Adeno-associated virus gene delivery targeting corneal-projecting trigeminal ganglion neurons
7 affiliations
- 1Center for Excellence in the Neurosciences, University of New England, Biddeford, ME, United States
- 2Graduate School of Biomedical Science and Engineering, University of Maine, Orono, ME, United States
- 3College of Optometry, University of Houston, Houston, TX, United States
- 4Department of Ophthalmology, Jules Stein Eye Institute, University of California, Los Angeles, Los Angeles, CA, United States
- 5Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA, United States
- 6Department of Neurobiology, University of Pittsburgh, Pittsburgh, PA, United States
- 7Department of Immunology, University of Pittsburgh, Pittsburgh, PA, United States
Abstract
Corneal innervation is essential for ocular surface homeostasis and protection, yet methods for selectively labeling and manipulating cornea-projecting neurons remain limited. Conventional retrograde tracers can identify cornea-projecting cell bodies in the trigeminal ganglion (TG), but they do not enable the expression of genetically encoded effector proteins, sensors, or recombinases. We describe and validate intrastromal adeno-associated virus (AAV) delivery as an approach for labeling cornea-projecting TG neurons in mice. A 1–2-μL viral injection was delivered into the corneal stroma and labeling was assessed 2–12 weeks later in cleared whole TG, TG sections, corneal whole mounts, and thick brainstem sections. Multiple AAV vectors, including tdTomato-, eGFP-, or mCherry-expressing vectors and Cre-expressing vectors in Ai14 tdTomato reporter mice, produced consistent neuronal labeling concentrated in the dorsomedial ophthalmic division of the TG. Native tdTomato fluorescence was readily detected, and eGFP labeling could be enhanced using immunolabeling with fluorophore-enhancing antibodies, increasing detection of labeled somata and axonal fibers. AAV-mediated labeling in the TG substantially overlapped with WGA and Fast Blue tracing from the cornea, although AAV labeled fewer neurons overall. Cre-expressing AAV vectors activated reporter expression in Ai14 mice, demonstrating compatibility with transgenic reporter strategies. Labeled axons were visualized at both peripheral and central projection sites. At the ocular surface, reporter expression was correlated to the extent of neuronal transduction. Central projections were observed in subnucleus principalis, the interpolaris/caudalis transition zone, and caudal subnucleus caudalis extending into upper cervical spinal cord. AAV transduction resulted in no gross immune activation. These findings indicate that intrastromal AAV delivery provides a versatile approach for anatomical tracing and genetic access to cornea-projecting neurons, enabling expression of genetically encoded tools for optogenetic and chemogenetic control, activity monitoring, and targeted genetic manipulation.
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BibTeX
@article{reynolds2026adenoassociated,
title = {{Adeno-associated virus gene delivery targeting corneal-projecting trigeminal ganglion neurons}},
author = {Samuel S. Reynolds and Md Zeeshan Rasheed and Ken Niyonkuru and Sachin Parikh and Kyle Whyland and Brooklynn M. Merrill and William Perez and Sreya Mitra and Tomasz Zal and Hongmin Yun and Brian Davis and Anthony J. St. Leger and Anna Matynia and Ian D. Meng},
journal = {Frontiers in Cellular Neuroscience},
year = {2026},
month = oct,
doi = {10.3389/fncel.2026.1925806},
url = {https://doi.org/10.3389/fncel.2026.1925806},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Adeno-associated virus gene delivery targeting corneal-projecting trigeminal ganglion neurons
AU - Samuel S. Reynolds
AU - Md Zeeshan Rasheed
AU - Ken Niyonkuru
AU - Sachin Parikh
AU - Kyle Whyland
AU - Brooklynn M. Merrill
AU - William Perez
AU - Sreya Mitra
AU - Tomasz Zal
AU - Hongmin Yun
AU - Brian Davis
AU - Anthony J. St. Leger
AU - Anna Matynia
AU - Ian D. Meng
PY - 2026
DA - 2026/10/22
JO - Frontiers in Cellular Neuroscience
DO - 10.3389/fncel.2026.1925806
UR - https://doi.org/10.3389/fncel.2026.1925806
AB - Corneal innervation is essential for ocular surface homeostasis and protection, yet methods for selectively labeling and manipulating cornea-projecting neurons remain limited. Conventional retrograde tracers can identify cornea-projecting cell bodies in the trigeminal ganglion (TG), but they do not enable the expression of genetically encoded effector proteins, sensors, or recombinases. We describe and validate intrastromal adeno-associated virus (AAV) delivery as an approach for labeling cornea-projecting TG neurons in mice. A 1–2-μL viral injection was delivered into the corneal stroma and labeling was assessed 2–12 weeks later in cleared whole TG, TG sections, corneal whole mounts, and thick brainstem sections. Multiple AAV vectors, including tdTomato-, eGFP-, or mCherry-expressing vectors and Cre-expressing vectors in Ai14 tdTomato reporter mice, produced consistent neuronal labeling concentrated in the dorsomedial ophthalmic division of the TG. Native tdTomato fluorescence was readily detected, and eGFP labeling could be enhanced using immunolabeling with fluorophore-enhancing antibodies, increasing detection of labeled somata and axonal fibers. AAV-mediated labeling in the TG substantially overlapped with WGA and Fast Blue tracing from the cornea, although AAV labeled fewer neurons overall. Cre-expressing AAV vectors activated reporter expression in Ai14 mice, demonstrating compatibility with transgenic reporter strategies. Labeled axons were visualized at both peripheral and central projection sites. At the ocular surface, reporter expression was correlated to the extent of neuronal transduction. Central projections were observed in subnucleus principalis, the interpolaris/caudalis transition zone, and caudal subnucleus caudalis extending into upper cervical spinal cord. AAV transduction resulted in no gross immune activation. These findings indicate that intrastromal AAV delivery provides a versatile approach for anatomical tracing and genetic access to cornea-projecting neurons, enabling expression of genetically encoded tools for optogenetic and chemogenetic control, activity monitoring, and targeted genetic manipulation.
SN - 1662-5102
ER -