Abstract The blood-retinal barrier (BRB) has evolutionarily adapted as a protective border for th... more Abstract The blood-retinal barrier (BRB) has evolutionarily adapted as a protective border for the cell structures found in this very important sensory organ responsible for vision. The inner BRB with its endothelium, pericytes, Muller glia cells, and astrocytes are the first barrier between the blood and the retina, controlling nutrients and metabolites supply, as well as preventing toxic molecules to enter the retinal space. The outer BRB with its retinal pigment epithelium and the Bruch’s membrane serve as additional barrier and at the same time a front line for the most intense phagocytosis and intra- and transcellular transport in the human body, as well as the place of nearly highest oxidative stress versus antioxidative and angiogenic versus antiangiogenic processes. The barrier molecules in physiology and disease are discussed in this chapter (zonula occludens (ZO)-1, occludin, collagens). Finally, a brief overview of the tissue engineering and three-dimensional organoids with implications for future artificial generation of tissue and barriers in the eye is given. Understanding the structural and molecular components of the BRB and their implications in physiology and disease, as well as tissue engineering can shed light on the current state-of-the-art in basic and applied ophthalmology research and novel developments in therapy.
Comparative histochemical and biochemical studies on acid beta-galactosidase activity in the rabb... more Comparative histochemical and biochemical studies on acid beta-galactosidase activity in the rabbit eye after various experimental injuries were performed using the same sensitive fluorogenic substrate beta-galactoside-4-trifluoromethylumbelliferyl (HFC). The aim of the study was to examine whether the severity of corneal damage corresponds with the level of the enzyme activity in the tear fluid. As until recently the substrate beta-galactoside-4-HFC had not been used for the histochemical detection of acid beta-galactosidase in the cornea, results obtained with this substrate in a fluorescent method were compared in parallel cryostat sections with results obtained using the substrate 5-bromo-4-chloro-3-indoxyl beta-galactoside in the indigogenic method (previously shown to be very sensitive for the detection of acid beta-galactosidase activity in the cornea). Both methods revealed similar localization and changes in enzyme activity; using beta-galactoside-4-HFC an acceptable cellular localization was achieved. For the measurement of acid beta-galactosidase activity in the tear fluid a semiquantitative biochemical method was elaborated using filter paper punches with the substrate (beta-galactoside-4-HFC) soaked with tears and incubated at 37 degrees C. The time of the first appearance of a greenish-yellow fluorescence (enzyme positivity) was recorded by UV lamp and compared with the appearance of fluorescence in calibrated punches containing known acid beta-galactosidase activities. The results show that beta-galactoside-4-HFC is useful for the biochemical assessment of acid beta-galactosidase activity in the tear fluid. Comparing histochemical and biochemical results, it can be concluded that increased enzymatic activity in tears parallels the severity of corneal damage. Further studies are necessary to evaluate whether the detection of acid beta-galactosidase activity in tears might be useful for diagnostic purposes in humans.
Ozone depletion leads to an increase in UV rays of solar radiation reaching the surface of the Ea... more Ozone depletion leads to an increase in UV rays of solar radiation reaching the surface of the Earth which is harmful to biological systems. Of the eye, the cornea is directly open to increased amount of UV rays of which mainly UVB rays are capable to induce reactive oxygen species damaging the cells. Previous studies showed that the irradiation of the cornea with UVB rays leads to morphological as well as metabolic disturbances of the cornea. Also, corneal hydration and corneal light absorption are increased after UVB rays. These changes were observed after five days of repeated irradiation of the cornea with UVB rays. The aim of the present paper was to examine how early the changes of corneal hydration and light absorption occur after UVB irradiation. The rabbit corneas were irradiated with UVB rays for one, two, three or four days. Corneal light absorption was examined spectrophotometrically and corneal hydration measured by pachymeter (as corneal thickness). Results show that changes of corneal hydration and light absorption appear early after UVB irradiation and increase along with the number of irradiations. In conclusion, irradiation of the rabbit cornea with UVB rays leads to harmful changes of its optical properties.
