A comparison of optophysiological biomarkers of photoreceptor stress and phototoxicity in BALB/cJ, B6 (Cg)-Tyrc-2J/J, and C57Bl/6J mouse strains.

Ophthalmic imaging instruments, including the confocal scanning laser ophthalmoscope and spectral-domain optical coherence tomography system, originally intended for revealing ocular microstructures in the human eye, have been deployed by vision researchers to evaluate the eyes of numerous small and large animal species for more than two decades. In this study, we have used these two instruments to obtain imaging data sequentially from the retinas of three prominent, widely used experimental mouse models to document changes induced by two contrasting vivarium lighting conditions. Mice studied include albino BALB/cJ and B6(Cg)-Tyrc-2J/J and pigmented C57Bl/6J. Mice were reared under dim light conditions until ~8 weeks of age where they underwent baseline imaging. Following, mice were returned to the dim vivarium or relocated to the top rack cage position in a standard vivarium. Mice were then followed for several months by ocular imaging to catalog the retinal dynamics as a function of long-term dim vs. elevated, standard vivarium lighting exposure levels. Upon exposure to elevated light levels, B6(Cg)-Tyrc-2J/J underwent similar changes as BALB/cJ in regard to photoreceptor outer segment shortening, photoreceptor layer proximal aspect hyperreflective changes, and the development of retinal infoldings and autofluorescent sub-retinal inflammatory monocyte infiltrate. Noteworthy, however, is that infoldings and infiltrate occurred at a slower rate of progression in B6(Cg)-Tyrc-2J/J vs. BALB/cJ. The photoreceptor outer nuclear layer thickness of BALB/cJ degenerated steadily following elevated light onset. In contrast, B6(Cg)-Tyrc-2J/J degeneration was unremarkable for many weeks before experiencing a noticeable change in the rate of degeneration that was concomitant with a plateau and decreasing trend in number of retinal infoldings and monocyte infiltrate. Pathological changes in C57Bl/6J mice were unremarkable for all imaging biomarkers assessed with exception to autofluorescent sub-retinal inflammatory monocyte infiltrate, which showed significant accumulation in dim vs. elevated light exposed mice following ~1 year of observation. These data were evaluated using Spearman's correlation and Predictive Power Score matrices to determine the best imaging optophysiological biomarkers for indicating vivarium light stress and light-induced photoreceptor degeneration. This study suggests that changes in proximal aspect hyperreflectivity, outer segment shortening, retinal infoldings and autofluorescent sub-retinal inflammatory monocyte infiltrate are excellent indicators of light stress and light-induced degeneration in albino B6(Cg)-Tyrc-2J/J and BALB/cJ mouse strains.

Deficiency of CC chemokine ligand 2 and decay-accelerating factor causes retinal degeneration in mice.

CC chemokine ligand 2 (CCL2) recruits macrophages to reduce inflammatory responses. Decay-accelerating factor (DAF) is a membrane regulator of the classical and alternative pathways of complement activation. In view of the link between complement genes and retinal diseases, we evaluated the retinal phenotype of C57BL/6J mice and mice lacking Ccl2 and/or Daf1 at 12 months of age, using scanning laser ophthalmoscopic imaging, electroretinography (ERG), histology, immunohistochemistry, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis. In comparison to C57BL/6J mice, mutant mice had an increased number of autofluorescent foci, with the greatest number in the Ccl2(-/-)/Daf1(-/-) retina. ERG amplitudes in Ccl2(-/-)/Daf1(-/-), Ccl2(-/-) and Daf1(-/-) mice were reduced, with the greatest reduction in Ccl2(-/-)/Daf1(-/-) mice. TUNEL-positive cells were not seen in C57BL/6J retina, but were prevalent in the outer and inner nuclear layers of Ccl2(-/-)Daf1(-/-) mice and were present at reduced density in Ccl2(-/-) or Daf1(-/-) mice. Cell loss was most pronounced in the outer and inner nuclear layers of Ccl2(-/-)/Daf1(-/-) mice. The levels of the endoplasmic reticulum chaperone GPR78 and transcription factor ATF4 were significantly increased in the Ccl2(-/-)/Daf1(-/-) retina. In comparison to the C57BL/6J retina, the phosphorylation of NF-κB p65, p38, ERK and JNK was significantly upregulated while SIRT1 was significantly downregulated in the Ccl2(-/-)/Daf1(-/-) retina. Our results suggest that loss of Ccl2 and Daf1 causes retinal neuronal death and degeneration which is related to increased endoplasmic reticulum stress, oxidative stress and inflammation.

The BALB/c mouse: Effect of standard vivarium lighting on retinal pathology during aging.

