared to pre and post treatment at 3, 6 and 12 months for each group and between both groups. RESULTS: Analysis of densitometry measurements found higher corneal densitometry after CXL which peaks at three months post treatment in both groups. There was significant diversity in corneal densitometry measurements in the stromal zone 0-2 and 2-6 mm for all layers except the posterior layer for both groups (P<0.05). Significantly increased densitometry value was found higher in the juvenile group at six months in the central (P=0.006) and posterior (P=0.004) layers for zone 0-2 mm. The same layers differed significantly also in the 2-6 mm zone in all layers (P=0.01). One year post treatment the same significant increased densitometry level was seen in the juvenile group in the 0-2 mm zone of the central (P=0.007) and posterior layers (P=0.01), as was the 2-6 mm zone (P=0.04). However, no significant difference was found between pre and post treatment for best corrected visual acuity (BCVA), central corneal thickness (CCT) and thinnest area between both groups. A significant difference was found between pre and post treatment for best corrected visual acuity (BCVA), in the adult group at 6 and 12 months post-treatment from pre-treatment (P=0.02, P=0.16) respectively. CONCLUSION: Corneal clarity post CXL treatment in the juvenile group differed significantly from the adult group. Both groups showed increased haze at 3 months post treatment but the adults showed improvement over the next 9 months. In contrast, the juvenile group showed higher densitometry readings at both 6 and 12 months post treatment in comparison to adult group. The reasons for this remain unclear.

Corneal imaging and densitometry measurements in keratoconus patients to monitor disease progression and treatment outcomes after contact lens or Intacs treatment.

AIM: The aim of this study was to compare the pre- and posttreatment corneal densitometry and corneal thickness value of keratoconus (KCN) patients managed via contact lenses (CLs) or by both intrastromal corneal rings and contact lenses. PATIENTS AND METHODS: This prospective study was performed at the Manchester Royal Eye Hospital, UK. Patients were recruited before treatment and followed up for 12 months. Data of corneal densitometry and corneal thickness were collected using the Oculus Pentacam at the pretreatment visit and posttreatment visit at 12 months. RESULTS: Corneal clarity significantly differs between both groups at pre treatment at zone 0-2 mm for the anterior layer (P=0.002). The same diversity is present at zone 2-6 mm for the anterior layer (P=0.003) and posterior layer (P=0.008). The corneal clarity diversity found was not statistically significant at 12 months post treatment (P>0.05). Corneal thickness was found to be statistically significantly different between pre treatment and post treatment for the CL group for central corneal thickness (CCT) and thinnest area (P=0.01 and P=0.02), respectively. DISCUSSION: This study shows that KCN management with Intacs was found to be effective in maintaining corneal clarity for a longer time than that with CL alone. On the other hand, corneal clarity reduces with disease progression in cases managed with CLs only. Analysis of Oculus Pentacam images provides an objective evaluation to monitor the corneal status after these different pathways of management.

Corneal clarity measurements in patients with keratoconus undergoing either penetrating or deep anterior lamellar keratoplasty.

PURPOSE: To compare the corneal clarity measurement between penetrating keratoplasty (PK) and deep anterior lamellar keratoplasty (DALK) in patients with keratoconus, using densitometry software for the Oculus Pentacam. METHODS: A retrospective comparative study was carried out at Manchester Royal Eye Hospital. Data were collected 12-18 months after corneal transplantation for keratoconus, including postoperative corneal densitometry, best corrected visual acuity (BCVA), central corneal thickness (CCT), and other relevant clinical details. RESULTS: Analysis of 37 keratoconus eyes from 36 patients found there was a significantly higher corneal densitometry measurement after DALK than PK. This was predominantly in the posterior layer of the concentric zone 0-2 mm of the cornea (P=0.0004). A significant correlation was found between postoperative BCVA and corneal densitometry in DALK groups at full thickness (P=0.03). This correlation was seen in the central 0-2 mm (P=0.03) and posterior 0-2 mm (P=0.04) zones. In addition, within the DALK group, a correlation was found between central corneal thickness and densitometry at full thickness 2-6 mm (P=0.007), central 0-2 (P=0.04), central 2-6 mm (P=0.01), and at posterior 2-6 mm (P=0.01) zones. CONCLUSION: This study showed that corneal densitometry measurement differs depending on the type of corneal transplantation used to treat keratoconus patients. Densitometry may have an important role to play in the final BCVA achieved by patients undergoing corneal transplantation for keratoconus. Analysis of Oculus Pentacam images provides an objective evaluation to monitor the cornea status after the surgery.

