Noninvasive, In Vivo Assessment of Mouse Retinal Structure Using Optical Coherence Tomography

BACKGROUND: Optical coherence tomography (OCT) is a novel method of retinal in vivo imaging. In this study, we assessed the potential of OCT to yield histology-analogue sections in mouse models of retinal degeneration. METHODOLOGY/PRINCIPAL FINDINGS: We achieved to adapt a commercial 3(rd) generation OCT system to obtain and quantify high-resolution morphological sections of the mouse retina which so far required in vitro histology. OCT and histology were compared in models with developmental defects, light damage, and inherited retinal degenerations. In conditional knockout mice deficient in retinal retinoblastoma protein Rb, the gradient of Cre expression from center to periphery, leading to a gradual reduction of retinal thickness, was clearly visible and well topographically quantifiable. In Nrl knockout mice, the layer involvement in the formation of rosette-like structures was similarly clear as in histology. OCT examination of focal light damage, well demarcated by the autofluorescence pattern, revealed a practically complete loss of photoreceptors with preservation of inner retinal layers, but also more subtle changes like edema formation. In Crb1 knockout mice (a model for Leber's congenital amaurosis), retinal vessels slipping through the outer nuclear layer towards the retinal pigment epithelium (RPE) due to the lack of adhesion in the subapical region of the photoreceptor inner segments could be well identified. CONCLUSIONS/SIGNIFICANCE: We found that with the OCT we were able to detect and analyze a wide range of mouse retinal pathology, and the results compared well to histological sections. In addition, the technique allows to follow individual animals over time, thereby reducing the numbers of study animals needed, and to assess dynamic processes like edema formation. The results clearly indicate that OCT has the potential to revolutionize the future design of respective short- and long-term studies, as well as the preclinical assessment of therapeutic strategies

Novel Rodent Models for Macular Research

BACKGROUND: Many disabling human retinal disorders involve the central retina, particularly the macula. However, the commonly used rodent models in research, mouse and rat, do not possess a macula. The purpose of this study was to identify small laboratory rodents with a significant central region as potential new models for macular research. METHODOLOGY/PRINCIPAL FINDINGS: Gerbillus perpallidus, Meriones unguiculatus and Phodopus campbelli, laboratory rodents less commonly used in retinal research, were subjected to confocal scanning laser ophthalmoscopy (cSLO), fluorescein and indocyanine green angiography, and spectral-domain optical coherence tomography (SD-OCT) using standard equipment (Heidelberg Engineering HRA1 and Spectralis™) adapted to small rodent eyes. The existence of a visual streak-like pattern was assessed on the basis of vascular topography, retinal thickness, and the topography of retinal ganglion cells and cone photoreceptors. All three species examined showed evidence of a significant horizontal streak-like specialization. cSLO angiography and retinal wholemounts revealed that superficial retinal blood vessels typically ramify and narrow into a sparse capillary net at the border of the respective area located dorsal to the optic nerve. Similar to the macular region, there was an absence of larger blood vessels in the streak region. Furthermore, the thickness of the photoreceptor layer and the population density of neurons in the ganglion cell layer were markedly increased in the visual streak region. CONCLUSIONS/SIGNIFICANCE: The retinal specializations of Gerbillus perpallidus, Meriones unguiculatus and Phodopus campbelli resemble features of the primate macula. Hence, the rodents reported here may serve to study aspects of macular development and diseases like age-related macular degeneration and diabetic macular edema, and the preclinical assessment of therapeutic strategies

Mural Cell SRF Controls Pericyte Migration, Vessel Patterning and Blood Flow

Background: Pericytes and vascular smooth muscle cells, collectively known as mural cells, are recruited through PDGFB (platelet-derived growth factor B)-PDGFRB (platelet-derived growth factor receptor beta) signaling. MCs are essential for vascular integrity, and their loss has been associated with numerous diseases. Most of this knowledge is based on studies in which MCs are insufficiently recruited or fully absent upon inducible ablation. In contrast, little is known about the physiological consequences that result from impairment of specific MC functions. Here, we characterize the role of the transcription factor SRF (serum response factor) in MCs and study its function in developmental and pathological contexts. Methods: We generated a mouse model of MC-specific inducible Srf gene deletion and studied its consequences during retinal angiogenesis using RNA-sequencing, immunohistology, in vivo live imaging, and in vitro techniques. Results: By postnatal day 6, pericytes lacking SRF were morphologically abnormal and failed to properly comigrate with angiogenic sprouts. As a consequence, pericyte-deficient vessels at the retinal sprouting front became dilated and leaky. By postnatal day 12, also the vascular smooth muscle cells had lost SRF, which coincided with the formation of pathological arteriovenous shunts. Mechanistically, we show that PDGFB-dependent SRF activation is mediated via MRTF (myocardin-related transcription factor) cofactors. We further show that MRTF-SRF signaling promotes pathological pericyte activation during ischemic retinopathy. RNA-sequencing, immunohistology, in vivo live imaging, and in vitro experiments demonstrated that SRF regulates expression of contractile SMC proteins essential to maintain the vascular tone. Conclusions: SRF is crucial for distinct functions in pericytes and vascular smooth muscle cells. SRF directs pericyte migration downstream of PDGFRB signaling and mediates pathological pericyte activation during ischemic retinopathy. In vascular smooth muscle cells, SRF is essential for expression of the contractile machinery, and its deletion triggers formation of arteriovenous shunts. These essential roles in physiological and pathological contexts provide a rationale for novel therapeutic approaches through targeting SRF activity in MCs

