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Ultrastructure of the normal corneal stroma. Transmission electron microscopy of the unwounded rabbit cornea. (A) The arrowhead indicates a keratocyte. Original magnification ×12.8K. (B) The uniform diameter and regular packing of the collagen fibrils associated with transparency. Note some groups of fibrils are seen longitudinally, whereas other groups are seen in cross-section. However, all the fibrils have identical diameter. Lamina densa (black arrowheads) and lamina lucida (immediately anterior to lamina densa) of the epithelial basement membrane (EBM) are well-delineated. In the most anterior stroma, besides fibrils cut in cross-section, many densities are noted (arrows). These likely represent structures that include anchoring fibrils that are part of the adhesion complex of the epithelium, which also includes hemidesmosomes (white arrowheads). Many of these densities in the anterior stroma likely also include exocytic vesicles produced by keratocytes to provide components such as nidogen-1, laminins, and perlecan to maintain the EBM. 21,22 Original magnification ×42K. (C) Higher magnification TEM shows the uniformity of the collagen fibrils, with some stromal lamellae cut tangentially, others cut obliquely, and some shown longitudinally. Arrows indicate lamina lucida of the EBM, and lamina densa is just posterior to lamina lucida in the EBM. Original magnification ×88K. Rabbits do not have Bowman's layer.
Source publication
Purpose:
To highlight the cellular, matrix, and hydration changes associated with opacity that occurs in the corneal stroma after injury.
Methods:
Review of the literature.
Results:
The regulated transition of keratocytes to corneal fibroblasts and myofibroblasts, and of bone marrow-derived fibrocytes to myofibroblasts, is in large part modula...
Context in source publication
Context 1
... exceptionally detailed review of the normal corneal stroma components and composition was published recently, 9 and only a few highlights relevant to transient haze and fibrosis are provided in this review. The stromal fibrils of the cornea (Fig. 3) are heterotypic (generated from ≥2 or more fibril-forming collagen types) fibrils 9 composed of fibril-forming collagen type I (80%-90%) (Fig. 4) and lesser amounts of regulatory fibril-forming collagen type V (10%-20%). Collagen V has been shown to regulate the nucleation of protofibril assembly, and thereby control the number of ...
Citations
... At the stage of corneal wound healing, myofibroblasts release a large amount of extracellular matrix (including collagen and fibrin) causing scarring, turbidity, and fibrosis [2]. A variety of detection methods have been used to study the physiological structure and pathological process of the cornea, such as observing the changes in cell morphology using a confocal microscope [5], detecting the components of proteins and cytokines through immunohistochemistry [6], and studying the ultrastructure and biomechanics of the cornea at the nanometer level [7]. The Raman spectrum is based on the Raman scattering effect. ...
Purpose
This study aimed to measure the Raman spectrum of the human corneal stroma lens obtained from small incision lenticule extraction surgery (SMILE) in Asian myopic eyes using a confocal Raman micro-spectrometer built in the laboratory.
Methods
Forty-three myopic patients who underwent SMILE with equivalent diopters between − 4.00 and − 6.00 D were selected, and the right eye data were collected. Corneal stroma lenses were obtained during surgery, and the Raman spectra were measured after air drying. The complete Raman spectrum of human myopic corneal stroma lens tissue was obtained within the range of 700–4000 cm⁻¹.
Results
Thirteen characteristic peaks were found, with the stronger peaks appearing at 937 cm⁻¹, corresponding to proline, valine, and the protein skeleton of the human myopic corneal stroma lens; 1243 cm⁻¹, corresponding to collagen protein; 1448 cm⁻¹, corresponding to the collagen protein and phospholipids; and 2940 cm⁻¹, corresponding to the amino acid and lipids, which was the strongest Raman peak.
Conclusion
These results demonstrated that Raman spectroscopy has much potential as a fast, cost-effective, and reliable diagnostic tool in the diagnosis and treatment of eye diseases, including myopia, keratoconus, and corneal infection.
