Biology subjects

Johnson, T. V.

Publications and source records attributed to Johnson, T. V..

6 recordsLinked to original sources

bioRxiv2026

Computational aberration-corrected volumetric imaging of single retinal cells in the living eye

The eye offers a unique non-invasive window for accessing single-cell level structures and functions of the central nervous system (CNS) throughout the retina. However, strong and space-varying ocular aberrations, along with limited volume rates, challenge large-scale cellular imaging in living eyes and stymie the full potential of possible biological and pathological studies in retina. Here, we present plenoptic illumination scanning laser ophthalmoscopy (PI-SLO), a 3D fluorescent retinal imaging modality that enables high-speed, widefield, volumetric single-cell imaging with low phototoxicity. By capturing multiple angular images of fluorescence signals from the entire volume, PI-SLO enables digital aberration correction and 3D imaging across a >20{o} FOV with >23 Hz volume rate. We leverage this structural and functional imaging modality to investigate three key aspects of CNS physiology through the living mouse retina, including: microglial process dynamics, vascular perfusion, and light evoked calcium fluxes in inner retinal neurons. PI-SLO is a versatile non-invasive platform for in vivo investigation of retinal and CNS physiology at the cellular level.

bioengineering↗

bioRxiv2025

Photodisruption of the inner limiting membrane promotes retinal engraftment of stem-cell derived retinal ganglion cells

Glaucoma is the leading cause of irreversible blindness, driven by the progressive loss of retinal ganglion cells (RGCs). Stem cell-derived RGC transplantation could revolutionize glaucoma treatment, but the inner limiting membrane (ILM) remains a major obstacle by hindering cell migration into the retina. Interestingly, the ILM represents a double-edged sword for RGC engraftment: on the one hand, it greatly hinders cell migration, whereas on the other hand, its presence during retinal development is necessary for neuronal migration and retinal lamination. As an alternative to current invasive and harmful strategies to disrupt the ILM, we introduce ILM photodisruption, a minimally invasive biophotonic method that can manipulate the integrity of the ILM with unprecedented precision. In this study, we have finetuned the technology in bovine and human organotypic retinal explants to create templated ILM pores, creating entryways for donor RGCs to enter the retina while preserving most of the membrane to confer guidance cues for their engraftment. Applying this technology, we were able to promote donor RGC survival, enhance cell spreading and facilitate integration into the retina. Overall, our findings demonstrate that ILM photodisruption effectively addresses a key barrier in RGC replacement, paving the way for advancing retinal regeneration toward clinical application.

pharmacology and toxicology↗

bioRxiv2025

Compact lens-based dual-channel adaptive optics scanning laser ophthalmoscopy for in-vivo three-dimensional retinal imaging in mice

Adaptive optics (AO) enables cellular-resolution retinal imaging, yet mirror-based AOSLO systems are constrained by off-axis aberrations that restrict high-quality imaging to narrow fields of view, limiting in vivo studies of dynamic, large-scale retinal processes. Clinical translation of regenerative cell therapy to neurologic disease is hampered by attrition of donor neurons following transplantation. We hypothesized that early innate neuroinflammatory responses to retinal ganglion cell (RGC) transplantation underlie early death of donor cells and that next-generation imaging technologies would provide evidence for microglial attack of grafted neurons. We present a compact refractive lens-based AOSLO system that achieves two-color fluorescence imaging across up to a 16{degrees} field of view in mouse retina. Dual-wavelength excitation (488 nm and 552 nm) enables visualization of two fluorescence labels, while AO correction improves axial resolution and depth fidelity, allowing robust separation of structures through anatomical layers in retina. Using this platform, we performed 3D time-lapse imaging of microglia and longitudinal imaging in an optic nerve crush model, revealing layer-dependent differences in microglial motility, early activation signatures, and large-scale redistribution longitudinally. The system enabled widefield visualization of injury-associated vascular changes and spatial coupling between microglia and vasculature. Finally, depth-resolved two-color imaging captured immune responses to intravitreally transplanted RGCs, including host-cell recruitment, rapid neurite retraction following local immune-cell contact, and microglial phagocytosis of donor RGCs. Together, these results demonstrate that refractive AOSLO enables in vivo observations of microvascular organization, neuroimmune dynamics, injury responses, and transplanted-cell behavior with spatiotemporal resolution. Our data also suggests that modulation of microglial reactivity may improve outcomes of RGC transplantation. Significance StatementWide-field, depth-resolved imaging is essential for understanding how different cell types interact across a large retinal area, yet existing AOSLO systems retain limited imaging fields due to a conventional optical design using reflective spherical mirrors. Our refractive large-FOV AOSLO overcomes this limitation, enabling simultaneous two-color, diffraction-limited 3D imaging across an up to 16{degrees} field in vivo. This platform reveals previously inaccessible biological phenomena--including layer-specific microglial dynamics after optic nerve injury, microvascular remodeling, and rapid microglial rejection of transplanted RGCs--providing critical insight into neuroimmune behavior and retinal repair mechanisms at single-cell and subcellular resolution.

bioengineering↗

bioRxiv2023

Regional Gene Expression in the Retina, Optic Nerve Head, and Optic Nerve of Mice with Experimental Glaucoma and Optic Nerve Crush

