scholarly journals A Fair Assessment of Evaluation Tools for the Murine Microbead Occlusion Model of Glaucoma

2021 ◽  
Vol 22 (11) ◽  
pp. 5633
Author(s):  
Marie Claes ◽  
Joana R. F. Santos ◽  
Luca Masin ◽  
Lien Cools ◽  
Benjamin M. Davis ◽  
...  
Keyword(s):  
Success Rate ◽  
Anterior Chamber Depth ◽  
Acquisition Time ◽  
Retinal Ganglion ◽  
Mild Phenotype ◽  
Future Studies ◽  
Threshold Response ◽  
Experimental Glaucoma ◽  
Occlusion Model

Despite being one of the most studied eye diseases, clinical translation of glaucoma research is hampered, at least in part, by the lack of validated preclinical models and readouts. The most popular experimental glaucoma model is the murine microbead occlusion model, yet the observed mild phenotype, mixed success rate, and weak reproducibility urge for an expansion of available readout tools. For this purpose, we evaluated various measures that reflect early onset glaucomatous changes in the murine microbead occlusion model. Anterior chamber depth measurements and scotopic threshold response recordings were identified as an outstanding set of tools to assess the model’s success rate and to chart glaucomatous damage (or neuroprotection in future studies), respectively. Both are easy-to-measure, in vivo tools with a fast acquisition time and high translatability to the clinic and can be used, whenever judged beneficial, in combination with the more conventional measures in present-day glaucoma research (i.e., intraocular pressure measurements and post-mortem histological analyses). Furthermore, we highlighted the use of dendritic arbor analysis as an alternative histological readout for retinal ganglion cell density counts.

scholarly journals The mito-QC Reporter for Quantitative Mitophagy Assessment in Primary Retinal Ganglion Cells and Experimental Glaucoma Models

2020 ◽  
Vol 21 (5) ◽  
pp. 1882
Author(s):  
Ines Rosignol ◽  
Beatriz Villarejo-Zori ◽  
Petra Teresak ◽  
Elena Sierra-Filardi ◽  
Xandra Pereiro ◽  
...  
Keyword(s):  
Retinal Ganglion Cells ◽  
Ganglion Cells ◽  
Ex Vivo ◽  
Iron Chelator ◽  
Stress Factors ◽  
Retinal Ganglion ◽  
Nerve Crush ◽  
Experimental Glaucoma

Mitochondrial damage plays a prominent role in glaucoma. The only way cells can degrade whole mitochondria is via autophagy, in a process called mitophagy. Thus, studying mitophagy in the context of glaucoma is essential to understand the disease. Up to date limited tools are available for analyzing mitophagy in vivo. We have taken advantage of the mito-QC reporter, a recently generated mouse model that allows an accurate mitophagy assessment to fill this gap. We used primary RGCs and retinal explants derived from mito-QC mice to quantify mitophagy activation in vitro and ex vivo. We also analyzed mitophagy in retinal ganglion cells (RGCs), in vivo, using different mitophagy inducers, as well as after optic nerve crush (ONC) in mice, a commonly used surgical procedure to model glaucoma. Using mito-QC reporter we quantified mitophagy induced by several known inducers in primary RGCs in vitro, ex vivo and in vivo. We also found that RGCs were rescued from some glaucoma relevant stress factors by incubation with the iron chelator deferiprone (DFP). Thus, the mito-QC reporter-based model is a valuable tool for accurately analyzing mitophagy in the context of glaucoma.

The metalloproteinase ADAM10 is required for retinal ganglion cell axon guidance in the developing visual systems

2007 ◽  
Vol 30 (4) ◽  
pp. 77
Author(s):  
Y. Y. Chen ◽  
C. L. Hehr ◽  
K. Atkinson-Leadbeater ◽  
J. C. Hocking ◽  
S. McFarlane
Keyword(s):  
Ganglion Cell ◽  
Axon Guidance ◽  
Retinal Ganglion Cell ◽  
Growth Cone ◽  
Expression Patterns ◽  
Optic Tract ◽  
Axon Growth ◽  
Proteolytic Processing ◽  
Retinal Ganglion