To evaluate the combined effect of intense pulsed light (IPL) therapy and meibomian gland express... more To evaluate the combined effect of intense pulsed light (IPL) therapy and meibomian gland expression on extracellular matrix metalloproteinase-9 (MMP-9) levels and clinical outcomes of moderate and severe meibomian gland dysfunction (MGD) treatment. Methods: This retrospective study was conducted on 45 eyes of 23 patients with moderate and severe MGD. Each eye underwent three IPL sessions and meibomian gland expression at 2-week intervals. In this study the evaluated parameters included tear film break-up time (TBUT), corneal and conjunctival fluorescein staining scores, biomicroscopic examination of lid margins and meibomian glands, ocular surface disease index (OSDI) questionnaire score, and extracellular MMP-9 levels using the immunoassay device before and two weeks after the last treatment session. Linear mixed model and generalized estimating equations model were used to evaluate possible differences. Results: There were significant improvements in TBUT (P < 0.001), SICCA ocular staining score (P = 0.008), Oxford staining score (P = 0.023), lid margin irregularity (P < 0.001 for upper and lower eyelids), lid thickness (P < 0.001 for upper and lower eyelids), meibomian gland plugging (P = 0.010 and P = 0.012 for upper and lower eyelids), meibum color (P = 0.044 and P < 0.001 for upper and lower eyelids), meibum consistency (P < 0.001 for upper and lower eyelids), MGD grade (P < 0.001), and OSDI questionnaire score (P < 0.001). Incidence of positive results for MMP-9 immunoassay significantly decreased from 84.0% to 56.0% (P = 0.031) after treatment. Conclusion: In patients with moderate to severe MGD, three sessions of IPL combined with meibomian gland expression improved objective findings, subjective symptoms, meibomian gland function, and MMP-9 immunoassay results. The results support the combination of IPL and meibomian gland expression for treating moderate to severe MGD.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Matrix metalloproteinases (MMPs) are proteolytic enzymes involved in tissue remodeling and wound ... more Matrix metalloproteinases (MMPs) are proteolytic enzymes involved in tissue remodeling and wound healing. These enzymes degrade and also synthesize components of the extracellular matrix. Overexpression of MMPs results in excessive extracellular matrix degradation and tissue destruction. In the cornea, destructive processes may lead to scarring and loss of vision. In this study MMPs (types 1, 2, 7, 8, 9 and 14) were examined immunohistochemically in the normal rabbit corneal epithelium and in epithelium irradiated in vivo with similar doses of UVB or UVA radiation (UVB rays 312 nm, UVA rays 365 nm, daily dose 1.01 J/cm(2) for four days). Results show that MMPs studied revealed low expression in the normal corneal epithelium, whereas after repeated UVB irradiation the expression of MMPs was significantly increased in the corneal epithelium, in ascending order: MMP-2, MMP-9, MMP-1, and MMP-7 with MMP-8. In contrast, compared to normal corneas, repeated UVA radiation did not significantly change the expression of MMPs in the irradiated corneal epithelium. MMP-14 was expressed at very low levels in all studied corneas, whereas no significant changes were detected upon UV exposure. In conclusion, UV radiation of shorter wavelength (UVB) induced an increase in expression of all MMPs except MMP-14. It is suggested that overexpression of MMPs in the corneal epithelium contributes to the damaging effect of UVB radiation to the cornea.
