Photoreceptor loss does not recruit neutrophils despite strong microglial activation.

In response to central nervous system (CNS) injury, tissue resident immune cells such as microglia and circulating systemic neutrophils are often first responders. The degree to which these cells interact in response to CNS damage is poorly understood, and even less so, in the neural retina which poses a challenge for high resolution imaging in vivo. In this study, we deploy fluorescence adaptive optics scanning light ophthalmoscopy (AOSLO) to study fluorescent microglia and neutrophils in mice. We simultaneously track immune cell dynamics using label-free phase-contrast AOSLO at micron-level resolution. Retinal lesions were induced with 488 nm light focused onto photoreceptor (PR) outer segments. These lesions focally ablated PRs, with minimal collateral damage to cells above and below the plane of focus. We used in vivo (AOSLO, SLO and OCT) imaging to reveal the natural history of the microglial and neutrophil response from minutes-to-months after injury. While microglia showed dynamic and progressive immune response with cells migrating into the injury locus within 1-day after injury, neutrophils were not recruited despite close proximity to vessels carrying neutrophils only microns away. Post-mortem confocal microscopy confirmed in vivo findings. This work illustrates that microglial activation does not recruit neutrophils in response to acute, focal loss of photoreceptors, a condition encountered in many retinal diseases.

Contributed Session II: Immune cell speed changes over 5 orders of magnitude in response to inflammation in the retina.

Inflammation in vascularized tissues is mediated by circulating immune cells that are recruited to damaged tissue. Immune cells undergo dramatic changes in speed and motility indicating the severity and staging of inflammation. Here, we characterize the spectrum of retinal leukocyte kinetics in response to an acute inflammatory stimulus using adaptive optics scanning light ophthalmoscopy (AOSLO) in living mice. C57BL/6J male mice were injected intravitreally with 1 µL lipopolysaccharide (LPS) and imaged at 6, 24 and 72 hours after LPS injection using phase contrast and fluorescence AOSLO. Speed of circulating leukocytes (n= 286 cells, 2 mice) was measured with 15kHz point-scan imaging using automated approach (Joseph et al. 2019). Rolling leukocytes (n=300 cells, 5 mice, 6 hrs after LPS) and extravasated cells (n=92 cells, 8 mice) were visualized with time-lapse imaging and manually tracked using ImageJ. Using our custom AOSLO, we observed leukocyte speeds spanning 5 orders of magnitude in the living retina. The fastest speeds were the circulating leukocytes (13,257.37 ± 7,086.41 µm/s). After LPS, leukocytes roll along the venular wall, where cell speed was 1000x slower (11.45 ± 7.45 µm/s.) When immune cells extravasated into the tissue, cell speed dropped further by 100x (0.3 ± 0.15 µm/s). Observed leukocyte speeds cluster around three distinct velocity bands that stratify the unique and purposeful behavior of these cells as they progress through the inflammatory cascade.

Contributed Session II: In vivo imaging of immune cell activity in primate retina after photoreceptor ablation.

The non-human primate (NHP) is the gold standard animal model for preclinical development of gene and cell based therapies for vision restoration. However, the ocular immune response to these interventions remains poorly understood. We conducted a proof of concept study using offset aperture adaptive optics scanning light ophthalmoscopy (AOSLO) to visualize cellular-scale changes in the primate retina following photoreceptor (PR) ablation. Ultrafast 730nm laser exposure at 26.6 - 32.5 J/cm2 was used to create six lesions in four NHPs. Offset aperture images focused on retinal vascular layers were collected with an offset distance of ~10 Airy Disk Diameters from 15 minutes up to three hours after PR ablation. We observed putative immune cells in and around vessels supplying the lesioned areas. Consistent with previous findings in murine models, cells within vessels adhered to the inner wall, exhibited crawling behavior, and had a diameter ranging from ~9.3 - 11.5 µm. Additionally, we observed the emergence of cellular-scale structures above the PR layer that originated in the center of the lesion 15 minutes post-insult and gradually radiated outward. Vascular perfusion was maintained in these regions. Our data suggest that offset aperture imaging offers cellular-scale, label free, in vivo assessment of the retinal response to insult in NHPs and could be employed to advance our understanding of the ocular immune response provoked by disease and therapeutic interventions.

