Genetically Encodable Contrast Agents for Optical Coherence Tomography

Optical coherence tomography (OCT) has gained wide adoption in biological research and medical imaging due to its exceptional tissue penetration, 3D imaging speed, and rich contrast. However, OCT plays a relatively small role in molecular and cellular imaging due to the lack of suitable biomolecular contrast agents. In particular, while the green fluorescent protein has provided revolutionary capabilities to fluorescence microscopy by connecting it to cellular functions such as gene expression, no equivalent reporter gene is currently available for OCT. Here, we introduce gas vesicles, a class of naturally evolved gas-filled protein nanostructures, as genetically encodable OCT contrast agents. The differential refractive index of their gas compartments relative to surrounding aqueous tissue and their nanoscale motion enables gas vesicles to be detected by static and dynamic OCT. Furthermore, the OCT contrast of gas vesicles can be selectively erased in situ with ultrasound, allowing unambiguous assignment of their location. In addition, gas vesicle clustering modulates their temporal signal, enabling the design of dynamic biosensors. We demonstrate the use of gas vesicles as reporter genes in bacterial colonies and as purified contrast agents in vivo in the mouse retina. Our results expand the utility of OCT to image a wider variety of cellular and molecular processes

Genetically Encodable Contrast Agents for Optical Coherence Tomography

Optical coherence tomography (OCT) has gained wide adoption in biological research and medical imaging due to its exceptional tissue penetration, 3D imaging speed, and rich contrast. However, OCT plays a relatively small role in molecular and cellular imaging due to the lack of suitable biomolecular contrast agents. In particular, while the green fluorescent protein has provided revolutionary capabilities to fluorescence microscopy by connecting it to cellular functions such as gene expression, no equivalent reporter gene is currently available for OCT. Here, we introduce gas vesicles, a class of naturally evolved gas-filled protein nanostructures, as genetically encodable OCT contrast agents. The differential refractive index of their gas compartments relative to surrounding aqueous tissue and their nanoscale motion enables gas vesicles to be detected by static and dynamic OCT. Furthermore, the OCT contrast of gas vesicles can be selectively erased in situ with ultrasound, allowing unambiguous assignment of their location. In addition, gas vesicle clustering modulates their temporal signal, enabling the design of dynamic biosensors. We demonstrate the use of gas vesicles as reporter genes in bacterial colonies and as purified contrast agents in vivo in the mouse retina. Our results expand the utility of OCT to image a wider variety of cellular and molecular processes

Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death

Glaucoma, a major cause of blindness worldwide, is a neurodegenerative optic neuropathy in which vision loss is caused by loss of retinal ganglion cells (RGCs). To better define the pathways mediating RGC death and identify targets for the development of neuroprotective drugs, we developed a high-throughput RNA interference screen with primary RGCs and used it to screen the full mouse kinome. The screen identified dual leucine zipper kinase (DLK) as a key neuroprotective target in RGCs. In cultured RGCs, DLK signaling is both necessary and sufficient for cell death. DLK undergoes robust posttranscriptional up-regulation in response to axonal injury in vitro and in vivo. Using a conditional knockout approach, we confirmed that DLK is required for RGC JNK activation and cell death in a rodent model of optic neuropathy. In addition, tozasertib, a small molecule protein kinase inhibitor with activity against DLK, protects RGCs from cell death in rodent glaucoma and traumatic optic neuropathy models. Together, our results establish a previously undescribed drug/drug target combination in glaucoma, identify an early marker of RGC injury, and provide a starting point for the development of more specific neuroprotective DLK inhibitors for the treatment of glaucoma, nonglaucomatous forms of optic neuropathy, and perhaps other CNS neurodegenerations

Live, die, or regenerate? New insights from multi-omic analyses

In this issue of Neuron, three studies establish new strategies to uncover mediators of retinal neuroprotection and optic nerve regeneration. Tian et al. (2022) carry out a multi-omics screen and identify transcriptional regulators of axon injury signaling leading to cell death; Jacobi et al. (2022) and Li et al. (2022) combine retrograde tracing and single-cell RNA-seq (scRNA-seq) to uncover molecular targets for axon regeneration

Aqueous shunts withmitomycin C versus aqueous shunts alone for glaucoma

10.1002/14651858.CD011875.pub2COCHRANE DATABASE OF SYSTEMATIC REVIEWS2019

Sheath-Preserving Optic Nerve Transection in Rats to Assess Axon Regeneration and Interventions Targeting the Retinal Ganglion Cell Axon.