Background: The growth cone interprets cues in its environment in order to reach its target. We want to identify molecules that regulate growth cone behaviour in the developing embryo. We investigated the role of A disintegrin and metalloproteinase 10 (ADAM10) in axon guidance in the developing visual system of African frog, Xenopus laevis. Methods: We first examined the expression patterns of adam10 mRNA by in situ hybridization. We then exposed the developing optic tract to an ADAM10 inhibitor, GI254023X, in vivo. Lastly, we inhibited ADAM10 function in diencephalic neuroepithelial cells (through which retinal ganglion cell (RGC) axons extend) or RGCs by electroporating or transfecting an ADAM10 dominant negative (dn-adam10). Results: We show that adam10 mRNA is expressed in the dorsal neuroepithelium over the time RGC axons extend towards their target, the optic tectum. Second, pharmacological inhibition of ADAM10 in an in vivo exposed brain preparation causes the failure of RGC axons to recognize their target at low concentrations (0.5, 1 μM), and the failure of the axons to make a caudal turn in the mid-diencephalon at higher concentration (5 μM). Thus, ADAM10 function is required for RGC axon guidance at two key guidance decisions. Finally, molecular inhibition of ADAM10 function by electroporating dn-adam10 in the brain neuroepithelium causes defects in RGC axon target recognition (57%) and/or defects in caudal turn (12%), as seen with the pharmacological inhibitor. In contrast, molecular inhibition of ADAM10 within the RGC axons has no effect. Conclusions: These data argue strongly that ADAM10 acts cell non-autonomously within the neuroepithelium to regulate the guidance of RGC axons. This study shows for the first time that a metalloproteinase acts in a cell non-autonomous fashion to direct vertebrate axon growth. It will provide important insights into candidate molecules that could be used to reform nerve connections if destroyed because of injury or disease. References Hattori M, Osterfield M, Flanagan JG. Regulated cleavage of a contact-mediated axon repellent. Science 2000; 289(5483):1360-5. Janes PW, Saha N, Barton WA, Kolev MV, Wimmer-Kleikamp SH, Nievergall E, Blobel CP, Himanen JP, Lackmann M, Nikolov DB. Adam meets Eph: an ADAM substrate recognition module acts as a molecular switch for ephrin cleavage in trans. Cell 2005; 123(2):291-304. Pan D, Rubin GM. Kuzbanian controls proteolytic processing of Notch and mediates lateral inhibition during Drosophila and vertebrate neurogenesis. Cell 1997; 90(2):271-80.

scholarly journals Optimization of spectral-domain optical coherence tomography with a supercontinuum source for in vivo motion detection of low reflective outer hair cells in guinea pig cochleae

2021 ◽  
Author(s):  
Fumiaki Nin ◽  
Samuel Choi ◽  
Takeru Ota ◽  
Zhang Qi ◽  
Hiroshi Hibino
Keyword(s):  
Optical Coherence Tomography ◽  
High Resolution ◽  
Guinea Pig ◽  
Hair Cells ◽  
Sensory Epithelium ◽  
Outer Hair Cells ◽  
Acquisition Time ◽  
Total Acquisition Time ◽  
Sd Oct

AbstractSound evokes sub-nanoscale vibration within the sensory epithelium. The epithelium contains not only immotile cells but also contractile outer hair cells (OHCs) that actively shrink and elongate synchronously with the sound. However, the in vivo motion of OHCs has remained undetermined. The aim of this work is to perform high-resolution and -accuracy vibrometry in live guinea pigs with an SC-introduced spectral-domain optical coherence tomography system (SD-OCT). In this study, to reveal the effective contribution of SC source in the recording of the low reflective materials with the short total acquisition time, we compare the performances of the SC-introduced SD-OCT (SCSD-OCT) to that of the conventional SD-OCT. As inanimate comparison objects, we record a mirror, a piezo actuator, and glass windows. For the measurements in biological materials, we use in/ex vivo guinea pig cochleae. Our study achieved the optimization of a SD-OCT system for high-resolution in vivo vibrometry in the cochlear sensory epithelium, termed the organ of Corti, in mammalian cochlea. By introducing a supercontinuum (SC) light source and reducing the total acquisition time, we improve the axial resolution and overcome the difficulty in recording the low reflective material in the presence of biological noise. The high power of the SC source enables the system to achieve a spatial resolution of 1.72 ± 0.00 μm on a mirror and reducing the total acquisition time contributes to the high spatial accuracy of sub-nanoscale vibrometry. Our findings reveal the vibrations at the apical/basal region of OHCs and the extracellular matrix, basilar membrane.

