Early biological responses in ocular tissue after SMILE and LASIK surgery.

We studied the early protein profile in the ocular tissue extracted after LASIK and SMILE surgery. SMILE and LASIK was performed in contralateral eyes and stromal tissue samples were collected from 10 eyes of 5 donors. The stromal tissue samples were analyzed using label free quantification approach and ITRAQ labelling approach in LC-MS/MS. Combined functional analysis revealed many differentially expressed proteins which were involved in important biological processes. About 117 unique differentially expressed proteins were identified using two different proteomic approaches. Collagens, proteoglycans, corneal crystallins were enriched and showed differential expression in SMILE and LASIK as compared to the non-surgical control. Apart from these, 14-3-3 class of proteins, Lysozyme (LYZ), Macrophage Migratory Inhibitory Factor protein (MIF), Pigment Epithelial Derived Factor (PEDF) were differentially expressed when compared between LASIK and SMILE. Peroxiredoxin 1 (PRDX1) expression was found to be reduced in LASIK as compared to SMILE. The expression of Lysozyme C and Macrophage Migratory Inhibitory Factor inflammatory response was found to be less in SMILE as compared to LASIK. Western blot validation of specific markers such as Collagen IV (COL4), Keratocan (KERA), Lumican (LUM), Aldehyde dehydrogenase 3 A1 (ALDH3A1), Lysozyme C (LYZC) confirmed the differences in the protein levels observed in SMILE and LASIK operated tissues as compared to non-surgical controls. In conclusion, this study revealed the early molecular changes occurring in the cornea resulting from these two surgical procedures which may have implications on managing post-operative complications.

Identification of novel predictive factors for post surgical corneal haze.

Molecular factors altered in corneas that develop haze post refractive surgery have been described, but pre-existing factors that predispose clinically normal corneas to aberrant fibrosis post surgery and the role of the corneal epithelium remains unknown. We analyzed the global gene expression in epithelium collected intraoperatively from subjects undergoing photorefractive keratectomy. Subjects were grouped into those that developed haze 12 months post surgery (n = 6 eyes; haze predisposed) and those that did not develop haze in a similar follow up duration (n = 11 eyes; controls). Ontological analysis of 1100 upregulated and 1780 downregulated genes in the haze predisposed group revealed alterations in pathways associated with inflammation, wnt signaling, oxidative stress, nerve functions and extra cellular matrix remodeling. Novel factors such as PREX1, WNT3A, SOX17, GABRA1and PXDN were found to be significantly altered in haze predisposed subjects and those with active haze(n = 3), indicating their pro-fibrotic role. PREX1 was significantly upregulated in haze predisposed subjects. Ectopic expression of PREX1 in cultured human corneal epithelial cells enhanced their rate of wound healing while its ablation using shRNA reduced healing compared to matched controls. Recombinant TGFß treatment in PREX1 overexpressing corneal cells led to enhanced αSMA expression and Vimentin phosphorylation while the converse was true for shPREX1 expressing cells. Our data identify a few novel factors in the corneal epithelium that may define a patient's risk to developing post refractive corneal haze.

Dysregulated Tear Fluid Nociception-Associated Factors, Corneal Dendritic Cell Density, and Vitamin D Levels in Evaporative Dry Eye.