Retinal ganglion cell (RGC) axons converge at the optic nerve head to convey visual information from the retina to the brain. Pathologies such as glaucoma, trauma, and ischemic optic neuropathies injure RGC axons, disrupt transmission of visual stimuli, and cause vision loss. Animal models simulating RGC axon injury include optic nerve crush and transection paradigms. Each of these models has inherent advantages and disadvantages. An optic nerve crush is generally less severe than a transection and can be used to assay axon regeneration across the lesion site. However, differences in crush force and duration can affect tissue responses, resulting in variable reproducibility and lesion completeness. With optic nerve transection, there is a severe and reproducible injury that completely lesions all axons. However, transecting the optic nerve dramatically alters the blood brain barrier by violating the optic nerve sheath, exposing the optic nerve to the peripheral environment. Moreover, regeneration beyond a transection site cannot be assessed without reapposing the cut nerve ends. Furthermore, distinct degenerative changes and cellular pathways are activated by either a crush or transection injury. The method described here incorporates the advantages of both optic nerve crush and transection models while mitigating the disadvantages. Hydrostatic pressure delivered into the optic nerve by microinjection completely transects the optic nerve while maintaining the integrity of the optic nerve sheath. The transected optic nerve ends are reapposed to allow for axon regeneration assays. A potential limitation of this method is the inability to visualize the complete transection, a potential source of variability. However, visual confirmation that the visible portion of the optic nerve has been transected is indicative of a complete optic nerve transection with 90-95% success. This method could be applied to assess axon regeneration promoting strategies in a transection model or investigate interventions that target the axonal compartments

Polymorphous low-grade adenocarcinoma with cavernous sinus involvement presenting as third nerve palsy.

PurposePolymorphous low-grade adenocarcinoma is a tumor of the salivary glands that typically localizes within the oral cavity. We present a case of isolated third cranial nerve palsy as the initial presentation of polymorphous low-grade adenocarcinoma involving the left cavernous sinus in a patient status post glaucoma surgery.ObservationsA 68-year-old woman status post glaucoma drainage device implantation in her left eye presented with an isolated left third nerve palsy ten weeks postoperatively. Differential diagnoses included microvascular ischemic neuropathy, postoperative ptosis, and compressive mass. MRI revealed a left cavernous sinus mass, and subsequent excisional biopsy revealed a diagnosis of polymorphous low-grade adenocarcinoma.ConclusionsThere are few cases reporting polymorphous low-grade adenocarcinoma originating from and extending beyond the nasopharynx. This report emphasizes an unexpected neuro-ophthalmic manifestation of this salivary gland tumor

Optic Nerve Engraftment of Neural Stem Cells.

PurposeTo evaluate the integrative potential of neural stem cells (NSCs) with the visual system and characterize effects on the survival and axonal regeneration of axotomized retinal ganglion cells (RGCs).MethodsFor in vitro studies, primary, postnatal rat RGCs were directly cocultured with human NSCs or cultured in NSC-conditioned media before their survival and neurite outgrowth were assessed. For in vivo studies, human NSCs were transplanted into the transected rat optic nerve, and immunohistology of the retina and optic nerve was performed to evaluate RGC survival, RGC axon regeneration, and NSC integration with the injured visual system.ResultsIncreased neurite outgrowth was observed in RGCs directly cocultured with NSCs. NSC-conditioned media demonstrated a dose-dependent effect on RGC survival and neurite outgrowth in culture. NSCs grafted into the lesioned optic nerve modestly improved RGC survival following an optic nerve transection (593 ± 164 RGCs/mm2 vs. 199 ± 58 RGCs/mm2; P < 0.01). Additionally, RGC axonal regeneration following an optic nerve transection was modestly enhanced by NSCs transplanted at the lesion site (61.6 ± 8.5 axons vs. 40.3 ± 9.1 axons, P < 0.05). Transplanted NSCs also differentiated into neurons, received synaptic inputs from regenerating RGC axons, and extended axons along the transected optic nerve to incorporate with the visual system.ConclusionsHuman NSCs promote the modest survival and axonal regeneration of axotomized RGCs that is partially mediated by diffusible NSC-derived factors. Additionally, NSCs integrate with the injured optic nerve and have the potential to form neuronal relays to restore retinofugal connections

Optic Nerve Engraftment of Neural Stem Cells.

PurposeTo evaluate the integrative potential of neural stem cells (NSCs) with the visual system and characterize effects on the survival and axonal regeneration of axotomized retinal ganglion cells (RGCs).MethodsFor in vitro studies, primary, postnatal rat RGCs were directly cocultured with human NSCs or cultured in NSC-conditioned media before their survival and neurite outgrowth were assessed. For in vivo studies, human NSCs were transplanted into the transected rat optic nerve, and immunohistology of the retina and optic nerve was performed to evaluate RGC survival, RGC axon regeneration, and NSC integration with the injured visual system.ResultsIncreased neurite outgrowth was observed in RGCs directly cocultured with NSCs. NSC-conditioned media demonstrated a dose-dependent effect on RGC survival and neurite outgrowth in culture. NSCs grafted into the lesioned optic nerve modestly improved RGC survival following an optic nerve transection (593 ± 164 RGCs/mm2 vs. 199 ± 58 RGCs/mm2; P < 0.01). Additionally, RGC axonal regeneration following an optic nerve transection was modestly enhanced by NSCs transplanted at the lesion site (61.6 ± 8.5 axons vs. 40.3 ± 9.1 axons, P < 0.05). Transplanted NSCs also differentiated into neurons, received synaptic inputs from regenerating RGC axons, and extended axons along the transected optic nerve to incorporate with the visual system.ConclusionsHuman NSCs promote the modest survival and axonal regeneration of axotomized RGCs that is partially mediated by diffusible NSC-derived factors. Additionally, NSCs integrate with the injured optic nerve and have the potential to form neuronal relays to restore retinofugal connections