PURPOSE. To investigate a novel strategy in constructing tissue-engineered corneal stromal equivalent based on amniotic membrane and keratocytes. METHODS. The ultrathin amniotic membrane (UAM) was laminated, with corneal stromal cells (CSCs) distributed between the space of the layered UAMs. Calcein AM staining was used to evaluate cellular viability, morphology, and arrangement. Immunostaining, qRT-PCR, and Western blot were performed to detect gene and protein expression in keratocytes. Optical coherence tomography visualized the cross sections and thickness of the UAM construction. The microstructure of the CSC-secreted extracellular matrix (ECM) was investigated by scanning electron microscopy and transmission electron microscopy (TEM). To evaluate the feasibility of the multilayer UAM-CSC lamination for surgery, the corneal substitute was used to perform lamellar keratoplasty. Slit lamp microscopy and corneal fluorescein staining were performed in postsurgery observation. RESULTS. The CSCs maintained their keratocyte phenotype and secreted well-organized ECM on the aligned UAM surface. The four-layer UAM-CSC lamination attained half thickness of the human cornea (250 6 18 lm) after 8 weeks' culture, which also showed promising optimal transparency. In TEM images, the CSC-generated ECM displayed stratified, multilayered lamellae with orthogonal fibril arrangement, which was similar to the human cornea microstructure. Furthermore, the stromal equivalent was successfully preformed in lamellar keratoplasty. Four weeks post surgery, the substitute was well integrated into the recipient cornea and completely epithelialized without myofibroblast differentiation. CONCLUSIONS. Our study established a novel 3D biomimetic corneal model to replicate the corneal stromal organization with multilayer UAM, which was capable of promoting the development of corneal stroma-like tissues in vitro, establishing a new avenue for basic research and therapeutic potential.
Citation: Li C, Chen P, Zhang J, et al. Enzyme-induced vitreolysis can alleviate the progression of diabetic retinopathy through the HIF-1a pathway. Invest Ophthalmol Vis Sci. 2013;54:4964-4970. DOI:10.1167/ iovs.12-11443 PURPOSE. We studied the mechanism by which complete posterior vitreous detachment by enzyme-induced vitreolysis alleviates the progression of diabetic retinopathy. METHODS.We enrolled 50 diabetic rats and 20 normal control rats in this study. The right eyes of these diabetic rats were treated with a hyaluronidase (5 U) plus plasmin (0.25 U) by intravitreous injection (ET group), while the left eyes of diabetic rats (DR group) and eyes of the normal rats (NC group) were injected with balanced saline. Eight months after intravitreous injection, the oxygen concentration in the vitreous was measured, and the expression levels of hypoxia inducible factor-1a (HIF-1a), vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), and basic fibroblast growth factor (bFGF) in retinas were determined by real-time PCR and Western blot. Clinical observation, visual electrophysiology tests, and scanning electron microscopy (SEM) also were performed on the rats.RESULTS. SEM results showed that the surface of retinas in the ET group had little vitreous cortex and the inner limiting membrane could be observed. The oxygen concentration of the midvitreous was higher in the ET group than other groups. A significantly high expression of HIF-1a, VEGF, and bFGF was detected in the retinas of the DR group compared to the ET and NC groups. In visual electrophysiology tests, amplitude decline and peak time latency were found in diabetic rats, and changes in the DR group were more obvious than in the ET group.CONCLUSIONS. Enzyme-induced vitreolysis can increase oxygen concentration in the vitreous, with reduced expression of HIF-1a, VEGF, and bFGF, and increased expression of PEDF in the retinas, thus alleviating the progression of diabetic retinopathy.
The aim of the present study was to evaluate the clinical results of pars plana vitrectomy (PPV) combined with the surgical enlargement of internal limiting membrane (ILM) peeling in patients who had previously undergone failed idiopathic macular hole (IMH) surgery. In the study, 134 eyes from 130 IMH patients who had received PPV combined with ILM peeling surgery (2 disk diameters) were analyzed. Within this cohort, 14 eyes had IMHs that were not closed, of which 13 eyes underwent a second surgery involving enlargement of the ILM peeling. The extent of the ILM peeling was increased to the vascular arcades of the posterior fundus in the secondary surgery. Of the 13 eyes that underwent secondary surgery, five were in stage III and nine were in stage IV. The second surgery successfully achieved IMH closure in 61.5% (8/13) of the eyes. The IMH was completely closed following surgery and the logMAR vision increased from 0.98 to 0.84 (P=0.013) in the 8 successfully treated cases. The surgical enlargement of ILM peeling closed the IMHs and improved vision in the majority of patients. In addition, the procedures were safe. Therefore, the results of the present study indicate that enlargement of ILM peeling may be an effective therapy for patients who have previously undergone the failed surgical correction of an IMH.
HIV-1-associated ocular complications, such as microvasculopathies, can lead to the loss of vision in HIV-1-infected patients. Even in patients under highly active antiretroviral therapy, ocular lesions are unavoidable. Ocular complications have been demonstrated to be closely related to the breakdown of the blood-retinal-barrier (BRB); however, the underlying mechanism is not clear. The data from this study indicated that the HIV-1 Tat protein induced the apoptosis of human retinal microvascular endothelial cells (HRMECs) and retinal pigmen epithelium (RPE) cells, which compose the inner BRB and the outer BRB, respectively. In addition, this study found that the activation of N-methyl-D-aspartate receptors (NMDARs) was involved in the apoptosis of RPE cells, but it caused no changes in HRMECs. Furthermore, both cell types exhibited enhanced expression of Bak, Bax and Cytochrome c. The inhibition of Tat activity protected against the apoptosis induced by NMDAR activation and prevented the dysregulation of Bak, Bax and Cytochrome c, revealing an important role for the mitochondrial pathway in HIV-1 Tat-induced apoptosis. Together, these findings suggest a possible mechanism and may identify a potential therapeutic strategy for HIV-1-associated ocular complications.
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