Purpose: The purpose of this study was to study the status and association among tear-soluble factors, corneal dendritic cell density, vitamin D, and signs and symptoms in dry eye disease (DED). Methods: A total of 33 control subjects and 47 evaporative dry eye patients were included in the study. DED diagnosis and classification was based on the 2017 Report of the Tear Film & Ocular Surface Society International Dry Eye Workshop (TFOS DEWS II). DED workup, including tear film break-up time (TBUT), Schirmer's test I (STI), corneal and conjunctival staining, ocular surface disease index (OSDI) scoring, and in vivo confocal microscopy (to assess corneal dendritic cell density [cDCD] and subbasal nerve plexus [SBNP] features) was performed in the study subjects. Tear fluid using Schirmer's strip and serum were collected from the subjects. Multiplex ELISA or single analyte ELISA was performed to measure 34 tear-soluble factors levels including vitamin D. Results: Significantly higher OSDI discomfort score, lower TBUT, and lower STI were observed in DED patients. cDCD was significantly higher in DED patients. No significant difference was observed in SBNP features. Tear fluid IL-1ß, IL-17A, MMP9, MMP10, MMP9/TIMP ratio, and VEGF-B were significantly higher in DED patients. Significantly lower tear fluid IL-2, IP-10, NPY, VEGF-A, and vitamin D was observed in DED patients. These dysregulated tear factors showed significant associations with DED signs and symptoms. Conclusions: Altered tear fluid soluble factors with potential to modulate nociception exhibited a distinct association with ocular surface discomfort status, TBUT, STI, and cDCD. This implies a functional relationship between the various tear-soluble factors and dry eye pathogenesis, indicating new molecular targets for designing targeted therapies.

Outcomes of Corneal Cross-Linking Correlate With Cone-Specific Lysyl Oxidase Expression in Patients With Keratoconus.

PURPOSE: To evaluate the correlation of visual and keratometry outcomes after corneal cross-linking (CXL) in patients with keratoconus with cone epithelium-specific gene expression levels. METHODS: Corneal epithelium was obtained from 35 eyes that underwent accelerated CXL (KXLII, 9 mW/cm for 10 min). Using corneal topography, epithelium over the cone and periphery was obtained separately from each subject. The ratio of gene expression for lysyl oxidase (LOX), matrix metalloproteinase 9 (MMP9), bone morphogenic protein 7, tissue inhibitor of metalloproteinase 1, collagen, type I, alpha 1, and collagen, type IV, alpha 1 (COL IVA1) from the cone and peripheral cornea was correlated with the outcome of cross-linking surgery. Patients were assessed for visual acuity, keratometry, refraction, and corneal densitometry before and 6 months after surgery. Based on the change in corneal flattening indicated by ΔKmax, the outcomes were classified as a higher response or lower response. RESULTS: Reduction in keratometric indices correlated with improved spherical equivalent after CXL. Preoperative levels of cone-specific LOX expression in cases with a higher response were significant (P = 0.001). COL IVA1, bone morphogenic protein 7, and tissue inhibitor of metalloproteinase 1 gene expressions were reduced in the cones of the subjects with a lower response. MMP9 levels were relatively lower in cases with a higher response compared with those with a lower response. CONCLUSIONS: Our study demonstrates that preoperative levels of molecular factors such as LOX, MMP9, and COL IVA1 aid in understanding CXL outcomes at the tissue level.

Corneal Biomechanical Changes and Tissue Remodeling After SMILE and LASIK.

Purpose: To evaluate transient corneal tissue healing and biomechanical changes between laser in situ keratomileusis (LASIK) and small incision lenticule extraction (SMILE) eyes. Methods: In each patient, one eye underwent LASIK and the other underwent SMILE. Optical coherence tomography (OCT) and dynamic Scheimpflug imaging (Corvis-ST) was used to assess tissue healing and biomechanics, respectively. Analyses of OCT scans yielded corneal speckle distribution (CSD) and Bowman's roughness index (BRI). Waveform analyses of deformation amplitude yielded corneal stiffness. Further, corneal force versus corneal deformation data helped compare the two procedures. Results: BRI increased and then decreased transiently after both treatments (P < 0.05). However, SMILE eyes had BRI similar to that of their preoperative state compared to LASIK eyes at 6-month follow-up. CSD indicated a marked increase in the number of bright pixels and a decrease in the number of dark pixels after SMILE (1-month follow-up) and LASIK eyes (3-month follow-up), respectively. CSD returned to near preoperative state thereafter, respectively. Corneal stiffness change from preoperative state was similar between LASIK and SMILE eyes. However, deformation at discrete values of corneal force indicated some recovery of biomechanical strength after SMILE, but not in LASIK eyes. Conclusions: BRI and CSD indicated earlier tissue healing in SMILE eyes than in LASIK. CSD results may indicate delayed cell death in LASIK eyes and increased light scatter due to interface fluid in SMILE eyes. Corneal biomechanical strength remodeled better in SMILE. This may indicate some hydration-related recovery.