Evaluation of corneal symmetry after UV corneal crosslinking for keratoconus.

PURPOSE: The purpose of this study was to assess UV corneal crosslinking (CXL) treatment outcomes for keratoconus by evaluating the corneal regularity in patients through follow-up using the Oculus Pentacam. PATIENTS AND METHODS: A total of 18 eyes from CXL patients with keratoconus were studied before and after CXL treatment, and six eyes from six patients who were not treated with CXL served as controls. Treated patients had Pentacam images taken before CXL treatment and regularly 3 months post treatment up to the 12th month. Controls were imaged during their first appointment and after 12 months. Symmetry and asphericity were evaluated and correlated with both best-corrected visual acuity (BCVA) and maximum K-readings. RESULTS: In the CXL-treated group, there was a significant improvement in the index of symmetrical variation (ISV) and keratoconus index (KI) at 3 months and in the index of height asymmetry (IHA) and minimum radius of curvature (Rmin) at 9 months post treatment. On the contrary, the untreated group's indices showed some significant worsening in ISV, KI, central keratoconus index (CKI), and Rmin. A novel finding in our study was a slight positive shift of anterior asphericity in the 6 mm, 7 mm, and 8 mm 3 months after treatment, which had a correlation with BCVA (R2=0.390, p=0.053) and a strong correlation with maximum K-reading (R2=0.690, p=0.005). However, the untreated group had no significant changes after 1 year. CONCLUSION: The corneal asymmetrical shape is associated with the spherical aberration alteration influenced by temporal evolution of surface ablation and increased corneal haze. However, insignificant changes in symmetry attest the stabilization effect on cornea postoperatively as compared with controls.

Corneal clarity measurements in healthy volunteers across different age groups: Observational study.

The aim of this study was to standardize and investigate the changes in corneal clarity with age. Densitometry software for the Oculus Pentacam was used to examine corneal clarity at different age groups.A total of 192 eyes from 97 healthy participants were included in this cohort comparative nonrandomized, cross-sectional study. An Oculus Pentcam was used to image the cornea of healthy participants grouped by age (between 10 and 70 years old). Data from the densitometry output have been used to determine clarity in concentric zones and different depths of the cornea.Corneal densitometry (CD) across all ages showed significant differences between groups when divided into the following layers: anterior, central, and posterior or divided into 0 to 2, 2 to 6, and 6 to 10 mm concentric zones (P < .05). The most striking decrease in clarity occurred with age in all 3 layers of the periphery (6-10 mm) (P < .05). In addition, we showed that the 10 to 19-year age group had lower clarity than the 20 to 30-age group (P < .05), and after 30 years, the cornea shows a steady progression of increased or decreased clarity.The values for CD, as well as for separate subdivisions based on layer and surface area, might provide a standard for use in further studies and clinical practice. This study established that relation between CD and age is differed when the cornea is divided into layers and zones. This study suggests that there are other factors that may play an essential role in corneal clarity as well as age.

Evaluation of Corneal Cross-Linking for Treatment of Fungal Keratitis: Using Confocal Laser Scanning Microscopy on an Ex Vivo Human Corneal Model.

