A simple dissection method for the isolation of mouse trabecular meshwork cells.

PURPOSE: The outflow pathway, especially trabecular meshwork (TM), plays an essential role in glaucoma, and the availability of TM cells is crucial for in vitro research. So far, the isolation of TM cells from mice has been anything but manageable due to the small size of the eye. Direct isolation using a stereomicroscope and forceps requires a high grade of dexterity. Indirect isolation is based on the phagocytic properties of TM cells and involves injecting magnetic microspheres into the anterior chamber of live mice followed by isolation. Therefore, a simpler, less expensive, and nonexperimental strategy for isolating mouse TM cells would be desirable. METHODS: After enucleation, the eyes were cut in half anterior-to-posteriorly. The lens and posterior segment were removed. Iris and the attached ciliary body were gently pulled backward and disconnected from the remaining tissue to expose the TM. By incising through the cornea anteriorly and posteriorly of the TM, the cornea/TM stripe could be isolated. The cornea/TM stripe was cultured with the pigmented side down in a 6-well. The outgrowing pigmented cells were analyzed by immunocytochemistry and mRNA expression for previously described TM cell markers. The phagocytic properties of the cells were additionally confirmed using fluorescent microspheres. RESULTS: Pigmented phagocytic cells were the first to grow out of the cornea/TM strips after approximately 4-7 days. Cells were positive for Collagen IV, Fibronectin1, Vimentin, and Actin alpha 2 and could phagocytize fluorescent microbeads. Cross-linked actin networks were visible after 9 days of exposure to TGFB2 (transforming growth factor-beta 2). Additionally, treatment with 500 nM Dexamethasone for one week increased myocilin expression, as previously reported for TM cells. In addition, we proved that this method can also be used in albino mice, which lack pigmentation of the trabecular meshwork. CONCLUSIONS: The isolated cells show phagocytic properties and specific expression of markers reported in TM cells. Therefore, our dissection-based method is inexpensive and reproducible for isolating TM cells in mice.

Characterization and Classification of Postoperative Cysts After Strabismus Surgery: Clinical, Histological, and Anterior Segment OCT Analysis in a Large German Cohort.

INTRODUCTION: In this work, we provide a detailed characterization of a rare complication-subconjunctival cyst formation after strabismus surgery-in a large German cohort. METHODS: We conducted a retrospective analysis of 822 consecutive patients who underwent strabismus surgery between 2015 and 2022. The patients received comprehensive eye and orthoptic examinations preoperatively, at 1 day, and at 3 months postoperatively. Cysts were analyzed with slit-lamp examination, anterior segment optical coherence tomography (AS-OCT), and histopathological subsumption. RESULTS: Nineteen cases of postoperative cysts were observed (2.3%), 12 of which underwent surgical revision. Clinical evaluation including slit-lamp and AS-OCT as well as histological analysis resulted in a classification of three types of cysts: type 1, which is a single hyporeflective cyst, type 2, which is a multilobular hyporeflective cyst, and type 3, a dense hyperreflective granulomatous-like cyst. Eta (η) correlation ratio analysis could show a correlation between time of clinical appearance and type of cyst (Eta = 0.63). Most cysts developed within 20 days after surgery. Not only did cysts more frequently affect the medial rectus muscle, which in most cases underwent a shortening procedure (11/19 tucks, 4/19 resections) for intermittent exotropia (X(T)), but the cyst also formed earlier than in the lateral rectus muscle (Eta = 0.45). No correlation could be shown between the type of surgical procedure and time of cyst occurrence (Eta = 0.1). Patient age and cyst type correlated strongly (Eta = 0.47). The underlying type of strabismus did not correlate with the type of cyst observed. CONCLUSIONS: Our cases showed a strong positive correlation to the type of strabismus (X(T)), age (young patients), and the procedure (tuck/resection). We introduce a grading system for postoperative cysts after strabismus surgery, complementing histopathology and slit-lamp aspects with AS-OCT information.