BALB/cJ mice housed under normal vivarium lighting conditions can exhibit profound retinal abnormalities, including retinal infoldings, autofluorescent inflammatory cells, and photoreceptor degeneration. To explore the sensitivity of the outer retina to cyclic lighting during aging, a cohort of BALB/cJ mice was evaluated with Scanning Laser Ophthalmoscopy (SLO), Spectral-Domain Optical Coherence Tomography (OCT) and conventional histopathology. Mice were bred and reared in a low-illuminance (extracage/intracage: 13 lx/1 lx) vivarium under cyclic light (14 h light: 10 h dark). Retinal imaging (around postnatal day 70) was performed to screen for any pre-existing abnormalities and to establish a baseline. Mice with normal retinas were separated into groups (A, B, C) and placed on bottom (Groups A & B) or top (Group C) of the cage racks where cage illumination was <10 & 150 lx respectively. Experimental groups B & C were imaged multiple times over a 17 month period. Mice from group A (controls) were imaged only once post-baseline at various times for comparison to groups B & C. Mice were assessed by histology at 8, 15, 20, 36, and 56 weeks and immunohistochemistry at 15 weeks post-baseline. SLO and OCT retinal images were measured and the resulting trends displayed as a function of age and light exposure. Retinal lesions (RL) and autofluorescent foci (AFF) were identified with histology as photoreceptor layer infoldings (IF) and localized microglia/macrophages (MM), respectively. Few RL and AFF were evident at baseline. Retinal infoldings were the earliest changes followed by subjacent punctate autofluorescent MM. The colocalization of IF and MM suggests a causal relationship. The incidence of these pathological features increased in all groups relative to baseline. OCT imaging revealed thinning of the outer nuclear layer (ONL) in all groups at 1 year relative to baseline. ONL thinning followed an exponential rate of change but the decay constant varied depending on intensity of illumination of the groups. Advanced age and top row illuminance conditions resulted in significant photoreceptor cell loss as judged by decreased thickness of the ONL. Photoreceptor loss was preceded by both retinal infoldings and the presence of autofluorescent inflammatory cells in the outer retina, suggesting that these changes are early indicators of light toxicity in the BALB/cJ mouse.

Retinal vasculature of adult zebrafish: in vivo imaging using confocal scanning laser ophthalmoscopy.

Over the past 3 decades the zebrafish (Danio rerio) has become an important biomedical research species. As their use continues to grow additional techniques and tools will be required to keep pace with ongoing research using this species. In this paper we describe a novel method for in vivo imaging of the retinal vasculature in adult animals using a commercially available confocal scanning laser ophthalmoscope (SLO). With this instrumentation, we demonstrate the ability to distinguish diverse vascular phenotypes in different transgenic GFP lines. In addition this technology allows repeated visualization of the vasculature in individual zebrafish over time to document vascular leakage progression and recovery induced by intraocular delivery of proteins that induce vascular permeability. SLO of the retinal vasculature was found to be highly informative, providing images of high contrast and resolution that were capable of resolving individual vascular endothelial cells. Finally, the procedures required to acquire SLO images from zebrafish are non-invasive, simple to perform and can be achieved with low animal mortality, allowing repeated imaging of individual fish.

Retinal regeneration following OCT-guided laser injury in zebrafish.

PURPOSE: Establish a focal injury/regeneration model in zebrafish using laser photocoagulation guided by optical coherence tomography (OCT). METHODS: Adult zebrafish were imaged by OCT and confocal scanning laser ophthalmoscopy (cSLO) in room air through a contact lens. Using a beam combiner, 532-nm laser photocoagulation was applied using the OCT C-scan image for targeting. Laser spots of 42 to 47 mW were delivered to the retina. At multiple intervals post injury, fish were imaged using both OCT and cSLO to follow the progression of each lesion. Histologic sections and TUNEL staining were performed to monitor the injury response. RESULTS: Round lesions (26057 ± 621 µm(2)) localized to the outer retina were successfully applied. Laser application was visualized by real-time OCT and lesions were detectable by both OCT and cSLO in vivo. Lesion size increased 1 day post lesion then decreased in size. Histologic sections showed focal areas of damage localized primarily to the outer retina. By 3 weeks, the damaged areas had regenerated and a fully laminated structure was re-established. However, subtle changes can still be detected by OCT, cSLO imaging, and histology. Infrared darkfield imaging was more sensitive than OCT at revealing subtle changes in regenerated areas. CONCLUSIONS: Optical coherence tomography-guided laser photocoagulation is a useful tool for inducing localized lesions and studying retinal regeneration in zebrafish. This novel method will allow us to characterize the cellular and molecular changes that take place at the interface between normal and damaged tissue. Regeneration can be observed using high-resolution OCT and cSLO imaging in vivo.