Microvesicle release from inner segments of healthy photoreceptors is a conserved phenomenon in mammalian species.

Many inherited visual diseases arise from mutations that affect the structure and function of photoreceptor cells. In some cases, the pathology is accompanied by a massive release of extracellular vesicles from affected photoreceptors. In this study, we addressed whether vesicular release is an exclusive response to ongoing pathology or a normal homeostatic phenomenon amplified in disease. We analyzed the ultrastructure of normal photoreceptors from both rod- and cone-dominant mammalian species and found that these cells release microvesicles budding from their inner segment compartment. Inner segment-derived microvesicles vary in their content, with some of them containing the visual pigment rhodopsin and others appearing to be interconnected with mitochondria. These data suggest the existence of a fundamental process whereby healthy mammalian photoreceptors release mistrafficked or damaged inner segment material as microvesicles into the interphotoreceptor space. This release may be greatly enhanced under pathological conditions associated with defects in protein targeting and trafficking. This article has an associated First Person interview with the first author of the paper.

Pre-retinal delivery of recombinant adeno-associated virus vector significantly improves retinal transduction efficiency.

Intravitreal injection is the most widely used injection technique for ocular gene delivery. However, vector diffusion is attenuated by physical barriers and neutralizing antibodies in the vitreous. The 13-lined ground squirrel (13-LGS), as in humans, has a larger relative vitreous body volume than the more common rodent models such as rats and mice, which would further reduce transduction efficiency with the intravitreal injection route. We report here a "pre-retinal" injection approach that leads to detachment of the posterior hyaloid membrane and delivers vector into the space between vitreous and inner retina. Vectors carrying a ubiquitously expressing mCherry reporter were injected into the deep vitreous or pre-retinal space in adult wild-type 13-LGSs. Then, adeno-associated virus (AAV)-mediated mCherry expression was evaluated with non-invasive imaging, immunofluorescence, and flow cytometry. Compared to deep vitreous delivery, pre-retinal administration achieved pan-retinal gene expression with a lower vector dose volume and significantly increased the number of transduced cone photoreceptors. These results suggest that pre-retinal injection is a promising tool in the development of gene therapy strategies in animal models and is a potential approach for use in human research, particularly in younger individuals with an intact posterior hyaloid membrane and stable vitreous.

Ultrathin micromolded 3D scaffolds for high-density photoreceptor layer reconstruction.

Polymeric scaffolds are revolutionizing therapeutics for blinding disorders affecting the outer retina, a region anatomically and functionally defined by light-sensitive photoreceptors. Recent engineering advances have produced planar scaffolds optimized for retinal pigment epithelium monolayer delivery, which are being tested in early-stage clinical trials. We previously described a three-dimensional scaffold supporting a polarized photoreceptor monolayer, but photoreceptor somata typically occupy multiple densely packed strata to maximize light detection. Thus, patients with severe photoreceptor degeneration are expected to extract greater benefits from higher-density photoreceptor delivery. Here, we describe the microfabrication of a biodegradable scaffold patterned for high-density photoreceptor replacement. The "ice cube tray" structure optimizes mechanical properties and cell-to-biomaterial load, enabling production of a multicellular photoreceptor layer designed for outer retinal reconstruction. Our approach may also be useful in the production of a multitude of micro- and nanoscale structures for multilayered cell delivery in other tissues.

Measuring the spatial distribution of multiply scattered light using a de-scanned image sensor for examining retinal structure contrast.

An optical platform is presented for examining intrinsic contrast detection strategies when imaging retinal structure using ex vivo tissue. A custom microscope was developed that scans intact tissue and collects scattered light distribution at every image pixel, allowing digital masks to be applied after image collection. With this novel approach at measuring the spatial distribution of multiply scattered light, known and novel methods of detecting intrinsic cellular contrast can be explored, compared, and optimized for retinal structures of interest.

Electroretinogram of the Cone-Dominant Thirteen-Lined Ground Squirrel during Euthermia and Hibernation in Comparison with the Rod-Dominant Brown Norway Rat.

