Inner Membrane Layer Research Articles

Drusen in the macula and parapapillary region.

To examine histological characteristics and differences between drusen beneath the retinal pigment epithelium (small hard drusen) located in the macula and located in the parapapillary region. We histomorphometrically examined human eyes enucleated due to uveal melanomas or secondary angle-closure glaucoma. The study included 106 eyes (age, 62.6 ± 15.2years) with macular drusen (n = 7 globes) or parapapillary drusen (n = 29 eyes) and 70 eyes without drusen. In all drusen, periodic-acid-Schiff-positive material was located between the RPE basal membrane and the inner collagenous layer of Bruch's membrane (BM). Macular drusen as compared with parapapillary drusen had lower height (15.2 ± 10.1µm versus 34.3 ± 19.8µm; P = 0.003), while both groups did not differ significantly in basal drusen width (74.0 ± 36.3µm versus 108.7 ± 101.0µm; P = 0.95). Eyes with macular drusen and eyes without drusen did not differ significantly in BM thickness (2.74 ± 0.44µm versus 2.55 ± 0.88µm; P = 0.57) or in RPE cell density (35.4 ± 10.4 cells/480µm versus 32.8 ± 7.5 cells/480µm; P = 0.53), neither in the drusen region nor in the drusen vicinity, while BM thickness (4.60 ± 1.490µm; P < 0.001) and RPE cell density (56.9 ± 26.8 cells/480µm; P = 0.005) were higher at the parapapillary drusen. Eyes with macular drusen, eyes with parapapillary drusen, and eyes without drusen did not differ significantly in choriocapillaris density (all P > 0.10) and thickness (all P > 0.35). Limitations of the study, among others, were a small number and size of drusen examined, diseases leading to enucleation, lack of serial sections, limited resolution of light microscopy, and enucleation-related and histological preparation-associated artefacts. The findings of this study, also taking into account its methodological limitations, suggest that macular drusen and parapapillary drusen shared the morphological feature of periodic-acid-Schiff-positive material between the RPE basal membrane and BM and that they did not vary significantly in choriocapillaris thickness and density. RPE cell density and BM thickness were higher in parapapillary drusen than in macular drusen.

Preparation and characterization of unidirectional water-transported bilayer PLA/ZnO-PAN/SPA nanofibrous membrane for wound healing

Wound infection caused by excessive exudate leads to overhydration of wounds and prevents wound healing. Thus, there is a need for a wound dressing to be developed that can unidirectionally export excess biological fluid while inhibiting bacterial infection. Herein, a nanofibrous bilayer membrane with a unidirectional water transport function was fabricated by electrospinning as a novel wound dressing, which can export excess biological fluid efficiently and further inhibit bacterial infection. The inner layer of the bilayer nanofiber membrane is polylactic acid (PLA)-zinc oxide (ZnO) nanofibers, which are hydrophobic and antibacterial. The outer layer is polyacrylonitrile (PAN)-sodium polyacrylate (SPA) nanofibers, which are hydrophilic and liquid-absorbent. The bilayer nanofibrous membrane's morphology, mechanics, thermal stability, wettability, water absorption, gas permeability, and unidirectional water transport ability were characterized and evaluated. The study showed that the nanofibrous bilayer membrane exhibited excellent physicochemical properties and biological performance, and its mechanical properties can reach 4.6 MPa. In addition, when the thickness of the hydrophobic layer is 3 µm and the thickness of the hydrophilic layer is 44 µm, the double-layer fiber membrane can effectively export the excess biological fluid in 5.09 s and prevent the wound from wetting again. The nanofibrous bilayer membrane with excellent water absorption, air permeability, thermal stability, and mechanical strength provides a feasible solution for chronic wound healing.