High-resolution structural and functional retinal imaging in the awake behaving mouse.

The laboratory mouse has provided tremendous insight to the underpinnings of mammalian central nervous system physiology. In recent years, it has become possible to image single neurons, glia and vascular cells in vivo by using head-fixed preparations combined with cranial windows to study local networks of activity in the living brain. Such approaches have also succeeded without the use of general anesthesia providing insights to the natural behaviors of the central nervous system. However, the same has not yet been developed for the eye, which is constantly in motion. Here we characterize a novel head-fixed preparation that enables high-resolution adaptive optics retinal imaging at the single-cell level in awake-behaving mice. We reveal three new functional attributes of the normal eye that are overlooked by anesthesia: 1) High-frequency, low-amplitude eye motion of the mouse that is only present in the awake state 2) Single-cell blood flow in the mouse retina is reduced under anesthesia and 3) Mouse retinae thicken in response to ketamine/xylazine anesthesia. Here we show key benefits of the awake-behaving preparation that enables study of retinal physiology without anesthesia to study the normal retinal physiology in the mouse.

In Vivo Capillary Structure and Blood Cell Flux in the Normal and Diabetic Mouse Eye.

Purpose: To characterize the early structural and functional changes in the retinal microvasculature in response to hyperglycemia in the Ins2Akita mouse. Methods: A custom phase-contrast adaptive optics scanning light ophthalmoscope was used to image retinal capillaries of 9 Ins2Akita positive (hyperglycemic) and 9 Ins2Akita negative (euglycemic) mice from postnatal weeks 5 to 18. A 15 kHz point scan was used to image capillaries and measure red blood cell flux at biweekly intervals; measurements were performed manually. Retinal thickness and fundus photos were captured monthly using a commercial scanning laser ophthalmoscope/optical coherence tomography. Retinal thickness was calculated using a custom algorithm. Blood glucose and weight were tracked throughout the duration of the study. Results: Elevated blood glucose (>250 mg/dL) was observed at 4 to 5 weeks of age in Ins2Akita mice and remained elevated throughout the study, whereas euglycemic littermates maintained normal glucose levels. There was no significant difference in red blood cell flux, capillary anatomy, lumen diameter, or occurrence of stalled capillaries between hyperglycemic and euglycemic mice between postnatal weeks 5 and 18. Hyperglycemic mice had a thinner retina than euglycemic littermates (p < 0.001), but retinal thickness did not change with duration of hyperglycemia despite glucose levels that were more than twice times normal. Conclusions: In early stages of hyperglycemia, retinal microvasculature structure (lumen diameter, capillary anatomy) and function (red blood cell flux, capillary perfusion) were not impaired despite 3 months of chronically elevated blood glucose. These findings suggest that hyperglycemia alone for 3 months does not alter capillary structure or function in profoundly hyperglycemic mice.

Imaging the dynamics of individual processes of microglia in the living retina in vivo.

Microglia are an essential population of resident immune cells in the central nervous system (CNS) and retina. These microscopic cells possess sub-cellular processes that make them challenging to image due to limited resolution and contrast. The baseline behavior of microglial processes in the living retina has been poorly characterized, and yet are essential to understanding how these cells respond under conditions of health, development, stress and disease. Here we use in vivo adaptive optics scanning light ophthalmoscopy combined with time-lapse imaging and quantification of process motility, to reveal the detailed behavior of microglial cells in a population of healthy mice. We find microglial processes to be dynamic at all branch-levels, from primary to end-protrusions. Cell-processes remodel at average speeds of 0.6 ± 0.4 µm/min with growth and deletion bursts of 0-7.6 µm/min. Longitudinal imaging in the same mice showed cell-somas to remain stable over seconds to minutes, but show migration over days to months. In addition to characterizing in vivo process motility and Sholl analysis using a microglial reporter mouse, we also demonstrate that microglia can be imaged without fluorescent labels at all. Phase-contrast imaging using safe levels of near-infrared light successfully imaged microglia soma and process remodeling with micron-level detail noninvasively, confirmed by simultaneous imaging of fluorescent microglial cells in transgenic mice. This label-free approach provides a new opportunity to investigate CNS immune system noninvasively without requiring transgenic or antibody labeling which could have off-target effects of changing normal microglial behavior. Additionally, CNS microglia study can now be conducted without the need for cranial window surgery which have the potential to change their behavior due to local or systemic inflammation.