PurposeHuntington′s disease (HD) belongs to the hereditary neurodegenerative disorder that is cau... more PurposeHuntington′s disease (HD) belongs to the hereditary neurodegenerative disorder that is caused by an expansion of a polyglutamine (polyQ) domain in the protein of huntingtin (Htt). Since mutant Htt (mtHtt) and especially their small proteolytic fragments are very toxic to all HD cells (particularly those of neuroectodermal origin such as neurons or retinal pigment epithelial cells), it has been suggested that upregulated proteolysis of mtHtt plays a crucial role in the HD pathogenesis including damage of the retina. Therefore, the purpose of the present study was to investigate the possible participation of the proteolytic enzymes from the group of caspases, matrix metalloproteinases (MMP), calpains in HD pathology of retinal pigment epithelial cells.MethodsIn this study we used wild type (WT) and transgenic minipigs for N‐terminal part of the human mtHtt (TgHD) (548aaHTT‐145Q, F2 generation age 36 and 48 months, respectively). Proteases were examined in cultured retinal pigment epithelial cells immunocytochemically (ICC) or biochemically by western blotting (WB) using the following primary antibodies: anti‐caspase‐3, anti‐caspase‐8, anti‐calpain‐5, anti‐MMP‐9.ResultsUsing biochemical and immunocytochemical analysis, we detected increased expressions of caspase‐3, caspase‐8, matrix metalloproteinase‐9, calpain‐5, and probably its multiple proteolytic cleavage products (generated from mtHtt) in cultured RPE cells of TgHD minipigs in comparison to WT animals.ConclusionsIncreased expression of proteolytic enzymes in TgHD RPE cells can contribute to the retinal damage during development of HD.
Retinal pigment epithelium (RPE) is a critical cell monolayer forming the blood-retina-barrier (B... more Retinal pigment epithelium (RPE) is a critical cell monolayer forming the blood-retina-barrier (BRB) and a permeable bridge between the choriocapillaris and the retina. RPE is also crucial in maintaining photoreceptor function and for completing the visual cycle. Loss of the RPE is associated with the development of degenerative diseases like age-related macular degeneration (AMD). To treat diseases like AMD, pluripotent stem cell-derived RPE (pRPE) has been recently explored extensively as a regenerative module. pRPE like other ectodermal tissues requires specific lineage differentiation and long-term in vitro culturing for maturation. Therefore, understanding the differentiation process of RPE could be useful for stem cell based RPE derivation. Developing pRPE-based transplants and delivering them into the subretinal space is another aspect that has garnered interest in the last decade. In this review, we discuss the basic strategies currently employed for stem cell-based RPE deri...
The retinal pigment epithelium (RPE) forms an important cellular monolayer, which contributes to ... more The retinal pigment epithelium (RPE) forms an important cellular monolayer, which contributes to the normal physiology of the eye. Damage to the RPE leads to the development of degenerative diseases, such as age-related macular degeneration (AMD). Apart from acting as a physical barrier between the retina and choroidal blood vessels, the RPE is crucial in maintaining photoreceptor (PR) and visual functions. Current clinical intervention to treat early stages of AMD includes stem cell-derived RPE transplantation, which is still in its early stages of evolution. Therefore, it becomes essential to derive RPEs which are functional and exhibit features as observed in native human RPE cells. The conventional strategy is to use the knowledge obtained from developmental studies using various animal models and stem cell-based exploratory studies to understand RPE biogenies and developmental trajectory. This article emphasises such studies and aims to present a comprehensive understanding of ...
The review intends to overview a wide range of nanostructured natural, synthetic and biological m... more The review intends to overview a wide range of nanostructured natural, synthetic and biological membrane implants for tissue engineering to help in retinal degenerative diseases. Herein, we discuss the transplantation strategies and the new development of material in combination with cells such as induced pluripotent stem cells (iPSC), mature retinal cells, adult stem cells, retinal progenitors, fetal retinal cells, or retinal pigment epithelial (RPE) sheets, etc. to be delivered into the subretinal space. Retinitis pigmentosa and age-related macular degeneration (AMD) are the most common retinal diseases resulting in vision impairment or blindness by permanent loss in photoreceptor cells. Currently, there are no therapies that can repair permanent vision loss, and the available treatments can only delay the advancement of retinal degeneration. The delivery of cell-based nanostructure scaffolds has been presented to enrich cell survival and direct cell differentiation in a range of ...