Purpose: Some previous reports have established the use of photoactivated chromophore-induced corneal cross-linking (PACK-CXL) in treating fungal keratitis. The results of these case reports have often been conflicting. To systematically study the effect of PACK-CXL in the management of Fusarium keratitis, we have developed an ex vivo model of human corneal infection using eye-banked human corneas. Methods: Sixteen healthy ex vivo human corneas were divided into four study groups: (1) untreated control, (2) cross-linked, (3) infected with fungal spores, and (4) infected with fungal spores and then cross-linked. All infected corneas were inoculated with Fusarium oxysporum spores. The PACK-CXL procedure was performed 24 hours post inoculation for group 4. For PACK-CXL treatment, the corneas were debrided of epithelium; then 1% (wt/vol) isotonic riboflavin was applied dropwise at 5-minute intervals for 30 minutes and during the course of UV-A cross-linking for another 30 minutes. The corneas were imaged using a confocal microscope at 48 hours post inoculation, and the Fusarium hyphal volume and spore concentration were calculated. Results: The infected and then cross-linked group had a significantly lower volume of Fusarium hyphae, compared to the infected (P = 0.001) group. In the infected and then cross-linked group there was significant inhibition of Fusarium sporulation compared with the infected (P = 0.007) group. Conclusions: A model of human corneal infection was successfully developed for investigation of the effects of PACK-CXL on fungal keratitis. A treatment regimen of combined UV-A/riboflavin-induced corneal cross-linking appears to be a valuable approach to inhibit the growth and sporulation of Fusarium and suppress the progression of fungal keratitis.

Regeneration of Corneal Epithelium With Dental Pulp Stem Cells Using a Contact Lens Delivery System.

PURPOSE: The corneal epithelium is sloughed off surface of the eye by the action of blinking and is continually replaced by division and maturation of the limbal stem cells (LSCs). In the case of injury or disease, LSCs can be lost or damaged to a point at which the corneal epithelial layer is no longer maintained. leading to LSC deficiencies (LSCDs). When this occurs, the opaque conjunctiva overgrows the anterior surface of the eye, leading to vision impairment or loss. Dental pulp stem cells (DPSCs) are promising candidates as autologous LSC substitutes. In this study, contact lenses (CLs) are used as a novel medical device to deliver DPSCs onto corneal surface to enhance corneal epithelium regeneration. METHODS: Dental pulp stem cells labeled with green fluorescent Qtracker 525 were seeded onto the pretreated CLs, allowed to adhere, then delivered to debrided human corneas. Expression of KRT3, 12, 13, and 19 was investigated by immunostaining, then standard and confocal microscopy. RESULTS: Dental pulp stem cells were successfully isolated, labeled, and delivered to the corneal surface using CLs. Following removal of CLs, confocal microscopy showed that the DPSCs had migrated onto the cornea. Coexpression of KRT12 and green fluorescent Qtracker 525 confirmed that the DPSCs had transdifferentiated into corneal epithelial progenitors. Delimitation of KRT 19 and green fluorescence provides evidence that Qtracker 525-labeled DPSCs establish a barrier to the invasion of the cornea by conjunctiva. CONCLUSIONS: In this study we show that DPSCs, delivered using CLs, can be used to enhance repair and regeneration of the human corneal epithelium.

Biomechanical Changes of Collagen Cross-Linking on Human Keratoconic Corneas Using Scanning Acoustic Microscopy.

PURPOSE: To assess the biomechanical changes of collagen cross-linking on keratoconic corneas in vitro. METHODS: Six keratoconic corneal buttons were included in this study. Each cornea was divided into two halves, where one half was cross-linked and the other half was treated with riboflavin only and served as control. The biomechanical changes of the corneal tissue were measured across the stroma using scanning acoustic microscopy (SAM). RESULTS: In the cross-linked corneas, there was a steady decrease in the magnitude of speed of sound from the anterior region through to the posterior regions of the stroma. The speed of sound was found to decrease slightly across the corneal thickness in the control corneas. The increase in speed of sound between the cross-linked and control corneas in the anterior region was by a factor of 1.039×. CONCLUSION: A higher speed of sound was detected in cross-linked keratoconic corneal tissue when compared with their controls, using SAM. This in vitro model can be used to compare to the cross-linking results obtained in vivo, as well as comparing the results obtained with different protocols.

Biomechanical changes after repeated collagen cross-linking on human corneas assessed in vitro using scanning acoustic microscopy.