Dual-layer hollow fibre haemodialysis membrane for effective uremic toxins removal with minimal blood-bacteria contamination

Bacterial endotoxin contamination in dialysate may pass through haemodialysis membrane and cause a silent chronic microinflammation to kidney patients. Dual-layer hollow fibre (DLHF) membranes with dual function, biocompatible adsorptive and antibacterial effects were developed to solve the problem of incompatible membrane and endotoxin contamination. All membranes were fabricated via the co-extrusion dry-wet phase inversion technique. In this study, silica/α-mangostin nanoparticle was incorporated into the inner layer of membrane to enhance the biocompatibility of the membrane while maintaining its adsorption capacity. Activated carbon (AC) was incorporated in the outer layer of membrane for improved antibacterial property. The DLHF membranes were characterised based on its morphology and surface hydrophilicity. The performance of the DLHF membranes was evaluated in terms of permeability, urea, and creatinine removal capabilities, bovine serum albumin (BSA) rejection and antibacterial properties. The dense and small pore size on the outer layer of AC created a smoother surface for the DLHF membrane. The BSA rejection of the DLHF membranes was enhanced by 6–8% compared to that of unmodified single layer hollow fibre membrane. Silica/α-mangostin nanoparticle in the inner layer of membrane enhanced the removal of urea and creatinine by chemisorption. Result also showed that the incorporation of AC in the outer layer of DLHF membrane successfully filtered bacteria by bacteria entrapment. DLHF membrane with the combination of silica/α-mangostin nanoparticle in the inner layer and AC in the outer layer possessed the higher bacteria inhibition into blood compartment against Escherichia coli and Staphylococcus aureus, with removal rate of 68% and 75%, respectively, and better urea and creatinine removal by 60.57% and 75.18%, respectively, compared to single-layer PSf based membrane. The development of co-adsorptive biocompatible DLHF membrane can play an important role in improvement of kidney patient life.

Retinal Pigment Epithelial Detachment in Age-Related Macular Degeneration.

Retinal pigment epithelial detachment is defined as a separation of the retinal pigment epithelium from the inner collagenous layer of Bruch’s membrane. It is a common manifestation in both dry and wet types of age-related macular degeneration. This review aims to provide a comprehensive guide to the pathophysiology, clinical and imaging characteristics, natural course and treatment of the various types of pigment epithelial detachments in order to assist in diagnosis and management of this important feature of age-related macular degeneration.

Renal leukocyte chemotactic factor 2 (ALECT2)-associated amyloidosis in Chinese patients

Background: Leukocyte chemotactic factor 2 (ALECT2) amyloidosis is one of the recently described types of amyloidosis. In this study, we reported the first large case series of renal ALECT2 amyloidosis in Chinese patients.Methods: We studied the prevalence, clinical characteristics, renal pathology, outcome and genetic features among seven patients diagnosed with renal ALECT2 amyloidosis at Peking University First Hospital of China from 2000 to 2018.Results: Seven patients were diagnosed with ALECT2 amyloidosis, representing 1.9% of the renal biopsy-proven amyloidosis cases. The mean age at diagnosis was 68 years without gender preference. The patients mainly manifested with varying impaired kidney function with a mean estimated glomerular filtration rate of 42.7 mL/min/1.73 m2 (range 8.0–80.5) and proteinuria at 3.9 g/24 h (range 0.4–11.3). There were four ALECT2 amyloidosis patients with concurrent membranous nephropathy (MN), who presented a higher proteinuria (6.4 ± 4.0 g/24 h vs. 2.0 ± 1.8 g/24 h) and higher frequency of nephrotic syndrome (50% vs. 0) than patients with isolated ALECT2 amyloidosis. Renal biopsy showed strongly congophilic amyloid deposits distributed mainly in the renal cortical interstitium, as well as the glomerular mesangium, the inner layer of glomerular basement membrane (GBM), and vascular walls. Two patients with concurrent ALECT2 amyloidosis and MN showed mild amyloid deposits, which have not been identified as ALECT2 amyloidosis by IHC and LMD/MS methods. All patients were corroborated by immunoelectron microscopy to exhibit the specific location of LECT2 in the amyloid fibrils. Genetic analysis revealed no mutations but homozygosity for the G allele encoding valine at position 40 in the mature protein in all patients. Except for one patient who died 8 years later after he was diagnosed with ALECT2 amyloidosis, the others presented with relatively stable renal function during the mean follow-up period of 12.5 months.Conclusions: ALECT2 amyloidosis was the third most common type of renal amyloidosis in Chinese patients from a single centre. The majority of ALECT2 amyloidosis patients were of Han ethnicity, with a high rate of concurrent MN. The recognition and accurate diagnosis of renal ALECT2 amyloidosis should be considered in Chinese patients.

