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Shuo Zhang;
Paul Lyuboslavsky;
Jendayi Azeezah Dixon;
Micah A. Chrenek;
Jana T. Sellers;
Jessica M. Hamm;
P Michael Iuvone;
Christophe P. Ribelayga;
Zhijing Zhang;
Yun Z. Le
PURPOSE. The present study tested the hypothesis that connexin-36 (Cx36) and gap junctions between photoreceptor cells contribute to the circadian rhythm of the photopic electroretinogram (ERG) b-wave amplitude. METHODS. Cone-specific disruption of Cx36 was obtained in mice with a floxed Gjd2 gene and human red/green pigment promoter (HRGP)-driven Cre recombinase. Standard ERG, spectral-domain optical coherence tomography (SD-OCT) and histochemical methods were used. RESULTS. HRGPcreGjd2fl/fl mice had a selective reduction in Cx36 protein in the outer plexiform layer; no reduction in Cx36 was observed in the inner plexiform layer. Cx36 disruption had no effect on the number of cones, the thickness of the photoreceptor layer, or the scotopic ERG responses. However, there was a reduction of the photopic ERG circadian rhythm, with b-wave amplitudes in the day and the night locked in the daytime, light-adapted state. In HRGPcreGjd2+/+ and Gjd2fl/fl controls, the circadian rhythm of light-adapted ERG persisted, similar to that in wild type mice. CONCLUSIONS. Cx36 regulation contributes to the circadian rhythm of light-adapted ERG; in the absence of photoreceptor gap junctions, mice appear to be in a fully light-adapted state regardless of the time of day. The higher amplitudes and reduced circadian regulation of the b-wave of HRGPcreGjd2fl/fl mice may be due to increased synaptic strength at the cone to ON bipolar cell synapse due to electrotonic isolation of the terminals lacking gap junctions.
Ocular injection (intravitreal, subretinal, or into the anterior space) is an efficient approach to deliver many classes of drugs, cells, and other treatments to various cell types of the eye. In particular, subretinal injection is efficient since delivered agents accumulate as there is no dilution due to transport processes or diffusion and because the volume of the interphotoreceptor space (IPS) is minimal (10–20 microliters in the human eye, less than 1 microliter in the mouse eye). We previously reported methods using subretinal injection and electroporation to deliver DNA to photoreceptor and retinal pigment epithelium (RPE) cells in retinas of live mice(1–3). Here we detail further optimization of that approach and additionally report its use in delivering DNA expression plasmids to the corneal endothelium.
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Kenkichi Baba;
Ilaria Piano;
Polina Lyuboslavsky;
Micah A. Chrenek;
Jana T. Sellers;
Shuo Zhang;
Claudia Gargini;
Li He;
Gianluca Tosini;
P Michael Iuvone
The mammalian retina contains an autonomous circadian clock system that controls many physiological functions within this tissue. Previous studies on young mice have reported that removal of the key circadian clock gene Bmal1 from the retina affects the circadian regulation of visual function, but does not affect photoreceptor viability. Because dysfunction in the circadian system is known to affect cell viability during aging in other systems, we compared the effect of Bmal1 removal from the retina on visual function, inner retinal structure, and photoreceptor viability in young (1 to 3 months) and aged (24 to 26 months) mice. We found that removal of Bmal1 from the retina significantly affects visual information processing in both rod and cone pathways, reduces the thickness of inner retinal nuclear and plexiform layers, accelerates the decline of visual functions during aging, and reduces the viability of cone photoreceptors. Our results thus suggest that circadian clock dysfunction, caused by genetic or other means, may contribute to the decline of visual function during development and aging.
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Nathaniel F. Henneman;
Stephanie L. Foster;
Micah A. Chrenek;
Jana T. Sellers;
Charles B. Wright;
Robin H. Schmidt;
John Nickerson;
Jeffrey Boatright
Purpose: To investigate whether treatment with xanthohumol (XN), the principal prenylated chalconoid from Humulus lupulus (hops), is protective in a mouse model of light-induced retinal degeneration (LIRD).
