Related Experiment Video
Updated: Mar 28, 2026
An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury
Published on: December 31, 2017
In vivo cellular imaging of various stress/response pathways using AAV following axonal injury in mice
Kosuke Fujita1, Koji M Nishiguchi2, Yu Yokoyama3
1Department of Retinal Disease Control, Graduate School of Medicine, Tohoku University, Sendai, 980-8574, Japan.
Abstract:
Glaucoma, a leading cause of blindness worldwide, is instigated by various factors, including axonal injury, which eventually leads to a progressive loss of retinal ganglion cells (RGCs). To study various pathways reportedly involved in the pathogenesis of RGC death caused by axonal injury, seven pathways were investigated. Pathway-specific fluorescent protein-coded reporters were each packaged into an adeno-associated virus (AAV). After producing axonal injury in the eye, injected with AAV to induce RGC death, the temporal activity of each stress-related pathway was monitored in vivo through the detection of fluorescent RGCs using confocal ophthalmoscopy. We identified the activation of ATF6 and MCP-1 pathways involved in endoplasmic reticulum stress and macrophage recruitment, respectively, as early markers of RGC stress that precede neuronal death. Conversely, inflammatory responses probed by NF-κB and cell-death-related pathway p53 were most prominent in the later phases, when RGC death was already ongoing. AAV-mediated delivery of stress/response reporters followed by in vivo cellular imaging is a powerful strategy to characterize the temporal aspects of complex molecular pathways involved in retinal diseases. The identification of promoter elements that are activated before the death of RGCs enables the development of pre-emptive gene therapy, exclusively targeting the early phases of diseased cells.
Insights
Researchers identified early markers of retinal ganglion cell (RGC) stress in glaucoma using AAV-mediated reporters. This advance aids in developing pre-emptive gene therapy for this leading cause of blindness.
Area of Science:
- Ophthalmology and Neuroscience
- Molecular and Cellular Biology
Background:
- Glaucoma is a leading cause of global blindness, characterized by progressive retinal ganglion cell (RGC) death due to axonal injury.
- Understanding the molecular pathways involved in RGC pathogenesis is crucial for developing effective treatments.
Purpose of the Study:
- To investigate the temporal activity of seven key stress-related pathways implicated in RGC death following axonal injury.
- To identify early molecular markers of RGC stress preceding neuronal demise.
Main Methods:
- Adeno-associated virus (AAV) vectors encoding pathway-specific fluorescent reporters were developed.
- Axonal injury was induced in the eye, followed by AAV injection to label active pathways in RGCs.
- In vivo confocal ophthalmoscopy was used to monitor the temporal activation of fluorescent reporters in RGCs.
Main Results:
- Activation of the ATF6 (endoplasmic reticulum stress) and MCP-1 (macrophage recruitment) pathways were identified as early markers of RGC stress.
- The NF-κB (inflammatory response) and p53 (cell death) pathways showed peak activity during later stages of ongoing RGC death.
- AAV-mediated reporter delivery combined with in vivo imaging effectively characterized the temporal dynamics of molecular pathways in retinal disease.
Conclusions:
- ATF6 and MCP-1 pathway activation precede RGC death, offering potential early diagnostic markers for glaucoma.
- The study demonstrates the utility of AAV-based reporters for dissecting temporal molecular events in retinal diseases.
- Identifying early-activated promoter elements can facilitate the development of targeted pre-emptive gene therapies for RGC protection.