Thermosensitive Polyacrylonitrile/Polyethylene Oxide/Polyacrylonitrile Membrane Separators for Prompt and Safer Thermal Lithium-Ion Battery Shutdown

With the rapid development of high-power and high-energy density lithium-ion batteries, thermal safety has become a critical issue. Due to the fast microstructure change triggered by heat, thermosensitive polymers have aroused increasing attention as thermal shutdown material candidates to prevent the thermal runaway of lithium-ion batteries. In this work, a sandwich-structured polyacrylonitrile/polyethylene oxide/polyacrylonitrile (PAN/PEO/PAN) fibrous membrane with prompt thermal shutdown properties and high thermal stability is prepared using electrospinning technique. The inner layer of the fusible PEO fibrous membrane impart the composite separator with a prompt thermal shutdown function at 80 °C. Meanwhile, the outer layer of the heat-resistant PAN membrane remains thermally stable, even at 200 °C. The large buffering temperature difference (120 °C) between the thermal shutdown temperature and the thermally stable temperature of the composite separator is wider than most of the reported separators and effectively enhances the thermal safety of the lithium-ion batteries. In addition, the PAN/PEO/PAN separator exhibits a higher porosity, better electrolyte wettability, larger ionic conductivity, and lower interfacial resistance compared with a commercial polypropylene/polyethylene/ polypropylene (PP/PE/PP) separator. Moreover, the coin cells assembled by the PAN/PEO/PAN separator presents stable cycle performance and excellent rate capability. Therefore, the novel PAN/PEO/PAN membrane shows great potential as a promising candidate for much safer lithium-ion battery separators.

Middle ear mucosal compartm

Mucosal folds of middle ear are not a significant barrier in spread of infection cholesteatoma spread towards the least resistance and usually follow the folds. The mucosa of the middle ear is continuous with that of the pharynx via Eustachian tube. It covers the ossicles, muscles, nerves and forms the inner layer of tympanic membrane. Mucosal fold are of two types Composite Fold – these are of ligament with lining mucosa. Duplicate Fold – Fusion of two expanding sacs in absence of any interposing structure. Epitympanic Diaphragm it is an oblique dividing septum between the posterior superior attic and anteroinferior mesotympanium. It comprises of anterior malleolar ligament, lateral malleolar ligament, posterior malleolar ligament, lateral incudal fold, tensor tympani fold and posterior incudal ligament fold. Tympanic Isthimus is 2.5 mm elongated narrow space in epitympanic diaphragm present naturally and provides ventilation. Key message is tensor tympani fold and anterior Pouch of VonTroltsch is responsible for ventilation of anterior compartment. It is extremely important to understand and restore the functional anatomy, proper gas exchange and mucosal clearance from the middle ear compartment. The obstruction site is at tympanic isthimus. It is crucial to visualise and clearance of disease to restore ventilation. In surgical procedures of ear just removal of tensor tympani fold along with resection of Cog provides good results by providing ventilation of attic, mastoid air cells and a successful tympanoplasty.

Temperature Responsive Shape-Memory Scaffolds with Circumferentially Aligned Nanofibers for Guiding Smooth Muscle Cell Behavior.

Structural simulation of the smooth muscle layer plays an important role in tissue engineering of blood vessels for the replacement of damaged arteries. However, it is difficult to construct small-diameter tubular scaffolds to homogenously locate and align smooth muscle cells (SMCs). In this work, novel temperature responsive shape-memory scaffolds are designed for SMC culturing. The scaffolds are composed of an outer layer of poly(lactide-glycolide-trimethylene carbonate) (PLGATMC) for programming the deformation from planar to small-diameter tubular shape and an inner layer of aligned nanofibrous membrane of poly(lactide-glycolide)/chitosan (PLGA/CS) to regulate cell adhesion, proliferation, and morphology. The SMC behaviors and functions are dependent on the PLGA/CS ratios of membranes, and the scaffold with PLGA/CS 7:3 membrane exhibits the most suitable ability to regulate SMC behavior. The PLGA/CS@PLGATMC scaffold can be deformed into a temporary planar at 20 °C for convenient seeding and attachment of SMCs and then immediately self-rolled into 3D tube at 37 °C. The proposed strategy offers a practical approach for the development of small-diameter vascular scaffolds from 2D planar into 3D tubular shape by self-rolling.