Waveform analysis of deformation amplitude and deflection amplitude in normal, suspect, and keratoconic eyes.

PURPOSE: To evaluate the performance of waveform-derived variables in distinguishing normal, suspect, and keratoconic eyes. SETTING: Narayana Nethralaya Eye Hospital, Bangalore, India. DESIGN: Retrospective case series. METHODS: Scheimpflug tomography (Pentacam) and dynamic Scheimpflug analysis (Corvis ST) of 253 normal (253 patients) eyes and 205 keratoconic eyes (205 patients) were evaluated. Among the 205 patients, 62 had keratoconus in 1 eye, while the unaffected eye was suspect. From deformation amplitude, deflection amplitude and whole-eye movement were extracted. A biomechanical model was used to derive a linear (kc [constant]) and nonlinear measure (kc [mean]) of corneal stiffness. Multivariate logistic regression was performed to determine sensitivity and specificity. The analysis was validated in another dataset of 59 normal, 45 suspect, and 160 keratoconic eyes. RESULTS: Deformation amplitude maximum, applanation 1 time and deformation amplitude, applanation 2 time, kc (constant), kc (mean), and deflection amplitude maximum were significantly different between normal and keratoconic eyes (P < .001). The deformation characteristics of the suspect eyes were similar to those of the keratoconic eyes, particularly grade 1 (P > .05). The kc (constant) and kc (mean) had the highest area under curve (>0.98), sensitivity, and specificity greater than 90% and 91%, respectively. Logistic regression using kc (constant) and kc (mean) improved the area to 1.0, with a sensitivity and specificity equal to 99.6% and 100%, respectively. In the validation dataset, the same cutoff yielded a sensitivity, specificity, and accuracy of 99.5%, 100%, and 99.6%, respectively. CONCLUSION: Corneal stiffness and waveform analyses could be reliable differentiators of suspect and keratoconic eyes from normal eyes.

Customized Corneal Cross-linking Using Different UVA Beam Profiles.

PURPOSE: To evaluate the performance of different customized corneal cross-linking (CXL) methods. METHODS: This was a single-center interventional, prospective, longitudinal case series. Four different customized CXL methods were evaluated in keratoconic eyes: (1) uniform (uniform intensity ultraviolet-A [UVA] beam [9 mW/cm2] for 10 minutes) (n = 12 eyes); (2) sector axial map (sector-based UVA irradiation) (n = 12 eyes); (3) ring axial map (concentric rings of UVA beam intensity centered at the steepest curvature of the anterior axial map) (n = 12 eyes); and ring tangential map (same as the ring axial map but centered at the steepest curvature of the anterior tangential map) (n = 14 eyes). Peak UVA energy density in the sector and ring axial map (and ring tangential map) protocols did not exceed 15.0 and 10.8 J/cm2, respectively. A 0.1% riboflavin solution was applied after epithelium removal. Corneal tomography and visual acuity were assessed before and 6 months after CXL. RESULTS: Average and peak energy density was lowest in the ring tangential protocol and highest in the sector axial map group (P < .001). Treated area was lowest in the ring tangential map group and highest in the uniform group (P < .001). Decrease in curvature was similar among the uniform, sector axial map, and ring axial map groups (P < .05). The ring tangential map group had the greatest decrease in curvature per unit energy dose to the cornea (P < .05). Improvement in uncorrected (0.081 ± 0.056 logMAR) and corrected (0.041 ± 0.026 logMAR) distance visual acuity per unit energy density was greatest in the ring tangential map group (P > .05). CONCLUSIONS: When normalized to the average energy density, the ring tangential map protocol appeared to provide maximum flattening and improvement in visual acuity. Further studies with larger sample sizes are needed to validate the findings of this pilot study. [J Refract Surg. 2017;33(10):676-682.].

Oxidative stress induces dysregulated autophagy in corneal epithelium of keratoconus patients.

