Jesse Schallek

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.

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.

Imaging translucent cell bodies in the living mouse retina without contrast agents

The transparency of most retinal cell classes typically precludes imaging them in the living eye; unless invasive methods are used that deploy extrinsic contrast agents. Using an adaptive optics scanning light ophthalmoscope (AOSLO) and capitalizing on the large numerical aperture of the mouse eye, we enhanced the contrast from otherwise transparent cells by subtracting the left from the right half of the light distribution in the detector plane. With this approach, it is possible to image the distal processes of photoreceptors, their more proximal cell bodies and the mosaic of horizontal cells in the living mouse 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.

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.

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.

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.

Label free measurement of retinal blood cell flux, velocity, hematocrit and capillary width in the living mouse eye

Measuring blood cell dynamics within the capillaries of the living eye provides crucial information regarding the health of the microvascular network. To date, the study of single blood cell movement in this network has been obscured by optical aberrations, hindered by weak optical contrast, and often required injection of exogenous fluorescent dyes to perform measurements. Here we present a new strategy to non-invasively image single blood cells in the living mouse eye without contrast agents. Eye aberrations were corrected with an adaptive optics camera coupled with a fast, 15 kHz scanned beam orthogonal to a capillary of interest. Blood cells were imaged as they flowed past a near infrared imaging beam to which the eye is relatively insensitive. Optical contrast of cells was optimized using differential scatter of blood cells in the split-detector imaging configuration. Combined, these strategies provide label-free, non-invasive imaging of blood cells in the retina as they travel in single file in capillaries, enabling determination of cell flux, morphology, class, velocity, and rheology at the single cell level.

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.