Deep Corneal Layers Research Articles

Dual drug-loaded coaxial nanofibers for the treatment of corneal abrasion

Corneal abrasion is a scratch wound on the surface of the anterior segment of the eye, which can predispose a patient to corneal infection and scarring, particularly if the cut penetrates to the deep corneal layers. Here we investigate a novel approach to co-administer an anti-scarring agent and an antibiotic, both being incorporated into one dosage form so as to accelerate wound closure and to treat any associated infection. More specifically, we have used electrospun fibers as a means of incorporating the two drugs into distinct compartments via coaxial electrospinning. Samples were characterised using a range of imaging, spectroscopic and thermal methods, while an HPLC assay has been developed to allow measurement of the concentration of both drug components in both the initial fibers and on release. Fibers loaded with pirfenidone in the hydrophobic polymer, PLGA, as the outer layer and moxifloxacin in the hydrophilic polymer PVP as the inner layer were successfully prepared, with smooth and non-porous surfaces and a mean diameter of circa 630nm. TEM image demonstrated clear distinctive layers (a core and a shell), suggesting the successful preparation of the drug-loaded coaxial fibers, supported by HPLC entrapment studies, while fluorescence microscopy confirmed the presence of the moxifloxacin within the fibers. The fibers were capable of extending the release of both drugs, hence raising the possibility of a single daily dose of the drug-loaded coaxial fibers for the treatment of corneal abrasion.

Clinical diversity in macular corneal dystrophy: an optical coherence tomography study.

To characterise the corneal deposits of macular corneal dystrophy and correlate with high-resolution optical coherence tomography (OCT). A total of 23 eyes of 15 patients were evaluated for clinical features on slit lamp biomicroscopy, and high-resolution OCT was performed to correlate the clinical findings. The deposits were characterised based upon their location and level in the corneal layers. Mean age was 31.5 (Range 20-67) years. The stromal deposits were restricted to central 8mm in 9 eyes; in the rest of the 14 eyes, the deposits were seen in both central and peripheral cornea. In one patient, no such distinction could be made due to diffuse nature of the deposits throughout the cornea with sparing of 1-2mm of the cornea internal to the limbus. The central deposits were in the anterior stromal layers, while the peripheral deposits were in the deep stromal corneal layers and non-contiguous with the anterior stromal deposits. In one patient aged 67years, the peripheral deposits in deep corneal layers were more prominent than the central anterior stromal deposits and were associated with a significant thickening of Descemet membrane. MCD exhibits a clinically diverse presentation as revealed on the clinical and optical coherence tomography study. Immunophenotype and genotype-phenotype correlation may further help in understanding various clinical presentations of MCD.

Second-harmonic generation microscopy of photocurable polymer intrastromal implants in ex-vivo corneas.

A custom adaptive-optics (AO) multiphoton microscope was used to visualize the corneal stroma after the insertion of a photocurable polymer material. A lamellar pocket was created and a certain amount of polymer in liquid form was injected. This turned into a rigid film after UV irradiation. Intact eyes were used as control. Tomographic and regular second harmonic generation (SHG) microscopy images were recorded from both control and corneas with polymer implants. In control corneas, the SHG signal decreased uniformly with depth. However, treated corneas exhibited an abrupt loss of SHG signal at the implant location. The use of AO increased the SHG levels and improved the visualization of the stroma, not only at deeper corneal layers but also beneath the implant. Moreover, the absence of SHG signal from the implant allowed its geometrical characterization (thickness and location). This technique offers a potential tool for non-invasive analysis of morphological changes in the cornea after surgery or treatment, and might be useful in future clinical environments.