The effect of microvascular obstruction on the myocardial microstructure: a diffusion tensor imaging study

2021 ◽  
Vol 22 (Supplement_1) ◽  
Author(s):  
A Das ◽  
K Kelly ◽  
M Aldred ◽  
I Teh ◽  
CK Stoeck ◽  
...  
Keyword(s):  
Microvascular Obstruction ◽  
Diffusion Tensor ◽  
Spin Echo ◽  
Mean Diffusivity ◽  
Imaging Study ◽  
Free Breathing ◽  
Helix Angle ◽  
Acquisition Time ◽  
Heart Research

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Heart Research UK Background Diffusion tensor cardiac magnetic resonance (DT-CMR) imaging allows for characterising myocardial microstructure in-vivo using mean diffusivity (MD), fractional anisotropy (FA), secondary eigenvector angle (E2A) and helix angle (HA) maps. Following myocardial infarction (MI), alterations in MD, FA and HA proportions have previously been reported. E2A depicts the contractile state of myocardial sheetlets, however the behaviour of E2A in infarct segments, and all DTI markers in areas of microvascular obstruction (MVO) is also not fully understood.  Purpose We performed spin echo DTI in patients following ST-elevation MI (STEMI) in order to investigate acute changes in DTI parameters in remote and infarct segments both with and without MVO. Method Twenty STEMI patients (16 men, 4 women, mean age 59) had acute (5 ± 2d) 3T CMR scans. CMR protocol included: second order motion compensated (M012) free-breathing spin echo DTI (3 slices, 18 diffusion directions at b-values 100s/mm2[3], 200s/mm2[3] and 500s/mm2[12], reconstructed resolution was 1.66x1.66x8mm); cine and late gadolinium enhancement (LGE) imaging. Average MD, FA, E2A HA parameters were calculated on a  16 AHA segmental level. HA maps were described by dividing values into left-handed HA (LHM, -90° < HA < -30°), circumferential HA (CM, -30° < HA < 30°), and right-handed HA (RHM, 30° < HA < 90°) and reported as relative proportions. Segments were defined as infarct (positive for LGE) and remote (opposite to the infarct).  Results DTI acquisition was successful in all patients (acquisition time 13 ± 5mins). Ten patients had evidence of MVO on LGE images. MD was significantly higher in infarct regions in comparison to remote; MVO-ve infarct segments had significantly higher MD than MVO + ve infarct segments (MD remote= 1.46 ± 0.12x10-3mm2/s, MD MVO + ve = 1.59 ± 0.12x10-3mm2/s, MD MVO-ve  = 1.75 ± 0.12x10-3mm2/s, ANOVA p < 0.01). FA was reduced in infarct segments in comparison to remote; MVO-ve infarct segments had significantly lower FA than MVO + ve infarct segments (FAremote= 0.37 ± 0.02, FA MVO + ve = 0.31 ± 0.02 x 10-3mm2/s, MD MVO-ve =0.25 ± 0.02, ANOVA p < 0.01). E2A values were significantly lower in infarct segments compared to remote; MVO + ve infarct segments had significantly lower values than MVO-ve. (E2A remote= 57.4 ± 5.2°, E2A MVO-ve = 46.8 ± 2.5°, E2A MVO + ve = 36.8 ± 3.1°, ANOVA p < 0.001). RHM% (corresponding to subendocardium) was significantly lower in infarct segments compared to remote; MVO + ve infarct segments had significantly lower RHM% than MVO-ve. (RHM remote= 37 ± 3%, RHM RHM MVO-ve= 28 ± 7%, MVO + ve= 8 ± 5%, ANOVA p < 0.001). Conclusion The presence of MVO results in a decrease in MD and increase in FA in comparison to surrounding infarct segments. However, the reduction in E2A and right-handed myocytes on HA in infarct segments is further exacerbated by the presence of MVO. Further study is required to investigate the underlying mechanisms for such alterations in signal intensity. Abstract Figure. A case of transmural septal MI with MVO

scholarly journals Early Functional Impairment in Experimental Glaucoma Is Accompanied by Disruption of the GABAergic System and Inceptive Neuroinflammation