Purpose: The majority of small animal species used in research are nocturnal, with retinae that are anatomically and functionally dissimilar from humans, complicating their use as disease models. Herein we characterize the retinal structure and electrophysiological function of the diurnal, cone-dominant 13-lined ground squirrel (13-LGS) retina during euthermia and in hibernation. Methods: Full-field electroretinography (ERG) was performed in 13-LGS and Brown Norway (BN) rat models to establish baseline values for retinal function in each species, including following intravitreal injection of pharmacologic agents to selectively block the contributions of ON- and OFF-bipolar cells. The effect of hibernation-associated retinal remodeling on electrophysiological function was assessed in 13-LGS during torpor and emergence, with correlative histology performed using transmission electron microscopy. Results: Under light-adapted conditions, the a-, b-, and d-wave amplitude of the 13-LGS was significantly greater than that of the BN rat. Retinal function was absent in the 13-LGS during hibernation and correlated to widespread disruption of photoreceptor and RPE structure. Remarkably, both retinal function and structure recovered rapidly on emergence from hibernation, with ERG responses reaching normal amplitude within 6 hours. Conclusions: ERG responses for both BN rats and 13-LGS reflect the relative proportions of cone photoreceptors present within the retinae, indicating that the cone-dominant 13-LGS may be a potentially useful model for studying human central retinal function and disease. That retinal remodeling and restoration of electrophysiological function occurs rapidly on emergence from hibernation implies the 13-LGS may also be a useful tool for studying aspects of retinal physiology and recovery from injury.

Diurnal rodents as pertinent animal models of human retinal physiology and pathology.

This presentation will survey the retinal architecture, advantages, and limitations of several lesser-known rodent species that provide a useful diurnal complement to rats and mice. These diurnal rodents also possess unusually cone-rich photoreceptor mosaics that facilitate the study of cone cells and pathways. Species to be presented include principally the Sudanian Unstriped Grass Rat and Nile Rat (Arvicanthis spp.), the Fat Sand Rat (Psammomys obesus), the degu (Octodon degus) and the 13-lined ground squirrel (Ictidomys tridecemlineatus). The retina and optic nerve in several of these species demonstrate unusual resilience in the face of neuronal injury, itself an interesting phenomenon with potential translational value.

Noninvasive Imaging and Correlative Histology of Cone Photoreceptor Structure in the Pig Retina.

PURPOSE: To evaluate different methods of studying cone photoreceptor structure in wild-type (WT) and transgenic pigs carrying the human rhodopsin P23H mutant gene (TgP23H). METHODS: For in vivo imaging, pigs were anesthetized with tiletamine-zolazepam and isoflurane and given lidocaine-bupivacaine retrobulbar injections. Stay sutures and a custom head mount were used to hold and steer the head for adaptive optics scanning light ophthalmoscopy (AOSLO). Six WT and TgP23H littermates were imaged at postnatal day 30 (P30), P90, and P180 with AOSLO and optical coherence tomography (OCT), and two additional sets of littermates were imaged at P3 and P15 with OCT only. AOSLO imaging and correlative differential interference contrast microscopy were performed on a P240 WT pig and on WT and TgP23H littermates at P30 and P180. RESULTS: AOSLO cone density generally underestimates histology density (mean difference ± SD = 24.8% ± 21.4%). The intensity of the outer retinal hyperreflective OCT band attributed to photoreceptors is attenuated in TgP23H pigs at all ages. In contrast, AOSLO images show cones that retain inner and outer segments through P180. At retinal locations outside the visual streak, TgP23H pigs show a heterogeneous degenerating cone mosaic by using two criteria: variable contrast on a split detector AOSLO and high reflectivity on a confocal AOSLO. CONCLUSIONS: AOSLO reveals that the cone mosaic is similar to ex vivo histology. Its use as a noninvasive tool will enable observation of morphologic changes that arise in the cone mosaic of TgP23H pigs over time. TRANSLATIONAL RELEVANCE: Pigs are widely used for translational studies, and the ability to noninvasively assess cellular changes in the cone mosaic will facilitate more detailed investigations of new retinal disease models as well as outcomes of potential therapies.

Interocular symmetry, intraobserver repeatability, and interobserver reliability of cone density measurements in the 13-lined ground squirrel.