Electron microscopic evaluation of the surfaces of variable depth lenticules in SMILE cases

AbstractPurpose Evaluating lenticule surfaces using electron microscopy in deeper corneal layers and comparing them to the superficial ones. This aims at determining the possibility of preserving the stronger superficial layers in the cornea by removing deeper lenticules in SMILE cases while attaining the same visual results and with the same ease.Methods 40 eyes underwent SMILE refractive procedure using femtosecond laser by VisuMax ( Zeiss Meditech). 2 groups were used, one with lenticules at 100 µm and the other with lenticules at 160 µm. Lenticules were immediately processed (fixed, dehydrated, dried and mounted with gold coating). JSM‐5300 scanning electron microscope was used for observations.Results Deeper lenticules had more irregular surfaces and were more difficult to dissect. Comparing the central and peripheral thickness of lenticules to the intended thickness was significantly more accurate in the superficial group. The visual results of the two groups were comparable. Surface irregularities were resulting from the break of bridges between the femtosecond cavities.Conclusion Energy settings still needs further adjustment to be able to create deeper predictable lenticules in SMILE cases

Nanoparticles for delivery of therapeutical nucleic acids to corneal endothelium

AbstractCorneal endothelium is as crucial for maintenance of corneal function as difficult it is to be accessed with any treatment that doesn’t involve invasive surgical procedures. Hovewer it is possible to administer therapeutic agents by topical application, their effect on deeper corneal layers and further fate remains of our control. This study has been designed to employ nanoparticles in order to improve our chances in therapy of corneal dystrophies as well as to prevent endothelial cell loss after transplantation of the cornea. Variety of nanoparticles serves a potential solution to problem of targeting the corneal endothelium, when properly functionalized to assure precise targeting. Widely proven as efficient carrier for nucleic acids may enable any gene therapy or gene silencing to be conducted with minimized risk of uncontrolled transfection. When to concern both growing need for corneal transplantations and shortage of tissue, efficient therapy to protect graft from failure or even to protect patient’s own cornea becomes essential.

Keratocyte Density after Laser-Assisted Subepithelial Keratectomy with Mitomycin C

To study the effects of laser-assisted subepithelial keratectomy (LASEK) with mitomycin C (MMC) on the keratocyte population. Prospective, nonrandomized, interventional, comparative case series. Fifty-six eyes treated at Vissum Santa Hortensia, Madrid, Spain, were included in the study. We compared 28 eyes treated with LASEK with intraoperative 0.02% MMC versus 28 non-treated eyes. Keratocyte density was measured 3 months after the surgery in the anterior, mid, and posterior stroma and was compared with the corresponding layers in the control eyes. The anterior layer in the LASEK group was compared with 2 layers in the control group: the most anterior stromal layer and the 80 μm-deep layer, because that was the mean ablation depth performed in eyes that underwent LASEK. We found a statistically significantly lower keratocyte population in the most anterior stromal layer after LASEK with MMC compared with both the most anterior stromal layer and the 80 μm-deep layer in controls. On the contrary, the treated group showed a significantly higher keratocyte density in both the mid stroma and the deep stroma. The comparison between the average densities through the entire cornea showed a significantly higher keratocyte population in the LASEK with MMC group. LASEK with MMC seems to cause a decrease in the anterior stromal cells 3 months after the surgery compared with nonoperated corneas. There seems to be a compensating proliferation of keratocytes in the deeper corneal layers, suggesting that the ability of keratocytes to repopulate the cornea is maintained after the surgical procedure.