... Interestingly, a strong positive correlation between CD values 5y postoperatively and 6.8 mm optic zone was observed in our study. Water, stromal matrix, stromal cells, and corneal nerves all take parts in maintenance of corneal transparency [19] . ...
AIM: To investigate the long-term changes of corneal densitometry (CD) and its contributing elements after small incision lenticule extraction (SMILE). METHODS: Totally 31 eyes of 31 patients with mean spherical equivalent of -6.46±1.50 D and mean age 28.23±7.38y were enrolled. Full-scale examinations were conducted on all patients preoperatively and during follow-up. Visual acuity, manifest refraction, axial length, corneal thickness, corneal higher-order aberrations, and CD were evaluated. RESULTS: All surgeries were completed successfully without complications or adverse events. Ten-year safety index was 1.17±0.20 and efficacy 1.04±0.28. CD value of 0–6 mm zones in central layer was statistically significantly lower 10y postoperatively, compared with preoperative values (0–2 mmΔ=-1.62, 2–6 mmΔ=-1.24, P<0.01). There were no correlations between CD values and factors evaluated. CONCLUSION: SMILE is a safe and efficient procedure for myopia on a long-term basis. CD values get lower 10y postoperatively, whose mechanism is to be further discussed.
... This patient developed a post-PRK stromal haze leading to an inferior elevation which resembles the topographic appearance of keratoconus. This is caused whether due to thickening and elevation at the site of haze formation or by haze-related contractive forces exerted on the cornea [16]. ...
Objective
To raise awareness of conditions that can tomographically mimic corneal ectasia and describe the actions required to avoid misdiagnosis.
Methods
We report a retrospective case series of seven patients presenting at two tertiary care centers in Israel with a presumed diagnosis of keratoconus or post-refractive ectasia. Upon further examination, the ectasia diagnosis was reconsidered and eventually ruled out.
Results
Included were ten eyes of seven patients. Cases included bilateral diffuse Salzmann’s nodular degeneration, ophthalmoplegia with strabismus which precluded proper fixation during the acquisition of tomography images, two cases of incorrect Pentacam parameter settings, a patient with a history of hyperopic laser-assisted in situ keratomileusis (LASIK) treatment in one eye and myopic LASIK in the fellow eye, a case of old post-photorefractive keratectomy (PRK) stromal haze, and a patient with posterior polymorphous corneal dystrophy.
Conclusions
Tomography patterns mimicking corneal ectasia can appear in patients without ectatic pathology. The comprehensive ophthalmologist should be aware of such cases as they may substantially alter the treatment course and prognosis of these patients.
... Corneal opacities can result from trauma, chemical injuries, infections, complicated surgeries, or genetic corneal disorders, such as corneal dystrophies. 1 Keratoplasty and Keratoprothesis are expensive and lengthy surgical procedures. Postoperative visual outcomes will be affected by the presence of posterior segment abnormalities. 2 Ultrasonography of the eye is useful in detecting the structural posterior segment abnormalities, such as retinal detachment or vitreous hemorrhage, but it shows no information about the functional abnormalities like macular scars or optic neuropathy. ...
Purpose
This study compared flash visual evoked potentials (VEPs) parameter differences between eyes with unilateral opaque media and their normal contralateral counterparts.
Patients and Methods
We included 178 patients with unilateral media opacity and normal contralateral eye in each patient. The diseased eyes had normal ultrasonographic findings and were candidates for keratoplasty or anterior segment reconstruction. The patients underwent electrophysiological testing by the flash VEPs recording with the use of 1.4Hz (transient) and 8Hz (flicker) frequencies.
Results
The medians of P2 implicit times were significantly higher in eyes with media opacity than in normal eyes, with median differences between both eyes of −6.7 ms. Furthermore, the median N2P2 amplitude was 9.70 µv with media opacity compared with 11.00 µv in the normal eyes, with a significant difference of 1.3 (p = 0.002). The flash VEPs median parameters recorded at a frequency of 8-Hz showed significant differences in medians of spectral plots of 0.6 µv between normal and abnormal eyes. The same for the signal-to-noise ratio, where the difference in medians was 3 dB. Analysis of the difference between both eyes (normal–abnormal) in each patient showed in P2 implicit time a difference range of −49 to 54.7 ms with a median difference of −3 ms. The difference in amplitudes of recorded N2P2 showed a median of 2.13 µv. There was a statistically significant but weak negative correlation between N2P2 amplitude (r spearman = −0.173, p = 0.021) and the grades of corneal opacity.