Oxidative stress is one of the key factors that contributes to the pathogenesis of keratoconus (KC). Macroautophagy is a vital cellular mechanism that is activated in response to oxidative stress. The aim of this study was to understand the role of the autophagic lysosomal pathway in the oxidative damage of KC corneal epithelium and the human corneal epithelial cell line (HCE).The corneal epithelium was collected from 78 KC patients undergoing corneal cross-linking or topography guided photorefractive keratectomy. We performed microarray, qPCR and western blot analysis for the expression of autophagy markers on this epithelium from patients with different clinical grades of KC. A differential expression pattern of autophagy related markers was observed in the diseased corneal cone-specific epithelium compared to matched peripheral epithelium from KC patients with increasing clinical severity. Human corneal epithelial cells exposed to oxidative stress were analyzed for the expression of key proteins associated with KC pathogenesis and the autophagic pathway. Oxidative stress led to an increase in reactive oxygen species and an imbalanced expression of autophagy markers in the HCE cells. Further, reduced levels of Akt/p70S6 Kinase, which is a known target of the mTOR pathway was observed in HCE cells under oxidative stress as well as in KC epithelium. Our results suggest that an altered expression of proteins suggestive of defective autophagy and is a consequence of oxidative damage. This could play a possible role in the pathogenesis of KC.

In vivo confocal microscopy and tear cytokine analysis in post-LASIK ectasia.

AIM: Corneal keratectasia is one of the complications associated with laser in situ keratomileusis (LASIK) that results in vision impairment. The pathogenesis of post-LASIK ectasia (PLE) remains underexplored. We report the tear cytokine profile and in vivo confocal microscopy (IVCM) findings in eyes with PLE. METHODS: This retrospective study included age-matched 7 (14 eyes) post-LASIK controls (PLCs) and 6 (12 eyes) PLE subjects. Corneal topography was used to categorise the subjects into PLC and PLE groups. Ocular Surface Disease Index (OSDI) scores obtained were based on standard questionnaire and IVCM images were used to determine corneal dendritic cells density (DCD) and sub-basal nerve plexus morphology. Inflammatory cytokines/chemokines in the tears were quantified using flow cytometry based cytometric bead array. RESULTS: Pentacam-based scores, OSDI scores and corneal DCD were significantly (p<0.05) higher in patients with PLE compared with PLC. Discomfort-related subscale of OSDI score exhibited a positive correlation with total corneal DCD in the PLE cohort. The fold difference of chemokine (C-C motif) ligand/monocyte chemotactic protein-1 (CCL2/MCP1) (3.4±0.6) was found to be significantly (p<0.05) higher in the PLE cohorts and a positive correlation between CCL2/MCP1 levels and total corneal DCD was also observed in the PLE cohort. CONCLUSION: The current study found a significant difference in the tear film cytokine profile between normal and PLE eyes. Presence of increased corneal dendritic cells and altered tear cytokines suggests an ongoing inflammatory response in PLE.

Application of high resolution OCT to evaluate irregularity of Bowman's layer in asymmetric keratoconus.

Irregularity of the Bowman's layer (BL) in keratoconus (KC, sample size (n) = 28) and forme fruste keratoconus (FFKC, n = 18) eyes was evaluated. Subjects underwent high resolution OCT imaging (Bioptigen Inc., USA) and corneal tomography (Pentacam v1.20r41, OCULUS Optikgeräte GmbH, Germany). Anterior edge of the BL was segmented. A Bowman's roughness index (BRI) was defined as the sum of the enclosed areas between segmented edge and a smooth 3rd order polynomial fit to the edge. BRI was compared with corneal aberrations, Keratoconus percentage index (KISA), Cone location magnitude index (CLMI) and Belin-Ambrosio enhanced ectasia display overall deviation index (BAD-D). Area under the curve (AUC) was determined with logistic regression (LR). Mean BRI (×10-3 ) was 2.12 mm2 , 1.81 mm2 and 1.7 mm2 in normal (n = 26), FFKC and KC eyes, respectively (p < 0.001). BAD-D (0.79) and BRI (0.74) had the best AUC for FFKC. By combining BAD-D and BRI, the AUC improved to 0.85 (p = 0.01). For KC eyes, KISA (0.94), CLMI (0.88), BAD-D (0.96) and aberrations had comparable AUC (p > 0.05). However, LR with BRI and other indices didn't improve the AUC in KC eyes (p > 0.05). BRI was significantly reduced in FFKC and KC eyes. It improved the detection of FFKC but not KC eyes.