Femtosecond laser‐assisted penetrating re‐keratoplasty

Editor, Penetrating keratoplasty has usually been performed taking a trephine for the sagittal excision of the corneal donor button and for the preparation of the recipient corneal bed. The disadvantages of this sagittal cutting technique are a lack of rotational stability and a lack of sagittal stability. With the introduction of the femtosecond laser into clinical ophthalmology (Jonas & Vossmerbaeumer 2004; Hoffart et al. 2008; Mastropasqua et al. 2008; Por et al. 2008; Price & Price 2008), a new cutting technique has emerged that offers the possibility of creating non-circular and angled incisions. According to preliminary results, this new technique can markedly reduce postoperative keratoplasty and enhance the overall stability of the wound because of the increased and angled surface of the interface between the graft and the host tissue. Because the femtosecond laser is based on an optical principle with the laser beam penetrating the corneal tissue, it necessitates sufficient transparency of the corneal stroma to allow the laser beam to penetrate to the deep corneal layer and Descemet’s membrane. Previous studies have shown that an intrastromal corneal oedema caused by a corneal endothelial insufficiency has not been an obstacle for the femtosecond laser beam (Hoffart et al. 2008; Por et al. 2008; Price & Price 2008). However, so far it has been unclear whether dense intracorneal scars, particularly after previous corneal surgery, may prevent the use of the femtosecond laser for keratoplasty. Therefore, in this article I report on the feasibility of the technique for a patient who underwent femtosecond laser keratoplasty after a previous keratoplasty had failed. A 48-year-old patient had undergone conventional trephine-guided penetrating keratoplasty as treatment of keratoconus in 1991. The diameter of the graft trephined from the endothelial side was 8.0 mm; the diameter of the excised corneal button in the host cornea was 7.8 mm. The graft was centred onto the optical axis. With an initially clear corneal graft, best-corrected visual acuity was 20/20. Seventeen years after the keratoplasty, the graft started to develop intrastromal oedema, particularly in the late afternoon, because of a progressive age-related insufficiency of the corneal endothelium of the graft. Using a commercially available femtosecond laser (Advanced Medical Optics; Intralase, Santa Ana, California, USA), a penetrating rekeratoplasty was carried out. A top-hat cutting configuration (Price & Price 2008) was chosen. The diameters of the superficial and deep parts were 7.0 mm and 8.5 mm, respectively; a lamellar cut was made at a depth of 250 μm. The lamellar overlapped the superficial part and the deep part by 0.3 mm, so that its outer diameter was 8.8 mm and its inner diameter was 6.7 mm. As per the previous corneal graft, the regraft was centred onto the optical axis. Despite the dense corneal opacities in the zone of the interface between the previous graft and the host cornea, the femtosecond laser was able to cut the corneal tissue unremarkably. The central corneal button precut by the laser could be removed without further use of a knife or scissors. The graft was fixated with a double running suture with buried knots in the interface (Fig. 1). Three months after the intervention, visual acuity was 20/20 with a regular corneal astigmatism of 2.0 dioptres (Fig. 1). Clinical photograph showing an eye 3 months after a femtosecond laser-assisted penetrating homologous keratoplasty 17 years after a previous penetrating keratoplasty. The scar of the interface between the former graft and the recipient cornea (remnants of the former scar: arrows) did not impede the femtosecond laser cutting of the cornea. These results suggest that the femtosecond laser is able to perform incisions in the superficial and deep corneal layers including Descemet’s membrane through dense intracorneal opacities such as scars in the interface after a previous penetrating keratoplasty. A previous keratoplasty is not a contradiction against a femtosecond laser-assisted keratoplasty.

In vivo confocal microscopy of patients with amiodarone-induced keratopathy.

To describe the corneal findings in patients with amiodarone-induced keratopathy by means of in vivo confocal microscopy. Twenty-two eyes of 11 patients (eight men and three women) receiving amiodarone therapy and 20 eyes of 10 healthy sex-and age-matched control subjects were selected for confocal microscopic examination. The patients were examined by use of a scanning slit corneal confocal microscope (Confoscan 2.0). Five complete scans of the entire cornea were performed for each eye with a total examination time of less than 5 minutes. All patients receiving amiodarone showed the presence of high reflective, bright intracellular inclusions in the epithelial layers. These findings were more evident within the basal cell layers. In the eyes with advanced keratopathy (stages 2 and 3), bright microdots were detectable within the anterior and posterior stroma and on the endothelial cell layer. In the anterior stroma, the keratocyte density in the treated group was reduced compared with values of the control group (p < 0.001), and a markedly irregular aspect of the stromal nerve fibers was found. The main characteristic of this nerve irregularity was represented by the clew-shaped appearance of the nerve trunks. Detailed examination of corneal structure by confocal microscopy shows that amiodarone keratopathy in long-term treated patients presents some findings that are consistent with higher toxicity than was expected and that involve the deep corneal layers.