BACKGROUND: The 13-lined ground squirrel (13-LGS) possesses a cone-dominant retina that is highly amenable to non-invasive high-resolution retinal imaging. The ability for longitudinal assessment of a cone-dominant photoreceptor mosaic with an adaptive optics scanning light ophthalmoscope (AOSLO) has positioned the 13-LGS to become an accessible model for vision research. Here, we examine the interocular symmetry, repeatability, and reliability of cone density measurements in the 13-LGS. METHODS: Thirteen 13-LGS (18 eyes) were imaged along the vertical meridian with a custom AOSLO. Regions of interest were selected superior and inferior to the optic nerve head, including the cone-rich visual streak. Non-confocal split-detection was used to capture images of the cone mosaic. Five masked observers each manually identified photoreceptors for 26 images three times and corrected an algorithm's cell identification outputs for all 214 images three times. Intraobserver repeatability and interobserver reliability of cone density were characterized using data collected from all five observers, while interocular symmetry was assessed in five animals using the average values of all observers. The distribution of image quality for all images in this study was assessed with open-sourced software. RESULTS: Manual identification was less repeatable than semi-automated correction for four of the five observers. Excellent repeatability was seen from all observers (ICC = 0.997-0.999), and there was good agreement between repeat cell identification corrections in all five observers (range: 9.43-25.71 cells/degree2). Reliability of cell identification was significantly different in two of the five observers, and worst in images taken from hibernating 13-LGS. Interocular symmetry of cone density was seen in the five 13-LGS assessed. Image quality was variable between blur- and pixel intensity-based metrics. CONCLUSIONS: Interocular symmetry with repeatable cone density measurements suggest that the 13-LGS is well-suited for longitudinal examination of the cone mosaic using split-detection AOSLO. Differences in reliability highlight the importance of observer training and automation of AOSLO cell detection. Cone density measurements from hibernating 13-LGS are not repeatable. Additional studies are warranted to assess other metrics of cone health to detect deviations from normal 13-LGS in future models of cone disorder in this species.

Noninvasive imaging of the tree shrew eye: Wavefront analysis and retinal imaging with correlative histology.

Tree shrews are small mammals with excellent vision and are closely related to primates. They have been used extensively as a model for studying refractive development, myopia, and central visual processing and are becoming an important model for vision research. Their cone dominant retina (∼95% cones) provides a potential avenue to create new damage/disease models of human macular pathology and to monitor progression or treatment response. To continue the development of the tree shrew as an animal model, we provide here the first measurements of higher order aberrations along with adaptive optics scanning light ophthalmoscopy (AOSLO) images of the photoreceptor mosaic in the tree shrew retina. To compare intra-animal in vivo and ex vivo cone density measurements, the AOSLO images were matched to whole-mount immunofluorescence microscopy. Analysis of the tree shrew wavefront indicated that the optics are well-matched to the sampling of the cone mosaic and is consistent with the suggestion that juvenile tree shrews are nearly emmetropic (slightly hyperopic). Compared with in vivo measurements, consistently higher cone density was measured ex vivo, likely due to tissue shrinkage during histological processing. Tree shrews also possess massive mitochondria ("megamitochondria") in their cone inner segments, providing a natural model to assess how mitochondrial size affects in vivo retinal imagery. Intra-animal in vivo and ex vivo axial distance measurements were made in the outer retina with optical coherence tomography (OCT) and transmission electron microscopy (TEM), respectively, to determine the origin of sub-cellular cone reflectivity seen on OCT. These results demonstrate that these megamitochondria create an additional hyper-reflective outer retinal reflective band in OCT images. The ability to use noninvasive retinal imaging in tree shrews supports development of this species as a model of cone disorders.

Evaluating seasonal changes of cone photoreceptor structure in the 13-lined ground squirrel.

Cone photoreceptors of the 13-lined ground squirrel (13-LGS) undergo reversible structural changes during hibernation, including cone outer segment disc degeneration and inner segment mitochondria depletion. Here, we evaluated cone structure with adaptive optics scanning light ophthalmoscopy (AOSLO) before, during, and after hibernation. Also, intra-animal comparisons of cone structure were made at distinct physiological states (pre-hibernation, torpor, interbout euthermia, and post-hibernation) with AOSLO and transmission electron microscopy. Our results indicate that the 13-LGS cone mosaic is only transiently affected by structural remodeling during hibernation. Outer segment remodeling starts during torpid states during a period of fall transition in room temperature, with more severe structural changes during bouts of torpor in cold temperature. Cones return to euthermic-like structure during brief periods of interbout euthermia and recover normal waveguiding properties as soon as 24 h post-hibernation. Cone structure is visible with split-detector AOSLO throughout hibernation, providing evidence that intact outer segments are not necessary to visualize cones with this technique. Despite the changes to cone structure during hibernation, cone density and packing remained unchanged throughout the seasonal cycle. Pairing non-invasive imaging with ultrastructural assessment may provide insight to the biological origins of cone photoreceptor signals observed with AOSLO.

Acid Ceramidase Deficiency in Mice Leads to Severe Ocular Pathology and Visual Impairment.