Long-Term Multimodal Imaging Analysis of Selective Retina Therapy Laser Lesions.

This study evaluates the long-term effects of selective retina therapy (SRT) on the retinal pigment epithelium (RPE) and neuroretina in patients with central serous chorioretinopathy. SRT was performed on 36 patients using a Nd:YLF-Laser at 527 nm (R:GEN®, Lutronic, Goyang-Si, Republic of Korea). A total of 994 titration spots were examined using up to three years' multimodal imaging. Leakage in fluorescein angiography (FA) was observed after SRT in 523 lesions and resolved after one month. SRT lesions were not visible clinically, but appeared as brightly reflective areas in infrared and multicolor images. Normal morphology was observed in optical coherence tomography (OCT) immediately after SRT. After one month, thickening of the RPE and interdigitation zone changes were seen and disappeared after 539 ± 308 days. No RPE atrophies occurred during the observation period. Decreased fundus autofluorescence (FAF) was mostly observed directly after SRT followed by increased FAF at one month, which faded over time. A significant decrease in the number of visible lesions in the FA and FAF was observed within the three-year follow-up. OCT findings are consistent with animal studies showing SRT-related defect closure by hypertrophy and migration of neighboring cells without RPE atrophy or photoreceptor damage. This suggests that SRT is a safe treatment option for macular diseases and does not lead to retinal atrophy.

Breaking a Dogma: High-Throughput Live-Cell Imaging in Real-Time with Hoechst 33342.

Automated high-throughput live cell imaging (LCI) enables investigation of substance effects on cells in vitro. Usually, cell number is analyzed by phase-contrast imaging, which is reliable only for a few cell types. Therefore, an accurate cell counting method, such as staining the nuclei with Hoechst 33342 before LCI, will be desirable. However, since the mid-1980s, the dogma exists that Hoechst can only be used for endpoint analyses because of its cytotoxic properties and the potentially phototoxic effects of the excitation light. Since microscopic camera sensitivity has significantly improved, this study investigates whether this dogma is still justified. Therefore, exposure parameters are optimized using a 4× objective, and the minimum required Hoechst concentration is evaluated, allowing LCI at 30-min intervals over 5 days. Remarkably, a Hoechst concentration of only 57 × 10-9 m significantly inhibits proliferation and thus impairs cell viability. However, Hoechst concentrations between 7 × 10-9  and 28 × 10-9 m can be determined, which are neither cytotoxic nor impacting cell viability, proliferation, or signaling pathways. The method can be adapted to regular inverted fluorescence microscopes and allows, for example, to determine the cytotoxicity of a substance or the transduction efficiency, with the advantage that the analysis can be repeated at any desired time point.

MiR-302d inhibits TGFB-induced EMT and promotes MET in primary human RPE cells.

PURPOSE: Transforming growth factor-beta (TGFB)-mediated epithelial-mesenchymal transition (EMT) plays a crucial role in the pathogenesis of retinal fibrosis, which is one of the leading causes of impaired vision. Current approaches to treating retinal fibrosis focus, among other things, on inhibiting the TGFB signaling pathway. Transient expression of microRNAs (miRNAs) is one way to inhibit the TGFB pathway post-transcriptionally. Our previous study identified the miRNA miR-302d as a regulator of multiple TGFB-related genes in ARPE-19 cells. To further explore its effect on primary cells, the effect of miR-302d on TGFB-induced EMT in primary human retinal pigment epithelium (hRPE) was investigated in vitro. METHODS: hRPE cells were extracted from patients receiving enucleation. Transfection of hRPE cells with miR-302d was performed before or after TGFB1 stimulation. Live-cell imaging, immunocytochemistry staining, Western blot, and ELISA assays were utilized to identify the alterations of cellular morphology and EMT-related factors expressions in hRPE cells. RESULTS: hRPE cells underwent EMT by TGFB1 exposure. The transfection of miR-302d inhibited the transition with decreased production of mesenchymal markers and increased epithelial factors. Meanwhile, the phosphorylation of SMAD2 activated by TGFB1 was suppressed. Moreover, miR-302d expression promoted TGFB1-induced fibroblast-like hRPE cells to revert towards an epithelial stage. As confirmed by ELISA, miR-302d reduced TGFB receptor 2 (TGFBR2) and vascular endothelial growth factor A (VEGFA) levels 48 hours after transfection. CONCLUSIONS: The protective effect of miR-302d might be a promising approach for ameliorating retinal fibrosis and neovascularization. MiR-302d suppresses TGFB-induced EMT in hRPE cells via downregulation of TGFBR2, even reversing the process. Furthermore, miR-302d reduces the constitutive secretion of VEGFA from hRPE cells.

