Browsing by Subject "Cornea"
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- PublicationOpen AccessAlteration in epithelium and stroma of post-INTACS cornea: Ultrastructure and 3D transmission electron tomography(2026) Omar Kirat2; Adrian Smedowski; Aljohara Alkanaan; Fahad Almoqbel; Turki Almubrad; Saeed Akhtar; Biología Celular e Histología; Universidad de Murcia, Departamento de Biologia Celular e HistiologiaThis study was conducted to investigate the ultrastructure of the epithelium and stroma of post INTACS corneas. INTACS were surgically inserted into the corneas of three patients to remodel the keratoconus shape. INTACS were inserted with the IntraLase femtosecond laser. Patients 1, 2, and 3 returned to the clinic 8, 6, and 9 years, respectively, after surgery due to their deteriorating vision. The lamellae above the INTACS were removed surgically and processed for light and electron microscopy. 2D and 3D digital images of lamellae, collagen fibrils (CFs), and proteoglycans (PGs) were captured by a bottom-mounted camera and analysed using iTEM software. The epithelium and stromal lamellae had degenerated. A large number of aggregates of microfilaments were present in the Bowman’s layer (BW) and throughout the stroma. In the stroma, just above the INTACS insertion, lamellae were completely disorganised and running randomly in the large electron-lucent spaces. The CF diameter was significantly smaller than in the normal cornea. 3D images showed microfibrils within the CF were less in the post-INTACS cornea than in the normal cornea. We believe that insertion of the INTACS disturbed the lamellar organisation and uniform distribution of CFs. This non-uniform CF distribution increased over time, resulting in vision impairment.
- ItemOpen AccessBioartificial human corneas generated by tissue engineering. A historical and technical review(2026) Pascual-Vicente Crespo; José-Manuel García; Maria-Carmen Sánchez-Quevedo; Antonio Campos; Miguel Alaminos; Biología Celular e HistologíaDifferent types of bioartificial corneas have been generated by tissue engineering through combining cells, biomaterials, and bioactive molecules. Orthotypical corneal cells can be obtained from corneal biopsies, and include epithelial, stromal, and endothelial cells, whereas heterotypical cells are obtained from alternative cell sources with corneal differentiation potential, such as mesenchymal stem cells. In turn, two main types of biomaterials have been applied to corneal tissue engineering: those generated by the de-cellularization of natural tissues and biomaterials generated de novo using synthetic or natural biomaterials, especially collagen, fibrin, and agarose. Cells and biomaterials are combined with bioactive factors, inducing cell proliferation and differentiation. A review of previous studies revealed that most bioartificial corneas were not able to fulfill the complex requirements required for clinical translation, which include a thorough preclinical characterization, generation of the tissue as an advanced therapy medicinal product, a clinical research phase, and a final authorization by the European Medicines Agency or another competent regulatory agency. Most authorized products correspond to partial corneal substitutes consisting of one cell type associated or not with a scaffold, and only one product consisting of a human bioartificial cornea containing a fibrin-agarose scaffold and two corneal cell lineages (epithelial and stromal cells) called NANOULCOR was evaluated in patients in the context of an advanced therapy medicinal product. These findings confirm the existence of a bottleneck between basic and clinical research and suggest the need to implement novel clinical studies to develop new therapies that can improve the results of current corneal therapies.
- PublicationOpen AccessEffect of regenerative factor rich plasma, P substance and fetal calf serum on the growth of epithelial cells in the cornea. Comparative experimental study(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Márquez de Aracena del Cid, R.; Pérez Ordoñez, A.Purpose. The goal of this study was to evaluate the experimental effectiveness of Regenerative Factor Rich Plasma (RFRP) of human blood versus Fetal Bovim Serum (FBS) and neuropeptide Substance P (SP) on corneal epithelium cell proliferation. Method. Rabbit corneal epithelium cell (CCL-60) growth was compared between different RFRP fractions, FBS and with the neuropeptide Substance P. The ability of the RFRP fractions and SP to revert the inhibitory effect of the CsA was also evaluated. Results. All groups showed an increase (p<0.001) in corneal epithelial cell growth compared with the control group. The maximum capacity of cell growth was obtained with dilutions of 50% in the FBS, RFRP-I, RFRP -II, RFRP-III groups and with 100nM of SP. The highest growth was observed with 50% FBS, RFRP-I and RFRP-II. The group with SP and RFRP-III had significantly lower growth (p<0.001). When the NK1 receptor antagonist CsA was added at a dose of IC50, we found a significant decrease in cell growth (p<0.001) in all culture conditions, including the control group. The decrease was similar in all groups, but was especially pronounced in RFRP-II. RFRP I, II and III promoted growth more than SFB 10%. Conclusion. The RFRP of human blood promotes the growth of corneal epithelial cells in a significantly more efficient manner than FSB and SP. RFRP can be effective both in cell cultures and stem cell cultures.