The critical factors affecting typical organophosphate flame retardants to mimetic biomembrane: An integrated in vitro and in silico study

Organophosphate flame retardants (OPFRs) have been reported to induce cytotoxicity in a structure-dependent manner. The toxic effects may be due to the damage of biomembrane integrity and/or the interference of membrane signal pathway. In this study, the damages of fifteen typical OPFRs (chlorinated phosphates, alkyl phosphates, aryl phosphates, and alkoxy phosphates) to mimetic biomembrane were determined by the electrochemical impedance spectroscopy (EIS). The molecular structure descriptors that characterized the action mechanisms were screened by stepwise regression. The six molecular descriptors (MATS7e, DLS_05, Mor19m, Mor22v, Mor12v and MATS8m) were screened to study the actions between OPFRs and mimetic biomembrane. A quantitative structure-activity relationship (QSAR) model was developed by the partial least squares (PLS) method. Statistical results indicated that the QSAR model had good robustness and mechanism interpretability. The distribution of atomic electronegativities (MATS7e) and atomic masses in three dimensional spaces (Mor19m) were the key factors influencing the actions between OPFRs and simulated biofilms. The compounds with strong electron-withdrawing property could invade the inner layer of membrane and destroy its integrity. High levels of steric hindrance could impair the damage capacity caused by electronegativity. Moreover, drug-like index (DLS_05), spatial structures of particle (Mor22v, Mor12v) and atomic masses (MATS8m) also affected the actions. The results revealed the mechanism of the actions of OPFRs with simulated biofilms and elucidated the key structural characteristics affecting the actions of OPFRs, which could provide theoretical basis for ecological risk assessment of OPFRs.

Sorsby fundus dystrophy: Insights from the past and looking to the future

Sorsby fundus dystrophy (SFD), an autosomal dominant, fully penetrant, degenerative disease of the macula, is manifested by symptoms of night blindness or sudden loss of visual acuity, usually in the third to fourth decades of life due to choroidal neovascularization (CNV). SFD is caused by specific mutations in the Tissue Inhibitor of Metalloproteinase-3, (TIMP3) gene. The predominant histo-pathological feature in the eyes of patients with SFD are confluent 20-30m thick, amorphous deposits found between the basement membrane of the retinal pigment epithelium (RPE) and the inner collagenous layer of Bruch's membrane. SFD is a rare disease but it has generated significant interest because it closely resembles the exudative or "wet" form of the more common age-related macular degeneration (AMD). In addition, in both SFD and AMD donor eyes, sub-retinal deposits have been shown to accumulate TIMP3 protein. Understanding the molecular functions of wild-type and mutant TIMP3 will provide significant insights into the patho-physiology of SFD and perhaps AMD. This review summarizes the current knowledge on TIMP3 and how mutations in TIMP3 cause SFD to provide insights into how we can study this disease going forward. Findings from these studies could have potential therapeutic implications for both SFD and AMD.

Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model.

PurposeExtracellular deposits containing hydroxyapatite, lipids, proteins, and trace metals that form between the basal lamina of the RPE and the inner collagenous layer of Bruch's membrane are hallmarks of early AMD. We examined whether cultured RPE cells could produce extracellular deposits containing all of these molecular components.MethodsRetinal pigment epithelium cells isolated from freshly enucleated porcine eyes were cultured on Transwell membranes for up to 6 months. Deposit composition and structure were characterized using light, fluorescence, and electron microscopy; synchrotron x-ray diffraction and x-ray fluorescence; secondary ion mass spectroscopy; and immunohistochemistry.ResultsApparently functional primary RPE cells, when cultured on 10-μm-thick inserts with 0.4-μm-diameter pores, can produce sub-RPE deposits that contain hydroxyapatite, lipids, proteins, and trace elements, without outer segment supplementation, by 12 weeks.ConclusionsThe data suggest that sub-RPE deposit formation is initiated, and probably regulated, by the RPE, as well as the loss of permeability of the Bruch's membrane and choriocapillaris complex associated with age and early AMD. This cell culture model of early AMD lesions provides a novel system for testing new therapeutic interventions against sub-RPE deposit formation, an event occurring well in advance of the onset of vision loss.