Conclusion
Preoperative flash VEPs recording is useful for evaluating visual function in patients with unilateral media opacity to determine the future benefits of keratoplasty or anterior segment reconstruction. There are no correlations between the flash VEP parameters or the difference between the 2 eyes and the grades of corneal opacity.
... Although scar formation is a natural response that maintains the structural integrity of injured corneas, corneal scar tissue may cause severe problems depending on its extent and location, including irregular astigmatism, opacification, and vision impairment [10]. These problems are because scar tissue lacks the crucial organization of the native cornea tissue's fibrillar architecture, leading to corneal opacity [11]. ...
... Consequently, scientists have developed and tested novel experimental methods to reduce corneal scarring [13]. For instance, gene therapy techniques with viral-vector-based gene delivery were proposed to target profibrotic TGFβ1-dependent pathways [11,13]. Moreover, researchers have considered repurposing some drugs approved for other disorders and applying them to limit corneal scarring. ...
... While the orthogonal arrangement of layers formed by parallel collagen fibrils interlacing with PGs facilitates corneal transparency, the disturbed fibrillar architecture of corneal scars alters it [11]. Consequently, we studied crucial fibril morphology and organization parameters in the scar neotissue and adjoining regions to determine the effects of ACA-mediated attenuation of neofibrillogenesis during corneal wound healing. ...
Highly organized collagen fibrils interlacing with proteoglycans form the crucial architecture of the cornea and facilitate its transparency. Corneal scarring from accidental injury, surgery, or infection alters this highly organized tissue, causing severe consequences, including blindness. There are no pharmacological or surgical methods to effectively and safely treat excessive corneal scarring. Thus, we tested the anticorneal scarring utility of a rationally designed anticollagen antibody (ACA) whose antifibrotic effects have already been demonstrated in nonocular models. Utilizing a rabbit model with an incisional corneal wound, we analyzed ACA’s effects on forming collagen and proteoglycan-rich extracellular matrices in scar neotissue. We used microscopic and spectroscopic techniques to quantify these components and measure crucial parameters characterizing the structure and organization of collagen fibrils. Moreover, we analyzed the spatial distribution of collagen and proteoglycans in normal and healing corneas. Our study demonstrated significant changes in the quality and quantity of the analyzed molecules synthesized in scar neotissue. It showed that these changes extend beyond incision margins. It also showed ACA’s positive impact on some crucial parameters defining proper cornea structure. This pilot study provides a stepping stone for future tests of therapeutic approaches that target corneal extracellular scar matrix assembly.
... Moreover, it is widely thought that regenerated EBM controls TGF-β1 infiltration into the stroma [13,15,24]. TGF-β1 is a well known key fibrotic modulator in mediating the development and maintenance of myofibroblasts [25,26]. Opaque myofibroblasts secrete large amounts of extracellular matrix, resulting in corneal scarring [27,28]. ...
Purpose:
To evaluate the relationship between basement membrane (BM) regeneration and the spatiotemporal expression of TGF-β1 during wound healing in rabbits with corneal perforating injury.
Methods:
Forty-two rabbits were randomly allocated into 7 experimental groups, with 6 rabbits per group at each time point. The central cornea of the left eye was injured with 2.0 mm trephine to establish the perforating injury model. Six rabbits that received no treatment were used as controls. The cornea was evaluated at 3 days, 1-3 weeks, and 1-3 months after injury with a slit lamp for haze levels. Real-time quantitative polymerase chain reaction (qRT-PCR) was performed to quantify the relative expression of TGF-β1 and α-SMA mRNA. Immunofluorescence (IF) was used to assess TGF-β1 and alpha-smooth actin (α-SMA) expression and localization. BM regeneration was assessed using transmission electron microscopy (TEM).