Laser Vision Correction with Q Factor Modification for Keratoconus Management.

PURPOSE: To evaluate the outcomes of corneal laser ablation with Q factor modification for vision correction in patients with progressive keratoconus. METHODS: In this prospective study, 50 eyes of 50 patients were divided into two groups based on Q factor (>-1 in Group I and ≤-1 in Group II). All patients underwent a detailed ophthalmic examination including uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), subjective acceptance and corneal topography using the Pentacam. The topolyzer was used to measure the corneal asphericity (Q). Ablation was performed based on the preoperative Q values and thinnest pachymetry to obtain a target of near normal Q. This was followed by corneal collagen crosslinking to stabilize the progression. RESULTS: Statistically significant improvement (p ≤ 0.05) was noticed in refractive, topographic, and Q values posttreatment in both groups. The improvement in higher-order aberrations and total aberrations were statistically significant in both groups; however, the spherical aberration showed statistically significant improvement only in Group II. CONCLUSION: Ablation based on the preoperative Q and pachymetry for a near normal postoperative Q value appears to be an effective method to improve the visual acuity and quality in patients with keratoconus.

Lower Vitamin D Level and Distinct Tear Cytokine Profile Were Observed in Patients with Mild Dry Eye Signs but Exaggerated Symptoms.

PURPOSE: Dry eye is associated with inflammation, pain, and discomfort. Vitamin D is known to modulate immune responses and pain. This study investigates the level of serum vitamin D and tear-inflammatory proteins with relation to exaggerated symptoms in patients with mild dry eye. METHODS: Patients with mild dry eye signs (Dry Eye Workshop [DEWS] severity grade 1) but with exaggerated symptoms and healthy controls (n = 19, each) were recruited for this cross-sectional study. Schirmer's Test I (mm), tear film break-up time (TBUT; secs), and ocular surface disease index (OSDI) score were recorded. Serum vitamin D level and tear cytokine levels were measured. RESULTS: The mean OSDI score in the patient cohort (46 ± 3) was significantly higher than controls (8.4 ± 1.6). TBUT was lower (7.6 ± 0.3 secs) in patients compared with controls (11.0 ± 0.9 secs). Mean Schirmer's Test I value in patients (19.3 ± 1.4 mm) was lower than in controls (30.6 ± 1.9 mm). An inverse correlation was observed between serum vitamin D levels and OSDI score (r = -0.569; P = 0.01). Significantly higher levels of interleukin (IL)-17A/F, interferon (IFN)-γ, monocyte chemotactic protein (MCP)-1, intercellular adhesion molecule (ICAM)-1, IL-4, IL-10, and decreased IL-2 concentrations was observed in the tears of patients compared with controls (P < 0.05). CONCLUSION: Decreased serum vitamin D was associated with exaggerated symptoms in dry eye patients with mild dry eye signs. In addition, altered tear cytokine profile was also observed in these patients. TRANSLATIONAL RELEVANCE: Vitamin D measurements would aid in the diagnosis and management of dry eye.

Differential Molecular Expression of Extracellular Matrix and Inflammatory Genes at the Corneal Cone Apex Drives Focal Weakening in Keratoconus.