Label-free imaging of immune cell dynamics in the living retina using adaptive optics.

Our recent work characterized the movement of single blood cells within the retinal vasculature (Joseph et al. 2019) using adaptive optics ophthalmoscopy. Here, we apply this technique to the context of acute inflammation and discover both infiltrating and tissue-resident immune cells to be visible without any labeling in the living mouse retina using near-infrared light alone. Intravital imaging of immune cells can be negatively impacted by surgical manipulation, exogenous dyes, transgenic manipulation and phototoxicity. These confounds are now overcome, using phase contrast and time-lapse videography to reveal the dynamic behavior of myeloid cells as they interact, extravasate and survey the mouse retina. Cellular motility and differential vascular responses were measured noninvasively and in vivo across hours to months at the same retinal location, from initiation to the resolution of inflammation. As comparable systems are already available for clinical research, this approach could be readily translated to human application.

In vivo imaging of corneal nerves and cellular structures in mice with Gabor-domain optical coherence microscopy.

Gabor-domain optical coherence microscopy (GDOCM) demonstrated in vivo corneal imaging with cellular resolution and differentiation in mice over a field of view of 1 mm2. Contact and non-contact imaging was conducted on six healthy and six hyperglycemic C57BL/6J mice. Cellular resolution in the 3D GDOCM images was achieved after motion correction. Corneal nerve fibers were traced and their lengths and branches calculated. Noncontact, label-free imaging of corneal nerves has clinical utility in health and disease, and in transplant evaluation. To the authors' knowledge, this is the first report of in vivo 3D corneal imaging in mice with the capability to resolve nerve fibers using a non-contact imaging modality.

Imaging Retinal Activity in the Living Eye.

Retinal function has long been studied with psychophysical methods in humans, whereas detailed functional studies of vision have been conducted mostly in animals owing to the invasive nature of physiological approaches. There are exceptions to this generalization, for example, the electroretinogram. This review examines exciting recent advances using in vivo retinal imaging to understand the function of retinal neurons. In some cases, the methods have existed for years and are still being optimized. In others, new methods such as optophysiology are revealing novel patterns of retinal function in animal models that have the potential to change our understanding of the functional capacity of the retina. Together, the advances in retinal imaging mark an important milestone that shifts attention away from anatomy alone and begins to probe the function of healthy and diseased eyes.

Retinal ischemia induces α-SMA-mediated capillary pericyte contraction coincident with perivascular glycogen depletion.

Increasing evidence indicates that pericytes are vulnerable cells, playing pathophysiological roles in various neurodegenerative processes. Microvascular pericytes contract during cerebral and coronary ischemia and do not relax after re-opening of the occluded artery, causing incomplete reperfusion. However, the cellular mechanisms underlying ischemia-induced pericyte contraction, its delayed emergence, and whether it is pharmacologically reversible are unclear. Here, we investigate i) whether ischemia-induced pericyte contractions are mediated by alpha-smooth muscle actin (α-SMA), ii) the sources of calcium rise in ischemic pericytes, and iii) if peri-microvascular glycogen can support pericyte metabolism during ischemia. Thus, we examined pericyte contractility in response to retinal ischemia both in vivo, using adaptive optics scanning light ophthalmoscopy and, ex vivo, using an unbiased stereological approach. We found that microvascular constrictions were associated with increased calcium in pericytes as detected by a genetically encoded calcium indicator (NG2-GCaMP6) or a fluoroprobe (Fluo-4). Knocking down α-SMA expression with RNA interference or fixing F-actin with phalloidin or calcium antagonist amlodipine prevented constrictions, suggesting that constrictions resulted from calcium- and α-SMA-mediated pericyte contractions. Carbenoxolone or a Cx43-selective peptide blocker also reduced calcium rise, consistent with involvement of gap junction-mediated mechanisms in addition to voltage-gated calcium channels. Pericyte calcium increase and capillary constrictions became significant after 1 h of ischemia and were coincident with depletion of peri-microvascular glycogen, suggesting that glucose derived from glycogen granules could support pericyte metabolism and delay ischemia-induced microvascular dysfunction. Indeed, capillary constrictions emerged earlier when glycogen breakdown was pharmacologically inhibited. Constrictions persisted despite recanalization but were reversible with pericyte-relaxant adenosine administered during recanalization. Our study demonstrates that retinal ischemia, a common cause of blindness, induces α-SMA- and calcium-mediated persistent pericyte contraction, which can be delayed by glucose driven from peri-microvascular glycogen. These findings clarify the contractile nature of capillary pericytes and identify a novel metabolic collaboration between peri-microvascular end-feet and pericytes.