Methods: Mice (129S2/SvPasCrl) were intraperitoneally injected with vehicle or XN prior to toxic light exposure and every 3 days thereafter. Retinal function was assessed by electroretinograms at 1, 2, and 4 weeks following toxic light exposure. Visual acuity was tested by optokinetic tracking 1 week and 4 weeks after toxic light exposure. Retina sections were stained with hematoxylin and eosin for morphologic analysis or by TUNEL. Redox potentials were assessed in retinal tissue by measuring levels of cysteine (CYS), cystine (CYSS), glutathione (GSH), and glutathione disulfide (GSSG) using HPLC with fluorescence detection.
Results: Toxic light significantly suppressed retinal function and visual acuity, severely disrupted the photoreceptor cell layer, and significantly decreased the number of nuclei and increased the accumulation of TUNEL-labeled cells in the outer nuclear layer. These effects were prevented by XN treatment. Treatment with XN also maintained GSSG and CYSS redox potentials and the total CYS pool in retinas of mice undergoing toxic light exposure.
Conclusions: XN treatment partially preserved visual acuity and retinal function in the LIRD mouse. Preservation of retinal CYS and of GSSG and CYSS redox potentials may indicate that XN treatment induces an increased antioxidant response, but further experiments are needed to verify this potential mechanism. To our knowledge, this is the first study to report protective effects of XN in a model of retinal degeneration.
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Timothy Olsen;
Roy B. Dyer;
Fukutaro Mano;
Jeffrey Boatright;
Micah A. Chrenek;
Daniel Paley;
Kathy Wabner;
Jenn Schmit;
Ju Byung Chae;
Jana T. Sellers;
Ravinder J. Singh;
Timothy S. Wiedmann
Purpose
To determine local ocular tissue levels of the bile acid, tauroursodeoxycholic acid (TUDCA), in the pig model using oral, intravenous (IV), intravitreal injection (IVitI) and low- and high-dose suprachoroidal, sustained-release implants (SCI-L or SCI-H).
Methods
Forty-six pigs (92 globes) were included in the study. TUDCA was delivered orally in 5 pigs, IV in 4, IVitI in 6, SCI-L in 17, and SCI-H in 14. Testing timeframes varied from the same day (within minutes) for IV; 1 to 6 days, oral; and 1 to 4 weeks, IVitI and SCI. Enucleated globes were dissected, specimens from specific tissues were separated, and TUDCA was extracted and quantified using mass spectrometry.
Results
The highest TUDCA tissue levels occurred after IV delivery in the macula (252 ± 238 nM) and peripheral retina (196 ± 171 nM). Macular choroid and peripheral choroid levels were also high (1032 ± 1269 and 1219 ± 1486 nM, respectively). For IVitI delivery, macular levels at day 6 were low (0.5 ± 0.5 nM), whereas peripheral choroid was higher (15.3 ± 16.7 nM). Neither the SCI-L nor SCI-H implants delivered meaningful macular doses (≤1 nM); however, peripheral retina and choroid levels were significantly higher. Bile acid isoforms were found in the serum specimens.
Conclusions
The highest TUDCA tissue levels in the pig model were obtained using IV delivery. Oral delivery was associated with reasonable tissue levels. Local delivery (IVitI and SCI) was able to achieve measurable local ocular tissue levels.
Translational Relevance
Diffusional kinetics from the suprachoroidal space follow the choroidal blood flow, away from the macula and toward the periphery.