Farber disease (FD) is a debilitating lysosomal storage disorder characterized by severe inflammation and neurodegeneration. FD is caused by mutations in the ASAH1 gene, resulting in deficient acid ceramidase (ACDase) activity. Patients with ACDase deficiency exhibit a broad clinical spectrum. In classic cases, patients develop hepatosplenomegaly, nervous system involvement, and childhood mortality. Ocular manifestations include decreased vision, a grayish appearance to the retina with a cherry red spot, and nystagmus. That said, the full effect of ACDase deficiency on the visual system has not been studied in detail. We previously developed a mouse model that is orthologous for a known patient mutation in Asah1 that recapitulates human FD. Herein, we report evidence of a severe ocular pathology in Asah1P361R/P361R mice. Asah1P361R/P361R mice exhibit progressive retinal and optic nerve pathology. Through noninvasive ocular imaging and histopathological analyses of these Asah1P361R/P361R animals, we revealed progressive inflammation, the presence of retinal dysplasia, and significant storage pathology in various cell types in both the retina and optic nerves. Lipidomic analyses of retinal tissues revealed an abnormal accumulation of ceramides and other sphingolipids. Electroretinograms and behavioral tests showed decreased retinal and visual responses. Taken together, these data suggest that ACDase deficiency leads to sphingolipid imbalance, inflammation, dysmorphic retinal and optic nerve pathology, and severe visual impairment.

Assessment of Outer Retinal Remodeling in the Hibernating 13-Lined Ground Squirrel.

Purpose: We examined outer retinal remodeling of the euthermic and torpid cone-dominant 13-lined ground squirrel (13-LGS) retina using optical coherence tomography (OCT) imaging and histology. Methods: Retinas and corneas of living 13-LGSs were imaged during euthermic and torpid physiological states using OCT. Retinal layer thickness was measured at the visual streak from registered and averaged vertical B-scans. Following OCT, some retinas were collected immediately for postmortem histologic comparison using light microscopy, immunofluorescence, or transmission electron microscopy. Results: Compared to OCT images from euthermic retinae, OCT images of torpid retinae revealed significantly thicker inner and outer nuclear layers, as well as increases in the distances between outer retinal reflectivity bands 1 and 2, and bands 3 and 4. A significant decrease in the distance between bands 2 and 3 also was seen, alongside significant thinning of the choriocapillaris and choroid. OCT image quality was reduced in torpid eyes, partly due to significant thickening of the corneal stroma during this state. Conclusions: The torpid retina of the hibernating 13-LGS undergoes structural changes that can be detected by OCT imaging. Comparisons between in vivo OCT and ex vivo histomorphometry may offer insight to the origin of hyperreflective OCT bands within the outer retina of the cone-dominant 13-LGS.

Seasonal and post-trauma remodeling in cone-dominant ground squirrel retina.

With a photoreceptor mosaic containing ∼85% cones, the ground squirrel is one of the richest known mammalian sources of these important retinal cells. It also has a visual ecology much like the human's. While the ground squirrel retina is understandably prominent in the cone biochemistry, physiology, and circuitry literature, far less is known about the remodeling potential of its retinal pigment epithelium, neurons, macroglia, or microglia. This review aims to summarize the data from ground squirrel retina to this point in time, and to relate them to data from other brain areas where appropriate. We begin with a survey of the ground squirrel visual system, making comparisons with traditional rodent models and with human. Because this animal's status as a hibernator often goes unnoticed in the vision literature, we then present a brief primer on hibernation biology. Next we review what is known about ground squirrel retinal remodeling concurrent with deep torpor and with rapid recovery upon re-warming. Notable here is rapidly-reversible, temperature-dependent structural plasticity of cone ribbon synapses, as well as pre- and post-synaptic plasticity throughout diverse brain regions. It is not yet clear if retinal cell types other than cones engage in torpor-associated synaptic remodeling. We end with the small but intriguing literature on the ground squirrel retina's remodeling responses to insult by retinal detachment. Notable for widespread loss of (cone) photoreceptors, there is surprisingly little remodeling of the RPE or Müller cells. Microglial activation appears minimal, and remodeling of surviving second- and third-order neurons seems absent, but both require further study. In contrast, traumatic brain injury in the ground squirrel elicits typical macroglial and microglial responses. Overall, the data to date strongly suggest a heretofore unrecognized, natural checkpoint between retinal deafferentiation and RPE and Müller cell remodeling events. As we continue to discover them, the unique ways by which ground squirrel retina responds to hibernation or injury may be adaptable to therapeutic use.