Abstract The blood-retinal barrier (BRB) has evolutionarily adapted as a protective border for th... more Abstract The blood-retinal barrier (BRB) has evolutionarily adapted as a protective border for the cell structures found in this very important sensory organ responsible for vision. The inner BRB with its endothelium, pericytes, Muller glia cells, and astrocytes are the first barrier between the blood and the retina, controlling nutrients and metabolites supply, as well as preventing toxic molecules to enter the retinal space. The outer BRB with its retinal pigment epithelium and the Bruch’s membrane serve as additional barrier and at the same time a front line for the most intense phagocytosis and intra- and transcellular transport in the human body, as well as the place of nearly highest oxidative stress versus antioxidative and angiogenic versus antiangiogenic processes. The barrier molecules in physiology and disease are discussed in this chapter (zonula occludens (ZO)-1, occludin, collagens). Finally, a brief overview of the tissue engineering and three-dimensional organoids with implications for future artificial generation of tissue and barriers in the eye is given. Understanding the structural and molecular components of the BRB and their implications in physiology and disease, as well as tissue engineering can shed light on the current state-of-the-art in basic and applied ophthalmology research and novel developments in therapy.
Comparative histochemical and biochemical studies on acid beta-galactosidase activity in the rabb... more Comparative histochemical and biochemical studies on acid beta-galactosidase activity in the rabbit eye after various experimental injuries were performed using the same sensitive fluorogenic substrate beta-galactoside-4-trifluoromethylumbelliferyl (HFC). The aim of the study was to examine whether the severity of corneal damage corresponds with the level of the enzyme activity in the tear fluid. As until recently the substrate beta-galactoside-4-HFC had not been used for the histochemical detection of acid beta-galactosidase in the cornea, results obtained with this substrate in a fluorescent method were compared in parallel cryostat sections with results obtained using the substrate 5-bromo-4-chloro-3-indoxyl beta-galactoside in the indigogenic method (previously shown to be very sensitive for the detection of acid beta-galactosidase activity in the cornea). Both methods revealed similar localization and changes in enzyme activity; using beta-galactoside-4-HFC an acceptable cellular localization was achieved. For the measurement of acid beta-galactosidase activity in the tear fluid a semiquantitative biochemical method was elaborated using filter paper punches with the substrate (beta-galactoside-4-HFC) soaked with tears and incubated at 37 degrees C. The time of the first appearance of a greenish-yellow fluorescence (enzyme positivity) was recorded by UV lamp and compared with the appearance of fluorescence in calibrated punches containing known acid beta-galactosidase activities. The results show that beta-galactoside-4-HFC is useful for the biochemical assessment of acid beta-galactosidase activity in the tear fluid. Comparing histochemical and biochemical results, it can be concluded that increased enzymatic activity in tears parallels the severity of corneal damage. Further studies are necessary to evaluate whether the detection of acid beta-galactosidase activity in tears might be useful for diagnostic purposes in humans.
Ozone depletion leads to an increase in UV rays of solar radiation reaching the surface of the Ea... more Ozone depletion leads to an increase in UV rays of solar radiation reaching the surface of the Earth which is harmful to biological systems. Of the eye, the cornea is directly open to increased amount of UV rays of which mainly UVB rays are capable to induce reactive oxygen species damaging the cells. Previous studies showed that the irradiation of the cornea with UVB rays leads to morphological as well as metabolic disturbances of the cornea. Also, corneal hydration and corneal light absorption are increased after UVB rays. These changes were observed after five days of repeated irradiation of the cornea with UVB rays. The aim of the present paper was to examine how early the changes of corneal hydration and light absorption occur after UVB irradiation. The rabbit corneas were irradiated with UVB rays for one, two, three or four days. Corneal light absorption was examined spectrophotometrically and corneal hydration measured by pachymeter (as corneal thickness). Results show that changes of corneal hydration and light absorption appear early after UVB irradiation and increase along with the number of irradiations. In conclusion, irradiation of the rabbit cornea with UVB rays leads to harmful changes of its optical properties.
