Degeneration of photoreceptors, retinal cells including retinal pigment epithelium, and retinal ganglion cells characterizes visual disorders involving retinal dysfunction. The efficient and targeted gene delivery into retinal cells is critical for treatment of a variety of visual disorders. Here, we report use of an OCT guided, spatially targeted, near-infrared laser microirradiation platform to successfully deliver genes encoding ambient-light activatable multi-characteristic opsin (MCO) to retinal cells in non-human primates. The efficacy of gene delivery was evaluated by fundus imaging of the reporter (mCherry) fluorescence, and immunohistochemistry. Functional improvement was measured by red-ERG which was attributed to MCO’s broadband activation spectrum.
We combine the real-time structural imaging capability of OCT with laser microirradiation for causing layer-specific dystrophies to mimic Retinitis Pigmentosa and dry-AMD model. In addition, we integrated an ERG-module for functional characterization after laser-injury to monitor the disease progression as well as to evaluate therapeutic efficacy. Here, we show creation of local atrophies with our combined OCT-Laser-ERG system in animal models and measurement of different cone and rod responses to focal stimulation light of different wavelengths. By varying the different mode of laser microirradiation and focal adjustment to the targeted depth, we demonstrate layer specific primary RPE injury.
We hypothesized that PEDF gene transduction in retina can provide single-dose treatment to prevent ganglion cell damage. Here, we present OCT guided ultrafast laser based non-viral targeted delivery PEDF-encoding genes to retina for neuroprotection. The ultrafast laser gene delivery showed layer-specific reliable expression of PEDF gene in retina without any detectable damage. Monitoring of IOP and electroretinogram after ultrafast laser transfection showed no adverse changes. The ultrafast laser transfection of large PEDF genes in retina exhibited significant therapeutic benefit in an injury model. Absence of any immune response in retina subsequent to ultrafast-laser transfection provides unique opportunity for repeated dosing.
Herein, we report use of near-infrared low-coherent light for non-contact, label-free in-vivo detection of retinal activity in response to visual illumination. Our multifractal phase-OCT employ phase/multifractal analysis to decipher layer specific cellular activity during visual stimulation to assess the functional state of retina. Our OCT-based interferometric technique coupled with in-depth multifractal analysis differentiated retinal activities between wild-type and mice with retinal dystrophy. Our findings open up possibility of clinical translation of multifractal phase-OCT for non-contact label free evaluation of retina health, progression of retinal dystrophies, and as well as for monitoring functional recovery after therapy.
The ability to both induce and monitor neural activity at cellular resolution is necessary to comprehend the activation paradigm in sensory processing and for the development of effective neuromodulation treatments. Our lab has produced an actuator-sensor construct via the hybridization of an optogenetic actuator (bMCOII) and bioluminescent Ca2+ sensor (GeNL) which allows continuous monitoring of neural activity with high spatiotemporal resolution. Modeling revealed that the construct is bound to the cell membrane through 14 transmembrane helices with the Ca2+ -bioluminescence indicator domain 20-40 Å inside the cytoplasm (membrane localization confirmed by imaging). The construct can be used to stimulate neural activity with very low intensity (10 μW/mm2 ) light but unlike fluorescent methods, requires no excitation light. Activation of the opsin causes influx of Ca2+ by opening MCOII-channels via trans-cis isomerization of all-trans-retinal. When these ions bind the indicator domain there is an increase in bioluminescence intensity. We observed significant correlation between the magnitude and kinetics of induced electrical activities and Ca2+ -bioluminescence. Longterm (>14 hours) recording of evoked neural activity in the visual cortices of murine models allowed the quantification of the strength of sensory activation. Additionally, through Artificial Intelligence-based Neural Activation Parameters, the Ca2+ -bioluminescence signals were used to map network activity patterns. We also observed delayed, secondary Ca2+ - bioluminescence responses murine visual cortices. These may be astrocyte-mediated responses to direct optogenetic and indirect visual stimulation. Our technique will enable the development of a scalable, and modular interface system which can be expanded to monitor and modulate a variety of neurological activities.
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