PURPOSE: In this study, we elucidated the differential expression of a set of local molecular factors in ectatic cone area of the cornea to uncover a functional cause for focal corneal weakening characteristic of the keratoconus (KC) disease. METHODS: All human corneal samples were collected after approval of Institutional Ethics Committee and informed consent. Keratoconus patients were classified based on clinical parameters, topographical features, and structural deformity. Epithelial cells were collected from KC patients (n = 66) undergoing corneal cross-linking procedures from cone apex and periphery. Nonectatic refractive surgery patients (n = 23) served as controls. The ratio of epithelial gene expression in cone and periphery of each eye was estimated by quantitative PCR and correlated with clinical data. Similar cone versus periphery analysis was done from the KC stroma and from KC patients with Bowman's layer (BL) breach observed by anterior segment optical coherence tomography (OCT). RESULTS: Epithelium from the cone apex of KC patients had elevated levels of inflammatory factors TNF-α, IL-6, and matrix metalloproteinase 9 (MMP-9) but reduced Lysyl oxidase (LOX) and Collagen IVA1, which also demonstrated correlation with corneal curvature and deformity parameters. Stromal gene expression from KC patients showed trends similar to epithelium. Epithelium collected from the cone apex of BL breached KC patients showed significantly elevated MMP-9, TNF-α, and IL-6 levels but reduced IL-10, tissue inhibitor of metalloproteinases 1 (TIMP-1), and Collagen IVA1 expression. CONCLUSIONS: This study provides the first evidence that altered corneal epithelial and stromal expression of specific genes at the corneal cone apex drives focal structural weakness in KC.

Quantitative Evaluation of Microdistortions in Bowman's Layer and Corneal Deformation after Small Incision Lenticule Extraction.

PURPOSE: To quantitatively evaluate microdistortions in Bowman's layer and change in corneal stiffness after small incision lenticule extraction (SMILE). METHODS: This was a prospective, longitudinal, and interventional study. Thirty eyes of 30 patients were screened preoperatively and underwent SMILE for treatment of myopia with astigmatism. Visual acuity, refraction, optical coherence tomography (OCT; Bioptigen, Inc., Morrisville, NC) imaging of the layer and air-puff applanation (Corvis-ST, OCULUS Optikgeräte Gmbh, Germany) was performed before and after surgery (1 day, 1 week, and 1 month). The Bowman's Roughness Index (BRI) was defined as the enclosed area between the actual and an ideal smooth layer to quantify the microdistortions. A viscoelastic model was used to quantify the change in corneal stiffness using applanation. RESULTS: Uncorrected distance visual acuity improved (P < 0.001) and refractive error decreased (P < 0.0001) after SMILE. BRI increased from preoperative levels (1.81 × 10-3 mm2) to 1 week (3.14 × 10-3 mm2) after SMILE (P < 0.05) and then decreased up to a month (2.43 × 10-3 mm2; P < 0.05). Increase in the magnitude of the index correlated positively with refractive error (P = 0.02). However, corneal stiffness reduced after SMILE (105.86 ± 1.4 N/m versus 97.97 ± 1.21 N/m at 1 month, P = 0.001). The decrease in corneal stiffness did not correlate with refractive error (P = 0.61). CONCLUSIONS: BRI correlated positively the magnitude of refractive error. However, decrease in corneal stiffness, assessed by air-puff applanation, may not be related to microdistortions after SMILE. TRANSLATIONAL RELEVANCE: An objective method of quantification of Bowman's layer microdistortions using OCT was developed to monitor corneal wound healing and improve lenticule extraction methods.

Correlation between tear fluid and serum vitamin D levels.

BACKGROUND: Vitamin D deficiency is associated with a range of systemic diseases including ocular disorders. The objective of this study is to measure tear vitamin D levels and investigate the correlation between serum and tear vitamin D levels. METHODS: A total of 48 healthy volunteers without any systemic and ocular disease were recruited for this observational cohort study. Serum was collected using clot activator coated Vacutainer® Plus tubes. Tear fluid was collected using Schirmer's strips. Serum and tear total 25-hydroxyvitamin D levels were measured by competitive chemiluminescent ELISA and the correlation between the levels were studied. RESULTS: The measured serum 25-hydroxyvitamin D level ranged between 3.3 and 27.5 ng/ml (Mean ± SEM, 9.4 ± 0.7 ng/ml; Median 8.4 ng/ml). Significantly (p < 0.0001) higher level of 25-hydroxyvitamin D was detected in the tears (Mean ± SEM, 17.0 ± 1.6 ng/ml; Range 3.2-45.8 ng/ml; Median, 16.3 ng/ml) compared to serum. An average of ~2 fold (Mean ± SEM, 1.9 ± 0.2; Range 0.4-5.8; Median, 1.7) higher 25-hydroxyvitamin D was observed in tears compared to serum in the subjects. In addition, a positive correlation was observed between serum and tear 25-hydroxyvitamin D levels (r = 0.5595; p < 0.0001). CONCLUSIONS: A higher level of 25-hydroxyvitamin D was observed in the tear fluid compared to that of the serum. It would be beneficial to consider tear vitamin D levels to study its role with reference to ocular surface diseases.