To evaluate the combined effect of intense pulsed light (IPL) therapy and meibomian gland express... more To evaluate the combined effect of intense pulsed light (IPL) therapy and meibomian gland expression on extracellular matrix metalloproteinase-9 (MMP-9) levels and clinical outcomes of moderate and severe meibomian gland dysfunction (MGD) treatment. Methods: This retrospective study was conducted on 45 eyes of 23 patients with moderate and severe MGD. Each eye underwent three IPL sessions and meibomian gland expression at 2-week intervals. In this study the evaluated parameters included tear film break-up time (TBUT), corneal and conjunctival fluorescein staining scores, biomicroscopic examination of lid margins and meibomian glands, ocular surface disease index (OSDI) questionnaire score, and extracellular MMP-9 levels using the immunoassay device before and two weeks after the last treatment session. Linear mixed model and generalized estimating equations model were used to evaluate possible differences. Results: There were significant improvements in TBUT (P < 0.001), SICCA ocular staining score (P = 0.008), Oxford staining score (P = 0.023), lid margin irregularity (P < 0.001 for upper and lower eyelids), lid thickness (P < 0.001 for upper and lower eyelids), meibomian gland plugging (P = 0.010 and P = 0.012 for upper and lower eyelids), meibum color (P = 0.044 and P < 0.001 for upper and lower eyelids), meibum consistency (P < 0.001 for upper and lower eyelids), MGD grade (P < 0.001), and OSDI questionnaire score (P < 0.001). Incidence of positive results for MMP-9 immunoassay significantly decreased from 84.0% to 56.0% (P = 0.031) after treatment. Conclusion: In patients with moderate to severe MGD, three sessions of IPL combined with meibomian gland expression improved objective findings, subjective symptoms, meibomian gland function, and MMP-9 immunoassay results. The results support the combination of IPL and meibomian gland expression for treating moderate to severe MGD.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Matrix metalloproteinases (MMPs) are proteolytic enzymes involved in tissue remodeling and wound ... more Matrix metalloproteinases (MMPs) are proteolytic enzymes involved in tissue remodeling and wound healing. These enzymes degrade and also synthesize components of the extracellular matrix. Overexpression of MMPs results in excessive extracellular matrix degradation and tissue destruction. In the cornea, destructive processes may lead to scarring and loss of vision. In this study MMPs (types 1, 2, 7, 8, 9 and 14) were examined immunohistochemically in the normal rabbit corneal epithelium and in epithelium irradiated in vivo with similar doses of UVB or UVA radiation (UVB rays 312 nm, UVA rays 365 nm, daily dose 1.01 J/cm(2) for four days). Results show that MMPs studied revealed low expression in the normal corneal epithelium, whereas after repeated UVB irradiation the expression of MMPs was significantly increased in the corneal epithelium, in ascending order: MMP-2, MMP-9, MMP-1, and MMP-7 with MMP-8. In contrast, compared to normal corneas, repeated UVA radiation did not significantly change the expression of MMPs in the irradiated corneal epithelium. MMP-14 was expressed at very low levels in all studied corneas, whereas no significant changes were detected upon UV exposure. In conclusion, UV radiation of shorter wavelength (UVB) induced an increase in expression of all MMPs except MMP-14. It is suggested that overexpression of MMPs in the corneal epithelium contributes to the damaging effect of UVB radiation to the cornea.