Purpose: We previously reported that modest running exercise protects photoreceptors in mice undergoing light-induced retinal degeneration and in the rd10 mouse model of autosomal recessive retinitis pigmentosa (arRP). We hypothesized that exercise would protect against other types of retinal degeneration, specifically, in autosomal dominant inherited disease. We tested whether voluntary running wheel exercise is protective in a retinal degeneration mouse model of class B1 autosomal dominant RP (adRP). Methods: C57BL/6J mice heterozygous for the mutation in I307N rhodopsin (Rho) (also known as RHOTvrm4/+, or Tvrm4) are normal until exposed to brief but bright light, whereupon rod photoreceptor degeneration ensues. I307N Rho mice were given access to free spinning (active) or locked (inactive) running wheels. Five weeks later, half of each cohort was treated with 0.2% atropine eye drops and exposed to white LED light (6,000 lux) for 5 min, then returned to maintenance housing with wheels. At 1 week or 4 weeks after induction, retinal and visual function was assessed with electroretinogram (ERG) and optomotor response (OMR). In vivo retinal morphology was assessed with optical coherence tomography (OCT), and fundus blue autofluorescence assessed using a scanning laser ophthalmoscope. The mice were then euthanized, and the eyes fixed for paraffin sectioning or flatmounting. The paraffin sections were stained with hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) to assess retina morphology and apoptosis. Half of the flatmounts were stained for ZO-1 and α-catenin to assess RPE cell structure and stress. (We previously reported that translocation of α-catenin from cell membranes into the cytosol indicates RPE cell stress.) The remaining flatmounts were stained for ZO-1 and Iba-1 to assess the RPE cell size and shape, and inflammatory responses. Results: In vivo measures revealed that induction of the I307N Rho degeneration decreased retinal and visual function, decreased the thickness of the retina and photoreceptor layers, and increased the number of blue autofluorescence spots at the level of the photoreceptor-RPE interface. Post-mortem analyses showed that induction caused loss of photoreceptors in the central retinal region, and increased TUNEL labeling in the outer nuclear layer (ONL). The RPE was disrupted 1 week after induction, with changes in cell size and shape accompanied by increased α-catenin translocation and Iba-1 staining. These outcomes were partially but statistically significantly prevented in the exercised mice. The exercised mice that underwent induced I307N Rho degeneration exhibited retinal function and visual function measures that were statistically indistinguishable from that of the uninduced mice, and compared to the unexercised induced mice, had thicker retina and photoreceptor layers, and decreased numbers of subretinal autofluorescent spots. Post-mortem, the retina sections from the exercised mice that had undergone induced I307N Rho degeneration exhibited numbers of photoreceptors that were statistically indistinguishable from those of uninduced mice. Similarly, exercise largely precluded a degeneration-induced increase in TUNEL-positive cells in the ONL. Finally, the RPE of the exercised mice appeared normal, with a regular cell shape and size, and little to no alpha-catenin translocation or Iba-1 immunosignal. Conclusions: Voluntary wheel running partially protected against retinal degeneration and inflammation, and RPE disruption in a model of inducible adRP. This is the first report of exercise protection in an adult adRP animal model. It is also the first report of an RPE phenotype in the I307N Rho mouse. These findings add to a growing literature reporting that modest whole-body exercise is protective across a wide range of models of retinal damage and disease, and further highlights the potential for this accessible and inexpensive therapeutic intervention in the ophthalmic clinic.
Glaucoma etiology often includes retinal ganglion cell (RGC) death associated with elevated intraocular pressure (IOP). However, even when IOP is managed well, disease can progress. It is thus important to develop therapeutic approaches that directly protect RGCs in an IOP-independent manner. Compromised nicotinamide adenine dinucleotide (NAD+) metabolism occurs in neurode-generative diseases, including models of glaucoma. Here we report testing the protective effects of prophylactically systemically administered nicotinamide riboside (NR), a NAD+ precursor, in a mouse model of acute RGC damage (optic nerve crush (ONC)), and in a chronic model of RGC degeneration (ocular hypertension induced by intracameral injection of microbeads). For both models, treatment enhanced RGC survival, assessed by counting cells in retinal flatmounts immunostained for Brn3a+. In the ONC model, treatment preserved RGC function, as assessed by pattern elec-troretinogram, and suppressed retinal inflammation, as assessed by immunofluorescence staining of retinal fixed sections for glial fibrillary acidic protein (GFAP). This is the first study to demonstrate that systemic treatment with NR is protective in acute and chronic models of RGC damage. The protection is significant and, considering that NR is highly bioavailable in and well-tolerated by humans, may support the proposition of prospective human subject studies.