Glucose concentration profiles of normal and ultraviolet radiation-exposed rabbit corneas

The passage of glucose within the cornea has been thought to occur by passive diffusion processes. However, corneal glucose concentration profiles have not been established to support this notion. While microfluorometric methods of metabolite assay typically have been used as a means of assessing regional brain metabolism, this unique methodology of tissue isolation and metabolite determination has not previously been applied to the cornea. Since this technique permits metabolite quantification on microgram-sized tissue samples, a co-ordinated corneal glucose concentration profile can be obtained. Tissue preparation consisted of liquid nitrogen freezing, cryo-sectioning, and freeze-drying, with storage at −20°C. The sections were thawed under vacuum pump, subsectioned, weighed, and assayed for glucose concentration (by dry weight). This study established a glucose concentration profile of the epithelium, anterior stroma, midstroma, posterior stroma, and endothelium for the normal pigmented rabbit cornea. A glucose concentration profile for UV radiation-exposed rabbit corneas also was documented. The UV radiation glucose profile data indicate the presence of an active transport mechanism capable of delivering glucose into the corneal epithelium against a concentration gradient. The presence of a transport system that ‘pulls’ glucose through the deeper corneal layers thus would make epithelial integrity important for the maintenance of overall corneal viability.

Experimental studies with a high-density silicone oil for giant retinal tear.

Fluorsilicone oil of high density (1.3 g/cm3) injected into the vitreous cavity after vitrectomy filled the rabbit eyeball in an anterior-posterior direction and unfolded a giant retinal tear (produced by peripheral retinotomy). Fluorsilicone oil injected into the anterior chamber causes a localized opacification of the cornea. Histologically, a fibroplastic metaplasia of the deep corneal layers with a newly formed posterior collagenous layer was found. Histologic findings were identical in fellow eyes in which polydimethylsilicone was used as a control substance.

Zur Ultrastruktur der fleckf�rmigen Hornhautdystrophie (Groenouw II): Untersuchung der parazentralen tiefen Hornhautschichten

Electron microscopic studies proved, that in cases of macular corneal dystrophy (Groenouw II) abnormal deposits of acid mucopolysaccharide are demonstrable in the different layers of the cornea in a polymorphic form. Because of the investigations now performed it can be established, that the opacities of the paracentral, deep corneal layers result from both dispersed vesicular and plaque-like deposits of storage material in the border area of the stroma and Descemet's membrane.

Structural changes in chronic herpetic keratitis. Studied by light and electron microscopy.

Herpesvirus was found by electron microscopy in the corneal stroma of five of 19 specimens obtained from patients with chronic herpetic keratitis. Structural changes involved the deeper layers of these corneas as well as the more superficial levels. Virus proliferation in the deep corneal layers may account for some forms of stromal inflammation in chronic herpetic keratitis.

On certain changes in the degree of corneal tissue differentiation under experimental conditions

Circular burn of the corneal surface reduces the differentiation and induces proliferative growth in the central area in the form of a “polyp”. Transplants from the cornea with incomplete development, mature rapidly, after being placed between the superficial layers of the corneal stroma. Nerve branches growing into the transplant are visible in the preparations impregnated according to Campos. In the same transplants placed between the deep corneal layers, where the nervous apparatus is poor developed, the degree of differentiation is decreased and follows the type of the tissue culture in vivo. It is suggested that the degree of the tissue differentiation and its functional adequacy are maintained by the nervous apparatus, whereas denervation provokes tissue “autonomism”, i.e., reduction of differentiation and acceleration of tissue growth.