PURPOSE: To explore the biomechanical changes induced by repeated cross-linking using scanning acoustic microscopy (SAM). METHODS: Thirty human corneas were divided into three groups. In group A, five corneas were cross-linked once. In group B, five corneas were cross-linked twice, 24 hours apart. In group C, five corneas were cross-linked three times, 24 hours apart. The contralateral controls in all groups had similar treatment but without UV-A. The speed of sound, which is directly proportional to the square root of the tissue's elastic modulus, was assessed using SAM. RESULTS: In group A, the speed of sound of the treated corneas was 1677.38 ± 10.70 ms(-1) anteriorly and 1603.90 ± 9.82 ms(-1) posteriorly, while it was 1595.23 ± 9.66 ms(-1) anteriorly and 1577.13 ± 8.16 ms(-1) posteriorly in the controls. In group B, the speed of sound of the treated corneas was 1746.33 ± 23.37 ms(-1) anteriorly and 1631.60 ± 18.92 ms(-1) posteriorly, while it was 1637.57 ± 22.15 ms(-1) anteriorly and 1612.30 ± 22.23 ms(-1) posteriorly in the controls. In group C, the speed of sound of the treated corneas was 1717.97 ± 18.92 ms(-1) anteriorly and 1616.62 ± 17.58 ms(-1) posteriorly, while it was 1628.69 ± 9.37 ms(-1) anteriorly and 1597.68 ± 11.97 ms(-1) posteriorly in the controls. The speed of sound in the anterior (200 × 200 µm) region between the cross-linked and control corneas in groups A, B, and C was increased by a factor of 1.051 (P = 0.005), 1.066 (P = 0.010), and 1.055 (P = 0.005) respectively. However, there was no significant difference among the cross-linked corneas in all groups (P = 0.067). CONCLUSIONS: A significant increase in speed of sound was found in all treated groups compared with the control group; however, the difference among the treated groups is not significant, suggesting no further cross-links are induced when collagen cross-linking treatment is repeated.

Biomechanical properties of human corneas following low- and high-intensity collagen cross-linking determined with scanning acoustic microscopy.

PURPOSE: To assess and compare changes in the biomechanical properties of the cornea following different corneal collagen cross-linking protocols using scanning acoustic microscopy (SAM). METHODS: Ten donor human corneal pairs were divided into two groups consisting of five corneal pairs in each group. In group A, five corneas were treated with low-fluence (370 nm, 3 mW/cm(2)) cross-linking (CXL) for 30 minutes. In group B, five corneas were treated with high-fluence (370 nm, 9 mW/cm(2)) CXL for 10 minutes. The contralateral control corneas in both groups had similar treatment but without ultraviolet A. The biomechanical properties of all corneas were tested using SAM. RESULTS: In group A, the mean speed of sound in the treated corneas was 1677.38 ± 10.70 ms(-1) anteriorly and 1603.90 ± 9.82 ms(-1) posteriorly, while it was 1595.23 ± 9.66 ms(-1) anteriorly and 1577.13 ± 8.16 ms(-1) posteriorly in the control corneas. In group B, the mean speed of sound of the treated corneas was 1665.06 ± 9.54 ms(-1) anteriorly and 1589.89 ± 9.73 ms(-1) posteriorly, while it was 1583.55 ± 8.22 ms(-1) anteriorly and 1565.46 ± 8.13 ms(-1) posteriorly in the untreated control corneas. The increase in stiffness between the cross-linked and control corneas in both groups was by a factor of 1.051×. CONCLUSIONS: SAM successfully detected changes in the corneal stiffness after application of collagen cross-linking. A higher speed-of-sound value was found in the treated corneas when compared with the controls. No significant difference was found in corneal stiffness between the corneas cross-linked with low- and high-intensity protocols.

Scanning acoustic microscopy for mapping the microelastic properties of human corneal tissue.