Tear biomarkers for keratoconus.

Keratoconus is a progressive corneal thinning, ectatic condition, which affects vision. Recent advances in corneal topography measurements has helped advance proper diagnosis of this condition and increased research and clinical interests in the disease etiopathogenesis. Considerable progress has been achieved in understanding the progression of the disease and tear fluid has played a major role in the progress. This review discusses the importance of tear fluid as a source of biomarker for keratoconus and how advances in technology have helped map the complexity of tears and thereby molecular readouts of the disease. Expanding knowledge of the tear proteome, lipidome and metabolome opened up new avenues to study keratoconus and to identify probable prognostic or diagnostic biomarkers for the disease. A multidimensional approach of analyzing tear fluid of patients layering on proteomics, lipidomics and metabolomics is necessary in effectively decoding keratoconus and thereby identifying targets for its treatment.

Correlation of Clinical and Biomechanical Outcomes of Accelerated Crosslinking (9 mW/cm2 in 10 minutes) in Keratoconus with Molecular Expression of Ectasia-Related Genes.

PURPOSE: To assess visual, keratometry, densitometry, and corneal deformation outcomes after accelerated crosslinking (CXL) and its association with gene expression of extracellular matrix proteins. METHODS: 33 eyes underwent accelerated CXL (9 mW/cm2 for 10 minutes) after epithelium removal. Refraction, visual acuity, keratometry, corneal densitometry, and deformation (Corvis-ST) were assessed before and 6 months after surgery. Epithelium-collected intraoperative was analyzed with qPCR to determine whether the molecular state of disease [lysyl oxidase (LOX), matrix metalloproteinase 9 (MMP 9), transforming growth factor beta (TGFß), tumor necrosis factor-α (TNFα), interleukin 10 (IL10), interleukin (IL6), collagens (COL IA1 and COL IVA1)] had any bearing on the outcome. RESULTS: Corrected distance visual acuity (CDVA) remained unchanged (p > 0.05). Cylinder (p = 0.0003) and spherical equivalent error (p = 0.02) reduced significantly after CXL. Keratometry and cone location magnitude index (CLMI) were unchanged after CXL (p > 0.05). Corneal densitometry was significantly altered only in the central 0-2 mm region (p = 0.009). A new measure of corneal deformation, named corneal stiffness, was also stable after CXL (p > 0.05). The preoperative level of different proteins did not influence the clinical outcomes described above (p > 0.05). CONCLUSION: Accelerated CXL appears to be safe and provides biomechanical stability. Keratometry and refraction remained stable after CXL, with significant improvement in cylindrical error. Molecular expression profile of the keratoconic epithelium did not influence the clinical outcomes.

Corneal Densitometry: Repeatability in Eyes With Keratoconus and Postcollagen Cross-Linking.

PURPOSE: To assess the repeatability of densitometry, a measure of corneal haze, in the control (group I), keratoconic (group II), and postcollagen cross-linking (CXL, group III) eyes as measured on Scheimpflug imaging. METHODS: Densitometry values for 160 eyes of 160 patients (50 eyes of 50 patients in group I, 50 eyes of 50 patients in group II, and 60 eyes of 60 patients in group III) were obtained for the 0- to 2-mm, 2- to 6-mm, and 6- to 10-mm zones of the anterior (up to 120 µm), posterior (posterior 60 µm), and central (between the anterior and posterior) cornea. The repeatability of these values was assessed by within-subject standard deviation, coefficient of repeatability, and coefficient of variation. RESULTS: Range of within-subject standard deviation and coefficient of variation in the control group (0.2%-0.5% and 2%-4%, respectively) was significantly better (less variable) than those in the keratoconus group (0.4%-0.6% and 3%-5%). The same parameters in the post-CXL group (0.8%-3.8% and 7%-15%) were significantly worse (more variable) than that in the other 2 groups. The repeatability measures of densitometry were significantly worse in the central 0- to 2-mm zone compared with the other 2 zones and for the anterior region compared with the central and posterior regions of cornea in all the 3 groups. CONCLUSIONS: Consequent to the low repeatability in post-CXL eyes, densitometry should be used with caution to gauge response to treatment and visual outcomes in treated keratoconus eyes.