- PublicationOpen AccessEnzyme histochemistry of corneal wound healing(Murcia : F. Hernández, 1998) Cejkova, J.The usefulness of enzyme histochemical methods for the localization of enzymes as catalysts of molecular interactions in the cells and tissues of healing corneal wounds is shown in rabbits. The current data on corneal wound healing in humans as well as in rabbits with particular reference to serine proteases are reviewed. Some inflammatory mediators are also discussed. Plasmin is a serine protease which is absent (or present only in very low concentration) in the tear fluid, and its activity appears under various pathological conditions in humans or following experimental injuries in rabbits. The role of increasing plasmin activity in the tear fluid in the depending upon the severity of corneal injury is evaluated. Great attention is devoted to conditions leading to long-lasting elevated levels of plasmin activity in the tear fluid correlated with corneal ulceration. The differences between the histochemical pattern of untreated corneas or corneas treated with some serine protease inhibitors are shown, and the efficacy of these drugs is discussed in light of present knowledge.
- PublicationOpen AccessImpairment of thrombospondin-1 expression during epithelial wound healing in corneas of vitamin A-deficient mice(Murcia : F. Hernández, 2005) Uno, K.; Kuroki, M.; Hayashi, H.; Uchida, H.; Oshima, K.The purpose of this study is to investigate the expression of thrombospondin-1 (TSP-1), a multifunctional extracellular matrix protein, during reepithelialization in wounded corneas of vitamin Adeficient mice. Epithelial defects were created in the corneas of normal and Vitamin A-deficient mice with a microgrinder. Wounded corneas were stained with fluorescein and photographed for evaluation of reepithelialization. Histological examination and immunohistochemical analysis of TSP-1 expression were also performed on the specimens from wounded corneas. In vitamin A-deficient mice, re-epithelialization of the wounded corneal epithelium was significantly delayed compared with that in normal mice. TSP-1 was detectable neither in the unwounded corneal epithelium of normal mice nor in that of vitamin A-deficient mice. In normal mice, linear staining of TSP-1 was observed on the wounded corneal surface and stroma at 30 min and 8 h to 16 h, respectively, after abrasion, and this TSP-1 expression disappeared at 36 to 48 h, when reepithelialization was completed. In contrast, no TSP-1 staining was observed in the wounded corneas of vitamin A-deficient mice, except for the endothelial cells, throughout the wound healing process. Histological examination revealed a progressive increase in polymorphonuclear neutrophil infiltration in the stroma of the corneas of vitamin A-deficient mice during the healing process. These findings suggest that vitamin A may modulate the expression of TSP-1 in the corneas to accelerate the re-epithelialization of wounded corneas.
- PublicationOpen AccessIrradiation of the rabbit cornea with UVB rays stimulates the expression of nitric oxide synthases-generated nitric oxide and the formation of cytotoxic nitrogen-related oxidants(Murcia : F. Hernández, 2005) Cejkova, J.; Ardan, T.; Cejka, C.; Kovaceva, J.; Zídek, Z.Until now, the role of nitric oxide (NO) in cornea irradiated with UVB rays remains unknown. Therefore, we investigated nitric oxide synthase isomers (NOS), enzymes that generate NO, nitrotyrosine (NT), a cytotoxic byproduct of NO, and malondialdehyde (MDA), a byproduct of lipid peroxidation, in rabbit corneas repeatedly irradiated with UVB rays (312 nm, 1x daily for 6 days, the dose per day 1.01 J/cm2) using immunohistochemical methods. The biochemical measurement of nitrite and nitrate has been used for the indirect investigation of NO concentration in the aqueous humor. Results show that in contrast to normal corneas, where of the NOS isomers only endothelial nitric oxide synthase (NOS3) was expressed in a significant amount (in the epithelium and endothelium), in irradiated corneas all NOS isomers (also brain nitric oxide synthase, NOS1, and inducible nitric oxide synthase, NOS2) as well as an indirect measure of ONOO-formation and MDA were gradually expressed, first in the epithelium, the endothelium and the keratocytes beneath the epithelium and finally in the cells of all corneal layers and the inflammatory cells that invaded the corneal stroma. This was accompanied by an elevated concentration of NO in the aqueous humor. In conclusion, repeated irradiation with UVB rays evoked the stimulation of NO production, peroxynitrite formation (demonstrated by NT residues) and lipid peroxidation (evaluated by MDA staining).
