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1.  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/mm2 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.
We provide the first high-resolution images of retinal pericytes in the living animal. Adaptive optics ophthalmoscopy provided subcellular resolution allowing for identification of fluorescent NG2-positive cells on capillaries, venules, and arterioles in vivo. Ex vivo imaging confirmed in vivo data.
doi:10.1167/iovs.13-12581
PMCID: PMC3869420  PMID: 24150762
diabetic retinopathy; neurovascular coupling; adaptive optics; capillaries; microvascular network
2.  Prevalence and distribution of Aggregatibacter actinomycetemcomitans and its cdtB gene in subgingival plaque of Chinese periodontitis patients 
BMC Oral Health  2014;14:37.
Background
Aggregatibacter actinomycetemcomitans (A.actinomycetemcomitans) is an important periodontal pathogen that can participate in periodontitis and other non-oral infections. The cytolethal distending toxin (Cdt) is among the virulence factors produced by this bacterium. This study was to elucidate the distribution of A.actinomycetemcomitans and the prevalence of its cdtB gene in Chinese subjects.
Methods
A total of 255 subgingival samples were obtained from 30 subjects. Samples were collected from periodontal healthy sites as well as shallow, moderate and deep pockets. The absolute quantity of A.actinomycetemcomitans and cdtB gene were determined by real-time polymerase chain reaction.
Results
A.actinomycetemcomitans was detected in 92 of 105 (87.6%) samples of aggressive periodontitis (AgP) patients, in 73 of 79 (92.4%) samples of chronic periodontitis ( CP) patients and in 5 of 71 (7.0%) samples of periodontal healthy subjects. The cdtB gene was detected in 72 sites (78.3%) with AgP infected with A.actinomycetemcomitans, 54 sites (74.0%) with CP infected with A.actinomycetemcomitans and none in healthy sites infected with A.actinomycetemcomitans. In addition, quantity of A.actinomycetemcomitans and cdt gene in samples from deep pockets were significant larger than moderate, shallow and healthy sites (P < 0.05). In comparison to CP, AgP patients were infected with increased numbers of cdt genotype in deep pockets (P < 0.05).
Conclusion
This study suggests that the cdtB gene are prevalent in A.actinomycetemcomitans, and the distribution of cdt genotype strain may be correlated with AgP and serious periodontal inflammation.
doi:10.1186/1472-6831-14-37
PMCID: PMC4002197  PMID: 24725913
Aggregatibacter actinomycetemcomitans; Cytolethal distending toxin; Subgingival plaque; Real-time PCR
3.  Derivation, culture and retinal pigment epithelial differentiation of human embryonic stem cells using human fibroblast feeder cells 
Purpose
Retinal pigment epithelium cells derived from human embryonic stem cells (ESCs) could be useful for restoring retinal function in age-related macular degeneration. However the use of non-human feeder cells to support the growth of ESCs for clinical applications raises the concern of possible contamination because of direct contact between animal and human cells.
Methods
In this study, we produced human ESCs using human fibroblast feeder layers isolated from foreskin and abdominal tissues. Using this system, human ESCs differentiated into retinal pigment epithelium cells in differentiation medium.
Results
Seven human ESC lines were established from 18 blastocysts. These human ESCs showed normal morphology, expressed all expected cell surface markers, had the ability to form embryoid bodies upon culture in vitro and teratomas after injection into SCID mice, and differentiated further into derivatives of all three germ layers. Under conditions of committed differentiation, these human ESCs could differentiate into retinal pigment epithelium cells after 2 months in culture.
Conclusions
The results of this study demonstrated that human foreskin/abdominal fibroblasts have the potential to support the derivation and long-term culture of human ESCs, which can then be used to generate retinal pigment epithelium cells with characteristic morphology and molecular markers. This technique avoids the concerns of contamination from animal feeder layers during human ESC derivation, culture and differentiation, and will thus facilitate the development of retinal pigment epithelium cell transplantation therapy.
doi:10.1007/s10815-012-9802-2
PMCID: PMC3430777  PMID: 22661130
Human embryonic stem cell; Human foreskin fibroblast feeder layer; Human abdominal fibroblast feeder layer; Retinal pigment epithelium differentiation
4.  In vivo two-photon imaging of the mouse retina 
Biomedical Optics Express  2013;4(8):1285-1293.