Large colloid drusen analyzed with structural en face optical coherence tomography

Drusen are extracellular deposits between the basal lamina of the retinal pigment epithelium (RPE) and the inner collagenous layer of Bruch's membrane. Large colloid drusen (LCD) are located below the RPE and are characterized by multiple, large, dome-shaped RPE detachments, with marked attenuation of the ellipsoid zone overlaying the drusen. This report presents the structural en face optical coherence tomography (OCT) findings of LCD and relates them to findings from fluorescein and indocyanine green angiography. We describe the case of a 55-year-old woman who presented with the chief complaint of a 5-year history of progressively worsening vision. Her best-corrected visual acuities were 20/40 and 20/400 in the right eye and the left eye, respectively. Fundus examination showed large bilateral, symmetrical, sub-retinal, yellowish lesions compatible with LCD. We describe the structural en face OCT characteristics and angiographic findings from this patient.

Hydrophilization of polysulfone hollow fiber membranes via addition of polyvinylpyrrolidone to the bore fluid

The novel method of modification of hollow fiber membranes from polysulfone is proposed. The method involves the use of aqueous solutions of polyvinylpyrrolidone (PVP) as a bore fluid in the dry-jet wet spinning process. Increasing PVP concentration (1–5wt%) in the bore fluid was found to yield in the efficient hydrophilization of the inner skin layer of the hollow fiber membrane. The contact angle of the skin layer was found to decrease from 76° down to 54° when the PVP concentration in the bore fluid is 1wt% and down to 45° – at 5wt%. The FTIR-spectra studies reveal the presence of PVP only on the inner surface of the modified hollow fiber membrane while outer surface was found to represent unmodified polysulfone. Scanning electron microscopy and atomic force microscopy studies reveal a difference in the structure of the initial and modified membranes. Introduction of PVP into the bore fluid leads to the marked changes in membrane pure water permeability and rejection. When the bore fluid contains 5wt% of PVP, the rejection coefficient for PVP (Mn=40 000gmol−1) was found to increase from 29% to 85%, and the pure water flux – to decrease from 370 to 130Lm−2 h−1bar−1 compared with the initial membrane. Antifouling characteristics against human serum albumin are shown to be significantly improved for the modified hollow fiber membranes compared with the initial membranes.

Thrombolysis of plasmin-ΔK on photochemically induced branch retinal vein occlusion after intravitreal injection in rats