PurposeHuntington′s disease (HD) belongs to the hereditary neurodegenerative disorder that is cau... more PurposeHuntington′s disease (HD) belongs to the hereditary neurodegenerative disorder that is caused by an expansion of a polyglutamine (polyQ) domain in the protein of huntingtin (Htt). Since mutant Htt (mtHtt) and especially their small proteolytic fragments are very toxic to all HD cells (particularly those of neuroectodermal origin such as neurons or retinal pigment epithelial cells), it has been suggested that upregulated proteolysis of mtHtt plays a crucial role in the HD pathogenesis including damage of the retina. Therefore, the purpose of the present study was to investigate the possible participation of the proteolytic enzymes from the group of caspases, matrix metalloproteinases (MMP), calpains in HD pathology of retinal pigment epithelial cells.MethodsIn this study we used wild type (WT) and transgenic minipigs for N‐terminal part of the human mtHtt (TgHD) (548aaHTT‐145Q, F2 generation age 36 and 48 months, respectively). Proteases were examined in cultured retinal pigment epithelial cells immunocytochemically (ICC) or biochemically by western blotting (WB) using the following primary antibodies: anti‐caspase‐3, anti‐caspase‐8, anti‐calpain‐5, anti‐MMP‐9.ResultsUsing biochemical and immunocytochemical analysis, we detected increased expressions of caspase‐3, caspase‐8, matrix metalloproteinase‐9, calpain‐5, and probably its multiple proteolytic cleavage products (generated from mtHtt) in cultured RPE cells of TgHD minipigs in comparison to WT animals.ConclusionsIncreased expression of proteolytic enzymes in TgHD RPE cells can contribute to the retinal damage during development of HD.
Retinal pigment epithelium (RPE) is a critical cell monolayer forming the blood-retina-barrier (B... more Retinal pigment epithelium (RPE) is a critical cell monolayer forming the blood-retina-barrier (BRB) and a permeable bridge between the choriocapillaris and the retina. RPE is also crucial in maintaining photoreceptor function and for completing the visual cycle. Loss of the RPE is associated with the development of degenerative diseases like age-related macular degeneration (AMD). To treat diseases like AMD, pluripotent stem cell-derived RPE (pRPE) has been recently explored extensively as a regenerative module. pRPE like other ectodermal tissues requires specific lineage differentiation and long-term in vitro culturing for maturation. Therefore, understanding the differentiation process of RPE could be useful for stem cell based RPE derivation. Developing pRPE-based transplants and delivering them into the subretinal space is another aspect that has garnered interest in the last decade. In this review, we discuss the basic strategies currently employed for stem cell-based RPE deri...
The retinal pigment epithelium (RPE) forms an important cellular monolayer, which contributes to ... more The retinal pigment epithelium (RPE) forms an important cellular monolayer, which contributes to the normal physiology of the eye. Damage to the RPE leads to the development of degenerative diseases, such as age-related macular degeneration (AMD). Apart from acting as a physical barrier between the retina and choroidal blood vessels, the RPE is crucial in maintaining photoreceptor (PR) and visual functions. Current clinical intervention to treat early stages of AMD includes stem cell-derived RPE transplantation, which is still in its early stages of evolution. Therefore, it becomes essential to derive RPEs which are functional and exhibit features as observed in native human RPE cells. The conventional strategy is to use the knowledge obtained from developmental studies using various animal models and stem cell-based exploratory studies to understand RPE biogenies and developmental trajectory. This article emphasises such studies and aims to present a comprehensive understanding of ...
The review intends to overview a wide range of nanostructured natural, synthetic and biological m... more The review intends to overview a wide range of nanostructured natural, synthetic and biological membrane implants for tissue engineering to help in retinal degenerative diseases. Herein, we discuss the transplantation strategies and the new development of material in combination with cells such as induced pluripotent stem cells (iPSC), mature retinal cells, adult stem cells, retinal progenitors, fetal retinal cells, or retinal pigment epithelial (RPE) sheets, etc. to be delivered into the subretinal space. Retinitis pigmentosa and age-related macular degeneration (AMD) are the most common retinal diseases resulting in vision impairment or blindness by permanent loss in photoreceptor cells. Currently, there are no therapies that can repair permanent vision loss, and the available treatments can only delay the advancement of retinal degeneration. The delivery of cell-based nanostructure scaffolds has been presented to enrich cell survival and direct cell differentiation in a range of ...
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