2021 ◽  
Vol 22 (14) ◽  
pp. 7581
Author(s):  
Oliver W. Gramlich ◽  
Cheyanne R. Godwin ◽  
David Wadkins ◽  
Benjamin W. Elwood ◽  
Markus H. Kuehn
Keyword(s):  
Axon Guidance ◽  
Gene Expression Analysis ◽  
Adenoviral Vectors ◽  
Significant Loss ◽  
Aminobutyric Acid ◽  
Complement Cascade ◽  
Retinal Ganglion ◽  
Functional Changes ◽  
Experimental Glaucoma ◽  
Iop Elevation

Glaucoma is a leading cause of irreversible blindness worldwide, and increased intraocular pressure (IOP) is a major risk factor. We aimed to determine if early functional and molecular differences in the glaucomatous retina manifest before significant retinal ganglion cell (RGC) loss is apparent. Adenoviral vectors expressing a pathogenic form of myocilin (Ad5.MYOC) were used to induce IOP elevation in C57BL/6 mice. IOP and pattern electroretinograms (pERG) were recorded, and retinas were prepared for RNA sequencing, immunohistochemistry, or to determine RGC loss. Ocular injection of Ad5.MYOC leads to reliable IOP elevation, resulting in significant loss of RGC after nine weeks. A significant decrease in the pERG amplitude was evident in eyes three weeks after IOP elevation. Retinal gene expression analysis revealed increased expression for 291 genes related to complement cascade, inflammation, and antigen presentation in hypertensive eyes. Decreased expression was found for 378 genes associated with the γ-aminobutyric acid (GABA)ergic and glutamatergic systems and axon guidance. These data suggest that early functional changes in RGC might be due to reduced GABAA receptor signaling and neuroinflammation that precedes RGC loss in this glaucoma model. These initial changes may offer new targets for early detection of glaucoma and the development of new interventions.

scholarly journals Endothelin 1-induced retinal ganglion cell death is largely mediated by JUN activation

2020 ◽  
Vol 11 (9) ◽  
Author(s):  
Olivia J. Marola ◽  
Stephanie B. Syc-Mazurek ◽  
Gareth R. Howell ◽  
Richard T. Libby
Keyword(s):  
Transcription Factor ◽  
Molecular Mechanisms ◽  
Ganglion Cells ◽  
Retinal Vessel ◽  
Vessel Diameter ◽  
Retinal Ganglion ◽  
Retinal Ganglion Cell Death ◽  
Ganglion Cell Death

Abstract Glaucoma is a neurodegenerative disease characterized by loss of retinal ganglion cells (RGCs), the output neurons of the retina. Multiple lines of evidence show the endothelin (EDN, also known as ET) system is important in glaucomatous neurodegeneration. To date, the molecular mechanisms within RGCs driving EDN-induced RGC death have not been clarified. The pro-apoptotic transcription factor JUN (the canonical target of JNK signaling) and the endoplasmic reticulum stress effector and transcription factor DNA damage inducible transcript 3 (DDIT3, also known as CHOP) have been shown to act downstream of EDN receptors. Previous studies demonstrated that JUN and DDIT3 were important regulators of RGC death after glaucoma-relevant injures. Here, we characterized EDN insult in vivo and investigated the role of JUN and DDIT3 in EDN-induced RGC death. To accomplish this, EDN1 ligand was intravitreally injected into the eyes of wildtype, Six3-cre+Junfl/fl (Jun−/−), Ddit3 null (Ddit3−/−), and Ddit3−/−Jun−/− mice. Intravitreal EDN1 was sufficient to drive RGC death in vivo. EDN1 insult caused JUN activation in RGCs, and deletion of Jun from the neural retina attenuated RGC death after EDN insult. However, deletion of Ddit3 did not confer significant protection to RGCs after EDN1 insult. These results indicate that EDN caused RGC death via a JUN-dependent mechanism. In addition, EDN signaling is known to elicit potent vasoconstriction. JUN signaling was shown to drive neuronal death after ischemic insult. Therefore, the effects of intravitreal EDN1 on retinal vessel diameter and hypoxia were explored. Intravitreal EDN1 caused transient retinal vasoconstriction and regions of RGC and Müller glia hypoxia. Thus, it remains a possibility that EDN elicits a hypoxic insult to RGCs, causing apoptosis via JNK-JUN signaling. The importance of EDN-induced vasoconstriction and hypoxia in causing RGC death after EDN insult and in models of glaucoma requires further investigation.

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