Though in vivo two-photon imaging has been demonstrated in non-human primates, improvements in the signal-to-noise ratio (SNR) would greatly improve its scientific utility. In this study, extrinsic fluorophores, expressed in otherwise transparent retinal ganglion cells, were imaged in the living mouse eye using a two-photon fluorescence adaptive optics scanning laser ophthalmoscope. We recorded two orders of magnitude greater signal levels from extrinsically labeled cells relative to previous work done in two-photon autofluorescence imaging of primates. Features as small as single dendrites in various layers of the retina could be resolved and predictions are made about the feasibility of measuring functional response from cells. In the future, two-photon imaging in the intact eye may allow us to monitor the function of retinal cell classes with infrared light that minimally excites the visual response.
doi:10.1364/BOE.4.001285
PMCID: PMC3756587  PMID: 24009992
(330.4460) Ophthalmic optics and devices; (180.4315) Nonlinear microscopy; (170.0110) Imaging systems
5.  Adaptive optics retinal imaging in the living mouse eye 
Biomedical Optics Express  2012;3(4):715-734.
Correction of the eye’s monochromatic aberrations using adaptive optics (AO) can improve the resolution of in vivo mouse retinal images [Biss et al., Opt. Lett. 32(6), 659 (2007) and Alt et al., Proc. SPIE 7550, 755019 (2010)], but previous attempts have been limited by poor spot quality in the Shack-Hartmann wavefront sensor (SHWS). Recent advances in mouse eye wavefront sensing using an adjustable focus beacon with an annular beam profile have improved the wavefront sensor spot quality [Geng et al., Biomed. Opt. Express 2(4), 717 (2011)], and we have incorporated them into a fluorescence adaptive optics scanning laser ophthalmoscope (AOSLO). The performance of the instrument was tested on the living mouse eye, and images of multiple retinal structures, including the photoreceptor mosaic, nerve fiber bundles, fine capillaries and fluorescently labeled ganglion cells were obtained. The in vivo transverse and axial resolutions of the fluorescence channel of the AOSLO were estimated from the full width half maximum (FWHM) of the line and point spread functions (LSF and PSF), and were found to be better than 0.79 μm ± 0.03 μm (STD)(45% wider than the diffraction limit) and 10.8 μm ± 0.7 μm (STD)(two times the diffraction limit), respectively. The axial positional accuracy was estimated to be 0.36 μm. This resolution and positional accuracy has allowed us to classify many ganglion cell types, such as bistratified ganglion cells, in vivo.
doi:10.1364/BOE.3.000715
PMCID: PMC3345801  PMID: 22574260
(170.4460) Ophthalmic optics and devices; (110.1080) Active or adaptive optics; (330.7324) Visual optics, comparative animal models
6.  Chloroquine-induced autophagic vacuole accumulation and cell death in glioma cells is p53 independent 
Neuro-Oncology  2010;12(5):473-481.
Glioblastoma (GBM) is a high-grade central nervous system malignancy and despite aggressive treatment strategies, GBM patients have a median survival time of just 1 year. Chloroquine (CQ), an antimalarial lysosomotropic agent, has been identified as a potential adjuvant in the treatment regimen of GBMs. However, the mechanism of CQ-induced tumor cell death is poorly defined. We and others have shown that CQ-mediated cell death may be p53-dependent and at least in part due to the intrinsic apoptotic death pathway. Here, we investigated the effects of CQ on 5 established human GBM lines, differing in their p53 gene status. CQ was found to induce a concentration-dependent death in each of these cell lines. Although CQ treatment increased caspase-3–like enzymatic activity in all 5 cell lines, a broad-spectrum caspase inhibitor did not significantly attenuate death. Moreover, CQ caused an accumulation of autophagic vacuoles in all cell lines and was found to affect the levels and subcellular distribution of cathepsin D, suggesting that altered lysosomal function may also play a role in CQ-induced cell death. Thus, CQ can induce p53-independent death in gliomas that do not require caspase-mediated apoptosis. To potentially identify more potent chemotherapeutics, various CQ derivatives and lysosomotropic compounds were tested on the GBM cells. Quinacrine and mefloquine were found to be more potent than CQ in killing GBM cells in vitro and given their superior blood–brain barrier penetration compared with CQ may prove more efficacious as chemotherapeutic agents for GBM patients.
doi:10.1093/neuonc/nop048
PMCID: PMC2940627  PMID: 20406898
glioblastoma; chloroquine; autophagy; p53
7.  Optical properties of the mouse eye 
Biomedical Optics Express  2011;2(4):717-738.