Imaging single-cell blood flow in the smallest to largest vessels in the living retina.

Tissue light scatter limits the visualization of the microvascular network deep inside the living mammal. The transparency of the mammalian eye provides a noninvasive view of the microvessels of the retina, a part of the central nervous system. Despite its clarity, imperfections in the optics of the eye blur microscopic retinal capillaries, and single blood cells flowing within. This limits early evaluation of microvascular diseases that originate in capillaries. To break this barrier, we use 15 kHz adaptive optics imaging to noninvasively measure single-cell blood flow, in one of the most widely used research animals: the C57BL/6J mouse. Measured flow ranged four orders of magnitude (0.0002-1.55 µL min-1) across the full spectrum of retinal vessel diameters (3.2-45.8 µm), without requiring surgery or contrast dye. Here, we describe the ultrafast imaging, analysis pipeline and automated measurement of millions of blood cell speeds.

Capillary pericytes express α-smooth muscle actin, which requires prevention of filamentous-actin depolymerization for detection.

Recent evidence suggests that capillary pericytes are contractile and play a crucial role in the regulation of microcirculation. However, failure to detect components of the contractile apparatus in capillary pericytes, most notably α-smooth muscle actin (α-SMA), has questioned these findings. Using strategies that allow rapid filamentous-actin (F-actin) fixation (i.e. snap freeze fixation with methanol at -20°C) or prevent F-actin depolymerization (i.e. with F-actin stabilizing agents), we demonstrate that pericytes on mouse retinal capillaries, including those in intermediate and deeper plexus, express α-SMA. Junctional pericytes were more frequently α-SMA-positive relative to pericytes on linear capillary segments. Intravitreal administration of short interfering RNA (α-SMA-siRNA) suppressed α-SMA expression preferentially in high order branch capillary pericytes, confirming the existence of a smaller pool of α-SMA in distal capillary pericytes that is quickly lost by depolymerization. We conclude that capillary pericytes do express α-SMA, which rapidly depolymerizes during tissue fixation thus evading detection by immunolabeling.

Vision science and adaptive optics, the state of the field.

Adaptive optics is a relatively new field, yet it is spreading rapidly and allows new questions to be asked about how the visual system is organized. The editors of this feature issue have posed a series of question to scientists involved in using adaptive optics in vision science. The questions are focused on three main areas. In the first we investigate the use of adaptive optics for psychophysical measurements of visual system function and for improving the optics of the eye. In the second, we look at the applications and impact of adaptive optics on retinal imaging and its promise for basic and applied research. In the third, we explore how adaptive optics is being used to improve our understanding of the neurophysiology of the visual system.

Morphology and topography of retinal pericytes in the living mouse retina using in vivo adaptive optics imaging and ex vivo characterization.