Background Retinal vein occlusion is a common retinal vascular diseases.Thromblysis and anticoagulation therapies are main approaches.However, systemic thrombolysis is relatively inefficient, and it often enhances the risk of hemorrhage. Objective This study was to investigate the therapeutic effects of PLM-ΔK, a kringle deficiency mutant of plasmin, on photochemically induced branch retinal vein occlusion (BRVO) after intravitreal injection. Methods BRVO models were established by the combination of caudal vein injection of Rose Bengal with argon laser radiation of periphery area of retinal veins in SD rats.Forty model rats were randomized into balance salt solution (BSS) group and 0.01 U, 0.02 U, 0.03 U PLM-ΔK group, and 10 μl corresponding drug was intravtreally injected 12 hours after modeling.Ophthalmoscopy and fundus fluorescein angiography (FFA) were performed to observe the change of retinal veins.The animals were sacrificed 3 days after intravitreal injection, and hematoxylin and eosin staining was used for the histopathological and ultrastructural examination of retinas.The retina of the rats was isolated for the stretched preparation of retina.The expressions of fibronectin (FN) and laminin (LN) in eyeball wall were assayed by immunofluorescence technology.The use and care of the animals complied with Statement of the Association for Research in Vision and Ophthalmology. Results The revascularization of over 2 retinal veins was found in 0, 3, 6 and 8 rats in the BBS group and 0.01 U, 0.02 U, 0.03 U PLM-ΔK group 3 days after intravitreal injection, respectively, showing a significant difference among the groups (χ2=9.635, P=0.022), and the rat number with revascularization in 0.01 U PLM-ΔK group was not significantly different from that in BSS group (Z=-1.558, P=0.119), but the difference between 0.03 U PLM-ΔK group and 0.01 U PLM-ΔK group was significant (Z=-2.762, P=0.006). In the third day after intravitreal injection, retinal vein thrombus were found in the BSS group under the light microscope, and angiogenesis was seen on the retinal flatmount nuclear.In the 0.03 U PLM-ΔK group, posterior vitreal detachment was exhibited under the light microcope, and no retinal new vessel and cell damage were seen.FN was strongly expressed in the inner limiting membrane (ILM) layer, photocyte layer, outer limiting membrane (OLM) layer, choroid and scleral layer, and LN was expressed mainly in the ILM, OLM and scleral layer in the BSS group.However, the expression intensities of FN and LN were obviously weakened in the 0.03 U PLM-ΔK group. Conclusions Intravitreal injection of PLM-ΔK can enhance the reperfusion of occluded branch retinal vein and serve as a potential therapeutic drug for BRVO.Also it can permeate into choroid after intravitreal injection to degradate FN and LN. Key words: Retinal vein occlusion/therapy; Fibrinolytic agents/administration d Intravitreal injection; Thrombolytic therapy/methods; Disease models, animal; SD rats

The cause and influence of sequentially assembling higher and lower deacetylated chitosans on the membrane formation of microcapsule.

Alginate-chitosan (AC) microcapsules with desired strength and biocompatibility are preferred in cell-based therapy. Sequential assembly of higher and lower deacetylated chitosans (C1 and C2 ) on alginate has produced AC1 C2 microcapsule with improved membrane strength and biocompatibility. In this article, the assembly and complexation processes of two cationic chitosans on anionic alginate were concerned, and the cause and influence of sequentially assembling chitosans on AC1 C2 microcapsules membrane formation were evaluated. It was found that C1 complexation was the key factor for deciding the membrane thickness of AC1 C2 microcapsule. Specifically, the binding amount of C2 positively related to the binding amount of C1 , which suggested the first layer by C1 complexation on alginate had no obvious resistance on the sequential cationic C2 complexation. Further analyses demonstrated that outward migration of alginate molecules and inward diffusion of both chitosans under electrostatic interaction contributed to the sequential coating of C2 on first C1 layer. Moreover, C2 complexation through the surface to inner layer of membrane helped smoothen the first layer by C1 complexation that displayed a synergy role on the formation of AC1 C2 microcapsule membrane. Therefore, the two chitosans played different roles and synergistically contributed to membrane properties that can be easily regulated with membrane complexation time.

Modulation of MEK/ERK and PI3K/Akt pathways to the expression of DDR2 and MMP-13 in choroidal neovascularization