Changes in Intraocular Pressure following Narcosis With Medetomidine, Midazolam, and Fentanyl in Association With Initial Intraocular Pressure in Mice.

PURPOSE: This article describes the development of decreased intraocular pressure (IOP) under general anesthesia with medetomidine, midazolam, and fentanyl in mice with normal and elevated IOP. METHODS: IOP was measured using the iCare Tonolab rebound tonometer. Twelve 3-4 months-old male and female C57BL/6J mice were randomized to a control group with physiological IOP and a high IOP group with experimentally induced ocular hypertension using tarsal injections of dexamethasone-21-acetate. For anesthesia, medetomidine and midazolam were used, subgroups additionally received fentanyl. IOP was measured every 2.5 min for 30 min. RESULTS: Control group differed with 14.89 mmHg (SEM: 0.58) significantly (p = 0.0002) from the high IOP group with initial 20.44 mmHg (SEM: 0.75). All groups showed a significant (p < 0.05) decrease in IOP under general anesthesia. There was no significant difference in IOP development and decrease between the group additionally receiving fentanyl and the group without fentanyl. The decrease in IOP was highly dependent on the initial value, with the high IOP group showing a greater decrease. After 10 min, no significant difference in IOP could be detected between the high IOP and control group. CONCLUSIONS: In mice, general anesthesia with medetomidine and midazolam leads to a declining IOP over time. Adding fentanyl to the anesthesia did not alter these effects. The decline is time-dependent and IOP-dependent.

Amitriptyline functionally antagonizes cardiac H2 histamine receptors in transgenic mice and human atria.

We have previously shown that histamine (2-(1H-imidazol-4-yl)ethanamine) exerted concentration-dependent positive inotropic effects (PIE) or positive chronotropic effects (PCE) on isolated left and right atria, respectively, of transgenic (H2R-TG) mice that overexpress the human H2 histamine receptor (H2R) in the heart; however, the effects were not seen in their wild-type (WT) littermates. Amitriptyline, which is still a highly prescribed antidepressant drug, was reported to act as antagonist on H2Rs. Here, we wanted to determine whether the histamine effects in H2R-TG were antagonized by amitriptyline. Contractile studies were performed on isolated left and right atrial preparations, isolated perfused hearts from H2R-TG and WT mice and human atrial preparations. Amitriptyline shifted the concentration-dependent PIE of histamine (1 nM-10 µM) to higher concentrations (rightward shift) in left atrial preparations from H2R-TG. Similarly, in isolated perfused hearts from H2R-TG and WT mice, histamine increased the contractile parameters and the phosphorylation state of phospholamban (PLB) at serine 16 in the H2R-TG mice, but not in the WT mice. However, the increases in contractility and PLB phosphorylation were attenuated by the addition of amitriptyline in perfused hearts from H2R-TG. In isolated electrically stimulated human atria, the PIE of histamine that was applied in increasing concentrations from 1 nM to 10 µM was reduced by 10-µM amitriptyline. In summary, we present functional evidence that amitriptyline also acts as an antagonist of contractility at H2Rs in H2R-TG mouse hearts and in the human heart which might in part explain the side effects of amitriptyline.