PURPOSE: To assess the feasibility of applying scanning acoustic microscopy (SAM) on UV cross-linked corneal tissue for mapping and analyzing its biomechanical properties. MATERIALS AND METHODS: Five corneal pairs (10 corneas) were used. In each pair, one cornea was cross-linked (epithelium removed, riboflavin application for 45 min and UVA irradiation for 30 min) and the contralateral control cornea was epithelial debrided and treated only with riboflavin for 45 min. Histological sections were prepared and their mechanical properties were examined using SAM. A line profile technique and 2D analysis was used to analyze the mechanical properties of the corneas. Then the corneal paraformaldehyde and unfixed sections were examined histologically using hematoxylin and eosin (H&E) staining. RESULTS: In the frozen fresh corneal tissue, the speed of sound of the treated corneas was 1672.5 ± 36.9 ms(-1), while it was 1584.2 ± 25.9 ms(-1) in the untreated corneas. In the paraformaldehyde fixed corneal tissue, the speed of sound of the treated corneas was 1863.0 ± 12.7 ms(-1), while it was 1739.5 ± 30.4 ms(-1) in the untreated corneas. The images obtained from the SAM technique corresponded well with the histological images obtained with H&E staining. CONCLUSION: SAM is a novel tool for examining corneal tissue with a high spatial resolution, providing both histological and mechanical data.

Functional role of CD105 in TGF-beta1 signalling in murine and human endothelial cells.

BACKGROUND: Angiogenesis is important in health and several disease states. CD105 is a proliferation-associated and hypoxia-inducible transmembrane protein abundantly expressed in angiogenic endothelial cells. CD105 is a receptor for transforming growth factors (TGF)-beta1 and -beta3. The exact mechanisms for CD105 regulation of vascular development have not been fully elucidated. MATERIALS AND METHODS: In this study, an antisense approach to create a murine and a human stably transfected endothelial cell line expressing a reduction in CD105 protein was used. RESULTS: We showed that inhibition of CD105 in cultured murine and human endothelial cells enhanced the ability of TGF-beta1 to suppress growth and migration, and influenced TGF-beta1 promoter activity. TGF-beta1 not only reduced the length of the capillary-like structures, but also caused mortality in CD105-deficient murine antisense cells compared to control cultures. To determine whether CD105 affected TGF-beta1-induced gene expression, a luciferase assay in transiently transfected cells with p3TP-Lux promoter constructs was performed. Both murine and human antisense transfectants showed a significant increase in p3TP-Lux promoter activity. Further studies on the functional importance of CD105 was undertaken in irradiated normoxic and hypoxic cells. The levels of pro- and anti-apoptotic markers were also evaluated. There was an increase in pro-apoptotic marker (p53), but a reduction in anti-apoptotic marker (Bcl-2) in CD105-deficient cells. CONCLUSION: These results provide direct evidence that CD105 antagonises the inhibitory effects of TGF-beta1 on human and murine vascular endothelial cells and that normal cellular levels of CD105 are required for the formation of new blood vessels.

Apoptotic gene expression in Paget's disease: a possible role for Bcl-2.

Paget's disease of bone is characterized by an increase in both the size and the number of bone-resorbing osteoclasts. An important regulator of osteoclast activity is the process of apoptosis, and any aberration in this process could lead to increased osteoclasis. Analysis using human apoptosis cDNA expression arrays revealed that the apoptotic suppressor, Bcl-2, showed a marked increase in expression in Pagetic bone. In situ hybridization (ISH) and computer-assisted image analysis confirmed that the levels of Bcl-2 transcripts were significantly (p<0.0001) increased in Pagetic osteoclasts. The Bcl-2:Bax transcript ratios were similarly elevated. These findings were confirmed by immunohistochemistry. The Bcl-2 gene promoter sequence from 20 Pagetic patients and controls was analysed. Single nucleotide mutations were identified in three of the Paget's patients and one of the controls. Luciferase reporter analysis showed that the mutations induced a basal 12-fold increase and hydrogen peroxide-induced 19-fold increase in luciferase expression, compared with the normal construct. It is concluded that in Paget's disease, there is an increase in the expression of genes that are involved in the inhibition of apoptosis, notably Bcl-2. The increase in Bcl-2 may be explained in some patients by mutations in the Bcl-2 gene promoter. These results provide a potential explanation for the dramatic increase in osteoclasis seen in patients with Paget's disease.