Results:
After injury, dense haze appeared at 1 month and then gradually faded. The relative expression of TGF-β1 mRNA peaked at 1 week and then decreased until 2 months. The relative α-SMA mRNA expression reached its peak at 1 week, then reached a small peak again at 1 month. IF results showed that TGF-β1 was initially detected in the fibrin clot at 3 days and then in the entire repairing stroma at 1 week. TGF-β1 localization gradually diminished from the anterior region to the posterior region at 2 weeks to 1 month, and it was nearly absent at 2 months. The myofibroblast marker α-SMA was observed in the entire healing stroma at 2 weeks. Localization of α-SMA gradually disappeared from the anterior region at 3 weeks to 1 month, remaining only in the posterior region at 2 months and disappearing at 3 months. Defective epithelial basement membrane (EBM) was first detected at 3 weeks after injury, then gradually repaired, and was nearly regenerated at 3 months. A thin and uneven Descemet's membrane (DM) was initially detected at 2 months after injury, then gradually regenerated to some extent, but remained abnormal at 3 months.
Conclusions:
In the rabbit corneal perforating injury model, EBM regeneration was observed earlier than DM. At 3 months, complete EBM regeneration was observed, while the regenerated DM was still defective. TGF-β1 was distributed throughout the entire wound area in the early stages and then decreased from the anterior to the posterior region. α-SMA exhibited a similar temporospatial expression to TGF-β1. EBM regeneration may play a key role in low expression of TGF-β1 and α-SMA in the anterior stroma. Meanwhile, incomplete DM regeneration may contribute to the sustained expression of TGF-β1 and α-SMA in the posterior stroma.
... The normal unwounded corneal stroma is populated by relatively quiescent keratocytes that secrete ECM proteins and enzymes to maintain ECM homeostasis within the stroma [23]. After an injury (e.g., trauma, infection, or surgery), keratocytes transition to an active repair phenotype (corneal fibroblasts), which may further trans-differentiate into myofibroblasts if exposed to adequate and ongoing levels of TGF beta-1 and/or TGF beta-2 [24][25][26]. In addition, bone marrow-derived fibrocytes that infiltrate into the injured tissue also undergo TGF beta-driven differentiation into myofibroblasts in the corneal stroma [16,27]. ...
... Further evidence was provided in a rabbit corneal wound healing model in situ. In rabbits treated with −3D PRK, the anterior stroma was predominantly occupied by corneal fibroblast cells, although fibrocytes were also likely present in the subepithelial stroma [16,25]. The strong association between the fibroblasts and BM regeneration further supports the important role of corneal fibroblasts in epithelial BM reassembly after injury. ...
Purpose:
To understand which cell types, either alone or in combination, contribute to the assembly of the epithelial basement membrane (BM) during corneal wound healing.
Methods:
A 3D corneal organotypic model and an in situ rabbit photorefractive keratectomy (PRK) model were used in this study. The 3D corneal organotypic model was established by culturing the rabbit corneal epithelial cells with either corneal fibroblasts or myofibroblasts embedded in collagen type I for 18 days. Corneal fibroblasts were isolated from fresh rabbit corneas, and the myofibroblasts were derived either directly from bone marrow or differentiated from corneal fibroblasts. Immunocytochemistry for alpha-smooth muscle actin (SMA), vimentin, desmin, and vinculin markers confirmed well-differentiated myofibroblasts. Immunohistochemistry was performed in cryofixed sections for BM markers, including laminin alpha-5, laminin beta-3, perlecan, nidogen-1, and collagen type IV. Specimens were also examined with transmission electron microscopy (TEM). Corneas were collected from rabbits after -3 diopter (D) PRK at different time points after surgery, with four corneas at each time point in each group. Cryofixed corneal sections were stained for vimentin, alpha-SMA, and nidogen-1.