- PublicationOpen AccessRepair in avascular tissues, fibrosis in the transparent structures of the eye and thrombospondin 1(Murcia : F. Hernández, 1999) Hiscott, P.; Armstrong, D.; Batterbury, M.; Kaye, S.Wound repair is a process which is normally dependent on the vasculature of the damaged tissue. However, the transparent structures of the eye (e.g. central cornea, lens, vitreous) are avascular and yet are still subject to repair and fibrosis. Moreover, the resulting ophthalmic scars often remain avascular. Since this type of ocular scarring may result in blindness, it is the subject of intense research. An aspect of avascular ophthalmic fibrosis which has attracted attention is the question concerning early wound healing components that are usually derived from blood constituents. One such molecule is the glycoprotein thrombospondin 1. Thrombospondin 1 is thought to be a key regulator of cell behaviour in early wound repair and appears to be derived totally from platelet cc-granules during repair of incisional skin wounds. It has been shown that the ocular cells involved in avascular repair processes, and which are thus responsible for healing in the absence of platelet-derived thrombospondin 1, are capable of synthesizing the protein themselves. It is suggested that cells involved in ophthalmic repair processes produce thrombospondin 1 in the absence of the platelet-derived molecule. Local synthesis of thrombospondin 1 may represent a therapeutic target in the management of ophthalmic fibrosis.
- PublicationOpen AccessThe deep-sea teleost cornea, a comparative study of gadiform fishes(Murcia : F. Hernández, 1998) Collin, S.P.; Collin, H.B.The corneal structure of three deep-sea species of teleosts (Gadiformes, Teleostei) from different depths (250-4000 m) and photic zones are examined at the leve1 of the light and electron microscopes. Each species shows a similar but complex arrangement of layers with a cornea split into dermal and scleral components. The dermal cornea comprises an epithelium overlying a basement membrane and a dermal stroma with sutures and occasional keratocytes. Nezumia aequalis is the only species to possess a Bowman's layer, although it is not well-developed. The scleral cornea is separated from the dermal cornea by a mucoid layer and, in contrast to shallow-water species, is divided into three main layers; an anterior scleral stroma, a middle or iridescent layer and a posterior scleral stroma. The iridescent layer of collagen and intercalated cells or cellular processes is bounded by a layer of cells and the posterior scleral stroma overlies a Descemet's membrane and an endothelium. In the relatively shallow-water Microgadus proximus, the keratocytes of the dermal stroma, the cells of the iridescent layer and the endothelial cells al1 contain aligned endoplasmic reticulum, which may elicit an iridescent reflex. No alignment of the endoplasmic reticulum was found in N. aequalis or Coryphanoides (Nematonurus) armatus. The relative differences between shallow-water and deep-sea corneas are discussed in relation to the constraints of light, depth and temperature.
- PublicationOpen AccessThe presence of lysyl oxidase-like enzymes in human control and keratoconic corneas(Universidad de Murcia. Departamento de Biología Celular e Histología, 2016) Dudakova, Lubica; Sasaki, Takako; Liskova, Petra; Palos, Michalis; Jirsova, KaterinaPurpose: Lysyl oxidases, a family comprising lysyl oxidase (LOX) and four LOX-like enzymes (LOXL1-4), catalyse the cross-linking of elastin and collagen fibrils. Keratoconus (KC) is characterized by progressive thinning leading to irregular astigmatism, resulting in significant visual impairment. Although the pathogenesis of KC remains unclear, one of the current hypotheses is based on alterations in the organization and structure of collagen fibrils. To extend existing general knowledge about cross-linking enzymes in the human cornea, in the present study we have focused on the detection of LOXL enzymes. Method: The localization and distribution of LOXL1-4 were assessed in cryosections of 7 control donors (three males and three females; 25-68 years; mean age 46±17.6 years) and 8 KC corneas (5 males and 3 females; 25-46 years; mean age 31.3±7.5 years) using indirect fluorescent immunohistochemistry (IHC). The specimens were examined using an Olympus BX51 microscope (Olympus Co., Tokyo, Japan) at a magnification of 200-1000x. Western blot analysis of 4 control and 4 KC corneas was performed for all tested enzymes. Results: All four LOX-like enzymes were present in all layers of control corneas as well as in the limbus and conjunctiva. Almost no differences between control and pathological specimens were found for LOXL1. A lower staining intensity of LOXL2 was found using IHC and Western blot analysis in KC specimens. Decreases of the signal and small irregularities in the staining were found in the epithelium, keratocytes and extracellular matrix, where a gradual anterior-posterior weakening of the signal was observed. LOXL3 IHC staining was lower in the corneal stromal extracellular matrix and keratocytes of KC samples. No prominent differences were detected using IHC for LOXL4, but a slight decrease was observed in KC corneas using Western blot analysis. Conclusion: We presume that the decrease of LOXL2 in KC corneas is more likely a consequence of the associated pathological processes (activation of stromal cells due to tissue weakening and consequent structural changes) than a direct cause leading to KC development. At this time, we are unable to provide a coherent explanation for the observed decrease of LOXL3 and LOXL4 in KC corneas.