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Salma Ferdous;
Kristie L. Liao;
Isabelle D. Gefke;
Vivian R. Summers;
Wenfei Wu;
Kevin J Donaldson;
Yong-Kyu Kim;
Jana T. Sellers;
Jendayi A. Dixon;
Debresha A. Shelton;
Shanu Markand;
Somin M. Kim;
Nan Zhang;
Jeffrey Boatright;
John Nickerson
PURPOSE. The purpose of this study was to extend our understanding of how aging affects normal retina function and morphology in wild-type C57BL/6J mice, by analyzing electrophysiological recordings and in vivo and post mortem anatomy. METHODS. Electroretinograms (ERGs), spectral domain optical coherence tomography (SD-OCT), and confocal scanning laser ophthalmoscope (cSLO) in vivo images were obtained from mice between the ages of 2 and 32 months in four groups: group 1 (<0.5 years), group 2 (1.0–1.5 years), group 3 (1.5–2.0 years), and group 4 (>2.0 years). Afterward, mouse bodies and eyes were weighed. Eyes were stained with hematoxylin and eosin (H&E) and cell nuclei were quantified. RESULTS. With aging, mice showed a significant reduction in both a- and b-wave ERG amplitudes in scotopic and photopic conditions. Additionally, total retina and outer nuclear layer (ONL) thickness, as measured by SD-OCT images, were significantly reduced in older groups. The cSLO images showed an increase in auto-fluorescence at the photoreceptor-RPE interface as age increases. H&E cell nuclei quantification showed significant reduction in the ONL in older ages, but no differences in the inner nuclear layer (INL) or ganglion cell layer (GCL). CONCLUSIONS. By using multiple age groups and extending the upper age limit of our animals to approximately 2.65 years (P970), we found that natural aging causes negative effects on retinal function and morphology in a gradual, rather than abrupt, process. Future studies should investigate the exact mechanisms that contribute to these gradual declines in order to discover pathways that could potentially serve as therapeutic targets.
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Xian Zhang;
Nathaniel F. Henneman;
Preston E. Girardot;
Jana T. Sellers;
Micah A. Chrenek;
Ying Li;
Jiaxing Wang;
Charles Brenner;
John Nickerson;
Jeffrey Boatright
Purpose
Maintaining levels of nicotinamide adenine dinucleotide (NAD+), a coenzyme critical for cellular energetics and biosynthetic pathways, may be therapeutic in retinal disease because retinal NAD+ levels decline during retinal damage and degeneration. The purpose of this study was to investigate whether systemic treatment with nicotinamide riboside (NR), a NAD+ precursor that is orally deliverable and well-tolerated by humans, is protective in a mouse model of light-induced retinal degeneration.
Methods
Mice were injected intraperitoneally with vehicle or NR the day before and the morning of exposure to degeneration-inducing levels of light. Retinal function was assessed by electroretinography and in vivo retinal morphology and inflammation was assessed by optical coherence tomography. Post mortem retina sections were assessed for morphology, TUNEL, and inflammatory markers Iba1 and GFAP. Retinal NAD+ levels were enzymatically assayed.
Results
Exposure to degeneration-inducing levels of light suppressed retinal NAD+ levels. Mice undergoing light-induced retinal degeneration exhibited significantly suppressed retinal function, severely disrupted photoreceptor cell layers, and increased apoptosis and inflammation in the outer retina. Treatment with NR increased levels of NAD+ in retina and prevented these deleterious outcomes.
Conclusions
This study is the first to report the protective effects of NR treatment in a mouse model of retinal degeneration. The positive outcomes, coupled with human tolerance to NR dosing, suggest that maintaining retinal NAD+ via systemic NR treatment should be further explored for clinical relevance.