Results:
The formation of an epithelial BM with expression of laminin alpha-5, laminin beta-3, perlecan, nidogen-1, and collagen IV was observed at the interface between the corneal epithelial cells and corneal fibroblasts. TEM images further confirmed the presence of epithelial BM in organotypic cultures of epithelial cells and corneal fibroblasts. No epithelial BM was observed in cultures of corneal epithelial cells and myofibroblasts (cornea or bone marrow derived), corneal epithelial cells alone, or corneal fibroblasts alone. In rabbit corneas after -3D PRK, a strong association was observed between the regenerating epithelial BM and the presence of corneal fibroblasts at the site of epithelial BM generation.
Conclusions:
The corneal epithelial BM assembly is mediated by epithelial cells in coordination with corneal fibroblasts during wound healing.
... This structure cancelled the scattered light, while the transmitted light was not disturbed, making the tissue transparent. The reason for corneal opacity was that the arrangement of the collagen fibers changed (Wilson et al., 2022), and the secondary reflected waves interfered with each other. This hypothesis has some defects, but it can still explain many phenomena. ...
Background: To investigate the correlation between corneal biomechanical characteristics (in vitro and in vivo) and corneal densitometry in myopia.
Methods: The Pentacam (Oculus, Wetzlar, Germany) corneal densitometry (CD) and Corvis ST (Oculus, Wetzlar, Germany) exams were conducted prior to surgery for myopic patients who were intended to undergo small-incision lenticule extraction (SMILE). CD values (grayscale units, GSUs), and in vivo biomechanical parameters were obtained. The stromal lenticule was subjected to a uniaxial tensile test to obtain the elastic modulus E in vitro. We exam the correlations among in vivo, in vitro biomechanical characteristics and CD values.
Results: In this study, 37 myopic patients (63 eyes) were included. The mean age of participants was 25.14 ± 6.74 years (range:16–39 years). The mean CD values of the total cornea, anterior layer, intermediate layer, posterior layer, 0–2 mm region and 2–6 mm region were 15.03 ± 1.23 GSU, 20.35 ± 1.98 GSU, 11.76 ± 1.01 GSU, 10.95 ± 0.83 GSU, 15.57 ± 1.12 GSU and 11.94 ± 1.77 GSU, respectively. Elastic modulus E (in vitro biomechanical indicator) was negatively correlated with intermediate layer CD (r = −0.35, p = 0.01) and 2–6 mm region CD (r = −0.39, p = 0.00). A negative correlation was also found between 0-2 mm central region CD and in vivo biomechanical indicator SP-HC (r = −0.29, p = 0.02).
Conclusion: In myopic patients, densitometry is negatively correlated with biomechanical properties both in vivo and in vitro. With an increase in CD, the cornea deformed more easily.
... When this arrangement is disturbed in corneal scarring there is deposition of abnormal collagen in irregular manner, conversion of keratocytes to myofibroblasts leading to opacity and contraction of the tissue. [25][26][27][28] In addition to the density and depth of the opacity, this can also affect the regularity of the refracting surface leading to lower and HOAs which collectively affect the quality of the vision. 16,17,21 The lower order aberrations can be corrected with glasses, beyond which the irregular surface induces various HOAs which were previously grouped under irregular astigmatism and are usually significant from the third to sixth order. ...
Purpose
To explore the relationship between the density, depth, and surface irregularity of superficial corneal scarring and vision.
Methods
This prospective imaging study included 19 patients with unilateral corneal scarring post-microbial keratitis. Each eye underwent an assessment of uncorrected visual acuity (UCVA), best spectacle-corrected visual acuity (BSCVA), contact lens corrected visual acuity (CLCVA), and Scheimpflug and anterior segment optical tomography imaging. Regression analysis was performed to detect the association between density, depth of scarring and the surface irregularity in terms of higher order aberrations (HOA), and keratometry and UCVA, CLCVA and the difference between BSCVA and CLCVA.
