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An in vitro pressure model towards studying the response of primary retinal ganglion cells to elevated hydrostatic pressures.

ABSTRACT: Glaucoma is a leading cause of blindness characterized by progressive degeneration of retinal ganglion cells (RGCs). A well-established risk factor for the development and progression of glaucoma is elevation of intraocular pressure (IOP). However, how elevated IOP leads to RGC degeneration remains poorly understood. Here, we fabricate a facile, tunable hydrostatic pressure platform to study the effect of increased hydrostatic pressure on RGC axon and total neurite length, cell body area, dendritic branching, and cell survival. The hydrostatic pressure can be adjusted by varying the height of a liquid reservoir attached to a three-dimensional (3D)-printed adapter. The proposed platform enables long-term monitoring of primary RGCs in response to various pressure levels. Our results showed pressure-dependent changes in the axon length, and the total neurite length. The proportion of RGCs with neurite extensions significantly decreased by an average of 38?±?2% (mean?±?SEM) at pressures 30?mmHg and above (p?

SUBMITTER: Wu J

PROVIDER: S-EPMC6588599 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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An in vitro pressure model towards studying the response of primary retinal ganglion cells to elevated hydrostatic pressures.

Wu Jing J Mak Heather Kayew HK Chan Yau Kei YK Lin Chen C Kong Cihang C Leung Christopher Kai Shun CKS Shum Ho Cheung HC

Scientific reports 20190621 1

Glaucoma is a leading cause of blindness characterized by progressive degeneration of retinal ganglion cells (RGCs). A well-established risk factor for the development and progression of glaucoma is elevation of intraocular pressure (IOP). However, how elevated IOP leads to RGC degeneration remains poorly understood. Here, we fabricate a facile, tunable hydrostatic pressure platform to study the effect of increased hydrostatic pressure on RGC axon and total neurite length, cell body area, dendri ...[more]

PMID: 31227762

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Project description:High pressure is the most important factor inducing retinal ganglion cell (RGC) apoptosis. However, the underlying mechanisms remain obscure. The present study investigated the effects of different levels of hydrostatic pressure (HP) on RGCs and the potential mechanisms involved. Primary cultured rat RGCs were exposed to five levels of HP (0, 20, 40, 60 and 80 mmHg) for 24 h. Morphological changes in RGCs were observed. The viability and apoptosis rate of RGCs were detected using a Cell Counting Kit‑8 assay and Annexin V‑fluorescein isothiocyanate/propidium iodide flow cytometry, respectively. Western blotting, reverse transcription‑quantitative polymerase chain reaction and immunofluorescence were used to detect the expression and mRNA levels of nerve growth factor (NGF), protein kinase B (AKT), apoptosis signal‑regulating kinase 1 (ASK1), forkhead box O1 (FoxO1) and cAMP response element binding protein (CREB). In the 0‑ and 20‑mmHg groups, there were no apoptotic morphological changes. In the 40 mmHg group, parts of the cell were shrunken or disrupted. In the 60 mmHg group, neurite extension was weakened and parts of the cells were disintegrating or dying. In the 80 mmHg group, the internal structures of the cells were not visible at all. The apoptosis rates of RGCs were significantly higher and the viability rates significantly lower under 40, 60 and 80 mmHg compared with under 0 or 20 mmHg (all P<0.01). The expression and mRNA levels of NGF, AKT and CREB decreased in a dose‑dependent manner in the 40‑, 60‑ and 80‑mmHg groups (all P<0.05), but those of ASK1 and FoxO1 increased in a dose‑dependent manner (all P<0.05). Interestingly, the alterations to the expression and mRNA levels of CREB were significantly larger compared with the changes in ASK1 or FoxO1 in the 40‑, 60‑ and 80‑mmHg groups (all P<0.01). The results of the present study demonstrate that elevated HP of 40, 60 or 80 mmHg reduces viability and induces apoptosis in RGCs, which may occur through effects on the NGF/ASK1/FoxO1 and NGF/AKT/CREB pathways, of which the latter is more strongly affected.

Project description:Many studies on retinal injury and repair following elevated intraocular pressure suggest that the survival ratio of retinal neurons has been improved by various measures. However, the visual function recovery is far lower than expected. The homeostasis of retinal synapses in the visual signal pathway is the key structural basis for the delivery of visual signals. Our previous studies found that complicated changes in the synaptic structure between retinal neurons occurred much earlier than obvious degeneration of retinal ganglion cells in rat retinae. The lack of consideration of these earlier retinal synaptic changes in the rescue strategy may be partly responsible for the limited visual function recovery with the types of protective methods for retinal neurons used following elevated intraocular pressure. Thus, research on the modulatory mechanisms of the synaptic changes after elevated intraocular pressure injury may give new light to visual function rescue. In this study, we found that thrombospondin 2, an important regulator of synaptogenesis in central nervous system development, was distributed in retinal macroglia cells, and its receptor ?2?-1 was in retinal neurons. Cell cultures including mixed retinal macroglia cells/neuron cultures and retinal neuron cultures were exposed to elevated hydrostatic pressure for 2 h. The expression levels of glial fibrillary acidic protein (the marker of activated macroglia cells), thrombospondin 2, ?2?-1 and presynaptic proteins were increased following elevated hydrostatic pressure in mixed cultures, but the expression levels of postsynaptic proteins were not changed. SiRNA targeting thrombospondin 2 could decrease the upregulation of presynaptic proteins induced by the elevated hydrostatic pressure. However, in retinal neuron cultures, elevated hydrostatic pressure did not affect the expression of presynaptic or postsynaptic proteins. Rather, the retinal neuron cultures with added recombinant thrombospondin 2 protein upregulated the level of presynaptic proteins. Finally, gabapentin decreased the expression of presynaptic proteins in mixed cultures by blocking the interaction of thrombospondin 2 and ?2?-1. Taken together, these results indicate that activated macroglia cells may participate in alterations of presynaptic proteins of retinal neurons following elevated hydrostatic pressure, and macroglia-derived thrombospondin 2 may modulate these changes via binding to its neuronal receptor ?2?-1.