Background It is estimated that discoidin domain receptor 2 (DDR2) and matrix metalloproteinase-13 (MMP-13) play an important role in the development of tumor angiogenesis. However, their effects on choroidal neovascularizaiton (CNV) have not been clarified yet. Objective This study was to observe the expression pattern of DDR2 and MMP-13 in CNV and to further investigate the regulation role of MEK/ERK and PI3K/Akt pathways to the expression of DDR2 and MMP-13 in CNV. Methods CNV models were established in 78 Brown Norway (BN) rats by retinal photocoagulation with 532 nm laser. Then the animals were randomly divided into the normal control group (n=7), the model control group (n=39), PD98059 (MEK1 inhibitor) group (n=16) and LY294002 (PI3K inhibitor) group (n=16), and 5 mmol/L PD98059 or 5 mmol/L LY294002 3 μl was intravitreally injected 1 day and 7 days after photocoagulation in the PD98059 group or LY294002 group. The expression of DDR2 and MMP-13 mRNA and proteins in the CNV area were detected by using reverse transcription PCR (RT-PCR), and the expression levels of p-ERK/ERK and p-Akt/Akt protein were detected by Western blot assay. CNV thickness was measured by pathological examination 14 days after photocoagulation, and the changes of CNV thickness, the expression levels of DDR2 and MMP-13 in CNV were compared among the model control group, PD98059 group and LY294002 group. Results Three days after photocoagulation, the cells within the lasered lesions proliferated, then CNV formed 7 days after photocoagulation and became stable 14 days after photocoagulation. Immunohistochemistry staining indicated that DDR2 was weakly expressed in the cells of ganglion cell layer, inner nuclear layer and vascular endothelial cells; while MMP-13 was strongly expressed in the cells of the inner limiting membrane layer, photoreceptor layer and sclera layer. Both DDR2 and MMP-13 were strongly expressed in CNV area. Double immunofluorescence staining revealed that MMP-13 and DDR2 co-expression in CNV area. RT-PCR revealed that the relative DDR2 mRNA levels at 1 day, 3 days, 7 days and 14 days after photocoagulation were 55. 22±4. 03, 47. 74±2. 23, 14. 82±4. 56 and 5. 59±2. 47 respectively, while the relative MMP-13 mRNA levels were 25. 54±3. 83, 43. 51±4. 36, 10. 90±4. 00 and 5. 23±3. 23 respectively. Compared with the normal control group, the expression of DDR2 and MMP-13 were significantly increased (all at P 0. 05). Both PD98059 and LY294002 treatment were able to attenuate the thickness of CNV to 57. 21% and 50. 34% at day 14 after photocoagulation and further decrease the expression levels of DDR2 and MMP-13 in CNV (all at P<0. 05). Conclusions The expressions of DDR2 and MMP-13 up-regulate in laser-induced CNV. MEK/ERK and PI3K/Akt pathways suppress the development of CNV by regulating the expression of DDR2 and MMP-13. Key words: Discoindin domain receptor 2; Choroid/pathology; Neovascularization; Matrix metalloproteinase-13; Disease model; Rat, Brown Norway

Grazing Incidence X-ray Diffraction and Brewster Angle Microscopy studies on domain formation in phosphatidylethanolamine/cholesterol monolayers imitating the inner layer of human erythrocyte membrane

In this work the properties of monomolecular films composed of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE) and cholesterol, differing in lipid proportion, were investigated in the context of domain formation in the inner leaflet of membrane. To perform comprehensive analysis of the studied model systems the Langmuir monolayer experiments were performed in combination with Brewster angle microscopy (BAM) and Grazing Incidence X-ray Diffraction (GIXD) techniques. The analysis of the collected data proved non-ideal behavior of the investigated films. It was found that cholesterol at its lower concentration in the system (10%) is of disturbing influence on SOPE film. Further addition of cholesterol into phospholipids film (33, 50, and 67% of cholesterol) induces an ordering effect on SOPE acyl chains and provokes the formation of sterol-poor and sterol-rich domains which stoichiometry is independent of monolayer composition. The foregoing findings allow one to conclude that in cytosolic leaflet of membrane the lipids may segregate into domains of various cholesterol contents which depending on their composition may play different roles in membrane functioning.

Comparison of growth of human fetal RPE cells on electrospun nanofibers and etched pore polyester membranes