PURPOSE: To noninvasively image retinal pericytes in the living eye and characterize NG2-positive cell topography and morphology in the adult mouse retina. METHODS: Transgenic mice expressing fluorescent pericytes (NG2, DsRed) were imaged using a two-channel, adaptive optics scanning laser ophthalmoscope (AOSLO). One channel imaged vascular perfusion with near infrared light. A second channel simultaneously imaged fluorescent retinal pericytes. Mice were also imaged using wide-field ophthalmoscopy. To confirm in vivo imaging, five eyes were enucleated and imaged in flat mount with conventional fluorescent microscopy. Cell topography was quantified relative to the optic disc. RESULTS: We observed strong DsRed fluorescence from NG2-positive cells. AOSLO revealed fluorescent vascular mural cells enveloping all vessels in the living retina. Cells were stellate on larger venules, and showed banded morphology on arterioles. NG2-positive cells indicative of pericytes were found on the smallest capillaries of the retinal circulation. Wide-field SLO enabled quick assessment of NG2-positive distribution, but provided insufficient resolution for cell counts. Ex vivo microscopy showed relatively even topography of NG2-positive capillary pericytes at eccentricities more than 0.3 mm from the optic disc (515 ± 94 cells/mm(2) of retinal area). CONCLUSIONS: We provide the first high-resolution images of retinal pericytes in the living animal. Subcellular resolution enabled morphological identification of NG2-positive cells on capillaries showing classic features and topography of retinal pericytes. This report provides foundational basis for future studies that will track and quantify pericyte topography, morphology, and function in the living retina over time, especially in the progression of microvascular disease.

Retinal intrinsic optical signals in a cat model of primary congenital glaucoma.

PURPOSE: To examine the impact of reduced inner retinal function and breed on intrinsic optical signals in cats. METHODS: Retinal intrinsic optical signals were recorded from anesthetized cats with a modified fundus camera. Near infrared light (NIR, 700-900 nm) was used to illuminate the retina while a charge-coupled device (CCD) camera captured the NIR reflectance of the retina. Visible stimuli (540 nm) evoked patterned changes in NIR retinal reflectance. NIR intrinsic signals were compared across three subject groups: two Siamese cats with primary congenital glaucoma (PCG), a control Siamese cat without glaucoma, and a control group of seven normally pigmented cats. Intraocular pressure (IOP), pattern electroretinogram, and optical coherence tomography measurements were evaluated to confirm the inner retinal deficit in PCG cats. RESULTS: Stimulus-evoked, NIR retinal reflectance signals were observed in PCG cats despite severe degeneration of the nerve fiber layer and inner retinal function. The time course, spectral dependence, and spatial profile of signals imaged in PCG cats were similar to signals measured from normal and Siamese control cats. CONCLUSIONS: Despite increased IOP, reduced nerve fiber layer thickness and ganglion cell function, intrinsic optical signals persist in cats affected with PCG. The mechanisms giving rise to intrinsic signals remain despite inner retinal damage. Signal strength was reduced in all Siamese cats compared to controls, suggesting that reduced intrinsic signals in PCG cats represent a difference between breeds rather than loss of ganglion cells. These results corroborated previous findings that retinal ganglion cells are not the dominant source of intrinsic optical signals of the retina.

Blood contrast agents enhance intrinsic signals in the retina: evidence for an underlying blood volume component.

PURPOSE: To examine the extent to which neurovascular coupling contributes to stimulus-evoked intrinsic signals in the retina. METHODS: The retinas of five adult cats were examined in vivo. Animals were anesthetized and paralyzed for imaging stability. The retinas were imaged through a modified fundus camera capable of presenting patterned visual stimuli simultaneous with a diffuse near infrared (NIR). RESULTS: Injections of nigrosin increased signal strength by as much as 36.3%, and indocyanine green (ICG) increased signal magnitudes by as much as 38.1%. In both cases, intrinsic signals maintained a colocalized pattern of activation corresponding to the visual stimulus presented. The time course of the evoked signals remained unaltered. The spectral dependency of signal enhancement mirrored the absorption spectra of the injected dyes. CONCLUSIONS: The data are consistent with a neurovascular coupling effect in the retina. Patterned visual stimuli evoke colocalized NIR reflectance changes. The patterned decrease in reflectance was enhanced after nigrosin or ICG was injected into the systemic circulation. These findings suggest stimulus-evoked changes in blood volume underlie a component of the retinal intrinsic signals.