Project description:PURPOSE: To investigate age-associated changes in retinal ganglion cell (RGC) response to elevated intraocular pressure (IOP), and to explore the mechanism underlying these changes. Specifically, the effect of aging on inhibitor of apoptosis (IAP) gene family expression was investigated in glaucomatous eyes. METHODS: IOP was induced unilaterally in 82 Wistar rats using the translimbal photocoagulation laser model. IOP was measured using a TonoLab tonometer. RGC survival was evaluated in 3-, 6-, 13-, and 18-month-old animals. Changes in the RNA profiles of young (3-month-old) and old glaucomatous retinas were examined by PCR array for apoptosis; changes in selected genes were validated by real-time PCR; and changes in selected proteins were localized by immunohistochemistry. RESULTS: There were no significant IOP differences between the age groups. However, there was a natural significant loss of RGCs with aging and this was more prevalent in glaucomatous eyes. The number of RGCs in glaucomatous eyes decreased from 669±123 RGC/mm² at 3 months to 486±114 RGC/mm² at 6 months and 189±46.5 RGC/mm² at 18 months (n=4-8, p=0.048, analysis of variance). The PCR array revealed different changes in proapoptotic and prosurvival genes between young and old eyes. The two important prosurvival genes, IAP-1 and X-linked IAP (XIAP), acted in opposite directions in 3-month-old and 15-month-old rats, and were significantly decreased in aged glaucomatous retinas, while their expression increased significantly in young glaucomatous eyes. P53 levels did not vary between young glaucomatous and normal fellow eyes, but were reduced with age. B-cell leukemia/lymphoma 2 (Bcl-2) family members and tumor necrosis factor (TNF)-? expression were unaffected by age. Immunohistochemistry results suggested that the sources of changes in IAP-1 protein expression are RGCs and glial cells, and that most XIAP secretion comes from RGCs. CONCLUSIONS: Decreased IAP-1 and XIAP gene expression in aged eyes may predispose RGCs to increased vulnerability to glaucomatous damage. These findings suggest that aging impairs the endogenous neuroprotective mechanism of RGCs evoked by elevated IOP.

Project description:Quantitative information on the dynamics of multiple molecular processes in individual live cells under controlled stress is central to the understanding of the cell behavior of interest and the establishment of reliable models. Here, the dynamics of the apoptosis regulator intracellular Ca(2+), apoptosis effector caspase-3/7, and morphological changes, as well as temporal correlation between them at the single cell level, are examined in retinal gangling cell line (differentiated RGC-5 cells) undergoing apoptosis at elevated hydrostatic pressure using a custom-designed imaging platform that allows long-term real-time simultaneous imaging of morphological and molecular-level physiological changes in large numbers of live cells (beyond the field-of-view of typical microscopy) under controlled hydrostatic pressure. This examination revealed intracellular Ca(2+) elevation with transient single or multiple peaks of less than 0.5 hour duration appearing at the early stages (typically less than 5 hours after the onset of 100 mmHg pressure) followed by gradual caspase-3/7 activation at late stages (typically later than 5 hours). The data reveal a strong temporal correlation between the Ca(2+) peak occurrence and morphological changes of neurite retraction and cell body shrinkage. This suggests that Ca(2+) elevation, through its impact on ion channel activity and water efflux, is likely responsible for the onset of apoptotic morphological changes. Moreover, the data show a significant cell-to-cell variation in the onset of caspase-3/7 activation, an inevitable consequence of the stochastic nature of the underlying biochemical reactions not captured by conventional assays based on population-averaged cellular responses. This real-time imaging study provides, for the first time, statistically significant data on simultaneous multiple molecular level changes to enable refinements and testing of models of the dynamics of mitochondria-mediated apoptosis. Further, the platform developed and the approach has direct significance to the study of a variety of signaling pathway phenomena.

Dataset Information

An in vitro pressure model towards studying the response of primary retinal ganglion cells to elevated hydrostatic pressures.

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An in vitro pressure model towards studying the response of primary retinal ganglion cells to elevated hydrostatic pressures.

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