To investigate and compare the growth of human fetal retinal pigment epithelial (RPE) cells seeded onto electrospun polyamide nanofibers (EPN) or etched pore polyester (EPP), and, further, to explore their possible use as prosthetic Bruch's membrane. Human fetal RPE cells were planted onto the EPN, EPP and plastic (control) substrates in Transwells. The cultures were assessed with respect to cell attachment at 2, 4, 8 hours and proliferation at 1, 4, 8 days after seeding. Growth and morphology of the cells were monitored under the phase contrast microscope, and the phenotype was identified by immunofluorescence staining with antibodies against tight junction protein ZO-1. Strips of single EPP coated with nothing or EPP coated with EPN was differently implanted into the subretinal space of two P21 RCS rats for two weeks and the histologic slides of the retina were assessed. Cultured human fetal RPE cells were attached to either EPN or EPP substrates (with seeding on plastic substrate as control). After 8 h, the numbers of adherent cells in the EPN, EPP and control groups were 1.23*10(5)/cm(2), 1.70*10(5)/cm(2), and 1.64*10(5)/cm(2), respectively. The number of RPE cells attached to EPN was obviously less than that to both EPP and control (P<0.05). On the first day, the proliferation of cells on EPN was less than that of EPP and control (P<0.05); but by the 8th day in culture, the proliferation of cells on EPN had increased and was higher than proliferation on both EPP and control (P<0.05). All of the RPE cells cultured on EPN and EPP substrates were in monolayer, and the EPN-attached cells resembled the inner collagenous layer of Bruch's membrane. Immunofluorescence staining showed that the RPE cells cultured on EPN and EPP substrates adopted a higher expression of ZO-1 than that on the plastic control substrate. Subretinal implantation of either EPP alone or EPP as a carrier for free EPN for 2 weeks in P21RCS rats resulted in an expected encapsulation and loss of photoreceptor layer. No toxicity or other adverse reaction was observed in the vicinity of the transplant. EPN and EPP could maintain human fetal RPE cell attachment and proliferation. Both EPN and EPP appeared to be grossly tolerance and biocompatible with subretinal implantation. EPN represents an intriguing prospect for prosthetic Bruch's membrane replacement because of its similarity in structure to native Bruch's membrane.

The interactions between cholesterol and phospholipids located in the inner leaflet of humane erythrocytes membrane (DPPE and DPPS) in binary and ternary films—The effect of sodium and calcium ions

The studies on the influence of cholesterol on phospholipids accumulated in inner leaflet of membrane are performed rather rarely, especially in the presence of electrolytes, which are present in membrane environment. Therefore, in this work the interactions between cholesterol and saturated phosphatidylethanolamine (PE) and phosphatidylserine (PS) were studied in binary (phospholipid/cholesterol) and ternary (PS/PE/cholesterol) monolayers in the presence and absence of sodium and calcium ions. The composition of ternary films was estimated to reflect the proportion of PSs to PEs in inner layer of human erythrocyte membrane. The influence of electrolytes on pure PS and PE films was also analyzed. It was found that both sodium and calcium ions affect the condensation of DPPS films, and influence the interactions in DPPS/cholesterol monolayers. On the other hand, no effect of these ions on DPPE films as well as on DPPE/cholesterol interactions in the mixed systems was observed. The results obtained for ternary mixtures prove that in the presence of Na + the interactions between the lipids are more favorable than in the absence of these ions. This is in contrast to the effect of Ca 2+. All the results were thoroughly analyzed in the context of the structure of polar heads of the investigated phospholipids.

Modification of native collagen with cell‐adhesive peptide to promote RPE cell attachment on Bruch's membrane

Current efforts to reverse loss of visual function due to Age-related Macular Degeneration point to the restoration of the Retinal Pigment Epithelial (RPE) layer. Restoration of the RPE layer involves replacing lost RPE cells as well as addressing the degeneration of the underlying Bruch's membrane (BM). To advance the potential of using donor BM, we present a strategy to achieve specific and controllable modification of the inner collagenous layer (ICL) of the Bruch's membrane. In particular, interaction between a collagen binding peptide (CBP) sequence with exposed collagen fibers on the ICL surface is utilized to anchor bioactive molecules. Here, a cell-adhesion sequence is added to the collagen binding sequence to promote attachment and survival of ARPE-19. First, the binding specificity of the CBP sequence is verified with a fluorescent binding assay. Subsequently, the effect of modification using the peptide is studied qualitatively using confocal fluorescent imaging and quantitatively through a cell proliferation assay. Results of these experiments indicate that the peptide sequence binds specifically to collagen fibers. Additionally, modification using the peptide enhanced cell adhesion, allowing large uniform cell networks to be formed on the surface. Furthermore, modification with the peptide also delayed the onset of apoptosis on adherent cells.