Results
The mean logMAR UCVA, BSCVA and CLCVA were 0.76, 0.35 and 0.28 respectively. The corneal scars had a mean thickness of 391.8 ± 64.7158.7 ± 61µ and density of 65.73 ± 24.46 GSU. Bivariate analysis model for UCVA showed an association with Z4 2 secondary astigmatism (p = 0.02), Z4 4 Quadrafoil (p = 0.01), combined Coma Z3 ± 1(p = 0.03), and combined HOA Z3-Z6(p = 0.045), out of which Z4Z44 Quadrafoil (p = 0.04) was most significant with multivariate analysis. Bivariate analysis for BCVA-CLVA showed association with Z3Z31 coma horizontal (p = 0.04), Z3Z33 oblique trefoil (p = 0.02), Z4Z40 primary spherical aberration(p = 0.008) aand Z5 (-5) Z5-5 (p = 0.007) out of which Z3Z31 horizontal coma (p = 0.04) and Z4Z40 spherical aberration (p = 0.009) were significant on multivariate analysis. Change in densitometry, corneal thickness, epithelial:stromal reflectivity ratio, scar depth and keratometry did not show any significant association with UCVA, BSCVA-CLCVA or CLCVA.
Conclusion
In superficial corneal stromal scarring, deranged surface irregularity parameters like higher-order aberrations affect the final visual acuity more than the depth or density of the opacity.
... Corneal fibroblasts express little, if any, keratocan, either in situ or in vitro [1][2][3]. Corneal fibroblasts ( Figure 3) develop from resident keratocytes that are stimulated by transforming growth factor (TGF) beta-1 and/or TGF beta-2, platelet-derived growth factor (PDGF), and possibly other yet to be identified cytokines or growth factors [10][11][12][13]. This conversion also involves integrin signaling [10]. ...
... Therefore, the large amounts of collagen type IV produced by the corneal fibroblasts, much of which is not associated with the corneal basement membranes (BM) [9,15,16], down-regulates the effects of TGF beta-1 and TGF beta-2 that enter the stroma from the tears, epithelium, corneal endothelium and/or aqueous humor, and are activated after injury. A major impact of that non-BM collagen type IV, therefore, is to moderate the pro-fibrotic effects of TGF beta by regulating (decreasing) myofibroblast development from the corneal fibroblasts themselves and bone marrow-derived fibrocytes [19,20], the two bestcharacterized precursors to myofibroblasts in the corneal stroma [1,4,8,9,[12][13][14]19,20]. This non-BM collagen type IV production by corneal fibroblasts may be a major determinant of why many relatively low-level corneal injuries, such as abrasions or lower correction photorefractive keratectomy (PRK) surgeries, generate corneal fibroblasts but not myofibroblasts in the corneal stroma [8,21]. ...
... Biomolecules 2022, 12, x FOR PEER REVIEW 5 of 14 by transforming growth factor (TGF) beta-1 and/or TGF beta-2, platelet-derived growth factor (PDGF), and possibly other yet to be identified cytokines or growth factors [10][11][12][13]. This conversion also involves integrin signaling [10]. ...
Mesenchymal cells (keratocytes, corneal fibroblasts, and myofibroblasts), as well as mesenchymal progenitor bone marrow-derived fibrocytes, are the major cellular contributors to stromal fibrosis after injury to the cornea. Corneal fibroblasts, in addition to being major progenitors to myofibroblasts, also have anti-fibrotic functions in (1) the production of non-basement membrane collagen type IV that binds activated transforming growth factor (TGF) beta-1 and TGF beta-2 to downregulate TGF beta effects on cells in the injured stroma, (2) the production of chemokines that modulate the entry of bone marrow-derived cells into the stroma, (3) the production of hepatocyte growth factor and keratinocyte growth factor to regulate corneal epithelial healing, (4) the cooperation with the epithelium or corneal endothelium in the regeneration of the epithelial basement membrane and Descemet’s membrane, and other functions. Fibrocytes also serve as major progenitors to myofibroblasts in the corneal stroma. Thus, mesenchymal cells and mesenchymal cell progenitors serve Yin and Yang functions to inhibit and promote tissue fibrosis depending on the overall regulatory milieu within the injured stroma.