An In Vivo Confocal Microscopic Study of Corneal Nerve Morphology in Unilateral Keratoconus.

Purpose. To study the corneal nerve morphology and its importance in unilateral keratoconus. Materials and Methods. In this prospective cross-sectional study, 33 eyes of 33 patients with keratoconus in one eye (Group 3) were compared with the other normal eye of the same patients (Group 2) and 30 eyes of healthy patients (Group 1). All patients underwent detailed ophthalmic examination followed by topography with Pentacam HR and in vivo confocal microscopy (IVCM). Five images obtained with IVCM were analyzed using an automated CCmetrics software version 1.0 for changes in subbasal plexus of nerves. Results. Intergroup comparison showed statistically significant reduction in corneal nerve fiber density (CNFD) and length (CNFL) in Group 3 as compared to Group 1 (p < 0.001 and p = 0.001, resp.) and Group 2 (p = 0.01 and p = 0.02, resp.). Though corneal nerve fiber length, diameter, area, width, corneal nerve branch density, and corneal total branch density were found to be higher in decentered cones, only the corneal nerve branch density (CNBD) was found to be statistically significant (p < 0.01) as compared to centered cones. Conclusion. Quantitative changes in the corneal nerve morphology can be used as an imaging marker for the early diagnosis of keratoconus before the onset of refractive or topography changes.

Safety and Efficacy of Epi-Bowman Keratectomy in Photorefractive Keratectomy and Corneal Collagen Cross-Linking: A Pilot Study.

PURPOSE: The purpose of this study is to compare the efficacy and safety of Epi-Bowman Keratectomy (EBK) using Epi-clear™ epikeratome with a mechanical scraper for corneal epithelium debridement during photorefractive keratectomy (PRK) and corneal collagen cross-linking (CXL). METHODOLOGY: Sixty eyes of 30 patients undergoing PRK and 44 eyes of 44 patients undergoing CXL were included in this study. In the PRK group, one eye of each patient underwent EBK and the other eye epithelial debridement with a mechanical scraper. In the CXL group, 22 eyes of 22 patients underwent EBK and the other 22 eyes with a mechanical scraping to remove the epithelium. Intra-operative spectral domain optical coherence tomography (SD OCT; Bioptigen Inc, Durham, NC) was performed to evaluate the integrity of Bowman's membrane following epithelial removal. The time taken for epithelial removal, post-operative pain score using the Wong-Baker's pain scale, time for epithelial healing, and the epithelial profile using the Optovue (Optovue Inc. Fremont, CA) during the healing was observed and analyzed between the two groups and subgroups. RESULTS: Intra-operative SD OCT showed a smooth and undamaged Bowman's membrane when EBK was performed. Post-operative pain was significantly less (p < 0.01 in the PRK group and p < 0.001 in the CXL group) with faster epithelial healing (p < 0.001 in the PRK group and p < 0.0001 in the CXL group) in the EBK subgroup as compared with the mechanical scraper subgroup. Epithelial profiling during the healing phase showed an edematous epithelium in the initial 2 weeks in the mechanical scraper subgroup while the EBK subgroup showed minimal epithelial edema lasting up to a week with regularized and smooth corneal epithelium healing. CONCLUSION: Epi-Bowman Keratectomy appears to be an effective and safe method of corneal epithelial debridement with negligible damage to Bowman's membrane and the surrounding epithelium leading to early healing thereby reducing the post-operative pain and complications.