A major risk factor for glaucomatous optic neuropathy is the level of intraocular pressure (IOP), which can lead to retinal ganglion cell axon injury and cell death. The optic nerve has a rostral unmyelinated portion at the optic nerve head followed by a caudal myelinated region. The unmyelinated region is differentially susceptible to IOP-induced damage in rodent models and in human glaucoma. While several studies have analyzed gene expression changes in the mouse optic nerve following optic nerve injury, few were designed to consider the regional gene expression differences that exist between these distinct areas. We performed bulk RNA-sequencing on the retina and on separately micro-dissected unmyelinated and myelinated optic nerve regions from naive C57BL/6 mice, mice after optic nerve crush, and mice with microbead-induced experimental glaucoma (total = 36). Gene expression patterns in the naive unmyelinated optic nerve showed significant enrichment of the Wnt, Hippo, PI3K-Akt, and transforming growth factor {beta} pathways, as well as extracellular matrix-receptor and cell membrane signaling pathways, compared to the myelinated optic nerve and retina. Gene expression changes induced by both injuries were more extensive in the myelinated optic nerve than the unmyelinated region, and greater after nerve crush than glaucoma. Changes three and fourteen days after injury largely subsided by six weeks. Gene markers of reactive astrocytes did not consistently differ between injury states. Overall, the transcriptomic phenotype of the mouse unmyelinated optic nerve was significantly different from immediately adjacent tissues, likely dominated by expression in astrocytes, whose junctional complexes are inherently important in responding to IOP elevation.

molecular biology↗

bioRxiv2022

Internal limiting membrane disruption facilitates engraftment of transplanted human stem cell derived retinal ganglion cells

Optic neuropathies cause irreversible vision loss as retinal ganglion cells (RGCs) die. Transplantation of pluripotent stem cell (PSC)-derived RGCs offers one potential therapeutic avenue to restore vision in patients suffering from optic neuropathy if the donor neurons survive long-term in the recipient eye and develop synaptic connections within the retinal inner plexiform layer (IPL) and subcortical visual centers (1). Thus far, attempts at intravitreal RGC transplantation have been hampered by sequestration on the epiretinal surface without engraftment into the retinal parenchyma. In mouse retinal explant cultures, enzymatic digestion of the retinal internal limiting membrane (ILM) promotes migration of transplanted RGCs into the recipient retina (2). Herein, we examined donor RGC survival and engraftment in living, immunosuppressed mice, rats, and rhesus macaques and in post-mortem human retinal explant cultures. Using 3 separate human PSC lines and 3 independent methods of ILM disruption, we demonstrate that the ILM is a barrier to the retinal engraftment of intravitreally delivered human PSC-derived RGCs. ILM disruption is associated with greater donor RGC survival over 2-8 weeks and enables migration of donor neuronal somas into the endogenous RGC layer where cells elaborate dendrites into the IPL and extend axons that follow the course of the endogenous retinal nerve fiber layer into the optic nerve head. Critically, ILM disruption enables donor RGCs to synaptically integrate into IPL circuits, conferring light responsivity. These findings have important implications for enabling neuronal replacement therapies to restore vision in patients with optic neuropathy. SIGNIFICANCE STATEMENTRetinal ganglion cell (RGC) replacement and optic nerve regeneration through transplantation of stem cell-derived RGCs holds potential for restoring vision lost to optic neuropathies. Here we demonstrate that intravitreally transplanted human RGCs laminate the epiretinal surface without projecting neurites into the retinal parenchyma. However, enzymatic, developmental and surgical disruption of the internal limiting membrane not only improves graft survival, but also enables structural and functional engraftment, with dendrites that stratify the inner plexiform layer, axons that grow into the optic nerve head, and acquired responsivity to light. These observations identify a translatable approach to enable transplantation-based RGC replacement for the treatment of optic neuropathy.

neuroscience↗

bioRxiv2020

Structural engraftment and topographic spacing of transplanted human stem cell-derived retinal ganglion cells

Retinal ganglion cell (RGC) replacement and optic nerve regeneration hold potential for restoring vision lost to optic neuropathy. Following transplantation, RGCs must integrate into the neuroretinal circuitry in order to receive afferent visual signals for processing and transmission to central targets. To date, the efficiency of RGC retinal integration following transplantation has been limited. We sought to characterize spontaneous interactions between transplanted human embryonic stem cell-derived RGCs and the recipient mature mammalian retina, and to identify and overcome barriers to the structural integration of transplanted neurons. Using an in vitro model system, following transplantation directly onto the inner surface of organotypic mouse retinal explants, human RGC somas form compact clusters and extend bundled neurites that remain superficial to the neural retinal tissue, hindering any potential for afferent synaptogenesis. To enhance integration, we explored methods to increase the cellular permeability of the internal limiting membrane (ILM). Digestion of extracellular matrix components using proteolytic enzymes was titrated to achieve disruption of the ILM while minimizing retinal toxicity and preserving endogenous retinal glial reactivity. Such ILM disruption is associated with dispersion rather than clustering of transplanted RGC bodies and neurites, and with a marked increase in transplanted RGC neurite extension into retinal parenchyma. The ILM appears to be a barrier to afferent retinal connectivity by transplanted RGCs and its circumvention may be necessary for successful functional RGC replacement through transplantation.

neuroscience↗