- PublicationOpen AccessThe spectrum of cytokeratins expressed in the adult human cornea, limbus and perilimbal conjunctiva(Murcia: F. Hernández, 2011) Merjava, Stanislava; Neuwirth, Ales; Tanzerova, Michaela; Jirsova, KaterinaThe aim of this study was to detect a spectrum of cytokeratins (CK) present in the adult human cornea, limbus and perilimbal conjunctiva. Cryosections from seven corneo-scleral discs were fixed, and indirect immunofluorescent staining was performed using antibodies directed against CK1-CK10 and CK13-CK20. The percentage of positive cells was calculated in the epithelium of the cornea, limbus and perilimbal conjunctiva. Quantitative real time RT-PCR (qRT-PCR) was used to detect CK6 and CK18 expression in the corneal and conjunctival epithelium. The most intense staining present throughout the cornea was observed for CK3, CK5 and CK14; CK19 was found at the corneal periphery only. CK4 and CK10/13 revealed mild to moderate positivity mostly in the superficial layers of the cornea. The suprabasal cell layers of all examined areas showed a strong positivity for CK16. A heterogeneous staining pattern with a centrifugal decrease in the signal was observed for CK8 and CK18. CK5/6, CK14 and CK19 were present in the limbus, where a positive signal for CK3 was observed in the suprabasal and superficial cells only. In contrast to the cornea, CK15 appeared in the basal and suprabasal layers of the limbus. The perilimbal conjunctiva showed strong immunostaining for CK10/13, CK14 and CK19. A moderate signal for CK7 was detected in the superficial layers of the conjunctiva. qRT-PCR confirmed CK6 and CK18 expression in the corneal and conjunctival epithelium. The detailed characterization of the corneal, limbal and perilimbal conjunctival epithelium under normal circumstances may be useful for characterizing the changes occurring under pathological conditions.
- PublicationOpen AccessTransfer of mesenchymal stem cells and cyclosporine A on alkali-injured rabbit cornea using nanofiber scaffolds strongly reduces corneal neovascularization and scar formation(Universidad de Murcia. Departamento de Biología Celular e Histología, 2016) Cejka, Čestmír; Cejkova, Jitka; Trosan, Peter; Zajicova, Alena; Sykova, Eva; Holan, VladimirThe aim of this study was to examine whether nanofiber scaffolds seeded with rabbit bone marrow mesenchymal stem cells (MSCs nanofibers) transferred onto the damaged corneal surface and covered with cyclosporine A (CsA)-loaded nanofiber scaffolds (CsA nanofibers) enable healing of the rabbit cornea injured with 1N NaOH. The healing of damaged corneas was examined morphologically, immunohistochemically and biochemically on day 24 after the injury. Compared to untreated injured corneas, where corneal ulceration or large corneal thinning or even perforation were developed, injured corneas treated with drug free nanofibers healed without profound disturbances in a majority of cases, although with fibrosis and scar formation. In injured corneas treated with CsA nanofibers or MSCs nanofibers, the development of scar formation was reduced. Best healing results were obtained with a combination of MSCs and CsA nanofibers (MSCs-CsA nanofibers). Corneas healed with highly restored transparency. Neovascularization highly expressed in untreated injured corneas and reduced in corneas treated with CsA nanofibers or MSCs nanofibers, was suppressed in corneas treated with MSCs-CsA nanofibers. The levels of matrix metalloproteinase 9, inducible nitric oxide synthase, interleukin 6, α-smooth muscle actin, tumor growth factor β and vascular endothelial growth factor were significantly decreased in these corneas as compared to untreated corneas, where the levels of the above mentioned markers were high. In conclusion, MSCsCsA nanofibers were effective in the treatment of severe alkali-induced corneal injury.