Noninvasive functional imaging of the retina reveals outer retinal and hemodynamic intrinsic optical signal origins.

We have adapted intrinsic signal optical imaging of neural activity to the noninvasive functional imaging of the retina. Results to date demonstrate the feasibility and potential of this new method of functional assessment of the retina. In response to visual stimuli, we have imaged reflectance changes in the retina that are robust and spatially colocalized to the sites of stimulation. However, the technique is in its infancy and many questions as to the underlying mechanisms remain. In particular, the source and nature of the activity-dependent intrinsic optical signals in the retina need to be characterized and their anatomic origins determined. The studies described here begin to address these issues. The evidence indicates that the imaged signals are driven by the outer retinal layers and have a dominant hemodynamic component.

Stimulus-evoked intrinsic optical signals in the retina: pharmacologic dissection reveals outer retinal origins.

PURPOSE: To elucidate the anatomic origins of stimulus-evoked intrinsic optical signals in the mammalian retina by using selective pharmacologic blockade of specific retinal layers. METHODS: Four adult cats were used to investigate the stimulus-evoked intrinsic signals. The retinas were visually stimulated with a liquid crystal display (LCD) integrated into a modified fundus camera. The evoked signals in the near infrared (NIR) were recorded with a digital camera to image the changes in the optical reflectance of the retinas. Variants of the electroretinogram (pattern ERG and long-pulse ERG) were also recorded as additional measures of retinal function. Specific retinal layers were inactivated via intravitreal injections of the voltage-gated sodium channel blocker, tetrodotoxin (TTX), the metabotropic glutamate receptor (mGluR6) agonist, 2-amino-4-phosphonobutyric acid (APB), and/or the ionotropic glutamate receptor antagonist cis-2,3 piperidinedicarboxylic acid (PDA). The stimulus-evoked intrinsic signals were imaged before and after drug injection. RESULTS: ERG recordings and tests of the consensual pupillary response confirmed the effectiveness of each drug. Yet despite the pharmacologic blockade of the inner retina (TTX) and postreceptoral retinal circuitry (APB and PDA), the stimulus-evoked intrinsic signals remained essentially unaltered from preinjection conditions. Similarly, the time course of the signal did not appreciably shift in time or shape. CONCLUSIONS: The findings demonstrate that stimulus-evoked intrinsic signals persist after injection of APB, PDA, and TTX, drugs that work to suppress inner and postreceptoral retinal circuitry. The persistence of the intrinsic signals after administration of these drugs indicates that the dominant intrinsic signals are likely to arise from the outer retina.

Stimulus-evoked intrinsic optical signals in the retina: spatial and temporal characteristics.

PURPOSE: To characterize the properties of stimulus-evoked retinal intrinsic signals and determine the underlying origins. METHODS: Seven adult cats were anesthetized and paralyzed to maximize imaging stability. The retina was stimulated with a liquid crystal display (LCD) integrated into a modified fundus camera (Topcon, Tokyo, Japan). The LCD presented patterned visual stimuli while the retina was illuminated with near infrared (NIR) light. The peristimulus changes in the NIR reflectance of the retina were recorded with a digital camera. RESULTS: Two stimulus-evoked reflectance signals in the NIR were observed: a positive signal, corresponding to a relative increase in reflectance, and a negative signal, corresponding to a relative decrease in reflectance. When presented with a positive-contrast stimulus, the negative reflectance signals showed a tight spatial coupling with the stimulated region of retina, whereas the positive signals arose in an adjacent region of the retina. Signals remained spatially confined to the stimulated region even when stimuli of much longer duration were used. In addition, the positive and negative signal polarities reversed when the stimulus contrast was inverted. Both signals showed a rise time on the order of seconds, similar to those observed in the mammalian neocortex. The spectral dependency of the signals on illumination was similar to the absorbance spectra of hemoglobin and the oximetric relationship. CONCLUSIONS: The findings characterize the basic properties of stimulus-evoked intrinsic signals of the retina. These signals were generally similar to the more extensively studied cortical signals. Collectively, the data suggest a hemodynamic component to the intrinsic optical signals of the retina.