Research Article
Volume 12 Issue 11 - 2021
Light-Sheet Microscopy of the Optic Nerve Reveals Axonal Degeneration and Microglial Activation in NMDA-Induced Retinal Injury
Yonju Ha1*, Lorenzo F Ochoa2, Olivia Solomon2,3, Shuizhen Shi1, Paula P Villarreal2, Shengguo Li1, Seth Buscho1, Gracie Vargas2*, Wenbo Zhang1,2*
1Department of Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston, Texas, USA
2Departments of Neuroscience, Cell Biology and Anatomy, University of Texas Medical Branch, Galveston, Texas, USA
3Human Pathophysiology and Translational Medicine Graduate Program, University of Texas Medical Branch, Galveston, Texas, USA
*Corresponding Author: Wenbo Zhang, Department of Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston, Texas, USA, Gracie Vargas, Department of Neuroscience, Cell Biology and Anatomy, University of Texas Medical Branch, Galveston, Texas, USA and Yonju Ha Department of Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston, Texas, USA.
Received: October 15, 2021; Published: October 28, 2021


Purpose: Optic nerve degeneration is a feature of neurodegenerative eye diseases and causes irreversible vision loss. Therefore, understanding the degeneration patterns of the optic nerve is critical to identify potential therapeutic targets for optic neuropathy. However, the traditional method of analyzing optic nerve degeneration by histology has the limitation of losing information about spatiotemporal tissue changes. Light sheet fluorescence microscopy (LSFM) is a fluorescence microscopy technique that allows capturing 3D images rapidly with high spatial optical resolution. In this study, we evaluated the application of LSFM on the optic nerve in a mouse model of N -methyl-D-aspartate (NMDA)-induced retinal injury.

Methods: NMDA or vehicle was injected to the vitreous of Thy1-CFP mice that express cyan fluorescent protein (CFP) in retinal ganglion cells (RGCs) and their axons. At 7 days after the injection, the retina and optic nerve were collected and immunostained with anti-Iba1 antibody. Retinal flatmounts were observed using confocal microscopy. The immunostained optic nerve was optically cleared with 2,2′-Thiodiethanol and mounted for LSFM imaging.

Results: We found significant loss of CFP-expressing RGCs and axon degeneration in retinal flatmounts at 7 days after NMDA injection. These data verified that NMDA induces the loss of RGCs and their axons, NMDA excitotoxicity induced microglial activation and leukostasis, including increased microglia number, the transformation of microglial morphology to amoeboid or rounded shape, and the increase of attached leukocytes to the vessel wall. Using LSFM, we observed that CFP-expressing nerve fiber was well organized and arranged in parallel in vehicle-treated optic nerve. In contrast, in NMDA-treated eyes, optic nerve showed axonal swelling and fragmentation, and loss of axonal density from the proximal to the distal regions. Furthermore, LSFM enabled the observation of microglial phenotypic transformation in the entire optic nerve. In NMDA-treated eyes, microglia displayed larger soma and shorter process with higher Iba1 expression within the entire optic nerve than those in the optic nerve of vehicle-treated eyes.

Conclusion: In summary, we demonstrated the applicability of LSFM to acquire 3D images of the optic nerve and revealed the complex spatial relationships between the axons and microglia within the entire optic nerve by single acquisition. We successfully obtained high-resolution 3D images of NMDA-induced optic neuropathy, including the clues for optic nerve degeneration such as axon swelling, axonal fragmentation, and microglial activation. Our study suggests that LSFM could be a useful technique to investigate the pathology of the optic nerve in neurodegenerative diseases.

Keywords: Mouse; Retina; Retinal Ganglion Cells; Microglia; Optic Nerve; NMDA; Light Sheet Fluorescence Microscopy


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Citation: Wenbo Zhang., et al. “Light-Sheet Microscopy of the Optic Nerve Reveals Axonal Degeneration and Microglial Activation in NMDA-Induced Retinal Injury”. EC Ophthalmology 12.11 (2021): 23-31.

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