A key role for cyclic nucleotide gated (CNG) channels in cGMP-related retinitis pigmentosa

The rd1 natural mutant is one of the first and probably the most commonly studied mouse model for retinitis pigmentosa (RP), a severe and frequently blinding human retinal degeneration. In several decades of research, the link between the increase in photoreceptor cGMP levels and the extremely rapid cell death gave rise to a number of hypotheses. Here, we provide clear evidence that the presence of cyclic nucleotide gated (CNG) channels in the outer segment membrane is the key to rod photoreceptor loss. In Cngb1(-/-) x rd1 double mutants devoid of regular CNG channels, cGMP levels are still pathologically high, but rod photoreceptor viability and outer segment morphology are greatly improved. Importantly, cone photoreceptors, the basis for high-resolution daylight and colour vision, survived and remained functional for extended periods of time. These findings strongly support the hypothesis of deleterious calcium (Ca2+)-influx as the cause of rapid rod cell death and highlight the importance of CNG channels in this process. Furthermore, our findings suggest that targeting rod CNG channels, rather than general Ca2+-channel blockade, is a most promising symptomatic approach to treat otherwise incurable forms of cGMP-related RP

Immunofluorescence labeling of retinal sections revealed distinct differences in cone cell distribution and vessel density.

<p>(<b>A–F</b>) Areas within the visual streak region are shown in the right panel, areas from dorsal peripheral retina in the left panel. (<b>A</b>) Isolectin IB4-FITC staining of G. perpallidus retina reveals the sparse vessel distribution within the streak region; (<b>B</b>) Peanut agglutinin staining of P. campbelli retina illustrates increased cone densities within the streak region. (<b>C</b>) SWS cone staining of M. unguiculatus retina reveals enhanced SWS cone density within the streak area compared to dorsal periphery. Rods and MWS cones are evenly distributed across the retina as shown by (<b>D</b>) rod transducin (GNAT1) staining in M. unguiculatus, (<b>E</b>) rod opsin staining, and (F) MWS cone opsin staining in G. perpallidus. (<b>A–F</b>) Nuclei were contrasted with DAPI (in blue).</p

<i>In vivo</i> fundus imaging.

<p>cSLO fundus images of (<b>A</b>) human, (<b>B</b>) mouse, and (<b>C</b>) Meriones unguiculatus. (<b>A–C</b>) D = dorsal, V = ventral. (<b>A</b>) Native infrared (λ = 830 nm) SLO image of one researcher's right eye illustrates the sparse vascularization of the macular region. (<b>B</b>) Fluorescein angiography (FLA) (488 nm, barrier filter at 500 nm) reveals the evenly distributed retinal blood vessels in mice. (<b>C</b>) FLA image of Meriones unguiculatus visualizes the horizontal sparse vascular band denoting the specialized retinal region dorsal to the optic disc.</p

Detailed analysis of the retinal blood vessel distribution <i>in vivo</i> using cSLO and <i>in vitro</i> using collagen IV staining.

<p>(<b>A–C</b>) G. perpallidus, (<b>D–F</b>) M. unguiculatus, and (<b>G–I</b>) P. campbelli. Collagen IV stained retinal wholemounts (<b>A</b>, <b>D</b>, <b>G</b>) illustrate the entire visual streak region. Magnifications of wholemounts (<b>B</b>, <b>E</b>, <b>H</b>) and corresponding in vivo cSLO data (<b>C</b>, <b>F</b>, <b>I</b>) show that retinal vessels ramify and narrow into a sparse capillary net at the border of the respective area.</p

<i>In vivo</i> morphological analysis and retinal layering with cSLO angiography and SD-OCT imaging.

<p>(<b>A–C</b>) Gerbillus perpallidus, (<b>D–F</b>) Meriones unguiculatus, and (<b>G–I</b>) Phodopus campbelli. cSLO en-face imaging of the vascular pattern using (<b>A</b>, <b>D</b>, <b>G</b>) ICG angiography mode (795 nm with barrier filter at 800 nm) depicts both retinal and choroidal structures. Black line depicts OCT scan direction. (<b>B</b>, <b>E</b>, <b>H</b>) FLA displays large retinal vessels and capillaries. Besides, choroidal vessels are visible in the visual steak region as different retinal layering allows light at λ = 488 nm to better penetrate the RPE/choriocapillary complex. (<b>C</b>, <b>F</b>, <b>I</b>) In vivo cross-sectional SD-OCT images show an increased retinal thickness in the visual streak region, indicating a specialized retinal region (arrows). ONL, outer nuclear layer; RPE, retinal pigment epithilium.</p

OCT assessment of light-induced murine retinal degeneration.

<p>A) OCT section across the central retina, containing adjacent damaged and non-damaged areas. B) Demarcation of the damaged area <i>in vivo</i> by SLO autofluorescence (AF) imaging based on fluorescent photoreceptor debris (marked by an asterisk). C) Detail of the transition zone between damaged and non-damaged retina in a). The asterisk marks the damaged area as in B). D), E) Comparison of the representation of light-induced retinal damage in histology and OCT. The arrowhead in the OCT image points towards a site of retinal edema.</p