The Shack-Hartmann wavefront sensor (SHWS) spots upon which ocular aberration measurements depend have poor quality in mice due to light reflected from multiple retinal layers. We have designed and implemented a SHWS that can favor light from a specific retinal layer and measured monochromatic aberrations in 20 eyes from 10 anesthetized C57BL/6J mice. Using this instrument, we show that mice are myopic, not hyperopic as is frequently reported. We have also measured longitudinal chromatic aberration (LCA) of the mouse eye and found that it follows predictions of the water-filled schematic mouse eye. Results indicate that the optical quality of the mouse eye assessed by measurement of its aberrations is remarkably good, better for retinal imaging than the human eye. The dilated mouse eye has a much larger numerical aperture (NA) than that of the dilated human eye (0.5 NA vs. 0.2 NA), but it has a similar amount of root mean square (RMS) higher order aberrations compared to the dilated human eye. These measurements predict that adaptive optics based on this method of wavefront sensing will provide improvements in retinal image quality and potentially two times higher lateral resolution than that in the human eye.
doi:10.1364/BOE.2.000717
PMCID: PMC3072116  PMID: 21483598
(170.4460) Medical optics and biotechnology: Ophthalmic optics and devices; (330.5370) Vision, color, and visual optics: Physiological optics; (330.4300) Vision system - noninvasive assessment; (110.1080) Active or adaptive optics; (330.7324) Vision, color, and visual optics: Visual optics, comparative animal models
8.  In vivo imaging of microscopic structures in the rat retina 
Purpose
The ability to resolve single retinal cells in rodents in vivo has applications in rodent models of the visual system and retinal disease. We have characterized the performance of a fluorescence adaptive optics scanning laser ophthalmoscope (fAOSLO) that provides cellular and subcellular imaging of rat retina in vivo.
Methods
Green fluorescent protein (eGFP) was expressed in retinal ganglion cells of normal Sprague Dawley rats via intravitreal injections of adeno-associated viral vectors. Simultaneous reflectance and fluorescence retinal images were acquired using the fAOSLO. fAOSLO resolution was characterized by comparing in vivo images with subsequent imaging of retinal sections from the same eyes using confocal microscopy.
Results
Retinal capillaries and eGFP-labeled ganglion cell bodies, dendrites, and axons were clearly resolved in vivo with adaptive optics (AO). AO correction reduced the total root mean square wavefront error, on average, from 0.30 μm to 0.05 μm (1.7-mm pupil). The full width at half maximum (FWHM) of the average in vivo line-spread function (LSF) was ∼1.84 μm, approximately 82% greater than the FWHM of the diffraction-limited LSF.
Conclusions
With perfect aberration compensation, the in vivo resolution in the rat eye could be ∼2× greater than that in the human eye due to its large numerical aperture (∼0.43). While the fAOSLO corrects a substantial fraction of the rat eye's aberrations, direct measurements of retinal image quality reveal some blur beyond that expected from diffraction. Nonetheless, subcellular features can be resolved, offering promise for using AO to investigate the rodent eye in vivo with high resolution.
doi:10.1167/iovs.09-3675
PMCID: PMC2873188  PMID: 19578019
9.  In-vivo imaging of retinal nerve fiber layer vasculature: imaging - histology comparison 
BMC Ophthalmology  2009;9:9.
Background
Although it has been suggested that alterations of nerve fiber layer vasculature may be involved in the etiology of eye diseases, including glaucoma, it has not been possible to examine this vasculature in-vivo. This report describes a novel imaging method, fluorescence adaptive optics (FAO) scanning laser ophthalmoscopy (SLO), that makes possible for the first time in-vivo imaging of this vasculature in the living macaque, comparing in-vivo and ex-vivo imaging of this vascular bed.
Methods
We injected sodium fluorescein intravenously in two macaque monkeys while imaging the retina with an FAO-SLO. An argon laser provided the 488 nm excitation source for fluorescence imaging. Reflectance images, obtained simultaneously with near infrared light, permitted precise surface registration of individual frames of the fluorescence imaging. In-vivo imaging was then compared to ex-vivo confocal microscopy of the same tissue.
Results
Superficial focus (innermost retina) at all depths within the NFL revealed a vasculature with extremely long capillaries, thin walls, little variation in caliber and parallel-linked structure oriented parallel to the NFL axons, typical of the radial peripapillary capillaries (RPCs). However, at a deeper focus beneath the NFL, (toward outer retina) the polygonal pattern typical of the ganglion cell layer (inner) and outer retinal vasculature was seen. These distinguishing patterns were also seen on histological examination of the same retinas. Furthermore, the thickness of the RPC beds and the caliber of individual RPCs determined by imaging closely matched that measured in histological sections.
Conclusion
This robust method demonstrates in-vivo, high-resolution, confocal imaging of the vasculature through the full thickness of the NFL in the living macaque, in precise agreement with histology. FAO provides a new tool to examine possible primary or secondary role of the nerve fiber layer vasculature in retinal vascular disorders and other eye diseases, such as glaucoma.
doi:10.1186/1471-2415-9-9
PMCID: PMC2744910  PMID: 19698151

Results 1-9 (9)